Professional Documents
Culture Documents
Difcobblmanual 2nded
Difcobblmanual 2nded
Second Edition
Editors
ISBN 0-9727207-1-5
All rights reserved
Printed in the United States of America
AOAC is a trademark and Performance Tested Methods is a service mark of AOAC International. ATCC is a
trademark of the American Type Culture Collection. CHROMagar is a trademark of Dr. A. Rambach. Bacto, BiTek
and Difco are trademarks of Difco Laboratories, Inc., subsidiary of Becton, Dickinson and Company.
Unless otherwise noted, BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company.
2009 BD.
Contents
Preface ................................................................................................................................................................v
About This Manual............................................................................................................................................vii
History of BD Diagnostics..................................................................................................................................ix
Section I: Monographs........................................................................................................................................1
History of Microbiology and Culture Media ...................................................................................................3
Microorganism Growth Requirements..............................................................................................................4
Functional Types of Culture Media...................................................................................................................5
Culture Media Ingredients – Agars....................................................................................................................6
Culture Media Ingredients – Peptones and Hydrolysates...................................................................................7
Media Sterilization..........................................................................................................................................13
Quality Control Organisms............................................................................................................................15
Typical Analyses..............................................................................................................................................17
Section II: General Technical Information.........................................................................................................19
Dehydrated Culture Media.............................................................................................................................21
Prepared Plated Media....................................................................................................................................25
Prepared Tubed, Bottled and Mycoflask™ Media.............................................................................................27
Section III: Culture Media and Ingredients........................................................................................................31
Section IV: Reference Guide............................................................................................................................635
Culture Media for Specific Groups of Microorganisms.................................................................................637
Application Tables........................................................................................................................................644
Agar Selection Guide.................................................................................................................................644
Antimicrobial Effectiveness Testing............................................................................................................645
Antimicrobial Residue Testing...................................................................................................................646
Bionutrient Selection Guide.......................................................................................................................647
Cosmetic Testing........................................................................................................................................648
Environmental Sampling............................................................................................................................650
Food, Dairy and Beverage Testing..............................................................................................................651
Food Testing for E. coli O157:H7 using BBL™ CHROMagar™ O157 . .....................................................655
Food Testing for Listeria using BBL™ CHROMagar™ Listeria...................................................................656
Food Testing for Salmonella using BBL™ CHROMagar™ Salmonella.........................................................657
Food Testing for Staphylococcus aureus using BBL™ CHROMagar™ Staph aureus....................................658
Molecular Genetics Selection Guide...........................................................................................................659
Pharmaceutical Testing per USP.................................................................................................................660
USP Chapter <61>: Microbial Enumeration Tests......................................................................................661
USP Chapter <62>: Tests for Specified Organisms.....................................................................................662
Veterinary Testing......................................................................................................................................663
Water/Wastewater Testing..........................................................................................................................666
Water Testing for Enterococcus using BBL™ mEI Agar..............................................................................669
Water Testing for E. coli using BBL™ Modified mTEC Agar.......................................................................670
Water Testing for Total Coliforms and E. coli using BBL™ MI Agar...........................................................671
Product Tables..............................................................................................................................................672
Culture Media – Antimicrobial Susceptibility Testing................................................................................672
Culture Media – General-Purpose..............................................................................................................672
Culture Media – Supplements/Selective Agents..........................................................................................673
Peptones and Hydrolysates (product listing)..............................................................................................674
Peptones by Category................................................................................................................................675
Typical Analyses – Peptones and Hydrolysates...........................................................................................676
Product Index...................................................................................................................................................679
iii
Copyright 2009 by
Becton, Dickinson and Company
Sparks, Maryland 21152 USA
iv
Preface
We are pleased to present the second edition of the Difco & BBL Manual. The first edition, published in 2003, replaced the
Difco Manual, 11th ed. (1998) and the Manual of BBL Products and Laboratory Procedures, 6th ed. (1988), manuals which
were first published in 1927 and 1948, respectively. The Difco & BBL Manual is devoted exclusively to culture media and
associated reagents offered by BD as Difco™ and BBL™ brands of dehydrated culture media and BBL™ brand prepared plated,
tubed and bottled media. In this manual, over 170 years of combined media experience is brought together in an educational
and reference text.
For the second edition of the Difco & BBL Manual, we have added new products, removed discontinued products and incorpo-
rated general updates throughout. Of special note, the descriptions for media affected by the harmonization of the pharmacopeias
(United States, European and Japanese) have been updated accordingly.
The reader is advised that these products are for use by or under the supervision of microbiologists and other professionals
qualified by training and experience to handle pathogenic microorganisms and samples and specimens containing or suspected to
contain them. Also, it is expected that the user will be thoroughly familiar with the intended uses of the formulations presented
and will follow test procedures outlined in the applicable official compendia and standard texts or the procedure manual of the
using laboratory.
In addition to providing this manual as an educational resource, BD Diagnostics offers an array of educational materials and
services:
• BD Bionutrients™ Technical Manual – a manual dedicated to products used in cell culture and microbial fermentation
production.
• BD LabO™ – a newsletter providing the latest microbiology news and ideas to the clinical laboratory.
• Technical and Product Support – a dedicated group of specialists available to answer questions about any of our products
or procedures.
• Our web site, www.bd.com/ds
Grateful acknowledgement is made of the encouragement and support received from microbiologists throughout the world. Our
appreciation is extended, also, to those who have contributed their talents and time to the creation of this manual and its
predecessors. It is our desire to continue to extend our services to the advancement of microbiology and related sciences.
vii
This manual includes international catalog numbers and standard methods icons. Under “Availability,” catalog numbers are provided
for prepared plated media manufactured in Europe, Japan and Mexico that are comparable to the formulations manufactured
in the United States. In addition, icons denoting media listed in “official” and “standard methods” publications are provided.
These icons represent:
AOAC Official Methods of Analysis of AOAC International1
BAM Bacteriological Analytical Manual (FDA)2
CCAM The Compendium of Analytical Methods (Canada)3
COMPF Compendium of Methods for the Microbiological Examination of Foods (APHA)4
EP European Pharmacopoeia5
EPA Manual for the Certification of Laboratories Analyzing Drinking Water (EPA)6
ISO International Standards Organization7
JP Japanese Pharmacopoeia8
SMD Standard Methods for the Examination of Dairy Products (APHA)9
SMWW Standard Methods for the Examination of Water and Wastewater (APHA)10
USDA USDA/FSIS Microbiology Laboratory Guidebook11
USP The United States Pharmacopeia12
For many prepared media, icons are provided denoting the listing of these media in selected publications describing microbio-
logical procedures:
BS12 Bailey & Scott’s Diagnostic Microbiology, 12th ed.13
CMPH2 Clinical Microbiology Procedures Handbook (ASM)14
MCM9 Manual of Clinical Microbiology, 9th ed. (ASM)15
CLSI Clinical and Laboratory Standards Institute16-18
Because procedures specified in these publications may differ from one another and from those included in this manual, these
publications should be consulted when adherence to specific procedures is preferred or required.
As new information becomes available between printings of this manual, individual product descriptions will be updated and
available on our web site at www.bd.com/ds/DifcoBBLManual.
Technical inquiries about BD products should be directed to BD Diagnostics Technical Services in the United States at 800-638-
8663 or consult www.bd.com/support/contact/international.asp for your local BD Diagnostics office.
1. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC International, Gaithersburg, Md. <http://www.eoma.aoac.org/>
2. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC International, Gaithersburg, Md. <http://www.cfsan.fda.gov/~ebam/bam-toc.html>
3. Health Canada. The compendium of analytical methods, online. Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada. <http://www.hc-sc.gc.ca/fn-an/res-rech/analy-
meth/microbio/index-eng.php>
4. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods, 4th ed. American Public Health Association, Washington, D.C.
5. European Directorate for the Quality of Medicines and Healthcare. 2008. The European pharmacopoeia, 6th ed., Supp. 1, 4-1-2008, online. European Directorate for the Quality of Medicines and Healthcare,
Council of Europe, 226 Avenue de Colmar BP907-, F-67029 Strasbourg Cedex 1, France. <http://online6.edqm.eu/ep600>
6. U.S. Environmental Protection Agency. 2005. Manual for the certification of laboratories analyzing drinking water: criteria and procedures quality assurance, 5th ed. Office of Ground Water and Drinking Water,
Technical Support Division, USEPA, Cincinnati, Ohio.
7. International Organization for Standardization. 2008. International Organization for Standardization, Geneva, Switzerland. <http://www.iso.org/iso/home.htm>
8. Japanese Ministry of Health, Labour and Welfare. 2006. The Japanese pharmacopoeia, 15th ed., online. Japanese Ministry of Health, Labour and Welfare. <http://jpdb.nihs.go.jp/jp15e/JP15.pdf>
9. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy products, 17th ed. American Public Health Association, Washington, D.C.
10. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wasterwater, 21st ed., online. American Public Health Association, Washington, D.C. <http://www.standardmethods.
org/Store/index.cfm>
11. U.S. Department of Agriculture. Microbiology laboratory guidebook, online. Food Safety and Inspection Service, USDA, Washington, D.C. <http://www.fsis.usda.gov/Science/Microbiological_Lab_Guide-
book/>
12. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The national formulary 26, Supp. 1, 8-1-08, online. United State Pharmacopeial Convention, Inc., Rockville, Md. <www.
uspnf.com/uspnf/login>
13. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby, Inc., St. Louis, Mo.
14. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed. American Society for Microbiology, Washington, D.C.
15. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed. American Society for Microbiology, Washington, D.C.
16. Clinical and Laboratory Standards Institute. 2006. Approved standard: M2-A9. Performance standards for antimicrobial disk susceptibility tests, 9th ed. CLSI, Wayne, Pa.
17. Clinical and Laboratory Standards Institute. 2006. Approved standard: M7-A7. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 7th ed. CLSI, Wayne, Pa.
18. Clinical and Laboratory Standards Institute. 2007. Approved standard: M11-A7. Methods for antimicrobial susceptibility testing of anaerobic bacteria, 7th ed. CLSI, Wayne, Pa.
viii
History
BD Diagnostics – A Tradition of Excellence
Difco Laboratories, originally known as Ray Chemical, was founded in 1895. The company
produced high quality enzymes, dehydrated tissues and glandular products. Ray Chemical
acquired the Digestive Ferments Company, a company that specialized in producing digestive
enzymes for use as bacterial culture media ingredients. This merger lead to the preparation of
a line of peptones, beginning with Bacto™ Peptone, and dehydrated culture media. In 1913,
the company moved to Detroit, Michigan, and dropped the Ray Chemical name.
In 1934, the Digestive Ferments Company chose the acronym “Difco” to rename the company.
The focus of Difco Laboratories was to develop new and improved bacteriological culture
media, many of which were adopted as “standard” formulations in water, dairy, food, pharma-
ceutical and other industrial microbiology laboratories. Difco Laboratories grew through the
acquisition, in 1974, of Lee Laboratories, one of the largest manufacturers of bacteriological
antisera. The Paul A. Smith Company, later known as Pasco, which manufactured a semi-
automated instrument used for bacterial identification and susceptibility testing, was acquired
Original Difco Laboratories
Manufacturing facility. in 1983.
BD’s original microbiology products division, acquired in 1955, was founded in 1935 as a partnership between Theodore J.
Carski and Dr. Einar Leifson, employees of the Johns Hopkins Hospital in Baltimore, Maryland. Named the Baltimore Biologi-
cal Laboratory, the laboratory undertook a study of the preparation of peptones, and it began production of three new culture
media: Selenite-F Enrichment, Desoxycholate Agar and Desoxycholate-Citrate Agar. The acronym “BBL” was often used and
became the brand name for products offered by the company.
The Baltimore Biological Laboratory received increased impetus from the inventions and encouragement of Dr. John Brewer.
Brewer’s early pipetting machines were produced by the company, and the Brewer anaerobic jar, the forerunner of the GasPak™
jar, made the performance of routine anaerobic bacteriology practical and safe.
New discoveries rapidly followed. Incorporation of the reducing chemical sodium thioglycollate resulted in the introduction of
thioglycollate media for the cultivation of anaerobes. Other new formulations were added resulting in the development of a full
line of culture media. Many of these media utilize peptones of known derivation, such as Trypticase™ Peptone, a pancreatic
digest of casein, and Phytone™ Peptone, a papaic digest of soybean meal, ingredients which are employed in Trypticase Soy Agar,
Trypticase Soy Broth and many other media.
In 1952, the formulation of the U.S. version of Lowenstein-Jensen Medium was introduced, launching the prepared tubed
media line. In 1960, the line of prepared culture media was completed by introducing commercially-prepared plated media.
Over the years, BD’s microbiology division grew through a series of mergers and acquisitions. In 1972 and 1979, BD purchased
two more Baltimore-based microbiology companies – Hynson Wescott and Dunning (HW&D) and Johnston Laboratories, Inc.,
respectively. HW&D brought with it the Macro-Vue™ RPR, RUBAscan™ and CMVscan™ card test kits and the Directigen™
line of immunomicrobiology systems for the direct non-growth-dependent detection of antigens in patient specimens. Johnston
Laboratories was the developer and manufacturer of the BACTEC™ line of automated blood culturing and detection systems.
The BACTEC System, launched in 1968, was the first automated bacterial detection system to appear on the market. In 2009,
BACTEC celebrated its 40th anniversary with the launch of its tenth generation – the BD BACTEC FX Blood Culture System.
The media lines were strengthened by the acquisition in 1987 of GIBCO Laboratories of Madison, Wisconsin. Many specialty
media formulations were added to the existing BBL™ brand prepared plated and tubed media product lines.
In 1989, when Marion Laboratories decided to divest its Marion Scientific division’s microbiology product lines, it selected BD’s
microbiology division as the new provider of products such as the Bio-Bag™ Environmental systems and Culturette™ Toxin CD
test kit for rapid Clostridium difficile testing.
ix
In 1992, the division acquired the worldwide microbiology business of Roche Diagnostic Systems, adding the SEPTI-CHEK™
blood culture bottles and Enterotube™ and Oxi/Ferm™ identification products.
In June 1997, BD announced the acquisition of Difco Laboratories, Inc. The merger of this subsidiary with BD’s Microbiology Systems
division brought together the leading providers of microbiology products to industrial and clinical microbiology laboratories
worldwide, with a combined total of over 170 years of culture media experience. Both businesses comprise BD Diagnostics –
Diagnostic Systems, which is headquartered in Sparks, Maryland, near the city of Baltimore.
The dawning of the twenty-first century heralded the arrival of several new products. In 2000, the BD ProbeTec™ ET Amplified
DNA Assays for Chlamydia trachomatis and Neisseria gonorrhoeae were launched. Utilizing proprietary homogeneous strand
displacement amplification (SDA) technology, these assays were the first real-time DNA amplification tests on the market. This
accomplishment was followed by the launch of the BD Phoenix™ Automated Microbiology System in 2004 – a fully automated
system for the rapid identification and antimicrobial susceptibility testing of bacteria. Not to be outdone, between 2000 and 2006
the prepared media product line introduced seven new chromogenic media formulations – BBL™ CHROMagar™ prepared plated
media for Candida, Salmonella, Staphylococcus aureus, Listeria, E. coli O157 and MRSA as well as CHROMagar Orientation
for detection of urinary tract pathogens.
Eager to expand its commitment to the prevention of healthcare-associated infections (HAIs), in 2006 BD acquired GeneOhm
Sciences, Inc., a pioneer in the development of molecular diagnostics for methicillin-resistant Staphylococcus aureus (MRSA)
and Clostridium difficile (Cdiff). The BD GeneOhm™ MRSA and BD GeneOhm Cdiff assays produce results in hours not days,
allowing these infections to be controlled before outbreaks occur.
Finally, 2009 will mark the opening of a new, dedicated AF2™ (Animal-Free/Antibiotic-Free) Facility for the cGMP production of
cell culture media and supplements. This new plant will manufacture products that are controlled for animal-origin component
raw materials to the tertiary level, the most stringent level of control available. The AF2 Facility will provide a new standard for
safety and quality for cell culture media that significantly reduces current risks associated with mixed-use plants.
BD Diagnostics offers a total spectrum of microbiology laboratory products from dehydrated culture media to fully automated
instrumentation for the rapid identification of clinically relevant bacteria. BD Diagnostics continues to focus on the missions and
needs of both industrial and clinical microbiology laboratories.
The businesses that now constitute BD Diagnostics were founded by entrepreneurs whose ideas, diligence and foresight have
contributed to the creation of BD as one of the world’s leaders in the health care field. Through its products and services, BD is
committed to “helping all people live healthy lives.”
A “booming” development of microbiology began after material. This study and other similar studies started a
World War II. Molecular biology, biotechnology and the biotechnology revolution that has gained momentum over
study of genetics were fields of extraordinary growth. By the years.
1941, the study of microbiology and genetics came together
In the 1980s, instrumentation entered the microbiology
when Neurospora crassa, a red bread mold, was used to
laboratory. Manual procedures could be replaced by fully
study microbial physiology. The study of bacterial genetics
automated instruments for bacterial identification, suscep-
moved dramatically forward during the 1940s following the
tibility testing and blood culture procedures. Immunoassays
discovery of antibiotic resistance. The birth of molecular biology
and probe technologies broadened the capabilities of the
began in 1953 after the publication by Watson and Crick of
microbiologist.
the structure of DNA.
Significant advances continued in the 1990s. The use of
In 1953, viruses were defined by Luria as “submicroscopic
chromogenic substrates in culture media was shown to
entities, capable of being introduced into specific living
enhance microbial identification capabilities directly from the
cells and of reproducing inside such cells only.” The work
culture medium. In 1995, J. Craig Venter, Claire Fraser and
of John Enders on culturing viruses lead to the development
Hamilton Smith published the DNA sequence of the first
of vaccines. Enders demonstrated that a virus could be
free-living organism, Haemophilus influenzae.
grown in chick embryos and would lose its ability to cause
disease after successive generations. Using this technique, Salk With rapid advances in technologies and instrumentation,
developed the polio vaccine. the basic culture media and ingredients listed in this Manual
remain some of the most reliable and cost effective tools
One organism that has made a great contribution to
in microbiology today.
molecular biology is Escherichia coli. In 1973, Herbert
Boyer and Stanley Cohen produced recombinant DNA References
through plasmid transformation. The researchers found that 1. Marti-Ibanez (ed.). 1962. The epic of medicine. Clarkson N. Potter, Inc., New York, N.Y.
2. Wainwright and Lederberg. 1992. In Lederberg (ed.), Encyclopedia of microbiology, vol 2.
the foreign gene not only survived, but copied the genetic Academic Press Inc., New York, N.Y.
include Trypticase™ Soy Agar (nonenriched) and Trypticase Soy Many culture media formulations combine the properties of
Agar with 5% Sheep Blood (enriched). selectivity and differentiation due to their inclusion of a variety
A selective medium favors the recovery of specific types or of chemicals. Chromogenic media fall into this category. In
genera of microorganisms and inhibits other members of a addition to selective agents, these unique formulations contain
mixed flora. Selectivity is usually achieved with a chemical chromogenic substrates which release differently colored
agent, dye or antibiotic. Group A Selective Strep Agar with compounds upon degradation by specific enzymes. This en-
5% Sheep Blood (ssA™) is an example of a selective medium zymatic reaction produces distinct colony colors that allows
as it preferentially isolates group A streptococci from throat cul- for easy differentiation and identification. For example, on
tures due to the inclusion of a combination of selective agents. BBL™ CHROMagar™ Staph aureus medium, Staphylococcus
Culture media differ in their degree of selectivity, and the highly aureus produces mauve-colored colonies while most gram-
selective ones may inhibit some strains of the organisms the positive organisms, if not inhibited, produce white, blue, green
recovery of which is desirable. For example, when attempting or blue-green (aqua) colonies. Gram-negative organisms and
isolation of enterics, several media possessing varying degrees yeasts are partially to completely suppressed.
of selectivity should be utilized. The isolation of microorganisms from clinical materials
A differential medium is one which possesses certain ingredi- frequently requires the use of enriched broth media in
ents that enable presumptive identification of a specific genus addition to the selective, differential and nonselective plated
or species either from a pure or mixed culture. Identification media normally used for primary isolation. The use of liquid
is usually based on the organism’s appearance; i.e., colony “back up” media reduces the possibility of completely
color or the presence of a precipitate. For example, TSI Agar missing an etiological agent that is present in low numbers,
(Triple Sugar Iron Agar) is a differential medium for gram- slow growing on plated media, susceptible to selective agents,
negative enteric organisms on the basis of their ability to or sensitive to unfavorable incubation conditions.
ferment dextrose, lactose and sucrose and to produce sulfides.
• Low and regular content of electronegative groups that Gelidium, resulting in a liquid agar that is purified. The liquid
could cause differences in diffusion of electropositive agar is first gelled, causing the soluble and suspended contami-
molecules (e.g., antibiotics, nutrients) nants to be trapped, then washed from the agar, eliminating the
• Freedom from toxic substances (bacterial inhibitors) contaminants. Detailed manufacturing methods are described
• Freedom from hemolytic substances that might interfere in references.2,5
with normal hemolytic reactions in culture media
Product Applications
• Freedom from contamination by thermophilic spores
For specific product applications, refer to the product descrip-
Agars are normally used in final concentrations of 1-2% for ton for “Agars.”
solidifying culture media. Smaller quantities of agar (0.05-
0.5%) are used in culture media for motility studies (0.5% w/v) References
and growth of anaerobes (0.1%) and microaerophiles.2 1. Tseng. 1946. In Alexander (ed.). Colloid Chemistry. Reinhold Publishing Corp., New York, N. Y.
2. Selby and Selby. 1959. In Whistler (ed.)., Industrial gums. Academic Press Inc., New York, N.Y.
3. Hitchens and Leikind. 1939. J. Bacteriol. 37:485.
The Manufacturing Process 4.
5.
Koch. 1882. Berl. Klin. Wochenschr. 19:221.
Armisen. 1991. Hydrobiol. 221:157.
The finest Gelidium marine algae from world sources is selected. 6.
7.
Hesse. 1894. Mitt. a. d. Kaiserl. Gesh. Berlin 2:182.
United States Pharmacopeial Convention, Inc. 2008. The United States Pharmacopeia 31/The
Through a variety of processes, the agar is extracted from the national forumulary 26, Supp. 1, 8-1-08, online. The United States Pharmacopeial Convention,
Inc., Rockville, Md.
The unique characteristics of each BD peptone product de- filtered to remove the insoluble materials and to clarify and
pends on the quality and source of the protein starting mate- concentrate the product. Vacuum-evaporation may be used
rial, the quality and source of the enzyme, and the method of for rapid concentration. The peptone syrup, which contains
hydrolysis used to make the peptone. The starting materials approximately 67% solids, may undergo further processing
for peptones vary from animal to vegetable. Protein sources for pH adjustment, pasteurization, and/or filtration. The final
include meat, casein and whey (milk proteins), gelatin, soy- drying step of the process further concentrates the peptone by
bean, yeast and grains.2 Enzyme sources include animal organs spray-drying or by pan-drying in vacuum ovens, which readies
(pancreatin and pepsin), papaya (papain), fig (ficin), pineapple the material for packaging.
(bromelain) and microbes.2
Ultrafiltration
Protein Hydrolysis Ultrafiltration (UF) is a membrane filtration process used
Acid hydrolysis is a harsh process, usually carried out at high to separate or concentrate constituents of protein solutions
temperatures, which attacks all peptide bonds in the protein based on molecular weight. BD offers several peptone and
substrate, destroying some of the individual amino acids liber- yeast extract products that are ultrafiltered using a 10k Dalton
ated. Tryptophan is usually totally lost in an acid hydrolysis. Molecular Weight Cut Off (MWCO) membrane. The result
Cystine, serine and threonine are partially broken down and of using the 10k Da MWCO is a retentate containing mol-
asparagine and glutamine are converted to their acidic forms. ecules over 10k Da MW, which may include fats, larger MW
Vitamins are mostly destroyed by acid hydrolysis. Salts may be polypeptides and proteins, and a permeate that contains salts,
formed during neutralization of an acid hydrolysis, resulting sugars, peptides, smaller polypeptides and other compounds
in a product with high salt content. of less than 10k Da MW.
Proteolytic enzymes hydrolyze proteins more gently than acids, In peptone manufacture, ultrafiltration is used to create a
do not require the high temperature and usually require specific product that is low in endotoxin, the toxin-containing lipo-
peptide bonds. The material that results from a proteolytic polysaccharide part of the cell wall shed from viable gram-
digestion is a mixture of amino acids and polypeptides of vary- negative bacteria and released when gram-negative bacteria
ing lengths. The enzyme pepsin will cut an amino acid chain die. Endotoxins will cause illness in humans, so they are
where there is a phenylalanine or leucine bond.3 Papain will considered contaminants that must be avoided or minimized in
cut the chain adjacent to arginine, lysine and phenylalanine,3 the preparation of pharmaceutical products. The ultrafiltration
and pancreatin shows activity at arginine, lysine, tyrosine, step takes place before drying in the peptone manufacturing
tryptophan, phenylalanine and leucine bonds.3 process.
Microbial proteases, proteolytic enzymes secreted by microor-
ganisms, are becoming more widely used in peptone produc-
Meat Peptones
Meat peptones are proteins from animal sources that have been
tion. Proteases from bacterial, algal, fungal and yeast sources
hydrolyzed, or broken down into amino acids and peptides,
cover a wide variety of enzyme activities, can be produced in
to provide nitrogen for microorganisms. Meat peptones can
large scale and usually require only simple purification.4
be tailored to specific nutritive needs of microorganisms by
controlling the quality and origin of the protein, the quality
Peptone Manufacture
and source of the enzyme used to digest the protein, and the
Most peptones are manufactured similarly, with steps for
methods used for hydrolysis, concentration and drying the
hydrolysis and downstream processing. The basic steps of pep-
peptone. Sources of animal protein include meat from muscu-
tone manufacture include: hydrolysis/digestion, centrifugation,
lar tissue or offal (waste parts, entrails) and gelatin. Muscular
filtration, concentration and drying. Protein and demineralized
tissue and offal are utilized fresh, frozen or dried. Gelatin is
water are combined to form a thick suspension of protein
extracted by boiling collagen, the fibrous protein found in
material in large-capacity digestion vessels, which are stirred
connective tissue, bone and cartilage.
continuously throughout the digestion process. The protein
suspension is then adjusted to the optimum pH for the specific A variety of proteolytic enzymes, or proteases, may be used
enzyme chosen for the hydrolysis. For example, pepsin is most to accomplish enzymatic hydrolysis of animal protein. Pepsin
effective at pH 2.0 and trypsin shows maximum activity at and trypsin are widely used for animal peptone manufacture.
pH 8.5.2 Enzyme is added when the pH and temperature are Pepsin is isolated from porcine or other animal stomach.
optimal. The amount of enzyme necessary, time for digestion, Trypsin, along with chymotrypsin, carboxypeptidase A,
and control of pH and temperature are dependent on the carboxypeptidase B, and elastase, are enzymes isolated from
desired degree of hydrolysis. animal pancreas.
Once the predetermined degree of protein digestion is achieved,
the enzymatic activity must be halted; the suspension is either
Casein Peptones
Casein and whey peptones are hydrolysates of bovine milk
heated to inactivate the enzymes or neutralized to inactivate
proteins. Milk is a complex material, consisting of water,
acids or bases. The protein slurry is then centrifuged and/or
lactose, lipids, salts and proteins.
Casein (80%) and whey (20%) are the fundamental protein Yeast Products
components in milk. After the cream, or fat, has been removed Yeast extract is defined in the USP as “a water-soluble, peptone-
from bovine milk, hydrochloric or sulfuric acid is added in like derivative of yeast cells (Saccharomyces).”10 Yeast extract
order to precipitate out casein, the insoluble portion.5,6 The is readily available in the U.S. as a spray-dried powder. In
casein recovered is known as acid casein and is insoluble in Europe, pharmaceutical companies use it as a liquid or paste,
water. Generally, the acid casein is dissolved in a suitable as well as in the powdered form.
hydroxide such as NaOH, to make it soluble in water. The
Yeast extract is used by the health food industry as an inex-
resulting sodium caseinate is then used as the basis for hydro-
pensive source for many of their vitamins, and has long been
lyzed caseins. Sodium caseinate typically consists of 87% to
recognized as a major source of B-complex vitamins. Yeast
90% protein.7 Casein, which can make up to 3% of the total
extract, as a substrate in a media formulation, supplies not
components in bovine milk, is one of the most nutritive of the
only vitamins, but also proteins, carbohydrates and some
milk proteins, as it contains all of the common amino acids
micronutrients.
and is rich in the essential ones.
There are many kinds of yeast extract. The two principle sources
The soluble supernatant material separated from milk after
of yeast extract are “brewer’s” yeast and “baker’s” yeast.
casein precipitates is whey, also called milk plasma. Whey
Brewer’s yeast is a by-product from the brewing industry. It
contains the lactalbumin and lactoglobulin proteins and is
requires de-bittering (removal of hop resins) before it is suitable
a by-product of casein (and cheese) production. Whey pro-
for fermentation use.2 A wide variety of strains and growth
tein concentrates and isolates are recovered using various
processes have been used in the manufacture of the brewer’s
separation technologies such as ion exchange and filtration;
yeast, thus precluding any consistency of the final product.
lactalbumin is recovered by heat denaturing and then separa-
tion.5 Whey peptones are manufactured using the process of Baker’s yeast (Saccharomyces cerevisiae) is defined as a primary
enzymatic hydrolysis on the proteins isolated from whey. The yeast because the yeast is grown for the specific purpose of
whey peptones contain free amino acids and peptides, as well being used as a substrate in a bioprocess or as a food prod-
as carbohydrates, vitamins and minerals. uct/flavoring. Manufacture of baker’s yeast is a reproducible
and controlled process. The yeast organism is grown on a
Casein peptones are manufactured by either acid or enzymatic
molasses-based medium optimized for the specific yeast.11
hydrolysis. Many acids can be utilized in the acid hydrolysis
Commercial yeast fermentations are always fed-batch type
of casein, but hydrochloric acid is most commonly used in
fermentations lasting from 12-20 hours.12 Commercial baker’s
the process. This process leads to complete hydrolysis of
yeast manufacturers have found that the more highly aerated
the casein to amino acids and other compounds of relative
a culture, the higher the final product yield.12
chemical simplicity.
The process of manufacturing baker’s yeast extract is unique
The manufacturing process for an enzymatic hydrolysis of
compared to the manufacture of other peptones. Yeast extract
casein is not as destructive as an acid hydrolysis. The ca-
is an autolysate. Cell hydrolysis is performed by the endog-
sein is not broken down as completely into its constituent
enous enzymes of the Saccharomyces organism. Autolysis is
components. In many cases, this makes for a more nutri-
usually begun by either a controlled temperature shock or, for
tious hydrolysate, especially for those organisms that prefer
the food industry, an osmotic shock, which causes the yeast
peptides to amino acids. Enzymes from the pancreas are
cells to expire. The temperature shock is not high enough to
utilized to manufacture these peptones. While the pancreas
inactivate the proteases of the yeast cell, which proceed to
contains a battery of enzymes from the protease, lipase and
degrade the cell. Autolysis can proceed from 10 to 60 hours.
amylase groups, it is the proteases that are used in the hydro-
lysis of casein. These proteases only have the ability to digest After autolysis, soluble material is separated from insoluble
proteins into peptides; they cannot break the protein down by means of centrifugation and several filtration steps.12 The
into its component amino acids. As a result, pancreatic digests final filtration product is concentrated and then spray dried,
of casein, as opposed to acid hydrolysates of casein, produce or can be left in the concentrated paste form, which contains
peptones that contain greater amounts of peptides. approximately 60-80% solids.
