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Important Notice
● Product labels/package inserts take precedence over the formula or instructions listed in the manual.
● Generic names may be substituted for trade names in the ingredient list, providing more information
regarding animal origin, i.e. “pancreatic digest of casein” instead of Casitone.
● During 1999, catalog numbers will be changed to meet new UCC/EAN128 labeling requirements.
Please visit us at www.bd.com/microbiology for more information.
Table of Contents
Introduction.................................................................................................ix
Monographs .................................................................................................1
Copyright 1998 by
Difco Laboratories,
Division of Becton Dickinson and Company
Sparks, Maryland 21152 USA
Foreword
This edition of the DIFCO MANUAL, the eleventh published since 1927, has been extensively revised and
rewritten. The purpose of the Manual is to provide information about products used in microbiology. The
Manual has never been intended to replace any official compendium or the many excellent standard text
books of scientific organizations or individual authors.
Difco is perhaps best known as the pioneer in bacteriological culture media. Numerous times one will find
the trademarks Difco® or Bacto® preceding the names of materials used by scientists in their published
papers. Because Difco products have been readily available worldwide longer than any others, Difco
products have become the common-language reagents of the microbiological community. Standardized
products readily available worldwide are essential for corroborative studies demanded by rigorous science.
Recommendation and approval have been extended to our products by the authors of many standard text
books and by the committees on methods and procedures of scientific societies throughout the world. Difco
products continue to be prepared according to applicable standards or accepted formulae. It is expected that
they will be used only by or under the supervision of microbiologists or other professionals qualified by
training and experience to handle pathogenic microorganisms. Further, it is expected that the user will be
throughly familiar with the intended uses of the formulations and will follow the test procedures outlined in
the applicable official compendia and standard text books or procedures manual of the using laboratory.
Grateful acknowledgment is made of the support we have received from microbiologists throughout the
world. It is our desire to continue and extend our services to the advancement of microbiology and related
sciences.
Difco Laboratories
Division of Becton Dickinson and Company
Early mycologists, A. de Bary and O. Brefeld, and bacteriologist, Almost without exception whenever bacteria occur in nature, and this
R. Koch and J. Schroeter, pioneered investigations of pure culture is particularly true of the pathogenic forms, nitrogenous compounds
techniques for the colonial isolation of fungi and bacteria on solid and carbohydrates are present. These are utilized in the maintenance
media. Koch, utilizing state-of-the-art clear liquid media which he of growth and for the furtherance of bacterial activities. So complex is
solidified with gelatin, developed both streak and pour plate methods the structure of many of these substances, however, that before they
for isolating bacteria. Gelatin was soon replaced with agar, a solidifying can be utilized by bacteria they must be dissimilated into simpler
agent from red algae. It was far superior to gelatin in that it was compounds then assimilated into cellular material. Such metabolic
resistant to microbial digestion and liquefaction. alterations are affected by enzymatic processes of hydrolysis, oxidation,
reduction, deamination, etc., and are the result of bacterial activities of
The capability of Koch to isolate disease-producing bacteria on solidi- primary and essential importance. These changes are ascribed to the
fied culture media was further advanced by manipulating the cultural activity of bacterial enzymes which are both numerous and varied. The
environment using meat extracts and infusions so as to reproduce, as processes involved, as well as their end-products, are exceedingly
closely as possible, the infected host’s tissue. The decade immediately complex; those of fermentation, for example, result in the production
following Koch’s epoch-making introduction of solid culture media of such end-products as acids, alcohols, ketones, and gases including
for the isolation and growth of bacteria ranks as one of the brightest in hydrogen, carbon dioxide, methane, etc. The study of bacterial
the history of medicine because of the number, variety, and brilliance of metabolism, which defines the organized chemical activities of a cell,
the discoveries made in that period. These discoveries, which, as Koch has led to the understanding of both catabolic or degradative activities
himself expressed it, came “as easily as ripe apples fall from a tree,” and anabolic or synthetic activities. From these studies has come a
were all dependent upon and resulted from the evolution of correct better understanding of the nutritional requirements of bacteria, and in
methods for the in vitro cultivation of bacteria. turn, the development of culture media capable of producing rapid and
The fundamental principles of pure culture isolation and propagation luxuriant growth, both essential requisites for the isolation and study
still constitute the foundation of microbiological practice and research. of specific organisms.
Nevertheless, it has become more and more apparent that a successful Studies to determine the forms of carbon, hydrogen, and nitrogen which
attack upon problems unsolved is closely related to, if not dependent could most easily be utilized by bacteria for their development were
upon, a thorough understanding of the subtle factors influencing originally carried on by Naegeli1 between 1868 and 1880, and were
bacterial metabolism. With a suitable culture medium, properly published by him in the latter year. Naegeli’s report covered the use of
used, advances in microbiology are more readily made than when a large variety of substances including carbohydrates, alcohols, amino
either the medium or method of use is inadequate. The microbiologist acids, organic nitrogen compounds, and inorganic nitrogen salts.
of today is, therefore, largely concerned with the evolution of methods
for the development and maintenance of microbial growth upon which The first reference to the use of peptone for the cultivation of microor-
an understanding of their unique and diversified biological and ganisms is that made by Naegeli in the report referred to above, when
biochemical characteristics can be investigated. To this end, microbi- in 1879, he compared peptone and ammonium tartrate. Because of its
ologists have developed innumerable enrichment culture techniques content amino acids and other nitrogenous compounds which are
for the isolation and cloning of microorganisms with specific nutri- readily utilized by bacteria, peptone soon became one of the most
tional requirements. These organisms and their unique characteristics important constituents of culture media, as it still remains. In the light
have been essential to progress in basic biological research and modern of our present knowledge, proteins are known to be complex compounds
applied microbiology. composed of amino acids joined together by means of the covalent
peptide bond linkage. When subjected to hydrolysis, proteins yield
The study of microorganisms is not easy using microscopic single cells. polypeptides of various molecular sizes, metapeptones, proteoses,
It is general practice to study pure cultures of a single cell type. In the peptones and peptides, down to the level of simple amino acids. The
laboratory, microbiological culture media are utilized which contain intermediate products should be considered as classes of compounds,
various nutrients that favor the growth of particular microorganisms in rather than individual substances, for there exists no sharp lines of
pure cultures. These media may be of simple and defined chemical demarcation between the various classes. One group shades by
composition or may contain complex ingredients such as digests of imperceptible degrees into the next. All bacteriological peptones, thus,
plant and animal tissue. In particular, the cultivation of bacteria is are mixtures of various products of protein hydrolysis. Not all the
products of protein decomposition are equally utilizable by all media. Bushby and Hitchings35 have shown that the antimicrobial ac-
bacteria. In their relation to proteins, bacteria may be divided into two tivities of trimethoprim and sulfamethoxazole are influenced consider-
classes; those which decompose naturally occurring proteins, and those ably by the thymine and thymidine found in peptones of culture media.
which require simpler nitrogenous compounds such as peptones and In this brief discussion of certain phases of bacterial nutrition, we have
amino acids. attempted to indicate the complexity of the subject and to emphasize
The relation of amino acids to bacterial metabolism, and the ability of the importance of continued study of bacterial nutrition. Difco Labo-
bacteria to use these compounds, have been studied by many workers. ratories has been engaged in research closely allied to this problem in
Duval,2,3 for example, reports that cysteine and leucine are essential in its broader aspects since 1914 when Bacto Peptone was first introduced.
the cultivation of Mycobacterium leprae. Kendall, Walker and Day4 Difco dehydrated culture media, and ingredients of such media, have
and Long5 reported that the growth of M. tuberculosis is dependent won universal acceptance as useful and dependable laboratory adjuncts
upon the presence of amino acids. Many other workers have studied the in all fields of microbiology.
relation of amino acids to the growth of other organisms, as for example,
Hall, Campbell, and Hiles6 to the meningococcus and Streptococcus; References
Cole and Lloyd7 and Cole and Onslow8 to the gonococcus; and Jacoby 1. Sitz’ber, math-physik. Klasse Akad. Wiss. Muenchen, 10:277,
and Frankenthal9 to the influenza bacillus. More recently Feeley, et 1880.
al. 34 demonstrated that the nonsporeforming aerobe, Legionella 2. J. Exp. Med., 12:46, 1910.
pneumophila requires L-cysteine . HCI for growth on laboratory media. 3. J. Exp. Med., 13:365, 1911.
Indispensable as amino acids are to the growth of many organisms,
4. J. Infectious Diseases, 15:455, 1914.
certain of them in sufficient concentration may exert an inhibitory
effect upon bacterial development. 5. Am. Rev. Tuberculosis, 3:86, 1919.
6. Brit. Med. J., 2:398, 1918.
From the data thus far summarized, it is apparent that the problem
of bacterial metabolism is indeed complicated, and that the phase 7. J. Path. Bact., 21:267, 1917.
concerned with bacterial growth and nutrition is of the utmost practical 8. Lancet, II:9, 1916.
importance. It is not improbable that bacteriological discoveries such 9. Biochem, Zelt, 122:100, 1921.
as those with Legionella pneumophila await merely the evolution of 10. Centr. Bakt., 1:29:617, 1901.
suitable culture media and methods of utilizing them, just as in the past
11. Indian J. Med. Research, 5:408, 1917-18.
important discoveries were long delayed because of a lack of similar
requirements. Bacteriologists are therefore continuing to expend much 12. Compt. rend. soc. biol., 78:261, 1915.
energy on the elucidation of the variations in bacterial metabolism, 13. J. Bact., 25:209, 1933.
and are continuing to seek methods of applying, in a practical way, the 14. Ann. de L’Inst., Pasteur, 12:26, 1898.
results of their studies. 15. Indian J. Med. Research, 7:536, 1920.
While the importance of nitrogenous substances for bacterial growth 16. Sperimentale, 72:291, 1918.
was recognized early in the development of bacteriological technique, 17. J. Med. Research, 43:61, 1922.
it was also realized, as has been indicated, that bacteria could not
18. Can. J. Pub. Health, 32:468, 1941.
always obtain their nitrogen requirements directly from protein. It
is highly desirable, in fact essential, to supply nitrogen in readily 19. Centr. Bakt., 1:77:108, 1916.
assimilable form, or in other words to incorporate in media proteins 20. J. Bact., 29:515, 1935.
which have already been partially broken down into their simpler and 21. Brit. J. Exp. Path., 27:335, 1936.
more readily utilizable components. Many laboratory methods, such 22. Brit. J. Exp. Path., 27:342, 1936.
as hydrolysis with alkali,10 acid,11,12,13 enzymatic digestion,8,14,15,16,17,18 23. J. Bact., 36:499, 1938.
and partial digestion of plasma10 have been described for the preparation
of protein hydrolysates. 24. J. Immunol., 37:103, 1939.
25. J. Immunol., 40:21, 1941.
The use of protein hydrolysates, particularly gelatln and casein, has
led to especially important studies related to bacterial toxins by 26. Proc. Soc. Expl. Biol. Med., 47:284, 1941.
Mueller, et al.20-25 on the production of diphtheria toxin; that of Tamura, 27. J. Immunol., 40:449, 1941.
et al.25 of toxin of Clostridium welchii; that of Bunney and Loerber27,28 28. J. Immunol., 40:459, 1941.
on scarlet fever toxin, and of Favorite and Hammon29 on Staphylococcus 29. J. Bact., 41:305, 1941.
enterotoxin. In addition, the work of Snell and Wright30 on the 30. J. Biol. Chem., 139:675, 1941.
microbiological assay of vitamins and amino acids was shown to
be dependent upon the type of protein hydrolysate utilized. Closely 31. J. Biol. Chem., 119:121, 1937.
associated with research on this nature are such studies as those of 32. J. Bact., 34:163, 1940.
Mueller31,32 on pimelic acid as a growth factor for Corynebacterium 33. J. Bact., 41:441, 1941.
diphtheriae, and those of O’Kane33 on synthesis of riboflavin by 34. J. Clin. Microbiol., 8:320, 1978.
staphylococci. More recently, the standardization of antibiotic suscep- 35. Brit. J. Pharmacol., 33:742, 1968.
tibility testing has been shown to be influenced by peptones of culture
demonstrated that a virus could be grown in chick embryos and would With rapid advances in technologies and instrumentation, the basic
lose its ability to cause disease after successive generations. Using culture media and ingredients listed in this Manual remain some of the
this technique, Salk developed the polio vaccine. most reliable and cost effective tools in microbiology today.
One organism that has made a great contribution to molecular
biology is Escherichia coli. In 1973, Herbert Boyer and Stanley Cohen
References
produced recombinant DNA through plasmid transformation. 1. Marti-Ibanez, F. 1962. Baroque medicine, p. 185-195. In
The researchers found that the foreign gene not only survived, but F. Marti-Ibanez (ed.). The epic of medicine. Clarkson N. Potter,
copied the genetic material. This study and similar others started a Inc., New York, N.Y.
biotechnology revolution that has gained momentum over the years. 2. Wainwright, M., and J. Lederberg. 1992. History of
In the 1980s, instrumentation entered the microbiology laboratory. microbiology, p. 419-437. In J. Lederberg (ed.), Encyclopedia of
Manual procedures could be replaced by fully automated instruments microbiology, vol 2. Academic Press Inc., New York, N.Y.
for bacterial identification, susceptibility testing and blood culture
procedures. Immunoassays and probe technologies are broadening the
capabilities of the microbiologist.
Selective Agents include Bile Salts, dyes and antimicrobial agents. • Absorption of the oxygen by chemicals;
Bile Salts and desoxycholate are selective for the isolation of • Inoculation into the deep layers of solid media or under a layer
gram-negative microorganisms, inhibiting gram-positive cocci. of oil in liquid media.
Dyes and indicators are essential in the preparation of differential and Many microorganisms require an environment of 5-10% CO2. Levels
selective culture media. In these formulations, dyes act as bacteriostatic greater than 10% are often inhibitory due to a decrease in pH as
agents, inhibitors of growth or indicators of changes in acidity or carbonic acid forms. Culture media vary in their susceptibility to form
alkalinity of the substrate. toxic oxidation products if exposed to light and air.
Antimicrobial agents are used in media to inhibit the growth of bacteria, Water Activity
yeasts and fungi. Proper moisture conditions are necessary for continued luxuriant
Solidifying agents, including agar, gelatin and albumin, can be added growth of microorganisms. Organisms require an aqueous environment
to a liquid medium in order to change the consistency to a solid and must have “free” water. “Free” water is not bound in complex
or semisolid state. structure and is necessary for transfer of nutrients and toxic waste
products. Evaporation during incubation or storage results in loss of
“free” water and reduction of colony size or total inhibition of
Environmental Factors in Culture Media
organism growth.
Atmosphere
Most bacteria are capable of growth under ordinary conditions of oxygen
Protective Agents and Growth Factors
tension. Obligate aerobes require the free admission of oxygen, while Calcium carbonate, soluble starch and charcoal are examples of
anaerobes grow only in the absence of atmospheric oxygen. Between protective agents used in culture media to neutralize and absorb toxic
these two groups are the microaerophiles, which develop best under metabolites produced by bacterial growth.
partial anaerobic conditions, and the facultative anaerobes, which are NAD (V factor) and hemin (X factor) are growth factors required by
capable of growing in the presence or absence of oxygen. Anaerobic certain bacteria; e.g., Haemophilus species, and for enhanced growth
conditions for growth of microorganisms are obtained in a number of ways: of Neisseria species.
• Addition of small amounts of agar to liquid media; Surfactants, including Tween® 80, lower the interfacial tension around
• Addition of fresh tissue to the medium; bacteria suspended in the medium. This activity permits more rapid
• Addition of a reducing substance to the medium; e.g., sodium entry of desired compounds into the bacterial cell and can increase
thioglycollate, thioglycollic acid and L-cystine; bacterial growth.
• Displacement of the air by carbon dioxide;
trapped in the frozen water. The ice is then washed from the agar,
eliminating the contaminants. The Ice Agar process results in greater
consistency and freedom from interposing contaminants when used
in microbiological procedures.
Product Applications
Bacto Agar is optimized for beneficial calcium and magnesium
content. Detrimental ions such as iron and copper are reduced. Bacto
Agar is recommended for clinical applications, auxotrophic studies,
bacterial and yeast transformation studies and bacterial molecular
genetics applications.7,8
Agar Flake is recommended for use in general bacteriological
purposes. The quality is similar to Bacto Agar. The flakes are more
wettable than the granules found in Bacto Agar.
Agar is derived from a group of red-purple marine algae as pictured above. Agar Granulated is qualified to grow recombinant strains of
Escherichia coli (HB101) and Saccharomyces cerevisiae. Agar
Granulated may be used for general bacteriological purposes where
For Difco Agars, Gelidium is the preferred source of agar. The most clarity is not a strict requirement. This agar was developed to
address the special needs of the Biotechnology Industry for large
important properties of agar are:5
scale applications.
• Good transparency in solid and gel forms to allow identification
of colony type; Noble Agar is the purest form of Difco agar. It is washed extensively
and bleached to remove extraneous material. The result is a white
• Consistent lot-to-lot gel strength that is sufficient to withstand
powder in dry form, clear and colorless in solution and when solidified
the rigors of streaking but not so stiff that it affects diffusion
in plates. This agar is suitable for immunodiffusion studies, for use in
characteristics;
some electrophoretic applications and as a substrate for mammalian
• Consistent gelling (32-40°C) and melting (approximately and plant tissue culture.
85°C) temperatures, a property known as hysteresis;
Agar Technical is suitable for many general bacteriological
• Essential freedom from metabolically useful chemicals such as applications. This agar is not as highly processed as other Difco agars
peptides, proteins and fermentable hydrocarbons; and has lower technical specifications. This agar is not recommended
• Low and regular content of electronegative groups that could for growth of fastidious organisms.
cause differences in diffusion of electropositive molecules
(e.g., antibiotics, nutrients); References
• Freedom from toxic substances (bacterial inhibitors); 1. C. K. Tsend. 1946. In J. Alexander (ed.). 6:630. Colloid
• Freedom from hemolytic substances that might interfere with Chemistry. Reinhold Publishing Corp., New York, N. Y.
normal hemolytic reactions in culture media; 2. Selby, H. H., and T. A. Selby. 1959. Agar. In Whister (ed.).,
• Freedom from contamination by thermophilic spores. Industrial gums. Academic Press Inc., New York, NY.
3. Hitchens, A. P., and M. C. Leikind. 1939. The introduction
Agars are normally used in final concentrations of 1-2% for
of agar-agar into bacteriology. J. Bacteriol. 37:485-493.
solidifying culture media. Smaller quantities of agar (0.05-0.5%) are
used in culture media for motility studies (0.5% w/v) and growth 4. Koch, R. 1882. Die Atiologie der Turberkulose. Berl. Klin.
of anaerobes (0.1%) and microaerophiles.2 Wochenschr. 19:221- 230.
5. Armisen, R. 1991. Agar and agarose biotechnological applications.
The Manufacturing Process Hydrobiol. 221:157-166.
Difco Laboratories selects the finest Gelidium marine algae from world 6. Hesse, W. 1894. Uber die quantitative Bestimmung der in der
sources and requires algae harvested from water where the tempera- Luft enthaltenen Mikroorganismen. Mitt. a. d. Kaiserl. Gesh.
ture is both constant and temperate. Bacto Agar and Agar Granulated Berlin 2:182-207.
are produced from an Ice Agar purification process. Agar is insoluble 7. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular
in cold water but is colloidally dispersible in water above 90°C.2 When cloning, a laboratory manual, 2nd ed. Cold Spring Harbor
an agar gel is frozen, the agar skeleton contracts toward the center of Laboratory Press, New York, N.Y.
the mass as a membrane, leaving ice as a separate phase.2 8. Schiestl, R. H., and R. D. Geitz. 1989. High efficiency
Through a variety of processes, the agar is extracted from the Gelidium, transformation of intact yeast cells using single stranded nucleic
resulting in a liquid agar that is purified. The liquid agar is first gelled acids as a carrier. Current Genetics 16:339-346.
and then frozen, causing the soluble and suspended contaminants to be
A typical analysis was performed on Difco peptones and hydrolysates cocci. Bile is derived from the liver. The liver detoxifies bile salts by
to aid in the selection of products for research or production conjugating them to glycine or taurine. A bile salt is the sodium salt of
needs when specific nutritional characteristics are required. The a conjugated bile acid. Bile Salts and Bile Salts No. 3 contain bile
specifications for the typical analysis include: extract standardized to provide inhibitory properties for selective
• Physical characteristics media. Bile Salts No. 3 is a modified fraction of bile acid salts,
providing a refined bile salt. Bile Salts No. 3 is effective at less than
• Nitrogen content
one-third concentration of Bile Salts.
• Amino acids
• Inorganics Casamino Acids
• Vitamins Casamino Acids, Technical
Casamino Acids, Vitamin Assay
• Biological testing
Casamino Acids, Casamino Acids, Technical and Casamino Acids,
The quality of peptones and culture media ingredients is truly assessed
Vitamin Assay are derived from acid hydrolyzed casein. Casein is a
by their ability to support adequate growth of various microorganisms
milk protein and a rich source of amino acid nitrogen. Casamino
when incorporated into the medium. 6 The nature of peptones,
Acids, Casamino Acids, Technical and Casamino Acids, Vitamin
infusions and extracts will then play a major role in the growth
Assay are added to media primarily because of their organic nitrogen
performance properties of the medium and, in turn, advance the
science of microbiology.6 and growth factor components; their inorganic components also play
a vital role.9 Casamino Acids is recommended for use with microbio-
Media Ingredients logical cultures that require a completely hydrolyzed protein as a
nitrogen source. In Casamino Acids, hydrolysis is carried on until all
Autolyzed Yeast the nitrogen in the casein is converted to amino acids or other com-
Autolyzed Yeast is a desiccated product containing both the soluble pounds of relative chemical simplicity. The hydrolysis of Casamino
and insoluble portions of autolyzed bakers’ yeast. Autolyzed Yeast is Acids, Technical is carried out as in the preparation of Casamino
recommended for the preparation of yeast supplements used in the Acids, but the sodium chloride and iron content have not been decreased
microbiological assay of riboflavin and pantothenic acid.7,8, Autolyzed to the same extent. Casamino Acids, Vitamin Assay is an acid digest of
Yeast provides vitamins, nitrogen, amino acids and carbon in casein specially treated to markedly reduce or eliminate certain
microbiological culture media. vitamins. It is recommended for use in microbiological assay media
and in growth promotion studies.
Beef
Beef Heart for Infusion Casein Digest
Beef and Beef Heart for Infusion provide nitrogen, amino acids and Casein Digest is an enzymatic digest of casein, providing a distinct
vitamins in microbiological culture media. Beef is desiccated, source of amino acids for molecular genetics media. Casein Digest is
powdered, fresh lean beef, prepared especially for use in beef infusion used as a nitrogen and amino acid source for microbiological culture
media. Large quantities of beef are processed at one time to secure a media. Casein Digest is similar to N-Z Amine A. This product is
uniform and homogenous product. Beef Heart for Infusion is prepared
digested under conditions different from other enzymatic digests
from fresh beef heart tissue and is recommended for preparing heart
of casein, including Tryptone and Casitone.
infusion media. Beef Heart for Infusion is processed from large
volumes of raw material, retaining all the nutritive and growth Casitone
stimulating properties of fresh tissues.
Casitone is a pancreatic digest of casein. Casitone is recommended
Beef Extract for preparing media where an enzymatic hydrolyzed casein is
Beef Extract, Desiccated desired. Casein is a rich source of amino acid nitrogen. This product
Beef Extract and Beef Extract, Desiccated are replacements for is used to support the growth of fastidious microorganisms and its
infusion of meat. Beef Extract and Beef Extract, Desiccated provide high tryptophan content makes it valuable for detecting indole
nitrogen, vitamins, amino acids and carbon in microbiological culture production.
media. Beef Extract is standard in composition and reaction and
generally used to replace infusion of meat. In culture media, Beef Fish Peptone No. 1
Extract is usually employed in concentration of 0.3%. Beef Extract, Fish Peptone No. 1 is a non-mammalian, non-animal peptone used
Desiccated, the dried form of Beef Extract, was developed to provide a as a nitrogen source in microbiological culture media. Fish Peptone
product for ease of use in handling. Beef Extract is in the paste form. No. 1 is a non-bovine origin peptone, to reduce Bovine Spongiform
The products are to be used in a 1 for 1 substitution. Encephalopathy (BSE) risk. This peptone was developed by Difco
Laboratories for pharmaceutical and vaccine production and can
Bile Salts replace any peptone, depending on the organism and production
Bile Salts No. 3 application.
Bile Salts and Bile Salts No. 3 are used as selective agents for the
isolation of gram-negative microorganisms, inhibiting gram-positive
Gelatin Peptamin
Gelatin is a protein of uniform molecular constitution derived chiefly Peptamin, referred to as Peptic Digest of Animal Tissue, complies
by the hydrolysis of collagen.10 Collagens are a class of albuminoids with the US Pharmacopeia XXIII (USP). 11 Peptamin provides
found abundantly in bones, skin, tendon, cartilage and similar tissues nitrogen, amino acids, vitamins and carbon in microbiological culture
of animals. 10 Gelatin is used in culture media to detect gelatin media. Diluting and rinsing solutions, Fluid A and Fluid D, contain
liquifaction by bacteria and as a nitrogen and amino acid source. 0.1% Peptamin.
Soytone References
Soytone is an enzymatic digest of soybean meal. The nitrogen source 1. Nash, P., and M. M. Krenz. 1991. Culture Media, p. 1226-1288.
in Soytone contains the naturally occurring high concentrations of vi- In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg,
tamins and carbohydrates of soybean. and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed.
American Society for Microbiology, Washington, D.C.
TC Lactalbumin Hydrolysate
2. De Feo, J. 1986. Properties and applications of hydrolyzed
TC Yeastolate
proteins. ABL. July/August, 44-47.
TC Lactalbumin Hydrolysate is an enzymatic digest of lactalbumin for
3. Naegeli. 1880. Sitz’ber, math-physik. Klasse Akad. Wiss.
use as an enrichment in tissue culture media. Lactalbumin is a protein
Muenchen. 10:277.
derived after removal of casein from milk. TC Yeastolate is a
desiccated, clarified, water soluble portion of autolyzed fresh yeast 4. Klinger, I. J. 1917. The effect of hydrogen ion concentration on
prepared and certified for use in tissue culture procedures. the production of precipitates in a solution of peptone and its
TC Yeastolate is a source of vitamin B complex. relation to the nutritive value of media. J. Bacteriol. 2:351-353.
5. Bridson, E. Y. 1990. Media in microbiology. Rev. Med. Microbiol.
Tryptone Peptone 1:1-9.
Tryptone Peptone is a pancreatic digest of casein used as a nitrogen 6. Alvarez, R. J., and M. Nichols. 1982. Formulating microbio-
source in culture media. Casein is the main protein of milk and is a rich logical culture media-a careful balance between science and art.
source of amino acid nitrogen. Tryptone Peptone is rich in tryptophan, Dairy Food Sanitation 2:356- 359.
making it valuable for use in detecting indole production.12 The ab- 7. J. Ind. Eng. Chem., Anal. Ed. 1941. 13:567.
sence of detectable levels of carbohydrates in Tryptone Peptone makes
it a suitable peptone in differentiating bacteria on the basis of their 8. J. Ind. Eng. Chem., Anal. Ed. 1942. 14:909.
ability to ferment various carbohydrates. 9. Nolan, R. A., and W. G. Nolan. 1972. Elemental analysis of
vitamin-free casamino acids. Appl. Microbiol. 24:290-291.
Tryptose 10. Gershenfeld, L., and L. F. Tice. 1941. Gelatin for bacteriological
Tryptose is a mixed enzymatic hydrolysate with distinctive nutritional use. J. Bacteriol. 41:645-652.
properties. The digestive process of Tryptose results in assorted 11. United States Pharmacopeial Convention. 1995. The United
peptides, including those of higher molecular weight. Tryptose was States pharmacopeia, 23rd ed. The United States Pharmacopeial
originally developed as a peptone particularly adapted to the growth Convention. Rockville, MD.
requirements of Brucella.
12. J. Bacteriol. 1933. 25:623.
Yeast Extract
Yeast Extract, Technical
Yeast Extract and Yeast Extract, Technical are water soluble portions
of autolyzed yeast containing vitamin B complex. Yeast Extract is
an excellent stimulator of bacterial growth and used in culture media.
The autolysis is carefully controlled to preserve the naturally
occurring B-complex vitamins. Yeast Extract is generally employed
in the concentration of 0.3-0.5%, with improved filterability at
20%. Yeast Extract, Technical is used in bacterial culture media when
a standardized yeast extract is not essential. Yeast Extract, Technical
was developed to demonstrate acceptable clarity and growth
promoting characteristics. Yeast Extract and Yeast Extract, Technical
also provide vitamins, nitrogen, amino acids and carbon in
microbiological culture media.
Media Preparation
The preparation of culture media from dehydrated media requires • Quantities of media in excess of two liters may require an
accuracy and attention to preparation. The following points are extended autoclave time to achieve sterilization. Longer
included to aid the user in successful and reproducible preparation sterilization cycles can cause nutrient concentration changes
of culture media. and generation of inhibitory substances.
Key
A Deteriorated Dehydrated Medium D Incorrect Weighing G Repeated Remelting
B Improperly Washed Glassware E Incomplete Mixing H Dilution by a Too Large Inoculum
C Impure Water F Overheating
2. Demonstrate that the critical control equipment and • Carmelization or darkening of the medium;
instrumentation are capable of operating within the prescribed • Loss of nutritive value;
parameters for the process equipment.
• Loss of selective or differential properties.
3. Perform replicate cycles representing the required operational
range of the equipment and employing actual or simulated There are certain media (e.g., Hektoen Enteric Agar and Violet Red
product. Demonstrate that the processes have been carried out Bile Agar) that should not be autoclaved. To dissolve these media
within the prescribed protocol limits and, finally, that the formulation, heat to boiling to dissolve completely. It is important
probability of microbial survival in the replicate processes to follow all label directions for each medium. Media supplements
completed is not greater than the prescribed limits. should be sterile and added aseptically to the sterilized medium,
usually at 45-55°C.
4. Monitor the validated process during routine operation.
Periodically as needed, requalify and recertify the equipment.
Dry Heat Sterilization1
5. Complete the protocols and document steps 1-4, above.
Dry heat is employed for materials such as metal instruments that could
For a complete discussion of process validation, refer to appropriate be corroded by moist heat, powders, ointments and dense materials
references. that are not readily penetrated by steam. Because dry heat is effective
Ensuring that the temperature is recorded correctly is vital. The only at considerably higher temperatures and longer times than moist
temperature must reach all parts of the load and be maintained for the heat, dry heat sterilization is restricted to those items that will
desired length of time. Recording thermometers are employed for the withstand higher temperatures. The dry heat time for sterilization is
chamber and thermocouples may be buried inside the load. 120 minutes at 160°C.
For best results when sterilizing culture media, plug tubes or flasks
of liquids with nonabsorbent cotton or cap loosely. Tubes should be
Chemical Sterilization1
placed in racks or packed loosely in baskets. Flasks should never be Chemical sterilization employs gaseous and liquid sterilants for
more than two-thirds full. It is important to not overload the autoclave certain medical and industrial instruments. The gases include ethylene
chamber and to place contents so that there is a free flow of steam oxide, formaldehyde and beta-propiolactone. The liquid sterilants
around the contents. After sterilizing liquids, the chamber pressure must include glutaraldehyde, hydrogen peroxide, peracetic acid, chlorine
be reduced slowly to atmospheric pressure. This allows the liquid to dioxide and formaldehyde. Chemical sterilization is not employed in
cool below the boiling point at atmospheric pressure before opening the preparation of culture media. For a complete discussion of this
the door to prevent the solution from boiling over. topic, consult appropriate references.
In autoclave operation, all of the air in the chamber must be expelled Radiation Sterilization1
and replaced by steam; otherwise, “hot spots” and “cold spots” will
Radiation sterilization is an optional treatment for heat-sensitive
occur. Pressure-temperature relations of a properly operated autoclave
materials. This includes ultraviolet light and ionizing radiation.
are shown in the table below.
Ultraviolet light is chemically active and causes excitation of atoms
Pressure-Temperature Relations in Autoclave 4 within the microbial cell, particularly the nucleic acids, producing
(Figures based on complete replacement of air by steam) lethal mutations. This action stops the organism from reproducing. The
PRESSURE IN POUNDS TEMPERATURE (°C) TEMPERATURE (°F)
range of the ultraviolet spectrum that is microbiocidal is 240-280 nm.
There is a great difference in the susceptibility of organisms to
5 109 228 ultraviolet radiation; Aspergillus niger spores are 10 times more
10 115 240 resistant than Bacillus subtilis spores, 50 times more resistant than
15 121 250 Staphylococcus aureus and Escherichia coli, and 150 times more
20 126 259 resistant than influenza virus.
25 130 267 Because most materials strongly absorb ultraviolet light, it lacks
penetrating power and its applications are limited to surface treatments.
30 135 275
Much higher energy, 100 to millions of times greater, is generated by
ionizing radiations. These include gamma-rays, high energy X-rays and
Over-sterilization or prolonged heating will change the composition of high energy electrons.
the medium. For example, carbohydrates are known to break down in Ionizing radiation, unlike ultraviolet rays, penetrates deeply into
composition upon overheating. Over-sterilizing media can cause a atoms, causing ionization of the electrons. Ionizing radiation may
number of problems, including: directly target the DNA in cells or produce active ions and free radicals
• Incorrect pH; that react indirectly with DNA.
• A decrease in the gelling properties of agar; Gamma radiation is used more often than x-rays or high-energy
electrons for purposes of sterilization. Gamma rays are generated by
• The development of a nontypical precipitate;
radioactive isotopes, cobalt-60 being the usual source. Gamma Testing Sterilizing Agents1,5
radiation requires many hours of exposure for sterilization. Validation Sterilization by moist heat (steam), dry heat, ethylene oxide and ioniz-
of a gamma irradiation procedure includes:4 ing radiation is validated using biological indicators. The methods of
• Establishment of article materials compatibility; sterilization and their corresponding indicators are listed below:
• Establishment of product loading pattern and completion of dose
mapping in the sterilization container;
STERILIZATION METHOD BIOLOGICAL INDICATOR
• Establishment of timer setting;
• Demonstration of the delivery of the required sterilization dose. Steam Bacillus stearothermophilus
The advantages of sterilization by irradiation include low chemical Dry heat Bacillus subtilis var. niger
reactivity, low measurable residues, and few variables to control.3 Ethylene oxide Bacillus subtilis var. globigii
Gamma irradiation is used for treating many heat-sensitive products Filtration Pseudomonas diminuta
that can also be treated by gaseous sterilization, including medical
materials and equipment, pharmaceuticals, biologicals, certain
prepared media and laboratory equipment. For moist heat sterilization, paper strips treated with chemicals that
change color at the required temperature may be used.
Sterilization by Filtration1,3
Filtration is a useful method for sterilizing liquids and gases. Filtration The heat-resistant spores of B. stearothermophilus are dried on paper
excludes microorganisms rather than destroying them. Two major types treated with nutrient medium and chemicals. After sterilization, the
of filters may be used, depth filters and membrane filters. strips are incubated for germination and growth, and a color change
indicates whether they have or have not been activated. Spore strips
The membrane filter screens out particles, while the depth filter should be used in every sterilization cycle.
entraps them. Membrane filters depend largely on the size of the pores
to determine their screening effectiveness. Electrostatic forces are also Glossary1,6
important. A membrane filter with an average pore size of 0.8 µm will
Bioburden is the initial population of living microorganisms in the
retain particulate matter as small as 0.05 µm. For removing bacteria, a
product or system being considered.
pore size of 0.2 µm is commonly used. For retention of viruses and
mycoplasmas, pore sizes of 0.01-0.1 µm are recommended. Cocci and Biocide is a chemical or physical agent intended to produce the death
bacilli range in size from about 0.3 to 1 µm in diameter. Most viruses of microorganisms.
are 0.02-0.1 µm, with some as large as 0.25 µm. Calibration is the demonstration that a measuring device produces
Rating the pore size of filter membranes is by a nominal rating that results within specified limits of those produced by a reference
reflects the capability of the filter membrane to retain microorganisms standard device over an appropriate range of measurements.
of size represented by specified strains. Sterilizing filter membranes Death rate is the rate at which a biocidal agent reduces the number
are membranes capable of retaining 100% of a culture of 10 7 of cells in a microbial population that are capable of reproduction.
microorganisms of a strain of Pseudomonas diminuta (ATCC® 19146) This is determined by sampling the population initially, during
per square centimeter of membrane surface under a pressure of and following the treatment, followed by plate counts of the surviving
not less than 30 psi. These filter membranes are nominally rated 0.22 microorganisms on growth media.
µm or 0.2 µm. Bacterial filter membranes (also known as analytical
filter membranes), which are capable of retaining only larger D value stands for decimal reduction time and is the time required in
microorganisms, are labeled with a nominal rating of 0.45 µm. minutes at a specified temperature to produce a 90% reduction in the
number of organisms.
Membrane filters are used for the commercial production of a number
of pharmaceutical solutions and heat-sensitive injectables. Serum Microbial death is the inability of microbial cells to metabolize and
for use in bacterial and viral culture media are often sterilized by reproduce when given favorable conditions for reproduction.
filtration, as well as some sugars that are unstable when heated. Process validation is establishing documented evidence that a
Membrane filtration is useful in testing pharmaceutical and medical process does what it purports to do.
products for sterility.
Sterility Assurance Level is generally accepted when materials
1 are processed in the autoclave and attain a 10-6 microbial survivor
Sterility Assurance
Sterility Assurance is the calculated probability that a microorganism probability; i.e., assurance of less than one chance in one million that
will survive sterilization. It is measured as the SAL, Sterility viable microorganisms are present in the sterilized article.3
Assurance Level, or “degree of sterility”. For sterility assurance, Sterilization process is a treatment process from which the probability
Bacillus stearothermophilus which contains steam heat-resistant spores of microorganism survival is less than 10-6, or one in a million.
is employed with steam sterilization at 121°C.
Thermal Death Time and Thermal-Chemical Death Time are terms 3. The United States Pharmacopeia (USP XXIII) and The
referring to the time required to kill a specified microbial population National Formulary (NF 18). 1995. Sterilization and
upon exposure to a thermal or thermal-chemical sterilizing agent sterility assurance of compendial articles, p. 1976-1980.
under specified conditions. A typical thermal death time value with United States Pharmacopeial Convention Inc., Rockville, MD.
highly resistant spores is 15 minutes at 121°C for steam sterilization. 4. Perkins, J. J. 1969. Principles and methods of sterilization
in health sciences, 2nd ed. Charles C. Thomas, Springfield, IL.
References
5. Leahy, T. J. 1986. Microbiology of sterilization processes. In F. J.
1. Block, S. 1992. Sterilization, p. 87-103. Encyclopedia of Carleton and J. P. Agalloco (ed.), Validation of aseptic
microbiology, vol. 4. Academic Press, Inc., San Diego, CA. pharmaceutical processes. Marcel Dekker, Inc. New York, N.Y.
2. Cote, R. J., and R. L. Gherna. 1994. Nutrition and media,
6. Simko, R. J. 1986. Organizing for validation. In F. J. Carleton and
p. 155-178. In P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R.
J. P. Agalloco (ed.), Validation of aseptic pharmaceutical processes.
Krieg (ed.), Methods for general and molecular bacteriology.
Marcel Dekker, Inc., New York, N.Y.
American Society for Microbiology, Washington, D.C.
OR Results
1. Adjust an overnight culture to a 0.5 McFarland. For general-purpose media, sufficient, characteristic growth and
2. Plate 0.01 ml of the specimen to confirm a colony count of typical colony morphology should be obtained with all test strains.
1-2 x 108 CFU/ml. If using a frozen culture, confirm the appropriate For selective media, growth of designated organisms is inhibited and
density. adequate growth of desired organisms is obtained. Color and hemolytic
reaction criteria must be met.
To Test Cultural Response
Reference
Non-Selective Media
National Committee for Clinical Laboratory Standards. 1996.
Dilute the cell suspension 1:100 in normal saline or purified water. Quality assurance for commercially prepared microbiological culture
Inoculate each plate with 0.01 ml to give 1-2 x 104 CFU/plate. Reduce media, 2nd ed. Approved standard. M22-A2, vol. 16, no. 16. National
the inoculum ten fold, if necessary, to obtain isolated colonies. Committee for Clinical Laboratory Standards, Wayne, PA.
Selective Media and Tubed Media
Dilute the cell suspension 1:10 in normal saline or purified water. Streak
each plate with 10.01 ml of the suspension to provide 1-2 x 105 CFU/
plate. Reduce the inoculum ten fold, if necessary, to avoid
overwhelming some selective media.
Typical Analysis
“Typical” chemical compositions have been determined on media Nitrogen
ingredients. The typical analysis is used to select products for research Total Nitrogen: Total nitrogen is usually measured by the
or production needs when specific nutritional characteristics are Kjeldhal digestion or titration method. Not all organic nitrogen is
required. The specifications for the typical analysis include: nutritive. Percent (%) nitrogen x 6.25 ≈ % proteins, peptides or amino
• Physical characteristics, acids present.
• Nitrogen content, Amino Nitrogen: The amino nitrogen value shows the extent of
• Amino acids, protein hydrolysis by measuring the increase in free amino groups.
This is a nutritionally meaningful value.
• Inorganics,
• Vitamins, and pH
• Biological testing. Changes in pH from specified values, either after storage or processing,
indicate deterioration. These changes are usually accompanied by
All values are presented as weight/weight; % = g/100 g.
darkening of the end product. Hydrolysates vary in their pH resistance
Glossary according to their inherent buffering (phosphate) capacity.
Ash Phosphates
The higher the ash content, the lower the clarity of the prepared High-phosphate ingredients may be unsuitable for pH indicator media
ingredient. The ash content includes sodium chloride, sulfate, phos- due to the inherent buffering of phosphates. However, phosphates do
phates, silicates and metal oxides. Acid-insoluble ash is typically aid in gas production, which can be enhanced by deliberate addition of
from silicates found in animal fodder. sodium phosphate.
maximal at low concentrations, 0.1- 0.2 mg/l, and inhibited at high presumptive identification only. Consult Reference 1 and other appro-
concentrations). Chelating agents (e.g., citrate) may be added to priate references for complete identification of Salmonella.1,3,9,11-14
culture media to sequester trace metals and clarify the media.
References
Antigenic Schema for Salmonella 1. McWhorter-Murlin, A. C., and F. W. Hickman-Brenner. 1994.
Identification and serotyping of Salmonella and an update of the
Update of the Kauffmann-White Schema1 Kauffmann-White Scheme. Centers for Disease Control and
The Centers for Disease Control has modified the Kauffmann-White Prevention, Atlanta, GA.
antigenic schema originally proposed by Ewing.1-3 The updated schema
2. Kauffmann, F. 1969. Enterobacteriaceae, 2nd ed. Munksgaard,
are used with Difco Salmonella Antisera as an aid in the serological
Copenhagen.
identification of Salmonella.
3. Ewing, W. H. 1986. Edwards and Ewing’s identification of
All of the Salmonella serovars belong to two species, S. bongori Enterobacteriaceae, 4th ed. Elsevier Science Publishing Co. Inc.,
containing 18 serovars and S. enterica containing the remaining New York, NY.
2300-plus serovars which are divided among six subspecies.1 The six
4. Penner, J. L. 1988. International committee on systematic
subspecies of S. enterica are:
bacteriology taxonomic subcommittee on Enterobacteriaceae. Int.
S. enterica subsp. enterica (I or 1) J. Syst. Bacteriol. 38:223-224.
S. enterica subsp. salamae (II or 2) 5. LeMinor, L., and M. Y. Popoff. 1987. Request for an opinion.
S. enterica subsp. arizonae (IIIa or 3a) Designation of Salmonella enterica sp. nov., nom. rev., as the type
S. enterica subsp. diarizonae (IIIb or 3b) and only species of the genus Salmonella. Int. J. Syst. Bacteriol.
S. enterica subsp. houtenae (IV or 4) 37:465-468.
S. enterica subsp. indica (VI or 6) 6. Wayne, L. G. 1991. Judicial Commission of the International
The legitimate species name for the above strains is S. choleraesuis. Committee on Systematic Bacteriology. Int. J. Syst. Bacteriol.
However, this name may be confused with the serotype named 41:185-187.
“choleraesuis.” At the International Congress for Microbiology in 1986, 7. Wayne, L. G. 1994. Actions of the Judicial Commission of the
the International Subcommittee for Enterobacteriaceae agreed to adopt International Committee on Systematic Bacteriology on requests
the species name “S. enterica.”4 LeMinor and Popoff5 published a for opinions published between January 1985 and July 1993. Int.
request to the Judicial Commission to use S. enterica as the official J. Syst. Bacteriol. 44:177.
species name. The Judicial Commission ruled that S. choleraesuis is 8. Old, D. C. 1992. Nomenclature of Salmonella. J. Med. Microbiol.
the legitimate name.6,7 S. enterica is used in many countries and is 37:361-363.
favorably accepted as the species name.3,8 The Centers for Disease 9. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
Control has adopted this designation until the problem of naming this R. H. Yolken. 1995. Manual of clinical microbiology, 6th ed.
species is resolved.1 American Society for Microbiology, Washington, D.C.
Nomenclature and classification of these bacteria are ever changing.9 10. Farmer, J. J., III, A. C. McWhorter, D. J. Brenner, and
Salmonella and the former Arizona should be considered a single G. D. Morris. 1984. The Salmonella-Arizona group of
genus, Salmonella.10 All serovars in subspecies enterica are named. Enterobacteriaceae: nomenclature, classification and reporting.
Serovars in other subspecies (except some in subspecies salamae and Clin. Microbiol. Newsl. 6:63-66.
houtenae) are not named. It is recommended that laboratories report 11. Isenberg, H. D. (ed.) 1992. Clinical microbiology procedures hand-
named Salmonella serovars by name and unnamed serovars by book, vol. 2. American Society for Microbiology, Washington, D.C.
antigenic formula and subspecies. For the most recent information on 12. Holt, J. G., N. R. Krieg, P. H. Sneath, J. T. Staley,
nomenclature, consult appropriate references.1,3,9,10,12 S. T. Williams. 1994. Bergey’s manual of determinative
Serotypes of Salmonella are defined based on the antigenic structure bacteriology, 9th ed. Williams & Wilkins, Baltimore, MD.
of both somatic or cell wall (O) antigens and flagellar (H) antigens. 13. Andrews, W. H., G. A. June, P. Sherrod, T. S. Hammack,
The antigenic formula gives the O antigen(s) first followed by the and R. M. Amaguana. 1995. Food and drug administration
H antigen(s). The major antigens are separated by colons and the bacteriological analytical manual, 8th edition. AOAC International,
components of the antigens separated by commas. For example, the Gaithersburg, MD.
antigenic formula for Salmonella typhimurium is Salmonella 14. Russell, S. F., J. D’Aoust, W. H. Andrews, and J. S. Bailey. 1992.
1,4,5,12:i:1,2. This means that the strain has O antigen factors 1,4,5 Salmonella. In C. Vanderzant and D. F. Splittstoesser (eds.),
and 12, the flagella phase 1 antigen I, and flagella phase 2 antigens Compendium of methods for the microbiological examination
1 and 2. of foods, 3rd ed. American Public Health Association,
Complete identification of Salmonella requires cultural isolation, Washington, D.C.
biochemical characterization and serotyping. Any serological results
obtained before biochemical identification must be considered as
Agar
Bacto Agar . Agar Flake . Agar, Granulated . Agar Noble
®
Agar Bacteriological Technical Agar Bacteriological Technical is a solidifying agent used in pre-
paring microbiological culture media. Although Agar Bacteriological
Intended Use Technical has wider quality control parameters than other
Bacto® Agar is a solidifying agent in which extraneous matter, bacteriological agars, solubility, gelation temperature and solidity are
pigmented portions and salts have been reduced to a minimum. Bacto® carefully monitored to permit its use.
Agar is used in preparing microbiological culture media.
Summary and Explanation
Agar Flake is a solidifying agent used in preparing microbiological Agar is a phycocolloid extracted from a group of red-purple marine
culture media. algae (Class Rhodophyceae) including Gelidium, Pterocladia and
Agar, Granulated is a solidifying agent used in preparing microbio- Gracilaria. Gelidium is the preferred source for Difco agars. Impurities,
logical culture media. debris, minerals and pigment are reduced to specified levels
Agar Noble is a solidifying agent that is essentially free of impurities. during manufacture.
It is used in electrophoretic and nutritional procedures and in preparing Agar was first suggested for microbiological purposes in 1881 by
microbiological culture media when increased purity is required. Fannie Hesse.1, 2 By the early 1900s, agar became the gelling agent of
choice over gelatin because agar remains firm at growth temperatures
Cultural Response
Prepare the agar formulation of Nutrient Broth (0003) or LB Broth, Miller (0446) by adding 1.5% agar.
Sterilize and pour plates. Inoculate with 100-1,000 CFU of the indicated test organisms and incubate
at 35 ± 2°C for 18-24 hours. Record recovery.
BACTO® AGAR AGAR, AGAR AGAR BACTERIOLOGICAL
AGAR FLAKE GRANULATED NOBLE TECHNICAL
Nutrient Broth with:
Escherichia coli ATCC® 25922* Good Good Good Good
Staphylococcus aureus ATCC® 25923* Good Good Good Good
LB Broth, Miller with:
Escherichia coli ATCC® 33694 (HB101) Good
Saccharomyces cerevisiae ATCC® 9763 Good
*These cultures are available as Bactrol™ Disks and should be Can of
used as directed in the Bactrol Disks Technical Information. Bacto Agar
AGAR
for many pathogens. Agar is also generally resistant to a breakdown BACTO® AGAR, AGAR BACTERIOLOGICAL
AGAR GRANULATED NOBLE TECHNICAL
by bacterial enzymes. The use of agar in microbiological media
significantly contributed to the advance of microbiology, paving the Biological Testing (CFU/g)
way for pure culture isolation and study. Spore Count <1,000 <1,000 <1,000 4,300
Standard Plate Count <1,000 <1,000 <1,000 2,725
Agar is a gel at room temperature, remaining firm at temperatures
as high as 65°C.3 Agar melts at approximately 85°C, a different Inorganics (%)
temperature from that at which it solidifies, 32-40°C. This property is Calcium 0.179 0.133 0.015 0.110
known as hysteresis. Agar is generally resistant to shear forces; however, Chloride 0.021 <0.005 <0.050 0.172
different agars may have different gel strengths or degrees of stiffness. Cobalt <0.001 <0.001 <0.001 <0.001
Copper <0.001 <0.001 <0.001 <0.001
Agar is typically used in a final concentration of 1-2% for solidifying Iron 0.002 0.003 <0.001 0.002
culture media. Smaller quantities (0.05-0.5%) are used in media for Lead <0.001 <0.001 <0.001 <0.001
motility studies (0.5% w/v) and for growth of anaerobes (0.1%) and Magnesium 0.068 0.041 0.002 0.093
microaerophiles.3 Manganese <0.001 <0.001 <0.001 <0.001
Specifications for bacteriological grade agar include good clarity, Nitrate <0.005 <0.005 <0.050 <0.005
controlled gelation temperature, controlled melting temperature, good Phosphate <0.005 0.010 <0.050 0.015
diffusion characteristics, absence of toxic bacterial inhibitors, and Potassium 0.121 0.079 0.022 0.124
relative absence of metabolically useful minerals and compounds. Sodium 0.837 0.776 0.335 0.932
Sulfate 1.778 1.710 0.663 0.367
Product Applications Sulfur 0.841 0.868 0.333 0.646
Bacto® Agar is optimized for beneficial calcium and magnesium Tin <0.001 <0.001 <0.001 <0.001
content. Detrimental ions such as iron and copper are reduced. Bacto® Zinc <0.001 <0.001 <0.001 <0.001
Agar is recommended for clinical applications, auxotrophic studies, Precautions
bacterial and yeast transformation studies, and bacterial molecular
1. For Laboratory and Manufacturing Use.
genetics applications.4, 5
2. Follow proper established laboratory procedures in handling and
Agar Flake is recommended for general bacteriological purposes. The disposing of infectious materials.
quality is similar to Bacto® Agar. However, the flakes are more easily
wetted than the granules found in Bacto® Agar. Storage
Agar, Granulated is qualified for culturing recombinant strains of Store dehydrated agar below 30°C. Dehydrated agar is very hygroscopic.
Escherichia coli (HB101) and Saccharomyces cerevisiae. Agar, Keep container tightly closed.
Granulated may be used for general bacteriological purposes where
clarity is not a strict requirement.
Expiration Date
The expiration date applies to the product in its intact container when
Noble Agar is extensively washed and bleached. This agar should be stored as directed. Do not use the product if it fails to meet specifications
used for applications where extreme clarity and high purity are required. for identity and performance.
Noble Agar is suitable for immunodiffusion, some electrophoretic
applications, and as a substrate for mammalian or plant tissue culture. Procedure
Agar Bacteriological Technical is suitable for many bacteriological Materials Provided
applications. This agar is not highly processed, has broader technical Bacto® Agar
specifications than other Difco agars, and is not recommended for Agar Flake
growth of fastidious organisms. Agar, Granulated
Agar Noble
Typical Analysis Agar Bacteriological Technical
AGAR
BACTO® AGAR, AGAR BACTERIOLOGICAL
AGAR GRANULATED NOBLE TECHNICAL Materials Required But Not Provided
Physical Characteristics Materials vary depending on the application.
Ash (%) 3.6 3.4 1.3 4.1
Method of Preparation
Color lt. beige lt. beige off white lt beige
Texture granular granular fine granular
Method of preparation varies depending on the application.
free-flowing free-flowing granular free-flowing Specimen Collection and Preparation
free flowing
Clarity, 1.5% Soln (NTU) 4.3 5.3 3.7 26.2 Obtain and process specimens according to the techniques and
Loss on Drying (%) 17.3 12.2 16.0 18.2 procedures established by laboratory policy.
pH, 1.5% Soln 6.5 6.6 5.7 6.9 Test Procedure
Gel Strength (g/cm2) 600 560 700 613 See appropriate references for specific procedures using Bacto® Agar,
Gelation Point(°C) 35°C 35°C 35°C 36°C Agar Flake, Agar, Granulated, Agar Noble or Agar Bacteriological
Melting Point (°C) 88°C 88°C 87°C 88°C Technical.
Bacto 2xYT ®
research.1-3 The components of 2xYT provide nitrogen and growth
factors that allow bacteriophage to reproduce in large quantities
without exhausting the host. E. coli grows more rapidly in this rich
Intended Use medium because it provides amino acids, nucleotide precursors,
Bacto 2xYT is used for cultivating recombinant strains of Escherichia coli. vitamins and other metabolites that the cell would otherwise have to
synthesize.2
Summary and Explanation
2xYT is a nutritionally rich growth medium designed for growth of Principles of the Procedure
recombinant strains of Escherichia coli. This medium is also used for
Tryptone and Yeast Extract provide the necessary nutrients and cofactors
propagation of M13 bacteriophage for sequencing and phage display
required for excellent growth of E. coli. Sodium Chloride is included to
provide a suitable osmotic environment.
In 1972 Andrews and Presnell developed A-1 Medium. A-1 Medium Method of Preparation
recovers E. coli from estuarine water in 24 hours instead of 72 hours, 1. Suspend 31.5 grams in 1 liter distilled or deionized water.
and in greater numbers without the preenrichment step.2 Using a
2. Heat to boiling to dissolve completely.
3-hour preincubation step for the enumeration of coliforms in
chlorinated wastewater gave results that were statistically comparable 3. Dispense into tubes containing inverted fermentation vials.
to those obtained in the two-step MPN technique.3 4. Autoclave at 121°C for 10 minutes.
A-1 Medium can be used in a single-step procedure for the detection NOTE: For 10 ml water samples, prepare double-strength medium to
of fecal coliforms in source water, seawater, treated wastewater and ensure the ingredient concentrations are not reduced below those of
foods. Prior enrichment in a presumptive medium is not required.4 the standard medium.4
A-1 Medium conforms to standard methods for the isolation of fecal
coliforms in water and foods.4,5,6
Specimen Collection and Preparation
Obtain and process specimens according to the procedures established
Principles of the Procedure by laboratory policy or standard methods.4,5,6
Tryptone provides the nitrogen, vitamins, minerals and amino acids in
Test Procedure
A-1 Medium. Lactose is the carbon source and, in combination with
Salicin, provides energy for organism growth. Sodium Chloride 1. Inoculate tubes of A-1 Medium as directed in standard methods.4,5,6
maintains the osmotic balance of the medium. Triton X-100 is a surfactant. 2. Incubate at 35 ± 0.5°C for 3 hours.
3. Transfer tubes to a water bath at 44.5 ± 0.2°C and incubate for an
Formula additional 21 ± 2 hours.
A-1 Medium 4. Maintain water level in bath above level of liquid in inoculated tubes.
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g Results5
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Gas production in the inverted vial, or dissolved gas that forms fine
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g bubbles when slightly agitated, is a positive reaction indicating the
Bacto Salicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g presence of fecal coliforms. Calculate fecal coliform densities using
Triton X-100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ml MPN tables from standard methods.
Final pH 6.9 ± 0.1 at 25°C
Limitations of the Procedure
Precautions 1. Since the nutritional requirements of organisms vary, some strains may
1. For Laboratory Use. be encountered that fail to grow or grow poorly on this medium.
2. Follow proper established laboratory procedures in handling and 2. Fecal coliform counts are usually greater than E. coli counts.5
disposing of infectious materials. 3. Interpretation of test procedure using A-1 Medium requires
Storage understanding of the microflora of the specimen.5
Store the dehydrated medium below 30°C. The dehydrated medium is References
very hygroscopic. Keep container tightly closed.
1. Andrews, W. H., C. D. Diggs, and C. R. Wilson. 1975.
Store prepared medium in the dark at room temperature for no longer Evaluation of a medium for the rapid recovery of Escherichia coli
than 7 days.4 from shellfish. Appl. Microbiol. 29:130- 131.
Expiration Date 2. Andrews, W. H., and M. W. Presnell. 1972. Rapid recovery of
Escherichia coli from estuarine water. Appl. Microbiol.
The expiration date applies to the product in its intact container when
23:521-523.
stored as directed. Do not use a product if it fails to meet the specifications
for identity and performance. 3. Standridge, and Delfino. 1981. Appl. Environ. Microbiol. 42:918.
4. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). 1995.
Procedure Standard methods for the examination of water and wastewater,
Materials Provided 19th ed. American Public Health Association, Washington, D.C.
A-1 Medium 5. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992.
Compendium of methods for the microbiological examination of
Materials Required But Not Provided food, 3rd ed. American Public Health Association, Washington, D.C.
Glassware
6. Association of Official Analytical Chemists. 1995. Bacteriological
Fermentation vials
analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
Autoclave
Incubator (35°C) Packaging
Waterbath (44.5°C) A-1 Medium 500 g 1823-17
Test tubes
Distilled or deionized water
Bacto AC Broth
® Several early studies reported on the wide variety of organisms able to
grow on AC Medium.3,4,5 AC Broth is suitable for use in the detection
of obligately aerobic contaminants in biologicals and other products.
Bacto AC Broth w/o Dextrose AC Broth and AC Broth w/o Dextrose are also useful in the isolation
and cultivation of many common pathogenic and saprophytic aerobes.6
Intended Use The media can be used to test the sterility of biologicals and solutions
Bacto AC Broth is used for cultivating a wide variety of microorganisms that do not contain mercurial preservatives. Fluid Thioglycollate
and for the sterility testing of turbid or viscous solutions and other Medium should be employed for the sterility testing of solutions
materials not containing mercurial preservatives. containing mercurial preservatives.
Bacto AC Broth w/o Dextrose is used, with the addition of a carbohydrate, AC Broth w/o Dextrose has the same formula as AC Broth except that
for cultivating a wide variety of microorganisms. the dextrose is omitted, allowing for the addition of other carbohydrates
if desired.
Summary and Explanation
AC Broth and AC Broth w/o Dextrose possess growth-promoting
Principles of the Procedure
properties for voluminous growth of a wide variety of microorganisms. Proteose Peptone No. 3, Beef Extract, and Malt Extract provide
Christensen1 and Malin and Finn2 reported that AC Medium does not the carbon and nitrogen sources required for good growth of a wide
exhibit the toxicity shown by media containing sodium thioglycollate. variety of organisms. Vitamins and cofactors required for growth as
well as additional sources of nitrogen and carbon are provided by Yeast
Extract. Dextrose is included in AC Broth as a carbon energy source.
Ascorbic Acid is added to clarify the solution.
User Quality Control
Identity Specifications Formula
AC Broth AC Broth
Dehydrated Appearance: Light tan, free-flowing, homogeneous. Formula Per Liter
Solution: 3.4% solution, soluble in distilled or Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 20 g
deionized water. Solution is medium Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
to dark amber, clear to very slightly Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
opalescent. Bacto Malt Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Prepared Tubes: Light to medium amber, clear to very Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
slightly opalescent. Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g
Reaction of 3.4% Final pH 7.2 ± 0.2 at 25°C
Solution at 25°C: pH 7.2 ± 0.2
AC Broth w/o Dextrose
AC Broth w/o Dextrose Formula Per Liter
Dehydrated Appearance: Light tan, free-flowing, homogeneous. Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 20 g
Solution: 2.92% solution, soluble in distilled or Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
deionized water. Solution is medium Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
to dark amber, clear to very slightly Bacto Malt Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
opalescent. Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g
Prepared Tubes: Medium to dark amber, clear to very Final pH 7.2 ± 0.2 at 25°C
slightly opalescent.
Reaction of 2.92% Precautions
Solution at 25°C: pH 7.2 ± 0.2 1. For Laboratory Use.
Cultural Response 2. Follow proper established laboratory procedure in handling and
Prepare AC Broth or AC Broth w/o Dextrose per label disposing of infectious materials.
directions. Inoculate and incubate at 35 ± 2°C for 18-48 hours.
INOCULUM
Storage
ORGANISM ATCC® CFU GROWTH Store the dehydrated media below 30°C. The dehydrated media are
Corynebacterium diphtheriae very hygroscopic. Keep container tightly closed.
Type mitis 8024 100-1,000 good
Streptococcus pneumoniae 6305 100-1,000 good AC Broth
Streptococcus pyogenes 19615* 100-1,000 good Store prepared medium at 15-30°C. After prolonged storage, reheat in
flowing steam or a boiling water bath for a few minutes to drive off
The cultures listed are the minimum that should be used for
performance testing. dissolved gases. Cool without agitation.
*This culture is available as Bactrol™ Disks and should be used AC Broth w/o Dextrose
as directed in Bactrol Disks Technical Information.
Store prepared medium at 15-30°C.
preparation of stock cultures. The others are used to prepare inoculum 3. Vedamuthu, E. R., M. Raccach, B. A. Glatz, E. W. Seitz, and M.
in APT Broth for assay as needed. Following incubation at 35-37°C for S. Reddy. 1992. Acid-producing microorganisms, p. 225-238. In
24-48 hours, store stock cultures at 2-8°C. C. Vanderzant, and D. F. Splittstoesser (ed.), Compendium of
methods for the microbiological examination of foods, 3rd ed.
Results American Public Health Association, Washington, D.C.
Refer to appropriate references and procedures for results.
Packaging
References APT Agar 500 g 0654-17
1. Evans, J. B., and C. F. Niven, Jr. 1951. Nutrition of the 2 kg 0654-07
heterofermentative lactobacilli that cause greening of cured meat 10 k 0654-08
products. J. Bact. 62:599-603.
APT Broth 500 g 0655-17
2. Deibel, R. H., J. B. Evans, and C. F. Niven, Jr. 1957.
Microbiological assay for thiamine using Lactobacillus
viridescens. J. Bact. 74:818-821.
Intended Use sole source of carbon. Many strains of E. coli are able to use acetate as
a carbon source, whereas typical cultures of Shigella are unable to grow.
Bacto Acetate Differential Agar is used for differentiating microorganisms
of the Shigella genus from those of the Escherichia genus. Trabulsi and Ewing 2 developed Acetate Differential Agar by
substituting sodium acetate for sodium citrate in their basal medium,
Also Known As Simmons Citrate Agar. They demonstrated that none of the Shigella
Acetate Differential Agar is also known as Sodium Acetate Agar. tested grew on the Acetate Differential Agar. A large percentage of
E. coli strains, belonging to various O antigen groups, did use the
Summary and Explanation acetate within 2 to 7 days of incubation.
Although classified taxonomically as different species for clinical The majority of Salmonella, Citrobacter, Klebsiella, Enterobacter and
reasons, Shigella species and E. coli are essentially the same genus and Serratia groups use acetate and grow on Acetate Differential Agar
species. Their DNA relatedness is high, they are difficult to differentiate within 1 to 7 days. Proteus and Providencia groups, however, fail
biochemically, and they cross-react serologically.1 One way they can to grow on the medium. Several standard methods list Acetate
be differentiated is by using a medium containing sodium acetate as a
Bacto Glycerol
organic nitrogen. Sodium Propionate is a substrate used in anaerobic
Intended Use fermentation. Dipotassium Phosphate provides buffering capability to
Bacto Actinomycete Isolation Agar is used with added glycerol for maintain pH balance. Magnesium Sulfate and Ferrous Sulfate provide
isolating and cultivating actinomycetes from soil and water. sources of sulfates and metallic ions. Bacto Agar is the solidifying
agent. The added Glycerol is a source of carbon.
Bacto Glycerol is used in preparing microbiological culture media.
Formula
Summary and Explanation
Actinomycete Isolation Agar
Although some genera are important to human medicine, most of the
actinomycetes are part of the indigenous flora of soil, water, and Formula Per Liter
vegetation. Actinomycetes may impart a musty odor to water or a Sodium Caseinate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
muddy flavor to fish.2 Actinomycetes can cause massive growths which Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g
will form a thick foam in the activated sludge process, causing a Sodium Propionate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 g
disruption in wastewater treatment. 3,4 Actinomycetes are gram Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
positive, acid-fast cells, growing as filaments that may branch and may Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g
form irregularly shaped rods and cocci. Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Olsen1 formulated Actinomycete Isolation Agar for isolating and
Final pH 8.1 ± 0.2 at 25°C
cultivating actinomycetes from soil and water. The formula,
supplemented with Glycerol, is a highly purified fermentable alcohol Glycerol
used occasionally for differentiating certain bacteria and in media for Not applicable
isolating and culturing fastidious bacteria.
Precautions
Principles of the Procedure 1. For Laboratory Use.
Actinomycete Isolation Agar contains Sodium Caseinate which is a 2. MAY BE IRRITATING TO EYES, RESPIRATORY SYSTEM
source of nitrogen. Asparagine is an amino acid and a source of AND SKIN. (US) Avoid contact with skin and eyes. Do not breathe
dust. Wear suitable protective clothing. Keep container tightly closed.
FIRST AID: In case of contact with eyes, rinse immediately with
plenty of water and seek medical advice. After contact with skin,
User Quality Control wash immediately with plenty of water. If inhaled, remove to fresh
Identity Specifications air. If not breathing, give artificial respiration. If breathing is
Actinomycete Isolation Agar difficult, give oxygen. Seek medical advice. If swallowed seek
Dehydrated Appearance: Light beige, free-flowing, homogeneous. medical advice immediately and show this container or label.
Solution: 2.2% solution, soluble in distilled or 3. Follow proper established laboratory procedures in handling and
deionized water on boiling. Solution disposing of infectious materials.
is light to medium amber, opalescent
to opaque with precipitation. Storage
Prepared Medium: Medium amber, opalescent. Store the dehydrated medium below 30°C. The dehydrated medium is
Reaction of 2.2% very hygroscopic. Keep container tightly closed.
Solution with 0.5% Store Glycerol at 15-30°C.
Glycerol at 25°C: pH 8.1 ± 0.2
Cultural Response Expiration Date
Prepare Actinomycete Isolation Agar per label directions The expiration date applies to the product in its intact container when
with the addition of 0.5% Glycerol. Inoculate and incubate at stored as directed. Do not use a product if it fails to meet specifications
30 ± 2°C for up to 72 hours. for identity and performance.
INOCULUM
ORGANISM ATCC® CFU GROWTH
Procedure
Streptomyces achromogenes 12767 100-1,000 good
Streptomyces albus 3004 100-1,000 good Materials Provided
Streptomyces lavendulae 8664 100-1,000 good Actinomycete Isolation Agar
Glycerol
The cultures listed are the minimum that should be used for
performance testing.
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g 2. Process each sample using procedures appropriate for that
Sodium Cholate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g sample.1,2
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03 g
Crystal Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002 g Test Procedure1,2
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g 1. Pre-enrich the sample in Lactose Broth. If the sample is insoluble
Final pH 7.4 ± 0.2 at 25°C in water, add 0.1 ml of polysorbate 20 or polysorbate 80 to the
Lactose Broth.
Precautions 2. Homogenize the mixture and incubate at 35 ± 2°C for 2-5 hours.
1. For Laboratory Use. 3. Transfer 1 ml of enriched Lactose Broth to 100 ml of EE Broth
2. Follow proper, established laboratory procedures in handling and Mossel (Enterobacteriaceae Enrichment Broth-Mossel).
disposing of infectious materials. 4. Incubate at 35 ± 2°C for 24-48 hours.
Storage 5. Subculture all enrichment broth cultures showing growth onto
Agar Medium No. F.
Store the dehydrated medium below 30°C. The dehydrated medium is
very hygroscopic. Keep container tightly closed. Store the prepared 6. Incubate at 35 ± 2°C for 18-24 hours.
medium at 2-8°C. 7. Examine plates for the presence of presumptive Enterobacteriaceae
colonies.
Expiration Date Results
The expiration date applies to the product in its intact container when Colonies of the family Enterobacteriaceae are reddish-purple in color
stored as directed. Do not use a product if it fails to meet specifications and are generally surrounded by a zone of precipitated bile salt. Growth
for identity and performance. of gram-positive organisms is markedly to completely suppressed.
Procedure Further biochemical testing is necessary to confirm the presence and
identification of Enterobacteriaceae. Consult appropriate references
Materials Provided for further information on identification of Enterobacteriaceae.3,4
Agar Medium No. F
References
Materials Required But Not Provided 1. DAB, 10th Edition. 1991. V.2 Biology, V.2.1.8 Proving Certain
Lactose Broth Microorganisms, VIII.10 Media (Microbiological Pollution),
Enterobacteriaceae Enrichment Broth Mossel (EE Broth Mossel) Frankfurt/Main.
Flasks with closures
2. British Pharmacopoeia, Volume II, Appendix XVI. 1988.
Distilled or deionized water
HMSO, London.
Incubator (35°C)
Polysorbate 20 or Polysorbate 80 3. Farmer, J. J. 1995. Enterobacteriaceae: introduction and
identification. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
Method of Preparation Tenover, and R. H. Yolken (ed.). Manual of clinical microbiology,
1. Suspend 51.5 grams in 1 liter distilled or deionized water. 6th ed. American Society for Microbiology, Washington, D.C.
2. Heat to boiling to dissolve completely. 4. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
3. Sterilize by steaming for 30 minutes. Do Not Autoclave. Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
St. Louis, MO.
Specimen Collection and Preparation
1. Collect samples in sterile containers and transport immediately to Packaging
the laboratory following recommended guidelines.1,2 Agar Medium No. F 500 g 0666-17
Summary and Explanation All amino acid assay media contain the following formula. Omit the
particular amino acid to be assayed from the medium.
Amino Acid Assay Media are prepared for use in the microbiological
assay of amino acids. Three types of media are used for this purpose: Formula Per Liter
1. Maintenance Media: For carrying the stock culture to preserve the Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 g
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g
viability and sensitivity of the test organism for its intended purpose.
Ammonium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 g
2. Inoculum Media: To condition the test culture for immediate use. Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 g
3. Assay Media: To permit quantitation of the amino acid under test. Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 g
They contain all the factors necessary for optimal growth of the test Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g
organism except the single essential amino acid to be determined. Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Amino Acid Assay Media are prepared according to the formulations Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 mg
of Steel et al.1 They are used in the microbiological assay of amino Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
acids using Pediococcus acidilactici ATCC® 8042 as the test organism. Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Guanine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Principles of the Procedure Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Lysine Assay Medium, Methionine Assay Medium and Cystine Assay Xanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Thiamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg
Medium contain all the factors essential for the growth of Pediococcus
Pyrodoxine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg
acidilactici ATCC® 8042, except the amino acid under assay. The
Pyridoxamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . 600 mg
addition of the amino acid in specified increasing concentrations gives
Pyridoxal Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 600 mg
a growth response by the test organism. Calcium Pantothenate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg
Formula Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg
Lysine Assay Medium, Methionine Assay Medium, or p-Aminobenzoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 µg
Cystine Assay Medium Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 µg
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 µg
Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g
DL-Alanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g
User Quality Control Bacto Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 g
Identity Specifications L-Aspartic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g
L-Proline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g
Lysine Assay Medium, Methionine Assay Medium,
or Cystine Assay Medium DL-Serine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g
DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 mg
Dehydrated Appearance: White to off-white, homogeneous,
may have a tendency to clump. L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g
L-Glutamic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 g
Solution: 5.25% (single strength) and 10.5% L-Histidine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . 0.124 g
(double strength) solution, soluble
DL-Phenylalanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g
in distilled or deionized water upon
boiling. Solution (single strength) is DL-Threonine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g
light to medium amber, clear to L-Tyrosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g
slightly opalescent, may have a DL-Valine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
slight precipitate. L-Lysine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
DL-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g
Prepared Medium: Single strength-light to medium
amber, clear. DL-Isoleucine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
DL-Leucine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
Reaction of 5.25% L-Arginine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . 0.484 g
Solution at 25°C: pH 6.7 ± 0.2
Final pH 6.7 ± 0.2 at 25ºC
Cultural Response
Prepare Lysine Assay Medium, Methionine Assay Medium and Precautions
Cystine Assay Medium per label directions. These media will 1. For Laboratory Use.
support the growth of Pediococcus acidilactici ATCC® 8042 2. Great care to avoid contamination of media or glassware must be
when supplemented with the appropriate amino acid. Test taken in microbiological assay procedures. Extremely small
Lysine Assay Medium by creating a standard curve using amounts of foreign material may be sufficient to give erroneous
L-Lysine at 0 to 300 µg per 10 ml. Test Methionine Assay
Medium by creating a standard curve using DL-Methionine at results. Scrupulously clean glassware free from detergents and
0 to 60 µg per 10 ml. Test Cystine Assay Medium by creating other chemicals must be used. Glassware is heated to 250°C for at
a standard curve using L-Cystine at 0 to 50 µg per 10 ml. least 1 hour to burn off any organic residues that might be present.
3. Methionine Assay Medium and Cystine Assay Medium
The test organism listed is the minimum used for performance testing.
IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM
AND SKIN. Avoid contact with skin and eyes. Do not breathe dust.
Wear suitable protective clothing. Keep container tightly closed. 3. Dispense 5 ml amounts into tubes, evenly dispersing the precipitate.
TARGET ORGAN(S): Kidney, Bladder. 4. Add standard or test samples.
FIRST AID: In case of contact with eyes, rinse immediately with 5. Adjust tube volume to 10 ml with distilled or deionized water.
plenty of water and seek medical advice. After contact with skin, 6. Autoclave at 121°C for 10 minutes.
wash immediately with plenty of water. If inhaled, remove to fresh
air. If not breathing, give artificial respiration. If breathing is diffi- Specimen Collection and Preparation
cult, give oxygen. Seek medical advice. If swallowed seek medical Assay samples are prepared according to references given in the specific
advice immediately and show this container or label. assay procedure. The samples should be diluted to approximately the
4. Take precautions to keep sterilizing and cooling conditions uniform same concentration as the standard solution.
throughout the assay.
Test Procedure
5. Follow proper established laboratory procedures in handling and
disposing of infectious materials. Stock Culture and Inoculum
Stock cultures of Pediococcus acidilactici ATCC® 8042 are prepared by
Storage stab inoculation into tubes of Lactobacilli Agar AOAC or Micro Assay
Store the dehydrated media at 2-8ºC. The dehydrated medium is very Culture Agar. Incubate cultures at 35-37°C for 24 hours. Store stock
hygroscopic and may be stored in a container with calcium chloride cultures at 2-8°C. Make transfers at monthly intervals in triplicate.
or other desiccant. Keep container tightly closed. The inoculum for assay is prepared by subculturing the test organism
into 10 ml Lactobacilli Broth AOAC or Micro Inoculum Broth. Incubate
Expiration Date at 35-37ºC for 16-24 hours. After incubation, centrifuge the cells under
The expiration date applies to the product in its intact container when aseptic conditions and decant the liquid supernatant. Wash the cells
stored as directed. Do not use a product if it fails to meet specifications 3 times with 10 ml sterile 0.85% NaCl solution. After the third wash,
for identity and performance. resuspend the cells in 10 ml sterile 0.85% NaCl solution. Dilute the
10 ml cell suspension with the appropriate amount of sterile 0.85%
Procedure NaCl solution. (See Table 1 below.) One drop of the diluted inoculum
Materials Provided suspension is used to inoculate each of the assay tubes.
Lysine Assay Medium or
Methionine Assay Medium or Amino Acid Solution
Cystine Assay Medium Prepare stock solutions of each amino acid as described in Table 1. If
the DL form is used, twice the concentration of the amino acid is required.
Materials Required But Not Provided Prepare the stock solutions fresh daily.
Glassware Increasing amounts of the standard or the unknown and sufficient distilled
Autoclave or deionized water to give a total volume of 10 ml per tube, are added
Stock culture of Pediococcus acidilactici ATCC® 8042 to the tubes containing 5 ml of the rehydrated medium. The appropriate
Sterile tubes, optically standardized volumes of the standards and their final concentrations are listed in the table.
Centrifuge
Measure the growth response turbidimetrically or titrimetrically.
Spectrophotometer (660 nm)
Turbidimetric readings are made after incubation at 35-37°C for 16-20
L-Lysine HCl
hours. Titrimetric readings are made after incubation at 35-37°C for 72 hours.
DL-Methionine
L-Cystine It is essential that a standard curve be constructed each time an assay is
Sterile 0.85% NaCl run. Conditions of autoclaving and temperature of incubation that
influence the standard curve readings cannot always be duplicated.
Method of Preparation
Lysine Assay Medium, Methionine Assay Medium, and Cystine Results
Assay Medium 1. Prepare a standard concentration response curve by plotting the
1. Suspend 10.5 grams in 100 ml distilled or deionized water. response readings against the amount of standard in each tube, disk
or cup.
2. Boil for 2-3 minutes to dissolve completely.
Table 1. Preparation of inoculum dilution, amino acid stock and working solution.
PREPARATION OF STANDARD VOLUME OF STANDARD FINAL
INOCULUM DILUTION PREPARATION OF WORKING SOLUTION WORKING AMINO ACID
(CELL SUSPENSION + AMINO ACID STOCK SOLUTION (STOCK SOLUTION) + SOLUTION CONCENTRATION
ASSAY MEDIUM TEST CULTURE (STERILE 0.85% NaCI) (AMINO ACID) + (DISTILLED H2O) (DISTILLED H2O) (ml/10 ml TUBE) µg/10 ml
Cystine Assay Pediococcus acidilactici 1 ml + 19 ml L-cystine 1 g + 100 ml + 1 ml 1 ml + 99 ml 0, 0.5, 1, 1.5, 0.0, 5, 10, 15, 20,
Medium ATCC® 8042 HCl heated, then cooled, 2, 2.5, 3, 4, 5 25, 30, 40, 50
add up to 1,000 ml
Lysine Assay Pediococcus acidilactici 1 ml + 19 ml L-lysine 6 g + 1,000 ml 1 ml + 99 ml 0, 0.5, 1, 1.5, 0.0, 30, 60, 90, 120,
Medium ATCC® 8042 2, 2.5, 3, 4, 5 150, 180, 240, 300
Methionine Assay Pediococcus acidilactici 1 ml + 19 ml DL-methionine 1.2 g + 1,000 ml 1 ml + 99 ml 0, 0.5, 1, 1.5, 0.0, 6, 12, 18, 24,
Medium ATCC® 8042 2, 2.5, 3, 4, 5 30, 36, 48, 60
2. Determine the amount of amino acid at each level of assay solution 4. For successful results of these procedures, all conditions of the
by interpolation from the standard curve. assay must be followed precisely.
3. Calculate the concentration of amino acid in the sample from the
average of these volumes. Use only those values that do not vary References
more than ±10% from the average. Use the results only if two thirds 1. Steel, Sauberlich, Reynolds, and Baumann. 1949. J. Biol.
of the values do not vary more than ±10%. Chem. 177:533.
The use of standardized culture media and careful control of all test quantitative determination of antibiotics in body fluids, animal feeds
conditions are fundamental requisites in the microbiological assay of and other materials.
antibiotics in order to achieve satisfactory test results.
Turbidimetric Assay
Principles of the Procedure The turbidimetric method is based on the inhibition of growth of a
microbial culture in a fluid medium containing a uniform solution of an
Cylinder Plate Assay antibiotic.1 Turbidimetric determinations have the advantage of requir-
This method is based on the diffusion of an antibiotic solution from a ing a short incubation period, providing test results after 3 or 4 hours.
cylinder placed on the surface of an inoculated agar medium. The However, the presence of solvents or other inhibitory materials may
diameter of a zone of inhibition after incubation depends, in part, on influence turbidimetric assays more markedly than cylinder plate assays.
the concentration or activity of the antibiotic. This method is used in Use of this method is appropriate only when test samples are clear.
the assay of commercial preparations of antibiotics, as well as in the
Precautions
User Quality Control 1. For Laboratory Use.
Identity Specifications 2. Follow proper established laboratory procedures in handling and
Dehydrated Appearance: Beige to pink, free-flowing, disposing of infectious materials.
homogeneous.
Solution: 1.75% solution, soluble in distilled Storage
or deionized water on warming, Store the dehydrated medium below 30°C. The dehydrated medium is
orange-red, clear. very hygroscopic. Keep container tightly closed.
Prepared Medium: Orange-red, clear.
Reaction of 1.75% Expiration Date
Solution at 25°C: pH 7.2 ± 0.2 The expiration date applies to the product in its intact container when
stored as directed. Do not use a product if it fails to meet specifications
Cultural Response for identity and performance.
Prepare the medium per label directions. Inoculate test
organisms into tubes with fermentation vials and incubate at Procedure
30 ± 2°C for 18-48 hours.
INOCULUM Materials Provided
ORGANISM ATCC® CFU GROWTH ACID GAS
Aseptic Commissioning Medium
Bacillus cereus 14579 100-1,000 good + –
Enterobacter aerogenes 13048* 100-1,000 good + + Materials Required But Not Provided
Escherichia coli 25922* 100-1,000 good +** + or –
Flasks with closures
Staphylococcus aureus 25923* 100-1,000 good + –
Distilled or deionized water
The cultures listed are the minimum that should be used for
performance testing. Method of Preparation
*These cultures are available as Bactrol™ Disks and should be used 1. Suspend 17.5 grams in 1 liter distilled or deionized water.
as directed in Bactrol Disks Technical Information. 2. Heat gently to dissolve completely.
**May revert to alkaline after prolonged incubation. 3. Autoclave at 121°C for 15 minutes.
Intended Use B12 Assay Medium USP is used in the microbiological assay of vitamin
Bacto B12 Assay Medium USP is used for determining vitamin B12 B12 according to the procedures of the Vitamin B12 Activity Assay in
concentration by the microbiological assay technique. USP1 and the Cobalamin (Vitamin B12 Activity) Assay in AOAC.2
Lactobacillus delbrueckii subsp. lactis ATCC® 7830 (Lactobacillus
Also Known As leichmannii) is the test organism used in this procedure.
USP is an abbreviation for United States Pharmacopeia.
Principles of the Procedure
Summary and Explanation B12 Assay Medium USP is a vitamin B12-free dehydrated medium
Vitamin Assay Media are used in the microbiological assay of vitamins. containing all other nutrients and vitamins essential for the cultivation
Three types of media are used for this purpose: of L. delbrueckii subsp. lactis ATCC® 7830. To obtain a standard curve,
1. Maintenance Media: For carrying the stock culture to preserve the USP Cyanocobalamin Reference is added in specified increasing
viability and sensitivity of the test organism for its intended purpose; concentrations giving a growth response that can be measured
2. Inoculum Media: To condition the test culture for immediate use; titrimetrically or turbidimetrically.
3. Assay Media: To permit quantitation of the vitamin under test.
Formula Precautions
B12 Assay Medium USP 1. For Laboratory Use.
Formula Per Liter 2. Great care must be taken to avoid contamination of media or glass-
Bacto Vitamin Assay Casamino Acids . . . . . . . . . . . . . . . . 15 g ware in microbiological assay procedures. Extremely small
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g amounts of foreign material may be sufficient to give erroneous
Bacto Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g results. Scrupulously clean glassware free from detergents and
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g other chemicals must be used. Glassware must be heated to 250°C
Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 g for at least 1 hour to burn off any organic residues that might be present.
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g 3. Take precautions to keep sterilization and cooling conditions uniform
DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g throughout the assay.
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Guanine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
4. Follow proper established laboratory procedures in handling and
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg disposing of infectious materials.
Xanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg
Storage
Thiamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg Store the dehydrated medium at 2-8°C. The dehydrated medium is very
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 µg hygroscopic. Keep container tightly closed.
Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg
p-Aminobenzoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg
Expiration Date
Calcium Pantothenate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg The expiration date applies to the product in its intact container when
Pyridoxine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mg stored as directed. Do not use a product if it fails to meet specifications
Pyridoxal Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mg for identity and performance.
Pyridoxamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . 800 µg
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 µg Procedure
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g Materials Provided
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
B12 Assay Medium USP
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg Materials Required But Not Provided
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg Glassware
Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Autoclave
Sorbitan Monooleate Complex . . . . . . . . . . . . . . . . . . . . . . . 2 g
Stock culture of Lactobacillus delbrueckii subsp. lactis ATCC® 7830
Final pH 6.0 ± 0.1 at 25°C Lactobacilli Agar AOAC or B12 Culture Agar USP
Lactobacilli Broth AOAC or B12 Inoculum Broth USP
Sterile 0.85% saline
User Quality Control Distilled or deionized water
Spectophotometer or nephelometer
Identity Specifications B12 Culture Agar USP
Dehydrated Appearance: Very light to light beige, homogeneous, B12 Inoculum Broth USP
with a tendency to clump. Cyanocobalamin USP (vitamin B12)
Solution: 4.25% (single strength) or 8.5%
(double strength) solution, soluble Method of Preparation
in distilled or deionized water on 1. Suspend 8.5 grams in 100 ml distilled or deionized water.
boiling for 2-3 minutes. Light amber,
clear, may have a slight precipitate 2. Heat to boiling for 2-3 minutes to dissolve completely.
(single strength). 3. Distribute 5 ml amounts into tubes, evenly dispersing the precipitate.
Prepared Medium: (Single strength) very light to light 4. Add standard or test samples.
amber, clear, may have a slight 5. Adjust tube volume to 10 ml with distilled or deionized water.
precipitate.
6. Autoclave at 121°C for 5 minutes.
Reaction of 4.25%
Solution at 25°C: pH 6.0 ± 0.1 Specimen Collection and Preparation
Cultural Response Assay samples are prepared according to references given in the
specific assay procedures. For assay, the samples should be diluted to
Prepare B12 Assay Medium USP per label directions. Prepare
a standard curve using USP Cyanocobalamin Reference approximately the same concentration as the standard solution.
Standard at levels of 0.0 to 0.25 ng per 10 ml. The medium Test Procedure
supports the growth of L. delbrueckii subsp. lactis ATCC® 7830
when supplemented with cyanocobalamin (vitamin B12). Follow assay procedures as outlined in USP1 or AOAC.2 Use levels
of B12 in the preparation of the standard curve according to these
references. It is essential that a standard curve be constructed each time more than ±10% from the average and use the results only if two
an assay is run. Autoclave and incubation conditions can influence the thirds of the values do not vary more than ±10%.
standard curve reading and cannot always be duplicated.
Generally satisfactory results are obtained with B12 at the following Limitations of the Procedure
levels: 0.0, 0.025, 0.05, 0.075, 0.1, 0.125, 0.15, 0.2 and 0.25 ng per 1. The test organism used for inoculating an assay medium must be
assay tube (10 ml). cultured and maintained on media recommended for this purpose.
Stock cultures of L. delbrueckii subsp. lactis ATCC ® 7830 are 2. For successful results to these procedures, all conditions of the
prepared by stab inoculation into 10 ml of B12 Culture Agar USP or assay must be followed precisely.
Lactobacilli Agar AOAC. After 16-24 hours incubation at 35-37°C, the 3. Aseptic technique should be used throughout the assay procedure.
cultures are kept refrigerated. The inoculum for assay is prepared by 4. The use of altered or deficient media may cause mutants having
subculturing a stock culture of L. delbrueckii subsp. lactis into different nutritional requirements and will not give a satisfactory
10 ml of B12 Inoculum Broth USP. For a complete discussion on B12 response.
Culture Agar USP and B12 Inoculum Broth USP, refer to USP.1
References
Results 1. The United States Pharmacopeial Convention. 1995. The United
1. Prepare a standard concentration response curve by plotting the States pharmacopeia, 23rd ed. The United States Pharmacopeial
response readings against the amount of standard in each tube, disk Convention Inc., Rockville, MD.
or cup. 2. Association of Official Analytical Chemists. 1995. Official
2. Determine the amount of vitamin at each level of assay solution by methods of analysis of AOAC International, 16th ed. AOAC
interpolation from the standard curve. International, Arlington, VA.
3. Calculate the concentration of vitamin in the sample from the
average of these volumes. Use only those values that do not vary
Packaging
B12 Assay Medium USP 100 g 0457-15
B12 Inoculum Broth USP 3. Great care must be taken to avoid contamination of media or glass-
Formula Per Liter ware in microbiological assay procedures. Extremely small
Tomato Juice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 ml amounts of foreign material may be sufficient to give erroneous
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 7.5 g results. Scrupulously clean glassware free from detergents and
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 g other chemicals must be used.
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Sorbitan Monooleate Complex . . . . . . . . . . . . . . . . . . . . . . 0.1 g Storage
Potassium Phosphate Dibasic . . . . . . . . . . . . . . . . . . . . . . . . 2 g Store the dehydrated media at 2-8°C. The dehydrated media are very
Final pH 6.8 ± 0.1 at 25°C hygroscopic. Keep containers tightly closed.
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
Cultural Response
BG Sulfa Agar
Prepare BG Sulfa Agar per label directions. Inoculate and incubate
the plates at 35 ± 2°C for 18-48 hours.
INOCULUM COLOR OF
ORGANISM ATCC® CFU GROWTH COLONIES/MEDIUM
Enterococcus faecalis 29212* 1,000-2,000 none –/no change
Escherichia coli 25922* 100-1,000 none to poor yellow-green
Salmonella 14028* 100-1,000 good pink-white/red
typhimurium
SBG Enrichment and SBG Sulfa Enrichment
Prepare SBG Enrichment and SBG Sulfa Enrichment per label
directions. Inoculate tubes with the test organisms. Incubate
inoculated medium at 35 ± 2°C for 18-24 hours. After incubation,
subculture onto prepared plates of MacConkey Agar.
INOCULUM COLONY COLOR
ORGANISM ATCC® CFU GROWTH ON MACCONKEY
Escherichia coli 25922* 100-1,000 none to poor pink, if any
Salmonella 14028* 100-1,000 good colorless
typhimurium
Shigella sonnei 9290 100-1,000 poor to fair colorless
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used
as directed in Bactrol Disks Technical Information.
IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN. Specimen Collection and Preparation
Avoid contact with skin and eyes. Do not breathe dust. Wear For information about specimen preparation and inoculation of food
suitable protective clothing. Keep container tightly closed. samples, consult appropriate references.7,12
TARGET ORGAN(S): Kidney, Liver, Spleen.
FIRST AID: In case of contact with eyes, rinse immediately with Results
plenty of water and seek medical advice. After contact with skin, BG Sulfa Agar
wash immediately with plenty of water. If inhaled, remove to fresh The typical Salmonella colonies appear as pink-white to red opaque
air. If not breathing, give artificial respiration. If breathing is colonies surrounded by a brilliant red medium. The few lactose and/or
difficult, give oxygen. Seek medical advice. If swallowed, induce sucrose fermenting organisms that grow are readily differentiated due
vomiting; seek medical advice immediately and show this container to the formation of a yellow-green colony surrounded by an intense
or label. yellow-green zone. BG Sulfa Agar is not suitable for the isolation of
3. Follow proper established laboratory procedures in handling and S. typhi or Shigella; however, some strains of S. typhi may grow forming
disposing of infectious materials. red colonies.
7. Federal Register. 1996. Pathogen reduction; hazard analysis 12. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compen-
and critical point (HACCP) systems; final rule. Fed. Regis. dium of methods for the microbiological examination of food,
61:38917-38925. 3rd ed. American Public Health Association, Washington, D.C.
8. Osborn, W. W., and J. L. Stokes. 1955. Appl. Microbiol. 3:217. 13. MacFaddin, J. F. 1985. Media for isolation-cultivation-identification-
9. Brooks, J. and D. J. Taylor. 1955.Rep. Rd. Invest., Bd. 60, H. M. maintenance of medical bacteria, Vol. 1. Williams & Wilkins,
S. O. London, England. Baltimore, MD.
10. Forsythe, R. H., J. C. Ayres, and J. L. Radlo. 1953. Factors
affecting the microbiological populations of shell eggs. Food
Packaging
Technol. 7:49. BG Sulfa Agar 500 g 0717-17
11. Stadelman, W. J., A. I. Ikeme, R. A. Roop, and S. E. Simmons. SBG Enrichment 500 g 0661-17
1982. Thermally processed hard-cooked eggs. Poultry Science SBG Sulfa Enrichment 500 g 0715-17
61:388.
Bacto EY Tellurite Enrichment EY Tellurite Enrichment is also known as Egg Yolk Tellurite
Enrichment.
Intended Use Summary And Explanation
Bacto Baird-Parker Agar Base is used with Bacto EY Tellurite
Enrichment in isolating and enumerating staphylococci in foods and The formulation of Baird-Parker Agar was published in 1962.1 It is a
other materials. selective medium for isolation and presumptive identification of
coagulase-positive staphylococci.
Also Known As
Baird-Parker is also known as Egg Tellurite Glycine Pyruvate Agar
(ETGPA) based on its composition. Uninoculated Staphylococcus aureus
plate ATCC® 25923
Coagulase-negative staphylococci produce poor or no growth. If growth examination of dairy products, 16th ed. American Public Health
occurs, colonies are black; clear or opaque zones are rare. Association, Washington, D.C.
Most other organisms are inhibited or grow poorly. If growth occurs, 4 Association of Official Analytical Chemists. 1995. Bacteriological
colonies are light to brown-black with neither clear nor opaque zones. analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
5. Andrews, W. H. 1995. Microbial Methods, p. 1-119. Official
Limitations of the Procedure methods of analysis of AOAC International, 16th ed. AOAC
Baird-Parker Agar is selective for coagulase-positive staphylococci but International. Arlington, VA.
other bacteria may grow. Microscopic examination and biochemical 6. United States Pharmacopeial Convention. 1995. The United
tests will differentiate coagulase-positive staphylococci from other States Pharmacopeia, 23rd ed. The United States Pharmacopeial
microorganisms. Convention, Rockville, MD.
References 7. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). 1995.
Recreational waters, p. 9.26-9.27. In Standard methods for the
1. Baird-Parker, A. C. 1962. An improved diagnostic and selective
examination of water and wastewater, 19th ed. American Public
medium for isolating coagulase-positive staphylococci. J. Appl.
Health Association, Washington, D.C.
Bacteriol. 25:12-19.
2. Lancette, G. A., and S. R. Tatini. 1992. Staphylococcus aureus, Packaging
p. 533-550. In C. Vanderzant, and D. F. Splittstoesser (ed.), Com- Baird-Parker Agar Base 100 g 0768-15
pendium of methods for the microbiological examination of foods, 500 g 0768-17
3rd ed. American Public Health Association, Washington, D.C. 2 kg 0768-07
3. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig. 10 kg 0768-08
1993. Pathogens in milk and milk products, p. 103-212. In EY Tellurite Enrichment 6 x 100 ml 0779-73
R. T. Marshall, (ed.), Standard methods for the microbiological
Bacto Beef Extract, Desiccated may be relied upon for biochemical studies, particularly fermentation
reactions, because of its independence from fermentable substances
that would interfere with the accuracy of such determinations.
Intended Use Beef Extract, Desiccated, the dried form of Beef Extract, was developed
to provide a product for ease of use in handling. Beef Extract is in the
Bacto Beef Extract and Bacto Beef Extract, Desiccated are used in paste form. The products are to be used in a one for one substitution,
preparing microbiological culture media. however variations tend to be formulation specific and require actual
performance testing.
Summary and Explanation
Beef Extract is prepared and standardized for use in microbiological Principles of the Procedure
culture media, where it is generally used to replace infusions of meat. Beef Extract and Beef Extract, Desiccated are replacements for infusion
Culture media containing Beef Extract have been recommended for of meat. Beef Extract and Beef Extract, Desiccated provide nitrogen,
use in the bacteriological examination of water, milk and other materials vitamins, amino acids and carbon in several formulations of microbio-
where having media of uniform composition is important. logical culture media.
Beef Extract has been employed by many investigators. Bedell and
Lewis1 used it in their medium for the study of non-sporulating Typical Analysis
BEEF EXTRACT BEEF EXTRACT, DESICCATED
anaerobes of the intestinal tract. Hutner2 used a medium containing
Physical Characteristics
Beef Extract as a stock broth in the study of nutritional needs of
Ash (%) 24.1 10.2
streptococci. Beef Extract is the formula of Potato Infusion Agar for Clarity, 1% Soln (NTU) 116.8 1.7
the cultivation of Brucella. Fletcher Medium Base, Starch Agar, Filterability (g/cm2) 0.1 0.6
Dextrose Agar, Dextrose Broth and CLED Agar all contain Beef Loss on Drying (%) 77.2 2.5
Extract to enhance the growth of bacteria. Antibiotic Assay media pH, 1% Soln 5.4 6.9
specified by US Pharmacopeia3 includes Beef Extract in the formula.
Carbohydrate (%)
Several media containing Beef Extract are recommended in standard
Total 0.2 <0.1
methods for multiple applications.4,5,6
Nitrogen Content (%)
In culture media, Beef Extract is usually employed in concentrations Total Nitrogen 11.2 14.0
of 0.3%. Concentrations may vary slightly according to the requirements Amino Nitrogen 3.8 2.2
of individual formulas, but do not often exceed 0.5%. Beef Extract AN/TN 33.8 15.7
BEEF EXTRACT BEEF EXTRACT, DESICCATED BEEF EXTRACT BEEF EXTRACT, DESICCATED
Materials Required But Not Provided 2. Formula allowances may be required due to the lower sodium
Materials vary depending on the medium being prepared. chloride concentration of Beef Extract, Desiccated.
4. It is recommended that BiGGY Agar be prepared fresh, just prior 4. Warren, N. G., and K. C. Hazen. 1995. Candida, Cryptococcus,
to use.1,2 and other yeasts of medical importance, p. 723-737. In P. R. Murray,
5. Do not use slants because the reactions are unsatisfactory.1,2 E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken (ed.).,
Manual of clinical microbiology, 6th ed. American Society for
References Microbiology, Washington, D.C.
1. Nickerson, W. J. 1947. Biology of pathogenic fungi. The Chronica 5. MacFaddin, J. D. 1985. Media for isolation-cultivation-
Botanica Co., Waltham, MA. identification-maintenance of medical bacteria, vol. 1, p. 65-68.
2. Nickerson, W. J. 1953. Reduction of inorganic substances by Williams & Wilkins, Baltimore, MD.
yeasts. I. Extracellular reduction of sulfite by species of Candida.
J. Infect. Dis. 93:43. Packaging
3. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey & BiGGY Agar 100 g 0635-15
Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc., 500 g 0635-17
St. Louis, MO.
Also Known As
Bacto Bile Esculin Agar Bile Esculin Agar is also known as Bile Esculin Medium (BEM). The
spelling, aesculin, is often seen in literature.
Facklam and Moody.2 Rochaix3 first noted the value of esculin hydrolysis Precautions
in the identification of enterococci. Meyer and Schönfeld4 added bile 1. For Laboratory Use.
to the esculin medium and demonstrated that 61 of 62 enterococci
strains were able to grow and hydrolyze esculin, while the other 2. Bile Esculin Agar Base:
streptococci could not. IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM
AND SKIN. Avoid contact with skin and eyes. Do not breathe dust.
Molecular taxonomic studies of the genus Streptococcus have placed Wear suitable gloves and eye/face protection. Use only in well
enterococci, previously considered group D streptococci, in the distinct ventilated areas. Keep container tightly closed.
genus Enterococcus.6 Streptococci with Lancefield group D antigen
FIRST AID: In case of contact with eyes, rinse immediately with
include the nonhemolytic species Streptococcus bovis.7 The ability to
plenty of water and seek medical advice. After contact with skin,
hydrolyze esculin in the presence of bile is a characteristic of enterococci
wash immediately with plenty of water. If inhaled, remove to fresh
and group D streptococci.
air. If not breathing, give artificial respiration. If breathing is diffi-
Swan1 compared the use of an esculin medium containing 40% bile cult, give oxygen. Seek medical advice. If swallowed seek medical
salts with the Lancefield serological method of grouping. He reported advice immediately and show this container or label.
that a positive reaction on the bile esculin medium correlated with a Bacto Bile Esculin Agar:
serological group D precipitin reaction. Facklam and Moody,2 in IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM
a comparative study of tests used to presumptively identify group D AND SKIN. Avoid contact with skin and eyes. Do not breathe dust.
streptococci, found that the bile esculin test provided a reliable means Wear suitable protective clothing. Keep container tightly closed.
of identifying group D streptococci and differentiating them from TARGET ORGAN(S): Lungs.
non-group D streptococci. Facklam5 further confirmed the usefulness
FIRST AID: In case of contact with eyes, rinse immediately with
of Bile Esculin Agar in another study differentiating enterococci/group
plenty of water and seek medical advice. After contact with skin,
D streptococci from non-group D streptococci.
wash immediately with plenty of water. If inhaled, remove to fresh
Lindell and Quinn8 showed that the medium is also useful in the air. If not breathing, give artificial respiration. If breathing is diffi-
differentiation of the Klebsiella-Enterobacter-Serratia group from other cult, give oxygen. Seek medical advice. If swallowed seek medical
Enterobacteriaceae. Edberg et al.9 recommended the medium for routine advice immediately and show this container or label.
testing of the Enterobacteriaceae in order to differentiate 3. Follow proper established laboratory procedure in handling and
Klebsiella-Enterobacter-Serratia spp. Bile Esculin Agar is listed in standard disposing of infectious materials.
procedures for the microbiological examination of food products.10-13
Storage
Principles of the Procedure Store the dehydrated medium below 30°C. The dehydrated medium is
Organisms positive for esculin hydrolysis hydrolyze the glycoside very hygroscopic. Keep container tightly closed.
esculin to esculetin and dextrose. The esculetin reacts with the ferric citrate
to form a dark brown or black complex. Oxgall (bile) is used to inhibit Expiration Date
gram-positive bacteria other than enterococci. Beef Extract and Bacto The expiration date applies to the product in its intact container when
Peptone provide the carbon and nitrogen sources required for growth of a stored as directed. Do not use a product if it fails to meet specifications
wide variety of organisms. Bacto Agar is the solidifying agent. for identity and performance.
Formula Procedure
Bile Esculin Agar Materials Provided
Formula Per Liter Bile Esculin Agar Base
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g Bile Esculin Agar
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g Materials Required But Not Provided
Bacto Oxgall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g Glassware
Ferric Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g Autoclave
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g Incubator (35°C)
Final pH 6.6 ± 0.2 at 25°C Esculin (to be added to Bile Esculin Agar Base)
Bile Esculin Agar Base Filter-sterilized horse serum (optional)
Petri dishes
Formula Per Liter
Tubes with closures
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Method of Preparation
Bacto Oxgall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g 1. Suspend the specidied amount of medium in 1 liter distilled or
Ferric Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g deionized water:
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Bile Esculin Agar Base - 63 grams
Final pH 6.6 ± 0.2 at 25°C
Bile Esculin Agar - 64 grams
2. Heat to boiling to dissolve completely. beider zum Streptococcus lactis. Zentralbl. Bakteriol. Parasitenkd.
3. Bile Esculin Agar Base, only: Add 1 gram (or another desired Infektionskr. Hyg. Abt. I Orig. 99:402-416.
amount) of Esculin and mix thoroughly. 5. Facklam, R. R. 1973. Comparison of several laboratory media for
4. Autoclave at 121°C for 15 minutes. Overheating may cause presumptive identification of enterococci and group D streptococci.
darkening of the media. Appl. Microbiol. 26:138.
5. Cool to 50-55°C. 6. Schleifer, K. H., and R. Kilpper-Balz. 1987. Molecular and
6. If desired, aseptically add 50 ml of filter-sterilized horse serum. chemotaxonomic approaches to the classification of streptococci,
Mix thoroughly. enterococci and lactococci: a review. Syst. Appl. Microbiol. 10:1-19.
7. Dispense as desired. 7. Ruoff, K. L. 1995. Streptococcus. P. R. Murray, E. J. Baron, M. A.
Pfaller, F. C. Tenover and R. H. Yolken (eds.), Manual of clinical
Specimen Collection and Preparation microbiology, 6th ed. American Society for Microbiology,
Refer to appropriate references for specimen collection and preparation. Washington, D.C.
8. Lindell, S. S., and P. Quinn. 1975. Use of bile-esculin agar for rapid
Test Procedure differentiation of Enterobacteriaceae. J. Clin. Microbiol. 1:440.
See appropriate references for specific procedures. 9. Edberg, S. C., S. Pittman, and J. M. Singer. 1977. Esculin
Results hydrolysis by Enterobacteriaceae. J. Clin. Microbiol. 6:111.
Refer to appropriate references and procedures for results. 10. Bacteriological Analytical Manual. 1995. 8th ed. AOAC
International, Gaithersburg, MD.
Limitations of the Procedure 11. Vanderzant, C., and D. F. Splittstoesser (eds.). 1992. Compen-
1. The bile esculin test was originally formulated to identify dium of methods for the microbiological examination of foods,
enterococci. However, the properties of growth on 40% bile media 3rd ed. American Public Health Association, Washington, D.C.
and esculin hydrolysis are characteristics shared by most strains of 12. Marshall, R. T. (ed.) 1992. Standard methods for the examination
Group D streptococci.14 The bile esculin test should be used in of dairy products, 16th ed. American Public Health Association,
combination with other tests to make a positive identification. Washington, D.C.
Facklam14 and Facklam et al.15 recommend a combination of the bile 13. Atlas, R. M. 1995. Handbook of microbiological media for the
esculin test and salt tolerance (growth in 6.5% NaCl). Streptococcus examination of food. CRC Press, Boca Raton, FL.
bovis will give a positive reaction on Bile Esculin Agar, but unlike
14. Facklam, R. 1972. Recognition of group D streptococcal species
Enterococcus spp., it cannot grow on 6.5% NaCl or at 10°C.16
of human origin by biochemical and physiological tests. Appl.
2. Bile Esculin Agar should be considered a differential medium, but Microbiol. 23:1131.
with the addition of sodium azide (which inhibits gram-negative
15. Facklam, R. R., J. F. Padula, L. G. Thacker, E. C. Wortham,
bacteria) the medium can be made more selective (see Bile Esculin
and B. J. Sconyers. 1974. Presumptive identification of group A,
Azide Agar).
B, and D streptococci. Appl. Microbiol. 27:107.
3. Occasional viridans strains will be positive on Bile Esculin Agar
or will display reactions that are difficult to interpret.17 Of the 16. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey
viridans group, 5 to 10% may be able to hydrolyze esculin in the & Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book,
presence of bile.16 Inc. St. Louis, MO.
4. Use a light inoculum when testing Escherichia coli on Bile Esculin 17. Ruoff, K. L., S. I. Miller, C. V. Garner, M. J. Ferraro, and S. B.
Agar. Wasilauskas18 suggests that the time required for an isolate Calderwood. 1989. Bacteremia with Streptococcus bovis and
to hydrolyze esculin is directly proportional to the size of the Streptococcus salivarius: clinical correlates of more accurate
inoculum. For a tabulation of those Enterobacteriaceae that can identification of isolates. J. Clin. Microbiol. 27:305-308.
hydrolyze esculin, refer to Farmer.19 18. Wasilauskas, B. L. 1971. Preliminary observations on the rapid
differentiation of the Klebsiella-Enterobacter-Serratia group on
References bile-esculin agar. Appl. Microbiol. 21:162.
1. Swan, A. 1954. The use of bile-esculin medium and of Maxted’s 19. Farmer, J. J., III. 1995. Enterobacteriaceae. P. R. Murray, E. J.
technique of Lancefield grouping in the identification of enterococci Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken (eds.), Manual
(group D streptococci). J. Clin. Pathol. 7:160. of clinical microbiology, 6th ed. American Society for Microbiology,
2. Facklam, R. R., and M. D. Moody. 1970. Presumptive identifi- Washington, D.C.
cation of group D streptococci: The bile-esculin test. Appl.
Microbiol. 20:245. Packaging
3. Rochaix, A. 1924. Milieux a leculine pour le diagnostid Bile Esculin Agar Base 500 g 0878-17
differentieldes bacteries du groups strepto-entero-pneumocoque. Bile Esculin Agar 100 g 0879-15
Comt. Rend. Soc. Biol. 90: 771-772. 500 g 0879-17
4. Meyer, K., and H. Schönfeld. 1926. Über die Untersheidung des Esculin 10 g 0158-12
Enterococcus vom Streptococcus viridans und die Beziehunger
Expiration Date exhibit growth on the medium (less than 1 mm, white-gray
The expiration date applies to the product in its intact container when colonies), but they will show no action on the esculin.2
stored as directed. Do not use a product if it fails to meet specifications 2. Other than the enterococci, Listeria monocytogenes consistently
for identity and performance. blackens the medium around colonies. After 18-24 hours, there may
be a reddish to black-brown zone of hydrolysis surrounding pinpoint
Procedure Listeria colonies. After 48 hours, white-gray pigmented colonies will
Materials Provided be seen. Listeria do not attain the same degree of esculin hydrolysis
displayed by enterococci in this short incubation period.2
Bile Esculin Azide Agar
Materials Required But Not Provided References
Glassware 1. Isenberg, H. D. 1970. Clin. Lab. Forum. July.
Autoclave 2. Isenberg, H. D., D. Goldberg, and J. Sampson. 1970. Laboratory
Incubator (35°C) studies with a selective enterococcus medium. Appl. Microbiol.
Petri dishes 20:433.
Horse Serum, filter sterilized (optional) 3. Swan, A. 1954. The use of bile-esculin medium and of Maxted’s
technique of Lancefield grouping in the identification of
Method of Preparation enterococci (group D streptococci). J. Clin. Pathol. 7:160.
1. Suspend 57 grams in 1 liter distilled or deionized water. 4. Facklam, R. R., and M. D. Moody. 1970. Presumptive
2. Boil to dissolve completely. identification of group D streptococci: The bile-esculin test.
3. Autoclave at 121°C for 15 minutes. Overheating may cause Appl. Microbiol. 20:245.
darkening of the medium. 5. Sabbaj, J., V. L. Sutter, and S. M. Finegold. 1971. Comparison
4. If desired, aseptically add 50 ml of filter-sterilized horse serum. of selective media for isolation of presumptive group D
Mix thoroughly. streptococci from human feces. Appl. Microbiol. 22:1008.
Specimen Collection and Preparation 6. Brodsky, M. H., and D. A. Schiemann. 1976. Evaluation of Pfizer
selective enterococcus and KF media for recovery of fecal
Refer to appropriate references for specimen collection and preparation. streptococci from water by membrane filtration. Appl. Environ.
Test Procedure Microbiol. 31:695-699.
For isolation of group D streptococci, inoculate the sample onto a small 7. Jensen, B. J. 1996. Screening specimens for vancomycin-resistant
area of one quadrant of a Bile Esculin Azide Agar plate and streak for Enterococcus. Laboratory Medicine 27:53-55.
isolation. This will permit development of discrete colonies. Incubate 8. Schleifer, K. H., and R. Kilpper-Balz. 1987. Molecular and
at 35°C for 18-24 hours. Examine for colonies having the characteristic chemotaxonomic approaches to the classification of streptococci,
morphology of group D streptococci. enterococci and lactococci: a review. Syst. Appl. Microbiol. 10:1-19.
9. Ruoff, K. L. 1995. Streptococcus. In P. R. Murray, E. J. Baron,
Results M. A. Pfaller, F. C. Tenover, and R. H. Yolken (eds.), Manual of
Group D streptococci grow readily on this medium and hydrolyze clinical microbiology, 6th ed. American Society for Microbiology,
esculin, resulting in a dark brown color around the colonies after Washington, D.C.
18-24 hours incubation.
Packaging
Limitations of the Procedure Bile Esculin Azide Agar 100 g 0525-15
1. Staphylococcus aureus and Staphylococcus epidermidis may 500 g 0525-17
2 kg 0525-07
Standard Curve 2. Determine the amount of vitamin at each level of assay solution by
It is essential that a standard curve be constructed each time an assay is interpolation from the standard curve.
run. Autoclave and incubation conditions can influence the standard 3. Calculate the concentration of vitamin in the sample from the
curve reading and cannot always be duplicated. The standard curve is average of these volumes. Use only those values that do not vary
obtained by using biotin at levels of 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 more than ±10% from the average. Use the results only if two
and 1 ng per assay tube (10 ml). thirds of the values do not vary by more than ±10%.
The concentration of biotin required for the preparation of the
standard curve may be prepared by dissolving 0.1 gram of d-Biotin
Limitations of the Procedure
or equivalent in 1,000 ml of 25% alcohol solution (100 µg per ml). 1. The test organism used for inoculating an assay medium must be
Dilute the stock solution by adding 2 ml to 98 ml of distilled water. cultured and maintained on media recommended for this
This solution is diluted by adding 1 ml to 999 ml distilled water, giving purpose.
a solution of 2 ng of biotin per ml. This solution is further diluted by 2. Aseptic technique should be used throughout the assay procedure.
adding 10 ml to 90 ml distilled water, giving a final solution of 0.2 ng 3. The use of altered or deficient media may cause mutants having
of biotin per ml. Use 0.0, 0.5, 1, 1.5, 2, 2.5, 3, 4 and 5 ml of this final different nutritional requirements that will not give a satisfactory
solution. Prepare the stock solution fresh daily. response.
Biotin Assay Medium may be used for both turbidimetric and titrimetric 4. For successful results to these procedures, all conditions of the as-
analysis. Before reading, the tubes are refrigerated for 15-30 minutes say must be followed precisely.
to stop growth. Turbidimetric readings should be made after 16-20
hours at 35-37°C. Titrimetric determinations are made after 72 hours References
incubation at 35-37°C. The most effective assay range, using Biotin 1. Federal Register. 1992. Tests and methods of assay of antibiotics
Assay Medium, has been found to be between 0.1 ng and 1 ng biotin. and antibiotic-containing drugs. Fed. Regist. 21:436.100-436.106.
For a complete discussion of antibiotic assay methodology, refer to 2. United States Pharmacopeial Convention. 1995. The United
appropriate procedures outlined in the references.1,2 States pharmacopeia, 23rd ed. Biological tests and assay,
p. 1690-1696. The United States Pharmacopeial Convention,
Results Rockville, MD.
Calculations
1. Prepare a standard concentration response curve by plotting the Packaging
response readings against the amount of standard in each tube, Biotin Assay Medium 100 g 0419-15
disk or cup.
For food testing, the use of Bismuth Sulfite Agar is specified for the Formula
isolation of pathogenic bacteria from raw and pasteurized milk, cheese Bismuth Sulfite Agar
products, dry dairy products, cultured milks, and butter.1,13-15 The use Formula Per Liter
of Bismuth Sulfite Agar is also recommended for use in testing clinical Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
specimens.16,17 In addition, Bismuth Sulfite Agar is valuable when Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
investigating outbreaks of Salmonella spp., especially S. typhi.18-20 Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bismuth Sulfite Agar is used for the isolation of S. typhi and other Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 g
Salmonella from food, feces, urine, sewage and other infectious Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 g
materials. The typhoid organism grows luxuriantly on the medium, Bismuth Sulfite Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
forming characteristic black colonies, while gram-positive bacteria
Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025 g
and members of the coliform group are inhibited. This inhibitory
action of Bismuth Sulfite Agar toward gram-positive and coliform Final pH 7.7 ± 0.2 at 25°C
organisms permits the use of a much larger inoculum than possible
with other media employed for similar purposes in the past. The use
Precautions
of larger inocula greatly increases the possibility of recovering the 1. For Laboratory Use.
pathogens, especially when they are present in relatively small 2. HARMFUL. MAY CAUSE SENSITIZATION BY INHALA-
numbers. Small numbers of organisms may be encountered in the early TION. IRRITATING TO EYES, RESPIRATORY SYSTEM AND
course of the disease or in the checking of carriers and releases. SKIN. Avoid contact with skin and eyes. Do not breathe dust. Wear
suitable protective clothing. Keep container tightly closed.
Principles of the Procedure FIRST AID: In case of contact with eyes, rinse immediately with
In Bismuth Sulfite Agar, Beef Extract and Bacto Peptone provide plenty of water and seek medical advice. After contact with skin,
nitrogen, vitamins and minerals. Dextrose is an energy source. wash immediately with plenty of water. If inhaled, remove to fresh
Disodium phosphate is a buffering agent. Bismuth sulfite indicator and air. If not breathing, give artificial respiration. If breathing is
brilliant green are complementary in inhibiting gram-positive bacteria difficult, give oxygen. Seek medical advice. If swallowed seek
and members of the coliform group, while allowing Salmonella to medical advice immediately and show this container or label.
grow luxuriantly. Ferrous sulfate is for H2S production. When H2S is 3. Follow proper established laboratory procedure in handling and
present, the iron in the formula is precipitated, giving positive cultures disposing of infectious materials.
the characteristic brown to black color with metallic sheen. Agar is a
solidifying agent. Uninoculated Salmonella typhi
plate ATCC® 19430
Storage Results
Store the dehydrated medium below 30°C. The dehydrated medium The typical discrete S. typhi surface colony is black and surrounded by
is very hygroscopic. Keep container tightly closed. Store prepared a black or brownish-black zone which may be several times the size of
plates at 2-8°C. the colony. By reflected light, preferably daylight, this zone exhibits a
distinctly characteristic metallic sheen. Plates heavily seeded with
Expiration Date S. typhi may not show this reaction except near the margin of the mass
The expiration date applies to the product in its intact container when inoculation. In these heavy growth areas, this organism frequently
stored as directed. Do not use a product if it fails to meet specifications appears as small light green colonies. This fact emphasizes the
for identity and performance. importance of inoculating plates so that some areas are sparsely populated
with discrete S. typhi colonies. Other strains of Salmonella produce black
Procedure to green colonies with little or no darkening of the surrounding medium.
Materials Provided Generally, Shigella spp. other than S. flexneri and S. sonnei are
Bismuth Sulfite Agar inhibited. Shigella flexneri and Shigella sonnei strains that do grow on
this medium produce brown to green, raised colonies with depressed
Materials Required But Not Provided centers and exhibit a crater-like appearance.
Flasks with closures
E. coli is partially inhibited. Occasionally a strain will be encountered
Distilled or deionized water
that will grow as small brown or greenish glistening colonies. This
Bunsen burner or magnetic hot plate
color is confined entirely to the colony itself and shows no metallic
Waterbath (45-50°C) sheen. A few strains of Enterobacter aerogenes may develop on this
Petri dishes medium, forming raised, mucoid colonies. Enterobacter colonies
Incubator (35°C) may exhibit a silvery sheen, appreciably lighter in color than that
Method of Preparation produced by S. typhi. Some members of the coliform group that
1. Suspend 52 grams in 1 liter distilled or deionized water. produce hydrogen sulfide may grow on the medium, giving colonies
similar in appearance to S. typhi. These coliforms may be readily
2. Heat to boiling no longer than 1-2 minutes to dissolve. Avoid differentiated because they produce gas from lactose in differential
overheating. DO NOT AUTOCLAVE. media, for example, Kligler Iron Agar or Triple Sugar Iron Agar. The
3. Cool to 45-50°C in a waterbath. hydrolysis of urea, demonstrated in Urea Broth or on Urea Agar Base,
4. Gently swirl flask to evenly disperse the flocculent precipitate. may be used to identify Proteus sp.
Dispense into sterile Petri dishes. To isolate S. typhi for agglutination or fermentation studies, pick
NOTE: Best results are obtained when the medium is dissolved characteristic black colonies from Bismuth Sulfite Agar and subculture
and used immediately. The melted medium should not be allowed them on MacConkey Agar. The purified colonies from MacConkey
to solidify in flasks and remelted. Current references suggest Agar may then be picked to differential tube media such as
that the prepared plated medium should be aged for one day Kligler Iron Agar, Triple Sugar Iron Agar or other satisfactory
before use.13,21 differential media for partial identification. All cultures that give
reactions consistent with Salmonella spp. on these media should be
Specimen Collection and Preparation
confirmed biochemically as Salmonella spp. before any serological
1. Collect specimens or food samples in sterile containers or with testing is performed. Agglutination tests may be performed from the
sterile swabs and transport immediately to the laboratory following fresh growth on the differential tube media or from the growth on
recommended guidelines.1,13-20 nutrient agar slants inoculated from the differential media. The growth
2. Process each specimen, using procedures appropriate for that on the differential tube media may also be used for inoculating
specimen or sample.1,13-20 carbohydrate media for fermentation studies.
Test Procedure Limitations of the Procedure
For isolation of Salmonella spp. from food, samples are enriched and 1. It is important to streak for well isolated colonies. In heavy growth
selectively enriched. Streak 10 µl of selective enrichment broth onto areas, S. typhi appears light green and may be misinterpreted as
Bismuth Sulfite Agar. Incubate plates for 24-48 hours at 35°C. negative growth for S. typhi.22
Examine plates for the presence of Salmonella spp. Refer to appropriate
2. S. typhi and S. arizonae are the only enteric organisms to exhibit
references for the complete procedure when testing food samples.1,13-15
typical brown zones on the medium. Brown zones are not produced
For isolation of Salmonella spp. from clinical specimens, inoculate by other members of the Enterobacteriaceae. However, S. arizonae
fecal specimens and rectal swabs onto a small area of one quadrant of is usually inhibited.22
the Bismuth Sulfite Agar plate and streak for isolation. This will 3. Colonies on Bismuth Sulfite Agar may be contaminated with
permit the development of discrete colonies. Incubate plates at 35°C. other viable organisms; therefore, isolated colonies should be
Examine at 24 hours and again at 48 hours for colonies resembling subcultured to a less selective medium (e.g., MacConkey Agar).22
Salmonella spp.
4. Typical S. typhi colonies usually develop within 24 hours;
For additional information about specimen preparation and inoculation however, all plates should be incubated for a total of 48 hours to
of clinical specimens, consult appropriate references.16-20 allow growth of all typhoid strains.22
5. DO NOT AUTOCLAVE. Heating this medium for a period longer 13. Andrews, W. H., G. A. June, P. S. Sherrod, T. S. Hammack, and
than necessary to just dissolve the ingredients destroys its selectivity. R. M. Amaguana. 1995. Salmonella, p. 5.01-5.20. In FDA
bacteriological analytical manual, 8th ed. AOAC International,
References Gaithersburg, MD.
1. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig.
14. Flowers, R. S., J. D’Aoust, W. H. Andrews, and J. S. Bailey.
1993. Pathogens in milk and milk products, p. 103-212. In
1992. Salmonella, p. 371-422. In Vanderzant, C. and D. F.
Marshall, R. T. (ed.), Standard methods for the examination of
Splittstoesser (ed.), Compendium of methods for the microbiological
dairy products, 16th ed. American Public Health Association,
examination of foods, 3rd ed. American Public Health Association,
Washington, D.C.
Washington, D.C.
2. Wilson, W. J., and E. M. Blair. 1926. A combination of bismuth
15. Andrews, W. H. (ed). 1995. Microbiological Methods, p. 17.1-17.119.
and sodium sulphite affording an enrichment and selective
In Cunniff, P. (ed.), Official methods of analysis of AOAC
medium for the typhoid-paratyphoid groups of bacteria. J. Pathol.
International, 16th ed. AOAC International, Arlington, VA.
Bacteriol. 29:310.
16. Washington, J. A. 1981. Initial processing for culture of
3. Wilson, W. J., and E. M. Blair. 1927. Use of a glucose bismuth
specimens, p. 91-126. Laboratory procedures in clinical microbi-
sulphite iron medium for the isolation of B. typhosus and
ology, p. 749. Springer-Verlag New York Inc. New York, NY.
B. proteus. J. Hyg. 26:374-391.
17. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994.
4. Wilson, W. J., and E. M. Blair. 1931. Further experience of the
Microorganisms encountered in the gastrointestinal tract,
bismuth sulphite media in the isolation of Bacillus typhosus and
p. 234-248. Bailey & Scott’s diagnostic microbiology, 9th ed.
Bacillus paratyphosus from faeces, sewage and water. J. Hyg.
31:138-161. Mosby-Year Book, Inc. St. Louis, MO.
5. Wilson, W. J. 1923. Reduction of sulphites by certain bacteria in 18. Gray, L. D. 1995. Escherichia, Salmonella, Shigella and Yersinia,
media containing a fermentable carbohydrate and metallic salts. p. 450-456. In Murray, P. R., E. J. Baron, M. A. Pfaller, F. C.
J. Hyg. 21:392. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology,
6th ed. American Society for Microbiology, Washington, D.C.
6. Wilson, W. J. 1928. Isolation of B. typhosus from sewage and
shellfish. Brit. Med. J. 1:1061. 19. Citron, F. 1992. Initial processing, inoculation, and incubation of
aerobic bacteriology specimens, p. 1.4.1-1.4.19. In Isenberg, H. D.
7. Cope, E., and J. Kasper. 1937. A comparative study of methods (ed.), Clinical microbiology procedures handbook, vol. 1. American
for the isolation of typhoid bacilli from the stool of suspected
Society for Microbiology, Washington, D.C.
carriers. Proceedings of local branches of the Society of American
Bacteriologists. J. Bacteriol. 34:565. 20. Grasnick, A. 1992. Processing and interpretation of bacterial
fecal cultures, p. 1.10.1-1.10.25. In Isenberg, H. D. (ed.), Clinical
8. Cope, E. J., and J. A. Kasper. 1938. Cultural methods for the
microbiology procedures handbook, vol. 1. American Society for
detection of typhoid carriers. Am. J. Public Health 28:1065-1068.
Microbiology, Washington, D.C.
9. Gunther, M. S., and L. Tuft. 1939. A comparative study of media
21. D’Aoust, J. Y. 1977. Effect of storage conditions on the
employed in the isolation of typhoid bacilli from feces and urine.
performance of bismuth sulfite agar. J. Clin. Microbiol. 5:122-124.
J. Lab. Clin. Med. 24:461-471.
22. MacFaddin, J. F. 1985. Media for isolation-cultivation-
10. Green, C. E., and P. J. Beard. 1938. Survival of E. typhi in sewage
identification-maintenance of medical bacteria, Vol. 1. Williams
treatment plant processes. Am. J. Public Health 28:762-770.
& Wilkins, Baltimore, MD.
11. Hajna, A. A., and C. A. Perry. 1938. A comparative study of
selective media for the isolation of typhoid bacilli from stool Packaging
specimens. J. Lab. Clin. Med. 23:1185-1193. Bismuth Sulfite Agar 100 g 0073-15
12. United States Pharmacopeial Convention. 1995. The United 500 g 0073-17
States pharmacopeia, 23rd ed. The United States Pharmacopeial 10 kg 0073-08
Convention, Rockville, MD.
Also Known As
Bacto Blood Agar Base No. 2 Blood Agar Base is abbreviated as BAB, and may be referred to as
Infusion Agar.
Intended Use
Bacto Blood Agar Base is used for isolating and cultivating a wide Summary and Explanation
variety of microorganisms and, with added blood, for cultivating Blood agar bases are typically supplemented with 5-10% sheep,
fastidious microorganisms. rabbit or horse blood for use in isolating, cultivating and determining
Bacto Blood Agar Base No. 2 is used for isolating and cultivating hemolytic reactions of fastidious pathogenic microorganisms. Without
fastidious microorganisms with or without added blood. enrichment, blood agar bases can be used as general purpose media.
In 1919, Brown1 experimented with blood agar formulations for the is the nitrogen source for Blood Agar Base No. 2 while Yeast Extract
effects of colony formation and hemolysis; the growth of pneumococci and Liver Digest provide essential carbon, vitamin, nitrogen and
was noticeably influenced when the medium contained peptone amino acids sources. Both media contain Sodium Chloride to maintain
manufactured by Difco. osmotic balance and Bacto Agar as a solidifying agent. Blood
Blood Agar Base is a modification of Huntoon’s2 “Hormone” Medium Agar Bases are relatively free of reducing sugars, which have been
with a slight acidic composition. Norton3 found the pH of 6.8 to be reported to adversely influence the hemolytic reactions of
advantageous in culturing streptococci and pneumococci. Blood Agar beta-hemolytic streptococci.7
Base No. 2 is a nutritionally rich medium for maximum recovery of Supplementation with blood (5-10%) provides additional growth
fastidious microorganisms. factors for fastidious microorganisms and is the basis for
Blood Agar Base media are specified in Standard Methods4,5,6 for determining hemolytic reactions. Hemolytic patterns may vary
food testing. with the source of animal blood or type of base medium used.8
Chocolate agar for isolating Haemophilus and Neisseria species can
Principles of the Procedure be prepared from Blood Agar Base No. 2 by supplementing the
Blood Agar Base formulations have been prepared using specially medium with 10% sterile defibrinated blood (chocolatized).
selected raw materials to support good growth of a wide variety of
fastidious microorganisms.
Infusion from Beef Heart and Tryptose provide nitrogen, carbon,
amino acids and vitamins in Blood Agar Base. Proteose Peptone No. 3
Staphylococcus aureus Streptococcus pyogenes
ATCC® 25923 ATCC® 19615
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
an equal volume of human or rabbit blood. The modified medium is Expiration Date
prepared according to the formula recommended by the American The expiration date applies to the product in its intact container when
Public Health Association.3 Eldering and Kendrick4 reported that the stored as directed. Do not use a product if it fails to meet specifications
addition of 1% proteose peptone or neopeptone increased growth of for identity and performance.
B. pertussis, thereby increasing the yield of vaccine.
The genus Bordetella consists of four species: Bordetella pertussis, Procedure
B. parapertussis, B. bronchiseptica and B. avium.5 All Bordetella are Materials Provided
respiratory pathogens, residing on the mucous membranes of the Bordet Gengou Agar Base
respiratory tract. B. pertussis and B. parapertussis are uniquely human
pathogens. B. pertussis is the major cause of whooping cough or Materials Required But Not Provided
pertussis. B. parapertussis is associated with a milder form of the Glassware
disease. 6 B. bronchiseptica is an opportunistic human pathogen Autoclave
associated with both respiratory and non-respiratory infections, often Incubator (35°C)
occurring in patients having close contact with animals. 5 Waterbath (45-50°C)
B. bronchiseptica has not been reported to cause pertussis. There have Sterile defibrinated blood
been no reports of recovery of B. avium from humans.5 Sterile Petri dishes
The “cough plate” method for the diagnosis of whooping cough was Method of Preparation
originally reported by Chievitz and Meyer.7 This technique is no longer 1. Suspend 30 grams in 1 liter distilled or deionized water containing
recommended. Nasopharyngeal washings or a nasopharyngeal swab 10 grams of glycerol.
(calcium alginate on a wire handle) should be collected within the first 2. Heat to boiling to dissolve completely.
week of paroxysmal coughing.8
3. Autoclave at 121°C for 15 minutes.
Principles of the Procedure 4. Cool to 45-50°C. Aseptically add 15% sterile defibrinated sheep
Infusion from Potato provides nitrogen, vitamins and amino acids. or rabbit blood. Mix well.
Glycerol is a carbon source. Sodium Chloride maintains the osmotic 5. Dispense into sterile Petri dishes.
balance of the medium. Bacto Agar is a solidifying agent. The addition Specimen Collection and Preparation9
of blood provides essential growth requirements for Bordetella species.
Specimens should be obtained during the early phases of the disease
Many factors will inhibit growth of B. pertussis, including fatty acids and prior to the convalescent stage and antimicrobial therapy. The
present in nasal secretions or cotton from the collection swab. Starch, specimen of choice is duplicate nasopharyngeal swabs. Direct plating
present from the Potato Infusion, absorbs fatty acids. of the specimen at bedside is recommended; when this is not possible,
Modified Bordet Gengou medium, enriched with 15-20% blood, yields submerge both swabs into Regan-Lowe transport medium.
typical B. pertussis growth. The colonies appear small, white, opaque
Test Procedure9
and surrounded by a characteristic zone of hemolysis that is not sharply
1. Roll one of the swabs over the primary inoculation area of the
defined but merges diffusely into the medium. The zone of hemolysis
Bordet Gengou plate and streak for isolation. Return the swab to
is usually absent if 30% or more blood is added to the medium and
the transport medium. Incubate the transport medium for 48 hours.
cannot be seen on charcoal-containing media.9 Sterile, defibrinated
Plate the swabs onto a duplicate set of media.
sheep or rabbit blood can be used in preparing the medium.
2. Incubate the culture plates at 35°C for 5-7 days in a moist chamber.
Formula Increased CO 2 is not recommended. Growth of B. pertussis
appears in 3-5 days. Other bordetellae species can appear in 1-3 days.
Bordet Gengou Agar Base
3. Nasopharyngeal specimens may contain staphylococci that produce
Formula Per Liter
a diffusible substance inhibitory to B. pertussis growth. For these
Potato, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 g
specimens, use a plating medium with methicillin (2.5 µg/ml) or
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g cephalexin (40 µg/ml) and a medium without antimicrobics.
Final pH 6.7 ± 0.2 at 25°C 4. Isolates suspected of being B. pertussis should be confirmed by
using a specific antiserum in either the slide agglutination or
Precautions fluorescent antibody staining techniques.10
1. For Laboratory Use. Results
2. Follow proper established laboratory procedures in handling and For a complete discussion on the isolation and identification of
disposing of infectious materials. Bordetella species refer to the appropriate procedures outlined in
the references.
Storage
Store the dehydrated medium below 30°C. The dehydrated medium is Limitations of the Procedure
very hygroscopic. Keep container tightly closed. 1. Since the nutritional requirements of organisms vary, some strains may
be encountered that fail to grow or grow poorly on this medium.
2. Some Haemophilus species will grow on Bordetella isolation 5. Marcon, M . J. 1995. Bordetella, p. 566-573. In P. R. Murray, E. J.
media and may cross-react with B. pertussis antisera. It may be Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual
prudent to rule out X and V factor dependence. of clinical microbiology, 6th ed. American Society for Microbiology,
Washington, D.C.
References 6. Linneman, C. C., and E. B. Pery. 1977. Bordetella parapertussis:
1. MacFaddin, J. F. 1985. Media for isolation-cultivation- recent experience and a review of the literature. Am. J. Dis. Child.
identification- maintenance of medical bacteria, vol. 1, p. 86-92. 131:560-563.
Williams & Wilkins, Baltimore, MD. 7. Chievitz, J., and A. H. Meyer. 1916. Recherches sur la
2. Bordet, J., and D. Gengou. 1906. Le microbe de la coqueluche. coqueluche. Ann. Inst. Pasteur 30:503.0
Ann. Inst. Pasteur 20:731. 8. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994.
3. Kendrick, P. L., E. Eldering, and W. L. Bradford. 1970. Bailey & Scott’s diagnostic microbiology, 9th ed. Mosby-Year
Whooping cough, p.106-117. In H. L. Bodily, E. L. Updyke, and Book, Inc., St. Louis, MO.
J. O. Mason (ed.), Diagnostic procedures for bacterial, mycotic and 9. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures hand-
parasitic infections, 5th ed. American Public Health Association, book, vol. 1. American Society for Microbiology, Washington, D.C.
New York, NY. 10. Koneman, E. W. 1988. Color atlas and textbook of diagnostic
4. Eldering, E., and P. L. Kendrick. 1936. Some practical microbiology, 3rd ed. J. B. Lippincott Company, Washington, D.C.
considerations in B. pertussis vaccine preparation. Am. J. Public
Health 24:309. Packaging
Bordet Gengou Agar Base 100 g 0048-15
500 g 0048-17
Also Known As Clostridium Difficile Agar. The complete medium is based on the
Brain Heart Infusion is abbreviated as BHI. formula of Willey and Bartlett5 and recommended for use in the isolation
of Clostridium difficile from fecal specimens. C. difficile is the major
Summary and Explanation cause of antibiotic-associated diarrhea and pseudomembranous colitis.6
In 1919, Rosenow 1 devised an excellent medium for culturing Brain Heart CC Agar is prepared with chloramphenicol and cyclo-
streptococci by supplementing dextrose broth with brain tissue. heximide (Actidione) according to the formulation of Ajello et al.7 and
Hayden2 revised Rosenow’s procedure by adding crushed marble to McDonough et al.8 These selective agents restrict growth of bacteria
the medium and reported favorable growth of organisms from dental and saprophytic fungi. Brain Heart CC Agar is used in the isolation of
pathogens. Brain Heart Infusion is a modification of the media fungi that cause systemic disease, such as Histoplasma capsulatum
described by Rosenow1 and Hayden2 in which infusion from calf and Blastomyces dermatiditis.
brains has replaced the brain tissue and disodium phosphate has Brain Heart Infusion media are specified in several standard methods
replaced the calcium carbonate buffer. references for food testing.9,10,11 Standard Methods for the Examination
Brain Heart Infusion Agar is used for cultivating a variety of fastidious of Water and Wastewater recommends Brain Heart Infusion media in
microorganisms, fungi and yeasts. This medium is used in combination tests for the verification of fecal streptococci.12
with penicillin and streptomycin. Roseburg, Epps and Clark3 reported Brain Heart Infusion is recommended by the National Committee for
that the isolation and cultivation of Actinomyces israelii was enhanced Clinical Laboratory Standards (NCCLS) for the preparation of inocula
on Brain Heart Infusion with 2% agar compared with 1% dextrose
used in antimicrobial susceptibility tests.13
infusion agar. Howell4 used Brain Heart Infusion with the addition
of 2% Bacto Agar and 10% sterile defibrinated horse blood for the Brain Heart Infusion w/o Dextrose is a basal medium used with added
cultivation of Histoplasma capsulatum. carbohydrates for fermentation studies.