Temperature, pH, addition of other enzymes, type of medium
Soy Peptones substrate for the growth of the Saccharomyces and duration
Soy peptones are all enzymatic digests of soy flour. Soy con- of autolysis are all variations that create the large variety of
tains several heat labile protease inhibitors.8 The most common yeast extracts available.
way of eliminating these factors is to heat or toast the defatted
soy beans in a processing plant under controlled conditions. Peptone Performance
Soy flour, the principle substrate in a soy peptone, is rich in The raw materials and manufacturing conditions for protein
high-quality protein, carbohydrates, calcium and B vitamins.9 hydrolysis are controlled to produce consistent peptone prod-
The enzymes used in the digestion of the soy flour are typically ucts. Ingredients used for peptone manufacture, including the
from animal-free sources or from microorganisms that have protein, agent of hydrolysis, and any buffering agents used, are
been grown in animal-free media.
selected based on specific purity and quality standards. The that peptone is present from the beginning of the run, others
conditions of the hydrolysis, such as the amount of enzyme perform best when the peptone is added as a feed later in the
used, the time for digestion, and the pH and temperature process. In some cases, optimal performance is achieved when
at which hydrolysis is conducted, determine the degree of the process begins with one peptone then another is added as
hydrolysis and the quality of the hydrolysate. Therefore, a feed later in the production run.
these conditions must be carefully controlled throughout BD AutoNutrient™ Media Design Service (AMDS)
the manufacturing process. Purification, concentration and
Performing thorough optimizations can require significant
drying steps are carefully regulated due to their bearing on
time and resources, so the decision is often made to eliminate
the characteristics of a peptone. Finally, each batch of protein
many potentially critical design points. To address this need
hydrolysate is tested for an array of physical, chemical, analyti-
and ensure the identification of the optimal media formulation
cal and growth support tests to ensure product quality and
that meets production goals, BD offers the AutoNutrient™
lot-to-lot consistency.
Media Design Service (AMDS). The BD team of dedicated,
Peptones/hydrolysates are available as original premium experienced scientists works with each customer in a highly
Bacto™ formulations, or as Difco™, BBL™ or BiTek™ brands. collaborative process to develop a media formulation that
The BiTek brand was developed for production-grade products satisfies the requirements. Through the AMDS program, BD
where a premium product is not required. offers a library of 45 diverse, chemically defined media, as well
as a number of peptones designed specifically for the biophar-
Applications maceutical industry. AMDS partners with each customer from
Cell Culture initial screens through final scale up to ensure an optimized
In the biopharmaceutical industry, the requirement for high- process at each step.
performance animal-free processes has prompted a greater Fermentation
focus on media and process optimization.13 Serum supple-
Fermentation media formulations are of two types: defined
mentation has traditionally been used to compensate for base
and complex. Defined media are made by the addition of
media deficiencies. However, the regulatory issues and high
chemically defined ingredients to water for injection (WFI) or
costs associated with using serum have led to a widespread
distilled water. Complex media are made with peptone digests
effort to find animal free or chemically defined alternatives.
or extracts of plant or animal origin.18
While chemically defined media are ideal, they often do not
meet the expected production goals. The advantages of chemically defined media are greater
reproducibility, cleaner downstream processing steps and
Through additional work, it has been observed that peptone
simplicity in the analysis of the end product. The disadvantages
supplementation, when appropriately applied, can exceed
are lower yields and greater expense, especially if the list of
the performance of serum while meeting stringent regulatory
media components include growth factors and vitamins.19
requirements. Additionally, downstream processing require-
The advantages of complex media are that they are relatively
ments are greatly reduced due to the lack of contaminating
inexpensive, they support a wide variety of growth from a
serum proteins, thereby reducing processing time and costs.
large group of microorganisms, they promote growth of the
In order to achieve this type of success, a complete process
more fastidious organisms that will not grow on a chemically-
optimization must occur where the base media, peptone
defined medium, they stimulate toxin production and they
supplementation, and feed strategy are empirically determined
routinely produce higher yields than many defined media.
through the use of a methodical optimization strategy.14
The disadvantages of complex media are that the downstream
The benefits of peptone supplementation in cell culture ap- processing may be more difficult and reproducibility can
plications have been well documented for many years. Due sometimes be compromised. When developing a new medium
to the complex composition of peptones, they provide a wide formulation, care should be taken in choosing the peptones
range of benefits to the cells. In some cases, peptides of various for the new formulation. Individual experimentation with a
lengths have resulted in increased cell performance.15 Others variety of peptones is suggested to select the optimum peptone
have benefited from free amino acids and other low molecular or combination of peptones.
weight nutrients.16 Since the nutritional requirements for each
With the continuing emergence of new confirmed cases
cell line are different, it is important to identify a peptone that
of BSE/TSE, a prime directive for the development of new
will meet the unique requirements of a particular cell line.
fermentation products has been to either source raw materials
Blends of peptones should also be considered, as synergistic
from a country free from BSEs or reformulate the media
effects have been observed in some processes when multiple
using animal-free components.20 BD began offering animal
peptones were used.17 Significantly higher antibody yields can
free alternatives to classical media formulations in 1997. In
be achieved with the identification of a peptone that meets the
1998, Select APS™ (alternative protein source) products were
specific requirements of the cell line.
introduced. These non-animal formulations provide growth
Determining how to apply the peptone is essential to achiev- support characteristics comparable, and in some cases superior,
ing the increased performance. While some processes require to classical animal formulations.
10
11
12
12. Reed and Nagodawithana. Yeast technology, 2nd ed. Van Nostrand Reinhold, New York, N.Y. 18. Demain and Solomon. 1986. Manual of industrial microbiology and biotechnology. American Society
13. Jerums and Yang. 2005. Bioproc. Int. 3:38-44. for Microbiology, Washington, D.C.
14. Burteau, Verhoeye, Mols, Ballez, Agathos, and Schneider. 2003. In Vitro Cell Dev. Biol. Anim. 19. Flickinger and Drew (ed.). 1999. Encyclopedia of bioprocess technology, fermentation, biocatalysis
39:291-296. and bioseparation. John Wiley & Sons, Inc. New York, N.Y.
16. Heidemann, Zhang, Qi, Rule, Rozales, Park, Chuppa, Raq, Michaels, Konstantinov, and Naveh. 20. Sommer. 1996. 9th International Symposium on Yeasts, Sydney, Australia. <www.ohly.de/sommer.
2000. Cytotechnology. 32:157-167. htm.>.
17. Kuchibhatla, Hunt, Holdread, and Brooks. 2004. Presented at IBC. Sept. 2004. 21. Nolan and Nolan. 1972. Appl. Microbiol. 24:290.
22. How malt is made. Briess Malting Company. 2 Dec 2002. <www.briess.com/Homebre
Media Sterilization
Sterilization is any process or procedure designed to entirely that the probability of microbial survival in the replicate
eliminate viable microorganisms from a material or medium. processes completed is not greater than the prescribed
Sterilization should not be confused with disinfection, sani- limits.
tization, pasteurization or antisepsis which are intended to 4. Monitor the validated process during routine operation.
inactivate microorganisms, but may not kill all microorganisms Periodically as needed, requalify and recertify the equip-
present. Sterilization can be accomplished by the use of heat, ment.
chemicals, radiation or filtration.1 5. Complete the protocols and document steps 1-4, above.
13
Over-sterilization or prolonged heating will change the compo- surface treatments. Much higher energy, 100 to millions of
sition of the medium. For example, carbohydrates are known to times greater, is generated by ionizing radiations. These include
break down in composition upon overheating. Over-sterilizing gamma-rays, high energy X-rays and high energy electrons.
media can cause a number of problems, including:
Ionizing radiation, unlike ultraviolet rays, penetrates deeply
• Incorrect pH into atoms, causing ionization of the electrons. Ionizing
• A decrease in the gelling properties of agar radiation may directly target the DNA in cells or produce
• The development of a nontypical precipitate active ions and free radicals that react indirectly with DNA.
• Carmelization or darkening of the medium Gamma radiation is used more often than x-rays or high-
• Loss of nutritive value energy electrons for purposes of sterilization. Gamma rays are
• Loss of selective or differential properties generated by radioactive isotopes, cobalt-60 being the usual
source. Gamma radiation requires many hours of exposure
There are certain media (e.g., Hektoen Enteric Agar and Violet for sterilization. Validation of a gamma irradiation procedure
Red Bile Agar) that should not be autoclaved. To dissolve these includes:4
media formulations, heat to boiling to dissolve completely. It is
important to follow all label directions for each medium. Media • Establishment of article materials compatibility
supplements should be sterile and added aseptically to the steril- • Establishment of product loading pattern and completion
ized medium, usually at 45-55°C. of dose mapping in the sterilization container
• Establishment of timer setting
Dry Heat Sterilization1 • Demonstration of the delivery of the required sterilization
Dry heat is employed for materials such as metal instruments dose
that could be corroded by moist heat, powders, ointments
and dense materials that are not readily penetrated by steam. The advantages of sterilization by irradiation include low
Because dry heat is effective only at considerably higher chemical reactivity, low measurable residues, and few variables
temperatures and longer times than moist heat, dry heat to control.3 Gamma irradiation is used for treating many
sterilization is restricted to those items, unlike culture media, heat-sensitive products that can also be treated by gaseous
that will withstand higher temperatures. The dry heat time sterilization, including medical materials and equipment,
for sterilization is 120 minutes at 160°C. pharmaceuticals, biologicals and laboratory equipment. BD
utilizes gamma irradiation in the manufacturing of BBL™
Chemical Sterilization1 Sterile Pack media for environmental monitoring of critical
Chemical sterilization employs gaseous and liquid sterilants for environments.
certain medical and industrial instruments. The gases include
ethylene oxide, formaldehyde and beta-propiolactone. The Sterilization by Filtration1,3
liquid sterilants include glutaraldehyde, hydrogen peroxide, Filtration is a useful method for sterilizing liquids and gases.
peracetic acid, chlorine dioxide and formaldehyde. Chemical Filtration excludes microorganisms rather than destroying
sterilization is not employed in the preparation of culture them. Two major types of filters may be used, depth filters
media due to unfavorable affects upon performance. For a and membrane filters.
complete discussion of this topic, consult appropriate refer- The membrane filter screens out particles, while the depth
ences. filter entraps them. Membrane filters depend largely on the
size of the pores to determine their screening effectiveness.
Radiation Sterilization1 Electrostatic forces are also important. A membrane filter with
Radiation sterilization is an optional treatment for heat-sensi- an average pore size of 0.8 µm will retain particulate matter as
tive materials. This includes ultraviolet light and ionizing small as 0.05 µm. For removing bacteria, a pore size of 0.2 µm
radiation. is commonly used. For retention of viruses and mycoplasmas,
Ultraviolet light is chemically active and causes excitation of pore sizes of 0.01-0.1 µm are recommended. Cocci and bacilli
atoms within the microbial cell, particularly the nucleic acids, range in size from about 0.3 to 1 µm in diameter. Most viruses
producing lethal mutations. This action stops the organism are 0.02-0.1 µm, with some as large as 0.25 µm.
from reproducing. The range of the ultraviolet spectrum that Rating the pore size of filter membranes is by a nominal rating
is microbiocidal is 240-280 nm. There is a great difference that reflects the capability of the filter membrane to retain
in the susceptibility of organisms to ultraviolet radiation; microorganisms of size represented by specified strains.
Aspergillus niger spores are 10 times more resistant than Sterilizing filter membranes are membranes capable of
Bacillus subtilis spores, 50 times more resistant than Staphy- retaining 100% of a culture of 107 microorganisms of a
lococcus aureus and Escherichia coli, and 150 times more strain of Pseudomonas diminuta (ATCC™ 19146) per square
resistant than influenza virus. centimeter of membrane surface under a pressure of not
Because most materials strongly absorb ultraviolet light, it less than 30 psi. These filter membranes are nominally rated
lacks penetrating power and its applications are limited to 0.22 µm or 0.2 µm. Bacterial filter membranes (also known
14
as analytical filter membranes), which are capable of retain- Calibration is the demonstration that a measuring device
ing only larger microorganisms, are labeled with a nominal produces results within specified limits of those produced
rating of 0.45 µm. by a reference standard device over an appropriate range of
measurements.
Membrane filters are used for the commercial production
of a number of pharmaceutical solutions and heat-sensitive Death rate is the rate at which a biocidal agent reduces the
injectables. Serum for use in bacterial and viral culture media number of cells in a microbial population that are capable of
are often sterilized by filtration, as well as some sugars that are reproduction. This is determined by sampling the population
unstable when heated. Membrane filtration is useful in testing initially, during and following the treatment, followed by plate
pharmaceutical and medical products for sterility. counts of the surviving microorganisms on growth media.
D value stands for decimal reduction time and is the time
Sterility Assurance1 required in minutes at a specified temperature to produce a
Sterility Assurance is the calculated probability that a micro-
90% reduction in the number of organisms.
organism will survive sterilization. It is measured as the SAL,
Sterility Assurance Level, or “degree of sterility”. For sterility Microbial death is the inability of microbial cells to metabolize
assurance, Bacillus stearothermophilus which contains steam and reproduce when given favorable conditions for reproduc-
heat-resistant spores is employed with steam sterilization at tion.
121°C. Process validation is establishing documented evidence that a
process does what it purports to do.
Testing Sterilizing Agents1,5
Sterilization by moist heat (steam), dry heat, ethylene oxide Sterility Assurance Level is generally accepted when materials
and ionizing radiation is validated using biological indicators. are processed in the autoclave and attain a 10-6 microbial
The methods of sterilization and their corresponding indica- survivor probability; i.e., assurance of less than one chance
tors are listed below: in one million that viable microorganisms are present in the
sterilized article.3
Sterilization Method Biological Indicator Sterilization process is a treatment process from which the
Steam Bacillus stearothermophilus probability of microorganism survival is less than 10-6, or
Dry heat Bacillus subtilis var. niger one in a million.
Ethylene oxide Bacillus subtilis var. globigii
Thermal Death Time and Thermal-Chemical Death Time are
Filtration Pseudomonas diminuta
terms referring to the time required to kill a specified microbial
population upon exposure to a thermal or thermal-chemical
For moist heat sterilization, paper strips treated with chemicals sterilizing agent under specified conditions. A typical thermal
that change color at the required temperature may be used. death time value with highly resistant spores is 15 minutes at
The heat-resistant spores of B. stearothermophilus are dried 121°C for steam sterilization.
on paper treated with nutrient medium and chemicals. After
sterilization, the strips are incubated for germination and References
1. Block. 1992. Sterilization. Encyclopedia of microbiology, vol. 4. Academic Press, Inc., San Diego,
growth, and a color change indicates whether they have or Calif.
2. Cote and Gherna. 1994. In Gerhardt, Murray, Wood and Krieg (ed.), Methods for general and
have not been activated. Spore strips should be used in every molecular bacteriology. American Society for Microbiology, Washington, D.C.
3. The United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The
sterilization cycle. national formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc.,
Rockville, Md.
4. Perkins. 1969. Principles and methods of sterilization in health sciences, 2nd ed. Charles C.
Glossary1,6 Thomas, Springfield, Ill.
5. Leahy. 1986. In Carleton and Agalloco (ed.), Validation of aseptic pharmaceutical processes. Marcel
Bioburden is the initial population of living microorganisms Dekker, Inc. New York, N.Y.
6. Simko. 1986. In Carleton and Agalloco (ed.), Validation of aseptic pharmaceutical processes. Marcel
in the product or system being considered. Dekker, Inc., New York, N.Y.
15
16
Typical Analyses
“Typical” chemical compositions have been determined on may reduce stability. In the presence of high moisture and
the peptones and hydrolysates used in microbiological culture high ambient temperatures, chemical interactions will cause
media. The typical analysis is used to select products for darkening of the product and falling pH. These characteristics
research or production needs when specific nutritional charac- indicate product deterioration. Loss on Drying determinations
teristics are required. The specifications for the typical analysis were based on the method described in the United States
include: Pharmacopoeia1 (with some modifications to the procedure).
• Nitrogen content NaCl: The sodium chloride (NaCl) content may reflect
• Physical characteristics significant pH adjustments during processing; e.g., acid
• Inorganics hydrolysates (see Ash). Sodium Chloride was determined by
silver nitrate/potassium thiocyanate titration method.
• Amino acids
pH: pH was measured in a 2% solution after autoclaving
For typical peptone and hydrolysate analyses, refer to the and equilibrating to room temperature. Hydrolysates vary
table in the Reference Guide section of this manual. To obtain in their pH resistance according to their inherent buffering
Certificates of Analysis on specific lots of peptones and (phosphate) capacity.
hydrolysates, visit http://regdocs.bd.com.
Inorganics
Glossary and Methods Elemental analysis (calcium, magnesium, potassium, sodium)
Nitrogen Content was determined by ICP (Inductively Coupled Plasma) using a
Total nitrogen (TN) was measured using a modified Kjeldhal Thermo Jarrell Ash instrument.
digestion method. Not all organic nitrogen is nutritive. Phosphate, chloride and sulfate percentages were determined
Percent (%) nitrogen × 6.25 ≅ % proteins, peptides or amino by ion chromatography.
acids present. Amino Acids
The amino nitrogen (AN) value shows the extent of protein Free Amino Acids are defined as amino acids that are not part
hydrolysis by measuring the increase in free amino groups. of a protein or peptide chain. The amino acids were measured
This is a nutritionally meaningful value. using the Waters AccQ•Tag™* Method. The AccQ•Tag™
Method is based on the derivatizing reagent 6-aminoquinolyl-
The AN/TN ratio gives an estimate of the degree of protein N-hydroxysuccinimide-activated heterocyclic carbamate.
hydrolysis.
Total Amino Acids were measured by the same method as
Physical Characteristics
the Free Amino Acids after an acid hydrolysis at 110°C for
Ash: The higher the ash content, the lower the clarity of the 20 hours. Cysteine and tryptophan are destroyed during the
prepared ingredient. Ash values refer to the non-combustible hydrolysis. The cysteine and tryptophan values are not
portion of the sample and roughly correspond to the min- reported for Total Amino Acids.
eral content of the sample. The ash content includes sodium
chloride, sulfate, phosphates, silicates and metal oxides. Reference
Acid-insoluble ash is typically from silicates found in animal 1. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The national
formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc., Rockville,
fodder. Ash values were measured after a minimum of 4 hours Md.
* AccQ•Tag is a trademark of Waters Corporation.
heating at 600°C.
Moisture (Loss on Drying): Lower moisture levels (<5%) are
preferred. Higher moisture levels in dehydrated ingredients
17
18
19
20
21
Dehydrated culture media are hygroscopic. When bottles of VIII. Materials Provided
dehydrated media have been opened for initial use, they should Dehydrated Culture Medium
be tightly closed as soon as possible to protect them from Supplements and Enrichments (if applicable)
hydration.
Provision should be made for rotating the stock of dehydrated IX. Materials Required But Not Provided
media to ensure product freshness, obviating the use of aged Glassware
materials, and discarding of media which is outdated. Measuring scale
pH meter
V. Expiration Date Autoclave
The expiration date applies to the products in their intact Purified water, pH 5.5-7.5
containers when stored as directed. Do not use a product if it Water bath
fails to meet specifications for identity and performance. Incubator
Sterile Petri dishes or tubes
VI. Product Deterioration Ancillary culture media, reagents and laboratory equipment
Verify that the physical characteristics of the powder are typical. as required.
Hydration can lead to caking and/or microbial contamination
which render the culture medium unusable. X. Procedures
Rehydration
VII. Specimen/Sample Collection and The procedure employed for dissolving dehydrated culture
Transport media very often determines the clarity and performance of
The success of a microbiological isolation procedure does the finished product. Homogeneity of the solution and minimal
not depend solely on the quality of the culture media utilized. heat exposure are important considerations.
Proper specimen/sample collection and transport are crucial
Prior to use, examine the dehydrated material. Caked or
steps in the isolation process. A variety of transport systems
discolored material should not be used for the preparation of
and holding media have been devised to prolong the
culture media batches.
survival of microorganisms when a significant delay is expected
between collection and definitive culturing. Add the precise amount of powdered material to approximately
Clinical Specimens one-half of the volume of purified water. After thorough mix-
For clinical specimens, BBL™ CultureSwab™ and Port-A-Cul™ ing, add the remainder of the water with care being taken to
collection and transport products are available. For transport wash down the sides of the container (preferably an Erlenmeyer
and growth of the pathogenic Neisseria, Transgrow, Gono-Pak flask that is at least 2-3× the volume of medium). Dissolution
and JEMBEC™* systems containing Modified Thayer-Martin is enhanced by allowing agar preparations to stand for 5 min-
(MTM II), Martin-Lewis or GC-Lect™ Agars are recom- utes with occasional agitation prior to boiling. Formulae that
mended. Specimens should be obtained before antimicrobial do not contain agar, gelatin or cystine will dissolve without
therapy has been administered. Provision must be made for heating, but heat is required to dissolve others so that they
prompt delivery to the laboratory. can be dispensed and sterilized. When heating is required,
heat should be applied gently and the preparation agitated as
The clinical laboratory must be furnished with sufficient required to prevent scorching. However, care should be taken
patient information to enable the microbiologist to select the to avoid media eruptions that may occur when agitating a
most suitable media and appropriate techniques. For detailed flask of medium which is at or very near the boiling point. Boil
information, appropriate references should be consulted.
* JEMBEC is a trademark of Miles Scientific.
as briefly as possible to obtain solution; 1 minute is usually
sufficient. Exposure for longer periods can darken the medium
Industrial Samples
and severely reduce its growth promotion properties. In no
Sterile containers should be used to collect samples. For case should powdered medium be added to water and immedi-
environmental monitoring, samples can be collected using ately placed into an autoclave since layering and separation of
BD Sterile Pack Swabs. ingredients, precipitate formation and darkening are likely to
Samples must represent the mass of the material being occur with diminution of performance.
examined. Samples may require special handling, includ- Sterilization
ing refrigeration, to prevent the direct contamination of the
Follow label directions for length and temperature of steriliza-
sample by microorganisms and the subsequent growth of such
tion. The recommended sterilization times assume a volume of
contaminants during sampling, transportation and storage
one liter (1000 mL) or less. For larger volumes, the steriliza-
before examination. For detailed information, appropriate
tion time should be extended but the temperature should not
references should be consulted.
22
be raised. When larger volumes are used, validation studies When they have cooled, tighten the caps of media that are
should be performed to determine the optimum sterilization contained in screw-capped tubes or bottles; store all tubes and
cycle for each unique container size/volume combination. bottles under appropriate conditions, usually at ambient room
Autoclave media containing carbohydrates at a temperature temperature. The shelf life of some media, such as Lowenstein-
not exceeding 116-118°C to avoid caramelization of the carbo- Jensen Medium, may be prolonged by refrigeration.
hydrate. It is important that physical parameters of the sterilizer Prepared media that have been refrigerated should be removed
and the efficacy of kill be monitored frequently through the use from refrigeration and equilibrated to room temperature prior
of calibration instrumentation and biological indicators. to inoculation to allow water of condensation to evaporate or
Do not autoclave media that should not be heat-sterilized. dissipate and to avoid temperature shock to the inoculum.
There are numerous formulations available that can merely be
dissolved and used directly. The performance of such media XI. User Quality Control
is seriously impaired by subjecting them to heat. For media prepared in-house, each lot of every medium must
be tested. When qualifying a new lot of culture medium,
Adding Enrichments and Supplements
always test the new lot of medium in parallel with an approved
Enrichments and supplements tend to be heat sensitive. Cool lot of medium. Quality control organisms must be maintained
the medium to 45-55°C in a water bath prior to adding enrich- appropriately and inoculum preparation should be performed
ments or supplements. Ensure adequate mixing of the basal according to published guidelines (refer to the monograph
medium with enrichments or supplements by swirling to mix “Quality Control Organisms”). Maintain appropriate records
thoroughly. and report deficiencies to the manufacturer.
Sterile broths may be cooled to room temperature before Comments on BD User Quality Control
adding enrichment. In the product descriptions, the User Quality Control section
pH contains procedures for identity (Identity Specifications) and
Commercial dehydrated culture media are designed to fall performance (Cultural Response). Differences in the Identity
within the specified pH range after steam sterilization. Specifications and Cultural Response testing for media offered
as both Difco and BBL brands may reflect differences in the
For filter sterilization, adjust the pH, if necessary, prior to
development and testing of media for industrial and clinical
filtering.
applications, per the referenced publications.
Measure pH at room temperature (25°C). Avoid excessive
For Identity Specifications, the expected appearance of the
pH adjustments.
powder, and of the solution following the addition of the
Dispensing and Storage of Prepared Media powder to purified water and boiling, when appropriate, are
After sterilization, media prepared in the laboratory should be indicated. The prepared (finished) medium appearance and
removed from the autoclave as soon as the pressure has fallen final pH, both determined at 25°C, are specified.
to zero. Hastening the opening of the autoclave before zero For Cultural Response, test organisms, inocula and results
pressure is reached can result in the ejection of media from the are provided. Except for those media which are tested with
sterilization vessels with considerable loss of contents. fresh cultures (undiluted agar or broth cultures), the number
For preparing plates, cool the medium to 50-55°C prior to of colony forming units (CFU) per plate is provided. Under
dispensing to reduce water evaporation. Ensure gentle mixing Recovery, growth on Difco and BBL plated media may be
during dispensing and dispense quickly. If using an automatic reported as None, Poor (growth in quadrant one), Fair (growth
plate dispenser, dispense general-purpose media before dis- in quadrants one and two) and Good (growth in quadrants
pensing selective media. Invert filled and cooled (solidified) three and four). Similar terms may be used for tubed media.
Petri dish bottoms over their off-set lids and allow to sit for The cultures listed are the minimum that should be used for
1-2 hours to obtain a dry surface. It is advisable to use freshly performance testing.
poured plated media on the day of preparation. Alternatively, For media referenced in Chapters <61> and <62> of the
plates should be placed in the refrigerator as soon as they recently harmonized United States Pharmacopeia, the “User
have solidified (agar side up) and several representative plates Quality Control” section contains the information required
incubated at 35 ± 2°C as a sterility check. If storage of plates to verify that these media were tested according to the USP,
is for more than several days, it is recommended that they EP and JP — for both dehydrated culture media and prepared
be wrapped in plastic or otherwise protected to prevent culture media products. In other words, the media listed under
moisture loss. Most media, and especially those containing “Availability” and identified with a staff mark (†), have been
dyes or indicators, should be protected from light during tested and meet USP, EP and JP performance specifications
storage. where applicable.
23
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
24
25
• Biosafety Level 3 practices are applicable to laboratories most suitable media and appropriate techniques. For detailed
working with agents with a potential for respiratory information, appropriate references should be consulted.
transmission and which may cause serious and potential * JEMBEC is a trademark of Miles Scientific.
26
macerate and immerse the electrode. Alternatively, a pH agents in selective media may inhibit some strains of the
electrode designed for flat surfaces may be used. desired species or permit growth of a species they were designed
to inhibit, especially if the species are present in large numbers
XII. Limitations of the Procedure in the specimen/sample. Cultures of specimens/samples grown
Some diagnostic tests may be performed with the primary plate on selective media should, therefore, be compared with
(e.g., BBL™ CHROMagar™ media). However, a pure culture is specimens/samples cultured on nonselective media to obtain
recommended for the majority of biochemical tests and other additional information and help ensure recovery of potential
identification procedures. Consult appropriate references for pathogens and other significant organisms.
further information.
Since the nutritional requirements of organisms vary, a single
XIII. References
1. U.S. Public Health Service, Centers for Disease Control and Prevention, and National Institutes of
medium is rarely adequate for detecting all organisms of potential Health. 2007. Biosafety in microbiological and biomedical laboratories, 5th ed. HHS Publication
No. (CDC) 93-8395. U.S. Government Printing Office, Washington, D.C.
significance in a clinical specimen or industrial sample. The 2. Clinical and Laboratory Standards Institute. 2004. Approved standard: M22-A3, Quality control for
commercially prepared microbiological culture media, 3rd ed. CLSI, Wayne, Pa.
27
variety of media for isolation and cultivation of tubercle open bench provided the potential for producing splashes
bacilli and fungi is offered. The Mycoflask bottle’s special or aerosols is low.
features include: • Biosafety Level 3 practices are applicable to laboratories
• A deep offset well to contain a deep culture bed working with agents with a potential for respiratory
transmission and which may cause serious and potential
• Surface area of 5.4 cm2 for cultivation
lethal infection. All laboratory manipulations should be
• Horizontal incubation to flood agar surface with inoculum
performed in a biological safety cabinet or other enclosed
• Flat-sided bottle which keeps bottle stable, reduces equipment to protect personnel and the environment from
chances of breakage exposure to potentially infectious aerosols.
• Tightly fitted screw caps which prevent the loss of moisture • Biosafety Level 4 practices are applicable for work with
even when incubation is extended to 8 weeks or more highly dangerous agents which may be transmitted via
Mycoflask media are packaged in specially designed trays for the aerosol route, for which there is no available vaccine
ease in handling and incubation; these Unit Boxes contain or therapy and for which specialized equipment and
10 bottles. The Shelf-Pack contains 10 Unit Boxes (10 × 10 facilities are required.
bottles). Consult the reference for specific recommendations on the
practices, equipment and facilities of the four biosafety levels.1
II. Formulae
Formulae for BBL and Difco brands of prepared tubed, bottled IV. Storage Instructions
and Mycoflask media are included in product inserts or the On receipt, media should be stored according to label instruc-
BBL™ Quality Control and Product Information Manual for tions. Freezing and overheating must be avoided. Allow the
Plated and Tubed Media or on product carton labels (bottled medium to warm to room temperature before inoculation.
media).
V. Expiration Date
III. Warnings and Precautions The expiration date applies to intact tubes and bottles stored
These media are For in vitro Diagnostic Use or For Labora- as directed. Do not open until ready to use. Tubed and bottled
tory Use as labeled. media stored as labeled until just prior to use may be inoculated
Directions for use should be read and followed carefully. up to the expiration date and incubated for recommended
incubation times, including up to 6 weeks for mycology
Care should be exercised in opening tubes and bottles with
tight caps to avoid injury due to breakage of glass. media and up to 8 weeks for mycobacteriology media.
Observe aseptic techniques and established precautions against VI. Product Deterioration
microbiological hazards throughout all procedures, since it Do not use tubes or bottles if they show evidence of microbial
must be assumed that all specimens/samples collected might contamination, discoloration, drying, cracking or other signs
contain infectious microorganisms. The use of a biohazard of deterioration.
cabinet is recommended when working with pure cultures and
specimens/samples suspected to contain fungi or mycobacteria. VII. Specimen/Sample Collection and
After use, prepared tubes, bottles or Mycoflask bottles, speci- Transport
men/sample containers and other contaminated materials must The success of a microbiological isolation procedure does
be sterilized before discarding. not depend solely on the quality of the culture media utilized.
To minimize the risk in microbiology laboratories of working Proper specimen/sample collection and transport are crucial
with infectious microorganisms and specimens and samples steps in the isolation process. A variety of transport systems
suspected to contain them, the United States Department of and holding media have been devised to prolong the survival
Health and Human Services has published guidelines for han- of microorganisms when a significant delay is expected
dling these agents and materials.1 The guidelines describe four between collection and definitive culturing.
biosafety levels, some of which are mentioned in this manual Clinical Specimens
in association with specific microorganisms:
For clinical specimens, BBL™ CultureSwab™ and Port-A-Cul™
• Biosafety Level 1 is applicable when work is done with collection and transport products are available. For transport
defined and characterized strains of viable organisms and growth of the pathogenic Neisseria, Transgrow, Gono-Pak
not known to consistently cause disease in healthy adult and JEMBEC™* systems containing Modified Thayer-Martin
humans. (MTM II), Martin-Lewis or GC-Lect™ Agars are recommended.
• Biosafety Level 2 practices are applicable to laborato- Specimens should be obtained before antimicrobial therapy
ries in which work is done with the broad spectrum has been administered. Provision must be made for prompt
of indigenous moderate-risk agents that are associated delivery to the laboratory.
with human disease; activities can be performed on the
28
The clinical laboratory must be furnished with sufficient XI. User Quality Control
patient information to enable the microbiologist to select the Quality control procedures for BBL™ brand prepared tubed
most suitable media and appropriate techniques. For detailed and Mycoflask media are included in product inserts or the
information, appropriate references should be consulted. BBL™ Quality Control and Product Information Manual for
* JEMBEC is a trademark of Miles Scientific.
Plated and Tubed Media.
Industrial Samples
If a culture medium being subjected to quality-control testing
Sterile containers should be used to collect samples. For is a formulation to which the Clinical and Laboratory Standards
environmental monitoring, samples can be collected using
Institute (CLSI) standard, Quality Control for Commercially
BD Sterile Pack Swabs.
Prepared Microbiological Culture Media,2 applies, American
Samples must represent the mass of the material being Type Culture Collection (ATCC™) control strains specified by
examined. Samples may require special handling, including the document are utilized; additional ATCC and other organ-
refrigeration, to prevent the direct contamination of the ism strains may be also employed. If no standard exists for the
sample by microorganisms and the subsequent growth of such particular medium, the organisms used represent strains from
contaminants during sampling, transportation and storage before our stock culture collection. Except for CLSI-specified strains,
examination. For detailed information, appropriate references cultures employed in testing procedures may be added or
should be consulted. changed from time to time as strains are found that provide
a greater challenge. Clinical isolates are included periodically
VIII. Materials Provided for various formulations so as to check performance with
Prepared tubed or bottled medium. “wild” strains.
IX. Materials Required But Not Provided An uninoculated tube of medium always should be incubated
Ancillary culture media, reagents and laboratory equipment with the inoculated tubes for purposes of comparison (e.g.,
as required. color changes, turbidity) following the incubation period. This
procedure should be adopted both for quality control and test
X. Procedures specimen evaluations.
In some tubes, the agar may become distorted during ship- A single electrode of sufficiently small size to fit into the tubes
ment (e.g., semisolid formulations used for motility studies). should be used to determine the pH potentiometrically of
Additionally, media (including all thioglycollate-containing tubed and Mycoflask media. The tip of the electrode should
media) may become oxidized within the tube or bottle during be placed below the surface of broth media, and positioned
shipment. These can be restored to their proper condition by in the central portion of the agar mass in semisolid or solid
bringing to 100°C in a boiling water bath and resolidifying media. Warm all media to room temperature (25°C) prior to
in the appropriate position; screw caps should be slightly measuring pH.
loosened prior to boiling. The boiling also serves to reduce
media intended for the cultivation of anaerobic organisms; XII. Limitations of the Procedure
caps should be tightened during cooling to room temperature. Some diagnostic tests may be performed with the primary
Consult product label for specific instructions. culture. However, a pure culture is recommended for bio-
Tubed media in deeps (pour tubes) must be boiled, cooled to chemical tests and other identification procedures. Consult
45-50°C, poured into sterile Petri dishes and allowed to harden appropriate references for further information.
for a minimum of 30 minutes prior to use. Since the nutritional requirements of organisms vary, a single
NOTE: Use of a microwave oven to melt tubed and bottled medium is rarely adequate for detecting all organisms of
media is not recommended. potential significance in a clinical specimen or industrial
sample. The agents in selective media may inhibit some strains
Cultures requiring prolonged incubation, for example, myco-
of the desired species or permit growth of a species they were
bacteria and fungi, should be incubated with caps tightly closed
designed to inhibit, especially if the species are present in
to prevent dehydration and consequent inhibition of growth.
large numbers in the specimen/sample. Cultures of specimens/
The United States Pharmacopeia requires that Fluid samples grown on selective media should, therefore, be com-
Thioglycollate Medium and Soybean-Casein Digest Medium pared with specimens/samples cultured on nonselective media
(Tryptic Soy Broth and Trypticase™ Soy Broth) be incubated to obtain additional information and help ensure recovery of
under aerobic conditions. Aerobic conditions can be main- potential pathogens and other significant organisms.
tained during incubation by insertion of a venting needle that
is left in place during incubation or by incubating the tubes Culture media sometime contain dead organisms derived
or bottles with the caps slightly loosened. from medium constituents, which may be visible in smears of
culture media. Other sources of dead organisms visible upon
29
Gram staining include staining reagents, immersion oil, glass XIII. References
slides and the specimens used for inoculation. If there is 1. U.S. Public Health Service, Centers for Disease Control and Prevention, and National Institutes of
Health. 2007. Biosafety in microbiological and biomedical laboratories, 5th ed. HHS Publication
uncertainty about the validity of the Gram stain, the culture No. (CDC) 93-8395. U.S. Government Printing Office, Washington, D.C.
2. Clinical and Laboratory Standards Institute. 2004. Approved standard: M22-A3, Quality control for
should be reincubated for another hour or two and the test commercially prepared microbiological culture media, 3rd ed. CLSI, Wayne, Pa.
repeated before a report is given.
30
32
Formula
Difco™ A-1 Medium
Approximate Formula* Per Liter
Tryptone ................................................................... 20.0 g
Lactose ....................................................................... 5.0 g
Sodium Chloride ......................................................... 5.0 g
Salicin ......................................................................... 0.5 g
Triton X-100 ................................................................ 1.0 mL
*Adjusted and/or supplemented as required to meet performance criteria.
33
A7 Agar, Modified
Intended Use The medium is particularly useful for detection and identifica-
A7 Agar, Modified is used for the cultivation, identification tion of U. urealyticum in primary cultures of urine and urethral
and differentiation of Ureaplasma urealyticum and other exudates. The medium, by incorporation of a direct test for
members of the genus Ureaplasma from other members of the urease in colonies of Ureaplasma, provides for a simple and rapid
Mycoplasmatales. differentiation of the genus from other Mycoplasmatales.
Procedure
Using an extract from swabs, or the specimen itself, streak the
surface of the medium. Incubate plates in a moist anaerobic
atmosphere supplemented with CO2 (BD GasPak™ EZ container
or equivalent system) at 35-37° C. Incubate plates for 48 hours
or longer. Examine plates for colonies of U. urealyticum with
the culture plate inverted on the microscope stage, under low
power (100×).2
Expected Results
Isolated colonies should give results consistent with those of
the quality control stains. Colonies of U. urealyticum are small
(usually 16-18 mm), dark, golden brown or deep brown with a
light background color of the medium. Species of Ureaplasma
34
AC Broth
Intended Use Principles of the Procedure
AC Broth is used for cultivating a wide variety of microorganisms Peptone, beef extract and malt extract provide the carbon and
and for the sterility testing of turbid or viscous solutions and nitrogen sources required for good growth of a wide variety of
other materials not containing mercurial preservatives. organisms. Vitamins and cofactors required for growth as well
as additional sources of nitrogen and carbon are provided by
Summary and Explanation yeast extract. Dextrose is a carbon energy source. Ascorbic acid
AC Broth possesses growth-promoting properties for a wide is added to clarify the solution.
variety of microorganisms. Christensen 1 and Malin and
Finn2 reported that AC Medium does not exhibit the toxicity Formula
shown by media containing sodium thioglycollate. Several early Difco™ AC Broth
studies reported on the wide variety of organisms able to grow Approximate Formula* Per Liter
on AC Medium.3-5 AC Broth is suitable for use in the detection Proteose Peptone No. 3.............................................. 20.0 g
Beef Extract.................................................................. 3.0 g
of obligately aerobic contaminants in biologicals and other Yeast Extract................................................................ 3.0 g
products. AC Broth is also useful in the isolation and cultiva- Malt Extract................................................................. 3.0 g
tion of many common pathogenic and saprophytic aerobes.6 Dextrose...................................................................... 5.0 g
Ascorbic Acid............................................................... 0.2 g
The medium can be used to test the sterility of biologicals and *Adjusted and/or supplemented as required to meet performance criteria.
solutions that do not contain mercurial preservatives. Fluid
Thioglycollate Medium should be employed for the sterility Directions for Preparation from
testing of solutions containing mercurial preservatives. Dehydrated Product
1. Dissolve 34 g of the powder in 1 L of purified water.
User Quality Control 2. Autoclave at 121°C for 15 minutes.
3. Store prepared medium at 15-30°C.
Identity Specifications 4. After prolonged storage, reheat in flowing steam or a boiling
Difco™ AC Broth
water bath for a few minutes to drive off dissolved gases.
Dehydrated Appearance: Light tan, free-flowing, homogeneous.
Cool without agitation.
Solution: 3.4% solution, soluble in purified water. Solution
is medium to dark amber, clear to very slightly 5. Test samples of the finished product for performance using
opalescent. stable, typical control cultures.
Prepared Appearance: Light to medium amber, clear to very slightly
opalescent. Procedure
Reaction of 3.4%
Solution at 25°C: pH 7.2 ± 0.2
See appropriate references for specific procedures.
35
References Availability
1. Christensen. 1944. Paper read at New York Meeting. American Public Health Association.
2. Malin and Finn. 1951. J. Bacteriol. 62:349.
Difco™ AC Broth
3. Reed and Orr. 1943. J. Bacteriol. 45:309. Cat. No. 231710 Dehydrated – 500 g
4. Schneiter, Dunn and Caminita. 1945. Public Health Rep. 60:789.
5. Kolb and Schneiter. 1950. J. Bacteriol. 59:401.
6. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria,
vol. 1. Williams & Wilkins, Baltimore, Md.
36
References
1. Arret and Kirshbaum. 1959. J. Milk Food Technol. 22:329.
2. Richardson (ed.). 1985. Standard methods for the examination of dairy products, 15th ed.
American Public Health Association. Washington, D.C.
37
Directions for Preparation from triplicate at monthly intervals. One of the transfers is saved
Dehydrated Product for the preparation of stock cultures. The others are used
Difco™ APT Agar to prepare inoculum in APT Broth for assay as needed.
1. Suspend 61.2 g of the powder in 1 L of purified water. Mix Following incubation at 35-37°C for 24-48 hours, store stock
thoroughly. cultures at 2-8°C.
2. Heat with frequent agitation and boil for 1 minute to
completely dissolve the powder.
Expected Results
Refer to appropriate references and procedures for results.
3. Autoclave at 121°C for 15 minutes. Avoid overheating.
4. Test samples of the finished product for performance using
References
stable, typical control cultures. 1. Evans and Niven. 1951. J. Bacteriol. 62:599.
2. Deibel, Evans and Niven. 1957. J. Bacteriol. 74:818.
Difco APT Broth
™
3. Hall, Ledenbach and Flowers. 2001. In Downes and Ito (ed.), Compendium of methods for the
microbiological examination of foods, 4th ed. American Public Health Association, Washington,
1. Suspend 46.2 g of the powder in 1 L of purified water. Mix D.C.
thoroughly.
2. Warm slightly to completely dissolve the powder. Availability
3. Autoclave at 121°C for 15 minutes. Avoid overheating. Difco™ APT Agar
4. Test samples of the finished product for performance using compf usda
Acetamide Agar
Intended Use Expected Results
Acetamide Agar is used in the differentiation of nonfermentative Deamination of the acetamide is indicated by a pronounced
gram-negative bacteria, particularly Pseudomonas aeruginosa. purplish-red color of the medium.
Complete identification requires determination of the Gram
Summary and Explanation reaction, cellular morphology, biochemical reactions, etc.
Assimilation studies by Gilardi and others using basal mineral
Appropriate references may be consulted for further infor-
media showed that acetamide was utilized by a wide variety
mation.10, 11
of nonfermenting organisms.1,2 However, few organisms are
reported to deaminate acetamide.3,4 A variety of media
Limitations of the Procedure
formulations have been developed to determine the ability of
Some strains deaminate acetamide slowly and may require as
various nonfermenting gram-negative organisms to deaminate
long as 7 days to yield a positive test result.
acetamide for purposes of identification.5-8 The formulation of
this medium is the one recommended in Standard Methods Only about 37% of apyocyanogenic strains of P. aeruginosa
for the Examination of Water and Wastewater.9 will produce a positive reaction. Therefore, this test should
not be relied upon as a sole criterion for identification.11
Principles of the Procedure
The ability to deaminate acetamide (acylamidase activity) has References
1. Gilardi. 1974. Antonie van Leewenhoek. J. Microbiol. Serol. 39:229.
been found to be most actively accomplished by P. aeruginosa, 2. Stainier, Palleroni and Doudoroff. 1966. J. Gen. Microbiol. 43:159.
3. Pickett and Pedersen. 1970. Can. J. Microbiol. 16:351.
Comamonas acidovorans, Achromobacter xylosoxidans subsp. 4. Pickett and Pedersen. 1970. Can. J. Microbiol. 16:401.
xylosoxidans (Alcaligenes xylosoxidans) and Alcaligenes faecalis 5.
6.
Hedberg. 1969. Appl. Microbiol. 17:481.
Smith and Dayton. 1972. Appl. Microbiol. 24:143.
(odorans).8 Deamination of acetamide produces ammonia 7. Buhlmann, Vischer and Bruhin. 1961. J. Bacteriol. 82:787.
8. Oberhofer and Rowen. 1974. Appl. Microbiol. 28:720.
which increases the pH of the medium causing a corresponding 9. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C.
color change from yellow-orange to purplish-red. 10. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology,
9th ed. Williams & Wilkins, Baltimore, Md.
11. Murray, Baron, Pfaller, Tenover and Yolken (ed.). 1999. Manual of clinical microbiology, 7th ed.
Procedure American Society for Microbiology, Washington, D.C.
few cannot use the acetate as a source of carbon. The blue color
Cultural Response
of the bromthymol blue is due to the production of alkaline Difco™ Acetate Differential Agar
products from the utilization of the sodium acetate. Prepare the medium per label directions. Inoculate with fresh cultures and
incubate at 35 ± 2°C for 2-7 days. Acetate utilization is indicated by a color
Formula change of the slant from green to blue.
Difco™ Acetate Differential Agar ORGANISM ATCC™ RECOVERY acetate UTILIZATION
Approximate Formula* Per Liter Escherichia coli 25922 Good Positive (blue)
Sodium Acetate........................................................... 2.0 g
Shigella sonnei 25931 Poor to good Negative (green)
Magnesium Sulfate...................................................... 0.1 g
Sodium Chloride.......................................................... 5.0 g
Monoammonium Phosphate........................................ 1.0 g
Dipotassium Phosphate................................................ 1.0 g
Bromthymol Blue......................................................... 0.08 g
Agar.......................................................................... 20.0 g
*Adjusted and/or supplemented as required to meet performance criteria.
39
References Availability
1. Koser. 1923. J. Bacteriol. 8:493. Difco™ Acetate Differential Agar
2. Simmons. 1926. J. Infect. Dis. 39:209.
3. Trabulsi and Ewing. 1962. Public Health Lab. 20:137. BAM COMPF SMD
4. Ewing. 1986. Edwards and Ewing’s identification of Enterobacteriaceae, 4th ed. Elsevier Science
Publishing Co., Inc., New York, N.Y. Cat. No. 274210 Dehydrated – 500 g
5. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology,
9th ed. Williams & Wilkins, Baltimore, Md. BBL™ Acetate Differential Agar
6. Farmer. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology,
7th ed. American Society for Microbiology, Washington, D.C. BAM COMPF SMD
Cat. No. 221375 Prepared Slants – Pkg. of 10
Acidicase™ Peptone
(See Casamino Acids)
Actinomyces Broth
Intended Use Formula
Actinomyces Broth is used as a liquid medium or, with the BBL™ Actinomyces Broth
addition of 7 or 20 g/L of agar, as a semisolid or solid medium, Approximate Formula* Per Liter
Heart Muscle, Infusion from (solids).............................. 2.0 g
respectively, for the maintenance or cultivation of Actinomyces Pancreatic Digest of Casein........................................ 17.0 g
species. Yeast Extract.............................................................. 10.0 g
Sodium Chloride.......................................................... 5.0 g
Dipotassium Phosphate.............................................. 13.0 g
Summary and Explanation Monopotassium Phosphate.......................................... 2.0 g
Actinomyces Broth is a basic medium modified from the Dextrose...................................................................... 5.0 g
Actinomyces Maintenance Medium of Pine and Watson.1 It Ammonium Sulfate...................................................... 1.0 g
L-Cysteine HCl............................................................. 1.0 g
is recommended for use in the growth and maintenance of
Soluble Starch.............................................................. 1.0 g
members of the genus Actinomyces.2 Magnesium Sulfate...................................................... 0.2 g
Calcium Chloride......................................................... 0.01 g
Principles of the Procedure *Adjusted and/or supplemented as required to meet performance criteria.
40
41
Agars
Bacto™ Agar • Agar, Grade A • Agar, Granulated
Agar, Technical • Agar, Noble • Agarose • Agar, Select
Intended Use used in media for motility studies (0.5% w/v) and for growth
Bacto Agar is a solidifying agent in which extraneous matter,
™
of anaerobes (0.1%) and microaerophiles.3
pigmented portions and salts have been reduced to a minimum. The use of small amounts of agar in media for sterility testing
Bacto Agar is used in preparing microbiological culture media. was recommended by Falk et al.4 and has been incorporated
Agar, Grade A is a high-grade agar, specially processed for into Fluid Thioglycollate Medium for sterility testing by
microbiological purposes. It is routinely used as a solidifying standard procedures.5
agent in microbiological media. Specifications for bacteriological grade agar include good
Agar, Granulated is a solidifying agent used in preparing clarity, controlled gelation temperature, controlled melting
microbiological culture media. temperature, good diffusion characteristics, absence of toxic
bacterial inhibitors and relative absence of metabolically useful
Agar, Technical is a solidifying agent used in preparing
minerals and compounds.
microbiological culture media. Although Agar, Technical has
wider quality control parameters than other bacteriological
Principles of the Procedure
agars, solubility, gelation temperature and solidity are carefully
Bacto Agar is optimized for beneficial calcium and magne-
monitored to permit its use.
sium content. Detrimental ions such as iron and copper are
Agar, Noble is a solidifying agent that is essentially free of reduced. Bacto Agar is recommended for clinical applications,
impurities. It is used in electrophoretic and nutritional procedures auxotrophic studies, bacterial and yeast transformation
and in preparing microbiological culture media when studies and bacterial molecular genetics applications.6,7
increased purity is required.
Grade A Agar is a select grade of agar containing essential
Agarose is a complex galactose polysaccharide of near neutral minerals for bacterial growth. When utilized as an ingredient,
charge. It is specially prepared and is intended mainly for use most media formulations demonstrate improved growth and
in gel electrophoresis. test reactions.
Agar, Select is recommended for molecular genetics testing. Granulated Agar is qualified for culturing recombinant strains
of Escherichia coli (HB101) and Saccharomyces cerevisiae.
Summary and Explanation Agar, Granulated may be used for general bacteriological
Agar is a phycocolloid extracted from a group of red-purple purposes where clarity is not a strict requirement.
marine algae (Class Rhodophyceae) including Gelidium,
Technical Agar is suitable for many bacteriological applications.
Pterocladia and Gracilaria. Gelidium is the preferred source for
This agar is not highly processed, has broader technical speci-
agars. Impurities, debris, minerals and pigment are reduced to
fications than other agars and is not recommended for growth
specified levels during manufacture.
of fastidious organisms.
Agar was first suggested for microbiological purposes in
Noble Agar is extensively washed and bleached. This agar
1881 by Fannie Hesse.1,2 By the early 1900s, agar became the
should be used for applications where extreme clarity and high
gelling agent of choice over gelatin because agar remains firm
purity are required. Noble Agar is suitable for immunodiffusion,
at growth temperatures for many pathogens. Agar is also
some electrophoretic applications, and as a substrate for
generally resistant to a breakdown by bacterial enzymes. The
mammalian or plant tissue culture.
use of agar in microbiological media significantly contributed
to the advance of microbiology, paving the way for pure Agarose is the low sulfate, neutral gelling fraction of agar.
culture isolation and study. During the fractionation of agar, the agarose-portion is
separated from the highly charged polysaccharides (high
Agar is a gel at room temperature, remaining firm at tempera-
sulfate, nongelling portion), purified and dried. Because of its
tures as high as 65°C.3 Agar melts at approximately 85°C, a
method of preparation, Agarose is considerably purer than the
different temperature from that at which it solidifies, 32-40°C.
special kinds of agar, with respect to ionic groups, rendering
This property is known as hysteresis. Agar is generally resistant
it more valuable for gel electrophoresis.8 In addition to
to shear forces; however, different agars may have different
high chemical purity, Agarose must exhibit certain physical
gel strengths or degrees of stiffness.
properties; e.g., high gel strength and high gel clarity.8 The
Agar is typically used in a final concentration of 1-2% for suggested concentration for use is 0.5-1.2%.
solidifying culture media. Smaller quantities (0.05-0.5%) are
Select Agar is a key ingredient used in molecular genetics work
for determining bacteriophage lambda titers.
42
Cultural Response
Prepare the agar formulation of Nutrient Broth or LB Broth, Miller by adding 1.5% agar. Inoculate with 102-103 CFU of the indicated test organisms and incubate
at 35 ± 2°C for 18-24 hours (18-72 hours for LB Broth, Miller). Record recovery.
Procedure References
See appropriate references for specific procedures using Bacto™ 1.
2.
Hesse. 1894. Mitt. a.d. Kaiserl. Gesh. Berlin 2:182.
Hitchens and Leikind. 1939. J. Bacteriol. 37:485.
Agar, Grade A Agar, Granulated Agar, Technical Agar, Noble 3. Selby and Selby. 1959. Agar. In Whister (ed.), Industrial gums. Academic Press Inc., New York, N.Y.
4. Falk, Bucca and Simmons. 1939. J. Bacteriol. 37:121.
Agar, Agarose or Select Agar. 5. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The
national formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc.,
Rockville, Md.
Expected Results 6. Sambrook, Fritsch and Maniatis. 1989. Molecular cloning, a laboratory manual, 2nd ed. Cold
Spring Harbor Laboratory Press, New York, N.Y.
Refer to appropriate references and procedures for results. 7. Schiestl and Geitz. 1989. Current Genetics 16:339.
8. Guiseley and Renn. 1975. Agarose: purification, properties, and biomedical applications. Marine
Colloids, Inc. Rockland, Maine.
43
Typical Analyses
BACTO™ BBL™ AGAR, DIFCO™ AGAR, DIFCO™ AGAR, DIFCO™ AGAR, BBL™ BBL™ AGAR,
AGAR GRADE A GRANULATED TECHNICAL NOBLE AGAROSE SELECT
Physical Characteristics
Concentration (%) 1.5 1.5 1.5 1.5 1.5 1.0 1.5
Ash (%) 3.6 3.0-6.5 3.4 4.1 1.3 < 1.0 2.0-6.5
Clarity (NTU)* 4.3 < 10 5.3 26.2 3.7 < 10 N/A
Color (430 nm, adsorbance) N/A < 0.2 N/A N/A N/A < 0.2 N/A
Loss on Drying (%) 17.3 < 10 12.2 18.2 16.0 < 10 N/A
pH 6.5 5.5-7.5 6.6 6.9 5.7 6.0-7.0 5.5-7.5
Gel Strength (g/cm2) 600 600-800 560 613 700 800-1200 N/A
Gelation Point (°C) 35 35-39 35 36 35 35-39 33-38
Melting Point (°C) 88 80-90 88 88 87 80-90 80-90
Resistivity (ohms) N/A N/A N/A N/A N/A > 50,000 N/A
-mr (electrophoretic)** N/A N/A N/A N/A ≤ 0.55 < 0.25 N/A
Inorganics (%)
Calcium 0.179 0.23 0.133 0.110 0.015 0.03 N/A
Chloride 0.021 N/A < 0.005 0.172 < 0.050 N/A N/A
Cobalt < 0.001 N/A < 0.001 < 0.001 < 0.001 N/A N/A
Copper < 0.001 N/A < 0.001 < 0.001 < 0.001 N/A N/A
Iron 0.002 < 0.0060 0.003 0.002 < 0.001 < 0.0050 N/A
Lead < 0.001 N/A < 0.001 < 0.001 < 0.001 N/A N/A
Magnesium 0.068 0.10 0.041 0.093 0.002 0.01 N/A
Manganese < 0.001 N/A < 0.001 < 0.001 < 0.001 N/A N/A
Nitrate < 0.005 N/A < 0.005 < 0.005 < 0.050 N/A N/A
Phosphate < 0.005 0.02 0.010 0.015 < 0.050 0.08 N/A
Potassium 0.121 0.03 0.079 0.124 0.022 0.015 N/A
Sodium 0.837 1.8 0.776 0.932 0.335 < 0.1 N/A
Sulfate 1.778 N/A 1.710 0.367 0.663 N/A N/A
Sulfur 0.841 0.7 0.868 0.646 0.333 0.1 N/A
Tin < 0.001 N/A < 0.001 < 0.001 < 0.001 N/A N/A
Zinc < 0.001 N/A < 0.001 < 0.001 < 0.001 N/A N/A
44
45
46
Cultural Response
Difco™ Lysine Assay Medium or Cystine Assay Medium
Prepare the medium per label directions. These media support the growth of Pediococcus acidilactici ATCC™ 8042 when prepared in single strength and supple-
mented with the appropriate amino acid. Lysine Assay Medium should produce a standard curve when tested with L-Lysine at 0.0 to 300 µg per 10 mL. Cystine
Assay Medium should produce a standard curve when tested with L-Cystine at 0 to 50 µg per 10 mL. Incubate tubes with caps loosened at 35-37°C for 16-20
hours. Read the percent transmittance at 660 nm.
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 + purified H2O) purified H2O) (ml/10 mL tube) µg/10 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
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
47
48
49
Anaerobic Agar
Intended Use bacteremia.5,6 Anaerobic bacteria are the predominant flora
Anaerobic Agar is used for cultivating anaerobic microorgan- colonizing the skin and mucous membranes of the body. 3
isms. Anaerobes vary in their sensitivity to oxygen and nutritional
requirements.2 Anaerobic bacteria lack cytochromes and thus
Summary and Explanation are unable to use oxygen as a terminal electron acceptor.3
Brewer1 described a special Petri dish cover that allowed
surface growth of anaerobes and microaerophiles without Principles of the Procedure
anaerobic equipment. The microorganisms were grown on Peptone provides the nitrogen, vitamins and amino acids in
an agar-based medium having a low oxidation-reduction Anaerobic Agar. Dextrose is a carbon source. Sodium chloride
potential. Anaerobic Agar is a modification of Brewer’s maintains the osmotic equilibrium. Sodium thioglycollate
original formula. This medium is suitable for standard plating and sodium formaldehyde sulfoxylate are reducing agents.
procedures used in cultivating anaerobic bacteria.2-4 Methylene blue serves as an indicator of anaerobiosis with
a blue color indicating the presence of oxygen. Agar is the
Anaerobic bacteria cause a variety of infections in humans,
solidifying agent.
including otitis media, oral infections, endocarditis, meningitis,
wound infections following bowel surgery or trauma, and
50
51
52
DEHYDRATED PREPARED
SOLUTION PH AT 25° C
APPEARANCE APPEARANCE
Difco™ Antibiotic Tan, free-flowing, 1.75% solution, soluble in purified water upon Light to medium
7.0 ± 0.05
Medium 3 homogeneous. boiling. Solution is light to medium amber, clear. amber, clear.
BBL™ Antibiotic Assay Fine, homogeneous, 1.75% solution, soluble in purified water upon Pale to light,
Broth (Antibiotic free of extraneous boiling. Solution is very pale to light, yellow to tan, yellow to tan, clear 7.0 ± 0.2
Medium 3) material. clear to slightly hazy. to slightly hazy.
Light tan, 2.65% solution, soluble in purified water upon Light amber, very
Difco™ Antibiotic
free-flowing, boiling. Solution is light amber, very slightly slightly to slightly 6.55 ± 0.05
Medium 4
homogeneous. opalescent. opalescent.
Light tan, 2.55% solution, soluble in purified water upon Light to medium
Difco™ Antibiotic
free-flowing, boiling. Solution is light to medium amber, very amber, slightly 7.9 ± 0.1
Medium 5
homogeneous. slightly to slightly opalescent. opalescent.
Light tan, 2.55% solution, soluble in purified water upon Light to medium
Difco™ Antibiotic
free-flowing, boiling. Solution is light to medium amber, very amber, slightly 5.85 ± 0.05
Medium 8
homogeneous. slightly to slightly opalescent. opalescent.
Light to medium
Light beige, 5.0% solution, soluble in purified water upon
Difco™ Antibiotic amber, slightly
free-flowing, boiling. Solution is light to medium amber, slightly 7.25 ± 0.05
Medium 9 opalescent with slight
homogeneous. opalescent, may have a slight flocculent precipitate.
flocculent precipitate.
Beige,
5.2% solution, soluble in purified water upon Light to medium
Difco™ Antibiotic homogeneous,
boiling. Solution is light to medium amber, very amber, slightly 7.25 ± 0.05
Medium 10 moist with a
slightly to slightly opalescent. opalescent.
tendency to clump.
BBL™ Sabouraud
Fine, homogeneous, 3.0% solution, soluble in purified water upon Light to medium,
Liquid Broth,
free of extraneous boiling. Solution is light to medium, yellow to tan, yellow to tan, 5.7 ± 0.1
Modified (Antibiotic
material. clear to slightly hazy. clear to slightly hazy.
Medium 13)
Continued
53
Cultural Response
Difco™ Antibiotic Medium 1 Antibiotic Medium 9
Difco™ Antibiotic Medium 2 Antibiotic Medium 10
Prepare the medium per label directions. Inoculate by the pour plate method Prepare the medium per label directions. Inoculate by the pour plate
and incubate at 35 ± 2°C for 18-24 hours. method and incubate at 35 ± 2°C for 40-48 hours.
Organism ATCC™ Inoculum CFU RECOVERY Organism ATCC™ Inoculum CFU RECOVERY
Staphylococcus aureus 6538P 30-300 Good Bordetella bronchiseptica 4617 30-300 Good
Antibiotic Medium 5
Antibiotic Medium 8
Prepare the medium per label directions. Inoculate by the pour plate
method and incubate at 35 ± 2°C for 18-24 hours.