Brain Heart Infusion Agar can be used with Clostridium Difficile Modifications of BHI media include:14
Antimicrobic Supplement CC, a selective supplement containing • Brain Heart Infusion Agar with penicillin (20,000 U) and
lyophilized cycloserine and cefoxitin, for the preparation of streptomycin (40 mg) for the selective isolation of pathogenic
fungi from specimens heavily contaminated with bacteria and Dextrose is a carbon energy source that facilitates organism growth.
saprophytic fungi; Sodium Chloride maintains the osmotic balance of the medium. Diso-
• Brain Heart Infusion with 3% sodium chloride for the isolation of dium Phosphate is a buffering agent. Bacto Agar is a solidifying agent.
Vibrio parahaemolyticus; The nutritionally rich broth formulation of Brain Heart Infusion
• Brain Heart Infusion with agar, yeast extract, sodium chloride, supports growth of a variety of microorganisms, as does the medium
inactivated horse serum and penicillin for the cultivation of when supplemented with agar and/or blood. BHI (broth) is often used
fastidious fungi; as a blood culture medium and as a basal medium for metabolic tests,
• Brain Heart Infusion with casein to support the growth of particularly for identifying streptococci.15 BHI with 0.5% Polysor-
Serratia marcescens; bate 80 can be used for detecting Mycobacterium avium-intracellulare
• Brain Heart Infusion with 0.7% agar to support the growth of complex organisms and M. tuberculosis from blood cultures.15
staphylococcal species for the production of enterotoxin; and, Brain Heart Infusion Agar is used in the aminoglycoside and
• Brain Heart Infusion with rabbit serum and yeast extract for the vancomycin screen test for resistant enterococci.16 BHI Agar with
cultivation of Mycoplasma equirhinis. 5-10% sheep blood and chloramphenicol (16 µg/ml) and gentamicin
(5 µg/ml) will inhibit the growth of bacteria while allowing growth of
Principles of the Procedure dimorphic fungi.15 This agar can be used as a primary plating medium
Infusion from Beef Heart, Calf Brains and Proteose Peptone provide
nitrogen, carbon, sulfur and vitamins in Brain Heart Infusion media.
for the growth of fungi since it has been shown to yield better recovery Brain Heart Infusion W/PAB and Agar
than the previously recommended Sabouraud Dextrose Agar.15 In Formula Per Liter
Brain Heart CC Agar, chloramphenicol is used as a broad-spectrum Calf Brains, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 200 g
antibiotic to inhibit a wide range of bacteria; cycloheximide inhibits Beef Heart, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 250 g
saprophytic fungi. Sheep blood provides essential growth factors for Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
fastidious fungi. Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
Clostridium Difficile Agar (Brain Heart Infusion Agar supplemented Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
with 5% sheep blood or 7% horse blood and Clostridium Difficile
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Antimicrobic Supplement CC) improves the growth and recovery of
p-Aminobenzoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g
C. difficile. Clostridium Difficile Agar markedly to completely inhibits
Final pH 7.4 ± 0.2 at 25°C
most aerobic and anaerobic enteric organisms other than C. difficile.5
The final concentration of cycloserine and cefoxitin in Clostridium Brain Heart CC Agar
Difficile Agar is 250 mcg/ml and 10 mcg/ml, respectively. Formula Per Liter
Brain Heart CC Agar can be supplemented with sheep blood (5-10%) Calf Brains, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 200 g
for enrichment and gentamicin (5 mg/l) for additional selectivity.14 Beef Heart, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 250 g
McDonough et al17 demonstrated that the temperature of incubation Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
affects the sensitivity of some pathogenic fungi to antibiotics. Incubate
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
the medium containing antibiotics at room temperature. The specimen
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
source and the type of fungus suspected will indicate the isolation Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
medium to be used. Both an antimicrobic-containing medium and Chloramphenicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 mg
a non-selective medium should be used on primary isolates with Cycloheximide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 mg
incubation at both 25°C and 37°C. Final pH 7.4 ± 0.2 at 25°C
Brain Heart Infusion w/PAB and Agar contains p-aminobenzoic acid
(0.05 g/l) to neutralize sulfonamides in the blood of patients receiving Clostridium Difficile Antimicrobic Supplement CC
this therapy. This formulation will also inactivate streptomycin in the Formula per 5 ml
ratio of 10 ml of medium to 100 units of streptomycin. The addition of Cycloserine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 mg
Cefoxitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mg
0.1% agar to Brain Heart Infusion w/PAB and Agar provides optimum
conditions for aerobic organisms, microaerophiles and obligate anaerobes. Precautions
Formula 1. For Laboratory Use.
Brain Heart Infusion 2. Brain Heart CC Agar: HARMFUL. HARMFUL BY INHALATION
Formula Per Liter AND IF SWALLOWED. POSSIBLE RISK OF IRREVERSIBLE
Calf Brains, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 200 g EFFECTS. POSSIBLE RISK OF HARM TO THE UNBORN
Beef Heart, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 250 g CHILD. Do not breathe dust. In case of accident or if you feel
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g unwell, seek medical advice immediately (show the label where
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g possible). Wear suitable protective clothing. Keep container tightly
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g closed. TARGET ORGAN(S): Eyes/Ears, Cardiovascular, Muscles,
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g Blood, Lymph Glands, Nerves, Urogenital.
Final pH 7.4 ± 0.2 at 25°C FIRST AID: In case of contact with eyes, rinse immediately with
Brain Heart Infusion Agar plenty of water and seek medical advice. After contact with skin,
Formula Per Liter wash immediately with plenty of water. If inhaled, remove to fresh
Calf Brains, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 200 g air. If not breathing, give artificial respiration. If breathing is
Beef Heart, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 250 g difficult, give oxygen. Seek medical advice. If swallowed, induce
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g vomiting; seek medical advice immediately and show this container
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g or label.
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g 3. Brain Heart CC Agar: Avoid overheating or holding the medium
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g in the melted state. Doing so tends to reduce the selective properties
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g of the medium.
Final pH 7.4 ± 0.2 at 25°C
4. When testing human serum, treat all specimens as infectious agents.
Brain Heart Infusion w/o Dextrose 5. Follow proper established laboratory procedures in handling and
Formula Per Liter disposing of infectious materials.
Calf Brains, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 200 g
Beef Heart, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 250 g Storage
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Store the dehydrated medium below 30°C. The dehydrated medium is
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
very hygroscopic. Keep container tightly closed.
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Final pH 7.4 ± 0.2 at 25°C Store Clostridium Difficile Antimicrobic Supplement CC at 2-8°C.
8. McDonough, E. S., L. K. Georg, L. Ajello, and S. Brinkman. 17. McDonough, E. S., L. Ajello, L. K. Georg, and S. Brinkman.
1960. Growth of dimorphic human pathogenic fungi on media 1960. In vitro effects of antibiotics on yeast phase of Blastomyces
containing cycloheximide and chloramphenicol. Mycopathol. dermatitidis and other fungi. J. Lab. Clin. Med. 55:116-119.
Mycol. Appl. 13:113. 18. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures
9. Cunnif, P. (ed). 1995. Official Methods of Analysis AOAC handbook, vol. 1. American Society for Microbiology, 6th ed.
International, 16th ed. AOAC International, Arlington, VA. American Society for Microbiology, Washington, D.C.
10. Association of Official Analytical Chemists. 1995. Bacteriological 19. Georg, L. K., L. Ajello, and C. Papageorge. 1954. Use of
analytical manual, 8th ed. AOAC International, Gaithersburg, MD. cycloheximide in the selective isolation of fungi pathogenic to man.
11. Vanderzant, C., and D. F. Splittstoesser (ed). 1992. Compen- J. Lab Clin. Med. 44:422-428.
dium of methods for the microbiological examination of food, 20. Onderdonk, A. B., and S. D. Allen. 1995. Clostridium, p. 574 -586.
3rd ed. American Public Health Association, Washington, D.C. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H.
12. Greenberg, A. E., L. S. Clesceri, and A. D. Eaton (ed.). 1995. Yolken (ed.). Manual of clinical microbiology, 6th ed. American
Membrane filter techniques, 9,72-74. Standard methods for the Society for Microbiology, Washington, D.C.
examination of water and wastewater, 19th ed. American Public
Health Association, Washington, D.C. Packaging
Brain Heart Infusion 100 g 0037-15
13. National Committee for Clinical Laboratory Standards. 1994.
500 g 0037-17
M11-A3, Vol. 13, No. 26, Methods for antimicrobial susceptibility
2 kg 0037-07
testing of anaerobic bacteria. National Committee for Clinical 10 kg 0037-08
Laboratory Standards, Villanova, PA.
14. Atlas, R. M. 1993. Handbook of microbiological media, p. 147-153, Brain Heart Infusion Agar 100 g 0418-15
CRC Press, Boca Raton, FL. 500 g 0418-17
2 kg 0418-07
15. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey
& Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc. Brain Heart CC Agar 500 g 0483-17
St. Louis, MO. Brain Heart Infusion w/PAB and Agar 500 g 0499-17
16. Swenson, J. M., N. C. Clark, M. J. Ferraro, D. F. Sahm, Brain Heart Infusion w/o Dextrose 10 kg 0502-08
G. Doern, M. A. Pfaller, L. B. Reller, M. P. Weinstein,
R. J. Zabransky, and F. C. Tenover. 1994. Development of a Clostridium Difficile Antimicrobic
standardized screening method for detection of vancomycin- Supplement CC 6 x 5 ml 3194-57*
resistant enterococci. J. Clin. Microbiol. 32:1700-1704. *Store at 2-8°C
Intended Use
User Quality Control Bacto Brain Heart Infusion, Porcine is used for cultivating a wide
Identity Specifications variety of microorganisms.
Dehydrated Appearance: Light tan, free-flowing, homogeneous.
Solution: 3.7% solution, soluble in distilled
Also Known As
or deionized water. Light to medium Brain Heart Infusion, Porcine is abbreviated as BHI, Porcine.
amber, clear.
Reaction of 3.7% Summary and Explanation
Solution at 25°C: pH 7.4 ± 0.2 Rosenow1 devised an excellent medium for culturing streptococci by
supplementing Dextrose Broth with brain tissue. Hayden,2 revising
Cultural Response Rosenow’s procedure by adding crushed marble to the medium,
Prepare medium per label directions. Inoculate tubes with test
organisms and incubate at 35 ± 2°C for 18-48 hours. reported favorable growth of organisms from dental pathogens. Brain
INOCULUM Heart Infusion (0037) is a modification of the media described
ORGANISM ATCC® CFU GROWTH by Rosenow1 and Hayden.2 Infusion from calf brains has replaced the
Neisseria meningitidis 13090* 100-1,000 fair brain tissue and Disodium Phosphate has replaced the Calcium
Streptococcus pneumoniae 6305 100-1,000 good Carbonate buffer.
Streptococcus pyogenes 19615* 100-1,000 good
Brain Heart Infusion, Porcine was developed as an alternative to Brain
The cultures listed are the minimum that should be used for Heart Infusion formula, and replaces calf brains and beef heart with
performance testing. porcine brains and heart. Brain Heart Infusion, Porcine was developed
*These cultures are available as Bactrol™ Disks and should be used for pharmaceutical and vaccine production and can replace the
as directed in Bactrol Disks Technical Information.
traditional BHI depending on organism and production application.
BHI, Porcine was formulated with no bovine components to minimize Materials Required But Not Provided
Bovine Spongiform Encephalopathy (BSE) risk. Glassware
The nutritionally rich formula of BHI is used to grow a variety of Autoclave
microorganisms The original Brain Heart Infusion media are specified Incubator
in standard methods for multiple applications.3,4,5,6 Waterbath (optional)
Principles of the Procedure Method of Preparation
Infusion from pork brains, infusion from pork heart and Pork Peptone 1. Dissolve 37 grams in 1 liter distilled or deionized water.
No. 2 provides nitrogen, carbon, sulfur and vitamins in Brain Heart 2. Autoclave at 121°C for 15 minutes.
Infusion, Porcine. Dextrose is the carbon energy source to facilitate 3. Dispense as desired.
organism growth. Sodium Chloride maintains the osmotic balance of
the medium. Disodium Phosphate is the buffering agent. Specimen Collection and Preparation
Obtain and process specimens according to the techniques and
Formula procedures established by laboratory policy.
Brain Heart Infusion, Porcine
Formula Per Liter Test Procedure
Pork Brains, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 200 g See appropriate references for specific procedures using Brain
Pork Heart, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 250 g Heart Infusion.
Bacto Pork Peptone No. 2 . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g Results
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Refer to appropriate references and procedures for results.
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Final pH 7.4 ± 0.2 at 25°C References
1. Rosenow, E. C. 1919. Studies on elective localization. J. Dent.
Precautions Res. 1:205-249.
1. For Laboratory Use. 2. Hayden, R. L. 1923. Elective localization in the eye of bacteria
2. Follow proper established laboratory procedures in handling and from infected teeth. Arch. Int. Med. 32:828-849.
disposing of infectious materials. 3. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compendium
of methods for the microbiological examination of food, 3rd ed.
Storage American Public Health Association, Washington, D.C.
Store the dehydrated medium below 30°C. The dehydrated medium is 4. Association of Official Analytical Chemists. 1995. Bacteriological
very hygroscopic. Keep container tightly closed. analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
Expiration Date 5. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). 1995.
Standard methods for the examination of water and wastewater,
The expiration date applies to the product in its intact container when 19th ed. American Public Health Association, Washington, D.C.
stored as directed. Do not use a product if it fails to meet specifications
for identity and performance. 6. Cunnif, P. (ed).1995. Official methods of analysis, AOAC
International, 16th ed. AOAC International, Arlington, VA.
Procedure Packaging
Materials Provided Brain Heart Infusion, Porcine 500 g 0561-17
Brain Heart Infusion, Porcine
is the carbon source, and Sodium Chloride maintains osmotic Expiration Date
equilibrium. Sodium Thioglycollate and Sodium Formaldehyde The expiration date applies to the product in its intact container when
Sulfoxylate are the reducing agents. Resazurin serves as an stored as directed. Do not use a product if it fails to meet specifications
indicator of anaerobiosis with a pink color indicating the presence of for identity and performance.
oxygen. Bacto Agar is the solidifying agent.
Procedure
Formula Materials Provided
Brewer Anaerobic Agar Brewer Anaerobic Agar
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Materials Required But Not Provided
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 10 g Glassware
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Autoclave
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Incubator (35°C)
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Waterbath (45-50°C) (optional)
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g Sterile Petri dishes
Sodium Thioglycollate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g Brewer Anaerobic Petri dish covers (optional)
Sodium Formaldehyde Sulfoxylate . . . . . . . . . . . . . . . . . . . 1 g
Resazurin, Certified . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002 g
Method of Preparation
1. Suspend 58 grams in 1 liter distilled or deionized water.
Final pH 7.2 ± 0.2 at 25°C
2. Heat to boiling to dissolve completely.
Precautions 3. Autoclave at 121°C for 15 minutes. Cool to 45-50°C.
1. For Laboratory Use. 4. Dispense as desired.
2. Follow proper established laboratory procedures in handling and Specimen Collection and Preparation
disposing of infectious materials.
Anaerobic bacteria are overlooked or missed unless the specimen is
Storage properly collected and transported to the laboratory.2 Obtain and
Store the dehydrated medium below 30°C. The dehydrated medium is process specimens according to the techniques and procedures
very hygroscopic. Keep container tightly closed. established by institutional policy.
Test Procedure
Standard Petri Dishes:2
1. Inoculate a properly obtained specimen onto the medium, and
User Quality Control streak to obtain isolated colonies.
Identity Specifications 2. Immediately incubate anaerobically at 35 ± 2°C.
Dehydrated Appearance: Light beige, free-flowing, homogeneous. 3. Examine at 24 hours if incubating plates in an anaerobic chamber.
Solution: 5.8% solution, soluble in distilled or Examine at 48 hours if incubating plates in an anaerobic jar or
deionized water on boiling. Light pouch, or if using Brewer anaerobic dish cover.
amber, slightly opalescent while hot,
turning red on aeration and cooling. 4. Extended incubation may be necessary to recover some anaerobes.
Prepared Medium: Light pink ring at outer edge, light Brewer Anaerobic Agar Plates:
amber in center, slightly opalescent. 1. Dispense 50-60 ml of Brewer Anaerobic Agar into a standard
Reaction of 5.8% Petri dish. For best results use porous tops to obtain a dry surface.
Solution at 25°C pH 7.2 ± 0.2 2. Inoculate the surface of the medium by streaking; avoid the edges
of the plates.
Cultural Response 3. Replace the standard Petri dish lid with a sterile Brewer anaerobic
Prepare Brewer Anaerobic Agar per label directions. Inoculate dish cover. The cover should not rest on the Petri dish bottom.
the plates using the streak method. Incubate plates at 35 ± 2°C The inner glass ridge should seal against the uninoculated
anaerobically for 40-48 hours.
periphery of the agar. It is essential that the sealing ring inside the
INOCULUM
ORGANISM ATCC® CFU GROWTH cover is in contact with the medium. This seal must not be broken
Bacteroides fragilis 25285* 100-1,000 good before the end of the incubation period. A small amount of air is
Clostridium beijerinckii 17795 100-1,000 good caught over the surface of the medium, and the oxygen in this space
Clostridium perfringens 12924 100-1,000 good reacts with the reducing agents to form an anaerobic environment.
4. Incubate aerobically as desired.
The cultures listed are the minimum that should be used for
performance testing. For a complete discussion on anaerobic and microaerophilic
*This culture is available as Bactrol™ Disks and should be used as bacteria from clinical specimens, refer to the appropriate
directed in Bactrol Disks Technical Information. procedures outlined in the references.2,3,4 For the examination of
anaerobic bacteria in food refer to standard methods.7,8,9
yellow-green zone. Brilliant Green Agar is not suitable for the 4. Galton, M. M., and M. S. Quan. 1944. Salmonella isolated in
isolation of S. typhi or Shigella; however, some strains of S. typhi may Florida during 1943 with the combined enrichment method of
grow forming red colonies. Kauffmann. Am. J. Public Health 34:1071.
5. Broh-Kahn, R. H. 1946. The laboratory diagnosis of enteric
Limitations of the Procedure infections caused by the Salmonella-Shigella group. Military
1. Colonies of Salmonella spp. vary from red-pink-white depending Surgeon 99:770-776.
on length of incubation and strain.11
6. Tate, C. R., and R. G. Miller. 1990. Modification of brilliant
2. Medium is normally orangish-brown in color; however, on green agar by adding sodium novobiocin to increase selectivity for
incubation, it turns bright red but returns to normal color at Salmonella. The Maryland Poultryman 4:7-11.
room temperature.11
7. Federal Register. 1993. Chicken Disease Caused by Salmonella
3. Studies by Taylor12 showed that slow lactose fermenters, Proteus,
enteritidis; proposed rule. Fed. Regis. 58:41048-41061.
Citrobacter, and Pseudomonas may grow on Brilliant Green
Agar as red colonies. 8. Pezzlo, M. (ed.). 1992. Aerobic bacteriology, p. 1.0.1-1.20.47. In
H. D.Isenberg, (ed.), Clinical microbiology procedures handbook,
4. In routine examination of clinical specimens or other materials for
vol. 1. American Society for Microbiology, Washington, D.C.
the gram-negative intestinal pathogens, other primary plating
media such as MacConkey Agar, and fluid enrichments such as 9. Gray, L. D. 1995. Escherichia, Salmonella, Shigella and Yersinia,
Tetrathionate Broth and Selenite Broth, should be used with p. 450-456. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
Brilliant Green Agar. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology,
5. S. typhi does not grow adequately on this medium. Shigella spp. do 6th ed. American Society for Microbiology, Washington, D.C.
not grow.11 10 United States Pharmacopeial Convention. 1995. The United
States pharmacopeia, 23rd ed. The United States Pharmacopeial
References Convention, Rockville, MD.
1. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig. 11. MacFaddin, J. F. 1985. Media for isolation-cultivation-
1993. Pathogens in milk and milk products, p. 103-212. In R. T. identification-maintenance of medical bacteria, vol. 1. Williams &
Marshall, (ed.). Standard methods for the examination of Wilkins, Baltimore, MD.
dairy products, 16th ed. American Public Health Association,
12. Taylor, W. I. 1965. Isolation of shigellae. I. Xylose lysine
Washington, D.C.
agars: New media for isolation of enteric pathogens. Am. J. Clin.
2. Kristensen, M., V. Lester, and A. Jurgens. 1925. On the use of Pathol. 44:471.
trypsinized casein, brom thymol blue, brom cresol purple,
phenol red and brilliant green for bacteriological nutrient media. Packaging
Br. J. Exp. Pathol. 6:291. Brilliant Green Agar 100 g 0285-15
3. Kauffmann, F. 1935. Weitere Erfahrungen mit den kombinierten 500 g 0285-17
Anreicherungsverfahren für Salmonellabacillen. Z. Hyg.
Infektionskr. 117:26.
Intended Use materials3 and meat and meat products.4 It is recommended by the British
Bacto Brilliant Green Agar Modified is used for isolating Salmonella Poultry Meat Society5 for the examination of poultry and poultry products.
from water, sewage and foodstuffs. The recommended procedures include using complementary selective
culture media and techniques to increase the likelihood of isolating
Summary and Explanation multiple serotypes of Salmonella from samples.6
Kampelmacher1 proposed the formula for a selective medium to isolate
Salmonella from pig feces and minced meat. Brilliant Green Agar
Principles of the Procedure
Modified is more selective than Desoxycholate Citrate Agar and other Brilliant Green Agar Modified contains Beef Extract and Bacto Peptone
brilliant green media, and inhibits the growth of Pseudomonas as sources of carbon, nitrogen, vitamins and minerals. Yeast Extract
aeruginosa and Proteus sp. which may resemble Salmonella. Salmonella supplies B-complex vitamins which stimulate bacterial growth. Lactose
cholerasuis grows well on Brilliant Green Agar Modified, but poorly and Sucrose are carbohydrate sources. In the presence of Phenol Red, a
on Desoxycholate Citrate Agar.2 pH indicator, nonlactose and/or nonsucrose-fermenting Salmonella will
produce red colonies. Brilliant Green inhibits gram positive organisms
Brilliant Green Agar Modified is recommended for the isolation of and many gram negative bacteria, except Salmonella. Bacto Agar is a
Salmonella, other than Salmonella typhi, from water and associated solidifying agent.
Intended Use air. If not breathing, give artificial respiration. If breathing is difficult,
Bacto Brilliant Green Bile Agar is used for isolating, differentiating give oxygen. Seek medical advice. If swallowed seek medical
and enumerating coliform bacteria. advice immediately and show this container or label.
3. Follow proper established laboratory procedures in handling and
Also Known As disposing of infectious materials.
Brilliant Green Bile Agar (BGBA) is also known as Brilliant Green
Agar2 (BGA). Storage
Store the dehydrated medium below 30°C. The dehydrated medium is
Summary and Explanation very hygroscopic. Keep container tightly closed.
Noble and Tonney1 described Brilliant Green Bile Agar for determining
This medium is light sensitive. Protect from exposure to direct sunlight.
the relative density of coliform bacteria in water and sewage. The
medium is particularly useful in selectively isolating Salmonella spp. Expiration Date
from other coliform bacteria. The American Public Health Association
The expiration date applies to the product in its intact container when
(APHA) specifies a qualitative procedure to isolate and identify
stored as directed. Do not use a product if it fails to meet specifications
Salmonella spp. from water and wastewater using concentration,
for identity and performance.
enrichment and selective growth.2
Cultural Response
Prepare m Brilliant Green Broth per label directions. Inoculate using the
membrane filter technique and incubate at 35 ± 2°C in a humid atmosphere
for 18-24 hours.
INOCULUM
ORGANISM ATCC® CFU GROWTH COLOR OF COLONY
Escherichia coli 25922* 20-80 good yellow
Salmonella enteritidis 13076 20-80 good pink to red
Salmonella typhimurium 14028* 20-80 good pink to red
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in
Bactrol Disks Technical Information.
Intended Use Bergère et al,3 who reported that their medium could be used for detecting
Bacto Bryant and Burkey Medium is used for detecting and and enumerating C. tyrobutyricum spores in milk and dairy products.3-5
enumerating spores of lactate-fermenting Clostridium in milk and
dairy products. Principles of the Procedure
Tryptone, Yeast Extract, Beef Extract Desiccated and L-Cysteine
Summary and Explanation Hydrochloride provide nutrients and cofactors required for good
Bryant and Burkey Medium is based on the lactate fermentation media growth of clostridia. Selectivity of this medium is achieved through
described by Rosenberger1 and Bryant and Burkey2, as modified by the addition of Sodium Acetate, which is also the principal promoter of
Formula Procedure
Bryant and Burkey Medium Materials Provided
Formula Per Liter Bryant and Burkey Medium
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Materials Required But Not Provided
Bacto Beef Extract, Desiccated . . . . . . . . . . . . . . . . . . . . . 7.5 g Flasks with closures
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Distilled or deionized water
L-Cysteine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g Autoclave
Sodium Lactate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Incubator (35 ± 2°C)
Resazurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0025 g
Method of Preparation
Final pH 5.9 ± 0.2 at 25°C
NOTE: This product contains sodium lactate; it is not necessary to add
Precautions sodium lactate during preparation.
1. For Laboratory Use. 1. Dissolve 38 grams in 1 liter of distilled or deionized water.
2. Follow proper, established laboratory procedures in handling and 2. Dispense 10 ml amounts into tubes.
disposing of infectious materials. 3. Autoclave at 121°C for 15 minutes.
Specimen Collection and Preparation
1. Collect food samples in sterile containers and transport immediately
User Quality Control to the laboratory following recommended guidelines.
Identity Specifications 2. Process each food sample using procedures appropriate for that
Dehydrated Appearance: Tan, free-flowing, homogeneous. sample.
Solution: 3.8% solution, soluble in distilled or Test Procedure
deionized water. Solution is light to
medium amber, clear when hot, Three-tube Most Probable Number (MPN) Method
becoming red upon cooling. 1. Before use, heat tubes to boiling for 10 minutes to regenerate
Reaction of 3.8% anaerobic conditions. Note: This step is required only with tubes
Solution at 25°C: pH 5.9 ± 0.2 stored under aerobic conditions. Tubes stored under anaerobic
conditions or freshly sterilized tubes do not need additional heating.
Cultural Response 2. Prepare 10-fold dilutions of the sample and inoculate triplicate
Prepare Bryant and Burkey Medium per label directions. tubes of Bryant and Burkey Medium with 1 ml of each sample
Inoculate using Most Probable Number (MPN) method and dilution.
incubate at 35 ± 2°C for 6 days.
3. Pour approximately 2 ml of melted paraffin (60-65°C), previously
ATCC® OR GAS
ORGANISM STRAIN INOCULUM GROWTH PRODUCTION autoclaved at 121°C for 20 minutes, into each tube.
Clostridium CNRZ 500 MPN method good >1 cm of gas 4. Heat the tubes at 75°C for 15 minutes to kill vegetative cells and
tyrobutyricum activate spores; allow to cool to room temperature.
Clostridium CNRZ 510 MPN method good >1 cm of gas 5. Incubate tubes at 35°C for 6 days.
tyrobutyricum
Clostridium CNRZ 608 MPN method good >1 cm of gas 6. Examine tubes for growth and gas production after 48 hours of
tyrobutyricum incubation and daily for up to 6 days.
Clostridium 25755 MPN method good >1 cm of gas Results
tyrobutyricum
Tubes showing both growth and production of gas (indicated by upward
The cultures listed are the minimum that should be used for movement of the paraffin more than 1 cm) are considered positive for
performance testing. the presence of lactate-fermenting clostridial spores. Determine the
spore count using the Most Probable Number (MPN) method.
enumerate total heterotrophs and hydrocarbon degradation Wear suitable protective clothing. Keep container tightly closed.
by microorganisms during bioremediation of Prince William Sound TARGET ORGAN(S): Blood, Liver, Nerves.
following the Exxon Valdez oil spill.3,4 FIRST AID: In case of contact with eyes, rinse immediately with
plenty of water and seek medical advice. After contact with skin,
Principles of the Procedure wash immediately with plenty of water. If inhaled, remove to fresh
Magnesium Sulfate, Calcium Chloride, and Ferric Chloride provide air. If not breathing, give artificial respiration. If breathing is
trace elements necessary for bacterial growth. Potassium Nitrate is a difficult, give oxygen. Seek medical advice. If swallowed seek
nitrogen source, while Monopotassium Phosphate and Ammonium medical advice immediately and show this container or label.
Phosphate Dibasic provide buffering capability. 3. Follow proper established laboratory procedure in handling and
disposing of infectious materials.
Formula
Bushnell-Haas Broth Storage
Formula Per Liter Store the dehydrated medium below 30°C. The dehydrated medium is
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g very hygroscopic. Keep container tightly closed. Store the prepared
Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g medium at 2-8°C.
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Ammonium Phosphate Dibasic . . . . . . . . . . . . . . . . . . . . . . 1 g Expiration Date
Potassium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g The expiration date applies to the product in its intact container when
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g stored as directed. Do not use a product if it fails to meet specifications
Final pH 7.0 ± 0.2 at 25°C for identity and performance.
Precautions Procedure
1. For Laboratory Use. Materials Provided
2. IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM Bushnell-Haas Broth
AND SKIN. Avoid contact with skin and eyes. Do not breathe dust.
Materials Required But Not Provided
Glassware
User Quality Control Autoclave
Incubator (25-30°C)
Identity Specifications
Dehydrated Appearance: Beige with pink tint, free-flowing, Method of Preparation
homogeneous. 1. Dissolve 3.27 grams in 1 liter distilled or deionized water.
Solution: 0.327% solution not soluble in 2. Dispense as desired and autoclave at 121°C for 15 minutes.
distilled or deionized water, white 3. Cool to 45-50ºC.
precipitate remains. Solution, after
autoclaving, is colorless to very NOTE: A precipitate that is white prior to sterilization and turns yellow
light amber, clear supernatant over to orange after sterilization is normal.
yellow-orange precipitate.
Prepared Medium: Colorless to very light amber, clear Specimen Collection and Preparation
supernatant over yellow-orange 1. Collect samples in sterile containers or with sterile swabs and
precipitate. transport immediately to the laboratory.
Reaction of 0.327% Test Procedure
Solution at 25°C: pH 7.0 ± 0.2
1. Inoculate the collected sample directly into the broth.
Cultural Response 2. Overlay the broth with a sterile hydrocarbon source.
Prepare Bushnell-Haas Broth per label directions. Inoculate in 3. Incubate aerobically at 25-30°C.
duplicate with the test organisms. Add sterile mineral oil (the
hydrocarbon source) to one set. Incubate at 25-30°C 4. Examine tubes daily for growth for up to one week.
for up to 1 week. Results
RECOVERY
ORGANISM ATCC® INOCULUM PLAIN w/Hydrocarbon Organisms capable of degrading hydrocarbons should show growth in
Pseudomonas aeruginosa 9027 100-1,000 none to poor good the Bushnell-Haas Broth supplemented with a hydrocarbon source.
Pseudomonas aeruginosa 10145 100-1,000 none to poor good
Pseudomonas aeruginosa 14207 100-1,000 none to poor good Limitations of the Procedure
Pseudomonas aeruginosa 27853* 100-1,000 none to poor good 1. Because the nutritional requirements of organisms vary, some strains
The cultures listed are the minimum that should be used for may be encountered that fail to grow or grow poorly in this medium.
performance testing.
*The cultures are available as Bactrol™ Disks and should be used References
as directed in Bactrol Disks Technical Information. 1. Bushnell, L. D., and H. F. Haas. 1941. The utilization of certain
hydrocarbons by microorganisms. J. Bacteriol. 41:653-673.
2. Allred, R. C., R. J. DeGray, R. W. Edwards, H. G. Hedrick, D. 4. Brown, E. J., and J. F. Braddock. 1990. Sheen Screen,
E. Klemme, M. Rogers, M. Wulf, and H. Hodge. 1963. Proposed a miniaturized most-probable-number method for enumeration
procedures for microbiological examination of fuels. SIM Special of oil-degrading microorganisms. Appl. Environ. Microbiol.
Publications, Number 1. Merck, Sharp & Dohme Research 56:3895-3896.
Laboratories, Rahway, NJ.
3. Bragg, J. R., J. C. Roffall, and S. McMillen. 1990. Column flow Packaging
studies of bioremediation in Prince William Sound. Exxon Bushnell-Haas Broth 500 g 0578-17
Production Research Co., Houston, TX. 10 kg 0578-08
Summary and Explanation infusion or brucella agar, enriched with 5 to 7% horse or rabbit blood,
The genus Campylobacter was proposed in 1963 for Vibrio fetus, a will support the growth of H. pylori.1
species not exhibiting true characteristics of Vibrionaceae.1 In 1977, The Skirrow formulation is recommended for clinical specimens.1
Skirrow succeeded in isolating C. jejuni from fecal samples. Skirrow Campylobacter Agar Base is specified for food testing in Standard
used a selective medium, incubated at 42°C in an atmosphere of Methods.3,4
5% oxygen, 10% carbon dioxide and 85% nitrogen. Skirrow
confirmed this organism as a major etiologic agent of human enteritis,1 Principles of the Procedure
an infection acquired through ingestion of water or food contaminated Campylobacter Agar Base is a nutritionally rich medium based on
with the microorganism. Blood Agar Base No. 2, rather than on Brucella Agar, to support more
The Skirrow formulation includes blood agar supplemented with luxuriant Campylobacter growth because Trimethoprim is more active
vancomycin, polymyxin B and trimethoprim for the selective isolation in Blood Agar Base No. 2. Supplementation of the base with antimi-
of C. fetus subsp. jejuni.2 Blaser et al. further Incorporated cephalothin crobial agents as described by Skirrow2 and Blaser et al.5,6 provides for
and amphotericin B to improve inhibition of normal enteric flora. markedly reduced growth of normal enteric bacteria and improved
recovery of C. fetus supsp. jejuni from fecal specimens. Growth of
In 1983, spiral-shaped organisms resembling campylobacteria were
fungi is markedly to completely inhibited with Campylobacter
isolated from the human stomach. The discovery sparked renewed
Antimicrobic Supplement B due to the presence of amphotericin B.
interest in the etiology of human type B gastritis.1 After genetic analysis,
the genus Helicobacter was created and most attention focused on
H. pylori. Specimens of gastric biopsies, brushings, or aspirates are
used for the detection of H. pylori. Chocolate agar and brain heart Uninoculated Campylobacter fetus
plate subsp. jejuni
ATCC® 33291
Formula Do not use a product if it fails to meet specifications for identity and
Campylobacter Agar Base performance.
Formula Per Liter
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 15 g
Procedure
Liver Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g Materials Provided
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Campylobacter Agar Base
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Campylobacter Antimicrobic Supplement B
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 g Campylobacter Antimicrobic Supplement S
Campylobacter Antimicrobic Supplement B Materials Required But Not Provided
Ingredients per vial 10 ml vial 5 ml vial Specimen collection containers or sterile rectal swabs
Vancomycin . . . . . . . . . . . . . . . . . . . . . . . 10 mg 5 mg Microaerophilic environment system
Polymyxin B . . . . . . . . . . . . . . . . . . . . 2,500 units 1,250 units Bunsen burner or incinerator
Trimethoprim . . . . . . . . . . . . . . . . . . . . . . 5 mg 2.5 mg Sterile defibrinated blood or sterile lysed horse blood
Cephalothin . . . . . . . . . . . . . . . . . . . . . . . 15 mg 7.5 mg Inoculating loops
Amphotericin B . . . . . . . . . . . . . . . . . . . . 3 mg 1 mg
Incubator (42°C)
Campylobacter Antimicrobic Supplement S Sterile Petri dishes
Ingredients per vial 10 ml vial 5 ml vial
Vancomycin . . . . . . . . . . . . . . . . . . . . . . . 10 mg 5 mg Method of Preparation
Polymyxin B . . . . . . . . . . . . . . . . . . . . 2,500 units 1,250 units Campylobacter Agar Base:
Trimethoprim . . . . . . . . . . . . . . . . . . . . . . 5 mg 2.5 mg 1. Suspend 39.5 grams of Campylobacter Agar Base in 1 liter of
Precautions distilled or deionized water.
1. For Laboratory Use. 2. Heat to boiling to dissolve completely.
2. Campylobacter Antimicrobic Supplement B 3. Autoclave at 121°C for 15 minutes.
HARMFUL. IRRITATING TO EYES, RESPIRATORY SYSTEM 4. Cool to 45-50°C. Aseptically add 5-7% sterile lysed horse blood
AND SKIN. MAY CAUSE SENSITIZATION BY INHALATION (final concentration) or 10% sterile defibrinated sheep blood
AND SKIN CONTACT. POSSIBLE RISK OF IRREVERSIBLE (final concentration).
EFFECTS. MAY CAUSE HARM TO THE UNBORN CHILD. 5. Aseptically add 1% rehydrated Campylobacter Antimicrobic
Avoid contact with skin and eyes. Do not breathe dust. Wear suitable Supplement B or Campylobacter Antimicrobic Supplement S
protective clothing. Keep container tightly closed. Target Organs: (10 ml per liter or 5 ml per 500 ml of basal medium). Mix well.
Blood, Kidneys, Ears, Bone Marrow. 6. Dispense 20 ml amounts into 90 mm Petri dishes.
Campylobacter Antimicrobic Supplement S Campylobacter Antimicrobic Supplement B
HARMFUL. IRRITATING TO EYES, RESPIRATORY SYSTEM Campylobacter Antimicrobic Supplement S
AND SKIN. MAY CAUSE SENSITIZATION BY INHALATION 1. Aseptically rehydrate the lyophilized supplement with 5 or 10 ml
AND SKIN CONTACT. MAY CAUSE HARM TO THE UNBORN of sterile distilled or deionized water, depending on label directions.
CHILD. Avoid contact with skin and eyes. Do not breathe dust. 2. Invert the vial gently several times to dissolve the powder. Use
Wear suitable protective clothing. Keep container tightly closed. within 24 hours of rehydration.
Target Organs: Kidneys, Ears, Bone Marrow.
Specimen Collection and Preparation
FIRST AID: In case of contact with eyes, rinse immediately with
Fecal specimens should be collected in sterile containers or with a
plenty of water and seek medical advice. After contact with skin,
sterile rectal swab and transported immediately to the laboratory for
wash immediately with plenty of water. If inhaled, remove to fresh
processing. If the specimen cannot be inoculated onto appropriate
air. If not breathing, give artificial respiration. If breathing is diffi-
media within four hours after collection, the specimen should be
cult, give oxygen. Seek medical advice. If swallowed seek medical
maintained or transported in Cary-Blair Transport Medium.1
advice immediately and show this container or label.
3. Follow proper established laboratory procedures in handling and Test Procedure
disposing of infectious materials. 1. Inoculate the specimen directly onto the surface of the prepared
Campylobacter Agar plate and streak for isolation.
Storage 2. Incubate at 42°C under a microaerophilic atmosphere containing
Store the dehydrated medium below 30°C. The dehydrated medium is 5-6% oxygen and 3-10% carbon dioxide. Consult appropriate
very hygroscopic. Keep container tightly closed. references for specific information on establishing a microaerophilic
Store lyophilized and rehydrated Campylobacter Antimicrobic Supple- environment.1,3,7
ments B and S at 2-8°C. Use the rehydrated supplement within 24 hours.
Results
Expiration Date The colonies of Campylobacter species appear as non-hemolytic, flat
The expiration date applies to the product in its intact container when and gray with an irregular edge or raised and round with a mucoid
stored as directed. appearance. Some strains may appear tan or slightly pink. Swarming
or spreading may be observed on moist surfaces. Growth of normal 4. Association of Official Analytical Chemists. 1995. Bacteriological
enteric bacteria is markedly to completely inhibited. Growth of fungi analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
is markedly to completely inhibited on Campylobacter Agar Blaser. 5. Blaser, M. J., V. Berkowitz, F. M. LaForce, J. Cravens, L. B.
Colonies are selected for further biochemical characterization. Reller, and W. L. Wang. 1979. Campylobacter enteritis: clinical
Identification is based on a positive oxidase reaction and characteristic and epidemiologic features. Ann. Intern. Med. 91:179-185.
darting motility in a wet mount.1 For further differentiation into 6. Blaser, M. J., J. Cravens, B. W. Powers, and W. L. Wang. 1978.
species and biotypes, test for catalase activity, urease, hydrogen Campylobacter enteritis associated with canine infection. Lancet
sulfide production, nitrate reduction, hippurate, indoxyl acetate, DNA (ii):979-980.
hydrolysis and susceptibility to cephalothin and nalidixic acid.1 7. Koneman E. W., S. D. Allen, W. M. Janda, P. C.
Schreckenberger, W. C. Winn. 1983. Color atlas and textbook
Limitations of the Procedure of diagnostic microbiology, 5th ed. J. B. Lippencott-Raven
1. Campylobacter Agar prepared with either Campylobacter Antimi- Publishers. Washington, D.C.
crobic Supplement S or Campylobacter Antimicrobic Supplement
B is selective primarily for Campylobacter species. Biochemical Packaging
testing using a pure culture is necessary for complete identification. Campylobacter Agar Base 2 kg 1820-07
Consult appropriate references for further information.1,3,7 Campylobacter Agar Kit Blaser 3279-32
2. Growth of Campylobacter fetus subsp. intestinalis may be To prepare: 6 x 1 liter
dramatically inhibited on Campylobacter Agar Blaser due to the Campylobacter Agar Base 6 x 39.5 grams
presence of cephalothin. The use of Campylobacter Agar Skirrow Campylobacter
and incubation at 35°C is suggested when isolating this organism Antimicrobic Supplement B 6 x 10 ml
from mixed populations. Campylobacter Agar Kit Blaser 3279-40
3. Some strains of C. fetus subsp. jejuni may be encountered that fail To prepare: 6 x 500 ml
to grow or grow poorly on prepared Campylobacter Agar. Campylobacter Agar Base 6 x 19.75 grams
4. Some strains of normal enteric organisms may be encountered that Campylobacter
are not inhibited or only partially inhibited on Campylobacter Agar. Antimicrobic Supplement B 6 x 5 ml
Campylobacter Agar Kit Skirrow 3280-32
References
To prepare: 6 x 1 liter
1. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
R. H. Yolken (ed.). Manual of clinical microbiology, 6th ed. Campylobacter Agar Base 6 x 39.5 grams
American Society for Microbiology, Washington, D.C. Campylobacter
Antimicrobic Supplement S 6 x 10 ml
2. Skirrow, M. D. 1977. Campylobacter enteritis: A “new” disease.
Br. Med. J. 2:9-11. Campylobacter Agar Kit Skirrow 3280-40
To prepare: 6 x 500 ml
3. Vanderzant, C., and D. F. Splittstoesser (ed). 1992. Compen-
Campylobacter Agar Base 6 x 19.75 grams
dium of methods for the microbiological examination of food,
Campylobacter
3rd ed. American Public Health Association, Washington, D.C. Antimicrobic Supplement S 6 x 5 ml
Neomycin is added to the medium in a concentration of 500 µg/ml. Materials Required But Not Provided
Neomycin and brom cresol green act as selective agents to inhibit Glassware
bacteria in Candida BCG Agar Base.4 Autoclave
Incubator (30°C)
Formula Waterbath (45-55°C)
Candida BCG Agar Base Neomycin (500 µg/ml)
Formula Per Liter Sterile Petri dishes
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g Method of Preparation
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g 1. Suspend 66 grams in 1 liter distilled or deionized water.
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g 2. Heat to boiling to dissolve completely.
Brom Cresol Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g
3. Autoclave at 121°C for 15 minutes.
Final pH 6.1 ± 0.1 at 25°C
4. Cool the medium to 50-55°C. Add sterile neomycin (500 µg/ml).
Precautions Mix well.
1. For Laboratory Use. Specimen Collection and Preparation
2. Follow proper established laboratory procedures in handling and Obtain and process specimens according to the techniques and procedures
disposing of infectious materials. established by laboratory policy.
Storage Test Procedure
Store the dehydrated medium below 30°C. The dehydrated medium is Refer to the scheme for yeast identification.3 For a complete discussion
very hygroscopic. Keep container tightly closed. on the isolation and identification of Candida species refer to the
procedures described in the appropriate references.2,3,5
Expiration Date
The expiration date applies to the product in its intact container when Results
stored as directed. Do not use a product if it fails to meet specifications Identification of Candida species on the basis of colony morphology
for identity and performance. on Candida BCG Agar follows:
C. albicans: Colonies appear as blunt cones 4.5-5.5 mm diameter with
Procedure smooth edges and surfaces; coarse feathery growths may arise from
Materials Provided the center of the colony base to penetrate the medium. The color of
Candida BCG Agar Base
Uninoculated Candida albicans
plate ATCC® 10231
the base and surface of the colonies is yellowish to bluish green green over much of the colony, being more intense in the base than
with the intensity diminishing from a gray green center spot to the surface which is modified by a thin grayish film of cells; the
paleness at the edge, although some strains may show a distinct intensity in color fades abruptly leaving a broad pale edge.
green outer ring. C. guilliermondii: Colonies appear as low cones 4.0-5.0 mm in
C. stellatoidea: Colonies appear convex 4.0-5.0 mm in diameter, with diameter with very smooth edges and highly glossy surfaces; there
smooth edges and smooth to irregular surfaces; there is a fine central maybe a weak, fine feathered submerged growth. Both base and
basal feathery growth penetrating the medium. The color of both surface of the colony tend to have blue centers of medium intensity
base and surface of colonies is yellow to green, the intensity of which fading into a pale edge; however the surface may be blue green
may or may not diminish from center to border but is usually light. with the central third lightened with gray.
C. tropicalis: Colonies appear convex or as low cones 4.5-5.0 mm in C. glabrata: Colonies are smooth and convex, 4.6-5.0 mm diameter;
diameter with smooth to undulate edges, and smooth to granular or the surface color pattern is pale green in the center which becomes
ridged surfaces; deeply stained feathery growth arises from several medium green at the edge, and the base has the same color pattern
points in the base of the colony to form an effusive cloud. The but of less intensity.
color of the submerged growth is normally an intense blue green
compared with that of the base which is much lighter; the surface Limitations of the Procedure
is uniformly pale and may be yellowish green to green, reflecting a 1. Since the nutritional requirements of yeast vary, some strains may
lower pH than observed of the base. be encountered that fail to grow or grow poorly on this medium.
C. pseudotropicalis: Colonies appear convex, 4.5-5.5 mm in diameter References
with undulate to smooth edges, and smooth surfaces; occasionally
1. Harold, W., and M. Snyder. 1968. Personal Communication.
the surface is membranous but all colonies are shiny in appearance,
and there is feathering growth emerging from several points in the 2. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
base of the colony. The color of a large central area in the base of the Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
colony is a medium green, which diminishes in intensity toward the St. Louis, MO.
edge; a similar distribution of color occurs on the surface, but this 3. Warren, N. G., and K. C. Hazen. 1995. Candida, Cryptococcus,
green is bright in hue and is never grayed as it is with C. tropicalis. and other yeasts of medical importance, p. 723-737. In P. R.
C. krusei: Colonies appear as low cones 4.5-5.0 m in diameter with Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken
pseudohyphal edges, which may be weakly contractile or spreading, (ed.), Manual of clinical microbiology, 6th ed. American Society
and have dull surfaces. There is abundant lightly colored growth for Microbiology, Washington, D.C.
penetrating the medium from the base of the colony. The base of 4. MacFaddin, J. D. 1985. Media for isolation-cultivation-
the colony is a medium blue green in the center diminishing in identification-maintenance of medical bacteria, vol. 1, p. 136-137.
intensity to paleness at the edge; the surface is usually a light green Williams & Wilkins, Baltimore, MD.
to yellow green without much concentration in any part.
5. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures
C. parapsilosis: Colonies appear as convex to low cones 3.5-4.5 mm handbook. American Society for Microbiology, Washington, D.C.
in diameter with smooth or slightly spreading edges, but vary from
smooth to granular or rough surfaces; there is no submerged Packaging
growth. The color for both base and surface of the colony is blue Candida BCG Agar Base 500 g 0835-17
Storage light following incubation at 30°C for 18-24 hours. Non-C. albicans
Store dehydrated medium below 30°C. The dehydrated medium is very isolates do not fluoresce.
hygroscopic. Keep container tightly closed.
Limitations of the Procedure
Expiration Date 1. Strains of Candida albicans have been reported that are false
The expiration date applies to the product in its intact container when negative for fluorescence on this medium.2
stored as directed. Do not use a product if it fails to meet specifications 2. Strains of C. parapsilosis, C. krusei, and C. pulcherrima that fluoresce
for identity and performance. on this medium may be encountered.2 These strains may be distin-
guished from C. albicans based on germ tube formation in serum.2,5
Procedure 3. Since the nutritional requirements of organisms vary, some strains
Materials Provided may be encountered that fail to grow or grow poorly on this medium.
Candida Isolation Agar
References
Materials Required but not Provided 1. Fung, D. Y. C., and C. Liang. 1988. A new fluorescent agar for
Glassware the isolation of Candida albicans. Bull. Inf. Lab. Serv. Vet. (France)
Autoclave 29/30:1-2.
2. Goldschmidt, M. C., D. Y. C. Fung, R. Grant, J. White, and T.
Method of Preparation
Brown. 1991. New aniline blue dye medium for rapid identification
1. Suspend 41.1 grams in 1 liter distilled or deionized water.
and isolation of Candida albicans. J. Clin. Micro. 29:1095-1099.
2. Heat to boiling to dissolve completely. 3. Miller, J. M., and H. T. Holmes. 1995. Specimen collection and
3. Autoclave at 121°C for 15 minutes. handling, p. 19- 32. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
Specimen Collection and Preparation Tenover, and R. H. Yolken, (ed.), Manual of clinical microbiology,
1. Specimens should be collected in sterile containers or with sterile 6th ed. American Society for Microbiology, Washington, D.C.
swabs and transported immediately to the laboratory according to 4. Splittstoesser, D. F., and C. Vanderzant (ed.). 1992. Compen-
recommended guidelines.3,4 dium of methods for the microbiological examination of foods,
3rd ed. American Public Health Association, Washington, D.C.
Test Procedure
5. Murray, P. R, E. J. Baron, M. A. Pfaller, F. C. Tenover, and
1. Process each specimen as appropriate for that specimen and inocu-
R. H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
late directly onto the surface of the medium. Streak for isolation.
American Society for microbiology, Washington, D.C.
2. Incubate plates aerobically at 30°C for 18-72 hours.
3. Examine plates for growth after 18-72 hours of incubation. Packaging
Candida Isolation Agar 500 g 0507-17
Results
Colonies of C. albicans fluoresce yellow-green under long wave UV Candida albicans
ATCC® 10231
recommended for use in microbiological assay media and in studies of Vitamins (µg/g)
the growth requirements of microorganisms. Vitamin Assay Casamino Biotin <0.1 PABA <5.0
Acids is commonly used as the amino acid source in early phases of Choline (as Choline Chloride) 160.0 Pantothenic Acid <0.1
nutrition work.9 Sarett10 used Vitamin Assay Casamino Acids as the Cyanocobalamin <0.1 Pyridoxine <0.1
acid hydrolyzed casein in his studies on p-aminobenzoic acid and Folic Acid <0.1 Riboflavin 1.8
p-teroylglutamic acid as growth factors for Lactobacillus species. Inositol <100.0 Thiamine 1.2
Nicotinic Acid <20.0 Thymidine <30.0
Several media containing Casamino Acids are specified in standard
methods for multiple applications.11,12,13 Biological Testing (CFU/g)
Coliform negative Standard Plate Count 950
Principles of the Procedure Salmonella negative Thermophile Count 25
Spore Count 390
Casamino Acids, Casamino Acids, Technical and Vitamin Assay
Casamino Acids are acid hydrolyzed casein. Casein is milk protein, Precautions
and a rich source of amino acid nitrogen. Casamino Acids, Casamino 1. For Laboratory Use.
Acids, Technical and Vitamin Assay Casamino Acids provide nitrogen,
vitamins, carbon and amino acids in microbiological culture media. 2. Follow proper established laboratory procedures in handling and
Although Casamino Acids, Casamino Acids, Technical, and Vitamin disposing of infectious materials.
Assay Casamino Acids are added to media primarily because of their
organic nitrogen and growth factor components, their inorganic
Storage
components also play a vital role.14 Store the dehydrated product below 30°C. The dehydrated product is
very hygroscopic. Keep container tightly closed.
Formula
Casamino Acids is a dehydrated acid hydrolyzed casein in which
Expiration Date
Sodium Chloride and Iron are present in low concentrations permitting The expiration date applies to the product in its intact container when
toxin production. stored as directed. Do not use a product if it fails to meet specifications
for identity and performance.
Casamino Acids, Technical is a dehydrated acid hydrolyzed casein.
The Sodium Chloride and Iron content have not been reduced to same Procedure
extent as Casamino Acids.
Materials Provided
Vitamin Assay Casamino Acids is an acid hydrolyzed casein used to
Casamino Acids
prepare media for microbiological assay of vitamins.
Casamino Acids, Technical
Typical Analysis Vitamin Assay Casamino Acids
Physical Characteristics Materials Required But Not Provided
Ash (%) 24.4 Loss on Drying (%) 4.5 Materials vary depending on the medium being prepared.
Clarity, 1% Soln (NTU) 0.5 pH, 1% Soln 6.4
Filterability (g/cm2) 2.9 Method of Preparation
Nitrogen Content (%) Refer to the final concentration of Casamino Acids, Casamino Acids,
Total Nitrogen 10.5 AN/TN 83.8 Technical or Vitamin Assay Casamino Acids in the formula of the
Amino Nitrogen 8.8 medium being prepared. Add Casamino Acids, Casamino Acids,
Amino Acids (%) Technical or Vitamin Assay Casamino Acids as required.
Alanine 3.26 Lysine 5.71
Arginine 2.20 Methionine 1.28 Specimen Collection and Preparation
Aspartic Acid 4.76 Phenylalanine 2.11 Obtain and process specimens according to the techniques and procedures
Cystine 0.16 Proline 6.17 established by laboratory policy.
Glutamic Acid 15.30 Serine 2.19
Glycine 1.31 Threonine 2.41 Test Procedure
Histidine 1.66 Tryptophan <0.01 See appropriate references for specific procedures using Casamino
Isoleucine 3.34 Tyrosine 0.47 Acids, Casamino Acids, Technical or Vitamin Assay Casamino Acids.
Leucine 5.47 Valine 4.30
Inorganics (%) Results
Calcium <0.001 Phosphate 3.325 Refer to appropriate references and procedures for results.
Chloride 7.400 Potassium 0.410
Cobalt <0.001 Sodium 8.710 References
Copper <0.001 Sulfate 0.045 1. Mueller and Miller. 1941. J. Immunol. 40:21.
Iron <0.001 Sulfur 0.420
Lead <0.001 Tin <0.001 2. Mueller and Johnson. 1941. J. Immunol. 40:33.
Magnesium 0.002 Zinc <0.001 3. Mueller. 1939. J. Immunol. 37:103.
Manganese <0.001
4. Klarman and Wright. 1945. Soap and San. Chem. 21:113. 13. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). 1995.
5. Straus, Dingle and Finland. 1941. J. Immunol. 42:331. Standard methods for the examination of water and wastewater,
19th ed. American Public Health Association, Washington, D.C.
6. Mueller and Hinton. 1941. Proc. Soc. Exp. Biol. Med. 48:330.
14. Nolan, R. A., and W. G. Nolan. 1972. Elemental analysis of
7. Levin. 1943. J. Bacteriol. 46:233.
vitamin-free casamino acids. Appl. Microbiol. 24:290-291.
8. Wolf. 1945. J. Bacteriol. 49:463.
9. Nolan, R. A. 1971. Amino acids and growth factors in vitamin-free Packaging
casamino acids. Mycol. 63:1231-1234. Casamino Acids 100 g 0230-15
500 g 0230-17
10. Sarett. 1947. J. Biol. Chem. 171:265.
2 kg 0230-07
11. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compendium 10 kg 0230-08
of methods for the microbiological examination of food, 3rd. ed.
Casamino Acids, Technical 500 g 0231-17
American Public Health Association, Washington, D.C.
10 kg 0231-08
12. Association of Official Analytical Chemists. 1995. Bacteriological
analytical manual, 8th ed. AOAC International, Gaithersburg, MD. Vitamin Assay Casamino Acids 100 g 0288-15
500 g 0288-17
Bacto Casitone®
Summary and Explanation
Casitone, is recommended for preparing media where an enzymatic
Intended Use hydrolyzed casein is desired. Casitone is used to support the growth of
Bacto Casitone is used in preparing microbiological culture media. fastidious microorganisms. The high tryptophan content of Casitone
makes it valuable for use in detecting indole production.
Dubos Broth and Dubos Oleic Agar media that support the growth of
User Quality Control Mycobacterium tuberculosis contain Casitone. Media used for the
enumeration of coliforms in water, m Endo Agar and m Endo Broth
Identity Specifications MF®, use Casitone as a nitrogen source. Several Thioglycollate media
Dehydrated Appearance: Tan, free-flowing granules. used for detecting microorganisms in normally sterile materials,
Solution: 1%, 2% and 10% solutions are include Casitone as a nitrogen and amino acid source.
soluble in distilled or deionized water.
Casitone is recommended for preparing media for sterility testing
1%-Light amber, clear, no precipitate. according to US Pharmacopeia XXIII (USP).1 Several media containing
2%-Light to medium amber, clear, Casitone are specified in standard methods2,3,4,5 for multiple applications.
may have a slight precipitate.
10%-Medium to dark amber, clear Principles of the Procedure
to very slightly opalescent, may
have a precipitate. Casitone is a pancreatic digest of casein. Casein is the main protein of
milk, and a rich source of amino acid nitrogen.
Reaction of 1%
Solution at 25°C: pH 6.8 - 7.4
Precautions
Cultural Response 1. For Laboratory Use.
All solutions are prepared with the pH adjusted to 7.2 - 7.4. 2. Follow proper established laboratory procedures in handling and
TEST SOLUTION ORGANISM ATCC® RESULT INOCULUM disposing of infectious materials.
Fermentable 2% Escherichia 25922* negative –
Carbohydrates coli Storage
Indole 0.1% Escherichia 25922* positive – Store the dehydrated product below 30°C. The dehydrated product is
Production coli
very hygroscopic. Keep container tightly closed.
Acetylmethylcarbinol 0.1% Enterobacter 13048* positive –
Production aerogenes
Expiration Date
Hydrogen Sulfide 1% Salmonella 6539 positive –
Production typhi The expiration date applies to the product in its intact container when
Toxicity 2%w/0.5% Escherichia 25922* good 100-1,000 stored as directed. Do not use a product if it fails to meet specifications
NaCl & coli growth for identity and performance.
1.5% Agar
Toxicity 2%w/0.5% Staphylococcus 25923* good 100-1,000 Typical Analysis
NaCl & aureus growth
1.5% Agar Physical Characteristics
Ash (%) 7.0 Loss on Drying (%) 3.7
The cultures listed are the minimum that should be used for Clarity, 1% Soln (NTU) 0.6 pH, 1% Soln 7.2
performance testing. Filterability (g/cm2) 1.7
*These cultures are available as Bactrol™ Disks and should be used
as directed in Bactrol Disks Technical Information. Carbohydrate (%)
Total 0.2
Intended Use Casman Agar Base with rabbit blood can be used for the cultivation
Bacto Casman Medium Base is used with blood in isolating fastidious and maintenance of Gardnerella vaginalis.4
microorganisms under reduced oxygen tension.
Principles of the Procedure
Summary and Explanation Proteose Peptone No.3, Tryptose and Beef Extract provide nitrogen,
In 1947, Casman1,2,3 described an infusion-free medium enriched with vitamins and amino acids. Nicotinamide enhances growth of
5% blood for fastidious microorganisms incubated anaerobically. This N. gonorrhoeae and H. influenzae by impeding the removal of
medium replaced labor intensive formulas containing fresh meat coenzyme (V factor) by nucleotidase from the enriched blood. The
infusion and unheated and heated blood.1 Casman adjusted the small amount of Dextrose is added to enhance growth of pathogenic
medium after experiments revealed that nicotinamide disrupted the cocci. Sodium chloride maintains the osmotic balance of the medium.
action of a blood enzyme that inactivates V factor (NAD).2 Using Para-aminobenzoic acid is a preservative. Corn starch is added to
unheated human blood in the formula, Haemophilus influenzae grew ensure that any toxic metabolites produced are absorbed, to neutralize
well and Neisseria was inhibited. The concentration of nicotinamide glucose inhibition of beta-hemolysis 4 and to enhance growth of
was lowered to support growth of Neisseria species.2,3 Neisseria species. Agar Noble is a solidifying agent.
Formula Procedure
Casman Medium Base Materials Provided
Formula Per Liter Casman Medium Base
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Materials Required But Not Provided
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g Glassware
Nicotinamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g Autoclave
p-Aminobenzoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g Incubator (35°C)
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g Waterbath (45-50°C) (optional)
Corn Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g Sterile defibrinated blood
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Sterile water-lysed blood
Agar Noble . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 g
Final pH 7.3 ± 0.2 at 25°C Method of Preparation
1. Suspend 43 grams in 1 liter distilled or deionized water.
Precautions 2. Heat to boiling to dissolve completely.
1. For Laboratory Use. 3. Autoclave at 121°C for 15 minutes. Cool to 50°C.
2. Follow proper established laboratory procedures in handling and 4. Add 5% sterile blood and 0.15% sterile water-lysed blood solution
disposing of infectious materials. (one part blood to three parts water). Omit water-lysed blood if
sterile blood is partially lysed.
Storage
5. Dispense into sterile Petri dishes or as desired.
Store the dehydrated medium below 30°C. The dehydrated medium is
very hygroscopic. Keep container tightly closed. Specimen Collection and Preparation
Obtain and process specimens according to the techniques and
Expiration Date procedures established by laboratory policy.
The expiration date applies to the product in its intact container when
stored as directed. Do not use a product if it fails to meet specifications
for identity and performance. Uninoculated plate Neisseria gonorrhoeae
with enrichment CDC 116
with enrichment
Test Procedure using pure cultures are recommended for complete identification.
For a complete discussion on the isolation and identification Consult appropriate references for further information.5,6
of fastidious microorganisms, refer to the procedures described in 6. Improper specimen collection, environment, temperature, CO2
appropriate references.5,6 level, moisture and pH can adversely affect the growth and
viability of the organism.
Results
Refer to appropriate references and procedures for results. References
1. Casman, E. P. 1947. A noninfusion blood agar base for Neisseriae,
Limitations of the Procedure Pneumococci and Streptococci. Am. J. Clin. Pathol. 27:281.
1. Since the nutritional requirements of organisms vary, some strains may 2. Casman, E. P. 1942. J. Bacteriol. 43:33.
be encountered that fail to grow or grow poorly on this medium. 3. Casman, E. P. 1947. J. Bacteriol. 53:561.
2. Nicotinamide in concentrations greater than 0.005% inhibits growth 4. MacFaddin, J. D. 1985. Media for isolation-cultivation-
of some N. gonorrhoeae strains; however, only slight stimulation identification-maintenance medical bacteria, vol. 1, p. 141-143.
of growth of H. influenzae occurs with this amount.1 Williams & Wilkins, Baltimore, MD.
3. Hemolytic reactions of some strains have been shown to be 5. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
affected by differences in animal blood.6 R. H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
4. Atmosphere of incubation has been shown to influence hemolytic American Society for Microbiology, Washington, D.C.
reactions of beta-hemolytic streptococci.6 6. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures hand-
5. Casman Medium Base is intended for use with supplementation. book, vol.1. American Society for Microbiology. Washington, D.C.
Although certain diagnostic tests may be performed directly on this
medium, biochemical and, if indicated, immunological testing Packaging
Casman Medium Base 500 g 0290-17
Storage References
Store the dehydrated medium below 30°C. The dehydrated medium is 1. King, E. O., M. K. Ward, and E. E. Raney. 1954. Two simple
very hygroscopic. Keep container tightly closed. media for the demonstration of pyocyanin and fluorescein. J. Lab.
Clin. Med. 44:301.
2. Brown, V.I., and E. J. L. Lowbury. 1965. Use of an improved 7. The United States Pharmacopeia. 1995. Microbiological Limits
Cetrimide Agar Medium and of culture methods for Pseudomonas Tests, The United States pharmacopeia, 23rd ed. United States
aeruginosa. J. Clin. Pathol. 18:752. Pharmacopeial Convention, Rockville, MD.
3. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. 8. MacFaddin, J. D. 1985. Media for isolation-cultivation-
Nonfermentative gram-negative bacilli and coccobacilli, identification-maintenance of medical bacteria, vol. 1, p. 146-149.
p. 386-405. Bailey & Scott’s diagnostic microbiology, 9th ed. Williams & Wilkins, Baltimore, MD.
Mosby-Year Book, Inc., St. Louis, MO. 9. Goto, S., and S. Enomoto. 1970. Nalidixic acid cetrimide agar:
4. Gilligan, P. H. 1995. Pseudomonas and Burkholderia, p. 509-519. A new selective plating medium for the selective isolation of
In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and Pseudomonas aeruginosa. Jpn. J. Microbiol. 14:65.
R. H. Yolken (ed.)., Manual of clinical microbiology, 6th ed. 10. Lowbury, E. J. L., and A. G. Collins. 1955. The use of a new
American Society for Microbiology, Washington, D.C. cetrimide product in a selective medium for Pseudomonas
5. Robin, T., and J. M. Janda. 1984. Enhanced recovery of aeruginosa. J. Clin. Pathol. 8:47.
Pseudomonas aeruginosa from diverse clinical specimens on a
Packaging
new selective agar. Diag. Microbiol. Infect. Dis. 2:207.
Cetrimide Agar Base 100 g 0854-15
6. Association of Official Analytical Chemists. 1995. Bacteriological 500 g 0854-17
analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
Glycerol 100 g 0282-15
500 g 0282-17
respiratory pathogens, residing on the mucous membranes of the 2. Follow proper established laboratory procedures in handling and
respiratory tract. B. pertussis is the major cause of whooping cough disposing of infectious materials.
or pertussis. B. parapertussis is associated with a milder form of
the disease.3 B. bronchiseptica is an opportunistic human pathogen Storage
associated with both respiratory and non-respiratory infections, Store the dehydrated medium below 30°C. The dehydrated medium is
often occurring in patients having close contact with animals. 2 very hygroscopic. Keep container tightly closed.
B. bronchiseptica has not been reported to cause pertussis. There
have been no reports of recovery of B. avium from humans.2 Expiration Date
Charcoal Agar supplemented with Horse Blood is used for the The expiration date applies to the product in its intact container when
cultivation and isolation of Haemophilus influenzae.4 stored as directed. Do not use a product if it fails to meet specifications
for identity and performance.
Principles of the Procedure
Infusion from Beef Heart and Bacto Peptone provide the nitrogen,
Procedure
carbon and amino acids in Charcoal Agar. Yeast Extract is a vitamin Materials Provided
source. Sodium Chloride maintains osmotic balance. Bacto Agar is a Charcoal Agar
solidifying agent. Soluble Starch absorbs toxic metabolites. Norit SG,
charcoal, provides growth requirements and selective properties. Materials Required But Not Provided
Glassware
Formula Autoclave
Charcoal Agar Incubator (35°C)
Formula Per Liter Waterbath (45-50°C)
Beef Heart, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 500 g Sterile Petri dishes
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Method of Preparation
Bacto Soluble Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g 1. Suspend 62.5 grams in 1 liter distilled or deionized water.
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 g 2. Heat to boiling to dissolve completely.
Norit SG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 g
3. Autoclave at 121°C for 15 minutes.
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 g
4. Mix thoroughly during dispensing to uniformly distribute
Final pH 7.3 ± 0.2 at 25°C
the charcoal.
Precautions
1. For Laboratory Use.
Uninoculated Bordetella bronchiseptica
plate ATCC® 4617
2. Heat to boiling to dissolve completely. 3. Hemolytic reactions of some strains of group D streptococci have
3. Autoclave at 121°C for 15 minutes. Cool to 45-50°C. been shown to be affected by differences in animal blood. Such
4. To prepare blood agar, aseptically add 5% sterile defibrinated blood strains are ß-hemolytic on horse, human and rabbit blood agar and
to the medium at 45-50°C. Mix well. α-hemolytic on sheep blood agar.4
5. Dispense into sterile Petri dishes. 4. Colonies of Haemophilus haemolyticus are ß-hemolytic on horse
and rabbit blood agar and must be distinguished from colonies
Specimen Collection and Preparation of ß-hemolytic streptococci using other criteria. The use of sheep
Collect specimens in sterile containers or with sterile swabs and blood has been suggested to obviate this problem since sheep
transport immediately to the laboratory in accordance with blood is deficient in pyridine nucleotides and does not support
recommended guidelines outlined in the references. growth of H. haemolyticus.6
Test Procedure 5. Atmosphere of incubation has been shown to influence hemolytic
1. Process each specimen as appropriate and inoculate directly onto reactions of ß-hemolytic streptococci.4 For optimal performance,
the surface of the medium. Streak for isolation with an inoculating incubate blood agar media under increased CO2 or anaerobic conditions.
loop, then stab the agar several times to deposit ß-hemolytic References
streptococci beneath the agar surface. Subsurface growth will
1. Ellner, P. D., C. J. Stoessel, E. Drakeford, and F. Vasi. 1966.
demonstrate the most reliable hemolytic reactions due to the
activity of both oxygen-stable and oxygen-labile streptolysins.4 A new culture medium for medical bacteriology. Am. J. Clin.
Pathol. 45:502-504.
2. Incubate plates aerobically, anaerobically or under conditions
of increased CO 2 (5-10%) in accordance with established 2. Vanderzant, C. and D. F. Splittstoesser (ed.). 1992. Compen-
laboratory procedures. dium of methods for the microbiological examination of foods,
3rd ed. American Public Health Association, Washington, D.C.
Results 3. Casman, E. P. 1947. A noninfusion blood agar base for neisseriae,
1. Examine plates for growth and hemolytic reactions after 18-24 and pneumococci and streptococci. Am. J. Clin Pathol. 17:281-289.
48 hours of incubation. Four types of hemolysis on blood agar
4. Ruoff, K. L. 1995. Streptococcus, p. 299-305. In P. R. Murray,
media can be described:5
E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.).
a. Alpha-hemolysis (α) is the reduction of hemoglobin to Manual of clinical microbiology, 6th ed. American Society for
methemoglobin in the medium surrounding the colony, causing Microbiology, Washington, D.C.
a greenish discolorization of the medium.
5. Isenberg, H. D. (ed). 1992. Clinical microbiology procedures hand-
b. Beta-hemolysis (ß) is the lysis of red blood cells, producing a
clear zone surrounding the colony. book, vol.1. American Society for Microbiology, Washington, D.C.
c. Gamma-hemolysis (γ) indicates no hemolysis. No destruction 6. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
of red blood cells occurs and there is no change in the medium. Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.
d. Alpha-prime-hemolysis (ά) is a small zone of complete hemolysis St. Louis, MO.
that is surrounded by an area of partial lysis. Packaging
Limitations of the Procedure Columbia Blood Agar Base 500 g 0792-17
2 kg 0792-07
1. Blood agar base media are intended for use with blood 10 kg 0792-08
supplementation. Although certain diagnostic tests may
be performed directly on these media, biochemical and, if Columbia Blood Agar Base EH 500 g 0790-17
indicated, immunological testing using pure cultures is 2 kg 0790-07
recommended for complete identification. Consult appropriate 10 kg 0790-08
references for further information. Columbia Blood Agar Base No. 2 500 g 0793-17
2. Since the nutritional requirements of organisms vary, some strains 2 kg 0793-07
may be encountered that fail to grow or grow poorly on this medium. 10 kg 0793-08
buffered with Tris. Corn Starch is omitted to reduce opalescence.1 Expiration Date
Cysteine is the reducing agent. Magnesium and Iron are added to The expiration date applies to the product in its intact container when
facilitate organism growth. stored as directed. Do not use a product if it fails to meet specifications
for identity and performance.
Formula
Columbia Broth Procedure
Formula Per Liter Materials Provided
Bacto Pantone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Bitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Columbia Broth
Tryptic Digest of Beef Heart . . . . . . . . . . . . . . . . . . . . . . . . 3 g Materials Required But Not Provided
L-Cysteine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g Glassware
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Autoclave
Magnesium Sulfate Anhydrous . . . . . . . . . . . . . . . . . . . . 0.1 g Incubator (35°C)
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g Waterbath (45-50°C)
Sodium Carbonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 g Sterile tubes
Tris (Hydroxymethyl) Aminomethane . . . . . . . . . . . . . . 0.83 g Sodium polyanetholesulfonate (SPS)
Tris (Hydroxymethyl) Aminomethane HCl . . . . . . . . . . . 2.86 g
Final pH 7.5 ± 0.2 at 25oC Method of Preparation
1. Dissolve 35 grams in 1 liter distilled or deionized water.
Precautions 2. Warm slightly if necessary to dissolve completely.
1. For Laboratory Use.
3. OPTIONAL: Sodium polyanetholesulfonate (SPS) may be added
2. Follow proper established laboratory procedures in handling and at this time with agitation to ensure a uniform solution. The culture
disposing of infectious materials. medium should contain 0.025 to 0.05% SPS.
Storage 4. Distribute in suitable containers. Autoclave at 121°C for 15 minutes.
Store the dehydrated medium below 30°C. The dehydrated medium is 5. Allow to cool to room temperature before using.
very hygroscopic. Keep container tightly closed.
Specimen Collection and Preparation
Obtain and process specimens according to the techniques and procedures
User Quality Control established by laboratory policy.
Identity Specifications Test Procedure
Dehydrated Appearance: Light beige, free-flowing homogeneous. Process clinical specimens from different body sites as described in
Solution: 3.5% solution, soluble in distilled Clinical Microbiology Procedures Handbook,2 Manual of Clinical
or deionized water on warming. Microbiology3 or according to laboratory procedures.
Solution is light amber, clear to
very slightly opalescent, may have Results
a slight amount of fine precipitate. Refer to appropriate references and procedures for results.
Prepared Medium: Light amber, clear to very slightly
opalescent, may have a slight Limitations of the Procedure
amount of fine precipitate.
1. Since the nutritional requirements of organisms vary, some strains
Reaction of 3.5% may be encountered that fail to grow or grow poorly on this medium.
Solution at 25°C: pH 7.5 ± 0.2
2. Neisseria spp. may be inhibited by SPS in Columbia Broth. The
Cultural Response addition of 1.2% gelatin may counteract the inhibitory effect, but
Prepare Columbia Broth per label directions. Inoculate and SPS may also inhibit other organisms.2
incubate at 35 ± 2°C under appropriate conditions for
18-48 hours. Incubate Bacteroides fragilis anaerobically. 3. Opalescence in Columbia Broth cannot always be relied upon as
INOCULUM evidence of bacterial growth in the bottle.
ORGANISM ATCC® CFU GROWTH
4. It is possible for significant numbers of viable bacteria to be present
Neisseria meningitidis 13090 100-1,000 good in an inoculated and incubated blood culture bottle without the
Staphylococcus aureus 25923* 100-1,000 good
usual signs of bacterial growth.
Streptococcus pyogenes 19615* 100-1,000 good
Bacteroides fragilis 25285 100-1,000 good References
Pseudomonas aeruginosa 27853 100-1,000 good
1. Morello, J. A., and P. D. Ellner. 1969. New medium for blood
The cultures listed are the minimum that should be used for cultures. Appl. Microbiol. 17:68-70.
performance testing.
*These cultures are available as Bactrol™ Disks and should be used
2. Isenberg, H. D. (ed). 1992. Clinical microbiology procedures hand-
as directed in Bactrol Disks Technical Information. book, vol.1. American Society for Microbiology, Washington, D.C.
Procedure References
1. Cooke. 1922. J. Bact. 7:339.
Materials Provided
Cooke Rose Bengal Agar 2. Dixon, D. M., and R. A. Fromtling. 1995. Morphology, taxonomy,
Antimicrobic Vial A and classification of the fungi, p. 699-708. In P. R. Murray, E. J.
Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.). Manual
Materials Required but not Provided of clinical microbiology, 6th ed. American Society for Microbiology,
Glassware Washington, D.C.
Autoclave 3. Waksman. 1954. Antibiotics and Chemotherapy 4:657.
Sterile Petri dishes 4. Taplin, Azias, Rebell, and Blank. 1969. Arch. Dermatol. 99:203.
Incubator
Waterbath (optional) Packaging
Method of Preparation Cooke Rose Bengal Agar 500 g 0703-17
Antimicrobic Vial A Antimicrobic Vial A 6 x 10 ml 3333-60
carbohydrate serum media. Robertson5 substituted beef heart for brain Formula
tissue and found successful results. Cooked Meat Medium is prepared Cooked Meat Medium
according to the formulation of Robertson.5 Formula Per Liter
The capacity of Cooked Meat Medium to detoxify metabolic products Beef Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454 g
of microorganisms makes it an excellent maintenance and Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
growth medium. A study of various formulations used to grow and Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
maintain clinical isolates of anaerobic bacteria found Chopped Meat Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Broth superior.6 Final pH 7.2 ± 0.2 at 25°C
Cooked Meat Medium’s ability to initiate growth in a small inoculum Precautions
makes it valuable for the primary culture of clinical specimens. Cooked 1. For Laboratory Use.
Meat Medium can be supplemented with vitamin K1 (1% alcohol
2. Follow proper established laboratory procedures in handling and
solution) and hemin (1% solution) for clinical isolates. 7 This disposing of infectious materials.
modification is used as a general enrichment for anaerobes, and as a
backup for anaerobic jar or chamber failure.7 Storage
Chopped Meat Carbohydrate Medium and Chopped Meat Glucose Store the dehydrated medium below 30°C. The dehydrated medium is
Medium is used for cultivation and maintenance of anaerobic very hygroscopic. Keep container tightly closed.
bacteria. 7,8,9 Cooked Meat Medium is recommended in the
Bacteriological Analytical Manual10 for use in the examination of
Expiration Date
The expiration date applies to the product in its intact container when
Clostridium botulinum from food and in the Compendium of
stored as directed. Do not use a product if it fails to meet specifications
Methods for the Microbiological Examination of Foods.11
for identity and performance.
Principles of the Procedure Procedure
Beef Heart and Proteose Peptone provide the nitrogen, vitamins and
Materials Provided
amino acids in Cooked Meat Medium. Sodium Chloride maintains
the osmotic balance of the medium. The low concentration of Cooked Meat Medium
Dextrose is sufficient as the energy source, but not high enough to Materials Required But Not Provided
accumulate toxic metabolites. This formulation provides an effective Glassware
maintenance medium. Autoclave
Solid meat particles provide favorable growth conditions for Incubator (35°C)
anaerobes due to the reducing action of -SH (sulfhydryl) groups of Distilled or deionized water
muscle protein.2,3,4 Sulfhydryl groups are more accessible in denatured Sterile tubes with closures
proteins, therefore the use of cooked meat particles is preferred.9
Method of Preparation 2. Tarozzi, G. 1905. Uber ein leicht in aerober Weise ausfuhrbares
1. Suspend 12.5 grams in 100 ml distilled or deionized water (1.25 g Kulturmittel von einigen bis jetzt fuu strenge Anaeroben
per 10 ml). gehlatenen Keimen. Zentralb. Bakteriol. 38:619.
2. Let stand until all particles are thoroughly wetted and form an 3. von Hibler, E. 1899. Beitrage zur Kenntnis der durch anaerobe
even suspension. Spaltpilze erzeugen Infektions-krankheiten der Tiere und des
3. Autoclave at 121°C for 15 minutes. Reduce pressure slowly. Menschen etc. Centr. Bakteriol. 25: 513,594,631.
4. Cool without agitation. 4. von Hibler, E. 1908. Untersuchungen uber die pathogenen
Anaerobier, Jena: Verlag Fischer.
5. If not used within 24 hours, reheat (100°C) prior to use to drive off
dissolved oxygen. 5. Robertson, M. 1916. Notes upon certain anaerobes isolated from
wounds. J. Pathol. Bacteriol. 20:327.
Specimen Collection and Preparation
6. Claros, M. C., D. M. Citron, and E. J. C. Goldstein. 1995.
Obtain and process specimens according to the techniques and Survival of anaerobic bacteria in various thioglycollate and chopped
procedures established by institutional policy. meat broth formulations. J. Clin. Microbiol. 33:2505-2507.
Test Procedure 7. Isenberg, H. E. (ed.). 1992. Clinical microbiology procedures
1. Inoculate specimen well into the meat particles (bottom of the handbook. American Society for Microbiology, Washington, D.C.
tube). Tissue specimens should be ground prior to inoculation. 8. Atlas, R. M. 1993. Handbook of microbiological media,
2. Growth is indicated by turbidity and/or the presence of gas bubbles. p. 224-226. CRC Press, Boca Raton, FL.
3. For a complete discussion on the isolation and identification 9. MacFaddin, J. D. 1985. Media for isolation-cultivation-
of aerobic and anaerobic bacteria, refer to appropriate procedures identification- maintenance of medical bacteria, vol. 1, p. 240-246.
outlined in the references. Williams & Wilkins, Baltimore, MD.
Results 10. Association of Official Analytical Chemists. 1995.
Bacteriological analytical manual, 8th ed. AOAC International,
Refer to appropriate references and procedures for results.
Gaithersburg, MD.
Limitations 11. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992.
1. Since the nutritional requirements of organisms vary, some Compendium of methods for the microbiological examination of food,
strains may be encountered that fail to grow or grow poorly on 3rd ed. American Public Health Association, Washington, D.C.
this medium. Packaging
References Cooked Meat Medium 100 g 0267-15
500 g 0267-17
1. Smith, T. 1890. Centr. Bakteriol. 7:509.
10 kg 0267-15r
2. Follow proper established laboratory procedure in handling and 3. Autoclave at 121°C for 15 minutes.
disposing of infectious materials.
Specimen Collection and Preparation
Storage 1. Specimens should be collected in sterile containers or with sterile
Store dehydrated medium below 30°C. The dehydrated medium is very swabs and transported immediately to the laboratory according to
hygroscopic. Keep container tightly closed. recommended guidelines.5
The low agar content of Cystine Tryptic Agar provides a suitable Storage
environment for motility studies. Motility determination aids in the Store the dehydrated medium below 30°C. The dehydrated medium is
identification of bacteria. CTA can also be used as a maintenance medium very hygroscopic. Keep container tightly closed.
for stock cultures.3,4 This formula will support the growth of fastidious
organisms, e.g., Streptococcus pneumonia and Corynebacterium species.4 Expiration Date
The expiration date applies to the product in its intact container when
Principles of the Procedure stored as directed. Do not use a product if it fails to meet specifications
Tryptose provides the nitrogen, vitamins and amino acids in Cystine for identity and performance.
Tryptic Agar. L-Cystine and Sodium Sulfite are added to this formula
to stimulate growth. Sodium Chloride maintains the osmotic balance Procedure
of the medium. Phenol Red is the pH indicator. Bacto Agar maintains Materials Provided
an Eh potential which facilitates anaerobic growth, and aids in disper- Cystine Tryptic Agar
sion of reducing substances and CO2 formed in the environment.5 The
agar is also used for the determination of motility. Materials Required But Not Provided
Glassware
Formula Autoclave
Cystine Tryptic Agar Incubator (35°C)
Formula Per Liter Waterbath (50-55°C) (optional)
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g Sterile 5-10% carbohydrate solution
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g Sterile tubes
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Method of Preparation
Sodium Sulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
1. Suspend 28.5 grams in 1 liter of distilled or deionized water.
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.017 g 2. Heat to boiling to dissolve completely.
Final pH 7.3 ± 0.2 at 25°C 3. Autoclave at 121°C for 15 minutes.
4. To prepare fermentation medium, use one of the following methods:
Precautions A. Add 5-10 grams carbohydrate before sterilization.
1. For Laboratory Use. or B. Dissolve 28.5 grams medium in 900 ml water, sterilize
2. Follow proper established laboratory procedures in handling and and aseptically add 100 ml sterile of carbohydrate solution.
disposing of infectious materials.
Specimen Collection and Preparation 2. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
Obtain and process specimens according to the techniques and Scott’s Diagnostic Microbiology, 9th ed. Mosby-Year Book, Inc.,
procedures established by laboratory policy. St. Louis, MO.
3. Myers, R. M., and G. Koshy. 1961. Beta-hemolytic streptococci
Test Procedure in survey throat cultures in an Indian population. Am. J. Public
For a complete discussion on motility and carbohydrate fermentation Health 51:1872.
studies refer to procedures described in appropriate references.2,6,7 4. Alford, J. A., G. E. Wiese, and J. J. Gunter. 1955. Heat
Results resistance in Corynebacterium and the relationship of the genus to
1. Fermentation of the test carbohydrate is observed when acid is Microbacterium. J. Bacteriol. 69:516.
formed and the medium turns from red to yellow. 5. MacFaddin, J. D. 1985. Media for isolation-cultivation-
2. Motility of an organism is evident as a haze of growth extending identification-maintenance of medical bacteria, vol. 1, p. 254-259,
into the agar from the stab line.2 802-804. Williams & Wilkins, Baltimore, MD.
6. Isenberg, H. D. (ed.). 1995. Clinical microbiology procedures
Limitations of the Procedure handbook, American Society for Microbiology, Washington, D.C.
1. Since the nutritional requirements of organisms vary, some strains 7. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
may be encountered that fail to grow or grow poorly on this medium. R. H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
2. CTA requires a heavy inoculum.5 American Society for Microbiology, Washington, D.C.
3. Prolonged incubation may lead to changes in pH indicator or 8. Faur, Y. C., M. H. Weisburd, and M. E. Wilson. 1975.
abnormal lactose/sucrose reactions with Neisseria pathogens.8,9 Carbohydrate fermentation plate medium for confirmation of
4. Neisseria species usually produce acid only in the area of stabs Neisseria species. J. Clin. Microbiol. 1:294.
(upper third). If there is a strong acid (yellow color) throughout the 9. Applebaum, P. C., and R. B. Lawrence. 1979. Comparison of
medium, a contaminating organism may be present. If in doubt three methods for identification of pathogenic Neisseria species.
about a tube containing a Neisseria species, a Gram stain and J. Clin. Microbiol. 9:598.
oxidase test should be performed on the growth.5
References Packaging
1. Vera, H. D. 1948. A simple medium for identification and mainte- Cystine Tryptic Agar 500 g 0523-17
nance of the gonococcus and other bacteria. J. Bacteriol. 55:531.
Bacto Czapek Solution Agar contains essential ions. Magnesium Sulfate and Ferrous Sulfate sources
of cations. Bacto Agar is the solidifying agent in Czapek Solution Agar.
Storage Results
Store the dehydrated medium below 30°C. The dehydrated medium is Typical coliforms that rapidly ferment sucrose and/or lactose will form
very hygroscopic. Keep container tightly closed. red, opaque colonies. Shigella and Salmonella species will produce
colorless to slightly pink, transparent colonies.
Expiration Date
The expiration date applies to the product in its intact container when Limitations of the Procedure
stored as directed. Do not use a product if it fails to meet specifications 1. Since the nutritional requirements of organisms vary, some
for identity and performance. strains may be encountered that fail to grow or grow poorly on
Procedure this medium.
2. DO NOT AUTOCLAVE MEDIUM. DO NOT OVERHEAT.
Materials Provided
DCLS Agar 3. DCLS Agar is intended for selective use and should be inoculated
in parallel with nonselective media.
Materials Required But Not Provided 4. Colonies suspected of being enteric pathogens must be confirmed
Glassware biochemically and, if required, serologically.
Incubator (35°C)
Waterbath (45-50°C) (optional) References
Sterile Petri dishes 1. Leifson, E. 1935. New culture media based on sodium
desoxycholate for the isolation of intestinal pathogens and for
Method of Preparation
the enumeration of colon bacilli in milk and water. J. Pathol.
1. Suspend 49.5 grams in 1 liter distilled or deionized water. Bacteriol. 40:581-599.
2. Heat to boiling to dissolve completely. DO NOT AUTOCLAVE. 2. Holt-Harris, J. E., and O. Teague. 1916. A new culture medium
3. Cool to 50-55°C. for the isolation of Bacillus typhosus from stools. J. Infect.
4. Dispense into sterile Petri dishes, or as desired. Dis. 18:596-601.
5. Allow prepared medium to dry for about 2 hours with the covers 3. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
partially removed. R. H. Yolken (ed.). 1995. Manual of clinical microbiology,
Specimen Collection and Preparation 6th ed. American Society for Microbiology, Washington, D.C.
Obtain and process specimens according to the techniques and 4. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures
procedures established by laboratory policy. handbook, vol.1. American Society for Microbiology,
Washington, D.C.
Test Procedure
For a complete discussion on the isolation and identification of enteric Packaging
pathogens from clinical specimens, refer to the procedures described DCLS Agar 500 g 0759-17
in appropriate references.3,4
organisms. Yeast Extract provides vitamins and cofactors required for D/E Neutralizing Broth
growth and additional nitrogen and carbon. Dextrose is a source of Formula Per Liter
fermentable carbohydrate. Sodium Thioglycollate neutralizes Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
mercurials. Sodium Thiosulfate neutralizes iodine and chlorine. Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Sodium Bisulfite neutralizes formaldehyde and gluteraldehyde. Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Lecithin neutralizes quaternary ammonium compounds and Polysorbate Sodium Thioglycollate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
80 neutralizes phenols, hexachlorophene, formalin and, with lecithin, Sodium Thiosulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 g
ethanol.11 Brom Cresol Purple is used as a colorimetric indicator to Sodium Bisulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
demonstrate the production of acid from the fermentation of dextrose. Polysorbate 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Lecithin (Soy Bean) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 g
D/E Neutralizing Agar uses Bacto Agar as a solidifying agent. Bacto Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g
Formula Final pH 7.6 ± 0.2 at 25° C
D/E Neutralizing Agar Precautions
Formula Per Liter 1. For Laboratory Use.
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
2. D/E Neutralizing Agar
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
D/E Neutralizing Broth
Sodium Thioglycollate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g HARMFUL. MAY CAUSE SENSITIZATION BY INHALA-
Sodium Thiosulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 g TION. IRRITATING TO EYES, RESPIRATORY SYSTEM AND
Sodium Bisulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g SKIN. Avoid contact with skin and eyes. Do not breathe dust. Wear
Polysorbate 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g suitable protective clothing. Keep container tightly closed.
Lecithin (Soy Bean) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 g FIRST AID: In case of contact with eyes, rinse immediately with
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g plenty of water and seek medical advice. After contact with skin,
Bacto Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g wash immediately with plenty of water. If inhaled, remove to fresh
Final pH 7.6 ± 0.2 at 25° C
Cultural Response
D/E Neutralizing Agar: Neutralization Test
Prepare medium per label directions. Inoculate 50 ml of D/E Neutralizing Agar with 0.1 ml of a heavy suspension of each test organism
and dispense into 150 x 15 mm Petri dishes of D/E Neutralizing Agar and Plate Count Agar. Place 1/2 inch sterile blank disks on each
plate. Dispense 0.1 ml of each disinfectant solution onto two disks per medium. Incubate at 35 ± 2°C for 40-48 hours. D/E Neutralizing
Agar should exhibit no zones of inhibition or zones significantly smaller than those found on Plate Count Agar.
continued on following page
Procedure
Materials Provided
D/E Neutralizing Agar
D/E Neutralizing Broth
Materials Required but not Provided
Glassware
Distilled or deionized water
Autoclave
Method of Preparation
D/E Neutralizing Agar
1. Suspend 54 grams in 1 liter of distilled or deionized water.
2. Heat to boiling to dissolve.
3. Autoclave at 121°C for 15 minutes.
D/E Neutralizing Broth
Uninoculated Bacillus subtilis Escherichia coli 1. Suspend 39 grams in 1 liter of distilled of deionized water.
tube ATCC® 6633 ATCC® 25922
2. Heat to dissolve.
3. Autoclave at 121°C for 15 minutes.
Specimen Collection and Preparation
Refer to appropriate references for specimen collection and preparation.
Test Procedure
D/E Neutralizing Agar and D/E Neutralizing Broth are used in a
variety of procedures. Consult appropriate references for further
information.6
Results
Refer to appropriate references and procedures for results.
References
1. Engley, F. B., Jr., and B. P. Dey. 1970. A universal neutralizing
medium for antimicrobial chemicals. Presented at the Chemical
Specialties Manufacturing Association (CSMA) Proceedings, 56th
Mid-Year Meeting.
2. Dey, B. P., and F. B. Engley, Jr. 1983. Methodology for recovery
Pseudomonas aeruginosa Salmonella typhimurium Staphylococcus aureus of chemically treated Staphylococcus aureus with neutralizing
ATCC® 27853 ATCC® 14028 ATCC® 25923 medium. Appl. Environ. Microbiol. 45:1533-1537.
3. Dey, B. P., and F. B. Engley, Jr. 1978. Environmental sampling 6. Curry, A. S., J. G. Graf, and G. N. McEwen, Jr. (ed.). 1993.
devices for neutralization of disinfectants. Presented at the 4th CTFA Microbiology Guidelines. The Cosmetic, Toiletry and
International Symposium on Contamination Control. Fragrance Association, Washington, D.C.
4. Dey, B. P., and F. B. Engley, Jr. 1994. Neutralization of antimicro-
bial chemicals by recovery media. J. Microbiol. Methods 19:51-58. Packaging
5. Dey, B. P., and F. B. Engley, Jr. 1995. Comparison of Dey and D/E Neutralizing Agar 500 g 0686-17
Engley (D/E) Neutralizing medium to Letheen Medium and 10 kg 0686-08
Standard Methods Medium for recovery of Staphylococcus aureus D/E Neutralizing Broth 500 g 0819-17
from sanitized surfaces. J. Ind. Microbiol. 14:21-25.
creating a clear zone around the growth. Applying this principle, Smith, Precautions
Hancock and Rhoden6 modified DNase Test Agar with added methyl green 1. For Laboratory Use.
to detect staphylococci, streptococci and Serratia. When using DNase
2. Follow proper established laboratory procedure in handling and
Test Agar w/Methyl Green, acid does not have to be added to the plate.
disposing of infectious materials.
Mannitol fermentation can be determined simultaneously with DNase
production by adding 10 grams of mannitol and 0.025 grams of phenol Storage
red to the DNase Test Agar prior to sterilization.7 Store dehydrated medium below 30°C. The dehydrated medium is very
hygroscopic. Keep container tightly closed.
Principles of the Procedure
Tryptose is a source of nitrogen, amino acids and carbon. Deoxyribo- Expiration Date
nucleic Acid enables the detection of DNase that depolymerizes DNA. The expiration date applies to the product in its intact container when
Sodium Chloride provides essential ions while maintaining osmotic stored as directed. Do not use a product if it fails to meet specifications
balance. Methyl Green is a colorimetric indicator. Bacto Agar is a for identity and performance.
solidifying agent.
Procedure
Formula Materials Provided
DNase Test Agar DNase Test Agar
Formula Per Liter DNase Test Agar w/Methyl Green
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Deoxyribonucleic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g Materials Required but not Provided
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Glassware
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g Autoclave
Final pH 7.3 ± 0.2 at 25°C 1N Hydrochloric acid (DNase Test Agar)
DNase Test Agar w/Methyl Green
Method of Preparation
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
1. Suspend 42 grams of either DNase Test Agar or DNase Test Agar
Deoxyribonucleic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g w/Methyl Green in 1 liter distilled or deionized water.
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g 2. Heat to boiling to dissolve completely.
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g 3. Autoclave at 121°C for 15 minutes.
Methyl Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g
Final pH 7.3 ± 0.2 at 25°C
Uninoculated Staphylococcus aureus
plate ATCC® 25923
The cultures listed are the minimum that should be used for
performance testing.
*These cultures are available as Bactrol™ Disks and should be used as
directed in Bactrol Disks Technical Information.
Staphylococcus epidermidis
ATCC® 12228
Intended Use plenty of water and seek medical advice. After contact with skin,
Bacto DRBC Agar is used for the enumeration of yeasts and molds. wash immediately with plenty of water. If inhaled, remove to fresh
air. If not breathing, give artificial respiration. If breathing is
Also Known As difficult, give oxygen. Seek medical advice. If swallowed seek
Dichloran Rose Bengal Chloramphenicol Agar medical advice immediately and show this container or label.
3. Follow proper, established laboratory procedures in handling and
Summary and Explanation disposing of infectious materials.
DRBC Agar is based on the Dichloran Rose Bengal Chlortetracycline
(DRBC) Agar formula described by King, Hocking and Pitt.1 DRBC Storage
Agar conforms with APHA guidelines for the mycological examination 1. Store the dehydrated medium below 30°C. The powder is very
of foods, containing chloramphenicol rather than chlortetracycline as hygroscopic. Keep container tightly closed.
proposed by King, Hocking and Pitt.2 DRBC Agar is a selective 2. Protect medium from light.
medium that supports good growth of yeasts and molds. 3. Store prepared medium in the dark at 2-8°C.
Principles of the Procedure Expiration Date
Proteose Peptone No. 3 provides nitrogen, vitamins and minerals. The expiration date applies to the product in its intact container when
Dextrose is a carbohydrate source. Phosphate is a buffering agent. stored as directed. Do not use a product if it fails to meet specifications
Magnesium Sulfate is a source of divalent cations and sulfate. The for identity and performance.
antifungal agent, Dichloran, is added to the medium to reduce colony
diameters of spreading fungi. The pH of the medium is reduced from Procedure2,3
7.2 to 5.6 for improved inhibition of the spreading fungi.1 The Materials Provided
presence of Rose Bengal in the medium suppresses the growth of DRBC Agar
bacteria and restricts the size and height of colonies of the more
rapidly growing molds. The concentration of Rose Bengal is reduced Materials Required But Not Provided
from 50 µg/ml to 25 µg/ml as found in Rose Bengal Chloramphenicol Peptone Water
Agar for optimal performance with Dichloran. Chloramphenicol is Flasks with closures
included in this medium to inhibit the growth of bacteria present in Distilled or deionized water
environmental and food samples. Inhibition of growth of bacteria and Autoclave
restriction of spreading of more rapidly growing molds aids in the Incubator (25°C)
isolation of slow-growing fungi by preventing their overgrowth by more
rapidly growing species. In addition, Rose Bengal is taken up by yeast Method of Preparation
and mold colonies, which allows these colonies to be easily 1. Suspend 31.6 grams in 1 liter of distilled or deionized water.
recognized and enumerated. Reduced recovery of yeasts may be 2. Heat to boiling to dissolve completely.
encountered due to increased activity of Rose Bengal at pH 5.6.1 Bacto 3. Autoclave at 121°C for 15 minutes.
Agar is a solidifying agent.
Specimen Collection and Preparation
Formula Prepare sample for surface inoculation following recommended
DRBC Agar guidelines. 2,3 The use of 0.1% Peptone Water as the diluent is
Formula Per Liter recommended.
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Test Procedure
Potassium Phosphate Monobasic . . . . . . . . . . . . . . . . . . . . . 1 g 1. Inoculate 0.1 ml of appropriate decimal dilutions of the sample in
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g duplicate onto the surface of DRBC Agar plates. The plates should
Dichloran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002 g be dried overnight at room temperature. Spread the inoculum over
Rose Bengal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025 g the entire surface of the plate using a sterile, bent-glass rod.
Chloramphenicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g 2. Incubate plates upright at 22-25°C. Examine for growth of yeasts
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g and molds after 3, 4 and 5 days incubation.
Final pH 5.6 ± 0.2 at 25°C
Results
Precautions Colonies of molds and yeasts should be apparent within 5 days of
1. For Laboratory Use. incubation. Colonies of yeast appear pink due to the uptake of Rose
Bengal. Report the results as colony forming units per gram or milliliter
2. TOXIC. MAY CAUSE CANCER. POSSIBLE RISK OF HARM
of sample.
TO THE UNBORN CHILD. Avoid contact with skin and eyes. Do
not breathe dust. Wear suitable protective clothing. Keep container References
tightly closed. TARGET ORGAN(S): Blood, Nerves, Lymph
Glands, Eyes. 1. King, A. D., A. D. Hocking, and J. I. Pitt. 1979. Dichloran-rose
bengal medium for the enumeration and isolation of molds from
FIRST AID: In case of contact with eyes, rinse immediately with foods. Appl. and Environ. Microbiol. 37:959-964.
2. Mislivec, P. B., L. R. Beuchat, and M. A. Cousin. 1992. Yeasts 3. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compen-
and molds, p. 239-249. In C. Vanderzant, and D. F. Splittstoesser, dium of methods for the microbiological examination of foods,
(ed.). Compendium of methods for the microbiological examination 3rd ed. American Public Health Association. Washington, D.C.
of foods, 3rd ed. American Public Health Association.
Washington, D.C. Packaging
DRBC Agar 500 g 0587-17
The cultures listed above are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
3. Follow proper, established laboratory procedures in handling and Sample Collection and Preparation
disposing of infectious materials. Refer to appropriate references for specimen collection and preparation.
Storage Test Procedure2
Store the dehydrated medium below 30°C. The dehydrated medium is 1. Pre-enrich the sample in Demi-Fraser Broth. Incubate for 18-24 hours
very hygroscopic. Keep container tightly closed. at 35 ± 2°C. Subculture onto Oxford Medium or PALCAM Medium.
Store Fraser Broth Supplement at 2-8°C. 2. Transfer 0.1 ml of the pre-enrichment culture into 10 ml of Fraser
Store the prepared medium at 2-8°C. Broth and incubate for 48 hours at 37°C. Subculture onto Oxford
Medium or PALCAM Medium after 18-24 hours and again after
Expiration Date 42-48 hours of incubation.
The expiration date applies to the product in its intact container when 3. Examine Oxford Medium or PALCAM Medium plates for the
stored as directed. Do not use a product if it fails to meet specifications appearance of presumptive Listeria colonies.
for identity and performance. 4. Confirm the identity of all presumptive Listeria by biochemical
and/or serological testing.
Procedure
Results
Materials Provided
The presence of Listeria is presumptively indicated by the blackening
Demi-Fraser Broth Base of Demi-Fraser Broth after incubation for 24-48 hours at 35°C.
Fraser Broth Supplement Confirmation of the presence of Listeria is made following subculture
Materials Required But Not Provided onto appropriate media and biochemical/serological identification.
Glassware References
Autoclave
1. Fraser, J., and W. Sperber. 1988. Rapid detection of Listeria in
Fraser Broth food and environmental samples by esculin hydrolysis. Journal of
Oxford Medium Food Protection 51:762- 765.
PALCAM Medium
2. L’association française de normalisation (AFNOR). 1993. Food
Sterile tubes with closures
Microbiology- Detection of Listeria monocytogenes-Routine
Method of Preparation Method, V 08-055. AFNOR, Paris, France.
1. Dissolve 55 grams of Demi-Fraser Broth Base in 1 liter distilled or Packaging
deionized water.
Demi-Fraser Broth Base 500 g 0653-17-0
2. Autoclave at 121°C for 15 minutes. Cool to room temperature. 10 kg 0653-07-0
3. Aseptically add 10 ml Fraser Broth Supplement. Mix well.
Fraser Broth Supplement 6 x 10 ml 0211-60-2
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
Formula 2. Heat and boil briefly with frequent, careful agitation to dissolve
Desoxycholate Citrate Agar completely. Avoid overheating.
Formula Per Liter 3. DO NOT AUTOCLAVE.
Meat, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 g
Specimen Collection and Preparation
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Collect specimens according to recommended guidelines.
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g Test Procedure
Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
Sodium Desoxycholate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g 1. Inoculate specimen directly onto surface of medium.
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5 g 2. Incubate plates at 35 ± 2°C for 18-24 hours. Plates can be incubated
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g for an additional 24 hours if no lactose fermenters are observed.
Final pH 7.5 ± 0.2 at 25°C Results
Precautions Non-lactose fermenters produce transparent, colorless to light pink or
tan colored colonies with or without black centers. Lactose fermenters
1. For Laboratory Use.
produce a red colony with or without a bile precipitate.
2. Follow proper, established laboratory procedures in handling and
disposing of infectious materials. Limitations of the Procedure
Storage 1. Coliform strains may be encountered that will grow on this
medium, making it difficult to detect pathogens.
Store the dehydrated medium below 30°C. The dehydrated medium is
very hygroscopic. Keep container tightly closed. 2. Heavy inocula should be distributed over the entire surface of the
medium prevent complete masking of pathogens by coliform
Expiration Date organisms.
The expiration date applies to the product in its intact container when References
stored as directed. Do not use a product if it fails to meet specifications
for identity and performance. 1. MacFaddin, J. F. 1985. Media for isolation-cultivation-
identification-maintenance of medical bacteria, vol. 1, p. 269-275.
Procedure Williams & Wilkins, Baltimore, MD.
Materials Provided 2. Leifson, E. 1935. New culture media based on sodium
desoxycholate for the isolation of intestinal pathogens and for the
Desoxycholate Citrate Agar
enumeration of colon bacilli in milk and water. J. Pathol. Bacteriol.
Materials Required but not Provided 40: 581-599.
Glassware 3. Farmer III, J. J., and M. T. Kelly. 1991. Enterobacteriaceae. p.
Petri dishes 360-383. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D.
Distilled or deionized water Isenberg and H. J. Shadomy (ed.), Manual of clinical microbiology,
Incubator (35°C) 5th ed. American Society for Microbiology, Washington, D.C.
Intended Use normal intestinal flora by using citrates and sodium desoxycholate
Bacto Desoxycholate Lactose Agar is used for isolating and in specified amounts as inhibitors to gram-positive bacteria.
differentiating gram-negative enteric bacilli and for enumerating Standard Methods manuals for dairy3 and water4 specified a modification
coliforms from water, wastewater, milk and dairy products. of Desoxycholate Agar to contain less sodium desoxycholate and,
accordingly, be less inhibitory to gram-positive bacteria. This
Also Known As formulation, known as Desoxycholate Lactose Agar, was used in pour
NOTE: Deoxy-; alternate spelling.1 plate procedures for isolation and enumeration of coliforms in milk,
Summary and Explanation water and other specimens.
Desoxycholate Lactose Agar is a modification of Desoxycholate Principles of the Procedure
Agar formulated by Leifson.2 His original medium demonstrated
Bacto Peptone provides nitrogen and carbon for general growth
improved recovery of intestinal pathogens from specimens containing
requirements. Lactose is a fermentable carbohydrate. Sodium
Chloride maintains the osmotic balance of the medium. Sodium Expiration Date
Desoxycholate and Sodium Citrate inhibit growth of gram-positive The expiration date applies to the product in its intact container when
bacteria. Neutral Red is a pH indicator. Bacto Agar is a solidifying stored as directed. Do not use a product if it fails to meet specifications
agent. for identity and performance.
Differentiation of enteric bacilli is based on fermentation of lactose.
Bacteria that ferment lactose produce acid and, in the presence of Procedure
neutral red, form red colonies. Bacteria that do not ferment lactose Materials Provided
form colorless colonies. The majority of normal intestinal bacteria Bacto Desoxycholate Lactose Agar
ferment lactose (red colonies) while Salmonella and Shigella species
do not ferment lactose (colorless colonies). Materials Required but not Provided
Glassware
Formula Distilled or deionized water
Bacto Desoxycholate Lactose Agar Bunsen burner or heating plate
Formula Per Liter Incubator(35°C)
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Petri dishes
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Method of Preparation
Sodium Desoxycholate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
1. Suspend 42.5 grams in 1 liter distilled or deionized water.
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g 2. Boil 1 minute with frequent, careful agitation to dissolve
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g completely. Avoid overheating.
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03 g 3. DO NOT AUTOCLAVE.
Final pH 7.1 ± 0.2 at 25°C Specimen Collection and Preparation
Precautions Refer to appropriate references for specimen collection and preparation.
1. For Laboratory Use. Test Procedure
2. Follow proper established laboratory procedures in handling and See appropriate references for specific procedures.
disposing of infectious materials.
Results
Storage Refer to appropriate references and procedures for results.
Store the dehydrated medium below 30°C. The dehydrated medium is
very hygroscopic. Keep container tightly closed. Uninoculated Escherichia coli
plate ATCC® 25922
as blood agar bases. Bacto Agar is a solidifying agent. Waterbath (45-50°C) (optional)
Supplementation with 5% blood provides additional growth factors for Sterile defibrinated blood (optional)
fastidious microorganisms. Sterile Petri dishes
must be heat shocked to kill off vegetative cells and subcultured into Incubator (35°C)
DRCA to confirm the presence of sulfite-reducing clostridia. Ringer’s solution or 0.1% peptone water
2. Autoclave at 121°C for 15 minutes. • Gross contamination interfered with the growth of the
3. Cool below 50°C. mycobacteria.
4. Aseptically add 20 ml Dubos Medium Albumin and mix thoroughly. • Proper aerobic conditions and increased CO2 tension were not
provided during incubation.
5. Dispense into tubes.
2. Mycobacteria are strict aerobes and growth is stimulated by
Dubos Oleic Agar increased levels of CO2. Screw caps on tubes or bottles should
1. Suspend 4 grams Dubos Oleic Agar Base in 180 ml distilled or remain loose for a free exchange of CO2.
deionized water.
2. Heat to boiling to dissolve completely. References
3. Autoclave at 121°C for 15 minutes. 1. Musser, J. M. 1995. Antimicrobial agent resistance in Mycobacteria:
4. Cool to 50-55°C. molecular genetic insights. Clin. Microbiol. Rev. 8:496-514.
5. Aseptically add 20 ml Dubos Oleic Albumin Complex and 5,000 2. Klietmann, W. 1995. Resistance and susceptibility testing for
-10,000 units penicillin (25-50 units per ml medium). Mycobacterium tuberculosis. Clin. Microbiol. Newsletter 17:65-69.
6. Mix thoroughly. 3. Nolte, F. S., and B. Methcock. 1995. Mycobacterium, p. 400-437.
In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H.
7. Dispense into sterile tubes or plates. Yolken (ed.), Manual of clinical microbiology, 6th ed. American
Specimen Collection and Preparation7 Society for Microbiology, Washington, D.C.
1. Collect specimens in sterile containers and transport immediately 4. Am. Rev. Tuberculosis, 1950, 61:66.
to the laboratory following recommended guidelines. 5. J. Exp. Med., 1946, 83:409.
2. Process each specimen as appropriate for that specimen. 6. Am. Rev. Tuberc., 1947, 56:334.
Test Procedure 7. A. Rev. Tuberculosis, 1950, 61:563.
1. Inoculate the specimen onto/into the medium and incubate tubes 8. Am. J. Clin. Path., 1950, 20:678.
for up to eight weeks. 9. Isenberg, H. D. (ed.). 1994. Clinical microbiology procedures hand-
2. Examine tubes for growth. book, suppl. 1. American Society for Microbiology, Washington, D.C.
10. Tenover, F. C., J. T. Crawford, R. E. Huebner, L. J. Geiter,
Results C. R. Horsburgh, Jr., and R. C. Good. 1993. The resurgence of
Mycobacteria grow on the medium or in the broth. tuberculosis: is your laboratory ready? J. Clin. Microbiol. 31:767-770.
Limitations of the Procedure Packaging
1. Negative culture results do not rule out active infection by mycobac- Dubos Albumin Broth 20 tubes 1022-39
teria. Some factors that are responsible for unsuccessful cultures are: Dubos Broth Base 500 g 0385-17
• The specimen was not representative of the infectious material,
i.e., saliva instead of sputum. Dubos Medium Albumin 12 x 20 ml 0309-64
• The mycobacteria were destroyed during digestion and Dubos Oleic Agar Base 500 g 0373-17
decontamination of the specimen. Dubos Oleic Albumin Complex 12 x 20 ml 0375-64
Bacto m E Agar
®
Summary and Explanation
Enterococcus species are a subgroup of fecal streptococci that
Bacto Esculin Iron Agar includes E. faecalis, E. faecium, E. gallinarum, and E. avium. 1
Enterococci are differentiated from other streptococci by their
ability to grow in 6.5% sodium chloride, at pH 9.6, and at 10°C and
Intended Use 45°C.1 The enterococci portion of the fecal streptococcus group
Bacto m E Agar is used with nalidixic acid and triphenyl tetrazolium is a valuable bacterial indicator for determining the extent of fecal
chloride in isolating and differentiating enterococci from water by contamination of recreational surface waters.1
membrane filtration and in an in situ esculin test on Bacto Esculin Iron
Agar. Slanetz and Bartley2 first reported quantitating enterococci by the
membrane filter method in 1957. A wide range of levels of enterococci
Bacto Esculin Iron Agar is used for enumerating enterococci from water in water can be enumerated and detected because small or large
by membrane filtration based on esculin hydrolysis. volumes of water can be analyzed by the membrane filter technique.3
In 1961, Kenner et al. 4 described the KF method for detecting
Also Known As and quantitating fecal streptococci. In 1966, Isenberg et al.5 reported
Esculin Iron Agar is abbreviated as EIA. a plating procedure with differentiation based on esculin hydrolysis.
Levin, Fischer and Cabelli6 compared the KF method with Isenberg’s
plating method, and found the latter method resulted in better Assessment Closure and Health (BEACH) Program. m EI Agar
recovery of fecal streptococci. They developed m E Agar as a primary eliminates the necessity of transferring the incubated membrane to
isolation medium for enterococci, and Esculin Iron Agar as an Esculin Iron Agar.
in situ substrate test medium for identifying organisms capable of
hydrolyzing esculin.6 Principles of the Procedure
Two research projects by the Environmental Protection Agency (EPA) m E Agar is a highly selective and differential primary isolation
evaluated the relationships between swimming-associated illness medium that supports good growth of enterococci. Bacto Peptone
and the ambient densities of indicator bacteria. 7,8 The studies and Yeast Extract provides carbon, nitrogen, minerals, vitamins
demonstrated that enterococci have a better correlation with and other growth factors for organism growth. Sodium Chloride
swimming-associated illness for both marine and fresh waters than maintains the osmotic balance of the medium. Nalidixic Acid and
fecal coliforms. Escherichia coli has a correlation in fresh water equal Sodium Azide act as selective agents to inhibit gram negative
to enterococci but does not correlate as well in marine waters.7,8 This bacteria. Actidione ® inhibits fungi. At the concentration in the
suggests that enterococci may be better indicator organisms for some formula, 2,3,5 triphenyl tetrazolium chloride (TTC) dyes enterococci
recreational waters.7,8 colonies. TTC slightly inhibits growth of other microorganisms.
In addition, the elevated incubation temperature of 41°C inhibits
m E Agar and Esculin Iron Agar are prepared according to the some indigenous microbial flora. Esculin is hydrolyzed by enterococci
formulas specified in Standard Methods.1 These media are used in to form esculetin and dextrose. The esculetin reacts with the iron
the membrane filter technique for the isolation of fecal streptococcus salt (ferric ammonium citrate) contained in the medium to produce
and enterococcus groups.1 This procedure can be used to test marine a black to reddish brown complex that appears in the medium
and fresh water sources. surrounding the colonies. The production of black to reddish brown
m E Agar with the addition of 0.075% indoxyl B-D glucoside complex verifies the colonies as enteroccci and facilitates their
(m EI Agar) is recommended by the U.S. EPA as a one step procedure enumeration. Bacto Agar is the solidifying agent in the medium.
for the isolation and identification of enterococci in recreational
water.9 This method is used in the EPA Beaches Environmental m E Agar
Enterococcus faecalis
ATCC® 29212
Cultural Response
Prepare m E Agar per label directions and pour into 9 x 50 mm plates. Dilute the test organisms and filter through membrane filters.
Place the filters on m E Agar plates and incubate the plates in an upright position for 48 hours at 41 ± 0.5°C. Remove the filters and
place over prepared Esculin Iron Agar plates. After 20 minutes incubation at 41 ± 0.5°C, count colonies giving positive esculin
reaction (formation of black or reddish brown precipitate).
ORGANISM ATCC® INOCULUM CFU/10 ml GROWTH ON m E AGAR REACTION ON ESCULIN IRON AGAR
Enterococcus faecalis 29212* 20-60 good/pink to red colonies black or red/brown ppt
Enterococcus faecalis 33186 20-60 good/pink to red colonies black or red/brown ppt
Escherichia coli 25922* 20-60 marked to complete inhibition inhibited
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as BactrolTM Disks and should be used as directed in Bactrol Disks Technical Information.
Bacto EC Medium
®
Summary and Explanation
EC Medium was developed by Hajna and Perry1 in an effort to
Intended Use improve the methods for the detection of the coliform group and
Bacto EC Medium is used for differentiating and enumerating E. coli. This medium consists of a buffered lactose broth with the
coliforms in water, wastewater, shellfish and foods. addition of 0.15% Bile Salts No. 3. Growth of spore forming
bacteria and fecal streptococci is inhibited by the bile salts, while
Also Known As growth of E.coli is enhanced by its presence. The medium can be
EC Medium is also referred to as EC Broth. EC is an abbreviation for
Escherichia coli.
The cultures listed are the minimum that should be used for performance testing. Escherichia coli
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks ATCC® 25922
Technical Information.
Storage and fluorescence. Non-E. coli coliforms that grow may exhibit
Store the dehydrated medium below 30°C. The dehydrated medium is fluorescence but will not produce gas.
very hygroscopic. Keep container tightly closed.
Limitations of the Procedure
Expiration Date 1. Since the nutritional requirements of organisms vary, some strains may
be encountered that fail to grow or grow poorly on this medium.
The expiration date applies to the product in its intact container when
stored as directed. Do not use a product if it fails to meet specifications 2. Strains of E. coli that fail to grow in EC Medium with MUG, fail to
for identity and performance. produce gas, or fail to produce glucuronidase may infrequently be
encountered.
Procedure 3. Strains of Salmonella, Shigella and Yersinia that produce glucu-
Materials Provided ronidase may be encountered. These strains must be distinguished
EC Medium with MUG from E. coli on the basis of other parameters, i.e., gas production,
growth at 44.5°C.
Materials Required But Not Provided 4. The presence of endogenous glucuronidase in shellfish samples
Test tubes may cause false positive fluorescent reactions at the presumptive
Fermentation vials stage. To prevent this problem, the use of EC Medium with MUG
Sterile pipettes in the confirmatory stage has been recommended.5
Incubator 35°C, 44.5°C
Long-wave UV lamp References
Autoclave 1. Hajna and Perry. 1943. Am. J. Public Health 33:550.
2. Feng, P. C. S., and P. A. Hartman. 1982. Fluorogenic assays for
Method of Preparation immediate confirmation of Escherichia coli. Appl. Environ.
1. Suspend 37.1 grams in 1 liter distilled or deionized water. Microbiol. 43:1320-1329.
2. Warm slightly to dissolve completely. 3. Robison, B. J. 1984. Evaluation of a fluorogenic assay for detection
3. Dispense into test tubes containing inverted fermentation vials. of Escherichia coli in foods. App. Environ. Microbiol. 48:285-288.
4. Autoclave at 121°C for 15 minutes. 4. Moberg, L. J. 1985. Fluorogenic assay for rapid detection of
5. Before opening the autoclave, allow the temperature to drop below Escherichia coli in food. Appl. Environ. Microbiol. 50:1383-1387.
75°C to avoid entrapping air bubbles in the fermentation vials. 5. Koburger, J. A., and M. L. Miller. 1985. Evaluation of a
fluorogenic MPN procedure for determining Escherichia coli in
Specimen Collection and Preparation oysters. J. Food Prot. 48:244-245.
Collect food, water or other environmental samples in accordance with 6. Federal Register. 1991. National primary drinking water regulation;
recommended procedures.6,7,8 analytical techniques; coliform bacteria. Fed. Regist. 56:636-643.
Test Procedure 7. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). 1995.
Follow the methods and procedures as stated in appropriate references.6,7,8 Standard methods for the examination of water and wastewater,
19th ed. American Public Health Association, Washington, D.C.
Results 8. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compen-
Following incubation, observe tubes for growth, production of gas and dium of methods for the microbiological examination of food,
fluorescence. Positive gas production is demonstrated by displacement 3rd ed. American Public Health Association, Washington, D.C.
of the medium from the fermentation vial. Positive MUG reactions
exhibit a bluish fluorescence under long-wave (approximately 366 nm) Packaging
UV light. Typical strains of E. coli are positive for both gas production EC Medium with MUG 100 g 0022-15
500 g 0022-17
Phosphate are buffering agents. Brilliant Green and Oxgall are Expiration Date
selective agents. The expiration date applies to the product in its intact container when
stored as directed. Do not use a product if it fails to meet specifications
Formula for identity and performance.
EE Broth Mossel
Formula Per Liter Procedure
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Materials Provided
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
EE Broth Mossel
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 g
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g Materials Required But Not Provided
Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0135 g Glassware
Bacto Oxgall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g Incubator (35°C)
Final pH 7.2± 0.2 at 25°C Waterbath
Precautions Method of Preparation
1. For Laboratory Use. 1. Dissolve 45 grams in 1 liter distilled or deionized water.
2. IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM 2. Dispense 120 ml amounts into 250 ml flasks.
AND SKIN. Avoid contact with skin and eyes. Do not breathe dust. 3. Heat at 100°C (waterbath or flowing steam) for 30 minutes. Do
Wear suitable protective clothing. Keep container tightly closed. Not Autoclave.
FIRST AID: In case of contact with eyes, rinse immediately with
plenty of water and seek medical advice. After contact with skin,
Specimen Collection and Preparation
wash immediately with plenty of water. If inhaled, remove to fresh Obtain and process specimens according to the techniques and
air. If not breathing, give artificial respiration. If breathing is diffi- procedures established by laboratory policy.
cult, give oxygen. Seek medical advice. If swallowed seek medical Test Procedure
advice immediately and show this container or label.
1. Inoculate flasks of EE Broth Mossel with approximately 10 grams
3. Follow proper established laboratory procedures in handling and of homogenized food or other material to be tested.
disposing of infectious materials.
2. Shake the inoculated medium thoroughly for a few seconds to mix well.
Storage 3. Incubate for a total of 20-24 hours at 35-37°C. Shake the flasks
Store the dehydrated medium below 30°C. The dehydrated medium is after the first 3 hours of incubation.
very hygroscopic. Keep container tightly closed.
4. Prepare plates such as Violet Red Bile Agar for streaking. To ensure References
recovery of dextrose fermenters, add 1% dextrose before boiling. 1. Mossel, D. A. A., M. Vissar, and A. M. R. Cornellisen. 1963. The
5. Streak a loopful of the enrichment culture onto the prepared plates. examination of foods for Enterobacteriaceae using a test of the
6. Incubate the plates for 18-24 hours at 35-37°C. Examine for the type generally adopted for the detection of salmonellae. J. Appl.
presence of coliforms which appear pink to purplish-red on Violet Bacteriol. 26:444-452.
Red Bile Agar. The color of coliform colonies may vary if a different 2. Hartman, P. A., and S. A. Minnich. 1981. Automation for rapid
medium is used. identification of Salmonella in foods. J. Food Prot. 44:385-386.
For a complete discussion on Enterobacteriaceae in food testing, refer 3. Association of Official Analytical Chemists. 1995. Bacteriological
to procedures in Standard Methods.3,4 analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
Results 4. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992.
Acid production causes the color of EE Broth Mossel to become Compendium of methods for the microbiological examination
yellow. A negative reaction results in no color change and the medium of food, 3rd ed. American Public Health Association,
remains green. Washington, D.C.
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
or possessed dark centers with transparent, colorless peripheries. Materials Required But Not Provided
Lactose- or sucrose-negative colonies were colorless. The Eosin Flasks with closures
Methylene Blue Agar of Holt-Harris and Teague had definite Distilled or deionized water
advantages over the Fuchsin Sulfite Agar of Endo. The EMB Agar Bunsen burner or magnetic hot plate
formulation was more sensitive, more accurate, more stable, and gave Autoclave
an earlier differentiation between the lactose fermenters and lactose Waterbath (45-50)°C
and sucrose nonfermenters. Petri dishes
Two years after Holt-Harris and Teague had introduced their new Incubator (35°C)
medium, Levine 2 described an Eosin Methylene Blue Agar for
differentiating fecal and nonfecal coliforms. Levine’s medium Method of Preparation
differentiated salmonellae and other lactose nonfermenters from the 1. Suspend 36 grams in 1 liter distilled or deionized water.
coliform organisms. 2. Heat to boiling to dissolve completely.
EMB Agar is a combination of the Levine and the Holt-Harris and 3. Autoclave at 121°C for 15 minutes. Avoid overheating.
Teague formulae. EMB Agar is selective due to the presence of inhibitors 4. Cool to 45-50°C in a waterbath.
and differential based on the ability of some organisms to ferment 5. Dispense into sterile Petri dishes. Evenly disperse the precipitate
carbohydrates with the absorption of eosin and methylene blue. when dispensing.
EMB Agar is recommended for use in examining clinical specimens
for enteric pathogens.3,4,5 The medium enables the isolation and differ-
Specimen Collection and Preparation
entiation of gram-negative enteric bacilli. 1. Collect specimens in sterile containers or with sterile swabs and
transport immediately to the laboratory following recommended
Principles of the Procedure guidelines.3,4,5
Peptone is a source of nitrogen and other nutrients in the formulation. 2. For specific information about specimen preparation and inoculation
Eosin and methylene blue are dyes which combine to form a precipitate of clinical specimens, consult appropriate references.3,4,5
at an acid pH. The dyes act both as pH indicators and inhibitors. Test Procedure
Gram-positive bacteria are partially inhibited on the medium. Lactose For isolation of enteric pathogens from clinical specimens, inoculate
and Sucrose are fermentable carbohydrates. Phosphate acts as a buffer. fecal specimens and rectal swabs onto a small area of one quadrant
Bacto Agar is a solidifying agent. of the EMB Agar plate and streak for isolation. This will permit
Formula development of discrete colonies. Incubate plates at 35°C. Examine
plates at 24 hours and again at 48 hours for colonies with characteristic
EMB Agar morphologies associated with potential pathogens.
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Results
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Salmonella and Shigella colonies are translucent and amber colored or
Bacto Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g colorless. Coliforms that use lactose and/or sucrose produce blue-black
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g colonies with dark centers and greenish metallic sheen. Other coliforms
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5 g such as Enterobacter form mucoid, pink colonies. Strains of
Eosin Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g Enterococcus faecalis are partially inhibited on this medium and
Methylene Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.065 g appear as colorless colonies.
Final pH 7.2 ± 0.2 at 25°C
Limitations of the Procedure
Precautions 1. EMB Agar is only moderately inhibitory. Some staphylococci,
1. For Laboratory Use. streptococci and yeast may grow. They will appear as small,
2. Follow proper established laboratory procedure in handling and pinpoint colonies. Gram-negative nonfermenting bacilli may grow
disposing of infectious materials. and appear as non-lactose fermenters. Biochemical tests are necessary
for further identification to genus or species.6
Storage 2. Some strains of Salmonella and Shigella may not grow on EMB
Store the dehydrated medium below 30°C. The dehydrated medium is very Agar.6 It is recommended that a nonselective, differential medium
hygroscopic. Keep container tightly closed. Store prepared plates at 2-8°C. (MacConkey Agar or Hektoen Enteric Agar) and a selective medium
(Bismuth Sulfite Agar, SS Agar or Desoxycholate Citrate Agar) be
Expiration Date run in parallel with EMB Agar.
Expiration date applies to the product in its intact container when stored 3. Sterilization reduces the methylene blue, leaving the medium
as directed. Do not use a product if it fails to meet specifications for orange in color. The normal purple color of the medium may be
identity and performance. restored by gentle mixing. If the reduced medium is not shaken to
oxidize the methylene blue, a dark zone beginning at the top and
Procedure extending downward through the medium will gradually appear.
Materials Provided The sterilized medium normally contains a flocculent precipitate
EMB Agar which should not be removed. By cooling to 50°C and gently
mixing the medium before pouring it into plates, the flocculation 4. Pezzlo, M. (ed.). 1992. Aerobic bacteriology, p. 1.0.1-1.20.47. In
will be finely dispersed. H. D. Isenberg, (ed.), Clinical microbiology procedures handbook,
4. Greenish metallic sheen is not always present. The presence of the vol. 1. American Society for Microbiology, Washington, D.C.
greenish metallic sheen is not diagnostic for E. coli.6 5. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
5. Store and incubate EMB Agar plates in the dark. Visible light can Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
alter the ability of the medium to support microbial growth, St. Louis, MO.
especially of Proteus spp.7 6. MacFaddin, J. F. 1985. Media for isolation-cultivation-
identification-maintenance of medical bacteria, vol. 1. Williams &
References Wilkins, Baltimore, MD.
1. Holt-Harris, J. E., and O. Teague. 1916. A new culture medium
7. Girolami, R. L., and J. M. Stamm. 1976. Inhibitory effect of light
for the isolation of Bacillus typhosa from stools. J. Infect. Dis.
on growth-supporting properties of Eosin Methylene Blue Agar.
18:596-600.
Appl. Environ. Microbiol. 31:141.
2. Levine, M. M. 1918. Differentiation of E. coli and B. aerogenes
on a simplified Eosin-Methylene Blue Agar. J. Infect. Dis. 23:43. Packaging
3. Gray, L. D. 1995. Escherichia, Salmonella, Shigella and Yersinia, EMB Agar 100 g 0076-15
p. 450-456. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. 500 g 0076-17
Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 2 kg 0076-07
6th ed. American Society for Microbiology, Washington, D.C. 10 kg 0076-08
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in
Bactrol Disks Technical Information. Uninoculated Enterococcus faecalis
tube ATCC® 29212
Ethyl Violet inhibit gram-positive bacilli and gram-positive cocci other Expiration Date
than enterococci. Monopotassium and Dipotassium Phosphates buffer The expiration date applies to the product in its intact container when
the medium. Sodium Chloride provides osmotic balance. stored as directed. Do not use a product if it fails to meet specifications
for identity and performance.
Formula
EVA Broth Procedure
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Materials Provided
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g EVA Broth
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 g Materials Required but not Provided
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 g
Glassware
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Sodium Azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g Distilled or deionized water
Ethyl Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.00083 g Autoclave
Final pH 7.0 ± 0.2 at 25°C Incubator (35°C)
aerogenes. Later, he described the use of this medium in studies of Materials Required but not Provided
intestinal putrefaction. 2 In 1923, Reddish and Rettger3 used the Glassware
medium in their detailed study of Clostridium putrificum and, the Garden soil extract (AOAC, 1995, 6.3.05, A.[a.][1.])5
following year, in a study of other spore-forming anaerobes.4 Autoclave
AOAC5 recommends Egg Meat Medium to propagate and maintain
cultures of Clostridium for use in testing sporicidal activity of liquid Method of Preparation
and gaseous chemicals. 1. Suspend 1.5 grams in 15 ml garden soil extract.5 Let stand at least
15 minutes to form a thoroughly wetted, even suspension.
Principles of the Procedure 2. Autoclave at 121°C for 15 minutes. Allow to cool.
Beef muscle provides carbon, nitrogen, inorganic salts, vitamins, and 3. Avoid a rapid release of pressure after sterilization to prevent
a variety of other nutrients to support bacterial growth. Egg whites are expelling the medium from the test tubes.
a source of protein. Calcium carbonate helps neutralize acids and
displaces oxygen in the media. Specimen Collection and Preparation
Refer to appropriate references for specimen collection and preparation.
Formula
Egg Meat Medium Test Procedure
Formula per liter Refer to AOAC5 for detailed procedures to determine presence or absence of
Beef Muscle, from . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454 g sporicidal activity of disinfectants against specified spore-forming bacteria.
Egg White, from . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 eggs
Calcium Carbonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Results
Final pH 7.2 ± 0.2 at 25°C Refer to appropriate references and procedures for results.
Precautions References
1. For Laboratory Use. 1. Rettger, L. F. 1903. An experimental study of the chemical
2. Follow proper established laboratory procedure in handling and products of Bacillus coli communis and Bacillus lactis aerogenes.
disposing of infectious materials. Am. J. Physiol. 8:284.
2. Rettger, L. 1906. Studies on putrefaction. J. Biol. Chem. 2:71.
Storage 3. Reddish, G. F., and L. F. Rettger. 1923. Clostridium putrificum.
Store the dehydrated medium below 30°C. The powder is very II. Morphological, cultural and biochemical study. J. Bact. 8:375.
hygroscopic. Keep container tightly closed. 4. Reddish, G. F., and L. F. Rettger. 1924. A morphological,
cultural and biochemical study of representative spore-forming
Expiration Date anaerobic bacteria. J. Bact. 9:13.
The expiration date applies to the product in its intact container when
5. Association of Official Analytical Chemists. 1995. Official
stored as directed. Do not use a product if it fails to meet specifications
methods of analysis of AOAC International, 16th ed. AOAC
for identity and performance.
International, Arlington, VA.
Procedure Packaging
Materials Provided Egg Meat Medium 500 g 0042-17
Egg Meat Medium
Chloride maintains the osmotic balance of the medium, and ascorbic stored as directed. Do not use a product if it fails to meet the specifications
acid is added to create a proper environment for organism growth. for identity and performance.
Sodium Acetate is a selective agent against gram negative bacteria.
Procedure
Formula Materials Provided
Elliker Broth Elliker Broth
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Materials Required But Not Provided
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Glassware
Bacto Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g Autoclave
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Incubator (35°C)
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Waterbath (45-50°C)
Bacto Saccharose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Sterile tubes
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 g
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g Method of Preparation
Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g 1. Suspend 48.5 grams in 1 liter distilled or deionized water.
Final pH 6.8 ± 0.2 at 25°C 2. Heat to boiling to dissolve completely.
3. Autoclave at 121°C for 15 minutes.
Precautions 4. Dispense as desired.
1. For Laboratory Use.
2. Follow proper established laboratory procedures in handling and Specimen Collection and Preparation
disposing of infectious materials. Obtain and process specimens according to the techniques and
procedures established by laboratory policy.
Storage
Store the dehydrated medium below 30°C. The dehydrated medium is Test Procedure
very hygroscopic. Keep container tightly closed. For a complete discussion on the isolation and identification of
streptococci and lactobacilli, refer to standard methods in food testing.3,4,5
Expiration Date
The expiration date applies to the product in its intact container when Results
Refer to appropriate references and procedures for results.
User Quality Control Limitations of the Procedure
Identity Specifications 1. Since the nutritional requirements of organisms vary, some
Dehydrated Appearance: Light beige, free-flowing, strains may be encountered that fail to grow or grow poorly on
homogeneous. this medium.
Solution: 4.85% solution, soluble in distilled
or deionized water on boiling. References
Solution is light to medium amber, 1. Elliker, P. R., A. W. Anderson, and G. Hannesson. 1956. An agar
clear without significant precipitate. culture medium for lactic acid streptococci and lactobacilli. J. Dairy
Prepared Medium: Light to medium amber, clear Sci. 39:1611.
without significant precipitate. 2. McLaughlin, C. B. 1946. Readily prepared medium for cultivation
Reaction of 4.85% of lactobacilli. J. Bacteriol. 51:560.
Solution at 25°C: pH 6.8 ± 0.2 3. Frank, J. F., G. L. Christen, and L. B. Bullerman. 1993. Test for
groups of microorganisms, p. 271-286. In R. T. Marshall (ed.),
Cultural Response Standard methods for the examination of dairy products. 16th ed.
Prepare Elliker Broth per label directions. Inoculate and American Public Health Association, Washington, D.C.
incubate at 35 ± 2°C for 18-48 hours except Streptococcus
cremoris which is incubated at 30 ± 2°C. 4. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compen-
INOCULUM dium of methods for the microbiological association of food, 3rd.
ORGANISM ATCC® CFU GROWTH ed. American Public Health Association, Washington, D.C.
Lactobacillus casei 7469 100-1,000 good 5. Association of Official Analytical Chemists. 1995. Bacteriological
Lactobacillus delbrueckii subsp. lactis 8000 100-1,000 good analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
Streptococcus cremoris 9596 100-1,000 good
Packaging
The cultures listed are the minimum that should be used for
performance testing. Elliker Broth 500 g 0974-17
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Bacto Emerson YpSs Agar
®
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Intended Use Final pH 7.0 + 0.2 at 25°C
Bacto Emerson YpSs Agar is used for cultivating Allomyces and other fungi.
Precautions
Summary and Explanation 1. For Laboratory Use.
Emerson YpSs Agar is prepared according to the formula given by 2. Follow proper established laboratory procedures in handling and
Emerson.1 Emerson and Wilson2 used the medium at half strength for disposing of infectious materials.
streaking zygotes or zoospores to obtain single germlings.
Fungi are extremely successful organisms, as evidenced by their
Storage
ubiquity in nature.3 Of the estimated 250,000 species, fewer than 150 Store dehydrated medium below 30°C. The dehydrated medium is very
are known primary human pathogens.3 Opportunistic fungal pathogens hygroscopic. Keep container tightly closed.
are increasing at an impressive rate relating directly to the expanding
size of the immunocompromised to patient population.3
Expiration Date
The expiration date applies to the product in its intact container when
Principles of the Procedure stored as directed. Do not use a product if it fails to meet specifications
Yeast Extract provides a source of trace elements, vitamins and amino for identity and performance.
acids. Soluble Starch provides starch for hydrolysis, detoxification of
metabolic byproducts and as a carbon source. Dipotassium Phosphate Procedure
is a buffer. Magnesium Sulfate is a source of divalent cations and Materials Provided
sulfate. Bacto Agar is the solidifying agent. Emerson YpSs Agar
Formula Materials Required But Not Provided
Emerson YpSs Agar Glassware
Formula Per Liter Autoclave
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 g Sterile Petri dishes
Bacto Soluble Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g Waterbath (optional)
Method of Preparation
1. Suspend 40.5 grams in 1 liter distilled or deionized water.
User Quality Control
2. Heat to boiling to dissolve completely.
Identity Specifications 3. Autoclave at 121°C for 15 minutes.
Dehydrated Appearance: Light beige, free-flowing,
homogeneous. Specimen Collection and Preparation
Solution: 4.05% solution, soluble in distilled Obtain and process specimens according to the techniques and
or deionized water on boiling. procedures established by laboratory policy.
Solution is light to medium amber,
slightly opalescent, may have a Test Procedure
slight flocculent precipitate.
Refer to appropriate references for specific procedures on the isolation
Prepared Medium: Light to medium amber, slightly and cultivation of fungi.
opalescent, may have a slight
flocculent precipitate. Results
Reaction of 4.05% Refer to appropriate references and procedures for results.
Solution at 25°C: pH 7.0 ± 0.2
Cultural Response References
Prepare Emerson YpSs Agar per label directions. Inoculate and 1. Lloydia. 1941. 4:77.
incubate at 30 ± 2°C for 18-48 hours. 2. Mycologia. 1954. 46:393.
INOCULUM
ORGANISM ATCC® CFU GROWTH
3. Dixon, D. M., and R. A. Fromtling. 1995. Morphology, taxonomy,
Allomyces macrogynus 38327 100-300 good and classification of the fungi, p. 699-708. In Murray, P. R.,
Allomyces reticulatus 42465 100-300 good E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.).