Organism ATCC™ Inoculum CFU RECOVERY
Bacillus subtilis 6633 30-300 Good
BBL™ Antibiotic Assay Broth (Antibiotic Medium 3) Difco™ Antibiotic Medium 9 (Polymyxin Base Agar)
Approximate Formula* Per Liter Approximate Formula* Per Liter
Beef Extract.................................................................. 1.5 g Pancreatic Digest of Casein........................................ 17.0 g
Yeast Extract................................................................ 1.5 g Soy Peptone................................................................. 3.0 g
Pancreatic Digest of Gelatin......................................... 5.0 g Dextrose...................................................................... 2.5 g
Dextrose ..................................................................... 1.0 g Sodium Chloride.......................................................... 5.0 g
Sodium Chloride.......................................................... 3.5 g Dipotassium Phosphate................................................ 2.5 g
Dipotassium Phosphate................................................ 3.68 g Agar.......................................................................... 20.0 g
Monopotassium Phosphate.......................................... 1.32 g
Difco™ Antibiotic Medium 10 (Polymyxin Seed Agar)
Difco™ Antibiotic Medium 4 (Yeast Beef Agar) Approximate Formula* Per Liter
Approximate Formula* Per Liter Pancreatic Digest of Casein........................................ 17.0 g
Beef Extract.................................................................. 1.5 g Soybean Peptone......................................................... 3.0 g
Yeast Extract................................................................ 3.0 g Dextrose...................................................................... 2.5 g
Peptone....................................................................... 6.0 g Sodium Chloride.......................................................... 5.0 g
Dextrose...................................................................... 1.0 g Dipotassium Phosphate................................................ 2.5 g
Agar.......................................................................... 15.0 g Agar.......................................................................... 12.0 g
Polysorbate 80........................................................... 10.0 g
Difco™ Antibiotic Medium 5 (Streptomycin Assay Agar)
Same as Medium 2, except for the final pH after autoclaving. Difco™ Antibiotic Medium 11 (Neomycin Assay Agar)
Same as Medium 1, except for the final pH after autoclaving.
Difco™ Antibiotic Medium 8
Same as Medium 2, except for the final pH after autoclaving.
54
55
Difco™ Antibiotic Medium 19 sporulation of B. subtilis and may be used in preparing the
Approximate Formula* Per Liter spore suspension.
Beef Extract.................................................................. 2.4 g
Yeast Extract................................................................ 4.7 g When B. cereus var. mycoides is required, inoculate the organism
Peptone....................................................................... 9.4 g on Antibiotic Medium 1 and incubate at 30°C for 1 week. Wash
Dextrose.................................................................... 10.0 g
Sodium Chloride........................................................ 10.0 g and prepare the spores as for B. subtilis, above.
Agar.......................................................................... 23.5 g
Cylinder Plate Assay
*Adjusted and/or supplemented as required to meet performance criteria.
Use 20 × 100 mm glass or plastic Petri dishes with sufficient
Directions for Preparation from depth so that cylinders used in the assay will not be pushed
Dehydrated Product into the medium by the cover.
1. Suspend the powder in 1 L of purified water: Use stainless steel or porcelain assay cylinders having the
Difco™ Antibiotic Medium 1 – 30.5 g; following dimensions (± 0.1 mm): 8 mm outside diameter,
Difco™ Antibiotic Medium 2 – 25.5 g; 6 mm inside diameter and 10 mm long.2 Carefully clean the
Difco™ Antibiotic Medium 3 – 17.5 g; cylinders to remove all residues, using an occasional acid bath
BBL™ Antibiotic Assay Broth (i.e., with approximately 2N nitric acid or with chromic acid).2
(Antibiotic Medium 3) – 17.5 g; Four or six cylinders are generally used per plate, evenly spaced
Difco™ Antibiotic Medium 4 – 26.5 g; on a 2.8 cm radius.
Difco™ Antibiotic Medium 5 – 25.5 g;
Difco™ Antibiotic Medium 8 – 25.5 g; To assure accurate assays, work on a level surface to obtain
Difco™ Antibiotic Medium 9 – 50 g; uniformly thick base and seed layers in the Petri dish. Allow
Difco™ Antibiotic Medium 10 – 52 g; the base layer to solidify and then overlay the seed layer contain-
Difco™ Antibiotic Medium 11 – 30.5 g; ing a proper concentration of the test organism. The amount
Difco™ Antibiotic Medium 12 – 62.5 g; of medium in the layers varies for different antibiotics, with
BBL™ Sabourand Liquid Broth, Modified most assays specifying a 21 mL base layer and a 4 mL seed
(Antibiotic Medium 13) – 30 g; layer. In any case, dishes with flat bottoms are required to
Difco™ Antibiotic Medium 19 – 60 g. assure complete coverage of the bottom of the dish when small
Mix thoroughly. amounts of base medium are used. Tilt the plate to obtain even
2. Heat with frequent agitation and boil for 1 minute to com- coverage of the base layer by the seed layer and allow it
pletely dissolve the powder. to solidify in a level position. Plates should be used the same
3. Autoclave at 121°C for 15 minutes. day as prepared.
4. To raise the pH of Antibiotic Medium 11 to 8.3 ± 0.1, cool Turbidimetric Assay
the base to 45-50°C and add NaOH. Use glass or plastic test tubes (i.e., 16 × 125 mm or 18 × 150 mm)
5. Test samples of the finished product for performance using that are relatively uniform in length, diameter and thickness
stable, typical control cultures. and substantially free from surface blemishes.2 Tubes that
will be placed in the spectrophotometer should be matched
Procedure and free of scratches or blemishes.2 Clean the tubes thoroughly
Test Organism Preparation to remove all antibiotic residues and traces of cleaning solution
Maintain stock cultures on agar slants and make transfers and, prior to subsequent use, sterilize tubes that have been
at 1- or 2-week intervals. Prepare the inoculum for assay by previously used.2
washing growth from a fresh 24-48 hour agar slant using
Prepare working dilutions of the antibiotic reference standards
sterile purified water, saline or Antibiotic Medium 3 and
in specific concentrations. To a 1 mL quantity of each solution
further dilute the culture to obtain the desired organism
in a suitable tube, add 9 mL of inoculated broth, as required.
concentration. In some turbidimetric assays, an 18- to 24-hour
Prepare similar solutions of the assay materials containing
culture of the test organism in Antibiotic Medium 3, diluted to
approximately the same amounts of antibiotic activity and
obtain the optimal number of organisms, is used.
place in tubes. Incubate the tubes for 3-4 hours at the required
When Bacillus subtilis is used as the test organism, inoculate temperature, generally in a water bath. At the end of the
it on Antibiotic Medium 1 and incubate at 37°C for 1 week, incubation period, stop growth by adding 0.5 mL of 1:3
wash spores from the agar surface, and heat the spores at 56°C formalin. Determine the amount of growth by measuring light
for 30 minutes. Wash the spores three times in purified water, transmittance with a suitable spectrophotometer. Determine
heat again at 65°C for 30 minutes, and then dilute to the the concentration of the antibiotic by comparing the growth
optimal concentration. This inoculum preparation should obtained with that given by reference standard solutions.
produce a sharp zone in the assay.
For a complete discussion of antibiotic assay methods, refer to
Antibiotic Medium 1 modified by the addition of 300 mg appropriate procedures outlined in the references.2,3,7
manganese sulfate (MnSO4•H2O) per liter often aids the
56
57
Level 3 practices, containment equipment and facilities are Incubate the 0.003 M broth for 14 days, then remove and add
required for laboratory activities in the propagation and six drops of the 1 M sodium carbonate solution. Observe for
manipulation of cultures of M. tuberculosis and M. bovis. a color change.
Animal studies also require special procedures.
Expected Results
Procedure A change in the color of the medium to pink or red following
The test procedures are those recommended by the Centers the addition of sodium carbonate is a positive reaction. The
for Disease Control and Prevention (CDC) for primary medium remains colorless if the reaction is negative.
isolation from specimens containing mycobacteria. 4
N-Acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) solution References
is recommended as a gentle, but effective, digesting and 1.
2.
Whitehead, Wildy and Engbaek. 1953. J. Pathol. Bacteriol. 65: 451.
Kubica and Vestal. 1961. Am. Rev. Respir. Dis. 83: 728.
decontaminating agent. These reagents are provided in the 3.
4.
Kubica and Rigdon. 1961. Am. Rev. Respir. Dis. 83: 737.
Cernoch, Enns, Saubolle and Wallace. 1994. Cumitech 16A, Laboratory diagnosis of mycobacterioses.
BBL™ MycoPrep™ Specimen Digestion/Decontamination Kit. Coord. ed., Weissfeld. American Society for Microbiology, Washington, D.C.
5. Kent and Kubica. 1985. Public health mycobacteriology: a guide for the level III laboratory. USDHHS.
For detailed decontamination and culturing instructions, Centers for Disease Control, Atlanta, Ga.
6. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby, Inc.,
consult an appropriate reference.4-7 St. Louis, Mo.
7. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
Inoculate the broth with 0.1 mL of a 7-day liquid culture or American Society for Microbiology, Washington, D.C.
8. U.S. Public Health Service, Centers for Disease Control and Prevention, and National Institutes of
heavily inoculate with organisms cultured on a solid medium. Health. 2007. Biosafety in microbiological and biomedical laboratories, 5th ed. HHS Publication No.
(CD) 93-8395. U.S. Government Printing Office, Washington, D.C.
Incubate the tubes at 35°C in an aerobic atmosphere without
added CO2. Remove the 0.001 M broth after 3 days and add Availability
no more than six drops of 1 M sodium carbonate solution BBL™ Arylsulfatase Broth (0.001 M)
(10.6 g anhydrous Na2CO3 in 100 mL of water), and observe Cat. No. 295654 Prepared Tubes – Pkg. of 10*
for a color change. BBL™ Arylsulfatase Broth (0.003 M)
Cat. No. 297156 Prepared Tubes – Pkg. of 10*
*Store at 2-8°C.
58
Tube Technique.5
Cultural Response
Difco™ Azide Dextrose Broth
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-24
hours.
Organism ATCC™ Inoculum CFU RECOVERY
Enterococcus faecalis 19433 102-103 Good
Escherichia coli 25922 3×10 -10
2 3
Inhibition
Uninoculated Enterococcus faecalis
Tube ATCC™ 29212
60
Directions for Preparation from All Azide Dextrose Broth tubes showing turbidity after 24- or
Dehydrated Product 48-hours of incubation must be subjected to the Confirmed
1. Dissolve 34.7 g of the powder in 1 L of purified water for Test Procedure. Consult appropriate references for details
the preparation of single-strength broth for inoculation of of the Confirmed Test Procedure5 and further identification
samples of 10 mL or smaller. Use 69.4 g for 1 L of double-
strength broth for samples larger than 10 mL.
of Enterococcus.5,6
B
2. Autoclave at 121°C for 15 minutes. Limitations of the Procedure
3. Test samples of the finished product for performance using 1. Azide Dextrose Broth is used to detect presumptive
stable, typical control cultures. evidence of fecal contamination. Further biochemical
testing must be done for confirmation.
Procedure5 2. For inoculum sizes of 10 mL or larger, use double strength
1. Inoculate a series of Azide Dextrose Broth tubes with medium to prevent dilution of ingredients.5,6
appropriately graduated quantities of sample. Use sample
quantities of 10 mL or less. Use double-strength broth References
1. Rothe. 1948. Illinois State Health Department.
for 10 mL inocula. Consult an appropriate reference for 2. Mallmann and Seligmann. 1950. Am. J. Public Health 40:286.
3. Larkin, Litsky and Fuller. 1955. Appl. Microbiol. 3:98.
suggested sample sizes.5 4. Splittstoesser, Wright and Hucker. 1961. Appl. Microbiol. 9:303.
5. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
2. Incubate inoculated tubes at 35 ± 2°C for 20-48 hours. 21st ed., online. American Public Health Association, Washington, D.C.
3. Examine each tube for turbidity at the end of 24 ± 2 hours. 6. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol.
1. Williams & Wilkins, Baltimore, Md.
If no turbidity is evident, reincubate and read again at the
end of 48 ± 3 hours. Availability
Difco™ Azide Dextrose Broth
Expected Results EPA SMWW
A positive test is indicated by turbidity (cloudiness) in the broth. Cat. No. 238710 Dehydrated – 500 g
A negative test remains clear.
Formula with B12 at the following levels: 0.0, 0.025, 0.05, 0.075, 0.1,
Difco™ B12 Assay Medium 0.125, 0.15, 0.2 and 0.25 ng per assay tube (10 mL).
Approximate Formula* Per Liter
Vitamin Assay Casamino Acids................................... 15.0 g
Stock cultures of L. delbrueckii subsp. lactis ATCC 7830 are
Dextrose.................................................................... 40.0 g prepared by stab inoculation into 10 mL of B12 Culture Agar
Asparagine................................................................... 0.2 g or Lactobacilli Agar AOAC. After 16-24 hours incubation at
Sodium Acetate......................................................... 20.0 g
35-37°C, the cultures are kept refrigerated. The inoculum for
Ascorbic Acid............................................................... 4.0 g
L-Cystine...................................................................... 0.4 g assay is prepared by subculturing a stock culture of L. delbrueckii
DL-Tryptophan............................................................. 0.4 g subsp. lactis into 10 mL of B12 Inoculum Broth. For a complete
Adenine Sulfate......................................................... 20.0 mg discussion on B12 Culture Agar and B12 Inoculum Broth, refer
Guanine Hydrochloride.............................................. 20.0 mg
Uracil......................................................................... 20.0 mg to USP.1
Xanthine.................................................................... 20.0 mg
Riboflavin..................................................................... 1.0 mg Expected Results
Thiamine Hydrochloride............................................... 1.0 mg
Biotin ........................................................................ 10.0 µg 1. Prepare a standard concentration response curve by plotting
Niacin.......................................................................... 2.0 mg the response readings against the amount of standard in each
p-Aminobenzoic Acid................................................... 2.0 mg tube, disk or cup.
Calcium Pantothenate.................................................. 1.0 mg
Pyridoxine Hydrochloride.............................................. 4.0 mg
2. Determine the amount of vitamin at each level of assay
Pyridoxal Hydrochloride................................................ 4.0 mg solution by interpolation from the standard curve.
Pyridoxamine Hydrochloride..................................... 800.0 µg 3. Calculate the concentration of vitamin in the sample from
Folic Acid................................................................. 200.0 µg
the average of these values. Use only those values that do
Monopotassium Phosphate.......................................... 1.0 g
Dipotassium Phosphate................................................ 1.0 g not vary more than ±10% from the average and use the
Magnesium Sulfate...................................................... 0.4 g results only if two-thirds of the values do not vary more
Sodium Chloride........................................................ 20.0 mg than ±10%.
Ferrous Sulfate........................................................... 20.0 mg
Manganese Sulfate.................................................... 20.0 mg
Polysorbate 80............................................................. 2.0 g Limitations of the Procedure
*Adjusted and/or supplemented as required to meet performance criteria. 1. The test organism used for inoculating an assay medium must
be cultured and maintained on media recommended for this
Precautions purpose.
Great care must be taken to avoid contamination of media
2. For successful results to these procedures, all conditions of
or glassware in microbiological assay procedures. Extremely
the assay must be followed precisely.
small amounts of foreign material may be sufficient to give
3. Aseptic technique should be used throughout the assay
erroneous results. Scrupulously clean glassware free from
procedure.
detergents and other chemicals must be used. Glassware must
4. The use of altered or deficient media may cause mutants
be heated to 250°C for at least 1 hour to burn off any or-
having different nutritional requirements and will not give
ganic residues that might be present. Take precautions to keep
a satisfactory response.
sterilization and cooling conditions uniform throughout the
assay. References
1. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The
Directions for Preparation from national formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc.,
Rockville, Md.
Dehydrated Product 2. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
International, Gaithersburg, Md.
1. Suspend 8.5 g of the powder in 100 mL of purified water.
2. Heat with frequent agitation and boil for 2-3 minutes to Availability
completely dissolve the powder. Difco™ B12 Assay Medium
3. Dispense in 5 mL amounts into tubes, evenly dispersing AOAC USP
the precipitate. Cat. No. 245710 Dehydrated – 100 g*
4. Add standard or test samples. *Store at 2-8˚C.
Procedure
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 con-
structed each time an assay is run. Autoclave and incubation
conditions can influence the standard curve reading and cannot
always be duplicated. Generally satisfactory results are obtained
62
63
BCYE Agars
BCYE Agar Base • BCYE Agar • BCYE Differential Agar
BCYE Selective Agars (CCVC, PAC, PAV) • Legionella
Agar Base • Legionella Agar Enrichment
Intended Use BCYE Differential Agar is used for the presumptive identification
These media are used in qualitative procedures for isolation and differentiation of Legionella spp. based on colony morphol-
of Legionella species from clinical specimens and nonclinical ogy and color.5 This medium is based on the formulation of
(environmental) samples. Vickers et al.,6 and consists of the dyes bromcresol purple and
bromthymol blue added to BCYE Agar.
Summary and Explanation BCYE Selective Agar w/CCVC is a highly selective medium con-
BCYE Agar is based on Edelstein’s modification of previously sisting of BYCE Agar supplemented with cephalothin, colistin,
described media. In 1979, Feely et al. described Charcoal Yeast vancomycin and cycloheximide. This medium is based on the
Extract (CYE) Agar as a modification of an existing medium, formulation of Bopp et al.7 They obtained improved recovery
F-G Agar.1,2 They replaced the starch in the F-G Agar with of L. pneumophila by using the selective medium in conjunc-
activated charcoal and substituted yeast extract for casein tion with an acid wash treatment to reduce the contaminating
hydrolysate, resulting in better recovery of L. pneumophila. In microbial flora present in environmental water samples.
1980, Pasculle reported that CYE Agar could be improved by
buffering the medium with ACES Buffer.3 A year later, Edelstein BCYE Selective Agar with PAC was developed by Edelstein for
further increased the sensitivity of the medium by adding alpha- isolation of Legionella spp. from specimens containing mixed
ketoglutarate (BCYE Agar).4 flora.4 He found that BYCE Agar supplemented with poly-
myxin B, cefamandole and anisomycin enhanced the recovery of
Legionella Agar is a modification of the BCYE Agar formula L. pneumophila from contaminated clinical specimens. In
of Edelstein. In the formula, the concentration of ACES buffer conjunction with an acid wash treatment to reduce microbial
was reduced from 10.0 g/L to 6.0 g/L. flora, it also facilitated the recovery of the bacterium from
potable water.
64
Legionella pneumophila
ATCC™ 33152 Principles of the Procedure
These media consist of a base medium (BCYE) supplemented
with antibiotics or dyes. Antibiotics improve the recovery of
Legionella spp. by inhibiting the growth of contaminating
organisms. Dyes facilitate differentiation and identification of
Legionella spp.
The base media (BCYE Agar Base and Legionella Agar Base)
contain yeast extract to supply the nutrients necessary to sup-
port bacterial growth. L-cysteine HCl, ferric pyrophosphate
and alpha-ketoglutarate are incorporated to satisfy the specific
nutritional requirements of Legionella species. The activated
charcoal decomposes hydrogen peroxide, a toxic metabolic
product, and may also collect carbon dioxide and modify surface
tension. The addition of the buffer helps maintain the proper
pH for optimal growth of Legionella species.
Antibiotics incorporated in the various BCYE formulations have
different spectra of activity. Vancomycin inhibits gram-positive
bacteria; colistin and polymyxin B inhibit gram-negative bacte-
ria, except for Proteus spp.; and cephalothin and cefamandole
inhibit both gram-positive and gram-negative bacteria. Aniso-
mycin and cycloheximide are antifungal agents.
BCYE Selective Agar with PAV is similar to the Edelstein
formula, above, except that the concentration of polymyxin BCYE Differential Agar contains the dyes bromcresol purple and
B is reduced by half, and vancomycin is substituted for cefa- bromthymol blue to aid in the differentiation and identification
mandole. of Legionella species.
65
66
BG Sulfa Agar
SBG Sulfa Enrichment
Intended Use color with the formation of acid when lactose and/or sucrose
is fermented. Agar is the solidifying agent.
B
BG Sulfa Agar is used for isolating Salmonella.
SBG Sulfa Enrichment is used for enriching Salmonella prior Peptone provides the nitrogen, minerals and amino acids in
to isolation procedures. SBG Sulfa Enrichment. Yeast extract is the vitamin source.
D-Mannitol is the carbon source to stimulate organism growth.
Summary and Explanation The phosphates act as buffers in the enrichment. Sodium
Salmonellosis continues to be an important public health taurocholate, sodium selenite and brilliant green are the selec-
problem worldwide, despite efforts to control the prevalence tive agents. The selective agents are used to inhibit gram-positive
of Salmonella in domesticated animals. Infection with organisms and enteric bacteria other than Salmonella. Sodium
non-typhi Salmonella often causes mild, self-limiting illness. sulfapyridine is added to increase selectivity.
The illness results from consumption of raw, undercooked or
improperly processed foods contaminated with Salmonella. Formulae
Many of these cases of Salmonella-related gastroenteritis are Difco™ BG Sulfa Agar
due to improper handling of poultry products. Various poultry Approximate Formula* Per Liter
Yeast Extract................................................................ 3.0 g
products are routinely monitored for Salmonella before their Proteose Peptone No. 3.............................................. 10.0 g
distribution for human consumption, but in many instances, Lactose...................................................................... 10.0 g
contaminated food samples elude detection. Saccharose................................................................. 10.0 g
Sodium Sulfapyridine................................................... 1.0 g
BG (Brilliant Green) Sulfa Agar is a highly selective medium. Sodium Chloride.......................................................... 5.0 g
Agar.......................................................................... 20.0 g
Osborne and Stokes1 added 0.1% sodium sulfapyridine to Brilliant Green............................................................ 12.5 mg
Brilliant Green Agar to enhance the selective properties of Phenol Red.................................................................. 0.08 g
this medium for Salmonella. This formula is recommended as Difco™ SBG Sulfa Enrichment
a selective isolation medium for Salmonella following enrich- Approximate Formula* Per Liter
ment.2 It is also recommended for direct inoculation with Yeast Extract................................................................ 5.0 g
primary specimens for Salmonella isolation. Peptone....................................................................... 5.0 g
D-Mannitol.................................................................. 5.0 g
For food testing, BG Sulfa Agar has been used for detection of Sodium Taurocholate.................................................... 1.0 g
Sodium Sulfapyridine................................................... 0.5 g
Salmonella in low and high moisture foods.3,4 It has also been Sodium Selenite........................................................... 4.0 g
used for detecting Salmonella in feeds and feed ingredients.5 Dipotassium Phosphate................................................ 2.65 g
This medium is recommended when testing foods for Salmonella Monopotassium Phosphate.......................................... 1.02 g
Brilliant Green.............................................................. 5.0 mg
following USDA guidelines.6-8 *Adjusted and/or supplemented as required to meet performance criteria.
SBG (Selenite Brilliant Green) Sulfa Enrichment is prepared Uninoculated Salmonella Typhimurium
according to the formula described by Stokes and Osborne.9 Plate ATCC™ 14028
The researchers found that whole egg and egg yolk reduced
the selective properties of selenite brilliant green enrichment.1
They also found that the addition of sulfapyridine (SBG Sulfa
Enrichment) restored these selective properties.1
SBG Sulfa Enrichment is a selective enrichment for the isola-
tion of Salmonella species, especially from egg products. The
shell and the contents of the egg at the time of oviposition
are generally sterile or harbor very few microorganisms. Con-
tamination of the shell occurs afterwards from nesting material,
floor litter and avian fecal matter.10-12
67
68
5. Since BG Sulfa Agar is highly selective, it is recommended 9. Osborn and Stokes. 1955. Appl. Microbiol. 3:217.
10. Brooks and Taylor. 1955. Rep. Rd. Invest., Bd. 60, H. M. S. O. London, England.
that less selective media, such as MacConkey Agar, be used 11. Forsythe, Ayres and Radlo. 1953. Food Technol. 7:49.
12. Stadelman, Ikeme, Roop and Simmons. 1982. Poultry Sci. 61:388.
simultaneously. 13. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria,
vol. 1. Williams & Wilkins, Baltimore, Md.
6. SBG Sulfa Enrichment should be used in conjunction with a
selective prepared medium for bacterial identification.
Availability
Difco™ BG Sulfa Agar
B
References
1. Osborn and Stokes. 1955. Appl. Microbiol. 3:295. CCAM COMPF USDA
2. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods, Cat. No. 271710 Dehydrated – 500 g
4th ed. American Public Health Association, Washington, D.C.
3. D’Aoust, Maishment, Burgener, Conley, Loit, Milling and Purvis. 1980. J. Food Prot. 43:343.
4. D’Aoust. 1984. J. Food Prot. 47:588.
Difco™ SBG Sulfa Enrichment
5. D’Aoust, Sewell and Boville. 1983. J. Food Prot. 46:851. USDA
6. Moats. 1981. J. Food Prot. 44:375.
7. Federal Register. 1996. Fed. Regist. 61:38917. Cat. No. 271510 Dehydrated – 500 g
8. U.S. Department of Agriculture. Microbiology laboratory guidebook, online. Food Safety and Inspec-
tion Service, USDA, Washington, D.C.
BiGGY Agar
Intended Use species reduce the bismuth sulfite, resulting in pigmentation
BiGGY (Bismuth Sulfite Glucose Glycine Yeast) is a selective of colonies and, with some species, pigmentation in the
and differential medium used in the detection, isolation and surrounding medium.
presumptive identification of Candida species.
Principles of the Procedure
Summary and Explanation Candida species, through a process of substrate reduction,
BiGGY Agar is based on the formulation of Nickerson. Nickerson 1 produce sulfide and bismuth which combine to produce brown
developed the medium in 1953 following a study of sulfite to black pigmented colonies and zones of dark precipitate in the
reduction by Candida species. medium surrounding colonies of some species. Dextrose and
yeast extract provide the nutrients in the formulation.
Differentiation of Candida is based on growth patterns and
pigmentation of isolated colonies. The bismuth sulfite acts NOTE: A decrease in pH is normal and does not affect per-
as an inhibitory agent to suppress bacterial growth, which formance.
enables the recovery of isolated colonies of Candida. Candida
Identity Specifications
BBL™ BiGGY Agar
Dehydrated Appearance: Medium fine, homogeneous, free of extraneous
material.
Solution: 4.5% solution, soluble in purified water upon
boiling. Solution is light to medium, cream yellow,
hazy to cloudy.
Prepared Appearance: Light to medium, cream yellow, hazy to cloudy.
Reaction of 4.5%
Solution at 25°C: pH 6.8 ± 0.2
Cultural Response
BBL™ BiGGY Agar
Prepare the medium per label directions. Inoculate with fresh cultures and
incubate at 25 ± 2°C for 18-24 hours (3-5 days if necessary).
color of
ORGANISM ATCC™ recovery colonies/medium
Candida albicans 10231 Good Brown to black/–
Candida kefyr 8553 Good Reddish brown/–
Candida tropicalis 1369 Good Brown to black, metallic
sheen/Brown to black
Escherichia coli 25922 Partial to –/–
complete inhibition
69
70
and most gram-negative anaerobic organisms is obtained by Anaerobe 5% Sheep Blood Agar. All media should be pre-
the presence of gentamicin and oxgall. Differentiation of the reduced. Incubate immediately under anaerobic conditions
B. fragilis group is based on esculin hydrolysis, which produces (BD GasPak™ EZ anaerobic systems or alternative anaerobic
esculetin and dextrose. The esculetin reacts with the iron salt system) for at least 48 hours at 35 ± 2°C.
(ferric ammonium citrate) contained in the medium to produce
a dark brown to black complex that appears in the medium Expected Results B
surrounding colonies of members of the B. fragilis group. After 48 hours of incubation, colonies of the B. fragilis group
should be greater than 1 mm in diameter and appear gray,
Procedure circular, entire and raised. Most anaerobes other than the
As some strains of the B. fragilis group may not grow well B. fragilis group are inhibited. Esculin hydrolysis is indicated by
due to the selective properties of the medium, it is advisable a blackening of the medium around the colonies.
to include a nonselective blood agar medium, such as CDC
Limitation of the Procedure
Bacteroides fragilis
ATCC™ 25285 B. vulgatus may not hydrolyze esculin.2,3
References
1. Livingston, Kominos and Yee. 1978. J. Clin. Microbiol. 7:448.
2. Isenberg and Garcia (ed.), 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
American Society for Microbiology, Washington, D.C.
3. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology. 9th ed.
American Society for Microbiology, Washington, D.C.
4. Dowell, Lombard, Thompson and Armfield. 1977. Media for isolation, characterization and
identification of obligately anaerobic bacteria. CDC laboratory manual. Center for Disease
Control, Atlanta, Ga.
Availability
BBL™ Bacteroides Bile Esculin Agar (BBE)
BS12 CMPH2 MCM9
United States and Canada
Cat. No. 221836 Prepared Plates – Pkg. of 10*
Japan
Cat. No. 251972 Prepared Plates – Pkg. of 10*
BBL™ Bacteroides Bile Esculin Agar (BBE)//
CDC Anaerobe Laked Sheep Blood Agar with KV
CMPH2 MCM9
Cat. No. 297022 Prepared I Plate™ Dishes – Pkg. of 20*
297260 Prepared I Plate™ Dishes – Ctn. of 100*
*Store at 2-8°C.
71
in order to stimulate the growth of S. aureus without destroying 3. Autoclave at 121°C for 15 minutes.
the selectivity. The tellurite additive is toxic to egg yolk- 4. Cool to 45-50°C and aseptically add 50 mL of EY Tellurite
clearing strains other than S. aureus and imparts a black color Enrichment. Mix thoroughly but gently.
to the colonies. The egg yolk additive, in addition to being 5. Test samples of the finished product for performance using
an enrichment, aids in the identification process by demon- stable, typical control cultures.
strating lecithinase activity (egg yolk reaction). Glycine and
lithium chloride have inhibitory action for organisms other Procedure
than S. aureus. Food samples are macerated in suitable broth medium, diluted
as desired and the dilutions spread-inoculated onto the agar
Formulae surfaces, which should be dry when inoculated. Incubate plates
Difco™ Baird-Parker Agar Base aerobically for 24 hours at 35 ± 2°C. Consult references for
Approximate Formula* Per 950 mL detailed instructions.7
Pancreatic Digest of Casein........................................ 10.0 g
Beef Extract.................................................................. 5.0 g
Yeast Extract................................................................ 1.0 g Expected Results
Glycine....................................................................... 12.0 g Typical colonies of S. aureus are black, shiny, convex and
Sodium Pyruvate........................................................ 10.0 g surrounded by clear zones (egg yolk reaction) of approximately
Lithium Chloride.......................................................... 5.0 g
Agar.......................................................................... 20.0 g 2-5 mm. Coagulase-negative staphylococci generally do not
grow well; if some growth occurs, the typical clear zones are
Difco™ EY Tellurite Enrichment
Egg yolk emulsion containing potassium tellurite consists of 30%
absent. The majority of other organisms are inhibited but some
egg yolk suspension with 0.15% potassium tellurite. may grow sparsely, producing white to brown colonies with no
*Adjusted and/or supplemented as required to meet performance criteria. clearing of the egg yolk.
Identity Specifications
Difco™ Baird-Parker Agar Base
Dehydrated Appearance: Light tan, free-flowing, homogeneous.
Solution: 6.3 g/95 mL solution, soluble in purified water
upon boiling. Solution is light to medium amber,
very slightly to slightly opalescent.
Prepared Appearance (Final): Yellow, opaque.
Reaction of 6.3 g/95 mL
Solution at 25°C: pH 6.9 ± 0.1
Difco EY Tellurite Enrichment
™
Cultural Response
Difco™ Baird-Parker Agar Base with EY Tellurite
Enrichment
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C
for 24-50 hours.
Lecithinase
INOCULUM Colony PRODUCTION
ORGANISM ATCC™ CFU RECOVERY COLOR (HALOS)
Bacillus subtilis 6633 103 None to poor Brown –
Proteus mirabilis 25933 10
3
Good Brown –
Staphylococcus
aureus 25923 102–3×102 Good Black +
Staphylococcus
epidermidis 14990 102–3×102 Poor to good Black –
72
B
6. Baer. 1971. J. Assoc. Off. Anal. Chem. 54:732.
7. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC AOAC BAM CCAM COMPF ISO SMD SMWW USDA
International, Gaithersburg, Md. United States and Canada
Cat. No. 297214 Prepared Plates (complete) – Pkg. of 20*
Availability 297725 Prepared Plates (complete) – Ctn. of 100*
Difco™ Baird-Parker Agar Base Europe
Cat. No. 255084 Prepared Plates (complete) – Pkg. of 20*
AOAC BAM CCAM COMPF ISO SMD SMWW USDA
Cat. No. 276840 Dehydrated – 500 g Mexico
276810 Dehydrated – 2 kg Cat. No. 223950 Prepared Plates (complete) – Pkg. of 10*
*Store at 2-8°C.
73
74
the equivalent of 500 g of fresh heart tissue. Beef Heart for Principles of the Procedure
Infusion supplies the nutritional requirements for growth of Beef Heart for Infusion provides nitrogen, amino acids and
microorganisms in Heart Infusion media. vitamins in microbiological culture media.
One of the first media used for the cultivation of bacteria was a
Typical Analysis
liquid medium containing an infusion of meat. Huntoon1 used
Refer to Product Tables in the Reference Guide section of this B
fresh beef heart and Bacto Peptone to prepare a “hormone”
manual.
broth to retain growth-promoting substances. Highly pathogenic
organisms, such as meningococci and pneumococci, could be
Directions for Preparation from
grown on infusion medium without enrichments.1
Dehydrated Product
Beef Heart for Infusion is a component of Heart Infusion Infusions can be prepared using 50 g of Beef Heart for
media. Heart Infusion media are used in the mass production Infusion per liter of purified water. For best results, infuse
of microorganisms for vaccine production and are specified at 50° C for 1 hour. Heat the infusion to boiling for a few
in standard methods for other multiple applications.2-7 minutes to coagulate some of the proteins and filter. Add
peptone and remaining ingredients of the medium to the
User Quality Control filtrate. Adjust the pH to 7.5-7.8. Boil the medium and filter
Identity Specifications before autoclaving. Consult appropriate references for further
Difco™ Beef Heart for Infusion directions on preparation of specific products.
Dehydrated Appearance: Tan to medium brown, fine, homogeneous.
Solution: 5.0% solution, not completely soluble in purified Procedure
water. Solution, after filtration, is light to medium See appropriate references for specific procedures using Beef
amber, clear to slightly opalescent, may have a Heart for Infusion.2-4
precipitate.
Reaction of 5.0%
Solution at 25°C: pH 7.5-7.8 Expected Results
Refer to appropriate references and procedures for results.
Cultural Response
Difco™ Beef Heart for Infusion References
Prepare a 5% solution of Beef Heart for Infusion. Infuse for one hour 1. Huntoon. 1918. J. Infect. Dis. 23:168.
2. Ruoff. 1995. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology,
at 50 ± 2°C. Heat to boiling for 3-5 minutes and filter. Add 2% Pro- 6th ed. American Society for Microbiology, Washington, D.C.
teose Peptone No. 3, 0.5% sodium chloride and 0.005% dextrose to 3. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
the filtrate. Adjust pH to 7.5-7.8. Boil and filter before autoclaving. Inoculate tional, Gaithersburg, Md.
4. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
and incubate tubes at 35 ± 2°C for 18-48 hours. International, Gaithersburg, Md.
5. U.S. Environmental Protection Agency. 2000. Improved enumeration methods for the recreational
ORGANISM ATCC™ INOCULUM CFU RECOVERY water quality indicators: Enterococci and Escherichia coli. EPA-821/R-97/004. Office of Water, USEPA,
Washington, D.C.
Escherichia coli 25922 102-103 Good 6. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C.
Klebsiella pneumoniae 13883 102-103 Good 7. U.S. Department of Agriculture. Microbiology laboratory guidebook, online. Food Safety and Inspec-
Staphylococcus aureus 25923 102-103 Good tion Service, USDA, Washington, D.C.
75
Biosate™ Peptone
Intended Use digest of casein and yeast extract has been used successfully
Biosate Peptone is used as a component in microbiological as components in media which supported the hatching and
culture media or in fermentation applications. culture of Giardia spp. from cysts and the first-time culturing of
a nematode without the need of its symbiotic bacteria.3,4
Summary and Explanation
Biosate Peptone is a mixed hydrolysate comprised of casein Principles of the Procedure
and yeast extract at a ratio of 65:35. The synergistic effect of Biosate Peptone provides nitrogen, amino acids and vitamins
two or more types of hydrolysates is well documented and has in microbiological culture media. In addition, the yeast
been utilized for decades in culture media formulation. The extract component of the product provides proteins, carbohy-
combination of pancreatic digest of casein and yeast extract drates and some micronutrients.
provides nutritional benefits that are not provided by the
components alone. It has been reported that the combined use Typical Analysis
of these two peptones has shown improved toxin production Refer to Product Tables in the Reference Guide section of
in clostridia.1,2 Additionally, the combination of pancreatic this manual.
76
cultivation of L. plantarum ATCC 8014. The addition of a Biotin Assay Medium supplemented with 0.5 ng biotin. After
biotin standard in specified increasing concentrations gives 16-24 hours incubation at 35-37°C, the cells are centrifuged
a growth response by this organism that can be measured under aseptic conditions and the supernatant liquid decanted.
titrimetrically or turbidimetrically. The cells are washed three times with 10 mL sterile 0.85%
saline. After the third wash, the cells are resuspended in 10 mL
Formula sterile 0.85% saline and finally diluted 1:100 with sterile 0.85%
Difco™ Biotin Assay Medium saline. One drop of this suspension is used to inoculate each
Approximate Formula* Per Liter 10 mL assay tube.
Vitamin Assay Casamino Acids................................... 12.0 g
Dextrose.................................................................... 40.0 g Standard Curve
Sodium Acetate......................................................... 20.0 g
L-Cystine...................................................................... 0.2 g It is essential that a standard curve be constructed each time
DL-Tryptophan............................................................. 0.2 g an assay is run. Autoclave and incubation conditions can
Adenine Sulfate......................................................... 20.0 mg influence the standard curve reading and cannot always be
Guanine Hydrochloride.............................................. 20.0 mg
Uracil......................................................................... 20.0 mg
duplicated. The standard curve is obtained by using biotin at
Thiamine Hydrochloride............................................... 2.0 mg levels of 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 and 1 ng per assay
Riboflavin..................................................................... 2.0 mg tube (10 mL).
Niacin.......................................................................... 2.0 mg
Calcium Pantothenate.................................................. 2.0 mg The concentration of biotin required for the preparation of
Pyridoxine Hydrochloride.............................................. 4.0 mg the standard curve may be prepared by dissolving 0.1 gram of
p-Aminobenzoic Acid............................................... 200.0 µg
Dipotassium Phosphate................................................ 1.0 g d-Biotin or equivalent in 1,000 mL of 25% alcohol solution
Monopotassium Phosphate.......................................... 1.0 g (100 µg per mL). Dilute the stock solution by adding 2 mL
Magnesium Sulfate...................................................... 0.4 g to 98 mL of purified water. This solution is diluted by adding
Sodium Chloride........................................................ 20.0 mg
Ferrous Sulfate........................................................... 20.0 mg 1 mL to 999 mL purified water, giving a solution of 2 ng
Manganese Sulfate.................................................... 20.0 mg of biotin per mL. This solution is further diluted by adding
*Adjusted and/or supplemented as required to meet performance criteria. 10 mL to 90 mL purified water, giving a final solution of 0.2 ng
of biotin per mL. Use 0.0, 0.5, 1, 1.5, 2, 2.5, 3, 4 and 5 mL of
Precautions this final solution. Prepare the stock solution fresh daily.
Great care must be taken to avoid contamination of media or
glassware in microbiological assay procedures. Extremely small Biotin Assay Medium may be used for both turbidimetric and
amounts of foreign material may be sufficient to give erroneous titrimetric analysis. Before reading, the tubes are refrigerated
results. Scrupulously clean glassware free from detergents and for 15-30 minutes to stop growth. Turbidimetric readings
other chemicals must be used. Glassware must be heated to should be made after 16-20 hours at 35-37°C. Titrimetric
250°C for at least 1 hour to burn off any organic residues that determinations are made after 72 hours incubation at 35-37°C.
might be present. Take precautions to keep sterilization and The most effective assay range, using Biotin Assay Medium, has
cooling conditions uniform throughout the assay. been found to be between 0.1 ng and 1 ng biotin.
For a complete discussion of vitamin assay methodology, refer
Directions for Preparation from to appropriate procedures outlined in the reference.1
Dehydrated Product
1. Suspend 7.5 g of the powder in 100 mL of purified water. Expected Results
2. Heat with frequent agitation and boil for 2-3 minutes to 1. Prepare a standard concentration response curve by plotting
completely dissolve the powder. the response readings against the amount of standard in each
3. Dispense 5 mL amounts into tubes, evenly dispersing the tube, disk or cup.
precipitate. 2. Determine the amount of vitamin at each level of assay
4. Add standard or test samples. solution by interpolation from the standard curve.
5. Adjust tube volume to 10 mL with purified water. 3. Calculate the concentration of vitamin in the sample from
6. Autoclave at 121°C for 5 minutes. the average of these values. Use only those values that do
not vary more than ±10% from the average. Use the results
Procedure only if two-thirds of the values do not vary by more than
Stock Cultures ±10%.
Stock cultures of the test organism, L. plantarum ATCC 8014,
are prepared by stab inoculation of Lactobacilli Agar AOAC. Limitations of the Procedure
After 16-24 hours incubation at 35-37°C, the tubes are stored 1. The test organism used for inoculating an assay medium
in the refrigerator. Transfers are made weekly. must be cultured and maintained on media recommended for
this purpose.
Inoculum
2. Aseptic technique should be used throughout the assay
Inoculum for assay is prepared by subculturing from a stock procedure.
culture of L. plantarum ATCC 8014 to 10 mL of single-strength
78
79
Employing this medium in the routine laboratory examination Bismuth Sulfite Agar is valuable when investigating outbreaks of
of fecal and urine specimens, these same authors8 obtained Salmonella spp., especially S. Typhi.17-19
40% more positive isolations of S. Typhi than were obtained
Bismuth Sulfite Agar is used for the isolation of S. Typhi and
on Endo medium. Gunther and Tuft,9 employing various
other Salmonella from food, feces, urine, sewage and other
media in a comparative way for the isolation of typhoid from
infectious materials. The typhoid organism grows luxuriantly
stool and urine specimens, found Bismuth Sulfite Agar most
on the medium, forming characteristic black colonies, while
productive. On Bismuth Sulfite Agar, they obtained 38.4%
gram-positive bacteria and members of the coliform group
more positives than on Endo Agar, 33% more positives than
are inhibited. This inhibitory action of Bismuth Sulfite Agar
on Eosin Methylene Blue Agar, and 80% more positives than
toward gram-positive and coliform organisms permits the use
on the Desoxycholate media. These workers found Bismuth
of a much larger inoculum than possible with other media
Sulfite Agar to be superior to Wilson’s original medium.
employed for similar purposes in the past. The use of larger
Bismuth Sulfite Agar was stable, sensitive and easier to
inocula greatly increases the possibility of recovering the
prepare. Green and Beard,10 using Bismuth Sulfite Agar, claimed
pathogens, especially when they are present in relatively small
that this medium successfully inhibited sewage organisms.
numbers. Small numbers of organisms may be encountered in
The value of Bismuth Sulfite Agar as a plating medium after
the early course of the disease or in the checking of carriers
enrichment has been demonstrated by Hajna and Perry.11
and releases.
Since these earlier references to the use of Bismuth Sulfite
Agar, this medium has been generally accepted as routine for Principles of the Procedure
the detection of most Salmonella. The value of the medium is In Bismuth Sulfite Agar, beef extract and peptone provide
demonstrated by the many references to the use of Bismuth Sulfite nitrogen, vitamins and minerals. Dextrose is an energy source.
Agar in scientific publications, laboratory manuals and texts. Disodium phosphate is a buffering agent. Bismuth sulfite
indicator and brilliant green are complementary in inhibiting
For food testing, the use of Bismuth Sulfite Agar is specified
gram-positive bacteria and members of the coliform group,
for the isolation of pathogenic bacteria from raw and pasteur-
while allowing Salmonella to grow luxuriantly. Ferrous
ized milk, cheese products, dry dairy products, cultured milks
sulfate is included for detection of H2S production. When
and butter.1,12-14 The use of Bismuth Sulfite Agar is also recom-
H2S is present, the iron in the formula is precipitated, giving
mended for use in testing clinical specimens.15,16 In addition,
Identity Specifications
Difco™ Bismuth Sulfite Agar
Dehydrated Appearance: Light beige to light green, free-flowing, homo-
geneous.
Solution: 5.2% solution, soluble in purified water upon
boiling. Solution is light green, opaque with a
flocculent precipitate that can be dispersed by
swirling contents of flask.
Prepared Appearance: Light gray-green to medium green, opaque with
a flocculent precipitate.
Reaction of 5.2%
Solution at 25°C: pH 7.7 ± 0.2
Cultural Response
Difco™ Bismuth Sulfite Agar
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 40-48 hours.
INOCULUM COLONY
ORGANISM ATCC™ CFU RECOVERY COLOR
Enterococcus faecalis 29212 103 Marked to –
complete inhibition Salmonella
Escherichia coli 25922 103 Partial inhibition Brown to green Typhimurium
ATCC™ 14028
Salmonella enterica
subsp. enterica
serotype Typhi 19430 102-103 Good Black with sheen
Salmonella enterica Black or greenish-gray,
subsp. enterica may or may not
serotype Typhimurium 14028 102-103 Good have sheen
80
positive cultures the characteristic brown to black color with Generally, Shigella spp. other than S. flexneri and S. sonnei are
metallic sheen. Agar is the solidifying agent. inhibited. S. flexneri and S. sonnei strains that do grow on this
medium produce brown to green, raised colonies with depressed
Formula centers and exhibit a crater-like appearance.
Difco™ Bismuth Sulfite Agar
Approximate Formula* Per Liter
Escherichia coli is partially inhibited. Occasionally a strain
will be encountered that will grow as small brown or greenish
B
Beef Extract.................................................................. 5.0 g
Peptone..................................................................... 10.0 g glistening colonies. This color is confined entirely to the colony
Dextrose...................................................................... 5.0 g itself and shows no metallic sheen. A few strains of Enterobacter
Disodium Phosphate.................................................... 4.0 g
Ferrous Sulfate............................................................. 0.3 g aerogenes may develop on this medium, forming raised,
Bismuth Sulfite Indicator.............................................. 8.0 g mucoid colonies. Enterobacter colonies may exhibit a silvery
Agar.......................................................................... 20.0 g sheen, appreciably lighter in color than that produced by
Brilliant Green............................................................ 25.0 mg
*Adjusted and/or supplemented as required to meet performance criteria.
S. Typhi. Some members of the coliform group that produce
hydrogen sulfide may grow on the medium, giving colonies
Directions for Preparation from similar in appearance to S. Typhi. These coliforms may be readily
Dehydrated Product differentiated because they produce gas from lactose in dif-
1. Suspend 52 g of the powder in 1 L of purified water. Mix ferential media, for example, Kligler Iron Agar or Triple Sugar
thoroughly. Iron Agar. The hydrolysis of urea, demonstrated in Urea Broth
2. Heat with frequent agitation and boil for 1 minute to or on Urea Agar Base, may be used to identify Proteus sp.
completely dissolve the powder. DO NOT AUTOCLAVE. To isolate S. Typhi for agglutination or fermentation studies,
3. Evenly disperse the precipitate when dispensing. Use the pick characteristic black colonies from Bismuth Sulfite Agar and
medium the same day it is prepared. subculture them on MacConkey Agar. The purified colonies
4. Test samples of the finished product for performance using from MacConkey Agar may then be picked to differential tube
stable, typical control cultures. media such as Kligler Iron Agar, Triple Sugar Iron Agar or
other satisfactory differential media for partial identification. All
Procedure cultures that give reactions consistent with Salmonella spp. on
For isolation of Salmonella spp. from food, samples are these media should be confirmed biochemically as Salmonella
enriched and selectively enriched. Streak 10 µL of selective spp. before any serological testing is performed. Agglutination
enrichment broth onto Bismuth Sulfite Agar. Incubate plates tests may be performed from the fresh growth on the differential
for 24-48 hours at 35°C. Examine plates for the presence tube media or from the growth on nutrient agar slants inoculated
of Salmonella spp. Refer to appropriate references for the from the differential media. The growth on the differential tube
complete procedure when testing food samples.1,12-14 media may also be used for inoculating carbohydrate media for
For isolation of Salmonella spp. from clinical specimens, fermentation studies.
inoculate fecal specimens and rectal swabs onto a small area
of one quadrant of the Bismuth Sulfite Agar plate and streak Limitations of the Procedure
for isolation. This will permit the development of discrete 1. It is important to streak for well-isolated colonies. In heavy
colonies. Incubate plates at 35°C. Examine at 24 hours and again growth areas, S. Typhi appears light green and may be
at 48 hours for colonies resembling Salmonella spp. misinterpreted as negative growth for S. Typhi.20
2. S. Typhi and S. arizonae are the only enteric organisms to
For additional information about specimen preparation exhibit typical brown zones on the medium. Brown zones are
and inoculation of clinical specimens, consult appropriate not produced by other members of the Enterobacteriaceae.
references.15-19 However, S. arizonae is usually inhibited.20
3. Colonies on Bismuth Sulfite Agar may be contaminated with
Expected Results other viable organisms; therefore, isolated colonies should
The typical discrete S. Typhi surface colony is black and
be subcultured to a less selective medium (e.g., MacConkey
surrounded by a black or brownish-black zone which may be
Agar).20
several times the size of the colony. By reflected light, preferably
4. Typical S. Typhi colonies usually develop within 24 hours;
daylight, this zone exhibits a distinctly characteristic metallic
however, all plates should be incubated for a total of
sheen. Plates heavily seeded with S. Typhi may not show this
48 hours to allow growth of all typhoid strains.20
reaction except near the margin of the mass inoculation. In
5. DO NOT AUTOCLAVE. Heating this medium for a period
these heavy growth areas, this organism frequently appears as
longer than necessary to just dissolve the ingredients destroys
small light green colonies. This fact emphasizes the importance
its selectivity.
of inoculating plates so that some areas are sparsely populated
with discrete S. Typhi colonies. Other strains of Salmonella
produce black to green colonies with little or no darkening of
the surrounding medium.
81
References 17. Murray, Baron, Pfaller, Tenover and Yolken (ed.). 1999. Manual of clinical microbiology, 7th ed.
American Society for Microbiology, Washington, D.C.
1. Flowers, Andrews, Donnelly and Koenig. 1993. In Marshall (ed.), Standard methods for the examina- 18. Cintron. 1992. In Isenberg (ed.), Clinical microbiology procedures handbook, vol. 1. American
tion of dairy products, 16th ed. American Public Health Association, Washington, D.C. Society for Microbiology, Washington, D.C.
2. Wilson and Blair. 1926. J. Pathol. Bacteriol. 29:310. 19. Grasmick. 1992. In Isenberg (ed.), Clinical microbiology procedures handbook, vol. 1. American
3. Wilson and Blair. 1927. J. Hyg. 26:374. Society for Microbiology, Washington, D.C.
4. Wilson and Blair. 1931. J. Hyg. 31:138. 20. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol.
5. Wilson. 1923. J. Hyg. 21:392. 1. Williams & Wilkins, Baltimore, Md.
6. Wilson. 1928. Br. Med. J. 1:1061.
7. Cope and Kasper. 1937. J. Bacteriol. 34:565.
8. Cope and Kasper. 1938. Am. J. Public Health 28:1065.
9. Gunther and Tuft. 1939. J. Lab. Clin. Med. 24:461.
Availability
10. Green and Beard. 1938. Am. J. Public Health 28:762. Difco™ Bismuth Sulfite Agar
11. Hajna and Perry. 1938. J. Lab. Clin. Med. 23:1185.
12. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna- AOAC BAM CCAM COMPF SMD SMWW
tional, Gaithersburg, Md.
13. Andrews, Flowers, Silliker and Bailey. 2001. In Downes and Ito (ed.), Compendium of methods for the Cat. No. 273300 Dehydrated – 500 g
microbiological examination of foods, 4th ed. American Public Health Association, Washington, D.C.
14. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
Mexico
International, Gaithersburg, Md. Cat. No. 252612 Prepared Plates – Pkg. of 10*
15. Washington. 1981. Laboratory procedures in clinical microbiology. Springer-Verlag, New York, *Store at 2-8°C.
N.Y.
16. Baron, Peterson and Finegold. 1994. Bailey & Scott’s diagnostic microbiology, 9th ed. Mosby-Year
Book, Inc. St. Louis, Mo.
Identity Specifications
BBL™ Blood Agar Base (Infusion Agar)
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution: 4.0% solution, soluble in purified water upon
boiling. Solution is medium, yellow to tan, clear
to slightly hazy.
Prepared Appearance: Plain – Medium, yellow to tan, clear to slightly
hazy.
With 5% sheep blood – Cherry red, opaque.
Reaction of 4.0%
Solution at 25°C: pH 7.3 ± 0.2
Cultural Response
BBL™ Blood Agar Base (Infusion Agar)
Prepare the medium per label directions without (plain) and with 5%
defibrinated sheep blood (SB). Inoculate and incubate at 35 ± 2°C for
18-24 hours (incubate streptococci with 3-5% CO2).
INOCULUM recovery REcovery
ORGANISM ATCC™ CFU PLAIN WITH SB
Candida albicans 10231 30-300 N/A Good,
no hemolysis Streptococcus
Listeria Good, beta pneumoniae
ATCC™ 6305
monocytogenes 19115 30-300 N/A hemolysis
Pseudomonas
aeruginosa 10145 30-300 Good N/A
Shigella flexneri 12022 30-300 Good N/A
Staphylococcus Good, beta
aureus 25923 30-300 Good hemolysis
Streptococcus Good, alpha
pneumoniae 6305 30-300 Good hemolysis
Streptococcus Good, beta
pyogenes 19615 30-300 Good hemolysis
82
with Blood Agar Base, and found that sheep blood gave the Expected Results
clearest and most reliable colony and hemolysis characteristics Colonial morphology on blood agar containing 5% sheep blood
at both 24 and 48 hours.1 In the course of the investigation, is as follows:
about 1,300 isolations of streptococci were made with Blood
1. Hemolytic streptococci may appear as translucent or
Agar Base containing 5% sheep blood.
Blood Agar Base media are specified in standard methods for
opaque, grayish, small (1 mm), or large matte or mucoid B
(2-4 mm) colonies, encircled by a zone of hemolysis. Gram
food testing.2-4 Infusion Agar has been largely replaced as a blood stains should be made and examined to check the macro-
agar base by the Tryptic/Trypticase™ Soy Agar formulations, scopic findings. (Other organisms which may cause hemolysis
which contain milk and plant peptones in place of the variable include Listeria, various corynebacteria, hemolytic staphy-
infusion component. lococci, Escherichia coli and Pseudomonas.) Approximate
quantitation of the number of colonies of hemolytic
Principles of the Procedure streptococci may be helpful to the clinician.
Infusion from heart muscle, casein peptone and yeast extract pro- 2. Pneumococci usually appear as very flat, smooth, translucent,
vide nitrogen, carbon, amino acids and vitamins in Blood Agar grayish and sometimes mucoid colonies surrounded by a
Base. Medium contains sodium chloride to maintain osmotic narrow zone of “green” (alpha) hemolysis.
equilibrium and agar is the solidifying agent. 3. Staphylococci appear as opaque, white to gold-yellow
Supplementation with blood (5-10%) provides additional colonies with or without zones of beta hemolysis.
growth factors for fastidious microorganisms, and is the basis 4. Listeria may be distinguished by their rod shape in stains,
for determining hemolytic reactions. Hemolytic patterns may and by motility at room temperature. Small zones of beta
vary with the source of animal blood or type of base medium hemolysis are produced.
used.5 5. Other organisms representing minimal flora and clinically
significant isolates can also be expected to grow on this
Formula nonselective formulation.
BBL™ Blood Agar Base (Infusion Agar)
Approximate Formula* Per Liter Limitation of the Procedure
Heart Muscle, Infusion from (solids).............................. 2.0 g Colonies of Haemophilus haemolyticus are beta-hemolytic on
Pancreatic Digest of Casein........................................ 13.0 g
horse and rabbit blood agar and must be distinguished from
Yeast Extract................................................................ 5.0 g
Sodium Chloride.......................................................... 5.0 g colonies of beta-hemolytic streptococci using other criteria.6
Agar.......................................................................... 15.0 g The use of sheep blood has been suggested to obviate this
*Adjusted and/or supplemented as required to meet performance criteria.
problem since sheep blood is deficient in pyridine nucleotides
and does not support growth of H. haemolyticus.5
Directions for Preparation from
Dehydrated Product References
1. Suspend 40 g of the powder in 1 L of purified water. Mix 1. Snavely and Brahier. 1960. Am. J. Clin. Pathol. 33:511.
2. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
thoroughly. tional, Gaithersburg, Md.
2. Heat with frequent agitation and boil for 1 minute to 3. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
completely dissolve the powder. 4. Atlas. 1993. Handbook of microbiological media. CRC Press, Boca Raton, Fla.
5. Ruoff, Whiley and Beighton. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of
3. Autoclave at 121°C for 15 minutes. clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
6. Forbes, Sahm and Weissfeld (ed.). 1998. Bailey & Scott’s diagnostic microbiology, 10th ed. Mosby,
4. For preparation of blood agar, cool the base to 45-50°C and Inc., St. Louis, Mo.
aseptically add 5% sterile, defibrinated blood. Mix well.
5. Test samples of the finished product for performance using Availability
stable, typical control cultures. BBL™ Blood Agar Base (Infusion Agar)
BAM COMPF
Procedure Cat. No. 211037 Dehydrated – 500 g
Use standard procedures to obtain isolated colonies from 211038 Dehydrated – 5 lb (2.3 kg)
specimens. After streaking, stab the agar several times to
deposit beta-hemolytic streptococci beneath the agar surface.
Subsurface growth will display the most reliable hemolytic
reactions owing to the activity of both oxygen-stable and
oxygen-labile streptolysins.5
Since many pathogens require carbon dioxide on primary
isolation, plates may be incubated in an atmosphere containing
approximately 3-10% CO2. Incubate plates at 35 ± 2°C for
18-24 hours.
83
Identity Specifications
Difco™ Bordet Gengou Agar Base
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution: 3.0% solution, soluble upon boiling in purified
water containing 1% glycerol. Solution is light
to medium amber, opalescent, may have a slight
precipitate.
Prepared Appearance: Plain – Light to medium amber, opalescent, may
have a precipitate.
With 15% blood – Cherry red, opaque.
Reaction of 3.0%
Solution at 25°C: pH 6.7 ± 0.2
Cultural Response
Difco™ Bordet Gengou Agar Base
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 48-72 hours.
INOCULUM recovery WITH
ORGANISM ATCC™ CFU 15% RABBIT BLOOD
Bordetella bronchiseptica 4617 30-300 Good
Bordetella parapertussis 15311 30-300 Good Bordetella
parapertussis
Bordetella pertussis 8467 30-300 Good ATCC™ 15311
84
Bovine Albumin 5%
Intended Use Bovine Albumin can be added to normally sterile specimens,
Bovine Albumin 5% is used to enrich media for cultivating tissues and body fluids for direct inoculation onto culture
a large variety of microorganisms and tissue cells. Bovine media used for isolating mycobacteria. BSA is also used as an
albumin is also known as bovine serum albumin or BSA.1 enrichment when contaminated specimens are digested.
Bovine Albumin 5%, modified with added sodium chloride and
Summary and Explanation dextrose, is available as Dubos Medium Albumin.
Davis and Dubos2 recommended the use of bovine albumin
at a final concentration of 0.5% in liquid media for culturing Principles of the Procedure
Mycobacterium tuberculosis. In this study, bovine albumin Bovine Albumin 5% is a filter sterilized solution of bovine
neutralized the toxicity of fatty acids and permitted more albumin fraction V. BSA is suggested as a culture media
luxuriant growth of M. tuberculosis. enrichment because its buffering capacity and detoxifying
Ellinghausen and McCullough3 used bovine albumin fraction effect on specimen sediment. 1 Bovine Albumin 5% also
V at a final concentration of 1% in liquid, semisolid and solid increases adhesion of the specimen to solid media.1
media for culturing leptospires. Morton et al.4 demonstrated
that 1% bovine albumin stimulated growth of Mycoplasma Precautions5
(PPLO). 1. Biosafety Level 2 practices and procedures, containment
equipment and facilities are required for non-aerosol-
User Quality Control producing manipulations of clinical specimens such as
preparation of acid-fast smears. All aerosol-generating
Identity Specifications activities must be conducted in a Class I or II biological
Difco™ Bovine Albumin 5% safety cabinet.
Appearance: Light amber, clear to very slightly opalescent.
2. Biosafety Level 3 practices, containment equipment and
Reaction of
Solution at 25°C: pH 7.0 ± 0.2
facilities are required for laboratory activities in the propa-
gation and manipulation of cultures of M. tuberculosis and
Cultural Response M. bovis. Animal studies also require special procedures.
Difco™ Bovine Albumin 5%
Prepare Dubos Broth Base per label directions, substituting Bovine Procedure
Albumin 5% for Dubos Medium Albumin. Inoculate and incubate at Sterile Specimens for the Isolation of Mycobacteria1
35 ± 2°C under CO2 for up to 3 weeks.
Normally sterile tissues may be ground in 0.2% BSA and
Organism ATCC™ Inoculum CFU RECOVERY
inoculated directly in culture media. Concentrate body fluids
Mycobacterium
intracellulare 13950 102-103 Good
before inoculation because they normally contain only a small
Mycobacterium
number of mycobacteria. Centrifuge fluids at ≥ 3,000 × g and
tuberculosis H37Ra 25177 102-103 Good inoculate the sediment onto liquid or solid media. For a complete
85
Expected Results
All media should be examined closely for evidence of growth.
Refer to the procedure established by laboratory policy or
to appropriate references on typical growth patterns and
confirmation tests.
Identity Specifications
BBL™ Brain Heart CC Agar
B
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution: 5.2% solution, soluble in purified water upon
boiling. Solution is light to medium, yellow to tan,
clear to moderately hazy.
Prepared Appearance: Light to medium, yellow to tan, clear to moderately
hazy.
Reaction of 5.2%
Solution at 25°C: pH 7.4 ± 0.2
Cultural Response
BBL™ Brain Heart CC Agar
Prepare the medium per label directions. Inoculate with fresh cultures
and incubate at 25 ± 2°C under appropriate atmospheric conditions for
7 days.