Aspergillus niger 16404 100-300 good Manual of clinical microbiology, 6th ed. American Society for
Saccharomyces cerevisiae 9763 100-300 good Microbiology, Washington, D.C.
The cultures listed are the minimum that should be used for Packaging
performance testing.
Emerson YpSs Agar 500 g 0739-17
Formula
Bacto Endo Agar®
Endo Agar
Formula Per Liter
Intended Use Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Endo Agar is used for confirming the presence of coliform Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
organisms. Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Summary and Explanation Bacto Basic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
Endo1 developed a medium using a fuchsin sulfite indicator to differ- Sodium Sulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
entiate lactose fermenting and lactose non-fermenting organisms. Final pH 7.5 ± 0.2 at 25°C
Coliform organisms that ferment lactose produce red colonies and color
the surrounding medium. Typical reactions of this medium are not Precautions
caused by acid production but by the intermediate product 1. For Laboratory Use.
acetaldehyde, which reacts with sodium sulfite.2-3 2. HARMFUL. IRRITATING TO EYES, RESPIRATORY SYSTEM
Endo Agar was formerly a standard methods medium for the microbio- AND SKIN. POSSIBLE RISK OF IRREVERSIBLE EFFECTS.
logical examination of water 4 and dairy products.5 Avoid contact with skin and eyes. Do not breathe dust. Wear
suitable protective clothing. Keep container tightly closed.
Principles of the Procedure TARGET ORGAN(S): Liver, Thyroid.
Lactose-fermenting bacteria produce acetaldehyde. The aldehyde is FIRST AID: In case of contact with eyes, rinse immediately with
fixed by the sodium sulfite and in the presence of fuchsin forms red plenty of water and seek medical advice. After contact with skin,
colonies. A sheen is produced by rapid lactose fermenting organisms. wash immediately with plenty of water. If inhaled, remove to fresh
Lactose non-fermenting bacteria form clear, colorless colonies. air. If not breathing, give artificial respiration. If breathing is diffi-
Endo Agar contains Bacto Peptone as a source of carbon, nitrogen, cult, give oxygen. Seek medical advice. If swallowed seek medical
vitamins and minerals. Lactose is the carbohydrate source. Basic advice immediately and show this container or label.
Fuchsin in the presence of Sodium Sulfite produces the red colonies.
Bacto Agar is the solidifying agent.
Uninoculated Escherichia coli
plate ATCC® 25922
Formula FIRST AID: In case of contact with eyes, rinse immediately with
m Endo Agar LES plenty of water and seek medical advice. After contact with skin,
wash immediately with plenty of water. If inhaled, remove to fresh
Formula Per Liter
air. If not breathing, give artificial respiration. If breathing is
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 g
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 g
difficult, give oxygen. Seek medical advice. If swallowed seek
Thiopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 g medical advice immediately and show this container or label.
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 g 3. Follow proper established laboratory procedures in handling and
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4 g disposing of infectious materials.
Potassium Phosphate Dibasic . . . . . . . . . . . . . . . . . . . . . . . 3.3 g
Potassium Phosphate Monobasic . . . . . . . . . . . . . . . . . . . . . 1 g Storage
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 g Store the dehydrated medium below 30°C. The dehydrated medium is
Sodium Desoxycholate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g very hygroscopic. Keep container tightly closed.
Sodium Lauryl Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g
Sodium Sulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6 g Expiration Date
Bacto Basic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 g The expiration date applies to the product in its intact container when
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g stored as directed. Do not use a product if it fails to meet specifications
Final pH 7.2 ± 0.2 at 25°C for identity and performance.
Precautions Procedure
1. For Laboratory Use. Materials Provided
2. HARMFUL. IRRITATING TO EYES, RESPIRATORY SYSTEM m Endo Agar LES
AND SKIN. POSSIBLE RISK OF IRREVERSIBLE EFFECTS. Lauryl Tryptose Broth
Avoid contact with skin and eyes. Do not breathe dust. Wear suit- Materials Required but not Provided
able protective clothing. Keep container tightly closed. TARGET 95% Ethanol (Not denatured)
ORGAN(S): Liver, Thyroid. Glassware
Distilled or deionized water
Membrane filter apparatus
User Quality Control Membrane filter absorbent pads
Identity Specifications Petri dishes, 60 mm
Dehydrated Appearance: Purple, free-flowing, homogeneous. Incubator (35°C)
Solution: 5.1% solution, soluble in distilled
or deionized water containing Method of Preparation
2% ethanol on boiling. Solution m Endo Agar LES
is pinkish-red, slightly opalescent 1. Suspend 51 grams in 1 liter distilled or deionized water containing
to opalescent with precipitate. 20 ml ethanol (95% not denatured).
Prepared Medium: Rose colored, slightly opalescent, 2. Boil to dissolve completely. Do Not Autoclave.
with precipitate.
3. Dispense in 5-7 ml quantities into 60 mm sterile petri dishes.
Reaction of 5.1%
Solution at 25°C: pH 7.2 ± 0.2 Lauryl Tryptose Broth
1. Suspend 35.6 g in 1 liter of distilled or deionized water.
Cultural Response
2. Warm slightly to dissolve completely.
Prepare m Endo Agar LES per label directions. Use the
membrane filter technique to inoculate filters and preincubate 3. Autoclave at 121°C for 15 minutes.
on pads saturated with Lauryl Tryptose Broth (0241) at
35 ± 2°C for 1 1/2-2 hours. Transfer filters to plates of m Endo Specimen Collection and Preparation
Agar LES and incubate at 35 ± 2°C for 22 ± 2 hours. Collect samples and process according to recommended guidelines for
INOCULUM enumerating coliforms in water.2,4,5
ORGANISM ATCC® CFU GROWTH APPEARANCE
Escherichia 25922* 20-80 good red Test Procedure
coli with sheen 1. Place a membrane filter absorbent pad inside the cover.
Salmonella 14028 20-80 good pink
typhimurium 2. Add 1.8-2.0 ml Lauryl Tryptose Broth to each pad.
Staphylococcus 25923* 1,000-2,000 marked to – 3. Run the water sample through a membrane filter.
aureus complete inhibition 4. Place the filter, top side up, onto the pad containing Lauryl Tryptose
The cultures listed are the minimum that should be used for Broth. Use a rolling motion to avoid entrapping air bubbles.
performance testing. 5. Incubate at 35 ± 2°C for 11/2 -2 1/2 hours. Transfer the membrane
*These cultures are available as Bactrol™ Disks and should be used from the pad to the surface of the m Endo Agar LES medium in the
as directed in Bactrol Disks Technical Information.
petri dish bottom, keeping the side on which the bacteria have been
collected facing upward.
6. Leave the filter pad in the lid and incubate the plates in the inverted 3. Cowman, S., and R. Kelsey. 1992. Bottled water, p. 1031-1036.
position at 35 ± 2°C for 22 ± 2 hours. In C. Vanderzant, and D. F. Splittstoesser (ed.), Compendium of
7. Observe and count all colonies that are red and have a metallic sheen. methods for the microbiological examination of foods, 3rd ed.
American Public Health Association, Washington, D.C.
Results 4. Bordner, R., and J. Winter (ed.), 1978. Microbiological
All colonies that are red and have the characteristic metallic sheen are methods for monitoring the environment, water and wastes.
considered coliforms. The sheen may cover the entire colony, may EPA-600/8-78-017. Environmental Monitoring and Support
only be in the center or may appear only around the edges. Laboratory, Office of Research and Development, U. S.
Environmental Protection Agency, Cincinnati, OH.
Limitations of the Procedure 5. Environmental Protection Agency. 1992. Manual for the
1. Occasionally, noncoliform organisms may produce typical sheen certification of laboratories analyzing drinking water.
colonies. Coliform organisms may also occasionally produce EPA-814B-92-002. Office of Ground Water and Technical Support
atypical colonies (dark red or nucleated colonies without sheen). Division, U. S. Environmental Protection Agency, Cincinnati, OH.
It is advisable to verify both colony types.2
Packaging
References m Endo Agar LES 100 g 0736-15
1. McCarthy, J. A., J. E. Delaney, and R. J. Grasso. 1961. 500 g 0736-17
Measuring coliforms in water. Water Sewage Works. 108:238.
Lauryl Tryptose Broth 100 g 0241-15
2. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.), 1995. 500 g 0241-17
Standard methods for the examination of water and wastewater, 2 kg 0241-07
19th ed. American Public Health Association, Washington, D.C. 10 kg 0241-08
test organisms and be free from fermentable carbohydrates. Enteric FINAL ADD BEFORE ADD AFTER
CARBOHYDRATE CONCENTRATION AUTOCLAVING AUTOCLAVING
Fermentation Base is prepared according to the formula described by
Adonitol 0.5% X –
Edwards and Ewing.5,6
Arabinose 0.5% – X
Principles of the Procedure Cellobiose 0.5% – X
Dextrose (Glucose) 1% X –
Beef Extract and Peptone provide the carbon and nitrogen sources
Dulcitol 0.5% X –
required for good growth of a wide variety of organisms. Sodium Glycerol* 0.5% X –
Chloride maintains the osmotic balance of the medium. The Inositol 0.5% X –
microorganisms tested are differentiated by their ability to ferment a Lactose 1% – X
particular carbohydrate that has been added to the Enteric Fermentation Mannitol 1% X –
Base. The fermentation and resultant acid production are indicated by Salicin 0.5% X –
a change in color of the pH indicator (Andrade’s indicator) which is Sucrose 1% – X
also added to the Enteric Fermentation Base. Xylose 0.5% – X
Formula *Medium containing glycerol should be autoclaved for 10 minutes at 15 lbs pressure (121°C).
Enteric Fermentation Base 7. Dispense 3 ml amounts into test tubes containing inverted
Formula Per Liter fermentation vials (Durham tubes).
Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Specimen Collection and Preparation
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g 1. Collect specimens or food samples in sterile containers or with
Final pH 7.2 ± 0.1 at 25°C sterile swabs and transport immediately to the laboratory following
recommended guidelines.7,8,9
Precautions 2. Process each specimen, using procedures appropriate for that
1. For Laboratory Use. specimen or sample.7,8,9
2. Follow proper established laboratory procedure in handling and
Test Procedure
disposing of infectious materials.
For a complete discussion on identification of Enterobacteriaceae,
Storage refer to the appropriate procedures outlined in the references.5,6,10
Store the dehydrated medium below 30°C. The dehydrated medium is
very hygroscopic. Keep container tightly closed. Results
A positive result for gas includes production in at least 3% of the
Expiration Date volume of the fermentation tube. A positive reaction for gas production
The expiration date applies to the product in its intact container when is a change in color from light amber to dark pink or red.
stored as directed. Do not use a product if it fails to meet specifications
for identity and performance. Limitations of the Procedure
1. Negative tubes remain colorless and should be observed regularly
Procedure for a total of 30 days.
Materials Provided
Enteric Fermentation Base References
1. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
Materials Required But Not Provided Scott’s diagnostic microbiology, 9th edition. Mosby-Year Book,
Glassware Inc., St. Louis, MO.
Autoclave 2. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
Incubator (35°C) R. H. Yolken. (ed.). 1995. Manual of clinical microbiology,
Waterbath (45-50°C) 6th edition. ASM Press, Washington, D.C.
Choice of filter-sterilized carbohydrate(s) 3. Holt, J. G., N. R. Krieg, P. H. A. Sneath, J. T. Staley, and S. T.
Andrade’s Indicator (0.5 g Acid Fuchsin in 100 ml water plus 16 ml Williams. 1994. Bergey’s manual of determinative bacteriology,
of 1.0 N Sodium Hydroxide)
9th edition. Williams & Wilkins, Baltimore, MD.
Method of Preparation 4. Ewing, W. H. 1986. Edwards and Ewing’s identification of
1. Suspend 18 grams in 1 liter distilled or deionized water. Enterobacteriaceae, 4th edition. Elsevier Science Publishing Co.,
2. Add 10 ml Andrade’s Indicator. Inc., New York, NY
3. Heat to boiling to dissolve completely. 5. Edwards, P. R., and W. H. Ewing. 1972. Identification of
4. Autoclave at 121°C for 15 minutes. Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneapolis.
5. Cool to 45 to 50°C in a waterbath. 6. Balows, A., and W. J. Hausler. 1981. Diagnostic Procedures for
Bacteria, Mycotic and Parasitic Infections, 6th ed. American
6. Add appropriate amounts of sterile carbohydrates as indicated in
the table below. Public Health Association, Washington, D.C.
7. Baron, E. J., and S. M. Finegold. 1990. Bailey & Scott’s Diag- 10. Isenberg, H. D. (ed.). 1992. Conventional tests (18 to 24 hours) -
nostic Microbiology, 8th ed. C.V. Mosby Company, St. Louis, MO. Carbohydrate Fermentation Test, p. 1.19.28. Clinical microbiology
8. Gilligan, P. H. 1995. In P. R. Murray, E. J. Baron, M. A. Pfaller, procedures handbook, vol.1. American Society for Microbiology.
F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiol- Washington, D.C.
ogy, 6th ed. American Society of Microbiology, Washington, D.C.
9. Pezzlo, M. (ed.). 1994. Aerobic bacteriology, p. 1.0.0-1.20.47. In
Packaging
H. D. Isenberg (ed.), Clinical microbiology procedures handbook, Enteric Fermentation Base 500 g 1828-17
Vol. 1. American Society for Microbiology, Washington, D.C.
medium. Sodium Azide is the selective agent to suppress the growth of Magnifying lens
gram negative organisms. Bacto Agar is the solidifying agent. Distilled or deionized water
Triphenyltetrazolium Chloride (TTC) is the dye used as an indicator of
bacterial growth. TTC is reduced to the insoluble formazan inside the Method of Preparation
bacterial cell, resulting in the production of red colonies. 1. Suspend 42 grams in 1 liter distilled or deionized water.
2. Heat to boiling to dissolve completely.
Formula 3. DO NOT AUTOCLAVE.
m Enterococcus Agar 4. Dispense into 9 x 50 mm Petri dishes to a depth of 4-5 mm
Formula Per Liter (approximately 4-6 ml).
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Specimen Collection and Preparation
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g Collect water samples as described in Standard Methods for the
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 g Examination of Water and Wastewater, Section 9230 1 or by
Sodium Azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g laboratory policy.
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
2,3,5-Triphenyl Tetrazolium Chloride . . . . . . . . . . . . . . . . 0.1 g Test Procedure
Final pH 7.2 ± 0.2 at 25°C Membrane filtration procedure
1. Follow the membrane filtration procedure as described in Standard
Precautions Methods for the Examination of Water and Wastewater,
1. For Laboratory Use. Section 9230C.1
2. HARMFUL. HARMFUL BY INHALATION AND IF 2. Choose a sample size so that 20-60 colonies will result.
SWALLOWED. IRRITATING TO EYES, RESPIRATORY 3. Transfer the filter to agar medium in a Petri dish, avoiding air
SYSTEM AND SKIN. Avoid contact with skin and eyes. Do not bubbles beneath the membrane.
breathe dust. Wear suitable protective clothing. Keep container
4. Let plates stand for 30 minutes.
tightly closed. TARGET ORGAN(S): Cardiovascular, Lungs,
Nerves. 5. Invert plates and incubate at 35 ± 0.5°C for 48 hours.
FIRST AID: In case of contact with eyes, rinse immediately with Direct plating procedure
plenty of water and seek medical advice. After contact with skin, 1. Inoculate medium with a specimen using the streak plate method.
wash immediately with plenty of water. If inhaled, remove to fresh 2. Incubate plates at 35 ± 2°C for 24-48 hours.
air. If not breathing, give artificial respiration. If breathing is
difficult, give oxygen. Seek medical advice. If swallowed seek Results1
medical advice immediately and show this container or label. Count all light and dark red colonies as enterococci. Count colonies
3. Follow proper established laboratory procedures in handling and using a fluorescent lamp and a magnifying lens.
disposing of infectious materials.
Limitations of the Procedure
Storage 1. Since the nutritional requirements of organisms vary, some
Store the dehydrated medium below 30°C. The dehydrated medium is strains may be encountered that fail to grow or grow poorly on
very hygroscopic. Keep container tightly closed. this medium.
Bacto m FC Agar
®
Summary and Explanation
Geldreich et al.1 formulated a medium to enumerate fecal coliforms
Bacto m FC Broth Base (MFC) using the membrane filter (MF) technique without prior
enrichment. Fecal coliforms, i.e., those found in the feces of
warm-blooded animals, are differentiated from coliforms from
Bacto Rosolic Acid environmental sources by their ability to grow at 44.5 ± 0.5°C.2
Many Standard Methods membrane filtration procedures specify M-FC
medium for testing water. The American Public Health Association
Intended Use (APHA) specifies M-FC medium and incubation at 44.5 ± 0.5°C in the
Bacto m FC Agar and Bacto m FC Broth Base are used with Bacto fecal coliform membrane filter procedure, the delayed-incubation
Rosolic Acid in cultivating and enumerating fecal coliforms by the fecal coliform procedure, the two-layer agar method for recovering
membrane filter technique at elevated temperatures. injured fecal coliforms,2 and in the membrane filter method for fecal
coliforms in bottled water.3 The Association of Official Analytical
Also Known As Chemists (AOAC) specifies m-FC Agar for detecting total coliforms
M-FC Medium and fecal coliforms in foods.4
The U. S. Environmental Protection Agency specifies using M-FC Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
medium in fecal coliform methods for testing water by the direct MF Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 g
method or the delayed-incubation MF method.5,6 Bacto Bile Salts No. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Principles of the Procedure Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
m FC Agar and m FC Broth Base contain Tryptose and Proteose Aniline Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g
Peptone No. 3 as sources of carbon, nitrogen, vitamins and minerals. Final pH 7.4 ± 0.2 at 25°C
Yeast Extract supplies B-complex vitamins that stimulate bacterial m FC Broth Base
growth. Lactose is a carbohydrate. Bile Salts No. 3 inhibits growth of Formula Per Liter
gram-positive bacteria. m FC Agar contains Bacto Agar as the Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
solidifying agent. The differential indicator system combines Aniline Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . . 5 g
Blue and Rosolic Acid. Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Colonies of fecal coliforms are blue; non-fecal coliforms and other Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
organisms are gray to cream-colored. Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 g
Bacto Bile Salts No. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g
Formula Aniline Blue (Water Blue) . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g
m FC Agar Final pH 7.4 ± 0.2 at 25°C
Formula Per Liter Rosolic Acid
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Rosolic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g/vial
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . . 5 g
Intended Use
Bacto Fildes Enrichment is used to enrich media for cultivating
Haemophilus influenzae.
User Quality Control Also Known As
Identity Specifications Fildes Enrichment is also referred to as Fildes Broth.
Solution Appearance: Dark brown solution.
Summary and Explanation
Cultural Response
Fildes Enrichment, a sterile digest of sheep blood, is prepared according
Prepare Heart Infusion Agar per label directions. Aseptically
add 5% Fildes Enrichment to the cooled medium at 50-55°C to Fildes.1,2 This enrichment is recommended for use in liquid and solid
and pour plates. Inoculate and incubate at 35 ± 2°C under media for culturing Haemophilus influenzae and other fastidious
increased CO2 for 18-24 hours. microorganisms requiring blood derivatives for optimal growth.
INOCULUM
ORGANISM ATCC® CFU GROWTH Body fluid specimens likely to harbor Haemophilus species should be
Haemophilus influenzae 19418 100-1,000 good inoculated on 5% sheep blood agar, chocolate agar and a suitable
Haemophilus parainfluenzae 7901 100-1,000 good enrichment broth, such as Fildes Broth.3 Fildes-enriched gonococcal
medium, composed of gonococcal agar base containing 5% Fildes
The cultures listed are the minimum that should be used for Enrichment, 5% horse blood and 3 µg of vancomycin per ml, is used
performance testing.
for lymph node aspirates and lesion scrapings from patients with
suspected chancroid.3
Expiration Date
User Quality Control The expiration date applies to the product in its intact container when
Identity Specifications stored as directed. Do not use a product if it fails to meet specifications
Dehydrated Appearance: Beige, free-flowing, homogeneous. for identity and performance.
Solution: 2.5 g in 920 ml distilled or deionized
water, soluble upon boiling. Solution Procedure
is very light amber, clear to very Materials Provided
slightly opalescent without Fletcher Medium Base
significant precipitate.
Prepared Medium: Very light amber, very slightly to Materials Required But Not Provided
slightly opalescent without Glassware
significant precipitate.
Autoclave
Reaction of 2.5 g in Incubator (35°C)
920 ml distilled water: pH 7.9 ± 0.1 at 25°C
Waterbath (56°C)
Cultural Response Sterile normal rabbit serum
Prepare Fletcher Medium Base per label directions. Enrich
with sterile normal rabbit serum. Inoculate and incubate at Method of Preparation
30 ± 2°C for up to 5 days. 1. Suspend 2.5 grams in 920 ml distilled or deionized water.
ORGANISM ATCC® INOCULUM GROWTH 2. Heat to boiling to dissolve completely.
Leptospira interrogans 23605 2-3 drops good 3. Autoclave at 121°C for 15 minutes. Cool to 56°C.
serovar australis
Leptospira interrogans 23470 2-3 drops good 4. Aseptically add 80 ml sterile normal rabbit serum at 56°C. Mix well.
serovar canicola 5. Determine pH. If necessary, aseptically adjust to pH 7.9 ± 0.1 with
Leptospira kirschneri 23604 2-3 drops good 1 N HCl or 1 N NaOH.
serovar grippotyphosa
Specimen Collection and Preparation
The cultures listed are the minimum that should be used for
performance testing. Obtain and process specimens according to the techniques and procedures
established by laboratory policy. Blood, cerebrospinal fluid (CSF) and
urine are the specimens of choice for the recovery of leptospires from 2. Myers, D. M., V. M. Varela-Diaz, and A. A. Siniuk. 1973.
patients with leptospirosis.3 Long-term survival of Leptospira in a biphasic culture medium
containing charcoal. Am. Soc. Microbiol. 25:514-516.
Test Procedure7 3. Kaufmann, A. F., and R. S. Weyant. 1995. Leptospiraceae,
1. Aseptically dispense into sterile screw-cap tubes in 5-7 ml amounts. p. 621-625. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover
Store at room temperature overnight. and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed.
2. Inactivate the whole medium the day following its preparation by American Society for Microbiology, Washington, D.C.
placing the tubes in a water bath at 56°C for 1 hour. 4. Koneman, E. W., S. D. Allen, V. R. Dowell, Jr., W. M. Janda,
3. Allow the medium to cool before inoculation. H. M. Sommers, and W. C. Winn, Jr. 1988. Color atlas and
4. Growth is first seen in approximately 10 days at 35°C (2 to 4 weeks textbook of diagnostic microbiology, 3rd ed. J. B. Lippincott
at 25°C) as a cloud of minute granules that develop into Company, Philadelphia, PA.
microcolonies just below the surface. 5. Elliott, S. H. 1980. Discussion and clinical diagnosis of
5. Gram stain is not satisfactory. The microcolonies can be fixed Leptospirosis. J. Am. Med. Tech. 42:37-44.
with methanol and stained with Giemsa stain to show rod forms 6. Faine, S. (ed.). 1982. Guidelines for the control of Leptospirosis. W.
at the edges.9 H. O. Offset publication no. 67. World Health Organization, Geneva.
7. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures
Results
handbook. American Society for Microbiology, Washington, D.C.
Refer to appropriate references and procedures for results.
8. Kelley, P. W. 1992. Leptospirosis, p. 1295-1301. In S. L. Gorbach,
Limitations of the Procedure J. G. Bartlett, and N. R. Blacklow (ed.), Infectious diseases. The
W. B. Saunders Co., Philadelphia, PA.
1. Since the nutritional requirements of organisms vary, some
9. Weinman, D. 1981. Bartonellosis and anemias associated with
strains may be encountered that fail to grow or grow poorly on
bartonella-like structures, p. 235-248. In A. Balows, and W. J.
this medium.
Hausler, Jr. (ed.), Diagnostic procedures for bacterial, mycotic and
References parasitic infections, 6th ed. American Public Health Association,
Washington, D.C.
1. Fletcher, W. 1928. Recent work on leptospirosis, tsutsugamushi
disease and tropical typhus in the federated Malay States. Trans. Packaging
Roy. Soc. Trop. Med. Hyg. 21: 265-287. Fletcher Medium Base 500 g 0987-17
increasing concentrations gives a growth response that can be Materials Required But Not Provided
measured turbidimetrically or titrimetrically. Glassware
Autoclave
Formula Stock culture of Enterococcus hirae ATCC® 8043
Folic AOAC Medium Sterile tubes
Formula Per Liter Distilled or deionized water
Bacto Vitamin Assay Casamino Acids . . . . . . . . . . . . . . . . 10 g Folic Acid
L-Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 g 0.01 N NaOH
L-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g Dilute HCl
L-Cysteine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . 0.76 g
Spectrophotometer or Nephelometer
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg Method of Preparation
Guanine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg
1. Suspend 11 grams in 100 ml distilled or deionized water.
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg
Xanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg 2. Heat to boiling for 2-3 minutes.
p-Aminobenzoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg 3. Distribute 5 ml amounts into tubes, evenly dispersing the precipitate.
Pyridoxine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mg 4. Add standard or test samples.
Thiamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 400 µg 5. Adjust tube volume to 10 ml with distilled or deionized water.
Calcium Pantothenate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 µg
6. Autoclave at 121°C for 5 minutes.
Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 µg
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 µg Specimen Collection and Preparation
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg Assay samples are prepared according to references given in the
Glutathione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 mg specific assay procedures. For assays, the samples should be diluted to
Sorbitan Monooleate Complex . . . . . . . . . . . . . . . . . . . . . . 0.1 g approximately the same concentration as the standard solution.
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 g
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 g Test Procedure
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g Follow assay procedures as outlined in AOAC.1 It is essential that a
Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg standard curve be set up for each separate assay. Autoclaving and
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg incubation conditions that can influence the standard curve readings
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg cannot always be duplicated. The standard curve is obtained by using
Final pH 6.7 ± 0.1 at 25°C folic acid at levels of 0.0, 1, 2, 4, 6, 8 and 10 ng per assay tube (10 ml).
Folic AOAC Medium may be used for both turbidimetric and titrimetric
Precautions analysis. Turbidimetric readings should be taken after incubation at
1. For Laboratory Use. 35-37°C for 16-18 hours. Titrimetric determinations are best made
2. Follow proper established laboratory procedures in handling and following incubation at 35-37°C for 72 hours.
disposing of infectious materials. The folic acid required for the preparation of the standard curve may
3. Great care must be taken to avoid contamination of media or be prepared as follows:
glassware in microbiological assay procedures. Extremely small A. Dissolve 50 mg dried folic acid in about 30 ml 0.01N NaOH
amounts of foreign material may be sufficient to give erroneous and 300 ml distilled water.
results. Scrupulously clean glassware free from detergents and B. Adjust the pH reaction to 7.5 ± 0.5 with diluted HCl solution.
other chemicals must be used. Glassware is heated to 250°C for at Dilute to 500 ml with distilled water.
least 1 hour to burn off any organic residues that might be present.
C. Add 2 ml of the solution to 50 ml distilled water. Adjust the
4. Take precautions to keep sterilizing and cooling conditions uniform
pH reaction to 7.5 ± 0.5. Dilute to 100 ml with distilled water.
throughout the assay.
This yields a stock solution containing 2 mcg folic acid per ml.
Storage D. Prepare the stock solution fresh daily.
Store the dehydrated medium at 2-8°C. The dehydrated medium is very The standard solution for the assay is made by diluting 1 ml of this
hygroscopic. Keep container tightly closed. stock solution to 1 liter with distilled water. This solution contains 2 ng
folic acid per ml. Use 0.0, 0.5, 1, 2, 3, 4, and 5 ml per assay tube.
Expiration Date Some laboratories may wish to alter the concentration of folic acid
The expiration date applies to the product in its intact container when recommended above for the standard curve. This is permissible if the
stored as directed. Do not use a product if it fails to meet specifications concentration used is within the limits specified by AOAC.1
for identity and performance.
Results
Procedure 1. Prepare a standard concentration response curve by plotting the
Materials Provided response readings against the amount of standard in each tube,
Folic AOAC Medium disk or cup.
2. Determine the amount of vitamin at each level of assay solution by 3. The use of altered or deficient media may cause mutants
interpolation from the standard curve. having different nutritional requirements that will not give a
3. Calculate the concentration of vitamin in the sample from the satisfactory response.
average of these volumes. Use only those values that do not vary 4. For successful results of these procedures, all conditions of the
more than ±10% from the average. Use the results only if two thirds assay must be followed precisely.
of the values do not vary more than ±10%.
References
Limitations of the Procedure 1. Association of Official Analytical Chemists. 1995. Official
1. The test organism used for inoculating an assay medium must methods of analysis of AOAC international, 16th ed. AOAC
International, Arlington, VA.
be cultured and maintained on media recommended for this
purpose. Packaging
2. Aseptic technique should be used throughout the assay procedure. Folic AOAC Medium 100 g 0967-15*
*Store at 2-8°C
amounts of foreign material may be sufficient to give erroneous aseptic conditions and decant the supernatant. Wash the cells three
results. Scrupulously clean glassware free from detergents and times with 10 ml of sterile 0.85% saline. After the third wash, dilute
other chemicals must be used. Glassware must be heated to 250°C the cell suspension 1:100 with sterile 0.85% saline. Use one drop of
for at least 1 hour to burn off any organic residues that might be this latter suspension to inoculate each of the assay tubes.
present. It is essential that a standard curve be set up for each separate assay.
4. Take precautions to keep sterilization and cooling conditions Autoclaving and incubation conditions that influence the standard
uniform throughout the assay. curve readings cannot always be duplicated. The standard curve is
obtained by using folic acid at levels of 0.0, 2, 4, 6, 8 and 10 ng per
Storage 10 ml assay tube. Turbidimetric readings should be made after
Store the dehydrated medium at 2-8°C. The dehydrated medium is very incubation at 35-37°C for 18-24 hours. Refrigerate tubes for 15-30
hygroscopic. Keep container tightly closed. minutes to stop growth before reading.
Prepare the folic acid stock solution required for the standard curve as
Expiration Date follows:
The expiration date applies to the product in its intact container when 1. Dissolve 50 mg dried Folic Acid USP Reference Standard or
stored as directed. Do not use a product if it fails to meet specifications equivalent in about 30 ml of 0.01 N NaOH and 300 ml distilled
for identity and performance. water.
2. Adjust to pH 7.5 ± 0.5 with diluted HCl solution. Add distilled
Procedure water to give a volume of 500 ml.
Materials Provided 3. Add 2 ml of the solution from step 2 to 50 ml distilled water.
Folic Acid Assay Medium Adjust the pH to 7.5 ± 0.5 with HCl solution. Dilute to 100 ml with
distilled water to give a stock solution containing 2 mcg folic acid
Materials Required But Not Provided per ml. Prepare the stock solution fresh daily.
Glassware Prepare the standard solution for the assay by diluting 1 ml of this
Autoclave stock solution in 1 liter with distilled water. This solution contains 2 ng
Stock culture of Enterococcus hirae ATCC® 8043 folic acid per ml. Use 0.0, 0.5, 1, 2, 3, 4 and 5 ml per assay tube.
Sterile tubes Following incubation, place the tubes in the refrigerator for 15-30
Sterile 0.85% saline minutes to stop growth. The growth can be measured by a turbidimetric
Distilled or deionized water method and the curve constructed from the values obtained. The most
0.01 N NaOH effective assay range is between the levels of 2 and 10 ng folic acid per
Dilute HCl 10 ml tube.
Folic Acid USP
Lactobacilli Agar AOAC Results
Lactobacilli Broth AOAC 1. Prepare a standard concentration response curve by plotting the
Centrifuge response readings against the amount of standard in each tube, disk
Spectrophotometer or cup.
2. Determine the amount of vitamin at each level of assay solution by
Method of Preparation interpolation from the standard curve.
1. Suspend 7.5 grams in 100 ml distilled or deionized water. 3. Calculate the concentration of vitamin in the sample from the
2. Boil 2-3 minutes to dissolve completely. average of these volumes. Use only those values that do not vary
3. Dispense 5 ml amounts into tubes, evenly dispersing the precipitate. more than ±10% from the average. Use the results only if two thirds
of the values do not vary more than ±10%.
4. Add standard or test samples.
5. Adjust tube volume to 10 ml with distilled or deionized water. Limitations of the Procedure
6. Autoclave at 121°C for 10 minutes. 1. The test organism used for inoculating an assay medium must be
cultured and maintained on media recommended for this purpose.
Specimen Collection and Preparation 2. Aseptic technique should be used throughout the assay procedure.
Prepare assay samples according to references given in the specific 3. The use of altered or deficient media may cause mutants having
assay procedures. Dilute the samples to approximately the same different nutritional requirements that will not give a satisfactory
concentration as the standard solution. response.
Test Procedure 4. For successful results of these procedures, all conditions of the
assay must be followed precisely.
Prepare stock cultures of E. hirae ATCC® 8043 by stab inoculation of
Lactobacilli Agar AOAC. Incubate at 35-37°C for 24-48 hours. Store References
tubes in the refrigerator. Make transfers at monthly intervals. Prepare 1. Capps, Hobbs, and Fox. 1948. J. Bacteriol. 55:869.
the inoculum for assay by subculturing a stock culture of E. hirae
ATCC® 8043 into a tube containing 10 ml of Lactobacilli Broth AOAC. Packaging
After incubation at 35-37°C for 18-24 hours, centrifuge the cells under Folic Acid Assay Medium 100 g 0318-15
Bacto Folic Buffer A, Dried rhamnosus ATCC® 7469 is used as the test organism in this assay.
Folic Acid Casei Medium is prepared according to the formulation
described by Flynn, Williams, O’Dell and Hogan1 and modified by
Baker et al.2 and Waters and Mollin.3
Intended Use
Bacto Folic Acid Casei Medium is used for determining folic acid Total serum folic acid activity can vary depending on the disease state.
concentration by the microbiological assay technique. It has been reported that normal subjects have a mean serum folic acid
level of 9.9 ng per ml. Patients with uncomplicated pernicious anemia
Bacto Folic Buffer A, Dried is prepared for use with Folic Acid Casei have a mean serum folic acid level of 16.6 ng per ml while patients
Medium in the microbiological assay of serum folic acid. with megaloblastic anemia have levels less than 4.0 ng per ml.
Summary and Explanation Folic Buffer A, Dried is used for preparing both the standard and the
Vitamin Assay Media are prepared for use in the microbiological assay serum specimen in the microbiological assay of folic acid.
of vitamins. Three types of media are used for this purpose:
Principles of the Procedure
1. Maintenance Media: For carrying the stock culture to preserve the
Folic Acid Casei Medium is a folic acid-free dehydrated medium
viability and sensitivity of the test organism for its intended purpose;
containing all other nutrients and vitamins essential for the cultivation
2. Inoculum Media: To condition the test culture for immediate use; of L. casei subsp. rhamnosus ATCC® 7469. The addition of folic acid
3. Assay Media: To permit quantitation of the vitamin under test. in specified increasing concentrations gives a growth response that can
Folic Acid Casei Medium is prepared for the microbiological assay of be measured turbidimetrically.
folic acid, particularly folic acid in serum. Lactobacillus casei subsp.
Formula
Folic Acid Casei Medium
User Quality Control Formula Per Liter
Charcoal Treated Casitone . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Identity Specifications Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g
Folic Acid Casei Medium Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g
Dehydrated Appearance: Off-white, homogeneous, with a Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
tendency to clump. Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Solution: 4.7% (single strength) and 9.4% DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g
(double strength) solution, soluble L-Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 g
in distilled or deionized water upon L-Cysteine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
boiling 1-2 minutes. Single-strength
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg
solution is light amber, clear, may
have a slight precipitate. Guanine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg
Prepared Medium: Single-strength solution is very light
Xanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
amber, clear, may have a very slight
precipitate. Sorbitan Monooleate Complex . . . . . . . . . . . . . . . . . . . . . . 0.1 g
Glutathione (reduced) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mg
Reaction of 4.7%
Magnesium Sulfate, Anhydrous . . . . . . . . . . . . . . . . . . . . . 0.2 g
Solution at 25°C: 6.7 ± 0.1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Folic Buffer A, Dried Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Dehydrated Appearance: White to off-white, free-flowing, Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 mg
homogeneous. Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg
Solution: 1.54% solution, soluble in distilled p-Aminobenzoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg
or deionized water. Pyridoxine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mg
Solution Appearance: Colorless to very light amber, clear. Thiamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 400 µg
Reaction of 1.54% Calcium Pantothenate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 µg
Solution at 25°C: pH 6.1 ± 0.05 Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 µg
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 µg
Cultural Response Final pH 6.7 ± 0.1
Prepare Folic Acid Casei Medium per label directions. The
medium is tested by creating a standard curve using Folic Acid Folic Buffer A, Dried
at concentrations of 0 to 1.0 ng per 10 ml. This medium should Formula Per Liter
support the growth of L. casei subsp. rhamnosus ATCC® 7469 Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . 10.656 g
when prepared in single strength and supplemented with Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . 3.744 g
ascorbic acid and Folic Acid. Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Final pH 6.1 ± 0.05
Preparation of Serum Specimen interpolating the results with the values obtained on the standard curve,
1. Thaw the serum containing ascorbic acid. taking into consideration the dilutions of the samples.
2. Add 5 ml of the uniform sample to 45 ml rehydrated Folic Buffer
A, Dried. Limitations of the Procedure
3. Incubate the serum-buffer solution at 37°C for 90 minutes. Autoclave 1. The test organism used for inoculating an assay medium must
the incubated mixture at 121°C for 2.5 minutes. be cultured and maintained on media recommended for this
purpose.
4. Remove the coagulated protein by centrifuging and transfer the
clear supernatant to a clean dry tube. The clear solution is the 2. Aseptic technique should be used throughout the assay procedure.
sample to use in the folic acid assay. 3. The use of altered or deficient media may cause mutants having
Procedure for Total Folic Acid different nutritional requirements that will not give a satisfactory
response.
1. Use 0.5, 1.0, 1.5 ml or other volumes of the prepared serum
extracts as described above. 4. For successful results of these procedures, all conditions of the
assay must be followed precisely.
2. Fill each assay tube with 5 ml of rehydrated Folic Acid Casei
Medium and sufficient distilled or deionized water to give a total References
volume of 10 ml per tube.
1. Flynn, Williams, O’Dell, and Hogan. 1951. Anal. Chem. 23:180.
3. Autoclave tubes at 121°C for 5 minutes.
2. Baker, Herbert, Frank, Pasher, Hunter, Wasserman, and
4. Add 1 drop of inoculum described under Preparation of Stock Sobotka. 1959. Clin. Chem. 5:275.
Culture and Inoculum to each assay.
3. Waters and Molin. 1961. J. Clin. Pathol. 14:335.
5. Incubate at 35-37°C for 18-24 hours. Tubes are refrigerated for
15-30 minutes to stop growth before reading turbidimetrically. Packaging
Results Folic Acid Casei Medium 100 g 0822-15
The amount of folic acid in the test samples can be determined by Folic Buffer A, Dried 6 x 15.4 g 3246-33
Fraser Broth
Bacto Fraser Broth Base . Fraser Broth Supplement
®
Formula FIRST AID: In case of contact with eyes, rinse immediately with
Fraser Broth Base plenty of water and seek medical advice. After contact with skin,
Formula Per Liter wash immediately with plenty of water. If inhaled, remove to fresh
air. If not breathing, give artificial respiration. If breathing is
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
difficult, give oxygen. Seek medical advice. If swallowed seek
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
medical advice immediately and show this container or label.
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g Fraser Broth Supplement:
Sodium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . . . . 9.6 g IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM
Potassium Phosphate, Monobasic . . . . . . . . . . . . . . . . . . 1.35 g AND SKIN. Avoid contact with skin and eyes. Do not breathe mist.
Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g Wear suitable protective clothing. Keep container tightly closed.
Nalidixic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g
FIRST AID: In case of contact with eyes, rinse immediately with
Acriflavine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.024 g
plenty of water and seek medical advice. After contact with skin,
Lithium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
wash immediately with plenty of water. If inhaled, remove to fresh
Final pH 7.2 ± 0.2 at 25°C air. If not breathing, give artificial respiration. If breathing is
Fraser Broth Supplement difficult, give oxygen. Seek medical advice. If swallowed seek
Ingredients per 10 ml vial medical advice immediately and show this container or label.
Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g 3. Follow proper, established laboratory procedures in handling and
disposing of infectious materials.
Precautions
1. For Laboratory Use. Storage
2. Fraser Broth Base: Store the dehydrated medium below 30°C. The dehydrated medium is
HARMFUL. IRRITATING TO EYES, RESPIRATORY SYSTEM very hygroscopic. Keep container tightly closed.
AND SKIN. MAY CAUSE HARM TO THE UNBORN CHILD. Store Bacto Fraser Broth Supplement at 2-8°C.
Avoid contact with skin and eyes. Do not breathe dust. Wear Store the prepared medium at 2-8°C.
suitable protective clothing. Keep container tightly closed.
TARGET ORGAN(S): Blood, Kidneys, Nerves
Expiration Date 2. Poor growth and a weak esculin reaction may be seen after 40 hours
The expiration date applies to the product in its intact container when incubation for some enterococci.
stored as directed. Do not use a product if it fails to meet specifications
for identity and performance.
References
1. Murray, E. G. D., R. A. Webb, and M. B. R. Swann. 1926.
Procedure A disease of rabbits characterized by large mononuclear
Materials Provided leucocytosis caused by a hitherto undescribed bacillus Bacterium
monocytogenes (n. sp.). J. Path. Bact. 29:407- 439.
Fraser Broth Base
Fraser Broth Supplement 2. Monk, J. D., R. S. Clavero, L. R. Beuchat, M. P. Doyle, and
R. E. Brackett. 1994. Irradiation inactivation of Listeria
Materials Required But Not Provided monocytogenes and Staphylococcus aureus in low- and high-fat,
Flasks with closure frozen and refrigerated ground beef. J. Food Prot. 57:969-974.
Distilled or deionized water 3. Wehr, H. M. 1987. Listeria monocytogenes - a current dilemma
Bunsen burner or magnetic hot plate special report. J. Assoc. Off. Anal. Chem. 70:769-772.
Autoclave 4. Bremer, P. J., and C. M. Osborne. 1995. Thermal-death times of
Waterbath (45-50°C) Listeria monocytogenes in green shell mussels (Perna canaliculus)
Test tubes with closures prepared for hot smoking. J. Food Prot. 58:604-608.
Incubator (30°C) 5. Grau, F. H., and P. B. Vanderlinde. 1992. Occurrence, numbers,
Incubator (35°C) and growth of Listeria monocytogenes on some vacuum-packaged
processed meats. J. Food Prot. 55:4-7.
Method of Preparation
6. Patel, J. R., C. A. Hwang, L. R. Beuchat, M. P. Doyle, and
1. Suspend 55 grams of Fraser Broth Base in 1 liter of distilled or
R. E. Brackett. 1995. Comparison of oxygen scavengers for their
deionized water.
ability to enhance resuscitation of heat-injured Listeria
2. Heat to boiling to dissolve completely. monocytogenes. J. Food Prot. 58:244-250.
3. Autoclave at 121°C for 15 minutes. Cool to room temperature. 7. Fraser, J., and W. Sperber. 1988. Rapid detection of Listeria in
4. Aseptically add 10 ml Fraser Broth Supplement. Mix well. food and environmental samples by esculin hydrolysis. J. Food
5. Dispense into tubes. Prot. 51:762-765.
8. Lee, W. H., and D. McClain. 1994. Laboratory Communication
Specimen Collection and Preparation
No. 57 (revised February 8, 1994), U.S.D.A., F.S.I.S. Microbiology
Refer to appropriate references for specimen collection and preparation. Division, Bethesda, MD.
Test Procedure 9. Kramer, P. A., and D. Jones. 1969. Media selective for Listeria
To isolate Listeria monocytogenes from processed meats and poultry, monocytogenes. J. Appl. Bacteriol. 32:381-394.
the following procedure is recommended by the U.S.D.A.8 10. Donnelly, C. W., R. E. Bracket, D. Doores, W. H. Lee, and
J. Lovett. 1992. Listeria, p. 637-663. In C. Vanderzant and D. F.
1. Add 25 grams of test material to 225 ml of UVM Modified Listeria
Splittstoesser (ed.), Compendium of methods for the microbiological
Enrichment Broth and mix or blend thoroughly.
examination of foods, 3rd ed. American Public Health Association,
2. Incubate for 20-24 hours at 30°C. Washington, D.C.
3. Transfer 0.1 ml of the incubated broth to Fraser Broth. Incubate at 11. Swaminathan, B., J. Rocourt, and J. Bille. 1995. Listeria,
35°C for 26 ± 2 hours. p. 342-343. In P. R. Murray, E. J. Baron, M. A. Pfaller,
4. At 24 and 48 hours, streak the Fraser Broth culture to Modified F. C. Tenover, and R. H. Yolken (ed.). Manual of clinical
Oxford Agar. microbiology, 6th ed. American Society for Microbiology,
5. Incubate the Modified Oxford plates at 35°C for 24-48 hours. Washington, D.C.
12. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig.
Results
1993. Pathogens in milk and milk products. In R. T. Marshall (ed.),
1. Examine agar plates for suspect colonies. For further identifica- Standard methods for the examination of dairy products, 16th ed.
tion and confirmation of Listeria spp., consult appropriate American Public Health Association, Washington, D.C.
references.8,10,11,12
2. Rapid slide and macroscopic tube tests can be used for definitive Packaging
serological identification. Fraser Broth Base 500 g 0219-17
2 kg 0219-07
Limitations of the Procedure
1. Since Listeria species other than L. monocytogenes can grow on Fraser Broth Supplement 6 x 10 ml 0211-60*
these media, an identification of Listeria monocytogenes must be *Store at 2-8°C
confirmed by biochemical and serological testing.11,12
Martin and Lewis10 further improved selectivity of MTM by increasing glutamine, coenzyme, cocarboxylase, hematin and growth factors.
the concentration of vancomycin and replacing nystatin with Supplement VX is a sterile, defined lyophilized concentrate of essential
anisomycin for greater inhibition of yeasts; this is known as Martin- growth factors. Supplement VX supplies vitamins, amino acids,
Lewis (ML) Agar Medium. Transgrow Medium is a transport medium coenzymes, dextrose and other factors to improve the growth of
system incorporating either MTM or ML formulations.11 Haemophilus and Neisseria species.
Antimicrobic Vial CNV and Antimicrobic Vial CNVT are antimicrobial
Principles of the Procedure agents used as inhibitors in the selective media, Thayer-Martin
GC Medium Base is employed as the basal medium in the preparation Medium and Modified Thayer-Martin Medium.
of Chocolate Agar Enriched, Thayer-Martin Medium and Modified
Thayer-Martin Medium. Formula
Proteose Peptone No. 3 provides nitrogen, vitamins and amino acids in GC Medium Base
GC Medium Base. Corn Starch absorbs any toxic metabolites that are Formula Per Liter
produced, Potassium Phosphate, Dibasic and Monobasic buffer the Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 15 g
medium. Sodium Chloride maintains osmotic balance. Bacto Agar is Corn Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
a solidifying agent. Potassium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . . . 4 g
Chocolate Agar is prepared from GC Medium Base with the addition Potassium Phosphate, Monobasic . . . . . . . . . . . . . . . . . . . . 1 g
of 2% Hemoglobin. Hemoglobin provides hemin (X factor) required Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
for growth of Haemophilus and enhanced growth of Neisseria. Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
The growth rate of Neisseria and Haemophilus is improved with the Final pH 7.2 ± 0.2 at 25°C
addition of 1% Supplement B or VX, providing the growth factors
glutamine and cocarboxylase. Supplement B contains yeast concentrate, Uninoculated Neisseria gonorrhoeae
plate ATCC® 43069
Expiration Date
User Quality Control The expiration date applies to the product in its intact container when
Identity Specifications stored as directed. Do not use a product if it fails to meet specifications
Dehydrated Appearance: Off-white to light tan, free-flowing, for identity and performance.
homogeneous.
Solution: 3.9% solution, soluble in distilled
Procedure
or deionized water. Solution is light Materials Provided
amber, clear to very slightly GN Broth, Hajna
opalescent.
Prepared Medium: Light amber, clear to very slightly Materials Required but not Provided
opalescent. Glassware
Reaction of 3.9% Distilled or deionized water
Solution at 25°C: pH 7.0 ± 0.2 Autoclave
Incubator (35°C)
Cultural Response
Prepare GN Broth, Hajna per label directions. Inoculate the Method of Preparation
medium and incubate at 35 ± 2°C for 18-24 hours. 1. Dissolve 39 grams in 1 liter distilled or deionized water.
INOCULUM 2. Autoclave at 121°C for 15 minutes. Avoid overheating.
ORGANISM ATCC® CFU GROWTH
Escherichia coli 25922* 100-1,000 good Specimen Collection and Preparation
Salmonella typhimurium 14028* 100-1,000 good Refer to appropriate references for specimen collection and preparation.
Shigella flexneri 12022* 100-1,000 good
Enterococcus faecalis 19433* 1,000-2,000 none to poor Test Procedure
The cultures listed are the minimum that should be used for See appropriate references for specific procedures.
performance testing.
*These cultures are available as Bactrol™ Disks and should be Results
used as directed in Bactrol Disks Technical Information. Growth of gram-negative organisms, especially Salmonella and
Shigella species, is enhanced.
Bacto Gelatin
®
content. Gelatone is used as an ingredient in media for fermentation
studies and, by itself, to support growth of non-fastidious microorganisms.
Bacto Gelatone Principles of the Procedure
The melting point of a 12% concentration of Gelatin is between 28 and
Intended Use 30%C, which allows it to be used as a solidifying agent. Certain
Bacto Gelatin is used in preparing microbiological culture media. microorganisms elaborate gelatinolytic enzymes (gelatinases)
Bacto Gelatone is used in preparing microbiological culture media. which hydrolyze gelatin, causing liquefaction of a solidified
medium or preventing the gelation of a medium containing gelatin.
Also Known As Gelatin is also used as a source of nitrogen and amino acids.
Gelatone is also referred to as Gelatin Peptone. Gelatone is a peptone from gelatin obtained by digesting gelatin
with pancreatin.
Summary and Explanation
Gelatin is a protein of uniform molecular constitution derived chiefly Precautions
by the hydrolysis of collagen.1 Collagens are a class of albuminoids 1. For Laboratory Use.
found abundantly in bones, skin, tendon, cartilage and similar 2. Follow proper established laboratory procedures in handling and
animal tissues.1 disposing of infectious materials.
Koch1 introduced gelatin into bacteriology when he invented the
gelatin tube method in 1875 and the plate method in 1881. This Storage
innovation, a solid culture method, became the foundation for Store the dehydrated product below 30°C. The dehydrated product is
investigation of the propagation of bacteria.1 However, gelatin-based very hygroscopic. Keep container tightly closed.
media were soon replaced by media containing agar as the
solidifying agent. Expiration Date
Gelatin is used in culture media for determining gelatinolysis The expiration date applies to the product in its intact container when
(elaboration of gelatinases) by bacteria. Levine and Carpenter2 and stored as directed. Do not use a product if it fails to meet specifications
for identity and performance.
Levine and Shaw3 employed gelatin media in their studies of gelatin
liquefaction. Garner and Tillett4 used culture media prepared with Procedure
gelatin to study the fibrinolytic activity of hemolytic streptococci.
Materials Provided
Gelatin is a high grade gelatin in granular form which may be used as
Gelatin
a solidifying agent or may be incorporated into culture media for
various uses. Gelatin is used in Nutrient Gelatin, Motility Gelatone
GI Medium, Motility Medium S, Stock Culture Agar and Dextrose Materials Required But Not Provided
Starch Agar. Media containing gelatin are specified in Standard
Materials vary depending on the medium being prepared.
Methods5,6 for multiple applications.
Gelatone, a granular pancreatic digest of gelatin, is deficient in Method of Preparation
carbohydrates. It is distinguished by low cystine and tryptophan Preparation varies depending on the medium being prepared.
Specimen Collection and Preparation 4. Garner and Tillett. 1934. J. Exp. Med. 60:255.
Obtain and process specimens according to the techniques and 5. Association of Official Analytical Chemists. 1995.
procedures established by laboratory policy. Bacteriological analytical manual, 8th ed. AOAC International,
Gaithersburg, MD.
Test Procedure 6. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). 1995.
See appropriate references for specific procedures using Gelatin Standard methods for the examination of water and wastewater,
or Gelatone. 19th ed. American Public Health Association, Washington, D.C.
Results Packaging
Refer to appropriate references and procedures for results. Gelatin 100 g 0143-15
500 g 0143-17
References 10 kg 0143-08
1. Gershenfeld, L., and L. F. Tice. 1941. Gelatin for bacteriological
use. J. Bacteriol. 41:645-652. Gelatone 500 g 0657-17
2. Levine and Carpenter. 1923. J. Bacteriol. 8:297.
3. Levine and Shaw. 1924. J. Bacteriol. 9:225.
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
buffer the pH to near neutrality. Sodium Carbonate inactivates low levels HARM TO THE UNBORN CHILD. Avoid contact with skin and
of preservatives that are active at a more acidic pH (e.g., benzoic acid). eyes. Do not breathe dust. Wear suitable protective clothing. Keep
Chloramphenicol inhibits bacteria, including Pseudomonas aeruginosa container tightly closed. TARGET ORGAN(S): Blood, Eye/Ear,
and Serratia marcescens, that are potential contaminants of cosmetic Muscles, Nerves, Urogenital.
products. Polysorbate 80 neutralizes preservatives and sequesters FIRST AID: In case of contact with eyes, rinse immediately with
surfactants that may be present in residual amounts from the product plenty of water and seek medical advice. After contact with skin,
sample.2 Bacto Agar is the solidifying agent. wash immediately with plenty of water. If inhaled, remove to fresh
air. If not breathing, give artificial respiration. If breathing is
Formula difficult, give oxygen. Seek medical advice. If swallowed seek
HC Agar Base medical advice immediately and show this container or label.
Formula Per Liter
3. Follow proper established laboratory procedures in handling and
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
disposing of infectious materials.
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Storage
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 g Store the dehydrated medium below 30°C. The dehydrated medium is
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 g very hygroscopic. Keep container tightly closed.
Ammonium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 g
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.06 g Expiration Date
Chloramphenicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g The expiration date applies to the product in its intact container when
Sodium Carbonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g stored as directed. Do not use a product if it fails to meet specifications
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g for identity and performance.
Final pH 7.0 ± 0.2 at 25°C
Procedure
Precautions Materials Provided
1. For Laboratory Use. HC Agar Base
2. TOXIC. IRRITATING TO EYES, RESPIRATORY SYSTEM
AND SKIN. MAY CAUSE CANCER. POSSIBLE RISK OF Materials Required but not Provided
Polysorbate 80
Glassware
User Quality Control Sterile Petri dishes
Autoclave
Identity Specifications Incubator (27.5 ± 0.5°C)
Dehydrated Appearance: Very light to light beige,
free-flowing, homogeneous. Method of Preparation
Solution: 5.45% solution, soluble in distilled 1. Suspend 54.5 grams in 1 liter distilled or deionized water.
or deionized water on boiling.
Solution is medium to dark amber, 2. Heat to boiling to dissolve completely.
slightly opalescent to opalescent, 3. Add 20 ml Polysorbate 80.
may have a slight precipitate. 4. Autoclave at 121°C for 15 minutes.
Prepared Medium: Medium amber with yellow tint, 5. Dispense into petri dishes.
very slightly to slightly opalescent,
no significant precipitate. Specimen Collection and Preparation
Reaction of 5.45% Collect specimens in sterile containers or with sterile swabs and
Solution at 25°C: pH 7.0 ± 0.2 transport immediately to the laboratory in accordance with
Cultural Response recommended guidelines.1
Prepare HC Agar Base per label directions. Supplement with Test Procedure
Polysorbate 80. Inoculate and incubate the plates at 27.5 ± 0.5°C
for 65-72 hours. 1. Process each specimen as appropriate for that specimen and
INOCULUM inoculate directly onto the surface of the medium.1 Inoculate
ORGANISM ATCC® CFU GROWTH duplicate plates.
Aspergillus niger 16404 100-1000 good 2. Incubate plates aerobically at 27.5 ± 0.5°C.
Pseudomonas aeruginosa 10145 1,000-2,000 none to poor
Serratia marcescens 13880 1,000-2,000 none to poor 3. Examine plates for growth and recovery after 72 hours incubation.
4. Count mold colonies from duplicate plates and record average
The cultures listed are the minimum that should be used for count as mold count per gram or milliliter of sample.
performance testing.
Results References
Mold cultures should yield good growth and recovery. Bacteria should 1. Hitchins, A. D., T. T. Tran, and J. E. McCarron. 1995.
be inhibited. Microbiological methods in cosmetics, p. 23-01-23.12. In
Bacteriological analytical manual, 8th ed. AOAC International,
Limitations of the Procedure Gaithersburg, MD.
1. The 27.5 ± 0.5°C incubation temperature is critical for obtaining 2. Mead, C., and J. O’Neill. 1986. A three-day mold assay for
statistically significant mold counts after three days using cosmetics and toiletries. J. Soc. Cosmet. Chem. 37:49-57.
this medium.
2. Nutritional requirements of organisms vary. Some strains may be Packaging
encountered that fail to grow or grow poorly on this medium. HC Agar Base 500 g 0685-17
The cultures listed are the minimum that should be used for
performance testing.
Streptococcus pyogenes ATCC® 19615 *These cultures are available as Bactrol™ Disks and should be used as
All with blood on Heart Infusion Agar directed in Bactrol Disks Technical Information.
The cultures listed are the minimum that should be used for performance.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
Novobiocin (15 mg/liter) can be added to Hektoen Enteric Agar to Materials Required but not Provided
inhibit growth of Citrobacter and Proteus colonies, which may Glassware
resemble those of Salmonella.9 Autoclave
Incubator
Principles of the Procedure Petri dishes
Proteose Peptone is a source of nitrogen and other nutrients in Hektoen
Enteric Agar. Bile Salts and the dyes, brom thymol blue and acid Method of Preparation
fuchsin, inhibit gram-positive organisms. Lactose, saccharose and 1. Suspend 76 grams in 1 liter distilled or deionized water.
salicin are sources of fermentable carbohydrates. Ferric ammonium 2 Heat to boiling with frequent agitation to dissolve completely.
citrate, a source of iron, allows production of hydrogen sulfide (H2S) Do not overheat. DO NOT AUTOCLAVE.
from sodium thiosulfate. H2S-positive colonies have black centers.
Yeast Extract provides vitamins and cofactors required for growth and Specimen Collection and Preparation
additional nitrogen and carbon. Bacto Agar is used as a solidifying agent. Refer to appropriate references for specimen collection and preparation.
Formula Test Procedure
Hektoen Enteric Agar See appropriate references for specific procedures.
Formula Per Liter
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 g
Results
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g Refer to appropriate references and procedures for results.
Bacto Bile Salts No. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 g
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 g Limitations of the Procedure
Bacto Saccharose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 g 1. Do not autoclave this medium because excessive heat may alter
Bacto Salicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g the ingredients.
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g 2. Proteus species may resemble salmonellae or shigellae. Further
Sodium Thiosulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g testing should be conducted to confirm the presumptive identification
Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g of organisms isolated on this medium.
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 g
Brom Thymol Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.065 g References
Acid Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g 1. King, S., and W. I. Metzger. 1968. A new plating medium for the
Final pH 7.5 ± 0.2 at 25°C isolation of enteric pathogens. Appl. Microbiol. 16:577-578.
Precautions 2. King, S., and W. I. Metzger. 1968. A new plating medium for the
isolation of enteric pathogens. II. Comparison of Hektoen Enteric
1. For Laboratory Use.
Agar with SS and EMB Agar. Appl. Microbiol. 16:579-581.
2. IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM
3. Gray, L .D. 1995. Escherichia, Salmonella, Shigella, and Yersinia,
AND SKIN. Avoid contact with skin and eyes. Do not breathe dust.
p. 450-456. In Murray, P. R., E. J. Baron, M. A. Pfaller, F. C.
Wear suitable protective clothing. Keep container tightly closed.
Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology,
FIRST AID: In case of contact with eyes, rinse immediately with 6th ed. American Society for Microbiology, Washington, D.C.
plenty of water and seek medical advice. After contact with skin,
4. Centers for Disease Control. 1991. Summary of notifiable
wash immediately with plenty of water. If inhaled, remove to fresh
diseases. Morbid. Mortal. Weekly Rep. 40 (53):3.
air. If not breathing, give artificial respiration. If breathing is
difficult, give oxygen. Seek medical advice. If swallowed seek 5. Flowers, R. S., J-Y. D’Aoust, W. H. Andrews, and J. S. Bailey.
medical advice immediately and show this container or label. 1992. Salmonella, p. 371-422. In Vanderzant, C., and D. F.
Splittstoesser (ed.), Compendium of methods for the microbiological
3. Follow proper, established laboratory procedures in handling and
examination of foods, 3rd ed. American Public Health Association,
disposing of infectious materials.
Washington, D.C.
Storage 6. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig.
Store the dehydrated medium below 30°C. The dehydrated medium is 1993. Pathogens in milk and milk products, p. 103-212.
very hygroscopic. Keep container tightly closed. In Marshall, R. T. (ed.), Standard methods for the examination
of dairy products. 16th ed. American Public Health Association,
Expiration Date Washington, D.C.
The expiration date applies to the product in its intact container when 7. Andrews, W. H., G. A. June, P. S. Sherrod, T. S. Hammack, and
stored as directed. Do not use a product if it fails to meet specifications R. M. Amaguana. 1995. Salmonella, p. 5.01-5.20. In Bacterio-
for identity and performance. logical analytical manual, 8th ed. AOAC International,
Gaithersburg, MD.
Procedure 8. Association of Official Analytical Chemists. 1996 official
Materials Provided methods of analysis of AOAC International, Supplement March
Hektoen Enteric Agar 1996. AOAC International, Arlington, VA.
Bacto Hemoglobin
®
2. Follow proper, established laboratory procedures in handling and
disposing of infectious materials.
Intended Use Storage
Bacto Hemoglobin is used in preparing microbiological culture media. Store Hemoglobin below 30°C. The dehydrated ingredient is very
hygroscopic. Keep container tightly closed.
Summary and Explanation
Hemoglobin, an autoclavable preparation of beef blood, is prepared Expiration Date
according to the procedure described by Spray.1
The expiration date applies to the product in its intact container when
Hemoglobin is used with GC Medium Base is in the preparation of stored as directed. Do not use a product if it fails to meet specifications
Chocolate Agar Enriched, Thayer-Martin Medium and Modified for identity and performance.
Thayer-Martin Medium. Supplemented with Hemoglobin and Supplement
B or VX, the enriched media are used for the isolation and cultivation Procedure
of fastidious microorganisms, especially Neisseria and Haemophilus Materials Provided
species. With the exception of some laboratory-adapted strains of Hemoglobin
Haemophilus aphrophilus, Haemophilus species require either
exogenous hemin (X factor), nicotinamide adenine dinucleotide (NAD) Materials Required But Not Provided
(V factor), or both.2 Glassware
Autoclave
Principles of the Procedure GC Medium Base (for the cultivation of Neisseria and
Hemoglobin provides the hemin (X factor) required for growth of Haemophilus species)
Haemophilus and for enhanced growth of Neisseria species. Supplement B or VX, depending on the medium being prepared
Antimicrobic Vial CNV or CNVT, depending on the medium
Formula being prepared
Hemoglobin is obtained from beef blood, desiccated.
Method of Preparation
Precautions 1. Place 10 grams of Hemoglobin in a dry beaker.
1. For Laboratory Use. 2. Measure 500 ml distilled or deionized water.
3. Add the water in approximately 100 ml amounts, stirring well
after each addition. Use a spatula to break up clumps.
User Quality Control 4. Transfer to flasks, as desired, for autoclaving.
Identity Specifications 5. Autoclave at 121°C for 15 minutes.
Dehydrated Appearance: Dark brown, fine, free-flowing. 6. Cool to 45-50°C.
Solution: 2% solution, insoluble in distilled or 7. Swirl the flask to reestablish complete solution, then add to an equal
deionized water. Solution is chocolate
brown, opaque with a dispersed amount of double-strength sterile agar base cooled to 45-50°C.
precipitate. Test Procedure
Reaction of 2% For a complete discussion on the isolation and identification of
Solution at 25°C: pH 8.2 ± 0.2 Neisseria and Haemophilus species, refer to procedures outlined in
Cultural Response appropriate references.2,3,4
Prepare GC Medium enriched with 2% Hemoglobin and Results
Supplement B or VX per label directions. Inoculate and
incubate at 35 ± 2°C under 5-10% CO2 for 18-48 hours. Refer to appropriate references and procedures for results.
INOCULUM
ORGANISM ATCC® CFU GROWTH References
Haemophilus influenzae 10211 100-1,000 good 1. Spray. 1930. J. Lab Clin. Med. 16:166.
Neisseria gonorrhoeae 43069 100-1,000 good 2. Campos, J. M. 1995. Haemophilus, p. 556-565. In P. R. Murray,
The cultures listed are the minimum that should be used for E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.).
performance testing. Manual of clinical microbiology, 6th ed. American Society for
Microbiology, Washington, D.C.
cells in 10 ml 0.85% saline. Dilute 1 ml of the cell suspension in 1000 ml 2. Determine the amount of vitamin at each level of assay solution by
of sterile 0.85% saline. This diluted suspension is the inoculum. Use interpolation from the standard curve.
1 drop of inoculum suspension to inoculate each assay flask. 3. Calculate the concentration of vitamin in the sample from the
The concentrations of inositol required for the preparation of the standard average of these volumes. Use only those values that do not vary
curve may be prepared by dissolving 200 mg inositol in 100 ml more than ± 10% from the average. Use the results only if two
distilled water. Mix thoroughly. Dilute 1 ml of this solution with 999 ml thirds of the values do not vary more than ± 10%.
distilled water to make a final solution containing 2 µg inositol per ml.
Use 0.0, 0.5, 1, 2, 3, 4 and 5 ml per flask. Prepare this stock solution
Limitations of the Procedure
fresh daily. 1. The test organism used for inoculating an assay medium must be
grown and maintained on media recommended for this purpose.
It is essential that a standard curve be constructed each time an assay is
2. Aseptic technique should be used throughout the assay procedure.
run. Autoclave and incubation conditions can impact the standard curve
readings and cannot always be duplicated. The standard curve is 3. The use of altered or deficient media may cause mutants having
obtained by using inositol at levels of 0.0, 1, 2, 4, 6, 8 and 10 µg per different nutritional requirements that will not give a satisfactory
assay flask (10 ml). response.
4. For successful results of these procedures, all conditions of the
Following inoculation, flasks are incubated at 25-30°C for 20-24 hours.
assay must be followed precisely.
Place flasks in the refrigerator for 15-30 minutes to stop growth. Growth
is measured turbidimetrically using any suitable spectrophotometer. References
1. Atkin, Schultz, Williams, and Frey. 1943. End. & Eng. Chem.,
Results
Ann. Ed. 15:141.
1. Prepare a standard concentration response curve by plotting the
response readings against the amount of standard in each tube, disk Packaging
or cup. Inositol Assay Medium 100 g 0995-15
KL Virulence Agar
Bacto KL Virulence Agar . KL Virulence Enrichment
®
KL Antitoxin Strips
Intended Use Acids is derived from acid-hydrolyzed casein that has low sodium
chloride and iron concentrations. The low iron concentration is
Bacto KL Virulence Agar is used with Bacto KL Virulence
beneficial because iron is known to prevent the production of diph
Enrichment, Bacto Chapman Tellurite Solution 1% and Bacto KL
theria toxin when present in more than minute amounts. Glycerol
Antitoxin Strips in differentiating virulent (toxigenic) from nonvirulent
(glycerine) contains no heavy metals and is used by bacteria as a source
strains of Corynebacterium diphtheriae.
of carbon. Tween ® 80 improves growth of certain strains of
Also Known As Corynebacterium diphtheriae. Toxin produced by bacteria and diffused
into the medium is detected by precipitation with the antitoxin present
KL Virulence Agar conforms with Klebs-Loeffler Virulence Agar.
on the KL Antitoxin Strip. Chapman Tellurite Solution 1%
Summary and Explanation (1% potassium tellurite solution) inhibits gram-negative and most
gram-positive bacteria except Corynebacterium spp., Streptococcus
Elek2 was the first to describe the agar plate diffusion technique for
mitis, S. salivarius, enterococci, and possibly Staphylococcus
demonstrating the in vitro toxigenicity (virulence) of Corynebacterium
epidermidis. This permits direct testing of mixed primary cultures.
diphtheriae. King, Frobisher, and Parsons 3 expanded on Elek’s
technique and, by using a carefully standardized medium, obtained Formula
results in agreement with animal inoculation tests. These authors
demonstrated that Difco Proteose Peptone possessed properties KL Virulence Agar
essential for toxin production. Incorporating Difco Proteose Peptone Formula Per Liter
into the test medium assured consistent results. The authors used Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
rabbit, sheep and horse serum as enrichments, finding human serum to Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
be unsatisfactory. To overcome irregularities encountered in previous Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
formulations, Hermann, Moore, and Parsons1 refined the medium used Final pH 7.8 ± 0.2 at 25°C
for the in vitro KL Virulence Test, simplifying the basal medium and
KL Virulence Enrichment
developing a nonserous enrichment. The medium and enrichment
described by these authors have been standardized for use in the Formula Per 100 ml
Bacto Casamino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
KL Virulence Test.
Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ml
KL Virulence Agar and KL Virulence Enrichment are prepared Tween® 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ml
according to the formulation of Hermann, Moore and Parsons.1
KL Antitoxin Strips
Principles of the Procedure KL Antitoxin Strips are 1 x 7 cm filter paper strips containing antitoxin
to diphtheria toxin.
Proteose Peptone provides the carbon and nitrogen sources required
for good growth of a wide variety of organisms and for toxin Precautions
production. Sodium Chloride maintains the osmotic balance of the 1. For Laboratory Use.
medium. Bacto Agar is incorporated as a solidifying agent.
2. Follow proper established laboratory procedure in handling and
KL Virulence Enrichment, composed of Casamino Acids, Glycerol and
disposing of infectious materials.
Tween® 80, provides a source of nonserous enrichment. Casamino
Test Procedure 3. King, E. O., M. Frobisher, Jr., and E. I. Parsons. 1949. The
Inoculate the medium by streaking a loopful of a 24-hour culture in in vitro test for virulence of Corynebacterium diphtheriae.
a single line across the plate perpendicular to (right angle to) Am. J. Public Health 39:1314.
the antitoxin strip. (Do not touch the actual strip itself). As many as 4. Clarridge, J. E., and C. A. Spiegel. 1995. Corynebacterium and
eight cultures may be tested on a single plate.6 Place test isolates about miscellaneous irregular gram-positive rods, Erysipelothrix and
1 cm apart. Also inoculate a toxigenic (positive control) and a Gardnerella, p. 357-378. In P. R. Murray, E. J. Baron, M. A. Pfaller,
nontoxigenic (negative control) C. diphtheriae strain approximately F. C. Tenover, and R. H. Yolken (eds.), Manual of clinical
1 cm on either side of the test isolates.6 Incubate the inverted plates microbiology, 6th ed. American Society for Microbiology,
at 37°C for 72 hours. Examine at 24-, 48- and 72-hour intervals. Washington, D.C.
5. Krech, T., and D. G. Hollis. 1991. Corynebacterium and related
Results
organisms, p. 277-286. In A. Ballows, W. J. Hausler, Jr.,
Toxigenic (virulent) cultures of C. diphtheriae will show fine lines K. Herrmann, H. D. Isenberg, and H. J. Shadomy (eds.), Manual of
of precipitation at approximately 45° angles from the culture streak. clinical microbiology, 5th ed. American Society for Microbiology,
This line forms where toxin (from the bacteria) combines with Washington, D.C.
antitoxin from the strip. Primary precipitin lines form an arc of
6. MacFaddin, J. F. 1985. Media for isolation-cultivation-
identity with the precipitin line produced by an adjacent positive
identification-maintenance of medical bacteria, vol. 1, p. 410-414.
control strain.7 Nontoxigenic strains of C. diphtheriae will show no
lines of precipitation. Williams & Wilkins, Baltimore, MD.
7. Washington, J. A., Jr. 1981. Laboratory procedures in clinical
Limitations of the Procedure microbiology. Springer-Verlag, New York, NY.
1. Each test should include positive and negative controls.5 8. Lennette, E. H., A. Balows, W. J. Hausler, Jr., and J. P. Truant
2. False-positive reactions may be seen after 24 hours as weak bands (eds.). 1980. Manual of clinical microbiology, 3rd ed. American
near the antitoxin strip. These can be recognized when compared Society for Microbiology, Washington, D.C.
with the positive control.8 9. Branson, D. 1972. Methods in clinical bacteriology. Charles
3. Corynebacterium ulcerans and C. pseudotuberculosis may also C. Thomas, Springfield, IL.
produce lines of toxin-antitoxin.9
Packaging
References KL Virulence Agar 500 g 0985-17
1. Hermann, G. J., M. S. Moore, and E. I. Parsons. 1958. A KL Virulence Enrichment 12 x 20 ml 0986-64
substitute for serum in the diphtheria in vitro test. Am. J. Clin. KL Antitoxin Strips 12 strips 3101-30
Pathol. 29:181-183.
2. Elek, S. D. 1948. The recognition of toxicogenic bacterial strains Chapman Tellurite Solution 1% 6 x 1 ml 0299-51
6 x 25 ml 0299-66
in vitro. Brit. Med. J. 1:493.
Formula Procedure
Kligler Iron Agar Materials Provided
Formula Per Liter Kligler Iron Agar
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g Materials Required But Not Provided
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g Flasks with closures
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Distilled or deionized water
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bunsen burner or magnetic hot plate
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g Tubes with closures
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Autoclave
Sodium Thiosulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 g Incubator (35°C)
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 g
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.024 g Method of Preparation
Final pH 7.4 ± 0.2 at 25°C 1. Suspend 55 grams in 1 liter distilled or deionized water.
2. Heat to boiling to dissolve completely.
Precautions 3. Dispense into tubes with closures.
1. For Laboratory Use.
4. Autoclave at 121°C for 15 minutes. Cool in slanted position
2. Follow proper established laboratory procedure in handling and with deep butts.
disposing of infectious materials.
Specimen Collection and Preparation
Storage 1. Collect specimens or food samples in sterile containers or with
Store the dehydrated medium below 30°C. The dehydrated medium sterile swabs and transport immediately to the laboratory
is very hygroscopic. Keep container tightly closed. Store prepared
following recommended guidelines.6-10
tubes at 2-8°C.
2. Process each specimen, using procedures appropriate for that
Expiration Date specimen or sample.6-10
The expiration date applies to the medium in its intact container when
stored as directed. Do not use a product if it fails to meet specifications
for identity and performance.
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
Test Procedure 7. Tubes should be incubated with caps loosened to allow a free
1. Obtain a pure culture of the organism to be tested. Select exchange of air, which is necessary to enhance the alkaline
well-isolated colonies. condition on the slant.11
2. With an inoculating needle, pick the center of well-isolated References
colonies obtained from solid culture media. 1. Kligler, I. J. 1917. A simple medium for the differentiation of
3. Stab the center of the medium into the deep of the tube to within members of the typhoid-paratyphoid group. Am. J. Public Health.
3-5 mm from the bottom. 7:1042-1044.
4. Withdraw the inoculating needle, and streak the surface of the slant. 2. Russell, F. F. 1911. The isolation of typhoid bacilli from urine and
5. Loosen closure on the tube before incubating. feces with the description of a new double sugar tube medium. J.
6. Incubate at 35°C for 18-48 hours. Med. Res. 25:217.
7. Read tubes for acid production of slant/butt, gas, and hydrogen 3. Kligler, I. J. 1918. Modifications of culture media used in the
isolation and differentiation of typhoid, dysentery, and allied
sulfide reactions.
bacilli. J. Exp. Med. 28:319-322.
Results 4. Bailey, S. F., and L. R. Lacy. 1927. A modification of the Kligler
1. An alkaline slant-acid butt (red/yellow) indicates fermentation of lead acetate medium. J. Bacteriol. 13:183.
dextrose only. 5. Sulkin, S. E., and J. C. Willett. 1940. A triple sugar-ferrous
2. An acid slant-acid butt (yellow/yellow) indicates fermentation of sulfate medium for use in identification of enteric organisms.
dextrose and lactose. J. Lab. Clin. Med. 25:649-653.
3. An alkaline slant-alkaline butt (red/red) indicates that neither 6. Pezzlo, M. (ed.). 1992. Aerobic bacteriology, p. 1.0.0-1.20.47. In
dextrose nor lactose was fermented (non-fermenter). Isenberg, H.D. (ed.), Clinical microbiology procedures handbook,
4. Cracks, splits, or bubbles in the medium indicate gas production. vol. 1. American Society for Microbiology, Washington, D.C.
5. A black precipitate in the butt indicates hydrogen sulfide production. 7. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
Limitations of the Procedure St. Louis, MO.
1. H2S-producing organisms may produce a black precipitate to such 8. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
a degree that the reaction in the butt is completely masked. If H2S R. H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
is produced, dextrose is fermented even if it is not observed.11 American Society for Microbiology, Washington, D.C.
2. Further biochemical tests and serological typing must be performed 9. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compen-
for definite identification and confirmation of organisms. dium of methods for the microbiological examination of foods,
3rd ed. American Public Health Association, Washington, D.C.
3. Do not use an inoculating loop to inoculate a tube of Kligler Iron
Agar. While stabbing the butt, mechanical splitting of the medium 10. Elliot, E. L., C. A. Kaysner, L. Jackson, and M. L. Tamplin.
occurs, causing a false positive result for gas production.11 1995. V. cholerae, V. parahaemolyticus, V. vulnificus, and other
Vibrio spp. In FDA bacteriological analytical manual, 8th ed.
4. Best reactions are obtained on freshly prepared medium. AOAC International, Gaithersburg, MD.
5. A pure culture is essential when inoculating Kligler Iron Agar. If 11. MacFaddin, J. F. 1985. Media for isolation-cultivation-
inoculated with a mixed culture, irregular observations may occur. identification-maintenance of medical bacteria, Vol. 1. Williams
6. Hydrogen sulfide determinations using Kligler Iron Agar should & Wilkins, Baltimore, MD.
be limited to the members of the Enterobacteriaceae. Other
organisms may require more sensitive methods for detection of H2S Packaging
production.11 Kligler Iron Agar 500 g 0086-17
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto LB Agar, Lennox
®
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Final pH 7.0 ± 0.2 at 25°C
Intended Use Precautions
Bacto LB Agar, Lennox is used for maintaining and cultivating 1. For Laboratory Use.
recombinant strains of Escherichia coli. 2. Follow proper established laboratory procedure in handling and
disposing of infectious materials.
Summary and Explanation
LB Agar, Lennox, a nutritionally rich medium, was developed by Storage
Lennox for the growth and maintenance of pure cultures of recombinant Store the dehydrated medium below 30°C. The dehydrated medium is
strains of E. coli.1 These strains are generally derived from E. coli K12, very hygroscopic. Keep container tightly closed.
which are deficient in B vitamin production. This strain of E. coli Store prepared medium at 2-8°C.
has been further modified through specific mutation to create an
auxotrophic strain that is not capable of growth on nutritionally deficient Expiration Date
media. LB Agar, Lennox provides all the nutritional requirements of The expiration date applies to the product in its intact container when
these organisms. LB Agar, Lennox contains half the sodium chloride stored as directed. Do not use a product if it fails to meet specifications
level of the Miller formulation of LB Agar.2 This allows the researcher for identity and performance.
to select the optimal salt concentration for a specific strain.
Procedure
Principles of the Procedure Materials Provided
Peptides and peptones are provided by Tryptone. Vitamins (including LB Agar, Lennox
B vitamins) and certain trace elements are provided by Yeast Extract.
Sodium ions for transport and osmotic balance are provided by Materials Required But Not Provided
Sodium Chloride. Bacto Agar is the solidifying agent. Flasks with closures
Distilled or deionized water
Formula Bunsen burner or magnetic hot plate
LB Agar, Lennox Autoclave
Formula Per Liter Waterbath (45-50°C)
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Petri dishes
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Incubator (35°C)
Method of Preparation
User Quality Control 1. Suspend 35 grams in 1 liter of distilled or deionized water.
2. Heat to boiling to dissolve completely.
Identity Specifications
3. Autoclave at 121°C for 15 minutes.
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
4. Cool to 45-50°C in a waterbath.
Solution: 3.5% solution, soluble in distilled or
deionized water on boiling. Solution 5. Dispense into sterile Petri dishes.
is light beige, slightly opalescent. Specimen Collection and Preparation
Prepared Plates: Medium amber, very slightly to
slightly opalescent. Not applicable.
Reaction of 3.5% Test Procedure
Solution at 25°C: pH 7.0 ± 0.2 Consult appropriate references for recommended test procedures.2
Cultural Response Results
Prepare LB Agar, Lennox per label directions. Inoculate and After sufficient incubation, the medium should show growth as evi-
incubate at 35 ± 2°C for 18- 24 hours.
denced by formation of colonies and/or a confluent lawn of growth.
INOCULUM
ORGANISM ATCC® CFU GROWTH
Escherichia coli 23724 100-300 Good References
Escherichia coli 33694 100-300 Good 1. Lennox, E. S. 1955. Transduction of linked genetic characters of
Escherichia coli 33849 100-300 Good the host by bacteriophage P1. Virology 1:190.
Escherichia coli 39403 100-300 Good 2. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G.
Escherichia coli 47014 100-300 Good Seidman, J. A. Smith, and K. Struhl. 1994. Current protocols in
Escherichia coli 53868 100-300 Good molecular biology, vol. 1. Current Protocols, New York, N.Y.
The cultures listed are the minimum that should be used for
performance testing. Packaging
LB Agar, Lennox 500 g 0401-17
Storage
Bacto LB Agar, Miller
®
Store the dehydrated medium below 30°C. The dehydrated medium is
very hygroscopic. Keep container tightly closed. Store prepared
Intended Use medium at 2-8°C.
Bacto LB Agar, Miller is used for maintaining and propagating Expiration Date
Escherichia coli in molecular microbiology procedures. The expiration date applies to the product in its intact container when
stored as directed. Do not use a product if it fails to meet specifications
Summary and Explanation for identity and performance.
LB Agar, Miller is based on LB Medium as described by Miller for the
growth and maintenance of E. coli strains used in molecular microbiology Procedure
procedures.1-3 LB Agar, Miller is a nutritionally rich medium designed Materials Provided
for growth of pure cultures of recombinant strains. E. coli grows more LB Agar, Miller
rapidly on this rich medium because it provides the cells with amino
acids, nucleotide precursors, vitamins and other metabolites that the Materials Required But Not Provided
microorganism would otherwise have to synthesize.4 Flasks with closures
Distilled or deionized water
Principles of the Procedure Bunsen burner or magnetic hot plate
Peptides and peptones are provided by Tryptone. Vitamins (including Autoclave
B vitamins) and certain trace elements are provided by Yeast Extract. Waterbath (45-50°C)
Sodium ions for transport and osmotic balance are provided by sodium Sterile Petri dishes
chloride. Agar is added to the medium as a gelling agent. Incubator (35°C)
Precautions
Bacto LB Broth, Lennox
®
1. For Laboratory Use.
2. Follow proper established laboratory procedures in handling and
Intended Use disposing of infectious materials.
Bacto LB Broth, Lennox is used for maintaining and propagating
Escherichia coli in molecular microbiology procedures. Storage
Store the dehydrated medium below 30°C. The dehydrated medium is
Summary and Explanation very hygroscopic. Keep container tightly closed.
LB Broth, Lennox is a nutritionally rich medium designed for growth
of pure cultures of recombinant strains. The formula is based on Store the prepared medium at 2-8°C.
L Broth described by Lennox for the growth and maintenance of
E. coli strains used in molecular microbiology procedures.1 E. coli
Expiration Date
is grown to late log phase in LB Broth. Some plasmid vectors may The expiration date applies to the product in its intact container when
replicate to high copy numbers without selective amplification. Some stored as directed. Do not use a product if it fails to meet specifications
vectors may require selective amplification to reach high copy numbers. for identity and performance.
Chloramphenicol can be added to inhibit host synthesis and, as a Procedure
result, prevent replication of the bacterial chromosome.2
Materials Provided
LB Broth, Lennox contains ten times the sodium chloride level of Luria
Broth Base, Miller and one half of that found in LB Broth, Miller.3 LB Broth, Lennox
This allows the researcher to select the optimal salt concentration for a Materials Required But Not Provided
specific strain. If desired, the medium may be aseptically supplemented Flasks with closures
with glucose to prepare the complete medium described by Lennox. Tubes with closures
Principles of the Procedure Distilled or deionized water
Peptides and peptones are provided by Tryptone. Vitamins (including B Autoclave
vitamins) and certain trace elements are provided by Yeast Extract. Sodium Incubator (35°C)
ions for transport and osmotic balance are provided by Sodium Chloride. Method of Preparation
Formula 1. Suspend 20 grams in 1 liter of distilled or deionized water.
LB Broth, Lennox 2. Dispense into tubes with closures.
Formula Per Liter 3. Autoclave at 121°C for 15 minutes.
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g 4. Allow to cool below 45°C.
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
5. If desired, aseptically add 10 ml sterile 10% glucose solution and
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
mix thoroughly.
Final pH 7.0 ± 0.2 at 25°C
Specimen Collection and Preparation
Not applicable
User Quality Control
Identity Specifications Test Procedure
Dehydrated Appearance: Light beige, free-flowing, homogeneous. Consult appropriate references for recommended test procedures.1,2,3
Solution: 2.0% solution, soluble in distilled or Results
deionized water. Solution is very light
amber, clear to very slightly opalescent. Growth is evident in the form of turbidity.
Prepared Medium: Very light amber, clear to very
slightly opalescent. References
Reaction of 2.0% 1. Lennox, E. S. 1955. Transduction of linked genetic characters of
Solution at 25°C: pH 7.0 ± 0.2 the host by bacteriophage P1. Virology 1:190.
Cultural Response 2. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular
Prepare LB Broth, Lennox per label directions. Inoculate and cloning: a laboratory manual, 2nd ed. Cold Spring Harbor
incubate at 35 ± 2°C for 18-24 hours. Laboratory Press, Cold Spring Harbor, New York.
INOCULUM 3. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring
ORGANISM ATCC® CFU GROWTH
Harbor Laboratory, Cold Spring Harbor, New York.
Escherichia coli 53868 (DH5) 100-300 Good
Escherichia coli JM103 100-300 Good Packaging
Escherichia coli 33694 (HB101) 100-300 Good
LB Broth, Lennox 500 g 0402-17
The cultures listed are the minimum that should be used for 2 kg 0402-07
performance testing. 10 kg 0402-08
Precautions
Bacto LB Broth, Miller
®
1. For Laboratory Use.
2. Follow proper established laboratory procedure in handling and
Intended Use disposing of infectious materials.
Bacto LB Broth, Miller (Luria-Bertani) is used for maintaining and
propagating Escherichia coli in molecular microbiology procedures. Storage
Store the dehydrated medium below 30°C. The dehydrated medium is
Summary and Explanation very hygroscopic. Keep container tightly closed. Store prepared
LB Broth, Miller is based on LB Medium as described by Miller for the
medium at 2-8°C.
growth and maintenance of Escherichia coli strains used in
molecular microbiology procedures.1-3 LB Broth, Miller is a nutritionally
rich medium designed for growth of pure cultures of recombinant
Expiration Date
strains. Escherichia coli is grown to late log phase in LB Medium. The expiration date applies to the product in its intact container when
Some plasmid vectors replicate to a high copy number without stored as directed. Do not use a product if it fails to meet specifications
selective amplification. Some vectors do not replicate so freely, and for identity and performance.
need to be selectively amplified. Chloramphenicol has been added to
inhibit host synthesis and as a result, prevents replication of the Procedure
bacterial chromosome.4 Materials Provided
LB Broth, Miller contains twenty times the sodium chloride level of Luria LB Broth, Miller
Broth Base, Miller and twice the level found in LB Broth,
Lennox.3-5 This allows the researcher to select the optimal salt concentration Materials Required But Not Provided
for a specific strain. Flasks with closures
Distilled or deionized water
Principles of the Procedure Autoclave
Peptides and peptones are provided by Tryptone. Vitamins (including Incubator (35°C)
B vitamins) and certain trace elements are provided by Yeast Extract.
Sodium ions for transport and osmotic balance are provided by Method of Preparation
sodium chloride. 1. Dissolve 25 grams in 1 liter of distilled or deionized water.
Formula 2. Autoclave at 121°C for 15 minutes.
LB Broth, Miller Specimen Collection and Preparation
Formula Per Liter Refer to appropriate references for specimen collection and preparation.
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Test Procedure
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Consult appropriate references for recommended test procedures.3,4
Final pH 7.0 ± 0.2 at 25°C
Results
Growth should be evident by the appearance of turbidity in the medium.
User Quality Control
Identity Specifications References
Dehydrated Appearance: Off-white to beige, free-flowing, 1. Luria, S. E., and J. W. Burrous. 1955. Hybridization between
homogeneous. Escherichia coli and Shigella. J. Bacteriol. 74:461-476.
Solution: 2.5% solution; soluble in distilled or
deionized water. Solution is light 2. Luria, S. E., J. N. Adams, and R. C. Ting. 1960. Transduction of
amber, clear to very slightly opalescent. lactose- utilizing ability among stains of E. coli and S. dysenteriae
Prepared Tubes: Very light amber, clear to very and the properties of the transducing phage particles. Virology.
slightly opalescent. 12:348-390.
Reaction of 2.5% 3. Miller, J. H. 1972. Experiments in molecular genetics. Cold
Medium at 25°C: pH 7.0 ± 0.2 Spring Harbor Laboratory. Cold Spring Harbor, New York.
Cultural Response 4. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular
Prepare LB Broth, Miller per label directions. Inoculate the cloning: a laboratory manual, 2nd ed. Cold Spring Harbor
tubes and incubate at 35 ± 2°C for 18-24 hours. Laboratory, Cold Spring Harbor, New York.
INOCULUM 5. Lennox, E. S. 1955. Transduction of linked genetic characters of
ORGANISM ATCC® CFU GROWTH
the host by bacteriophage P1. Virology. 1:190-206.
Escherichia coli 33526 100-1,000 Good
The culture listed above is the minimum that should be used for Packaging
performance testing. LB Broth, Miller 500 g 0446-17
2 kg 0446-07
Lactobacilli Broth AOAC foreign material may be sufficient to give erroneous results.
Formula Per Liter Scrupulously clean glassware free from detergents and other
Peptonized Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g chemical must be used.
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Storage
Tomato Juice (100 ml) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Store the dehydrated medium below 2-8°C. The dehydrated medium is
Monobasic Potassium Phosphate . . . . . . . . . . . . . . . . . . . . . 2 g very hygroscopic. Keep container tightly closed.
Tween® 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Final pH 6.8 ± 0.2 at 25°C Expiration Date
The expiration date applies to the product in its intact container when
Precautions stored as directed. Do not use a product if it fails to meet specifications
1. For Laboratory Use. for identity and performance.
2. Follow proper established laboratory procedures in handling and
disposing of infectious materials. Procedure
3. Take care to avoid contamination of media or glassware used for Materials Provided
microbiological assay procedures. Extremely small amounts of Lactobacilli Agar AOAC
Lactobacilli Broth AOAC
5. Wash the cells by centrifuging and decanting the supernatant two 4. For a successful completion of these procedures, all conditions of
additional times unless otherwise indicated. the assay must be adhered to meticulously.
6. Dilute the washed suspension 1:100 with sterile 0.9% NaCl or
sterile single- strength basal assay medium or as indicated. Where
References
1. Association of Official Analytical Chemists. 1995. Official
applicable, inoculum concentration should be adjusted according
methods of analysis of AOAC International, 16th ed. AOAC
to limits specified in AOAC1 or US Pharmacopeia.5
International, Arlington, VA.
For a complete discussion on vitamin assay methodology refer to
2. Loy. 1958. J. AOAC. 4:61.
appropriate procedures outlined in the references.1,5
3. Mickle and Breed. 1925. Technical Bulletin 110, NY State
Results Agriculture Ex. Station.
Refer to appropriate references for vitamin assay results.1,5 4. Kulp, J. W. L., and V. White. 1932. Modified medium for plating
Lactobacillus acidophilus. Science 76:17.
Limitations of the Procedure 5. The United States Pharmacopeial Convention. 1995. The United
1. The test organism used for inoculating an assay medium must be States pharmacopeia, 23rd ed. The United States Pharmacopeial
cultured and maintained on media recommended for that purpose. Convention Inc., Rockville, MD.
2. Aseptic technique should be used throughout the vitamin assay
procedure. Packaging
3. The use of altered or deficient media may result in mutants with Lactobacilli Agar AOAC 100 g 0900-15*
different nutritional requirements that will not give a satisfactory Lactobacilli Broth AOAC 100 g 0901-15*
response. *Store at 2-8°C
non-Salmonella bacteria ferment lactose while Salmonella does not. of food samples for isolation of Salmonella. Consult standard references
As lactose-fermenting bacteria metabolize lactose, the pH of the for specific instructions for each type of material being tested.1,2,3,4
medium decreases, creating a bacteriostatic effect on competing 1. Transfer a 25 gram or 25 ml sample of test material into a container.
microorganisms. Add 225 ml of sterile Lactose Broth. Mix as necessary to make a
homogeneous suspension. Incubate at 35°C for 24 ± 2 hours.
Formula
2. Transfer 1 ml of suspension to appropriate enrichment broths,
Bacto Lactose Broth such as Tetrathionate Broth and Selenite Cystine Broth. Incubate
Formula Per Liter at 35°C for 24 ± 2 hours.
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g 3. Transfer a loopful of suspension to appropriate selective agar
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g media, such as Hektoen Enteric Agar, XLD Agar and Bismuth
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Sulfite Agar. Incubate at 35°C for 24 ± 2 hours.
Final pH 6.9 ± at 25°C
Results
Precautions Pre-enrichment, selective enrichment and selective plating increase
1. For Laboratory Use. the likelihood of isolating Salmonella from foods and other
2. Follow proper established laboratory procedure in handling and materials.
disposing of infectious materials.
References
Storage 1. Flowers, R. S., J. D’Aoust, W. H. Andrews, and J. S. Bailey.
Store the dehydrated medium below 30°C. The powder is very 1992. Salmonella, p. 371-442. In C. Vanderzant and D. F.
hygroscopic. Keep container tightly closed. Splittstoesser (ed.), Compendium of methods for the microbio-
Store the prepared medium at 2-8°C. logical examination of foods, 3rd ed. American Public Health
Association, Washington, D.C.
Expiration Date 2. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig.
The expiration date applies to the product in its intact container when 1993. Pathogens in milk and milk products, p. 103-212. In R. T.
stored as directed. Do not use a product if it fails to meet specifications Marshall (ed.), Standard methods for the microbiological examina-
for identity and performance. tion of dairy products, 16th ed. American Public Health Association,
Washington, D.C.
Procedure 3. Andrews, W. H., G. A. June, P. S. Sherrod, T. S. Hammack, and
Materials Provided R. M. Amaguana. 1995. Salmonella, p. 5.01-5.20. In Bacterio-
Lactose Broth logical analytical manual, 8th ed. AOAC International,
Gaithersburg, MD.
Materials Required but not Provided 4. Andrews, W. H. 1995. Microbial Methods, p. 1-119. In Official
Flasks with closures methods of analysis of AOAC International, 16th ed. AOAC
Distilled or deionized water International. Arlington, VA.
Autoclave 5. American Public Health Association. 1975. Standard methods
Incubator (35°C) for the examination of water and wastewater, 14th ed. American
Tubes with closures Public Health Association, Washington, D.C.
Fermentation vials 6. American Public Health Association. 1976. Compendium of
Disinfectant solution (for prepared Lactose Broth) methods for the microbiological examination of foods. American
Public Health Association, Washington, D.C.
Method of Preparation
7. American Public Health Association. 1978. Standard methods
Lactose Broth (dehydrated) for the examination of dairy products, 14th ed. American Public
1. Suspend 13 grams in 1 liter distilled or deionized water. Health Association, Washington, D.C.
2. Warm slightly to dissolve completely.
3. Dispense into tubes containing inverted fermentation vials.
Packaging
Lactose Broth 100 g 0004-15
4. Autoclave at 121°C for 15 minutes.
500 g 0004-17
Specimen Collection and Preparation 2 kg 0004-07
Refer to appropriate references for specimen collection and preparation. 10 kg 0004-07
Lactose Broth 10 x 90 ml 9070-73
Test Procedure
Lactose Broth is used in the pre-enrichment phase of the preparation
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Lactose Peptone Broth
®
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g
Final pH 7.4 ± 0.2 at 25°C
Intended Use
Bacto Lactose Peptone Broth is used for the detection of coliform Precautions
organisms in water. 1. For Laboratory Use.
2. Follow proper, established laboratory procedures in handling and
Summary and Explanation disposing of infectious materials.
Lactose Peptone Broth is based on the Lactose Peptone Broth formula
described in German Standard Methods and German Drinking Water Storage
Regulations.1 Lactose Peptone Broth is recommended as a non-selective Store the dehydrated medium below 30°C. The dehydrated medium is
broth enrichment and detection medium for E. coli and other coliform very hygroscopic. Keep container tightly closed. Store prepared
bacteria present in water. Lactose fermentation and gas production at medium at 2-8°C.
36 ± 1°C are used as the basis for this presumptive coliform test.
Expiration Date
Principles of the Procedure The expiration date applies to the product in its intact container when
Lactose Peptone Broth contains Tryptone and Soytone which provide stored as directed. Do not use a product if it fails to meet specifications
the carbon and nitrogen sources required for good growth of a wide for identity and performance.
variety of organisms. Lactose is provided as a source of fermentable
carbohydrate. Sodium Chloride is present in the medium to provide
Procedure
a suitable osmotic environment. Brom Cresol Purple is used as Materials Provided
a colorimetric indicator to show the production of acid from the Lactose Peptone Broth
fermentation of lactose.
Materials Required But Not Provided
Formula Flasks with closures
Distilled or deionized water
Lactose Peptone Broth
Durham tubes
Formula Per Liter Autoclave
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 g
Incubator (36 ± 1°C)
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Specimen Collection and Preparation
1. Collect samples in sterile containers and transport immediately to
User Quality Control the laboratory following recommended guidelines.1
Identity Specifications 2. Process each sample using procedures appropriate for that sample.1
Dehydrated Medium: Light beige, free-flowing,
homogeneous. Method of Preparation
Solution: 10.5% solution (triple-strength), 1. To prepare a triple strength solution, dissolve 105 grams in 1 liter
soluble in distilled or deionized of distilled or deionized water. To prepare a single strength
water. Solution is dark reddish-purple, solution, dissolve 35 grams in 1 liter of distilled or deionized water.
clear to slightly opalescent. 2. Dispense 50 ml into tubes or bottles containing a Durham tube.
Prepared Tubes: Dark reddish purple, clear to slightly 3. Autoclave at 121°C for 15 minutes.
opalescent.
Reaction of 10.5% Test Procedure1
Solution at 25°C: pH 7.4 ± 0.2 Direct Broth Method
1. Add 100 ml of sample to 50 ml of triple strength Lactose
Cultural Response
Peptone Broth.
Prepare Lactose Peptone Broth per label directions. Inoculate
and incubate at 35 ± 2°C for 24-48 hours. 2. Incubate at 36 ± 1°C for 24-48 hours.
INOCULUM LACTOSE GAS 3. Examine tubes or bottles for evidence of acid formation and gas
ORGANISM ATCC® CFU FERMENTATION PRODUCTION
production.
Escherichia coli 25922* 10-100 + +
Salmonella 14028* 100-1,000 – –
Membrane Filtration Broth Method
typhimurium 1. Filter 100 ml of sample through a sterile 0.45 micron membrane filter.
The cultures listed are the minimum that should be used for
2. Remove filter and place in 50 ml of single strength Lactose
performance testing. Peptone Broth.
*These cultures are available as Bactrol™ Disks and should be used 3. Incubate at 36 ± 1°C for 24-48 hours.
as directed in Bactrol Disks Technical Information.
4. Examine tubes or bottles for evidence of acid formation and gas
production.
Results References
Acid formation is demonstrated by a change in the color of the 1. DIN Deutsches Institut für Normung. 1991. e.V.: Deutsche
medium from reddish-purple to yellow. Gas production is demonstrated Einheitsverfahren zur Wasser-, Abwasser-und Schlammunter
by the displacement of the medium from the Durham tube. Production suchung: Mikrobiologische Verfahren (Gruppe k), Nachwels von
of both acid and gas is a presumptive indication of the presence of Escherichia coli und coliformen Keimen (K6). Reference Method
coliform organisms. DIN 38411.
Subculture presumptive positives onto Endo Agar and MacConkey 2. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and R.
Agar. Incubate at 35 ± 2°C for 24 hours. Examine plates for the H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
presence of typical coliform colonies. Further biochemical testing is American Society for Microbiology, Washington, D.C.
necessary to confirm the presence and identify coliforms. Consult 3. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
appropriate references for further information on identification Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
of coliforms.2,3 St. Louis, MO.
Limitations of the Procedure Packaging
1. Detection of coliform bacteria in Lactose Peptone Broth using this Lactose Peptone Broth 500 g 0665-17
method is only a presumptive test.
coliforms. Potassium Phosphates are the buffering agents, and PREPARATION OF LAURYL TRYPTOSE BROTH5
Sodium Chloride is used to maintain the osmotic balance of the
Dehydrated Lauryl
medium. Sodium Lauryl Sulfate is the selective agent used to inhibit Amount of Volume of Tryptose Broth
organisms other than coliforms. Inoculum Medium in Tube Medium+Inoculum Required
mL mL mL g/L
Formula 1 10 or more 11 or more 35.6
Lauryl Tryptose Broth 10 10 20 71.2
Formula Per Liter 10 20 30 53.4
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g 20 10 30 106.8
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g 100 50 150 106.8
Potassium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . 2.75 g 100 35 135 137.1
Potassium Phosphate, Monobasic . . . . . . . . . . . . . . . . . . 2.75 g 100 20 120 213.6
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Sodium Lauryl Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g Specimen Collection and Preparation
Final pH 6.8 ± 0.2 at 25°C Collect and process specimens according to laboratory policy or
standard methods.1,5,6,7,8,9
Precautions
1. For Laboratory Use. Test Procedure
2. Follow proper established laboratory procedures in handling and Follow the methods and procedures for the detection of coliform
disposing of infectious materials. organisms as described in standard methods.1,5,6,7,8,9
Storage Results
Store the dehydrated medium below 30°C. The dehydrated medium is After incubation of the tubes at 35 ± 2°C for 24 hours, examine for
very hygroscopic. Keep container tightly closed. turbidity and gas production. If no gas has formed in the inverted
tube, reincubate and reexamine after 48 hours.5,6
NOTE: Refrigerated Lauryl Tryptose Broth generally becomes cloudy
or forms precipitates. Incubate medium overnight at room temperature Turbidity of the medium accompanied by formation of gas within
(20°C) before use to clear the medium.5 48 hours is a positive presumptive test for the presence of
coliforms.5,6 The result should be confirmed by additional standard
Expiration Date testing.1,5,6,7,8,9
The expiration date applies to the product in its intact container when
stored as directed. Do not use a product if it fails to meet specifications
Limitations of the Procedure
for identity and performance. 1. Since the nutritional requirements of organisms vary, some
strains may be encountered that fail to grow or grow poorly on
Procedure this medium.
Materials Provided References
Lauryl Tryptose Broth 1. Christen, G. L., P. M. Davidson, J. S. McAllister, and L. A. Roth.
1992. Coliform and other indicator bacteria, p. 247-267. In R. T.
Materials Required But Not Provided Marshal (ed.). Standard methods for the examination of dairy
Glassware products. 16th ed. American Public Health Association,
Test tubes Washington, D.C.
Incubator (35°C) 2. Mallmann, W. L., and C. W. Darby. 1941. Uses of a lauryl
Fermentation vials sulphate tryptose broth for the detection of coliform organisms.
Autoclave Am. J. Public Health. 31:127.
Distilled or deionized water 3. Darby, C. W., and W. L. Mallmann. 1939. J. of Am. Water Works
Method of Preparation Assoc. 31:689.
1. Suspend 35.6 grams in 1 liter distilled or deionized water. 4. Perry and Hajna. 1944. Am. J. Public Health. 34:735.
2. Warm slightly to dissolve completely. 5. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). Standard
methods for the examination of water and wastewater, 19th ed.
3. Dispense into tubes containing inverted fermentation vials. American Public Health Association, Washington, D.C.
4. Autoclave at 121°C for 15 minutes. 6. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992.
NOTE: Lauryl Tryptose Broth may be prepared in single strength Compendium of methods for the microbiological examination of
when examining 1 ml or less of water as an inoculum. For inocula food, 3rd ed. American Public Health Association, Washington, D.C.
of 10 ml consult the table below. 7. Association of Official Analytical Chemists. 1995. Bacteriological
analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
Enrichment EMJH
Indirect exposure through contaminated water and soil accounts for
Intended Use most sporadic cases. Direct exposure occurs in pet owners, veterinarians
and persons working with livestock.3
Bacto Leptospira Medium Base EMJH is used with Bacto Leptospira
Enrichment EMJH in cultivating Leptospira. The basal medium and enrichment are prepared according to the
formulations described by Ellinghausen and McCullough4 as modified
Summary and Explanation by Johnson and Harris.5 They modified the formula by replacing rabbit
In 1816, Adolf Weil described the first recognized leptospiral serum medium with Tween 80-albumin. Leptospira Medium EMJH
infections in humans. 1 These cases were caused by Leptospira was used in cultivation studies of Leptospira.6
icterohaemorrhagiae and the disease was subsequently named Weil’s Leptospira Medium EMJH is recommended for the clinical isolation
Disease.1 Leptospirosis is a zoonotic disease, having its reservoir in of Leptospira.7,8
wild, domestic, and peridomestic animals. Infection usually results
from direct or indirect exposure to the urine of leptospiruric animals.2
2. Faine, S. (ed.). 1982. Guidelines for the control of leptospirosis. W. 6. Rule, P. L., and A. D. Alexander. 1986. Gellan gum as a substitute
H. O. Offset publication no. 67. World Health Organization, Geneva. for agar in leptospiral media. J. Clin. Microbiol. 23:500-504.
3. Kaufmann, A. F., and R. S. Weyant. 1995. Leptospiraceae, 7. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures
p.621-625. In Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover handbook. American Society for Microbiology, Washington, D.C.
and R. H. Yolken (ed.). Manual of clinical microbiology, 6th ed. 8. Koneman, E. W., S. D. Allen, V. R. Dowell, Jr., W. M. Janda,
American Society for Microbiology, Washington, D.C. H. M. Sommers, and W. C. Winn, Jr. 1988. Color atlas and
4. Ellinghausen, Jr., H. C., and W. G. McCullough. 1965. textbook of diagnostic microbiology, 3rd ed. J. B. Lippincott
Nutrition of Leptospira pomona and growth of 13 other Company, Philadelphia, PA.
serotypes: fractionation of oleic albumin complex (OAC) and a
medium of bovine albumin and polysorbate 80. Am. J. Vet.
Packaging
Leptospira Medium Base EMJH 500 g 0794-17
Research 26:45-51.
5. Johnson, R., and V. G. Harris. 1967. Differentiation of pathogenic Leptospira Enrichment EMJH 6 x 100 ml 0795-73*
of leptospires. J. Bacteriol. 94:27-31. *Store at 2-8°C
Storage Procedure
Store the dehydrated media at 2-8°C. The dehydrated media are very Materials Provided
hygroscopic. Keep containers tightly closed. Letheen Agar
Letheen Broth
Expiration Date
The expiration date applies to the product in its intact container when Materials Required But Not Provided
stored as directed. Do not use a product if it fails to meet specifications Glassware
for identity and performance. Distilled or deionized water|
Autoclave
Method of Preparation
1. Letheen Agar: Suspend 32 grams in 1 liter distilled or deionized
User Quality Control water. Boil to dissolve completely.
Identity Specifications Letheen Broth: Suspend 25.7 grams in 1 liter distilled or deionized
Letheen Agar water. Boil to dissolve completely.
Dehydrated Appearance: Tan, moist appearance, with a 2. Autoclave at 121°C for 15 minutes.
tendency to clump.
Solution: 3.2% solution, soluble in distilled or Specimen Collection and Preparation
deionized water on boiling. Solution Refer to appropriate references for specimen collection and preparation.
is light to medium amber, clear to
slightly opalescent, may have a Test Procedures
slight, fine precipitate. Letheen Agar and Letheen Broth are used in a variety of procedures.
Prepared Plates: Light to medium amber, slightly Please consult appropriate references for further information.3,4
opalescent, may have a slight
precipitate. Results
Reaction of 3.2% Refer to appropriate references and procedures for results.
Solution at 25°C: pH 7.0 ± 0.2
Limitations of the Procedure
Letheen Broth
1. The dehydrated Letheen Agar has a characteristic “brown sugar”
Dehydrated Appearance: Tan, moist appearance, with a
tendency to clump. appearance. This does not indicate deterioration.
Solution: 2.57% solution, soluble in distilled References
or deionized water on boiling. 1. Weber, G. R., and L. A. Black. 1948. Relative efficiency of
Solution is light to medium amber,
clear to slightly opalescent quaternary inhibitors. Soap and Sanit. Chem. 24:134-139.
(opalescent when hot), May have a 2. Quisno, R., I. W. Gibby, and M. J. Foter. 1946. A neutralizing
very slight precipitate. medium for evaluating the germicidal potency of the quaternary
Prepared Tubes: Light to medium amber, clear to ammonium salts. Am. J. Pharm. 118:320-323.
slightly opalescent, may have a 3. Association of Official Analytical Chemists. 1995. Official
slight precipitate. methods of analysis, 16th ed. Association of Official Analytical
Reaction of 2.57% Chemists, Washington, D.C.
Solution at 25°C: pH 7.0 ± 0.2 4. American Society for Testing Materials. 1991. Standard test
Cultural Response method for preservatives in water-containing cosmetics, E 640-78.
Prepare Letheen Agar per label directions. Using the pour plate Annual Book of ASTM Standards, Philadelphia, PA.
technique, inoculate and incubate at 35 ± 2°C for 40-48 hours. 5. Erlandson, A. L., Jr., and C. A. Lawrence. 1953. Inactivating
INOCULUM medium for hexachlorophene (G-11) types of compounds and some
ORGANISM ATCC® CFU GROWTH substituted phenolic disinfectants. Science 118:274-276.
Escherichia coli 11229 100-1,000 good
6. Brummer, B. 1976. Influence of possible disinfectant transfer on
Staphylococcus aureus 6538 100-1,000 good
Staphylococcus aureus plate counts after contact sampling. App.
Prepare Letheen Broth per label directions. Inoculate and Environ. Microbiol. 32:80-84.
incubate at 35 ± 2°C for 40- 48 hours. 7. Favero (chm.). 1967. Microbiological sampling of surfaces-a state
INOCULUM of the art report. Biological Contamination Control Committee,
ORGANISM ATCC® CFU GROWTH
American Association for Contamination Control.
Escherichia coli 11229 100-1,000 good
Staphylococcus aureus 6538 100-1,000 good Packaging
Salmonella typhi 6539 100-1,000 good Letheen Agar 500 g 0680-17
Letheen Broth 500 g 0681-17
Method of Preparation 2. The medium will support growth of most gram-negative bacilli but
1. Suspend 37.5 grams in 1 liter distilled or deionized water. some strains of Salmonella and Shigella may be inhibited.
2. Heat to boiling to dissolve completely. References
3. Autoclave at 121°C for 15 minutes. Avoid overheating. 1. Holt-Harris, J. E., and O. Teague. 1916. A new culture medium
4. Evenly disperse the precipitate when dispensing. for the isolation of Bacillus typhosa from stools. J. Infect. Dis. 18:596.
2. Levine, M. 1918. Differentiation of E. coli and A. aerogenes on a
Specimen Collection and Preparation
simplified eosin-methylene blue agar. J. Infect. Dis. 23:43-47.
Consult standard references for specific instructions for the type of
material being tested.4,5,6,7 3. Levine, M. 1921. Bacteria fermenting lactose-the significance in
water analysis. Bull. 62. Iowa State College Eng. Exp. Sta., Ames, Iowa.
Test Procedure 4. Hitchins, A. D., P. A. Hartman, and E. C. D. Todd. 1992.
Consult standard references for specific instructions for the type of Coliforms - Escherichia coli and its toxins, p. 325-369. In
material being tested.4,5,6,7 C. Vanderzant and D. F. Splittstoesser (ed.). Compendium of
methods for the microbiological examination of foods, 3rd ed.
Results
American Public Health Association, Washington, D.C.
E. coli colonies are typically dark-centered with or without a
metallic sheen. 5. Marshall, R. T. (ed.). 1993. Standard methods for the
Lactose fermenters: Blue-black to brownish colored microbiological examination of dairy products, 16th ed. American
colonies, may have dark centers Public Health Association, Washington, D.C.
with or without metallic sheen. 6. Hitchins, A. D., P. Feng, W. D. Watkins, S. R. Rippey, and
Lactose non-fermenters: Colorless or transparent, amber to L. A. Chandler. 1995. Escherichia coli and the coliform bacteria.
light-purple colonies. p. 4.01-4.29. In Bacteriological analytical manual, 8th ed. AOAC
International, Gaithersburg, MD.
Staphylococci and streptococci: Colorless pinpoint colonies.
7. Andrews, W. 1995. Microbial Methods, p. 1-119. In Official
Yeasts: Colorless, dull pinpoint colonies; methods of analysis of AOAC International, 16th ed. AOAC
may be “spidery” at edges. International, Arlington, VA.
Limitations of the Procedure Packaging
1. Levine EMB Agar is only moderately inhibitory. Some staphylococci, Levine EMB Agar 100 g 0005-15
streptococci, and yeasts may grow as small pinpoint colonies. 500 g 0005-17
Perform microscopic examination and biochemical tests to 2 kg 0005-07
identify to genus and species. 10 kg 0005-08
Intended Use Listeria species grow over a pH range of 5.0-9.6, and survive in food
Bacto Listeria Enrichment Broth is used to selectively enrich Listeria products with pH levels outside these parameters.7 Listeria spp. are
from food. microaerophilic, gram-positive, asporogenous, non-encapsulated,
non-branching, regular, short, motile rods. Motility is most pronounced
Summary and Explanation at 20°C.
First described in 1926 by Murray, Webb and Swann, 1 Listeria The most common contaminating bacteria found in food sources
monocytogenes is a widespread problem in public health and the food potentially containing Listeria are: streptococci, especially the enterococci,
industries. This organism can cause human illness and death, particularly micrococci and Bacillus species, Escherichia coli, Pseudomonas
in immunocompromised individuals and pregnant women.2 The first aeruginosa and Proteus vulgaris.8
reported food-borne outbreak of listeriosis was in 1985,3 and since then, Identification of Listeria is based on successful isolation of the
microbiological and epidemiological evidence from both sporadic and organism, biochemical characterization and serological confirmation.
epidemic cases of listeriosis has shown that the principal route of
transmission is via the consumption of foodstuffs contaminated with Listeria Enrichment Broth is based on the formula developed by Lovett
Listeria monocytogenes.4 et al.9 in which Tryptic Soy Broth is supplemented with Yeast Extract
for optimum growth of Listeria.
Implicated vehicles of transmission include turkey frankfurters,5
coleslaw, pasteurized milk, Mexican-style cheese, paté, and pickled Principles of the Procedure
pork tongue. The organism has been isolated from commercial dairy Tryptone, Soytone and Yeast Extract provide nitrogen, vitamins and
and other food processing plants, and is ubiquitous in nature, being minerals. Dextrose is a carbohydrate source. Sodium Chloride
present in a wide range of unprocessed foods and in soil, sewage, maintains the osmotic balance of the medium. Phosphate acts as a
silage and river water.6 buffer. Acriflavine HCl and Nalidixic Acid are added for selectivity
and Cycloheximide is used to inhibit growth of saprophytic fungi.
4. Bremer, P. J., and C. M. Osborne. 1995. Thermal-death times of 8. Kramer, P. A., and D. Jones. 1969. Media selective for Listeria
Listeria monocytogenes in green shell mussels (Perna canaliculus) monocytogenes. J. Appl. Bacteriol. 32:381-394.
prepared for hot smoking. J. Food Prot. 58:604-608. 9. Lovett, J., D. W. Frances, and J. M. Hunt. 1987. Listeria
5. Grau, F. H., and P. B. Vanderlinde. 1992. Occurrence, numbers, monocytogenes in raw milk: detection, incidence and pathogenicity.
and growth of Listeria monocytogenes on some vacuum-packaged J. Food Prot. 50:188-192.
processed meats. J. Food Prot. 55:4-7. 10. McBride, M. E., and K. F. Girard. 1960. A selective method for
6. Patel, J. R., C. A. Hwang, L. R. Beuchat, M. P. Doyle, and the isolation of Listeria monocytogenes from mixed bacterial
R. E. Brackett. 1995. Comparison of oxygen scavengers for their populations. J. Lab. Clin. Med. 55:153-157.
ability to enhance resuscitation of heat-injured Listeria
monocytogenes. J. Food Prot. 58:244-250. Packaging
7. Conner, D. E., R. E. Brackett, and L. R. Beuchat. 1986. Listeria Enrichment Broth 500 g 0222-17
Effect of temperature, sodium chloride, and pH on growth of Listeria 10 kg 0222-08
monocytogenes in cabbage juice. Appl. Environ. Microbiol. 52:59.
A = Acid reactions: 1. Pinkish-red medium. Uninoculated Bacillus subtilis Clostridium perfringens Lactobacillus casei
2. Due to fermentation of the carbohydrates lactose and glucose. tube ATCC® 6633 ATCC® 12924 ATCC® 7469
K = Alkaline reactions: 1. Blue medium.
2. No fermentation of carbohydrates.
3. Organism attacks nitrogenous substances present in medium. The breakdown of lactalbumin by proteolytic enzymes form ammonia or basic amines.
R = Reduction: 1. White medium.
2. The enzyme reductase removes oxygen from the litmus, resulting in a decolorized, milky white appearance.
3. Reduction usually begins at the bottom of the tube.
C = Clot or Curd: 1. Milk protein coagulation.
2. Coagulation due to either a precipitation of casein by acid formation or the conversion of casein to paracasein by the enzyme rennin resulting in a clear
watery fluid called “whey.”
D = Digestion: 1. Milk protein digested.
2. Clearing of medium and dissolution of clot by digestion of casein.
air. If not breathing, give artificial respiration. If breathing is Specimen Collection and Preparation
difficult, give oxygen. Seek medical advice. If swallowed seek Refer to appropriate references for specimen collection and preparation.
medical advice immediately and show this container or label.
3. Follow proper established laboratory procedure in handling and
Test Procedure
disposing of infectious materials. See appropriate references for specific procedures.
Storage Results
Store dehydrated medium below 30°C. The dehydrated medium is very Refer to appropriate references and procedures for results.
hygroscopic. Keep container tightly closed. Limitations of the Procedure
1. Although culture techniques are primary in the identification of
Expiration Date etiological agents of mycotic infections, they are not absolute.
The expiration date applies to the product in its intact container when Often identification must be accomplished by using one or more
stored as directed. Do not use a product if it fails to meet specifications of the following techniques to corroborate cultural findings: direct
for identity and performance. microscopic examination of the specimen; animal inoculation;
biochemical determination; or serological procedures.
Procedure
2. Do not use to culture Nocardia asteroides, Streptomyces, or any
Materials Provided other microorganism sensitive to streptomycin.3
Littman Oxgall Agar
References
Materials Required but not Provided 1. Littman, M. L. 1947. Culture medium for primary isolation of
Glassware fungi. Science 106:109-111.
Autoclave 2. Littman, M. L. 1948. Growth of pathogenic fungi on a culture
Streptomycin medium. Am. J. Clin. Pathol. 18:409-420.
Method of Preparation 3. MacFaddin, J. D. 1985. Media for isolation-cultivation-
identification-maintenance of medical bacteria, vol. 1, p. 445-447.
1. Suspend 55 grams in 1 liter distilled or deionized water. Williams & Wilkins, Baltimore, MD.
2. Heat to boiling to dissolve completely.
3. Autoclave at 121°C for 15 minutes. Packaging
Littman Oxgall Agar 500 g 0294-17
4. Cool to 46°C.
5. Add 30 mcg streptomycin per ml of medium. Uninoculated Saccharomyces cerevisiae
plate ATCC® 9763
Bacto Liver Infusion Broth gram-positive organisms.3 Five percent (5%) heated horse or rabbit
serum enhances growth of Brucella.4
Liver Infusion Agar at approximately half strength may be used to
Intended Use prepare Endamoeba medium for cultivating Endamoeba histolytica.5
Bacto Liver Infusion Agar is used for cultivating Brucella and other Liver Infusion Broth maintains a degree of anaerobiosis well
pathogenic organisms. suited to support growth of anaerobic microorganisms, especially
Bacto Liver Infusion Broth is used for cultivating a variety of Clostridium species.
organisms, particularly Brucella and anaerobes.
Principles of the Procedure
Summary and Explanation Infusion from Beef Liver and Proteose Peptone provide the nitrogen, amino
Brucellosis is a zoonotic disease with a domestic animal reservoir. acids, vitamins and carbon sources in Liver Infusion media. Sodium
Transmission by milk, milk products, meat and direct contact with chloride maintains the osmotic balance. Bacto Agar is a solidifying agent.
infected animals is the usual route of exposure.1 Formula
Most strains of Brucella will grow on chocolate or blood agar. Liver Infusion Agar
However, special media such as liver infusion, tryptose, tryptone or Formula Per Liter
brucella agar are preferred.2 The nutritive factors of Liver Infusion Beef Liver, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . 500 g
media permit luxuriant growth of Brucella and other fastidious pathogens. Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
User Quality Control Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Final pH 6.9 ± 0.2 at 25°C
Identity Specifications
Liver Infusion Agar Liver Infusion Broth
Dehydrated Appearance: Dark beige to light tan, free-flowing, Formula Per Liter
homogeneous. Beef Liver, infusion from . . . . . . . . . . . . . . . . . . . . . . . . . 500 g
Solution: 5.5% solution, in distilled or Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
deionized water on boiling, medium Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
to dark amber, slightly opalescent to Final pH 6.9 ± 0.2 at 25°C
opalescent.
Prepared Medium: Medium to dark amber, slightly Precautions
opalescent. 1. For Laboratory Use.
Reaction of 5.5% 2. Brucella species are classified as Biosafety Level 3 pathogens. All
Solution at 25°C pH 6.9 ± 0.2 manipulations with live cultures and antigens must be confined to
Liver Infusion Broth a Class II biological safety cabinet (BSC).2
Dehydrated Appearance: Tan, free-flowing, homogeneous. 3. Follow proper established laboratory procedures in handling and
Solution: 3.5% solution, soluble in distilled disposing of infectious materials.
or deionized water, medium to dark
amber, clear to very slightly Storage
opalescent with a few particles. Store the dehydrated media below 30°C. The dehydrated media are
Prepared Medium: Medium to dark amber, clear to very very hygroscopic. Keep containers tightly closed.
slightly opalescent with a few particles.
Reaction of 3.5% Expiration Date
Solution at 25°C pH 6.9 ± 0.2 The expiration date applies to the product in its intact container when
stored as directed. Do not use if it fails to meet specifications for
Cultural Response identity and performance.
Prepare Liver Infusion Agar and Liver Infusion Broth per label
directions. Inoculate prepared medium and incubate under Procedure
5-10% CO2 at 35 ± 2°C for 18-48 hours, or up to 72 hours if
necessary. Incubate Clostridium under anaerobic conditions. Materials Provided
INOCULUM Liver Infusion Agar
ORGANISM ATCC® CFU GROWTH
Liver Infusion Broth
Brucella abortus 4315 100-1,000 good
Brucella melitensis 4309 100-1,000 good Materials Required But Not Provided
Brucella suis 4314 100-1,000 good Glassware
Clostridium sporogenes 11437 100-1,000 good Autoclave
The cultures listed are the minimum that should be used for Incubator (35°C)
performance testing. Waterbath (45-50°C) (optional)
Sterile Petri dishes
Cleveland and Sanders,5 and Spector6 used Loeffler Blood Serum in Expiration Date
media for the cultivation of Endamoeba histolytica. Thompson 7 The expiration date applies to the product in its intact container when
hydrolyzed Loeffler Blood Serum with sodium hydroxide and added stored as directed. Do not use a product if it fails to meet specifications
it to a citrate agar for the isolation of C. diphtheriae. On Thompson’s7 for identity and performance.
medium, growth of diphtheria bacilli was stimulated while other
respiratory flora were inhibited. Procedure
Principles of the Procedure Materials Provided
Loeffler Blood Serum
Beef Blood Serum provides the nitrogen, vitamins and amino acids
necessary to support the growth of corynebacteria in Loeffler Blood Materials Required But Not Provided
Serum. Dextrose Broth is a source of fermentable carbohydrate and Glassware
maintains the osmotic equilibrium of the medium.
Autoclave
Formula Incubator
Loeffler Blood Serum Method of Preparation
Formula Per Liter 1. Suspend 80 grams in 1 liter distilled or deionized water warmed to
Beef Blood Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 parts 42-45°C. Check pH. Adjust to pH 7.1, if necessary.
Dextrose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 part 2. Dispense into tubes having screw caps or other tightly sealing
Final pH 7.1 ± 0.2 at 25°C closures. Slant the tubes in the autoclave. Close the door loosely.
3. Coagulate the medium in constantly flowing steam for 10 minutes.
Precautions Close the door tightly.
1. For Laboratory Use.
4. Autoclave at 121°C for 15 minutes.
2. Follow proper established laboratory procedures in handling and
5. Allow autoclave pressure to fall to zero before removing tubes.
disposing of infectious materials.
Specimen Collection and Preparation
Storage Both throat and nasopharyngeal specimens are necessary in cases
Store the dehydrated medium at 2-8%C. The dehydrated medium is of respiratory illness. If cutaneous diphtheria is suspected, collect
very hygroscopic. Keep container tightly closed. skin, throat and nasopharynx specimens. Sterile silica gel is recom-
mended for shipping clinical specimens when cultures are not
obtained on site.1
User Quality Control Test Procedure1
Identity Specifications 1. If the swab appears desiccated, was collected several days prior to
Dehydrated Appearance: Medium beige, homogeneous, receipt, or is received in silica gel, place it into Todd-Hewitt Broth
free-flowing. supplemented with 3% sterile rabbit blood. Incubate the culture
8% Solution: Soluble in distilled or deionized overnight, then inoculate onto isolation media.
water warmed to 42-45°C. 2. Inoculate the specimen onto cystine tellurite blood agar and blood
Prepared Medium: Coagulated in tubes - White to cream agar plates, and streak for isolation on a Loeffler Blood Serum
colored, slightly transparent at apex slant, leaving the swab on the slant during incubation.
of slant.
3. Incubate aerobically at 35°C.
Reaction of
8.0% Solution: pH 7.1 ± 0.2 at 25°C 4. After 2-4 hours, prepare and heat fix a smear from the Loeffler
Blood Serum slant. Flood the slide with Methylene Blue, Loeffler
Cultural Response for 1 minute, rinse with tap water, and blot dry. Examine the smear
Inoculate tubes with 30-300 CFU of test organism. Incubate for morphology typical of C. diphtheriae.
18-24 hours at 35 ± 2°C. Prepare slides from the growth, heat-fix,
and stain with Methylene Blue, Loeffler. Stained cells will 5. After 18-24 hours incubation, subculture onto a second plate of
contain bipolar granules, club cells and some cells with cystine tellurite blood agar.
general granulation.
Results
ORGANISM ATCC® GROWTH
Examine all plates at 24-48 hours for colonies typical of C. diphtheriae.
Corynebacterium diphtheriae type mitis 8024 good Subculture colonies that are catalase positive and exhibit typical
Corynebacterium diphtheriae type intermedius 8032 good morphology onto blood agar to provide growth for identification
Corynebacterium diphtheriae type gravis 8028 good procedures.
The cultures listed are the minimum that should be used for Definitive identification of a C. diphtheriae isolate as a true pathogen
performance testing. requires demonstration of toxin production.8
For a complete discussion on the collection, isolation and identification Gardnerella, p. 357-377. In P.R. Murray, E.J. Baron, M.A. Pfaller,
of Corynebacterium diptheriae and other Corynebacterium species, F.C. Tenover and R.H. Yolken (ed.), Manual of clinical microbiology,
refer to the appropriate procedures. 6th ed. American Society for Microbiology, Washington, D.C.
3. Loeffler, F. 1887. Darauf theilte HeuLoeffer en einem Zweiten
Limitations of the Procedure Vortrag die ergebnisse seiner weiteren untersuchungen uber die
1. Since the nutritional requirements of organisms vary, some strains may Diphtherie-Bacillen mit. Zentralbl. Bacteriol. 2:105.
be encountered that fail to grow or grow poorly on this medium. 4. MacFaddin, J. D. 1985. Media for isolation-cultivation-
2. Loeffler Blood Serum must be used in parallel with blood agar identification-maintenance of medical bacteria, p. 448-451,
and a tellurite- containing medium (cystine tellurite agar or Williams & Wilkins, Baltimore, MD.
modified Tinsdale medium) for selection and differentiation of 5. Cleveland, L. R., and E. P. Sanders. 1930. Encystation, multiple
Corynebacterium.2 fission without encystment, metacystic development, and
3. Metachromatic granules that take up methylene blue are variation in a pure line and nine strains of Entamoeba histolytica.
characteristic of Corynebacterium; however, other microorganisms Arch. Protistenkd. 70:223.
may also display stained granules (e.g., Propionibacterium, some 6. Spector, B. K. 1932. A comparative study of cultural and
Actinomyces, and pleomorphic streptococci strains) and resemble immunological methods of diagnosing infections with Entamoeba
corynebacteria. Additional culture, biochemical identification, histolytica. J. Prevent. Med. 6:117.
and toxigenicity tests must be performed for differentiation 7. Thompson. 1929. J. Infect. Dis. 45:163.
and identification.4 8. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.
References St. Louis, MO.
1. Isenberg, H. D. (ed.) 1992. Clinical microbiology procedures
handbook. American Society for Microbiology, Washington, D.C. Packaging
2. Clarridge, J. E., and C. A. Spiegel. 1995. Corynebacterium and Loeffler Blood Serum 500 g 0070-17*
miscellaneous irregular gram-positive rods, Erysipelothrix, and *Store at 2-8°C
Prepared Lowenstein Media molecular genetic insights. Clinical Microbiology Reviews 8:496-514.
Prepared media are ready to use. 2. Kleitmann, W. 1995. Resistance and susceptibility testing for
Mycobacterium tuberculosis. Clinical Microbiology Newsletter
Specimen Collection and Preparation 17:65-69.
Refer to appropriate references for specimen collection and preparation. 3. Nolte, F. S., and B. Methcock. 1995. Mycobacterium, p. 400-437.
Test Procedure In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H.
See appropriate references for specific procedures. Yolken (ed.), Manual of clinical microbiology, 6th ed. American
Society for Microbiology, Washington, D.C.
Results 4. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures hand-
Observe for colonies that may or may not be pigmented. Colony book, vol. 1. American Society for Microbiology, Washington, D.C.
morphology depends on the species isolated. 5. Enter. Bacteriol. Parasitnek. 1932. Abt. 1, 125:222.
Limitations of the Procedure 6. J. Bact. 1965. 90:829.
Negative culture results do not rule out an active mycobacterial
infection. Some factors responsible for unsuccessful cultures are:
Packaging
Lowenstein Medium Base 500 g 0444-17
1. The specimen was not representative of the infectious material, 2 kg 0444-07
i.e., saliva instead of sputum.
Lowenstein Medium, Gruft 20 tubes 1417-39
2. The mycobacteria were destroyed during digestion and decontami- 100 ubes 1417-79
nation of the specimen.
Lowenstein Medium, Jensen 20 tubes 1017-39
3. Gross contamination interfered with the growth of mycobacteria. 100 tubes 1017-79
4. Proper aerobic and increased CO2 tension were not provided 100 x 1 oz 1017-57
during incubation. Lowenstein Medium w/5% NaCl 20 tubes 1423-39
References Lowenstein Medium, Jensen Deeps 20 tubes 1289-76
1. Musser, J. M. 1995. Antimicrobial resistance in Mycobacteria:
Cultural Response
Lowenstein Medium Base; Lowenstein Medium, Gruft Tubes;
Lowenstein Medium, Jensen Tubes
Prepare Lowenstein Medium Base (as Lowenstein Medium, Jensen) per label
directions or use prepared tubes. Inoculate and incubate at 35°C under CO2 for
up to three weeks.
ORGANISM ATCC® INOCULUM CFU RECOVERY
†
Escherichia coli 25922* 1,000-2,000 partial inhibition
Mycobacterium fortuitum 6841 100-300 good
Mycobacterium intracellulare 13950 100-300 good
Mycobacterium kansasii 12478 100-300 good
Mycobacterium scrofulaceum 19981 100-300 good
Mycobacterium tuberculosis H37Ra 25177 100-300 good
†Tested on Lowenstein Medium, Gruft, only
Lowenstein Medium w/5% NaCl
Inoculate and incubate at 35°C under CO2 for up to three weeks.
ORGANISM ATCC® INOCULUM CFU RECOVERY
Mycobacterium smegmatis 14468 100-300 good
Mycobacterium tuberculosis 25177 100-300 inhibited
Uninoculated Mycobacterium
Lowenstein Medium, Jensen Deeps tube fortuitum
ATCC® 6841
Inoculate and incubate at 35°C under CO2 for 2 weeks. Cap loosely for 7 days,
then tighten cap and incubate 1 week longer. Add 1 ml of Tween-Hydrogen
Peroxide reagent to the 2-week culture. Measure the height of the column of
bubbles after 5 minutes. (Tween-Hydrogen Peroxide reagent is a 1:1 final
solution of 30% hydrogen peroxide in distilled water and sterile, cooled 10%
Tween® 80 in distilled water.) The cultures listed are the minimum that should be
used for performance testing.
ORGANISM ATCC® INOCULUM CFU RECOVERY
*These cultures are available as Bactrol™ Disks
Mycobacterium gordonae 14470 100-300 greater than 45 mm and should be used as directed in Bactrol Disks
Mycobacterium tuberculosis 25177 100-300 less than 45 mm Technical Information.
Formula Storage
Lysine Medium Store the dehydrated medium below 30°C. The dehydrated medium is
Formula Per Liter very hygroscopic. Keep container tightly closed.
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44.5 g
Potassium Dihydrogen Phosphate . . . . . . . . . . . . . . . . . . 1.78 g Expiration Date
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.89 g The expiration date applies to the product in its intact container when
Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.178 g stored as directed. Do not use a product if it fails to meet specifications
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.089 g for identity and performance.
Adenine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.00178 g
DL-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.000891 g Procedure
L-Histidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.000891 g Materials Provided
DL-Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.000891 g
Lysine Medium
Boric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0000089 g
Zinc Sulphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0000356 g Materials Required but not Provided
Ammonium Molybdate . . . . . . . . . . . . . . . . . . . . . 0.0000178 g
Glassware
Manganese Sulphate . . . . . . . . . . . . . . . . . . . . . . . . 0.0000356 g
Ferrous Sulphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0002225 g
Petri dishes
Inositol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g Distilled or deionized water
Calcium Pantothenate . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002 g Autoclave
Aneurine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0004 g Incubator (25°C)
Pyridoxine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0004 g 10% Lactic acid solution
p-Aminobenzoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0002 g 50% Potassium lactate solution
Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0004 g
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0002 g
Method of Preparation
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.000002 g 1. Suspend 6.6 grams in 100 ml distilled or deionized water containing
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.000001 g 1 ml 50% potassium lactate solution.
L-Lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g 2. Boil gently to dissolve completely.
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.5 g 3. Cool to 50°C.
Final pH 4.8 ± 0.2 at 25°C 4. Adjust to final pH using 10% lactic acid, if necessary.
Precautions Specimen Collection and Preparation
1. For Laboratory Use. Refer to appropriate references for specimen collection and preparation.
2. Follow proper established laboratory procedures in handling and
disposing of infectious materials. Test Procedure
1. Wash and centrifuge the sample of pitching yeast three times with
distilled water.
User Quality Control
2. Resuspend the pellet in distilled water to contain approximately
Identity Specifications 107 cells per ml.
Dehydrated Appearance: Light beige, free-flowing, homogeneous. 3. Spread 0.2 ml of the cell suspension over the surface of the
Solution: 6.6% solution containing 1 ml 50% prepared medium.
potassium lactate solution per 100 ml
medium, soluble in distilled or deionized 4. Incubate plates at 25°C. Examine daily for growth.
water on gentle boiling; very light amber, Results
very slightly to slightly opalescent
without significant precipitate. Count the number of colonies that develop and express the degree of
contamination as the number of wild cells per million cells of inoculum.
Prepared Medium: Very light amber, slightly opalescent
without significant precipitate. Limitations of the Procedure
Final reaction of pH-adjusted 1. Use of a test inoculum having less than 104 cells may permit growth
6.6% solution at 25°C: pH 4.8 ± 0.2
of brewing yeast cells that can be confused with growth of wild
Cultural Response yeasts. Use of an inoculum that exceeds 104 cells will restrict
Prepare Lysine Medium per label directions. Inoculate and growth of the unwanted brewing yeasts to tiny microcolonies.2
incubate at 25 ± 2°C for 72 hours.
INOCULUM References
ORGANISM ATCC® CFU GROWTH
1. Walters, L. S., and M. R. Thiselton. 1953. J. Inst. Brew.
Pichia fermentans 10651 100-1,000 good 59:401-404.
Saccharomyces pastorianus 2700 100-1,000 none to fair
2. Morris, E. O., and A. A. Eddy. 1957. J. Inst. Brew. 63:34-35.
The cultures listed are the minimum that should be used for
performance testing. Packaging
Lysine Medium 500 g 1894-17
M17 Agar M17 Agar: Suspend 48.25 grams in 950 ml distilled or deionized
Formula Per Liter water and boil to dissolve completely.
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g 2. Autoclave at 121°C for 15 minutes.
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g 3. Cool to 50°C.
Meat Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
4. Add 50 ml sterile 10% lactose solution. Mix well.
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g Specimen Collection and Preparation
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25 g Refer to appropriate references for specimen collection and preparation.
Disodium-ß-glycerophosphate . . . . . . . . . . . . . . . . . . . . . . 19 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 g Test Procedure
Final pH 6.9 ± 0.2 at 25°C See appropriate references for specific procedures.
Precautions Results
1. For Laboratory Use. Refer to appropriate references and procedures for results.
2. Follow proper established laboratory procedures in handling and
disposing of infectious materials. References
1. Reiter, B., and J. D. Oram. 1962. Nutritional studies on cheese
Storage starters. I. vitamin and amino acid requirements of single strain
Store the dehydrated medium below 30°C. The dehydrated medium is starters. J. Dairy Res. 29:63-77.
very hygroscopic. Keep container tightly closed. 2. Lowrie and Pearce. 1971. J. Dairy Sci. Technol. 6:166.
3. Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for
Expiration Date lactic streptococci and their bacteriophages. Appl. Microbiol.
The expiration date applies to the product in its intact container when 29:807-813.
stored as directed. Do not use a product if it fails to meet specifications 4. Shankar, P. A., and F. L. Davies. 1977. A note on the suppression
for identity and performance. of Lactobacillus bulgaricus in media containing ß-glycerophosphate
and application of such media to selective isolation of Streptococcus
Procedure thermophilus from yogurt. J. Soc. Dairy Tech. 30:28-30.
Materials Provided 5. International Dairy Federation. 1981. Identification and enu-
M17 Broth or meration of micro- organisms in fermented milks. Joint IDF/ISO/
M17 Agar AOAC Group E44.
Materials Required but not Provided 6. Vedamuthu, E. R., M. Raccach, B. A. Glatz, E. W. Seitz, and
Glassware M. S. Reddy. 1992. Acid-producing microorganisms, p. 225-238.
Petri Dishes (for M17 Agar) In C. Vanderzant, and D. F. Splittstoesser (ed.), Compendium of
methods for the microbiological examination of foods, 3rd ed.
Distilled or deionized water
American Public Health Association, Washington, D.C.
Autoclave
Incubators (30°C and 35°C) Packaging
Method of Preparation M17 Broth 500 g 1856-17
1. M17 Broth: Dissolve 37.25 grams in 950 ml distilled or deionized M17 Agar 500 g 1857-17
water.
Bacto M Broth ®
Bacteriological Analytical Manual3 (BAM) and reported excellent
agreement between the two. They found the ES procedure not only to
Intended Use be faster and less complicated but also as accurate and sensitive
as the BAM procedure.
Bacto M Broth is used for cultivating Salmonella in foods and feeds
by the accelerated enrichment serology (ES) procedure. M Broth also conforms to the testing standards recommended
by Compendium of Methods for the Microbiological Examination
Summary and Explanation of Foods 4 (APHA) for the isolation and identification of
M Broth, prepared according to the formula of Sperber and Diebel1, foodborne Salmonella.
contains all the nutrients necessary for good growth and flagella Both monoclonal and polyclonal enzyme immunoassay (EIA)
development of Salmonella. methods have been described in AOAC Official Methods of Analysis5
Fantasia, Sperber and Deibel2 compared the enrichment serology (ES)‘ using M Broth. These methods are screening procedures for the presence
procedure with the traditional procedure outlined in the of Salmonella and positive results must be confirmed by culture.
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in
Bactrol Disks Technical Information. Uninoculated Salmonella choleraesuis
tube ATCC® 12011
3. Inoculate one 10 ml tube of M Broth, tempered to 35°C, with immunoassay or DNA hybridization to detect Salmonella antigen
one drop from each of the above cultures. Incubate at 35 ± 2°C in test samples.
for 6-8 hours.
4. Prepare a formalin-salt solution by adding 4.2 grams of NaCl and References
3 ml of formalin to 100 ml of distilled water. Place one drop in 1. Sperber, W. H. and R. H. Deibel. 1969. Accelerated procedure
each of two Kahn tubes. for Salmonella detection in dried foods and feeds involving
5. Carefully insert a pipette about 1 inch below the surface of the only broth cultures and serological reactions. Appl. Microbiol.
M Broth culture and transfer 0.85 ml of culture to each of the above 17:533-539.
Kahn tubes containing formalin-salt solution. 2. Fantasia, L. D., W. H. Sperber, and R. H. Deibel. 1969.
6. Prepare a pooled antiserum by combining together 0.5 ml each Comparison of two procedures for detection of Salmonella in food,
of rehydrated Salmonella H Antiserum Poly D and Salmonella H feed, and pharmaceutical products. Appl. Microbiol. 17:540-541.
Antiserum z6 (Salmonella H Antisera Spicer-Edwards Set) in 3. Bacteriological Analytical Manual, 2nd ed. 1969. US HEW,
11.5 ml of 0.85% NaCl. Washington, D.C.
7. Add 0.1 ml pooled Salmonella H Antiserum to one of the Kahn 4. Flowers, R. S., J.- Y. D’Aoust, W. H. Andrews, and J. S. Bailey.
tubes (above). Add 0.1 ml 0.85% NaCl solution to the other tube. 1992. Salmonella, p. 371-422. In C. Vanderzant, and D. F.
Shake the tubes gently. Incubate in a 50°C water bath for 1 1/2 hours. Splittstoesser (ed.). Compendium of methods for the
Results microbiological examination of foods, 3rd ed. American Public
Agglutination in the Kahn tube containing antiserum indicates the Health Association, Washington, D.C.
presence of Salmonella. Agglutination in the Kahn tube containing 5. Association of Official Analytical Chemists. 1995. Official
0.85% NaCl solution (control tube) indicates a rough culture which methods of analysis of AOAC International, 16th ed. AOAC
should be streaked for isolation, passed through Motility GI Medium International, Arlington, VA.
to enhance flagella, and then retested with pooled antiserum.
Packaging
Alternative Testing Procedures M Broth 500 g 0940-17
Refer to AOAC International5 for screening procedures using enzyme 2 kg 0940-07
2. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and 4. MacFaddin, J. F. 1985. Media for isolation-cultivation-
R. H. Yolken. (ed.). 1995. Manual of clinical microbiology, identification-maintenance of medical bacteria, vol.1. Williams
6th ed. American Society of Microbiology, Washington, D.C. & Wilkins, Baltimore, MD.
3. Baron, E. J., and S. M. Finegold. 1990. Bailey and Scott’s Packaging
Diagnostic Microbiology, 8th ed. The C. V. Mosby Co.,
MIL Medium 500 g 1804-17
St. Louis, MO.
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
Refer to appropriate references for typical motility, indole production and ornithine decarboxylase activity of various members of the Enterobacteriaceae.3,4,5,6
Precautions
1. For Laboratory Use. Uninoculated Bacillus subtilis
plate ATCC® 6633
Procedure Results
Materials Provided Consult appropriate references.4,5,6
MYP Agar
Egg Yolk Enrichment 50% References
Antimicrobic Vial P 1. Mossel, D. A. A., M. J. Koopman, and E. Jongerius. 1967.
Enumeration of Bacillus cereus in foods. Appl. Microbiol.
Materials Required but not Provided 15:650-653.
Glassware 2. Donovan, K. O. 1958. A selective medium for Bacillus cereus in
Petri dishes milk. J. Appl. Bacteriol. 21:100-103.
Distilled or deionized water
Autoclave 3. Coliner, A. R. 1948. The action of Bacillus cereus and related spe-
Incubator (35°C) cies on the lechithin complex of egg yolk. J. Bacteriol. 55:777-785.
4. Jeffery, E. J., and S. M. Harmon. 1995. Bacillus cereus, p. 14.
Method of Preparation 01-14.08. In Bacteriological analytical manual, 8th ed. AOAC
MYP Agar International, Gaithersburg, MD.
1. Suspend 46 grams of MYP Agar in 900 ml distilled or deionized water. 5. Harmon, S. M., J. M. Goepfert, and R. W. Bennett. 1992.
2. Heat to boiling to dissolve completely. Bacillus cereus, p. 593-604. In C. Vanderzant, and D. F.
3. Dispense 225 ml into 500 ml flasks. Splittstoesser (ed.), Compendium of methods for the microbiological
4. Autoclave at 121°C for 15 minutes. Cool to 45-50°C. examination of foods, 3rd ed. American Public Health Association,
5. Aseptically add 12.5 ml Egg Yolk Enrichment 50% and 4.1 ml Washington, D.C.
rehydrated Antimicrobic Vial P (25,000 units of polymyxin B). 6. Andrews, W. 1995. Microbial methods, p. 1-119. In Official methods
Mix thoroughly. of analysis of AOAC International, 16th ed. AOAC International.
Antimicrobic Vial P Arlington, VA.
1. Rehydrate with 5 ml sterile water. Packaging
Specimen Collection and Preparation MYP Agar 500 g 0810-17
Consult appropriate references.4,5,6 Antimicrobial Vial P 6 x 10 ml 3268-60*
Test Procedure * Store at 2-8°C
Consult appropriate references.4,5,6
The cultures listed are the minimum that should be used as for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in
Uninoculated Escherichia coli
Bactrol Disks Technical Information. tube ATCC® 25922
Precautions Results
1. For in Laboratory Use. Lactose-fermenting organisms grow very well in MacConkey Broth
and produce acid, causing the medium to turn yellow. Gas is also
2. IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN.
(US) Avoid contact with skin and eyes. Do not breathe dust. Wear produced. Non-fermenting organisms produce good growth but will
suitable protective clothing. Keep container tightly closed. not produce acid or gas.
FIRST AID: In case of contact with eyes, rinse immediately with References
plenty of water and seek medical advice. After contact with skin,
1. MacConkey, A. 1901. Centr. Bakt. 29:740.
wash immediately with plenty of water. If inhaled, remove to fresh
air. If not breathing, give artificial respiration. If breathing is 2. MacConkey, A. 1905. Lactose-fermenting bacteria in faeces.
difficult, give oxygen. Seek medical advice. If swallowed seek J. Hyg. 5:333-379.
medical advice immediately and show this container or label. 3. MacConkey, A. 1908. Bile salt media and their advantage in some
3. Follow proper, established laboratory procedure in handling and bateriological examinations. J. Hyg. 8:322-334.
disposing of infectious materials. 4. Childs, E., and L. A. Allen. 1953. Improved methods for
determining the most probable number of Bacterium coli and of
Storage Streptococcus faecalis. J. Hyg. Camb. 51:468-477.
Store MacConkey Broth below 30°C. The powder is very hygoscopic.
Keep container tightly closed. Packaging
Store prepared medium at 2-8°C. MacConkey Broth 500 g 0020-17
Expiration Date
The expiration date applies to the product in its intact container when
stored as directed. Do not use a product if it fails to meet specifications
for identity and performance.
MacConkey Media
Bacto MacConkey Agar . Bacto MacConkey Agar Base
®
Also Known As
User Quality Control MacConkey Agar is also known as MAC.
Identity Specifications Summary and Explanation
MacConkey Agar MacConkey Agar is based on the bile salt-neutral red-lactose agar of
Dehydrated Appearance: Pink to pinkish beige, free-flowing,
homogenous. MacConkey.8
Solution: 5.0% solution, soluble in distilled or The original MacConkey medium was used to differentiate strains of
deionized water on boiling; reddish Salmonella typhosa from members of the coliform group. Formula
purple, very slightly to slightly modifications improved the growth of Shigella and Salmonella strains.
opalescent. These modifications included the addition of 0.5% sodium chloride,
Prepared Plates: Pinkish red, slightly opalescent. decreased agar content, and altered bile salts and neutral red concen-
Reaction of 5.0% trations. The formula improvements gave improved differential
Solution at 25°C: pH 7.1 ± 0.2 reactions between these enteric pathogens and the coliform group.
MacConkey Agar Base MacConkey Agar contains crystal violet and bile salts that inhibit
Dehydrated Appearance: Pink to pinkish beige, free-flowing, gram-positive organisms and allow gram-negative organisms to grow.
homogenous. Isolated colonies of coliform bacteria are brick red in color and may be
Solution: 4.0% solution, soluble in distilled or surrounded by a zone of precipitated bile. This bile precipitate is due
deionized water upon boiling; red, to a local pH drop around the colony due to lactose fermentation.
very slightly to slightly opalescent Colonies that do not ferment lactose (such as typhoid, paratyphoid and
without significant precipitate. dysentery bacilli) remain colorless. When lactose non-fermenters grow
Prepared Plates: Red, slightly opalescent without in proximity to coliform colonies, the surrounding medium appears as
precipitate. cleared areas.
Reaction of 4.0% MacConkey Agar Base is prepared without added carbohydrates,
Solution at 25°C: pH 7.1 ± 0.2 which permits their addition either individually or in combination. It is
MacConkey Agar CS recommended that carbohydrates such as sucrose or lactose be added
Dehydrated Appearance: Pinkish beige, homogenous, free-flowing. in a concentration of 1% to the basal medium.
Solution: 5.0% solution, soluble in distilled or MacConkey CS (“Controlled Swarming”) contains carefully selected
deionized water on boiling; reddish raw materials to reduce the swarming of Proteus species which could
purple in color, slightly opalescent, cause difficulty in isolating and enumerating other gram-negative
without significant precipitate.
bacilli.
Prepared Plates: Reddish purple, slightly opalescent,
without precipitate. MacConkey Agar w/o CV (Crystal Violet) is a differential medium
that is less selective than MacConkey Agar. The lack of crystal violet
Reaction of 5.0%
Solution at 25°C: pH 7.1 ± 0.2 permits the growth of Staphylococcus and Enterococcus. Staphylococci
continued on following page
produce pale pink to red colonies and enterococci produce compact
tiny red colonies either on or beneath the surface of the medium.
MacConkey Agar w/o Salt is a differential medium that restricts Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
the swarming of Proteus species to aid in the detection and isolation Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
of enteric microorganisms. In addition, this medium does not contain Bacto Bile Salts, No. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g
crystal violet, allowing Staphyloccocus and Enterococcus species Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
to grow. Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5 g
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03 g
Principles of the Procedure Bacto Crystal Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001 g
Bacto Peptone and Proteose Peptone are sources of nitrogen and Final pH 7.1 ± 0.2 at 25°C
other nutrients. Lactose is a fermentable carbohydrate. When lactose MacConkey Agar Base
is fermented, a local pH drop around the colony causes a color change Formula Per Liter
in the pH indicator (neutral red) and bile precipitation. Bile Salts, Bile Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 g
Salts No. 3 and Crystal Violet are selective agents that inhibit growth Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
of gram-positive organisms. Bacto Agar is a solidifying agent. Bacto Bile Salts, No. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Formula Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5 g
MacConkey Agar Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03 g
Formula Per Liter Bacto Crystal Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001 g
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 g Final pH 7.1 ± 0.2 at 25°C
MacConkey Agar CS
User Quality Control cont.
Formula Per Liter
MacConkey Agar w/o CV Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 g
Dehydrated Appearance: Pinkish beige, free-flowing, Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
homogenous. Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Solution: 5.2% solution, soluble in distilled Bacto Bile Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
or deionized water upon boiling; Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
reddish orange, clear to very slightly Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5 g
opalescent without significant
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03 g
precipitate.
Bacto Crystal Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001 g
Prepared Plates: Reddish orange, slightly opalescent
without significant precipitate. Final pH 7.1 ± 0.2 at 25°C
Reaction of 5.2% MacConkey Agar w/o CV
Solution at 25°C: pH 7.4 ± 0.2 Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
MacConkey Agar w/o Salt
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Dehydrated Appearance: Pinkish beige, free-flowing,
homogenous. Bacto Bile Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Solution: 4.7% solution, soluble in distilled
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g
or deionized water upon boiling;
reddish orange, slightly opalescent. Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 g
Prepared Plates: Reddish orange, slightly opalescent. Final pH 7.4 ± 0.2 at 25°C
Reaction of 4.7% MacConkey Agar w/o Salt
Solution at 25°C: pH 7.4 ± 0.2 Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Cultural Response Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Prepare MacConkey media per label directions. Inoculate and Bacto Bile Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
incubate at 35 ± 2°C for 18-24 hours. Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.075 g
MacConkey Agar Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 g
INOCULUM BILE
ORGANISM ATCC® CFU GROWTH APPEARANCE PPT. Final pH 7.4 ± 0.2 at 25°C
Enterococcus 29212* 1,000-2,000 markedly to – –
faecalis completely inhibited Precautions
Escherichia 25922* 100-1,000 good pink + 1. For Laboratory Use.
coli
2. For MacConkey Agar w/o CV
Proteus 12453 100-1,000 good colorless –
mirabilis IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM
Salmonella 14028* 100-1,000 good colorless –
AND SKIN. Avoid contact with skin and eyes. Do not breathe dust.
typhimurium Wear suitable protective clothing. Keep container tightly closed.
continued on following page
MacConkey Agar w/o Salt plenty of water and seek medical advice. After contact with skin,
IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM wash immediately with plenty of water. If inhaled, remove to fresh
AND SKIN. Avoid contact with skin and eyes. Do not breathe dust. air. If not breathing, give artificial respiration. If breathing is
Wear suitable protective clothing. Keep container tightly closed.
FIRST AID: In case of contact with eyes, rinse immediately with Uninoculated Escherichia coli
plate ATCC® 25922
difficult, give oxygen. Seek medical advice. If swallowed seek 5. Cool to 45-50°C and dispense into sterile Petri dishes.
medical advice immediately and show this container or label. 6. The surface of the medium should be dry when inoculated. Dry the
3. Follow proper established laboratory procedure in handling and plates for 1-2 hours with the lids slightly ajar.
disposing of infectious materials.
Specimen Collection and Preparation
Storage For a complete discussion on the isolation and identification of enteric
Store the dehydrated medium below 30°C. The dehydrated medium is organisms consult the appropriate references.
very hygroscopic. Keep container tightly closed.
Test Procedure
Expiration Date For procedures on the isolation and identification of enteric organisms
The expiration date applies to the product in its intact container when consult the appropriate references.
stored as directed. Do not use a product if it fails to meet specifications
for identity and performance. Results
Lactose-fermenting organisms grow as pink to brick-red colonies
Procedure with or with out a zone of precipitated bile. Non-lactose fermenting
Materials Provided organisms grow as colorless or clear colonies.
MacConkey Agar Swarming by Proteus spp. is reduced on MacConkey Agar CS and
MacConkey Agar Base MacConkey Agar w/o Salt.
MacConkey Agar CS On MacConkey Agar w/o CV and MacConkey Agar w/o Salt, staphy-
MacConkey Agar w/o CV lococci produce pale pink to red colonies and enterococci produce
MacConkey Agar w/o Salt tiny red colonies; these organisms are inhibited on MacConkey Agar
and MacConkey Agar CS.
Materials Required But Not Provided
Glassware Limitations of the Procedure
Autoclave 1. Although MacConkey media are selective primarily for gram-
35°C incubator negative enteric bacilli, biochemical and, if indicated, serological
50°C waterbath (optional) testing using pure cultures are recommended for complete
Carbohydrate (lactose, sucrose, etc.) (optional) identification. Consult appropriate references for further
information. 1,3
Method of Preparation
2. Due to the selective properties of MacConkey Agar CS, some
For MacConkey Agar, MacConkey Agar CS, MacConkey Agar strains of gram-negative enteric bacilli may be encountered that
w/o CV or MacConkey Agar w/o Salt: fail to grow or grow poorly on this medium. Some strains of gram-
1. Suspend the medium in 1 liter distilled or deionized water: positive organisms may be encountered that are not inhibited
MacConkey Agar 50 grams or only partially inhibited on this medium; some strains of
MacConkey Agar CS 50 grams enterococci may grow on MacConkey Agar CS after prolonged
MacConkey Agar w/o CV 52 grams incubation.
MacConkey Agar w/o Salt 47 grams 3. Incubation of MacConkey Agar plates under increased CO2 has
been reported to reduce the growth and recovery of a number of
2. Heat to boiling to dissolve completely. Avoid overheating.
strains of gram-negative bacilli.9
3. Autoclave at 121°C for 15 minutes. The media may be used with-
out autoclave sterilization if the plates are to be inoculated on the 4. For optimal performance, plates prepared from MacConkey Agar
day of preparation. CS should be incubated under aerobic conditions.
4. Cool to 45-50°C and dispense into sterile Petri dishes. References
5. The surface of the medium should be dry when inoculated. Dry the 1. Gray, L. D. 1995. Escherichia, Salmonella, Shigella and
plates for 1-2 hours with the lids slightly ajar. Yersinia, p. 450-456. In P. R. Murray, E. J. Baron, M. A, Pfaller,
For MacConkey Agar Base: F. C. Tenover, and R. H Yolken (ed.), Manual of clinical
1. Suspend 40 grams of medium in 1 liter distilled or deionized water. microbiology, 6th ed. American Society for Microbiology,
Washington, D.C.
2. Heat to boiling to dissolve completely. Avoid overheating.
2. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig
3. Add 10 grams lactose or other desired carbohydrate before or after
(H.M. Wehr, Tech. Comm.). 1992. Pathogens in milk and milk
sterilization, depending on heat lability.
products, p. 103-212. In R. T. Marshall, (ed.). Standard methods
4. Autoclave at 121°C for 15 minutes. The media may be used with- for the examination of dairy products. 16th ed., American Public
out autoclave sterilization if the plates are to be inoculated on the Health Association, Washington, D.C.
day of preparation. In this case, boiling the medium gently for 5
minutes is sufficient.
3. Hitchins, A. D., P. A. Hartman, and E. C. D. Todd. 1992. 9. Mazura-Reetz, G. T. Neblett, and J. M. Galperin. 1979.
Coliforms-Escherichia coli and its Toxins, p. 325-369. In C. MacConkey Agar: CO2 vs. ambient incubation. Abst. Ann. Mtg.
Vanderzant, and D. F. Splittstoesser (ed.), Compendium of methods American Society for Microbiology. C179.
for the microbiological examination of foods, 3rd ed. American
Public Health Association, Washington. D.C. Packaging
4. Food and Drug Administration. 1995. Bacteriological analytical MacConkey Agar 100 g 0075-15-3
manual, 8th ed. AOAC International. Gaithersburg, MD. 500 g 0075-17-1
5. Eaton, A. D., L. S. Clesceri, and A.E. Greenberg (ed.). 1995. 2 kg 0075-07-3
Standard methods for the examination of water and wastewater, 10 kg 0075-08-2
19th ed. American Public Health Association, Washington, D.C. MacConkey Agar Base 500 g 0818-17-3
6. United States Pharmacopeial Convention, Inc. 1995. The United MacConkey Agar CS 500 g 1818-17-1
States pharmacopeia, 23rd ed. The United States Pharmacopeial 2 kg 1818-07-3
Convention. Rockville, MD. 10 kg 1818-08-2
7. Association of Official Analytical Chemists. 1995. Official
MacConkey Agar w/o CV 500 g 0470-17-2
methods of analysis of AOAC International, 16th ed. AOAC
International, Arlington, VA. MacConkey Agar w/o Salt 500 g 0331-17-1
8. MacConkey, A. 1905. Lactose-fermenting bacteria in feces. 10 kg 0331-08-2
J. Hyg. 5:333-379.
MacConkey Sorbitol Agar is a modification of the formula given by 5. Dry plates for 1-2 hours with the lids slightly ajar. The surface of
Rappaport and Henig1 for isolating enteropathogenic Escherichia coli the medium should be dry when inoculated.
serotypes 011 and 055. The usefulness of this medium in detecting MacConkey Sorbitol Agar may be used without autoclave
E. coli 0157:H7, a human pathogen associated with hemorrhagic colitis, sterilization if the plates are to be used on the day of preparation.
has been described.2,3,4 Boil the medium 2-3 minutes before pouring into Petri dishes and
This medium employs d-sorbitol rather than lactose for isolating and dry before inoculation.
differentiating the enteropathogenic E. coli serotypes which tend to be
Specimen Collection and Preparation
sorbitol negative. This medium can be used for clinical and food testing.1,5,6
1. Collect specimens in sterile containers or with sterile swabs
Principles of the Procedure and immediately transport to the laboratory in accordance with
Bacto Peptone and Proteose Peptone are nitrogen sources in the recommended guidelines.
medium. D-Sorbitol is a fermentable carbohydrate. Many hemorrhagic 2. Process each specimen as appropriate for that specimen.
E. coli strains will not ferment d-sorbitol and appear as colorless 3. Inoculate the specimen onto medium appropriate for that specimen.
colonies on MacConkey Sorbitol Agar. Bile salts and crystal violet are 4. Incubate plates for 18-24 hours at 35 ± 2°C.
selective agents that inhibit growth of gram-positive organisms.
5. Examine plates.
Neutral red is a pH indicator. Bacto Agar is a gelling agent.
Test Procedure
Formula See appropriate references for specific procedures.
MacConkey Sorbitol Agar
Formula Per Liter Results
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.5 g Sorbitol-fermenting organisms produce pink colonies on MacConkey
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g Sorbitol Agar. Organisms that do not ferment sorbitol, such as
d-Sorbitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g E. coli 0157:H7, are colorless.
Bacto Bile Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Limitations of the Procedure
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g 1. The color of sorbitol-positive colonies can fade, making them hard
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03 g to distinguish from sorbitol-negative colonies.3
Bacto Crystal Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001 g
2. Upon prolonged incubation, strains of E. coli 0157:H7 can ferment
Final pH 7.1 ± 0.2 at 25°C sorbitol.3
Precautions 3. Strains of other organisms that do not ferment sorbitol may grow
on MacConkey Sorbitol Agar. It is necessary to select suspected
1. For Laboratory Use.
colonies for further identification.3
2. Follow proper, established laboratory procedure in handling and
4. The sole use of this medium can cause the microbiologist to miss
disposing of infectious materials.
other organisms that may be pathogenic.7
Storage 5. To isolate E. coli 0157:H7 from clinical specimens, inoculate fecal
Store the dehydrated medium below 30°C. The dehydrated medium is specimens and rectal swabs on a small area of one quadrant and
very hygroscopic. Keep container tightly closed. streak for isolation. This will permit development of discrete colonies.
6. Hitchins, A. D., P. Feng, W. D. Watkins, S. R. Rippey, and L. A. identification of Escherichia coli serotypes associated with cases
Chandler. 1995. Escherichia coli and the coliform bacteria. of diarrhea of the newborn. Public Health Lab. 12:75-81.
p. 4.01-4.29. In Bacteriological analytical manual, 8th ed. AOAC
International, Gaithersburg, MD. Packaging
7. Ewing, W. H., and P. R. Edwards. 1954. Isolation and preliminary MacConkey Sorbitol Agar 500 g 0079-17
The cultures listed are the minimum that should be used for
performance testing.
*These cultures are available as Bactrol™ Disks and should be
used as directed in Bactrol Disks Technical Information. Uninoculated Enterobacter aerogenes Escherichia coli
tube ATCC® 13048 ATCC® 25922
Storage Results
Store the dehydrated medium below 30°C. The dehydrated medium is Malonate utilization is indicated by a change in the color of the medium
very hygroscopic. Keep container tightly closed. from green to blue:
Positive: Blue
Expiration Date Negative: Green
The expiration date applies to the product in its intact container. Do not
use a product if it fails to meet specifications for identity and performance. Limitations of the Procedure
1. A slight bluing (blue-green) of the medium may occur after prolonged
Procedure incubation.6 In such cases, care should be taken in interpreting results.
Materials Provided
Malonate Broth References
1. Leifson, E. 1933. The fermentation of sodium malonate as a means
Materials Required But Not Provided of differentiating Aerobacter and Escherichia. J. Bacteriol. 26: 329.
Glassware 2. Bacteriological Analytical Manual. 1995. 8th ed. AOAC
Autoclave International. Gaithersburg, MD.
Incubator (35°C) 3. Vanderzant, C., and D. F. Splittstoesser (eds.). 1992. Compen-
Method of Preparation dium of methods for the microbiological examination of foods,
1. Dissolve 8 grams in 1 liter distilled or deionized water. 3rd ed. American Public Health Association, Washington, D.C.
2. Autoclave at 121°C for 15 minutes. 4. Marshall, R. T. (ed.). 1993. Standard methods for the examination
of dairy products, 16th ed. American Public Health Association,
3. Avoid introducing extraneous carbon and nitrogen.
Washington, D.C.
Specimen Collection 5. Edwards, P. R., and W. H. Ewing. 1962. Enterobacteriaceae. U.S.
Refer to appropriate references for specimen collection and preparation. Public Health Service Bulletin No. 734:19.
Test Procedure 6. Oberhofer, T. R. 1985. Manual of nonfermenting gram-negative
1. Inoculate tubes with a loopful of test organism. bacteria. Churchill Livingstone, New York, NY.
2. Incubate at 35 ± 2°C for 18-48 hours. Packaging
3. Examine tubes for a change in the color of the medium from Malonate Broth 100 g 0395-15
green to blue. 500 g 0395-17
Intended Use
User Quality Control Bacto Malonate Broth Modified is used for differentiating
Identity Specifications Enterobacteriaceae based on malonate utilization.
Dehydrated Appearance: Beige, homogeneous, free-flowing.
Solution: 0.93% solution, soluble in distilled Also Known As
or deionized water with agitation. Malonate Broth Modified conforms with Malonate Broth, Ewing.
Solution is green, clear.
Reaction of 0.93% Summary and Explanation
Solution at 25°C: pH 6.7 ± 0.2
Malonate Broth Modified is essentially Malonate Broth to which Yeast
Cultural Response Extract and Dextrose have been added.1 These additional ingredients
Prepare Malonate Broth Modified per label directions. initiate growth of some organisms that otherwise would fail to grow on
Inoculate the medium with a loopful of undiluted organism
and incubate at 35 ± 2°C for 18-48 hours. the unmodified medium and, thus, permit observation of those
COLOR OF organisms’ malonate activity.
ORGANISM ATCC® INOCULUM MEDIUM
Enterobacter aerogenes 13048* undiluted blue Malonate utilization by microorganisms is indicated by an increase
Escherichia coli 25922* undiluted green in alkalinity and development of a deep blue color in the medium.
Salmonella arizonae 13314 undiluted blue Malonate utilization forms a basis on which organisms can be
Salmonella typhimurium 14028* undiluted green differentiated when testing food products for Enterobacteriaceae.2,3,4
It is useful in the differentiation of Escherichia coli from the
The cultures listed are the minimum that should be used for
performance testing. Klebsiella-Enterobacter groups and is considered especially valuable
*These cultures are available as Bactrol™ Disks and should in the differentiation of Salmonella. The majority of salmonellae do
be used as directed in Bactrol Disks Technical Information. not utilize malonate whereas Salmonella arizonae does.4,5,6
Test Procedure 3. Fulmer, E. I., and M. J. Grimes. 1923. The growth of yeasts on
See appropriate references for specific procedures. synthetic agar media. Bacteriol., 8:585-588.
4. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compen-
Results
dium of methods for the microbiological examination of foods,
Refer to appropriate references and procedures for results. 3rd ed. American Public Health Association, Washington, D.C.
Limitations of the Procedure 5. Association of Official Agricultural Chemists. 1995. Official
1. Do not heat the medium after addition of acid, as this will methods of analysis, 16th ed. Association of Official Agricultural
hydrolyze the agar and reduce its solidifying properties. Chemists, Washington, D.C.
References Packaging
1. Abs. Bact., 3:6, 1919. Malt Agar 500 g 0024-17
2. Thom, C., and M. B. Church. 1926. The Aspergilli. Williams and 10 kg 0024-08
Wilkins Co., Baltimore, MD.
The cultures listed are the minimum that should be used. Results
Refer to appropriate references and procedures for results.
Procedure References
Materials Provided 1. Abs. Bact., 3:6, 1919.
Malt Extract Agar 2. Thom, C., and M. B. Church. 1926. The Aspergilli. Williams and
Malt Extract Broth Wilkins Co., Baltimore.
3. MacFaddin, J. 1985. Media for isolation-cultivation-
Materials Required But Not Provided identification-maintenance of medical bacteria, vol. 1. Williams
Glassware and Wilkins, Baltimore.
Autoclave
4. Association of Official Analytical Chemists. 1995. Bacteriological
Incubator
analytical manual, 8th ed. AOAC International. Gaithersburg, MD.
Method of Preparation
1. Malt Extract Agar: Suspend 33.6 grams in 1 liter of distilled or Packaging
deionized water. Heat to boiling to dissolve completely. Malt Extract Agar 500 g 0112-17
Malt Extract Broth: Dissolve 15 grams in 1 liter of distilled or 10 kg 0112-08
deionized water. Malt Extract Broth 500 g 0113-17
2. Autoclave at 121°C for 15 minutes. Avoid overheating which could 10 kg 0113-08
cause a softer medium.
Procedure Results
Materials Provided Staphylococci will grow on this medium while the growth of most other
bacteria will be inhibited. Coagulase-positive staphylococci will
Mannitol Salt Agar
produce luxuriant growth of yellow colonies with yellow zones around
Materials Required but not Provided them. Coagulase negative staphylococci will produce small red
Glassware colonies with no color change to the medium surrounding them.
Petri dishes
Distilled or deionized water
References
Autoclave 1. Chapman, G. H. The significance of sodium chloride in studies of
staphylococci. J. Bacteriol. 50:201.
Incubator (35°C)
2. Kloos, W. E., and T. L. Bannerman. 1995. Staphylococcus and
Method of Preparation Micrococcus. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
1. Suspend 111 grams in 1 liter distilled or deionized water. Tenover, and R. H. Yolken (ed.). Manual of clinical microbiology,
2. Heat to boiling to dissolve completely. 6th ed. American Society for Microbiology, Washington, D.C.
3. Autoclave at 121°C for 15 minutes. 3. Hitchins, A. D., T. T. Tran, and J. E. McCarron. 1995.
Microbiology methods for cosmetics, p. 23.01-23.12. In
Specimen Collection and Preparation Bacteriological analytical manual, 8th ed. AOAC International,
1. Collect specimens as appropriate for the specimen and transport Gaithersburg, MD.
immediately to the laboratory in accordance with recommended 4. United States Pharmacopeial Convention. 1995. The United
guidelines. States pharmacopeia, 23rd ed. The United States Pharmacopeial
2. Process each specimen as appropriate for that specimen. Convention. Rockville, MD.
Staphylococcus 25923* 100-1,000 good yellow colony The cultures listed are the minimum that should be used for
aureus with yellow zone performance testing.
Staphylococcus 12228* 100-1,000 good red colony with no *These cultures are available as Bactrol™ Disks and should
epidermidis zone of color change be used as directed in Bactrol Disks Technical Information.
Cultural Response Prepare Marine Broth 2216 per label directions. Dispense 50 ml
Prepare Marine Agar 2216 per label directions. Inoculate and amounts in 250 ml Erlenmeyer flasks. Inoculate and incubate at
incubate at 20-25°C for 40-72 hours. 20-25°C on a shaker for 40-72 hours.
ORGANISM ATCC® INOCULUM CFU GROWTH ORGANISM ATCC® INOCULUM CFU GROWTH
Vibrio fischeri 7744 100-1,000 good Vibrio fischeri 7744 100-1,000 good
Vibrio harveyi 14126 100-1,000 good Vibrio harveyi 14126 100-1,000 good
The cultures listed are the minimum that should be used for performance testing.
2. Gunter, S. E. 1954. Factors determining the viability of selected value of adding peptone to diluents used in the bacteriological
microorganisms in inorganic media. J. Bacteriol. 67:628-634. testing of bacon curing brines. J. Appl. Bacteriol. 26:493-497.
3. Straka, R. P., and J. L. Stokes. 1957. Rapid destruction of bacteria
in commonly used diluents and its eliminations. Appl. Microbiol. Packaging
5:21-25. Maximum Recovery Diluent 500 g 1897-17
4. Patterson, J. T., and J. A. Cassells. 1963. An examination of the 5 kg 1897-03
Intended Use from foods. With the addition of 50% egg yolk emulsion, C. perfringens
Bacto McClung Toabe Agar Base is used with Bacto Egg Yolk and a few other Clostridium species show the lecithinase reaction.
Enrichment 50% for isolating and detecting Clostridium perfringens C. perfringens is found in raw meats, poultry, dehydrated soups and
in foods based on the lecithinase reaction. sauces, raw vegetables and other foods and food ingredients, but
occurrences of food borne illness are usually associated with cooked
Summary and Explanation meat or poultry products.2 Spores of some strains that may resist heat
McClung and Toabe1 formulated a medium for isolating C. perfringens during cooking germinate and grow in foods that are not adequately
refrigerated.3 Enumerating the microorganism in food samples plays a
role in epidemiological investigation of outbreaks of food borne illness.2
User Quality Control Principles of the Procedure
Identity Specifications McClung Toabe Agar Base contains Proteose Peptone as a source of
McClung Toabe Agar Base carbon, nitrogen, vitamins and minerals. Dextrose is the carbohydrate
Dehydrated Appearance: Very light beige, free-flowing, source. Sodium Chloride maintains the osmotic balance of the medium.
homogeneous. Magnesium Sulfate provides divalent cations and sulfate. Sodium
Solution: 7.5% solution, soluble in distilled or Phosphate Dibasic and Potassium Phosphate Monobasic maintain
deionized water on boiling. Solution pH balance and provide a source of phosphates. Bacto Agar is the
is light amber, opalescent, with a solidifying agent. Egg Yolk Enrichment 50% provides egg yolk
precipitate. lecithin. Lecithinase-producing clostridia, such as C. perfringens,
Prepared Medium: Light yellow, smooth, opaque. hydrolyze the lecithin and produce opaque halos.
Reaction of 7.5%
Solution at 25°C: pH 7.6 ± 0.2 Formula
Egg Yolk Enrichment 50% McClung Toabe Agar Base
Appearance: Canary yellow, opaque liquid with Formula Per Liter
a resuspendable precipitate. Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
Cultural Response Sodium Phosphate Dibasic . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
McClung Toabe Agar Base with Egg Yolk Enrichment 50% Potassium Phosphate Monobasic . . . . . . . . . . . . . . . . . . . . . 1 g
Prepare McClung Toabe Agar Base with Egg Yolk Enrichment Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
50% per label directions. Inoculate and incubate the plates at Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g
35 ± 2°C anaerobically for 18-48 hours. Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 g
INOCULUM LECITHINASE Final pH 7.6 ± 0.2 at 25°C
ORGANISM ATCC® CFU GROWTH REACTION
Clostridium 12919 100-1,000 good opaque halo Egg Yolk Enrichment 50%
perfringens
Sterile concentrated egg yolk emulsion
Clostridium 12924 100-1,000 good opaque halo
perfringens Precautions
Staphylococcus 25923* 100-1,000 good opaque halo
aureus 1. For Laboratory Use.
Staphylococcus 14990 100-1,000 good none 2. Follow proper established laboratory procedures in handling and
epidermidis disposing of infectious materials.
The cultures listed are the minimum that should be used for Storage
performance testing.
*These cultures are available as Bactrol™ Disks and should be Store the dehydrated McClung Toabe Agar Base medium below
used as directed in Bactrol Disks Technical Information. 30°C. The dehydrated medium is very hygroscopic. Keep container
tightly closed.
Store the Egg Yolk Enrichment 50% at 2-8°C. Egg Yolk Enrichment 50%
1. Ready for use.
Expiration Date 2. Shake gently to resuspend precipitate.
The expiration date applies to the product in its intact container when
stored as directed. Do not use a product if it fails to meet specifications Specimen Collection and Preparation
for identity and performance. Refer to appropriate references for specimen collection and preparation.
The MR and VP tests are used to complete and confirm the identification by the mixed acid pathway and produce acidic end products (pH < 4.4),
of Escherichia coli. 4,5,6 such as lactic, acetic and formic acids. Other bacteria metabolize
pyruvate by the butylene glycol pathway and produce neutral end
Principles of the Procedure products (pH > 6.0), one of which is acetoin (acetylmethylcarbinol). In
MR-VP Medium contains Buffered Peptone as a carbon and nitrogen the MR test, the pH indicator methyl red detects acidic end products.7
source for general growth requirements. Dextrose is a fermentable In the VP test, acetoin is oxidized in the presence of oxygen and
carbohydrate. potassium hydroxide (KOH) to diacetyl, which produces a red color.8
Members of the Enterobacteriaceae convert glucose to pyruvate by
the Embden-Meyerhof pathway. Some bacteria metabolize pyruvate
The cultures listed are the minimum that should be used for
performance testing.
*These cultures are available as Bactrol™ Disks and should be
used as directed in Bactrol Disks Technical Information.
Alternate VP Tube Method 7. VP reagents must be added in the order and the amounts specified
1. Inoculate MR-VP Medium with the test organism and incubate at or a weak-positive or false-negative reaction may occur. A weak-
35°C for 24-48 hours. positive reaction may be masked by a copper-like color which may
2. Aseptically transfer 1 ml of the incubated MR-VP Medium culture form due to the reaction of KOH and α-naphthol.8
to a clean test tube. 8. Read the VP test within 1 hour of adding the reagents. The KOH
3. Add 15 drops of Voges-Proskauer Reagent A followed by 5 drops and α-naphthol may react to form a copper-like color, causing a
of Voges-Proskauer Reagent B. potential false-positive interpretation.8
4. Shake gently to aerate. 9. Due to the possible presence of acetoin, diacetyl or related
substances in certain raw materials,9 the use of media low in
5. Examine for the appearance of a red color within 20 minutes. these substances (such as MR-VP Medium) is recommended for
6. If the 24-hour test is negative, repeat the test with a 48-hour culture this test.
of the test organism. If equivocal results are obtained, repeat the
test with cultures incubated for 5 days at 25-30°C. References
Rapid Micro Method 1. Clark, W. M., and H. A. Lubs. 1915. The differentiation of bacteria
1. Inoculate 0.2 ml of MR-VP Medium with the test organism. of the colon- aerogenes family by the use of indicators. J. Infect.
Dis. 17:160-173.
2. Incubate for 4 hours at 35°C.
2. Voges, O., and B. Proskauer. 1898. Z. Hyg. 28:20-22.
3. Add 0.1 ml of 0.3% creatine solution.
3. Farmer, J. J., III. 1995. Enterobacteriaceae: Introduction and
4. Add 5 drops of Voges-Proskauer Reagent A followed by 2 drops of
identification, p. 438-449. In P. R. Murray, E. J. Baron, M. A.
Voges-Proskauer Reagent B.
Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical
5. Shake gently to aerate. microbiology, 6th ed. American Society for Microbiology,
6. Examine for the appearance of a red color within 20 minutes. Washington, D.C.
Results 4. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compen-
dium of methods for the microbiological examination of foods,
Methyl Red (MR) Test 3rd ed. American Public Health Association, Washington, D.C.
Positive: Bright red color.
5. Marshall, R. T. (ed.). 1993. Standard methods for the microbio-
Negative: Yellow-orange color. logical examination of dairy products, 16th ed. American Public
Note: If the test is negative, continue to incubate the broth without Health Association, Washington, D.C.
added reagent; repeat the test after an additional 18 to 24 hours incubation. 6 Association of Official Analytical Chemists. 1995. Bacterio-
Voges-Proskauer (VP) Test logical analytical manual, 8th ed. AOAC International,
Positive: Red color. Gaithersburg, MD.
Negative: No red color. 7. Isenberg, H. D. (ed.). 1994. Clinical microbiology procedures
handbook, sup. 1, 1.19.48. American Society for Microbiology,
Limitations of the Procedure Washington, D.C.
1. Results of the MR and VP tests need to be used in conjunction with 8. Isenberg, H. D. (ed.). 1994. Clinical microbiology procedures
other biochemical tests to differentiate genus and species within handbook, sup. 1, 1.19.58. American Society for Microbiology,
the Enterobacteriaceae. Washington, D.C.
2. A precipitate may form in the potassium hydroxide reagent solution. 9. Barritt, M. M. 1936. The intensification of the Voges-Proskauer
This precipitate has not been shown to reduce the effectiveness of reaction by the addition of alpha-naphthol. J. Pathol. 42:441-454.
the reagent.
3. Most members of the family Enterobacteriaceae give either a positive Packaging
MR test or a positive VP test. However, certain organisms such as MR-VP Medium 100 g 0016-15
Hafnia alvei and Proteus mirabilis may give a positive result for 500 g 0016-17
both tests. 2 kg 0016-07
4. Incubation time for the Methyl Red test cannot be shortened by SpotTest Voges-Proskauer
increasing the glucose concentration in the medium or by heavily Reagent A 50 x 0.75 ml 3558-26
inoculating the broth.7
SpotTest Voges-Proskauer
5. Incubate MR-negative tests for more than 48 hours and test again. Reagent B 50 x 0.75 ml 3559-26
(See Results section.)
6. Read the VP test at 48 hours. Increased incubation may produce
acid conditions in the broth that will interfere with reading the
results.8
Bacto Micro Inoculum Broth 2. Inoculum Media, which condition the test culture for immediate use; and,
3. Assay Media, which permit quantitation of the vitamin under test.
Assay media contain all factors necessary for optimal growth of
Intended Use the test organism except the single essential vitamin to be determined.
Bacto Micro Assay Culture Agar is used for cultivating lactobacilli
and other organisms used in microbiological assays. Micro Assay Culture Agar is used for maintaining stock cultures of
lactobacilli and other test microorganisms. This medium is also used
Bacto Micro Inoculum Broth is used for preparing the inoculum of for general cultivation of lactobacilli.
lactobacilli and other microorganisms used in microbiological assays
Micro Inoculum Broth is used for cultivating lactobacilli and
of vitamins and amino acids.
preparing the inoculum for microbiological assays.
Summary and Explanation Principles of the Procedure
Three types of media are used in the microbiological assay of vitamins:
Proteose Peptone No. 3 provides nitrogen and amino acids in both Micro
1. Maintenance Media, which preserve the viability and sensitivity of Assay Culture Agar and Micro Inoculum Broth. Yeast Extract is a vitamin
the test culture for its intended purpose; source. Dextrose is a carbon source. Monopotassium phosphate is a
buffering agent. Sorbitan monooleate complex (Micro Inoculum
User Quality Control Broth) and Polysorbate 80 (Micro Assay Culture Agar) act as emulsifiers.
Bacto Agar is a solidifying agent (Micro Assay Culture Agar).
Identity Specifications
Micro Assay Culture Agar Formula
Dehydrated Appearance: Light tan to tan, free-flowing, Micro Assay Culture Agar
homogeneous. Formula Per Liter
Solution: 4.7% solution, soluble in distilled or Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . . 5 g
deionized water on boiling; light to Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
medium amber, very slightly to Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
slightly opalescent without Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
significant precipitate. Polysorbate 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g
Prepared Medium: Light to medium amber, slightly Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
opalescent. Final pH 6.7 ± 0.2 at 25°C
Reaction of 4.7%
Solution at 25°C: pH 6.7 ± 0.2 Micro Inoculum Broth
Formula Per Liter
Micro Inoculum Broth
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . . 5 g
Dehydrated Appearance: Beige, homogeneous, free-flowing.
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Solution: 3.7% solution, soluble in distilled or Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
deionized water. Light to medium Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
amber in color, clear to very slightly Sorbitan Monooleate Complex . . . . . . . . . . . . . . . . . . . . . 0.1 g
opalescent without significant
precipitate. Final pH 6.7 ± 0.2 at 25°C
Prepared Medium: Light to medium amber, clear to Precautions
very slightly opalescent.
1. For Laboratory Use.
Reaction of 3.7%
Solution at 25°C: pH 6.7 ± 0.2 2. Follow proper established laboratory procedures in handling and
disposing of infectious materials.
Cultural Response 3. Take care to avoid contamination of media and glassware used
Prepare Micro Assay Culture Agar and Micro Inoculum Broth in microbiological assay procedures. Extremely small amounts
per label directions. Inoculate tubes with test organisms. of foreign material may be sufficient to give erroneous results.
Incubate Micro Assay Culture Agar at 35 ± 2°C for 18-48 hours,
incubate Micro Inoculum Broth at 35 ± 2°C for 18-24 hours. Scrupulously clean glassware free from detergents and other
INOCULUM
chemicals must be used.
ORGANISM ATCC® CFU GROWTH
Enterococcus hirae 804 100-1,000 good Storage
Lactobacillus casei subsp. rhamnosus 7469 100-1,000 good Store the dehydrated media below 30°C. The media are very
Lactobacillus delbrueckii subsp. lactis 7830 100-1,000 good hygroscopic. Keep containers tightly closed.
Lactobacillus plantarum 8014 100-1,000 good
Expiration Date
The cultures listed are the minimum that should be used for The expiration date applies to the product in its intact container when
performance testing.
stored as directed. Do not use a product if it fails to meet specifications
for identity and performance.
Procedure 6. Dilute the washed suspension 1:100 with sterile 0.9% single
strength basal assay medium or as indicated. Where applicable,
Materials Provided
adjust inoculum concentration according to limits specified in
Micro Assay Culture Agar
AOAC1 or US Pharmacopeia.2
Micro Inoculum Broth
Specimen Collection and Preparation
Materials Required But Not Provided Prepare samples for assay according to references given in the specific
Glassware assay procedure. Dilute assay samples to approximately the same
Autoclave concentration as the standard solution.
Incubator
Inoculating needle Test Procedure
0.9% NaCl For a complete discussion of vitamin assay methodology, refer to
appropriate procedures.1,2
Method of Preparation
Micro Assay Culture Agar Results
1. Suspend 47 grams in 1 liter distilled or deionized water. For test results on vitamin assay procedures, refer to appropriate
procedures.1,2
2. Heat to boiling to dissolve.
3. Dispense 10 ml amounts into 16-20 mm diameter tubes. Limitations of the Procedure
4. Autoclave at 121°C for 15 minutes. 1. Test organisms used in assay procedures must be cultured and
5. Agitate tubes prior to solidification to disperse the flocculent maintained on media recommended for this purpose.
precipitate. 2. Follow assay directions exactly. The age, preparation and size of
Micro Inoculum Broth inoculum are extremely important factors in obtaining a satisfactory
assay result.
1. Dissolve 37 grams in 1 liter distilled or deionized water.
3. Although other media and methods may be used successfully for
2. Dispense 10 ml amounts into tubes of 16-20 mm diameter.
maintaining cultures and preparing inocula, uniformly good results
3. Autoclave at 121°C for 15 minutes. will be obtained if the methods described are followed exactly.
Stock Cultures 4. Aseptic technique should be used throughout the microbiological
1. Prepare stock cultures in triplicate on Micro Assay Culture Agar, assay procedure.
inoculating tubes using a straight-wire inoculating needle. 5. The use of altered or deficient media may create mutants
2. Incubate tubes at 30-37°C for 18-24 hours. having different nutritional requirements. Such organisms will not
3. Store at 2-8°C. produce a satisfactory test response.
4. Transfer cultures at weekly or twice-monthly intervals. References
Assay Inoculum 1. Association of Official Analytical Chemists. 1995. Official methods
1. Subculture from a 16-24 hour stock culture of lactobacilli in Micro of analysis of AOAC International, 16th ed. AOAC International,
Assay Culture Agar into a 10 ml tube of Micro Inoculum Broth. Arlington, VA.
2. Incubate at 35-37°C for 16-24 hours or as specified in the assay 2. The United States Pharmacopeial Convention. 1995. The United
procedure. States pharmacopeia, 23rd ed. The United States Pharmacopeial
3. Centrifuge the culture and decant the supernatant. Convention Inc. Rockville, MD.
4. Resuspend cells in 10 ml of sterile 0.9% NaCl solution or sterile Packaging
single strength basal assay medium. Micro Assay Culture Agar 100 g 0319-15
5. Wash the cells by centrifuging and decanting the supernatant two 500 g 0319-17
additional times unless otherwise indicated.
Micro Inoculum Broth 500 g 0320-17
common terms for Microbial Content Test Agar. Tween 80® is also
Intended Use known as Polysorbate 80.
Bacto Microbial Content Test Agar is recommended for the detection
of microorganisms on surfaces sanitized with quaternary ammonium Summary and Explanation
compounds. Microbial Content Test Agar is a modification of Tryptic Soy Agar
with Lecithin and Tween 80. The formulation is recommended for
Also Known as determining the sanitation efficiency of containers, equipment and work
“Tryptic Soy Agar with Lecithin and Polysorbate 80” (TSALT) and areas (environmental monitoring). The Lecithin and Tween in the
“Casein Soy Peptone Agar with Polysorbate 80 and Lecithin” are formula inactivate some preservatives that may inhibit bacterial
Agar-base media:
User Quality Control cont.
1. Tend not to liquefy in the presence of contaminating proteolytic
Cultural Response organisms;3
Middlebrook 7H9 Broth with Middlebrook ADC Enrichment 2. Are recommended for specimens from nonsterile sites7 because
Prepare medium per label directions. Inoculate and incubate at colonies of mycobacteria can be viewed in a clear medium after
35 ± 2°C under approximately 10% CO2 for up to 21 days. 10-12 days incubation using a stereo microscope even if contami-
INOCULUM
ORGANISM ATCC® CFU GROWTH nating organisms are present;
Mycobacterium fortuitum 6841 100-300 good 3. Retain exact concentrations of added drugs because the medium is
Mycobacterium intracellulare 13950 100-300 good solidified with agar rather than by inspissation of the egg. Also,
Mycobacterium kansasii 12478 100-300 good there is less drug inactivation when egg ingredients are absent.
Mycobacterium tuberculosis 25177 100-300 good Middlebrook 7H10 Agar is prepared according to Middlebrook, Cohn,
Mycobacterium scrofulaceum 19981 100-300 good Dye, Russell and Levy.4 This medium contains a low concentration of
Middlebrook 7H10 Agar with Middlebrook OADC Enrichment malachite green, which may be preferable for primary isolation.
Mycobacteria 7H11 Agar with Middlebrook OADC Enrichment Mycobacteria 7H11 Agar is a modification of Middlebrook 7H10 Agar
Prepare medium per label directions or use prepared tubes. Special as recommended by Cohn, Waggoner and McClately.5 Cohn
Inoculate and incubate at 35 ± 2°C under approximately 10% et al. demonstrated that the addition of an enzymatic digest of casein
CO2 for up to 21 days. stimulates growth of the more fastidious strains of Mycobacterium
INOCULUM
ORGANISM ATCC® CFU GROWTH tuberculosis and provides improved susceptibility testing.
Escherichia coli 25922* 1,000-2,000 markedly
inhibited Principles of the Procedure
Mycobacterium fortuitum 6841 100-300 good L-Glutamic Acid, Ammonium Sulfate, Biotin, Sodium Citrate and
Mycobacterium intracellulare 13950 100-300 good Pyridoxine supply growth factors. Magnesium Sulfate, Ferric Ammo-
Mycobacterium kansasii 12478 100-300 good nium Sulfate, Zinc Sulfate and Copper Sulfate are sources of trace ions.
Mycobacterium scrofulaceum 25177 100-300 good Disodium Phosphate and Monopotassium Phosphate help to maintain
Mycobacterium tuberculosis 19981 100-300 good
the pH of the medium. Malachite Green inhibits contaminating
Middlebrook 7H10 Agar with Middlebrook OADC Enrichment organisms. Pancreatic Digest of Casein, a good source of nitrogen and
w/WR 1339 carbon, increases the recovery of isoniazid-resistant mycobacteria
Prepare medium per label directions. Inoculate and incubate at on Mycobacteria 7H11 Agar. Glycerol enhances the growth of
35 ± 2°C under approximately 10% CO2 for up to three weeks.
INOCULUM
Mycobacterium avium as well as other Mycobacterium species.7 Bacto
ORGANISM ATCC® CFU GROWTH Agar is a solidifying agent.
Mycobacterium tuberculosis 27294 100-1,000 good Middlebrook OADC and ADC Enrichments contain Dextrose and Oleic
The cultures listed are the minimum that should be used for Acid as carbon sources. Albumin Fraction V, Bovine and Catalase (Beef)
performance testing. are growth factors. WR 1339, Triton® encourages the demonstration
*This culture is available as a Bactrol™ Disk and should be used of cording in Mycobacterium tuberculosis.8
as directed in Bactrol Disks Technical Information.
Formula
Middlebrook 7H9 Broth
Formula Per Liter
Ammonium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
L-Glutamic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g
Pyridoxine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001 g
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0005 g
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . 0.04 g
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g
Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0005 g
Zinc Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001 g
Copper Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001 g
Final pH 6.6 ± 0.2 at 25%C
Middlebrook 7H10 Agar
Formula Per Liter
Ammonium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
Uninoculated Mycobacterium fortuitum Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g
tube ATCC® 6841 Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g
Mycobacteria 7H11 Agar 2. Kleitmann, W. 1995. Resistance and susceptibility testing for
1. Suspend 21 grams in 900 ml distilled or deionized water containing Mycobacterium tuberculosis. Clin. Microbiol. News. 17:65-69.
5 ml glycerol. 3. Nolte, F. S., and B. Methcock. 1995. Mycobacterium, p. 400-437.
2. Boil to dissolve completely. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H.
Yolken (ed.). Manual of clinical microbiology, 6th ed. American
3. Autoclave at 121°C for 15 minutes. Society for Microbiology, Washington, D.C.
4. Cool medium to 50-55°C. 4. Middlebrook, G., M. L. Cohn, W. B. Dye, W. B. Russell, Jr.,
5. Aseptically add 100 ml Middlebrook OADC Enrichment. Mix well. and D. Levy. 1960. Microbiologic procedures of value in
Specimen Collection and Preparation7 tuberculosis. Acta. Tubercul. Scand., 38:66.
5. Cohn, M. L., R. F. Waggoner, and J. K. McClatchy. 1968. The
1. Collect specimens in sterile containers and transport immediately
7H11 Medium for the cultivation of mycobacteria. Am. Rev.
to the laboratory following recommended guidelines.
Resp. Dis., 98:295.
2. Process each specimen as appropriate for that specimen.
6. Tenover, F. C., J. T. Crawford, R. E. Huebner, L. J. Geiter,
Test Procedure C. R. Horsburgh, Jr., and R. C. Good. 1993. The resurgence
1. Inoculate the specimen onto the medium. of tuberculosis: is your laboratory ready? J. Clin. Microbiol.
31:767-770.
2 Incubate tubes for up to eight weeks.
7. Isenberg, H. D. (ed.). 1994. Clinical microbiology procedures hand-
3 Examine tubes for growth. book, sup. 1. American Society for Microbiology, Washington, D.C.
Results 8. Am. Rev. Resp. Dis., 97:1133.
Observe for colonies that may or may not be pigmented. Colony Packaging
morphology is dependent on the species isolated.
Middlebrook 7H9 Broth 500 g 0713-17
Limitations of the Procedure Middlebrook 7H10 Agar 500 g 0627-17
Negative culture results do not rule out active infection by mycobacteria. 20 tubes 0627-39
Some factors responsible for unsuccessful cultures are: 100 tubes 0627-79
1. The specimen was not representative of the infectious material, Mycobacteria 7H11 Agar 500 g 0838-17
i.e., saliva instead of sputum; 20 tubes 0838-39
2. The mycobacteria were destroyed during digestion and decontami- 100 tubes 0838-79
nation of the specimen; Middlebrook ADC Enrichment 2 x 20 ml 0714-64
3. Gross contamination interfered with the growth of the mycobacteria; Middlebrook OADC Enrichment 12 x 20 ml 0722-64
4. Proper aerobic and increased CO2 tension were not provided 6 x 100 ml 0722-73
during incubation.
Middlebrook OADC Enrichment
References w/ WR 1339 6 x 20 ml 0801-63
1. Musser, J. M. 1995. Antimicrobial resistance in Mycobacteria: Glycerol 100 g 0282-15
molecular genetic insights. Clin. Microbiol. Rev. 8:496-514. 500 g 0282-17
Proteolytic bacteria will be surrounded by a clear zone from the Materials Required but not Provided
conversion of casein into soluble nitrogenous compounds.1 Flasks with closures
Distilled or deionized water
Formula Hot plate
Milk Agar Autoclave
Formula Per Liter Dilution tubes containing 1/4-strength Ringer’s solution
Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Petri dishes
Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g 1% hydrochloric acid or 10% acetic acid
Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Skim Milk Powder (antibiotic free) . . . . . . . . . . . . . . . . . . . 1 g Method of Preparation
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 g
1. Suspend 22 grams in 1 liter distilled or deionized water and boil
Final pH 6.9 ± 0.1 at 25°C gently to dissolve completely.
Precautions 2. Dispense 10-12 ml per tube. Cap loosely.
1. For In Laboratory Use. 3. Autoclave at 121°C for 15 minutes.
2. Follow proper established laboratory procedures in handling and 4. Pour Milk Agar into Petri dishes for the spread plate technique or
disposing of infectious materials. allow tubes to cool to 45°C for the pour plate technique.
Specimen Collection and Preparation
Storage
Refer to appropriate references for specimen collection and preparation.
Store the dehydrated medium below 30°C. The dehydrated medium is
very hygroscopic. Keep container tightly closed. Test Procedure
Total counts may be carried out using either pour plates or surface
Expiration Date
counting techniques.
The expiration date applies to the product in its intact container when
1. Prepare milk dilutions of 1/10, 1/100, 1/1,000 in 1/4-strength
stored as directed. Do not use a product if it fails to meet specifications
Ringer’s solution. Use this inoculum within 15 minutes.
for identity and performance.
2. Pour Plates: Pipette 1 ml of each dilution into Petri dishes.
Procedure Add 10-12 ml of molten Milk Agar, cooled to 45°C, and mix
thoroughly.
Materials Provided
Spread Plates: Spread 1 ml of milk dilution over the surface of
Milk Agar
the solidified medium in a Petri dish.
3. Incubate at 30°C for 72 hours.
5. Abbiss, J. S., J. M. Wilson, R. M. Blood, and B. Jarvis. 1981. A 7. Departments of the Environment, Health & Social Security,
comparison of Minerals Modified Glutamate Medium with other and P.H.L.S. 1982. The Bacteriological Examination of Drinking
media for the enumeration of coliforms in delicatessen foods. Water Supplies, Report on Public Health and Medical Subjects
J. Appl. Bact. 51:121-127. No. 71. HMSO, London.
6. Holbrook, R., J. M. Anderson, and A. C. Baird-Parker. 1980.
Modified Direct Plate Method for counting Escherichia coli in
Packaging
foods. Food Technol. in Aust. 32:78- 83. Minerals Modified Glutamate Broth 500 g 1850-17
4. Resuspend the cells in minimal medium and centrifuge. Delayed Enrichment Method
5. Decant the supernatant and resuspend the pellet in minimal Mutant colonies will grow as small colonies after the addition of the
medium to give a cell concentration of about 2 x 108 cells per ml. complete medium which diffuses through the Minimal Agar.
6. Irradiate the suspension with a low pressure mercury ultraviolet lamp Penicillin Method
for a sufficient time to give a cell survival of 1 x 104 cells per ml.
Mutant colonies grow after the addition of penicillin.
7. Incubate the suspension at room temperature for 4-18 hours in the
minimal medium with appropriate substances added to allow for B. subtilis Method
the growth of desired mutants. Mutant colonies grow on Nutrient Agar after the addition of penicillin.
8. Wash the culture in sterile minimal medium.
Limitations of the Procedure
9. Centrifuge and resuspend in the same medium.
1. Strains vary in their sensitivity to penicillin. Adjustments to
10. Dilute 1 to 10 with sterile minimal medium. the time of treatment and concentration of penicillin may be
11. Let stand for 60 minutes to starve the mutants. necessary.1
12. Add penicillin to give a concentration of 2,000 units per ml.
13. Incubate 15 minutes. References
14. Plate the culture on nutrient agar for colony isolation. 1. Lederberg, J. 1950. Isolation and characterization of biochemical
mutants of bacteria. Methods in Med. Res. 3:5-21.
15. Identify the nutrition mutants by transferring colonies by replicate
plating onto plates of minimal agar which has been supplemented 2. Davis. 1949. Proc. Nat’l Acad. Sci. 35:1.
with the appropriate nutritional substances. 3. Nester, Schafer, and Lederberg. 1963. Genetics 48:529.
Results Packaging
Random Technique Minimal Agar Davis 500 g 0544-17
Growth in the nutritionally complete medium and no growth in the Minimal Broth Davis w/o Dextrose 500 g 0756-17
Minimal Broth indicates a mutant.
Wear suitable protective clothing. Keep container tightly closed. Distilled or deionized water
FIRST AID: In case of contact with eyes, rinse immediately with Autoclave
plenty of water and seek medical advice. After contact with skin, Incubator (35°C)
wash immediately with plenty of water. If inhaled, remove to fresh Method of Preparation
air. If not breathing, give artificial respiration. If breathing is diffi-
1. Suspend 90 grams of Mitis Salivarius Agar in 1 liter distilled or
cult, give oxygen. Seek medical advice. If swallowed seek medical
deionized water.
advice immediately and show this container or label.
2. Heat to boiling to dissolve completely.
3. Follow proper established laboratory procedures in handling and
disposing of infectious materials. 3. Autoclave at 121°C for 15 minutes. Cool to 50-55°C.
4. Just prior to dispensing, add 1 ml Chapman Tellurite Solution 1%.
Storage 5. DO NOT HEAT THE COMPLETE MEDIUM.
Store the dehydrated medium below 30°C. The dehydrated medium is
very hygroscopic. Keep container tightly closed. Specimen Collection and Preparation
Store Chapman Tellurite Solution 1% at 15-30°C. Collect specimens according to recommended guidelines.
2. Molds will grow on the medium after two days incubation. 3. Chapman, G. H. 1944. The isolation of streptococci from mixed
3. Erysipelothrix rhusiopathiae produces colorless, circular, convex cultures. J. Bacteriol. 48:113.
colonies. 4. Chapman, G. H. 1946. The isolation and testing of fecal
4. Beta-hemolytic streptococci produce colonies that resemble S. mitis. streptococci. Am. J. Dig. Dis. 13:105.
5. Chapman, G. H. 1947. Relationship of nonhemolytic and
References viridans streptococci in man. Trans. N.Y. Acad. Sci. (Series 2) 10:45.
1. Facklam, R. R., and J. A. Washington II. 1991. Streptococcus
and related catalase-negative gram-positive cocci. p. 238-257. In 6. MacFaddin, J. F. 1985. Media for isolation-cultivation-
A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and identification-maintenance of medical bacteria, vol. 1, p. 522-526.
H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed. Williams & Wilkins, Baltimore, MD.
American Society for Microbiology. Washington, D.C.
Packaging
2. Facklam, R.R., and D. F. Sahm. 1995. Enterococcus, p. 308-314.
Mitis Salivarius Agar 500 g 0298-17
In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and
R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed. Chapman Tellurite Solution 1% 6 x 1 ml 0299-51
American Society for Microbiology, Washington, D.C. 6 x 25 ml 0299-66
Bacto Bile Salts No. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.12 g Materials Required But Not Provided
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
MacConkey Sorbitol Agar
Potassium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . . . 4 g
EMB Agar
Potassium Phosphate, Monobasic . . . . . . . . . . . . . . . . . . . 1.5 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Flasks with closures
Sterile distilled or deionized water
Final pH 6.9 ± 0.2 at 25°C
Autoclave
Novobiocin Antimicrobic Supplement Incubator (35°C)
Formula per 10 ml vial Incubator (42°C)
Sodium Novobiocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg Butterfield’s Phosphate Diluent
Phenol Red Sorbitol Agar with MUG
Precautions
1. For Laboratory Use. Method of Preparation
2. Modified EC Medium Rehydrate Novobiocin Antimicrobic Supplement with 10 ml sterile
IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN. distilled or deionized water.
(US) Avoid contact with skin and eyes. Do not breathe dust. Wear 1. Dissolve 36.6 grams Modified EC Medium in 1 liter of distilled or
suitable protective clothing. Keep container tightly closed. deionized water.
FIRST AID: In case of contact with eyes, rinse immediately with 2. Autoclave at 121°C for 15 minutes.
plenty of water and seek medical advice. After contact with skin, 3. Cool to room temperature.
wash immediately with plenty of water. If inhaled, remove to fresh 4. Aseptically add 10 ml rehydrated supplement to 1 liter sterile basal
air. If not breathing, give artificial respiration. If breathing is medium. Mix well.
difficult, give oxygen. Seek medical advice. If swallowed, seek
medical advice immediately and show this container or label. Specimen Collection and Preparation
Novobiocin Antimicrobic Supplement 1. Collect food samples in sterile containers and transport immediately
to the laboratory following recommended guidelines.
HARMFUL. HARMFUL BY INHALATION AND IF SWAL-
LOWED. (EC) MAY CAUSE ALLERGIC EYE, RESPIRATORY 2. Process each food sample using procedures appropriate for that
SYSTEM AND SKIN REACTION. Avoid contact with skin and sample.
eyes. Do not breathe dust. Wear suitable protective clothing. Keep Test Procedure
container tightly closed.
Many procedures and systems have been described for the use of
FIRST AID: In case of contact with eyes, rinse immediately with Modified EC Medium with Novobiocin in the selective and differential
plenty of water and seek medical advice. After contact with skin, enrichment of E. coli O157:H7 in meat and poultry samples. Please
wash immediately with plenty of water. If inhaled, remove to fresh consult appropriate references. 4-6 Listed below is the USDA’s
air. If not breathing, give artificial respiration. If breathing is recommended procedure for the enrichment and detection of E. coli
difficult, give oxygen. Seek medical advice. If swallowed, seek O157:H7 in meat and poultry samples using Modified EC Medium
medical advice immediately and show container or label. with Novobiocin.2-4
3. Follow proper, established laboratory procedures in handling and 1. Inoculate 25 grams of meat sample into 225 ml of Modified EC
disposing of infectious materials. Medium with Novobiocin in a stomacher bag. Blend or stomach as
Storage required (i.e., 2 minutes) for thorough mixing.
1. Store Modified EC Medium below 30°C. The dehydrated medium 2. Incubate at 35°C for 24 hours.
is very hygroscopic. Keep container tightly closed. 3. Dilute cultures 10-fold in Butterfield’s Phosphate Diluent and
2. Store Novobiocin Antimicrobic Supplement at 2-8°C. inoculate 0.1 ml of appropriate dilutions using a spread plate
technique onto MacConkey Sorbitol Agar (MSA) and MacConkey
3. Store prepared medium at 2-8°C. Sorbitol Agar with BCIG (MSA-BCIG) agar plates.
Expiration Date 4. Incubate plates at 42°C for 24 hours.
The expiration date applies to the product in its intact container when 5. Examine MSA plates for sorbitol-negative colonies (white)
stored as directed. Do not use a product if it fails to meet specifications and MSA-BCIG plates for sorbitol-negative, BCIG-negative
for identity and performance. colonies (white).
6. Subculture sorbitol-negative colonies to respective plates of EMB
Procedure Agar and Phenol Red Sorbitol Agar containing MUG (PRS-MUG).
Materials Provided 7. Incubate EMB and PRS-MUG Agar plates at 35°C for 18-24 hours.
Modified EC Medium Examine plates for sorbitol fermentation, MUG reaction
Novobiocin Antimicrobic Supplement (fluorescence), and typical E. coli growth on EMB Agar.
References Packaging
1. Okrend, A. J. G., and B. E. Rose. 1989. Isolation and identification Modified EC Medium 500 g 0340-17
of E. coli O157:H7 from meat. USDA Food Safety Inspection Novobiocin Antimicrobic Supplement 6x10 ml 3197-60*
Service. Rev. 3 of Laboratory Communication no. 38. E. coli O157:H7.
*Store at 2-8°C.
20 December 1989. U.S. Department of Agriculture, Washington, D.C.
Intended Use Listeria species grow over a pH range of 5.0-9.6 and survive in food
Bacto Modified Listeria Enrichment Broth is used for selectively products with pH levels outside these parameters.8 Listeria spp. are
enriching Listeria from raw and pasteurized milk according to the microaerophilic, gram-positive, asporogenous, non-encapsulated,
International Dairy Federation.1 non-branching, regular, short, motile rods. Motility is most pronounced
at 20°C. Many common food contaminants such as streptococci,
Summary and Explanation enterococci, Bacillus species, Escherichia coli, Pseudomonas
First described in 1926 by Murray, Webb and Swann, 2 Listeria aeruginosa and Proteus vulgaris interfere with the isolation of
monocytogenes is a widespread problem in public health and the food Listeria monocytogenes.9
industries. This organism can cause human illness and death, particularly Listeria Enrichment Broth is based on the formula developed by Lovett
in immunocompromised individuals and pregnant women.3 The first et al.10 in which Tryptic Soy Broth was supplemented with yeast
reported food-borne outbreak of listeriosis was in 1985.4 Since then, extract for optimum growth of Listeria. Modified Listeria Enrichment
microbiological and epidemiological evidence from both sporadic and Broth is a modification of Listeria Enrichment Broth in which the
epidemic cases of listeriosis has shown that the principal route of acriflavine content has been reduced from 15 mg to 10 mg per liter.
transmission is via the consumption of foodstuffs contaminated with This modification reflects the lower concentration specified by the
Listeria monocytogenes.5 International Dairy Federation1 for isolation of L. monocytogenes from
Implicated vehicles of transmission include turkey frankfurters,6 milk and milk products.
coleslaw, pasteurized milk, Mexican-style cheese, paté and pickled Identification of Listeria is based on successful isolation of the
pork tongue. The organism has been isolated from commercial dairy organism, biochemical characterization and serological confirmation.
and other food processing plants. It is ubiquitous in nature, being
present in a wide range of unprocessed foods and in soil, sewage, Principles of the Procedure
silage and river water.7 Modified Listeria Enrichment Broth contains Tryptone, Soytone and
Yeast Extract as nitrogen and vitamin sources. Dextrose provides an
energy source. Sodium Chloride maintains the osmotic balance of the
User Quality Control medium. Potassium Phosphate is a buffering agent. Cycloheximide is
incorporated to inhibit saprophytic fungi, while Nalidixic Acid inhibits
Identity Specifications
growth of gram-negative organisms. Acriflavine HCl is added at 10 mg
Dehydrated Appearance: Light beige, free flowing, homogeneous.
per liter to suppress growth of gram-positive bacteria.
Solution: 3.61% solution, soluble in distilled or
deionized water on boiling. Solution Formula
is light to medium yellowish-amber
with a faint green ring at the surface, Modified Listeria Enrichment Broth
clear to very slightly opalescent. Formula Per Liter
Prepared Tubes: Light yellowish-amber, clear to very Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 g
slightly opalescent. Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Reaction of 3.61% Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Solution at 25°C: pH 7.3 ± 0.2 Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Potassium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Cultural Response Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 g
Prepare Modified Listeria Enrichment Broth per label directions. Cycloheximide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g
Inoculate and incubate at 30 ± 2°C for 18-48 hours. Acriflavine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01 g
INOCULUM Nalidixic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.04 g
ORGANISM ATCC® CFU GROWTH
Enterococcus faecalis 29212* 2,000-10,000 partially suppressed Final pH 7.3 ± 0.2 at 25°C
at 18- 24 hours
Escherichia coli 25922* 2,000-10,000 marked to Precautions
complete inhibition 1. For Laboratory Use.
Listeria monocytogenes 19114 100-1,000 good 2. TOXIC. HARMFUL BY INHALATION AND IF SWALLOWED.
Saccharomyces 9080 2,000-10,000 marked to (EC) MAY CAUSE CANCER. POSSIBLE RISK OF HARM TO
pastorianus complete inhibition
THE UNBORN CHILD. Do not breathe dust. In case of accident
The cultures listed are the minimum that should be used for or if you feel unwell, seek medical advice immediately. (Show
performance. label where possible.) Wear suitable protective clothing. Keep
*These cultures are available as Bactrol™ Disks and should be container tightly closed. TARGET ORGAN(S): Blood, Cardiovas-
used as directed in Bactrol Disks Technical Information. cular, Face, Lungs, Nerves, Skin, Thorax.
FIRST AID: In case of contact with eyes, rinse immediately with Limitations of the Procedure
plenty of water and seek medical advice. After contact with skin, 1. Since the nutritional requirements of organisms vary, some strains
wash immediately with plenty of water. If inhaled, remove to fresh of Listeria may be encountered that fail to grow or grow poorly on
air. If not breathing, give artificial respiration. If breathing is diffi- this medium.
cult, give oxygen. Seek medical advice. If swallowed, induce 2. Modified Listeria Enrichment Broth is a partially selective medium.
vomiting; seek medical advice immediately and show this container Growth of some contaminating strains will be markedly but not
or label. totally inhibited.
3. Follow proper, established laboratory procedures in handling and
disposing of infectious materials. References
1. International Dairy Federation. 1990. Milk and milk products -
Storage detection of Listeria monocytogenes. IDF Provisional International
Store the dehydrated medium below 30°C. The dehydrated medium is Standard No. 143. International Dairy Federation, Brussels.
very hygroscopic. Keep container tightly closed. Store the prepared 2. Murray, E. G. D., R. A. Webb, and M. B. R. Swann. 1926.
medium at 2-8°C. A disease of rabbits characterized by large mononuclear
leucocytosis caused by a hitherto undescribed bacillus Bacterium
Expiration Date monocytogenes (n. sp.). J. Path. Bact., 29:407- 439.
The expiration date applies to the product in its intact container when
3. Monk, J. D., R. S. Clavero, L. R. Beuchat, M. P. Doyle, and
stored as directed. Do not use a product if it fails to meet specifications
R. E. Brackett. 1994. Irradiation inactivation of Listeria
for identity and performance.
monocytogenes and Staphylococcus aureus in low- and high-fat,
Procedure frozen and refrigerated ground beef. J. Food Prot. 57:969-974.
Materials Provided 4. Wehr, H. M. 1987. Listeria monocytogenes - a current dilemma
Special Report. J. Assoc. Off. Anal. Chem. 70:769-772.
Modified Listeria Enrichment Broth
5. Bremer, P. J., and C. M. Osborne. 1995. Thermal-death times of
Materials Required But Not Provided Listeria monocytogenes in green shell mussels (Perna canaliculus)
Flasks with closures prepared for hot smoking. J. Food Prot. 58:604-608.
Distilled or deionized water 6. Grau, F. H., and P. B. Vanderlinde. 1992. Occurrence, numbers,
Bunsen burner or magnetic hot plate and growth of Listeria monocytogenes on some vacuum-packaged
Test tubes with closures processed meats. J. Food Prot. 55:4-7.
Autoclave 7. Patel, J. R., C. A. Hwang, L. R. Beuchat, M. P. Doyle, and
Incubator (30°C) R. E. Brackett. 1995. Comparison of oxygen scavengers for their
ability to enhance resuscitation of heat-injured Listeria
Method of Preparation monocytogenes. J. Food Prot. 58:244-250.
1. Suspend 36.1 grams in 1 liter distilled or deionized water.
8. Donnelly, C. W., R. E. Bracket, D. Doores, W. H. Lee, and
2. Heat to boiling to dissolve completely. J. Lovett. 1992. Listeria, p. 637-663. In C. Vanderzant and D. F.
3. Autoclave at 121°C for 15 minutes. Splittstoesser (ed.), Compendium of methods for the microbiological
Specimen Collection and Preparation examination of foods, 3rd ed. American Public Health Association,
Washington, D.C.
1. Collect samples in sterile containers or with sterile swabs and
transport immediately to the laboratory following recommended 9. Kramer, P. A., and D. Jones. 1969. Media selective for Listeria
guidelines.1 monocytogenes. J. Appl. Bacteriol. 32:381-394.
2. For specific information about sample preparation and inoculation, 10. Lovett, J., D. W. Frances, and J. M. Hunt. 1987. Listeria
consult appropriate reference.1 monocytogenes in raw milk: detection, incidence and pathogenicity.
J. Food Prot. 50:188-192.
Test Procedure 11. Swaminathan, B., J. Rocourt, and J. Bille. 1995. Listeria,
For dairy samples, the IDF1 selective enrichment method is as follows: p. 342-343. In P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover,
1. Add 25 ml liquid or 25 grams of solid test material to 225 ml and R. H. Yolken (ed.). Manual of clinical microbiology, 6th ed.
Modified Listeria Enrichment Broth and mix or blend thoroughly. American Society for Microbiology, Washington, D.C.
2. Incubate for 48 hours at 30°C.1 12. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig.
3. At 48 hours, streak the Modified Listeria Enrichment Broth 1993. Pathogens in milk and milk products. In R. T. Marshall (ed.).
culture onto plates of Oxford Medium or Palcam Medium. Standard methods for the examination of dairy products, 16th ed.
4. Incubate the agar plates at 37°C for 48 ± 2 hrs. American Public Health Association, Washington, D.C.
Results Packaging
1. Examine agar plates for typical Listeria colonies. Modified Listeria Enrichment Broth 500 g 0205-17
2. Consult appropriate references for selection of biochemical or 10 kg 0205-08
serological tests for confirmation of Listeria sp.1,8,11,12
Specimen Collection and Preparation 3. Some isolates of Yersinia enterocolitica demonstrate motility at 35°C
Refer to appropriate references for specimen collection and preparation. while others may be nonmotile at 25°C.2 The motility of Proteus is
also temperature dependent. This effect of temperature on motility
Test Procedure needs to be taken into account when deciding on a testing regimen.
1. Inoculate with growth from an 18-24 hour pure culture. 4. Due to the temperature dependency of motility in some organisms,
2. If tubes are used, inoculate by stab inoculation. If plates are used, spot a negative test tube or plate should be incubated an additional
the inoculum on the surface or stab just below the medium surface. 5 days at a lower temperature of 22-25°C.3
3. Incubate at a temperature and duration appropriate for the suspected
organism being tested.
References
1. Jordan, E. O., M. E. Caldwell, and D. Reiter. 1934. Bacterial
4. Examine tubes or plates for growth and signs of motility.
motility. J. Bacteriol. 27:165.
Results 2. D’Amato, R. F., and K. M. Tomfohrde. 1981. Influence of
Motility is evidenced by the presence of diffuse growth away from the media on temperature-dependent motility test for Yersinia
line or spot of inoculation. Nonmotile organisms grow only along the enterocolitica. J. Clin. Microbiol. 14:347-348.
line of inoculation. 3. MacFaddin, J. F. 1985. Media for isolation-cultivation-
identification-maintenance of medical bacteria, vol. 1, Williams
Limitations of the Procedure & Wilkins, Baltimore, MD.
1. All weak or questionable motility test results should be confirmed
by flagella stain or by direct wet microscopy.2 Packaging
2. Some flagellar proteins are not synthesized at higher temperatures.3 Motility GI Medium 500 g 0869-17
Bacto Mueller Hinton Broth This unsupplemented medium has been selected by the National
Committee for Clinical Laboratory Standards (NCCLS) for several
Intended Use reasons:11
• It shows good batch-to-batch reproducibility.
Bacto Mueller Hinton Medium is used for antimicrobial susceptibility
testing of rapidly growing aerobic microorganisms by the disk • It is low in sulfonamide, trimethoprim, and tetracycline inhibitors.
diffusion technique. • It gives satisfactory growth of most non-fastidious pathogens.
Bacto Mueller Hinton Broth is for antimicrobial susceptibility testing • A large amount of data has been collected from antimicrobial
of aerobic microorganisms by broth dilution methods. susceptibility tests with this medium.
A variety of supplements can be added to Mueller Hinton Medium.
Also Know As For testing streptococci, supplementation with 5% defibrinated sheep
Mueller Hinton media are abbreviated as M-H Agar and M-H Broth. or horse blood is recommended.16 GC agar base with added 1% growth
supplement, is used for antimicrobial susceptibility testing of
Summary and Explanation Neisseria gonorrhoeae. Susceptibility testing of Haemophilus species
Mueller Hinton Medium duplicates the formula recommended by should be performed on Haemophilus Test Medium. Mueller Hinton
Mueller and Hinton1 for the primary isolation of Neisseria species. In Medium should be supplemented with 2% NaCl for testing methicillin
the development of a simple transparent medium containing heat stable or oxacillin against staphylococci.5 Mueller Hinton Medium with
ingredients, Mueller and Hinton selected pea meal extract agar.2 In their Rabbit Serum is used for the cultivation and maintenance of
modification, starch replaced the growth-promoting properties of Corynebacterium species.6
pea extract, acting as a “protective colloid” against toxic substances.
Tryptic digest of meat was substituted with casamino acids, technical. Principles of the Procedure
Bauer, Kirby, Sherris and Tuck 3 recommended Mueller Hinton Infusion from Beef and Casamino Acids, Technical provide nitrogen,
Medium for performing antibiotic susceptibility tests using a single vitamins, carbon and amino acids in Mueller Hinton media. Starch is
disk of high concentration. Mueller Hinton Medium is used in the disk added to absorb any toxic metabolites produced. Bacto Agar is the
diffusion method of susceptibility testing.7 Mueller Hinton Broth is solidifying agent.
used for determining minimal inhibitory concentrations (MICs).4 The use of a suitable medium is essential for testing the susceptibility
Mueller Hinton Medium complies with requirements of the World of microorganisms to sulfonamides and trimethoprim. Antagonism to
Health Organization.8 Mueller Hinton Medium is specified in the FDA sulfonamide activity is demonstrated by para-aminobenzoic acid
Bacteriological Analytical Manual 9 for food testing. (PABA) and its analogs. Reduced activity of trimethoprim, resulting in
Mueller Hinton Medium is the recommended medium for testing most smaller growth inhibition zones and innerzonal growth, is demonstrated
commonly encountered aerobic and facultatively anaerobic bacteria.10 on medium possessing high levels of thymidine. The PABA and
Cultural Response
Mueller Hinton Medium: Prepare, inoculate and dispense
antibiotic disks following the procedure described by NCCLS.7,11
The cultures listed should have zone sizes near the middle of the Typical test of Mueller Hinton Medium by agar diffusion method.
range of the concentration tested.7
Mueller Hinton Broth: Prepare and dispense into microdilution trays or microdilution tubes described by NCCLS.4 The cultures
listed should have MIC (endpoints) near the middle of the range of the concentration tested.4
ORGANISM ATCC®
Enterococcus faecalis 29212*
Escherichia coli 25922* The cultures listed are the minimum that should be used for performance testing.
Pseudomonas aeruginosa 27853* *These organisms are available as Bactrol™ Disks and should be used as
Staphylococcus aureus 25923* directed in Bactrol Disks Technical Information.
and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed. 15. Buschelman, B. J., R. N. Jones, and M. J. Bale. 1994. Effects of
American Society for Microbiology, Washington, D.C. blood medium supplements on activities of newer cephalosporins
11. National Committee for Clinical Laboratory Standards. 1993. tested against enterococci. J. Clin. Microbiol. 32:565-567.
Evaluating production lots of dehydrated Mueller-Hinton agar. 16. National Committee for Clinical Laboratory Standards. 1997.
Tentative standard M6-T. National Committee for Clinical Performance standards for antimicrobial disk susceptibility
Laboratory Standards, Villanova, PA. tests-sixth edition. Approved Standard. M2-A6, Volume 7,
12. Isenberg, H. E. (ed.). 1992. Clinical microbiology procedures No.1 National Committee for Clinical Laboratory Standards,
handbook, American Society for Microbiology, Washington, D.C. Wayne, PA.
13. Barry, A. L., G. H. Miller, C. Thornsberry, R. S. Hare,
R. N. Jones, R. R. Lorber, R. Ferraresi, and C. Cramer. 1987. Packaging
Influence of cation supplements on activity of netilmicin against Mueller Hinton Broth 100 g 0757-15
Pseudomonas aeruginosa in vitro and in vivo. Antimicrob. Agents 500 g 0757-17
Chemother. 31:1514-1518. 2 kg 0757-07
14. Barry, A. L., L. B. Reller, G. H. Miller, J. A. Washington, F. D. Mueller Hinton Medium 100 g 0252-15
Schoenknecht, L. R. Peterson, R. S. Hare, and C. Knapp. 1992. 500 g 0252-17
Revision of standards for adjusting the cation content of Mueller- 2 kg 0252-07
Hinton broth for testing susceptibility of Pseudomonas aeruginosa 10 kg 0252-08
to aminoglycosides. J. Clin. Microbiol. 30:585-589.
2. IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM 3. Transfer 10 ml samples to 100 ml Muller Kauffmann Tetrathionate
AND SKIN. Avoid contact with skin and eyes. Do not breathe dust. Broth and to 100 ml Selenite Brilliant Green Medium (0661).
Wear suitable protective clothing. Keep container tightly closed. 4. Incubate Muller Kauffmann Tetrathionate Broth at 42-43°C and
FIRST AID: In case of contact with eyes, rinse immediately with the Selenite Brilliant Green Enrichment at 37°C.
plenty of water and seek medical advice. After contact with skin, 5. Subculture broths after 18-24 hours and 48 hours onto Brilliant
wash immediately with plenty of water. If inhaled, remove to fresh Green Agar.
air. If not breathing, give artificial respiration. If breathing is diffi- 6. Incubate overnight.
cult, give oxygen. Seek medical advice. If swallowed seek medical 7. Examine for the growth of typical colonies of Salmonella spp.
advice immediately and show this container or label.
Sewage Polluted Natural Waters
3. Follow proper established laboratory procedures in handling and
disposing of infectious materials. This procedure is applicable to the isolation of Salmonella spp.
other than S. typhi.
Storage 1. Inoculate 25 ml aliquots of the sample into 25 ml of double strength
Store the dehydrated medium below 30°C. The dehydrated medium is Buffered Peptone Water (1810). Incubate at 37°C for 18 hours.
very hygroscopic. Keep container tightly closed. 2. Transfer 1 ml samples into 10 ml of Muller Kauffmann
Tetrathionate Broth.
Expiration Date 3. Incubate at 43°C for 48 hours.
The expiration date applies to the product in its intact container when 4. Subculture broths after 18-24 and 48 hours onto Brilliant Green
stored as directed. Do not use a product if it fails to meet specifications MacConkey Agar, prepared by adding 10 ml of a 0.33% (w/v)
for identity and performance. aqueous solution of brilliant green to MacConkey Agar (0331) to
give a final concentration of 0.033 g/l.
Procedure 5. Incubate at 37°C overnight.
Materials Provided 6. Examine for colonies typical of Salmonella spp.
Muller Kauffmann Tetrathionate Broth Base
Results
Materials Required but not Provided Salmonella spp. will produce red colonies with good growth.
Iodine solution (20 g iodine and 25 g potassium iodide in 100 ml water)
Brilliant Green solution (0.1 g Brilliant Green in 100 ml water) Limitations of the Procedure
Glassware 1. The complete medium is unstable and should be used immediately.
Distilled or deionized water It may be stored at 2-8°C in the dark for no more than seven days.
Autoclave 2. Due to the nutritional requirements and inhibitory characteristics
Incubator (43°C) of the organisms themselves, organisms other than salmonellae,
Buffered Peptone Water such as Morganella morganii and some Enterobacteriaceae may
Blender grow in the medium.
Tetrathionate Broth 3. Confirmatory tests, such as fermentation reactions and
Selenite Brilliant Green Medium seroagglutination should be carried out on all presumptive
Brilliant Green Agar Enrichment Salmonella colonies that are recovered.
Brilliant Green Agar
References
Method of Preparation-Single Strength 1. Muller, L. 1923. Un nouveau millieu d’enrichissement pour la
1. Suspend 105.8 grams in 1 liter distilled or deionized water. recherche du bacille typhique et des paratyphiques. C. R. Soc. Biol.
2. Boil gently. (Paris) 89:434-443.
3. Cool below 45°C. 2. Kauffmann, F. 1935. Weitere erfahrungen mit dem kombininierten
anreicherungsverfahren fur Salmonella bazillen. Ztschr. F. Hyg.
4. Add 19 ml iodine solution and 9.5 ml brilliant green solution.
117:26-32.
5. Dispense into sterile tubes, mixing well to evenly dispense the
3. International Organization for Standardization. Geneva. 1974.
calcium carbonate.
(Draft International Standard ISO/DIS 3565).
Specimen Collection and Preparation 4. A manual for recommended methods for the microbiological
Collect specimens according to recommended guidelines. examination of poultry and poultry products. 1982.
Test Procedure 5. P.H.L.S. Monograph Series No. 8. 1974.
Meat and Meat Products 6. Harvey, R. W. S., and T. H. Price. 1976. Isolation of salmonellae
1. Weigh 25 grams of the sample into a sterile blender jar and add from sewage- polluted river water using selenite F and Muller-
225 ml of Buffered Peptone Water (1810) and macerate for sufficient Kauffmann tetrathionate. J. Hyg. Camb. 77:333-339.
time to give 10,000-15,000 revolutions. Packaging
2. Transfer contents of the blender jar aseptically to a 500 ml flask. Muller Kauffmann
Incubate at 37°C ± 0.1°C for 16-20 hours. Tetrathionate Broth Base 500 g 1853-17
Formula
Bacto Mycobiotic Agar
®
Mycobiotic Agar
Formula Per Liter
Intended Use Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Mycobiotic Agar is used for isolating pathogenic fungi. Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Summary and Explanation Cycloheximide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
Numerous media, such as Sabouraud Dextrose Agar, Sabouraud Chloramphenicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g
Maltose Agar, Littman Oxgall Agar, Brain Heart Infusion Agar, and Final pH 6.5 ± 0.2 at 25°C
Malt Agar have been used widely in culturing pathogenic fungi. The
Sabouraud media and Malt Agar are somewhat selective in nature Precautions
due to low pH, which may suppress bacterial growth. It is well known 1. For Laboratory Use.
that media for isolated pathogenic fungi can also be made selective by 2. TOXIC. TOXIC BY INHALATION AND IF SWALLOWED.
the addition of antibiotics.1-8 POSSIBLE RISK OF IRREVERSIBLE EFFECTS. POSSIBLE
Mycobiotic Agar contains cycloheximide and chloramphenicol RISK OF HARM TO THE UNBORN CHILD. Do not breathe dust.
making it much more selective when compared to other fungal media. In case of accident or if you feel unwell, seek medical advice
This medium has proven useful in the isolation of the dermatophytes immediately (show the label where possible). Wear suitable
and other pathogenic fungi from clinical specimens.9 protective clothing. Keep container tightly closed. TARGET
ORGAN(S): Eyes/Ears, Blood, Cardiovascular, Lymph Glands,
Georg10 recommends the use of Mycobiotic Agar exclusively for Muscles, Nerves, Urogenital
isolating dermatophytes (because none of the dermatophytes are
FIRST AID: In case of contact with eyes, rinse immediately with
sensitive to cycloheximide or chloramphenicol) and in parallel to media
plenty of water and seek medical advice. After contact with skin, wash
without antibiotics for isolating fungi which cause systemic disease.
immediately with plenty of water. If inhaled, remove to fresh air. If
Principles of the Procedure not breathing, give artificial respiration. If breathing is difficult,
Soytone provides carbon and nitrogen sources. Dextrose is a source give oxygen. Seek medical advice. If swallowed, induce vomiting;
of carbon. Cycloheximide suppresses the growth of saprophytic seek medical advice immediately and show this container or label.
fungi. Chloramphenicol inhibits bacterial growth. Bacto Agar is the 3. Follow proper established laboratory procedure in handling and
solidifying agent. disposing of infectious materials.
Storage
User Quality Control Store dehydrated medium below 30°C. The dehydrated medium is very
hygroscopic. Keep container tightly closed.
Identity Specifications
Dehydrated Appearance: Light beige, free-flowing, Expiration Date
homogeneous. The expiration date applies to the product in its intact container when
Solution: 3.56% solution, soluble in distilled stored as directed. Do not use a product if it fails to meet specifications
or deionized water on boiling. for identity and performance.
Solution is light to medium amber,
slightly opalescent, with no Procedure
significant precipitate.
Materials Provided
Reaction of 3.56%
Solution at 25° C: pH 6.5 ± 0.2 Mycobiotic Agar
Mycological Media
Bacto Mycological Agar . Bacto Mycological Agar w/Low pH
®
Precautions Procedure
1. Mycological Agar: For Laboratory Use. Materials Provided
Mycological Agar w/Low pH: For Laboratory Use. Mycological Agar
2. Follow proper established laboratory procedure in handling and Mycological Agar w/Low pH
disposing of infectious materials.
Materials Required but not Provided
Storage Glassware
Store the dehydrated medium below 30°C. The dehydrated medium is Autoclave
very hygroscopic. Keep containers tightly closed. Antibacterial/antifungal agents
Bacto Neopeptone
®
that a peptone free from toxic factors can support the growth of
S. pneumococci from small inocula. Spray2 used Neopeptone in his
culture media for classification of sporulating anaerobes. Casman3
Intended Use reported Neopeptone to be best suited for use in infusion base. Eldering
Bacto Neopeptone is used in preparing microbiological culture media. and Kendrick4 reported good results with Neopeptone in cultivating
Bordetella pertussis.
Also Known As Neopeptone is valuable in culture media for the cultivation of
Neopeptone is also referred to as Special Peptone. pathogenic fungi. Growth of these microorganisms is rapid and
Summary and Explanation colonial formation is uniform and typical for the various types. Bacto
Sabouraud Dextrose Agar and Bacto Sabouraud Maltose Agar are
Neopeptone is particularly well suited to the growth requirements of
prepared with Neopeptone.
fastidious miroorganisms. Certain delicate strains of microorganisms
are highly susceptible to the effects of bacteriostatic substances Bacto Todd Hewitt Broth prepared with Neopeptone, is excellent for
frequently present in some peptones. The work of Dubos1 shows clearly growing Group A streptococci for serological typing. Several media
containing Neopeptone are specified in standard methods 5-7 for
multiple applications.
User Quality Control Principles of the Procedure
Identity Specifications Neopeptone is an enzymatic digest of protein. Neopeptone contains a
Dehydrated wide variety of peptide sizes in combination with vitamins, nucleotides,
Appearance: Tan, free-flowing granules. minerals and other carbon sources.
Solution: 1%, 2% and 10% solutions are soluble in
distilled or deionized water: Typical Analysis
1%-Very light to light amber, clear to very Physical Characteristics
slightly opalescent, may have a precipitate. Ash (%) 7.0 Loss on Drying (%) 3.2
2%-Light to medium amber, clear to very Clarity, 1% Soln (NTU) 1.2 pH, 1% Soln 7.4
slightly opalescent, may have a precipitate. Filterability (g/cm2) 0.3
10%-Medium to dark amber, slightly Carbohydrate (%)
opalescent to opalescent, may have a Total 0.8
precipitate.
Reaction of 1% Nitrogen Content (%)
Solution at 25°C: pH 6.9 - 7.5 Total Nitrogen 13.7 AN/TN (%) 23.8
Amino Nitrogen 3.3
Cultural Response Amino Acids (%)
All solutions are prepared with the pH adjusted to 7.2 - 7.4. Alanine 4.03 Lysine 5.16
TEST SOLUTION ORGANISM ATCC® RESULT Arginine 4.14 Methionine 2.00
Fermentable 2% Escherichia 25922* negative Aspartic Acid 6.19 Phenylalanine 8.67
Carbohydrates coli Cystine 0.26 Proline 6.73
Indole 0.1% Escherichia 25922* positive Glutamic Acid 13.22 Serine 4.22
Production coli Glycine 7.02 Threonine 3.69
Acetylmethylcar- 0.1% Enterobacter 13048* positive Histidine <0.01 Tryptophan 0.96
binol Production aerogenes
Isoleucine 0.36 Tyrosine 4.21
Hydrogen 1% Salmonella 6539 positive
Sulfide typhi Leucine 3.65 Valine 4.96
Toxicity 2% w/0.5% NaCl Escherichia 25922* good Inorganics (%)
& 1.5% Bacto Agar coli growth Calcium 0.012 Phosphate 2.209
Toxicity 2% w/0.5% NaCl Staphylococcus 25923* good Chloride 0.344 Potassium 0.149
& 1.5% Bacto Agar aureus growth Cobalt <0.001 Sodium 2.057
The cultures listed are the minimum that should be used for Copper <0.001 Sulfate 0.340
performance testing. Iron <0.001 Sulfur 0.657
*These cultures are available as Bactrol™ Disks and should be Lead <0.001 Tin <0.001
used as directed in Bactrol Disks Technical Information. Magnesium 0.006 Zinc <0.001
Manganese <0.001
Intended Use
Bacto Niacin Assay Medium is used for determining niacin concentra-
User Quality Control tion by the microbiological assay technique.
Identity Specifications Summary and Explanation
Dehydrated Appearance: Off-white, homogeneous, tendency Vitamin Assay Media are used in the microbiological assay of vitamins.
to clump. Three types of media are used for this purpose:
Solution: 3.75% (single strength) or 1. Maintenance Media: For carrying the stock culture to preserve the
7.5% (double strength) solution,
soluble in distilled or deionized viability and sensitivity of the test organism for its intended purpose;
water on boiling 2-3 minutes. 2. Inoculum Media: To condition the test culture for immediate use;
Single strength solution is very 3. Assay Media: To permit quantitation of the vitamin under test.
light amber, clear, may have a Niacin Assay Medium is prepared according to the formula described
slight precipitate.
by Snell and Wright,1 modified by Krehl, Strong and Elvehjem2 and
Prepared Medium: (Single strength) very light amber, Barton-Wright.3 Niacin Assay Medium is used in the microbiological
clear, may have a slight precipitate.
assay of nicotinic acid or nicotinamide (niacin) using Lactobacillus
Reaction of 3.75% plantarum ATCC® 8014 as the test organism. The medium complies
Solution at 25°C: pH 6.7 ± 0.2 with USP4 and AOAC.5
Cultural Response
Prepare Niacin Assay Medium per label directions. Prepare
Principles of the Procedure
a standard curve using nicotinic acid reference standards at Niacin Assay Medium is a dehydrated medium free from nicotinic acid
0.0 to 0.25 µg per 10 ml. The medium supports the growth and its analogs but containing all other nutrients and vitamins essential
of L. plantarum ATCC® 8014 when supplemented with for the cultivation of L. plantarum ATCC® 8014. The addition of nicotinic
nicotinic acid. acid or its analogs in specified increasing concentrations gives a growth
response that can be measured turbidimetrically or titrimetrically.
Formula Procedure
Niacin Assay Medium Materials Provided
Formula Per Liter Niacin Assay Medium
Bacto Vitamin Assay Casamino Acids . . . . . . . . . . . . . . . . 12 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g Materials Required But Not Provided
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g Glassware
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g Autoclave
DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g Stock culture of Lactobacillus plantarum ATCC® 8014.
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg Sterile tubes
Guanine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg Distilled or deionized water
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg Sterile 0.85% saline
Thiamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 200 µg Centrifuge
Calcium Pantothenate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 µg Spectrophotometer
Pyridoxine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . 400 µg Lactobacilli Broth AOAC or Micro Inoculum Broth
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400 µg Niacin
p-Aminobenzoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 µg
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 µg Method of Preparation
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g 1. Suspend 7.5 grams in 100 ml distilled or deionized water.
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g 2. Boil for 2-3 minutes.
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g
3. Dispense 5 ml amounts into tubes, evenly dispersing the precipitate.
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg 4. Add standard or test samples.
Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg 5. Adjust tube volumes to 10 ml with distilled or deionized water.
Final pH 6.7 ± 0.2 at 25°C 6. Autoclave at 121°C for 10 minutes.
distilled water, giving a stock solution of 50 µg per ml. Dilute the stock 3. The use of altered or deficient media may cause mutants having
solution by adding 1 ml to 999 ml distilled water (50 ng/ml). Use 0.0, different nutritional requirements that will not give a satisfactory
0.5, 1, 2, 3, 4 and 5 ml of the 50 ng/ml solution per tube. Other standard response.
concentrations may be used provided the standard falls within the limits 4. For successful results to these procedures, all conditions of the
specified by AOAC.5 assay must be followed precisely.
Results References
1. Prepare a standard concentration response curve by plotting the 1. Snell and Wright. 1941. J. Biol. Chem. 13:675.
response readings against the amount of standard in each tube, disk
2. Krehl, Strong, and Elvehjem. 1943. Ind. & Eng. Chem., Ann.
or cup. Ed., 15:471.
2. Determine the amount of vitamin at each level of assay solution by 3. Barton-Wright. 1944. J. Biochem. 38:314.
interpolation from the standard curve.
4. The United States Pharmacopeial Convention. 1995. The United
3. Calculate the concentration of vitamin in the sample from the States pharmacopeia, 23rd ed. The United States Pharmacopeial
average of these volumes. Use only those values that do not vary
Convention, Inc., Rockville, MD.
more than ±10% from the average and use the results only if two
thirds of the values do not vary more than ±10%. 5. Association of Official Analytical Chemists. 199. Official
methods of analysis of AOAC International, 16th ed. AOAC
Limitations of the Procedure International, Arlington, VA.
1. The test organism used for inoculating an assay medium must be
cultured and maintained on media recommended for this purpose.
Packaging
Niacin Assay Medium 100 g 0322-15
2. Aseptic technique should be used throughout the assay procedure.
Procedure References
1. MacFaddin, J. D. 1985. Media for isolation-cultivation-
Materials Provided
identification-maintenance medical bacteria, p.275-284, vol 1.
Nitrate Broth Williams & Wilkins, Baltimore, MD.
Materials Required but not Provided
Packaging
Glassware
Nitrate Broth 500 g 0268-17
Autoclave
2. American Public Health Association. 1923. Standard methods 6. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). 1995.
of water analysis, 5th ed. American Public Health Association, Standard methods for the examination of water and wastewater,
Washington, D.C. 19th ed. American Public Health Association, Washington, D.C.
3. American Public Health Association. 1923. Standard methods 7. Association of Official Analytical Chemists. 1995. Bacteriological
of milk analysis, 4th ed. American Public Health Association, analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
Washington, D.C. 8. Association of Official Analytical Chemists. 1995. Official
4. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compen- methods of analysis of AOAC International, 16th ed. AOAC
dium of methods for the microbiological examination of foods, International, Arlington, VA.
3rd ed. American Public Health Association, Washington, D.C. Packaging
5. Marshall, R. T. (ed.) 1993. Standard methods for the microbio- Nutrient Broth 100 g 0003-15-0
logical examination of dairy products, 16th ed. American Public 500 g 0003-17-8
Health Association, Washington, D.C. 2 kg 0003-07-0
10 kg 0003-08-9
Precautions Procedure
1. For Laboratory Use. Materials Provided
2. Follow proper established laboratory procedure in handling and NZCYM Broth
disposing of infectious materials. NZYM Broth
NZM Broth
Precautions No color change in both tubes of the set indicates the organism is
1. For Laboratory Use. nonsaccharolytic for the carbohydrate tested.
2. Follow proper established laboratory procedure in handling and
disposing of infectious materials.
Limitations of the Procedure
1. The acid reaction produced by oxidative organisms is apparent at
Storage the surface and gradually spreads throughout the medium. If the
Store the dehydrated medium below 30°C. The dehydrated medium is oxidation is weak or slow, however, an initial alkaline reaction
very hygroscopic. Keep container tightly closed. at the surface of the open tube may persist for several days and
eventually convert to an acid reaction.
Expiration Date 2. If an organism is unable to grow on OF Basal Medium, Cowan10
The expiration date applies to the product in its intact container when recommends adding either 2% serum or 0.1% yeast extract to each
stored as directed. Do not use a product if it fails to meet specifications carbohydrate tube.
for identity and performance. 3. Nonsaccharolytic organisms produce a slight alkalinity in the
open tube (blue-green color), while the covered tube will not
Procedure exhibit a color change (green).
Materials Provided
OF Basal Medium
References
1. Hugh, R. and E. Leifson. 1953. The taxonomic significance of
Materials Required But Not Provided fermentative versus oxidative metabolism of carbohydrates by
Glassware various gram negative bacteria. J. Bacteriol. 66:24-26.
Autoclave 2. Bacteriological Analytical Manual, 8th ed. 1995. AOAC
Sterile melted petrolatum (mineral oil) International. Gaithersburg, MD.
Carbohydrates of choice: Glucose (dextrose), Lactose, Maltose, 3. Vanderzant, C. and D. F. Splittstoesser (eds.). 1992. Compen-
Mannitol, Saccharose and/or Xylose dium of methods for the microbiological examination of foods,
Incubator (35°C) 3rd ed. American Public Health Association, Washington, D.C.
Test tubes with closures 4. Marshall, R. T. (ed.). 1992. Standard methods for the examination
of dairy products, 16th ed. American Public Health Association,
Method of Preparation
Washington, D.C.
1. Suspend 9.4 grams in 1 liter distilled or deionized water.
5. Oberhofer, T. R. 1985. Manual of nonfermenting gram-negative
2. Boil to dissolve completely. bacteria. Churchill Livingstone, New York, NY.
3. Autoclave at 121°C for 15 minutes. 6. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures
4. Add 1% carbohydrate before or after sterilization, depending on handbook, vol. 1. American Society for Microbiology,
heat lability. Washington, D.C.
5. Dispense in test tubes. 7. Knapp, J. S. and K. K. Holmes. 1983. Modified oxidation-
Specimen Collection and Preparation fermentation medium for detection of acid production from
carbohydrates by Neisseria spp. and Branhamella catarrhalis.
Refer to appropriate references for specimen collection and preparation. J. Clin. Microbiol. 18:56-62.
Test Procedure 8. Welch, D. F., M. J. Muszynski, C. H. Pai, M. J. Marcon, M. M.
1. Inoculate duplicate test tubes by stabbing the medium to the Hribar, P. H. Gilligan, J. M. Matsen, P. A. Ahlin, B. C. Hilman
desired depth with an inoculating needle. and S. A. Chartrand. 1987. Selective and differential medium for
recovery of Pseudomonas cepacia from the respiratory tracts of
2. Overlay one tube of each set with 2 ml sterile mineral oil.
patients with cystic fibrosis. J. Clin. Microbiol. 25:1730-1734.
3. Incubate at a temperature appropriate for the test organism for a
9. MacFaddin, J. F. 1985. Media for isolation-cultivation-
minimum of 48 hours.
identification-maintenance of medical bacteria, vol. 1. Williams
4. Examine the medium for gas production and a color change from & Wilkins, Baltimore, MD.
green to yellow.
10. Cowan, S. T. 1974. Cowan and Steel’s manual for the identification
Results of medical bacteria, 2nd ed. Cambridge University Press,
Alkaline reaction: green to blue medium Cambridge, MA.
Acid reaction: yellow medium
Packaging
A color change of the medium in both the plain and overlay tubes,
OF Basal Medium 100 g 0688-15
with or without gas production, indicates fermentation of the
500 g 0688-17
carbohydrate tested.
Mineral Oil 30 ml 6663-30
A color change in the plain tube, only, indicates oxidative metabolism
of the carbohydrate tested.
OGYE Agar
Bacto OGYE Agar Base . Bacto Antimicrobic Vial
®
Oxytetracycline
Intended Use OGYE Agar is specified as a standard methods medium for use with
Bacto OGYE Agar Base is for use with Bacto Antimicrobic Vial dairy products.2
Oxytetracycline in isolating and enumerating yeasts and molds in foods.
Principles of the Procedure
Also Known As OGYE Agar Base contains Yeast Extract to supply B-complex vitamins
OGY Agar Base 1 which stimulate bacterial growth. Dextrose is the carbon energy source.
OGYA (Oxytetracycline-Glucose-Yeast (Extract) Agar)1 Bacto Agar is the solidifying agent. Antimicrobic Vial Oxytetracycline
inhibits the growth of bacteria.
Summary and Explanation
Acidified agar may be used for enumerating yeasts and molds in foods
Formula
and dairy products. However, in some cases, antimicrobics better OGYE Agar Base
suppress bacterial growth and improve recovery of yeasts and molds.2, 3 Formula Per Liter
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Mossel et al.4, 5 described Oxytetracycline-Glucose Yeast Extract Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
(OGYE) Agar for selectively isolating and enumerating yeasts and Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 g
molds in foods. Mossel et al. demonstrated improved recovery compared
Final pH 7.0 ± 0.2 at 25°C
to acidified agar media.
Antimicrobic Vial Oxytetracycline
Oxytetracycline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 mg
4. Aseptically add 10 ml rehydrated Antimicrobic Vial Oxytetracy- 2. Frank, J. F., G. L. Christen, and L. B. Bullerman. 1993. Tests
cline to the medium. Mix well. for groups of microorganisms, p. 271-286. In R. T. Marshall (ed.),
Antimicrobic Vial Oxytetracycline Standard methods for the microbiological examination of
dairy products, 16th ed. American Public Health Association,
1. Aseptically add 10 ml sterile distilled or deionized to the Anti- Washington, D.C.
microbic Vial Oxytetracycline.
3. Mislivec, P. B., L. R. Beuchat, and M. A. Cousin. 1992. Yeasts
2. Shake to dissolve contents. and molds, p. 239-249. In C. Vanderzant, and D. F. Splittstoesser
Specimen Collection and Preparation (ed.), Compendium of methods for the microbiological
examination of foods, 3rd ed. American Public Health Association,
Refer to appropriate references for specimen collection and preparation. Washington, D.C.
Test Procedure 4. Mossel, D. A. A., A. M. C. Kleynen-Semmeling, H. M. Vincentie,
H. Beerens, and M. Catsaras. 1970. Oxytetracycline-Glucose-
See appropriate references for specific procedures. Yeast Extract Agar for selective enumeration of moulds and yeasts
Results in foods and clinical material. J. Appl. Bacteriol. 33:454-457.
Refer to appropriate references and procedures for results. 5. Mossel, D. A. A., M. Visser, and W. H. J. Mengerink. 1962. A
comparison of media for the enumeration of moulds and yeasts in
References food and beverages. Lab. Pract. 11:109-112.
1. MacFaddin, J. F. 1985. Media for isolation-cultivation- Packaging
identification-maintenance of medical bacteria, vol. 1. p. 579-582. OGYE Agar Base 500 g 1811-17
Williams & Wilkins, Baltimore, MD.
Antimicrobic Vial Oxytetracycline 10 ml 3267-59
The dehydrated Orange Serum Agar is very hygroscopic. Keep 3. Autoclave at 121°C for 15 minutes. Avoid overheating which could
container tightly closed. cause a softer medium.
Orange Serum Broth Concentrate 10X
Expiration Date
1. To prepare the single-strength medium, aseptically add 100 ml
The expiration date applies to the product in its intact container when
Orange Serum Concentrate 10X to 900 ml sterile distilled or
stored as directed. Do not use a product if it fails to meet specifications
deionized water and mix thoroughly.
for identity and performance.
2. Use aseptic technique to dispense 10 ml amounts into sterile test tubes.
Procedure Specimen Collection and Preparation
Materials Provided Refer to appropriate references for specimen collection and preparation.
Orange Serum Agar
Orange Serum Broth Concentrate 10X Test Procedure
Orange Serum Agar
Materials Required but not Provided 1. For plate count method, prepare serial 10-fold dilutions of the
For Orange Serum Agar: test material.
Flask with closure 2. Add 1 ml of test sample to sterile Petri dish.
Distilled or deionized water 3. Add 18-20 ml of sterile, molten agar (cooled to 45-50°C) and swirl
Autoclave plate gently to mix well.
For Orange Serum Broth Concentrate 10X: 4. Allow to solidify before incubating at 30°C for 48 hours. Plates
Sterile distilled or deionized water can be held up to 5 days.
Sterile test tubes
Orange Serum Broth Concentrate 10X
Sterile transfer pipettes, 10 ml
Orange Serum Broth Concentrate 10X is used for small samples to
Method of Preparation initiate growth.
Orange Serum Agar Results
1. Suspend 45.5 grams in 1 liter distilled or deionized water. Orange Serum Agar
2. Heat to boiling to dissolve completely. Record colony morphology for each type of growth.
Orange Serum Broth Concentrate 10X
User Quality Control Turbidity indicates growth.
PALCAM Medium
Bacto PALCAM Medium Base . Bacto PALCAM
®
Antimicrobic Supplement
achieved through the presence of Lithium Chloride, Polymyxin B
Sulfate and Acriflavine HCl, present in PALCAM Medium Base, and
Intended Use Ceftazidime, provided by PALCAM Antimicrobic Supplement. These
Bacto PALCAM Medium Base is used with Bacto PALCAM Antimi- agents effectively suppress growth of most commonly occurring
crobic Supplement in isolating and cultivating Listeria from foods. non-Listeria spp. of bacteria present in foods. The ceftazidime con-
centration is reduced from 20 mg/l to 8 mg/l for improved growth
Summary and Explanation and recovery of Listeria.
PALCAM Medium Base and PALCAM Antimicrobic Supplement are
Differentiation on PALCAM Medium is based on esculin hydrolysis
based on the PALCAM agar formulation of van Netten et al.,1 who
and mannitol fermentation. All Listeria spp. hydrolyze esculin
developed this selective and differential medium for use in the isolation
as evidenced by a blackening of the medium. This blackening by
and enumeration of Listeria spp. from food samples. PALCAM
esculin-hydrolyzing bacteria results from the formation of 6,7
medium is recommended by AFNOR for use in the detection of
dihydroxycoumarin, which reacts with ferric ions that are present in
L. monocytogenes in foods,2 and by the IDF as an additional plating
the medium as Ferric Ammonium Citrate. On occasion, organisms other
medium for the detection of Listeria spp. in milk and milk products.3
than Listeria, such as staphylococci or enterococci, may grow on this
PALCAM medium is recommended by Health Canada for the detection
medium. Mannitol and the pH indicator, Phenol Red, have been added
of L. monocytogenes in food and environmental samples.4
to differentiate mannitol-fermenting strains of these species from
Principles of the Procedure Listeria based on mannitol fermentation. Mannitol fermentation is
demonstrated by a color change in the colony and/or the surrounding
Good growth of Listeria spp. is obtained by including Columbia
medium from red or gray to yellow due to the production of acidic end
Blood Agar Base in PALCAM Medium Base. Columbia Blood Agar
products.
Base provides the nutrients and cofactors required for good to excel-
lent growth of Listeria. Selectivity of the complete medium is
Uninoculated Listeria monocytogenes
plate ATCC® 19114
Cultural Response
Prepare PALCAM Medium per label directions. Inoculate and incubate
at 35 ± 2°C for 40-48 hours under microaerophilic conditions.
ORGANISM ATCC® INOCULUM GROWTH
Escherichia coli 25922* 1,000-2,000 inhibited
Listeria monocytogenes 19114 100-1,000 good growth, gray-green The cultures listed are the minimum that should be used
colonies with black precipitate for performance testing.
Staphylococcus aureus 25923* 1,000-2,000 inhibited *These cultures are available as Bactrol™ Disks and should
Enterococcus faecalis 29212* 1,000-2,000 inhibited be used as directed in Bactrol Disks Technical Information.
Formula Procedure
PALCAM Medium Base Materials Provided
Formula Per Liter PALCAM Medium Base
Bacto Columbia Blood Agar Base . . . . . . . . . . . . . . . . . . . 39 g PALCAM Antimicrobic Supplement
Bacto Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g Materials Required But Not Provided
Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g Flasks with closures
Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g Sterile distilled or deionized water
Lithium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g Autoclave
Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08 g
Waterbath (45-50°C)
Acriflavine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005 g
Incubator (35°C)
Polymyxin B Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g Depending on testing method:
Final pH 7.2 ± 0.2 at 25°C Fraser Broth Base
PALCAM Antimicrobic Supplement Demi-Fraser Broth Base
Formula per 10 ml vial Fraser Broth Supplement
Ceftazidime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 mg Oxford Medium Base
Oxford Antimicrobic Supplement
Precautions Modified Oxford Antimicrobic Supplement
1. For Laboratory Use. Listeria Enrichment Broth
2. Follow proper, established laboratory procedures in handling and Modified Listeria Enrichment Broth
disposing of infectious materials. Tryptic Soy Agar with 0.6% Yeast Extract
3. PALCAM Medium Base: LPM Agar Base
HARMFUL. IRRITATING TO EYES, RESPIRATORY SYSTEM Moxalactam Antimicrobic Supplement
AND SKIN. MAY CAUSE HARM TO THE UNBORN CHILD.
Avoid contact with skin and eyes. Do not breathe dust. Wear suitable
Method of Preparation
protective clothing. Keep container tightly closed. TARGET 1. Suspend 68 grams PALCAM Medium Base in 1 liter distilled or
ORGAN(S): Blood, Kidneys, Nerves. deionized water and boil to dissolve completely.
FIRST AID: In case of contact with eyes, rinse immediately with 2. Autoclave at 121°C for 15 minutes. Cool to 45-50°C.
plenty of water and seek medical advice. After contact with skin, 3. Aseptically add 2 ml PALCAM Antimicrobic Supplement which
wash immediately with plenty of water. If inhaled, remove to fresh has been rehydrated with 10 ml sterile distilled or deionized water.
air. If not breathing, give artificial respiration. If breathing is diffi- Mix well.
cult, give oxygen. Seek medical advice. If swallowed seek medical
advice immediately and show this container or label. Specimen Collection and Preparation
4. PALCAM Antimicrobic Supplement: 1. Collect food samples in sterile containers and transport immediately
to the laboratory following recommended guidelines.
MAY CAUSE ALLERGIC EYE, RESPIRATORY SYSTEM AND
SKIN REACTION. (US) Avoid contact with skin and eyes. Do not 2. Process each food sample using procedures appropriate for that
breathe dust. Wear suitable protective clothing. Keep container sample.
tightly closed. Test Procedure
FIRST AID: In case of contact with eyes, rinse immediately with A number of methods and incubation conditions may be used for
plenty of water and seek medical advice. After contact with skin, detecting and isolating Listeria on PALCAM Medium. In their original
wash immediately with plenty of water. If inhaled, remove to fresh work, van Netten et al. recommended incubation at 37°C for 48 hours
air. If not breathing, give artificial respiration. If breathing is diffi- under microaerophilic conditions.1 AFNOR, HPB and IDF methods
cult, give oxygen. Seek medical advice. If swallowed seek medical for detecting Listeria in foods and dairy products are listed below.
advice immediately and show this container or label. Consult guidelines appropriate to your country and sample type.
Storage AFNOR Method for Foods2
Store PALCAM Medium Base below 30°C. The dehydrated medium 1. Pre-enrich the sample in Demi-Fraser Broth. Incubate at 30°C
is very hygroscopic. Keep container tightly closed. for 18-24 hours. Subculture onto Oxford Medium or PALCAM
Store PALCAM Antimicrobic Supplement at 2-8°C. Medium.
Store the rehydrated supplement and prepared medium at 2-8°C. 2. Transfer 0.1 ml of the pre-enrichment culture into 10 ml of Fraser
Broth and incubate at 37°C for 48 hours. Subculture onto Oxford
Expiration Date Medium or PALCAM Medium after 18-24 and 42-48 hours of
The expiration date applies to the product in its intact container when incubation.
stored as directed. Do not use a product if it fails to meet specifications 3. After the required incubation, examine for presumptive Listeria
for identity and performance. colonies.
4. Confirm the identity of each presumptive Listeria isolate by 5. Confirm the identity of each presumptive Listeria isolate by
biochemical and/or serological testing. biochemical and/or serological testing.
IDF Method for Milk and Milk Products3 Results
1. Enrich the sample in Modified Listeria Enrichment Broth. On PALCAM Medium, colonies of Listeria appear gray-green with
Incubate at 30 ± 1°C for 48 ± 2 hours. a black precipitate following inoculation and incubation at 35°C
2. Subculture onto Oxford Medium (and onto PALCAM Medium, if for 24-48 hours under aerobic or microaerophilic conditions.
desired). Incubate at 37 ± 1°C for 48 ± 2 hours. Confirmation of the presence of Listeria is made following
3. After the required incubation, examine for presumptive Listeria subculture onto appropriate media and biochemical/serological
colonies. identification.2,3 Colonies of mannitol-fermenting organisms such
4. Subculture five presumptive colonies (or all of the colonies if there as staphylococci, which may grow on this medium, appear yellow
are less than five) from each isolation medium onto Tryptic Soy with a yellow halo.
Agar with 0.6 % Yeast Extract.
5. Confirm the identity of each presumptive Listeria isolate by
References
biochemical and/or serological testing. 1. Van Netten, P., I. Perales, A. Van de Moosalijk, G. D. W.
Curtis, and D. A. A. Mossel. 1989. Liquid and solid selective
Health Canada Method for Foods and Environmental Samples4 differential media for the detection and enumeration of
1. Enrich the sample in Listeria Enrichment Broth (LEB). Incubate at L. monocytogenes and other Listeria spp. Int. J. of Food
30°C for 48 hours. Microbiol. 8:299-317.
2. Transfer 0.1 ml of the primary enrichment broth culture into 9.9 ml 2. L’association française de normalisation (AFNOR). 1993. Food
of modified Fraser Broth. Incubate at 35°C for 24-48 hours. (If Microbiology- Detection of Listeria monocytogenes-Routine
desired, the LEB culture may also be streaked onto Oxford Method, V 08-055. AFNOR, Paris.
Medium [OXA] and lithium chloride-phenylethanol-moxalactam 3. International Dairy Federation. 1990. Milk and milk products-
agar [LPM], modified Oxford medium [MOX] or PALCAM Detection of Listeria monocytogenes. IDF Provisional International
medium [PAL]. Incubate LPM at 30°C for 24-48 hours and OXA, Standard no. 143. International Dairy Federation, Brussels.
MOX and PAL at 35°C for 24-48 hours.)
4. Farber, J. M., D. W. Warburton, and T. Babiuk. 1994. Isolation
3. Examine modified Fraser broth for reactions. Subculture all positive
of Listeria monocytogenes from all food and environmental
cultures (black, dark brown or dark green) after 24 and 48 hours
samples. Health Protection Branch Ottawa, MFHPB-30.
of incubation onto OXA and LPM, MOX or PAL, streaking for
Polyscience Publications, Quebec.
isolation. Incubate LPM at 30°C for 24-48 hours and OXA, MOX
and PAL at 35°C for 24-48 hours. If desired, all negative modified Packaging
Fraser broth cultures (straw color) may be subcultured onto OXA
and LPM, MOX or PAL to facilitate recovery of esculin-negative PALCAM Medium Base 500 g 0636-17
strains of L. monocytogenes. 2 kg 0636-07
4. Examine for presumptive Listeria colonies. Examine LPM under PALCAM Antimicrobic Supplement 3 x 10 ml 0637-57*
oblique lighting positioned at a 45° angle relative to the surface of *Store at 2-8°C
the plate.
Bacto PKU Test Agar . Bacto PKU Test Agar w/o Thienylalanine
®
In the PKU Test procedure, PKU Test Agar containing thienylalanine Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
or PKU Test Agar w/o Thienylalanine with added thienylalanine are Ammonium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
inoculated with a suspension of B. subtilis ATCC® 6633. Filter paper Ammonium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
disks saturated with infant blood and control disks impregnated with Sodium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
known concentrations of L-phenylalanine (2,4,6,8,12 and 20 mg%) are Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g
Manganese Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005 g
applied to the surface of the medium. After incubation at 35°C for
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005 g
12-16 hours, the zones of growth around the test disks are compared
Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0025 g
to the zones around the control disks. A growth zone around the test B2 Thienylalanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0033 g
disk comparable to the zone around the 4 mg% or higher disk is a Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
presumptive positive indication of phenylketonuria. A positive result Final pH 7.0 ± 0.2 at 25°C
must be repeated using a duplicate test disk and a chemical or
spectrofluorometric procedure.7,8 PKU Test Agar w/o Thienylalanine
Formula Per Liter
Principles of the Procedure L-Glutamic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
PKU Test Agar and PKU Test Agar w/o Thienylalanine are defined DL-Alanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
minimal media containing the factors necessary for B. subtilis growth Bacto Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
under appropriate conditions. ß-2-thienylalanine is an inhibitor Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
of B. subtilis growth. PKU Test Agar contains the inhibitor, Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
ß-2-thienylalanine; PKU Test Agar w/o Thienylalanine does not, Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
requiring the user to add ß-2-thienylalanine to the medium. Ammonium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Phenylalanine supplied from a PKU-positive patient specimen will Ammonium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
overcome the inhibitory action of ß-2-thienylalanine. Sodium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g
Formula Manganese Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005 g
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005 g
PKU Test Agar
Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0025 g
Formula Per Liter Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
L-Glutamic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
Final pH 7.0 ± 0.2 at 25°C
DL-Alanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
Bacto Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g Bacillus Spore Suspension No. 2
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Standardized, stable suspension of Bacillus subtilis ATCC® 6633
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g containing 1.2 to 1.8 x 108 spores/ml.
Precautions
1. For Laboratory Use.
User Quality Control 2. PKU Test Agar
Identity Specifications PKU Test Agar w/o Thienylalanine
PKU Test Agar, PKU Test Agar w/o Thienylalanine HARMFUL. POSSIBLE RISK OF IRREVERSIBLE EFFECTS.
Dehydrated Appearance: Light beige to beige, free-flowing, IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN.
homogeneous. (US) Avoid contact with skin and eyes. Do not breathe dust. Wear
Solution: 5.0% solution, soluble in distilled or suitable protective clothing. Keep container tightly closed.
deionized water on boiling. Solution TARGET ORGAN(S): Skin, Lungs.
is light amber, very slightly to slightly FIRST AID: In case of contact with eyes, rinse immediately with
opalescent with a slight precipitate. plenty of water and seek medical advice. After contact with skin,
Prepared Medium: Light amber, very slightly to slightly wash immediately with plenty of water. If inhaled, remove to fresh
opalescent with a slight precipitate. air. If not breathing, give artificial respiration. If breathing is diffi-
Reaction of 5.0% cult, give oxygen. Seek medical advice. If swallowed seek medical
Solution at 25°C: pH 7.0 ± 0.2 advice immediately and show this container or label.
Subtilis Spore Suspension No. 2 3. Bacillus Spore Suspension No. 2
Appearance: White, opalescent, homogeneous CAUTION. While spore suspensions are not considered to be
suspension. pathogens, they are, nevertheless, live organisms. Never use mouth
Cultural Response pipetting. Always use some type of pipetting aid.
PKU Test Agar, PKU Test Agar w/o Thienylalanine 4. Follow proper established laboratory procedures in handling and
Prepare the final medium per label directions. Apply PKU disposing of infectious materials.
Standard Disks. Incubate at 35 + 2°C for 12-16 hours. Measure
zones of growth around each PKU Standard Disk. Zones of Storage
growth should increase in size comparable to the increasing Store the dehydrated medium below 30°C. The dehydrated medium is
concentration of phenylalanine in the Standard Disks. very hygroscopic. Keep container tightly closed.
Store Subtilis Spore Suspension No. 2 at 2-8°C.
Expiration Date 5. Compare zones of growth around the patient disks to those around
The expiration date applies to the product in its intact container when the control disks to determine the approximate concentration of
stored as directed. Do not use a product if it fails to meet specifications phenylalanine in the blood.
for identity and performance. Results
Procedure Growth zone diameters around the control disks are related to the
concentration of phenylalanine in the disks. A zone of growth may or
Materials Provided may not be present around the test disks depending on the presence or
PKU Test Agar absence of phenylalanine in the test specimen. The culture medium
PKU Test Agar w/o Thienylalanine outside the zones of growth will be comparable to an inoculated and
Subtilis Spore Suspension No. 2 incubated plate to which no disks have been applied.
Materials Required but not Provided Compare the zone of growth around a test disk to the zone around the
ß-2-thienylalanine (use with PKU Test Agar w/o Thienylalanine) standard disk containing 4 mg% phenylalanine. If the test zone is equal
PKU Standard Disks to or larger than the 4 mg% control zone, the test result is a presumptive
Blood test forms with Lancet positive and should be confirmed using a second sample. If the second
Disk test pattern for 150 mm Petri dish sample gives a similar result, determine the serum phenylalanine con-
150 mm Petri dishes centration by either a chemical10 or a spectrofluorometric procedure.
Forceps
Alcohol sponges
Limitations of the Procedure
1. Collect the blood sample with care. The sample must saturate the
Glassware
paper. Do not allow contact between the absorbent specimen card
Distilled or deionized water
and the collector’s hands.
Autoclave
Incubator (35°C) 2. Autoclaved samples must be dry before use.
3. PKU Test Agar must not be overheated. Bring to a boil and mix
Method of Preparation gently during heating. DO NOT AUTOCLAVE.
1. Suspend 50 grams in 1 liter distilled or deionized water. 4. Do not add spores if the temperature of the medium is above 55°C.
2. Heat to boiling to dissolve completely. Simmer for 5 minutes. Distribute the spores uniformly in the medium without creating
3. PKU Test Agar w/o Thienylalanine, only: Add 1 ml of 0.33% bubbles.
ß-2-thienylalanine solution per liter after simmering the medium; 5. Place the Petri dish on a horizontal surface while pouring the
mix thoroughly. medium to ensure an even depth of agar and a uniform distribution
4. Dispense 150 ml amounts into flasks. of spores throughout the plate.
5. Aseptically add 1 ml Subtilis Spore Suspension No. 2 to each 150 ml 6. Test results at the 4 and 6 mg% levels are questionable and should
aliquot at 50-55°C. Mix thoroughly to uniformly distribute the spores. be repeated with a second test sample and the results confirmed by
a quantitative procedure.
Specimen Collection and Preparation
1. Obtain the sample at least 48 hours after the first milk feeding. 7. Take care when opening ampules containing B. subtilis spores.
Autoclave the emptied ampules at 121-124°C for 20 minutes.
2. Collect a venous blood sample by heel puncture following estab-
lished collection technique.9 Obtain sufficient blood to fill each 8. Infants who are tested before 24 hours of age should have a repeat
circle by a single application of the specimen card to the drop of test performed by 2 weeks of age.
blood. Completely saturate the entire circle to ensure accuracy. 9. A negative test of an infant on antibiotics should be reconfirmed
Allow the blood sample to air dry. after antibiotic therapy is terminated. Antibiotics present in the
3. Punch a 1/4" disk from one of the blood spots and place it into a blood sample are usually inactivated by the autoclaving procedure,
labeled, clean, dry vial or place the entire specimen card on a wire but could be a source of error because some antibiotics will inhibit
rack in the autoclave. the growth of B. subtilis.11
4 Autoclave the patient disks for exactly three minutes at 121°C. 10. False-negative tests can result from the submission of an inadequate
Remove the disks promptly after the temperature has dropped sample, or if the patient has recently been exchange-transfused, or
below 100°C. Do not use the disks until they are dry. if the patient has an insufficient dietary protein load.11
5. Follow manufacturer’s instructions for preparation of the control disks. 11. False-positive results can occur.12
5. Demain, A. L. 1958. J. Bact. 75:517. 11. Nichols, Michael J. 1994. Tips on technology. MLO. 26:11-12.
6. Guthrie, R., and A. Susi. 1963. A simple phenylalanine method 12. Kirkman, H. N. , C. L. Carroll, E. G. Moore, et al. 1982.
for detecting phenylketonuria in large populations of newborn Fifteen-year experience with screening for phenylketonuria with an
infants. Pediatrics 32:338-343. automated fluorometric method. Am. J. Hum. Genet. 34:743-752.
7. Ambrose, J. A., et al. 1967. Clin. Chem. Acta. 15:493.
Packaging
8. Ambrose, J. A. 1969. Clin. Chem. 15:15.
PKU Test Agar 500 g 0980-17
9. National Committee for Clinical Laboratory Standards. 1992.
Blood collection on filter paper for neonatal screening programs, PKU Test Agar w/o Thienylalanine 500 g 0474-17
2nd ed.; Approved Standard. LA4-A2, vol. 12, no. 13. Wayne, PA. Subtilis Spore Suspension No. 2 25 x 1 0981-36
10. LaDu, B. N., and P. J. Michael. 1960. J. Lab. Clin. Med. 55:491. 100 x 1 0981-84
PPLO Media
Bacto PPLO Agar . Bacto PPLO Broth w/o CV . Bacto
®
2. Autoclave at 121°C for 15 minutes. Cool medium to 50-60°C. 4. Craven, R. B., R. P. Wenzel, A. M. Calhoun, J. O. Hendley,
4. Aseptically add 300 ml Mycoplasma Supplement or 300 ml B. H. Hamory and J. M. Gwaltney, Jr. 1976. Comparison of the
Mycoplasma Supplement S to the sterile medium. Mix well. sensitivity of two methods for isolation of Mycoplasma
pneumoniae. J. Clin. Microbiol. 4:225-226.
5. Dispense as desired.
5. Gregory, J. E., and K. R. Cundy. 1970. Mycoplasma recovery
Mycoplasma Supplement from the male genitourinary tract: voided urine versus the urethral
Mycoplasma Supplement S swab. Appl. Microbiol. 19:268- 270.
1. Rehydrate with 30 ml sterile distilled or deionized water. 6. Adler, H. E., and A. J. Da Massa. 1967. Use of formalinized
2. Rotate gently to dissolve. Mycoplasma gallisepticum antigens and chicken erythrocytes in
3. Add 30 ml (the contents of one vial) to 70 ml sterile PPLO Agar or hemagglutination and hemagglutination-inhibition studies. Appl.
PPLO Broth w/o CV. Microbiol. 15:245-248.
Specimen Collection and Preparation 7. Leland, D. S., M. A. Lapworth, R. B. Jones, and M. L. V. French.
1982. Comparative evaluation of media for isolation of Ureaplasma
Obtain and process specimens according to the techniques and urealyticum and genital Mycoplasma species. J. Clin. Microbiol.
procedures established by laboratory policy. 16:709-714.
Test Procedure 8. Hayflick, L. 1965. Tissue cultures and mycoplasmas. Tex. Rep.
For a complete discussion of the isolation and identification of Biol. Med. 23:285-303.
Mycoplasma spp. from clinical specimens, refer to appropriate 9. Chanock, R. M., L. Hayflick, and M. F. Barlie. 1962. Growth on
procedures outlined in the references.12,13,14 artificial medium of an agent associated with atypical pneumonia
and its identification as a pleuropneumonia-like organism. Proc.
Results Nat. Acad. Science 48:41.
PPLO Agar 10. Hayflick, L. 1968. Personal communication.
PPLO colonies are round with a dense center and a less dense periphery, 11. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
giving a “fried egg” appearance on PPLO Agar. Vacuoles, large bodies Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.
characteristic of Mycoplasma spp., are seen in the periphery. St. Louis, MO.
Colonies vary in diameter from 10 to 500 microns (0.01-0.5 mm) and 12. Kenny, G. E. 1985. Mycoplasmas, p. 407-411. In E. H. Lennette,
penetrate into the medium. A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.). Manual of
clinical microbiology, 4th ed. American Society for Microbiology,
Limitations of the Procedure Washington, D.C.
1. Since the nutritional requirements of organisms vary, some strains may 13. Taylor-Robinson, D. 1995. Mycoplasma and Ureaplasma,
be encountered that fail to grow or grow poorly on this medium. p. 652-661. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
2. Thallium acetate can partially inhibit some mycoplasmas.12 Tenover, and R. H. Yolken (ed.). Manual of clinical microbiology,
6th ed. American Society for Microbiology, Washington, D.C.
References
14. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures hand-
1. Morton, H. E., P. F. Smith, and P. R. Leberman. 1951. Venereal
book, vol. 1. American Society for Microbiology, Washington, D.C.
diseases. Am. J. Syphilis Gonorrh. 35:361.
2. Morton, H. E., and J. G. Lecce. 1953. Selective action of thallium Packaging
acetate and crystal violet for pleuropneumonia like organisms of PPLO Agar 500 g 0412-17
human origin. J. Bacteriol. 66:646-649. PPLO Broth w/o CV 500 g 0554-17
3. Chanock, R. M., W. D. James, H. H. Fox, H. C. Turner, 10 kg 0554-08
M. A. Mufson, and l. Hayflick. 1962. Growth of Eaton PPLO in
Mycoplasma Supplement 6 x 30 ml 0836-68
broth and preparation of complement fixing antigen. Soc. Exp.