ORGANISM ATCC™ recovery
Aspergillus brasiliensis
(niger) 16404 Partial to complete inhibition
Candida albicans 10231 Good
Escherichia coli 25922 Partial to complete inhibition
Trichophyton
mentagrophytes 9533 Good
Procedure Availability
Consult appropriate references for information about the BBL™ Brain Heart (Infusion) CC Agar
processing and inoculation of specimens.1,4 Cat No. 211057 Dehydrated – 500 g
296261 Prepared Plates (Deep Fill) – Pkg. of 20*
For isolation of fungi from potentially contaminated specimens, 297650 Prepared Slants (A Tubes) – Pkg. of 10*
a nonselective medium should be inoculated along with the 296106 Prepared Slants (C Tubes) – Ctn. of 100*
221834 Mycoflask™ Bottles – Pkg. of 10*
selective medium. Incubate at 25-30°C (plates in an inverted
position, agar side up, with increased humidity). For isola- BBL™ Brain Heart Infusion CC Agar with Sheep Blood
tion of fungi causing systemic mycoses, two sets of media Cat. No. 296178 Prepared Plates (Deep Fill) – Pkg. of 20*
should be inoculated, with one set incubated at 25-30°C and a BBL™ Brain Heart CC Agar with 10% Sheep Blood
duplicate set at 35 ± 2°C. and Gentamicin
Cat. No. 221842 Prepared Plates (Deep Fill) – Pkg. of 10*
All cultures should be examined at least weekly for fungal 296358 Prepared Slants (C Tubes) – Pkg. of 10*
growth and should be held for 4-6 weeks before being reported 295757 Prepared Slants (C Tubes) – Ctn. of 100*
as negative. BBL™ Brain Heart Infusion Agar with 10% Sheep Blood,
Gentamicin and Chloramphenicol
Expected Results BS12 CMPH2 MCM9
After sufficient incubation, examine cultures for fungal colonies Cat. No. 221841 Prepared Plates (Deep Fill) – Pkg. of 20*
296343 Prepared Slants (C Tubes) – Pkg. of 10*
exhibiting typical color and morphology. Biochemical tests 295756 Prepared Slants (C Tubes) – Ctn. of 100*
and serological procedures should be performed to confirm
findings. BBL™ Brain Heart Infusion (Sheep) Blood Agar with
Penicillin and Streptomycin
Cat. No. 296097 Prepared Plates (Deep Fill) – Pkg. of 20*
Limitation of the Procedure 297335 Prepared Slants (A Tubes) – Pkg. of 10*
Some fungi may be inhibited by antibiotics in this medium.5 *Store at 2-8°C.
References
1. Reisner, Woods, Thomson, Larone, Garcia and Shimizu. 1999. In Murray, Baron, Pfaller, Tenover and
Yolken (ed.), Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington,
D.C.
2. Kwon-Chung and Bennett. 1992. Medical mycology. Lea & Febiger, Philadelphia, Pa.
3. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
4. Merz and Roberts. 1995. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical
microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
5. Ajello, Georg, Kaplan and Kaufman. 1963. CDC laboratory manual for medical mycology. PHS
Publication No. 994, U.S. Government Printing Office, Washington, D.C.
87
88
Solution: 3.5% solution, soluble in purified water upon Dehydrated Appearance: Fine, homogeneous, free of extraneous
boiling. Solution is light to medium amber, material.
clear.
Solution: 3.8% solution, soluble in purified water
Prepared Appearance: Light to medium amber, clear. upon boiling. Solution is light to medium,
Reaction of 3.5% yellow to tan, clear to slightly hazy.
Solution at 25°C: pH 7.4 ± 0.2 Prepared Appearance: Light to medium, yellow to tan, clear to
slightly hazy.
Cultural Response Reaction of 3.7%
Bacto™ Brain Heart Infusion or Difco™ Brain Heart Solution at 25°C: pH 7.4 ± 0.2
Infusion without Dextrose
Prepare the medium per label directions. Inoculate and incubate at Cultural Response
35 ± 2°C for 18-48 hours. BBL™ Brain Heart Infusion or BBL™ Brain Heart
ORGANISM ATCC ™
INOCULUM CFU RECOVERY
Infusion Broth, Modified
Prepare the medium per label directions. Inoculate and incubate at
Neisseria meningitidis 13090 102-103 Good 35 ± 2°C under appropriate atmospheric conditions for 7 days
Streptococcus pneumoniae 6305 102-103 Good (incubate C. albicans at 20-27°C).
Streptococcus pyogenes 19615 102-103 Good INOCULUM REcovery REcovery
ORGANISM ATCC™ CFU BHI BHI, MODIFIED
Bacteroides fragilis 25285 ≤104 Good Good
BBL™ Brain Heart Infusion Broth, Modified
Candida albicans 10231 ≤103 Good Good
Approximate Formula* Per Liter
Brain Heart, Infusion from (solids)................................. 3.5 g Enterococcus faecalis 29212 ≤103 Good N/A
Peptic Digest of Animal Tissue.................................... 15.0 g Neisseria meningitidis 13090 ≤10 3
Good Good
Pancreatic Digest of Casein........................................ 10.0 g
Streptococcus pneumoniae 6305 ≤103 Good Good
Dextrose...................................................................... 2.0 g
Sodium Chloride.......................................................... 5.0 g Streptococcus pyogenes 19615 ≤103 Good Good
Disodium Phosphate.................................................... 2.5 g
*Adjusted and/or supplemented as required to meet performance criteria.
Liquid media for anaerobic incubation should be reduced prior 9. Clinical and Laboratory Standards Institute. 2006. Approved Standard: M7-A7, Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically, 7th ed. CLSI, Wayne, Pa.
to inoculation by placing the tubes, with caps loosened, under 10. Clinical and Laboratory Standards Institute. 2006. Approved Standard M2-A9, Performance standards
for antimicrobial disk susceptibility tests, 9th ed. CLSI, Wayne, Pa.
anaerobic conditions for 18-24 hours prior to use. An efficient
and easy way to obtain suitable anaerobic conditions is through Availability
the use of BD GasPak™ EZ anaerobic systems or an alternative Bacto™ Brain Heart Infusion
anaerobic system. Alternatively, liquid media may be reduced AOAC BAM CCAM CLSI CMPH2 COMPF EPA ISO MCM9
immediately prior to use by boiling with caps loosened and cooling SMD SMWW USDA
with tightened caps to room temperature before inoculation. Cat. No. 237400 Dehydrated – 100 g
237500 Dehydrated – 500 g
Before inoculating Hungate tubes, disinfect the septum of the cap. 237200 Dehydrated – 2 kg
To inoculate, insert needle of syringe containing specimen through 237300 Dehydrated – 1 kg
the septum and inject the specimen into the medium. Withdraw BBL™ Brain Heart Infusion
the needle slowly to avoid introducing air into the tube. AOAC BAM CCAM CLSI CMPH2 COMPF EPA ISO MCM9
For use in antimicrobial susceptibility testing, refer to appropri- SMD SMWW USDA
microorganisms utilize by fermentative action. The medium For isolation of fungi from potentially contaminated specimens,
is buffered through the use of disodium phosphate. a selective medium should be inoculated along with the nonse-
lective medium. Incubate the plates at 25-30°C in an inverted
When defibrinated sheep blood is added to the basal medium,
position (agar side up) with increased humidity. For isolation
it provides essential growth factors for the more fastidious
of fungi causing systemic mycoses, two sets of media should be
fungal organisms.
inoculated, with one set incubated at 25-30°C and a duplicate
Formulae set at 35 ± 2°C. All cultures should be examined at least weekly
Difco™ Brain Heart Infusion Agar
for fungal growth and should be held for 4-6 weeks before being
Approximate Formula* Per Liter reported as negative.
Calf Brains, Infusion from 200 g................................... 7.7 g BHI Agar slants primarily are used for the cultivation and
Beef Heart, Infusion from 250 g................................... 9.8 g
Proteose Peptone....................................................... 10.0 g maintenance of pure cultures of microorganisms.
Dextrose...................................................................... 2.0 g
Sodium Chloride.......................................................... 5.0 g Expected Results
Disodium Phosphate.................................................... 2.5 g
Agar.......................................................................... 15.0 g
After sufficient incubation, the plates should show isolated
colonies in streaked areas and confluent growth in areas of
BBL™ Brain Heart Infusion Agar
heavy inoculation. When culturing for fungi, examine plates
Approximate Formula* Per Liter
Brain Heart, Infusion from (solids)................................. 8.0 g for fungal colonies exhibiting typical color and morphology.
Peptic Digest of Animal Tissue...................................... 5.0 g Biochemical tests and serological procedures should be
Pancreatic Digest of Casein........................................ 16.0 g performed to confirm findings.
Dextrose...................................................................... 2.0 g
Sodium Chloride.......................................................... 5.0 g Slant cultures may be used as sources of inocula for additional
Disodium Phosphate.................................................... 2.5 g studies or for organism maintenance purposes.
Agar.......................................................................... 13.5 g
BBL™ Brain Heart Infusion Agar, Modified References
Approximate Formula* Per Liter 1. Creitz and Puckett. 1954. Am. J. Clin. Pathol. 24:1318.
Brain Heart, Infusion from (solids)................................. 3.5 g 2. Murray, Baron, Jorgensen, Landry and Pfaller, (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
Peptic Digest of Animal Tissue.................................... 15.0 g 3. Reisner, Woods, Thompson, Larone, Garcia and Shimizu. 1999. In Murray, Baron, Pfaller, Tenover
Pancreatic Digest of Casein........................................ 10.0 g and Yolken (ed.), Manual of clinical microbiology, 7th ed. American Society for Microbiology,
Dextrose...................................................................... 2.0 g Washington, D.C.
Sodium Chloride.......................................................... 5.0 g
Disodium Phosphate.................................................... 2.5 g Availability
Agar.......................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria. Difco™ Brain Heart Infusion Agar
AOAC BAM CCAM COMPF EPA MCM9 SMD SMWW USDA
Directions for Preparation from Cat. No. 241820 Dehydrated – 100 g
241830 Dehydrated – 500 g
Dehydrated Product 241810 Dehydrated – 2 kg
1. Suspend the powder in 1 L of purified water:
BBL™ Brain Heart Infusion Agar
Difco™ Brain Heart Infusion Agar – 52 g; AOAC BAM CCAM COMPF EPA MCM9 SMD SMWW USDA
BBL™ Brain Heart Infusion Agar – 52 g; Cat. No. 211065 Dehydrated – 500 g
BBL™ Brain Heart Infusion Agar, Modified – 53 g. 212166 Dehydrated – 5 lb (2.3 kg)
Mix thoroughly. United States and Canada
2. Heat with frequent agitation and boil for 1 minute to Cat. No. 221569 Prepared Plates (Deep Fill) – Pkg. of 20*
221570 Prepared Plates (Deep Fill) – Ctn. of 100*
completely dissolve the powder. 220838 Prepared Pour Tubes (20 mL) – Pkg. of 10
3. Autoclave at 121°C for 15 minutes. 221610 Prepared Slants (K Tubes) – Pkg of 10
4. Before use, agitate gently to distribute the precipitate 297283 Prepared Slants (A Tubes) – Pkg. of 10
uniformly throughout the medium. Europe
5. Test samples of the finished product for performance using Cat. No. 255003 Prepared Plates – Pkg. of 20*
Cultural Response
Bacto™ Brain Heart Infusion, Porcine
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 18-48 hours.
Organism ATCC™ INOCULUM CFU RECOVERY
Neisseria meningitidis 13090 102-103 Fair
Streptococcus pneumoniae 6305 102-103 Good
Streptococcus pyogenes 19615 102-103 Fair
93
94
2. Heat with frequent agitation and boil for 1 minute to com- Expected Results
pletely dissolve the powder. Examine tubes at intervals for up to 7 days for growth, which
3. Autoclave at 121°C for 15 minutes. is indicated by the presence of turbidity compared to an
4. Test samples of the finished product for performance using uninoculated control.
stable, typical control cultures.
If growth appears, cultures should be examined by Gram stain B
and subcultured onto appropriate media; e.g., a Trypticase™ Soy
Procedure
Agar with 5% Sheep Blood and/or Chocolate II Agar plate, Eosin
With liquid specimens, tubed media should be inoculated
Methylene Blue Agar, Levine, or MacConkey II Agar plates.
with 1-2 drops of the specimen using a sterile pipette. Swab
If anaerobes are suspected, subcultures should be incubated
specimens may be inserted into broth after inoculation of plated
anaerobically, as in a BD GasPak™ EZ anaerobic system.
media.
Liquid tubed media for anaerobic incubation should be Availability
reduced prior to incubation by placing the tubes, with caps Difco™ Brain Heart Infusion with PAB and Agar
loosened, under anaerobic conditions for 18-24 hours prior Cat. No. 249910 Dehydrated – 500 g
to use. An efficient and easy way to obtain suitable anaerobic BBL™ Brain Heart Infusion with PABA
conditions is through the use of BD GasPak™ EZ anaerobic Cat. No. 211069 Dehydrated – 500 g
system or an alternative anaerobic system. Alternatively, 220842 Prepared Tubes with 0.1% Agar, 20 mL
liquid media may be reduced immediately prior to use by (A Tubes) – Pkg. of 10
boiling with caps loosened and cooling with tightened caps to
room temperature before inoculation.
95
Directions for Preparation from the surface of the medium, and the oxygen in this space
Dehydrated Product reacts with the reducing agents to form an anaerobic
1. Suspend 58 g of the powder in 1 L of purified water. Mix environment.
thoroughly. 4. Incubate aerobically as desired.
2. Heat with frequent agitation and boil for 1 minute to com- For a complete discussion on anaerobic and microaerophilic
pletely dissolve the powder. bacteria from clinical specimens, refer to the appropriate
3. Autoclave at 121°C for 15 minutes. procedures outlined in the references.2-4 For the examination of
4. Test samples of the finished product for performance using anaerobic bacteria in food refer to standard methods.6-8
stable, typical control cultures.
Expected Results
Procedure Refer to appropriate references and procedures for results.
Standard Petri Dishes2
1. Inoculate a properly obtained specimen onto the medium, Limitations of the Procedure
and streak to obtain isolated colonies. 1. Clinical specimens must be obtained properly and trans-
2. Immediately incubate anaerobically at 35 ± 2°C. ported to the laboratory in a suitable anaerobic transport
3. Examine at 24 hours if incubating plates in an anaerobic container.2
chamber. Examine at 48 hours if incubating plates in an 2. The microbiologist must be able to verify quality control
anaerobic jar or pouch, or if using Brewer anaerobic dish of the medium and determine whether the environment is
cover. anaerobic.2
4. Extended incubation may be necessary to recover some 3. The microbiologist must perform aerotolerance testing on each
anaerobes. isolate recovered to ensure the organism is an anaerobe.2
Brewer Anaerobic Agar Plates
References
1. Dispense 50-60 mL of Brewer Anaerobic Agar into a 1. Brewer. 1942. Science 95:587.
2. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
standard Petri dish. For best results use porous tops to American Society for Microbiology, Washington, D.C.
obtain a dry surface. 3. Baron, Peterson and Finegold. 1994. Bailey & Scott’s diagnostic microbiology, 9th ed. Mosby-Year
Book, Inc., St. Louis, Mo.
2. Inoculate the surface of the medium by streaking; avoid the 4. Murray, Baron, Jorgensen, Landry and Pfaller, (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
edges of the plates. 5. Smith. 1975. The pathogenic anaerobic bacteria, 2nd ed. Charles C. Thomas, Springfield, Ill.
6. Wehr and Frank (ed.). 2004. Standard methods for the microbiological examination of dairy products,
3. Replace the standard Petri dish lid with a sterile Brewer 17th ed. American Public Health Association, Washington, D.C.
7. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
anaerobic dish cover. The cover should not rest on the tional, Gaithersburg, Md.
Petri dish bottom. The inner glass ridge should seal against 8. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
the uninoculated periphery of the agar. It is essential that
the sealing ring inside the cover is in contact with the Availability
medium. This seal must not be broken before the end of Difco™ Brewer Anaerobic Agar
the incubation period. A small amount of air is caught over Cat. No. 227920 Dehydrated – 500 g
96
B
Dehydrated Appearance: Pink, free-flowing, homogeneous.
2. Heat with frequent agitation and boil for 1 minute to
Solution: 5.8% solution, soluble in purified water upon
completely dissolve the powder. boiling. Solution is brownish-green, clear to very
3. Autoclave at 121°C for 15 minutes. slightly opalescent.
4. Test samples of the finished product for performance using Prepared Appearance: Orange-brown, very slightly to slightly opales-
stable, typical control cultures. cent.
Reaction of 5.8%
Solution at 25°C: pH 6.9 ± 0.2
Procedure
Use standard procedures to obtain isolated colonies from Cultural Response
specimens. A less selective medium and a nonselective medium Difco™ Brilliant Green Agar
should also be streaked to increase the chance of recovery Prepare the medium per label directions. Inoculate and incubate at
when the population of gram-negative organisms is low and 35 ± 2°C for 18-24 hours.
to provide an indication of other organisms present in the INOCULUM COLONY
ORGANISM ATCC™ CFU RECOVERY COLOR
specimen. Incubate plates, protected from light, at 35 ± 2°C
Escherichia coli 25922 ~104 Poor Yellow-green
for 18-24 hours. If negative after 24 hours, reincubate an
Salmonella enterica
additional 24 hours. subsp. enterica
serotype Enteritidis 13076 30-300 Good Red
Expected Results Salmonella enterica
Typical colonial morphology on Brilliant Green Agar is as subsp. enterica None to
serotype Typhi 19430 30-300 poor Red
follows:
Salmonella enterica
Salmonella (other than subsp. enterica
S. Typhi and S. Paratyphi)....... White to red, opaque colonies serotype Typhimurium 14028 30-300 Good Red
. ............................................ surrounded by red zones in the
. ............................................ medium Staphylococcus Marked
aureus 25923 ~104 inhibition –
S. Typhi and S. Paratyphi........ No growth to trace growth
Shigella.................................. No growth to trace growth
Escherichia coli and
Availability
Enterobacter/Klebsiella........... Yellow to greenish-yellow Difco™ Brilliant Green Agar
. ............................................ colonies surrounded by intense EP SMWW
. ............................................ yellow-green zones in medium Cat. No. 228530 Dehydrated – 500 g
Proteus.................................. No growth to trace growth BBL™ Brilliant Green Agar
Pseudomonas......................... Pink to red colonies EP SMWW
Gram-positive bacteria........... No growth to trace growth Cat. No. 295963 Prepared Plates – Pkg. of 20*
*Store at 2-8°C.
References
1. Kristensen, Lester and Jurgens. 1925. Br. J. Exp. Pathol. 6:291.
2. Kauffmann. 1935. Z. Hyg. Infektionskr. 117:26.
3. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C.
97
Identity Specifications
Difco™ Brilliant Green Agar Modified
Dehydrated Appearance: Pink, free-flowing, homogeneous.
Solution: 5.2% solution, soluble in purified water upon
boiling. Solution is orange-brown, clear to
slightly opalescent.
Prepared Appearance: Orange-brown, clear to slightly opalescent.
Reaction of 5.2%
Solution at 25°C: pH 6.9 ± 0.1
Cultural Response
Difco™ Brilliant Green Agar Modified
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 18-24 hours.
INOCULUM Colony
Organism ATCC™ CFU RECOVERY Color
Escherichia coli 25922 103 Complete to Green
partial inhibition
Proteus mirabilis 25933 103 Complete to Red
partial inhibition
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028 102-103 Good Red
Directions for Preparation from 1. Subculture from the broth at 18-24 hours and at 48 hours
onto Brilliant Green Agar Modified.
Dehydrated Product
2. Examine for typical colonies of Salmonella after overnight
1. Suspend 52 g of the powder in 1 L of purified water. Mix
incubation at 37°C.
thoroughly.
2. Heat with frequent agitation and boil for 1 minute to
Expected Results
completely dissolve the powder. DO NOT AUTOCLAVE.
Salmonella will produce red colonies.
3. Test samples of the finished product for performance using
stable, typical control cultures.
98
References
1. Guinee and Kampelmacher. 1962. Antonie van Leeuwenhoek 28:417.
2. Heard, Jennet and Linton. 1969. Br. Vet. J. 125:635.
3. H. M. S. O. 1982. Methods for the isolation and identification of salmonellae (other than Salmonella
typhi) from water and associated materials.
4. International Organisation for Standardization. 1974. Draft International Standard ISO/DIS 3565.
Geneva, Switzerland.
5. British Poultry Meat Society. 1982. A manual of recommended methods for the microbiological
examination of poultry and poultry products.
6. Harvey and Price. 1976. J. Hyg. Camb. 77:333.
Identity Specifications
Difco™ Brilliant Green Bile Agar
Dehydrated Appearance: Light purple, free-flowing, homogeneous (may
contain small dark particles).
Solution: 2.06% solution, soluble in purified water upon
boiling. Solution is bluish-purple, slightly opales-
cent.
Prepared Appearance: Blue with or without a tint of purple, slightly
opalescent.
Reaction of 2.06%
Solution at 25°C: pH 6.9 ± 0.2
Cultural Response
Difco™ Brilliant Green Bile Agar
Prepare the medium per label directions. Inoculate using the pour plate
technique and incubate at 35 ± 2°C for 18-24 hours.
Inoculum colony
ORGANISM ATCC™ CFU RECOVERY Color
Enterobacter aerogenes 13048 102-103 Good Pink
Escherichia coli 25922 102-103 Good Deep red with
bile precipitate Salmonella
Escherichia coli Enteritidis
Salmonella enterica ATCC™ 25922 ATCC™ 13076
subsp. enterica Colorless to
serotype Typhimurium 14028 102-103 Good light pink
Staphylococcus aureus 25923 103-2×103 Marked to –
complete inhibition
99
Dehydrated Product
Availability
1. Suspend 20.6 g of the powder in 1 L of purified water. Mix
Difco™ Brilliant Green Bile Agar
thoroughly.
COMPF
2. Heat with frequent agitation and boil for 1 minute to com- Cat. No. 214100 Dehydrated – 500 g
pletely dissolve the powder.
100
Cultural Response
Difco™ Brilliant Green Bile Broth 2%
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for
48 hours.
INOCULUM GAS
ORGANISM ATCC™ CFU RECOVERY PRODUCTION
Enterobacter aerogenes 13048 102-103 Good +
Enterococcus faecalis 19433 10 -2×10 Partial to
3 3
–
complete inhibition
Uninoculated Escherichia coli
Escherichia coli 25922 102-103 Good + Tube ATCC™ 25922
Staphylococcus aureus 25923 103-2×103 Marked to –
complete inhibition
References Availability
1. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C.
Difco™ Brilliant Green Bile Broth 2%
2. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy products, 17th ed. AOAC BAM CCAM COMPF EPA ISO SMD SMWW
American Public Health Association, Washington, D.C.
3. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna- Cat. No. 273000 Dehydrated – 100 g
tional, Gaithersburg, Md. 274000 Dehydrated – 500 g
4. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
International, Gaithersburg, Md.
271000 Dehydrated – 2 kg
5. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C. BBL™ Brilliant Green Bile Broth, 2%
AOAC BAM CCAM COMPF EPA ISO SMD SMWW
Cat. No. 221612 Prepared Tubes with Durham Tube – Pkg. of 10*
*Store at 2-8°C.
101
Expected Results
Salmonella species form pink to red colonies.
References
1. Murray, Baron, Pfaller, Tenover and Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
American Society for Microbiology, Washington, D.C.
2. Kauffmann. 1935. Z. Hyg. Infektionskr. 117:26.
3. Kabler and Clark. 1952. Am. J. Public Health 42:390.
4. Geldreich and Jeter. 1952. Abstr. Bacteriol. Proc. 52nd Gen. Meet. Soc. Am. Bacteriologists 1952.
Availability
Difco™ m Brilliant Green Broth
Cat. No. 249410 Dehydrated – 500 g
Salmonella
Typhimurium
ATCC™ 14028
102
Brucella Media
Brucella Agar • Brucella Agar with 5% Horse Blood
Brucella Broth B
Intended Use of several media suitable for use as the liquid medium component
Brucella Agar is a culture medium for the cultivation of Brucella of biphasic blood culture bottles.1,2 It is also used to cultivate
organisms. With the addition of 5% horse blood, the medium Campylobacter spp.3
is used in qualitative procedures for the isolation and cultivation
of nonfastidious and fastidious microorganisms from a variety Principles of the Procedure
of clinical and nonclinical specimens. Brucella Agar and Brucella Broth support the growth of
fastidious microorganisms due to their content of peptones,
Brucella Broth is used for the cultivation of Brucella species and
dextrose and yeast extract. The peptones supply organic
for the isolation and cultivation of a wide variety of fastidious
nitrogen. The yeast extract is a potent source of the B-complex
and nonfastidious microorganisms.
vitamins. Dextrose is utilized as an energy source. Sodium
bisulfite is a reducing agent, and sodium chloride maintains
Summary and Explanation
the osmotic equilibrium. Agar is the solidifying agent in
Brucella Agar was developed for the cultivation of Brucella
Brucella Agar.
species from diagnostic specimens, such as blood, and from
foods and other potentially contaminated material. Brucella In BBL™ Brucella Agar with 5% Horse Blood plates, the horse
Agar with 5% Horse Blood plates are particularly useful for blood supplies both the X and V factors which are growth
the cultivation of the more fastidious aerobic and anaerobic requirements for certain organisms; e.g., Haemophilus influen-
microorganisms, including streptococci, pneumococci, Listeria, zae.3 Sheep and human blood are not suitable for this purpose
Neisseria meningitidis and Haemophilus influenzae. because they contain enzymes that inactivate the nicotinamide
adenine dinucleotide (NAD) which is the V factor.4
Brucella Broth may be used for the isolation and cultivation
of a wide variety of microorganisms including nutritionally Defibrinated horse blood may give hemolytic reactions differ-
fastidious specimens. 1 This medium is recommended for ent than sheep blood.5 Some streptococci (e.g., group D) give
the cultivation of Brucella species and was recommended as one hemolytic reactions on horse blood but not on sheep blood
103
BBL™ Brucella Broth After incubation, most plates will show an area of confluent
Consists of the same ingredients without the agar. growth. Because the streaking procedure is, in effect, a
*Adjusted and/or supplemented as required to meet performance criteria. “dilution” technique, diminishing numbers of microorganisms
are deposited on the streaked areas. Consequently, one or more
Precautions7 of these areas should exhibit isolated colonies of the organ-
1. Biosafety Level 2 practices, containment equipment and isms contained in the specimen. Further, growth of each
facilities are recommended for activities with clinical speci- organism may be semi-quantitatively scored on the basis of
mens of human or animal origin containing or potentially growth in each of the streaked areas.
containing pathogenic Brucella spp.
Broth
2. Biosafety Level 3 practices, containment equipment and
facilities are recommended for all manipulations of cultures Growth in the tubes is indicated by the presence of turbidity
of the pathogenic Brucella spp. and for experimental compared with an uninoculated control.
animal studies. If growth appears, cultures should be examined by Gram
stain and subcultured onto appropriate media; e.g., Trypticase™
Directions for Preparation from Soy Agar with 5% Sheep Blood and/or Brucella Agar and
Dehydrated Product Chocolate II Agar, Eosin Methylene Blue Agar, Levine or
1. Suspend the powder in 1 L of purified water: MacConkey II Agar.
BBL™ Brucella Agar – 43 g;
BBL™ Brucella Broth – 28 g. References
Mix thoroughly. 1. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol.
1. Williams & Wilkins, Baltimore, Md.
2. For the agar, heat with frequent agitation and boil for 2. Moyer, Holcomb and Hausler. 1991. In Balows, Hausler, Herrmann, Isenberg, and Shadomy (ed.),
Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C.
1 minute to completely dissolve the powder. For the broth, 3. Chapin and Murray. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.). Manual of clinical
microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
heat slightly, if necessary, to obtain solution. 4. Krumweide and Kuttner. 1938. J. Exp. Med. 67:429.
5. Vera and Power. 1980. In Lennette, Balows, Hausler and Truant (ed.), Manual of clinical microbiology,
3. Autoclave at 121°C for 15 minutes. 3rd ed. American Society for Microbiology, Washington, D.C.
4. For preparation of blood plates, add 5 to 10% sterile de- 6. Vera. 1971. Health Lab. Sci. 8:176.
7. U.S. Public Health Service, Centers for Disease Control and Prevention, and National Institutes of
fibrinated blood to sterile agar which has been cooled to Health. 2007. Biosafety in microbiological and biomedical laboratories, 5th ed. HHS Publication No.
(CDC) 93-8395. U.S. Government Printing Office, Washington, D.C.
45-50°C.
5. Test samples of the finished product for performance using
Availability
stable, typical control cultures. BBL™ Brucella Agar
CCAM ISO USDA
Procedure Cat. No. 211086 Dehydrated – 500 g
Agar (without or with added blood) 221547 Prepared Plates with 5% Horse Blood –
Use standard procedures to obtain isolated colonies from Pkg. of 20*
221548 Prepared Plates with 5% Horse Blood –
specimens. Ctn. of 100*
Since many pathogens require carbon dioxide on primary isola- BBL™ Brucella Broth
tion, incubate plates at 35 ± 2°C for 24-72 hours in anaerobic CCAM ISO USDA
atmosphere supplemented with carbon dioxide. Cat. No. 211088 Dehydrated – 500 g
296185 Prepared Tubes (K Tubes), 5 mL –
Ctn. of 100
*Store at 2-8°C.
104
105
BS12 CMPH2 MCM9 Cat. No. 297840 Prepared Plates – Pkg. of 20*
United States and Canada BBL™ Brucella 5% Sheep Blood Agar with Hemin
Cat. No. 297848 Prepared Plates – Pkg. of 20* and Vitamin K1//Brucella Laked Sheep Blood Agar
297716 Prepared Plates – Ctn. of 100* with Kanamycin and Vancomycin
Europe Cat. No. 297849 Prepared I Plate™ Dishes – Pkg. of 20*
Cat. No. 255509 Prepared Plates – Ctn. of 20* *Store at 2-8°C.
Procedure
Using a sterile swab or inoculating loop, remove fresh growth Availability
from the plated or slanted medium and suspend in the broth BBL™ Brucella Broth with 20% Glycerol
Cat. No. 297466 Prepared Tubes – Pkg. of 10
maintenance medium to achieve the desired concentration
of viable cells. Freeze suspension immediately at –20°C or
below. Consult texts for detailed information about preparing
stock cultures of specific organisms.2-4
106
Bushnell-Haas Broth
Intended Use Potassium nitrate is a nitrogen source, while monopotassium
phosphate and diammonium hydrogen phosphate provide
Bushnell-Haas Broth is used for studying microbial utilization
of hydrocarbons. buffering capability. B
Summary and Explanation Formula
Bushnell-Haas Broth (Bushnell-Haas marine salts broth), Difco™ Bushnell-Haas Broth
prepared according to the formula described by Bushnell Approximate Formula* Per Liter
Magnesium Sulfate...................................................... 0.2 g
and Haas1, is used to evaluate the ability of microorganisms
Calcium Chloride......................................................... 0.02 g
to decompose hydrocarbons. It is formulated without a carbon Monopotassium Phosphate.......................................... 1.0 g
source which allows for the addition of alternative hydrocarbons Diammonium Hydrogen Phosphate.............................. 1.0 g
such as kerosene, light and heavy mineral oils, paraffin wax Potassium Nitrate......................................................... 1.0 g
Ferric Chloride.............................................................. 0.05 g
and gasoline. *Adjusted and/or supplemented as required to meet performance criteria.