Biol. Med. 110:884-889. Mycoplasma Supplement S 6 x 30 ml 0837-68
5. Baron, E. J., L. R. Peterson, S. M. Finegold. 1994. Bailey & R. H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc., American Society for Microbiology, Washington, D.C.
St. Louis, MO.
Packaging
6. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and Pagano Levin Base 500 g 0141-17
4. Take precaution to keep sterilization and cooling conditions uniform saline. After the third wash, resuspend the cells with sterile 0.85%
throughout the assay. saline and adjust to a turbidity of 40-45% transmittance when read on
5. Follow proper established laboratory procedures in handling and a spectrophotometer at 660 nm. Aseptically inoculate each assay tube
disposing of infectious materials. with one drop of the cell suspension.
Precautions
1. For Laboratory Use.
User Quality Control 2. Great care must be taken to avoid contamination of media or
Identity Specifications glassware in microbiological assay procedures. Extremely small
Dehydrated Appearance: Very light beige, homogeneous, amounts of foreign material may be sufficient to give erroneous
tendency to clump. results. Scrupulously clean glassware free from detergents and other
Solution: 3.65% (single strength) or 7.3% chemicals must be used. Glassware must be heated to 250°C for at
(double strength) solution, soluble least 1 hour to burn off any organic residues that might be present.
in distilled or deionized water on 3. Take precautions to keep sterilization and cooling conditions
boiling 2-3 minutes. Single-strength
solution is light amber, clear, may uniform throughout the assay.
have a slight precipitate. 4. Follow proper established laboratory procedures in handling and
Prepared Medium: (Single strength) light amber, clear, disposing of infectious materials.
may have a very slight precipitate.
Storage
Reaction of 3.65%
Solution at 25°C: pH 6.7 ± 0.1 Store the dehydrated medium at 2-8°C. The dehydrated medium is very
hygroscopic. Store in a container with calcium chloride or other desic-
Cultural Response cant. Keep container tightly closed.
Prepare Pantothenate Medium AOAC USP per label directions.
Test the medium by creating a standard curve using a Expiration Date
pantothenic acid reference standard at 0.0 to 0.05 µg per The expiration date applies to the product in its intact container when
10 ml. The medium supports the growth of Lactobacillus stored as directed. Do not use a product if it fails to meet specifications
plantarum ATCC® 8014 when prepared in single strength and
supplemented with pantothenic acid. for identity and performance.
Bacto Peptamin
®
Bacto Peptone®
Storage
User Quality Control Store the products below 30°C. The dehydrated product is very hygro-
Identity Specifications scopic. Keep container tightly closed.
Bacto Peptone
Dehydrated Appearance: Tan, free-flowing granules. Expiration Date
Solution: 1%, 2% and 10% solutions are soluble The expiration date applies to the product in its intact container when
in distilled or deionized water: stored as directed. Do not use a product if it fails to meet specifications
1%- Light amber, clear, no precipitate; for identity and performance.
2%- Light to medium amber, clear,
no precipitate; Procedure
10%- Medium to dark amber, clear Materials Provided
to very slightly opalescent, may Bacto Peptone
have a very slight precipitate.
Peptone Bacteriological Technical
Reaction of 1%
Solution at 25°C: pH 6.8-7.2 Materials Required But Not Provided
Peptone Bacteriological Technical Materials vary depending on the medium being prepared.
Dehydrated Appearance: Tan, free-flowing, granules.
Method of Preparation
Solution: 1%, 2% and 10% solutions are soluble
in distilled or deionized water: Refer to the final concentration of Bacto Peptone or Peptone Bacterio-
1%- Very light to light amber, clear; logical Technical in the formula of the medium being prepared. Add
2%- Light-medium amber, clear; Bacto Peptone or Peptone Bacteriological Technical as required.
10%- Medium-dark amber, clear to Specimen Collection and Preparation
very slightly opalescent.
Obtain and process specimens according to the techniques and
Reaction of 1%
Solution at 25°C: pH 6.3-7.6 procedures established by laboratory policy.
2. Follow proper established laboratory procedures in handling and 1% Phenol Red solution
disposing of infectious materials. Indole Test strips
Storage Method of Preparation
Store the dehydrated medium below 30°C. The dehydrated medium is 1. Dissolve 15 grams in 1 liter distilled or deionized water with
very hygroscopic. Keep container tightly closed. warming and frequent agitation.
2. Autoclave at 121°C for 15 minutes.
Expiration Date
The expiration date applies to the product in its intact container when For Determining Carbohydrate Fermentation Patterns
stored as directed. Do not use a product if it fails to meet specifications 1. Add 1.8 ml 1% phenol red solution to 1 liter rehydrated Peptone
for identity and performance. Water. Mix thoroughly.
2. Dispense into test tubes containing inverted Durham vials.
Procedure 3. Autoclave at 121°C for 15 minutes.
Materials Provided 4. Aseptically add sufficient sterile carbohydrate solution to yield a
Peptone Water 1% final concentration. Rotate each tube to thoroughly distribute
the carbohydrate.
Materials Required but not Provided
Glassware Specimen Collection and Preparation
Distilled or deionized water Obtain and process specimens according to the techniques and procedures
Autoclave established by laboratory policy.
Incubator (35°C) Test Procedure
Tubes with closures
For Performing Carbohydrate Fermentation
Fermentation tubes
Carbohydrate solutions 1. Inoculate tubes with test organism.
2. Incubate tubes at 35 ± 2°C for 18-48 hours.
3. Observe for color change.
User Quality Control For Performing the Indole Test
Identity Specifications 1. Using aseptic technique, suspend an Indole Test Strip 10 mm above
Dehydrated Appearance: Cream-white to light tan, the surface of a 24 or 48 hour culture.
free-flowing, homogeneous. 2. Incubate at 37°C for 5-30 minutes.
Solution: 1.5% solution, soluble in distilled or Results
deionized water on warming with
frequent agitation. Solution is light For Determining Carbohydrate Fermentation Patterns
amber, clear to very slightly opalescent. Acid is produced when carbohydrates are fermented. This is indicated
Reaction of 1.5% by a yellow color in the medium. Gas production is indicated by the
Solution at 25°C: pH 7.2 ± 0.2 presence of gas bubbles in the fermentation tube.
Cultural Response For performing the Indole Test
Growth/Indole Reaction Observe for the formation of a violet color on the strip which indicates
Prepare Peptone Water per label directions. Inoculate and a positive test for indole production.
incubate at 35 ± 2°C for 18-48 hours. Indole reaction is read
using Indole Test Strips (1627). Limitations of the Procedure
INOCULUM INDOLE 1. Medium is pink in color when hot but becomes colorless upon cooling.
ORGANISM ATCC® CFU GROWTH REACTION
2. Vibrio spp. should not be incubated longer than 18-20 hours. Longer
Escherichia coli 25922* undiluted good positive incubation may cause the development of suppressed forms.3
Carbohydrate Fermentation References
Prepare Peptone Water per label directions with the addition 1. MacFaddin, J. F. 1985. Media for isolation-cultivation-
of phenol red and dextrose. Inoculate and incubate at 35 ± 2°C
for 18-48 hours. identification-maintenance of medical bacteria, vol. 1, p. 610-612.
INOCULUM ACID Williams & Wilkins, Baltimore, MD.
ORGANISM ATCC® CFU GROWTH PRODUCTION
2. Balows, A., W. J. Hausler, K. L. Herrmann, H. D. Isenberg,
Escherichia coli 25922* 100-1,000 good positive and H. J. Shadomy (ed.). 1991. Manual of clinical microbiology,
Staphylococcus aureus 25923* 100-1,000 good positive 5th ed. American Society for Microbiology, Washington, D.C.
The cultures listed are the minimum that should be used for 3. Finegold, S. M., and W. Martin. 1982. Bailey and Scott’s
performance testing. diagnostic microbiology, 6th ed. St. Louis
*These cultures are available as Bactrol™ Disks and should
be used as directed in Bactrol Disks Technical Information. Packaging
Peptone Water 500 g 1807-17
Phenol Red Dextrose Broth, Phenol Red Lactose Broth, 2. Incubate at 35 ± 2°C for 4-18 hours with caps loosened.
Phenol Red Mannitol Broth, Phenol Red Saccharose Broth 3. Examine tubes for growth, acid production, and gas production
1. Suspend 21 grams of the appropriate Phenol Red Carbohydrate (if fermentation vials are used).
Broth in 1 liter distilled or deionized water and stir to
dissolve completely. Results
2. For better growth of fastidious organisms (such as streptococci, A yellow color of the medium indicates a positive reaction for
pneumococci, and gonococci) add 1 gram of Bacto Agar per liter carbohydrate fermentation. If fermentation vials are used, bubbles in
of medium and dissolve by boiling prior to sterilizing. the inverted vials are an indication of gas production. The presence of
a single bubble is recorded as positive for the production of gas.10
3. Dispense into tubes. To detect gas production, place inverted
fermentation tubes in the tubes of medium. Limitations of the Procedure
4. Autoclave at 121°C for 15 minutes. 1. The addition of some carbohydrates to the basal medium may
If the media are not used the same day they are sterilized, prior to use, result in an acid reaction. In this case, it is suggested that
place the medium in flowing steam or a boiling water bath for a few 0.1N sodium hydroxide be added drop by drop to restore the original
minutes to drive off dissolved gases. Allow to cool without agitation. color. Take care not to make the medium too alkaline for true
fermentation to occur within the usual incubation period.
Specimen Collection and Preparation
2. To ensure accuracy of interpretation, uninoculated control tubes
Refer to appropriate references for specimen collection and preparation.
and/or inoculated Phenol Red Broth Base control tubes should be
Test Procedure run in parallel with the fermentation tests.
1. Inoculate tubes with one drop of a diluted pure culture.
other Enterobacteriaceae based on the ability of Proteus and Materials Required But Not Provided
Providencia to deaminate phenylalanine to phenylpyruvic acid by Glassware
enzymatic activity.2 Bynae modified this method by incorporating Autoclave
phenylalanine in the medium used to grow the organisms. Ewing,
Incubator (35°C)
Davis and Reavis4 simplified the Bynae formulation by omitting
SpotTest™ Ferric Chloride Reagent (3557) or 8-12% ferric chloride
proteose peptone. Phenylalanine Agar is prepared according their
formula. 0.1 N HCl
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
Storage Results
Store the dehydrated medium at 2-8°C. The dehydrated medium is Examine plates for growth and hemolysis. Perform additional bio-
very hygroscopic. Keep container tightly closed. Store prepared chemical testing to identify the organism.
medium at 2-8°C.
Limitations of the Procedure
Expiration Date 1. Some gram-positive cocci may be slightly inhibited and may
The expiration date applies to the product in its intact container when require further incubation (to 48 hours) for sufficient growth to be
stored as directed. Do not use a product if it fails to meet specifications evident.6
for identity and performance. 2. Subculture gram-positive colonies onto Tryptic Soy Agar (TSA),
Procedure Selenite Broth and other biochemical media for definitive
identification.6
Materials Provided
3. Pseudomonas aeruginosa is not inhibited on this medium.7
Phenylethanol Agar
Materials Required But Not Provided References
Glassware 1. Brewer, J. H., and B. D. Lilley. 1949. Paper presented at the
Autoclave December meeting of the Maryland Association of Medical and
Incubator (35°C) Public Health Laboratories.
Waterbath (45-50°C) (optional) 2. Lilley, B. D., and J. H. Brewer. 1953. The selective antibacterial
Sterile defibrinated blood (optional) action of phenylethylalcohol. J. Pharm. Assoc. 42:6.
3. Baron, E. J., and S. M. Finegold. 1990. Bailey & Scott’s
Method of Preparation diagnostic microbiology, 8th ed. The C.V. Mosby Company,
1. Suspend 35.5 grams in 1 liter distilled or deionized water. St. Louis, MO.
2. Boil to dissolve completely. 4. Isenberg, H. D. 1992. Clinical microbiology procedures handbook.
3. Autoclave at 121°C for 15 minutes. American Society for Microbiology, Washington, D.C.
4. OPTIONAL: To prepare blood agar, aseptically add 5% sterile 5. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
defibrinated blood to the cooled medium at 45-50°C. Mix well. R. H. Yolken (ed.). 1995. Manual of clinical microbiology,
Specimen Collection and Preparation 6th ed. ASM Press, Washington, D.C.
1. Collect specimens or food samples in sterile containers or with 6. MacFaddin, J. F. 1985. Media for isolation-cultivation-
sterile swabs and transport immediately to the laboratory following identification-maintenance of medical bacteria, vol. 1. Williams &
recommended guidelines.3-5 Wilkins, Baltimore, MD.
2. Process each specimen using procedures appropriate for that 7. Washington, J. A., Jr. 1981. Laboratory procedures in clinical
specimen or sample.3-5 microbiology. Springer-Verlag, New York.
Test Procedure
1. Inoculate plates with test specimens. Streak to obtain isolated
Packaging
colonies. Phenylethanol Agar 100 g 0504-15
2. Incubate plates at 35 ± 2°C under 5-10% CO2 for 18-24 hours and, 500 g 0504-17
if necessary, 40-48 hours. 2 kg 0504-07
Phytohemagglutinins
Bacto Phytohemagglutinin M . Bacto Phytohemagglutinin P
®
dog, cat, chicken, duck, mouse, rat, sheep, horse, pig, frog and guinea Phytohemagglutinin P is a sterile, desiccated, purified, highly potent
pig. Phytohemagglutinin has been used to obtain the plasma suspension protein phytohemagglutinin from which the polysaccharide moiety has
of trypanosomes from the blood of infected rats.5 been removed.
Mitogenic Activity Precautions
Nowell6 discovered that phytohemagglutinin M initiates mitosis in
1. For Laboratory Use.
cultures of lymphocytes isolated from peripheral blood. Later,
phytohemagglutinin P was also shown to possess this property. The 2. Observe universal blood and body fluid precautions in the handling
application of this technique is important in the characterization of and disposing of specimens.18.19
chromosomes. A procedure using phytohemagglutinin-stimulated 3. Practice the following routine laboratory safety procedures:
lymphoblasts has been used to cultivate human immunodeficiency Do not pipette by mouth.
virus type 1 (HIV-1) from infected individuals by cocultivation Use aseptic technique and established laboratory procedures in
cultures.7 Human T-lymphocytes have been activated by phytohemag- handling and disposing of infectious materials.
glutinin to the blastic killer-cell state in preparation for in-vivo
immunotherapy trials in donor cancer patients.8 Storage
A simplified procedure for lymphocyte mitogenesis was developed by Store desiccated Phytohemagglutinin M and Phytohemagglutinin P at
Moorhead, Nowell, Mellman, Batipps and Hungerford,9 in which the 2-8°C. The rehydrated solutions are stable for at least 2 weeks at -20°C.
cultures were routinely allowed to incubate for 3 days (65-70 hours).
Their method incorporated the hypotonic treatment developed by
Expiration Date
Hughes10 and Hsu and Pomerat.11 The flame drying of slides by Scherz12 The expiration date applies to the product in its intact container when
and the staining procedure by Rothfels and Siminovitch13 were helpful stored as directed. Do not use a product if it fails to meet specifications
contributions in this procedure. Staining of chromosomes by one of for identity and performance.
many methods produces characteristic bands. For more information
on chromosome staining, please refer to appropriate references.14-17
Procedure
Materials Provided
Principles of the Procedure Phytohemagglutinin M
Both Phytohemagglutinin M and P will agglutinate the erythrocytes of Phytohemagglutinin P
all human blood types, and those of animals. The rehydrated P-form
has approximately 40 times more hemagglutinating potency than the Materials Required But Not Provided
M-form. Both forms will also stimulate the lymphocytes of peripheral Sterile syringe
blood to undergo mitosis in vitro. Sterile test tube
30 units sterile heparin dissolved in 0.85% sterile saline
Reagents RPMI Medium #1640
Phytohemagglutinin M is a stable, nontoxic, desiccated 700 units of Penicillin
mucophytohemagglutinin. 700 µg Streptomycin
Colchicine (10-5 Molar)
Hanks Balanced Salt Solution
User Quality Control Methanol, reagent grade
Identity Specifications Glacial Acetic Acid, reagent grade
Deionized water
Phytohemagglutinin M or P
Lyophilized Appearance: White, porous lyophilized cake. Giemsa Stain
Pipettes, 0.1 ml, 1 ml, 5 ml
Solution Appearance: Contents of 1 vial, soluble in 5 ml
sterile distilled or deionized water Water aspirator
within 2 minutes. Solution is Pasteur pipettes
colorless, clear to slightly opalescent. Centrifuge
Performance Response Incubator, 35°C
When reconstituted with 5 ml sterile distilled or deionized Microscope slides
water, 0.1 ml Phytohemagglutinin M or 0.01 ml Phytohemag- Microscope (12.5X eyepiece with 10X low power, 40X high dry, and
glutinin P is added to 7 ml RPMI #1640 Medium containing 100X oil immersion objectives)
the lymphocytes from 5 ml heparinized human blood. The
mitogenicity test is performed using the above components Reagent Preparation
and procedures with 4 samples of human blood. A mitotic Phytohemagglutinin M or the sterile Phytohemagglutinin P is rehydrated
index of at least 75 should be obtained from the lymphocytes by adding 5 ml of sterile distilled or deionized water, or equivalent,
of each of the four samples of blood. A total of at least 400 and rotating gently to mix contents thoroughly. The solutions are
should be obtained from the sum of all four cultures.
approximately 1% in 0.85% saline. Both solutions contain approximately
50 mg protein per 5 ml.
Specimen Collection and Preparation 14. Add 3 ml of warm (35 ± 2°C) distilled water, in 1 ml portions,
For each culture, a 5 ml sample of blood is adequate. Draw the blood with momentary agitation after each addition to produce a
with a sterile syringe and immediately place in a sterile screw-capped hypotonic solution.
test tube containing 30 units of sterile heparin and mix thoroughly. 15. Incubate the suspension at 35 ± 2°C for 10 minutes only. The
Dissolve the heparin in 1 ml of a sterile 0.85% saline solution before exposure of the cells to this hypotonic, diluted Hanks Balanced
collecting the specimen. Start the agglutination and mitotic procedures Salt Solution should not exceed 10 minutes.
immediately, or they may be postponed for at least 24 hours, if the 16. Centrifuge the lymphocyte solution at 600-800 rpm for 6-8 minutes.
specimen is stored at 2-8°C. 17. Carefully aspirate off the supernatant.
Observe aseptic technique from the collection of the blood sample 18. Add slowly, without disturbing the button of cells, 4 ml of freshly
until the addition of the colchicine. prepared fixative consisting of 1 part glacial acetic acid and 3 parts
methanol (reagent grade only).
Test Procedure
19. Let the cells soak in the fixative for 15-30 minutes. Cells should be
Lymphocyte Separation and Inoculation
treated gently during this stage of fixation. At this point, cells may
1. Transfer 5 ml of blood containing 30 units of heparin to a sterile be stored overnight at 2-8°C.
screw-capped test tube under aseptic conditions.
20. Resuspend with the Pasteur pipette.
2. Add either 0.1 ml of rehydrated Phytohemagglutinin M or 0.0025 ml
21. Centrifuge at 600-800 rpm for 6-8 minutes, and carefully remove
of Phytohemagglutinin P to the 5 ml of heparinized blood, and mix
the supernatant by aspiration.
the contents by inverting several times.
22. Resuspend the cells in 4 ml fresh fixative with the Pasteur pipette,
3. Let the erythrocytes agglutinate at 25°C for 15-30 minutes.
and centrifuge at 600-800 rpm for 6-8 minutes. Repeat this step
4. Centrifuge the tube at 500 rpm for 2 minutes. Excessive centrifug- again if necessary to disperse clumps of cells.
ing must be avoided to prevent sedimentation of the lymphocytes.
23. Carefully aspirate the supernatant.
5. Transfer the hazy plasma-lymphocyte suspension (about 2 ml) by
24. Add 0.5-1.0 ml of fresh fixative to the button of cells and resus-
means of a sterile Pasteur pipette to 7 ml of a culture medium
pend with the Pasteur pipette to get a hazy suspension.
consisting of RPMI #1640 Medium, 700 units of Penicillin, 700
µg Streptomycin, and either 0.1 ml of Phytohemagglutinin M, Preparation of Slides
if the erythrocytes have been agglutinated with the M-form, or 0.01 25. Label clean microscope slides and place them in clean, chilled
ml of Phytohemagglutinin P, if the erythrocytes have been aggluti- distilled water.
nated with the P-form. The optimal concentration of lymphocytes 26. In rapid succession, shake the excess water off a chilled slide, wipe
in the culture is 1.0-1.2 X 106 per ml. If Phytohemagglutinin P and the water off its underside, add 3-4 drops of the cell suspension by
aseptic conditions are used, the antibiotics may be omitted. means of the Pasteur pipette, tip the slide several times to spread
Incubation of Culture the suspension, and ignite the fixative by bringing it momentarily
6. Incubate the culture in a vertical position at 35 ± 2°C with in contact with a flame. When the fixative is burned off, wave the
occasional swirling for 3-4 days. Care should be taken to maintain slide vigorously to hasten drying. The slide should not get hot, but
proper incubation temperature. A significant increase in mitotic drying should be accomplished as rapidly as possible.
index is often obtained by incubating 4 days instead of 3. It is very Staining of Slides
important to always maintain the proper pH range in the culture. Slides may be stained with Giemsa, orcein or other stains according to
The phenol red indicator should not become more acidic than a the method of Rothfels and Siminovitch.14 The procedure using
light amber nor more alkaline than a light pink. If the indicator Giemsa is given below.
becomes amber, loosen the cap for an hour or so to allow the
escape of CO2. This precaution is often most necessary at the 27. Dilute the 1 ml of stock Giemsa Stain (20X stock) with 19 ml of
beginning and end of the incubation. distilled water. The 1 ml of stock Giemsa Stain should be used the
same day it is diluted 20-fold with water.
7. End the mitosis by the addition of 1 ml of 10å5 molar colchicine,
and continue the incubation at 35 ± 2°C for another 4-6 hours. The 28. Place the slides in a small staining dish or Petri dish and cover
exposure of cells to the colchicine should not be less than 4 hours them with 20 ml of the staining solution for 10-20 minutes.
or more than 6 hours. 29. Rinse the slides gently in distilled water and air dry.
Harvesting and Fixation of Cells 30. Examine the slides under the microscope. The mitotic spreads may
be scanned at a total magnification of 125X, examined more closely
8. Transfer the entire culture to a graduated conical centrifuge tube
at 500X, or photographed under oil immersion at 1,000X. Slides
(15 ml) and centrifuge for 6-8 minutes at 600-800 rpm.
may be protected by cover slips and made permanent by conven-
9. Carefully aspirate off the supernatant fluid. tional procedures.
10. Add 5 ml of warm (35 ± 2°C) Hanks Balanced Salt Solution and Alternatively, the chromosomes may be treated by staining procedures
resuspend the cells in the centrifuge tube with a Pasteur pipette. to show G-banding. Refer to appropriate references for alternative
11. Centrifuge at 600-800 rpm for 6-8 minutes. staining procedures.18
12. Carefully aspirate off the supernatant with the pipette and add 1 ml
of Hanks Balanced Salt Solution.
Results
A mitotic index of at least 30 may be expected from the lymphocytes
13. Resuspend the packed cells with the Pasteur pipette.
from the heparinized peripheral blood of a healthy individual.
Limitations of the Procedure 10. Hughes, A. 1952. Some effects of abnormal tonicity on dividing
1. For mitotic investigations, avoid the following: cells in chick tissue cultures. Quart. J. Microscopic Sci. 93:207.
› Anticoagulants containing oxalates or phenols 11. Hsu, T. C., and C. M. Pomerat. 1953. Mammalian chromosomes
in vitro II. A method for spreading the chromosomes of cells in
› Cytotoxic antibiotics, drugs or heavy metals (Penicillin and
tissue culture. J. Hered. 44:23-29.
Streptomycin are acceptable.)
12. Scherz, R. G. 1962. Blaze drying, by igniting the fixative, for
› Hypertonic and hypotonic media except for the intentional
improved spreads of chromosomes in leukocytes. Stain. Tech.
swelling of the chromosomes
37:386.
› Irradiation of the patient or culture, which can produce “breaks”
13. Rothfels, K. H., and L. Siminovitch. 1958. An air-drying technique
in the chromosomes.
for flattening chromosomes in mammalian cells grown in vitro.
› Some plastic materials cause cytotoxic effects. Stain Tech. 33:73-77.
References 14. Bird, B. R., and F. T. Forrester. 1981. Basic Laboratory Tech-
1. Li, J. G., and E. E. Osgood. 1949. A method for the rapid niques in Cell Culture. U. S. Department of Health and Human
separation of leukocytes and nucleated erythrocytes from blood Services, CDC, Atlanta, GA.
or marrow with a phytohemagglutinin from red beans (Phaseolus 15. Freshney, R. I. 1983. Culture of animal cells: A manual of basic
vulgaris). Blood 4:670-675. technique. Alan R. Liss, Inc., New York, NY.
2. Takikawa, K., T. Ito, J. Kato, T. Yoshida, H. Kondo, and 16. Jones Brando, L. V. 1995. Cell culture systems, p. 158-165. In
I. Miyata. 1957. Studies on the isolation of granules and P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H.
mitrochondria of leukocytes. Acta Haemat. 18:179-184. Yolken (eds.), Manual of clinical microbiology, 6th ed. American
3. Chen, H. P., and G. K. Palmer. 1958. A method for isolating Society for Microbiology, Washington, D.C.
leukocytes. Am. J. Clin. Pathol. 30:567-569. 17. Gustashaw, K. M. 1991. Chromosome stains. In M. J. Barch (ed.),
4. Skoog, W. A., and W. S. Beck. 1956. Studies on the fibrinogen, The ACT Cytogenetics Laboratory Manual, 2nd ed. The Associa-
dextran, and phytohemagglutinin methods of isolating leukocytes. tion of Cytogenetic Technologists, Raven Press, Ltd., New York,
Blood 11:436-54. NY.
5. Yaeger, R. G. 1960. A method of isolating trypanosomas from 18. Centers for Disease Control. 1988. Update: universal precautions
blood. J. Parasitol. 46:288. for prevention of transmission of human immunodeficiency virus,
hepatitis B virus, and other blood borne pathogens in health-care
6.. Nowell, P. C. 1960. Phytohemagglutinin: an initiator of mitosis in
settings. Morbidity and Mortality Weekly Reports 37:377-382,
cultures of normal human leukocytes. Cancer Research 20:462-468.
387-388.
7. Clarke, L. M. (ed.). 1992. Viruses, Rickettsiae, Chlamydiae, and
19. Occupational Safety and Health Administration, U.S.
Mycoplasmas, p. 8.1.1-8.26.21. In H. D. Isenberg, (ed.), Clinical
Department of Labor. 1991. 29 CFR part 1910. Occupational
microbiology procedures manual, vol. 2. American Society for
exposure to blood borne pathogens; final rule. Federal Register
Microbiology, Washington, D.C.
56:64175-64182.
8. Frenster, J. H. 1976. Phytohemagglutinin-activated autochthonous
lymphocytes for systemic immunotherapy of human neoplasms. Packaging
Ann. NY. Acad. Sci. 277:45- 51. Phytohemagglutinin M 5 ml 0528-56*
9. Moorhead, P. S., P. C. Nowell, W. J. Mellman, D. M. Batipps, 6 x 5 ml 0528-57*
and D. A. Hungerford. 1960. Chromosome preparations of Phytohemagglutinin P 5 ml 3110-56*
leukocytes cultured from human peripheral blood. Exp. Cell. 6 x 5 ml 3110-57*
Res. 20:613.
*Store at 2-8°C
Standard Methods for the Examination of Dairy Products,2 Compendium Expiration Date
of Methods for the Microbiological Examination of Foods3 and the The expiration date applies to the product in its intact container product
Association of Official Analytical Chemists (AOAC)4 and the FDA when stored as directed. Do not use a product if it fails to meet
Bacteriological Analytical Manual.5 specifications for identity and performance.
Principles of the Procedure Procedure
Plate Count Agar contains Tryptone and Yeast Extract which provide
Materials Provided
the carbon and nitrogen sources required for growth of a wide variety
Plate Count Agar
of organisms. Dextrose is a source of fermentable carbohydrate
Standard Methods Agar
(energy source). Bacto Agar is a solidifying agent.
Materials Required but not Provided
Formula Glassware
Plate Count Agar
Distilled or deionized water
Standard Methods Agar
Autoclave
Formula Per Liter Waterbath (optional)
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g Method of Preparation
Bacto Dextrose (Glucose) . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g Plate Count Agar
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
1. Suspend 23.5 grams in 1 liter of distilled or deionized water.
Final pH 7.0 ± 0.2 at 25°C
2. Heat to boiling to dissolve completely.
Precautions 3. Autoclave at 121°C for 15 minutes.
1. For Laboratory Use.
Standard Methods Agar (prepared)
2. Follow proper, established laboratory procedures in handling and
disposing of infectious materials. 1. Loosen the caps on the bottles prior to heating.
2. Heat the medium in the autoclave for 7 minutes to melt the agar. A
Storage small solidified mass remains that can be melted by swirling the hot
Store Plate Count Agar below 30°C. The powder is very hygroscopic. agar. Cycle time depends on the number of bottles in the chamber.
Keep container tightly closed.
Uninoculated Pasteurized milk
Store Standard Methods Agar at 15-30°C. plate
Specimen Collection and Preparation of methods for the microbiological examination of foods, 3rd ed.
Obtain and process specimens according to the techniques and pro- American Public Health Association, Washington, D.C.
cedures established by laboratory policy. 4. Association of Official Agricultural Chemists. 1995. Official
methods of analysis, 16th ed. Association of Official Agricultural
Test Procedure Chemists, Washington, D.C.
1. Perform serial dilutions on samples (food, water) to be tested 5. Bandler, R., M. E. Stack, H. A. Koch, V. H. Tournas, and P. B.
using the heterotrophic (standard) plate count method. Select Mislivec. 1995. Yeasts, molds, and mycotoxins, p. 18.01-18.03.
dilutions that will yield plates with counts of 30-300 colonies. Bacteriological analytical manual, 8th ed. AOAC International,
2. Dispense a portion of each test dilution (e.g., 0.1 ml, 1.0 ml) into Arlington VA.
separate sterile Petri dishes. 6. Buchbinder, L., Y. Baris, and L. Goldstein. 1953. Further
3. Add 10-12 ml of tempered (45°C) Plate Count Agar to Petri dishes studies on new milk-free media for the standard plate count of dairy
containing test dilutions. products. Am. J. Public Health 43:869- 872.
4. Swirl the dishes to thoroughly mix the agar and test dilution. 7. Buchbinder, L., Y. Baris, E. Alff, E. Reynolds, E. Dillon,
5. Allow plates to cool and solidify. V. Pessin, L. Pincus, and A. Strauss.1951. Studies to formulate
6. Incubate at 32 ± 1°C for 48 hours. new media for the standard plate count of dairy products. Pub
Health Rep. 66:327-340.
Results 8. Prickett, P. S. 1928. Thermophillic and thermoduric microor-
Count colonies on all plates containing 30-300 colonies. Calculate ganisms with special reference to species isolated from milk: V.
bacterial count per milliliter of sample by multiplying the average Description of spore-forming types. Technical Bulletin. NY
number of colonies per plate by the reciprocal of the dilution used. State Agri. Exp. Station 147:5-58.
Report the count as CFU/ml. 9. Breed, R. S., P. A. Down, G. C. Supplee, P. S. Prickett, and G. J.
References Hucker. 1932. Methods for use in the bacteriological examination
of dry milk and related powders. J. Dairy Sci. 15:383-389.
1. Greenberg, A. E., L. S. Clesceri, and A. D. Eaton (ed.). 1992.
Standard methods for the examination of water and wastewater, Packaging
18th ed. American Public Health Association, Washington, D.C. Plate Count Agar 100 g 0479-15
2. Marshall, R. T. (ed.).1993. Standard methods for the examination 500 g 0479-17
of dairy products, 16th ed. American Public Health Association, 2 kg 0479-07
Washington, D.C. 10 kg 0479-08
3 Vanderzant, C., and D. F. Splittstoesser (ed.).1992. Compendium Standard Methods Agar 10 x 500 ml 9081-80
Intended Use
User Quality Control Bacto m Plate Count Broth is used for enumerating microorganisms
Identity Specifications by membrane filtration.
Dehydrated Appearance: Light beige to beige, free flowing
homogeneous. Also Known As
Solution: 1.7% solution, soluble in distilled m Plate Count Broth is also referred to as m TGY Broth, m Tryptone
or deionized water; light to medium Glucose Yeast Broth, or m Standard Methods Broth.
amber, clear to slightly opalescent,
may have a very slight precipitate. Summary and Explanation
Reaction of 1.7% m Plate Count Broth is a nonselective general-purpose medium
Solution at 25°C: pH 7.0 ± 0.2 for determining bacterial counts from food and water samples using
Cultural Response the membrane filtration procedure. This medium has the same
Prepare m Plate Count Broth per label directions. Inoculate formulation as Plate Count Agar except that agar has been omitted
and incubate the plates at 35 ± 2°C for 18-24 hours. and the ingredients are employed in twice the concentration as in the
INOCULUM solid medium.1
ORGANISM ATCC® CFU RECOVERY
Escherichia coli 25922* 20-80 good to excellent Principles of the Procedure
Staphylococcus aureus 25923* 20-80 good to excellent
Yeast Extract is a source of trace elements, vitamins and amino acids.
The above cultures are the minimum used for performance testing. Tryptone provides carbon and nitrogen for bacterial metabolism.
*These organisms are available as Bactrol™ Disks and are to be Dextrose is a fermentable carbohydrate and carbon source.
used as directed in the Bactrol Disks Technical Information.
Sodium chloride maintains the osmotic balance of the medium. Waterbath (45-50°C)
Bacto Agar is the solidifying agent. Sterile Petri dishes
Formula Method of Preparation
Potato Infusion Agar 1. Suspend 49 grams in 1 liter distilled or deionized water containing
Formula Per Liter 2% Glycerol.
Potatoes, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . . 200 g 2. Heat to boiling to dissolve completely.
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g 3. Autoclave at 121°C for 15 minutes. Cool to 45-50°C.
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g 4. Dispense into sterile Petri dishes or as desired.
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Specimen Collection and Preparation
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g Specimens should be collected in sterile containers or with sterile swabs
Final pH 6.8 ± 0.2 at 25°C and transported immediately to the laboratory in accordance with
recommended guidelines.
Precautions
1. For Laboratory Use.
Test Procedure
1. Incubate plates at 35 ± 2°C in 5-10% CO2 for 10 days.1 For a
2. Follow proper established laboratory procedures in handling and
complete discussion on the inoculation and identification of
disposing of infectious materials.
Brucella spp., consult appropriate references.
3. Brucella spp. are classified as Biosafety Level 3 pathogens. All
manipulations with live cultures and antigens must be confined Results
to a Class II biological safety cabinet (BSC).1 Refer to appropriate references and procedures for results.
Storage Limitations
Store the dehydrated medium below 30°C. The dehydrated medium is 1. Since the nutritional requirements of organisms vary, some strains may
very hygroscopic. Keep container tightly closed. be encountered that fail to grow or grow poorly on this medium.
2. Best results are obtained on freshly prepared medium with a
Expiration Date moist surface.
The expiration date applies to the product in its intact container when
stored as directed. Do not use a product if it fails to meet specifications References
for identity and performance. 1. Moyer, N. P., and L. A. Holcomb. 1995. Brucella, p. 549-555. In
Murray, P.R., E.J. Baron, M.A. Pfaller, F.C. Tenover, and R.H.
Procedure Yolken (ed.), Manual of clinical microbiology, 6th ed. American
Material Provided Society for Microbiology, Washington, D.C.
Potato Infusion Agar 2. Baron, E. J., L. R. Peterson and S. M. Finegold. 1994. Bailey &
Scott’s Diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
Material Required But Not Provided St. Louis, MO.
Glassware
Autoclave Packaging
Incubator (35°C) Potato Infusion Agar 500 g 0051-17
source in the formula. The Potassium Phosphates provide buffering Expiration Date
capacity; Sodium Chloride maintains the osmotic balance of the medium. The expiration date applies to the product in its intact container when
Sodium Lauryl Sulfate is the selective agent, inhibiting many organisms stored as directed. Do not use a product if it fails to meet specifica-
except coliforms. Brom Cresol Purple is used as an indicator dye; tions for identity and performance.
lactose-fermenting organisms turn the medium from purple to yellow
with or without gas production. Procedure
Formula Materials Provided
Presence-Absence Broth
Presence-Absence Broth (single-strength)
Formula Per Liter Materials Required But Not Provided
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g Glassware
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Screw-cap dilution bottle with capacity > 150 ml
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.46 g Incubator (35°C)
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.83 g
Potassium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . 1.35 g Method of Preparation
Potassium Phosphate, Monobasic . . . . . . . . . . . . . . . . . . 1.35 g 1. To prepare triple-strength medium, suspend 91.5 grams in 1 liter
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.46 g distilled or deionized water.
Sodium Lauryl Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g 2. Warm gently to dissolve completely.
Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0085 g
3. Dispense 50 ml amount into screw-cap 250 ml milk dilution
Final pH 6.8 ± 0.2 at 25°C bottles.
Precautions 4. Autoclave at 121°C for 12 minutes, with the total autoclave time
not to exceed 30 minutes.
1. For Laboratory Use.
5. Cool to room temperature.
2. Follow proper established laboratory procedures in handling and
disposing of infectious materials. Specimen Collection and Preparation
Collect water samples as described in recommended procedures.1,9
Storage
Store the dehydrated medium below 30°C. The dehydrated medium is
very hygroscopic. Keep container tightly closed.
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
2. Follow proper established laboratory procedures in handling and 2. Heat to boiling to dissolve completely.
disposing of infectious materials. 3. Autoclave at 121°C for 15 minutes. Cool to 50-60°C.
Storage 4. Aseptically add 500 ml sterile 2% Hemoglobin solution. Mix well.
Store the dehydrated medium below 30°C. The dehydrated medium is 5. Add 10 ml of Supplement B or Supplement VX. Mix thoroughly.
very hygroscopic. Keep container tightly closed. 6. Dispense into sterile Petri dishes or as desired.
Specimen Collection and Preparation
Expiration Date
Obtain and process specimens according to the techniques and
The expiration date applies to the product in its intact container when
procedures established by laboratory policy.
stored as directed. Do not use a product if it fails to meet specifications
for identity and performance. Test Procedure
For a complete discussion of the isolation and identification of
Procedure Haemophilus or Neisseria spp., refer to the procedures outlined in the
Materials Provide references.4,5,6
Proteose No. 3 Agar
Results
Materials Required But Not Provided Refer to appropriate references and procedures for results.
Glassware
Autoclave Limitations of the Procedure
Incubator (35°C) 1. Since the nutritional requirements of organisms vary, some
Waterbath (45-50°C) strains may be encountered that fail to grow or grow poorly on
Hemoglobin (2%) this medium
Supplement B or Supplement VX 2. Proteose No. 3 Agar is intended for use with supplementation.
Sterile Petri dishes Although certain diagnostic tests may be performed directly on
this medium, biochemical and, if indicated, immunological testing
Method of Preparation using pure cultures are recommended for complete identification.
1. Suspend 45 grams in 500 ml liter distilled or deionized water. Consult appropriate references for further information.
References
User Quality Control 1. Carpenter, C. M., M. A. Bucca, T. C. Buck, E. P. Casman, C.
W. Christensen, E. Crowe, R. Drew, J. Hill, C. E. Lankford, H.
Identity Specifications
E. Morton, L. R. Peizer, C. S. Shaw, and J. D. Thayer. 1949.
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Evaluation of twelve media for the isolation of the gonococcus.
Solution: 9% (double strength) solution, soluble Am. J. Syphil. Gonorrh. Vener. Dis. 33:164
in distilled or deionized water upon
boiling with frequent agitation. Light 2. Lankford, C. E., V. Scott, M. F. Cox, and W. R. Cooke. 1943.
to medium amber in color, opalescent Some aspects of nutritional variation of the gonococcus.
with a slight flocculent precipitate. J. Bacteriol. 45:321.
Prepared Medium 3. Lankford, C. E., and E. E. Snell. 1943. Glutamine as growth factor
(Single-strength): Light amber, opalescent with a slight for certain strains of Neisseria gonorrhoeae. J. Bacteriol. 45:410.
flocculent precipitate, firmly solid.
4. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures hand-
Reaction of 9% book, vol.1. American Society for Microbiology, Washington, D.C.
Solution at 25°C: pH 7.3 + 0.2
5. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
Cultural Response R. H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
Prepare Proteose Agar No. 3 per label directions. Inoculate American Society for Microbiology, Washington, D.C.
and incubate at 35 ± 2°C under approximately 5-10% CO2 6. Baron, E. J., L. R. Petersons, and S. M. Finegold. 1994. Bailey
for 18-48 hours.
INOCULUM & Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
ORGANISM ATCC® CFU GROWTH St. Louis, MO.
Haemophilus influenzae 10211 100-1,000 good
Neisseria gonorrhoeae 43070 100-1,000 good Packaging
Neisseria meningitidis 13102 100-1,000 good Proteose No. 3 Agar 500 g 0065-17
Neisseria sicca 9913* 100-1,000 good
Hemoglobin 2% Solution 6 x 100 ml 3248-73
The cultures listed are the minimum that should be used for
performance testing. Supplement B w/Reconstituting Fluid 6 x 10 ml 0276-60
*These cultures are available as Bactrol™ Disks and should be used 100 ml 0276-72
as directed in Bactrol Disks Technical Information. Supplement VX w/Reconstituting Fluid 6 x 10 ml 3354-60
100 ml 3354-72
Proteose Peptones
Bacto Proteose Peptone . Bacto Proteose Peptone No. 2
®
Procedure Results
Materials Provided Refer to appropriate references and procedures for results.
Proteose Peptone
Proteose Peptone No. 2
References
Proteose Peptone No. 3 1. Hewitt. 1930. Biochem. J. 24:984.
H2S Test Strips 2. Bunney. 1930. J. Immunol. 20:71.
Indole Test Strips 3. Kirkbride, Berthelsen and Clark. 1931. J. Immunol. 21:1.
KL Antitoxin Strips 4. Hazen and Heller. 1932. J. Bacteriol. 23:195.
KL Virulence Enrichment 5. Kirkbride and Wheeler. 1926. J. Immunol. 11:477.
Materials Required But Not Provided 6. Nelson. 1927. J. Infect. Dis. 41:9.
Materials vary depending on the medium being prepared. 7. Kneeland and Dawes. 1932. J. Exp. Med. 55:735.
8. Hanks and Rettger. 1932. J. Immunol. 22:283.
Method of Preparation
9. Bunney and Thomas. 1936. J. Immunol. 31:95.
Refer to the final concentration of Proteose Peptone, Proteose Peptone
No. 2 or Proteose Peptone No. 3 in the formula of the medium being Packaging
prepared. Add as required.
Proteose Peptone 500 g 0120-17
Specimen Collection and Preparation 10 kg 0120-08
Obtain and process specimens according to the techniques and procedures Proteose Peptone No. 2 500 g 0121-17
established by laboratory policy. 10 kg 0121-08
Test Procedure Proteose Peptone No. 3 500 g 0122-17
2 kg 0122-07
See appropriate references for specific procedures using Proteose
Peptone, Proteose Peptone No. 2 or Proteose Peptone No. 3. 10 kg 0122-08
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
elaborate only one of the two. When Pseudomonas Agar F and Materials Required But Not Provided
Pseudomonas Agar P are used together, they provide for easy and rapid Glassware
identification of most Pseudomonas strains as specified in the FDA Autoclave
Bacteriological Analytical Manual.3 Incubator (35°C)
Principles of the Procedure Sterile Petri dishes
Tubes with closures
Pseudomonas Agar F
Bacto Glycerol
Tryptone and Proteose Peptone No. 3 provide carbon and nitrogen
sources required for good growth and also aid in fluorescein production. Method of Preparation
Phosphate stimulates fluorescein production and has an inhibitory effect 1. Suspend the medium in 1 liter distilled or deionized water containing
on pyocyanin. Dipotassium Phosphate increases the phosphorus 10 grams of Glycerol:
content over that supplied by the peptones. Magnesium Sulfate provides
Pseudomonas Agar F - 38 grams;
necessary cations for the activation of fluorescein production. Bacto
Agar is a solidifying agent. Glycerol, added during preparation of the Pseudomonas Agar P - 46.4 grams.
medium, is a carbon source. 2. Boil to dissolve completely.
Pseudomonas Agar P 3. Autoclave at 121°C for 15 minutes.
Bacto Peptone provides the carbon and nitrogen sources required for Specimen Collection and Preparation
good growth. Glycerol is a carbon source. Magnesium Chloride and Refer to appropriate references for specimen collection and preparation.
Potassium Sulfate stimulate pyocyanin production. Bacto Agar is a
solidifying agent. Test Procedure
1. Obtain the inoculum from a pure 18-24 hour culture of Pseudomonas.
Formula
2. Inoculate plates or agar slants by streaking the surface.
Pseudomonas Agar F
Formula Per Liter 3. Incubate at 35 ± 2°C for 18-24 hours.
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Results
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 10 g Examine colonies under ultraviolet light (Wood’s lamp).4 Take care
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g when using UV illumination because it may have a bactericidal effect.
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g Be sure there is good growth before placing the culture under UV light.
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Pseudomonas Agar F: Positive result is indicated by a light, bright
Final pH 7.0 ± 0.2 at 25°C
greenish-yellow color diffusing into the agar with a fluorescent zone
Pseudomonas Agar P surrounding the growth.
Formula Per Liter Pseudomonas Agar P: Positive result is indicated by a blue pigment
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g that diffuses into the agar.
Magnesium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 g
Potassium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Limitations of the Procedure
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g 1. Occasionally, a Pseudomonas culture is encountered that will
Final pH 7.0 ± 0.2 at 25°C produce small amounts of pigment in the medium. When this
happens, a yellow-green color will appear on Pseudomonas Agar F
Precautions or a blue-green color on Pseudomonas Agar P. If a blue-green color
1. For Laboratory Use. occurs on Pseudomonas Agar P, confirmation of the presence of
2. Follow proper established laboratory procedure in handling and pyocyanin can be made by extraction with chloroform (CHCl3).4
disposing of infectious materials. 2. The formation of nonpigmented colonies does not completely rule
Storage out a Pseudomonas aeruginosa isolate.
Store the dehydrated medium below 30°C. The dehydrated medium is 3. A pyocyanin-producing Pseudomonas strain will usually also
very hygroscopic. Keep container tightly closed. Store the prepared produce fluorescein. It must, therefore, be differentiated from other
media at 2-8°C. simple fluorescent pseudomonads by other means. Temperature can
be a determining factor as most other fluorescent strains will not
Expiration Date grow at 35°C. Rather, they grow at 25-30°C.4
The expiration date applies to the product in its intact container when
stored as directed. Do not use a product if it fails to meet specifications References
for identity and performance. 1. King, E. O., M. K. Ward, and D. E. Raney. 1954. Two simple
media for the demonstration of pyocyanin and fluorescein. J. Lab.
Procedure Clin. Med. 44:301.
Materials Provided 2. The United States Pharmacopeia. 1995. The United States phar-
Pseudomonas Agar F macopeia, 23rd ed. United States Pharmacopeial Convention,
Pseudomonas Agar P Rockville, MD.
acid is produced during the fermentation of the added carbohydrate. In Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Purple Agar Base, the Bacto Agar serves as a solidifying agent. Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g
Formula Final pH at 25°C 6.8 ± 0.2
Purple Broth Base
Formulas Per Liter
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 10 g
Method of Preparation 2. Medium is slightly acid (pH 6.8) and positive reactions may be
1. Suspend 28 grams in 1 liter distilled or deionized water. slower than with phenol red carbohydrate medium.5
2. Heat to boiling to dissolve completely. References
3. Autoclave at 121°C for 15 minutes. Cool to 45-50°C. 1. Wurtz. 1897. Technique Bacteriologique, Masson, Paris.
4. Dispense into sterile tubes.
2. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
Specimen Collection and Preparation Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.
Refer to appropriate references for specimen collection and preparation. St. Louis, MO.
3. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
Test Procedure R. H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
For a complete discussion on the expected reactions of specific American Society for Microbiology, Washington, D.C.
Enterobacteriaceae species, refer to Manual of Clinical Microbiology,3
Clinical Microbiology Procedures Handbook 4 and Bailey & Scott’s 4. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures
Diagnostic Microbiology.2 handbook, vol 1. American Society for Microbiology, Washington, D.C.
5. MacFaddin, J. D. 1985. Media for isolation-cultivation-
Results identification-maintenance of medical bacteria, vol 1. Williams
Refer to appropriate references and procedures for results. & Wilkins, Baltimore, MD.
Limitations of the Procedure Packaging
1. Since the nutritional requirements of organisms vary, some strains may Purple Lactose Agar 500 g 0082-17
be encountered that fail to grow or grow poorly on this medium.
Following inoculation, incubate the tubes on a shaker (about 100 rpm) 2. Aseptic technique should be used throughout the assay procedure.
at 25-30°C for 22 hours. Steam in the autoclave for 5 minutes to stop 3. The use of altered or deficient media may cause mutants having
growth. Measure the growth turbidimetrically using a spectrophotometer different nutritional requirements that will not give a satisfactory
at any specific wavelength between 540 and 660 nm. response.
Results 4. For successful results of these procedures, all conditions of the
1. Prepare a standard concentration response curve by plotting the response assay must be followed precisely.
readings against the amount of standard in each tube, disk or cup.
2. Determine the amount of vitamin at each level of assay solution by References
interpolation from the standard curve. 1. Campling, and Nixon. 1954. J. Physiol. 126:71.
3. Calculate the concentration of vitamin in the sample from the 2. Hurley. 1960. J. AOAC. 43:43.
average of these volumes. Use only those values that do not vary 3. Parrish, Loy, and Kline. 1956. J. AOAC. 39:157.
more than ±10% from the average and use the results only if two
thirds of the values do not vary more than ±10%. Packaging
Pyridoxine Y Medium 100 g 0951-15*
Limitations of the Procedure
1. The test organism used for inoculating an assay medium must be *Store at 2-8°C
grown and maintained on a medium recommended for this purpose.
Results Packaging
R2A Agar 100 g 1826-15
Count colonies on spread or pour plates demonstrating 30-300
500 g 1826-17
colonies per plate or 20-200 colonies when using the membrane
2 kg 1826-07
filter method. Compute bacterial count per ml of sample by multiplying
the average number of colonies per plate by the reciprocal of the
appropriate dilution.
Report counts as colony forming units (CFU) per ml and report variables
of incubation such as temperature and length of time.
MSRV medium showed that a semi-solid medium in Petri dishes could Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.34 g
be used as a rapid and sensitive means of isolating motile Salmonella Potassium Dihydrogen Phosphate . . . . . . . . . . . . . . . . . . 1.47 g
from food products following pre-enrichment or selective enrichment.1,2 Magnesium Chloride Anhydrous . . . . . . . . . . . . . . . . . . 10.93 g
The semisolid medium allows motility to be detected as halos of growth Malachite Green Oxalate . . . . . . . . . . . . . . . . . . . . . . . . 0.037 g
around the original point of inoculation. Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 g
Final pH 5.2 ± 0.2 at 25°C
The medium is recommended by the European Chocolate Manufacturer’s
Association. A collaborative study performed with support of the Novobiocin Antimicrobic Supplement
American Cocoa Research Institute (ACRI) and the Canadian Chocolate Formula per 10 ml
Manufacturer’s Association (CCMA) resulted in first action adoption of Sodium Novobiocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
the MSRV method by AOAC International.3
Precautions
MSRV Medium may be used as a plating medium for isolating
Salmonella spp. (other than S. typhi and S. paratyphi type A) from 1. For Laboratory Use.
stool specimens with high sensitivity and specificity.4,5 2. Rappaport-Vassiliadis (MSRV) Medium Semisolid Modification
IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM
Principles of the Procedure AND SKIN. Avoid contact with skin and eyes. Do not breathe dust.
Rappaport-Vassiliadis (MSRV) Medium Semisolid Modification Wear suitable protective clothing. Keep container tightly closed.
contains Tryptose and Casein Hydrolysate as carbon and nitrogen TARGET ORGAN(S): Nerves, Kidneys.
sources for general growth requirements. Magnesium Chloride raises Novobiocin Antimicrobic Supplement
the osmotic pressure in the medium. Novobiocin (Novobiocin HARMFUL. HARMFUL BY INHALATION AND IF SWAL-
Antimicrobic Supplement) and Malachite Green inhibit organisms LOWED. (EC) MAY CAUSE ALLERGIC EYE, RESPIRATORY
other than Salmonella. The low pH of the medium combined with the SYSTEM AND SKIN REACTION. Avoid contact with skin and
Novobiocin, Malachite Green and Magnesium Chloride select for highly eyes. Do not breathe dust. Wear suitable protective clothing. Keep
resistant Salmonella spp. Bacto Agar is the solidifying agent. container tightly closed.
Formula FIRST AID: In case of contact with eyes, rinse immediately with
plenty of water and seek medical advice. After contact with skin,
Rappaport-Vassiliadis (MSRV) Medium Semisolid Modification
wash immediately with plenty of water. If inhaled, remove to fresh
Formula per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.59 g
Casein Hydrolysate (Acid) . . . . . . . . . . . . . . . . . . . . . . . . 4.59 g Uninoculated Salmonella typhimurium
plate ATCC® 14028
Method of Preparation influence the standard curve readings, cannot be duplicated exactly
1. Suspend 4.8 grams in 100 ml of distilled or deionized water. from assay to assay. The standard curve is obtained by using Riboflavin
2. Boil 2-3 minutes to dissolve completely. USP Reference Standard or equivalent at levels of 0.0, 0.025, 0.05,
0.075, 0.1, 0.15, 0.2 and 0.3 µg riboflavin per assay tube (10 ml).
3. Dispense 5 ml amounts into tubes, evenly dispersing the precipitate.
4. Add standard or test samples. The concentration of riboflavin required for the preparation of the
standard curve may be prepared by dissolving 0.1 g of Riboflavin USP
5. Adjust tube volume to 10 ml with distilled or deionized water.
Reference Standard or equivalent in 1,000 ml of distilled water by
6. Autoclave at 121°C for 10 minutes. heating, giving a stock solution of 100 µg per ml. Dilute the stock
Specimen Collection and Preparation solution by adding 1 ml to 999 ml distilled water. Use 0.0, 0.25, 0.5,
Assay samples are prepared according to references given in the 0.75, 1, 1.5, 2 and 3 ml of the diluted stock solution per tube. Prepare
specific assay procedures. For assays, the samples should be diluted to the stock solution fresh daily.
approximately the same concentration as the standard solution. Results
Test Procedure 1. Prepare a standard concentration response curve by plotting the
1
Follow assay procedures as outlined in AOAC. Levels of riboflavin response readings against the amount of standard in each tube,
used in the determination of the standard curve should be prepared disk or cup.
according to this reference or according to the following procedure. 2. Determine the amount of vitamin at each level of assay solution by
interpolation from the standard curve.
Stock Cultures
Stock cultures of L. casei subsp. rhamnosus ATCC® 7469 are prepared 3. Calculate the concentration of vitamin in the sample from the
by stab inoculation into 10 ml of Lactobacilli Agar AOAC. After average of these volumes. Use only those values that do not vary
24-48 hours incubation at 35-37°C, the stock cultures are kept in the more than ±10% from the average and use the results only if two
refrigerator. Transfers are made at monthly intervals in triplicate. thirds of the values do not vary by more than ±10%.
Inoculum Limitations of the Procedure
Inoculum for assay is prepared by subculturing a stock culture of 1. The test organism used for inoculating an assay medium must be
L. casei subsp. rhamnosus ATCC® 7469 into 10 ml of Lactobacilli Broth cultured and maintained on media recommended for this purpose.
AOAC or Micro Inoculum Broth. Following incubation for 16-24 hours 2. Aseptic technique should be used throughout the assay procedure.
at 35-37°C, the culture is centrifuged under aseptic conditions and the
3. The use of altered or deficient media may cause mutants having dif-
supernatant liquid decanted. After washing 3 times with 10 ml sterile
ferent nutritional requirements that will not give a satisfactory response.
0.85% saline, the cells are resuspended in 10 ml sterile 0.85% saline.
The cell suspension is then diluted with sterile 0.85% saline, to a 4. For successful results of these procedures, all conditions of the
turbidity of 35-40% transmittance when read on the spectrophotometer assay must be followed precisely.
at 660 nm. One drop of this latter suspension is then used to inoculate 5. Maintain pH below 7.0 to prevent loss of riboflavin.
each of the assay tubes.
References
Riboflavin Assay Medium may be used for both turbidimetric and
1. Snell and Strong. 1939. Ind. and Eng. Chem. 11:346.
titrimetric determinations. Turbidimetric readings should be made
after 18-24 hours incubation at 35-37°C, where as titrimetric 2. Association of Analytical Chemists. 1996. U.S. Food and Drug
determinations are best made after 72 hours incubation at 35-37°C. Administration methods or the microbiological analysis of selected
Using Riboflavin Assay Medium, the most effective assay range is nutrients. AOAC Internationl, Gaithersburg, MD.
between 0.025 and 0.15 µg riboflavin.
Packaging
Standard Curve Riboflavin Assay Medium 100 g 0325-15*
It is essential that a standard curve be constructed each time an assay is
run. Conditions of autoclaving and temperature of incubation, which *Store at 2-8°C
used for obtaining a specimen and then rolled on the surface of a rice Procedure
extract agar plate; a cover glass was then applied to the agar, covering Materials Provided
most of the inoculum.
Rice Extract Agar
Principles of the Procedure Materials Required But Not Provided
The Rice Extract provides the sole source of nutrients in the medium. Glassware
This lack of nutrients together with the oxygen-deficient culture Autoclave
conditions (covering the inoculum with a cover glass) creates a deficient Distilled or deionized water
environment that induces the formation of specific morphological
forms (chlamydospores and pseudomycelia in particular) in some Method of Preparation
yeasts. The addition of Tween 80 further stimulates chlamydospore 1. Suspend 25 grams in 1 liter distilled or deionized water.
formation due to its content of oleic acids. Bacto Agar is incorporated 2. Heat to boiling to dissolve completely.
into the medium as a solidifying agent. 3. Autoclave at 121°C for 15 minutes.
Formula 4. Aseptically dispense medium into sterile Petri dishes.
Rice Extract Agar Specimen Collection and Preparation
Formula Per Liter Refer to appropriate references for specimen collection and preparation.
White Rice, Extract from . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g Test Procedure
Final pH 7.1 ± 0.2 at 25°C 1. Inoculate the plates by cutting through the surface of the agar with
an inoculating wire.
Precautions 2. Cover the inoculated area with a sterile cover slip.
1. For Laboratory Use. 3. Invert plates and incubate at 23-25°C for 18-72 hours.
2. Follow proper established laboratory procedure in handling and 4. Examine for chlamydospores microscopically using approximately
disposing of infectious materials. 100X magnification and by focusing upon the line of inoculation.
Storage Results
Store the dehydrated medium below 30°C. The dehydrated medium is After 24 to 48 hours most strains of C. albicans and C. stellatoidea
very hygroscopic. Keep container tightly closed. will have formed typical chlamydospores.5
Antimicrobic Supplement C
Intended Use and Dawson7 used Rose Bengal in a neutral pH medium for the selective
isolation of fungi from soil samples. Chloramphenicol, streptomycin,
Bacto Rose Bengal Agar Base is used with Bacto Rose Bengal Antimi-
oxytetracycline and chlortetracycline have been used for the improved,
crobic Supplement C in isolating and enumerating yeasts and molds.
selective isolation and enumeration of yeasts and molds from soil,
Also Known As sewage and foodstuffs.4,8,9,10,11
Rose Bengal Agar is also known as Rose Bengal Chloramphenicol Agar Rose Bengal Agar Base supplemented with Rose Bengal Antimicrobic
and Rose Bengal-Malt Extract Agar. Supplement C is a modification of the Rose Bengal Chlortetracycline
Agar formula of Jarvis.11 Instead of chlortetracycline, chloramphenicol
Summary and Explanation is employed in this medium as a selective supplement. Of the antibiotics
A number of methods have been described for the selective isolation most frequently employed in media of neutral pH, chloramphenicol
of fungi from environmental materials and foodstuffs containing mixed is recommended because of its heat stability and broad antibacterial
populations of fungi and bacteria. The use of media with an acid pH spectrum.12 Rose Bengal Agar is recommended in standard methods for
that selectively inhibits the growth of bacteria and thereby promotes the enumeration of yeasts and molds from foodstuffs and water.12,13,14,15
the growth of fungi has been widely employed.1,2,3 A number of inves-
tigators have reported, however, that acidified media may actually Principles of the Procedure
inhibit fungal growth,4,5 fail to completely inhibit bacterial growth,5 Soytone provides the carbon and nitrogen sources required for good
and have little effect in restricting the size of mold colonies.6 Smith growth of a wide variety of organisms. Dextrose is an energy source.
Monopotassium Phosphate provides buffering capability. Magnesium 2. Rose Bengal Antimicrobic Supplement C
Sulfate provides necessary trace elements. Rose Bengal is included as TOXIC. MAY CAUSE CANCER. MAY CAUSE HERITABLE
a selective agent that inhibits bacterial growth and restricts the size GENETIC DAMAGE. POSSIBLE RISK OF HARM TO THE
and height of colonies of the more rapidly growing molds. The restric- UNBORN CHILD. MAY CAUSE SENSITIZATION BY INHA-
tion in growth of molds aids in the isolation of slow-growing fungi by LATION AND SKIN CONTACT. Wear suitable protective cloth-
preventing overgrowth by more rapidly growing species. Rose Bengal ing, gloves and eye/face protection. In case of accident or if you
is taken up by yeast and mold colonies, thereby facilitating their feel unwell, seek medical advice immediately. (Show label where
recognition and enumeration. Rose Bengal Antimicrobic Supplement C possible.) Do not breathe dust. Keep container tightly closed.
is a lyophilized antimicrobic supplement containing chloramphenicol Target Organs: Blood, Bone Marrow.
which inhibits bacteria. Bacto Agar is the solidifying agent. FIRST AID: In case of contact with eyes, rinse immediately with
Formula plenty of water and seek medical advice. After contact with skin,
wash immediately with plenty of water. If swallowed seek medical
Rose Bengal Agar Base advice immediately and show this container or label. If inhaled,
Formula Per Liter remove to fresh air. If not breathing, give artificial respiration. If
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g breathing is difficult, give oxygen. Seek medical advice.
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
3. Follow proper established laboratory procedure in handling and
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
disposing of infectious materials.
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
Rose Bengal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g Storage
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g Store Rose Bengal Agar Base dehydrated below 30°C. The dehydrated
Final pH 7.2 ± 0.2 at 25°C medium is very hygroscopic. Keep container tightly closed. Store the
Rose Bengal Antimicrobic Supplement C prepared medium at 2-8°C.
Formula Per 2 ml Vial Store Rose Bengal Antimicrobic Supplement C at 2-8°C. Do not open
Chloramphenicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g or rehydrate vials until ready to use. Store rehydrated vials at 2-8°C
and use within 24 hours.
Precautions
1. For Laboratory Use. Uninoculated Aspergillus niger
plate ATCC® 1015
Expiration Date that fail to grow or grow poorly on the complete medium; similarly,
The expiration date applies to the products in their intact containers some strains of bacteria may be encountered that are not inhibited
when stored as directed. Do not use a product if it fails to meet speci- or only partially inhibited.
fications for identity and performance. 3. Care should be taken not to expose this medium to light since photo-
degradation of rose bengal yields compounds that are toxic to fungi.
Procedure
Materials Provided References
1. Waksman, S. A. 1922. A method for counting the number of fungi
Rose Bengal Agar Base
in the soil. J. Bacteriol. 7:339-341.
Rose Bengal Antimicrobic Supplement C
2. Koburger, J. A. 1976. Yeasts and molds, p. 225-229. In M. L. Speck
Materials Required But Not Provided (ed.), Compendium of methods for the microbiological examination
Glassware of foods. American Public Health Association, Washington, D.C.
Autoclave 3. Mossel, D. A. A., M. Visser, and W. H. J. Mengerink. 1962. A
Incubator (25°C) comparison of media for the enumeration of moulds and yeasts in
Sterile Petri dishes foods and beverages. Lab Practice 11:109-112.
Ethanol (reagent grade) 4. Martin, J. P. 1950. Use of acid, rose bengal and streptomycin in
Bent glass rods the plate method for estimating soil fungi. Soil Sci. 69:215-232.
Method of Preparation 5. Koburger, J. A. 1972. Fungi in foods. IV. Effect of plating
1. Rose Bengal Antimicrobic Supplement C: To rehydrate, asepti- medium pH on counts. J. Milk Food Technol. 35:659-660.
cally add 2 ml of ethanol per vial of dehydrated supplement and 6. Tyner, L. E. 1944. Effect of media compositions on the numbers
invert several times to dissolve the powder. of bacterial and fungal colonies developing in Petri plates. Soil
2. Rose Bengal Agar Base: To rehydrate, suspend 16 grams in 500 ml Sci. 57:271-274.
distilled or deionized water. 7. Smith, N. R., and V. T. Dawson. 1944. The bacteriostatic action
3. Heat to boiling to dissolve completely. of rose bengal in media used for the plate counts of soil fungi. Soil
Sci. 58:467-471.
4. Sterilize the basal medium at 121°C for 15 minutes and then cool
to 45-50°C. 8. Cooke, W. B. 1954. The use of antibiotics in media for the isolation of
fungi from polluted water. Antibiotics and Chemotherapy 4:657-662.
5. Aseptically add 2 ml of the rehydrated Rose Bengal Antimicrobic
Supplement C to 500 ml of cooled agar base. Mix thoroughly. 9. Papavizas, G. C., and C. B. Davey. 1959. Evaluation of various
media and antimicrobial agents for isolation of soil fungi. Soil Sci.
6. Dispense into sterile Petri dishes and allow to dry overnight at room
88:112-117.
temperature (21-25°C).
10. Overcast, W. W., and D. J. Weakley. 1969. An aureomycin-rose
Specimen Collection and Preparation bengal agar for enumeration of yeast and mold in cottage cheese.
Collect specimens in sterile containers and transport immediately to J. Milk Technol. 32:442-445.
the laboratory in accordance with recommended guidelines. 12,13 11. Jarvis, B. 1973. Comparison of an improved rose bengal-
Prepare samples for dilution plating inoculation. It is recommended that chlortetracycline agar with other media for the selective isolation
yeast and molds be enumerated by a surface spread-plate technique and enumeration of molds and yeasts in foods. J. Appl. Bact.
rather than with pour plates.12 The spread-plate technique provides 36:723-727.
maximal exposure of cells to atmospheric oxygen and eliminates heat 12. Mislivec, P. B., L. R. Beuchat, and M. A. Cousin. 1992. Yeasts
stress from molten agar.12 and Molds. In C. Vanderzant and D. F. Splittstoesser (eds.),
Test Procedure Compendium of methods for the microbiological examination of
1. Inoculate 0.1 ml of appropriate dilutions in duplicate on the solidified foods, 3rd ed. American Public Health Assoc., Washington, D.C.
agar. Spread over the entire surface using a sterile bent glass rod. 13. Marshall, R. T. (ed.). 1993. Standard methods for the examination
2. Incubate plates at 25-30°C for up to 7 days. of dairy products, 16th ed. American Public Health Assoc.,
Washington, D.C.
Results 14. Eaton, A.D., L.S. Clesceri, and A.E. Greenberg (ed.). 1995.
Colonies of yeast appear pink due to the uptake of rose bengal. Count Standard methods for the examination of water and wastewater,
plates containing 15 to 150 colonies and report the counts as colony 19th ed. American Public Health Association, Washington, D.C.
forming units (CFU) per gram or ml of sample.
15. MacFaddin, J. F. 1985. Media for isolation-cultivation-
Limitations of the Procedure identification-maintenance of medical bacteria. Williams
& Wilkins, Baltimore, MD.
1. Although this medium is selective primarily for fungi, microscopic
examination is recommended for presumptive identification. Packaging
Biochemical testing using pure cultures is required for complete Rose Bengal Agar Base 500 g 1831-17
identification. 10 kg 1831-08
2. Due to the selective properties of this medium and the type of Rose Bengal Antimicrobic
specimen being cultured, some strains of fungi may be encountered Supplement C 6 x 2 ml 3352-54
Bacto SF Medium
®
inhibitor, sodium azide. Second, it detects whether an organism can
ferment the carbohydrate, dextrose, producing a pH color change.
Intended Use Principles of the Procedure
Bacto SF Medium is used for isolating and cultivating fecal Tryptone is a source of carbon, nitrogen, vitamins and minerals.
streptococci from milk, water, sewage and feces. Dextrose is a fermentable carbohydrate. Sodium Chloride maintains
the osmotic balance of the medium. Sodium Azide inhibits cytochrome
Also Known As oxidase of gram-negative bacteria. Brom Cresol Purple is a pH
Streptococcus Faecalis Medium indicator. Phosphates buffer the medium.
Summary and Explanation Group D enterococci will grow in the presence of azide and ferment
Hajna and Perry1 specified the formulation of SF Broth, a medium glucose. This produces an acid pH that changes the color of the
that is selective for fecal streptococci when incubated at 45.5°C. medium from purple to yellow.
SF Broth has been used for testing water and other materials for fecal Formula
contamination.2,3,4 Detection of fecal streptococci is used as an indicator
of pollution. SF Medium
Formula Per Liter
SF medium is used to differentiate Group D enterococci from Group D Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
non-enterococci and other Streptococcus spp. that are not Group D. Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
SF Medium is differential in two ways. First, it differentiates based on Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 g
whether an organism has the ability to grow in the presence of the Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g
6. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compen- 8. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (eds.). 1995.
dium of methods for the microbiological examination of foods, Standard methods for the examination of water and wastewater,
3rd ed. American Public Health Association, Washington, D.C. 19th ed. American Public Health Association, Washington, D.C.
7. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig. 1993.
Pathogens in milk and milk products, p. 103-212. In R. T. Marshall, Packaging
(ed.), Standard methods for the examination of dairy products, 16th SF Medium 500 g 0315-17
ed. American Public Health Association, Washington, D.C.
SFP Agar
Bacto SFP Agar Base . Bacto Egg Yolk Enrichment 50%
Bacto Antimicrobic Vial K . Bacto Antimicrobic Vial P
Intended Use Summary and Explanation
Bacto SFP Agar Base is used with Bacto Egg Yolk Enrichment 50%, Shahidi Ferguson Perfringens (SFP) Agar Base is prepared according
Bacto Antimicrobic Vial P and Bacto Antimicrobic Vial K in detecting to the formulation of Shahidi and Ferguson.1 With the addition of 50%
and enumerating Clostridium perfringens in foods. egg yolk emulsion, both the lecithinase reaction and the sulfite
reaction can identify Clostridium perfringens. The selectivity of the
Also Known As medium is due to the added kanamycin and polymixin B.
Tryptose Sulfite Cycloserine (TSC) Agar
Uninoculated Clostridium perfringes
plate ATCC® 12919
C. perfringens is found in raw meats, poultry, dehydrated soups and Antimicrobic Vial P
sauces, raw vegetables and other foods and food ingredients, but MAY BE HARMFUL IF ABSORBED OR INTRODUCED
occurrences of food borne illness are usually associated with cooked THROUGH SKIN. (US) MAY CAUSE ALLERGIC EYE, RESPI-
meat or poultry products.2 Spores of some strains that may resist heat RATORY SYSTEM AND SKIN REACTION. (US) Avoid contact
during cooking germinate and grow in foods that are not adequately with skin and eyes. Do not breathe dust. Wear suitable protective
refrigerated.3 Enumerating the microorganism in food samples plays a clothing. Keep container tightly closed.
role in the epidemiological investigation of outbreaks of food borne FIRST AID: In case of contact with eyes, rinse immediately with
illness.2 plenty of water and seek medical advice. After contact with skin,
SFP Agar (with added kanamycin and polymixin B) is comparable to wash immediately with plenty of water. If inhaled, remove to fresh
Tryptose Sulfite Cycloserine (TSC) Agar, which uses cycloserine as air. If not breathing, give artificial respiration. If breathing is diffi-
the inhibitory component.2, 4 cult, give oxygen. Seek medical advice. If swallowed seek medical
advice immediately and show this container or label.
Principles of the Procedure 3. Follow proper established laboratory procedures in handling and
SFP Agar Base contains Tryptose and Soytone as sources of carbon, disposing of infectious materials.
nitrogen, vitamins and minerals. Yeast Extract supplies B-complex
vitamins which stimulate bacterial growth. Ferric Ammonium Storage
Citrate and Sodium Sulfite are H2S indicators. Clostridia reduce sulfite Store SFP Agar Base below 30°C. The dehydrated medium is very
to sulfide, which reacts with iron to form a black iron sulfide precipi- hygroscopic. Keep container tightly closed.
tate. Antimicrobic Vial P contains Polymyxin B and Antimicrobic Store Egg Yolk Enrichment 50%, Antimicrobic Vial K and Antimicrobic
Vial K contains Kanamycin; both are inhibitors to organisms other Vial P at 2-8°C.
than Clostridium spp. Egg Yolk Enrichment 50% provides egg yolk
lecithin which some clostridia hydrolyze. Bacto Agar is the Expiration Date
solidifying agent. The expiration date applies to the product in its intact container when
stored as directed. Do not use a product if it fails to meet specifications
Formula for identity and performance.
SFP Agar Base
Formula Per Liter Procedure
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Materials Provided (one of the following)
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
SFP Agar Base
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g Egg Yolk Enrichment 50%
Sodium Bisulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g Antimicrobic Vial K
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g Antimicrobic Vial P
Final pH 7.6 ± 0.2 at 25°C Materials Required but not Provided
Egg Yolk Enrichment 50% Glassware
Sterile concentrated egg yolk emulsion Petri dishes
Antimicrobic Vial K Distilled or deionized water
25,000 mcg Kanamycin per 10 ml vial Autoclave
Antimicrobic Vial P Incubator, anaerobic (35°C)
30,000 units Polymyxin B per 10 ml vial
Method of Preparation
Precautions SFP Agar Base
1. For Laboratory Use. Base Layer:
2. Antimicrobic Vial K 1. Suspend 47 grams in 900 ml distilled or deionized water.
HARMFUL. MAY CAUSE ALLERGIC EYE, RESPIRATORY 2. Heat to boiling to dissolve completely.
SYSTEM AND SKIN REACTION. (US) MAY CAUSE HARM 3. Autoclave at 121°C for 15 minutes. Cool to 50°C.
TO THE UNBORN CHILD. Avoid contact with skin and eyes. Do 4. Add 100 ml Egg Yolk Enrichment 50%, 10 ml of rehydrated
not breathe dust. Wear suitable protective clothing. Keep container Antimicrobic Vial P (30,000 units polymyxin B sulfate) and 4.8 ml
tightly closed. rehydrated Antimicrobic Vial K (12 mg kanamycin).
FIRST AID: In case of contact with eyes, rinse immediately with
5. Mix thoroughly.
plenty of water and seek medical advice. After contact with skin,
wash immediately with plenty of water. If inhaled, remove to fresh Cover Layer:
air. If not breathing, give artificial respiration. If breathing is diffi- 1. Suspend 47 grams in 1 liter distilled or deionized water.
cult, give oxygen. Seek medical advice. If swallowed seek medical 2. Prepare as above, except omit Egg Yolk Enrichment 50%.
advice immediately and show this container or label.
Precautions
Bacto SOB Medium ®
1. For Laboratory Use.
2. MAY BE IRRITATING TO EYES, RESPIRATORY SYSTEM
Intended Use AND SKIN. (US) Avoid contact with skin and eyes. Do not breathe
dust. Wear suitable protective clothing. Keep container tightly closed.
Bacto SOB Medium is used for cultivating recombinant strains of
Escherichia coli. FIRST AID: In case of contact with eyes, rinse immediately with
plenty of water and seek medical advice. After contact with skin,
Summary and Explanation wash immediately with plenty of water. If inhaled, remove to fresh
air. If not breathing, give artificial respiration. If breathing is diffi-
SOB Medium was developed by Hanahan1 as a nutritionally rich growth
cult, give oxygen. Seek medical advice. If swallowed seek medical
medium for preparation and transformation of competent cells. Trans-
advice immediately and show this container or label.
formation requires making perforations in the bacterium (i.e., making the
cells “competent”) to allow the introduction of foreign DNA into the cell. 3. Follow proper established laboratory procedure in handling and
To survive this process, competent cells need a rich, isotonic environment. disposing of infectious materials.
SOC Medium, used in the final stage of transformation, may be Storage
prepared by aseptically adding 20 ml of a filter-sterilized 20% solution Store the dehydrated medium below 30°C. The dehydrated medium is
of glucose (dextrose) to the sterile SOB Medium. This addition provides very hygroscopic. Keep container tightly closed.
a readily available source of carbon and energy in a form E. coli can Store prepared medium at 2-8°C.
use in mending the perforations and for replication.2
Expiration Date
Principles of the Procedure The expiration date applies to the product in its intact container when
Tryptone and Yeast Extract provide sources of nitrogen and growth stored as directed. Do not use a product if it fails to meet specifications
factors which allow the bacteria to recover from the stress of transfor- for identity and performance.
mation and grow well. Sodium Chloride and Potassium Chloride
provide a suitable osmotic environment. Magnesium Sulfate is a source Procedure
of magnesium ions required in a variety of enzymatic reactions, Materials Provided
including DNA replication. SOB Medium
Bacto SS Agar®
dysentery that has been reported to have fatality rates of up to 20%.
Most cases of shigellosis are individual cases due to person-to-person
transmission. When associated with outbreaks, the disease usually is
Intended Use transmitted by contaminated food and/or water.1
Bacto SS Agar is used for isolating Salmonella and some Shigella. SS Agar is a modification of the Desoxycholate Citrate Agar described
by Leifson.2 SS Agar was found to be superior to other media for the
Also Known As isolation of Salmonella and Shigella spp.3 Ewing and Bruner found
SS Agar is also known as Salmonella-Shigella Agar. SS Agar to have the advantage that large amounts of inoculum could
Summary and Explanation be used when isolating Salmonella or Shigella from clinical samples.4
Caudill5 reported on the satisfactory use of SS Agar in isolation of
Salmonellosis continues to be an important public health problem
Shigella organisms. Hormaeche and his co-workers6 used SS Agar with
worldwide, despite efforts to control the prevalence of Salmonella in
other media for isolation of Shigella as the causative agent of infantile
domesticated animals. Infection with non-typhi Salmonella often
summer diarrhea.
causes mild, self-limiting illness. Typhoid fever, caused by S. typhi, is
characterized by fever, headache, diarrhea, and abdominal pain, and The use of SS Agar is recommended for testing clinical specimens for
can produce fatal respiratory, hepatic, splenic, and/or neurological dam- the presence of Salmonella and some Shigella spp.1,7 For food testing,
age.1 These illnesses result from the consumption of raw, undercooked consult appropriate references on the use of SS Agar.8
or improperly processed foods contaminated with Salmonella.
Principles of the Procedure
Shigella spp. cause classic bacillary dysentery (shigellosis), which is a In SS Agar, Bacto Bile Salts No. 3 and Brilliant Green are
descending intestinal illness characterized by abdominal pain, fever, complementary in inhibiting gram-positive bacteria, most coliform
and watery diarrhea. Shigella dysenteriae can cause a severe form of bacteria, and the swarming phenomenon of Proteus spp., while allowing
Salmonella spp. to grow. Sodium thiosulfate and ferric citrate allow difficult, give oxygen. Seek medical advice. If swallowed seek
the detection of hydrogen sulfide by the production of colonies with medical advice immediately and show this container or label.
black centers. Lactose is the carbohydrate present in SS Agar. Neutral 3. Follow proper established laboratory procedure in handling and
red and brilliant green are present as pH indicators. disposing of infectious materials.
Formula Storage
SS Agar Store the dehydrated medium below 30°C. The dehydrated medium is
Formula Per Liter very hygroscopic. Keep container tightly closed. Store prepared plates
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g at 2-8°C.
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Expiration Date
Bacto Bile Salts No. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5 g The expiration date applies to the product in its intact container when
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5 g stored as directed. Do not use a product if it fails to meet specifications
Sodium Thiosulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5 g for identity and performance.
Ferric Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5 g
Procedure
Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.33 mg Materials Provided
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025 g SS Agar
Final pH 7.0 ± 0.2 at 25°C Materials Required But Not Provided
Flasks with closures
Precautions Distilled or deionized water
1. For Laboratory Use. Bunsen burner or magnetic hot plate
2. IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM Waterbath (45-50°C)
AND SKIN. Avoid contact with skin and eyes. Do not breathe dust. Petri dishes
Wear suitable protective clothing. Keep container tightly closed. Incubator (35°C)
FIRST AID: In case of contact with eyes, rinse immediately with
plenty of water and seek medical advice. After contact with skin, Method of Preparation
wash immediately with plenty of water. If inhaled, remove to fresh 1. Suspend 60 grams in 1 liter distilled or deionized water.
air. If not breathing, give artificial respiration. If breathing is
Uninoculated Salmonella typhimurium
plate ATCC® 14028
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
2. Heat to boiling for no more than 2-3 minutes to dissolve completely. 2. A few nonpathogenic organisms may grow on SS Agar. These
Avoid overheating. DO NOT AUTOCLAVE. organisms can be differentiated by their ability to ferment lactose.10
3. Cool to 45-50°C in a waterbath. References
4. Dispense into sterile Petri dishes. Allow the surface of the medium 1. Gray, L. D. 1995. Escherichia, Salmonella, Shigella and Yersinia,
to air dry for two hours by leaving the lids ajar. p. 450-456. In Murray, P. R., E. J. Baron, M. A. Pfaller, F. C.
Specimen Collection and Preparation Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology,
1. Collect specimens or food samples in sterile containers or with 6th ed. American Society for Microbiology, Washington, D.C.
sterile swabs and transport immediately to the laboratory following 2. Leifson, E. 1935. New culture media based on sodium desoxycholate
recommended guidelines.1,7,8 for the isolation of intestinal pathogens and for the enumeration of
2. Process each specimen, using procedures appropriate for that colon bacilli in milk and water. J. Pathol. Bacteriol. 40:581.
specimen or sample.1,7,8 3. Rose, H. M., and M. H. Kolodny. 1942. The use of SS
(Shigella-Salmonella) Agar for the isolation of Flexner Dysentery
Test Procedure bacilli from the feces. J. Lab. Clin. Med. 27:1081-1083.
For isolation of Salmonella and Shigella spp. from clinical specimens,
4. Ewing, W. H., and D. W. Bruner. 1947. Selection of Salmonella
inoculate fecal samples and rectal swabs onto one quadrant of a SS Agar
and Shigella cultures for serologic classification. Am. J. Clin.
plate and streak for isolation. This will permit the development of
Pathol. 17:1-12.
discreet colonies. Incubate plates at 35°C. Examine at 24 hours and
again at 48 hours for colonies resembling Salmonella or Shigella spp. 5. Caudill, F. W., R. E. Teague, and J. T. Duncan. 1942. A rural
Note: SS Agar is inhibitory to some strains of Shigella spp. For shiga dysentery epidemic. JAMA 119:1402-1406.
additional information about specimen preparation and inoculation of 6. Hormaeche, E., N. L. Surraco, C. A. Peluffo, and P. L. Aleppo.
clinical specimens, consult appropriate references.1,7 1943. Causes of infantile summer diarrhea. Am. J. Dis. Child.
For testing food samples, consult appropriate references.8 66:539-551.
7. Pezzlo, M. (ed.). 1992. Aerobic bacteriology, p. 1.0.1-1.20.47. In
Results Isenberg, H. D. (ed.), Clinical microbiology procedures handbook,
Enteric organisms are differentiated by their ability to ferment lactose. vol. 1. American Society for Microbiology, Washington, D.C.
Salmonella and Shigella spp. are lactose non-fermenters and form 8. Vanderzant, C., and D. F. Splittstoesser (ed.) 1992. Compen-
colorless colonies on SS Agar. Salmonella spp. that are H2S positive dium of methods for the microbiological examination of foods,
produce colonies with black centers. Some Shigella spp. are inhibited 3rd ed. American Public Health Association, Washington, D.C.
on SS Agar.
9. Taylor, W. I., and B. Harris. 1965. Isolation of shigellae. II.
Coliforms are partially inhibited on SS Agar. E. coli produces pink to Comparison of plating media and enrichment broths. Am. J. Clin.
red colonies and may have some bile precipitation. Colonies of Pathol. 44:476.
Enterobacter aerogenes appear cream to pink in color. Citrobacter and 10. MacFaddin, J. F. 1985. Media for isolation-cultivation-
Proteus spp. may grow on SS Agar and produce colonies with gray to identification-maintenance of medical bacteria, Vol. 1. Williams
black centers due to H2S production. Enterococcus faecalis is partially & Wilkins, Baltimore, MD.
inhibited on SS Agar; colonies of E. faecalis are colorless.
Packaging
Limitations of the Procedure
SS Agar 100 g 0074-15
1. SS Agar is a highly selective medium. For this reason, it is not
500 g 0074-17
recommended as the sole medium for primary isolation of
2 kg 0074-07
Shigella.1,2,9 Some strains of Shigella may not grow.
10 kg 0074-08
Sabouraud Media
Bacto Sabouraud Agar Modified . Bacto Sabouraud Dextrose Agar
®
3. Follow proper established laboratory procedures in handling and 2. Sorrells, K. M., M. L. Speck, and J. A. Warren. 1970.
disposing of infectious materials. Pathogenicity of Salmonella gallinarum after metabolic injury by
freezing. Appl. Microbiol. 19:39- 43.
Storage
3. Gray, L. D. 1995. Escherichia, Salmonella, Shigella and Yersinia,
Store the dehydrated medium below 30°C. The dehydrated medium is
p. 450-456. In Murray, P. R., E. J. Baron, M. A. Pfaller, F. C.
very hygroscopic. Keep container tightly closed.
Tenover and R. H. Yolken (ed.), Manual of clinical microbiology,
Expiration Date 6th ed. American Society for Microbiology, Washington, D.C.
The expiration date applies to the product in its intact container when 4. Leifson, E. 1936. New selenite selective enrichment medium for
stored as directed. Do not use a product if it fails to meet specifications the isolation of typhoid and paratyphoid (Salmonella) bacilli. Am.
for identity and performance. J. Hyg. 24:423.
5. Guth, F. 1916. Centr. Bakt. I Abt. Orig. 77:487.
Procedure
6. Weiss, K. F., J. C. Ayres, and A. A. Kraft. 1965. Inhibitory action
Material Provided of selenite on Escherichia coli, Proteus vulgaris, and Salmonella
Selenite Broth thompson. J. Bacteriol. 90:857-862.
Materials Required But Not Provided 7. Rose, M. J., N. K. Enriki, and J. A. Alford. 1971. Growth and
survival of enterobacteria in selenite-cystine broth containing
Glassware
thiosulfate. J. Food Sci. 36:590-593.
Distilled or deionized water
Incubator 8. Association of Official Analytical Chemists. 1995. Official
Waterbath (45-50°C) (optional) methods of analysis of AOAC International, 16th ed. AOAC
Sterile tubes International, Arlington, VA.
9. United States Pharmacopeial Convention. 1995. The United
Method of Preparation States pharmacopeia, 23rd ed. The United States Pharmacopeial
1. Dissolve 23 grams in 1 liter distilled or deionized water. Convention. Rockville, MD.
2. Heat to boiling to pasteurize. 10. MacFaddin, J. F. 1985. Media for isolation-cultivation-
3. Avoid overheating. DO NOT AUTOCLAVE. identification- maintenance of medical bacteria, p. 701-705, vol 1,
Williams & Wilkins, Baltimore, MD.
Specimen Collection and Preparation
Obtain and process specimens according to the techniques and 11. Russell, S. F., J.-Y. D’Aoust, W. H. Andrews, and J. S. Bailey.
procedures established by institutional policy. 1992. Salmonella, p. 371-422. In Vanderzant, C., and D. F.
Splittstoesser (ed.) Compendium of methods for the microbiological
Test Procedure examination of foods, 3rd ed. American Public Health Association,
For a complete discussion on the isolation and identification Washington, D.C.
of Salmonella species refer to the appropriate procedures outlined 12. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures
in the references. handbook, vol. 1, American Society for Microbiology,
Results Washington. D.C.
Refer to appropriate references and procedures for results. 13. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
R. H. Yolken (ed.) 1995. Manual of clinical microbiology, 6th ed.
Limitations of the Procedure American Society for Microbiology, Washington, D. C.
1. Since the nutritional requirements of organisms vary, some strains
may be encountered that fail to grow or grow poorly on this medium. Packaging
Selenite Broth 100 g 0275-15
References 500 g 0275-17
1. Hartman, P. A., and S. A. Minnich. 1981. Automation for rapid 10 kg 0275-08
identification of salmonellae in foods. J. Food Prot. 44:385-386.
Cystine Broth is formulated to allow the proliferation of Salmonella and TARGET ORGAN(S): Lungs, Kidneys, Spleen, Liver.
while inhibiting the growth of competing non-Salmonella bacteria.2 FIRST AID: In case of contact with eyes, rinse immediately with
plenty of water and seek medical advice. After contact with skin,
Principles of the Procedure wash immediately with plenty of water. If skin irritation persists,
Selenite Cystine Broth contains Tryptone as a source of carbon, seek medical advice. If inhaled, remove to fresh air. If not
nitrogen, vitamins and minerals. Lactose is the carbohydrate. Sodium breathing, give artificial respiration. If breathing is difficult, give
Acid Selenite inhibits gram-positive bacteria and most enteric oxygen. Seek medical advice. If swallowed, induce vomiting; seek
gram-negative bacteria except Salmonella. L-cystine is a reducing agent. medical advice immediately and show this container or label.
Formula 3. Follow proper established laboratory procedures in handling and
disposing of infectious materials.
Selenite Cystine Broth
Formula Per Liter Storage
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Store the dehydrated medium below 30°C. The dehydrated medium is
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 g
very hygroscopic. Keep container tightly closed.
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Sodium Acid Selenite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 g Expiration Date
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01 g
The expiration date applies to the product in its intact container when
Final pH 7.0 ± 0.2 at 25°C stored as directed. Do not use a product if it fails to meet specifications
for identity and performance.
Precautions
1. For Laboratory Use. Procedure
2. VERY TOXIC. FATAL IF INHALED OR SWALLOWED. (US)
Materials Provided
VERY TOXIC BY INHALATION AND IF SWALLOWED. (EC)
DANGER OF CUMULATIVE EFFECTS. (EC) IRRITATING TO Selenite Cystine Broth
EYES, RESPIRATORY SYSTEM AND SKIN. Avoid contact with Materials Required but not Provided
skin and eyes. Do not breathe dust. Wear suitable protective Glassware
clothing. Keep container tightly closed. Distilled or deionized water
Autoclave
Incubator (35°C)
Tetrathionate Broth
User Quality Control Bismuth Sulfite Agar
Identity Specifications XLD Agar
Dehydrated Appearance: Off-white, free-flowing, homogeneous. Hektoen Enteric Agar
Solution: 2.3% solution, soluble in distilled or MacConkey Agar
deionized water on boiling.
Method of Preparation
Prepared Medium: Very light amber, clear to very
slightly opalescent, may have a slight 1. Suspend 23 grams in 1 liter distilled or deionized water.
precipitate. 2. Heat to boiling to dissolve completely.
Reaction of 2.3% 3. Dispense into tubes to a depth of 60 mm.
Solution at 25°C: pH 7.0 ± 0.2 4. DO NOT AUTOCLAVE. Use immediately.
Cultural Response Specimen Collection and Preparation
Prepare Selenite Cystine Broth per label directions. Inoculate Collect specimens according to recommended guidelines.
and incubate the tubes at 35 ± 2°C for 24 ± 2 hours and
subculture on MacConkey Agar plates. Test Procedure4,5
INOCULUM
ORGANISM ATCC® CFU GROWTH APPEARANCE 1. Prepare sample according to food type.
Escherichia 25922* 100-1,000 partial to pink with bile 2. Inoculate into recommended pre-enrichment broth.
coli complete precipitate 3. Transfer 1 ml of mixture to 10 ml Selenite Cystine Broth and to
inhibition
Salmonella
10 ml Tetrathionate Broth.
typhimurium 14028* 100-1,000 good colorless 4. Incubate at 35°C for 24 ± 2 hours.
Shigella sonnei 9290* 100-1,000 fair to good colorless 5. Mix and streak 3 mm loopful (10 µl) of sample from both broths
The cultures listed are the minimum that should be used for onto Bismuth Sulfite Agar, Xylose Lysine Desoxycholate Agar,
performance testing. Hektoen Enteric Agar or MacConkey Agar.
*These cultures are available as Bactrol™ Disks and should be used 6. Incubate plates at 35°C for 24 ± 2 hours.
as directed in Bactrol Disks Technical Information.
7. Examine plates for the presence of colonies that are typical for
Salmonella spp.
6. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compen- and critical point (HACCP) systems; final rule. Fed. Regis.
dium of methods for the microbiological examination of foods, 61:38917-38925.
3rd ed. American Public Health Association, Washington, D.C. 10. MacFaddin, J. F. 1985. Media for isolation-cultivation-
7. FDA Bacteriological Analytical Manual, 8th ed. AOAC identification-maintenance of medical bacteria, Vol. 1. Williams
International, Gaithersburg, MD. & Wilkins, Baltimore, MD.
8. Association of Official Analytical Chemists. 1995. Official
methods of analysis of AOAC International, 16th ed. AOAC Packaging
International, Arlington, VA. Simmons Citrate Agar 100 g 0091-15
9. Federal Register. 1996. Pathogen reduction; hazard analysis 500 g 0091-17
Formula 3. Rotate the inoculated tubes to mix the inoculum uniformly with
Snyder Test Agar the medium and allow to solidify in an upright position.
Formula Per Liter 4. Incubate at 35°C. Observe color at 24, 48 and 72 hours.
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g Alban Modication
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g 1. Collect enough unstimulated saliva to just cover the medium in the
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g tube. When specimen collection is difficult, dip a sterile cotton
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g swab into the saliva under the tongue or rub on tooth surfaces and
Brom Cresol Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g place the swab just below the surface of the medium.
Final pH 4.8 ± 0. 2 at 25°C 2. Incubate the inoculated tubes and an uninoculated control at 35°C.
Precautions 3. Examine tubes daily for four days.
1. For Laboratory Use. 4. Observe daily color change compared to control tube.
2. Follow proper established laboratory procedures in handling and Results
disposing of infectious materials. Snyder Procedure
Storage Observe tubes for a change in color of the medium from bluish-green
(control) to yellow. A positive reaction is a change in color so that
Store the dehydrated medium below 30°C. The dehydrated medium is
green is no longer dominant. Record as ++ to ++++. A negative
very hygroscopic. Keep container tightly closed.
reaction is no change in color or only a slight change. Green is still
Expiration Date dominant. Record as 0 to +.
The expiration date applies to the product in its intact container when Interpretation:
stored as directed. Do not use a product if it fails to meet specifications HOURS INCUBATION
CARIES ACTIVITY 24 48 72
for identity and performance.
Marked Positive – –
Procedure Moderate Negative Positive –
Slight Negative Negative Positive
Materials Provided Negative Negative Negative Negative
Snyder Test Agar
Data summarizing the correlation between the Snyder colorimetric test
Materials Required but not Provided and Lactobacillus counts on specimens of saliva collected routinely
Glassware are tabulated.
Petri dishes
Distilled or deionized water Alban Modification
Autoclave a. No color change
Incubator (35°C) b. Color beginning to change to yellow from top of medium down (+)
Waterbath (45°C) c. One half of medium yellow (++)
Cotton Swab
d. Three fourths of medium yellow (+++)
Paraffin
e. The entire medium is yellow (++++)
Method of Preparation The final report is a composite of the daily readings, for example;
1. Suspend 65 grams in 1 liter distilled or deionized water. – + ++ +++. The readings indicate the rapidity and amount of acid
2. Heat to boiling to dissolve completely. production.
3. Autoclave at 121°C for 15 minutes. Limitations of the Procedure
Specimen Collection and Preparation 1. The data indicate only what is happening at the time the specimen
Specimens should be collected preferably before breakfast, lunch, or was collected.
dinner, and before the teeth are brushed. This procedure can be done 2. At least two specimens collected with 2-4 days must be obtained
just before lunch or dinner. to establish a base-line or reference point.
3. Only when two or more specimens have been cultured can any
Test Procedure reliability or prediction be obtained.
Snyder Procedure 3,4 4. The clinician must study enough cases by use of periodic laboratory
1. Collect specimens of saliva in a sterile container while patient is data to establish the value of significance for the purpose intended.
chewing paraffin for 3 minutes.
2. Shake specimens thoroughly and transfer 0.2 ml to a tube of sterile
References
Snyder Test Agar melted and cooled to 45°C. (Prepared medium 1. MacFaddin, J. F. 1985. Media for isolation-cultivation-
in tubes is heated in a boiling water bath for 10 minutes and identification-maintenance of medical bacteria, p. 713-715. vol. 1.
cooled to 45°C. Williams & Wilkins, Baltimore, MD.
2. Lewis, D. W., and A. I. Ismail. 1995. Periodic health examination, 4. Snyder. 1941. J. Am. Dent. Assoc. 28:44.
1995 update: 2. Prevention of dental caries. Canadian Medical 5. Alban. 1970. J. Dent. Res. 49:641.
Association Journal 152:836- 846.
3. Snyder. 1941. J. Dent. Res. 20:189. Packaging
Snyder Test Agar 500 g 0247-17
Bacto Soytone®
SOLUTION OF
Carbohydrate (%)
TEST SOYTONE ORGANISM ATCC® RESULT Total 24.0
Growth 2% w/0.5% Brucella suis 4314 good growth Nitrogen Content (%)
Response NaCl and
1.5% agar Total Nitrogen 9.4 AN/TN 33.0
Amino Nitrogen 3.1
Growth 2% w/0.5% Escherichia 25922* good growth
Response NaCl and coli Amino Acids (%)
1.5% agar Alanine 2.46 Lysine 3.45
Growth 2% w/0.5% Staphylococccus 25923* good growth Arginine 3.82 Methionine 0.86
Response NaCl and aureus Aspartic Acid 7.27 Phenylalanine 2.46
1.5% agar
Cystine 1.45 Proline 2.92
The cultures listed are the minimum that should be used for Glutamic Acid 12.76 Serine 2.87
performance testing. Glycine 2.51 Threonine 2.17
*These culture are available as Bactrol™ Disks and should be used as Histidine 1.24 Tryptophan 0.47
directed in Bactrol Disks Technical Information. Isoleucine 2.37 Tyrosine 1.93
Leucine 4.03 Valine 2.65
Inorganics (%) stored as directed. Do not use a product if it fails to meet specifications
Calcium 0.055 Phosphate 0.820 for identity and performance.
Chloride 0.165 Potassium 2.220
Cobalt <0.001 Sodium 3.404 Procedure
Copper <0.001 Sulfate 2.334 Materials Provided
Iron 0.008 Sulfur 1.660
Soytone
Lead <0.001 Tin <0.001
Magnesium 0.161 Zinc 0.001 Materials Required But Not Provided
Manganese <0.001 Materials vary depending on the medium being prepared.
Vitamins (µg/g)
Biotin 0.2 PABA 9.0
Method of Preparation
Choline (as Choline Chloride) 2200.0 Pantothenic Acid 13.0 Refer to the final concentration of Soytone in the formula of the me-
Cyanocobalamin <0.1 Pyridoxine 11.0 dium being prepared. Add Soytone as required.
Folic Acid 3.0 Riboflavin <0.1 Specimen Collection and Preparation
Inositol 2100.0 Thiamine 1.2
Nicotinic Acid 19.1 Thymidine 113.2 Obtain and process specimens according to the techniques and procedures
established by laboratory policy.
Biological Testing (CFU/g)
Coliform negative Standard Plate Count 38 Test Procedure
Salmonella negative Thermophile Count <3 See appropriate references for specific procedures using Soytone.
Spore Count 10
Results
Precautions Refer to appropriate references and procedures for results.
1. For Laboratory Use.
2. Follow proper established laboratory procedures in handling and Packaging
disposing of infectious materials. Soytone 500 g 0436-17
10 kg 0436-08
Storage
Store Soytone below 30°C. The product is very hygroscopic. Keep con-
tainer tightly closed.
Expiration Date
The expiration date applies to the product in its intact container when
Lipase Reagent
Lipase Reagent, a mixture of tributyrin and Polysorbate 80, is
recommended as the lipid source. Other lipoidal emulsions may be
Intended Use prepared from cottonseed meal, cream, Wesson® oil and olive oil. A
Bacto Spirit Blue Agar is for use with Bacto Lipase Reagent or other satisfactory emulsion can be prepared by dissolving 10 grams gum
lipid source for detecting and enumerating lipolytic microorganisms. acacia or 1 ml Tween 80® in 400 ml warm distilled water, adding 100
ml cottonseed or olive oil and agitating vigorously to emulsify.
Summary and Explanation
In 1941, Starr1 described a lipid emulsion medium for detecting Principles of the Procedure
lipolytic (lipase-producing) microorganisms to which he added the dye, Spirit Blue Agar contains Tryptone as a source of carbon, nitrogen,
spirit blue. Other dyes as indicators of lipolysis were toxic to many vitamins and minerals. Yeast Extract supplies B-complex vitamins
microorganisms. Spirit blue did not have toxic effects. When testing which stimulate bacterial growth. Spirit Blue is the indicator of lipolysis.
samples of dairy products, air and sewage on Spirit Blue Agar, Starr Bacto Agar is the solidifying agent.
obtained accurate counts of lipolytic microorganisms and total Lipase Reagent contains tributyrin, a true fat and the simplest triglyceride
microbial counts on the same medium. occurring in natural fats and oils. It is a good substrate when testing for
Lipolytic microorganisms, such as psychrotrophic bacteria, molds or lipolytic microorganisms because some microorganisms that hydrolyze
yeasts, can adversely affect the flavor of milk and high fat dairy tributyrin will not hydrolyze other triglycerides or fats containing
products. Spirit Blue Agar is a recommended medium for testing milk longer chain fatty acids.2
and dairy products.2
Formula Procedure
Spirit Blue Agar Materials Provided
Formula Per Liter Spirit Blue Agar
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g Lipase Reagent
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g Materials Required but not Provided
Spirit Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.15 g Glassware
Final pH 6.8 ± 0.2 at 25°C Petri dishes
Lipase Reagent Distilled or deionized water
A ready-to-use lipid suspension, containing a mixture of Autoclave
tributyrin and Polysorbate 80. Incubator (35°C)
Intended Use
Bacto Staphylococcus Medium 110 is used for isolating and differentiating adding 7.5% NaCl to Phenol Red Mannitol Agar to make a selective
staphylococci based on mannitol fermentation, pigment formation and isolation medium for staphylococci using a high salt content. Further
gelatinase activity. studies by Chapman5 led to the development of Staphylococcus
Medium 110. This medium is included in standard methods procedures
Also Known As for selectively isolating pathogenic staphylococci from foods.6
Staphylococcus Medium 110 is also known as Staphylococcus Agar
No. 110 (Staphy-110, S-110) and Stone Gelatin Agar.1 Principles of the Procedure
Staphylococcus Medium 110 contains Tryptone as a source of carbon,
Summary and Explanation nitrogen, vitamins and minerals. Yeast Extract supplies B-complex
Stone2 described a culture medium on which food-poisoning staphylococci vitamins which stimulate bacterial growth. Sodium Chloride, in high
gave a positive gelatinase test. Chapman, Lieb and Curcio3 later concentration, inhibits most bacteria other than staphylococci. Lactose
reported that pathogenic staphylococci strains typically ferment and D-Mannitol are the carbohydrates. Gelatin is included for testing
mannitol, form pigment and produce gelatinase. Chapman4 suggested liquefaction. Bacto Agar is the solidifying agent.
DL-Histidine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 g plenty of water and seek medical advice. After contact with skin,
L-Lysine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.85 g wash immediately with plenty of water. If inhaled, remove to fresh
L-Tyrosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.21 g air. If not breathing, give artificial respiration. If breathing is
DL-Threonine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g difficult, give oxygen. Seek medical advice. If swallowed seek
DL-Valine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g medical advice immediately and show this container or label.
L-Leucine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 g
DL-Isoleucine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.44 g
3. Follow proper established laboratory procedure in handling and
Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.06 g disposing of infectious materials.
DL-Serine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 g
DL-Alanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.43 g
Storage
L-Glutamic Acid HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 g Store the dehydrated medium below 30°C. The dehydrated medium is
L-Aspartic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.45 g very hygroscopic. Keep container tightly closed.
DL-Phenylalanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.26 g
DL-Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g Expiration Date
L-Proline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g The expiration date applies to the product in its intact container when
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g stored as directed. Do not use a product if it fails to meet specifications
Potassium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g for identity and performance.
Magnesium Sulfate Anhydrous Reagent . . . . . . . . . . . . . 0.05 g
Potassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g Procedure
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 g Materials Provided
Thiamine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01 g
Nicotinamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01 g
Synthetic Broth AOAC
Final pH 7.1 ± 0.1 at 25°C Materials Required but not Provided
Glassware
Precautions Distilled or deionized water
1. For Laboratory Use. Autoclave
2. MAY BE IRRITATING TO EYES, RESPIRATORY SYSTEM Incubator (35°)
AND SKIN. (US) Avoid contact with skin and eyes. Do not breathe 20 x 150 mm tubes with closures
dust. Wear suitable protective clothing. Keep container tightly Sterile 10% dextrose solution
closed.
FIRST AID: In case of contact with eyes, rinse immediately with Method of Preparation
1. Suspend 17 grams in 1 liter distilled or deionized water.
2. Boil for 1-2 minutes.
3. Dispense 10 ml amounts into 20 x 150 mm culture tubes.
User Quality Control
4. Autoclave at 121°C for 20 minutes.
Identity Specifications 5. Before inoculating, aseptically add 0.1 ml sterile 10% dextrose
Dehydrated Appearance: White, homogeneous, free-flowing. solution to each tube.
Solution: 1.7% solution, soluble in distilled or
deionized water on boiling. Solution Specimen Collection and Preparation
is colorless and clear with no Refer to appropriate references for specimen collection and preparation.
precipitate.
Prepared Medium: Colorless and clear with no Test Procedure
precipitate. See appropriate references for specific procedures.
Reaction of 1.7% Results
Solution at 25°C: pH 7.1 ± 0.1
Refer to appropriate references and procedures for results.
Cultural Response
Prepare Synthetic Broth AOAC per label directions. Inoculate Limitations of the Procedure
and incubate the tubes at 35 ± 2°C for 18-24 hours. Not applicable
APPROXIMATE
ORGANISM ATCC® INOCULUM CFU GROWTH References
Pseudomonas aeruginosa 15442 100 good 1. Association of Official Analytical Chemists. 1995. Official
Salmonella choleraesuis 10708 100 good methods of analysis of AOAC International, 16th ed. AOAC
Salmonella typhi 6539 100 good International, Arlington, VA.
Staphylococcus aureus 6538 100 good
Packaging
The cultures listed are the minimum that should be used for
performance testing. Synthetic Broth AOAC 500 g 0352-17
10 kg 0352-08
Bacto m T7 Agar
® Standard Methods procedures to recover injured total coliform bacteria
from treated water specify m T7 Agar.9 Stressed organisms can be
present in treated drinking water and wastewater, saline waters and
Intended Use relatively clean surface waters.9
Bacto mT7 Agar is used for recovering injured coliforms from treated
water by membrane filtration. Principles of the Procedure
The ingredients of m T7 Agar support growth of injured coliforms.
Summary and Explanation Proteose Peptone No. 3 provides nitrogen and amino acids. Yeast
Selective media used with the membrane filter method do not Extract is a vitamin source and Lactose provides carbon. Tergitol 7
adequately recover injured coliforms.1,2,3,4 McFeters et al. studied the and Polyoxyethylene Ether W-1 are selective agents at optimal
influences of diluents, media and procedures in recovering injured concentrations that will not affect recovery of injured coliforms. Brom
coliform bacteria and found improved recovery using Tergitol 7 Agar.5 Cresol Purple and Brom Thymol Blue are indicators of lactose
LeChevallier et al. modified Tergitol 7 Agar and developed a new fermentation. The combination of dyes provides a good differential
medium, m T7 Agar, for improved recovery of injured coliforms from reaction as well as additional inhibition to noncoliform bacteria. Bacto
drinking water.6 In a later study, LeChevallier et al.7 evaluated mT7 Agar is a solidifying agent.
Agar as a fecal coliform medium and found optimum recovery using Penicillin G (1.0 µg/ml), aseptically added to the medium after auto-
preincubation at 37°C for 8 hours followed by incubation at 44.5°C for claving, prevents growth of gram-positive cocci without interfering
12 hours.7 The authors found that incorporation of 0.1 µg of penicillin G with recovery of coliforms.8
per ml, aseptically added to the medium after autoclaving, prevented
growth of gram-positive cocci that may break through. Later, they Formula
found that 1.0 µg/ml of penicillin G provided far better inhibition of m T7 Agar
gram-positive organisms without interfering with the recovery of Formula Per Liter
coliforms. LeChevallier and McFeters reported the work of five col- Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . . 5 g
laborating laboratories testing coliform recovery from contaminated Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
surface water and sewage samples.8 They found m T7 Agar to be more Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
effective than m Endo Agar in recovering coliforms. Tergitol 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 ml
Polyoxyethylene Ether W-1 . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Brom Thymol Blue . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g
User Quality Control Bacto Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g
Identity Specifications Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Dehydrated Appearance: Yellow-green to blue-green, Final pH 7.4 ± 0.2 at 25°C
free-flowing, homogeneous, may
have a slightly moist appearance Precautions
and/or tendency to form soft lumps. 1. For Laboratory Use.
Solution: 4.86% solution, soluble in distilled 2. Follow proper established laboratory procedures in handling and
or deionized water upon boiling. disposing of infectious materials.
Reddish purple, slightly opalescent
without significant precipitate. Storage
Prepared Medium: Reddish purple, slightly opalescent Store the dehydrated medium below 30°C. The dehydrated medium is
without significant precipitate.
very hygroscopic. Keep container tightly closed.
Reaction of 4.86%
Solution at 25°C: pH 7.4 ± 0.2 Store prepared plates containing penicillin G at 2-8°C and use within
1 week after preparation.6
Cultural Response
Prepare mT7 Agar per label directions. Inoculate with test Expiration Date
organisms diluted in 10 ml of water. Incubate at 35 ± 2°C for The expiration date applies to the product in its intact container when
8 hours and then at 44.5°C for an additional 12 hours. stored as directed. Do not use a product if it fails to meet specifications
INOCULUM COLONY
ORGANISM ATCC® CFU (approx.) GROWTH COLOR for identity and performance.
Escherichia coli 25922* 100 good yellow
Escherichia coli 13762 100 good yellow
Procedure
Enterococcus faecalis 19433 100 poor to fair – Materials Provided
Pseudomonas aeruginosa 27853* 100 poor to fair – m T7 Agar
The cultures listed are the minimum that should be used for Materials Required But Not Provided
performance testing.
*These cultures are available as Bactrol™ Disks and should be Glassware
used as directed in Bactrol Disks Technical Information. Autoclave
Distilled or deionized water
Membrane filter equipment 2. The procedure for enumerating fecal coliforms with m T7 Agar
Sterile 47 mm 0.45 µm gridded membrane filters requires two incubation temperatures.
Sterile Petri dishes 50 x 9 mm 3. The addition of penicillin G is required for better inhibition of
Pipettes gram-positive bacteria.
Stereoscopic microscope 4. m T7 Agar may recover other coliforms in addition to E. coli. Some
Dilution bottles drinking water samples contain so many non-coliform bacteria that
Incubator or waterbath (37°C and 45°C ) confluent growth may occur. Care must be taken to distinguish
Penicillin G (1.0 µg/ml) yellow colonies from background growth.9
Method of Preparation References
1. Suspend 48.6 grams in 1 liter distilled or deionized water. 1. Maxcy, R. B. 1970. Non-lethal injury and limitations of recovery
2. Heat to boiling to dissolve completely. of coliform organisms on selective media. J. Milk Food Technol.
3. Autoclave at 121°C for 15 minutes. 33:445-448.
4. To prepare a more selective medium, aseptically add 1.0 µg 2. Scheusner, D. L., F. F. Busta, and M. L. Speck. 1971. Inhibition
penicillin G per ml to the sterile medium cooled to 45°C. of injured Escherichia coli by several selective agents. Appl.
Microbiol. 21:46-49.
5. Dispense 4-5 ml amounts into 50 x 9 mm Petri dishes.
3. Grabow, W. O. K., and M. du Preez. 1979. Comparison of
Note: Stock solutions of 0.1 mg/ml of penicillin G (sodium salt) can mEndo LES, MacConkey and Teepol media for membrane filtration
be filter sterilized, frozen in aliquots, and stored for up to 6 months. counting of total coliform bacteria in water. Appl. Environ.
(One international or USP penicillin unit is equivalent to 0.6 µg of Microbiol. 38:351-358.
benzylpenicillin sodium).
4. Hoadley, A. W., and C. M. Cheng. 1974. Recovery of indicator
Specimen Collection and Preparation bacteria on selective media. J. Appl. Bacteriol. 37:45-57.
Water samples should be collected as described in Standard Methods 5. McFeters, G. A. , S. C. Cameron, and M. W. LeChevallier. 1982.
for the Examination of Water and Wastewater.9 Influence of diluents, media and membrane filters on detection of
injured waterborne coliform bacteria. Appl. Environ. Microbiol.
Test Procedure 43:97-103.
For a complete discussion of stressed organisms in water testing, refer 6. LeChevallier, M. W., S. C. Cameron, and G. A. McFeters. 1983.
to the membrane filter procedure for the coliform group as described New medium for improved recovery of coliform bacteria from
in Standard Methods for the Examination of Water and Wastewater.9 drinking water. Appl. Environ. Microbiol. 45:484-492.
Incubate inoculated plates at 37°C for 8 hours and then at 44.5°C for 7. LeChevallier, M. W., P. E. Jajanoski, A. K. Camper, and G. A.
an additional 12 hours. This procedure has been found to produce McFeters. 1984. Evaluation of m-T7 agar as a fecal coliform
consistently higher fecal coliform counts with mT 7 Agar.7 bacteria from drinking water. Appl. Environ. Microbiol. 48:371-375.
8. LeChevallier, M. W., and G. A. McFeters. 1985. Enumerating
Results injured coliforms in drinking water. Research and Technology.
After incubation, count all yellow, smooth, convex colonies as J. AWWA. 77:81-87.
coliforms with the aid of a stereoscopic microscope. 9. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). 1995.
Standard methods for the examination of water and wastewater,
Limitations of the Procedure 19th ed. American Public Health Association, Washington, D.C.
1. Since the nutritional requirements of organisms vary, some strains
may be encountered that fail to grow or grow poorly on this Packaging
medium. mT7 Agar 100 g 0018-15
Formula Procedure
TAT Broth Base Materials Provided
Formula Per Liter TAT Broth Base (dehydrated)
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g TAT Broth (prepared)
Azolectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Final pH 7.2 ± 0.2 at 25°C Materials Required But Not Provided
Tween® 20 (for dehydrated TAT Broth Base)
TAT Broth Glassware
Formula Per Liter Autoclave
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g Waterbath (50-60°C)
Azolectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Sterile test tubes
Tween® 20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 ml
Final pH 7.2 ± 0.2 at 25°C Method of Preparation
TAT Broth Base (dehydrated)
Precautions 1. Suspend 25 grams in 960 ml distilled or deionized water.
1. For Laboratory Use. 2. Add 40 ml Tween® 20.
2. Follow proper established laboratory procedures in handling and 3. Heat to 50-60°C.
disposing of infectious materials. 4. Let stand 15-30 minutes with occasional agitation to dissolve completely.
Storage 5. Autoclave at 121°C for 15 minutes.
Store the dehydrated medium below 30°C. The dehydrated medium is 6. Dispense as desired.
very hygroscopic. Keep container tightly closed. TAT Broth (prepared)
Store the prepared medium at 15-30°C. 1. In an area adjacent to the clean room, remove bottles from their boxes.
2. Follow careful aseptic technique when uncapping bottles for testing.
User Quality Control Specimen Collection and Preparation
Obtain and process specimens according to the techniques and
Identity Specifications procedures established by laboratory policy.
TAT Broth Base
Dehydrated Appearance: Beige, free-flowing, homogeneous. Test Procedure
Solution: 2.5% solution with 4% Tween® 20; 1. Add one gram or one ml of an undiluted sample to 40 ml of
solution is light amber, clear to very complete medium and agitate to obtain an even suspension.
slightly opalescent with a very slight 2. Incubate tubes at 35 ± 2°C for 18-48 hours.
precipitate.
For a complete discussion on sterility testing refer to appropriate
Prepared Medium: Light amber, clear to very slightly procedures in USP.2
opalescent.
Reaction of 2.5% Results
Solution w/ 4% Tween® Tubes or bottles exhibiting growth should be subcultured for identification.
20 at 25°C: pH 7.2 ± 0.2
Limitations of the Procedure
Cultural Response 1. Since the nutritional requirements of organisms vary, some strains may
TAT Broth be encountered that fail to grow or grow poorly on this medium.
Prepare TAT Broth Base per label directions or use prepared
TAT Broth. Inoculate and incubate at 35 ± 2°C for 18-48 hours. References
INOCULUM 1. Orth, D. S. 1993. Handbook of cosmetic microbiology. Marcel
ORGANISM ATCC® CFU GROWTH
Pseudomonas aeruginosa 27853* 100-1,000 good
Dekker, Inc., New York, N.Y.
Salmonella typhi 6539 100-1,000 good 2. The United States Pharmacopeial Convention. 1995. The United
Staphylococcus aureus 25923* 100-1,000 good States pharmacopeia, 23rd ed. Microbial limits tests, p. 1681-1686.
The United States Pharmacopeial Convention Inc., Rockville, MD.
The cultures listed are the minimum that should be used for
performance testing. Packaging
*These cultures are available as Bactrol™ Disks and should be used
as directed in Bactrol Disk Technical Information. TAT Broth Base 500 g 0984-17
TAT Broth 10 x 90 ml 9072-73
2. Process each specimen, using procedures appropriate for that sample.7 incubation. A negative reaction is no color change after 48 hours
3. Test actively metabolizing 3-4 week old pure cultures of of incubation.
Mycobacterium, or an 18-24 hour isolate of Moraxella spp.
Carefully exclude underlying culture medium. References
1. Wayne, L. G., J. R. Doubek, and R. L. Russell. 1964. Classifi-
Test Procedure cation and identification of mycobacteria. 1. Tests employing
1. Prepare and sterilize 13 x 75 mm screw cap test tubes containing Tween 80 as substrate, Am. Rev. Respir. Dis. 90:588-597.
1 ml distilled or deionized water. Cool to room temperature.
2. Kubica, G. P., and W. E. Dye. 1967. Laboratory methods for
2. Add two drops TB Hydrolysis Reagent, taking care not to touch clinical and public health, mycobacteriology, p. 44. National
the glass dropper, which could contaminate the reagent and cause Communicable Disease Center, Atlanta, Georgia.
aberrant test results.
3. Runyon, E. H., A. G. Karlson, G. P. Kubica, and L. G. Wayne.
3. Transfer one loopful of test culture to the tube. Thoroughly emulsify 1974. Mycobacterium, p. 165. In E. H. Lennette, E. H. Spaulding,
the culture in the reagent. and J. P Truant (ed.), Manual of clinical microbiology, 2nd ed.
4. When testing Mycobacterium spp.: American Society for Microbiology, Washington, D.C.
a. use a known positive (M. kansasii ATCC® 12478) and negative 4. Kubica, G. P. 1973. Differential identification of mycobacteria.
(uninoculated tube and M. scrofulaceum) control in parallel with Am. Rev. Respir. Dis. 107:9-21.
the test culture to ascertain the validity of test results.
5. Wayne, L. G., et al. 1974. Highly reproducible techniques for use in
b. incubate at 35 ± 2°C in the dark with caps tight for 5-10 days.
systematic bacteriology in the genus Mycobacterium: tests for pigment,
c. read tubes at 5 and 10 days for any change in color in a strong urease, resistance to sodium chloride, hydrolysis of Tween 80, and
light against a white background. beta-galactosidase. Int. J. Syst. Bacteriol. 24:412-419.
5. When testing Moraxella and Neisseria spp.:
6. Weiner, M., and P. D. Penha. 1990. Evaluation of Bacto TB
a. use Moraxella catarrhalis ATCC® 25238 for a positive control, Hydrolysis Reagent (Tween 80) for the identification of
and Neisseria sicca ATCC® 9913 as a negative control. Branhamella catarrhalis. J. Clin. Microbiol. 28:126-127.
b. incubate at 35 ± 2°C in the dark with caps tight for 18-48 hours.
7. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures hand-
c. read tubes at 24 and 48 hours. book, vol. 2. American Society for Microbiology, Washington, D.C.
6. Do not shake the tubes. Examine the liquid, not the sedimented 8. Strain, B. A., and D. M. Grochel. 1995 Laboratory safety and
cells. Compare the color of the liquid with the control tube color. infectious waste management, p. 75-85. In P. R. Murray, E. J.
7. Record results. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual
8. Upon completion of the test, follow proper established laboratory of clinical microbiology, 6th ed. American Society for Microbiology,
procedures in disposing of infectious materials. Washington, D.C.
Results 9. Kent, P. T., and G. P. Kubica. 1985. Public Health
For Mycobacterium spp. - A positive reaction is indicated by a color Mycobacteriology, p. 5-20. Centers for Disease Control,
change of the solution from amber to pink or red in 5 days or less. A Atlanta, Georgia.
doubtful reaction is a color change in 5 to 10 days. A negative reaction
is no color change after 10 days.
Packaging
TB Hydrolysis Reagent 5 ml 3192-56*
For Moraxella and Neisseria spp. - A positive reaction is a color
change of the solution from amber to red or pink after 24 hours of *Store at 2-8°C
The cultures listed are the minimum that should be used for performance testing.
*This culture is available as BactrolTM Disks and should be used as directed in Bactrol Disks Technical Information.
ability to grow at elevated temperatures and produce indole from Brom Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08 g
tryptophane.1,2 The determination of indole production in conjunction Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
with the most-probable-number procedure often requires the use of Final pH 7.3 + 0.2 at 25°C
another medium and additional incubation time.
The membrane filter procedure has been recognized by Standard
Precautions
Methods for the Examination of Water and Wastewater as an alternate 1. For Laboratory Use.
test procedure.3 In 1981, Dufour et al. developed a simple, accurate, 2. Follow proper established laboratory procedures in handling and
nonlethal membrane filter technique for the rapid enumeration of disposing of infectious materials.
E. coli.4 This medium, m TEC Agar, quantifies E. coli within 24 hours
without requiring subculture and identification of isolates. The authors
Storage
reported that they were able to recover E. coli from marine, estuarine Store the dehydrated medium below 30°C. The dehydrated medium is
and fresh water samples. very hygroscopic. Keep container tightly closed.
2. MAY BE IRRITATING TO EYES, RESPIRATORY SYSTEM Materials Required But Not Provided
AND SKIN. (US) MAY CAUSE HARM TO THE UNBORN Glassware
CHILD. (US) Avoid contact with skin and eyes. Do not breathe Autoclave
dust. Wear suitable protective clothing. Keep container tightly Waterbath (50-55°C)
closed. TARGET ORGAN(S): Blood, Kidneys, Nerves. Incubator (35°C)
FIRST AID: In case of contact with eyes, rinse immediately with
plenty of water and seek medical advice. After contact with skin, Method of Preparation
wash immediately with plenty of water. If inhaled, remove to fresh 1. Suspend 60 grams of TPEY Agar Base in 900 ml distilled or
air. If not breathing, give artificial respiration. If breathing is deionized water.
difficult, give oxygen. Seek medical advice. If swallowed seek 2. Heat to boiling to dissolve completely.
medical advice immediately and show this container or label. 3. Autoclave at 121°C for 15 minutes. Cool to 50-55°C.
3. Follow proper established laboratory procedure in handling and 4. Aseptically add 100 ml of EY Tellurite Enrichment warmed to
disposing of infectious materials. room temperature and 10 ml of rehydrated Antimicrobic Vial P.
Mix thoroughly.
Storage
Store the dehydrated medium below 30°C. The dehydrated medium is Alternatively, use 100 ml of a 30% egg yolk emulsion, 10 ml of
very hygroscopic. Keep container tightly closed. Chapman Tellurite Solution 1% and 0.4 ml of filter sterilized 1%
polymyxin B solution.
Expiration Date 5. Pour 15-17 ml amounts into sterile Petri dishes.
The expiration date applies to the product in its intact container when Specimen Collection and Preparation
stored as directed. Do not use a product if it fails to meet specifications
Refer to appropriate references for specimen collection and preparation.
for identity and performance.
Test Procedure
Procedure Consult appropriate references.3
Materials Provided
TPEY Agar Base Results
EY Tellurite Enrichment Coagulase-positive staphylococci form black or dark-gray colonies due
Antimicrobic Vial P (Polymyxin B) to the reduction of colorless tellurite to free tellurium. Three types of
egg yolk precipitation reactions are produced by coagulase-positive
staphylococci:
User Quality Control 1. a discrete zone of precipitated egg yolk around and beneath the
Identity Specifications colonies;
Dehydrated Appearance: Light tan, free-flowing, homogeneous. 2. a clear zone or halo surrounding the colonies with a possible zone
Solution: 60 grams per 900 ml solution, soluble of precipitate beneath the colonies; and,
in distilled or deionized water on 3. no zone or halo around the colonies, but a precipitate beneath the
boiling. Prior to adding enrichment, colonies.
solution is light to medium amber,
opalescent. Limitations of the Procedure
Prepared Medium: Yellowish-beige, opaque. 1. Mannitol-positive and/or tellurite-positive staphylococcal strains
Reaction of 6.0% that are coagulase-negative are occasionally found. Definitive
Solution at 25°C: pH 7.2 ± 0.2 identification of S. aureus, therefore, should be based primarily on
the coagulase reaction, with mannitol fermentation and tellurite
Cultural Response reduction being used only for confirmation.3,5
Prepare TPEY Agar Base per label directions. Inoculate and 2. The prepared medium becomes less inhibitory to coagulase-negative
incubate at 35 ± 2°C for 18-48 hours.
strains of staphylococci if it is stored for longer than one week.2
INOCULUM COLONY
ORGANISM ATCC® CFU GROWTH APPEARANCE HALO† 3. Graves and Frazier4 showed that Bacillus spp. able to grow
Escherichia 25922* 1,000-2,000 marked to on TPEY Agar produce an antibiotic that inhibits growth of
coli complete inhibition – – staphylococci.
Staphylococcus 25923* 100-1,000 good black +
aureus References
Staphylococcus 14990 100-1,000 poor to fair black – 1. Crisley, F. D., R. Angelotti, and M. J. Foter. 1964. Multiplica-
epidermidis
tion of Staphylococcus aureus in synthetic cream fillings and pies.
†Zone of precipitation/clearing around the colony. Public Health Rep. 79:369.
The cultures listed are the minimum that should be used for 2. Crisley, F. D., J. T. Peeler, and R. Angelotti. 1965. Comparative
performance testing.
*These cultures are available as Bactrol™ Disks and should be
evaluation of five selective and differential media for the detection
used as directed in Bactrol Disks Technical Information. and enumeration of coagulase-positive staphylococci in foods.
Appl. Microbiol. 13:140.
3. Koneman, E. W., S. D. Allen, V. R. Dowell, Jr., and 5. MacFaddin, J. F. 1985. Media for isolation-cultivation-
H. M. Sommers. 1979. Color atlas and textbook of diagnostic identification-maintenance of medical bacteria, Vol. 1. Williams &
microbiology, 2nd ed. J. B. Lippincott Company, Philadelphia, PA. Wilkins, Baltimore, M.D.
4. Graves, R. R., and W. C. Frazier. 1963. Food microorganisms
influencing the growth of Staphylococcus aureus. Appl. Microbiol. Packaging
11:513. TPEY Agar Base 500 g 0556-17
Bacto TSA Blood Agar Base . Bacto Tryptic Soy Blood Agar
®
5. Dispense into sterile tubes while keeping suspension well mixed. 4. Hajna, A. A., and S. R. Damon. 1956. New enrichment and
6. Do not heat the medium after adding iodine. plating medium for the isolation of Salmonella and Shigella
organisms. Appl. Microbiol. 4:341.
Specimen Collection and Preparation 5. Kauffman, F. 1930. Ein kombiniertes Anreicherungsverfahren fur
Obtain and process specimens according to the techniques and proce- Typhus-und-Paratyphusbazillen. Zentralb. Bakteriol. Parasitenkd.
dures established by laboratory policy. For a complete discussion on Infektionskr. Hyg. Abr. I Orig. 113:148.
the collection, isolation and identification of Salmonella, refer to the 6. Knox, R., P. H. Gell, and M. R. Pollack. 1942. Selective media
appropriate procedures outlined in the references. for organisms of the Salmonella group. J. Pathol. Bacteriol.
Results 54:469-483.
Refer to appropriate references and procedures for results. 7. Catalano, C. R., and S. J. Knable. 1994. Incidence of
Salmonella in Pennsylvania egg processing plants and destruction
References by high pH. J. Food Prot. 57:587-591.
1. Hartman, P. A., and S. A. Minnich. 1981. Automation for rapid 8. Russell, S. F., J.-Y. D’Aoust, W. H. Andrews, and J. S. Bailey.
identification of salmonellae in foods. J. Food Prot. 44:385-386. 1992. Salmonella, p. 371-422. In C. Vanderzant, and D. F.
2. Sorrells, K. M., M. L. Speck, and J. A. Warren. 1970. Splittstoesser (ed.). Compendium of methods for the microbiological
Pathogenicity of Salmonella gallinarum after metabolic injury by examination of foods, 3rd ed. American Public Health Association,
freezing. Appl. Microbiol. 19:39- 43. Washington, D.C.
3. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, Packaging
and R. H. Yolken (ed.). 1995. Manual of clinical microbiology, TT Broth Base Hajna 500 g 0491-17
6th ed. American Society for Microbiology, Washington, D.C. 2 kg 0491-07
Procedure Results
Materials Provided Refer to appropriate references and procedures for results.
Tellurite Blood Solution
Limitations of the Procedure
Materials Required But Not Provided 1. Definitive identification of a strain of C. diphtheriae as a true
Materials vary depending on the medium being prepared. pathogen requires demonstration of toxin production.3
Method of Preparation References
1. Shake Tellurite Blood Solution before use. 1. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures
2. Refer to the final concentration of Tellurite Blood Solution in the handbook. American Society for Microbiology, Washington, D.C.
formula of the medium being prepared. Add Tellurite Blood 2. Clarridge, J. E., and C. A. Spiegel. 1995. Corynebacterium and
Solution as required. miscellaneous irregular gram-positive rods, Erysipelothrix, and
Specimen Collection and Preparation1 Gardnerella, p. 357-377. In P. R. Murray, E. J. Baron, M. A. Pfaller,
Both throat and nasopharyngeal specimens are necessary in cases of F. C. Tenover, and R. H. Yolken (ed.). Manual of clinical microbiol-
respiratory illness. If cutaneous diphtheria is suspected, collect skin, ogy, 6th ed. American Society for Microbiology, Washington, D.C.
throat and nasopharynx specimens. Use sterile silica gel for shipping 3. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
clinical specimens when cultures are not taken locally. Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
Test Procedure St. Louis, MO.
For a complete discussion of the collection, isolation and identification Packaging
of C. diphtheriae and other Corynebacterium spp., refer to Tellurite Blood Solution 6 x 25 ml 0139-66
appropriate procedures in the references.1,2
Solution 1% Uninoculated
plate
Staphylococcus aureus
ATCC® 25923
Salmonella typhimurium
ATCC® 14028
Cultural Response
Prepare medium per label directions. Inoculate Tergitol 7 Agar plates
with test organisms. Inoculate Tergitol 7 Broth tubes and leave caps
loosened. Incubate at 35 ± 2°C for 18-48 hours.
INOCULUM ACID
ORGANISM ATCC® CFU GROWTH PRODUCTION
Enterococcus faecalis 19433 100-1,000 none to poor N/A
Escherichia coli 25922* 100-1,000 good +
Salmonella typhimurium 14028* 100-1,000 good –
+ = positive, yellow colony or medium
– = negative, blue colony or medium as directed.
The cultures listed are the minimum that should be used for performance
testing.
*These cultures are available as Bactrol™ Disks and should be used
as directed in Bactrol Disks Technical Information.
extra Tryptone and Yeast Extract in the medium allows higher plasmid Procedure
yield per volume. Glycerol is used as the carbohydrate source in this Materials Provided
formulation. Unlike glucose, glycerol is not fermented to acetic acid.
Terrific Broth
Principles of the Procedure Materials Required But Not Provided
Tryptone and Yeast Extract provide necessary nutrients and cofactors Flasks with closures
for excellent growth of recombinant strains of E. coli. The Yeast Extract Distilled or deionized water
concentration is increased to allow for elevated cell yields. Autoclave
Potassium Phosphates are added to provide potassium for cellular Incubator (35°C)
systems and prevent cell death due to a drop in pH. Glycerol is added Glycerol
as a carbon and energy source.
Method of Preparation
Formula 1. Dissolve 47.6 grams in 1 liter of distilled or deionized water. Add
Terrific Broth 4 ml of Glycerol to the medium.
Formula Per Liter 2. Autoclave at 121°C for 15 minutes.
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 g
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 g
Specimen Collection and Preparation
Potassium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . . 9.4 g Refer to appropriate references for specimen collection and preparation.
Potassium Phosphate, Monobasic . . . . . . . . . . . . . . . . . . . 2.2 g Test Procedure
Final pH 7.2 ± 0.2 at 25°C Consult appropriate references for recommended test procedures.1,2
Precautions Results
1. For Laboratory Use. Growth is evident in the form of turbidity.
2. Follow proper established laboratory procedure in handling and
disposing of infectious materials. References
1. Tartoff, K. D., and C. A. Hobbs. 1987. Improved media for
Storage growing plasmid and cosmid clones. Bethesda Research
Store the dehydrated medium below 30°C. The dehydrated medium is Laboratories Focus 9:12.
very hygroscopic. Keep container tightly closed. 2. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular
Store prepared medium at 2-8°C. cloning: a laboratory manual, 2nd ed. Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.
Expiration Date
The expiration date applies to the product in its intact container when Packaging
stored as directed. Do not use a product if it fails to meet specifications Terrific Broth 500 g 0438-17
for identity and performance. Glycerol 100 g 0282-15
500 g 0282-17
Intended Use m Tetrathionate Broth Base has the same formulation as Tetrathionate
Broth Base, except that calcium carbonate has been omitted.1
Bacto m Tetrathionate Broth Base is used for selectively enriching
Salmonella by membrane filtration prior to isolation procedures. Principles of the Procedure
Proteose Peptone provides nitrogen, vitamins, amino acids and carbon
Summary and Explanation in m Tetrathionate Broth Base. Selectivity is achieved by the combination
Salmonella spp. cause many types of infections, from mild self-limiting of sodium thiosulfate and tetrathionate, which suppresses commensal
gastroenteritis to life-threatening typhoid fever.2 The most common intestinal organisms.3 Tetrathionate is formed in the medium by the
form of Salmonella disease is self- limiting gastroenteritis with fever addition of iodine and potassium iodide solution. Organisms containing
lasting less than two days and diarrhea lasting less than 7 days.2 the enzyme tetrathionate reductase will proliferate in the medium. Bile
Tetrathionate Broth, in single strength and without calcium carbonate, Salts, a selective agent, is added to suppress coliform bacteria and
was used by Kabler and Clark1 for the preliminary enrichment of inhibit gram-positive organisms.
Salmonella other than S. typhi. Their investigation found that
approximately 80% of Salmonella species recovered were from mixed Formula
cultures and that most coliforms were suppressed. The presence of m Tetrathionate Broth Base
calcium carbonate in the medium gave poor, erratic results. The Formula Per Liter
authors1 reported favorable results for enrichment of S. typhimurium Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
in the membrane filtration technique. This study used a 3-hour Bacto Bile Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
preliminary incubation on pads saturated with Tetrathionate Broth Sodium Thiosulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 g
followed by 15 hours incubation on m Brilliant Green Broth. Final pH 8.0 ± 0.2 at 25°C
Autoclave Results
Incubator (55°C) Growth is evident in the form of turbidity.
Petri dishes
Limitations of the Procedure
Method of Preparation Microorganisms other than B. coagulans may grow on this medium.
1. Suspend 39 grams in 1 liter distilled or deionized water. Perform microscopic examination and biochemical tests to identify to
2. Heat to boiling to dissolve completely. genus and species if necessary.
3. Autoclave at 121°C for 15 minutes. Avoid overheating which could
cause a softer medium.
References
1. Stern, Hegarty, and Williams. 1942. Food Research 7:186.
4. Cool to room temperature.
2. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compendium
Specimen Collection and Preparation of methods for the microbiological examination of foods, 3rd ed.
Refer to appropriate references for specimen collection and preparation. American Public Health Association, Washington, D.C.
Test Procedure Packaging
Consult appropriate references for recommended test procedures.1,2 Thermoacidurans Agar 500 g 0303-17
Storage
User Quality Control Store the dehydrated media at 2-8°C. The dehydrated medium is very
Identity Specifications hygroscopic. Keep container tightly closed.
Thiamine Assay Medium
Dehydrated Medium: Beige, homogeneous, tendency to Expiration Date
clump. The expiration date applies to the product in its intact container when
Solution: 4.25% (single strength) and 8.5% stored as directed. Do not use a product if it fails to meet specifications
(double strength) solution, soluble in for identity and performance.
distilled or deionized water on boiling
2-3 minutes. 4.25% solution is light Procedure
amber, clear, may have a slight precipitate.
Materials Provided
Prepared Medium: 4.25% solution is light amber, clear,
may have a slight precipitate. Thiamine Assay Medium
Thiamine Assay Medium LV
Reaction of 4.25%
Solution at 25°C: pH 6.5 ± 0.2 Materials Required But Not Provided
Thiamine Assay Medium LV Glassware
Dehydrated Appearance: Beige, homogeneous, tendency to Autoclave
clump. Spectrophotometer or nephelometer
Solution: 4.2% (single strength) and 8.4% Centrifuge
(double strength) solution, soluble in Incubator, 30°C and 35°C
distilled or deionized water on boiling
2-3 minutes. 4.2% solution is light Sterile tubes and caps
amber, clear, may have a slight Sterile 0.85% NaCl
precipitate. Stock culture of Lactobacillus viridescens ATCC® 12706 or
Prepared Medium: 4.2% solution is light amber, clear, Stock culture of Lactobacillus fermentum ATCC® 9338
may have a slight precipitate. Lactobacilli Agar AOAC
Reaction of 4.2% Micro Assay Culture Agar
Solution at 25°C: pH 6.0 ± 0.2 APT Agar
Lactobacilli Broth AOAC
Cultural Response
Micro Inoculum Broth
Thiamine Assay Medium APT Broth
Prepare single-strength Thiamine Assay Medium per label
directions. Prepare a standard curve using a thiamine hydro- Thiamine hydrochloride
chloride reference standard at 0.0 to 0.05 µg per 10 ml.
Inoculate with Lactobacillus fermentum ATCC® 9338 and Method of Preparation
incubate with caps loosened at 35-37°C for 16-18 hours. Read 1. Suspend the medium in 100 ml distilled or deionized water:
percent transmittance using a spectrophotometer at 660 nm. Thiamine Assay Medium - 8.5 grams;
Thiamine Assay Medium LV Thiamine Assay Medium LV - 8.4 grams.
Prepare single-strength Thiamine Assay Medium LV per label 2. Boil 2-3 minutes to dissolve completely.
directions. Prepare a standard curve using a thiamine hydro-
chloride reference standard at 0.0 to 25.0 µg per 10 ml. 3. Dispense 5 ml amounts into tubes, evenly dispersing the precipitate.
Inoculate with Lactobacillus viridescens ATCC® 12706 and 4. Add standard or test samples.
incubate with caps loosened at 30 ± 2°C for 16-20 hours. Read 5. Adjust tube volume to 10 ml with distilled or deionized water.
percent transmittance using a spectrophotometer at 660 nm.
6. Autoclave at 121°C for 5 minutes.
Specimen Collection and Preparation A standard curve should be run with each assay because conditions of
Prepare assay samples according to references given in the specific heating and incubation temperature that influence the standard curve
assay procedures. The samples should be diluted to approximately the readings cannot always be duplicated.
same concentration as the standard solution. The standard curve for Thiamine Assay Medium LV is obtained by
using thiamine at levels of 0.0, 1, 2.5, 5, 7.5, 10, 15, 20 and 25 ng of
Test Procedure thiamine hydrochloride per 10 ml tube. This is obtained by using 0.0,
Thiamine Assay Medium 0.2, 0.5, 1, 1.5, 2, 3, 4 and 5 ml of the standard solution, which contains
Prepare stock cultures of the test organism, Lactobacillus fermentum 5 ng (0.005 µg) thiamine hydrochloride per ml. The most effective
ATCC® 9338, by stab inoculation on Lactobacilli Agar AOAC or assay range is between 2.5 and 20 ng per tube.
Micro Assay Culture Agar. After 24-48 hours incubation at 35-37°C, The solution for preparing the standard curve for Thiamine Assay
keep the tubes in the refrigerator. Make transfers in triplicate at monthly Medium LV may be prepared as follows:
intervals. 1. Dissolve 50 mg of thiamine hydrochloride in 500 ml distilled
Prepare the inoculum by subculturing a stock culture of the test water (100 µg/ml).
organism in 10 ml of Lactobacilli Broth AOAC or Micro Inoculum 2. Add 1 ml of the solution in Step 1 to 99 ml distilled water (1 µg/ml).
Broth. After 16-18 hours incubation at 35-37°C, centrifuge the cells 3. Add 1 ml of the solution in Step 2 to 199 ml distilled water to give
under aseptic conditions and decant the supernatant liquid. Wash the a final concentration of 5 ng (0.005 µg) per ml.
cells three times with 10 ml sterile 0.85% NaCl. After the third wash,
Following incubation of L. viridescens ATCC® 12706 at 30 ± 2°C for
resuspend the cells in 10 ml sterile 0.85% NaCI. Add 0.5 ml of this
16-20 hours, the growth response is measured turbidimetrically.
suspension to 100 ml sterile 0.85% NaCl. Use one drop of the resulting
suspension to inoculate the assay tubes. Results
A standard curve should be run with each assay because conditions of Thiamine Assay Medium and Thiamine Assay Medium LV
heating and incubation temperature that influence the standard curve 1. Prepare a standard concentration response curve by plotting the
readings cannot always be duplicated. response readings against the amount of standard in each tube, disk
The tubes for the Thiamine Assay Medium standard curve contain or cup.
0.0, 0.005, 0.01, 0.015, 0.02, 0.03, 0.04 and 0.05 µg of thiamine 2. Determine the amount of vitamin at each level of assay solution by
hydrochloride per 10 ml tube. The most effective assay range for interpolation from the standard curve.
Thiamine Assay Medium is between 0.005 and 0.03 µg thiamine.
3. Calculate the concentration of vitamin in the sample from the
Prepare the stock solution of thiamine required for the preparation of average of these volumes. Use only those values that do not vary
the standard curve in Thiamine Assay Medium as follows: more than ±10% from the average and use the results only if two
1. Dissolve 0.1 gram of thiamine hydrochloride in 1,000 ml of thirds of the values do not vary more than ±10%.
distilled water (100 µg/ml).
2. Add 1 ml of the solution in Step 1 to 99 ml distilled water (1 µg/ml). Limitations of the Procedure
3. Add 1 ml of the solution in Step 2 to 99 ml distilled water to give a 1. The test organism used for inoculating an assay medium must be
final concentration of 10 ng (0.010 µg/ml). Use 0.0, 0.5, 1, 1.5, 2, cultured and maintained on media recommended for this purpose.
3, 4 and 5 ml of this final solution per tube. Prepare fresh stock 2. Aseptic technique should be used throughout the microbiological
solution daily. assay procedure.
After 20-24 hours incubation at 35-37°C, L. fermentum ATCC® 9338 is 3. The use of altered or deficient media may cause mutants having
capable of using the pyrimidine and thiazole moieties of the thiamine different nutritional requirements which will not give a satisfactory
molecule. It is essential that the growth response be measured turbidi- response.
metrically prior to this time. Incubate the tubes at 35-37°C for 16-18 4. For successful results, all conditions of the assay must be followed
hours, then place in the refrigerator for 15-30 minutes to stop growth. exactly.
The growth can then be measured by any suitable nephelometric method.
Thiamine Assay Medium LV References
Prepare stock cultures of the test organism, L. viridescens ATCC® 12706, 1. Sarett and Cheldelin. 1944. J. Biol. Chem. 155:153.
by stab inoculation on APT Agar or Lactobacilli Agar AOAC. After 2. Deibel, Evans, and Niven. 1957. Paper presented 57th general
24-48 hours incubation at 30 ± 2°C, keep the tubes in the refrigerator. meet. Soc. Am. Bacteriol. Detroit, MI.
Make transfers in triplicate at monthly intervals. 3. Evans and Niven. 1951. J. Bacteriol. 62:599.
Prepare the inoculum by subculturing a stock culture of the test organism 4. Diebel, Evans, and Niven. 1955. Bacteriol. Proc.
to 10 ml APT Broth or Lactobacilli Broth AOAC. After 16-20 hours
incubation at 30 ± 2°C, centrifuge the cells under aseptic conditions Packaging
and decant the supernatant liquid. Wash the cells three times with 10 ml Thiamine Assay Medium 100 g 0326-15
sterile 0.85% NaCl. After the third wash, resuspend the cells in 10 ml Thiamine Assay Medium LV 100 g 0808-15
sterile 0.85% NaCl. Add 1 ml of this cell suspension to 100 ml sterile
0.85% NaCl. Use one drop of this suspension to inoculate the assay tubes.
Thioglycollate Media
Bacto Fluid Thioglycollate Medium . Bacto NIH Thioglycollate
®
of sodium polyanetholesulfonate (SPS), are used in the inoculum of preservatives, making thioglycollate media useful in testing material
blood cultures specifically, for the isolation of anaerobes.18 which contains heavy metals.
The methodologies for the multiple applications using thioglycollate Resazurin or Methylene Blue are oxidation indicators. In the oxidized
medium are outlined in the references. state, methylene blue appears green, resazurin turns pink. In the
reduced state both compounds are colorless. Bacto Agar eliminates the
Principles of the Procedure need for seals because it retards dispersion of CO2, diffusion of oxygen
Thioglycollate media support the growth of a large variety of fastidious and reducing substances. 12 Substituting K Agar and Potassium
microorganisms having a wide range of growth requirements. The Chloride for Bacto Agar and Sodium Chloride in Fluid Thioglycollate
nitrogen source, provided by Casitone, Infusion from Beef, Proteose Medium w/K Agar produces a medium with greater clarity to facilitate
Peptone, Beef Extract, Pancreatic Digest of Casein varies with the earlier visual recognition of growth.
formula. Yeast Extract is added as a source of vitamins. Dextrose is included in the formulations because many organisms show
Sodium Thioglycollate, Thioglycollic Acid and L-Cystine lower the earlier and more vigorous growth. Sodium chloride is used to maintain
oxidation-reduction potential of the medium by removing oxygen to the osmotic balance of the media. Potassium Chloride and Dipotassium
maintain a low Eh. By creating an environment with a low Eh, the Phosphate are used as buffering agents.
reducing agents prevent the accumulation of peroxides which can be
toxic to some organisms. The sulfhydryl groups (-SH) of these
compounds also neutralize the antibacterial effect of mercurial
18. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Cultivation Fluid Thioglycollate Medium
and isolation of viable pathogens, p. 91,. Bailey & Scott’s diagnostic w/ Beef Extract 500 g 0697-17
microbiology, 9th ed. Mosby-Year Book, Inc. St. Louis, MO. 10 kg 0697-08
Packaging Fluid Thioglycollate Medium
Fluid Thioglycollate Medium 100 g 0256-15 w/K Agar 500 g 0607-17
500 g 0256-17 2 kg 0607-07
2 kg 0256-07 Thioglycollate Medium w/o Dextrose 500 g 0363-17
10 kg 0256-08
Thioglycollate Medium w/o Dextrose
NIH Thioglycollate Broth 500 g 0257-17 or Indicator 500 g 0432-17
Brewer Thioglycollate Medium 500 g 0236-17 Thioglycollate Medium w/o Indicator 500 g 0430-17
10 kg 0236-08
Tinsdale Agar
Bacto Tinsdale Base . Bacto Tinsdale Enrichment Desiccated
®
2. Clarridge, J. E., and C. A. Spiegel. 1995. Corynebacterium and 5. Moore, M. S., and E. I. Parsons. 1958. A study of a modified
miscellaneous irregular gram-positive rod, Erysipelothrix, and Tinsdale’s medium for the primary isolation of Corynebacterium
Gardnerella, p. 357-377. In P. R. Murray, E. J. Baron, M. A. diphtheriae. J. Infect. Dis. 102:88.
Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical 6. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
microbiology, 6th ed. American Society for Microbiology, Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
Washington, D.C. St. Louis, MO.
3. Tinsdale, G. F. W. 1947. A new medium for the isolation and 7. Bailey, R. W., and E. G. Scott. 1966. Diagnostic microbiology,
identification of C. diphtheriae based on the production of hydrogen 2nd ed., p. 213. The C. V. Mosby Company, St. Louis, MO.
sulphide. J. Pathol. Bacteriol. 59:461-466.
4. Billings, E. 1956. An investigation of Tinsdale Tellurite medium: Packaging
its usefulness and mechanisms of halo-formation. M.S. thesis. Tinsdale Base 500 g 0786-17
University of Michigan, Ann Arbor, MI. Tinsdale Enrichment Desiccated 6 x 15 ml 0342-33
Storage 2. Todd Hewitt Broth cannot be used unbuffered for bile solubility
Store the dehydrated medium below 30°C. The dehydrated medium is testing10
very hygroscopic. Keep container tightly closed.
References
Expiration Date 1. Todd, E. W., and L. F. Hewitt. 1932. A new culture medium for
The expiration date applies to the product in its intact container when the production of antigenic streptococcal haemolysin. J. Pathol.
stored as directed. Do not use a product if it fails to meet specifications Bacteriol. 35:973.
for identity and performance. 2. Updyke, E. L., and M. I. Nickle. 1954. A dehydrated medium for
the preparation of type specific extracts of group A
Procedure streptococci. Appl. Microbiol. 2:117.
Materials Provided 3. Elliott. 1945. J. Exp. Med. 81:573.
Todd Hewitt Broth 4. Moody, M. D., A. C. Siegel, B. Pittman, and C. C. Winter. 1963.
Materials Required But Not Provided Fluorescent- antibody identification of group A streptococci from
throat swabs. Am. J. Public Health, 53:1083.
Glassware
Autoclave 5. Facklam, R. R., and R. B. Carey. 1985. Streptococci and
Incubator (35°C) Aerococci, p. 154-175. In, E. H.Lennette, A. Balows, W. J.
Waterbath (45-50°C) (optional) Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical
Sterile tubes microbiology, 4th ed. American Society for Microbiology,
Washington, D.C.
Method of Preparation 6. Bourbeau, P. P., B. J. Heiter, J. P. Anhalt, and D. W. Naumovitz.
1. Suspend 30 grams in 1 liter distilled or deionized water. 1993. Comparison of direct specimen testing utilizing testpack
2. Autoclave at 121°C for 15 minutes. strep A with testing of specimens following a two-hour broth
3. Cool to room temperature. enrichment. Diagn. Microbiol. Infect. Dis. 17:93-96.
Specimen Collection and Preparation 7. MacFaddin, J. D. 1985. Media for isolation-cultivation-
Obtain and process specimens according to the techniques and procedures identification-maintenance medical bacteria, p.772-775. vol. 1.
established by laboratory policy. Williams & Wilkins, Baltimore, MD.
8. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
Test Procedure R. H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th
For a complete discussion on the isolation, identification and serological ed. American Society for Microbiology, Washington, D.C.
procedures of fastidious microorganisms, refer to the procedures
9. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures
described in appropriate references.4,5,8,9
handbook, American Society for Microbiology, Washington, D.C.
Results
Refer to appropriate references and procedures for results. Packaging
Todd Hewitt Broth 100 g 0492-15
Limitations of the Procedure 500 g 0492-17
1. Since the nutritional requirements of organisms vary, some strains may 2 kg 0492-07
be encountered that fail to grow or grow poorly on this medium. 10 kg 0492-08
Tomato Juice Agar Special is recommended for the direct plate count Principles of the Procedure
of lactobacilli from saliva and for cultivation of other acidophilic Tomato Juice Agar and Tomato Juice Agar Special
microorganisms. The number of lactobacilli in saliva is an index of
Tomato Juice is a source of carbon, protein and nutrients. Peptone
a predisposition to dental caries as described by Jay.5, 6 Many dentists
provides a source of nitrogen, amino acids and carbon. Peptonized Milk
use the direct count of lactobacilli for the diagnosis of caries.
contains lactose as an energy source. Bacto Agar is a solidifying agent.
The acidic pH of Tomato Juice Agar Special encourages growth of
Tomato Juice Broth
lactobacilli while inhibiting growth of accompanying bacteria. This
medium is more selective for lactobacilli than Tomato Juice Agar. Tomato Juice is a source of carbon, protein and nutrients. Yeast Extract
is a source of trace elements, vitamins and amino acids. Dipotassium
Tomato Juice Broth is recommended for use in cultivating and Phosphate and Monopotassium Phosphate provide buffering
isolating yeasts, lactobacilli and other aciduric microorganisms from capability. Magnesium Sulfate, Ferrous Sulfate and Manganese
clinical specimens and foods.
Cultural Response
Tomato Juice Agar Tomato Juice Broth
Prepare Tomato Juice Agar per label directions. Inoculate using Prepare Tomato Juice Broth per label directions. Inoculate and
the pour plate technique and incubate at 35 ± 2°C for 40-48 hours. incubate at 35 ± 2°C for 18-72 hours.
INOCULUM INOCULUM
ORGANISM ATCC® CFU GROWTH ORGANISM ATCC® CFU GROWTH
Lactobacillus acidophilus 4356 100-1,000 good Lactobacillus casei 9595 100-1,000 good
Lactobacillus casei 9595 100-1,000 good Lactobacillus delbrueckii 4797 100-1,000 good
Lactobacillus delbrueckii 4797 100-1,000 good Saccharomyces carlsbergensis 9080 100-1,000 good
Tomato Juice Agar Special Saccharomyces cerevisiae 9763 100-1,000 good
Prepare Tomato Juice Agar Special per label directions. Inoculate
and incubate at 35 ± 2°C for 18-48 hours (72 hours if necessary).
INOCULUM The cultures listed are the minimum that should be used for
ORGANISM ATCC® CFU GROWTH performance testing.
Lactobacillus acidophilus 4356 100-1,000 good
Lactobacillus casei 9595 100-1,000 good
Lactobacillus delbrueckii 4797 100-1,000 good
Procedure Packaging
Materials Provided Tomato Juice Agar 500 g 0031-17
Tomato Juice Agar Tomato Juice Agar Special 500 g 0389-17
Tomato Juice Agar Special
Tomato Juice Broth 500 g 0517-17
Tomato Juice Broth 10 kg 0517-08
Materials Required but not Provided
Glassware
Transport Media
Bacto Transport Medium Amies . Bacto Transport Medium
®
with skin and eyes. Do not breathe dust. Wear suitable protective 3. Label the bottle or vial and send to the laboratory with minimum
clothing. Keep container tightly closed. delay. Specimens may be refrigerated until ready for shipment.
FIRST AID: In case of contact with eyes, rinse immediately with 4. Submit to laboratory within 24 hours for culture and analysis.
plenty of water and seek medical advice. After contact with skin,
Results
wash immediately with plenty of water. If inhaled, remove to fresh
air. If not breathing, give artificial respiration. If breathing is Survival of bacteria in a transport medium depends on many factors
difficult, give oxygen. Seek medical advice. If swallowed seek including the type and concentration of bacteria in the specimen, the
medical advice immediately and show this container or label. formulation of the transport medium, the temperature and duration of
transport, and inoculation to appropriate culture media within 24 hours.
3. Follow proper established laboratory procedure in handling and
disposing of infectious materials. Optimal growth and typical morphology can only be expected following
direct inoculation and appropriate cultivation.
Storage
Store dehydrated media below 30°C. The dehydrated media are very Limitations of the Procedure
hygroscopic. Keep containers tightly closed. 1. Specimens taken from transport media will not exhibit the optimal
or comparative growth as expected from direct inoculation and
Expiration Date cultivation. These media do, however, provide an adequate degree
The expiration date applies to the product in its intact container when of preservation for those specimens which cannot be forwarded
stored as directed. Do not use a product if it fails to meet specifications immediately to the laboratory for prompt evaluation.
for identity and performance. 2. Viability of cells will diminish over time and some degree of
multiplication or growth of contaminants can occur during
Procedure prolonged periods of transit. This is particularly true of fecal
Materials Provided specimens that contain substantial numbers of coliform organisms.
Transport Medium Amies, Transport Medium Amies w/o Charcoal, 3. The condition of the specimen received by the laboratory for
Transport Medium Stuart, or Cary-Blair Transport Medium. culture is a significant variable in recovery and final identification
of the suspect pathogen. An unsatisfactory specimen (overgrown
Materials Required but not Provided by contaminants, containing non-viable organisms, or having the
Glassware number of pathogens greatly diminished) can lead to erroneous or
Autoclave inconclusive results.
Distilled or deionized water
Incubator References
1. Moffett, M., J. L. Young, and R. D. Stuart. 1948. Centralized
Method of Preparation gonococcus culture for dispersed clinics; the value of a new
1. Transport Medium Amies: Suspend 20 grams in 1 liter distilled transport medium for gonococci and trichomonas. Brit. Med. J.
or deionized water. Invert vials just before solidification to 2:421-424.
uniformly distribute the charcoal. 2. Stuart, R. D., S. R. Toshach, and T. M. Patsula. 1954. The
Transport Medium Amies w/o Charcoal: Suspend 10 grams in problem of transport of specimens for culture of gonococci. Can.
1 liter distilled or deionized water. J. Public Health 45:73-83.
Transport Medium Stuart: Suspend 14.1 grams in 1 liter 3. Stuart, R. D. 1946. The diagnosis and control of gonorrhea by
distilled or deionized water. bacteriological cultures. Glasgow M. J. 27:131-143.
Cary-Blair Transport Medium: Suspend 12.7 grams in 1 liter 4. Stuart, R. D. 1959. Transport medium for specimens in public
distilled or deionized water. health bacteriology. Public Health Reports 74:431-438.
2. Heat to boiling to dissolve completely. 5. Cary, S. G., and E. B. Blair. 1964. New transport medium for
3. Dispense into 6-8 ml capacity screw-cap vials to within 5mm of shipment of clinical specimens. J. Bacteriol. 88:96-98.
the top. Cap tightly. 6. Cary, S. G., M. S. Matthew, M. H. Fusillo, and C. Harkins.
4. Autoclave at 121°C for 15 minutes. 1965. Survival of Shigella and Salmonella in a new transport
medium. Am. J. Clin. Path. 43:294- 296.
Specimen Collection and Preparation 7. Neuman, D. A., M. W. Benenson, E. Hubster, and Thi Nhu Tuan.
Refer to appropriate references for specimen collection and primary 1971. N. Am. J. Clin. Path. 57:33-34.
isolation technique recommendations.10,11,12 8. Kelly, M. T., F. W. Hickman-Brenner, and J. J. Farmer III. 1991.
Test Procedure Vibrio, p. 384- 395. In A. Balows, W. J. Hausler, Jr., K. L.
Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of
1. Insert specimen swab(s) into the upper third of the medium in the
clinical microbiology, 5th ed.. American Society for Microbiology,
transport container.
Washington D.C.
2. Cut or break off the protruding portion of the swab stick. Tightly
9. Amies, C. R. 1967. A modified formula for the preparation of
screw the lid on the bottle or vial.
Stuart’s transport medium. Can. J. Public Health 58:296-300.
10. Miller, J. M., and H. T. Holmes. 1995. Specimen collection, 12. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures
transport, and storage, p. 19-31. In P. R. Murray, E. J. Baron, handbook. American Society for Microbiology, Washington, D.C.
M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of
clinical microbiology, 6th ed. American Society of Microbiology, Packaging
Washington, D.C. Transport Medium Amies 500 g 0996-17
11. Isenberg, H. D., F. D. Schoenknecht, and A. von Graevenitz. Transport Medium Amies w/o Charcoal 500 g 0832-17
1979. Cumitech 9, Collection and processing of bacteriological Transport Medium Stuart 500 g 0621-17
specimens. Coord. Ed., S. J. Rubin. American Society for
Microbiology, Washington, D.C. Cary-Blair Transport Medium 500 g 0505-17
Trichophyton Agars
Bacto Trichophyton Agar 1 . Trichophyton Agar 2 . Trichophyton
®
* T. equinum will grow to a 4+ reaction on Trichophyton Agar 5, which is not commercially available from Difco Laboratories. Trichophyton Agar 5 is equivalent
to Trichophyton Agar 1 (product code 0877) with added nicotinic acid (200 µg per liter).
Group 3 includes T. violaceum. It seldom produces microconidia but formulations for Trichophyton Agars 2,3 and 4, Inositol and Dextrose
does develop characteristically pigmented colonies. T. violaceum has a provide carbon; Thiamin is present for organisms requiring the
similar nutritional pattern as T. tonsurans; however, it grows very vitamin for growth.
slowly even in the presence of thiamine and produces a glabrous colony
Trichophyton Agars 6 and 7
without spores. T. tonsurans grows rapidly in the presence of thiamine
and shows numerous microconidia. Ammonium Nitrate is a source of nitrogen. Dextrose provides carbon.
Monopotassium Phosphate provides buffering capability. Magnesium
Group 4 includes T. megninii and T. equinum. Both can be identified Sulfate is a source of divalent cations and sulfate. Bacto Agar is a
solely from nutritional requirements. T. megninii requires histidine, solidifying agent. Histidine Hydrochloride is present in Trichophyton
as indicated on Trichophyton Agar 6 in Table 1. T. equinum requires Agar 7 for organisms requiring the amino acid histidine.
nicotinic acid, as indicated in the table.
T. gallinae is differentiated morphologically as well as culturally from Formula
T. megninii and T. equinum. T. gallinae grows on Trichophyton Agar 1 Trichophyton 1
or 6 without added vitamins. Formula Per Liter
Bacto Vitamin Assay Casamino Acids . . . . . . . . . . . . . . . . 2.5 g
Principles of the Procedure Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g
Trichophyton Agars 1, 2, 3 and 4 Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . 1.8 g
Vitamin Assay Casamino Acids is the source of nutrients. Dextrose Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g
provides carbon. Monopotassium Phosphate provides buffering Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
capability. Magnesium Sulfate is a source of divalent cations and Final pH 6.8 ± 0.2 at 25°C
sulfate. Bacto Agar is a solidifying agent. Where included in the
4. McGinnis, M. R., and L. Pasarell. 1992. Mycology, p. 6.11.6-6.11.7. Trichophyton Agar 2 500 g 0874-17
In H. D. Isenberg (ed.), Clinical Microbiology Procedures Hand- Trichophyton Agar 3 500 g 0965-17
book, vol. 1. American Society for Microbiology, Washington, D.C.
Trichophyton Agar 4 500 g 0197-17
Packaging Trichophyton Agar 6 500 g 0524-17
Trichophyton Agar 1 500 g 0877-17 Trichophyton Agar 7 500 g 0955-17
A = acid reaction (yellow) K = alkaline reaction (no color change) The cultures listed are the minimum that should be used for
+gas = cracks, splits or bubbles –gas = no cracks, splits or bubbles performance testing.
in medium in medium *These cultures are available as Bactrol™ Disks and should
+H2S = black precipitate in butt –H2S = no black precipitate in butt be used as directed in Bactrol Disks Technical Information.
Triple Sugar Iron Agar is recommended for differentiation of enteric Bunsen burner or magnetic hot plate
gram negative bacilli from clinical specimens,7,8,9 dairy samples10, and Tubes with closures
food samples.11,12,13,14 Its use is also recommended for microbial limits Inoculating needle
testing of Escherichia coli and Salmonella spp.15 Autoclave
Incubator (35°C)
Principles of the Procedure
Beef Extract, Yeast Extract, Bacto Peptone, and Proteose Peptone Method of Preparation
provide nitrogen, vitamins, and minerals. Triple Sugar Iron Agar 1. Suspend 65 grams in 1 liter distilled or deionized water.
contains three carbohydrates (dextrose, lactose and sucrose). When 2. Heat to boiling to dissolve completely.
these carbohydrates are fermented, the resulting production of acid 3. Dispense into tubes with closures.
is detected by the phenol red indicator. The color changes that result 4. Autoclave at 121°C for 15 minutes. Cool in slanted position
are yellow for acid production and red for alkalinization. Sodium with deep butts.
thiosulfate is reduced to hydrogen sulfide. Hydrogen sulfide then
reacts with an iron salt yielding the typical black iron sulfide. Sodium Specimen Collection and Preparation
chloride maintains the osmotic balance of the medium. Bacto Agar is 1. Collect specimens or food samples in sterile containers or with
a solidifying agent. sterile swabs and transport immediately to the laboratory
following recommended guidelines.7-14
Formula 2. Process each specimen, using procedures appropriate for that
Triple Sugar Iron Agar specimen or sample.7-14
Formula Per Liter
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Test Procedure
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g 1. Obtain a pure culture of the organism to be tested. Select
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g well-isolated colonies.
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g 2. With an inoculating needle, pick the center of well-isolated
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g colonies obtained from solid culture media.
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g 3. Stab the center of the medium into the deep of the tube to within
Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g 3 - 5 mm from the bottom.
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
4. Withdraw the inoculating needle, and streak the surface of the slant.
Sodium Thiosulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 g 5. Loosen closure on the tube before incubating.
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 g 6. Incubate at 35°C for 18-24 hours.
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.024 g 7. Read tubes for acid production of slant/butt, gas, and hydrogen
Final pH 7.4 ± 0.2 at 25°C sulfide reactions.
Precautions Results
1. For Laboratory Use. 1. An alkaline slant-acid butt (red/yellow) indicates fermentation of
2. Follow proper established laboratory procedure in handling and dextrose only.
disposing of infectious materials. 2. An acid slant-acid butt (yellow/yellow) indicates fermentation of
dextrose, lactose and/or sucrose.
Storage 3. An alkaline slant-alkaline butt (red/red) indicates that neither
Store the dehydrated medium below 30°C. The dehydrated medium dextrose nor lactose was fermented (non-fermenter).
is very hygroscopic. Keep container tightly closed. Store prepared 4. Cracks, splits, or bubbles in the medium indicate gas production.
tubes at 2-8°C.
5. A black precipitate in the butt indicates hydrogen sulfide production.
Expiration Date Limitations of the Procedure
The expiration date applies to the product in its intact container 1. Hydrogen sulfide production may be evident on Kligler Iron Agar
when stored as directed. Do not use a product if it fails to meet but negative on Triple Sugar Iron Agar. Studies by Bulmash and
specifications for identity and performance. Fulton15 showed that the utilization of sucrose could suppress the
enzymatic mechanisms responsible for H2S production. Padron
Procedure and Dockstader16 found that not all H2S-positive Salmonella are
Materials Provided positive on TSI.
Triple Sugar Iron Agar 2. Sucrose is added to TSI to eliminate some sucrose-fermenting
non-lactose fermenters such as Proteus and Citrobacter spp.17
Materials Required But Not Provided
3. Further biochemical tests and serological typing must be performed
Flasks with closures
for definite identification and confirmation of organisms.
Distilled or deionized water
4. Do not use an inoculating loop to inoculate a tube of Triple Sugar 9. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
Iron Agar. While stabbing the butt, mechanical splitting of the R. H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
medium occurs, causing a false positive result for gas production.17 American Society for Microbiology, Washington, D.C.
5. A pure culture is essential when inoculating Triple Sugar Iron Agar. 10. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig.
If inoculated with a mixed culture, irregular observations may occur. 1993. Pathogens in milk and milk products, p. 103-212. In R. T.
6. Tubes should be incubated with caps loosened. This allows a free Marshall (ed.), Standard methods for the examination of dairy
exchange of air, which is necessary to enhance the alkaline products, 16th ed. American Public Health Association,
condition on the slant.17 Washington, D.C.
11. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992.
References Compendium of methods for the microbiological examination
1. Russell, F. F. 1911. The isolation of typhoid bacilli from urine and
of foods, 3rd ed. American Public Health Association,
feces with the description of a new double sugar tube medium.
Washington, D.C.
J. Med. Res. 25:217.
12. FDA Bacteriological Analytical Manual, 8th ed. AOAC
2. Kligler, I. J. 1917. A simple medium for the differentiation of
International, Gaithersburg, M.D.
members of the typhoid-paratyphoid group. Am. J. Public Health
7:1042-1044. 13. Association of Official Analytical Chemists. 1995. Official
3. Kligler, I. J. 1918. Modifications of culture media used in the methods of analysis of AOAC International, 16th ed. AOAC
isolation and differentiation of typhoid, dysentery, and allied International, Arlington, VA.
bacilli. J. Exp. Med. 28:319-322. 14. Federal Register. 1996. Pathogen reduction; hazard analysis
4. Krumwiede, C. and L. Kohn. 1917. A triple sugar modification and critical point (HACCP) systems; final rule. Fed. Regis.
of the Russell Double Sugar medium. J. Med. Res. 37:225. 61:38917-38925.
5. Sulkin, S. E., and J. C. Willett. 1940. A triple sugar-ferrous 15. Bulmash, J. M. and M. D. Fulton. 1964. Discrepant tests for
sulfate medium for use in identification of enteric organisms. hydrogen sulfide. J. Bacteriol. 88:1813.
J. Lab. Clin. Med. 25:649-653. 16. Padron, A. P. and W. B. Dockstader. 1972. Selective medium
6. Hajna, A. A. 1945. Triple-sugar iron agar medium for the for hydrogen sulfide production. Appl. Microbiol. 23:1107.
identification of the intestinal group of bacteria. J. Bacteriol. 17. MacFaddin, J. F. 1985. Media for isolation-cultivation-
49:516-517. identification-maintenance of medical bacteria, vol. 1. Williams &
7. Pezzlo, M. (ed.). 1994. Aerobic bacteriology, p. 1.0.0-1.20.47. Wilkins, Baltimore, MD.
In H. D. Isenberg, (ed.), Clinical microbiology procedures hand-
book, vol. 1. American Society for Microbiology, Washington, D.C. Packaging
8. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey & Triple Sugar Iron Agar 100 g 0265-15
Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc., 500 g 0265-17
St. Louis, MO. 2 kg 0265-07
TSA is recommended in multiple water and wastewater applications.4 2. Follow proper established laboratory procedure in handling and
Tryptic Soy Agar conforms to the formula specified by US disposing of infectious materials.
Pharmacopeia for use in Microbiological Tests 5, Antimicrobial
Preservatives-Effectiveness and Microbial Limits Test. Storage
Store the dehydrated medium below 30°C. The dehydrated medium is
Clinically, Tryptic Soy Agar is used in the differentiation of
very hygroscopic. Keep container tightly closed.
Haemophilus species, because it does not contain the X and V
factors required for Haemophilus growth. With the addition of Expiration Date
Differentiation Disks V, X and VX, Haemophilus growth can be
The expiration date applies to the product in its intact container
observed around the appropriate disk.
when stored as directed. Do not use a product if it fails to meet
Tryptic Soy Agar is a nutritious base to which a variety of supplements specifications for identity and performance.
can be added. Addition of 5% sterile, defibrinated sheep, horse or
rabbit blood provides an excellent general purpose medium that Procedure
allows the growth of yeast species, staphylococci, enterococci and
gram-negative bacilli.6 TSA blood agars may be used to determine
Materials Provided
hemolytic reactions of bacteria. TSA supplemented with lecithin and Tryptic Soy Agar
polysorbate 80 is the formula for Microbial Content Test Agar Materials Required But Not Provided
(also known as TSALT) used in environmental monitoring.7 For
Glassware
the examination of foods, Tryptic Soy Agar is supplemented
with 3% NaCl for the isolation of Vibrio species and halophilic Autoclave
microorganisms.8 Tryptic Soy Agar supplemented with 0.6% yeast Incubator (35°C)
extract is used for the isolation of Listeria monocytogenes and Waterbath (45-50°C) (optional)
cultivation of a wide variety of heterotrophic microorganisms. 8 Defibrinated blood (optional)
Addition of colistin and nalidixic acid to TSA is used for the
selective isolation of gram-positive cocci. 9 Gunn et al. 10 used Method of Preparation
trimethoprim and sulfamethoxazole (SxT) supplementation to inhibit 1. Suspend 40 grams in 1 liter distilled or deionized water.
normal flora on throat specimens, allowing Groups A and B 2. Heat to boiling to dissolve completely.
streptococci to grow well. Addition of iron salt and sodium thiosulfate
3. Autoclave at 121°C for 15 minutes. Cool to room temperature.
to TSA aids in the identification of non-fermentating gram-negative
bacilli, and with nitro blue tetrazolium (0.5% aqueous solution) 4. OPTIONAL: To prepare blood agar, aseptically add 5% sterile
allows for the selective isolation of Corynebacterium diphtheriae.11 defibrinated blood to the medium at 45-50°C. Mix well.
Chocolate Agar for culturing Haemophilus influenzae and related Specimen Collection and Preparation
organisms may be prepared by adding 1% Hemoglobin and Refer to appropriate references for specimen collection and preparation.
Supplement B to Tryptic Soy Agar.
The methodologies for multiple applications using Tryptic Soy Agar Test Procedure
are outlined in the references. See appropriate references for specific procedures.
5. The United States Pharmacopeia. 1995. Microbiological tests, 10. Gunn, B. A., D. K. Ohashi, C. A. Gaydos, and E. S. Holt. 1977.
p. 1681-1686. The United States pharmacopeia, 23rd ed. United Selective and enhanced recovery of group A and B streptococci
States Pharmacopeial Convention, Rockville, MD. from throat cultures with sheep blood containing sulfamethoxazole
6. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. and trimethoprim. J. Clin. Microbiol. 5:650-655.
Etiological agents recovered from clinical material, p. 244. Bailey 11. MacFaddin, J. D. 1985. Media for isolation-cultivation-
& Scott’s Diagnostic Microbiology, 9th ed. Mosby-Year Book, Inc., identification- maintenance of medical bacteria, vol. 1, p. 794-802.
St. Louis, MO. Williams & Wilkins, Baltimore, MD.
7. Orth, D. S. 1993. Handbook of cosmetic microbiology. Marcel
Dekker, Inc., New York, NY. Packaging
8. Harmon, S. M., D. A. Kautter, D. A. Golden, and E. J. Tryptic Soy Agar 100 g 0369-15
Rhodehamel. 1995. Bacteriological analytical manual, 8th ed. 500 g 0369-17
AOAC International, Arlington, VA. 2 kg 0369-07
9. Ellner, P. 1966. J. of Clin. Pathol. 45:502-504. 10 kg 0369-08
Principles of the Procedure 2. Follow proper established laboratory procedures in handling and
Tryptone and Soytone are nitrogen sources in Tryptic Soy Broth and disposing of infectious materials.
Tryptic Soy Broth w/o Dextrose. Dextrose is a carbon energy source Storage
that facilitates organism growth. Sodium chloride maintains osmotic Store the dehydrated medium below 30°C. The dehydrated medium is
balance, while dipotassium phosphate is a buffering agent. very hygroscopic. Keep container tightly closed.
Dextrose is omitted from the formula for Tryptic Soy Broth w/o
Dextrose to permit use of the medium in fermentation studies. The Expiration Date
carbohydrate concentration used most frequently in fermentation The expiration date applies to the product in its intact container when
reactions is 0.5% or 1%. stored as directed. Do not use a product if it fails to meet specifications
for identity and performance.
Formula
Tryptic Soy Broth Procedure
Formula Per Liter Materials Provided
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 g Tryptic Soy Broth
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g Tryptic Soy Broth w/o Dextrose
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Materials Required But Not Provided
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g Glassware
Final pH 7.3 ± 0.2 at 25°C Autoclave
Incubator (35°C)
Tryptic Soy Broth w/o Dextrose Sterile tubes
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 g Method of Preparation
Bacto Soyton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g 1. Suspend the medium in 1 liter distilled or deionized water:
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Tryptic Soy Broth - 30 grams;
Dipostassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Tryptic Soy Broth w/o Dextrose - 27.5 grams.
Final pH 7.3 ± 0.2 at 25°C
2. Dispense as desired.
Precautions 3. Autoclave at 121°C for 15 minutes. Cool to room temperature.
1. For Laboratory Use.
Specimen Collection and Preparation 9. Federal Register. 1992. Detection of viable bacteria and fungi
Obtain and process specimens according to the techniques and except in live vaccine. Fed. Regist. 21:113.26.
procedures established by laboratory policy. 10. Greenberg, A. E., L. S. Clesceri, and A. D. Eaton (eds). 1992.
Coliphage detection, 9,22-23. Standard methods for the
Test Procedure examination of water and wastewater, 18th ed. American Public
Methodologies for the multiple applications using Tryptic Soy Broth Health Association, Washington, D.C.
and Tryptic Soy Broth w/o Dextrose are outlined in the references.
11. Curry, A. S., G. G. Joyce, and G. N. McEwen, Jr. 1993. CTFA
Results Microbiology guidelines. The Cosmetic, Toiletry, and Fragrance
Refer to appropriate references and procedures for results. Association, Inc., Washington, D.C.
References 12. Association of Official Analytical Chemists. 1995. Bacteriological
1. United States Pharmacopeial Convention. 1995. The United analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
States pharmacopeia, 23rd ed. The United States Pharmacopeial 13. Cunnif, P. 1995. Official methods of analysis AOAC International,
Convention, Rockville, MD. 16th ed. AOAC International, Arlington, VA.
2. Federal Register. 1992. General biological products standards. 14. National Committee for Clinical Laboratory Standards. 1994.
Fed. Regist. 21:610.12. Performance standards for antimicrobial disk susceptibility tests,
3. McCullough, N. B. 1949. Laboratory tests in the diagnosis of M2-A5, vol. 13, no. 24. National Committee for Clinical
brucellosis. Amer. J. of Public Health 39:866-869. Laboratory Standards, Villanova, PA.
4. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. 15. Isenberg, H. D. (ed.). 1992. Processing and interpretation of blood
Microorganisms encountered in the blood, p. 205. Bailey & Scott’s cultures, p. 1.7.1-1.7.2. Clinical microbiology procedures hand-
diagnostic microbiology, 9th ed. Mosby-Year Book, Inc., book, vol. 1. American Society for Microbiology, Washington, D.C.
St. Louis, MO. 16. MacFaddin, J. D. 1985. Media for isolation-cultivation-
5. Garrison, R. G. 1961. Studies of the respiratory activity of identification-maintenance of medical bacteria, p. 797, vol. 1.
Histoplasma capsulatum. J. of Infect. Dis. 108:120-124. Williams & Wilkins, Baltimore, MD.
6. Hedgecock, L. W. 1971. Effect of vaccines prepared from Packaging
Histoplasma capsulatum and other yeast on experimental Tryptic Soy Broth 100 g 0370-15
tuberculosis. J. Bacteria. 82:115- 123. 500 g 0370-17
7. Mashimo, P. A., and S. A. Ellison. 1959. Simple method for the 2 kg 0370-07
isolation of anaerobic oral vibrios. J. Bacteria. 78:636-639. 10 kg 0370-08
8. Sherman, J. M., and P. Stark. 1961. Streptococci which grow at Tryptic Soy Broth w/o Dextrose 500 g 0862-17
high temperatures. J. Bacteria. 22:275-285. 10 kg 0862-08
Intended Use
Bacto Tryptone Peptone
®
Bacto Tryptone Peptone is used in preparing microbiological culture
media.
Also Known As
User Quality Control Tryptone Peptone is also referred to as Peptone C, Peptone 50 and
Tryptone T.
Identity Specifications
Dehydrated Appearance: Light beige, free-flowing, Summary and Explanation
homogeneous powder. Tryptone Peptone is a pancreatic digest of casein used as a nitrogen
Solution: 1%, 2% and 10% solutions are soluble source in culture media formulated for isolating and cultivating fas-
in distilled or deionized water: tidious and nonfastidious bacteria and fungi.
1%-Very light to light amber, clear Tryptone Peptone was developed by Difco Laboratories while investi-
without precipitate;
gating a peptone particularly suitable for the elaboration of indole by
2%-Light to medium amber, clear
without precipitate; bacteria. The high tryptophane content of Tryptone Peptone makes it
10%-Medium to dark amber, clear to valuable for use in detecting indole production.1,2,3 The absence of de-
slightly opalescent, may have a tectable levels of carbohydrates in Tryptone Peptone makes it a suit-
slight precipitate. able peptone in differentiating bacteria on the basis of their ability to
Nitrogen (Kjeldahl Method): 11.4-13.9% ferment various carbohydrates.
Amino Nitrogen Several media containing Tryptone Peptone are specified in standard
(Modified Sorensen Method): 4.0-6.6% methods4,5,6,7 for multiple applications.
Reaction of 2%
Solution at 25°C: pH 6.9-7.4 Principles of the Procedure
continued on following page
Tryptone Peptone is a pancreatic digest of casein especially rich in tryp-
tophane. Casein, a milk protein, is a rich source of amino acid nitrogen.
The Difco Manual 529
Tryptone Peptone Section II
4. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compendium 7. Marshall, R. T. (ed.). 1993. Standard methods for the examination
of methods for the microbiological examination of food, 3rd ed. of dairy products, 16th ed. American Public Health Association,
American Public Health Association, Washington, D.C. Washington, D.C.
5. Association of Official Analytical Chemists. 1995. Bacteriological
analytical manual , 8th ed. AOAC International, Gaithersburg, MD. Packaging
6. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). 1995. Tryptone Peptone 100 g 0123-15
Standard methods for the examination of water and wastewater, 500 g 0123-17
19th ed. American Public Health Association, Washington, D.C. 2 kg 0123-07
10 kg 0123-08
Reaction of 1.8%
Solution at 25°C: pH 7.0 ± 0.2
Cultural Response
m TGE Broth
Tryptone Glucose Extract Agar Prepare mTGE Broth per label directions. Inoculate using the
Prepare Tryptone Glucose Extract Agar per label directions in membrane filter technique and incubate in a humid atmosphere
parallel with a reference control. Inoculate with pasteurized at 35 ± 2°C for 18-24 hours.
and raw milk samples using the pour plate technique and
ORGANISM ATCC® INOCULUM CFU GROWTH
incubate at 32 ± 1°C for 47-49 hours. Recovery of bacteria
from the milk samples should be comparable for both the test Escherichia coli 25922* 100-1,000 good
and reference lots. Staphylococcus aureus 25923* 100-1,000 good
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
Method of Preparation Examine and record the tubes of Brilliant Green Bile 2% tubes
1. Suspend 15 grams in 1 liter of distilled or deionized water. containing gas. Add 0.5 ml SpotTest™ Indole Reagent Kovacs to the
Tryptone Water tubes. Observe tubes for the formation of a red ring at
2. Autoclave at 121°C for 15 minutes.
the top of medium indicating indole production. Record the tubes for
Specimen Collection and Preparation positive indole production. Determine the MPN (Most Probable
1. Collect food and water samples in sterile containers and transport Number) of E. coli present in the sample based on the number of tubes
immediately to the laboratory following recommended guidelines.1-4 that are positive for both gas and indole. Consult the appropriate
2. Process each food and water sample using procedures appropriate 3-tube MPN table.2
for that sample.1-4 Indole Determination Using Pure Cultures
Test Procedure Examine tubes for the formation of a red ring at the top of the tube
indicating indole production.
Presumptive Test For E. coli in Meats and Meat Products1
1. Suspend one part sample in 9 parts diluent. Homogenize sample. Limitations of the Procedure
2. Dilute homogenate in duplicate using serial 10-fold dilutions to 1. Detection of E. coli in meats using Tryptone Water is a pre-
10-6 using 1 ml of material to be diluted to 9 ml of diluent. Mix sumptive test. If confirmatory testing is required, please consult
each dilution thoroughly. appropriate references.
3. Transfer 1 ml of homogenate (step 1) to 6 tubes containing 10 ml 2. Indole testing is recommended as an aid in the differentiation of
Brilliant Green Bile 2% and group in 2 sets of 3 tubes each. If the microorganisms based on indole production. For complete
number of coliforms is expected to be high, transfer 10 ml of identification of the organism, further biochemical evaluation
homogenate into 6 tubes containing double strength Brilliant Green is necessary.
Bile 2%.
4. Transfer 1 ml from each of the dilutions prepared in step 2 into References
each of 3 tubes containing 10 ml Brilliant Green Bile 2%. 1. International Organization for Standardization: Meat and
5. Incubate Brilliant Green Bile 2% tubes (prepared in steps 3 and 4) meat products. Detection and enumeration of presumptive coliform
at 30 ± 1°C for 48 ± 2 hours. Subculture all tubes containing gas, bacterial and presumptive E. coli (Reference method ISO/DIS
using 1 drop of inoculum, into Tryptone Water and Brilliant Green 3811-1979).
Bile 2%. 2. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compen-
6. Incubate tubes prepared in step 5 at 44 ± 1°C for 48 ± 2 hours. dium of methods for the microbiological examination of foods,
Indole Determination Using Pure Cultures 3rd ed. American Public Health Association, Washington, D.C.
1. Inoculate Tryptone Water using a light inoculum of an 18-24 hour 3. Greenberg, A. E., L. S. Clesceri, and A. D. Eaton (ed.). 1995.
pure culture. Standard Methods for the Examination of Water and Wastewater,
2. Incubate the tubes at 35 ± 2°C with loosened caps for 18-24 hours. 19th ed. American Public Health Association, Washington, D.C.
3. Add 0.5 ml of SpotTest™ Indole Reagent Kovacs directly to the 4. MacFaddin, J. F. Biochemical Test for Identification of Medical
tube and agitate. Allow tubes to stand for 5-10 minutes. Bacteria, 3rd ed. 1985. Williams and Wilkens, Baltimore, MD.
Results Packaging
Presumptive Test For E. coli in Meats and Meat Products Tryptone Water 500 g 0644-17
Bacto Tryptose
®
of these organisms.1 Culture media prepared with Tryptose were
superior to the meat infusion peptone media previously used for the
Intended Use cultivation of Brucella, streptococci, pneumococci, meningococci and
other fastidious bacteria.2,3
Bacto Tryptose is an enzymatic digest of protein for use in preparing
microbiological culture media. An agar medium prepared with 2% Tryptose and 0.5-0.8% sodium
chloride, without tissue infusion, is an excellent blood agar base. The
Also Known As growth of organisms on Tryptose Blood Agar Base is luxuriant and the
Tryptose is also referred to as Polypeptone™ Peptone. zones of hemolysis produced are distinct and clear. Huddleson1 used a
broth containing 2% Tryptose as an enrichment medium in the
Summary and Explanation isolation of Brucella from clinical specimens.
Tryptose is a mixed enzymatic hydrolysate with distinctive nutritional
properties. Tryptose was originally developed as a peptone particularly Principles of the Procedure
adapted to the growth requirements of Brucella. An agar medium Tryptose is a mixed enzymatic hydrolysate with distinctive nutritional
containing Tryptose, sodium chloride and dextrose, without liver or properties. The digestive process in Tryptose results in assorted
other infusions, was shown to be an excellent medium for propagation peptides, including those of higher molecular weight.
4. Colonies of Haemophilus haemolyticus are beta-hemolytic on horse 3. Ruoff, K. L. 1995. Streptococcus, p. 299-305. In P. R. Murray,
and rabbit blood agar, and must be distinguished from colonies on E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.),
beta-hemolytic streptococci using other criteria. The use of sheep Manual of clinical microbiology, 6th ed. American Society for
blood has been suggested to obviate this problem since sheep blood Microbiology, Washington, D.C.
is deficient in pyridine nucleotides and does not support growth of 4. Harmon, S. M., D. A. Kautter, D. A. Golden, and E. J.
H. haemolyticus.6 Rhodehamel. 1995. FDA Bacteriological analytical manual, 8th
5. The atmosphere of incubation has been shown to influence ed. AOAC International, Arlington, VA.
hemolytic reactions of beta-hemolytic streptococci.3 For optimal 5. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures hand-
performance, incubate blood agar base media under increased CO2 book, vol. 1. American Society for Microbiology, Washington, D.C.
or anaerobic conditions. 6. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
6. Hemolytic patterns may vary with the source of animal blood or Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
type of base medium used.3 St. Louis, MO.
References Packaging
1. Casman, E. P. 1942. A dehydrated medium to supplement meat Tryptose Blood Agar Base 500 g 0232-17
infusion as a base for blood agar. J. Bacteriol. 43:33. 2 kg 0232-07
2. Casman, E. P. 1947. A noninfusion blood agar base for neisseriae, Tryptose Blood Agar Base 500 g 0662-17
pneumococci and streptococci. Am. J. Clin. Pathol. 17:281-289. w/ Yeast Extract
Tryptose Phosphate Broth is valuable in tissue culture procedures, Materials Required But Not Provided
as shown by Ginsberg, Gold and Jordan. 2 The proteose content Glassware
of Tryptose Phosphate Broth is considered to be a stimulating factor Autoclave
for cells. Tryptose Phosphate Broth is specified in the FDA Incubator (35°C)
Bacteriological Analytical Manual for cell culture procedures.3 Tubes with closures
Bacto Agar (optional)
Principles of the Procedure
Tryptose provides carbon and nitrogen. Dextrose is a carbon source. Method of Preparation
Sodium Chloride maintains osmotic balance. Buffering capacity is 1. Dissolve 29.5 grams in 1 liter distilled or deionized water.
provided by Disodium Phosphate. 2. If a medium containing 0.1% agar is desired, add 1 gram of Bacto
The addition of 0.1-0.2% agar to Tryptose Phosphate Broth facilitates Agar. Heat to boiling to dissolve completely.
anaerobic growth and aids in dispersion of reducing substances and 3. Dispense as desired.
CO2 formed in the environment.4 The low agar concentration provides 4. Autoclave at 121°C for 15 minutes.
suitable conditions for both aerobic growth in the upper zone and for
microaerophilic and anaerobic growth in the lower zone. Specimen Collection and Preparation
Obtain and process specimens according to the techniques and
Formula procedures established by laboratory policy.
Tryptose Phosphate Broth Test Procedure
Formula Per Liter
See appropriate references for specific procedures.
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g Results
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Refer to appropriate references and procedures for results.
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Final pH 7.3 ± 0.2 at 25°C Limitations of the Procedure
1. Since the nutritional requirements of organisms vary, some strains may
Precautions be encountered that fail to grow or grow poorly on this medium.
1. For Laboratory Use.
2. Follow proper established laboratory procedures in handling and References
disposing of infectious materials. 1. Waisbren, B. A., M. S. Carr, and J. Dunnett. 1951. The tube
dilution method of determining bacterial sensitivity to antibiotics.
Storage Am. J. Clin. Pathol. 21:884.
Store the dehydrated medium below 30°C. The dehydrated medium is 2. Ginsberg, Gold, and Jordan. 1955. Proc. Soc. Exp. Biol. Med. 89:66.
very hygroscopic. Keep container tightly closed. 3. Harmon, S. M., D. A. Kautter, D. A. Golden, and E. J.
Rhodehamel. 1995. FDA Bacteriological analytical manual,
Expiration Date 8th ed. AOAC International, Arlington, VA.
The expiration date applies to the product in its intact container when 4. MacFaddin, J. D. 1985. Media for isolation-cultivation-
stored as directed. Do not use a product if it fails to meet specifications identification-maintenance of medical bacteria, vol. 1, p. 802-804.
for identity and performance. Williams & Wilkins, Baltimore, MD.
Procedure Packaging
Materials Provided Tryptose Phosphate Broth 500 g 0060-17
Tryptose Phosphate Broth 2 kg 0060-07
10 kg 0060-08
in the medium, it is selective for growth of microorganisms that have Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 g
adapted themselves to existent conditions in the brewery. The pres- Final pH 6.3 ± 0.2 at 25°C
ence of hop constituents and alcohol inhibits growth of many airborne
microorganisms not adapted to this environment.3 Precautions
UBA Medium supports growth of Lactobacillus, Pediococcus, 1. For Laboratory Use.
Acetobacter and yeast strains which may be found contaminating the 2. Follow proper established laboratory procedure in handling and
wort and beer. disposing of infectious materials.
Intended Use The most common contaminating bacteria found in food sources
Bacto UVM Modified Listeria Enrichment Broth is used for rapidly potentially containing Listeria are: streptococci, especially the
isolating Listeria monocytogenes. enterococci, micrococci and Bacillus species, Escherichia coli,
Pseudomonas aeruginosa and Proteus vulgaris.8
Summary and Explanation Identification of Listeria is based on successful isolation of the
First described in 1926 by Murray, Webb and Swann, 1 Listeria organism, biochemical characterization and serological confirmation.
monocytogenes is a widespread problem in public health and the food
industries. This organism can cause human illness and death, UVM Modified Listeria Enrichment Broth is a modification of the
particularly in immunocompromised individuals and pregnant women.2 formula described by Donnelly and Baigent.9 It is used for selective
The first reported food-borne outbreak of listeriosis was in 1985,3 and enrichment of Listeria spp. from food7,11 and clinical specimens.10
since then, microbiological and epidemiological evidence from both
sporadic and epidemic cases of listeriosis has shown that the principal
Principles of the Procedure
route of transmission is via the consumption of foodstuffs Tryptose, Beef Extract and Yeast Extract in UVM Modified Listeria
contaminated with Listeria monocytogenes.4 Enrichment Broth provide nitrogen, vitamins and minerals. Sodium
chloride maintains the osmotic balance of the medium. Phosphate acts
Implicated vehicles of transmission include turkey frankfurters,5
as a buffering agent. Nalidixic acid inhibits growth of gram-negative
coleslaw, pasteurized milk, Mexican-style cheese, paté and pickled
organisms. Acriflavine hydrochloride inhibits many gram-positive
pork tongue. The organism has been isolated from commercial dairy
bacteria. Esculin is hydrolyzed by Listeria species.
and other food processing plants and is ubiquitous in nature, being
present in a wide range of unprocessed foods and in soil, sewage, Formula
silage and river water.6
UVM Modified Listeria Enrichment Broth
Listeria species grow over a pH range of 5.0-9.6 and survive in food Formula Per Liter
products with pH levels outside these parameters.7 Listeria spp. are Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
microaerophilic, gram-positive, asporogenous, non-encapsulated, Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
non-branching, regular, short, motile rods. Motility is most pronounced Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
at 20°C. Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Sodium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . . . . 9.6 g
Potassium Phosphate, Monobasic . . . . . . . . . . . . . . . . . . 1.35 g
User Quality Control Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Identity Specifications Nalidixic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g
Dehydrated Appearance: Beige, free-flowing, homogeneous. Acriflavine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.012 g
Solution: 5.2% solution, soluble in distilled or Final pH 7.2 ± 0.2 at 25°C
deionized water on boiling. Solution
is light to medium amber with a faint Precautions
bluish-green ring at the surface, very 1. For Laboratory Use.
slightly opalescent with a fine
precipitate. 2. MAY BE IRRITATING TO EYES, RESPIRATORY SYSTEM
Reaction of 5.2% AND SKIN. (US) Avoid contact with skin and eyes. Do not breathe
Solution at 25°C: pH 7.2 ± 0.2 dust. Wear suitable protective clothing. Keep container tightly
closed.
Cultural Response FIRST AID: In case of contact with eyes, rinse immediately with
Prepare UVM Modified Listeria Enrichment Broth per label plenty of water and seek medical advice. After contact with skin,
directions. Inoculate tubes and incubate at 35 ± 2°C for wash immediately with plenty of water. If inhaled, remove to fresh
18-48 hours.
INOCULUM air. If not breathing, give artificial respiration. If breathing is
ORGANISM ATCC® CFU GROWTH difficult, give oxygen. Seek medical advice. If swallowed seek
Escherichia faecalis 29212* 1,000-2,000 suppressed at medical advice immediately and show this container or label.
18-24 hours
Escherichia coli 25922* 1,000-2,000 marked to 3. Follow proper established laboratory procedure in handling and
complete inhibition disposing of infectious materials.
Listeria monocytogenes 19114 100-1,000 good
Storage
The cultures listed are the minimum that should be used for
performance testing. Store the dehydrated medium below 30°C. The dehydrated medium is
*These cultures are available as Bactrol™ Disks and should be used very hygroscopic. Keep container tightly closed. Store the prepared
as directed in Bactrol Disks Technical Information. medium at 2-8°C.
References Packaging
1. Murray, E. G. D., R. A. Webb, and M. B. R. Swann. 1926. UVM Modified Listeria
A disease of rabbits characterized by large mononuclear Enrichment Broth 500 g 0223-17
leucocytosis caused by a hitherto undescribed bacillus Bacterium 2 kg 0223-07
monocytogenes (n. sp.). J. Path. Bact. 29:407-439. 10 kg 0223-08
Intended Use common way to detect the production of urease by yeasts.5 Urea Agar
Bacto Urea Agar Base, when combined with Bacto Agar, is used for Base Concentrate has also been used in differentiating mycobacteria
differentiating microorganisms based on urease activity. species.6
Bacto Urea Agar Base Concentrate is a sterile 10X solution of Urea Urea Broth, prepared according to the formula of Stuart, Van Stratum
Agar Base which, when combined with Bacto Agar, is used for preparing and Rustigian7 is a highly buffered urea medium that provides all the
Urea Agar. essential growth requirements for Proteus. Stuart et al.7 noted that by
Bacto Urea Broth is used for differentiating microorganisms, particularly decreasing the amount of buffer in their standard medium to one-tenth
Proteus species, based on urease production. or one-hundredth of the original concentration, the incubation time for
Proteus could be decreased from 12-48 hours to 2-4 hours. When the
Bacto Urea Broth Concentrate is a sterile 10X solution of Urea Broth amount of buffer is decreased, however, other organisms capable of
ready to use as recommended. It is suggested for laboratories that urease production give a positive test. Rustigian and Stuart8 used urea
require only small amounts of medium. decomposition as a limiting characteristic for the identification of
Also Known As Proteus strains from other members of the family Enterobacteriaceae.
Ferguson and Hook9 reported that urease production could be used to
Urea Agar Base is also known as Urea Agar Base, Christensen or
differentiate between members of the Proteus and Salmonella groups.
Christensen’s Urea Agar.
The medium is positive for Proteus, Morganella morganii, Providencia
Urea Broth is also referred to as Stuart’s Urea Broth. rettgeri and a few Providencia stuartii strains.
Summary and Explanation The detection of urease production is an important differential test in
Christensen1 devised a urea agar medium containing peptone and microbiology and is outlined in standard references.10-16
dextrose that had a reduced buffer content. The medium supported a
more vigorous growth of many of the gram-negative enteric bacilli and
Principles of the Procedure
readily permitted observation of urease production. Bacto Peptone provides carbon and nitrogen required for good growth
of a wide variety of organisms. Yeast Extract provides vitamins and
Ewing2 used Urea Agar as a differential medium in the examination of cofactors required for growth and as an additional source of nitrogen
many cultures from stool specimens. Urea Agar may be used as a and carbon. Dextrose is included as an energy source. Sodium Chloride
screening medium (along with Triple Sugar Iron Agar) for the selection maintains the osmotic balance of the medium. Potassium Phosphate,
of Salmonella and Shigella cultures for serologic classification.3 Qadri Monobasic and Potassium Phosphate, Dibasic provide buffering
et al.4 developed a spot test for the rapid detection of urease activity capability. Urea provides a source of nitrogen for those organisms
by applying diluted Urea Agar Base Concentrate to filter paper and producing urease. This is indicated by a color change of the pH indicator,
inoculating the paper with a loopful of 24-48 hour culture. Urease- Phenol Red, from yellow (pH 6.8) to red to pink-red (pH 8.1).
positive results were obtained within 2 minutes. When combined with
results of other rapid screening methods, Urea Agar is the most
13. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and 17. MacFaddin, J. F. 1985. Media for isolation-cultivation-
R. H. Yolken. 1995. Manual of clinical microbiology, 6th ed. ASM identification-maintenance of medical bacteria. Williams &
Press, Washington, D.C. Wilkins, Baltimore, MD.
14. Bacteriological Analytical Manual, 8th ed. 1995. AOAC
International, Gaithersburg, MD.
Packaging
Urea Agar Base 100 g 0283-15
15. Oberhofer, T. R. 1985. Manual of nonfermenting gram-negative 500 g 0283-17
bacteria. Churchill Livingstone, New York, NY.
16. Ewing, W. H. 1986. Edwards and Ewing’s Identification of Urea Agar Base Concentrate 10X 12 x 10 ml 0284-61
Enterobacteriaceae, 4th ed. Elsevier Science Publishing Co., Inc., Urea Broth 500 g 0272-17
New York, NY. Urea Broth Concentrate 12 x 10 ml 0280-61
Bacto VJ Agar
® To isolate coagulase-positive, mannitol fermenting staphylococci,
Vogel and Johnson3 modified Tellurite-Glycine Agar by Zebovitz
et al.1 by increasing the mannitol content and adding a pH indicator.
Intended Use Vogel-Johnson (VJ) Agar selects and differentiates the coagulase-
Bacto VJ Agar is used with Bacto Chapman Tellurite Solution 1% for positive staphylococci which ferment mannitol and reduce tellurite.2
isolating coagulase-positive, mannitol-fermenting staphylococci. VJ Agar is specified as a standard methods medium for cosmetics,4,5
Also Known As pharmaceutical articles6 and nutritional supplements.6
VJ Agar is also known as Vogel and Johnson Agar, Modification of Principles of the Procedure
Tellurite-Glycine Agar1, and Tellurite-Glycine-Phenol Red Agar Base2
VJ Agar contains Tryptone as a source of carbon, nitrogen, vitamins
Summary and Explanation and minerals. Yeast Extract supplies B-complex vitamins which
Coagulase-positive staphylococci, primarily Staphylococcus aureus, stimulate bacterial growth. Mannitol is the carbohydrate. Chapman
are among the microorganisms that can cause spoilage or chemical Uninoculated Staphylococcus aureus
changes in cosmetic products.4 palate ATCC® 25923
Tellurite Solution 1% contains Potassium Tellurite which, along with Petri dishes
Lithium Chloride and Glycine, inhibits most microorganisms except Distilled or deionized water
the staphylococci. Phenol Red is the pH indicator. Bacto Agar is the Autoclave
solidifying agent. Incubator (35°C)
Formula Method of Preparation
Bacto VJ Agar VJ Agar
Formula Per Liter 1. Suspend 60 grams in 1 liter distilled or deionized water.
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g 2. Heat to boiling to dissolve completely.
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g 3. Autoclave at 121°C for 15 minutes. Cool to 45-50°C.
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g 4. Add 20 ml Chapman Tellurite Solution 1%. Mix well.
Lithium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Specimen Collection and Preparation
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g Refer to appropriate references for specimen collection and preparation.
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025 g
Test Procedure
Final pH 7.2 ± 0.1 at 25°C
See appropriate references for specific procedures.
Precautions Results
1. For Laboratory Use. Coagulase-positive strains of S. aureus reduce tellurite and form black
2. HARMFUL. IRRITATING TO EYES, RESPIRATORY SYSTEM colonies on the medium. These strains typically ferment mannitol and
AND SKIN. MAY CAUSE HARM TO THE UNBORN CHILD. exhibit yellow halos around the black colonies.
Avoid contact with skin and eyes. Do not breathe dust. Wear
suitable protective clothing. Keep container tightly closed. References
TARGET ORGAN(S): Blood, Kidneys, Nerves. 1. Zebovitz, E., J. B. Evans, and C. F. Niven, Jr. 1955. Tellurite-
FIRST AID: In case of contact with eyes, rinse immediately with Glycine Agar: a selective plating medium for the quantitative
plenty of water and seek medical advice. After contact with skin, detection of coagulase-positive staphylococci. J. Bacteriol. 70:686.
wash immediately with plenty of water. If inhaled, remove to fresh
2. MacFaddin, J. F. 1985. Media for isolation-cultivation-
air. If not breathing, give artificial respiration. If breathing is
identification-maintenance of medical bacteria, vol. 1, p. 846-849.
difficult, give oxygen. Seek medical advice. If swallowed seek
medical advice immediately and show this container or label. Williams & Wilkins, Baltimore, MD.
3. Follow proper established laboratory procedures in handling and 3. Vogel, R. A., and M. Johnson. 1960. A modification of the
disposing of infectious materials. Tellurite-Glycine medium for use in the identification of
Staphylococcus aureus. Public Health Lab. 18:131.
Storage 4. Hitchins, A. D., T. T. Tran, and J. E. McCarron. 1995.
Store the dehydrated VJ Agar below 30°C. The dehydrated medium is Microbiological methods for cosmetics, p. 23.01-23.11. In
very hygroscopic. Keep container tightly closed. Bacteriological analytical manual, 8th ed. AOAC International,
Gaithersburg, MD.
Expiration Date
The expiration date applies to the product in its intact container when 5. Curry, A. S., J. G. Graf, and G. N. McEwen, Jr. (ed.). 1993.
stored as directed. Do not use a product if it fails to meet specifications CTFA microbiology guidelines. The Cosmetic, Toiletry, and
for identity and performance. Fragrance Association, Washington, D.C.
6. United States Pharmacopeial Convention. 1995. The United
Procedure States pharmacopeia, 23rd ed. The United States Pharmacopeial
Materials Provided Convention. Rockville, MD.
VJ Agar
Packaging
Materials Required but not Provided VJ Agar 100 g 0562-15
Chapman Tellurite Solution 1% 500 g 0562-17
Glassware
Principles of the Procedure stored as directed. Do not use product if it fails to meet specifications
Violet Red Bile Agar (VRBA) contains Bacto Peptone to provide for identity and performance.
carbon and nitrogen sources for general growth requirements.
Yeast Extract supplies B-complex vitamins which stimulate
Procedure
bacterial growth. Bile Salts No. 3 and Crystal Violet inhibit most Materials Provided
gram-positive microorganisms. Lactose is the carbohydrate source Violet Red Bile Agar
and Neutral Red is the pH indicator. Bacto Agar is the solidifying agent.
Materials Required but not Provided
Formula Flask with closure
Violet Red Bile Agar Distilled or deionized water
Formula Per Liter Autoclave
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g Incubator (35°C or 32°C for dairy products)
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 g Method of Preparation
Bacto Bile Salts No. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g
1. Suspend 41.5 grams in 1 liter distilled or deionized water.
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g 2. Heat to boiling to dissolve completely. Do not boil for more than
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g 2 minutes. DO NOT AUTOCLAVE.
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03 g Specimen Collection and Preparation
Bacto Crystal Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002 g
Refer to appropriate references for specimen collection and preparation.
Final pH 7.4 ± 0.2 at 25°C
Test Procedure
Precautions Presumptive test for coliforms using solid medium:
1. For Laboratory Use. 1. Transfer a 1 ml aliquot of test sample to a Petri dish.
2. Follow proper established laboratory procedure in handling and 2. Add 10 ml of Violet Red Bile Agar (at 48°C) and swirl to mix.
disposing of infectious materials.
3. Allow medium to solidify before incubating at 35°C for 18 to
Storage 24 hours; use 32°C for dairy products.
Store the dehydrated medium below 30°C. The dehydrated medium is 4. Examine for purple-red colonies, 0.5 mm in diameter (or larger),
very hygroscopic. Keep container tightly closed. surrounded by a zone of precipitated bile acids.
Expiration Date 5. Continue with confirmatory testing of typical coliform colonies.1.2,3
The expiration date applies to the product in its intact container when Uninoculated Enterobacter aerogenes
plate ATCC® 13048
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
Results References
Lactose fermenters: Purple-red colonies, with or without a zone of 1. Christen, G. L., P. M. Davidson, J. S. McAllister, and
precipitate around the colonies L. A. Roth. 1993. Coliform and other indicator bacteria, p. 247-252.
Lactose non-fermenters: Colorless to transparent colonies In Marshall, R. T. (ed.). Standard methods for the microbiological
Gram-positive cocci: Colorless, pinpoint colonies examination of dairy products, 16th ed. American Public Health
Association, Washington, D.C.
Limitations of the Procedure 2. Hitchins, A. D., P. A. Hartman, and E. C. D. Todd. 1992.
1. Violet Red Bile Agar may not be completely inhibitory to gram- Coliforms - Escherichia coli and its toxins, p. 325-369. In
positive organisms. Perform Gram stain and biochemical tests as Vanderzant, C., and D. F. Splittstoesser (ed.). Compendium of
necessary to identify isolates. methods for the microbiological examination of foods, 3rd ed.
2. The medium will grow gram-negative bacilli other than members American Public Health Association, Washington, D.C.
of the Enterobacteriaceae. Perform biochemical tests to identify 3. Hitchins, A. D., P. Feng, W. D. Watkins, S. R. Rippey, and
isolates to genus and species. L. A. Chandler. 1995. Escherichia coli and the coliform bacteria,
3. Boiling the medium for longer than 2 minutes can decrease the p. 4.01-4.29. In Bacteriological analytical manual, 8th ed. AOAC
ability to support growth. International, Gaithersburg, MD.
4. Plates of Violet Red Bile Agar should not be incubated longer than
24 hours because microorganisms that are only partially Packaging
inhibited may grow after extended incubation. Violet Red Bile Agar 100 g 0012-15
500 g 0012-17
5. For optimum performance, prepare and use the medium within
2 kg 0012-07
24 hours.
Materials Required but not Provided under long-wave fluorescent light, MUG-positive colonies are
Glassware surrounded by a bluish fluorescent halo. MUG-negative colonies lack
Petri dishes the fluorescent halo.
Distilled or deionized water E. coli colonies are red surrounded by a zone of precipitated bile and
Autoclave fluoresce blue under long-wave UV light.
Incubator (32°C) Salmonella and Shigella strains that produce glucuronidase may be
Waterbath (45°C) encountered infrequently but these are generally lactose negative and
Method of Preparation appear as colorless colonies which may fluoresce.
1. Suspend 41.6 grams in 1 liter distilled or deionized water. Limitations of the Procedure
2. Heat to boiling and boil no more than 2 minutes to dissolve 1. Glucuronidase-negative strains of E. coli have been encountered.5,6,7
completely. DO NOT AUTOCLAVE. Similarly, glucuronidase-positive strains of E. coli that do not
3. Cool to 45°C. fluoresce have been reported.8
4. Dispense into sterile Petri dishes. 2. Strains of Salmonella and Shigella that produce glucuronidase may
Specimen Collection and Preparation infrequently be encountered.9 These strains must be distinguished
from E. coli on the basis of other parameters, e. g., gas production,
Collect specimens in sterile containers or with sterile swabs and trans- lactose fermentation or growth at 44.5°C.
port immediately to the laboratory in accordance with recommended
guidelines.1,2,3 3. Since the nutritional requirements of organisms vary, some strains
may be encountered that fail to grow or grow poorly on this medium.
Test Procedure
1. Process each specimen as appropriate for that specimen.1,2,3
References
1. Christen, G. L., P. M. Davidson, J. S. McAllister, and L. A.
2. Incubate plates at 35°C for 22-26 hours.
Roth. 1993. Coliform and other indicator bacteria, p. 247-269. In
3. Examine plates for growth and fluorescence. R. T. Marshall (ed.). Standard methods for the microbiological
Results examination of dairy products, 16th ed. American Public
Coliform organisms form purplish-red colonies that are generally Health Association, Washington, D.C.
surrounded by a reddish zone of precipitated bile. When examined
Uninoculated Escherichia coli
plate ATCC® 25922
2. Hitchins, A. D., P. A. Hartman, and E. C. D. Todd. 1992. 6. Hansen, W., and E. Yourassowsky. 1984. Detection of
Coliforms-Escherichia coli and its toxins, p. 325-369. In ß-glucuronidase in lactose fermenting members of the family
C. Vanderzant and D. F. Splittstoesser (ed.). Compendium of Enterobacteriaceae and its presence in bacterial urine cultures.
methods for the microbiological examination of foods, 3rd ed. J. Clinical Microbiol. 20:1177-1179.
American Public Health Association, Washington, D.C. 7. Kilian, M., and P. Bulow. 1976. Rapid diagnosis of
3. Hitchins, A. D., P. Feng, W. D. Watkins, S. R. Rippey, and Enterobacteriaceae. Acta Pathol. Microbiol. Scand. Sect. B
L. A. Chandler. 1995. Escherichia coli and the coliform bacteria, 84:245-251.
p. 4.01-4.29. In Bacteriological analytical manual, 8th ed. AOAC 8. Mates, A., and M. Shaffer. 1989. Membrane filtration
International, Gaithersburg, MD. differentiation of E. coli from coliforms in the examination of
4 Feng, P. C. S., and P. A. Hartman. 1982. Fluorogenic assays for water. J. Appl. Bacteriology 67:343-346.
immediate confirmation of Escherichia coli. Appl. Environ. 9. Damare, J. M., D. F. Campbell, and R. W. Johnston. 1985.
Microbiol. 43:1320-1329. Simplified direct plating method for enhanced recovery of
5. Chang, G. W., J. Brill, and R. Lum. 1989. Proportion of Escherichia coli in food. Journal of Food Science 50:1736-1746.
ß-D-glucuronidase- negative Escherichia coli in human fecal Packaging
samples. Appl. Environ. Microbiol. 55:335-339.
Violet Red Bile Agar with MUG 500 g 0029-17
some foods, it is desirable to detect Enterobacteriaceae rather than the Materials Required but not Provided
coliform bacteria.1,2 Glassware
Enterobacteriaceae are glucose-fermenting bacteria. Mossel et al 3 Petri dishes
modified Violet Red Bile Agar, suggested by MacConkey4, that contains Distilled or deionized water
lactose by adding glucose to improve the recovery of Enterobacteriaceae. Autoclave
Later work by Mossel et al.5,6 demonstrated that lactose could be omitted, Incubator (35°C)
resulting in the formulation known as Violet Red Bile Glucose Agar.
Method of Preparation
Principles of the Procedure 1. Suspend 41.5 grams in 1 liter distilled or deionized water.
Violet Red Bile Glucose Agar contains Bacto Peptone as a source of 2. Heat to boiling and boil for no more than 2 minutes to dissolve
carbon, nitrogen, vitamins and minerals. Yeast Extract supplies B-complex completely.
vitamins which stimulate bacterial growth. Glucose is a carbohydrate. 3. DO NOT AUTOCLAVE.
Bile Salts No. 3 and Crystal Violet inhibit gram positive bacteria. Glucose
fermenters produce red colonies with red-purple halos in the presence Specimen Collection and Preparation
of Neutral Red, a pH indicator. Bacto Agar is a solidifying agent. Refer to appropriate references for specimen collection and preparation.
Procedure In the preparation of the standard curve, further dilutions of this stock
Materials Provided solution (1 µ/ml) are made as follows:
Vitamin B12 Assay Medium A. Add 1 ml stock solution to 99 ml distilled water (1 ml = 10 ng).
B. Add 1 ml of the solution from step A to 199 ml distilled water
Materials Required But Not Provided (1 ml = 0.05 ng).
Glassware An acceptable standard curve can be obtained by using the USP
Autoclave Cyanocobalamin Reference Standard at levels of 0.0, 0.025, 0.05, 0.1,
Stock culture of Lactobacillus delbrueckii subsp. lactis 0.15, 0.2 and 0.25 ng per assay tube. This is accomplished by adding 0,
ATCC® 4797 or 7830 0.5, 1, 2, 3, 4 and 5 ml of the 0.05 ng/ml solution per assay tube and
Sterile 0.85% saline sufficient distilled or deionized water to make 10 ml volume per tube.
Distilled or deionized water
Centrifuge A standard concentration is used which, after incubation, gives a
0.1 N NaOH transmittance value at the 5 ml level of not less than that which
Cyanocobalamin USP corresponds to a dry cell weight of 1.25 mg (see USP2 for method of
Spectrophotometer or nephalometer calibration of a spectrophotometer and determination of dry cell
Lactobacilli Agar AOAC or B12 Culture Agar USP weight). For the titrimetric method, a standard concentration should be
Lactobacilli Broth AOAC or B12 Inoculum Broth USP used which, after incubation, will give a titration at the 5 ml level of
8-12 ml 0.1N sodium hydroxide.
Method of Preparation Inoculate and incubate at 35-37°C for 18-24 hours. For turbidimetric
1. Suspend 7.6 grams in 100 ml distilled or deionized water. determinations, place tubes in a refrigerator at 2-8°C for 15-20 minutes
2. Boil 2-3 minutes. to stop growth. The growth can be measured by a nephelometric
3. Dispense 5 ml amounts into tubes, evenly dispersing the precipitate. method. Titrimetric determinations of growth are made after incubation
at 37°C for 72 hours. The curve is then constructed from the
4. Add standard or test samples.
values obtained.
5. Adjust tube volume to 10 ml with distilled or deionized water.
6. Autoclave at 121°C for 5 minutes. Results
1. Prepare a standard concentration response curve by plotting the
Specimen Collection and Preparation
response readings against the amount of standard in each tube, disk
Assay samples are prepared according to references given in the or cup.
specific assay procedures. For assays, the samples should be diluted to
2. Determine the amount of vitamin at each level of assay solution by
approximately the same concentration as the standard solution.
interpolation from the standard curve.
Test Procedure 3. Calculate the concentration of vitamin in the sample from the
Stock cultures of the test organism, L. delbrueckii subsp. lactis average of these volumes. Use only those values that do not vary
ATCC® 4797 or 7830, are prepared by stab inoculation of Lactobacilli more than ±10% from the average. Use the results only if two thirds
Agar AOAC or B12 Culture Agar USP. Following incubation at 37°C of the values do not vary more than ±10%.
for 24-48 hours, the tubes are stored in the refrigerator. Transfers are
made at 2 week intervals. Limitations of the Procedure
1. The test organism used for inoculating an assay medium must be
Inoculum for the assay is prepared by subculturing a stock of
cultured and maintained on media recommended for this purpose.
L. delbrueckii subsp. lactis ATCC 4797 or 7830 into a tube containing
10 ml of Lactobacilli Broth AOAC or B12 Inoculum Broth USP. After 2. Aseptic technique should be used throughout the assay procedure.
incubation at 35-37°C for 18-24 hours, the cells are centrifuged under 3. The use of altered or deficient media may cause mutants having
aseptic conditions and the supernatant liquid decanted. The cells are different nutritional requirements that will not give a satisfactory
washed by resuspending in 10 ml of sterile 0.85% saline solution and response.
centrifuging. The washing is repeated for a total of 3 times. Finally the 4. For successful results of these procedures, all conditions of the
cells are resuspended in 10 ml of sterile 0.85% saline. The cell suspension assay must be followed precisely.
is then diluted 1:100 with sterile 0.85% saline. One drop is used to
inoculate each assay tube. References
It is essential that a standard curve be constructed each time an assay is 1. Capps, Hobbs, and Fox. 1949. J. Biol. Chem. 178:517.
run. Conditions of autoclaving and temperature of incubation that 2. The United States Pharmacopeial Convention. 1995. The United
influence the standard curve readings cannot always be duplicated. States pharmacopeia, 23rd ed. The United States Pharmacopeial
Convention Inc. Rockville, MD.
The concentrations required for the preparation of the standard curve
are obtained by adding sufficient 25% ethanol to an accurately weighed Packaging
amount of USP Cyanocobalamin Reference Standard (resulting in a Vitamin B12 Assay Medium 100 g 0360-15*
solution containing 1.0 µg of cyanocobalamin per ml). This stock
solution is stored in the refrigerator and should be used within 60 days. *Store at 2-8°C
Lactobacillus fermentum
ATCC® 9338
WL Nutrient Broth
Prepare WL Nutrient Broth per label directions. Inoculate and
incubate for 40-48 hours at 35 ± 2°C for bacteria and at 30 ± 2°C
for yeasts.
INOCULUM
ORGANISM ATCC® CFU GROWTH ACID GAS
Escherichia coli 25922* 100-1,000 fair to good + +
Lactobacillus fermentum 9338 100-1,000 fair to good + sl. +
Saccharomyces cerevisiae 9763 100-1,000 good + +
Acid + = positive, yellow Acid – = negative, no color change
WL Differential Medium
Prepare WL Differential Medium per label directions. Inoculate
Escherichia coli Lactobacillus Saccharomyces and incubate for 40-48 hours at 35 ± 2°C for bacteria and at
ATCC® 25922 fermentum cerevisiae
ATCC® 9338 ATCC® 9763 30 ± 2°C for yeasts.
INOCULUM
ORGANISM ATCC® CFU GROWTH
The cultures listed are the minimum that should be used for Escherichia coli 25922* 100-1,000 good
performance testing. Lactobacillus fermentum 9338 500-1,000 good
*These cultures are available as Bactrol™ Disks and should be used as Saccharomyces cerevisiae 9763 1,000-2,000 inhibited
directed in Bactrol Disks Technical Information.
4. Bhat, P., and D. Rajan. 1975. Comparative evaluation of Marshall, (ed.), Standard methods for the examination of dairy
desoxycholate citrate medium and xylose lysine desoxycholate products, 16th ed. American Public Health Association,
medium in the isolation of shigellae. Am. J. Clin. Pathol. 64:399-404. Washington, D.C.
5. MacFaddin, J. F. 1985. Media for isolation-cultivation- 10. Andrews, W. H., G. A. June, P. S. Sherrod, T. S. Hammack,
identification-maintenance of medical bacteria, vol. 1. Williams and R. M. Amaguana. 1995. Salmonella, p. 5.01-5.20. In
& Wilkins, Baltimore, MD. Bacteriological Analytical Manual, 8th ed. AOAC International,
6. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and Gaithersburg, MD.
R. H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed. 11. Association of Official Analytical Chemists. 1996. Official
American Society for Microbiology, Washington, D.C. methods of analysis of AOAC International, Supplement March
7. United States Pharmacopeial Convention. 1995. The United 1996. AOAC International, Arlington, VA.
States pharmacopeia, 23rd ed. The United States Pharmacopeial
Convention, Rockville, MD. Packaging
8. Smith, J. L., and R. L. Buchanan. 1992. Shigella, p. 423-431. In XL Agar Base 500 g 0555-17
C. Vanderzant and D. F. Splittstoesser (ed.), Compendium of XLD Agar 100 g 0788-15
methods for the microbiological examination of food, 3rd ed. 500 g 0788-17
American Public Health Association, Washington, D.C. 2 kg 0788-07
9. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig. 10 kg 0788-08
1993. Pathogens in milk and milk products, p. 103-212. In R. T.
The cultures listed are the minimum that should be used for performance testing.
*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.
Summary and Explanation AND SKIN. Avoid contact with skin and eyes. Do not breathe dust.
Numerous media have been developed for isolating and differentiating Wear suitable protective clothing. Keep container tightly closed.
enteric pathogens. The majority were designed to recover a broad FIRST AID: In case of contact with eyes, rinse immediately with
spectrum of enteric pathogens.1 Consequently, overgrowth of nuisance plenty of water and seek medical advice. After contact with skin,
or contaminating organisms can be a major problem when recovery of wash immediately with plenty of water. If inhaled, remove to fresh
a specific organism or species is desired. This is particularly true for air. If not breathing, give artificial respiration. If breathing is diffi-
Salmonella isolation media where overgrowth of Proteus, Providencia cult, give oxygen. Seek medical advice. If swallowed seek medical
and Pseudomonas can dramatically interfere with the detection and advice immediately and show this container or label.
isolation of Salmonella. XLT4 Agar Supplement
In 1990, Miller and Tate described a new medium, XLT4 Agar, for CORROSIVE. CAUSES BURNS. HARMFUL BY INHALA-
isolating Salmonella.1 The authors established the selectivity of XLT4 TION, IN CONTACT WITH SKIN AND IF SWALLOWED. Avoid
Agar using pure cultures of a variety of enteric organisms. They also contact with skin and eyes. Do not breathe mist. Wear suitable
evaluated its sensitivity in detecting and isolating Salmonella using protective clothing, gloves and eye/face protection. Keep container
fecal-contaminated farm samples containing high numbers of competing tightly closed.
bacteria. In follow-up studies, Miller2,3 and Tate4 reported that XLT4 FIRST AID: In case of contact with eyes, rinse immediately with
Agar significantly improved the recovery of non-typhi Salmonella from plenty of water and seek medical advice. After contact with skin,
chicken and farm environmental drag-swab samples. wash immediately with plenty of water. If inhaled, remove to fresh
XLT4 Agar can be used clinically to screen stool samples for non- air. If not breathing, give artificial respiration. If breathing is diffi-
typhoid Salmonella.5,6 cult, give oxygen. Seek medical advice. If swallowed seek medical
advice immediately and show this container or label.
Principles of the Procedure 3. Follow proper established laboratory procedure in handling and
XLT4 Agar Base contains Proteose Peptone No. 3 as a source of complex disposing of infectious materials.
nitrogen compounds. Yeast Extract is added as a source of vitamins
and other cofactors. Differentiation of Salmonella from other organisms Storage
that also grow on this medium is based on fermentation of Xylose, Store XLT4 Agar Base below 30°C. The dehydrated medium is very
Lactose and Sucrose, decarboxylation of Lysine, and the production of hygroscopic. Keep container tightly closed. Store prepared medium
hydrogen sulfide. Hydrogen sulfide production is detected by the addition at 2-8°C.
of ferric ions. Sodium Thiosulfate is added as a source of inorganic Store XLT4 Agar Supplement at 15-30°C.
sulfur. Sodium Chloride maintains the osmotic balance of the medium.
Bacto Agar is the solidifying agent. Phenol Red is added as an indicator Expiration Date
of pH changes resulting from fermentation and decarboxylation The expiration date applies to the product in its intact container when
reactions. XLT4 Agar Supplement is added to inhibit growth of stored as directed. Do not use a product if it fails to meet specifications
non-Salmonella organisms. for identity and performance.
Formula Procedure
XLT4 Agar Base Materials Provided
Formula Per Liter XLT4 Agar Base
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 1.6 g XLT4 Agar Supplement
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g Materials Required But Not Provided
L-Lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Flasks with closures
Bacto Xylose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.75 g
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 g
Distilled or deionized water
Bacto Saccharose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 g
Bunsen burner or magnetic hot plate
Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 g Waterbath (45-50°C)
Sodium Thiosulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.8 g Petri dishes
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g Incubator (35°C)
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 g Method of Preparation
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08 g 1. Suspend 59 grams of XLT4 Agar Base in 1 liter distilled or
Final pH 7.4 ± 0.2 at 25°C deionized water.
XLT4 Agar Supplement 2. Add 4.6 ml XLT4 Agar Supplement.
A 27% solution (approximate) of the surfactant 7-ethyl-2-methyl-4-undecanol 3. Heat to boiling to dissolve completely. Avoid overheating. DO
hydrogen sulfate, sodium salt, formerly produced by Union Carbide under NOT AUTOCLAVE. Cool to 45-50°C in a waterbath.
the tradename of Tergitol 4.
4. Dispense into sterile Petri dishes.
Precautions Specimen Collection and Preparation
1. For Laboratory Use. 1. Collect specimens in sterile containers or with sterile swabs and
2. XLT4 Agar Base transport immediately to the laboratory following recommended
IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM guidelines.7-10
2. Process each specimen, using procedures appropriate for that 2. Miller, R. G., C. R. Tate, E. T. Mallinson, and J. A. Schemer.
sample.7,8,10,11 1991. Xylose-Lysine-Tergitol 4: An improved selective agar medium
Test Procedure for the isolation of Salmonella. Poultry Science 70:2429-2432.
1. Inoculate a suitable Salmonella enrichment broth (such as 3. Miller, R. G., C. R. Tate, E. T. Mallinson, and J. A. Schemer.
Tetrathionate Broth) and incubate at 35°C for 18-24 hours. 1992. Erratum. Xylose-Lysine-Tergitol 4: An improved selective
agar medium for the isolation of Salmonella. Poultry Science
2. Following enrichment, subculture onto XLT4 Agar. Streak for
71:398.
isolation.
3. Incubate plates aerobically at 35 ± 2°C. Examine for growth after 4. Tate, C. R., R. G. Miller, and E. T. Mallinson. 1992. Evaluation
18-24 and 48 hours incubation. of two isolation and two non-isolation methods for detecting
naturally occurring salmonellae from broiler flock environmental
Results drag-swab samples. J. Food Prot. 55:964-967.
Typical Salmonella colonies (H2S-positive) appear black or black- 5. Dusch, H., and M. Altwegg. 1994. Evaluation of Xylose-Lysine-
centered with a yellow periphery after 18-24 hours of incubation. Upon Tergitol 4 (XLT4) Agar and Modified Semisolid Rappaport
continued incubation, the colonies become entirely black or pink to Vassiliadis (MSRV) Medium for the isolation of non-typhoid
red with black centers. salmonellae from stool samples. Abstr. Annu. Meet. Am. Soc.
Colonies of H2S-negative Salmonella strains appear pinkish-yellow. Microbiol. C5:557.
Most Citrobacter colonies that grow on this medium are yellow without 6. Dusch, H., and M. Altwegg. 1995. Evaluation of five new plating
evidence of blackening. Growth of Enterobacter aerogenes and media for the isolation of Salmonella species. J. Clin. Microbiol.
Escherichia coli is markedly inhibited; colonies that do grow appear 33:802-804.
yellow without evidence of blackening. Growth of Proteus, 7. Andrews, W. H., G. A. June, P. S. Sherrod, T. S. Hammack,
Pseudomonas, Providencia, Alteromonas putrefaciens, Yersinia and R. M. Amaguana. 1995. Salmonella. In FDA Bacteriological
enterocolitica and Acinetobacter calcoaceticus is markedly to analytical manual, 8th ed. AOAC International, Arlington, VA.
completely inhibited on XLT4 Agar. Shigella species are partially 8. Vanderzant, C., and D. F. Splittstoesser (ed.) 1992. Compen-
inhibited and colonies appear red. dium of methods for the microbiological examination of foods,
Limitations of the Procedure 3rd ed. American Public Health Association, Washington, D.C.
1. XLT4 Agar is intended for detecting and isolating Salmonella based 9. Miller, J. M., and H. T. Holmes. 1995. Specimen collection,
on selectivity and colonial characteristics. Presumed Salmonella transport, and storage, p. 19-31. In P. R. Murray, et al. (ed). Manual of
colonies must be confirmed by biochemical and/or immunological clinical microbiology, 6th ed. American Society for Microbiology,
methods. Consult appropriate references for further information.5,7,8,12 Washington, D.C.
2. Since the nutritional requirements of organisms vary, some strains 10. Pezzlo, Marie (ed). 1992. Aerobic bacteria. In H. D. Isenberg (ed),
of Salmonella may be encountered that fail to grow or grow poorly Clinical microbiology procedures handbook, vol. 1. American
on this medium. Society for Microbiology, Washington, D.C.
3. Non-Salmonella strains that are not completely inhibited on this 11. Forbes, B. A., and P. A. Granato. 1995. Processing specimens for
medium may be encountered and must be differentiated from bacteria, p. 265-281. In P. R. Murray, et al. (ed), Manual of clinical
Salmonella. Consult appropriate references.7,8,10,12 microbiology, 6th ed. American Society for Microbiology, Wash-
4. Freshly inoculated plates and plates held over several days may ington, D.C.
develop multicolored, metallic looking crystals/flecks on the 12. Gray, L. D. 1995. Escherichia, Salmonella, Shigella and Yersinia.
surface. These crystals/flecks do not interfere with the performance In P. R. Murray, et al. (ed), Manual of clinical microbiology, 6th
of the medium. ed. American Society for Microbiology, Washington, D.C.
References Packaging
1. Miller, R. G., and C. R. Tate. 1990. XLT4: A highly selective XLT4 Agar Base 500 g 0234-17
plating medium for the isolation of Salmonella. The Maryland
XLT4 Agar Supplement l00 ml 0353-72
Poultryman April:2-7.
1 cm deep on the surface of the inoculated broth. Incubate the culture such as sodium propionate and diphenyl may be added to YM Agar
until growth appears and then streak onto YM Agar to obtain isolated to eliminate molds and permit the enumeration of yeasts to
yeast colonies. To isolate fermentative and oxidative strains, place mixed populations.
acidified inoculated YM Broth on a rotary shaker for 1 or 2 days. This
favors yeast recovery while preventing the sporulation of molds. Principles of the Procedure
Media selectivity may be enhanced through acidification or through Yeast Extract is a source of trace elements, vitamins and amino acids.
addition of selective agents. YM Broth may be acidified prior to Malt Extract is a source of carbon, protein and nutrients. Bacto Peptone
sterilization. YM Agar should be sterilized without pH adjustment is an additional source of carbon and provides nitrogen and amino
and sterile acid added to the sterile molten medium cooled to acids. Dextrose provides carbon. Bacto Agar is a solidifying agent.
45-50°C. Acidified YM Agar should not be heated. Antibiotics may Formula
be aseptically added to the sterile media. Other fungistatic materials,
YM Agar
Formula Per Liter
User Quality Control Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Bacto Malt Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Identity Specifications Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
YM Agar Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Dehydrated Appearance: Beige, free-flowing, homogeneous. Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Solution: 4.1% solution, soluble in distilled or Final pH 6.2 ± 0.2 at 25°C
deionized water on boiling. Solution
is light to medium amber, very YM Broth
slightly opalescent, without Formula Per Liter
significant precipitate. Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Prepared Plates: Light to medium amber, slightly Bacto Malt Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
opalescent without precipitate. Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Reaction of 4.1% Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Solution at 25°C: pH 6.2 ± 0.2 Final pH 6.2 ± 0.2 at 25°C
YM Broth
Dehydrated Appearance: Light beige, free-flowing, homogeneous. Precautions
Solution: 2.1% solution, soluble in distilled or 1. For Laboratory Use.
deionized water. Solution is light to 2. Follow proper established laboratory procedure in handling and
medium amber, clear to very slightly disposing of infectious materials.
opalescent without significant
precipitate. Storage
NOTE: At pH adjusted to 3.0-4.0, medium Store dehydrated medium below 30°C. The dehydrated medium is very
becomes slightly opalescent. hygroscopic. Keep container tightly closed.
Prepared Tubes: Light - medium amber, clear to very
slightly opalescent without significant Expiration Date
precipitate. The expiration date applies to the product in its intact container when
Reaction of 2.1% stored as directed. Do not use a product if it fails to meet specifications
Solution at 25°C: pH 6.2 ± 0.2 for identity and performance.
Cultural Response Procedure
Prepare 2 sets of YM Agar plates or YM Broth tubes (one set
pH 6.2, one set adjusted to pH 3.0-4.0) per label directions. Materials Provided
Inoculate and incubate at 30 ± 2°C for 18-72 hours. YM Agar
INOCULUM GROWTH GROWTH YM Broth
ORGANISM ATCC® CFU PH 3.0-4.0 PH 6.2
Aspergillus niger 16404 100-1,000 good good
Materials Required but not Provided
Candida albicans 10231 100-1,000 good good Glassware
Escherichia coli 25922* 100-1,000 markedly to good Autoclave
completely inhibited Antibiotics
Lactobacillus casei 7469 100-1,000 poor to fair good Sterile 10% HCl, Tartaric Acid or 10% Citric Acid
Saccharomyces
cerevisiae 9763 100-1,000 good good Method of Preparation
YM Agar
The cultures listed are the minimum that should be used for
performance testing. 1. Suspend 41 grams in 1 liter distilled or deionized water.
*These cultures are available as Bactrol™ Disks and should be used 2. Heat to boiling to dissolve completely.
as directed in Bactrol Disks Technical Information. 3. Autoclave at 121°C for 15 minutes. Cool to 45-50°C.
4. Dispense into sterile Petri dishes.
YM Broth colony forming units (CFU) per volume of sample. Record YM Broth
1. Dissolve 21 grams in 1 liter distilled or deionized water. results as growth or no growth.
2. Autoclave at 121°C for 15 minutes. Limitations of the Procedure
Optional (for Agar or Broth): If desired, acidify the medium 1. Acidified YM Agar should not be overheated.
to pH 3.0-4.0 by adding sterile 10% HCl, Tartaric Acid or
10% Citric Acid. Selective agents, e.g., penicillin (20 units per ml References
final concentration) or streptomycin (40 micrograms per ml final 1. 1951. U. S. Dept. Agricult. Tech. Bull. No. 1029.
concentration) may be added to the medium after sterilization 2. 1939. J. Tropical Med. Hyg. 42:176.
using aseptic technique.
3. Jong, S. C., and M. J. Edwards. 1991. American Type Culture
Test Procedure Collection Catalog of filamentous fungi, 18th ed. American Type
1. Inoculate YM Agar plates or YM Broth tubes with sample to evaluated Collection, Rockville, MD.
for the presence of yeasts, molds, or aciduric microorganisms.
2. Incubate at 30 ± 2°C for 18-72 hours.
Packaging
YM Agar 500 g 0712-17
Results YM Broth 500 g 0711-17
Examine the plates or tubes for growth. Record YM Agar results as 10 kg 0711-08
Also Known As
User Quality Control YPD media are also known as Yeast Extract-Peptone-Glucose media
Identity Specifications and may be abbreviated as YEPD.
YPD Agar Summary and Explanation
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution: 6.5% solution, soluble in distilled or General methods in yeast genetics specify using yeast extract-
deionized water on boiling. Solution peptone-glucose (YPD) medium for cultivating Saccharomyces
is light to medium amber, very cerevisiae and other yeasts.1 Yeasts grow well on a minimal medium
slightly to slightly opalescent. containing only glucose and salts. The addition of protein and yeast
Prepared Medium: Light to medium amber, slightly cell extract hydrolysates allow faster growth so that during exponential
opalescent. or log-phase growth, the cells divide every 90 minutes.1
Reaction of 6.5%
Solution at 25°C: pH 6.5 ± 0.2 Principles of the Procedure
YPD Broth YPD Agar and YPD Broth contain Bacto Peptone as a source of
Dehydrated Appearance: Beige, free-flowing, homogeneous. carbon, nitrogen, vitamins and minerals. Yeast Extract supplies B-complex
Solution: 5.0% solution, soluble in distilled or vitamins which stimulate bacterial growth. Dextrose is the carbohydrate
deionized water. Solution is light to source. YPD Agar contains Bacto Agar as the solidifying agent.
medium amber, clear to very slightly
opalescent. Formula
Prepared Medium: Light to medium amber, clear to very YPD Agar
slightly opalescent.
Formula Per Liter
Reaction of 5.0%
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Solution at 25°C: pH 6.5 ± 0.2
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Cultural Response Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Prepare YPD Agar or YPD Broth per label directions. Inoculate Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
and incubate the plates or tubes at 25 ± 2°C for 42-48 hours. Final pH 6.5 ± 0.2 at 25°C
INOCULUM
ORGANISM ATCC® CFU GROWTH YPD Broth
Kluyveromyces lactis 8563 100-1,000 good Formula Per Liter
Saccharomyces cerevisiae 18790 100-1,000 good Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Saccharomyces pastorianus 9080 100-1,000 good Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
The cultures listed are the minimum that should be used for Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
performance testing. Final pH 6.5 ± 0.2 at 25°C
Procedure Results
Growth of colonies on the agar or in the broth (turbidity).
Materials Provided
YPD Agar References
YPD Broth 1. Ausubel, F. M. , R. Brent, R. E. Kingston, D. D. Moore, J. G.
Materials Required but not Provided Seidman, J. A. Smith, and K. Struhl. 1994. Current Protocols in
Molecular Biology., Current Protocols, Brooklyn, NY.
Glassware
Distilled or deionized water Packaging
Autoclave YPD Agar 500 g 0427-17
Incubator (25°C) 2 kg 0427-07
Method of Preparation YPD Broth 500 g 0428-17
2 kg 0428-07
YPD Agar
Yeast Extract
Bacto Yeast Extract . Bacto Yeast Extract, Technical
®
Principles of the Procedure is harvested, washed and resuspended in water, where is undergoes
Yeast Extract is typically prepared by growing baker’s yeast, autolysis, i.e., self digestion using yeast’s enzymes. Yeast Extract is
Saccharomyces sp., in a carbohydrate-rich plant medium. The yeast the total soluble portion of this autolytic action. The autolytic activity
is stopped by a heating step. The resulting Yeast Extract is then filtered
clear and subsequently made a powder by the spray drying process.
User Quality Control Yeast Extract, Yeast Extract, Technical and Autolyzed Yeast provide
Identity Specifications vitamins, nitrogen, amino acids and carbon in microbiological culture
Yeast Extract media.
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 1% solution - soluble in distilled or Typical Analysis
deionized water. Solution is light to Yeast Extract
medium amber, clear, may have a
very slight precipitate. Physical Characteristics
2% solution-medium amber, clear, Ash (%) 11.2 Loss on Drying (%) 3.1
may have a very slight precipitate. Clarity, 1% Soln (NTU) 1.5 pH, 1% Soln 6.7
Reaction of 1% Filterability (g/cm2) 2.7
Solution at 25°C: pH 6.6 ± 0.2 Carbohydrate (%)
Yeast Extract, Technical Total 17.5
Dehydrated Appearance: Light to medium beige, free-flowing, Nitrogen Content (%)
homogeneous. Total Nitrogen 10.9 AN/TN 55.0
Solution: 1% solution - soluble in distilled or Amino Nitrogen 6.0
deionized water. Solution is light to
medium amber in color, clear to Amino Acids (%)
very slightly opalescent, may have Alanine 5.36 Lysine 5.15
a precipitate. Arginine 3.02 Methionine 1.05
Autolyzed Yeast Aspartic Acid 6.69 Phenylalanine 2.53
Dehydrated Appearance: Medium to dark brown, homogenous, Cystine 0.74 Proline 2.60
free-flowing. Glutamic Acid 14.20 Serine 2.84
Solution: 1% solution - not completely soluble Glycine 3.25 Threonine 2.95
in distilled or deionized water upon Histidine 1.20 Tryptophan 1.36
boiling. Solution is amber, opaque, Isoleucine 3.23 Tyrosine 1.20
may have a precipitate. Leucine 4.69 Valine 3.79
Reaction of 1% Inorganics (%)
Solution at 25°C: pH 4.9-6.3 Calcium 0.013 Phosphate 3.270
Cultural Response Chloride 0.380 Potassium 3.195
Cobalt <0.001 Sodium 1.490
Yeast Extract
Copper <0.001 Sulfate 0.091
Prepare a solution containing 1% Yeast Extract and 0.5%
Iron <0.001 Sulfur 0.634
sodium chloride. Adjust the pH to 7.2 ± 0.2 using dilute NaOH.
Inoculate tubes with the test organisms and incubate at 35 ± 2°C Lead <0.001 Tin <0.001
for 18-48 hours. Magnesium 0.075 Zinc 0.011
INOCULUM Manganese <0.001
ORGANISM ATCC® CFU GROWTH
Neisseria meningitidis 13090* 100-1,000 fair to good Vitamins (µg/g)
Staphylococcus aureus 25923* 100-1,000 good Biotin 3.3 PABA 763.0
Streptococcus pneumoniae 6305 100-1,000 good Choline (as Choline Chloride) 300.0 Pantothenic Acid 273.7
Cyanocobalamin <0.1 Pyridoxine 43.2
Yeast Extract, Technical Folic Acid 1.5 Riboflavin 116.5
Prepare a solution containing 2% Yeast Extract with 0.5% Inositol 1400.0 Thiamine 529.9
sodium chloride. Adjust the pH to 7.2 ± 0.2 using dilute NaOH. Nicotinic Acid 597.9 Thymidine 17.5
Inoculate tubes with the test organisms and incubate at 35 ± 2°C
for 18-24 hours. Biological Testing (CFU/g)
INOCULUM Coliform negative Standard Plate Count 60
ORGANISM ATCC® CFU GROWTH
Salmonella negative Thermophile Count <5
Escherichia coli 25922* 100-1,000 good
Spore Count 9
Streptococcus pyogenes 19615* 100-1,000 good
The cultures listed are the minimum that should be used for Precautions
performance testing. 1. For Laboratory Use.
*These cultures are available as Bactrol™ Disks and should be used 2. Follow proper established laboratory procedures in handling and
as directed in Bactrol Disks Technical Information.
disposing of infectious materials.
Storage References
Store the dehydrated ingredient below 30°C. The dehydrated ingredient 1. Prickett. 1928. Tech. Bull. 147. NY Agr. Exp. Sta.
is very hygroscopic. Keep container tightly closed. 2. Hutner. 1938. J. Bacteriol. 35:429.
Expiration Date 3. Partansky and McPherson. 1940. Ind. Eng. Chem., Anal. Ed. 12:443.
The expiration date applies to the product in its intact container when 4. Snell and Strong. 1939. Ind. Eng. Chem., Anal. Ed. 11:346.
stored as directed. Do not use a product if it fails to meet specifications 5. Kawaguchi, T., T. Asahi, T. Satoh, T. Uozumi, and T. Beppu.
for identity and performance. 1984. B-factor, an essential regulatory substance inducing the pro-
duction of rifamycin in a Nocardia sp. J. Antibiot. 37:1587-1594.
Procedure
6. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compen-
Materials Provided dium of methods for the microbiological examination of food, 3rd
Yeast Extract ed. American Public Health Association, Washington, D.C.
Yeast Extract, Technical 7. Association of Official Analytical Chemists. 1995. Bacteriological
Autolyzed Yeast
analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
Materials Required But Not Provided 8. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). 1995.
Materials vary depending on the medium being prepared. Standard methods for the examination of water and wastewater,
19th ed. American Public Health Association, Washington, D.C.
Method of Preparation
Refer to the final concentration of Yeast Extract, Yeast Extract, Technical 9. Marshall, R. T. (ed.). 1993. Standard methods for the examination
or Autolyzed Yeast in the formula of the medium being prepared. Add of dairy products, 16th ed. American Public Health Association,
Yeast Extract, Yeast Extract, Technical or Autolyzed Yeast as required. Washington, D.C.
10. J. Ind. Eng. Chem., Anal. Ed. 1941. 13:567.
Specimen Collection and Preparation
11. J. Ind. Eng. Chem., Anal. Ed. 1942. 14:909.
Obtain and process specimens according to the techniques and
procedures established by laboratory policy. Packaging
Test Procedure Yeast Extract 100 g 0127-15
See appropriate references for specific procedures using Yeast Extract, 500 g 0127-17
Yeast Extract, Technical or Autolyzed Yeast. 2 kg 0127-07
10 kg 0127-08
Results
Yeast Extract, Technical 500 g 0886-17
Refer to appropriate references and procedures for results.
10 kg 0886-08
Limitations of the Procedure Autolyzed Yeast 500 g 0229-17
1. Since the nutritional requirements of organisms vary, some strains may 10 kg 0229-08
encountered that fail to grow or grow poorly on prepared medium.
5. Koburger, J. A. 1970. Fungi in foods: 1. Effect of inhibitor and 7. Overcase, W. W., and D. J. Weakley. 1969. An aureomycin-rose
incubation temperature on enumeration. J. Milk Food Technol. bengal agar for enumeration of yeast and mold in cottage cheese.
33:433-434. J. Milk Food Technol. 32:442- 445.
6. Koburger, J. A. 1973. Fungi in foods: 5. Response of natural Packaging
populations to incubation temperatures between 12 and 32°C. Yeast Extract Glucose
J. Milk Food Technol. 36:434- 435. Chloramphenicol Agar 500 g 1900-17
5 kg 1900-03
Yeast Media
Bacto Yeast Morphology Agar . Bacto Yeast Carbon Base
®
Yeast Carbon Base contains all essential nutrients and vitamins necessary Carbon Source
for the cultivation of yeasts except a source of nitrogen. Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Yeast Nitrogen Base contains all essential nutrients and vitamins Amino Acids
L-Histidine Monohydrochloride . . . . . . . . . . . . . . . . . . . . 10 mg
necessary for the cultivation of yeasts except a source of carbohydrate.
LD-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Yeast Nitrogen Base w/o Amino Acids contains all essential vitamins LD-Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
and inorganic salts necessary for the cultivation of yeasts except histidine, Vitamins
methionine, tryptophane and a source of carbohydrate. Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 µg
Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate contains Calcium Pantothenate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400 µg
all essential nutrients and vitamins necessary for the cultivation of yeasts Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 µg
except amino acids and a source of nitrogen and carbohydrate. Inositol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2,000 µg
Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400 µg
Formula p-Aminobenzoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 µg
Yeast Morphology Agar Pyridoxine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . 400 µg
Formula per Liter Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 µg
Nitrogen Sources Thiamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 400 µg
Ammonium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 g
Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g Uninoculated Candida albicans
plate ATCC® 10231
Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate Yeast Carbon Base, Yeast Nitrogen Base, Yeast Nitrogen Base
1. Prepare a 10X solution by suspending 1.7 grams of Yeast Nitrogen w/o Amino Acids, Yeast Nitrogen Base w/o Amino Acids and
Base w/o Amino Acids and Ammonium Sulfate and nitrogen and Ammonium Sulfate
carbon sources, as required, in 100 ml distilled or deionized water. Measure growth turbidimetrically at 660 nm wavelength using a spec-
2. Filter sterilize the solution. trophotometer. Turbidimetric readings on assay tubes should be com-
parable to the control.
3. Store at 2-8°C.
4. Prepare the final medium by aseptically pipetting 0.5 ml of the Limitations of the Procedure
10X solution into 4.5 ml sterile distilled water. 1. Because the nutritional requirements of organisms vary, some strains
5. Mix the solution thoroughly by shaking before inoculation. may be encountered that fail to grow or grow poorly on a medium.
Specimen Collection and Preparation 2. Yeasts grown on a rich medium may carry a reserve of nitrogen in
the form of protein. Possible errors due to this reserve are eliminated
Obtain and process specimens according to the techniques and by making two serial transfers in the complete medium. When the
procedures established by laboratory policy. first transfer is seven days old, the culture is shaken and one loopful
Procedure is transferred to a second tube of the complete medium containing
the same source of nitrogen. If a positive test is obtained when the
Yeast Morphology Agar second culture is seven days old, the organism being tested assimilates
Inoculate plates using the Dolman technique (as follows) described by this particular nitrogen source.
Wickerham and Rettger.1 This is an excellent method for studying the
hyphae of filamentous yeasts. References
1. Warren, N. G., and K. C. Hazen. 1995. Candida, Cryptococcus,
1. Near one side of the plate (from the relative positions of 10 o’clock and other yeasts of medical importance, p. 723-737. In P. R.
to 2 o’clock), lightly inoculate a single streak taken from a slant Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken
culture. (ed.). Manual of clinical microbiology, 6th ed. American Society
2. In addition to the single streak, inoculate two points near the other for Microbiology, Washington, D.C.
side of the plate (at the 4 o’clock and 8 o’clock positions). 2. Wickerham, L. J. 1951. Taxonomy of yeasts. Technical bulletin
3. Cover a central section of the streak inoculation and one point No. 1029, U. S. Dept Agriculture.
inoculation with cover glasses, as follows: 3. Wickerham, L. J. 1939. J. Tropical Med. Hyg. 42:176.
a. With forceps, remove a cover glass from absolute alcohol, drain 4. Wickerham, L. J. 1946. A critical evaluation of the nitrogen
momentarily, and burn off excess alcohol by passing over a low assimilation tests commonly used in the classification of yeasts.
flame. J. Bacteriol. 52:293-301.
b When the cover glass has cooled, place one edge on the agar 5. Wickerham, L. J. 1948. J. Bacteriol. 56:363.
and allow it to fall across the central portion of the inoculated 6. Wickerham, L. J. 1943. J. Bacteriol. 46:501.
streak. Place a second cover glass over one point inoculation.
7. Guenter. Personal Communication.
4. Incubate at 25-30°C for 6-7 days.
8. Sherman F., G. R. Fink, and J. B. Hicks. 1986. Methods in yeast
5. After incubation, observe with a high dry objective. genetics. Cold Spring Harbor Laboratory Press, Cold Spring
Yeast Carbon Base, Yeast Nitrogen Base, Yeast Nitrogen Base Harbor, N.Y.
w/o Amino Acids, Yeast Nitrogen Base w/o Amino Acids and 9. Brownstein, B. H., G. A. Silverman, R. D. Little, D. T. Burke,
Ammonium Sulfate S. J. Korsmeyer, D. Schlessinger, and M. V. Olson. 1989.
1. Inoculate the prepared tubed medium very lightly with the test Isolation of single-copy human genes from a library of yeast
organism. artificial chromosomes clones. Science. 244:1348-1351.
2. Incubate at 25°C for 6-7 days. 10. Warren, N. G., and H. J. Shadomy. 1991. Yeasts of medical
3. After incubation (6-7 days and, if necessary, 20-24 days), shake importance, p. 617- 629. In A. Balows, W. J. Hausler, Jr., K. L.
the tubes to suspend growth. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.). Manual of
clinical microbiology, 5th ed. American Society for Microbiology,
4. Read for growth.
Washington, D.C.
Carbon Assimilation Test
Refer to the procedure described in the Manual of Clinical Microbiology.10 Packaging
Yeast Morphology Agar 100 g 0393-15
Nitrogen Assimilation Test
Yeast Carbon Base 100 g 0391-15
Refer to the procedure described in the Manual of Clinical Microbiology.10
Yeast Nitrogen Base 100 g 0392-15
Results Yeast Nitrogen Base w/o Amino Acids 100 g 0919-15
Yeast Morphology Agar 2 kg 0919-07
Using the high-dry objective, observe for hyphae of filamentous 10 kg 0919-08
yeasts. Yeast Nitrogen Base w/o Amino Acids 100 g 0335-15
and Ammonium Sulfate 10 kg 0335-08
Antimicrobic Supplement CN and Salmonella-Shigella Agar, Schiemann found that CIN Agar provided
better inhibition of normal enteric organisms and provided improved
direct recovery of Y. enterocolitica from feces.3 Schiemann later modified
Intended Use his original formula by substituting 0.5 grams of deoxycholate for
Bacto Yersinia Selective Agar Base is used with Bacto Yersinia the bile salts mixture and by reducing the content of novobiocin to
Antimicrobic Supplement CN in isolating and cultivating 2.5 mg/liter for improved growth of strains of Y. enterocolitica
Yersinia enterocolitica. serogroup 0:8.5 The concentration of cefsulodin in the antimicrobic
Also Known As supplement was reduced from that described by Schiemann to further
improve growth and recovery of Y. enterocolitica.
Yersinia Selective Agar is also known as CIN Agar, Modified or
Cefsulodin-Irgasan-Novobiocin Agar, Modified. Principles of the Procedure
Selectivity of Yersinia Selective Agar Base is due to the presence of
Summary and Explanation bile salts, crystal violet and Irgasan®, which markedly inhibit growth
Yersinia enterocolitica is a significant enteric pathogen6 and can be
of gram-positive and many gram-negative organisms. Supplementation
food- or water- borne.7 with Yersinia Antimicrobic Supplement CN (Cefsulodin and Novobiocin)
Yersinia Selective Agar is a selective and differential medium that improves inhibition of normal enteric organisms. Differentiation is
supports good growth of Y. enterocolitica and some other Yersinia species. based on mannitol fermentation. Organisms capable of fermenting
The formulation is based on the Cefsulodin-Irgasan-Novobiocin (CIN)
Uninoculated Yersinia enterocoliticia
Agar formulation of Schiemann.2-5 In comparison with MacConkey Agar plate ATCC® 9610
mannitol produce a pH decrease around the colony which allows Materials Required But Not Provided
absorption of neutral red, giving the colony a red color. Due to the Glassware
localized pH decrease, a zone of precipitated bile may also be present. Distilled or deionized water
Organisms that do not metabolize mannitol to acid end products will Incubator (22-25°C or 30 ± 2°C)
form colorless, translucent colonies. Autoclave
Sterile Petri dishes
Formula
Yersinia Selective Agar Base Method of Preparation
Formula Per Liter Yersinia Antimicrobic Supplement CN
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g 1. To rehydrate the supplement, aseptically add 10 ml sterile distilled
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 g or deionized water to the vial.
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
2. Invert the vial gently several times to dissolve the powder.
Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Sodium Deoxycholate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g Yersinia Selective Agar Base
Sodium Cholate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g 1. Suspend 59.5 grams in 1 liter distilled or deionized water and boil
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g to dissolve completely.
Sodium Pyruvate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g 2. Autoclave at 121°C for 15 minutes. Avoid overheating.
Magnesium Sulfate Heptahydrate . . . . . . . . . . . . . . . . . . . 10 mg
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5 g
3. Cool the medium to 45-50°C. Aseptically add 10 ml rehydrated
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 mg Yersinia Antimicrobic Supplement CN to the medium. Mix well.
Crystal Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg 4. Dispense into Petri dishes.
Irgasan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mg
Specimen Collection and Preparation
Yersinia Antimicrobic Supplement CN All specimens should be collected in sterile containers in accordance
Formula Per 10 ml Vial with recommended guidelines and should be transported immediately
Cefsulodin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mg to the laboratory. For specific information on collection and storage of
Novobiocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 mg specimens consult appropriate references.
Precautions Test Procedure
1. For Laboratory Use.
For a complete discussion on the isolation and identification of Yersinia,
2. MAY CAUSE ALLERGIC EYE, RESPIRATORY SYSTEM AND consult appropriate references.
SKIN REACTION. (US) Avoid contact with skin and eyes. Do not
breathe dust. Wear suitable protective clothing. Keep container Results
tightly closed. Target Organs: Liver, Blood. Y. enterocolitica colonies appear translucent or translucent with dark
FIRST AID: In case of contact with eyes, rinse immediately with pink centers. Colony edges are entire or irregular. After 48 hours
plenty of water and seek medical advice. After contact with skin, incubation, colonies appear dark pink with a translucent border and
wash immediately with plenty of water. If inhaled, remove to fresh may be surrounded by a zone of precipitated bile.
air. If not breathing, give artificial respiration. If breathing is diffi- Growth of non-Yersinia organisms is markedly to completely inhibited.
cult, give oxygen. Seek medical advice. If swallowed seek medical
advice immediately and show this container or label. Limitations of the Procedure
3. Follow proper established laboratory procedure in handling and 1. Yersinia Selective Agar Base and Yersinia Antimicrobic Supplement
disposing of infectious materials. CN are intended for use in the preparation of Yersinia Selective Agar.
Although this medium is selective for Yersinia, biochemical testing
Storage using pure cultures is necessary for complete identification.
Store Yersinia Selective Agar Base dehydrated below 30°C. The
2. Due to the selective properties of the medium, some Yersinia strains
dehydrated medium is very hygroscopic. Keep container tightly closed
may be encountered that fail to grow or grow poorly on the complete
Store Yersinia Antimicrobic Supplement CN lyophilized and rehydrated medium. Some strains of normal enteric organisms may be encoun-
at 2-8°C. Do not open or rehydrate vials until ready to use. Use the tered that are not inhibited or are only partially inhibited on the
rehydrated product within 24 hours. complete medium, such as Citrobacter freundii, Serratia
liquefaciens and Enterobacter agglomerans.
Expiration Date 3. Growth of Yersinia frederiksenii, Y. kristensenii, Y. pseudotuberculosis
The expiration date applies to the product in its intact container when and Y. intermedia is not inhibited on the complete medium. Colonies
stored as directed. Do not use a product if it fails to meet specifications of these organisms must be differentiated from Y. enterocolitica on
for identity and performance. the basis of additional characteristics.
Procedure References
Materials Provided 1. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
Yersinia Selective Agar Base R. H. Yolken. (ed.). 1995. Manual of clinical microbiology,
Yersinia Antimicrobic Supplement CN 6th ed. American Society for Microbiology, Washington, D.C.
Packaging
Yersinia Selective Agar Base 500 g 1817-17
10 kg 1817-08
Yersinia Antimicrobic Supplement CN 6 x 10 ml 3196-60
2. Kleitmann, W. 1995. Resistance and susceptibility testing for R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed.
Mycobacterium tuberculosis. Clin. Microbiol. News 17:65-69. American Society for Microbiology, Washington, D.C.
3. Woodruff, C. E., D. Crombie, J. S. Woolley, E. Medlar, and 5. Isenberg, H. D. (ed.). 1994. Clinical microbiology procedures hand-
W. Steeken. 1946. Report of the committee on evaluation of book, sup. 1. American Society for Microbiology, Washington, D.C.
laboratory procedures. Am. Rev. Tuberc. 54:428-432.
4. Nolte, F. S., and B. Methcock. 1995. Mycobacterium, p. 400-437.
Packaging
In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and ATS Medium 100 tubes 1019-79*
HYcheck ™
pH at 25°C: 7.2 ± 0.2 7.3 ± 0.2 Aspergillus niger NCPF 2275 – good good
Candida albicans 2091 good good
Cultural Response (approx inoculum 30-300 CFU) Escherichia coli 25922* none to poor good
HYcheck D/E Neutralizing Agar Saccharomyces cerevisiae NCYC 1211 – good good
Inoculate and incubate at 35 ± 2°C for 18-48 hours. Serratia marcescens 8100 none to poor good
ORGANISM ATCC® GROWTH ON D/E AGAR
Staphylococcus aureus 25923* none to poor good
Streptococcus pyogenes 19615* none to poor good
Aspergillus niger NCPF 2275 – good
Bacillus subtilis 6633 good HYcheck for Yeasts and Molds with TTC
Candida albicans 2091 good
Escherichia coli 25922* good
Inoculate and incubate at 30 ± 2°C for 18-48 hours.
GROWTH GROWTH
Pseudomonas aeruginosa 27853* good ORGANISM ATCC® ON RBCA ON TSA W/TTC
Staphylococcus aureus 25923* good
Staphylococcus epidermidis 12228* good Aspergillus niger NCPF 2275 — good good
Candida albicans 2091 good good
HYcheck for Disinfection Control Escherichia coli 25922* none to poor good
Inoculate and incubate at 35 ± 2°C for 18-48 hours. Saccharomyces cerevisiae NCYC 1211 – good poor
GROWTH GROWTH Serratia marcescens 8100 none to poor good
ORGANISM ATCC® ON D/E ON TSA
Staphylococcus aureus 25923* none to poor poor
Aspergillus niger NCPF 2275 – good good
Streptococcus pyogenes 19615* none to poor good
Bacillus subtilis 6633 good good
Candida albicans 2091 good good
The cultures listed are the minimum that should be used for
Escherichia coli 25922* good good performance testing.
Pseudomonas /nosa 27853* good good
Staphylococcus aureus 25923* good good *These cultures are available as Bactrol™ Disks and should be used
Staphylococcus epidermidis 12228* good good as directed in Bactrol Disks Technical Information.
HYcheck for Disinfection Control has side one coated with fermentable carbohydrate. Sodium Thioglycollate neutralizes
D/E Neutralizing Agar (D/E) (see above), and side two coated with mercurials. Sodium Thiosulfate neutralizes iodine and chlorine.
Tryptic Soy Agar (TSA). In 1955, Leavitt et al.12 demonstrated that Sodium Bisulfite neutralizes formaldehyde and glutaraldehyde. Lecithin
Tryptic Soy Agar supports excellent growth of a both aerobic and neutralizes quaternary ammonium compounds and Polysorbate 80
anaerobic microorganisms. Tryptic Soy Agar is a general purpose neutralizes phenols, hexachlorophene, formalin and, with lecithin,
medium that is recommended in multiple water and wastewater ethanol. Brom Cresol Purple is a colorimetric indicator. Bacto Agar is
applications.13 a solidifying agent.
HYcheck for Enterobacteriaceae has side one coated with Violet Red Tryptic Soy Agar (TSA) - side two
Bile Glucose Agar and side two coated with Tryptic Soy Agar, a general Tryptone and Soytone provide nitrogen, vitamins and minerals. The
purpose growth medium. Violet Red Bile Glucose Agar is a selective natural sugars from the soybean promote bacterial growth. Sodium
medium used for the enumeration of Enterobacteriaceae in foods. Chloride maintains the osmotic balance of the medium. Bacto Agar is
Coliform bacteria have long been used as an index of fecal contamination a solidifying agent.
in waters, and their presence in milk is used as an index of sanitation in
milk processing.14 The presence of Enterobacteriaceae, coliforms, HYcheck for Enterobacteriaceae
Salmonellae, Klebsiella or Citrobacter, in raw foodstuffs is an indicator Violet Red Bile Glucose Agar (VRBGA) - side one
of fecal contamination. Their presence after processing may indicate a Yeast Extract provides vitamins, cofactors, nitrogen and carbon. Glucose
failure in the manufacturing process. provides a source of fermentable carbohydrate. Bacto Agar is a
HYcheck Plate Count Agar with TTC has both sides coated with solidifying agent.
Plate Count Agar with TTC (0.01% 2,3,5-Triphenyl Tetrazolium
Tryptic Soy Agar - side two
Chloride).
Tryptone and Soytone provide nitrogen, vitamins and minerals. The
HYcheck for Total Count has side one coated with Plate Count Agar natural sugars from the soybean promote bacterial growth. Sodium
and side two coated with Plate Count Agar with 0.01% TTC. Plate Chloride maintains the osmotic balance of the medium. Bacto Agar is
Count Agar is used for enumerating bacteria in water, wastewater, food a solidifying agent.
and dairy products.13,15-18 TTC is a redox indicator that is colorless in
the oxidized form. TTC is reduced to insoluble triphenylformazan by HYcheck Plate Count Agar (PCA) with TTC
certain actively metabolizing bacteria, resulting in a red color in the Tryptone and Yeast Extract provide carbon and nitrogen. Dextrose
presence of bacterial growth. provides a source of fermentable carbohydrate. TTC is a redox indicator.
There are two HYcheck products for yeasts and molds: 1) HYcheck Bacto Agar is a solidifying agent.
for Yeasts and Molds has side one coated with Rose Bengal HYcheck for Total Count
Chloramphenical Agar and side two coated with Tryptic Soy Agar;
2) HYcheck for Yeasts and Molds with TTC has side one coated Plate Count Agar - side one
with Rose Bengal Chloramphenical Agar and side two coated with Tryptone and Yeast Extract provide carbon and nitrogen. Dextrose provides
Tryptic Soy Agar with 0.01% TTC. Rose Bengal Chloramphenical Agar a source of fermentable carbohydrate. Bacto Agar is a solidifying agent.
is recommended in the selective isolation and enumeration of yeasts Plate Count Agar with TTC - side two
and molds from environmental materials and foodstuffs. The pH of the Tryptone and Yeast Extract provide carbon and nitrogen. Dextrose provides
medium is near neutrality for improved growth and recovery of acid a source of fermentable carbohydrate. TTC is a redox indicator. Bacto
sensitive strains. 19-21 Agar is a solidifying agent.
Principles of the Procedure HYcheck for Yeasts and Molds
HYcheck D/E Neutralizing Agar Rose Bengal Chloramphenicol Agar (RBCA) - side one
Tryptone provides carbon and nitrogen. Yeast Extract provides vitamins, Soytone provides carbon and nitrogen. Dextrose provides a source of
cofactors and additional nitrogen and carbon. Dextrose provides fermentable carbohydrate. Rose Bengal and Chloramphenicol inhibit
fermentable carbohydrate. Sodium Thioglycollate neutralizes bacterial growth and restrict size and height of rapidly growing mold
mercurials. Sodium Thiosulfate neutralizes iodine and chlorine. Sodium colonies. Bacto Agar is a solidifying agent.
Bisulfite neutralizes formaldehyde and glutaraldehyde. Lecithin Tryptic Soy Agar - side two
neutralizes quaternary ammonium compounds and Polysorbate 80 Tryptone and Yeast Extract provide carbon and nitrogen. Dextrose provides
neutralizes phenols, hexachlorophene, formalin and, with lecithin, a source of fermentable carbohydrate. Bacto Agar is a solidifying agent.
ethanol. Brom Cresol Purple is a colorimetric indicator. Bacto Agar is
a solidifying agent. HYcheck for Yeasts and Molds with TTC
HYcheck for Disinfection Control Rose Bengal Chloramphenicol Agar - side one
Soytone provides carbon and nitrogen. Dextrose provides a source of
D/E Neutralizing Agar (D/E) - side one fermentable carbohydrate. Rose Bengal suppresses bacterial growth
Tryptone provides carbon and nitrogen. Yeast Extract provides vitamins, and restricts size and height of rapidly growing mold colonies.
cofactors and additional nitrogen and carbon. Dextrose provides Chloramphenicol inhibits bacteria. Bacto Agar is a solidifying agent.
Precautions
1. Do not touch agar surface.
2. Do not use if there are signs of dehydration or contamination.
Storage
Store HYcheck slides at 2-15°C.
Expiration Date
The expiration date applies to the product in its intact container when
stored as directed. Do not use a product if it fails to meet specifications Candida albicans ATCC® 60193
for identity and performance. on Rose Bengal Chloramphenicol Agar
Procedure
103 104 105
Materials Provided
(One type is provided per package.)
HYcheck D/E Neutralizing Agar
HYcheck for Disinfection Control
HYcheck for Enterobacteriaceae
HYcheck Plate Count Agar with TTC
HYcheck for Total Count
HYcheck for Yeasts and Molds
HYcheck for Yeasts and Molds with TTC.
Test Procedure
Surfaces
1. Loosen cap and remove HYcheck slide from the container.
2. Examine for dehydration or contamination. Aspergillus niger ATCC® 1015
on Rose Bengal Chloramphenicol Agar
3. Hold terminal spike against surface to be tested.
4. Press down on the spike to bend the paddle around the hinge line.
5. Gently lower the slide and press agar into contact with the test surface. 103 104 105 106
6. Apply firm and even pressure on the test surface for a few seconds.
7. Repeat procedure using the second agar surface on an area adjacent
to the initial test site.
8. Replace slide in the container and close tightly.
9. Incubate in an upright position at indicated temperature.
Liquids
1. Loosen cap and remove HYcheck Slide from the container.
2. Examine for dehydration or contamination.
3. Immerse slide into test fluid so that agar surface becomes totally
covered (if insufficient liquid is available, pour over surface of
the slide).
4. Allow to drain.
Escherichia coli ATCC® 11229
5. Replace slide in the container and close tightly. on Tryptic Soy Agar with 0.01% TTC
6. Incubate in an upright position at indicated temperature.
Results Limitations of the Procedure
The following photos are reactions to Candida albicans, Aspergillus 1. Do not use the HYcheck Slide if it is contaminated or the agar
niger and Escherichia coli. medium is significantly dehydrated.
Packaging
Petragnani Medium 100 tubes 1010-79
6. Flood the slide with Acridine Orange Stain for 2 minutes. 2. Strugger, S. 1948. Fluorescence microscope examination of
7. Rinse thoroughly with tap water and allow to dry. bacteria in soil. Can. J. Research 26:188-193.
8. Smears may be initially examined at 100X to 400X magnification 3. Jones, J. F., and B. M. Simon. 1975. An investigation of errors in
using a fluorescent microscope. Findings should be confirmed by direct counts of aquatic bacteria by epifluorescence microscopy,
examination at 1000X with an oil immersion objective. with reference to a new method for dyeing membrane filters.
J. Appl. Bacteriol. 39:317-329.
Results 4. Kronvall, G., and E. Myhre. 1977. Differential staining of bacteria
Bacteria and fungi stain bright orange. The background appears in clinical specimens using acridine orange buffered at low pH.
black to yellow green. Human epithelial and inflammatory cells and Acta. Path. Microbiol. Scand. Sect. B 85:249-254.
tissue debris stain pale green to yellow. Activated leukocytes will 5. McCarthy, L. R., and J. E. Senne. 1980. Evaluation of acridine
stain yellow, orange or red depending on the level of activation and orange stain for detection of microorganisms in blood cultures.
the amount of RNA produced. Erythrocytes either do not stain or J. Clin. Microbiol. 11:281-285.
stain pale green. 6. Lauer, B. A., L. B. Reller, and S. Mirrett. 1981. Comparison of
acridine orange and Gram stains for detection of microorganisms
Limitations of the Procedure in cerebrospinal fluid and other clinical specimens. J. Clin.
1. Acridine Orange staining provides presumptive information on the Microbiol. 14:201-205.
presence and identification of microorganisms in the specimen. 7. Greenwood, J. R., and K. Kirk-Hillaire. 1981. Evaluation of
Because microorganisms seen in smears, including nonviable acridine orange stain for detection of Trichomonas vaginalis in
organisms, may arise from external sources (i.e., specimen collection vaginal specimens. J. Clin. Microbiol. 14:699.
devices, slides or water used for rinsing), all positive smears should 8. Forsum, U., and A. Hallén. 1979. Acridine orange staining of
be confirmed by culture. urethral and cervical smears for the diagnosis of gonorrhea. Acta.
2. Approximately 104 colony-forming units per ml are required for Dermatovener 59:281-282.
detection by the Acridine Orange staining method. 9. Rosendal, S., and A. Valdivieso-Garcia. 1981. Enumeration of
3. Acridine orange staining does not distinguish between gram-positive mycoplasmas after acridine orange staining. Appl. Environ.
and gram-negative organisms. The gram reaction may be determined Microbiol. 41:1000-1002.
by performing the Gram stain procedure directly over the acridine 10. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
orange stain after removing the immersion oil with xylene.12 R. H. Yolken (eds.). 1995. Manual of clinical microbiology,
4. Nuclei or granules from disintegrated, activated leukocytes may 6th ed. American Society for Microbiology, Washington, D.C.
resemble cocci at lower magnifications (e.g., 100X-400X). They 11. Kasten, F. H. 1967. Cytochemical studies with acridine orange
may be distinguished on the basis of morphology at higher magni- and the influence of dye contaminants in the staining of nucleic
fications (e.g., 1000X). acids. Internat. Rev. Cytol. 21:141- 202.
5. Certain types of debris may fluoresce in Acridine Orange stained 12. Baron, E. J., and S. M. Finegold. 1990. Bailey & Scott’s diagnostic
smears. This debris may be distinguished from microorganisms on microbiology, 8th ed. The C. V. Mosby Company, St. Louis, MO.
the basis of morphology when viewed at higher magnification.
Packaging
References Acridine Orange Stain 1 x 250 ml 3336-75
1. Strugger, S., and P. Hilbrich. 1942. Die fluoreszenzmikroskopische 6 x 250 ml 3336-76
unterscheidung lebender und toten bakterienzeillen mit hilfe des SpotTest™
akridinorangefärbung. Deut. Teirarztl. Wochscher. 50:121-130. Acridine Orange Stain 50 x 0.75 ml 3561-26
to characterize them, and the therapy to initiate while awaiting test Generally, the cell wall is nonselectively permeable. It is theorized that
results. during the Gram stain procedure, the cell wall of gram-positive cells
is dehydrated by the alcohol in the decolorizer and loses permeability,
Principles of the Procedure thus retaining the primary stain. However, the cell wall of
The Gram stain procedure consists of 4,5,6 : gram-negative cells has a higher lipid content and becomes more
1. Staining a fixed smear with crystal violet; permeable when treated with alcohol, resulting in loss of the primary stain.
2. Applying iodine as a mordant; The principles of the 3-Step Gram Stain procedure are identical to
3. Decolorizing the primary stain with alcohol/acetone; and, the 4-step procedure described above. However, the decolorizing and
4. Counterstaining with safranin or basic fuchsin. counterstaining steps have been combined into one reagent.
A crystal violet-iodine complex forms in the protoplast (not the cell The molecular basis for the Gram stain has not yet been determined.
wall) of all organisms stained by this procedure. Organisms able to
retain this dye complex after decolorization are classified as Formula
gram-positive while those that can be decolorized and Reagents are provided in two sizes, a 250 ml plastic dispensing bottle
counterstained are classified as gram-negative.2,4,5,6 with a dropper cap and a one-gallon container with a dispensing tap.
Standardization may include adjustment to meet performance
Positive Blood Culture Bottle
Specimen containing numerous
specifications.
gram-negative rods with shape and
size of enteric rods. The culture 3329-Gram Crystal Violet
grew Klebsiella pneumoniae.
PRIMARY STAIN
Aqueous solution of Crystal Violet.
3331-Gram Iodine
MORDANT
(Working solution prepared from Gram Diluent and Gram Iodine 100X)
Iodine Crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 g
Potassium Iodide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6 g
Ground Beef
Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 liter
Sample containing E. coli: H7
and Staphylococcus aureus.
3342-Stabilized Gram Iodine
MORDANT
Polyvinylpyrrolidone-Iodine Complex . . . . . . . . . . . . . . 100 g
Potassium Iodide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 g
Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 liter
3330-Gram Decolorizer
DECOLORIZER
Acetone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 ml
Upon disruption or removal of the cell wall, the protoplast of Isopropanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750 ml
gram-positive (as well as gram-negative) cells can be decolorized and 3332-Gram Safranin
the gram-positive attribute lost. Thus, the mechanism of the Gram
COUNTERSTAIN
stain appears to be related to the presence of an intact cell wall able to
Safranin O Powder (pure dye) . . . . . . . . . . . . . . . . . . . . . . . 4 g
act as a barrier to decolorization of the primary stain.