109
110
CDC Anaerobe 5% Sheep Blood Agar CDC Anaerobe Agar with Laked Sheep Blood and KV
Clostridium perfringens Bacteroides fragilis Porphyromonas levii Bacteroides fragilis
ATCC™ 13124 ATCC™ 25285 ATCC™ 29147 ATCC™ 25285
Procedure
Streak the specimen as soon as possible after it is received in the
laboratory. Minimize exposure to air. With liquid specimens,
media should be inoculated with one drop of the specimen.
Tissue specimens should be minced and then ground in sterile
broth, such as BBL Enriched Thioglycollate Medium, before
inoculation. Inoculation is then performed as for liquid speci-
mens. Swab specimens may be rolled onto the first quadrant
of plated media and then used to inoculate liquid media.
Alternatively, the swab may be “scrubbed” in a small volume
111
Incubate all cultures at 35 ± 2°C for a minimum of 24 hours BBL™ CDC Anaerobe 5% Sheep Blood Agar with PEA//
and up to 7 days. Anaerobe Laked Sheep Blood Agar with KV
Cat. No. 299611 Prepared I Plate™ Dishes – Pkg. of 20*
Record the relationship to oxygen as either obligate anaerobe *Store at 2-8°C.
or nonanaerobe (aerotolerant anaerobe, microaerophilic, or
facultative anaerobe).15
Colonies of the type(s) which proved to be obligate anaerobes
can be further studied using the corresponding broth cultures.
Organisms failing to grow on the aerobic subculture plates may
be presumed to be obligately anaerobic in terms of their oxygen
requirements.
112
CIN Agar
Yersinia Selective Agar Base
Yersinia Antimicrobic Supplement CN
Intended Use inhibition of normal enteric organisms. Organisms that do not
CIN (cefsulodin-Irgasan *-novobiocin) Agar supplemented with
™ metabolize mannitol to acid end products will form colorless,
cefsulodin and novobiocin is a differential and selective medium translucent colonies.
used in qualitative procedures for the isolation of Yersinia entero-
colitica from a variety of clinical and nonclinical specimens. Formulae C
*Irgasan is a trademark of Ciba-Geigy. Difco™ Yersinia Selective Agar Base
Approximate Formula* Per Liter
Summary and Explanation Peptone..................................................................... 17.0 g
Proteose Peptone......................................................... 3.0 g
CIN Agar, also known as Yersinia Selective Agar, was first Yeast Extract................................................................ 2.0 g
described by Schiemann as an alternative to MacConkey Mannitol.................................................................... 20.0 g
Agar and other commonly used media for isolation of Sodium Pyruvate.......................................................... 2.0 g
Sodium Chloride.......................................................... 1.0 g
Y. enterocolitica, a causative agent of gastroenteritis.1 CIN Agar Magnesium Sulfate Heptahydrate.............................. 10.0 mg
has been found to be far superior to MacConkey, SS, CAL or Y Sodium Desoxycholate................................................. 0.5 g
agars for the recovery of Y. enterocolitica.2 Sodium Cholate........................................................... 0.5 g
Irgasan™ . .................................................................... 4.0 mg
Agar.......................................................................... 13.5 g
Principles of the Procedure Crystal Violet............................................................... 1.0 mg
Fermentation of mannitol in the presence of neutral red results Neutral Red................................................................ 30.0 mg
in a characteristic “bull’s-eye” colony, colorless with red center. Difco™ Yersinia Antimicrobic Supplement CN
Selective inhibition of gram-negative and gram-positive organ- Formula Per 10 mL Vial
isms is obtained by means of crystal violet, sodium desoxycholate Cefsulodin................................................................... 4.0 mg
Novobiocin................................................................... 2.5 mg
and Irgasan (triclosan). Supplementation with Yersinia Antimi- *Adjusted and/or supplemented as required to meet performance criteria.
crobic Supplement CN (cefsulodin and novobiocin) improves
Identity Specifications
Difco™ Yersinia Selective Agar Base
Dehydrated Appearance: Light beige to light pinkish beige, free-flowing,
homogeneous.
Solution: 5.95% solution, soluble in purified water upon
boiling. Solution is reddish-purple, very slightly
to slightly opalescent.
Prepared Appearance: Reddish-orange, very slightly to slightly opales-
cent.
Reaction of 5.95%
Solution at 25°C: pH 7.4 ± 0.2
Difco™ Yersinia Antimicrobic Supplement CN
Dehydrated Appearance: Lyophilized, white, homogeneous cake.
Solution: Soluble on rehydration with 10 mL purified water.
Solution is colorless, clear.
Cultural Response
Difco™ Yersinia Selective Agar Base
Prepare the medium per label directions. Inoculate and incubate at 30 ± 2°C
for 18-48 hours.
ORGANISM ATCC™ INOCULUM CFU RECOVERY COLONY COLOR
Enterococcus faecalis 29212 103 Inhibition –
Escherichia coli 25922 103 Inhibition –
Proteus mirabilis 12453 10
3
Inhibition –
Pseudomonas aeruginosa 27853 103 Inhibition –
Yersinia enterocolitica 9610 102 Good Colorless with dark pink
centers, may have bile precipitate
113
CLED Agar
Intended Use cystine-dependent “dwarf colony” coliforms and by deletion
CLED Agar is used for the isolation, enumeration and presump- of sucrose.3 They designated the new medium as Cystine-
tive identification of microorganisms from urine. Lactose-Electrolyte-Deficient (CLED) medium and reported
it to be ideal for dip-inoculum techniques and for urinary
Summary and Explanation bacteriology in general.
In 1960, Sandys reported on the development of a new method of CLED Agar is recommended for use in plates or in urine
preventing the swarming of Proteus on solid media by restrict- dipsticks for detecting significant bacteriuria by quantita-
ing the electrolytes in the culture medium.1 Previous chemical tive culture of urine. For reliable results, inoculation of the
methods used to inhibit swarming by Proteus included the medium must occur as soon after collection as possible.
addition of chloral hydrate, alcohol, sodium azide, surface-active Confluent or semiconfluent growth of bacteria will occur on
agents, boric acid and sulfonamides to the culture medium.1 the surface of the dipstick medium when bacterial counts
This electrolyte-deficient medium of Sandys was modified by are greater than 105 per mL of urine, as confirmed by plates
Mackey and Sandys2 for use in urine culture by substituting inoculated by the calibrated-loop or duplicate-dilution
lactose and sucrose for the mannitol and increasing the pour-plate methods.4 Once the medium has been inoculated
concentrations of the bromthymol blue indicator and of the by immersion of the dipstick or by pouring the urine over the
agar. These two investigators further modified the medium by surface of the medium if only a small volume is available, the
the incorporation of cystine in order to enhance the growth of dipstick may be held 48 hours or longer, refrigerated or at room
114
Identity Specifications
BBL™ CLED Agar
Dehydrated Appearance: Fine, homogenous, free of extraneous material.
Solution: 3.6% solution, soluble in purified water upon
boiling. Solution is medium, yellow green to blue
green, clear to slightly hazy, with up to a large
amount of minute suspended insolubles.
Prepared Appearance: Medium, yellow green to blue green, clear to
slightly hazy.
Reaction of 3.6%
Solution at 25°C: pH 7.3 ± 0.2 C
Cultural Response
BBL™ CLED Agar
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 42-48 hours.
INOCULUM
ORGANISM ATCC™ CFU REcovery reaction
Enterococcus faecalis 29212 10 -10
3 4
Good Yellow
Escherichia coli 25922 103-104 Good Yellow
Klebsiella pneumoniae 33495 103-104 Good With or Enterococcus
without green to faecalis Escherichia coli
yellow reaction ATCC™ 29212 ATCC™ 25922
Pseudomonas aeruginosa 10145 103-104 Good With or without
blue reaction
Staphylococcus aureus 25923 103-104 Good Yellow
Proteus vulgaris 8427 10 -10
3 4
Good Blue
115
CTA Agar
Intended Use Formula
CTA Agar is primarily used for carbohydrate fermentation BBL™ CTA Agar
tests with corynebacteria and especially for differentiation of Approximate Formula* Per Liter
C. diphtheriae from related species. L-Cystine...................................................................... 0.5 g
Pancreatic Digest of Casein........................................ 20.0 g
Agar.......................................................................... 14.0 g
Summary and Explanation Sodium Chloride.......................................................... 5.0 g
CTA Medium™, a semi-solid formulation, was developed Sodium Sulfite.............................................................. 0.5 g
Phenol Red................................................................ 17.0 mg
by Vera and is widely used for fermentation and motility *Adjusted and/or supplemented as required to meet performance criteria.
determinations by a wide variety of microorganisms.1 CTA
Agar is the solid form of CTA Medium and, when employed Directions for Preparation from
as a plated medium and used in conjunction with BBL™ Taxo™ Dehydrated Product
carbohydrate discs, is useful in the speciation of Corynebac- 1. Suspend 40 g of the powder in 1 L purified water. Mix
terium isolates of medical importance.2 Supplemented with thoroughly.
carbohydrates and prepared as slants, it is used for the differen- 2. Heat with frequent agitation and boil for 1 minute to
tiation of Neisseria species.3 completely dissolve the powder.
3. Autoclave at 118°C for 15 minutes.
Principles of the Procedure 4. If desired, add 2 drops of sterile rabbit serum per tube
CTA Agar utilizes peptone as a carbohydrate-free source prior to solidification in order to enhance the recovery of
of nutrients. Inorganic salts are included in order to supply C. diphtheriae.
essential ions. Phenol red is an indicator of pH changes in the 5. Test samples of the finished product for performance using
medium surrounding the Taxo carbohydrates discs, which are stable, typical control cultures.
applied to the surface of inoculated plates.
Procedure
Inoculate a pure culture of the organism onto the surface of the
plated medium using a swab technique to inoculate the entire
surface. Taxo carbohydrate discs are then applied to the agar
surface using no more than four discs per plate.
116
C
v = variable reaction
Expected Results 5. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby, Inc.,
St. Louis, Mo.
Typical diphtheria bacilli ferment dextrose and maltose, but
not sucrose. Availability
BBL™ CTA Agar
Cat. No. 211094 Dehydrated – 500 g
Summary and Explanation For clostridia, bacilli, common micrococci, enteric bacilli and
This formulation was developed by Vera as a simple semi-solid other organisms not generally considered to be nutritionally
medium for the identification and maintenance of the gonococ- fastidious, the use of Trypticase Agar Base is recommended
cus and other bacteria.1 instead of this formulation.
The phenol red indicator changes from reddish-orange to 2. Heat with frequent agitation and boil for 1 minute to
yellow when the amount of acid produced by carbohydrate completely dissolve the powder.
fermentation is greater than the alkaline end products of 3. Autoclave at not over 118°C for 15 minutes.
peptone degradation. The color change with phenol red 4. To prepare fermentation medium, add 5-10 g of carbohy-
occurs around pH 6.8, near the original pH of the medium. drate before autoclaving or dissolve medium in 900 mL
water, autoclave, and aseptically add 100 mL sterile 5-10%
Formulae carbohydrate solution.
Difco™ Cystine Tryptic Agar 5. Test samples of the finished product for performance using
Approximate Formula* Per Liter stable, typical control cultures.
Tryptose..................................................................... 20.0 g
L-Cystine...................................................................... 0.5 g BBL™ CTA Medium™
Sodium Chloride.......................................................... 5.0 g
Sodium Sulfite.............................................................. 0.5 g 1. Suspend 28.5 g of the powder in 1 L of purified water. Add
Agar............................................................................ 2.5 g carbohydrate (0.5 to 1.0%) if desired, and adjust the pH if
Phenol Red................................................................ 17.0 mg necessary. Mix thoroughly.
BBL™ CTA Medium™ 2. Heat with frequent agitation and boil for 1 minute or until
Approximate Formula* Per Liter solution is complete.
Pancreatic Digest of Casein........................................ 20.0 g
L-Cystine...................................................................... 0.5 g 3. Tube and autoclave at not over 118°C for 15 minutes. Cool
Sodium Chloride.......................................................... 5.0 g in the upright position.
Sodium Sulfite.............................................................. 0.5 g 4. Store at room temperature. Do not refrigerate unless in tightly
Agar............................................................................ 2.5 g
Phenol Red................................................................ 17.0 mg closed, screw-capped tubes.
*Adjusted and/or supplemented as required to meet performance criteria. 5. Test samples of the finished product for performance using
stable, typical control cultures.
Directions for Preparation from
Dehydrated Product Procedure
Difco™ Cystine Tryptic Agar 1. Loosen caps, boil, tighten caps and cool before use.
1. Suspend 28.5 g of the powder in 1 L of purified water. Mix 2. Remove fresh colony growth from the surface of a suitable
thoroughly. culture medium; e.g., Chocolate Agar, not from a selective,
primary isolation plate.3
118
3. For fermentation tests with members of the genus Neis- 3. Neisseria species usually produce acid only in the area of
seria, only the surface of the tubed medium is inoculated. stabs (upper third). If there is a strong acid (yellow color)
For facultative organisms, such as streptococci and strictly throughout the medium, a contaminating organism may be
anaerobic organisms, inoculate by stabbing the center of the present. If in doubt about a tube containing a Neisseria spe-
medium with an inoculating needle to about 1/2 the depth cies, a Gram stain and oxidase test should be performed on
of the medium. the growth.10
4. Repeat for each tube to be inoculated.
5. Incubate at 35 ± 2°C with loosened caps aerobically or References
1. Vera. 1948. J. Bacteriol. 55:531.
anaerobically depending upon the organisms being tested; 2. Vera and Petran. 1954. Bull. Nat. Assoc. Clin. Labs. 5:90.
Neisseria should be incubated with tight caps4 especially if 3. Morello, Janda and Doern. 1991. In Balows, Hausler, Herrmann, Isenberg and Shadomy (ed.), Manual
of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C.
tubes must be incubated in a CO2 incubator,5,6 or with loose
caps in a non-CO2 incubator.7,8 Examine periodically up to
4. Kellogg. 1974. In Lennette, Spaulding and Truant (ed.), Manual of clinical microbiology, 2nd ed.
American Society for Microbiology, Washington, D.C.
5. Yu and Washington. 1985. In Washington (ed.), Laboratory procedures in clinical microbiology, 2nd
C
ed. Springer-Verlag, New York, N.Y.
24 hours for growth (turbidity), evidence of motility, and 6. Morse and Knapp. 1987. In Wentworth (ed.), Diagnostic procedures for bacterial infections, 7th ed.
acid production in carbohydrate-containing medium (yellow American Public Health Association, Washington, D.C.
7. Center for Disease Control. 1978. Laboratory methods in clinical bacteriology. CDC, Atlanta, Ga.
color in upper layer of medium). A few strains may require 8. Baron, Peterson and Finegold. 1994. Bailey & Scott’s diagnostic microbiology, 9th ed. Mosby-Year
Book, Inc., St. Louis, Mo.
incubation for up to 48-72 hours.9 9. Finegold and Martin. 1982. Bailey & Scott’s diagnostic microbiology, 6th ed. The C.V. Mosby
Company, St. Louis, Mo.
6. Many fastidious organisms, including Neisseria, Pasteurella, 10. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol.
1. Williams & Wilkins, Baltimore, Md.
streptococci, Brucella, corynebacteria and vibrios, may 11. Faur, Weisburd and Wilson. 1975. J. Clin. Microbiol. 1:294.
be readily cultivated in this medium, no added carbon 12. Applebaum and Lawrence. 1979. J. Clin. Microbiol. 9:598.
Campy-Cefex Agar
Intended Use Campylobacter species from chicken carcasses. Campy-Cefex
Campy-Cefex Agar* is a selective medium used for the primary Agar demonstrated easier differentiation of C. jejuni from
isolation and cultivation of Campylobacter species, especially other flora when compared to Campylobacter Cefoperazone
C. jejuni and C. coli, from poultry. Desoxycholate Agar and better selectivity than Campylobacter
* U.S. Patent No. 5,891,709 Brucella Agar (Campy BAP).1
In September 2005, Campy-Cefex Agar was adopted by the
Summary and Explanation National Advisory Committee on Microbiological Criteria for
In 1992, Stern et al. published on the development of Campy- Foods for the isolation of Campylobacter species from chicken
Cefex Agar, a selective-differential medium for the isolation of carcasses.2
119
Principles of the Procedure or spiral-shaped bacterial rods that may demonstrate a rapid
This medium consists of Brucella Agar, a general purpose me- corkscrew-like movement. Suspect colonies that demonstrate
dium that supports the growth of Campylobacter species. Laked the described colonial and microscopic morphology, and are
horse blood provides additional nutrients. Antimicrobial agents catalase and oxidase positive, can be presumptively identified
are incorporated to suppress the growth of normal fecal flora that as Campylobacter species.1,5
could mask the presence of C. jejuni. Cefoperazone is a cepha-
losporin antibiotic that suppresses the growth of gram-negative Limitations of the Procedure
enteric bacilli and some gram-positive species. Cycloheximide is 1. Since C. jejuni is thermophilic, it is important to incubate
used to suppress the growth of fungi. the plates at 42°C; otherwise growth will be delayed. Also,
the higher temperature improves selectivity by inhibiting the
Sample Collection and Handling normal flora.
For agrifood samples consult appropriate standard methods 2. For identification, organisms must be in pure culture. Mor-
for details on sample preparation and processing according to phological, biochemical, and/or serological tests should be
sample type.3,4 performed for final identification. Consult appropriate texts
for detailed information and recommended procedures.2-4
Procedure
References
Inoculate the sample as soon as possible after it is received in the 1. Stern, Wojton and Kwiatek. 1992. J. Food Protect. 55:514.
laboratory, by means of a swab, directly onto the agar surface 2. NACMCF Executive Secretariat. 2007. Analytical utility of Campylobacter methodologies. U.S.
Department of Agriculture, Food Safety and Inspection Service, Washington, D.C. J. Food Protect.
and streak the plate for isolation. Incubate inoculated plates, 70:241.
3. Ransom and Rose. 1998. Isolation, identification, and enumeration of Campylobacter jejuni/coli
protected from light, at 42°C in a reduced oxygen, increased from meat and poultry products. In Microbiology laboratory guidebook, 3rd ed., Food Safety and
Inspection Service, U.S. Department of Agriculture, Washington, D.C.
carbon dioxide atmosphere. This atmosphere can be achieved 4. Hunt, Abeyta and Tran. 2001. Chapter 7 Campylobacter. In Bacteriological analytical manual, online.
by using the BD GasPak™ EZ Campy Container System with U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition. Washington,
D.C.
sachets or the BD GasPak EZ Campy Pouch System with sachets. 5. Stern and Pretanik. 2006. Counts of Campylobacter spp. on U.S. broiler carcasses. J. Food Protect.
69:1034.
Examine plates after 36-48 hours incubation.1
Availability
Expected Results BBL™ Campy-Cefex Agar
Colonies of Campylobacter appear translucent. Direct ex- Cat. No. 215221 Prepared Plates – Pkg. of 20*
292487 Prepared Plates – Ctn. of 100*
amination using phase-contrast microscopy (x1000) can be
used to confirm typical morphology and motility – curved *Store at 2-8°C.
Campylobacter Agars
Campylobacter Agar Base • Campylobacter Agar
with 5 Antimicrobics and 10% Sheep Blood (Blaser)
Campy CSM Agar • Campy CVA Agar • Skirrows Medium
Campylobacter Antimicrobic Supplement Skirrow
Camplyobacter Antimicrobic Supplement Blaser
Intended Use blood-based selective media that differed in the numbers and
Campylobacter Agar Base, when supplemented with blood or types of antimicrobics.2-6 Bolton et al. reported that charcoal
other additives and antimicrobial agents, is used for the primary can effectively replace the blood in selective media for campy-
isolation and cultivation of Campylobacter jejuni subsp. lobacters.7
jejuni from human fecal specimens. Several prepared selective In 1978, Blaser et al. reported success in isolating C. jejuni
media formulations are provided for the same purpose. with a medium containing four antimicrobics incorporated
into Brucella Agar supplemented with 10% defibrinated sheep
Summary and Explanation blood.3,4 Subsequently, cephalothin was incorporated to
In 1972, Dekeyser et al. reported that C. jejuni was isolated from increase its ability to inhibit the normal bacterial flora associated
the feces of patients with diarrhea and acute gastroenteritis with fecal specimens.5
using a filtration technique and a blood-containing selective
medium with antimicrobics to suppress the normal enteric flora.1 In 1983, Reller et al. introduced an improved selective
Subsequently, Skirrow and other investigators reported similar medium containing cefoperazone, vancomycin and ampho-
120
tericin B (CVA) for isolation of C. jejuni.6 They reported Campy CVA Agar consists of Brucella Agar, a general-purpose
that this combination of antimicrobial agents provided better medium that supports the growth of Campylobacter species.
inhibition of normal fecal flora for easier detection of C. jejuni Defibrinated sheep blood provides additional nutrients. Anti-
than the selective blood agar plate developed previously. microbial agents are incorporated to suppress the growth of
normal fecal flora that could mask the presence of C. jejuni.
Karmali et al., in 1986, evaluated a blood-free, charcoal-based
Cefoperazone is a cephalosporin antibiotic that suppresses the
selective medium (designated CSM) in parallel with a Skirrow-
growth of gram-negative enteric bacilli and some gram-positive
type selective medium containing lysed horse blood. They
species. Vancomycin is a glycopeptide antibiotic that inhibits
reported that the quality of Campylobacter growth on CSM
many species of gram-positive bacteria. Amphotericin B is an
(luxuriant growth with smooth and effuse colonies) was similar
antifungal agent.
to that seen on blood-based media and was significantly more
selective than Skirrow medium.8
Formulae
C
Difco Campylobacter Agar Base
™
Principles of the Procedure
Approximate Formula* Per Liter
These media support the growth of Campylobacter species due Proteose Peptone No. 3............................................. 15.0 g
to their content of peptones, yeast extract and other digests, Liver Digest................................................................. 2.5 g
extracts and components specific for the individual formula- Yeast Extract............................................................... 5.0 g
Sodium Chloride......................................................... 5.0 g
tions provided. Campylobacter isolation relies, in addition, Agar......................................................................... 12.0 g
on a medium’s selectivity, which depends on the antimicrobial
Difco™ Campylobacter Antimicrobic Supplement Skirrow
agents in the medium, a microaerophilic environment and the Formula Per 5 mL Vial
incubation temperature of 42°C, which suppresses the growth Vancomycin................................................................ 5.0 mg
of most normal bacteria.9 Polymyxin B............................................................1250.0 units
Trimethoprim.............................................................. 2.5 mg
The antimicrobial agents required to make Skirrow’s and Blaser’s Difco™ Campylobacter Antimicrobic Supplement Blaser
formulations are provided as Campylobacter Antimicrobic Formula Per 5 mL Vial
Supplement Skirrow and Campylobacter Antimicrobic Supple- Vancomycin................................................................ 5.0 mg
ment Blaser, respectively. Polymyxin B............................................................1250.0 units
Trimethoprim.............................................................. 2.5 mg
Campylobacter Agar with 5 Antimicrobics and 10% Sheep Cephalothin................................................................ 7.5 mg
Blood supports the growth of Campylobacter species due to its Amphotericin B........................................................... 1.0 mg
*Adjusted and/or supplemented as required to meet performance criteria.
content of peptones, dextrose, yeast extract and blood. The pep-
tones supply nitrogenous compounds, carbon, sulfur and trace Directions for Preparation from
ingredients. Yeast extract is a source of the B-complex vitamins. Dehydrated Product
Dextrose is utilized as an energy source. Sheep blood supplies 1. Suspend 39.5 g of the powder in 1 L of purified water. Mix
additional nutrients. The incorporation of the antimicrobial thoroughly.
agents (amphotericin B, cephalothin, polymyxin B, trimethoprim 2. Heat with frequent agitation and boil for 1 minute to
and vancomycin) suppresses the growth of the normal micro- completely dissolve the powder.
bial flora in fecal specimens, thereby facilitating isolation of 3. Autoclave at 121°C for 15 minutes. Cool the medium to
C. jejuni. 45-50°C.
Skirrows Medium contains, in addition to the usual nutritional 4. Aseptically add 5-7% sterile lysed horse blood or 10%
components, laked horse blood, which supplies the X factor sterile defibrinated sheep blood. Mix thoroughly.
(heme) and other growth requirements. Vancomycin inhibits 5. To prepare Skirrow’s medium: aseptically rehydrate one vial
gram-positive bacteria, polymyxin B inhibits most gram- of Campylobacter Antimicrobic Supplement Skirrow with
negative bacilli except Proteus and trimethoprim is inhibitory 5 mL of sterile purified water. Rotate in an end-over-end mo-
for Proteus spp. tion to dissolve the contents completely. Store the rehydrated
vials at 2-8°C. Use within 24 hours after rehydration.
Campy CSM Agar consists of Columbia Agar Base supple-
To prepare Blaser’s medium: aseptically rehydrate one vial of
mented with activated charcoal, hematin, sodium pyruvate
Campylobacter Antimicrobic Supplement Blaser with 5 mL
and three antimicrobial agents (cefoperazone, cycloheximide
of sterile purified water. Rotate in an end-over-end motion
and vancomycin). The charcoal, hematin and sodium pyru-
to dissolve the contents completely. Store the rehydrated vials
vate improve the aerotolerance of Campylobacter species; it
at 2-8°C. Use within 24 hours after rehydration.
has been suggested that these supplements act as quenching
agents of photochemically-produced toxic oxygen derivatives.8 Aseptically add 1% of the desired antimicrobic supplement
Cefoperazone is a cephalosporin antibiotic that suppresses (10 mL of supplement to 1 L or 5 mL of supplement to
the growth of gram-negative enteric bacilli and some gram- 500 mL of medium base). Mix thoroughly, avoiding the for-
positive species. Vancomycin is a glycopeptide antibiotic that mation of air bubbles and dispense into sterile Petri dishes.
inhibits many species of gram-positive bacteria. Cycloheximide 6. Test samples of the finished product for performance using
is an antifungal agent. stable, typical control cultures.
121
Cultural Response
Difco™ Campylobacter Agar Base
Prepare the medium per label directions; e.g., with 10% sterile defibrinated
sheep blood and antimicrobic supplements (Skirrow or Blaser). Inoculate and
incubate at 42°C under microaerophilic conditions for 40-48 hours.
INOCULUM recovery RECOVERY
ORGANISM ATCC™ CFU Skirrow blaser
Campylobacter
jejuni subsp. jejuni 29428 102-103 Good Good
Campylobacter
jejuni subsp. jejuni 33291 102-103 Good Good
Candida albicans 10231 103 Good Inhibition
Enterococcus faecalis 33186 103 Inhibition Inhibition
Escherichia coli 25922 103 Inhibition Inhibition
translucent, grayish and has an irregular edge. Cat. No. 214890 Vial – 6 × 5 mL
C
BBL™ Campylobacter Agar with 5 Antimicrobics and
A small percentage of strains may appear tan or slightly pinkish. 11
10% Sheep Blood (Blaser)
Colonies tend to spread, especially when initially isolated from BS12 CMPH2 MCM9
fresh clinical specimens. United States and Canada
Cat. No. 221727 Prepared Plates – Pkg. of 20*
Limitations of the Procedure 221728 Prepared Plates – Ctn. of 100*
1. Due to the presence of 15 mg/L of cephalothin, growth of Europe
Cat. No. 254001 Prepared Plates – Pkg. of 20*
C. fetus subsp. fetus will be inhibited on Campylobacter 254069 Prepared Plates – Ctn. of 120*
Agar with 5 Antimicrobics and 10% Sheep Blood; therefore,
Japan
this medium is not recommended for the isolation or culture Cat. No. 251727 Prepared Plates – Pkg. of 20*
of this subspecies.
BBL™ Campy CSM Agar
2. Since C. jejuni is thermophilic, it is important to incubate
BS12 CMPH2 MCM9 SMWW
the plates at 42°C; otherwise, growth will be delayed. Also, Cat. No. 299614 Prepared Plates – Pkg. of 20*
the higher temperature improves selectivity by inhibiting the
BBL™ Campy CVA Agar
normal flora.
BS12 COMPF MCM9
Cat. No. 297246 Prepared Plates – Pkg. of 20*
References 297713 Prepared Plates – Ctn. of 100*
1. Dekeyser, Gossuin-Detrain, Butzler and Sternon. 1972. J. Infect. Dis. 125:390.
2. Skirrow. 1977. Br. Med. J. 2:9.
3. Blaser, Cravens, Powers and Wang. 1978. Lancet ii:979.
BBL™ Skirrows Medium
4. Blaser, Berkowitz, LaForce, Cravens, Reller and Wang. 1979. Ann. Intern. Med. 91:179. ISO SMWW
5. Wilson and Wang. October 13, 1979. Background and culture techniques for Campylobacter fetus
subsp. jejuni. Information flier, Campylobacter Laboratory, Veterans Administration Hospital, United States and Canada
Denver, Co. Cat. No. 297793 Prepared Plates – Pkg. of 20*
6. Reller, Mirrett and Reimer. 1983. Abstr. C274. Abstr. Annu. Meet. Am. Soc. Microbiol. 1983.
7. Bolton and Coates. 1983. J. Appl. Bacteriol. 54:115. Japan
8. Karmali, Simor, Roscoe, Fleming, Smith and Lane. 1986. J. Clin. Microbiol. 23:456.
9. Grasmick. 1992. In Isenberg (ed.), Clinical microbiology procedures handbook, vol. 1. American Society
Cat. No. 252111 Prepared Plates – Pkg. of 20*
for Microbiology, Washington, D.C. *Store at 2-8°C.
10. Karmali and Fleming. 1979. J. Clin. Microbiol. 10:245.
11. Kaplan. 1980. In Lennette, Balows, Hausler and Truant (ed.). 1980. Manual of clinical microbiology,
3rd ed. American Society for Microbiology, Washington, D.C.
123
broth medium was used alone.4 Luechterfeld et al. reported that GasPak EZ Campy systems. Alternatively, the atmosphere
the number of positives was not substantially increased by hold- can be achieved using evacuation of GasPak vented jars and
ing turkey fecal specimens at 4ºC overnight in Campylobacter replacement with cylinder gases,8 or by using the Fortner
Thioglycollate Medium.5 principle.9
Campylobacter Thioglycollate Medium has been recommended
Expected Results
as a holding medium when facilities for streaking and incubation
Plates of Campylobacter Agar with 5 Antimicrobics and 10%
are not immediately available.6
Sheep Blood inoculated from Campylobacter Thioglycollate
Medium with 5 Antimicrobics should be examined for the
Principles of the Procedure
presence of colonies of Campylobacter jejuni. These colonies will
Campylobacter Thioglycollate Medium is a selective holding
appear as small, mucoid, usually grayish in coloration, flat with
medium recommended for the isolation of C. jejuni from clinical
irregular edges and nonhemolytic at 24 and 48 hours.10
specimens. The incorporation of antimicrobial agents (i.e.,
amphotericin B, cephalothin, polymyxin B, trimethoprim and Colonies may be only barely visible at 18 and 24 hours. An
vancomycin) and refrigeration inhibits further multiplication alternate colonial morphology, which appears to be strain
of normal microbial flora in fecal specimens, thus facilitating related, consists of round colonies 1-2 mm in diameter, which
isolation of C. jejuni. are convex, entire and glistening.10 A small percentage of strains
may appear tan or slightly pinkish in coloration.7
Procedure Colonies tend to spread or swarm, especially when initially
1. Sample collection, storage and subculturing to plated isolated from fresh clinical specimens.
medium.7
Place rectal swab about 1 cm into the medium and twirl the NOTE: If plates are examined after 24 hours of incubation,
swab. Remove the swab or lower it to bottom of the tube treat plates as if they were anaerobic cultures; i.e., examine
and break the shaft of the swab with the lip of the tube to plates quickly and place them back into a reduced oxygen
allow easy access to the shaft. atmosphere immediately after examination.