Denatured Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 ml
Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 ml
3343-Gram Basic Fuchsin
User Quality Control
Run controls daily using 18-24 hour cultures of known gram- COUNTERSTAIN
positive and gram-negative microorganisms. It is very important Basic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08 g
that controls be included in each staining run, preferably on the Phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 g
same slide. When performing the Gram stain on a clinical Isopropyl Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 ml
specimen, particularly when the results will be used as a guide Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 993 ml
to the selection of a therapeutic agent, such a control system
furnishes assurance that the iodine solution is providing proper 3335-3-Step Gram Safranin-S
mordant activity and that decolorization was performed properly. DECOLORIZER/COUNTERSTAIN
Alcohol-based solution of safranin.*
ORGANISM* ATCC® EXPECTED RESULTS
Staphylococcus aureus 25923 gram-positive cocci 3341-3-Step Gram Safranin-T
Escherichia coli 25922 gram-negative rods DECOLORIZER/COUNTERSTAIN
* Available as Bactrol™ Disks.
Alcohol-based solution of safranin.*
* Patent Pending
Swabs 6. Blot with blotting paper or paper towel or allow to air dry.
Blotting paper 7. Examine the smear under an oil immersion lens.
Microscope with oil immersion lens
Bactrol™ Gram Slide Results
4-STEP 4-STEP 3-STEP TECHNIQUE
Bactrol™ Disks TECHNIQUE TECHNIQUE USING EITHER
USING USING GRAM SAFRANIN-S
1. Flood the fixed smear with primary stain (Gram Crystal Violet) and REACTION GRAM SAFRANIN BASIC FUCHSIN OR GRAM SAFRANIN-T
stain for 1 minute. Gram-positive Purple-black Bright purple to Purple-black
2. Remove the primary stain by gently washing with cold tap water. cells purple-black cells to purple cells
3. Flood the slide with mordant (either Gram Iodine or Stabilized Gram Gram-negative Pink to red Bright pink to Red-pink to
Iodine) and retain on the slide for 1 minute. cells fuchsia cells fuchsia cells
4. Remove the mordant by gently washing with tap water. Limitations of the Procedure
5. Decolorize (Gram Decolorizer) until solvent running from the slide 1. The Gram stain provides preliminary identification information
is colorless (30-60 seconds). only and is not a substitute for cultural studies of the specimen.
6. Wash the slide gently in cold tap water. 2. Prior treatment with antibacterial drugs may cause gram-positive
7. Flood the slide with counterstain (either Gram Safranin or Gram organisms from a specimen to appear gram-negative.
Basic Fuchsin) and stain for 30-60 seconds. 3. Use of an 18-24 hour culture is advisable for best results since
8. Wash the slide with cold tap water. fresh cells have a greater affinity than old cells for most dyes. This
9. Blot with blotting paper or paper towel or allow to air dry. is particularly true of many spore formers, which are strongly
10. Examine the smear under an oil immersion lens. gram-positive when examined in fresh cultures but which later
become gram-variable or gram-negative.
3-Step Staining Procedure 4. The Gram stain reaction, like the acid-fast reaction, is altered by
Materials Provided physical disruption of the bacterial cell wall or protoplast. The cell
walls of gram-positive bacteria interpose a barrier which prevents
3-Step Stabilized Iodine Technique leaching of the dye complex from the cytoplasm. Cell walls of
Gram Crystal Violet gram-negative bacteria contain lipids soluble in organic solvents,
Stabilized Gram Iodine which are then free to decolorize the cytoplasm. Therefore, a
3-Step Gram Safranin-S microorganism that is physically disrupted by excess heating will
not react to Gram staining as expected.
3-Step Traditional Iodine Technique
5. 3-Step Gram Safranin-S is intended for use with stabilized iodine.
Gram Crystal Violet 3-Step Gram Safranin-T is intended for use with traditional iodine.
Gram Iodine Unsatisfactory results may occur if other combinations of iodine
3-Step Gram Safranin-T and 3-Step Gram Safranin are used.
Materials Required but not Provided 6. Over time, a fine precipitate may develop in Gram Basic Fuchsin,
Microscope slides 3-Step Gram Safranin-S and 3-Step Gram Safranin-T. Product
Bunsen burner or methanol performance will not be affected.
Bacteriological loop References
Swabs
1. Fortschr. Med., 1884, 2:185
Blotting paper
Microscope with oil immersion lens 2. Donnelly, J. P. 1962. The secrets of Gram’s stain. Infec. Dis. Alert.
Bactrol™ Gram Slide 15:109-112.
Bactrol™ Disks 3. N.Y. Agr. Exp. Sta. Tech. Bull., 1923. 93.
1. Flood the fixed smear with primary stain (Gram Crystal Violet) 4. Bartholomew, J. W. 1962. Variables influencing results, and the
and stain for 1 minute. precise definition of steps in gram staining as a means of
standardizing the results obtained. Stain Technol. 37:139-155.
2. Remove the primary stain by gently washing with cold tap water.
5. Kruczak-Filipov, P., and R. G. Shively. 1992. Gram Stain
3. Flood the slide with mordant (Stabilized Gram Iodine or Gram
procedure, p. 1.5.1-1.5.18. In H.D. Isenberg (ed.), Clinical
Iodine [traditional formulation]) and retain on the slide for 1 minute.
Microbiology Procedures Handbook, vol. 1. American Society for
(Refer to LIMITATIONS OF THE PROCEDURE, #5.)
Microbiology, Washington, D.C.
4. Wash off the mordant with decolorizer/counterstain (3-Step Gram
6. Murray, P. R. (ed.). 1995. Manual of Clinical Microbiology, 6th
Safranin-S or 3-Step Gram Safranin-T). (NOTE: Do not wash off
ed. American Society of Microbiology, Washington, D.C.
iodine with water.) Add more decolorizer/counterstain solution to
the slide and stain 20-50 seconds. 7. Mangels, J. I., M. E. Cox, and L. H. Lindley. 1984. Methanol
fixation. An alternative to heat-fixation of smear. Diag. Microbiol.
5. Remove the decolorizer/counterstain solution by gently washing
Infect. Dis. 2:129-137.
the slide with cold tap water.
EFFECTS.EC Avoid contact with skin and eyes. Do not breathe mist. If inhaled, remove to fresh air. If not breathing, give artificial
Wear suitable protective clothing. Keep container tightly closed. respiration. If breathing is difficult, give oxygen. Seek medical
advice.
3314-TB Decolorizer TM
If swallowed seek medical advice immediately and show this
HIGHLY FLAMMABLE. CAUSES BURNS. Avoid contact with
container or label.
skin and eyes. Do not breathe mist. Wear suitable protective
clothing. Keep container tightly closed. Keep away from sources Storage
of ignition. No smoking. Store TB Stain Sets and reagents at 15-30°C. Reagents that have been
3315-TB Potassium Permanganate removed from the packing carton should be stored in the dark.
IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN. Expiration Date
Avoid contact with skin and eyes. Do not breathe mist. Wear suitable The expiration date applies to the product in its intact container when
protective clothing. Keep container tightly closed. stored as directed. Do not use a product if it fails to meet specifications
3316-TB Auramine M for identity and performance.
FLAMMABLE. IRRITATING TO EYES, RESPIRATORY SYS-
TEM AND SKIN. POSSIBLE RISK OF IRREVERSIBLE
Procedure
EFFECTS.US Avoid contact with skin and eyes. Do not breathe mist. Materials Provided
Wear suitable protective clothing. Keep container tightly closed. TB Stain Set K*
Keep away from sources of ignition. No smoking. TB Stain Set ZN*
TB Fluorescent Stain Set M*
3317-TB Auramine-Rhodamine T
TB Fluorescent Stain Set T*
FLAMMABLE. IRRITATING TO EYES, RESPIRATORY SYS- Bactrol™ TB Slides
TEM AND SKIN. TOXIC IN CONTACT WITH SKIN AND IF
*Individual reagents available separately. See Packaging.
SWALLOWED.EC CAUSES BURNS.EC POSSIBLE RISK OF
IRREVERSIBLE EFFECTS. Avoid contact with skin and eyes. Do Materials Required but not Provided
not breathe mist. Wear suitable protective clothing. Keep container Microscope slides–new or cleaned in acid dichromate solution
tightly closed. Keep away from sources of ignition. No smoking. Staining rack
3318-TB Decolorizer Microscope with oil immersion lens, OR
HIGHLY FLAMMABLE. HARMFUL BY INHALATION AND Fluorescent microscope
IF SWALLOWED. IRRITATING TO EYES, RESPIRATORY (See #8 of each fluorescent staining procedure for a complete
SYSTEM AND SKIN.US POSSIBLE RISK OF IRREVERSIBLE description of the appropriate assembly required.)
EFFECTS.US POSSIBLE RISK OF HARM TO THE UNBORN Specimen Collection and Preparation
CHILD.US Avoid contact with skin and eyes. Do not breathe mist. 1. Acid fast stains may be performed on any type of clinical specimen
Wear suitable protective clothing. Keep container tightly closed. suspected of containing mycobacteria.4-5 Smears from sputum and
Keep away from sources of ignition. No smoking. other respiratory tract secretions are usually made from concentrated
3319-TB Methylene Blue specimens. For procedures used in concentrating specimens for
FLAMMABLE. IRRITATING TO EYES, RESPIRATORY SYS- acid fast bacilli, please consult appropriate references.4-7
TEM AND SKIN. HARMFUL BY INHALATION AND IF 2. Apply a thin smear of the specimen directly on a clear microscope
SWALLOWED. POSSIBLE RISK OF IRREVERSIBLE slide.
EFFECTS.US Avoid contact with skin and eyes. Do not breathe 3. Allow smear to air dry.
vapors. Wear suitable protective clothing. Keep container tightly 4. Fix the smear to the slide by passing the slide through a low flame
closed. 2-3 times, avoiding excessive heat.
3321-TB Carbolfuchsin KF Test Procedure
FLAMMABLE. IRRITATING TO EYES, RESPIRATORY SYS- See appropriate references for specific procedures.
TEM AND SKIN. HARMFUL BY INHALATION AND IF
SWALLOWED. EC POSSIBLE RISK OF IRREVERSIBLE Kinyoun Stain
EFFECTS.US POSSIBLE RISK OF HARM TO THE UNBORN TB Stain Set K
CHILD.US Avoid contact with skin and eyes. Do not breathe mist. 1. Place slides on a staining rack and flood with TB Carbolfuchsin
Wear suitable protective clothing. Keep container tightly closed. KF for 4 minutes. Do not heat.
Keep away from sources of ignition. No smoking. 2 Wash gently in running water.
FIRST AID: 3. Decolorize with TB Decolorizer for 3-5 seconds, or until no more
In case of contact with eyes, rinse immediately with plenty of red color appears in washing.
water and seek medical advice. 4. Wash gently in running water.
After contact with skin, wash immediately with plenty of water. 5. Counterstain with either TB Brilliant Green K or TB Methylene
Blue (available separately) for 30 seconds.
6. Wash gently in running water. 2. Rapidly growing mycobacteria may retain acid-fast stains to a
7. Air dry. If using TB Methylene Blue, dry over gentle heat. varying degree. Most rapidly growing mycobacteria will not
fluoresce in fluorochrome-stained smears.4
Ziehl-Neelsen Stain
3. Organisms other than mycobacteria, such as Rhodococcus spp.,
TB Stain Set ZN Nocardia spp., Legionella micdadei, and the cysts of
1. Place slides on a staining rack and flood with TB Carbolfuchsin Cryptosporidium spp. and Isospora spp., may display various
ZN. Heat gently to steaming and allow to steam for 5 minutes. degrees of acid-fastness.4
2. Wash gently in running water. 4. When decolorizing with acid-alcohol, avoid under-decolorization.
3. Decolorize with TB Decolorizer for 3-5 seconds or until no more It is difficult to over-decolorize acid-fast organisms.
red color appears in washing. 5. During the counterstaining step with potassium permanganate,
4. Wash gently in running water. timing is critical. Quenching the fluorescing bacilli occurs when
5. Counterstain with either TB Methylene Blue or TB Brilliant counterstaining for a longer period of time.4
Green K for 30 seconds. 6. If fluorochrome stained slides cannot be observed immediately,
6. Wash gently in running water. they may be stored at 2-8°C in the dark for up to 24 hours. This is
7. Dry over gentle heat. required to prevent fading of the fluorescence.4
7. Prolonged counterstaining in non-fluorochrome stains may mask
Morse Stain the presence of acid-fast bacilli. Use of brilliant green may help to
Fluorescent Stain Set M minimize this problem.4
1. Place slides on a staining rack and flood with TB Auramine M for
15 minutes. References
2. Wash gently in running water. 1. Ziehl, F. 1882. Zur Färbung des Tuberkelbacillus. Dtsch. Med.
Wochenschr. 8:451.
3. Decolorize with TB Decolorizer TM for 30-60 seconds.
2. Neelsen, F. 1883. Ein Casuistischer Beitrag zur Lehre von der
4. Wash slides gently in running water.
Tuberkulose. Centralbl. Med. Wiss. 21:497-501.
5. Counterstain with TB Potassium Permanganate for 2 minutes.
3. National Tuberculosis Association. 1961. Diagnostic Standards
6. Wash gently in running water. and Classification of Tuberculosis. National Tuberculosis Associa-
7. Air dry. tion, New York, NY.
8. Examine under a microscope fitted, as described by Morse et al.,11 4. Master, R. N. 1992. Mycobacteriology, p. 3.0.1-3.16.4. In
with an incandescent bulb, a KG 1 heat filter, a 3-4 mm thick BG Isenberg, H. D. (ed.), Clinical 2microbiology procedures handbook,
excitation filter, an ordinary substage condenser and a No. 51 bright vol. 1. American Society for Microbiology, Washington, D.C.
field or GG barrier filter. 5. Nolte, F. S., and B. Metchock. 1995. Mycobacterium, p. 400-437.
Truant Stain In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H.
TB Fluorescent Stain Set T Yolken (eds.), Manual of clinical microbiology, 6th ed. American
1. Place slides on a staining rack and flood with TB Auramine- Society for Microbiology, Washington, D.C.
Rhodamine T that has been thoroughly shaken prior to use. Leave 6. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
undisturbed for 20-25 minutes at room temperature. Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
2. Wash gently in running water. St. Louis, MO.
3. Decolorize with TB Decolorizer TM for 2-3 minutes. 7. Kent, P. T., and G. P. Kubica. 1985. Public Health
Mycobacteriology: a guide for the Level III laboratory, p. 57-68.
4. Wash gently in running tap water.
U.S. Department of Health and Human Services, Centers for
5. Counterstain with TB Potassium Permanganate for 4-5 minutes. Disease Control, Atlanta, GA.
6. Wash gently in running water. 8. Taylor, R. D. 1966. Modification of the Brown and Brenn Gram
7. Blot lightly. Dry in air or very gently over a flame. Stain for the differential staining of gram-positive and gram-
8. Examine under a microscope fitted, as described by Truant et al.,13 negative bacteria in tissue sections. Am. J. Clin. Pathol. 46:472-4.
with 25X objective, an HBO L2 bulb heat filter, a BG 12 primary 9. Kinyoun, J. J. 1915. A note on Uhlenhuth’s method for sputum
filter and OG 1 barrier filter. examination for tubercle bacilli. Am. J. Pub. Health. 5:867-70.
Results 10. Kubica, G. P., and W. E. Dye. 1967. Laboratory methods for
Refer to appropriate references and procedures for results. clinical and public health Mycobacteriology. U.S.P.H. Serv.
Publication No., 1547; Superintendent of Documents, U.S.
Limitations of the Procedure Government Printing Office, Washington, D.C.
1. A positive staining reaction provides presumptive evidence of the 11. Fitzsimmons General Hospital. 1968. Mycobact. Lab. Methods.
presence of M. tuberculosis in the specimen. A negative staining Rept. No. 17., May, 1968.
reaction does not necessarily indicate that the specimen will be 12. Truant, J. P., W. A. Brett, and W. Thomas. 1962. Fluorescence
culturally negative for M. tuberculosis. For positive identification microscopy of tubercle bacilli stained with auramine and
of M. tuberculosis, cultural methods must be employed. rhodamine. Bull. Henry Ford Hosp. 10:287-296.
Bordetella Pertussis Antigen is a ready-to-use suspension of killed, are very small, white, glistening, convex, entire and usually exhibit
whole organisms adjusted to a density approximating two times a tiny zones of hazy hemolysis. Colonies of B. parapertussis are usually
McFarland Barium Sulfate Standard No. 3 (9 x 108 organisms per ml). larger than those of B. pertussis, may have a slightly brown color, and
Bordetella Pertussis Antigen contains 0.04% Thimerosal. When used do not have a glistening surface. For specific recommendations for
as described, each 5 ml vial contains sufficient reagent to perform culture and identification, consult appropriate references.3,5 Determine
approximately 140 slide tests. that a pure culture of the microorganism has been obtained and that
Because antigen density may vary, it is adjusted to ensure optimum biochemical test reactions are consistent with the identification of
performance when the antigen is standardized with hyperimmune sera the organism as Bordetella. After these criteria are met, serological
obtained from laboratory animals. identification can proceed.
7. Some Hemophilus species will grow on Bordetella isolation media Manual of clinical microbiology, 6th ed. American Society for
and may cross-react with B. pertussis antisera. Rule out X- and Microbiology, Washington, D.C.
V-factor dependence using Differentiation Disks V, X and VX.5 4. Wright, P. F. 1991. Pertussis in developing countries: definition
8. Shake the antigen vial well before use to obtain a smooth, uniform of the problem and prospects for control. Rev. Infect. Dis.
suspension. Occasionally, a Bordetella suspension may settle out 13:S228-S234.
during storage. 5. Pezzlo, M. 1992. Aerobic bacteriology, p. 1.0.1-1.20.47. In H. D.
9. Bordetella antigen will display irreversible autoagglutination if, at Isenberg (ed.) Clinical microbiology procedures handbook, vol. 1.
anytime during shipment or storage, it is subjected to freezing American Society for Microbiology, Washington, D.C.
temperatures. Do not allow to freeze. 6. Rose, N. R., H. Friedman, and J. L. Fahey (ed.). 1986. Manual
References of clinical laboratory immunology, 3rd ed. American Society for
1. Linneman, C. C., and E. B. Pery. 1977. Bordetella parapertussis: Microbiology, Washington, D.C.
recent experience and a review of the literature. Am. J. Dis. Child
131:560-563.
Packaging
Bordetella Pertussis Antiserum 1 ml 2309-50
2. Bass, J. W., and S. R. Stephenson. 1987. The return of pertussis.
Pediatr. Infect. Dis. J. 6:141-144. Bordetella Parapertussis Antiserum 1 ml 2310-50
3. Marcon, M. J. 1995. Bordetella, p. 566-573. In P. R. Murray, Bordetella Pertussis Antigen 5 ml 2585-56
E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.),
The rapid slide procedure is a screening test designed to detect contain approximately 2% packed cells and 20% glycerin, as well as
agglutinins. The tube test is a confirmatory procedure designed to 0.5% phenol and approximately 0.002% crystal violet and 0.005%
quantitate febrile agglutinins. It is, therefore, necessary that any brilliant green as preservatives. When used as described, each 5 ml vial
positive results obtained in the screening (slide test) of specimens be contains sufficient reagent for 20 slide tests.
confirmed by a tube test. The tube agglutination test is used clinically Brucella Abortus Antigen (Tube) is a ready-to-use suspension of
in the United States.9,10,11 Brucella abortus 1119-312 adjusted to a density approximating a
Certain organisms may share cross-reacting antigens leading to the McFarland Barium Sulfate Standard No. 3 (9 x 108 organisms per ml).
production of heterologous antibodies. These heterologous antibodies Brucella Abortus Antigen (Tube) contains 0.5% phenol as a preserva-
may react with one or more antigens in a febrile antibody test procedure, tive but does not contain dye. When used as directed, each 25 ml vial
causing low-level antibody titers that may not singly be indicative of contains sufficient reagent for 6 tests.
disease. Cross reactions between Brucella and Francisella tularensis, Because antigen density may vary, density is adjusted to ensure optimum
Yersinia enterocolitica and Vibrio cholerae can occur. performance when the antigen is standardized with hyperimmune sera
obtained from laboratory animals. Variation in antigen color intensity
Principles of the Procedure is normal and will not affect the outcome of the test.
Agglutination tests involving the use of Brucella antigens detect the
presence of antibodies that react with the test antigen. The serological Brucella Abortus Antiserum is a lyophilized, polyclonal rabbit anti-
procedure involves serially diluting the patient serum and then adding serum containing approximately 0.04% Thimerosal as a preservative.
a standard volume of antigen. The endpoint of the test is the last dilu- Brucella Abortus Antiserum is unabsorbed. Serological cross-reactions
tion of the serum that shows a specific amount of agglutination. The occur in unabsorbed sera from Brucella species because B. abortus, B.
end point, reported as a dilution of the serum, is called the patient’s suis and B. melitensis are antigenically related, containing common A
antibody “titer.” (abortus) and M (melitensis) substances. (Monospecific sera prepared
by absorption produce weak, unstable reagents that make interpretation
Reagents of agglutination results difficult.)
Brucella Abortus Antigen (Slide), Brucella Melitensis Antigen When rehydrated and used as described, each 3 ml vial of Brucella
(Slide) and Brucella Suis Antigen (Slide) are ready-to-use, chemically Abortus Antiserum contains sufficient reagent for 19 slide tests or
inactivated and stabilized suspensions of Brucella abortus 1119-3,12 30 tube tests.
Brucella melitensis and Brucella suis, respectively. The slide antigens
Febrile Negative Control is a standard protein solution containing
approximately 0.04% Thimerosal as a preservative. When used as
User Quality Control described, each 3 ml vial contains sufficient reagent for 32 slide tests.
Identity Specifications Precautions
Brucella Abortus Antigen (Slide), Brucella Suis Antigen 1. For In Vitro Diagnostic Use.
(Slide), Brucella Melitensis Antigen (Slide) 2. Brucella Abortus Antiserum
Appearance: Turquoise, blue-violet suspension.
The Packaging of This Product Contains Dry Natural Rubber.
Brucella Abortus Antigen (Tube) 3. Observe universal blood and body fluid precautions in the
Appearance: Light gray to white suspension. handling and disposing of specimens.13,14
Brucella Abortus Antiserum 4. Brucella Antigens are not intended for use in the immunization of
Lyophilized Appearance: Light gold to amber, button to humans or animals.
powdered cake. 5. Follow proper established laboratory procedure in handling and
Rehydrated Appearance: Light gold to amber, clear liquid. disposing of infectious materials.
Febrile Negative Control
Lyophilized Appearance: Colorless to light gold, button to Storage
powdered cake. Store Brucella Antigens (Slide) and (Tube) at 2-8°C.
Rehydrated Appearance: Colorless to light gold, clear liquid. Store lyophilized and rehydrated Brucella Abortus Antiserum at 2-8°C.
Performance Response Store lyophilized and rehydrated Febrile Negative Control at 2-8°C.
Rehydrate Brucella Abortus Antiserum and Febrile Negative
Control per label directions. Perform the slide or tube Expiration Date
agglutination test using Brucella Abortus Antigen (Tube), The expiration date applies to the product in its intact container when
Brucella Abortus (Slide), Brucella Suis (Slide), or Brucella stored as directed. Do not use a product if it fails to meet specifications
Melitensis (Slide). Dilute both positive and negative controls
in the same proportion as the patient’s serum and process in for identity and performance.
the same manner, following appropriate procedure.
Procedure
An antigen is considered satisfactory if it does not agglutinate
with the negative control and yields a 2+ reaction at a titer of Materials Provided
1:80 or more with the positive control. Brucella Abortus Antigen (Slide)
Brucella Suis Antigen (Slide)
Brucella Melitensis Antigen (Slide) 2. Positive control: Using a 0.2 ml serological pipette, dispense 0.08,
Brucella Abortus Antigen (Tube) 0.04, 0.02, 0.01 and 0.005 ml of Brucella Abortus Antiserum into a
Brucella Abortus Antiserum row of squares on the agglutination slide.
Febrile Negative Control 3. Negative control: Using a 0.2 ml serological pipette, dispense 0.08,
0.04, 0.02, 0.01 and 0.005 ml of Febrile Negative Control into a
Materials Required But Not Provided row of squares on the agglutination slide.
Slide Test 4. Antigen: Shake the vial of Brucella Antigen (Slide) well to ensure
Agglutination slides with 5 squares a smooth, uniform suspension. Dispense 1 drop (35 µl) of antigen
Applicator sticks in each drop of test serum, positive control and negative control.
Sterile 0.85% NaCl solution 5. Mix the rows of test and control serum, using a separate applicator
Serological pipettes, 0.2 ml stick for each row. Start with the most dilute mixture (0.005 ml)
Distilled or deionized water and work to the most concentrated (0.08 ml).
Tube Test 6. Rotate the slide for 1 minute and read for agglutination.
Culture tubes, 12 x 75 mm, and rack 7. The final dilutions in squares 1-5 correspond to tube dilutions of
Waterbath, 35-37°C 1:20, 1:40, 1:80, 1:160 and 1:320, respectively.
Serological pipettes, 1 ml and 5 ml
Tube Test
Sterile 0.85% NaCl solution
Distilled or deionized water 1. In a rack, prepare a row of 8 culture tubes (12 x 75 ml) for each test
serum, including a positive control row for the Brucella Abortus
Reagent Preparation Antiserum and an antigen control row for the Febrile Negative
Brucella Abortus Antigen (Slide) and (Tube), Brucella Suis Antigen Control Serum.
(Slide) and Brucella Melitensis Antigen (Slide) are ready to use. 2. Dispense 0.9 ml of sterile 0.85% NaCl solution in the first tube of
each row and 0.5 ml in the remaining tubes.
Equilibrate all materials to room temperature prior to performing
the tests. Ensure that all glassware and pipettes are clean and free of 3. Test serum: Using a 1 ml serological pipette, dispense 0.1 ml of
detergent residues. serum in the first tube in the row and mix thoroughly. Transfer
0.5 ml from tube 1 to tube 2 and mix thoroughly. Similarly, continue
Brucella Abortus Antiserum: To rehydrate, add 3 ml sterile 0.85% transferring 0.5 ml through tube 7, discarding 0.5 ml from tube 7
NaCl solution and rotate gently to completely dissolve the contents. after mixing. Proceed in like manner for each serum to be tested.
The rehydrated antiserum is considered a 1:2 working dilution. Tube 8 is the antigen control tube and contains only sterile 0.85%
Febrile Negative Control: To rehydrate, add 5 ml sterile distilled or NaCl solution.
deionized water and rotate gently to completely dissolve the contents. 4. Positive control: Using a 1 ml serological pipette, dispense 0.1 ml
Specimen Collection and Preparation of Brucella Abortus Antiserum in the first tube in the row and mix
thoroughly. Transfer 0.5 ml from tube 1 to tube 2 and mix
Collect a blood specimen by aseptic venipuncture. After the specimen thoroughly. Similarly, continue transferring 0.5 ml through tube 7,
has clotted, centrifuge to obtain the serum required for the test. Serum discarding 0.5 ml from tube 7 after mixing. Tube 8 is the antigen
specimens must be clear, free of hemolysis and show no visible control tube and contains only sterile 0.85% NaCl solution.
evidence of bacterial contamination (turbidity, hemolysis or particulate
5. Antigen Control: Shake the vial of Brucella Abortus Antigen
matter). Consult appropriate references for more information on the
(Tube) to ensure a smooth, uniform suspension. Add 0.5 ml of
collection of specimens.15,16
antigen to all 8 tubes in each row and shake the rack to mix the
Store serum specimens at room temperature for no longer than 4 hours; suspensions.
for prolonged storage, keep at 2-8°C for up to 5 days or maintain below 6. Final dilutions in tubes 1-7 are 1:20, 1:40, 1:80, 1:160, 1:320, 1:640
-20°C. Serum specimens must not be heated; heat may inactivate or and 1:1280, respectively.
destroy certain antibodies.
7. Incubate in a waterbath at 35-37°C for 48 ± 3 hours.
Slide Test 8. Remove from the waterbath. Avoid excessive shaking before
Use the slide test only as a screening test. Confirm positive results with reading the reactions, either when the tubes are in the waterbath
the tube test. or when removing them from the waterbath.
In some cases of brucellosis, sera may display a prozone reaction, the 9. Read and record results.
inability of an antigen to react at higher serum antibody concentrations. Results
It is advisable to run all 5 serum dilutions of the rapid slide test, rather 1. Read and record results as follows.
than just one dilution, to eliminate the possibility of missing positive
reactions due to the prozone phenomenon. 4+ 100% agglutination; background is clear to slightly hazy.
3+ 75% agglutination; background is slightly cloudy.
1. Test serum: Using a 0.2 ml serological pipette, dispense 0.08, 0.04,
2+ 50% agglutination; background is moderately cloudy.
0.02, 0.01 and 0.005 ml of each test serum into a row of squares on
the agglutination slide. 1+ 25% agglutination; background is cloudy.
– No agglutination.
2. Positive control: Should produce 2+ or greater agglutination at a antibody. A serum specimen with a prozone reaction shows no
1:80 dilution. agglutination because of excessively high antibody concentrations.
Negative control–Rapid Slide Test, only: Should produce no To avoid this occurrence, all 5 slide test serum dilutions should be run.
agglutination. The detection of antibodies in serum specimens may complete the
Antigen control–Macroscopic Tube Test, only: Should show no clinical picture of brucellosis. However, isolation of the causative
agglutination in tube #8 of each row. agent from patient specimens may be required. A definitive
If results for either the positive or the negative control are not as diagnosis must be made by a physician based on patient history,
specified, the test is invalid and results cannot be reported. physical examination and data from all laboratory tests.
Test serum: The titer is the highest dilution that shows 2+ 3. The accuracy and precision of the tests can be affected not only by
agglutination. test conditions but also by the subjectivity of the person reading
Refer to Table 1 and Table 2 for examples of test reactions. the endpoint.
3. The Rapid Slide Test is a screening test, only; results must be con- 4. Cross-reactions may occur due to antigenic similarities to other
firmed using the Macroscopic Tube Test. organisms. A definite serological relationship exists between
Brucella and Francisella tularensis. Cross-reactions may also
Table 1. Sample Rapid Slide Test reactions. occur between Brucella-positive sera and Proteus OX19 antigen,
Vibrio cholerae or Yersinia enterocolitica serotype 9.20
REACTIONS 5. While a single serum specimen showing a positive reaction at a
CORRELATED
SERUM (ml) TUBE DILUTION SPECIMEN 1 SPECIMEN 2 SPECIMEN 3
1:80 dilution suggests infection, it is not diagnostic. An antibody
0.08 1:20 3+ 4+ 4+ titer greater than 1:160 may occur in healthy individuals with a
0.04 1:40 2+ 4+ 3+ past history of the disease.
0.02 1:80 1+ 3+ 2+ 6. To test for a significant rise in antibody titer, at least two specimens
0.01 1:160 – 3+ + are necessary, an acute specimen obtained at the time of initial
0.005 1:320 – 1+ – symptoms and a convalescent specimen obtained 7 to 14 days later.
Serum titer 1:40 1:160 1:80 A two-dilution increase in titer is significant and suggests infection.
7. Prolonged exposure of reagents to temperatures other than those
specified is detrimental to the products.
Table 2. Sample Macroscopic Tube Test reactions. 8. Exposure to temperatures below 2°C can cause autoagglutination.
REACTIONS
Antigens must be smooth, uniform suspensions. Examine antigen
SERUM DILUTION SPECIMEN 1 SPECIMEN 2 SPECIMEN 3 vials for agglutination before use. Agglutinated suspensions are
not usable and should be discarded.
1:20 4+ 3+ 4+
9. Adhering to the recommended time and temperature of incubation
1:40 4+ 2+ 4+
is important when performing the tube test. For best results, locate
1:80 3+ 1+ 4+ the waterbath in an area free of mechanical vibration.
1:160 2+ – 4+
10. Serum specimens from patients suffering from acute brucellosis
1:320 1+ – 3+ demonstrate little or no antibody titer during the first 10 days of
1:640 – – 2+ the disease.
1:1280 – – 1+ 11. Serological interpretation of an agglutinin titer in vaccinated indi-
Serum titer 1:160 1:40 1:640 viduals should be avoided since antibody levels may persist for
years.
Interpretation 12. Individuals who have recovered from brucellosis may demonstrate
For a single serum specimen, a titer of 1:80 is a weak positive that a nonspecific agglutinin response upon infection with an etiologi-
suggests infection, but not necessarily a recent infection.3,17 cal agent of a heterologous febrile species.
A two-dilution increase in the titer of paired serum specimens (from the References
acute to the convalescent serum) is significant and suggests infection. 1. Moyer, N. P., and L. A. Holcomb. 1988. Brucellosis, p. 143-154.
A one-dilution difference is within the limits of laboratory error. In A. Balows, W. J. Hausler, Jr., M. Ohashi, and A. Tubano (ed.),
Past history in the use of Brucella suspensions has produced a pattern Laboratory diagnosis and infectious diseases: principles and
of titers that are considered “significant”. A titer of 1:80 is considered practice, vol. 1. Springer and Verlag, New York, NY.
a weakly positive result while most patients with acute undulant fever 2. Smith, L. D., and T. A. Ficht. 1990. Pathogenesis of Brucella.
demonstrate a titer of 1:160 or greater. Crit. Rev. Microbiol. 17:209-230.
Limitations of the Procedure18,19 3. Moyer, N. P., and L. A. Holcomb. 1995. Brucella, p. 549-555. In
P, R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H.
1. The slide test is intended for screening only and results should be
Yolken (ed.), Manual of clinical microbiology, 6th ed. American
confirmed by the tube test. Slide test dilutions are made to detect a
Society for Microbiology, Washington, D. C.
prozone reaction and do not represent true quantitation of the
4. Potasman, I., L. Even, M. Banai, E. Cohen, D. Angel, and 14. Occupational Safety and Health Administration, U.S.
M. Jaffe. 1991. Brucellosis: an unusual diagnosis for a sero- Department of Labor. 1991. 29 CFR part 1910. Occupational
negative patient with abscesses, osteomyelitis, and ulcerative exposure to bloodborne pathogens; final rule. Federal Register
colitis. Rev. Infect. Dis. 13:1039-1042. 56:64175-64182.
5. Young, E. J. 1983. Human brucellosis. Rev. Infect. Dis. 5:821-842. 15. Pezzlo, M. 1992. Aerobic bacteriology, p. 1.0.1-1.20.47. In H. D.
6. Arnow, P. M., M. Smaron, and V. Ormiste. 1984. Brucellosis in Isenberg (ed.), Clinical microbiology procedures handbook, vol.
a group of travelers to Spain. J. Am. Med. Assoc. 251:505-507. 1. American Society for Microbiology, Washington, D.C.
7. Centers for Disease Control. 1983. Brucellosis - Texas. Morbid. 16. Miller, J. M., and H. T. Holmes. 1995. Specimen collection, transport
Mortal. Weekly Rep. 32:548-553. and storage. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology,
8. Rose, N. R., H. Friedman, and J. L. Fahey, (ed.). 1986. Manual
6th ed. American Society for Microbiology, Washington, D.C.
of clinical immunology, 3rd ed. American Society for Microbiology,
Washington, D. C. 17. Hausler, W. J. Jr., and F. P. Knontz. 1970. Brucellosis. In H. L.
Bodily, E. L. Updyke, and J. O. Mason (ed.), Diagnostic
9. Moyer, N. P., G. M. Evans, N. E. Pigott, J. D. Hudson, C. E. procedures for bacterial, mycotic and parasitic infections, 5th ed.
Farshy, J. C. Feeley, and W. J. Hausler, Jr. 1987. Comparison of American Public Health Association, New York, NY.
serologic screening tests for brucellosis. J. Clin. Microbiol.
18. Alton, G. G., L. M. Jones, and D. E. Peitz. 1975. Laboratory
25:1969-1972.
techniques in brucellosis. World Health Organization Monogr.
10. Peiris, V., S., Fraser, M. Fairhurst, D. Weston, and Ser No. 55.
E. Kaczmarski. 1992. Laboratory diagnosis of Brucella infection:
19. McCullough, N. D. 1976. Immune response to Brucella, p. 304-
some pitfalls. Lancet 339:1415.
311. In N. R. Rose and H. Friedman (ed.), Manual of clinical
11. Young, E. J. 1991. Serologic diagnosis of human brucellosis: immunology. American Society for Microbiology, Washington, D.C.
analysis of 214 cases by agglutination tests and review of the
20. Ahovnen, P., E. Jansson, and K. Aho. 1969. Marked cross-
literature. Rev. Infect. Dis. 13:359-372. agglutination between brucellae and a subtype of Yersinia
12. Spink, W. W., N. D. McCullough, L. M. Hutchings, and C. K. enterocolitica. Acta Pathol. Microbiol. Scand. 75:291-295.
Mingle. 1954. A standardized antigen for agglutination technique
for human brucellosis. Report No. 3 of the National Research Packaging
Council, Committee on Public Health Aspects of Brucellosis. Am. Brucella Abortus Antigen (Slide) 5 ml 2909-56
J. Patho. 24:496-498. Brucella Abortus Antigen (Tube) 25 ml 2466-65
13. Centers for Disease Control. 1988. Update: universal precautions Brucella Melitensis Antigen (Slide) 5 ml 2916-56
for prevention of transmission of human immunodeficiency virus,
hepatitis B virus, and other bloodborne pathogens in health-care Brucella Suis Antigen (Slide) 5 ml 2915-56
settings. Morbidity and Mortality Weekly Reports 37:377-382, Brucella Abortus Antiserum 3 ml 2871-47
387-388. Febrile Negative Control 3 ml 3239-56
Intended Use germination upon seeding tissue from the bloodstream,3 protease
Bacto Candida Albicans Antiserum is used in the slide agglutination production,4 complement protein-binding receptor,5,6 surface variation
test for identifying Candida albicans. and hydrophobicity.7
Candida albicans will grow on Sabauroud Dextrose Agar as white to
Summary and Explanation cream-colored, butyrous colonies. C. albicans can be isolated from
Candida albicans is an opportunistic pathogen. Infection with this blood agar as a colony with short marginal extensions. Microscopically,
organism will usually arise from an endogenous source in a compromised C. albicans produces budding yeast cells, pseudohyphae or true
host. Candidiasis caused by C. albicans presents as superficial infections hyphae. The organism may be identified by the production of germ
of the skin, oral thrush, systemic and disseminated infections involving tubes or chlamydospores. Identification of Candida albicans includes
most internal organs, and mucocutaneous candidiasis.1 Vaginitis caused both biochemical and serological confirmation.8
by C. albicans is the most common type of yeast infection.
C. albicans, a saprophyte, appears in large numbers throughout the Principles of the Procedure
oral-gastrointestinal tract of many warm-blooded vertebrates.1 It is Serological confirmation requires that the microorganism (antigen)
rarely isolated from normal skin. Person-to-person transmission of react with its corresponding antibody. This in vitro reaction produces
candidiasis can occur. macroscopic clumping called agglutination. The desired homologous
Candida albicans appears to possess many virulence attributes that reaction is rapid, does not dissociate (has high avidity), and bonds
may promote successful parasitism. These attributes include rapid strongly (has high affinity).
3. Rough culture isolates do occur and will agglutinate spontaneously, 5. Calderone, R. A., L. Linehan, E. Wadsworth, and A. L.
causing agglutination of the negative control (autoagglutination). Sandberg. 1988. Identification of C3d receptors on Candida
Smooth colonies must be selected and tested in serological procedures. albicans. Infect. Immun. 252-258.
4. Agglutination reactions of 3+ or greater in the slide test are 6. Gilmore, B. J., E. M. Retsinas, J. S. Lorenz, and M. K.
interpreted as positive reactions. Cross-reactions resulting in a 1+ Hostetter. 1988. An iC3b receptor on Candida albicans:
or 2+ agglutination are likely since somatic antigens are shared structure, function, and correlates for pathogenicity. J. Infect.
among such organisms as Candida tropicalis, Candida kefyr and Dis. 257:38-46.
Candida stellatoidea. 7. Hazen, K. C., and P. M. Glee. Cell surface hydrophobicity and
5. Prolonged exposure of reagents to temperatures other than those medically important fungi. Curr. Top. Med. Mycol., in press.
specified is detrimental to the products. 8. Rosenthal, S. A., and D. Furnari. 1958. Slide agglutination as a
6. Discard any Candida Albicans Antiserum that is cloudy or has a presumptive test in the laboratory diagnosis of Candida albicans.
precipitate after rehydration or storage. J. Invest. Derm. 31:251-253.
References 9. Warren, N. G., and K. C. Hazen. 1995. Candida, Cryptococcus,
1. Ahearn, D. G., and R. L. Schlitzer. 1981. Yeast Infections, and other yeasts of medical importance. In P. R. Murray,
p. 991-1012. In A. Balows, and W. J. Hausler (ed.), Diagnostic E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.),
procedures for bacterial, mycotic and parasitic infections, 6th ed. Manual of clinical microbiology, 6th ed. American Society for
American Public Health Association, Washington, D.C. Microbiology, Washington, D.C.
2. Odds, F. C. 1988. Candida and candidosis, 2nd ed. Bailliere 10. Land, G. A. 1992. Mycology, p. 6.0.1.-6.12.4. In H. D. Isenberg
Tindall, London, England. (ed.), Clinical microbiology procedures handbook, vol. 1. American
3. Hazen, K. C., D. O. Brawner, M. H. Riesselman, J. E. Cutler, Society for Microbiology, Washington, D.C.
and M. A. Jutila. 1991. Differential adherence of hydrophobic and 11. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
hydrophilic Candida albicans yeast cells to mouse tissues. Infect. Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
Immun. 59:907-912. St. Louis, MO.
4. Kwon-Chung, K. J., D. Lehman, C. Good, and P. T. Magee.
1985. Genetic evidence for the role of extracellular proteinase in
Packaging
virulence of Candida albicans. Infect. Immun. 49:571-575. Candida Albicans Antiserum 3 ml 2281-47
of many warm-blooded vertebrates.16 It is rarely isolated from normal Principles of the Procedure
skin. Person-to-person transmission of candidiasis can occur. Usually, Coagulase Detection
candidiasis caused by C. albicans is endogenous in origin and develops S. aureus produces two types of coagulase, free and bound. Free co-
with stress or debilitation of the host.16 agulase is an extracellular enzyme produced when the organism is cul-
C. albicans is the species most commonly isolated from patients with tured in broth. Bound coagulase, also known as the clumping factor,
nearly all forms of candidiasis.17 This organism is an opportunistic patho- remains attached to the cell wall of the organism.
gen and appears to possess many virulence attributes that may promote In the tube test, free coagulase liberated from the cell acts on prothrombin
successful parasitism. These attributes include rapid germination in the coagulase plasma to give a thrombin-like product. This product
upon seeding tissue from the bloodstream,18 protease production,19 then acts on fibrinogen to form a fibrin clot.3
complement protein-binding receptor,20,21 and surface variation and
The tube test is performed by mixing an overnight broth culture or
hydrophobicity.22
colonies from a noninhibitory agar plate into a tube of rehydrated
C. albicans will grow on Sabouraud Dextrose Agar as white to cream coagulase plasma. The tube is incubated at 37°C. The formation of a
colored, creamy colonies. It can be isolated from blood agar as a colony clot in the plasma indicates coagulase production.
with short marginal extensions. Microscopically, C. albicans produces
budding yeast cells, pseudohyphae or true hyphae. Germ Tube Development
The germ tube test involves suspending suspected colonies of yeast in
One of the simplest and most valuable tests for the rapid presumptive a tube of Coagulase Plasma. The tube is incubated at 37°C for 2-4
identification of C. albicans is the germ tube test.23 Smith and Elliott hours. The cells are then observed microscopically for short, hyphal
recommended the use of rabbit coagulase plasma.2 The test is considered extensions from the yeast cells called germ tubes. Germ tubes are eas-
presumptive because not all isolates of C. albicans will be germ ily differentiated from blastoconidial germination; germ tubes have no
tube-positive and false positives may be obtained despite well-trained constriction at their juncture with the yeast cell while blastoconidial
staff.24 Ferrigno, Ramirez and Robison recommended testing for germ germination does produce a constriction. C. albicans usually produces
tube production with citrated plasma.25 germ tubes under specified test conditions within 2 hours. Other species
of Candida do not produce germ tubes, except for an occasional
isolate of Candida tropicalis.4
User Quality Control
Reagents
Identity Specifications Coagulase Plasma is lyophilized rabbit plasma to which sodium
Coagulase Plasma citrate has been added as the anticoagulant.
Lyophilized Appearance: Off-white to cream colored, dried
button or fluffy powder. Coagulase Plasma EDTA is lyophilized rabbit plasma to which EDTA
Rehydrated Appearance: Off-white to cream to light rose (ethylenediaminetetraacetic acid) has been added as the anticoagulant.
colored, opaque liquid. EDTA is not utilized by bacteria. Coagulase Plasma EDTA does not
give false-positive reactions with bacteria that utilize citrate.
Coagulase Plasma EDTA
Lyophilized Appearance: Off-white to cream colored, dried Precautions
button or fluffy powder. 1. For In Vitro Diagnostic Use.
Rehydrated Appearance: Off-white to cream to light rose 2. Follow proper established laboratory procedure in handling and
colored, opaque liquid. disposing of infectious materials.
Performance Response
Storage
Rehydrate Coagulase Plasma or Coagulase Plasma EDTA per
label directions. Perform the Coagulase Test or the Germ Tube Store unopened Coagulase Plasma and Coagulase Plasma EDTA at
Test procedure as described (see Test Procedure). 2-8°C.
GERM TUBE Store reconstituted plasma at 2-8°C for up to 5 days, or aliquot in 0.5 ml
ORGANISM ATCC® COAGULASE TEST DEVELOPMENT
amounts, freeze promptly and store at -20°C for up to 30 days. Do not
Staphylococcus aureus 25923* Clot in tube – thaw and refreeze.
Staphylococcus aureus 3647 Clot in tube –
Staphylococcus epidermidis 12228* No clot in tube – Expiration Date
Staphylococcus saprophyticus 15305 No clot in tube –
The expiration date applies to the product in its intact container when
Candida albicans 18804 – Germ tube
development stored as directed. Do not use a product if it fails to meet specifications
Candida tropicalis 750 – No germ tube for identity and performance.
development
Procedure
The cultures listed are the minimum that should be used for
performance testing. Materials Provided
*These cultures are available as Bactrol™ Disks and should be Coagulase Plasma
used as directed in Bactrol Disks Technical Information. Coagulase Plasma EDTA
Materials Required But Not Provided Alternatively, 2-4 colonies (1 loopful) taken directly from a
Bacteriological inoculating loop noninhibitory agar plate may be used as an inoculum instead of a
Sterile 1 ml pipettes broth culture.
Sterile Pasteur pipettes Germ Tube Development
Sterile serological pipettes, 1, 5, and 10 ml 1. Obtain a pure culture of the organism to be tested. Select
Incubator (37°C) well-isolated colonies grown on Sabouraud Dextrose Agar for
Sterile distilled or deionized water 48-72 hours.
Culture tubes, 12 x 75 mm
Timer Test Procedure
Waterbath (35-37°C) Coagulase Test
BHI broth or noninhibitory agar (Coagulase Detection) 1. Using a sterile 1 ml pipette, add 0.5 ml of rehydrated Coagulase
Sabouraud Dextrose Agar (Germ Tube Development) Plasma or Coagulase Plasma EDTA to a 12 x 75 mm test tube
supported in a rack.
Reagent Preparation
2. Using a sterile 1 ml serological pipette, add 2 drops of the overnight
Rehydrate Coagulase Plasma and Coagulase Plasma EDTA by adding broth culture of the test organism to the tube of plasma or, using a
sterile distilled or deionized water to the vial as indicated below. Mix sterile bacteriological loop, thoroughly emulsify 2-4 colonies
by gentle end-over-end rotation of the vial. (1 loopful) from a noninhibitory agar plate in the tube of plasma.
STERILE APPROXIMATE
PRODUCT SIZE DISTILLED WATER NUMBER OF TESTS 3. Mix gently.
3 ml 3 ml 6 4. Incubate in a waterbath at 35-37°C for up to 4 hours.
15 ml 15 ml 30 5. Examine the tube for coagulation hourly until a clot is evident or
25 ml 25 ml 50 until 4 hours have elapsed. If no clot has formed within 4 hours,
reincubate and examine after 24 hours.
Specimen Collection and Preparation Examine by gently tipping the tube. Avoid shaking or agitating the
1. Collect specimens or samples in sterile containers or with sterile tube, which could cause breakdown of the clot and, consequently,
swabs and transport immediately to the laboratory according to doubtful or false-negative test results.
recommended guidelines.1,3-8
6. Record results.
2. Process each specimen using procedures appropriate for that
sample.1,3-8 Germ Tube Test
1. Using a sterile 1 ml pipette, add 0.5 ml of the rehydrated Coagulase
Coagulase Detection Plasma (citrated) to a 12 x 75 mm test tube in a rack.
1. Obtain a pure culture of the organism to be tested. Select well- 2. Touch the tip of a sterile Pasteur pipette to a yeast colony growing
isolated colonies. on a Sabouraud Dextrose Agar plate.
2. Determine that the test culture has characteristics of S. aureus as 3. Gently emulsify the cells in the tube of rehydrated plasma.
listed below. Consult appropriate references for further identification
4. Incubate the mixture in a waterbath at 37°C for 2-4 hours.
of S. aureus.1,3-8
5. Examine 1 drop of the incubated mixture microscopically for
Morphology (media dependent):
germ tubes.
Blood Agar Base Opaque, yellow to orange,
w/5% Sheep Blood with hemolysis. 6. Record results.
DNase Test Agar Clearing of green dye. Results
w/Methyl Green
Mannitol Salt Agar Yellow to orange, surrounded Coagulase Test
by yellow zones. Any degree of clotting in Coagulase Plasma or Coagulase Plasma
Staphylococcus Medium 110 Yellow to orange. EDTA is considered a positive test.
Tellurite Glycine Agar Black. Germ Tube Test
VJ Agar Black, surrounded by The development of short, lateral hyphal filaments (germ tubes) on the
yellow zones. individual yeast cells with no constriction at the point of attachment is
Baird Parker Agar Grey to black shiny colonies considered a positive test.
surrounded by zones of clearing.
Gram Stain: Gram-positive cocci occurring Limitations of the Procedure
in grape-like clusters or, 1. The slide agglutination technique for determining the coagulase
occasionally, in chains.
activity of staphylococci is not recommended because false-posi-
Catalase Test: Positive. tive reactions may occur with some strains when animal plasmas
Mannitol Fermentation: Positive. are used. In addition, spontaneous agglutination may occur when
3. Using a bacteriological loop, transfer a well-isolated colony from rough cultures are used. Because 10-15% of S. aureus isolates may
a pure culture into a tube of sterile Brain Heart Infusion broth. yield a negative result when this test is employed, all negative slide
Incubate for 18-24 hours or until a dense growth is observed. reactions must be confirmed by the tube test.
2. Some species of organisms utilize citrate in their metabolism and 13. Gregson, D. B., D. E. Low, M. Skulnick, and A. E. Simor. 1988.
will yield false-positive reactions for coagulase activity. Normally, Problems with rapid agglutination methods for identification of
this would not cause problems since the coagulase test is performed Staphylococcus aureus when Staphylococcus saprophyticus is
almost exclusively on staphylococci. However, it is possible that being tested. J. Clin Microbiol. 26:1398-1399.
bacteria which utilize citrate may contaminate Staphylococcus 14. Hinnebusch, C., D. Glenn, and D. A. Bruckner. 1992. Potential
cultures on which the coagulase test is being performed. These misidentification of Staphylococcus species when using rapid iden-
contaminated cultures may, upon prolonged incubation, give tification tests that detect clumping factor. Abstr. General Meeting,
false-positive results due to citrate utilization.3 Amer. Soc. Microbiol., C-485, p. 498. American Society for Mi-
3. When checking results of the Coagulase Test, observe tubes hourly crobiology, Washington, D.C.
during the first 4 hours of incubation. Some strains of S. aureus 15. Bayliss, B. G., and E. R. Hall. 1965. Plasma coagulation by
produce fibrinolysin, which may lyse clots. If the tubes are not organisms other than Staphylococcus aureus. J. Bacteriol.
read until 24 hours of incubation, reversion to a false-negative may 89:101-104.
occur.26 16. Ahearn, D. G., and R. L. Schlitzer. 1981. Yeast Infections,
4. Do not use plasmas if a heavy precipitate or clot has formed before p. 991-1012. In A. Balows and W. J. Hausler (ed.), Diagnostic
inoculation. procedures for bacterial, mycotic and parasitic infections, 6th ed.
American Public Health Association, Washington, D.C .
References
17. Odds, F. C. 1988. Candida and candidiasis, 2nd ed. Bailliere
1. Kloos, W. E., and T. L. Bannerman. 1995. Staphylococcus and
Tindall, London, England.
Micrococcus, p. 282-298. In P. R. Murray, P. R., E. J. Baron, M. A.
Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical micro- 18. Hazen, K. C., D. O. Brawner, M. H. Riesselman, J. E. Cutler,
biology, 6th ed. American Society for Microbiology, Washington, D.C. and M. A. Jutila. 1991. Differential adherence of hydrophobic and
hydrophilic Candida albicans yeast cells to mouse tissues. Infect.
2. Smith, R., and L. P. Elliott. 1983. Are there better ways to Immun. 59:907-912.
diagnose candidiasis? Diag. Med. May-June:91-93.
19. Kwon-Chung, K. J., D. Lehman, C. Good, and P. T. Magee.
3. Pezzlo, M. (ed.). 1992. Aerobic bacteriology, p. 1.0.0.-1.20.47. In 1985. Genetic evidence for the role of extracellular proteinase in
H. D. Isenberg (ed.), Clinical microbiology procedures handbook, virulence of Candida albicans. Infect. Immun. 49:571-575.
vol. 1. American Society for Microbiology, Washington, D.C.
20. Calderone, R. A., L. Linehan, E. Wadsworth, and A. L.
4. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey & Sandberg. 1988. Identification of C3d receptors on Candida
Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc., albicans. Infect. Immun. 56:252-258.
St. Louis, MO.
21. Gilmore, B. J., E. M. Retsinas, J. S. Lorenz, and M. K.
5. Association of Official Analytical Chemists. 1995. Official Hostetter. 1988. An iC3b receptor on Candida albicans: structure,
methods of analysis of AOAC International, 16th ed. AOAC function, and correlates for pathogenicity. J. Infect. Dis. 257:38-46.
International, Arlington, VA.
22. Hazen, K. C., and P. M. Glee. Cell surface hydrophobicity and
6. FDA Bacteriological Analytical Manual. 1995. 8th ed. AOAC medically important fungi. Curr. Top. Med. Mycol., in press.
International, Gaithersburg, MD.
23. Warren, N. G., and K. C. Hazen. 1995. Candida, Cryptococcus,
7. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compen- and other yeasts of medical importance, p. 723-737. In P. R.
dium of methods for the microbiological examination of foods, Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken
3rd ed. American Public Health Association, Washington, D.C. (ed.), Manual of clinical microbiology, 6th ed. American Society
8. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig. 1993. for Microbiology, Washington, D.C.
Pathogens in milk and milk products, p. 103-212. In R. T. Marshall 24. Dealler, S. F. 1991. Candida albicans colony identification in
(ed.), Standard methods for the examination of dairy products, 5 minutes in a general microbiology laboratory. J. Clin Microbiol.
16th ed. American Public Health Association, Washington, D.C. 29:1081-1082.
9. Loeb, L. 1903. The influence of certain bacteria on the coagulation 25. Ferrigno, R. G., J. M. Ramirez, and D. Robison. 1983. EDTA
of the blood. J. Med. Res. 10:407-419. interference in germ-tube production. Diag. Med. 6:10.
10. Kloos, W. E., and J. H. Jorgensen. 1985. Staphylococci, p. 143- 26. Kloos, W. E., and D. W. Lambe, Jr. 1991. Staphylococcus, p. 222-
153. In E. H. Lennette, A Balows, W. J. Hausler, Jr., and H. J. 237. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D.
Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiol-
Society for Microbiology, Washington, D.C. ogy, 5th ed. American Society for Microbiology, Washington, D.C.
11. Hall, G. S., K. Pratt, G. Woods, and C. C. Knapp. 1988. Differ-
entiation of Staphylococcus aureus from other Micrococcaceae: Packaging
comparison of Staphaurex and the slide coagulase test with the Coagulase Plasma 6 x 3 ml 0286-46
tube coagulase test. Lab. Med. 19:817-820. 6 x 15 ml 0286-86
6 x 25 ml 0286-66
12. Smith, S. M., and C. Berezny. 1986. Comparative evaluation of
identification systems for testing methicillin-resistant strains of Coagulase Plasma EDTA 6 x 3 ml 0803-46
Staphylococcus aureus. J. Clin Microbiol. 24:173-176. 6 x 15 ml 0803-86
6 x 25 ml 0803-66
These strains may be used for Quality Control. All cultures should be Materials Required But Not Provided
serologically validated before use. Veal Infusion Agar
Motility GI Medium
Test tubes (12 x 75 mm) or other suitable test tubes and rack 4. E. Coli H Antiserum H7: Prepare a 1:500 dilution by adding
Sterile 0.85% NaCl solution 0.2 ml of rehydrated antiserum, which is already a 1:2 working
Formalin dilution, to 49.8 ml of 0.85% NaCl solution.
1 ml serological pipettes 5. Pipette 0.5 ml of the antiserum dilution into a test tube.
McFarland Standard No. 3 6. Test antigen: Add 0.5 ml to the above dilution and shake well.
Waterbath (50 ± 2°C) The resulting antiserum dilution will be 1:1,000.
Method of Preparation 7. Incubate the tube in a 50 ± 2°C waterbath for 1 hour and read for
E. Coli Antiserum: To rehydrate, add 3 ml sterile 0.85% NaCl solution agglutination.
and rotate gently to dissolve contents completely. The rehydrated Results
antiserum is considered a 1:2 working dilution. Observe test results with indirect lighting against a dark background.
Tube Technique for O Antigen Titration Record as follows.
1. To prepare pure cultures of the test organism, plate the organism 4+ 100% agglutination of cells; supernatant fluid is clear to
on Veal Infusion Agar and incubate at 35 ± 2°C for 16-18 hours. very slightly hazy.
2. Suspend some growth from the solid medium in 0.85% NaCl 3+ 75% agglutination of cells; supernatant fluid is slightly
cloudy.
solution to give a homogeneous suspension.
2+ 50% agglutination of cells; supernatant fluid is moderately
3. Heat the bacterial suspension in a boiling water bath for 30-60 cloudy.
minutes. The culture should be homogeneous. Precipitation
1+ 25% agglutination of cells; supernatant fluid is cloudy.
indicates a rough culture and the suspension should be discarded.
± Less than 25% agglutination of cells.
4. Allow the suspension to cool; dilute with 0.85% NaCl solution to
a density approximating that of a McFarland Barium Sulfate – No agglutination.
Standard No. 3. E. Coli O157: Cultures showing 2+ or greater agglutination at a
5. Add formalin to a final concentration of 0.5% by volume. dilution of 1:320 or greater are considered positive.
6. In a rack, prepare a row of 8 culture tubes (12 x 75 mm) for each E. Coli H7: Tubes showing 2+ or greater agglutination are considered
test suspension positive.
7. Dispense 0.9 ml of 0.85% NaCl solution in the first tube of each
row and 0.5 ml in the remaining tubes. Limitations of the Procedure
8. E. Coli O Antiserum O157: Prepare serial dilutions using the 1. Final identification of E. coli O157 is based on biochemical
rehydrated antiserum, which is already at a 1:2 working dilution. reactions and the presence of the O antigen.
Dispense 0.1 ml of antiserum in the first tube in the row and mix 2. The test organism must be identified to at least the genus level and,
thoroughly. Transfer 0.5 ml from tube 1 to tube 2 and mix in some cases, to the species level biochemically before serotyping
thoroughly. Similarly, continue transferring 0.5 ml through tube 7, E. coli.
discarding 0.5 ml from tube 7 after mixing. Proceed in like manner 3. If the antiserum is cloudy after rehydration, check its bacterial
for each suspension to be tested. Tube 8 is the antigen control tube purity and the pH of the saline. Discard any serum that is cloudy
and contains only sterile 0.85% NaCl solution. and/or has a precipitate unless it has been clarified and shown to
This procedure yields antiserum dilutions of 1:20-1:1280. react properly with known control cultures.
9. Heated bacterial suspension: Add 0.5 ml to each of the 8 tubes. 4. Adhere strictly to the time limitations in both tests.
Final antiserum dilutions are 1:40-1:2560. 5. Exposure of the organism or plate to heat from external sources (a
8. Incubate in a waterbath at 50 ± 2°C for 18-20 hours. Read for hot bacteriological loop, burner flame, light source, etc.) may
agglutination. result in either a culture that cannot be suspended readily or evapo-
Tube Agglutination Technique for H Antigen Detection ration and/or precipitation of the test mixture. These conditions
may cause false-positive reactions.
1. Prepare an actively motile culture of the suspect E. coli culture by
several successive transfers in Motility GI Medium. At least 2-3 6. The test culture must be checked in a saline control for smoothness.
passages through Motility GI Medium are necessary before Stock cultures and, sometimes, isolated cultures may be rough and
attempting to establish the presence and identity of H antigens. will agglutinate spontaneously in a normal serum. Therefore, it is
Fresh isolates of E. coli generally have poorly developed flagella. necessary to select smooth colonies for serological testing.
2. Inoculate a loopful of the Motility GI Medium culture into a 7 In E. coli serology, as in any serological test, known positive and
tube of Veal Infusion Broth. Incubate 6-8 hours at 35 ± 2°C or negative control cultures should be employed.
overnight, if necessary. 8. Antisera should not be subjected to repeated freezing and thawing.
3. Inactivate the culture by adding formalin to a final concentration Such treatment is detrimental to antibody content.
of 0.3% (0.3 parts formaldehyde per 100 parts of the Veal Infusion References
Broth culture). If necessary, adjust the density of the suspension
1. Furowicz, A. J., and F. Orskov. 1972. Two new Escherichia coli
with formalinized saline to approximate a McFarland Barium
antigens, O150 and O157, and one new K antigen, K93, in strains
Sulfate Standard No. 3. This broth culture will be used as the
isolated from veterinary diseases. Acta. Pathol. Microbiol. Scand.
test antigen in step 6.
Sect. B. 80:441-444.
2. Centers for Disease Control. 1993. Emerging infectious diseases. 9. Marrier, R., J. G. Wells, R. C. Swanson, W. Callahan, and
Morb. Mort. Wkly. 42:257-260. I. J. Mehlman. 1973. An outbreak of enteropathogenic E. coli
3. Glass, K. A., J. M. Leffelholtz, J. P. Ford, and M. P. Doyle. 1992. foodborne disease traced to imported French cheese. Lancet
Fate of Escherichia coli O157:H7 as affected by pH or sodium 2:1376-1378.
chloride and in fermented, dry sausage. Appl. Environ. Microbiol. 10. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig.
58:2513-2516. 1993. Pathogens in milk and milk products, p. 168-176. In R. T.
4. Padhye, N. V., and M. P. Doyle. 1992. Escherichia coli O157:H7 Marshall (ed.), Standard methods for the examination of dairy prod-
epidemiology, pathogenesis, and methods for detection in food. ucts, 16th ed. American Public Health Association. Washington, D.C.
J. Food Prot. 55:555-565. 11. Morbidity and Mortality Weekly Reports. November, 1982.
5. Riley, L. W., R. S. Remis, and S. D. Helgerson, et al. 1983. 12. Lior, H. 1994. Escherichia coli O157:H7 and verotoxigenic
Hemorrhagic colitis associated with a rare Escherichia coli Escherichia coli (VTEC). Dairy, Food, and Environ. Sanit. 14:378-382.
serotype. N. Engl. J. Med. 308:681-685. 13. Hitchins, A. D., P. Feng, W. D. Watkins, S. R. Rippey, and
6. Samadpour, M., L. Grimm, B. Desai, D. Alfi, J. E. Ongerth, L. A. Chandler. 1995. Escherichia coli and the coliform bacteria,
and P. I. Tarr. 1993. Molecular epidemiology of Escherichia coli p. 4.01-4.29. FDA bacteriological analytical manual, 8th ed. AOAC
O157:H7 strains using bacteriophage A -restriction fragment length International, Arlington, VA.
polymorphism analysis: application to a multistate foodborne out- 14. Hitchins, A. D., P. A. Hartman, and E. C. D. Todd. 1992.
break and a day care center cluster. J. Clin. Microbiol. 31:3179-3183. Coliforms - E. coli and its toxins, p. 325-369. ln C. Vanderzant and
7. Tarr, P. I. 1994. Review of 1993 Escherichia coli O157:H7 D. F. Splittstoesser (ed.), Compendium of methods for the micro-
outbreak: western United States. Dairy, Food, and Environmental biological examination of foods, 3rd ed. American Public Health
Sanitation 14:372-373. Association, Washington, D.C.
8. Gray, L. D. 1995. Escherichia, Salmonella, Shigella, and Yersinia, Packaging
p. 450-455. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
E. Coli O Antiserum O157 3 ml 2970-47
Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology,
6th ed. American Society for Microbiology, Washington, D.C. E. Coli H Antiserum H7 3 ml 2159-47
4. All glassware employed in the preparation, testing and storage 7. Onorato, I. M., and S. G. F. Wassilak. 1987. Laboratory diagnosis
of these reagents must be free of detergents or other harmful of pertussis: the state of the art. Pediatr. Infect. Dis. J. 6:145-151.
residues. 8. Kendrick, P. L., Eldering, G., and W. C. Eveland. 1961. Fluores-
5. The fluorescent antibody technique can provide only presumptive cent antibody techniques. Am. J. Diseases Children 101:149-154.
identification of B. pertussis or B. parapertussis. A negative result 9. Eldering, G., W. C. Eveland, and P. L. Kendrick. 1962.
should not be considered conclusive as this type of reaction may Fluorescent antibody staining and agglutination reactions in
occur when only a few organisms are present in the specimen. Bordetella pertussis cultures. J. Bacteriol. 83:745-749.
Final identification can be made only after consideration of cultural,
10. Holwerda, J., and G. Eldering. 1963. Culture and fluorescent
morphological and serological characteristics.
antibody methods in diagnosis of whooping cough. J. Bacteriol.
References 86:449-451.
1. Linneman, C. C., and E. B. Pery. 1977. Bordetella parapertussis: 11. Gilchrist, M. J. R. 1991. Bordetella, p. 471-477. In A. Balows, W.
recent experience and a review of the literature. Am. J. Dis. Child J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy
131:560-563. (ed.), Manual of clinical microbiology, 5th ed. American Society
2. Bass, J. W., and S. R. Stephenson. 1987. The return of pertussis. for Microbiology, Washington, D.C.
Pediatr. Infect. Dis. J. 6:141-144. 12. Pezzlo, M. 1992. Aerobic bacteriology, p. 1.0.1-1.20.47. In H. D.
3. Marcon, M. J. 1995. Bordetella, p. 566-573. In P. R. Murray, E. J. Isenberg (ed.) Clinical microbiology procedures handbook, vol. 1.
Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), American Society for Microbiology, Washington, D.C.
Manual of clinical microbiology, 6th ed. American Society for 13. Centers for Disease Control. 1988. Update: universal precautions
Microbiology, Washington, D. C. for prevention of transmission of human immunodeficiency virus,
4. Wright, P. F. 1991. Pertussis in developing countries: definition hepatitis B virus, and other bloodborne pathogens in health-care
of the problem and prospects for control. Rev. Infect. Dis. settings. Morbidity and Mortality Weekly Reports 37:377-382,
13:S228-S234. 387-388.
5. Strebel, P. M., S. L. Cochi, K. M. Farizo, B. J. Payne, S. D. 14. Occupational Safety and Health Administration, U.S. Depart-
Hanauer, and A. L. Baughman. 1993. Pertussis in Missouri: ment of Labor. 1991. 29 CFR, part 1910. Occupational exposure
evaluation of nasopharyngeal culture, direct fluorescent antibody to bloodborne pathogens; final rule. Federal Register 56:64175-
testing, and clinical case definitions in the diagnosis of pertussis. 64182.
Clin. Infect. Dis. 16:276-285.
Packaging
6. Halperin, S. A., R. Bortolussi, and A. J. Wort. 1989. Evaluation
FA Bordetella Pertussis 5 ml 2359-56
of culture, immunofluorescence and serology for the diagnosis of
pertussis. J. Clin Microbiol. 27:752-757. FA Bordetella Parapertussis 5 ml 2378-56
time because of oxidation of the glycerol and absorption of CO2 by air. If not breathing, give artificial respiration. If breathing is diffi-
the mounting fluid.3 cult, give oxygen. Seek medical advice. If swallowed seek medical
The Staining Tray provides a moist, dark incubation chamber for advice immediately and show this container or label.
FA conjugated slides. 3. Follow proper established laboratory procedure in handling and
disposing of infectious materials.
Principles of the Procedure
Slides are stained using fluorescent antibody procedures. After Storage
removal of excess conjugate or serum, a drop of the appropriate Store dehydrated FA Buffer, Dried below 30°C. Upon rehydration, store
FA Mounting Fluid pH 7.2 or FA Mounting Fluid pH 9 is added. A FA Buffer, Dried at 2-8°C.
cover slip is applied, taking care not to form bubbles when the cover Store FA Mounting Fluid pH 7.2 and FA Mounting Fluid pH 9 at
slip is added. 15-30°C.
Streptococci are facultatively anaerobic gram-positive cocci. They are 1:5 4+*
catalase negative and may be alpha, beta or non-hemolytic. Lancefield 1:10 4+
divided the streptococci into serological groups according to the group-
specific somatic carbohydrate they possessed.1,2,3 The Lancefield groups 1:20 4+
have quite different clinical significance. There may be biochemical 1:40 4+
and hemolytic differences within the same serological group. 1:80 2+
Moody, Ellis and Updyke4 showed that group-specific conjugates could
be prepared from the antiserum used in the Lancefield precipitin test. *4+ fluorescence is defined as brilliant yellow-green cocci with sharp
This led to the development of the direct fluorescent antibody technique cell outlines and nonstaining centers.
for the identification of Streptococcus groups. The Group A conjugate, In this example, the last 4+ fluorescence is found in a 1:40 dilution of
prepared according to the method of Moody, Ellis and Updyke4 and the conjugate. Use one dilution lower for a margin of safety. The
Moody, Siegel, Pittman and Winter5 , is used for the detection and working dilution, therefore, in this case is 1:20.
identification of group A Streptococcus.6-8
Precautions
Principles of the Procedure 1. For In Vitro Diagnostic Use.
The direct FA technique involves the preparation of a smear from the 2. The Packaging of This Product Contains Dry Natural Rubber.
clinical specimen on a glass slide. Smears are ethanol-fixed and stained
3. Follow proper established laboratory procedures in handling and
with a specific antibody labeled with a fluorescent marker (fluorescein
disposing of infectious materials.
isothiocyanate or FITC) directed against Group A Streptococcus. The
antigen-antibody reaction is then observed microscopically, using a Storage
suitable wave length of light compatible with the fluorescent marker
Store lyophilized FA Streptococcus Group A at 2-8°C.
employed.
Aliquots of the titered conjugate should be prepared in small vials,
frozen in the undiluted state and stored below -20°C for optimal stability.
Prepare only a sufficient amount of diluted conjugate for each day’s use.
User Quality Control
Expiration Date
Identity Specifications
The expiration date applies to the product in its intact container when
FA Streptococcus Group A stored as directed. Do not use a product if it fails to meet specifications
Lyophilized Appearance: Yellow button to powdered cake. for identity and performance.
Rehydrated Appearance: Yellow, clear solution.
Performance Response Procedure
Rehydrate FA Streptococcus Group A per label directions. Materials Provided
Perform the fluorescent antibody staining procedure using a FA Streptococcus Group A
known culture of Group A Streptococcus as the homologous
control. The positive control should produce a 4+ reaction Materials Required but not Provided
in the 1:20 or greater dilution of the conjugate with the FA Buffer, Dried
homologous antigen. FA Mounting Fluid pH 9
3. Lancefield, R. C. 1938. A micro precipitin-technique for 8. Estela, L. A., and H. E. Shuey. 1963. Comparison of fluorescent
classifying hemolytic streptococci, and improved methods for antibody, precipitin, and bacitracin disk methods in the identification
producing antisera. Proc. Soc. Exp. Biol. Med. 38:473-478. of group A streptococci. Amer. J. Clin. Pathol. 40:591-597.
4. Moody, M. D., E. C. Ellis, and E. L. Updyke. 1958. Staining 9. Miller, J. M., and H. T. Holmes. 1995. Specimen collection,
bacterial smears with fluorescent antibody. IV. Grouping transport, and storage, p. 19-32. In P. R. Murray, E. J. Baron, M. A.
streptococci with fluorescent antibody. J. Bacteriol. 75:553-560. Pfaller, F. C. Tenover, R. H. Yolken (ed.), Manual of clinical microbi-
5. Moody, M. D., A. C. Siegel, B. Pitmann, and C. C. Winter. 1963. ology, 6th ed. American Society for Microbiology, Washington, D. C.
Fluorescent-antibody identification of group A streptococci from 10. Isenberg, H. D. (ed.) 1992. Clinical microbiology procedures hand-
throat swabs. Am. J. Publ. Hlth. 53:1083-1092. book, vol. 2. American Society for Microbiology, Washington, D. C.
6. Warfield, M. A., R. H. Page, W. W. Zuelzer, and C. S. Stulberg. Packaging
1961. Immunofluorescence in diagnostic bacteriology. II. Identifi- FA Streptococcus Group A 5 ml 2318-56
cation of Group A Streptococci in throat smears. Am. J. Dis. Child.
101:160-163. FA Buffer, Dried 6 x 10 ml 2314-33
100 g 2314-15
7. Peeples, W. J., D. W. Spielman, and M. D. Moody. 1961. Field 10 kg 2314-08
application of fluorescent antibody technique for identification of
FA Mounting Fluid pH 9 6x5 ml 3340-57
group A streptococcus. Pub. Health Rep. 76:651-654.
Staining Tray 1 tray 5251-31
diluted beyond their titer and could no longer interfere with the test. by the Centers for Disease Control and Prevention (CDC). Other standard
However, testing a highly diluted serum decreased the sensitivity of treponemal tests include Fluorescent Treponemal Antibody-Absorption
the test. Low antibody titer, which occurs during primary syphilis, was Double Staining Test (FTA-ABS DS) and the Micro Hemagglutination
not detected. Assay for Antibodies to Treponema pallidum (MHA-TP).
Deacon and Hunter6 showed that appropriate absorption could eliminate Treponemal antigen tests, such as the FTA-ABS test, are used as con-
or block the reactivity of nonspecific antibodies. This absorption pro- firmatory tests in diagnostic problem cases, such as with patients for
duced the FTA-ABS test, an improved test using a 1:5 serum dilution.7 whom the clinical, historical or epidemiological evidence of syphilis
The FTA-ABS test is a standard diagnostic test for syphilis as defined disagrees with nontreponemal tests. The FTA-ABS test is more
sensitive than the VDRL test in primary, late latent and tertiary syphilis.
However, the persistent reactivity of the FTA-ABS test to a treated
User Quality Control case of syphilis, sometimes for life, minimizes its use for following
Identity Specifications response to therapy. Therefore, the FTA-ABS test is also unreliable in
detecting new untreated cases in epidemiological investigations. The
FTA Antigen test should not be used as a routine screening procedure.3,8
Lyophilized Appearance: White button to powdered cake.
The likelihood of obtaining a reactive FTA-ABS test result in various
Rehydrated Appearance: White to off-white, slightly stages of untreated syphilis has been reported as follows:2
opalescent liquid.
STAGE OF UNTREATED SYPHILIS % REACTIVE
FTA Serum Reactive Primary 84
Lyophilized Appearance: Off-white to light amber, button to
powdered cake. Secondary 100
Rehydrated Appearance: Light gold to slightly amber liquid. Latent 100
Tertiary (Late) 96
FTA Serum Non-Reactive
Lyophilized Appearance: Off-white to light amber, button to Principles of the Procedure
powdered cake.
Patient serum is diluted 1:5 in sorbent and layered on a microscope
Rehydrated Appearance: Light gold to slightly amber liquid.
slide fixed with T. pallidum. If the patient’s serum contains antibodies,
FTA Sorbent these antibodies will coat the treponemes on the slide. Fluorescein-
Lyophilized Appearance: Light amber to dark brown, button labeled anti-human immunoglobulin is added. It combines with the
to powdered cake. patient antibodies already adhering to the T. pallidum and produces
Rehydrated Appearance: Gold to brown liquid. fluorescein-stained spirochetes that can be observed with a fluorescent
microscope.7,9
FTA Sorbent Control:
Lyophilized Appearance: Off-white to light amber, button Reagents
to powdered cake.
FTA Antigen (also known as T. pallidum antigen) is a lyophilized,
Rehydrated Appearance: Light gold to slightly amber liquid.
standardized, killed suspension of Treponema pallidum (Nichols
FTA Human Globulin Antiglobulin (Rabbit) strain).
Lyophilized Appearance: Light yellow to yellow-orange, FTA Serum Reactive is lyophilized, standardized syphilitic human
button to powdered cake.
sera containing 0.02% Thimerosal as a preservative. It is used to make
Rehydrated Appearance: Yellow-green to yellow-orange Reactive Control Serum (4+) - Unabsorbed, Reactive Control Serum
liquid.
(4+) - Absorbed, and Minimally Reactive Control Serum (1+). It is
Control Pattern used as a positive control in the FTA-ABS test.
Rehydrate and dilute reagents per directions (see Reagent FTA Serum Non-Reactive is lyophilized, standardized, non-syphilitic
Preparation). Test as described. Tests failing to exhibit the human sera containing 0.02% Thimerosal as a preservative. It is used
following control results are unsatisfactory and should not to make Nonreactive Control Serum (N). It is used as a negative
be reported.8,13 control in the FTA-ABS test.
EXPECTED
SERUM TESTED FLUORESCENCE INTERPRETATION
FTA Sorbent is a lyophilized, standardized extract of the nonpathogenic
Reactive Control Serum - Unabsorbed 4+ Reactive Reiters treponeme (T. phagedenis) prepared from broth culture. It is used
Reactive Control Serum - Absorbed 3+ to 4+ Reactive to remove antibodies against nonpathogenic treponemes during prepa-
Minimally Reactive Control Serum 1+ Reactive ration of the test specimen, Reactive Control Serum (4+) - Absorbed
Nonreactive Control Serum N Nonreactive and Nonspecific Staining Control - Absorbed.
Nonspecific Serum Control - Unabsorbed 2+ to 4+ Reactive FTA Sorbent Control is lyophilized, standardized, non-syphilitic
Nonspecific Serum Control - Absorbed N to ± Nonreactive human sera containing 0.02% Thimerosal as a preservative. It is used
Nonspecific Staining Control - Unabsorbed N Nonreactive to make Nonspecific Control Serum - Unabsorbed, which demonstrates
Nonspecific Staining Control - Absorbed N Nonreactive at least 2+ nonspecific reactivity at a 1:5 dilution in FA Buffer, and
Nonspecific Control Serum - Absorbed, which demonstrates essentially
no reactivity at a 1:5 dilution in FTA Sorbent.
FA Human Globulin Antiglobulin (Rabbit) is lyophilized, fluo- FTA Sorbent Control 2-8°C
rescein-conjugated (FITC) antihuman globulin containing 0.02% FA Human Globulin 2-8°C in the dark
Thimerosal as a preservative. It is used to show the presence of Antiglobulin (Rabbit)
human syphilitic antibodies on the treponemal antigen. Tween® 80 15-30°C
Tween® 80 is Polysorbate 80, U.S.P. It is used to prepare 2% Tween 80, FA Buffer, Dried Below 30°C
which acts as a dispersing agent. FA Mounting Fluid pH 7.2 15-30°C
FA Buffer, Dried is phosphate buffered saline (PBS) which, upon Expiration Date
rehydration, yields a 0.85% NaCl solution buffered to pH 7.2. FA The expiration date applies to the product in its intact container when
Buffer is used in preparing Reactive Control Serum (4+) - Unabsorbed, stored as directed. Do not use a product if it fails to meet specifications
Minimally Reactive Control Serum (1+), Nonreactive Control Serum for identity and performance.
(N) and Nonspecific Staining Control - Unabsorbed.
Rehydrated FTA Antigen stored at 2-8°C is stable for 1 week.
FA Mounting Fluid pH 7.2 is standardized, reagent grade glycerin
adjusted to pH 7.2 for use in mounting specimens on slides to be viewed Rehydrated FA Buffer showing turbidity or mold growth should be
under the fluorescent microscope. discarded.
Discard 2% Tween 80 that exhibits a precipitate or pH change.
Precautions
1. For In Vitro Diagnostic Use. Procedure
2. FTA Serum Reactive Materials Provided
FTA Serum Non-Reactive FTA Antigen
FTA Sorbent Control FTA Serum Reactive
WARNING! POTENTIAL BIOHAZARDOUS REAGENTS. FTA Serum Non-Reactive
Each donor unit used in the preparation of these reagents was tested FTA Sorbent
by an FDA-approved method for the presence of the antibody to FTA Sorbent Control
human immunodeficiency virus (HIV) as well as for hepatitis B sur- FA Human Globulin Antiglobulin (Rabbit)
face antigen and found to be negative (were not repeatedly reactive). Tween® 80
Because no test method can offer complete assurance that HIV, FA Buffer, Dried
hepatitis B virus or other infectious agents are absent, these reagents FA Mounting Fluid, pH 7.2
should be handled at the Biosafety Level 2 as recommended for
any potentially infectious human serum or blood specimen.10,11 Materials Required But Not Provided
Timer
3. FTA Antigen
Serological pipettes, 0.2 ml, 5 ml, 1 ml
FTA Serum Reactive
Micropipettors delivering 10-200 µl
FTA Serum Non-Reactive
FTA Sorbent Test tubes, 12 x 75 mm
FTA Sorbent Control Water bath (56°C)
FA Human Globulin Antiglobulin (Rabbit) Vortex mixer
The Packaging of This Product Contains Dry Natural Rubber. Platinum loop, 2 mm, 26 gauge
Slides, plain or frosted, 1 x 3 inch, 1 mm thick, inscribed with 2 x 1
4. FA Buffer, Dried cm circles
MAY BE IRRITATING TO EYES, RESPIRATORY SYSTEM Staining dish with removable slide carriers
AND SKIN. (US) Avoid contact with skin and eyes. Do not breathe Slide board or holder
dust. Wear suitable protective clothing. Keep container tightly closed. Moisture chamber
FIRST AID: In case of contact with eyes, rinse immediately with Acetone
plenty of water and seek medical advice. After contact with skin, Bibulous paper
wash immediately with plenty of water. If inhaled, remove to fresh Distilled water
air. If not breathing, give artificial respiration. If breathing is diffi- Incubator, 35-37°C
cult, give oxygen. Seek medical advice. If swallowed seek medical Oil, Immersion
advice immediately and show this container or label. Cover slips, No. 1, 22 mm square
5. Observe universal blood and body fluid precautions in handling Fluorescent microscope assembly:
and disposing of specimens. Lamps: HBO-50, HBO-100, HBO-200 or Xenon
6. Follow proper established laboratory procedure in handling and XBO-150; 6X 5A Tungsten
disposing of infectious materials. Ocular: 10X
Objective: 10X, 40X (Fluorite)
Storage Filters: BG-12 or KP490, K515 or K530
Store unopened products as specified below: Condenser: Dark-field D1.20-1.40
FTA Antigen 2-8°C
FTA Serum Reactive 2-8°C Reagent Preparation
FTA Serum Non-Reactive 2-8°C FTA Antigen: Rehydrate with 1 ml distilled or deionized water and
FTA Sorbent 2-8°C rotate to completely dissolve the contents. This solution will yield
approximately 3.5 x 107 treponemes per ml. Mix thoroughly with a 4. During further testing, use the dilution that produces 1 doubling
disposable pipette and rubber bulb, drawing the suspension into and dilution lower than the 4+ endpoint. The 4+ endpoint is the highest
expelling it from the pipette 8-10 times to break treponemal clumps dilution of conjugate yielding 4+ fluorescence with the Reactive
and ensure an even distribution of treponemes. Confirm the even Control Serum (4+).
distribution by dark-field examination. Use FTA Antigen in its entirety FA Buffer, Dried: Dissolve 10 grams in 1 liter of distilled or deionized
to prepare antigen smears on the day it is rehydrated. Approximately water and rotate gently to completely dissolve the contents. Store at
200-300 slides may be prepared with 1 ml of antigen. 2-8°C. Use the solution if it is free of mold growth and turbidity.
To prepare FTA Antigen smears: Tween® 80: Heat the bottle of Tween 80 and a flask containing 98 ml
1. Wipe inscribed slides with clean gauze and, if necessary, alcohol FA Buffer to 56°C in a water bath. Add 2 ml of Tween 80 to the buffer
to remove dust particles. and rinse the pipette thoroughly in the buffer. Check the pH and adjust
2. Using a platinum wire loop (2 mm, 26 gauge), smear 1 loopful of to pH 7.2 with 1N NaOH. Discard if a precipitate develops or the pH
reconstituted FTA Antigen within the 2 circles. Air dry at room changes.
temperature for at least 15 minutes. Specimen Collection and Preparation
3. Immerse the dry slide into acetone for 10 minutes to fix the Test serum: Collect patient (test) serum according to recommended
treponemal antigen smear to the slide; air dry. Fix no more than procedures.2,3,8,9,13 Store specimens at room temperature for up to
50 slides per 200 ml of acetone. 4 hours or at 2-8°C for up to 5 days; serum specimens may be frozen at
4. Use slides immediately or store at or below -20°C after acetone or below -20°C.
fixation. Thaw before use; do not refreeze. Use within 1 year, but Test and control sera: Equilibrate the sera to room temperature, then
only if satisfactory results are obtained with test controls. heat at 56°C for 30 minutes. Reheat previously heated sera for 10
FTA Serum Reactive: Rehydrate with 5 ml distilled or deionized minutes on the day of testing. Cool to room temperature before testing.
water and rotate gently to completely dissolve the contents. Aliquot in Bacterial contamination or excessive hemolysis may render a specimen
0.4 ml amounts and store at or below -20°C. Do not refreeze thawed unsuitable for testing. Such specimens should not be tested.
aliquot. Approximately 12 tests may be obtained per 5 ml vial. This
serum should be heated at 56°C for 30 minutes before use. Test Procedure
This procedure conforms with those published by the U. S. Department
FTA Serum Non-Reactive: Rehydrate with 5 ml distilled or deionized
of Health, Education and Welfare14 and with subsequent procedures
water and rotate gently to completely dissolve the contents. Aliquot in published by the American Public Health Association.9,13
0.4 ml amounts and store at or below -20°C. Approximately 90-100
tests may be obtained per 5 ml vial. This serum should be heated at 1. FTA Antigen smears: Obtain previously prepared smears, thaw
56°C for 30 minutes before use. and dry if appropriate, and identify the frosted end of the slides to
correspond with each test and control serum to be tested.
FTA Sorbent: Rehydrate with 5 ml distilled or deionized water and
2. Prepare the following test and control sera in appropriately identified
rotate gently to completely dissolve the contents. Store at 2-8°C or
tubes no more than 30 minutes before testing and mix thoroughly
aliquot and store at -20°C. The quantity of FTA Sorbent used for each (at least 8 times):
test sample or serum is 0.2 ml. The quantity of FTA Sorbent needed for
Test Serum (1:5): Dilute 0.05 ml (50 µl) of heated (or reheated)
3 controls is 0.6 ml. Approximately 20-25 tests may be performed with
test serum in 0.2 ml (200 µl) FTA Sorbent.
5 ml of FTA Sorbent.
Reactive Control Serum (4+) - Unabsorbed: Dilute 0.05 ml (50 µl)
FTA Sorbent Control: Rehydrate with 0.5 ml distilled or deionized FTA Serum Reactive in 0.2 ml (200 µl) FA Buffer (PBS).
water and rotate gently to completely dissolve the contents. Aliquot in Reactive Control Serum (4+) - Absorbed: Dilute 0.05 ml (50 µl)
0.25 ml amounts and store at or below -20°C. For each test, 0.1 ml of FTA Serum Reactive in 0.2 ml (200 µl) FTA Sorbent.
FTA Sorbent Control is needed. Approximately 2 tests may be Minimally Reactive Control Serum (1+): Dilute FTA Serum
performed per 0.5 ml vial because of evaporation from heating. This Reactive, as indicated on the label, in FA Buffer (PBS) to yield a
serum should be heated at 56°C for 30 minutes before using. 1+ fluorescence. The minimal degree of fluorescence that can be
FA Human Globulin Antiglobulin (Rabbit): Rehydrate with 1 ml or reported as reactive is 1+ fluorescence.
5 ml distilled or deionized water, depending on label directions. Aliquot Nonreactive Control Serum (N) (1:40): Prepare a 1:40 dilution
in 0.5 ml amounts and store at or below -20°C. Each lot is supplied of FTA Serum Non-Reactive by adding 0.05 ml (50 µl) of serum
with a dilution titer. Since conditions and equipment differ from one to 1.95 ml FA Buffer (PBS).
laboratory to another, it is necessary to titer and test a new lot of conju- Nonspecific Serum Control - Unabsorbed (2+ nonspecif ic
gate with the fluorescent microscope assembly currently in use.3,8,13 reactivity): Dilute 0.05 ml (50 µl) FTA Sorbent Control in 0.2 ml
1. Prepare serial dilutions in 2% Tween 80, including the titer specified (200 µl) FA Buffer (PBS).
on the vial. Nonspecific Serum Control - Absorbed (nonreactive, - to ±):
Dilute 0.05 ml (50 µl) FTA Sorbent Control in 0.2 ml (200 µl)
2. Test each dilution per the Test Procedure with Reactive Control FTA Sorbent.
Serum (4+) and Nonspecific Staining Control.
Nonspecific Staining Control - Unabsorbed: Use 0.03 µl (30 ml)
3. Test a known lot of reagent using the Reactive Control Serum (4+), FA Buffer (PBS) undiluted.
Minimally Reactive Control Serum (1+) and Nonspecific Staining Nonspecific Staining Control - Absorbed: Use 0.03 ml (30 µl)
Control as controls of the reagents and test conditions. FTA Sorbent undiluted.
3. FTA Antigen smears: Cover the previously identified FTA Antigen for additional confirmatory tests. The final diagnosis depends on
smears with 0.03 ml (30 µl) of the corresponding test or control serum the clinical judgment of a specialist very experienced in sexually
prepared above, making certain that the entire smear is covered. transmitted diseases.2,3
4. Place the slides in a moist chamber to prevent evaporation and 2. The test should not be used to follow the response to therapy nor
incubate at 35-37°C for 30 minutes. can it be relied on to detect new, untreated cases in epidemiological
5. Place the slides in a slide carrier and rinse as follows: investigations.
• Rinse in running FA Buffer for 5 seconds. 3. “Atypical” fluorescence and false-positive results have been
• Soak in FA Buffer for 5 minutes. associated with patients having active systemic, discoid and
• Agitate by dipping in and out of the buffer 30 times. drug-induced varieties of lupus erythematosus 13-17 and other
• Repeat the soaking and agitation in fresh buffer. autoimmune diseases.
• Rinse in running distilled water for 5 seconds. 4. Elderly patients may exhibit unexplained FTA-ABS reactions.
• Gently blot dry with bibulous paper.
5. At times, deciding whether a reading is weak or vaguely visible
6. FA Human Globulin Antiglobulin (Rabbit): Dilute the antiglobulin
may be difficult. The ability to make this distinction is critical,
to its working titer (determined above) using 2% Tween 80 in
since a nonreactive (vaguely visible to none) serum is not retested.
FA Buffer.
7. FTA Antigen smears: Cover each test and control smear with References
approximately 0.03 ml (30 µl) of diluted FA Human Globulin 1. Creighton, E. T. 1990. Dark field microscopy for the detection
Antiglobulin (Rabbit). Spread uniformly to cover the entire smear. and identification of Treponema pallidum, p. 49-61. In S. A. Larsen,
8. Repeat steps 4 and 5. E. F. Hunter, and S. J. Kraus (ed.), Manual of tests for syphilis,
9. Mount the slides immediately using a small drop of FA Mounting 8th ed. American Public Health Association, Washington, D. C.
Fluid pH 7.2 and apply a cover slip, being careful not to trap air 2. Janda, W. M. (ed.). 1992. Immunology, p. 9.7.1-9.7.20. In H. D.
bubbles in the mounting fluid. Isenberg (ed.), Clinical microbiology procedures handbook,
10. Immediately examine the slides microscopically for intensity of vol. 2. American Society for Microbiology, Washington, D. C.
fluorescence using the microscope assembly described above. If it 3. Norris, S. J., and S. A. Larsen. 1995. Treponema and other
is necessary to delay reading, store the slides in the dark and read host-associated spirochetes, p. 636-651. In P. R. Murray, E. J.
within 4 hours. Results are valid only if the quality control pattern Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual
is satisfactory. of clinical microbiology, 6th ed. American Society for Microbiology,
11. Verify the presence of treponemes on the nonreactive control slides Washington, D. C.
by dark-field microscopy. 4. Deacon, W. E., V. H. Falcone, and A. Harris. 1957. A fluorescent
Results test for treponemal antibodies. Proc. Soc. Exp. Biol. and Med.
96:477-480.
Using the 1+ serum control as a reading standard, record the intensity
of fluorescence of the treponemes and report as follows. Retest all 5. Deacon, W. E., V. H. Falcone, and A. Harris. 1960. Fluorescent
specimens with an initial test fluorescence of 1+. When a specimen treponemal antibody test. A modification based on quantitation
initially read as 1+ yields a retest reading of 1+ or greater, it is reported (FTA-200). Proc. Soc. Exp. Biol. and Med. 103:827-829.
as reactive. All other results are reported as nonreactive. Retesting 6. Deacon, W. E., and E. M. Hunter. 1962. Treponemal antigens as
nonreactive specimens is not necessary. related to identification and syphilis serology. Proc. Soc. Exp. Biol.
and Med. 110:352-356.
Without historical or clinical evidence of treponemal infection, equivocal
test results (see below) suggest the need for testing a second specimen 7. Hunter, E. F., W. E. Deacon, and P. E. Meyer. 1964. An improved
obtained 1-2 weeks after the initial specimen. FTA test for syphilis; the absorption procedure (FTA-ABS). Publ.
INTENSITY OF INITIAL TEST RETEST
Hlth. Report 79:410-412.
FLUORESCENCE RESULT RESULT REPORT 8. Turgeon, M. L. 1990. Immunology and serology in laboratory
Moderate to strong 2+ to 4+ NA Reactive medicine. The C. V. Mosby Company, St. Louis, MO.
Equivalent to 1+ control 1+ >1+ Reactive 9. Wentworth, B. B., and F. N. Judson. 1984. Laboratory methods
1+ 1+ Reactive minimal* for the diagnosis of sexually transmitted diseases. American
1+ <1+ Nonreactive Public Health Association, Washington, D.C.
Visible staining but <1+ ± to <1+ NA Nonreactive 10. Centers for Disease Control. 1988. Update: universal precautions
None or vaguely visible, – NA Nonreactive for prevention of transmission of human immunodeficiency virus,
not distinct
hepatitis B virus, and other bloodborne pathogens in health-care
“Moth eaten” or “beaded” Atypical
settings. Morbid. Mortal. Weekly Rep. 37:377-382, 387-388.
*Equivocal result.
11. Occupational Safety and Health Administration, U. S. Depart-
Limitations of the Procedure ment of Labor. 1991. 29CFR, part 1910. Occupational exposure to
1. When the treponemal test results and the clinical opinion disagree, bloodborne pathogens; final rule. Federal Register 56:64175-64182.
repeat the treponemal test and obtain additional clinical and 12. Johnson, R. M. Letter. July 1, 1994. Department of Health
historical information. If the disagreement persists, send the specimen & Human Services, Public Health Service, Food and Drug
to a reference laboratory such as the local state health department Administration, Rockville, MD.
13. Larsen, S. A., E. F. Hunter, and S. J. Kraus. 1990. A manual of tests for 19. Anderson, B., and M. T. Stillman. 1978. False-positive FTA-ABS
syphilis. American Public Health Association, Washington, D.C. in hydralazine-induced lupus. J.A.M.A. 239:1392-1493.
14. U.S. Department of Health, Education and Welfare. 1969. Packaging
Manual of tests for syphilis; PHS Publication No. 411. US FA Buffer, Dried 6 x 10 g 2314-33
Government Printing Office, Washington, D.C. 100 g 2314-15
15. Kraus, S. J., J. R. Haserick, and M. A. Lantz. 1970. Fluorescent FA Human Globulin Antiglobulin 1 ml 2449-50
treponemal antibody absorption test reactions in lupus erythema- (Rabbit) 5 ml 2449-56
tosus. N. Engl. J. Med. 282:1287-1290. FA Mounting Fluid pH 7.2 6x5 ml 2329-57
16. Goldman, J. N., and M. A. Lantz. 1971. FTA-ABS and VDRL
FTA Antigen 1 ml 2344-50
slide test reactivity in a population of nuns. J.A.M.A. 217:53-55.
FTA Serum Non-Reactive 5 ml 2440-56
17. Shore, R. N., and J. A. Faricelli. 1977. Borderline and reactive
FTA-ABS results in lupus erythematosus. Arch. Dermatol. FTA Serum Reactive 5 ml 2439-56
113:37-41. FTA Sorbent 5 ml 3259-56
18. Monson, R. A. 1973. Biological false-positive FTA-ABS test in FTA Sorbent Control 6 x 0.5 ml 3266-49
drug-induced lupus erythematosus. J.A.M.A. 224:1028-1030. Tween® 80 6x5 ml 3118-57
Tween® 80
Heat Tween 80 and
FA Buffer to 56°C.
Add 2 ml Tween 80
to 98 ml FA Buffer.
Adjust to pH 7.2.
between Brucella species and Yersinia enterocolitica or Vibrio cholerae. 4. Febrile Antigens contain the following preservatives:
Antibodies produced in response to a Proteus infection can react with Brucella Abortus Antigen (Slide): 0.5% phenol, and approximately
Proteus OX19 and be misinterpreted as rickettsial antibodies. 0.002% crystal violet and 0.005% brilliant green.
The rapid slide test is the most widely used procedure employing Proteus OX19 Antigen (Slide): 0.25% formaldehyde, and approxi-
febrile antigens because of the simplicity with which the results may mately 0.002% crystal and 0.005% brilliant green.
be reported. Negative slide test reactions can usually be reported as Salmonella O Antigen Group D: 0.5% phenol, and approximately
such if all five serum dilutions have been used. Although the slide test 0.002% crystal violet and 0.005% brilliant green.
is not quantitative, running the series of dilutions is necessary to detect
agglutinin content of a serum that might be overlooked for a “prozone Salmonella H Antigens a: 0.5% formaldehyde, and approximately
phenomenon” where higher concentrations of the serum may yield 0.002% crystal violet and 0.005% brilliant green.
negative results, but a dilution of the serum is positive. This often Salmonella H Antigens b: 0.5% formaldehyde, and approximately
occurs in sera containing Brucella agglutinins and, to a lesser extent, 0.002% crystal violet and 0.005% brilliant green.
typhoid agglutinins. Salmonella H Antigens d: 0.5% formaldehyde, and approximately
The macroscopic tube test2 should be used to confirm the presence of 0.002% crystal violet and 0.005% brilliant green.
antibodies demonstrated by the slide technique and to quantitate their Antisera
titer in suspect sera. When quantitative determinations of Rickettsia or
1. Febrile Positive Control Polyvalent is lyophilized, polyclonal,
Brucella agglutinins are necessary, tube antigens are used.
polyvalent goat antisera containing approximately 0.04% Thimerosal
Principles of The Procedure as a preservative. It contains antibodies for all of the components
Agglutination tests involving the use of febrile antigens determine the of the Febrile Antigen Set. Each vial contains sufficient reagent for
presence of antibodies that react with the test antigen. The serological 32 slide tests or 50 tube tests using single antigens or for approxi-
procedure involves serially diluting the patient serum, then adding a mately 5 slide tests when using all of the antigens in the set.
standard volume of antigen. The end point of the test is the last dilution of 2. Febrile Negative Control is a lyophilized, standard protein solution
the serum that shows a specific amount of agglutination. The end point containing approximately 0.02% Thimerosal as a preservative.
converted to a dilution of the serum is called the patient’s antibody “titer.” Each vial contains sufficient reagent for 32 slide tests using single
antigens or for approximately 5 slide tests using all of the antigens
Reagents in the set.
Antigens
1. Febrile Antigens are ready-to-use, whole cell suspensions of the Precautions
organisms listed below. Proteus OX19 Antigen (Slide) contains 1. For In Vitro Diagnostic Use.
20% glycerin. 2. Observe universal blood and body fluid precautions in the handling
Brucella Abortus Antigen (Slide) - Brucella abortus and disposing of specimens.8,9
Proteus OX19 Antigen (Slide) - Proteus vulgaris OX19 3. Proteus OX19 Antigen (Slide)
Salmonella O Antigen Group D - Salmonella typhi O901 Salmonella H Antigen a
Salmonella H Antigen a - Salmonella paratyphi A Salmonella H Antigen b
Salmonella H Antigen b - Salmonella paratyphi B Salmonella H Antigen d
Salmonella H Antigen d - Salmonella typhi H901 POSSIBLE RISK OF IRREVERSIBLE EFFECTS. Avoid contact
2. Slide test: The Febrile Antigens (Brucella Abortus Antigen (Slide), with skin and eyes. Do not breathe mist. Wear suitable
Proteus OX19 Antigen (Slide), Salmonella O Antigen Group D, protective clothing. Keep container tightly closed. TARGET
Salmonella H Antigen a, Salmonella H Antigen b and Salmonella ORGAN(S): Eyes, Kidneys, Lungs, Skin.
typhi H901) are used in the slide test and contain sufficient FIRST AID: In case of contact with eyes, rinse immediately with
reagent for 20 slide tests. plenty of water and seek medical advice. After contact with skin,
Tube test: Salmonella O and H Antigens may also be used in the wash immediately with plenty of water. If inhaled, remove to fresh
tube test and contain sufficient reagent for 25 tube tests. air. If not breathing, give artificial respiration. If breathing is diffi-
Brucella Abortus Antigen (Slide) and Proteus OX19 Antigen (Slide) cult, give oxygen. Seek medical advice. If swallowed seek medical
are used only in the slide test. When confirmation of the slide test advice immediately and show this container or label.
and quantitation are required, Brucella Abortus Antigen (Tube) and 4. Follow proper established laboratory procedure in handling and
Proteus OX19 (Tube) may be purchased as separate products. disposing of infectious materials.
3. Antigen Density: Salmonella O and H Antigens are adjusted to a 5. Febrile Antigens are not intended for use in the immunization of
density approximating 20 times a McFarland Barium Sulfate humans or animals.
Standard No. 3 (1.8 x 1010 organisms per ml). These antigens are
used undiluted for the slide test and diluted 1:20 for the tube test. Storage
Because antigen density may vary, it is adjusted for optimum Store Febrile Antigens at 2-8°C.
performance when standardized with hyperimmune sera obtained Store lyophilized and rehydrated Febrile Positive Control Polyvalent
from laboratory animals. at 2-8°C.
Variation in antigen color intensity is normal and will not affect
Store lyophilized and rehydrated Febrile Negative Control at 2-8°C.
test performance.
Expiration Date A preliminary test using either the rapid slide test and/or the macro-
The expiration date applies to a product in its intact container when scopic tube test may be performed on the initial serum specimen and
stored as directed. Do not use a product if it fails to meet specifications reported to the physician at that time. An aliquot of the serum should
for identity and performance. be transferred to a sterile test tube, sealed tightly, and kept in the freezer.
When the second serum is obtained, it should be run in parallel with
Procedure the original specimen. In this manner, the original serum will serve as
Materials Provided a control and any difference in titer will be more credible, since the
bias associated with the performance of the test and determining the
Febrile Antigen Set:
endpoint will be reduced.
Brucella Abortus Antigen (Slide)
Proteus OX19 Antigen (Slide) Test Procedure
Salmonella O Antigen Group D Slide Test
Salmonella H Antigen a Use the slide test only as a screening test; confirm positive results
Salmonella H Antigen b with the tube test. Test each Febrile Antigen separately, repeating steps
Salmonella H Antigen d 1-6 for each Antigen.
Febrile Positive Control Polyvalent
1. Test Serum: Using a 0.2 ml serological pipette, dispense 0.08, 0.04,
Febrile Negative Control
0.02, 0.01 and 0.005 ml of each test serum into a row of squares on
Materials Required But Not Provided the agglutination slide.
Slide Test 2. Positive control: Using a 0.2 ml serological pipette, dispense 0.08,
Agglutination slides, 5 squares, 1” each 0.04, 0.02, 0.01 and 0.005 ml of Febrile Positive Control Polyva-
lent into a row of squares on the agglutination slide.
Applicator sticks
Sterile deionized water or equivalent 3. Negative control: Using a 0.2 ml serological pipette, dispense 0.08,
Serological pipettes, 0.2 ml 0.04, 0.02, 0.01 and 0.005 ml of Febrile Negative Control into a
row of squares on the agglutination slide.
Tube Test 4. Febrile Antigen: Gently shake the vial of antigen to ensure a
Culture tubes 12 x 75 mm and rack smooth, uniform suspension. Place one drop (35 µl) of antigen
Waterbath, 35-37°C and 50 ± 2°C suspension in each drop of test serum, positive control and
Refrigerator, 2-8°C negative control.
Serological pipettes, 1 ml and 5 ml 5. Mix each row of test and control serum, using a separate applicator
Sterile 0.85% NaCl solution stick for each row. Start with the most dilute mixture (0.005 ml)
Reagent Preparation and work to the most concentrated (0.08 ml).
Febrile Antigens are ready to use. 6. Rotate the slide for 1 minute and read for agglutination.
Febrile Positive Control Polyvalent: To rehydrate, add 5 ml sterile 7. The final dilutions in squares 1-5 correspond with tube dilutions of
distilled or deionized water and rotate gently to completely dissolve 1:20, 1:40, 1:80, 1:160, 1:320, respectively.
the contents. Results
Febrile Negative Control: To rehydrate, add 5 ml sterile deionized water, 1. Read and record results as follows.
or equivalent, and rotate gently to completely dissolve the contents. 4+ 100% agglutination; background is clear to slightly hazy.
Equilibrate all materials to room temperature before performing the 3+ 75% agglutination; background is slightly cloudy.
tests. Ensure that all glassware and pipettes are clean and free of 2+ 50% agglutination; background is moderately cloudy.
residues such as detergent.
1+ 25% agglutination; background is cloudy.
Specimen Collection and Preparation – No agglutination.
Collect a blood specimen by aseptic venipuncture. Serum is required 2. Positive control: Should show 2+ or greater agglutination at the
for the test. Store serum specimens at room temperature for no longer following dilutions:
than 4 hours; for prolonged storage, keep at 2-8°C for up to 5 days or Brucella Abortus Antigen 1:80
maintain at or below -20°C. Serum specimens must be clear, free of Proteus OX19 Antigen 1:160
hemolysis and show no visible evidence of bacterial contamination (tur- Salmonella O Antigen Group D 1:80
bidity, hemolysis or particulate matter). Refer to appropriate references Salmonella H Antigen a 1:80
for more information on collection of specimens.10,11 Serum specimens Salmonella H Antigen b 1:80
must not be heated. Heat may inactivate or destroy certain antibodies. Salmonella H Antigen d 1:80
An increase in titer over a period of time is the best indicator of active 3. Negative control: Should show no agglutination.
infection. The accuracy and precision of the tests can be affected not 4. Test specimens: The serum titer is that dilution which shows 2+
only by test conditions, but also by the subjectivity of the person reading or greater agglutination. See Table 1.
the endpoint.
Table 1. Sample Rapid Slide Test reactions. 9. Read and record results.
REACTIONS Results
CORRELATED
SERUM (ml) TUBE DILUTION SPECIMEN 1 SPECIMEN 2 SPECIMEN 3 1. Read and record results as follows.
0.08 1:20 3+ 4+ 4+ 4+ 100% agglutination; background is clear to slightly hazy.
0.04 1:40 2+ 4+ 3+ 3+ 75% agglutination; background is slightly cloudy.
0.02 1:80 1+ 3+ 2+ 2+ 50% agglutination; background is moderately cloudy.
0.01 1:160 – 3+ + 1+ 25% agglutination; background is cloudy.
0.005 1:320 – 1+ – – No agglutination.
Serum titer 1:40 1:160 1:80
2. Salmonella Antigens used in tube agglutination procedures detect
antibodies to either O (somatic) antigens or H (flagellar) antigens
Tube Test and these antibodies give different reactions. An O antigen and the
Salmonella O Antigen Group D and Salmonella H Antigens a, b and d corresponding antibody give a coarse, compact agglutination that
in the Febrile Antigen Set are used for both slide and tube agglutina- may be difficult to disperse. An H antigen and its corresponding
tion tests. Brucella Abortus Antigen (Slide) and Proteus OX19 Antigen antibody give a loose flocculent agglutination. Do not vigorously
(Slide) are intended only for slide tests. When confirmation of the slide shake tubes containing H antigens. Characteristic O and H aggluti-
test and quantitation is required, separate tube test antigens, Brucella nation is illustrated below.
Abortus Antigen (Tube) and Proteus OX19 (Tube), may be purchased
separately.
Each Febrile Antigen must be tested separately. Repeat steps 1-10 for
each antigen.
Prepare a 1:20 dilution of each antigen to be tested by adding 1 part of
antigen to 19 parts of sterile NaCl solution. Somatic “O” Agglutination
1. Prepare a row of 8 culture tubes (12 x 75 ml) for each test serum,
including a row for the Febrile Positive Control Polyvalent.
2. 0.85% NaCl solution: Dispense 0.9 ml in the first tube of each
row and 0.5 ml in the remaining tubes.
3. Test serum: Using a 1 ml serological pipette, dispense 0.1 ml of
test serum in the first tube in the row and mix thoroughly. Transfer
0.5 ml from tube 1 to tube 2 and mix thoroughly. Similarly, continue Flagellar “H” Agglutination
transferring 0.5 ml through tube 7, discarding 0.5 ml from tube 7
after mixing. Tube 8 is the antigen control tube and contains only 3. Positive control: Should show a 2+ or greater agglutination at the
sterile 0.85% NaCl solution. following dilutions:
4. Positive control: Using a 1 ml serological pipette, dispense 0.1 ml Brucella Abortus Antigen 1:80
of Febrile Positive Control Polyvalent in the first tube in the row Proteus OX19 Antigen 1:160
and mix thoroughly. Transfer 0.5 ml from tube 1 to tube 2 and mix Salmonella O Antigen Group D 1:80
thoroughly. Similarly, continue transferring 0.5 ml through tube 7, Salmonella H Antigens a, b and d 1:80
discarding 0.5 ml from tube 7 after mixing. Tube 8 is the antigen 4. Antigen control: Tube 8 of each row should show no agglutination.
control tube and contains only sterile 0.85% NaCl solution.
5. Test serum: The serum titer is that dilution which shows a 2+ or
5. Febrile Antigen: Add 0.5 ml of the diluted antigen suspension to greater agglutination. See Table 2.
all 8 tubes in each row and shake the rack to mix.
6. The final dilutions in tubes 1-7 are 1:20, 1:40, 1:80, 1:160, 1:320,
Table 2. Sample Macroscopic Tube Test reactions.
1:640 and 1:1280, respectively.
7. Incubate as specified (eg., in a waterbath or refrigerator): REACTIONS
SERUM DILUTION SPECIMEN 1 SPECIMEN 2 SPECIMEN 3
Brucella Abortus Antigen: 35-37°C for 48 ± 3 hours.
Proteus OX19 Antigen: 35-37°C for 2 hours, then at 1:20 4+ 3+ 4+
2-8°C for 22 ± 2 hours. 1:40 4+ 2+ 4+
Salmonella O Antigen Group D: 50 ± 2°C for 17 ± 1 hours. 1:80 3+ 1+ 4+
Salmonella H Antigens a: 50 ± 2°C for 1 hour. 1:160 2+ – 4+
Salmonella H Antigens b: 50 ± 2°C for 1 hour.
1:320 1+ – 3+
Salmonella H Antigens d: 50 ± 2°C for 1 hour.
1:640 – – 2+
8. Remove from incubation. Avoid excessive shaking before reading 1:1280 – – 1+
the reactions, either when the tubes are incubating or when
Serum titer 1:160 1:40 1:640
removing them from incubation.
Reagents Precautions
Francisella Tularensis Antigen (Slide) is a ready-to-use suspension 1. For In Vitro Diagnostic Use.
of Francisella tularensis containing 20% glycerin, as well as 0.5% 2. Francisella Tularensis Antigen (Tube)
phenol, approximately 0.2% crystal violet and approximately 0.5% POSSIBLE RISK OF IRREVERSIBLE EFFECTS. Avoid contact
brilliant green as preservatives. When used as described, each 5 ml vial with skin and eyes. Do not breathe mist. Wear suitable
contains sufficient reagent for 20 slide tests. protective clothing. Keep container tightly closed. Target Organs:
Francisella Tularensis Antigen (Tube) is a ready-to-use suspension Eyes, Kidneys, Lungs, Skin.
of Francisella tularensis adjusted to a density approximating a FIRST AID: In case of contact with eyes, rinse immediately with
McFarland Barium Sulfate Standard No. 3 (9 x 108 organisms per ml). plenty of water and seek medical advice. After contact with skin,
Francisella Tularensis Antigen (Tube) contains 0.5% formalin but does wash immediately with plenty of water. If inhaled, remove to fresh
not contain dye. When used as described, each 25 ml vial contains air. If not breathing, give artificial respiration. If breathing is diffi-
sufficient reagent for 6 tests. cult, give oxygen. Seek medical advice. If swallowed seek medical
Because antigen density may vary, density is adjusted to ensure optimum advice immediately and show this container or label.
performance when the antigen is standardized with hyperimmune sera 3. Observe universal blood and body fluid precautions in the
obtained from laboratory animals. Variation in antigen color intensity handling and disposing of specimens.7,8
is normal and will not affect the outcome of the test. 4. Biosafety level 2 precautions are recommended when handling
specimens suspected of containing F. tularensis.9
Francisella Tularensis Antiserum is a lyophilized, polyclonal rabbit
antiserum containing approximately 0.04% Thimerosal as a 5. Francisella Tularensis Antigens are not intended for use in the
preservative. When rehydrated and used as described, each 3 ml vial immunization of humans or animals.
contains sufficient reagent for 19 slide tests or 30 tube tests. 6. Follow proper established laboratory procedure in handling and
disposing of infectious materials.
Febrile Negative Control is a standard protein solution containing
approximately 0.02% Thimerosal as a preservative. When used as Storage
described, each 3 ml vial contains sufficient reagent for 32 slide tests.
Store Francisella Tularensis Antigens (Slide) and (Tube) at 2-8°C.
Store lyophilized and rehydrated Francisella Tularensis Antiserum at
2-8°C.
User Quality Control Store lyophilized and rehydrated Febrile Negative Control at 2-8°C.
Identity Specifications Expiration Date
Francisella Tularensis Antigen (Slide) The expiration date applies to the product in its intact container when
Appearance: Blue-violet suspension. stored as directed. Do not use a product if it fails to meet specifications
Francisella Tularensis Antigen (Tube) for identity and performance.
Appearance: Light gray to white suspension.
Francisella Tularensis Antiserum
Procedure
Lyophilized Appearance: Light gold to amber, button to Materials Provided
powdered cake. Francisella Tularensis Antigen (Slide)
Rehydrated Appearance: Light gold to amber, clear liquid. Francisella Tularensis Antigen (Tube)
Francisella Tularensis Antiserum
Febrile Negative Control
Febrile Negative Control
Lyophilized Appearance: Colorless to light gold, button to
powdered cake. Materials Required But Not Provided
Rehydrated Appearance: Colorless to light gold, clear liquid. Slide Test
Performance Response Agglutination slides with five 1-inch squares
Rehydrate Francisella Tularensis Antiserum and Febrile Applicator sticks
Negative Control per label directions. Perform the Rapid Sterile 0.85% NaCl solution
Slide Test using Francisella Tularensis Antigen (Slide) or the Serological pipettes, 0.2 ml
Macroscopic Tube Test using Francisella Tularensis Antigen Distilled or deionized water
(Tube). Dilute both positive and negative controls in the same
proportion as a patient serum and process in the same manner, Tube Test
following appropriate procedure. Culture tubes, 12 x 75 mm, and rack
An antigen is considered satisfactory if it fails to agglutinate Waterbath, 35-37°C
with the negative control and reacts to a titer of 1:160 or more Serological pipettes, 1 ml and 5 ml
with the positive control. Sterile 0.85% NaCl solution
Distilled or deionized water
Reagent Preparation ml from tube 1 to tube 2 and mix thoroughly. Similarly, continue
Francisella Tularensis Antigen (Slide) and Francisella Tularensis transferring 0.5 ml through tube 7, discarding 0.5 ml from tube 7
Antigen (Tube) are ready to use. after mixing. Proceed in like manner for each serum to be tested.
Equilibrate all materials to room temperature before performing the 4. Positive control: Using a 1 ml serological pipette, dispense 0.1 ml
tests. Ensure that all glassware and pipettes are clean and free of of Francisella Tularensis Antiserum in the first tube in the row and
detergent residues. mix thoroughly. Transfer 0.5 ml from tube 1 to tube 2 and mix
thoroughly. Similarly, continue transferring 0.5 ml through tube 7,
Francisella Tularensis Antiserum: To rehydrate, add 3 ml sterile 0.85% discarding 0.5 ml from tube 7 after mixing.
NaCl solution and rotate gently to dissolve the contents completely.
The rehydrated antiserum is considered a 1:2 working dilution. 5. Antigen control: Tube 8 is the antigen control tube and contains
only sterile 0.85% NaCl solution.
Febrile Negative Control: To rehydrate, add 5 ml sterile distilled or
6. Antigen: Shake the vial of Francisella Tularensis Antigen (Tube)
deionized water and rotate gently to dissolve the contents completely.
to ensure a smooth, uniform suspension. Add 0.5 ml of antigen to
Specimen Collection and Preparation all 8 tubes in each row and shake the rack to mix the suspensions.
Collect a blood specimen by aseptic venipuncture. After the specimen 7. Final dilutions in tubes 1-7 are 1:20, 1:40, 1:80, 1:160, 1:320, 1:640
has clotted, centrifuge to obtain the serum required for the test. Serum and 1:1280, respectively.
specimens must be clear, free of hemolysis and show no visible 8. Incubate in a waterbath at 35-37°C for 22 ± 2 hours.
evidence of bacterial contamination (turbidity, hemolysis or particulate 9. Remove from the waterbath. Avoid excessive shaking before read-
matter). Consult appropriate references for more information on ing the reactions, when the tubes are in the waterbath, or when
collection of specimens.2,10 removing them from the waterbath.
Store serum specimens at room temperature for no longer than 4 hours;
for prolonged storage, keep at 2-8°C for up to 5 days or maintain be- Results
low -20°C. Serum specimens must not be heated; heat may inactivate 1. Read and record results as follows.
or destroy certain antibodies. 4+ 100% agglutination; background is clear to slightly hazy.
Slide Test 3+ 75% agglutination; background is slightly cloudy.
Use the slide test only as a screening test. Confirm positive results with 2+ 50% agglutination; background is moderately cloudy.
the tube test. 1+ 25% agglutination; background is cloudy.
1. Test serum: Using a 0.2 ml serological pipette, dispense 0.08, 0.04, – No agglutination.
0.02, 0.01 and 0.005 ml of each test serum into a row of squares on 2. Positive control: Should produce 2+ or greater agglutination at a
an agglutination slide. 1:160 dilution.
2. Positive control: Using a 0.2 ml serological pipette, dispense 0.08, Negative control - Rapid Slide Test, only: Should produce no
0.04, 0.02, 0.01 and 0.005 ml of Francisella Tularensis Antiserum agglutination.
into a row of squares on the agglutination slide. Antigen control - Macroscopic Tube Test, only: Should produce
3. Negative control: Using a 0.2 ml serological pipette, dispense 0.08, no agglutination in tube #8 of each row.
0.04, 0.02, 0.01 and 0.005 ml of Febrile Negative Control into a
If results for either the positive or negative control are not as
row of squares on the agglutination slide.
specified, the test is invalid and results cannot be reported.
4. Antigen: Shake the vial of Francisella Tularensis Antigen (Slide)
Test serum: The titer is the highest dilution that shows 2+
thoroughly to ensure a smooth, uniform suspension. Dispense 1
agglutination.
drop (35 µl) of antigen in each drop of test serum, positive control
and negative control. Refer to Table 1 and Table 21 for examples of test reactions.
5. Mix each row of test serum and control serum, using a separate 3. The Rapid Slide Test is a screening test, only; results must be
applicator stick for each row. Start with the most dilute mixture confirmed using the Macroscopic Tube Test.
(0.005 ml) and work to the most concentrated (0.08 ml).
6. Rotate the slide for 1 minute and read for agglutination. Table 1. Sample Rapid Slide Test reactions.
7. The final dilutions in squares 1-5 correspond approximately to tube
REACTIONS
dilutions of 1:20, 1:40, 1:80, 1:160 and 1:320, respectively. CORRELATED
SERUM (ml) TUBE DILUTION SPECIMEN 1 SPECIMEN 2 SPECIMEN 3
Tube Test
0.08 1:20 3+ 4+ 4+
1. In a rack, prepare a row of 8 culture tubes (12 x 75 mm) for each
0.04 1:40 2+ 4+ 3+
test serum and a positive control row for the Francisella Tularensis
Antiserum. 0.02 1:80 1+ 3+ 2+
2. Dispense 0.9 ml of sterile 0.85% NaCl solution in the first tube of 0.01 1:160 – 3+ +
each row and 0.5 ml in the remaining tubes. 0.005 1:320 – 1+ –
3. Test serum: Using a 1 ml serological pipette, dispense 0.1 ml of Serum titer 1:40 1:160 1:80
serum in the first tube in the row and mix thoroughly. Transfer 0.5
Table 2. Sample Macroscopic Tube Test reactions. 8. Adhering to the recommended time and temperature of incubation
is important when performing this test. For best results, locate the
REACTIONS
waterbath in an area free of mechanical vibration.
SERUM DILUTION SPECIMEN 1 SPECIMEN 2 SPECIMEN 3
1:20 4+ 3+ 4+ References
1:40 4+ 2+ 4+ 1. Stewart, S. J. 1995. Francisella, p. 545-548. In P. R. Murray, E. J.
1:80 3+ 1+ 4+ Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.),
1:160 2+ – 4+ Manual of clinical microbiology, 6th ed. American Society for
1:320 1+ – 3+ Microbiology, Washington, D. C.
1:640 – – 2+ 2. Pezzlo, M. 1992. Aerobic bacteriology, p. 1.0.1-1.20.47. In H. D.
1:1280 – – 1+ Isenberg (ed.), Clinical microbiology procedures handbook,
Serum titer 1:160 1:40 1:640
vol. 1. American Society for Microbiology, Washington, D. C.
3. Holt, J. G., N. R. Krieg, P. H. Sneath, J. T. Staley, and S. T.
Williams. 1994. Bergey’s manual of determinative bacteriology,
Interpretation 9th ed. Williams & Wilkins, Baltimore, MD.
For a single serum specimen, a titer of 1:160 at 2+ or greater suggests 4. Miller, L. E., H. R. Ludke, J. E. Peacock, and R. H. Tomar.
infection.1 1991. Manual of laboratory immunology, 2nd ed. Lea & Febiger.
A 2-dilution increase in the titer of paired serum specimens (from the 5. Rose, N. R., H. Friedman, and J. L. Fahey (ed.). 1986. Manual
acute to the convalescent serum) is significant and suggests infection. of clinical immunology, 3rd ed. American Society for Microbiology,
A 1-dilution difference is within the limits of laboratory error. Washington, D. C.
6. Turgeon, M. L. 1990. Immunology and serology in laboratory
Limitations of the Procedure medicine. The C. V. Mosby Company, St. Louis, MO.
1. The slide test is intended for screening only and results should be
confirmed by the tube test. Slide test dilutions are made to detect a 7. Centers for Disease Control. 1988. Update: universal precautions
prozone reaction and do not represent true quantitation of the anti- for prevention of transmission of human immunodeficiency virus,
body. A serum specimen with a prozone reaction shows no hepatitis B virus, and other bloodborne pathogens in health-care
agglutination because of excessively high antibody concentrations. settings. Morbidity and Mortality Weekly Reports 37:377-382,
To avoid this occurrence, all five serum dilutions (slide test) should 387-388.
be run. 8. Occupational Safety and Health Administration, U.S.
2. The detection of antibodies in serum specimens may complete the Department of Labor. 1991. 29 CFR, part 1910. Occupational
clinical picture of tularemia. However, isolation of the causative exposure to bloodborne pathogens; final rule. Federal Register
agent from patient specimens may be required. A definitive diagno- 56:64175-64182.
sis must be made by a physician based on patient history, physical 9. U. S. Department of Health and Human Services. 1988.
examination and data from all laboratory tests. Biosafety in microbiological and biomedical laboratories,
3. Cross-reacting heterologous antibodies are responsible for many 2nd ed. U. S. Department of Health and Human Services
low titer reactions. Cross-reactions between antigens and antibodies publication no. 88-8395. U. S. Government Printing Office,
of Brucella species and Francisella tularensis can occur. Infections Washington, D. C.
with other organisms, vaccinations and a history of disease may 10. Miller, J. M., and H. T. Holmes. 1995. Specimen collection,
cause low antibody titers. Antimicrobial therapy may suppress transport and storage. In P. R. Murray, E. J. Baron, M. A.
antibody production. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical
4. While a single serum specimen showing a titer of 1:160 suggests microbiology, 6th ed. American Society for Microbiology,
infection, it is not diagnostic. Washington, D. C.
5. To test for a significant rise in antibody titer, at least two Packaging
specimens are necessary, an acute specimen obtained at the time Francisella Tularensis Antigen (Slide) 5 ml 2240-56
of initial symptoms and a convalescent specimen obtained 7 to
14 days later. A two-dilution increase in titer is significant and Francisella Tularensis Antigen (Tube) 5 ml 2251-56
suggests infection. 25 ml 2251-65
6. Prolonged exposure of reagents to temperatures other than those Francisella Tularensis Antiserum 3 ml 2241-47
specified is detrimental to the products. Febrile Negative Control 3 ml 3239-56
7. Exposure to temperatures below 2°C can cause antigen autoagglu-
tination. Antigens must be smooth, uniform suspensions.
Examine antigen vials for agglutination before use. Agglutinated
suspensions are not usable and should be discarded.
agglutination reaction should support the morphological and biochemical Testing the Isolate for Autoagglutination
identification of the microorganism. 1. From the test culture on chocolate agar, transfer a loopful of growth
Homologous reactions are rapid and strong. Heterologous reactions to a drop of sterile 0.85% NaCl solution on a clean slide and
are slow and weak. emulsify the organism.
2. Rotate the slide for one minute and then observe for agglutination.
Reagents 3. If agglutination (autoagglutination) occurs, the culture is rough and
Haemophilus Influenzae Antisera are lyophilized, polyclonal rabbit cannot be tested. Subculture to chocolate agar, incubate, and test
antisera containing approximately 0.02% Thimerosal as a preservative. the organism again as described in steps 1 and 2.
When rehydrated and used as described, each 1 ml vial of Haemophilus If no agglutination occurs, proceed with testing the organism.
Influenzae Antiserum contains sufficient reagent for 20 slide tests.
Test Procedure
Precautions Test culture isolates with Haemophilus Influenzae Poly for presumptive
1. For In Vitro Diagnostic Use. identification, then test with monospecific antisera.
2. The Packaging of This Product Contains Dry Natural Rubber. 1. Dispense 1 drop of the Haemophilus Influenzae Antiserum to be
3. Follow established laboratory procedure in handling and disposing tested on an agglutination slide.
of infectious materials. 2. Transfer a loopful of growth of the test organism to the drop of
Storage antiserum and mix thoroughly.
Store lyophilized and rehydrated Haemophilus Influenzae Antisera 3. Rotate the slide for one minute and read for agglutination.
at 2-8°C. 4. Repeat this procedure for known positive and negative control
cultures.
Expiration Date Results
The expiration date applies to the product in its intact container when
Observe test results and record agglutination as follows:
stored as directed. Do not use a product if it fails to meet specifications
for identity and performance. 4+ 100% agglutination; background is clear to slightly hazy.
3+ 75% agglutination; background is slightly cloudy.
Procedure 2+ 50% agglutination; background is moderately cloudy.
Materials Provided 1+ 25% agglutination; background is cloudy.
Haemophilus Influenzae Antiserum Poly – No agglutination.
Haemophilus Influenzae Antiserum Type a
Positive control: Should produce 3+ or greater agglutination.
Haemophilus Influenzae Antiserum Type b
Haemophilus Influenzae Antiserum Type c Negative control: Should produce no agglutination.
Haemophilus Influenzae Antiserum Type d Positive test result: Agglutination of 3+ or greater within one minute.
Haemophilus Influenzae Antiserum Type e
Haemophilus Influenzae Antiserum Type f Limitations of the Procedure
1. Correct interpretation of serological reactions depends on culture
Materials Required but not Provided purity as well as morphological characteristics and biochemical
Agglutination slides reactions that are consistent with identification of the microorganism
Applicator sticks as H. influenzae.
Sterile distilled or deionized water 2. Serological methods alone cannot identify the isolate as
Sterile 0.85% NaCl solution H. influenzae.
Reagent Preparation 3. Excessive heat from external sources (hot bacteriological loop,
burner flame, light source, etc.) may prevent a smooth suspension
Haemophilus Influenzae Antiserum: To rehydrate, add 1 ml sterile
of the microorganism or may cause evaporation or precipitation of
distilled or deionized water and rotate to completely dissolve the contents.
the test mixture. False-positive reactions may occur.
Equilibrate all materials to room temperature prior to performing 4. Rough culture isolates occur and will agglutinate spontaneously
the tests. Ensure that all glassware and pipettes are clean and free of causing agglutination of the negative control (autoagglutination).
detergent residues. Smooth colonies must be selected and tested in serological procedures.
Specimen Collection and Preparation 5. H. influenzae has antigenic similarities to several unrelated bacteria.
H. influenzae can be recovered from clinical specimens on chocolate Cross-reactions can occur between H. influenzae and strains
agar. For specific recommendations, consult appropriate references.1,5 of S. pneumoniae, Escherichia coli and several species of
Determine that a pure culture of the microorganism has been obtained Staphylococcus, Streptococcus and Bacillus.
and that biochemical test reactions are consistent with the identification 6. Haemophilus Influenzae Antisera have been tested using undiluted
of the organism as H. influenzae. After these criteria are met, serological cultures taken from agar media. These antisera have not been tested
identification can be performed. using antigen suspensions in NaCl solution or other diluents. If the
user employs a variation of the recommended procedure, each lot of clinical laboratory immunology, 3rd ed. American Society for
of antiserum must be tested with known control cultures to verify Microbiology, Washington, D.C.
that expected reactions are obtained under the modified procedure. 4. Cruse, J. M., and R. E. Lewis. 1995. Illustrated Dictionary of
7. Prolonged exposure of reagents to temperatures other than those Immunology, p. 253. CRC Press, Inc., Boca Raton, FL.
specified is detrimental to the products. 5. Pezzlo, M. 1992. Aerobic bacteriology, p. 1.0.1.-1.20.47. In
8. A rehydrated Haemophilus Influenzae Antiserum that is cloudy or H. D. Isenberg, (ed.), Clinical microbiology procedures handbook,
develops a precipitate during use should be discarded. vol. 1. American Society for Microbiology, Washington, D.C.
References Packaging
1. Campos, J. M. 1995. Haemophilus, p. 556-565. In P. R. Murray, Haemophilus Influenzae Antiserum Poly 1 ml 2237-50
E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Haemophilus Influenzae Antiserum Type a 1 ml 2250-50
Manual of clinical microbiology, 6th ed. American Society for
Microbiology, Washington, D. C. Haemophilus Influenzae Antiserum Type b 1 ml 2236-50
2. Pittman, M. 1931. Variation and type specificity in the bacterial Haemophilus Influenzae Antiserum Type c 1 ml 2789-50
species Hemophilus influenzae. J. Exp. Med. 53:471-495. Haemophilus Influenzae Antiserum Type d 1 ml 2790-50
3. Insel, R., and P. Anderson. 1986. Haemophilus influenzae Type b: Haemophilus Influenzae Antiserum Type e 1 ml 2791-50
assays for the capsular polysaccharide and for antipolysaccharide
antibody. In N. R. Rose, H. Friedman, and J. L. Fahey (ed.), Manual Haemophilus Influenzae Antiserum Type f 1 ml 2792-50
Because a microorganism (antigen) may agglutinate with an antibody REAGENT VIAL NUMBER OF TESTS
produced in response to another species, heterologous reactions are Listeria O Antiserum 1 ml 10 tube tests, 400 slide tests
possible. These are characterized as weak in strength or slow in formation. Listeria O Antigen (Slide) 5 ml 100 slide tests
Such unexpected and perhaps unpredictable reactions may lead to some Listeria O Antigen (Tube) 25 ml 5 tube tests
confusion in serological identification. A positive homologous agglu-
tination reaction should support the morphological and biochemical Precautions
identification of the microorganism. 1. For In Vitro Diagnostic Use.
Agglutination of the somatic antigen in the slide test appears as a firm 2. Listeria O Antiserum Type 1
granular clumping. Homologous reactions occur rapidly and are strong Listeria O Antiserum Type 4
(3+). Heterologous reactions form slowly and are weak. Listeria O Antiserum Poly
The Packaging of This Product Contains Dry Natural Rubber.
The agglutination of the somatic antigen in the tube tests appears as a
loose flocculation that can easily be resuspended. Homologous reactions 3. Listeria O Antigen Type 1 (Slide)
Listeria O Antigen Type 1 (Tube)
using Listeria O Antisera should exceed a titer of 2+ at 1:320. Listeria O Antigen Type 4 (Slide)
Listeria O Antigen Type 4 (Tube)
Reagents POSSIBLE RISK OF IRREVERSIBLE EFFECTS. (US) Avoid
Listeria O Antisera Types 1, 4, and Poly are lyophilized, polyclonal contact with skin and eyes. Do not breathe mist. Wear suitable
rabbit antisera containing approximately 0.04% Thimerosal as a protective clothing. Keep container tightly closed. TARGET
preservative. The antisera are prepared according to procedures ORGAN(S): Eyes, Kidneys, Lungs, Skin.
recommended by Gray.11 Listeria O Antisera Types 1 and 4 are specific
FIRST AID: In case of contact with eyes, rinse immediately with
for the respective serotypes of L monocytogenes while Listeria O
plenty of water and seek medical advice. After contact with skin,
Antiserum Poly contains agglutinins for L. monocytogenes serotypes
wash immediately with plenty of water. If inhaled, remove to fresh
1 and 4.
air. If not breathing, give artificial respiration. If breathing is diffi-
Listeria O Antigens Types 1 and 4 (Tube) and (Slide) are suspensions cult, give oxygen. Seek medical advice. If swallowed seek medical
of appropriate L. monocytogenes serotypes containing 0.3% formaldehyde advice immediately and show this container or label.
as a preservative. When used according to the suggested procedure, 4. Follow proper established laboratory procedure in handling and
the reagents will yield the following: disposing of infectious materials.
Storage
Store lyophilized and rehydrated Listeria O Antisera at 2-8°C.
Store Listeria O Antigen (Slide) and (Tube) at 2-8°C.
User Quality Control
Identity Specifications Expiration Date
Listeria O Antiserum Type 1 The expiration date applies to the product in its intact container when
Listeria O Antiserum Type 4 stored as directed. Do not use a product if it fails to meet specifications
Listeria O Antiserum Poly for identity and performance.
Lyophilized Appearance: Light gold to amber, button to
powdered cake. Procedure
Rehydrated Appearance: Light gold to amber, clear liquid. Materials Provided
Listeria O Antiserum Type 1
Listeria O Antigen Type 1 (Slide)
Listeria O Antigen Type 1 (Tube) Listeria O Antiserum Type 4
Listeria O Antigen Type 4 (Slide) Listeria O Antiserum Poly
Listeria O Antigen Type 4 (Tube) Listeria O Antigen Type 1 (Slide)
Appearance: White, liquid suspension. Listeria O Antigen Type 1 (Tube)
Listeria O Antigen Type 4 (Slide)
Performance Response Listeria O Antigen Type 4 (Tube)
Rehydrate Listeria O Antiserum per label directions. Perform
the slide or tube agglutination test using appropriate Listeria Materials Required But Not Provided
O Antigens (Slide) or (Tube). Rapid Slide Test
Slide test: An antiserum is considered satisfactory if it FA Buffer, Dried
demonstrates a 3+ or greater reaction at 1:80 with a 1:5 dilution Agglutination slides
of the homologous antigen. Applicator sticks
Macroscopic tube test: An antiserum is considered satisfactory Waterbath, 80-100°C
if it demonstrates a 3+ or greater reaction with the 1:320
dilution of the homologous antigen. Formaldehyde
Droppers
Macroscopic Tube Test 9. Positive control: Add one drop of homologous Listeria O Antigen
FA Buffer, Dried (Slide) to the second drop of antiserum.
McFarland Barium Sulfate Standard No. 3 10. Rotate the slide for 1-2 minutes and read for agglutination.
Culture tubes 12 x 75 mm and rack
Serological pipettes, 1 ml Slide Test Results
Waterbath, 50°C 1. Read and record results as follows.
Refrigerator, 2-8°C 4+ 100% agglutination; background is clear to slightly hazy.
Formaldehyde 3+ 75% agglutination; background is slightly cloudy.
2+ 50% agglutination; background is moderately cloudy.
Reagent Preparation 1+ 25% agglutination; background is cloudy.
Equilibrate all materials to room temperature before performing the – No agglutination.
tests. Ensure that all glassware and pipettes are clean and free of
residues such as detergents. 2. Positive control: Should show a 3+ or greater agglutination.
3. Negative control: Should show no agglutination. If agglutination
Listeria O Antisera: To rehydrate, add 1 ml sterile distilled or deionized
occurs, the culture is rough and cannot be tested. Subculture to a
water to each vial. Rotate gently to dissolve contents completely.
non-inhibitory medium, incubate and test the organism again.
Listeria O Antigens (Slide) and (Tube) are ready to use. 4. Test isolates: 3+ or greater agglutination is a positive result.
Specimen Collection and Preparation 5. A partial (less than 3+) or a delayed agglutination reaction should
From clinical specimens, Listeria can be recovered on selective be considered negative.
differential media such as McBride Listeria Agar, Oxford Agar, Macroscopic Tube Test
Modified Oxford Agar, LPM Agar or Palcam Medium. For specific 1. Test isolate: Suspend growth of the test organism from a solid agar
recommendations on isolation of Listeria from clinical specimens, medium in FA Buffer. Adjust to a density approximating that of a
consult appropriate references.10,12,13 Determine that a pure culture of McFarland Barium Sulfate Standard No. 3.
the microorganism has been obtained and that biochemical test reactions 2. Prepare a row of 9 culture tubes (12 x 75 mm) for each serum
are consistent with the identification of the organism as Listeria suspension to be tested, including the positive control.
monocytogenes. After these criteria are met, serological identification
3. Formalized FA Buffer: Dispense 0.9 ml formalized FA Buffer
can be performed.
(0.3 ml formaldehyde per 300 ml FA Buffer) to the first tube in
From food or dairy samples, Listeria can be recovered when samples each row and 0.5 ml to the remaining tubes.
are processed to recover injured microorganisms and prevent over- 4. Listeria O Antiserum: Using a 1 ml serological pipette, add 0.1 ml
growth of competing microorganisms. Consult appropriate references of the desired antiserum to tube 1 in each row and mix thoroughly.
for recommended procedures for the isolation of Listeria from Transfer 0.5 ml from tube 1 to tube 2 and mix thoroughly. In like
foods.7,14,16 Having followed an established protocol, isolate a pure cul- manner, continue transferring 0.5 ml through tube 8, discarding
ture of the microorganism and confirm that biochemical test 0.5 ml from tube 8 after mixing. Tube 9 is an antigen control tube.
reactions are consistent with the identification of the organism as Upon addition of the test suspension, final dilutions will be 1:20
Listeria monocytogenes. After these criteria are met the serological through 1:2560 for tubes 1 through 8, respectively.
identification can be performed.
5. Test Suspension: Add 0.5 ml of the test suspension to each of
Test Procedure 9 tubes.
Rapid Slide Test 6. Positive control: Add 0.5 ml of an appropriate Listeria O Antigen
1. FA Buffer, Dried: Rehydrate per label directions. to each of 9 tubes containing antiserum.
2. Test isolate: Suspend growth from a solid agar medium in FA Buffer. 7. Negative control: Add 0.5 ml of the test suspension to a tube
containing FA Buffer.
3. Heat the organism suspension at 80-100°C (in a waterbath) for
1 hour. 8. Shake the rack to mix. Incubate in a 50°C waterbath for 2 hours.
Refrigerate overnight. Read for agglutination the following
4. Centrifuge the suspension and remove the bulk of the supernatant morning.
fluid.
5. Resuspend the organism in the remaining portion of liquid. Tube Test Results
1. Read and record results as follows.
6. Listeria Antiserum: On an agglutination slide, dispense 2 separate
drops of the desired antiserum diluted 1:20 in NaCl solution. The 4+ 100% agglutination; background is clear to slightly hazy.
first drop will be used for the test isolate and the second for the 3+ 75% agglutination; background is slightly cloudy.
negative control. 2+ 50% agglutination; background is moderately cloudy.
7. Organism suspension: Add 1 drop of heated organism to the first 1+ 25% agglutination; background is cloudy.
drop of antiserum. – No agglutination.
8. Negative control: Dispense 1 drop of FA Buffer on the agglutination 2. Positive control: Should show 2+ or greater agglutination at 1:320.
slide. Add one drop of organism suspension from step 5. 3. Antigen control: Tube 9 of each row should show no agglutination.
4. If results of the positive control or antigen control are not as de- 6. Patel, J. R., C. A. Hwang, L. R. Beuchat, M. P. Doyle, and
scribed, the test is invalid and results cannot be read. R. E. Brackett. 1995. Comparison of oxygen scavengers for their
5. Test serum: The titer is that dilution which shows a 2+ or greater ability to enhance resuscitation of heat-injured Listeria
agglutination at 1:320. monocytogenes. J. Food Prot. 58:244-250.
7. Donnelly, C. W., R. E. Bracket, D. Doores, W. H. Lee, and
Limitations of the Procedure J. Lovett. 1992. Listeria, p. 637-663. In C. Vanderzant and
1. Serological techniques employing Listeria O Antisera serve as D. F. Splittstoesser (ed.), Compendium of methods for the
corroborative evidence for the identification of Listeria microbiological examination of foods, 3rd ed. American Public
monocytogenes. Final identification cannot be made without Health Association, Washington, D.C.
consideration of morphological, serological and biochemical 8. Kramer, P. A., and D. Jones. 1969. Media selective for Listeria
characterization. monocytogenes. J. Appl. Bacteriol. 32:381-394.
2. Excessive heat from external sources (hot bacteriological loop, 9. Seeliger, H. P. R., and K. Hohne. 1979. Serotyping of Listeria
burner flame, light source, etc.) may prevent making a smooth monocytogenes and related species, p. 31-49. In T. Bergen and
suspension of the microorganism or cause evaporation or precipi- J. R. Norris (ed.), Methods in microbiology, vol. 13. Academic
tation of the test mixture. False-positive reactions may occur. Press, London, England.
3. Rough culture isolates occur and will agglutinate spontaneously,
10. Swaminathan, B., J. Rocourt, and J. Bille. 1995. Listeria,
causing agglutination of the negative control (autoagglutination).
p. 342-343. In P. R. Murray, Baron, Ffaller, Tenover and Yolken
Smooth colonies must be selected and tested in serological
(ed.), Manual of clinical microbiology, 6th ed. American Society
procedures.
for Microbiology, Washington, D.C.
4. Agglutination reactions of 3+ or greater in the slide test are inter-
11. Gray, M. L., and A. H. Killinger. 1966. Listeria monocytogenes
preted as positive reactions. Cross-reactions resulting in a 1+ or 2+
infections. Bacteriol. Rev. 30:309-382.
agglutination are likely since there are somatic antigens shared
among different organisms such as staphylococci, enterococci and 12. Pezzlo, M. 1994. Aerobic bacteriology, p. 1.0.1.-1.20.47. In H. D.
Bacillus species.10 Isenberg (ed.), Clinical microbiology procedures handbook,
vol. 1. American Society for Microbiology, Washington, D.C.
5. Prolonged exposure of reagents to temperatures other than those
specified is detrimental to the products. 13. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
Scott’s diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
6. Exposure of Listeria O Antigens to temperatures below 2°C can
St. Louis, MO.
result in autoagglutination. Antigens must be smooth uniform
suspensions; examine antigen vials for agglutination before use. 14. Hitchins, A. D. 1995. Listeria monocytogenes, p. 10.01-10.13.
Suspensions with agglutination are not usable and should be In FDA Bacteriological analytical manual, 8th ed. AOAC
discarded. International, Arlington, VA.
7. It is important in this test to use the recommended time and 15. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig.
temperature of incubation. Also, care should be taken to make certain 1992. Pathogens in milk and milk products. In Marshall, R. T.,
that the waterbath is in a location free of mechanical vibration. (ed.), Standard methods for the examination of dairy products,
16th ed. American Public Health Association, Washington, D.C.
8. Discard any Listeria O Antiserum that is cloudy or has a precipitate
after rehydration or storage. Packaging
References Listeria O Antiserum Type 1 1 ml 2300-50
1. Murray, E. G. D., R. A. Webb, and M. B. R. Swann. 1926. A Listeria O Antiserum Type 4 1 ml 2301-50
disease of rabbits characterized by large mononuclear leucocytosis
Listeria O Antiserum Poly 1 ml 2302-50
caused by a hitherto undescribed bacillus Bacterium monocytogenes
(n. sp.). J. Path. Bact. 29:407-439. Listeria O Antigen Type 1 (Slide) 5 ml 2303-56
2. Monk, J. D., R. S. Clavero, L. R. Beuchat, M. P. Doyle, and Listeria O Antigen Type 1 (Tube) 25 ml 2305-65
R. E. Brackett. 1994. Irradiation inactivation of Listeria
Listeria O Antigen Type 4 (Slide) 5 ml 2304-56
monocytogenes and Staphylococcus aureus in low- and high-fat,
frozen and refrigerated ground beef. J. Food Prot. 57:969-974. Listeria O Antigen Type 4 (Tube) 25 ml 2306-65
3. Wehr, H. M. 1987. Listeria monocytogenes - a current dilemma FA Buffer, Dried 6 x 10 g 2314-33
special report. J. Assoc. Off. Anal. Chem. 70:769-772. 100 g 2314-15
4. Bremer, P. J., and C. M. Osborne. 1995. Thermal-death times of 10 kg 2314-08
Listeria monocytogenes in green shell mussels (Perna canaliculus)
prepared for hot smoking. J. Food Prot. 58:604-608.
5. Grau, F. H., and P. B. Vanderlinde. 1992. Occurrence, numbers,
and growth of Listeria monocytogenes on some vacuum-packaged
processed meats. J. Food Prot. 55:4-7.
Serological confirmation involves the reaction in which the microor- Neisseria Meningitidis Antiserum A
ganism (antigen) reacts with its corresponding antibody. This in vitro Neisseria Meningitidis Antiserum B
reaction produces macroscopic clumping called agglutination. The Neisseria Meningitidis Antiserum C
desired homologous reaction is rapid, has at least a 3+ reaction, does Neisseria Meningitidis Antiserum D
not dissociate (high avidity), and binds (high affinity). Neisseria Meningitidis Antiserum X
Because a microorganism (antigen) may agglutinate with an antibody Neisseria Meningitidis Antiserum Y
produced in response to another species, heterologous reactions are Neisseria Meningitidis Antiserum Z
possible. These are characterized as weak in strength or slow in for- Neisseria Meningitidis Antiserum Z´
mation. Such unexpected and, perhaps, unpredictable reactions may Neisseria Meningitidis Antiserum W135
lead to some confusion in serological identification. Therefore, a
positive homologous agglutination reaction should support the Materials Required But Not Provided
morphological and biochemical identification of the microorganism. Agglutination slides
Applicator sticks
Homologous reactions are rapid and strong. Heterologous reactions
are slow and weak. Sterile distilled or deionized water
Sterile 0.85% NaCl solution
Reagents Reagent Preparation
Neisseria Meningitidis Antisera are lyophilized, polyclonal rabbit
antisera containing approximately 0.02% Thimerosal as a preservative. Neisseria Meningitidis Antisera: To rehydrate, add 1 ml sterile distilled
Neisseria Meningitidis Antisera Poly and Group D are absorbed for or deionized water and rotate gently to completely dissolve the contents.
detection of Group D; Neisseria Meningitidis Antisera Poly 2, Z´, Equilibrate all materials to room temperature prior to performing the
W135, A, B, C, X, Y and Z are not absorbed for detection of Group D, tests. Ensure that all glassware and pipettes are clean and free of
which is rarely isolated. detergent residues.
Neisseria Meningitidis Antisera detect the following antigenic groups: Specimen Collection and Preparation
ANTISERUM ANTIGENIC GROUP(S) DETECTED
N. meningitidis can be recovered on blood agar or chocolate agar.
Poly A, B, C, D Determine that the test organism has the following characteristics of
Poly 2 X, Y, Z N. meningitidis:
W135 W135
Morphology: grayish-white, opaque, smooth,
A A butyrous, non-pigmented
B B
Gram stain: gram-negative diplococcus
C C
Oxidase: positive
D D
X X Catalase: positive
Y Y ONPG reaction: negative
Z Z, Z´ Nitrate reduction: negative
Z´ Z´ Glucose: positive
When rehydrated and used as described, each 1 ml vial of Neisseria Maltose: positive
Meningitidis Antiserum contains sufficient reagent for 20 slide tests. Sucrose: negative
Fructose: negative
Precautions Lactose: negative
1. For In Vitro Diagnostic Use.
Determine that a pure culture of the microorganism has been obtained
2. The Packaging of This Product Contains Dry Natural Rubber. and that biochemical test reactions are consistent with the identification
3. Follow proper established laboratory procedure in handling and of the organism as N. meningitidis. For more detailed information on
disposing of infectious materials. the biochemical identification of N. meningitidis, consult appropriate
Storage references.3,5 After these criteria are met, serological identification can
proceed.
Store lyophilized and rehydrated Neisseria Meningitidis Antisera at 2-8°C.
Testing the Isolate for Autoagglutination
Expiration Date
1. From the test culture on chocolate agar, transfer a loopful of growth to
The expiration date applies to the product in its intact container when
a drop of sterile 0.85% NaCl solution on a clean slide and emulsify
stored as directed. Do not use a product if it fails to meet specifications
the organism.
for identity and performance.
2. Rotate the slide for one minute and then observe for agglutination.
Procedure If agglutination (autoagglutination) occurs, the culture is rough and
Materials Provided cannot be tested. Subculture to chocolate agar, incubate, and test
Neisseria Meningitidis Antiserum Poly the organism again as described in steps 1 and 2.
Neisseria Meningitidis Antiserum Poly 2 If no agglutination occurs, proceed with testing the organism.
Choosing Antisera to Test 4. Rough culture isolates occur and will agglutinate spontaneously,
1. Test the organism first with Neisseria Meningitidis Antisera Poly, causing agglutination of the negative control (autoagglutination).
Poly 2 and Group W135. Smooth colonies must be selected and tested in serological procedures.
2. Depending on the reaction, continue testing as follows. 5. N. meningitidis Group A and N. meningitidis Group C may cross-
If agglutination occurs with Test with react due to the presence of common capsular polysaccharides.
Neisseria Meningitidis Antiserum: Neisseria Meningitidis Antiserum: 6. Group Z´ meningococci may agglutinate group Z antiserum. Group
Poly Groups A, B, C, D Z meningococci will not agglutinate group Z´ antiserum.
Poly 2 Groups X, Y, Z, Z´ (See NOTE.) 7. Neisseria Meningitidis Antisera have been tested using undiluted
Group W135 No further testing is required. cultures taken from agar media. These antisera have not been tested
using antigen suspensions in NaCl solution or other diluents. If the
NOTE: N. meningitidis Group Z´ organisms may agglutinate user employs a variation of the recommended procedure, each lot
monospecific Neisseria Meningitidis Antiserum Group Z. However, N. of antiserum must be tested with known control cultures to verify
meningitidis Group Z organisms will not agglutinate Neisseria that expected reactions are obtained under the modified procedure.
Meningitidis Antiserum Group Z´. The expected agglutination
reactions of Neisseria Meningitidis Antiserum Groups Z´ and Z with 8. Prolonged exposure of reagents to temperatures other than those
test organisms are: specified is detrimental to the products.
Neisseria Meningitidis Antiserum 9. A rehydrated Neisseria Meningitidis Antiserum that is cloudy or
Test Organism Group Z´ Group Z develops a precipitate during use should be discarded.
N. meningitidis Group Z´ 3+ +
References
N. meningitidis Group Z – 3+
1. Given, K. F., B. W. Thomas, and A. G. Johnston. 1977. Isolation
Test Procedure of Neisseria meningitidis from the urethra, cervix, and anal canal:
1. Neisseria Meningitidis Antiserum: Dispense 1 drop of the anti- further observations. Br. J. Vener. Dis. 53:109-112.
serum to be tested on an agglutination slide. 2. Janda, W. M., M. Bohnhoff, J. A. Morello, and S. A. Lerner.
2. Test isolate: Transfer a loopful of growth to the drop of antiserum 1980. Prevalence and site-pathogen studies of Neisseria
and mix thoroughly. meningitidis and N. gonorrhoeae in homosexual men. JAMA
244:2060-2064.
3. Rotate the slide for one minute and read for agglutination.
3. Knapp, J. S., and R. J. Rice. 1995. Neisseria and Branhamella,
4. Repeat this procedure for known positive and negative cultures.
p. 324-340. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
Results Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology,
1. Read and record results as follows. 6th ed. American Society for Microbiology, Washington, D.C.
4+ 100% agglutination; background is clear to slightly hazy. 4. Zollinger, W. D., B. L. Brandt, and E. C. Tramont. 1986.
3+ 75% agglutination; background is slightly cloudy. Immune response to Neisseria meningitidis, p. 346-352. In N. R.
Rose, H. Friedman, and J. L. Fahey (ed.), Manual of clinical labo-
2+ 50% agglutination; background is moderately cloudy.
ratory immunology, 3rd ed. American Society for Microbiology,
1+ 25% agglutination; background is cloudy.
Washington, D.C.
– No agglutination.
5. Pezzlo, M. 1994. Aerobic bacteriology, p. 1.0.1-1.20.47. In H. D.
2. Positive control: Should produce 3+ or greater agglutination. Isenberg (ed.), Clinical microbiology procedures handbook,
Negative control: Should produce no agglutination. vol. 1. American Society for Microbiology, Washington, D.C.
Test isolate: A positive test result is defined as agglutination of 3+
or greater within one minute. Packaging
Neisseria Meningitidis Antiserum Poly 1 ml 2232-50
Limitations of the Procedure
Neisseria Meningitidis Antiserum Poly 2 1 ml 2910-50
1. Correct interpretation of serological reactions depends on culture
purity, as well as morphological characteristics and biochemical Neisseria Meningitidis Antiserum Group A 1 ml 2228-50
reactions that are consistent with identification of the microorganism Neisseria Meningitidis Antiserum Group B 1 ml 2229-50
as N. meningitidis.
Neisseria Meningitidis Antiserum Group C 1 ml 2230-50
2. Serological methods alone cannot identify the isolate as
N. meningitidis. Organisms unrelated to Neisseria, yet capable of Neisseria Meningitidis Antiserum Group D 1 ml 2231-50
causing meningitis, and other species of Neisseria can cross-react Neisseria Meningitidis Antiserum Group X 1 ml 2880-50
with meningococcal antisera. Cultural isolation must precede
Neisseria Meningitidis Antiserum Group Y 1 ml 2881-50
serological examination.
3. Excessive heat from external sources (hot bacteriological loop, Neisseria Meningitidis Antiserum Group Z 1 ml 2891-50
burner flame, light source, etc.) may prevent a smooth suspension Neisseria Meningitidis Antiserum Group Z´ 1 ml 2252-50
of the microorganism or may cause evaporation or precipitation of Neisseria Meningitidis Antiserum Group W135 1 ml 2253-50
the test mixture. False-positive reactions may occur.
Principles of the Procedure FIRST AID: In case of contact with eyes, rinse immediately with
Agglutination tests involving the use of Proteus antigens determine plenty of water and seek medical advice. After contact with skin,
the presence of antibodies that react with the test antigen. The sero- wash immediately with plenty of water. If inhaled, remove to fresh
logical procedure involves serially diluting the patient serum and then air. If not breathing, give artificial respiration. If breathing is diffi-
adding a standard volume of an antigen. The end point of the test is the cult, give oxygen. Seek medical advice. If swallowed seek medical
last dilution of the serum that shows a specific amount of agglutination. advice immediately and show this container or label.
The end point converted to a dilution of the serum is called the patient’s 4. Proteus OX2 Antiserum
antibody “titer.” Proteus OX19 Antiserum
Proteus OXK Antiserum
Reagents The Packaging of This Product Contains Dry Natural Rubber.
Antigens 5. Follow proper established laboratory procedure in handling and
1. Proteus Antigens are ready to use, nonmotile strains of the disposing of infectious materials.
organisms listed below. Proteus Antigen (Slide) contains 20% 6. Proteus Antigens are not intended for use in the immunization of
glycerin. Each vial of Proteus Antigen (Slide) contains sufficient humans or animals.
reagent for 33 slide tests. Each vial of Proteus Antigen (Tube)
contains sufficient reagent for 6 tube tests. Storage
Proteus OX2 Antigen (Slide) and (Tube) - Proteus vulgaris OX2 Store Proteus OX2, OX19 and OXK Antigens (Slide) and (Tube) at
Proteus OX19 Antigens (Slide) and (Tube) - Proteus vulgaris OX19 2-8°C.
Proteus OXK Antigen (Slide) and (Tube) - Proteus mirabilis OXK Store lyophilized and rehydrated Proteus OX2, OX19 and OXK
2. Concentration of Antigen: Antigen density may vary because it is Antisera at 2-8°C.
adjusted for optimum performance when standardized with Store lyophilized and rehydrated Febrile Negative Control at 2-8°C.
hyperimmune sera obtained from laboratory animals.
Variation in color intensity is normal and will not affect test Expiration Date
performance. The expiration date applies to the product in its intact container when
3. Proteus antigens contain the following preservative(s): stored as directed. Do not use a product if it fails to meet specifications
Proteus OX2, OX19 and OXK Antigens (Slide): 0.5% for identity and performance.
formaldehyde, and approximately 0.002% crystal violet and
0.005% brilliant green.
Procedure
Proteus OX2, OX19 and OXK Antigens (Tube): 0.25% Materials Provided
formaldehyde. Proteus OX2 Antigen (Slide)
Proteus OX2 Antigen (Tube)
Antisera Proteus OX19 Antigen (Slide)
1. Proteus Antisera are lyophilized, polyclonal rabbit antisera Proteus OX19 Antigen (Tube)
containing approximately 0.04% Thimerosal as a preservative. Each Proteus OXK Antigen (Slide)
vial contains sufficient reagent for 19 slide tests or 30 tube tests. Proteus OXK Antigen (Tube)
2. Febrile Negative Control is a standard protein solution containing Proteus OX2 Antiserum
0.02% Thimerosal as a preservative. Each vial of Febrile Negative Proteus OX19 Antiserum
Control contains sufficient reagent for 32 slide tests. Proteus OXK Antiserum
Febrile Negative Control
Precautions
1. For In Vitro Diagnostic Use. Materials Required But Not Provided
2. Observe universal blood and body fluid precautions in the Slide test
handling and disposing of specimens.7,8 Agglutination slides, 5 squares, 1” each
3. Proteus OX2 Antigen (Slide) Applicator sticks
Proteus OX2 Antigen (Tube) Sterile 0.85% NaCl solution
Proteus OX19 Antigen (Slide) Sterile distilled or deionized water
Proteus OX19 Antigen (Tube) Serological pipettes, 0.2 ml
Proteus OXK Antigen (Slide)
Tube Test
Proteus OXK Antigen (Tube)
Culture tubes 12 x 75 mm and rack
POSSIBLE RISK OF IRREVERSIBLE EFFECTS. (US) Avoid
Waterbath, 35-37°C
contact with skin and eyes. Do not breathe mist. Wear suitable
Serological pipettes, 1 ml and 5 ml
protective clothing. Keep container tightly closed. TARGET
ORGAN(S): Eyes, Kidneys, Lungs, Skin. Sterile 0.85% NaCl solution
Sterile distilled or deionized water
Interpretation1 9. Rehydrated Proteus OX2, OX19 and OXK Antisera that is cloudy
Compare results: or has a precipitate during use should be discarded.
PROTEUS PROTEUS PROTEUS
DISEASE AGENT OX2 OX19 OXK
References
Epidemic Typhus* R. prowazekii + + –
1. Eisemann, C. S., and J. V. Osterman. 1986. Rickettsiae,
Murine Typhus* R. typhi + + –
p. 593-599. In N. R. Rose, H. Friedman, and J. L. Fahey, (ed.),
Scrub Typhus O. tsutsugamushi – – + Manual of clinical laboratory immunology, 3rd ed. American
Rocky Mountain Society for Microbiology, Washington, D.C.
Spotted Fever** R. rickettsii + + –
Other Spotted Fevers** Rickettsia sp. + + – 2. McDade, J. E. 1991. Rickettsiae, p. 1036-1044. In A. Balows (ed.),
Manual of clinical microbiology, 5th ed. American Society for
*In cases of epidemic and murine typhus, the strength of the antibody agglutination
with Proteus OX19 is usually stronger (4+) than the agglutination with Proteus Microbiology, Washington, D.C.
OX2 (2+). 3. Olson, J. G, and J. E. McDade. 1995. Rickettsia and Coxiella,
**In cases of spotted fevers, antibodies may agglutinate either or both strains of
Proteus OX19 or OX2, and the strength of agglutination may vary from 1+ to 4+. p. 678-685. In P. R. Murray, E. J. Baron, M A. Pfaller, F. C. Tenover,
and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed.
For a single serum specimen, a titer of 1:160 is suggestive of infection.
American Society for Microbiology, Washington, D.C.
A pair of serum specimens (acute and convalescent) showing a two- 4. Miller, L. E., H. R. Ludke, J. E. Peacock, and R. H. Tomar.
dilution difference in the titers is a significant increase in antibody 1991. Manual of laboratory immunology, 2nd ed. Lea & Febiger.
level and is suggestive of infection. A one dilution difference is within
5. Turgeon, M. L. 1990. Immunology and serology in laboratory
the limits of laboratory error.
medicine. The C. V. Mosby Company, St. Louis, MO.
Limitations of the Procedure 6. Weil, E., and A. Felix. 1916. Zur serologischen Diagnosis des
1. The slide test is for screening only and results should be con- Fleckfiebers. Wien. Klin. Wochenschr. 29:33-35.
firmed by performing the tube test. The slide test dilutions are 7. Centers for Disease Control. 1988. Update: universal precautions
made to detect a prozone reaction and do not represent true for prevention of transmission of human immunodeficiency virus,
quantitation of the antibody. A serum specimen with a prozone hepatitis B virus, and other bloodborne pathogens in health-care
reaction shows no agglutination because of excessively high settings. Morbidity and Mortality Weekly Reports 37:377-382,
antibody concentrations. To avoid this occurrence, all 5 serum 387-388.
dilutions (slide test) should be run. 8. Occupational Safety and Health Administration, U.S.
2. The detection of antibodies in serum specimens may complete the Department of Labor. 1991. 29 CFR, part 1910. Occupational
clinical picture of a patient having infection. However, the isolation exposure to bloodborne pathogens; final rule. Federal Register
of the causative agent from patient specimens may be required. A 56:64175-64182.
definitive diagnosis must be made by a physician based on patient 9. Pezzlo, M. 1992. Aerobic bacteriology, p. 1.0.1-1.20.47. In H. D.
history, physical examination and data from all laboratory tests. Isenberg (ed.), Clinical microbiology procedures handbook, vol.
3. Cross-reacting heterologous antibodies are responsible for many 1. American Society for Microbiology, Washington, D.C.
low titer reactions. Infections with other organisms, vaccinations 10. Miller, J. M., and H. T. Holmes. 1995. Specimen collection,
and past history of disease may result in low level of antibody titers. transport and storage. In P. R. Murray, E. J. Baron, M. A. Pfaller,
Antimicrobial therapy may suppress antibody production. F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical
The Weil-Felix test is not specific for rickettsial diseases. Rickettsia microbiology, 6th ed. American Society for Microbiology,
species cause cross-reacting antibodies, and infections with Washington, D.C.
Proteus species can also cause cross-reacting antibodies.
4. While a single serum specimen showing a titer of 1:160 suggests Packaging
infection, it is not diagnostic. Proteus OX2 Antigen (Slide) 5 ml 2243-56
5. To test for a significant rise in antibody titer, at least two specimens Proteus OX2 Antigen (Tube) 25 ml 2248-65
are necessary, an acute specimen obtained at time of initial symp-
Proteus OX19 Antigen (Slide) 5 ml 2234-56
toms and a convalescent specimen obtained 7 to 14 days later. A
two-dilution difference in titer is a significant increase in antibody Proteus OX19 Antigen (Tube) 25 ml 2247-65
level and is suggestive of infection. Proteus OXK Antigen (Slide) 5 ml 2244-56
6. The Weil-Felix test does not differentiate between epidemic and
Proteus OXK Antigen (Tube) 25 ml 2249-65
murine typhus.
7. Prolonged exposure of reagents to temperatures other than those Proteus OX2 Antiserum 3 ml 2245-47
specified is detrimental to the products. Proteus OX19 Antiserum 3 ml 2235-47
8. Exposure of the antigen reagents to temperatures below 2°C can Proteus OXK Antiserum 3 ml 2246-47
result in autoagglutination. Antigens must be smooth, uniform
suspensions. Examine antigen vials for agglutination before use. Febrile Negative Control 5 ml 3239-56
Suspensions with agglutination are not usable and should be discarded.
The use of Salmonella antisera in the serological identification of produced in response to some other species or serotype. These
Salmonella requires the use of quality control test suspensions to reactions occur slowly and are weak.
verify that the antisera are performing as expected. Most laboratories Heterologous reactions may be unexpected and unpredictable and may
are required to test antisera with positive and negative controls prior lead to confusion in serological identification. Therefore, only strongly
to use. 1,2 QC Antigens Salmonella are designed as homologous positive homologous agglutination reactions should be regarded as
controls for testing the efficacy of the Salmonella grouping antisera significant.
employed in routine laboratory procedures.
Reagents
Principles of the Procedure QC ANTIGEN ORGANISM USED FOR
HOMOLOGOUS
IDENTIFYING
Serological procedures that confirm the identification of an organism SALMONELLA ANTIGEN PREPARATION ANTIGEN(S)
are usually agglutination reactions. Agglutination reactions may be O Group A S. paratyphi A var. Durazzo, Factors 2, 12 2
either homologous or heterologous. Homologous reactions occur O Group B S. typhimurium Factors 1, 4, [5], 12 4, 5
between a microorganism (antigen) and the corresponding antibody. O Group C1 S. choleraesuis factors 6, 7 7
These reactions occur rapidly and are strong. Heterologous reactions O Group C2 S. newport factors 6, 8 8
occur when a microorganism (antigen) reacts with an antibody O Group D S. gallinarum factors 1, 9, 12 9
O Group E1 S. anatum factors 3, 10 10
O Group E2 S. newington factors 3, 15 15
User Quality Control cont. O Group E4 S. senftenberg factors 1, 3, 19 19
SALMONELLA QC ANTIGEN SALMONELLA O Group F S. rubislaw factor 11 11
ANTISERUM HOMOLOGOUS CONTROL(S)
O Group G1 S. poona factors [1], 13, 22, [36], [37] 22
Poly A-I & Vi A, B, D, E1, E2, E4, F,
G1, H, I, Vi O Group H S. carrau factors 6, 14, 24 14
Poly A A, B, D, E1, E2, E4 O Group I S. hvittingfoss factor 16 16
Poly B C1, C2, F, G1, H Vi Citrobacter ballerup O29 Vi
Poly C I Note: Brackets [ ] indicate that the antigen may be absent.
Group A Factors 1, 2, 12 A
Group B Factors 1, 4, 5, 12 B Underlining indicates that the O antigen has been lysogenized in that
Group B Factors 1, 4, 12, 27 B strain.
Group C1 Factors 6, 7 C1 These antigen suspensions are ready to use. QC Antigens Salmonella
Group C2 Factors 6, 8 C2 O are preserved with 0.5% phenol USP; QC Antigen Salmonella Vi
Group D1 Factors 1, 9, 12 D contains 0.01% Thimerosal. When used as described, each vial of QC
Group E Factors 1, 3, 10, 15, 19, 34 E 1, E 2 , E4 Antigen Salmonella has sufficient reagent for 20 slide tests.
Group E1 Factors 3, 10 E1
Febrile Negative Control is a lyophilized standard protein solution,
Group E2 Factors 3, 15 E2
Group E4 Factors 1, 3, 19 E4
containing approximately 0.04% Thimerosal as a preservative. When
Group F Factor 11 F
used as described, each vial of Febrile Negative Control has sufficient
Group G Factors 13, 22, 23, (36), (37) G1
reagent for 100 slide tests.
Group G1 Factors 13, 22, (36), (37) G1
Group H Factors 1, 6, 14, 24, 25 H
Precautions
Group I Factor 16 I 1. For In Vitro Diagnostic use.
Vi Vi 2. QC Antigens Salmonella
Factor 2 A The Packaging of This Product Contains Dry Natural Rubber.
Factor 4 B 3. Follow proper established laboratory procedure in handling and
Factors 4, 5 B disposing of infectious materials.
Factor 5 B
4. QC Antigens Salmonella are not to be used for immunization of
Factor 7 C1
humans or animals.
Factor 8 C2
Factor 9 D Storage
Factor 10 E1
Store QC Antigens Salmonella at 2-8°C.
Factor 15 E2
Factor 19 E4 Store lyophilized and rehydrated Febrile Negative Control at 2-8°C.
Factor 22 G1
Factor 14 H
Expiration Date
The expiration date applies to the product in its intact container when
Note: Parentheses ( ) indicate that the antigen is poorly developed or stored as directed. Do not use a product if it fails to meet specifications
agglutinates weakly. For a complete and current explanation of the
classification of Salmonella, consult appropriate references.3,4,5 for identity and performance.
Serovars of other subspecies of S. enterica (except some in the Complete identification of Salmonella requires cultural isolation,
subspecies salamae and houtenae) and those of S. bongori are not named biochemical characterization and serotyping. However well-defined
and are designated by their antigenic formula. For the most recent the serology of Salmonella, the use of serological procedures does not
information on nomenclature, consult appropriate references.1,7,10-13 supersede cultural isolation and biochemical characterization. Any
Results are for 48-hours incubation. Tests were performed at 35-37°C. serological results obtained before biochemical identification must be
considered as presumptive identification only. Consult to appropriate
Serotypes of Salmonella are defined based on the antigenic structure references for complete identification of Salmonella.1,2,3,8,11-14
of both somatic or cell wall (O) antigens and flagellar (H) antigens.
The antigenic formula lists the O antigen(s) first, followed by the Characterizing the Serotypes of Salmonella
H antigen(s). The major antigens are separated by colons and the Salmonella O Antigens: The somatic (O) heat-stable antigens are
components of the antigens separated by commas. For example, the identified first. The O antigens are numbered 1-67 using Arabic
antigenic formula for Salmonella Typhimurium is Salmonella numerals. The numbers are not completely continuous because certain
1,4,5,12:i:1,2. This means that the strain has O antigen factors 1,4,5 and strains were reclassified to other genera and the antigenic Arabic
12, the flagella phase 1 antigen i, and flagella phase 2 antigens 1 and 2. numbers were deleted from the schema.
Table 2a. Differentiation of Salmonella species, subspecies, and Table 2b. Differentiation of Salmonella species, subspecies and
some serovars.1,2 some serovars.1,2
Salmonella enterica Salmonella enterica
subsp. subsp. enterica
Salmonella subsp. subsp. subsp. subsp. subsp. salamae serovar serovar Serovar serovar Serovar
Test bongori arizonae enterica diaizonae houtenae indica Test Choleraesuis Gallinarum Paratyphi A Pullorum Typhi
Citrate, + + + + + [+] Citrate, + [–] – – – –
Simmons Simmons
H 2S + + + + + + H 2S + d + – + +
production production
Lysine + + + + + + Lysine + + + – + +
decarboxylase decarboxylase
Ornithine + + + + + + Ornithine + + – + + –
decarboxylase decarboxylase
Motility + + + + + + Motility + + – + – +
KCN, growth + – – – + – KCN, growth – – – – – –
Malonate – + – + – – Malonate + – – – – –
utilization utilization
D-Glucose, [+] + + + + + D-Glucose, + + – + + –
gas gas
L-arobinose, + + + + + + L-arobinose, + – [+] + + –
acid acid
Dulcitol, + – + – – d Dulcitol, + – + + – –
acid acid
Lactose, acid – [–] – [+] – [–] Lactose, acid – – – – – –
Maltose, acid + + + + + + Maltose, acid + + + + – +
Melibiose, [+] + + + + [+] Melibiose, – d – + – +
acid acid
L-Rhamnose, + + + + + + L-Rhamnose, + + – + + –
acid acid
D-Sorbitol + + + + + – D-Sorbitol + [+] – + [–] +
Trehalose, + + + + + + Trehalose, + – d + [+] +
acid acid
D-Xylose, + + + + + + D-Xylose, + + d – [+] [+]
acid acid
Mucate, acid + + + d – + Mucate, acid + – d – – –
Tartrate, – – + [–] d + Tartrate, d [+] + – – +
Jordans Jordans
ONPG + + – + – D ONPG [–] – – – – –
Serogroups represent the organization of Salmonella strains based on Using Salmonella Antisera
the antigen(s) shared in common and are designated by the letters A-Z. Salmonella O Antisera: The recommended serological Identification
After exhausting the alphabet, the serogroups were numbered beginning scheme begins with Salmonella O Antisera Poly A through Poly G,
with No. 51 (the serogroup Z organism having antigen No. 50). While which contain the following:
one somatic antigen identifies each serogroup, certain other antigens SALMONELLA POLY GROUP ANTISERA SOMATIC GROUPS PRESENT
may be shared among several serogroups. Bacto Salmonella O Antiserum Poly A A,B,D,E1,(E2,E3),*E4,L
Most organisms contain antigens in common that will cause cross- Bacto Salmonella O Antiserum Poly B C1,C2,F,G,H
reactions in an unabsorbed or “partially absorbed” antiserum. One Bacto Salmonella O Antiserum Poly C I,J,K,M,N,O
somatic antigen identifies a serogroup and is shared in common by Bacto Salmonella O Antiserum Poly D P,Q,R,S,T,U
all members of a given serogroup. For example, serogroup A is Bacto Salmonella O Antiserum Poly E V,W,X,Y,Z
represented by three members, Salmonella Paratyphi A (somatic antigens Bacto Salmonella O Antiserum Poly F 51–55
1,2,12), Salmonella Kiel (somatic antigens 1,2,12), and Salmonella Bacto Salmonella O Antiserum Poly G 56–61
Nitra (somatic antigens 2,12). All three members of this serogroup *Strains of groups E2 and E3 are lysogenized by phage 15, then by phage 34.
contain antigens 2 and 12 in common. Serogroup B is represented by These strains are now classified into group E1.3
many organisms consisting having different combinations of somatic
antigens 1,4,5,12 and 27. Serogroup D organisms contain somatic If agglutination occurs, use individual Salmonella O Group Factor
Antisera to determine the specific serogroup to which the isolate
antigens 1,9,12, etc.
belongs. For efficiency, test first with individual Salmonella O Group
In the above example, all three serogroups A, B, and D contain Factor Antisera.
antigens 1 and 12. An antiserum prepared from a 1,2,12 culture, if not
absorbed, will react with cultures of serogroups B and D in varying If agglutination does not occur with Poly A or B, test the isolate with
degrees depending on the concentration of the commonly shared 1 and Salmonella O Antiserum Vi. If positive, heat and retest. If agglutination
12 factors. This must be taken into consideration when choosing an does not occur with Salmonella O Antiserum Vi, the isolate is not
antiserum to be used in the examination of the salmonellae. likely to be Salmonella. Results should be examined. If questions
exist, the isolate should be sent to a reference laboratory.
Several different antisera are available. Some represent group antigens.
Others are single factor sera, which should be used when testing for an If agglutination does not occur with Poly C, D, E, F, and G, the isolate
identifiable antigen in a given serogroup. Such a single factor serum is is not likely to be Salmonella.
not called a “group” serum, though it contains the group identifiable
agglutinin. (It has been recommended by the CDC that the term “group” Table 3. Schema for using Salmonella O Antisera Poly Groups A, B,
be applied only to those sera possessing all the major agglutinins found C, D, E, F and G.
in that group.) Test with Salmonella O antisera Poly Groups A, B, C, D, E, F and G
In unabsorbed antisera, cross-reactions occur if strains sharing some Test Result + – with – with
“like” antigens are tested, even when they are in separate serogroups Poly A or B Poly C, D, E,
based on the major group antigen(s) they possess. Unabsorbed antisera F and G
are available as group antisera containing all factors in that group. Test with Individual Vi Antiserum
In absorbed antisera, cross reactions are less likely and are weaker. Salmonella O
Antisera
Absorbed antisera are available as factor specific antisera.
Test Result + with one + –
Flagellar Salmonella H Antigens: The flagellar (H) antigens are heat Salmonella O
labile and are usually associated with motility. Cultures are ordinarily Antiserum
flagellated and actively motile, although flagellated cultures can be (required)
nonmotile. H antigen characterization is done after the serogroup of Test Determine the Heat and retest Test isolate Test isolate
the strain is determined. The H antigens of Salmonella are designated Conclusion Salmonella H with individual is not a is not a
by letters of the alphabet, a-z, followed by z, z1, z2, etc., and by Arabic or Next Antigen Salmonella O Salmonella Salmonella
Action Antisera
numerals. H antigens exist in 2 phases, phase 1 and phase 2. Phase 1
antigens are expressed in letters a-z, etc., and the phase 2 antigens are
most often expressed in Arabic numerals. Older cultures may express Salmonella O Antiserum Poly A-I and Vi: This antiserum detects
both phases of a diphasic serotype, but recent clinical isolates more factors 1-16, 19, 22-25, 34 and Vi. This combination of factors
often express only one phase. Phase reversal may be necessary to represents the most frequently isolated Groups A-I and the Vi antigens
isolate both phases of a diphasic culture. Consult an appropriate and is used to screen possible Salmonella isolates.
reference for more detailed information.8 A positive reaction indicates that further serological testing is needed
A pure H antiserum cannot be prepared without some somatic content. to identify the isolate using Salmonella O Group Factor Antisera. The
However, since H antigens are highly antigenic, the serum derived most common serogroups are B, D and C1. For efficiency, first use
from motile cultures may be used at a dilution that reduces somatic the Salmonella O Group Factor Antisera for these serogroups.
agglutination below the detection level. If the isolate is positive with Salmonella O Antiserum Poly A-I and Vi
Salmonella Vi Antigen: The Vi Antigen is a heat-labile envelope but negative with Poly A-Poly G, test the isolate with Salmonella Vi
antigen that may surround a cell wall and mask somatic antigen activity. Antiserum. If positive with Salmonella Vi Antiserum, heat and retest
Microorganisms having the Vi Antigen will not agglutinate in O antisera. using individual Salmonella O Antisera. If negative with Salmonella
Vi Antiserum, the isolate is not likely to be Salmonella. Results Absorbed H antisera specific for single antigens or a complex of
should be examined. If questions exist, the isolate should be sent to a antigens can be used to identify the isolate further.
reference laboratory. Unabsorbed and Absorbed Salmonella H Antisera: Complete
A negative reaction indicates the isolate is not in serogroups A-I. If identification of a Salmonella isolate involves analysis of phase 1 and
the biochemical reactions are consistent with Salmonella, a serogroup phase 2 antigens using H antisera. For the complex pattern of analysis
other than A-I is possible. Further testing with antisera for other and procedures, consult appropriate references.8
serogroup antigens is necessary. Salmonella H Antisera Spicer-Edwards: Salmonella H Antisera
Spicer-Edwards is used for screening and identifying the most commonly
Table 4. Schema for using Salmonella O Antiserum Poly A-I & Vi.
encountered Salmonella using a combination of polyvalent and single
Test with Salmonella O Antisera Poly A-I and Vi complex antisera.
Test Result + –
Table 5. Identification of Salmonella H using Salmonella H Antisera
Test with Individual Salmonella O Antisera
Spicer-Edwards.
Test Result + –
Salmonella H Antisera
Test with Salmonella Vi Antiserum Spicer-Edwards
Test Result + – H Antigen(s) 1 2 3 4
that biochemical test reactions are consistent with the identification of 7. If the heated culture continues to react with Salmonella Vi Anti-
the organism as a Salmonella species. After these criteria are met, serum and not with the Salmonella O Antisera, the isolate may
serological identification can be performed. not be Salmonella. Test the isolate further to determine if it is
Food samples: Salmonella can be recovered when samples are correctly identified.
processed to recover injured microorganisms and prevent overgrowth 8. If an H antigen identification is required, proceed to the next
of competing microorganisms. Consult appropriate references for section.
recommended procedures for isolation of Salmonella from foods.13,14 9. When a negative reaction is obtained with Salmonella O Antise-
Determine that a pure culture of the microorganism has been obtained rum Poly A-I and Vi in the above procedure, the organism is pre-
and that biochemical test reactions are consistent with the identifica- sumptively negative for Salmonella that belong to serogroups A-I.
tion of the organism as a Salmonella species. After these criteria have Biochemical tests should be performed to confirm this negative
been met, serological identification can be performed. result. If biochemical tests prove the organism to be a Salmonella,
a serogroup beyond serogroup A-I is probably involved.
Slide Test Procedure
If the organism reacts with Poly A-I and Vi but does not react
Salmonella O and Vi Antisera with the specific somatic antisera, it should be checked with
Use this procedure to test the isolate with each selected antiserum. Salmonella Vi Antiserum by the above procedure.
1. Salmonella Antiserum: Add Dispense 1 drop (3 µl) of each
antiserum to be tested on an agglutination slide.
Tube Test Preparation
2. Negative control: Dispense 1 drop of 0.85% sterile NaCl solution 1. 0.6% formalized saline: Prepare by adding 6 ml formaldehyde
on an agglutination slide. per 1,000 ml of sterile 0.85% NaCl solution.
3. Test isolate: From a solid agar medium, transfer a portion of a 2. Test organism: It is often necessary to increase the motility of the
loopful of an isolated colony to each of the two reaction areas test organism. To accomplish this, make several consecutive
above and mix thoroughly. transfers in Motility GI Medium.
• Inoculate the tube slightly below the surface of the medium
4. Positive control: Dispense 1 drop of each Salmonella O Antiserum
using the stab method.
to be tested on an agglutination slide. Add 1 drop of an appropriate
QC Antigen Salmonella. • Incubate at 35-37°C for 18-20 hours.
• Transfer only those organisms that have migrated to the bottom
5. Rotate the slides for 1 minute and read for agglutination. Results
of the tube.
must be read within 1 minute.
• When the organism successfully travels 50-60 mm through the
Slide Test Results medium in 18-20 hours, it is ready for use.
1. Read and record results as follows: • An infusion broth such as Veal Infusion Broth is recommended
4+ 100% agglutination; background is clear to slightly hazy. for cultivating the motile Salmonella prior to testing. It should
be inoculated and incubated at 35°C for 24 hours. Brain Heart
3+ 75% agglutination; background is slightly cloudy.
Infusion Broth may be used with incubation at 35°C for 4-6 hours.
2+ 50% agglutination; background is moderately cloudy. If Tryptic Soy Broth is used, incubate at 35°C for 24 hours.
1+ 25% agglutination; background is cloudy. • Prepare the test organism suspension by using equal volumes
– No agglutination. of broth culture and 0.6% formalized saline. The final density
2. Positive control: Should show a 3+ or greater agglutination. of this test suspension should be that of a McFarland Barium
3. Negative control: Should show no agglutination. If agglutination Sulfate Standard No. 3.
occurs, the culture is rough and cannot be tested. Subculture to a 3. Positive control: Commercially prepared QC Salmonella H anti-
non-inhibitory medium, incubate and test the organism again. gens are not available. The user must maintain stock cultures of
4. Test isolates: 3+ or greater agglutination is a positive result. known serological identification for use in quality control. Prepare
5. A partial (less than 3+) or delayed agglutination reaction should the antigen by using known serotypes and following the procedure
be considered negative. described above. (See Test organism, above.)
6. If a positive reaction occurs with Salmonella Vi Antiserum, follow 4. Salmonella H Antisera: Rehydrated antisera is considered a 1:2
this procedure: working dilution. Prepare dilutions as follows and use on the day
prepared. Discard any unused portion.
• Prepare a dense suspension of the isolate from an agar medium
in 3-5 ml of sterile 0.85% NaCl solution. • Most Salmonella H Antisera: A 1:1,000 final dilution is used.
Prepare by adding 0.1 ml rehydrated antiserum to 25 ml of
• Heat in a boiling waterbath for 30-60 minutes and cool. The
0.85% NaCl solution.
suspension should not precipitate after heating. If this occurs,
select another colony for testing. • Salmonella H Antisera x, z 13, z 15 and z 28: A 1:500 final
dilution is used. Prepare by adding 0.1 ml rehydrated antiserum
• Centrifuge at 1,000 rpm for 10-15 minutes.
to 12.5 ml of 0.85% NaCl solution.
• Aspirate and discard the supernatant.
• Salmonella H Antiserum Poly a-z: A 1:100 final dilution is
• Resuspend the sediment in 0.5 ml sterile 0.85% NaCl solution. used. Prepare by adding 0.1 ml rehydrated antiserum to 2.5 ml
• Retest a drop of the sediment with Salmonella O Group of 0.85% NaCl solution.
Antisera, as outlined.
11. There may exist common antigens between various “O” serogroups 14. Russell, S. F., J. D’Aoust, W. H. Andrews, and J. S. Bailey. 1992.
of Salmonella. As an example, Salmonella O Antiserum Poly A Salmonella. In C. Vanderzant, C., and Splittstoesser, D.F. (eds.),
contains, among others, agglutinins for factor 1, since cultures Compendium of methods for the microbiological examination of
possessing factor 1 were used in immunization. It may be expected foods, 3rd ed. American Public Health Association, Washington, D.C.
that this polyvalent antiserum will react with cultures other than
those contained in “O” serogroups A, B, D, E, and L due to the Packaging
common 1 antigen (those organisms in Group G1, G2, H, R, T, etc., Salmonella H Antiserum a 3 ml 2820-47
which contain factor 1). Salmonella H Antiserum b 3 ml 2821-47
12. Salmonella O Antiserum Poly A-I & Vi has been prepared with Salmonella H Antiserum c 3 ml 2822-47
representative members of those somatic groups and has not been
absorbed. It is obvious that this serum may and will react with Salmonella H Antiserum d 3 ml 2823-47
higher O groups of Salmonella. Salmonella H Antiserum eh 3 ml 2273-47
Salmonella H Antiserum f 3 ml 2544-47
References
1. Holt, J. G., N. R. Krieg, P. H. Sneath, J. T. Staley, and S. T. Salmonella H Antiserum h 3 ml 2545-47
Williams. 1994. Bergey’s manual of determinative bacteriology, Salmonella H Antiserum I 3 ml 2824-47
9th ed. Williams & Wilkins, Baltimore, MD. Salmonella H Antiserum k 3 ml 2274-47
2. McWhorter-Murlin, A. C., and F. W. Hickman-Brenner. 1994. Salmonella H Antiserum m 3 ml 2546-47
Identification and serotyping of Salmonella and an update of the
Salmonella H Antiserum p 3 ml 2548-47
Kauffmann-White Scheme. Centers for Disease Control and
Prevention, Atlanta, GA. Salmonella H Antiserum r 3 ml 2275-47
3. Popoff, M. Y., and L. LeMinor. 1997. Antigenic Formulas of the Salmonella H Antiserum s 3 ml 2550-47
Salmonella Serovars. WHO Collaborating Centre for Reference Salmonella H Antiserum t 3 ml 2551-47
and Research on Salmonella. Institut Pasteur, Paris, France. Salmonella H Antiserum w 3 ml 2554-47
4. Penner, J. L. 1988. International committee on systematic
Salmonella H Antiserum x 3 ml 2555-47
bacteriology taxonomic subcommittee on Enterobacteriaceae. Int.
J. Syst. Bacteriol. 38:223-224. Salmonella H Antiserum y 3 ml 2276-47
5. LeMinor, L., and M. Y. Popoff. 1987. Request for an opinion. Salmonella H Antiserum z 3 ml 2277-47
Designation of Salmonella enterica sp. nov., nom. rev., as the type Salmonella H Antiserum z6 3 ml 2473-47
and only species of the genus Salmonella. Int. J. Syst. Bacteriol. Salmonella H Antiserum z10 3 ml 2279-47
37:465-468.
Salmonella H Antiserum z13 3 ml 2556-47
6. Wayne, L. G. 1991. Judicial Commission of the International Com-
mittee on Systematic Bacteriology. Int. J. Syst. Bacteriol. 41:185-187. Salmonella H Antiserum z15 3 ml 2557-47
7. Wayne, L. G. 1994. Actions of the Judicial Commission of the Salmonella H Antiserum z23 3 ml 2558-47
International Committee on Systematic Bacteriology on requests Salmonella H Antiserum z28 3 ml 2561-47
for opinions published between January 1985 and July 1993. Int. Salmonella H Antiserum z29 3 ml 2280-47
J. Syst. Bacteriol. 44:177.
Salmonella H Antiserum z32 3 ml 2562-47
8. Ewing, W.H. 1986. Edwards and Ewing’s Identification of
Enterobacteriaceae, 4th ed. Elisevier Science Publishing Co., Inc., Salmonella H Antiserum EN Complex 3 ml 2270-47
New York, NY. Salmonella H Antiserum G Complex 3 ml 2269-47
9. Old, D. C. 1992. Nomenclature of Salmonella. J. Med. Microbiol. Salmonella H Antiserum L Complex 3 ml 2271-47
37:361-363. Salmonella H Antiserum Z4 Complex 3 ml 2278-47
10. Farmer III, J. J., III, A. C. McWhorter, D. J. Brenner, and G.
Salmonella H Antiserum Poly a-z 3 ml 2406-47
D. Morris. 1984. The Salmonella-Arizona group of Enterobacte-
riaceae: nomenclature, classification and reporting. Clin. Salmonella H Antiserum 3 ml 2539-47
Microbiol. Newsl. 6:63-66. Poly A (a,b,c,d,i,z10,z29)
11. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and Salmonella H Antiserum Poly B 3 ml 2540-47
R. H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed. (eh,en,enx,enz15, and G Complex)
American Society for Microbiology, Washington, D.C. Salmonella H Antiserum 3 ml 2541-47
12. Isenberg, H. D. (ed.) 1992. Clinical microbiology procedures Poly C (k,l,r,y,z1,z4)
handbook, vol. 2. American Society for Microbiology, Salmonella H Antiserum 3 ml 2542-47
Washington, D.C. Poly D (z35,z36,z37,z38,z39,z41,z42)
13. Andrews, W. H., G. A. June, P. Sherrod, T. S. Hammack, Salmonella H Antiserum 3 ml 2543-47
and R. M. Amaguana. 1995. Food and drug administration Poly E (1 Complex, z6)
bacteriological analytical manual, 8th ed. AOAC International, Salmonella H Antiserum 3 ml 2474-47
Gaithersburg, MD. Single Factor 2
When writing an antigenic formula, list the O antigen(s) first followed 14. Andrews, W. H., G. A. June, P. Sherrod, T. S. Hammack,
by the H antigen(s). Separate the major antigens by colons and the and R. M. Amaguana. 1995. Food and drug administration
components of the antigens by commas. For example, the antigenic bacteriological analytical manual, 8th edition. AOAC International,
formula for Salmonella Typhimurium is Salmonella 1,4,5,12:i:1,2. This Gaithersburg, MD.
means that the strain has O antigen factors 1,4,5 and 12, the flagella 15. Russell, S. F., J. D’Aoust, W. H. Andrews, and J. S. Bailey. 1992.
phase 1 antigen i, and flagellar phase 2 antigens 1 and 2. Salmonella. In Vanderzant, C. and D. F. Splittstoesser, (eds.),
Complete identification of Salmonella requires cultural isolation, Compendium of methods for the microbiological examination of
biochemical characterization and serotyping. Any serological foods, 3rd ed. American Public Health Association, Washington, D.C.
results obtained before biochemical identification must be considered 16. Popoff, M, Y., and L. LeMinor. 1992. Antigenic formulas of
as presumptive identification only. Refer to appropriate references for the Salmonella serovars, 6 th revision. WHO Collaborating
complete identification of Salmonella.1,3,4,10,12-15 Centre for Reference and Research on Salmonella. Pasteur
The Kauffmann-White Scheme is presented in two forms. Appendix A Institute, Paris, France.
contains a list of Salmonella Serotypes by O Group. Appendix B 17. Rohde, R. 1979. Serological integration of all known Arizona
contains an alphabetical list of Salmonella Serotypes. species into the Kauffmann-White scheme. Zentralbl. Bakteriol.
Hyg, I. Abt. Orig. A. 243:148-176.
References 18. LeMinor, L. 1984. Genus III. Salmonella. In N. R. Krieg, (ed.),
1. McWhorter-Murlin, A. C. and F. W. Hickman-Brenner. 1994. Bergey’s manual of systematic bacteriology, vol. 1. The Williams
Identification and Serotyping of Salmonella and an update of the and Wilkins Co., Baltimore, MD.
Kauffmann-White Scheme. Centers for Disease Control and
Prevention, Atlanta, GA. Appendix A
2. Kauffmann, F. 1969. Enterobacteriaceae, 2nd ed. Munksgaard, Kauffmann-White Scheme
Copenhagen.
3. Ewing, W. H. 1986. Edwards and Ewing’s identification of List of Salmonella Serotypes by O Group
Enterobacteriaceae, 4th ed. Elsevier Science Publishing Co. Inc., (Updated 1994)
New York, NY.
4. Popoff, M. Y. and L. LeMinor. 1997. Antigenic Formulas of the Appendix A contains a list of Salmonella Serotypes by O Group. The
Salmonella Serovars. WHO Collaborating Centre for Reference serotypes are sorted by O group first, then by Phase I and Phase 2 of
and Research on Salmonella. Institut Pasteur, Paris, France. the H antigens. The z antigens do not appear in the correct numerical
5. Penner, J. L. 1988. International committee on systematic order - z4 will be listed after z10 because the 1 of 10 is read first, and
bacteriology taxonomic subcommittee on Enterobacteriaceae. Int. will appear after z29.
J. Syst. Bacteriol. 38:223-224. Key:
6. LeMinor, L. and M. Y. Popoff. 1987. Request for an opinion.
Designation of Salmonella enterica sp. nov., nom. rev., as the type IP Institut Pasteur. See reference #16 in
and only species of the genus Salmonella. Int. J. Syst. Bacteriol. Kauffmann-White Scheme monograph published
37:465-468. by WHO Collaborating Centre for Reference and
7. Wayne, L. G. 1991. Judicial Commission of the International
Research on Salmonella.
Committee on Systematic Bacteriology. Int. J. Syst. Bacteriol. Ar. “Arizona” antigenic formula
41:185-187. Rohde Refer to reference #17 in Kauffmann-White
8. Wayne, L. G. 1994. Actions of the Judicial Commission of the Scheme monograph by R. Rohde. He
International Committee on Systematic Bacteriology on requests incorporated all known Arizona serotypes
for opinions published between January 1985 and July 1993. Int. into the Kauffmann-White Scheme.
J. Syst. Bacteriol. 44:177. Bergey Refer to reference #18 in Kauffmann-White
9. Old, D. C. 1992. Nomenclature of Salmonella. J. Med. Microbiol. Scheme monograph by L. LeMinor for
37:361-363. “Bergey’s Manual of Systematic Bacteriology.”
10. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and Underlined
R. H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed. Numbers Numbers that are underlined in a serotype
American Society for Microbiology, Washington, D.C. represent somatic factors determined by phage
11. Farmer, J. J., III, A. C. McWhorter, D. J. Brenner, and conversion. They are present if the culture is
G. D. Morris. 1984. The Salmonella-Arizona group of lysogenized by the corresponding converting
Enterobacteriaceae: nomenclature, classification and reporting. phage.
Clin. Microbiol. Newsl. 6:63-66. [ ] O or H factors may be present or absent
12. Isenberg, H. D. (ed.) 1992. Clinical microbiology procedures hand- without relation to phage conversion.
book, vol. 2. American Society for Microbiology, Washington, D.C. ( ) O or H factor is weakly agglutinable.
13. Holt, J. G., N. R. Krieg, P. H. Sneath, J. T. Staley, S. T. R phases Abnormal specificities of H antigens that were
Williams. 1994. Bergey’s manual of determinative bacteriology, described by Kauffmann. They are uncommon.
9th ed. Williams & Wilkins, Baltimore, MD.
II C1 6,7 k [z6]
IIIa C1 6,7 (k) z:[z55] (Ar. 27:22:31:[37])
I C1 Thompson 6,7,14 [k] [1,5] IP combined Cardiff that contains H phase
R1,10 (6,7:k:R1,10) with Thompson.
I C1 Concord 6,7 l,v 1,2
I C1 Irumu 6,7 l,v 1,5
I C1 Mkamba 6,7 l,v 1,6
I C1 Kortrijk 6,7 l,v 1,7
I C1 Bonn 6,7 l,v e,n,x
I C1 Potsdam 6,7,14 l,v e,n,z15
I C1 Coromandel 6,7 l,v z35
IIIb C1 6,7 l,v z53 (Ar. 27:23:25)
I C1 Gdansk 6,7 l,v z6 IP combined Gelsenkirchen (6,7,14:l,v:z6)
with Gdansk to form Gdansk 6,7,14:l,v:z6.
I C1 Gelsenkirchen 6,7,14 l,v z6 IP combined Gelsenkirchen with Gdansk
(6,7:l,v:z6) to form Gdansk 6,7,14:l,v:z6.
Gelsenkirchen is now called Gdansk
var. O 14+ by IP.
I C1 Gabon 6,7 l,w 1,2
I C1 Colorado 6,7 l,w 1,5
II C1 6,7 l,w 1,5,7
II C1 6,7 l,w z42
I C1 Nessziona 6,7 l,z13 1,5
I C1 Kenya 6,7 l,z13 e,n,x
I C1 Strathcona 6,7 l,z13,z28 1,7
I C1 Makiso 6,7 l,z13,z28 z6
I C1 Neukoelin 6,7 l,z13,[z28] e,n,z15
II C1 Heilbron 6,7 l,z28 1,5:[z42]
II C1 6,7 l,z28 e,n,x
II C1 6,7 l,z28 z6
I C1 Haelsingborg 6,7 m,p,t,[u] –
I C1 Oranienburg 6,7 m,t – IP combined Theilallee (6,7,14:m,t:-) with
Oranienburg to form Oranienburg 6,7,14:m,t:-.
Oranienburg may possess H phase Rz57.
I C1 Thielallee 6,7,14 m,t – IP combined Thielallee with Oranienburg
(6,7:m,t:-) to form Oranienburg 6,7,14:m,t:-.
Theilallee is now called Oranienburg
var. O 14+ by IP.
II C1 6,7 m,t –
I C1 Winston 6,7 m,t 1,6
I C1 Oakey 6,7 m,t z64
I C1 Virchow 6,7 r 1,2
I C1 Infantis 6,7,14 r 1,5 Infantis may possess H phase Rz49.
I C1 Nigeria 6,7 r 1,6
I C1 Colindale 6,7 r 1,7
I C1 Papuana 6,7 r e,n,z15
I C1 Grampian 6,7 r l,w
I C1 Richmond 6,7 y 1,2
I C1 Bareilly 6,7,14 y 1,5
I C1 Oyonnax 6,7 y 1,6
I C1 Gatow 6,7 y 1,7
I C2 Labadi 8,20 d z6
II C2 6,8 d z6:z42
I C2 Bardo 8 e,h 1,2
I C2 Newport 6,8,20 e,h 1,2 Newport may possess H phase Rz50 or Rz58
or Rz78 or R1,12
I C2 Ferruch 8 e,h 1,5
I C2 Kottbus 6,8 e,h 1,5
I C2 Cremieu 6,8 e,h 1,[6]
I C2 Atakpame 8,20 e,h 1,7
I C2 Tshiongwe 6,8 e,h e,n,z15
I C2 Rechovot 8,20 e,h z6
I C2 Sandow 6,8 f,g e,n,z15
II C2 6,8 f,g,m,t [e,n,x]
I C2 Emek 8,20 g,m,s –
I C2 Chincol 6,8 g,m,[s] [e,n,x]
I C2 Reubeuss 8,20 g,m,t –
II C2 6,8 g,m,t 1,7
I C2 Alminko 8,20 g,s,t –
I C2 Nanergou 6,8 g,s,t –
I C2 Lindenburg 6,8 i 1,2
I C2 Bargny 8,20 i 1,5
I C2 Takoradi 6,8 i 1,5
I C2 Warnow 6,8 i 1,6
I C2 Malmoe 6,8 i 1,7
I C2 Bonariensis 6,8 i e,n,x
I C2 Aba 6,8 i e,n,z15
I C2 Cyprus 6,8 i l,w
I C2 Magherafelt 8,20 i l,w
I C2 Kentucky 8,20 i z6
I C2 Kallo 6,8 k 1,2
I C2 Blockley 6,8 k 1,5 Blockley may possess H phase Rz58
I C2 Haardt 8 k 1,5
I C2 Schwerin 6,8 k e,n,x
I C2 Charlottenburg 6,8 k e,n,z15
I C2 Litchfield 6,8 l,v 1,2
I C2 Pakistan 8 l,v 1,2
I C2 Loanda 6,8 l,v 1,5
I C2 Manchester 6,8 l,v 1,7
I C2 Holcomb 6,8 l,v e,n,x
II C2 6,8 l,v e,n,x
I C2 Edmonton 6,8 l,v e,n,z15
I C2 Amherstiana 8 l,(v) 1,6
I C2 Fayed 6,8 l,w 1,2
II C2 6,8 l,w z6:z42
I C2 Hiduddify 6,8 l,z13,z28 1,5
II C2 6,8 l,z28 e,n,x
I C2 Breukelen 6,8 l,z13,[z28] e,n,z15
I C2 Bassa 6,8 m,t -
I C2 Yokoe 8,20 m,t -
The Difco Manual 685
Salmonella, Antigenic Scheme Section V
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I F Pharr 11 b e,n,z15
I F Chiredzi 11 c 1,5
I F Woodinville 11 c e,n,x
II F 11 c e,n,z15
I F Ati 11 d 1,2
I F Gustavia 11 d 1,5
I F Chandans 11 d e,n,x:[r]
I F Pennsylvania 11 d e,n,z15
I F Findorff 11 d z6
I F Chingola 11 e,h 1,2
I F Adamstua 11 e,h 1,6
I F Redhill 11 e,h l,z13,z28
II F Grabouw 11 g,[m],s,t [z39]
I F Missouri 11 g,s,t –
IV F Mundsburg 11 g,z51 –
I F Aberdeen 11 i 1,2
I F Brijbhumi 11 i 1,5
I F Heerlen 11 i 1,6
I F Veneziana 11 i e,n,x
I F Pretoria 11 k 1,2
I F Abaetetuba 11 k 1,5
I F Sharon 11 k 1,6
I F Colobane 11 k 1,7
I F Kisarawe 11 k e,n,x,[z15]
I F Mannheim 11 k l,w
I F Amba 11 k l,z13,z28
IIIb F 11 k z53 (Ar. 17:29:25)
I F Stendal 11 l,v 1,2
I F Maracaibo 11 l,v 1,5
I F Fann 11 l,v e,n,x
I F Bullbay 11 l,v e,n,z15
IIIb F 11 l,v z53 (Ar. 17:23:25)
IIIb F 11 l,v z (Ar. 17:23:31). May possess H phase Rz56 (Ar. 38).
I F Glidji 11 l,w 1,5
I F Connecticut 11 l,z13,z28 1,5
I F Osnabrueck 11 l,z13,z28 e,n,x
II F Huila 11 l,z28 e,n,x
I F Moers 11 m,t –
II F Lincoln 11 m,t e,n,x
I F Senegal 11 r 1,5
I F Rubislaw 11 r [e,n,x]
I F Volta 11 r l,z13,z28
I F Euston 11 r,i e,n,x,z15
I F Solt 11 y 1,5
I F Jalisco 11 y 1,7
I F Herzliya 11 y e,n,x
I F Crewe 11 z 1,5
I F Maroua 11 z 1,7
II F 11 z e,n,x
698 The Difco Manual
Section V Salmonella, Antigenic Scheme
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
II F Soutpan 11 z z39
I F Nyanza 11 z z6
I F Wentworth 11 z10 1,2
I F Straengnaes 11 z10 1,5
I F Telhashomer 11 z10 e,n,x
I F Lene 11 z38 –
IIIa F 11 z4,z23 – (Ar. 17:1,2,5:-)
IV F Parera 11 z4,z23 –
I F Remete 11 z4,z23 1,6
I F Etterbeek 11 z4,z23 e,n,z15
I F Yehuda 11 z4,z24 –
IV F 11 z4,z32 –
I F Maastricht 11 z41 1,2
II G 13,23 – 1,6
I G Chagoua 1,13,23 a 1,5
II G 1,13,23 a 1,5
I G Mim 13,22 a 1,6
II G 13,22 a e,n,x
I G Wyldegreen 1,13,23 a l,w
I G Marshall 13,22 a l,z13,z28
II G Tygerberg 1,13,23 a z42
I G Atlanta 13,23 b – Atlanta was combined with Mississippi
(1,13,23:b:1,5). The name Atlanta has
been dropped.
I G Ibadan 13,22 b 1,5
I G Mississippi 1,13,23 b [1,5]
II G Acres 1,13,23 b [1,5]:z42
I G Bracknell 13,23 b 1,6
I G Oudwijk 13,22 b 1,6
I G Rottnest 1,13,22 b 1,7
I G Ullevi 1,13,23 b e,n,x
I G Vaertan 13,22 b e,n,x
I G Bahati 13,22 b e,n,z15
I G Durham 13,23 b e,n,z15
II G 1,13,22 b z42
I G Haouaria 13,22 c e,n,x,z15
I G Handen 1,13,23 d 1,2
I G Mishmarhaemek 1,13,23 d 1,5
I G Friedenau 13,22 d 1,6
I G Wichita 1,13,23 d 1,6 Wichita may possess H phase Rz37.
I G Grumpensis 1,13,23 d 1,7
II G 13,23 d e,n,x
I G Diguel 1,13,22 d e,n,z15
I G Telelkebir 13,23 d e,n,z15
II G 1,13,23 d e,n,z15
I G Putten 13,23 d l,w
I G Isuge 13,23 d z6
I G Tschangu 1,13,23 e,h 1,5
I G Willemstad 1,13,22 e,h 1,6
The Difco Manual 699
Salmonella, Antigenic Scheme Section V
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I I Saphra 16 y 1,5
I I Akuafo 16 y 1,6
I I Kikoma 16 y e,n,x
I I Avignon 16 y e,n,z15
I I Fortlamy 16 z 1,6
I I Lingwala 16 z 1,7
II I Louwbester 16 z [e,n,x]
I I Brevik 16 z e,n,[x],z15
II I 16 z z42
I I Bouake 16 z z6
I I Badagry 16 z10 1,5
IIIb I 16 z10 1,5,7 (Ar. 25:27:30)
I I Lisboa 16 z10 1,6
IIIb I 16 z10 e,n,x,z15 (Ar. 25:27:28)
I I Redlands 16 z10 e,n,z15
I I Angouleme 16 z10 z6
I I Saloniki 16 z29 –
II I 16 z29 1,5
II I Jacksonville 16 z29 [e,n,x]
I I Trier 16 z35 1,6
I I Dakota 16 z35 e,n,z15
IV I 16 z36 –
I I Naware 16 z38 –
I I Grancanaria 16 z39 [1,6] Grancanaria can be d-tartrate neg., dulcitol
neg., ONPG pos., and anaerogenic.
II I Haddon 16 z4,z23 –
IV I Ochsenzoll 16 z4,z23 –
I I Kibi 16 z4,z23 [1,6]
II I 16 z4,z24 –
IV I Chameleon 16 z4,z32 –
II I Woodstock 16 z42 1,[5],7
IIIb I 16 z52 z35 (Ar. 25:26:21)
II I 16 z6 1,6
I J Bonames 17 a 1,2
I J Jangwani 17 a 1,5
I J Kinondoni 17 a e,n,x
I J Kirkee 17 b 1,2
I J Dahra 17 b 1,5
II J Hillbrow 17 b e,n,x,z15
I J Bignona 17 b e,n,z15
II J 17 b z6
I J Victoriaborg 17 c 1,6
II J Woerden 17 c z39
I J Berlin 17 d 1,5
I J Niamey 17 d l,w
I J Jubilee 17 e,h 1,2
II J 17 e,n,x,z15 1,[5],7
II J Verity 17 e,n,x,z15 1,6
II J Bleadon 17 (f),g,t [e,n,x,z15] IP has dropped f.
The Difco Manual 705
Salmonella, Antigenic Scheme Section V
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
II J 17 g,m,s,t –
I J Lowestoft 17 g,s,t –
II J 17 g,t z39
I J Ahanou 17 i 1,7
IIIb J 17 i z35 (Ar. 12:33:21)
II J 17 k –
I J Irenea 17 k 1,5
I J Warri 17 k 1,7
I J Matadi 17 k e,n,x
I J Zaria 17 k e,n,z15
IIIb J 17 k z (Ar. 12:29:32)
I J Morotai 17 l,v 1,2
I J Michigan 17 l,v 1,5
I J Lancaster 17 l,v 1,7
I J Carmel 17 l,v e,n,x
IIIb J 17 l,v e,n,x,z15 (Ar. 12:23:28)
IIIb J 17 l,v z35 (Ar. 12:23:21)
I J Granlo 17 l,z28 e,n,x
I J Bama 17 m,t –
II J 17 m,t –
I J Lode 17 r 1,2
IIIb J 17 r z (Ar. 12:24:31)
II J 17 y –
I J Hadejia 17 y e,n,z15
I J Gori 17 z 1,2
I J Warengo 17 z 1,5
I J Tchamba 17 z e,n,z15
II J Constantia 17 z l,w:z42
I J Djibouti 17 z10 e,n,x
IIIb J 17 z10 e,n,x,z15 (Ar.12:27:28). May possess H phase Rz56 (Ar. 38).
IIIb J 17 z10 z (Ar. 12:27:31)
I J Kandla 17 z29 –
IIIa J 17 z29 – (Ar. 12:16,17,18:-)
IV J 17 z29 –
IIIa J 17 z36 – (Ar. 12:17,20:-)
IV J 17 z36 –
IIIa J 17 z4,z23 – (Ar. 12:1,2,5:- and 12:1,2,6:-)
IIIa J 17 z4,z23,z32 – (Ar. 12:1,6,7,9:-)
IIIa J 17 z4,z24 – (Ar. 12:1,3,11:-)
IIIa J 17 z4,z32 – (Ar. 12:1,6,7:- and 12:1,7,8:-)
I K Cotia 18 – 1,6
I K Brazos 6,14,18 a e,n,z15
I K Fluntern 6,14,18 b 1,5
I K Rawash 6,14,18 c e,n,x
I K Groenekan 18 d 1,5
I K Usumbura 6,14,18 d 1,7
I K Pontypridd 18 g,m –
IIIa K 18 g,z51 – (Ar. 7a,7b:13,14:-)
I K Memphis 18 k 1,5
706 The Difco Manual
Section V Salmonella, Antigenic Scheme
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
IV L 21 g,z51 –
I L Diourbel 21 i 1,2
IIIb L 21 i 1,5,7 (Ar. 22:33:30)
IIIb L 21 i e,n,x,z15 (Ar. 22:33:28)
IIIb L 21 k e,n,x,z15 (Ar. 22:29:28)
IIIb L 21 k z (Ar. 22:29:31)
IIIb L 21 l,v z (Ar. 22:23:31)
IIIb L 21 l,v z57 (Ar. 22:23:40)
I L Keve 21 l,w –
I L Jambur 21 l,z28 e,n,z15
II L 21 m,t –
I L Mountmagnet 21 r –
IIIb L 21 r z (Ar. 22:24:31)
I L Ibaragi 21 y 1,2
I L Ruiru 21 y e,n,x
II L 21 z – CDC does not have this.
IIIb L 21 z10 e,n,x,z15 (Ar. 22:27:28)
IIIb L 21 z10 z (Ar. 22:27:31)
IIIb L 21 z10 z53 (Ar. 22:27:25)
II L Wandsbek 21 z10 [z6]
IIIa L 21 z29 – (Ar. 22:16,17,18:-)
I L Gambaga 21 z35 e,n,z15
IV L 21 z36 –
I L Baguida 21 z4,z23 –
IIIa L 21 z4,z23 – (Ar. 22:1,2,5:- and 22:1,2,6:-)
IV L Soesterberg 21 z4,z23 –
II L Gwaai 21 z4,z24 –
IIIa L 21 z4,z24 – (Ar. 22:1,3,11:-)
IV L 21 z4,z32 –
IIIb L 21 z65 e,n,x,z15 (Ar. 22:32:28)
I M Solna 28 a 1,5
I M Dakar 28 a 1,6
I M Bakau 28 a 1,7
I M Seattle 28 a e,n,x
II M 28 a e,n,x
I M Honelis 28 a e,n,z15
I M Dibra 28 a z6
I M Moero 28 b 1,5
I M Ashanti 28 b 1,6
I M Bokanjac 28 b 1,7
I M Soumbedioune 28 b e,n,x
II M 28 b e,n,x
I M Langford 28 b e,n,z15
II M Kaltenhausen 28 b z6
I M Hermannswerder 28 c 1,5
I M Eberswalde 28 c 1,6
I M Halle 28 c 1,7
I M Dresden 28 c e,n,x
I M Wedding 28 c e,n,z15
708 The Difco Manual
Section V Salmonella, Antigenic Scheme
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I M Techimani 28 c z6
I M Amoutive 28 d 1,5
I M Hatfield 28 d 1,6
I M Mundonobo 28 d 1,7
I M Mocamedes 28 d e,n,x
I M Patience 28 d e,n,z15
I M Cullingworth 28 d l,w
I M Kpeme 28 e,h 1,7
I M Gozo 28 e,h e,n,z15
II M 28 e,n,x 1,7
I M Friedrichsfelde 28 f,g –
I M Yardley 28 g,m 1,6
I M Abadina 28 g,m [e,n,z15]
I M Croft 28 g,m,s [e,n,z15]
II M 28 g,m,t e,n,x
II M 28 g,m,t z39
II M Llandudno 28 g,[m],[s],t 1,5
I M Ona 28 g,s,t -
II M 28 g,s,t e,n,x
I M Doorn 28 i 1,2
I M Cotham 28 i 1,5
I M Volksmarsdorf 28 i 1,6
I M Dieuppeul 28 i 1,7
I M Warnemuende 28 i e,n,x
I M Kuessel 28 i e,n,z15
I M Douala 28 i l,w
I M Guildford 28 k 1,2
I M Ilala 28 k 1,5
I M Adamstown 28 k 1,6
I M Ikeja 28 k 1,7
I M Taunton 28 k e,n,x
I M Ank 28 k e,n,z15
I M Leoben 28 l,v 1,5
I M Vitkin 28 l,v e,n,x
I M Nashua 28 l,v e,n,z15
I M Ramsey 28 l,w 1,6
I M Catalunia 28 l,z13,z28 1,5
I M Penilla 28 l,z13,z28 e,n,z15
II M 28 l,z28 1,5
I M Fajara 28 l,z28 e,n,x
I M Morillons 28 m,t 1,6
II M 28 m,t [e,n,x]
I M Vinohrady 28 m,t [e,n,z15]
I M Bassadji 28 r 1,6
I M Kibusi 28 r e,n,x
II M Oevelgoenne 28 r e,n,z15
I M Fairfield 28 r l,w
I M Banco 28 r,i 1,7
I M Chicago 28 r,[i] 1,5
The Difco Manual 709
Salmonella, Antigenic Scheme Section V
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I M Kitenge 28 y e,n,x
I M Telaviv 28 y e,n,z15
I M Shomolu 28 y l,w
I M Selby 28 y z6
I M Vanier 28 z 1,5
II M 28 z 1,5
I M Doel 28 z 1,6
I M Ezra 28 z 1,7
I M Brisbane 28 z e,n,z15
II M Ceres 28 z z39
I M Rogy 28 z10 1,2
I M Farakan 28 z10 1,5
I M Libreville 28 z10 1,6
I M Malaysia 28 z10 1,7
I M Umbilo 28 z10 e,n,x
I M Luckenwalde 28 z10 e,n,z15
I M Moroto 28 z10 l,w
IIIb M 28 z10 z (Ar. 35:27:31)
IIIb M 28 z10 z:[z57] (Ar. 35:27:31:[40])
I M Djermaia 28 z29 –
II M 28 z29 1,5
II M 28 z29 e,n,x
I M Konolfingen 28 z35 1,6
I M Babili 28 z35 1,7
I M Santander 28 z35 e,n,z15
I M Aderike 28 z38 [e,n,z15]
I M Cannobio 28 z4,z23 1,5
I M Teltow 28 z4,z23 1,6
I M Babelsberg 28 z4,z23 [e,n,z15]
I N Overvecht 30 a 1,2
I N Zehlendorf 30 a 1,5
I N Guarapiranga 30 a e,n,x
I N Doulassame 30 a e,n,z15
II N Odijk 30 a z39
I N Louga 30 b 1,2
I N Aschersleben 30 b 1,5
I N Urbana 30 b e,n,x
I N Neudorf 30 b e,n,z15
II N 30 b z6
I N Zaire 30 c 1,7
I N Morningside 30 c e,n,z15
II N 30 c z39
I N Messina 30 d 1,5
I N Livulu 30 e,h 1,2
710 The Difco Manual
Section V Salmonella, Antigenic Scheme
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I O Ealing 35 g,m,s –
II O 35 g,m,s,t –
I O Ebrie 35 g,m,t –
I O Anecho 35 g,s,t –
I O Agodi 35 g,t –
II O 35 g,t 1,5
II O 35 g,t z42
IIIa O 35 g,z51 – (Ar. 20:13,14:-)
IIIb O 35 i e,n,x,z15 (Ar. 20:33:28)
I O Gambia 35 i e,n,z15
I O Bandia 35 i l,w
IIIb O 35 i z (Ar. 20:33:31)
IIIb O 35 i z35 (Ar. 20:33:21)
IIIb O 35 i z53 (Ar. 20:33:25)
IIIb O 35 k e,n,x,z15 (Ar. 20:29:28)
IIIb O 35 k z (Ar. 20:29:31)
IIIb O 35 k z53 (Ar. 20:29:25). May possess H phase
Rz50 (Ar.42).
IIIb O 35 (k) z (Ar. 20:22:31)
IIIb O 35 (k) z35 (Ar. 20:22:21)
IIIb O 35 l,v 1,5,7 (Ar. 20:23:30)
IIIb O 35 l,v e,n,x,z15 (Ar. 20:23:28)
IIIb O 35 l,v z35 (Ar. 20:23:21)
II O 35 l,z28 –
I O Monschaui 35 m,t -
II O 35 m,t -
IIIb O 35 r e,n,x,z15 (Ar. 20:24:28)
I O Massakory 35 r l,w
IIIb O 35 r z (Ar. 20:24:31)
IIIb O 35 r z35 (Ar. 20:24:21)
IIIb O 35 r z61 (Ar. 20:24:41)
I O Camberene 35 z10 1,5
I O Enschede 35 z10 l,w
IIIb O 35 z10 z35 (Ar. 20:27:21)
I O Ligna 35 z10 z6
I O Widemarsh 35 z29 –
IIIa O 35 z29 – (Ar. 20:16,17,18:-)
II O Utbremen 35 z29 e,n,x
IIIa O 35 z36 – (Ar. 20:17,20:-)
I O Haga 35 z38 –
I O Alachua 35 z4,z23 – Alachua may possess H phase Rz37 or Rz45.
IIIa O 35 z4,z23 – (Ar. 20:1,2,6:-)
I O Westphalia 35 z4,z24 –
IIIa O 35 z4,z32 – (Ar. 20:1,7,8:-)
IIIb O 35 z52 1,5,7 (Ar. 20:26:30)
IIIb O 35 z52 e,n,x,z15 (Ar. 20:26:28)
IIIb O 35 z52 z (Ar. 20:26:31)
IIIb O 35 z52 z35 (Ar. 20:26:21)
II P 38 b 1,2
712 The Difco Manual
Section V Salmonella, Antigenic Scheme
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I P Rittersbach 38 b e,n,z15
I P Sheffield 38 c 1,5
I P Kidderminster 38 c 1,6
II P Carletonville 38 d [1,5]
I P Thiaroye 38 e,h 1,2
I P Kasenyi 38 e,h 1,5
I P Korovi 38 g,m,[s] –
II P Foulpointe 38 g,t –
IIIa P 38 g,z51 – (Ar. 16:13,14:-)
IV P 38 g,z51 –
I P Mgulani 38 i 1,2
I P Lansing 38 i 1,5
IIIb P 38 i z (Ar. 16:33:31)
IIIb P 38 i z53 (Ar. 16:33:25)
I P Echa 38 k 1,2
I P Mango 38 k 1,5
I P Inverness 38 k 1,6
I P Njala 38 k e,n,x
IIIb P 38 k e,n,x,z15 (Ar. 16:29:28)
IIIb P 38 k z (Ar. 16:29:31)
IIIb P 38 k z53 (Ar. 16:29:25)
IIIb P 38 (k) 1,5,7 (Ar. 16:22:30)
IIIb P 38 (k) z (Ar. 16:22:31)
IIIb P 38 (k) z35 (Ar. 16:22:21). May possess H phase Rz56 (Ar. 38).
IIIb P 38 (k) z54 (Ar. 16:22:34)
IIIb P 38 (k) z55 (Ar. 16:22:37)
I P Alger 38 l,v 1,2
I P Kimberley 38 l,v 1,5
I P Roan 38 l,v e,n,x
IIIb P 38 l,v z (Ar. 16:23:31)
IIIb P 38 l,v z35 (Ar. 16:23:21)
IIIb P 38 l,v z53:[z54] (Ar. 16:23:25:[34])
I P Rothenburgsort 38 m,t –
I P Lindi 38 r 1,5
IIIb P 38 r 1,5,7 (Ar. 16:24:30)
I P Emmastad 38 r 1,6
IIIb P 38 r e,n,x,z15 (Ar. 16:24:28)
IIIb P 38 r z:[z57] (Ar. 16:24:31:[40])
IIIb P 38 r z35 (Ar. 16:24:21)
I P Freetown 38 y 1,5
I P Colombo 38 y 1,6
I P Perth 38 y e,n,x
I P Stachus 38 z –
I P Neunkirchen 38 z10 –
IIIb P 38 z10 z (Ar. 16:27:31)
IIIb P 38 z10 z53 (Ar. 16:27:25)
I P Klouto 38 z38 –
IIIa P 38 z4,z23 – (Ar. 16:1,2,6:-)
IV P 38 z4,z23 –
The Difco Manual 713
Salmonella, Antigenic Scheme Section V
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I U Milwaukee 43 f,g,[t] –
II U Mosselbay 43 g,m,[s],t [z42]
II U Veddel 43 g,t –
II U 43 g,t 1,5
IIIa U 43 g,z51 – (Ar. 21:13,14:-)
IV U 43 g,z51 –
II U 43 g,z62 e,n,x
I U Mbao 43 i 1,2
I U Voulte 43 i e,n,x
I U Thetford 43 k 1,2
I U Ahuza 43 k 1,5
IIIb U 43 k z (Ar. 21:29:31)
IIIb U 43 l,v z53:[Rz56] (Ar. 21:23:25:[38])
I U Sudan 43 l,z13 –
II U 43 l,z13,z28 1,5
IIIb U 43 r e,n,x,z15 (Ar. 21:24:28)
IIIb U 43 r z (Ar. 21:24:31)
IIIb U 43 r z53 (Ar. 21:24:25)
I U Farcha 43 y 1,2
I U Kingabwa 43 y 1,5
I U Ogbete 43 z 1,5
II U 43 z 1,5
I U Arusha 43 z e,n,z15
I U Adana 43 z10 1,5
I U Makiling 43 z29 –
IV U 43 z29 –
II U 43 z29 e,n,x
II U 43 z29 z42
I U Ahepe 43 z35 1,6
IIIa U 43 z36 – (Ar. 21:17,20:-)
IV U Volksdorf 43 z36,z38 –
I U Irigny 43 z38 –
IIIa U 43 z4,z23 – (Ar. 21:1,2,5:- and 21:1,2,6:-)
IV U Houten 43 z4,z23 –
IV U 43 z4,z23 –
IIIa U 43 z4,z24 – (Ar. 21:1,3,11:-)
IV U 43 z4,z24 –
IV U Tuindorp 43 z4,z32 –
II U Bunnik 43 z42 [1,5,7]
IIIb U 43 z52 z53 (Ar. 21:26:25)
I V Niakhar 44 a 1,5
I V Tiergarten 44 a e,n,x
I V Niarembe 44 a l,w
I V Sedgwick 44 b e,n,z15
I V Madigan 44 c 1,5
I V Quebec 44 c e,n,z15
I V Bobo 44 d 1,5
I V Kermel 44 d e,n,x
I V Fischerstrasse 44 d e,n,z15
The Difco Manual 719
Salmonella, Antigenic Scheme Section V
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I W Tornow 45 g,m,[s],[t] –
II W Perinet 45 g,m,t e,n,x,z15
I W Binningen 45 g,s,t –
IIIa W 45 g,z51 – (Ar. 11:13,14:-)
IV W 45 g,z51 –
I W Verviers 45 k 1,5
I W Casablanca 45 k 1,7
I W Cairns 45 k e,n,z15
I W Imo 45 l,v [e,n,z15]
I W Apapa 45 m,t –
II W 45 m,t 1,5
I W Kofandoka 45 r e,n,z15
II W 45 z 1,5
I W Yopougon 45 z e,n,z15
II W Klapmuts 45 z z39
I W Jodhpur 45 z29 –
IIIa W 45 z29 – (Ar. 11:16,18:-)
II W 45 z29 1,5
II W 45 z29 e,n,x
II W 45 z29 z42
I W Lattenkamp 45 z35 1,5
I W Balcones 45 z36 –
IV W 45 z36,z38 –
IIIa W 45 z4,z23 – (Ar. 11:1,2,5:-)
IV W 45 z4,z23 –
I W Transvaal 45 z4,z24 –
IIIa W 45 z4,z24 – (Ar. 11:1,3,11:-)
IIIa W 45 z4,z32 – (Ar. 11:1,7,8:-)
II X Bilthoven 47 a [1,5]
II X 47 a e,n,x,z15
I X Saka 47 b – IP combined Saka with Sya (47:b:z6) and
called it Sya.
I X Wenatchee 47 b 1,2
II X Phoenix 47 b 1,5
II X Khami 47 b [e,n,x,z15]
I X Sya 47 b z6
II X 47 b z6
IIIb X 47 c 1,5,7 (Ar. 28:32:30)
I X Kodjovi 47 c [1,6] Kodjovi may possess H phase Rz78.
IIIb X 47 c e,n,x,z15:[z57] (Ar. 23:32:28 and 28:32:28:[40])
IIIb X 47 c z (Ar. 28:32:31)
IIIb X 47 c z35 (Ar. 28:32:21)
II X 47 d e,n,x,z15
I X Stellingen 47 d [e,n,x]
II X Quimbamba 47 d z39
II X 47 e,n,x,z15 1,6
I X Sljeme 1,47 f,g –
I X Anie 47 (g),m,t – IP combined Anie with Mesbit
(47:m,t:[e,n,z15]) and called it Mesbit.
The Difco Manual 721
Salmonella, Antigenic Scheme Section V
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I X Bergen 47 i e,n,z15
IIIb X 47 i z (Ar. 28:33:31)
IIIb X 47 i z35 (Ar. 23:33:21 and 28:33:21)
IIIb X 47 i z53:[z57] (Ar. 23:33:25 and 28:33:25:[40])
I X Staoueli 47 k 1,2
I X Bootle 47 k 1,5
IIIb X 47 k 1,5,7 (Ar. 28:29:30)
I X Dahomey 47 k 1,6 Dahomey may possess H phase Rz58.
IIIb X 47 k e,n,x,z15 (Ar. 28:29:28)
I X Lyon 47 k e,n,z15
IIIb X 47 k z (Ar. 28:29:31)
IIIb X 47 k z35 (Ar. 23:29:21)
IIIb X 47 k z53 (Ar. 23:29:25)
IV X 47 l,v –
IIIb X 47 l,v 1,5,(7) (Ar. 23:23:30). May possess H phase Rz50 (Ar. 42).
II X 47 z29 e,n,x,z15
I X Bingerville 47 z35 e,n,z15
IV X 47 z36 –
I X Alexanderplatz 47 z38 –
IIIa X 47 z4,z23 – (Ar. 28:1,2,5:-)
I X Tabligbo 47 z4,z23 [e,n,z15]
I X Binche 47 z4,z23 l,w
I X Bere 47 z4,z23 [z6] Bere may possess H phase Rz45.
I X Tamberma 47 z4,z24 –
I X Quinhon 47 z44 –
IIIb X 47 z52 1,5,7 (Ar. 28:26:30)
IIIb X 47 z52 e,n,x,z15 (Ar. 28:26:28)
IIIb X 47 z52 z (Ar. 28:26:31)
IIIb X 47 z52 z35 (Ar. 28:26:21)
II X 47 z6 1,6
I Y Hisingen 48 a 1,5,7
IIIb Y 48 a z35 (Ar. 5:35:21). Not in 1992 IP, but is in Bergey.
II Y 48 a z39
II Y 48 a z6
V Y 48 b –
II Y 48 b [z6]
IIIb Y 48 c z (Ar. 29:32:31)
II Y Etosha 48 d 1,11 Etosha was not considered a new serotype by
Kauffmann and is not used.
II Y 48 d 1,2
II Y Hagenbeck 48 d z6
I Y Fitzroy 48 e,h 1,5
II Y Hammonia 48 e,n,x,z15 z6
II Y Erlangen 48 g,m,t –
IIIa Y 48 g,z51 – (Ar. 5:13,14:-)
IV Y Marina 48 g,z51 –
IIIb Y Sydney 48 i z Sydney was formerly in subspecies II, but it
it now combined with Arizona 5:33:31. The
name Sydney has been dropped.
IIIb Y 48 i z35:[z57] (Ar. 29:33:21:[40])
IIIb Y 48 i z53 (Ar. 5:33:25)
IIIb Y 48 i z61 (Ar. 5,29:33:41)
IIIb Y 48 i z:[z72] (Ar. 5,29:33:31:[z72]). CDC does not have
z72 strain.
IIIb Y 48 k 1,5,(7) (Ar. 5:29:30)
II Y 48 k e,n,x,z15
IIIb Y 48 k e,n,x,z15 (Ar. 5:29:28)
I Y Dahlem 48 k e,n,z15
IIIb Y 48 k z (Ar. 5,29:29:31)
IIIb Y 48 k z35:[Rz75] (Ar. [5:29:21:Rz75]). CDC does not have Rz75.
IIIb Y 48 k z53 (Ar. 5,29:29:25)
IIIb Y 48 (k) z53 (Ar. 5:22:25 and Ar. 5,29:22:25).
Called 5:22:25 by IP.
II Y Sakaraha 48 [k] z39
I Y Australia 48 l,v 1,5
The Difco Manual 723
Salmonella, Antigenic Scheme Section V
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
II Z 50 k e,n,x:z42
IIIb Z 50 k z (Ar. 9a,9b:29:31 and 9a,9c:29:31). Ar. 9a,9b
may possess H phase Rz50 (Ar. 42).
IIIb Z 50 k z35 (Ar. 9a,9b:29:21)
IIIb Z 50 k z53 (Ar. 9a,9b:29:25 and 9a,9c:29:25). IP and
Rohde only list the 9a,9c.
II Z Seaforth 50 k z6
IIIb Z 50 (k) z (Ar. 9a,9b:22:31)
IIIb Z 50 (k) z35 (Ar. 9a,9b:22:21)
I Z Fass 50 l,v 1,2
IIIb Z 50 l,v e,n,x,z15 (Ar. 9a,9b:23:28)
IIIb Z 50 l,v z (Ar. 9a,9b:23:31 and 9a,9c:23:31). IP only
lists 9a,9c.
IIIb Z 50 l,v z35 (Ar. 9a,9c:23:21)
II Z 50 l,w e,n,x,z15:z42
II Z 50 l,z28 z42
II Z Atra 50 m,t z6:z42
IIIb Z 50 r 1,5,(7) (Ar. 9a,9b:24:30)
IIIb Z 50 r e,n,x,z15 (Ar. 9a,9c:24:28)
IIIb Z 50 r z (Ar. 9a,9b:24:31 and 9a,9c:24:31).
IIIb Z 50 r z35 (Ar. 9a,9b:24:21). May possess H phase Rz58
(Ar. Rz58). This is not in IP book, but is on
Rohde’s list.
IIIb Z 50 r z53 (Ar. 9a,9b:24:25). May possess H phase Rz50
(Ar. 42). This is not in IP book, but is on
Rohde’s list.
I Z Dougi 50 y 1,6
II Z Greenside 50 z e,n,x
IIIb Z 50 z10 z (Ar. 9a,9c:27:31). May possess H phase Rz56
(Ar. 38).
IIIb Z 50 z10 z53 (Ar. 9a,9c:27:25)
II Z Hooggraven 50 z10 z6:z42
I Z Ivorycoast 50 z29 –
IIIa Z 50 z29 – (Ar. 9a,9b:16,18:-)
IIIa Z 50 z36 – (Ar. 9a,9b:17,20:-)
IIIa Z 50 z4,z23 – (Ar. 9a,9b:1,2,5:- and 9a,9b:1,2,6:-)
IV Z Flint 50 z4,z23 –
IIIa Z 50 z4,z23,z32 – (Ar. 9a,9b:1,2,10:-). Called 9a,9b:1,6,7:- by
IP and Rohde.
IIIa Z 50 z4,z24 – (Ar. 9a,9b:1,3,11:-)
IV Z 50 z4,z24 –
IIIa Z 50 z4,z32 – (Ar. 9a,9b:1,2,10; 9a,9b:1,6,7:-; and
9a,9b:1,7,8:-). 9a,9b:1,2,10:- and 9a,9b:1,7,8:-
used by IP and Rohde.
IV Z Bonaire 50 z4,z32 –
II Z Faure 50 z42 1,7
IIIb Z 50 z52 1,5,7 (Ar. 9a,9b:26:30 and 9a,9c:26:30)
IIIb Z 50 z52 z (Ar. 9a,9b:26:31 and 9a,9c:26:31)
IIIb Z 50 z52 z35 (Ar. 9a,9b:26:21 and 9a,9c:26:21)
IIIb Z 50 z52 z53 (Ar. 9a,9b:26:25 and 9a,9c:26:25)
II 51 Roggeveld 51 – 1,7
I 51 Tione 51 a e,n,x
The Difco Manual 725
Salmonella, Antigenic Scheme Section V
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
IV 51 51 b –
I 51 Karaya 51 b 1,5
II 51 51 c –
I 51 Gokul 1,51 d [1,5]
I 51 Meskin 51 e,h 1,2
II 51 51 g,s,t e,n,x
IIIa 51 51 g,z51 – (Ar. 1,2:13,14:-)
I 51 Kabete 51 i 1,5
I 51 Dan 51 k e,n,z15
IIIb 51 51 k z35 (Ar. 1,2:29:21)
I 51 Harcourt 51 l,v 1,2
I 51 Overschie 51 l,v 1,5
I 51 Dadzie 51 l,v e,n,x
IIIb 51 51 l,v z (Ar. 1,2:23:31)
I 51 Moundou 51 l,z28 1,5
II 51 Askraal 51 l,z28 [z6]
I 51 Lutetia 51 r,i l,z13,z28
I 51 Antsalova 51 z 1,5
I 51 Treforest 1,51 z 1,6
I 51 Lechler 51 z e,n,z15
I 51 Bergues 51 z10 1,5
II 51 51 z29 e,n,x,z15
IIIa 51 51 z4,z23 – (Ar. 1,2:1,2,5:- and 1,2:1,2,6:-)
IV 51 Harmelen 51 z4,z23 –
IIIa 51 51 z4,z24 – (Ar. 1,2:1,3,11:-)
IIIa 51 51 z4,z32 – (Ar. 1,2:1,7,8:-)
I 52 Uithof 52 a 1,5
I 52 Ord 52 a e,n,z15
I 52 Molesey 52 b 1,5
I 51 Flottbek 52 b [e,n,x]
II 52 52 c k
IIIb 52 52 c k (Ar. 31:32:29). This is not in IP book, but is
on Rohde’s list.
I 52 Utrecht 52 d 1,5
II 52 52 d e,n,x,z15
II 52 52 d z39 CDC does not have this.
I 52 Butare 52 e,h 1,6
I 52 Derkle 52 e,h 1,7
I 52 Saintemarie 52 g,t –
II 52 52 g,t –
I 52 Bordeaux 52 k 1,5
IIIb 52 52 k z35 (Ar. 31:29:21)
IIIb 52 52 k z53 (Ar. 31:29:25)
IIIb 52 52 (k) z35 (Ar. 31:22:21)
IIIb 52 52 l,v z53 (Ar. 31:23:25)
II 52 52 z z39
II 52 Wilhemstrasse 52 z44 1,5 IP combined Wilhemstrasse with Lobatsi
(52:z44:1,5,7). The name Wilhemstrasse has
been dropped.
I 54 Yerba 54 z4,z23 –
II 55 Tranoroa 55 k z39
II 56 Artis 56 b –
II 56 56 d –
II 56 56 e,n,x 1,7
II 56 56 l,v z39
II 56 56 l,z28 –
II 56 56 z z6
II 56 56 z10 e,n,x
IIIa 56 56 z29 – (Ar. 14:16,18:-)
IIIa 56 56 z4,z23 – (Ar. 14:1,2,5:- and 14:1,2,6:-)
IIIa 56 56 z4,z23,z32 - (Ar. 14:1,6,7,9:-)
II 57 57 a z42
I 57 Antonio 57 a z6
I 57 Maryland 57 b 1,7
I 57 Batonrouge 57 b e,n,z15
IIIb 57 57 c z:[z60] (Ar. 34:32:31:[44])
II 57 57 d 1,5
II 57 57 g,[m],s,t z42
II 57 57 g,t -
IIIb 57 57 i e,n,x,z15 (Ar. 34:33:28)
IIIb 57 57 i z (Ar. 34:33:31)
IIIb 57 57 k e,n,x,z15 (Ar. 34:29:28). CDC does not have this
and not on Rohde’s list.
IIIb 57 57 z10 z (Ar. 34:27:31)
II 57 Locarno 57 z29 z42
II 57 Manombo 57 z39 e,n,x,z15
IV 57 57 z4,z23 -
II 57 Tokai 57 z42 1,6:z53
II 58 58 a z6
II 58 58 b 1,5
II 58 58 c z6
II 58 58 d z6
IIIb 58 58 i e,n,x,z15 (Ar. 1,33:33:28)
IIIb 58 58 k z (Ar. 1,33:29:31)
IIIb 58 58 l,v e,n,x,z15 (Ar. 1,33:23:28)
IIIb 58 58 l,v z35 (Ar. 1,33:23:21)
II 58 Basel 58 l,z13,z28 1,5
II 58 58 l,z13,z28 z6
IIIb 58 58 r e,n,x,z15 (Ar. 1,33:24:28)
IIIb 58 58 r z (Ar. 1,33:24:31)
IIIb 58 58 r z53 (Ar. 1,33:24:25). May possess H phase Rz47
(Ar. 39) or Rz57 (Ar. 40) or Rz70 (Ar. Rz70).
II 58 58 z10 1,6
IIIb 58 58 z10 e,n,x,z15 (Ar. 1,33:27:28)
IIIb 58 58 z10 z53 (Ar. 1,33:27:25). May possess H phase
Rz50 (Ar. 42).
II 58 58 z10 z6
II 58 58 z39 e,n,x,z15
Appendix B
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I C2 Be 8,20 a [z6]
I H Beaudesert [1],6,14,[25] e,h 1,7
II B Bechuana 1,4,12,27 g,[m],t [1,5]
I E4 Bedford 1,3,19 l,z13,z28 e,n,z15
I C2 Belem 6,8 c e,n,x
I C2 Belfast 6,8 c 1,7
I C2 Bellevue 8 z4,z23 1,7
I I Bellville 16 e,n,x 1,(5),7
II K Beloha 18 z36 –
I E1 Benfica 3,10 b e,n,x
I R Benguella 40 b z6
I D2 Benin 9,46 y 1,7
I C2 Benue 6,8 y l,w
I X Bere 47 z4,z23 [z6] Bere may possess H phase Rz45.
I D2 Bergedorf 9,46 e,h 1,2
I X Bergen 47 i e,n,z15
I 51 Bergues 51 z10 1,5
I U Berkeley 43 a 1,5
I J Berlin 17 d 1,5
IV R Bern 1,40 z4,z32 –
I D1 Berta 1,9,12 [f],g,t –
I E1 Bessi 3,10 i e,n,x
I E4 Bethune 1,3,19 k 1,7
II 59 Betioky 59 k (z)
I E1 Biafra 3,10 z10 z6
I E4 Bida 1,3,19 c 1,6
I N Bietri 30 y 1,5
I J Bignona 17 b e,n,z15
I R Bijlmer 1,40 g,m –
II X Bilthoven 47 a [1,5]
I E4 Bilu (1),3,10,(19) f,g,t 1,(2),7
I X Binche 47 z4,z23 l,w
I X Bingerville 47 z35 e,n,z15
I W Binningen 45 g,s,t –
I E2 Binza 3,15 y 1,5 IP combined Binza and Thomasville
(3,15,34:y:1,5) with Orion (3,10:y:1,5) to
form Orion 3,10,[15],[15,34]:y:1,5. Binza is
now called Orion var. O 15+ by IP.
I C1 Birkenhead 6,7 c 1,6
I E1 Birmingham 3,10 d l,w
I B Bispebjerg 1,4,[5],12 a e,n,x
I B Bissau 4,12 c e,n,x
II D1 Blankenese 1,9,12 b z6
II J Bleadon 17 (f),g,t [e,n,x,z15] IP has dropped f.
I D1 Blegdam 9,12 g,m,q –
I H Blijdorp 1,6,14,25 c 1,5
I X Blitta 47 y e,n,x
I C2 Blockley 6,8 k 1,5 Blockley may possess H phase Rz58
II C1 Bloemfontein 6,7 b [e,n,x]:z42
I 51 Flottbek 52 b [e,n,x]
I K Fluntern 6,14,18 b 1,5
I W Fomeco 45 b e,n,z15
I I Fortlamy 16 z 1,6
I B Fortune 1,4,12,27 z10 z6
II P Foulpointe 38 g,t –
I D1 Franken 1,9,12 z60 z67
I I Frankfurt 16 i e,n,z15
I P Freetown 38 y 1,5
I E1 Freiburg 3,10 l,z13 1,2
II T Fremantle 42 (f),g,t –
I D2 Fresno 9,46 z38 –
I G Friedenau 13,22 d 1,6
I M Friedrichsfelde 28 f,g –
I D1 Frintrop 1,9,12 b 1,5
I E1 Fufu 3,10 z 1,5
II E1 Fuhlsbuettel 3,10 l,v z6
I E4 Fulda 1,3,19 l,w 1,5
I B Fulica 4,[5],12 a 1,5
I B Fyris 4,[5],12 l,v 1,2
I C1 Gabon 6,7 l,w 1,2
I I Gafsa 16 c 1,6
I C1 Galiema 6,7,14 k 1,2
I E1 Galil 3,10 a e,n,z15
I F Gallen 11 a 1,2
I D1 Gallinarum 1,9,12 – – Gallinarum must be identified biochemically.
I V Gamaba 1,44 g,m,[s] –
I L Gambaga 21 z35 e,n,z15
I O Gambia 35 i e,n,z15
I I Gaminara 16 d 1,7
I H Garba 1,6,14,25 a 1,5
I C1 Garoli 6,7 i 1,6
I O Gassi 35 e,h z6
I D2 Gateshead 9,46 g,s,t –
I E4 Gatineau 1,3,19 y 1,5
I C1 Gatow 6,7 y 1,7
I C2 Gatuni 6,8 b e,n,x
I E1 Gbadago 3,10,[15] c 1,5
I C1 Gdansk 6,7 l,v z6 IP combined Gelsenkirchen (6,7,14:l,v:z6)
with Gdansk to form Gdansk 6,7,14:l,v:z6.
I N Gege 30 r 1,5
I C1 Gelsenkirchen 6,7,14 l,v z6 IP combined Gelsenkirchen with Gdansk
(6,7:l,v:z6) to form Gdansk 6,7,14:l,v:z6.
Gelsenkirchen is now called Gdansk var.
O 14+ by IP.
I C1 Georgia 6,7 b e,n,z15
I T Gera 1,42 z4,z23 [1,6]
I D2 Geraldton 9,46 l,v 1,6
II C2 Germiston 6,8 m,t e,n,x
I L Ghana 21 b 1,6
The Difco Manual 743
Salmonella, Antigenic Scheme Section V
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I N Giessen 30 g,m,s –
II C1 Gilbert 6,7 z39 1,5,7
I E1 Give 3,10 l,v 1,7 IP combined Newbrunswick (3,15:l,v:1,7) and
Menhaden (3,15,34:l,v:1,7) with Give to form
Give 3,10,[15],[15,34]:[d],l,v:[d],1,7. Give may
possess H phase d; Rl,z40; or Rz77.
I C2 Giza 8,20 y 1,2
I I Glasgow 16 b 1,6
II F Glencairn 11 a z6:z42
I F Glidji 11 l,w 1,5
I C2 Glostrup 6,8 z10 e,n,z15
I B Gloucester 1,4,12,27 i l,w
I E4 Gnesta 1,3,19 b 1,5
I N Godesberg 30 g,m,[t] –
I E1 Goelzau 3,10 a 1,5 IP combined Clichy (3,15:a:1,5) with Goelzau
to form Goelzau 3,10,[15]:a:1,5.
I E2 Goerlitz 3,15 e,h 1,2 IP combined Goerlitz with Vejle (3,10:e,h:1,2)
to form Vejle 3,10,15:e,h:1,2. Goerlitz is now
called Vejle var. O 15+ by IP.
I D1 Goeteborg 9,12 c 1,5
I D1 Goettingen 9,12 l,v e,n,z15
II G Gojenberg 1,13,23 g,t 1,5
I 51 Gokul 1,51 d [1,5]
I C2 Goldcoast 6,8 r l,w
I C1 Goma 6,7 z4,z23 z6
I C1 Gombe 6,7,14 d e,n,z15
I M Good 21 f,g e,n,x
II G Goodwood 13,22 z29 e,n,x
I J Gori 17 z 1,2
I R Goulfey 1,40 k 1,5
I O Gouloumbo 35 c 1,5
I D1 Goverdhan 9,12 k 1,6
I M Gozo 28 e,h e,n,z15
II F Grabouw 11 g,[m],s,t [z39]
I C1 Grampian 6,7 r l,w
I I Grancanaria 16 z39 [1,6] Grancanaria can be d-tartrate neg., dulcitol
neg., ONPG pos., and anaerogenic.
I N Grandhaven 30 r 1,2
I J Granlo 17 l,z28 e,n,x
I U Graz 43 a 1,2
II Z Greenside 50 z e,n,x
I R Greiz 40 a z6
I K Groenekan 18 d 1,5
Group A 1,2,12
Group B 4,12; 1,4,5,12; or 1,4,12,27
Group C1 6,7,[Vi] or 6,7,14
Group C2 6,8 IP combined C2 and C3.
Group C3 8; or 8,20 IP combined with C2.
Group D1 1,9,12
Group D2 9,46
Group D3 1,9,12,46,27
Group E1 3,10
Group E2 3,15 IP combined E2 and E3 with E1.
Group E3 3,15,34 IP combined E2 and E3 with E1.
Group E4 1,3,19
Group F 11
Group G 13,22 or 13,23
Group H 6,14; 6,14,24; or 1,6,14,25
Group I 16
Group J 17
Group K 18
Group L 21
Group M 28
Group N 30
Group O 35
Group P 38
Group Q 39
Group R 40
Group S 41
Group T 42
Group U 43
Group V 44
Group W 45
Group X 47
Group Y 48
Group Z 50
Group 51 51
Group 52 52
Group 53 53
Group 54 54
Group 55 55
Group 56 56
Group 57 57
Group 58 58
Group 59 59
Group 60 60
Group 61 61
Group 62 62
Group 63 63
Group 65 65
Group 66 66
Group 67 67
I G Grumpensis 1,13,23 d 1,7
II R Grunty 1,40 z39 1,6
I N Guarapiranga 30 a e,n,x
I D2 Guerin 9,46 e,h z6
I M Guildford 28 k 1,2
I V Guinea 1,44 z10 [1,7]
I F Gustavia 11 d 1,5
The Difco Manual 745
Salmonella, Antigenic Scheme Section V
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
II L Gwaai 21 z4,z24 –
I T Gwale 1,42 k z6
I E4 Gwoza 1,3,19 a e,n,z15
I C2 Haardt 8 k 1,5
II D2 Haarlem 9,46 z e,n,x
I C2 Hadar 6,8 z10 e,n,x
II I Haddon 16 z4,z23 –
I J Hadejia 17 y e,n,z15
I B Haduna 4,12 l,z13,[z28] 1,6
I C1 Haelsingborg 6,7 m,p,t,[u] –
I T Haferbreite 42 k [1,6]
I O Haga 35 z38 –
II Y Hagenbeck 48 d z6
I B Haifa 1,4,[5],12 z10 1,2
I M Halle 28 c 1,7
I B Hallfold 1,4,12,27 c l,w
I E2 Halmstad 3,15 g,s,t – IP combined Halmstad and Canoga
(3,15,34:g,s,t:-) with Westhampton (3,10:g,s,t:-)
to form Westhampton 3,10,[15],[15,34]:g,s,t:-.
Halmstad is now called Westhampton
var. O 15+ by IP.
II D1 Hamburg 1,9,12 g,t – IP combined Hamburg with Manica
(1,9,12:g,m,s,t:z42) and Muizenberg
(9,12:g,m,s,t:1,5) to form S. II
1,9,12:g,m,[s],t:[1,5,7]:[z42].
I E2 Hamilton 3,15 Rz27 – IP combined Hamilton with Goerlitz
(3,15:e,h:1,2) and Vejle (3,10:e,h:1,2) to form
Vejle 3,10,15:e,h:1,2:Rz27. Hamilton is now
called Vejle var. Rz27+. The name Hamilton
has been dropped.
II Y Hammonia 48 e,n,x,z15 z6
I G Handen 1,13,23 d 1,2
I R Hann 40 k e,n,x
I I Hannover 16 a 1,2
I G Haouaria 13,22 c e,n,x,z15
I H Harburg [1],6,14,[25] k 1,5
I 51 Harcourt 51 l,v 1,2
I E1 Harleystreet 3,10 z 1,6
IV 51 Harmelen 51 z4,z23 –
I E1 Harrisonburg 3,10,[15],[15,34] z10 1,6
I C1 Hartford 6,7 y e,n,x Hartford may possess H phase Rz50 or Rz67.
I T Harvestehude 1,42 y z6
I M Hatfield 28 d 1,6
I B Hato 4,[5],12 g,m,s –
I G Havana 1,13,23 f,g,[s] – Havana may possess H phase Rz45 or Rz79.
I E4 Hayindogo 1,3,19 e,h 1,6
I F Heerlen 11 i 1,6
I Q Hegau 39 z10 –
I B Heidelberg 1,4,[5],12 r 1,2
II C1 Heilbron 6,7 l,z28 1,5:[z42]
II B Helsinki 1,4,12 z29 [e,n,x]
I Z Hemingford 50 d 1,5 Hemingford may possess H phase Rz82.
746 The Difco Manual
Section V Salmonella, Antigenic Scheme
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
II S Hennepin 41 d z6
I M Hermannswerder 28 c 1,5
I I Heron 16 a z6
I C2 Herston 6,8 d e,n,z15
I F Herzliya 11 y e,n,x
I B Hessarek 4,12,27 a 1,5
I H Heves 6,14,[24] d 1,5
I C2 Hidalgo 6,8 r,i e,n,z15
I C2 Hiduddify 6,8 l,z13,z28 1,5
II J Hillbrow 17 b e,n,x,z15
I D2 Hillegersberg 9,46 z35 1,5
I D2 Hillingdon 9,46 g,m –
I C1 Hillsborough 6,7 z41 l,w
I N Hilversum 30 k 1,2
I C2 Hindmarsh 8,20 r 1,5
I Y Hisingen 48 a 1,5,7
I C1 Hissar 6,7,14 c 1,2
I I Hithergreen 16 c e,n,z15
I E1 Hoboken 3,10 i l,w
I Q Hofit 39 i 1,5
I E1 Hoghton 3,10 l,z13,z28 z6
I C2 Holcomb 6,8 l,v e,n,x
I H Homosassa 1,6,14,25 z 1,5
I M Honelis 28 a e,n,z15
I E4 Hongkong 1,3,19 z z6
II Z Hooggraven 50 z10 z6:z42
I H Horsham 1,6,14,[25] l,v e,n,x
IV U Houten 43 z4,z23 –
I E1 Huddinge 3,10 z 1,7
II D1 Hueningen 9,12 z z39
I B Huettwilen 1,4,12 a l,w
II F Huila 11 l,z28 e,n,x
I I Hull 16 b 1,2
II 53 Humber 53 z4,z24 –
I E1 Huvudsta 3,10 b 1,7
I I Hvittingfoss 16 b e,n,x
I L Hydra 21 c 1,6
I G Ibadan 13,22 b 1,5
I L Ibaragi 21 y 1,2
I G Idikan 1,13,23 i 1,5
I E1 Ikayi 3,10,[15] c 1,6 Ikayi Var. O 15+ was described after E1 and
E2 were combined.
I M Ikeja 28 k 1,7
I M Ilala 28 k 1,5
I E3 Illinois 3,15,34 z10 1,5 IP combined Illinois and Manila (3,15:z10:1,5)
with Lexington (3,10:z10:1,5) to form
Lexington 3,10,[15],[15,34]:z10:1,5. Illinois is
now called Lexington var. O 15+, 34+ by IP.
I E4 Ilugun 1,3,10,19 z4,z23 z6
The Difco Manual 747
Salmonella, Antigenic Scheme Section V
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I P Kidderminster 38 c 1,6
I A Kiel 1,2,12 g,p –
I I Kikoma 16 y e,n,x
II B Kilwa 4,12 l,w e,n,x
I P Kimberley 38 l,v 1,5
I D1 Kimpese 9,12 z 1,6
I B Kimuenza 1,4,12,27 l,v e,n,x
I E4 Kindia 1,3,19 l,z28 e,n,x
I U Kingabwa 43 y 1,5
I B Kingston 1,4,12,27 g,s,t [1,2] IP combined Kingston with Joenkoeping
(4,5,12:g,s,t:-) to form Kingston
1,4,[5],12,27:g,s,t:[1,2]. Kingston may
possess H phase Rz27 or Rz43.
I J Kinondoni 17 a e,n,x
I E2 Kinshasa 3,15 l,z13 1,5 IP combined Kinshasa with Uganda
(3,10:l,z13:1,5) to form Uganda 3,10,[15]:l,z13:1,5.
Kinshasa is now called Uganda var. O 15+ by IP.
I E4 Kinson 1,3,19 y e,n,x
I G Kintambo 1,13,23 m,t –
I J Kirkee 17 b 1,2
I B Kisangani 1,4,[5],12 a 1,2
I F Kisarawe 11 k e,n,x,[z15]
I C1 Kisii 6,7 d 1,2
I M Kitenge 28 y e,n,x
I C1 Kivu 6,7 d 1,6
II W Klapmuts 45 z z39
I P Klouto 38 z38 –
II B Kluetjenfelde 4,12 d e,n,x
I X Kodjovi 47 c [1,6] Kodjovi may possess H phase Rz78.
I B Koenigstuhl 1,4,[5],12 z e,n,z15
I A Koessen 2,12 l,v 1,5
I W Kofandoka 45 r e,n,z15
I V Koketime 44 z38 –
I N Kokoli 30 z35 1,6
I Q Kokomlemle 39 l,v e,n,x
I D2 Kolar 9,46 b z35
I C2 Kolda 8,20 z35 1,2
II U Kommetje 43 b z42
I M Konolfingen 28 z35 1,6
I C2 Konstanz 8 b e,n,x
I C2 Korbol 8,20 b 1,5
I E4 Korlebu 1,3,19 z 1,5
I P Korovi 38 g,m,[s] –
I C1 Kortrijk 6,7 l,v 1,7
I C2 Kottbus 6,8 e,h 1,5
I C1 Kotte 6,7 b z35
I D1 Kotu 9,12 l,z28 1,6
I E4 Kouka 1,3,19 g,m,[t] –
I C1 Koumra 6,7 b 1,7
I M Kpeme 28 e,h 1,7
750 The Difco Manual
Section V Salmonella, Antigenic Scheme
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I C2 Phaliron 8 z e,n,z15
I F Pharr 11 b e,n,z15
II X Phoenix 47 b 1,5
I E1 Pietersburg 3,10,[15,34] z69 1,7
I C2 Pikine 8,20 r z6 Pikine was combined with Altona (8,20:r,[i]:z6).
The name Pikine has been dropped.
I I Pisa 16 i l,w
I C1 Planckendael 6,7 z4,z23 1,6
I V Ploufragan 1,44 z4,z23 e,n,z15
I D2 Plymouth 9,46 d z6
I H Poano 1,6,14,25 z l,z13,z28
I 54 Poeseldorf 8,20,54 i z6
I C1 Poitiers 6,7 z 1,5
I M Pomona 28 y 1,7 Pomona may possess H phases Rz60, Rz70 or Rz80.
I K Pontypridd 18 g,m –
I G Poona 1,13,22 z 1,6 Poona may possess H phase Rz59.
I C2 Portanigra 8,20 d 1,7
II T Portbech 42 l,v e,n,x,z15
I D1 Portland 9,12 z10 1,5
I E2 Portsmouth 3,15 l,v 1,6 IP combined Portsmouth with London
(3,10:l,v:1,6) to form London 3,10,[15]:l,v:1,6.
Portsmouth is now called London
var. O 15+ by IP.
I K Potengi 18 z –
I H Potosi 6,14 z36 1,5
I C1 Potsdam 6,7,14 l,v e,n,z15
I D2 Potto 9,46 i z6
I D1 Powell 9,12 y 1,7
I C2 Praha 6,8 y e,n,z15
I E1 Pramiso 3,10 c 1,7
I C2 Presov 6,8 b e,n,z15
I B Preston 1,4,12 z l,w
I F Pretoria 11 k 1,2
I D1 Pullorum 1,9,12 – – IP combined Pullorum with Gallinarum
(1,9,12:-:-). They must be identified
biochemically.
I G Putten 13,23 d l,w
I V Quebec 44 c e,n,z15
I D2 Quentin 9,46 d 1,6
II X Quimbamba 47 d z39
I X Quinhon 47 z44 –
I C2 Quiniela 6,8 c e,n,z15
I N Ramatgan 30 k 1,5
I M Ramsey 28 l,w 1,6
II T Rand 42 z e,n,x,z15
I G Raus 13,22 f,g e,n,x
I K Rawash 6,14,18 c e,n,x
I B Reading 1,4,[5],12 e,h [1,5]
I C2 Rechovot 8,20 e,h z6
I C1 Redba 6,7 z10 z6
760 The Difco Manual
Section V Salmonella, Antigenic Scheme
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I E1 Rutgers 3,10 Rl,z40 1,7 Rutgers has been dropped from the scheme
and the H phase Rl,z40 is now considered an
R phase of Give.
I E1 Ruzizi 3,10 l,v e,n,z15
I D1 Saarbruecken 1,9,12 a 1,7
I I Saboya 16 e,h 1,5
IV R Sachsenwald 1,40 z4,z23 –
I N Sada 30 z10 1,2
I 52 Saintemarie 52 g,t –
I B Saintpaul 1,4,[5],12 e,h 1,2
I X Saka 47 b – IP combined Saka with Sya (47:b:z6) and
called it Sya.
II Y Sakaraha 48 [k] z39
I I Salford 16 l,v e,n,x
I B Salinatis 4,12 d,e,h d,e,n,z15 IP states that Salinatis was combined with
Duisburg (1,4,12,27:d:e,n,z15). This is
incorrect; IP should have stated that it was
combined with Sandiego (4,[5],12:e,h:e,n,z15),
because Salinatis loses the d and becomes
Sandiego.
I I Saloniki 16 z29 –
I S Samaru 41 i 1,5
I E4 Sambre 1,3,19 z4,z24 –
I B Sandiego 4,[5],12 [e,h] e,n,z15
I C2 Sandow 6,8 f,g e,n,z15
I C2 Sanga 8 b 1,7
I D2 Sangalkam 9,46 m,t –
I I Sangera 16 b e,n,z15
I C1 Sanjuan 6,7 a 1,5
I M Sanktgeorg 28 r,[i] e,n,z15
I E4 Sanktmarx 1,3,19 e,h 1,7
I M Santander 28 z35 e,n,z15
I R Santhiaba 40 l,z28 1,6
I C2 Santiago 8,20 c e,n,x
I E4 Sao 1,3,19 e,h e,n,z15
I I Saphra 16 y 1,5
I H Sara 1,6,14,25 z38 [e,n,x]
I B Sarajane 1,4,[5],12,27 d e,n,x
II I Sarepta 16 l,z28 z42
I R Saugus 40 b 1,7
I H Schalkwijk 6,14,[24] i e,n,..
I B Schleissheim 4,12,27 b –
I E4 Schoeneberg 1,3,19 z e,n,z15
I C1 Schwabach 6,7 c 1,7
I B Schwarzengrund 1,4,12,27 d 1,7
I C2 Schwerin 6,8 k e,n,x
I I Sculcoates 16 d 1,5
II Z Seaforth 50 k z6
I M Seattle 28 a e,n,x
I V Sedgwick 44 b e,n,z15
II U Veddel 43 g,t –
I I Vegesack 16 b l,w
I E1 Vejle 3,10 e,h 1,2 IP combined Goerlitz (3,15:e,h:1,2) with
Vejle to form Vejle 3,10,[15]:e,h:1,2.
I B Vellore 1,4,12,27 z10 z35
I F Veneziana 11 i e,n,x
II J Verity 17 e,n,x,z15 1,6
I S Verona 41 i 1,6
I W Verviers 45 k 1,5
I D1 Victoria 1,9,12 l,w 1,5
I J Victoriaborg 17 c 1,6
I S Vietnam 41 b z6
II S Vietnam var. subsp. II 41 b –
I E4 Vilvoorde 1,3,19 e,h 1,5
I M Vinohrady 28 m,t [e,n,z15]
I C1 Virchow 6,7 r 1,2
I C2 Virginia 8 d [1,2]
I E4 Visby 1,3,19 b 1,6
I M Vitkin 28 l,v e,n,x
I V Vleuten 44 f,g –
I T Vogan 1,42 z38 z6
IV U Volksdorf 43 z36,z38 –
I M Volksmarsdorf 28 i 1,6
I F Volta 11 r l,z13,z28
I B Vom 1,4,12,27 l,[z13],[z28] e,n,z15
I U Voulte 43 i e,n,x
II G Vredelust 1,13,23 l,z28 z42
I G Vridi 1,13,23 e,h l,w
VI W Vrindaban 45 a e,n,x
I B Vuadens 4,12,27 z4,z23 z6
I I Wa 16 b 1,5
I D2 Waedenswil 9,46 e,h 1,5
I E1 Wagadugu 3,10 z4,z23 z6
I B Wagenia 1,4,12,27 b e,n,z15
II L Wandsbek 21 z10 [z6]
I Q Wandsworth 39 b 1,2
I D1 Wangata 1,9,12 z4,z23 [1,7]
I T Waral 1,42 m,t –
I J Warengo 17 z 1,5
I W Warmsen 45 d e,n,z15
I M Warnemuende 28 i e,n,x
I C2 Warnow 6,8 i 1,6
I H Warragul [1],6,14,[25] g,m –
I J Warri 17 k 1,7
I G Washington 13,22 m,t –
IV Z Wassenaar 50 g,z51 –
I S Waycross 41 z4,z23 [e,n,z15]
IV S Waycross var.
subsp.IV 41 z4,z23 –
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Section V Salmonella, Antigenic Scheme
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
I N Wayne 30 g,z51 –
I M Wedding 28 c e,n,z15
I I Welikade 16 l,v 1,7
I E1 Weltevreden 3,10 r z6 IP combined Lanka 3,15:r:z6 with Weltevreden
to form Weltevreden 3,10,[15]:r:z6.
I X Wenatchee 47 b 1,2
I F Wentworth 11 z10 1,2
I D2 Wernigerode 9,46 f,g –
I T Weslaco 42 z36 –
I D1 Westafrica 9,12 e,h 1,7
I I Westeinde 16 l,w 1,6
I E4 Westerstede 1,3,19 l,z13 [1,2]
I E1 Westhampton 3,10 g,s,t – IP combined Halmstad (3,15:g,s,t:-) and
Canoga (3,15,34:g,s,t:-) with Westhampton to
form Westhampton 3,10,[15],[15,34]:g,s,t:-.
Westhampton may possess H phase Rz37
or Rz43 or Rz45.
I E1 Westminster 3,10,[15] b z35 CDC has no 3,10:b:z35.
I I Weston 16 e,h z6
II E1 Westpark 3,10 l,z28 e,n,x
I O Westphalia 35 z4,z24 –
I E1 Weybridge 3,10 d z6
I G Wichita 1,13,23 d 1,6 Wichita may possess H phase Rz37.
I O Widemarsh 35 z29 –
I B Wien 1,4,12,27 b l,w
I C1 Wil 6,7 d l,z13,z28
I E3 Wildwood 3,15,34 e,h l,w IP combined Wildwood and Cambridge
(3,15:e,h:l,w) with Meleagridis (3,10:e,h:l,w)
to form Meleagridis 3,10,[15],[15,34]:e,h:l,w.
Wildwood is now called Meleagridis
var. O 15+, 34+ by IP.
I B Wilhelmsburg 1,4,[5],12,27 z38 [e,n,z15]
II 52 Wilhemstrasse 52 z44 1,5 IP combined Wilhemstrasse with Lobatsi
(52:z44:1,5,7). The name Wilhemstrasse has
been dropped.
I G Willemstad 1,13,22 e,h 1,6
I E1 Wilmington 3,10 b z6
I E1 Wimborne 3,10 k 1,2
II E1 Winchester 3,10 z39 1,[5],7
I Q Windermere 39 y 1,5
II W Windhoek 45 g,m,s,t 1,5
I C2 Wingrove 6,8 c 1,2
I B Winneba 4,12 r 1,6
I 54 Winnipeg 54 e,h 1,5
I C1 Winston 6,7 m,t 1,6
I E4 Winterthur 1,3,19 l,z13 1,6
I C2 Wippra 6,8 z10 z6
I I Wisbech 16 i 1,7
II J Woerden 17 c z39
I F Wohlen 11 b 1,6
The Difco Manual 769
Salmonella, Antigenic Scheme Section V
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
II B 4,12 b 1,5
II B 4,12 e,n,x 1,2,7
II B 4,[5],12 f,g,t z6,z42
II B 4,12 (f),g – Not in IP book
II B 4,12 g,m,t z39
II B 4,12 g,m,t – IP calls this monophasic var. of Bechuana.
II B 4,12 g,z62 –
II B 4,12,27 i z35
II B 1,4,12,27 k 1,6
II B 1,4,12,27 l,v e,n,x
II B 1,4,12,27 l,v z39
II B 4,12 l,z28 –
II B 1,4,12,27 z 1,5
II B 4,12 z 1,7
II B 4,12 z z39
II B 4,12 – 1,6
II C1 6,7,14 a 1,5
II C1 6,7 a z6
II C1 6,7 b z39
II C1 6,7 d z42
II C1 6,7 g,m,[s],t e,n,x
II C1 6,7 (g),m,[s],t [1,5]
II C1 6,7 g,[m],s,t [z42]
II C1 6,7 g,t e,n,x:z42
IV C1 6,7 g,z51 –
II C1 6,7 k [z6]
IIIa C1 6,7 (k) z:[z55] (Ar. 27:22:31:[37])
IIIb C1 6,7 l,v z53 (Ar. 27:23:25)
II C1 6,7 l,w 1,5,7
II C1 6,7 l,w z42
II C1 6,7 l,z28 e,n,x
II C1 6,7 l,z28 z6
II C1 6,7 m,t –
II C1 6,7 z z6
II C1 6,7 z z39
II C1 6,7 z4,z24 z42
II C1 6,7 z10 z35
II C1 6,7 z29 –
VI C1 6,7 z41 1,7
II C1 6,7 z42 e,n,x:1,6
II C1 6,7 – 1,6
II C2 6,8 a e,n,x
II C2 6,8 a z39
II C2 6,8 b 1,5
II C2 6,8 d z6:z42
II C2 6,8 f,g,m,t [e,n,x]
II C2 6,8 g,m,t 1,7
II E1 3,10 z4,z24 –
II E1 3,10 z29 e,n,x
II E1 3,10 z29 –
VI F 11 a 1,5
VI F 11 b 1,7
II F 11 c e,n,z15
IIIb F 11 k z53 (Ar. 17:29:25)
IIIb F 11 l,v z (Ar. 17:23:31). May possess H phase Rz56
(Ar. 38).
IIIb F 11 l,v z53 (Ar. 17:23:25)
II F 11 z e,n,x
IIIa F 11 z4,z23 – (Ar. 17:1,2,5:-)
IV F 11 z4,z32 –
II F 11 – 1,5
II G 1,13,23 a 1,5
II G 13,22 a e,n,x
II G 1,13,22 b z42
II G 13,23 d e,n,x
II G 1,13,23 d e,n,z15
II G 1,13,23 g,m,s,t 1,5
II G 1,13,23 g,m,s,t z42
II G 1,13,23 g,[s],t z42
IIIa G 1,13,23 g,51 – (Ar. 18:13,14:-)
V G 1,13,22 i –
II G 13,22 k 1,5:z42
II G 13,23 k z41
IIIb G 13,22 l,v 1,5,7 (Ar. 18:23:30)
II G 13,23 l,w e,n,x
II G 13,22 l,z28 1,5
II G 13,23 l,z28 1,5
II G 13,23 l,z28 z6
II G 13,22 m,t z42:z39
V G 13,22 r –
II G 13,22 z –
II G 1,13,23 z z42
IIIa G 13,22 z4,z23 – (Ar. 18:1,2,5:-)
IIIa G 13,23 z4,z23,z32 – (Ar. 18:1,6,7:-). CDC would call this 1,6,7,9.
II R 40 b –
II R 1,40 c z39
II R 1,40 e,n,x 1,[5],7
II R 1,40 e,n,x,z15 1,6
II R 1,40 g,t 1,5
II R 1,40 g,t [e,n,x]
II R 1,40 g,t e,n,x,z15
II R 40 g,t z39
II R 1,40 g,[m],[s],t z42
IIIb R 40 g,z51 [e,n,x,z15] (Ar. 10a,10b:13,14:[28])
IIIb R 40 i 1,5,7 (Ar. 10a,10b:33:30)
IIIb R 40 k z:z57 (Ar. 10a,10b:29:31:40)
II R 40 k z6
IIIb R 40 k z53 (Ar. 10a,10b:29:25)
IIIb R 40 l,v z (Ar. 10a,10b,(10c):23:31)
IIIb R 40 l,v z53 (Ar. 10a,10b:23:25)
II R 1,40 l,z28 1,5:z42
II R 1,40 l,z28 z39
II R 40 m,t z39
II R 1,40 m,t z42
IV R 40 m,t -
II R 1,40 z z6
II R 1,40 z z39
II R 40 z z42
IIIa R 40 z4,z23 – (Ar. 10a,10b:1,2,5:-; 10a,10b:1,2,5,6:-; and
10a,10b:1,2,6:-)
IIIa R 40 z4,z24 – (Ar. 10a,10b:1,3,11:-)
IV R 40 z4,z24 – Also called Degania var. subsp. IV.
IIIa R 40 z4,z32 – (Ar. 10a,10b:1,2,10:-; 10a,10c:1,2,10:-;
and 10a,10b:1,7,8:-)
IIIa R 40 z4,z32 –
II R 1,40 z6 1,5
IIIb R 40 z10 z35 (Ar. 10a,10b:27:21)
IIIa R 40 z29 – (Ar. 10a,10b:16,18:-)
V R 1,40 z35 –
IIIa R 40 z35 – (Ar. 10a,10b:17,20:-)
II R 40 z39 1,5:z42
II R 40 z39 1,7
II R 1,40 z42 1,6
II R 1,40 [z42] 1,(5),7
V R 40 z81 – H z81 was formerly H a in S. bongori.
II S 41 b [1,5]
VI S 41 b 1,7
IIIb S 41 c e,n,x,z15 (Ar. 13:32:28)
II S 41 c [z6]
II S 41 g,m,s,t z6
IIIa S 41 g,z51 – (Ar. 13:13,14:-)
II S 41 k 1,6
II S 41 k [z6]
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Section V Salmonella, Antigenic Scheme
SUBSPECIES O ANTIGEN GROUP SEROTYPE O ANTIGENS PHASE 1 PHASE 2 NOTE
II U 43 a z6
II U 43 d e,n,x,z15
II U 43 d z39
II U 43 d z42
II U 43 e,n,x,z15 1,(5),7
II U 43 e,n,x,z15 1,6
II U 43 g,t 1,5
IIIa U 43 g,z51 – (Ar. 21:13,14:-)
IV U 43 g,z51 –
II U 43 g,z62 e,n,x
IIIb U 43 k z (Ar. 21:29:31)
IIIb U 43 l,v z53:[Rz56] (Ar. 21:23:25:[38])
II U 43 l,z13,z28 1,5
IIIb U 43 r e,n,x,z15 (Ar. 21:24:28)
IIIb U 43 r z (Ar. 21:24:31)
IIIb U 43 r z53 (Ar. 21:24:25)
II U 43 z 1,5
IV U 43 z4,z23 –
IIIa U 43 z4,z23 – (Ar. 21:1,2,5:- and 21:1,2,6:-)
IIIa U 43 z4,z24 – (Ar. 21:1,3,11:-)
IV U 43 z4,z24 –
IV U 43 z29 –
II U 43 z29 e,n,x
II U 43 z29 z42
IIIa U 43 z36 – (Ar. 21:17,20:-)
IIIb U 43 z52 z53 (Ar. 21:26:25)
II V 1,44 e,n,x 1,6
II V 44 g,t z42
IV V 44 g,z51 –
II V 44 z4,z23 –
IIIa V 44 z4,z23 – (Ar. 1,3:1,2,5:- and 1,3:1,2,6:-)
IV V 44 z4,z23 –
IIIa V 44 z4,z23,z32 – (Ar. 1,3:1,6,7,9:-)
IIIa V 44 z4,z24 – (Ar. 1,3:1,3,11:-)
IV V 44 z4,z24 –
IIIa V 44 z4,z32 – (Ar. 1,3:1,2,10:- and 1,3:1,7,8:-). IP calls
z4,z23,z32, Ar. 1,2,10.
IV V 44 z29 –
II V 44 z29 e,n,x:z42
IV V 44 z36,[z38] –
V V 44 z39 –
IIIa W 45 g,z51 – (Ar. 11:13,14:-)
IV W 45 g,z51 –
II W 45 m,t 1,5
II W 45 z 1,5
IIIa W 45 z4,z23 – (Ar. 11:1,2,5:-)
IV W 45 z4,z23 –
IIIa W 45 z4,z24 – (Ar. 11:1,3,11:-)
II 53 53 z z6
IIIa 53 53 z4,z23 – (Ar. 1,4:1,2,5:- and 1,4:1,2,6:-)
IV 53 53 z4,z23 –
IIIa 53 53 z4,z23,z32 – (Ar. 1,4:1,6,7,9:-)
IIIa 53 53 z4,z24 – (Ar. 1,4:1,3,11:-)
IIIa 53 53 z4,z32 – (Ar. 1,4:1,6,7:-). IP combin