With solid stools, prepare a saline suspension, blend in a
mechanical mixer (i.e., vortex) and place five drops into References
1. Dekeyser, Gossuin-Detrain, Butzler and Sternon. 1972. J. Infect. Dis. 125:390
the medium about 1 cm below the surface. Alternatively, 2. Skirrow. 1977. Br. Med. J. 2:9.
3. Blaser, Cravens, Powers and Wang. 1978. Lancet 2:979
probe all areas of the stool with a swab and inoculate the 4. Blaser, Berkowitz, LaForce, Cravens, Reller and Wang. 1979. Ann. Intern. Med. 91:179.
5. Luechtefeld, Wang, Blaser and Reller. 1981. J. Clin. Microbiol. 13:438
medium as described for a rectal swab. With diarrheal 6. Reller, Wang and Blaser. 1979. Campylobacter enteritis: Campylobacter fetus subspecies jejuni. ASCP
stools, place five drops in the medium about 1 cm below Check Sample, Microbiology No. MB-99, Commission on Continuing Education, American Society
of Clinical Pathologists, Chicago, Ill.
the surface. 7. Kaplan. 1980. In. Lennette, Balows, Hausler and Truant (ed.), Manual of Clinical Microbiology, 3rd
ed. American Society for Microbiology, Washington, D.C.
Refrigerate inoculated Campylobacter Thioglycollate Medium 8. Nachamkin, 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical
microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
overnight and subculture the next day to Campylobacter Agar 9. Karmali and Fleming. 1979. J. Clin. Microbiol. 10:245.
10. Smibert. 1984. In Kreig and Holt (ed.), Bergey’s Manual™ of systematic bacteriology, vol. 1,
with 5 Antimicrobics and 10% Sheep Blood plates using a Williams & Wilkins, Baltimore, Md.
Pasteur pipette inserted about 2 cm below the surface of the
broth to continuously withdraw a sample as the tip is slowly Availability
drawn to the surface. Do not subculture onto nonselective BBL™ Campylobacter Thioglycollate Medium
media since the normal flora may still be viable. with 5 Antimicrobics
2. Incubation of plated medium. Cat. No. 221747 Prepared Tubes – Pkg. of 10
221748 Prepared Tubes – Ctn. of 100
Incubate plated medium at 42ºC in a reduced oxygen,
increased carbon dioxide atmosphere. This atmosphere can
be achieved by using one BBL™ CampyPak™ Plus disposable
gas generator envelope in a GasPak™ 100 jar, three envelopes
in a GasPak™ 150 jar or using the Bio-Bag™ Type Cfj or
124
Identity Specifications
Difco™ Candida BCG Agar Base
Dehydrated Appearance: Beige to blue-green, free-flowing, homoge-
neous.
Solution: 6.6% solution, soluble in purified water upon
boiling. Solution is blue-green to green-blue,
slightly opalescent to opalescent, may have a
precipitate.
Prepared Appearance: Blue-green to green-blue, slightly opalescent to
C
opalescent.
Reaction of 6.6%
Solution at 25°C: pH 6.1 ± 0.1
Cultural Response
Difco™ Candida BCG Agar Base
Prepare the medium per label directions. Inoculate and incubate at
30 ± 2°C for 24-72 hours.
INOCULUM COLOR OF
ORGANISM ATCC™ CFU RECOVERY MEDIUM
Candida albicans 10231 102-103 Good Yellow
Candida
Candida tropicalis 9968 10 -10
2 3
Good Yellow tropicalis
ATCC™ 3869
Escherichia coli 25922 103 Inhibition Green
medium retarded the growth of some species of Candida and 3. Autoclave at 121°C for 15 minutes.
completely inhibited the growth of others. To overcome this, 4. Add sterile neomycin (500 µg/mL) to the medium at
they replaced TTC with bromcresol green, a non-toxic indicator, 50-55°C. Mix well.
to develop Candida BCG Agar. Neomycin is incorporated to 5. Test samples of the finished product for performance using
inhibit gram-negative and some gram-positive bacteria. stable, typical control cultures.
125
Casamino Acids
Bacto™ Casamino Acids • Bacto™ Casamino Acids,
Technical • Casamino Acids, Vitamin Assay
Acidicase™ Peptone
Intended Use Bacto Casamino Acids, Technical is prepared according to the
Bacto Casamino Acids and Bacto Casamino Acids, Technical method suggested by Mueller1 for use in the preparation of
are used in preparing microbiological culture media. diphtheria toxin. Bacto Casamino Acids, Technical has been
used in a medium for primary isolation of gonococcus and
Casamino Acids, Vitamin Assay is used in vitamin assay
meningococcus, in agar-free media for the isolation of Neisseria,
procedures.
in a tellurite medium for the isolation of Corynebacterium and in
Acidicase Peptone is used as a nutritional supplement in the preparation of a medium for the testing of disinfectants.6-8
vitamin assay, susceptibility testing and other laboratory
Casamino Acids, Vitamin Assay is an acid digest of casein
media and microbial fermentation where the high salt content
specially treated to markedly reduce or eliminate certain vita-
will not interfere.
mins. It is recommended for use in microbiological assay media
and in studies of the growth requirements of microorganisms.
Summary and Explanation
Casamino Acids, Vitamin Assay is commonly used as the
Bacto Casamino Acids is an acid hydrolysate of casein, prepared
amino acid source in early phases of nutrition work.9 Casamino
according to the method described by Mueller and Miller.1
Acids, Vitamin Assay was used as the acid hydrolyzed casein
The method described reduces the sodium chloride and iron
in studies on p-aminobenzoic acid and p-teroylglutamic acid as
content of the hydrolyzed casein. This hydrolyzed casein,
growth factors for Lactobacillus species.10
supplemented with inorganic salts, growth factors, cystine,
maltose and an optimum amount of iron was used by Mueller Several media containing Casamino Acids are specified in
and Miller to prepare diphtheria toxin. Bacto Casamino Acids standard methods for multiple applications.11-16
duplicates this specially treated hydrolyzed casein. Acidicase Peptone is a hydrochloric acid hydrolysate of casein.
Bacto Casamino Acids, due to the nearly complete hydrolysis The manufacturing process produces a casein hydrolysate that
of casein and the low sodium chloride and iron content, makes has a high salt content of approximately 37% and nitrogen
an excellent supplement for many media formulations where content of approximately 8%. The hydrolysis of the casein, a
nitrogen requirements are minimal. This product has been milk protein rich in amino acid nitrogen, is carried out until all
recommended as a compromise for the replacement of pure the nitrogen is converted to amino acids or other compounds
amino acids in a defined medium for the growth of Lactobacillus, of relative simplicity. It is deficient in cystine, because casein
thus eliminating the complexity of preparation.2 Additionally, contains little cystine, and in tryptophan, which is destroyed
it has been successfully used, along with Tryptone Peptone, by the acid treatment.
in nutritional studies to determine a bacterium’s growth
requirement for peptides or amino acids.3,4 It also works well Principles of the Procedure
as a component in laboratory media. It has been utilized in Bacto Casamino Acids, Bacto Casamino Acids, Technical,
such diverse applications as TYI-S-33 media for the parasite Casamino Acids, Vitamin Assay and Acidicase Peptone are acid
Entamoeba histolytica and LCM medium for the growth of a hydrolyzed casein. Casein is milk protein and a rich source of
nematode-bacterium complex.5 amino acid nitrogen. Bacto Casamino Acids, Bacto Casamino
Acids, Technical, Casamino Acids, Vitamin Assay and Acidicase
Bacto Casamino Acids, Technical is an acid hydrolysate of
Peptone provide nitrogen, vitamins, carbon and amino acids
casein. The hydrolysis is carried out as in the preparation
in microbiological culture media. Although Bacto Casamino
of Bacto Casamino Acids, but the sodium chloride and iron
Acids, Bacto Casamino Acids, Technical, Casamino Acids,
content of this product have not been decreased to the same
Vitamin Assay and Acidicase Peptone are added to media
extent. Bacto Casamino Acids, Technical is recommended for
primarily because of their organic nitrogen and growth factor
use in culture media where amino acid mixtures are required
components, their inorganic components also play a vital role.17
for a nitrogen source and the sodium chloride content is slightly
increased. It is particularly valuable in studying the growth
requirements of bacteria.
126
C
Reaction of 2.0%
Solution at 25°C: pH 5.8-6.65 Salmonella enterica
subsp. enterica
Bacto Casamino Acids, Technical
™
serotype Typhi 19430 102-103 Good
Dehydrated Appearance: Very light beige, free-flowing, homogeneous.
Solution: 1.0% solution, soluble in purified water. Solution Difco™ Casamino Acids, Vitamin Assay
is colorless to very light amber, clear. Prepare various vitamin assay media using Casamino Acids, Vitamin Assay
to determine the vitamin content. It should not contain a vitamin content
Reaction of 1.0%
higher than 20% above the following values:
Solution at 25°C: pH 5.0-7.5
Vitamin B12 . ...................................................... 0.2 ng/g
Difco™ Casamino Acids, Vitamin Assay Biotin................................................................. 0.3 ng/g
Dehydrated Appearance: Light beige, free-flowing, homogeneous. Folic Acid........................................................... 3.3 ng/g
Solution: 3.0% solution, soluble in purified water upon Niacin................................................................. 0.17 µg/g
boiling. Solution is very light to light amber, clear, Pantothenate..................................................... 0.04 µg/g
may have a slight precipitate. Riboflavin........................................................... 0.1 µg/g
Thiamine............................................................ 0.1 µg/g
Reaction of 3.0%
Solution at 25°C: pH 6.5-8.5 BBL™ Acidicase™ Peptone
BBL Acidicase Peptone
™ ™ Prepare a sterile solution of 10.0 g of Acidicase Peptone, 2.5 g of sodium
chloride and 6.5 g of agar in 500 mL of purified water. Adjust final pH to
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
7.2-7.4. Inoculate and incubate plates at 35 ± 2°C for 2-3 days under ap-
Solution: 2.0% solution, soluble in purified water. Solution propriate atmospheric conditions.
is clear to slightly hazy.
ORGANISM ATCC™ INOCULUM CFU recovery
Reaction of 2.0%
Solution at 25°C: pH 6.5-7.5 Escherichia coli 25922 103-104 Good
Pseudomonas aeruginosa 27853 103-104 Good
Staphylococcus aureus 25923 10 -10
3 4
Good
Casein Agar
(See Nocardia Differentiation Media)
Casein Digest
Intended Use This product is digested under conditions different from other
Casein Digest is used in preparing microbiological culture enzymatic digests of casein, including Tryptone and Casitone.
media. Casein Digest is contained in the formulas of NZ media
(NZCYM Broth, NZYM Broth and NZM Broth), which are
Summary and Explanation used for cultivating recombinant strains of Escherichia coli.
Casein Digest, an enzymatic digest of casein similar to N-Z- E. coli grows rapidly in these rich media because they
Amine A, was developed for use in molecular genetics media. provide amino acids, nucleotide precursors, vitamins and
other metabolites that the cells would otherwise have to
User Quality Control synthesize.1 Consult appropriate references for recommended
Identity Specifications test procedures using NZ media.1,2
Difco™ Casein Digest
Dehydrated Appearance: Light beige, free-flowing, homogeneous. Principles of the Procedure
Solution: 1%, 2%, and 10% solutions, soluble in purified Casein Digest is a nitrogen and amino acid source for microbio-
water. Solutions are: 1%-Light to medium amber, logical culture media. Casein is raw milk protein, a rich source
clear; 2%-Medium amber, clear; 10%-Dark amber,
clear.
of amino acid nitrogen.
Reaction of 1%
Solution at 25°C: pH 7.0 ± 0.2 Procedure
See appropriate references for specific procedures using Casein
Cultural Response Digest.
Difco™ Casein Digest
Prepare NZM Broth per formula. Inoculate and incubate at 35 ± 2°C for Expected Results
18-72 hours.
Refer to appropriate references and procedures for results.
Organism ATCC™ Inoculum CFU RECOVERY
Bacillus subtilis 6633 102-103 Good References
Escherichia coli (HB101) 33694 102-103 Good 1. Ausubel, Brent, Kingston, Moore, Seidman, Smith and Struhl (ed.). 1994. Current protocols in molecular
biology, vol.1. Current Protocols, New York, N.Y.
Escherichia coli (JM107) 47014 10 -10
2 3
Good 2. Sambrook, Fritsch and Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.
Escherichia coli (DH5) 53868 102-103 Good
Saccharomyces cerevisiae* 9763 102-103 Good
Availability
Streptomyces avermitilis 31267 10 -10
2 3
Fair to good Difco™ Casein Digest
*Tested with addition of 0.5% dextrose.
Cat. No. 211610 Dehydrated – 500 g
128
of Analysis of AOAC International and meets specifications in applications. It has been used successfully with commonly used
the USP for pancreatic digest of casein.2,3 organisms, such as Escherichia coli,13 as well as uncommon
organisms, such as the diatom Nitzschia laevis.14
Bacto Tryptone was developed by Difco Laboratories while
investigating a peptone particularly suitable for the elabora- BiTek Tryptone is prepared similarly to Bacto Tryptone but
tion of indole by bacteria. It is also notable for the absence the final product goes through fewer refinement steps during
of detectable levels of carbohydrates. Bacto Tryptone has processing. This product provides some of the same benefits as
been used in conjunction with casamino acids in nutritional Bacto Tryptone in instances where a less refined hydrolysate
studies to determine amino acids vs. peptide utilization.4,5 It is can be utilized.
included in standard methods applications and is listed in the
reagent section of the USP as meeting the specifications for Principles of the Procedure
pancreatic digest of casein, a component in many of the media Bacto Casitone, Trypticase Peptone, Bacto Tryptone and BiTek C
listed.2,3,6-11 The European Pharmacopoeia also lists pancreatic Tryptone are pancreatic digests of casein. Casein is the main milk
digest of casein as a component in many of the recommended protein and a rich source of amino acid nitrogen.
media.12 Bacto Tryptone also works well in fermentation
Typical Analysis
Refer to Product Tables in the Reference Guide section of this
User Quality Control
NOTE: Differences in the Identity Specifications and Cultural Response testing manual.
for media offered as both Difco™ and BBL™ brands may reflect differences in
the development and testing of media for industrial and clinical applications, Directions for Preparation from
per the referenced publications.
Dehydrated Product
Identity Specifications Refer to the final concentration of Bacto Casitone, Trypticase Pep-
Bacto Casitone
™ tone, Bacto Tryptone and BiTek Tryptone in the formula of the
Dehydrated Appearance: Tan, free-flowing, granules. medium being prepared. Add appropriate product as required.
Solution: 1.0%, 2.0% and 10.0 % solutions, soluble in
purified water. 1.0% solution is light amber, Procedure
clear. 2.0% solution is light to medium amber,
clear, may have a slight precipitate. 10.0%
See appropriate references for specific procedures using Bacto
solution is medium to dark amber, clear to very Casitone, Trypticase Peptone, Bacto Tryptone and BiTek
slightly opalescent, may have a precipitate. Tryptone.
Reaction of 1.0%
Solution at 25°C: pH 6.8-7.4 Expected Results
Bacto Tryptone
™
Refer to appropriate references and procedures for results.
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 1.0%, 2.0% and 10.0% solutions, soluble in References
purified water. 1.0% solution is very light to light 1. Yannarell, Goldberg and Hjorth. 2001. J. Virol. Methods (in press).
amber, clear. 2.0% solution is light to medium 2. Horowitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
International, Gaithersburg, Md.
amber, clear. 10.0% solution is medium to dark 3. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The national
amber, clear to slightly opalescent,may have a formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc., Rockville, Md.
slight precipitate. 4. Takahashi and Yamada. 2000. J. Bacteriol. 182:4704.
5. Nagel, Oostra, Tramper and Rinzema. 1999. Process Biochem. 35: 69.
Reaction of 2.0% 6. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
Solution at 25°C: pH 6.5-7.5 4th ed. American Public Health Association, Washington, D.C.
7. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
BiTek Tryptone
™ tional, Gaithersburg, Md.
8. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
Dehydrated Appearance: Light beige, free-flowing, homogeneous. 21st ed., online. American Public Health Association, Washington D.C.
9. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy products, 17th ed.
Solution: 1.0%, 2.0% and 10.0% solutions, soluble in American Public Health Association, Washington D.C.
purified water. 1.0% solution is very light to light 10. U.S. Environmental Protection Agency. 2000. Improved enumeration methods for the recreational water
amber, clear. 2.0% solution is light to medium quality indicators: Enterococci and Escherichia coli. EPA-821/R-97/004. Office of Water, Washington,
D.C.
amber, clear. 10.0% solution is medium to dark 11. U.S. Department of Agriculture. 1998. Microbiology laboratory guidebook, 3rd ed. Food Safety and
amber, clear to slightly opalescent, may have a Inspection Service, USDA, Washington, D.C.
12. Council of Europe. 2008. European pharmacopoeia, 6th ed. Council of Europe, Strasbourg, France.
slight precipitate. 13. Sivakesavs, Chen, Hackett, Huang, Lam, Lam, Siu, Wong and Wong. 1999. Process Biochem.
Reaction of 2.0% 34:893.
14. Wen and Chen. 2001. Enzyme Microbia Technol. 29:341.
Solution at 25°C: pH 7.2 ± 0.2
BBL™ Trypticase™ Peptone
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution: 2.0% solution, soluble in purified water. Solution
is clear to slightly hazy.
Reaction of 2.0%
Solution at 25°C: pH 6.5-7.5
Continued
129
Cultural Response
Biochemical Reactions
Bacto™ Casitone, Bacto™ Tryptone or BiTek™ Tryptone
Prepare a sterile solution as directed below. Adjust final pH to 7.2-7.4. Inoculate and incubate at 35 ± 2°C for 18-48 hours.
TEST TEST SOLUTION ORGANISM ATCC™ INOCULUM CFU RESULT
Fermentable Carbohydrates 2% Escherichia coli 25922 ~107 Negative
Indole Production 0.1% Escherichia coli 29552 0.1 mL, undiluted Positive
Acetylmethylcarbinol Production 0.1% with 0.5% dextrose Enterobacter aerogenes 13048 0.1 mL, undiluted Positive
Hydrogen Sulfide Production 1% Salmonella enterica 14028 0.1 mL, undiluted Positive
subsp. enterica serotype Typhimurium
Growth Response
Bacto™ Casitone, Bacto™ Tryptone or BiTek™ Tryptone
Prepare a sterile solution with 2.0% Bacto Casitone, Bacto Tryptone or BiTek Tryptone, 0.5% sodium chloride and 1.5% agar. Adjust final pH to
7.2-7.4. Inoculate and incubate plates at 35 ± 2°C for 18-48 hours.
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Escherichia coli 25922 30-300 Good
Staphylococcus aureus 25923 30-300 Good
2. Prepare a sterile solution of chocolate peptone agar using Trypticase Peptone. Adjust final pH to 7.2-7.4. Inoculate and incubate plates at 35 ± 2°C
for 3 days with CO2.
ORGANISM ATCC™ INOCULUM CFU REcovery
Neisseria gonorrhoeae 19424 103-104 Good
130
132
Staphylococcus
aureus 25923 103- 2 × 103 Inhibition —
Pseudomonas
aeruginosa 9027 10-100 Growth N/A
Escherichia coli 8739 >100 No growth N/A
133
Availability Europe
Cat. No. 254419 Prepared Plates – Pkg. of 20*†
Difco™ Cetrimide Agar Base
Mexico (Cetrimide Agar)
AOAC BAM BS12 CCAM EP JP MCM9 USP
Cat. No. 252626 Prepared Plates (60 × 15 mm-style) –
Cat. No. 285420 Dehydrated – 500 g† Pkg. of 10*†
BBL™ Pseudosel™ Agar 257506 Prepared Plates – Pkg. of 10*†
AOAC BAM BS12 CCAM EP JP MCM9 USP Difco™ Glycerol
United States and Canada Cat. No. 228210 Bottle – 100 g
Cat. No. 297882 Prepared Plates – Pkg. of 10*† 228220 Bottle – 500 g
221344 Tubed Slants – Pkg. of 10 * Store at 2-8°C.
221345 Tubed Slants – Ctn. of 100 † QC testing performed according to USP/EP/JP performance specifications.
Cultural Response
Difco™ Chapman Stone Medium
Prepare the medium per label directions. Inoculate and incubate at 30 ± 2°C for 18-48 hours. Add bromcresol purple indicator to determine mannitol
fermentation (yellow = positive).
Organism ATCC™ INOCULUM CFU RECOVERY Halo (Gelatinase) Mannitol Fermentation
Escherichia coli 25922 102-103 Inhibition – –
Staphylococcus aureus 25923 102-103 Good + +
Staphylococcus epidermidis 12228 102-103 Good + –
134
Charcoal Agar
Intended Use The genus Bordetella consists primarily of four species:
Charcoal Agar is used for cultivating fastidious organisms, Bordetella pertussis, B. parapertussis, B. bronchiseptica and
especially Bordetella pertussis, for vaccine production and stock B. avium; additional species have recently been described.2 All
culture maintenance. Bordetella are respiratory pathogens, residing on the mucous
membranes of the respiratory tract. B. pertussis is the major
Summary and Explanation cause of whooping cough or pertussis. B. parapertussis is
Charcoal Agar is prepared according to the method of Mishulow, associated with a milder form of the disease.3 B. bronchisep-
Sharpe and Cohen.1 The authors found this medium to be an tica is an opportunistic human pathogen associated with both
efficient substitute for Bordet-Gengou Agar in the production respiratory and non-respiratory infections, often occurring in
patients having close contact with animals.2 B. bronchiseptica
of B. pertussis vaccines.
has not been reported to cause pertussis. There have been no
reports of recovery of B. avium from humans.2
Identity Specifications
Difco™ Charcoal Agar
Dehydrated Appearance: Gray, free-flowing, homogeneous.
Solution: 6.25% solution, soluble in purified water upon boil-
ing. Solution is black, opaque with a precipitate.
Prepared Appearance: Black, opaque.
Reaction of 6.25%
Solution at 25°C: pH 7.3 ± 0.2
Cultural Response
Difco™ Charcoal Agar
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C under 5-10% CO2 for 18-72 hours.
Organism ATCC™ INOCULUM CFU RECOVERY
Bordetella bronchiseptica 4617 102-103 Good
Bordetella parapertussis 15237 102-103 Good
Bordetella pertussis 8467 10 -10
2 3
Good
135
Charcoal Agar supplemented with Horse Blood is used for the 4. Mix thoroughly during dispensing to uniformly distribute
cultivation and isolation of Haemophilus influenzae.4 the charcoal.
5. Test samples of the finished product for performance using
Principles of the Procedure stable, typical control cultures.
Infusion from beef heart and peptone provide the nitrogen,
carbon and amino acids in Charcoal Agar. Yeast extract is a Procedure
vitamin source. Sodium chloride maintains osmotic balance. For a complete discussion on the isolation and maintenance of
Agar is the solidifying agent. Soluble starch and Norit SG, fastidious microorganisms refer to the procedures described in
charcoal, neutralize substances toxic to Bordetella species, such appropriate references.2,4,5
as fatty acids.
Expected Results
Formula
Refer to appropriate references and procedures for results.
Difco™ Charcoal Agar
Approximate Formula* Per Liter
Beef Heart, Infusion from 500 g................................. 12.0 g
Limitation of the Procedure
Peptone..................................................................... 10.0 g Charcoal has a tendency to settle out of the medium. Swirl
Sodium Chloride.......................................................... 5.0 g the flask gently when dispensing to obtain a uniform charcoal
Soluble Starch............................................................ 10.0 g
Yeast Extract................................................................ 3.5 g
suspension.4
Norit SG....................................................................... 4.0 g
Agar.......................................................................... 18.0 g References
*Adjusted and/or supplemented as required to meet performance criteria. 1. Mishulow, Sharpe and Cohen. 1953. Am. J. Public Health, 43:1466.
2. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
Directions for Preparation from 3. Linneman and Pery. 1977. Am. J. Dis. Child. 131:560.
4. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol
Dehydrated Product 1. Williams & Wilkins, Baltimore, Md.
5. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
1. Suspend 62.5 g of the powder in 1 L of purified water. Mix American Society for Microbiology, Washington, D.C.
thoroughly.
2. Heat with frequent agitation and boil for 1 minute to Availability
completely dissolve the powder. Difco™ Charcoal Agar
3. Autoclave at 121°C for 15 minutes. Cat. No. 289410 Dehydrated – 500 g
137
BBL™ Chocolate II Agar with Pyridoxal BBL™ Chocolate II Agar//Trypticase™ Soy Agar
Cat. No. 297259 Prepared Plates – Ctn. of 100* with 5% Sheep Blood (TSA II)
United States and Canada
BBL™ Chocolate II Agar//Martin-Lewis Agar Cat. No. 221302 Prepared I Plate™ Dishes – Pkg. of 20*
Cat. No. 297060 Prepared Bi-Plate Dishes – Pkg. of 20* 221303 Prepared I Plate™ Dishes – Ctn. of 100*
297245 Prepared Bi-Plate Dishes – Ctn. of 100*
Europe
BBL™ Chocolate II Agar//Modified Martin-Lewis Agar Cat. No. 251302 Prepared I Plate™ Dishes – Pkg. of 20*
Cat. No. 298513 Prepared Bi-Plate Dishes – Pkg. of 20* 251303 Prepared I Plate™ Dishes – Ctn. of 100*
298206 Prepared Bi-Plate Dishes – Ctn. of 100* BBL™ Chocolate II Agar//Trypticase™ Soy Agar
BBL Chocolate II Agar//Modified Thayer-Martin
™ with 5% Sheep Blood (TSA II)//MacConkey II Agar
(MTM II) Agar Cat. No. 299580 Prepared Y Plate™ Dishes – Ctn. of 100*
*Store at 2-8°C.
BS12 CMPH2 MCM9
Cat. No. 221623 Prepared Bi-Plate Dishes – Pkg. of 20*
138
CHROMagar™ Candida
Intended Use Principles of the Procedure
BBL™ CHROMagar™ Candida* medium is for the isolation Specially selected peptones supply the nutrients in BBL™
and differentiation of Candida albicans, C. tropicalis and CHROMagar™ Candida medium. The chromogen mix consists
C. krusei.1 Due to the differences in morphology and colors of of artificial substrates (chromogens), which release differently
the yeast colonies, this medium facilitates the detection of mixed colored compounds upon degradation by specific enzymes.
yeast cultures in specimens.2,3 It may also be used as a selective This permits the differentiation of certain species, or the detec-
isolation medium for other yeasts and for filamentous fungi tion of certain groups of organisms, with only a minimum of
instead of Sabouraud Dextrose Agar or similar media. confirmatory tests. Chloramphenicol inhibits most bacterial
*U.S. Patent Nos. 5,716,799 and 5,962,251 contaminants.
Availability
BBL™ CHROMagar™ Candida
United States and Canada
Cat. No. 254093 Prepared Plates – Pkg. of 20*
Europe
Cat. No. 254106 Prepared Plates – Ctn. of 120*
Japan
Cat. No. 251594 Prepared Plates – Pkg. of 20*
251367 Prepared Plates – Ctn. of 100*
Mexico
Candida
Cat. No. 252630 Prepared Plates – Pkg. of 10* albicans
*Store at 2-8°C. ATCC™ 10231
CHROMagar™ Listeria
Intended Use AOAC and ISO methods1-4 with no confirmatory biochemical
BBL CHROMagar Listeria* is a selective medium for the
™ ™ tests required for the identification of Listeria monocytogenes/
isolation, differentiation and identification of Listeria monocyto- L. ivanovii.
genes and L. ivanovii from food and environmental samples. Confirmatory testing of isolates from food matrices other than
BBL CHROMagar Listeria has been validated by the AOAC ™ those that have been validated, and from environmental samples,
Research Institute under the Performance Tested MethodsSM is recommended.
Program for the analysis of raw ground beef, smoked salmon, *U.S. Patent Pending
141
Listeria monocytogenes
ATCC™ 19114
enrichments and selective enrichments. Of the 265 food samples
tested, 140 were tested using BAM, USDA, or AOAC methods
and 125 were tested using ISO methods. BBL CHROMagar
Listeria produced a sensitivity of 99.3% and a specificity
of 100% as compared to the reference methods for all food
matrices. No false negatives were found in testing the food
matrices. No statistical difference was found in recovery using
the CHROMagar Listeria method compared to the reference
plated media based on Chi square analysis. Furthermore, in the
testing of raw ground beef and smoked salmon using ISO method
11290, time to recovery was shorter for BBL CHROMagar
Listeria compared to ALOA.9 Specifically, BBL CHROMagar
Listeria recovered 27 positive samples from the primary (Half
Fraser) and secondary (Fraser) broths after 24 hours of incuba-
tion compared to 3 positive samples detected on ALOA after 24
hours.9 Finally, known isolates were evaluated and CHROMagar
Listeria had a sensitivity and specificity of 100%. The results
of these studies demonstrate that BBL CHROMagar Listeria is
an effective medium for the recovery and detection of Listeria
monocytogenes in raw ground beef, smoked salmon, lettuce
and Brie cheese using FDA/BAM, USDA/FSIS, AOAC and ISO
Summary and Explanation methods.
Listeriosis is a foodborne illness caused by L. monocytogenes. In a separate study, BBL CHROMagar Listeria was compared
It is of particular concern for immunocompromised patients: to AOAC3 and Health Canada10 reference methods using 50
cancer, HIV, pregnant women, neonates and the elderly. Because natural and 150 spiked food samples comprising 50 different
of the severity of the disease, 20 deaths per 100 cases, listeriosis food types, including vegetables, milk, milk products, meat,
is a serious public health and agrifood industry concern. Illness seafood, poultry, ready-to-eat meats and mushrooms.11 Overall,
caused by L. monocytogenes has been associated with deli meats, the sensitivity and specificity for BBL CHROMagar Listeria
poultry, soft cheeses, ready-to-eat seafood, smoked fish, hot medium were 99% and 100%, respectively.11 An additional
dogs, salad greens and inadequately or unpasteurized milk.5,6 study was performed comparing BBL CHROMagar Listeria
L. ivanovii, rarely found in foods, is pathogenic to animals and to the USDA/FSIS Listeria isolation method using 63 poultry
some cases of human listeriosis have been associated with this and environmental samples. BBL CHROMagar Listeria pro-
organism.7 duced 100% (11/11) sensitivity and 100% (52/52) specificity.
BBL CHROMagar Listeria is intended for the isolation, differ- Modified Oxford medium was 100% sensitive; however, it
entiation and identification of L. monocytogenes and L. ivanovii lacked the ability to differentiate L. monocytogenes from other
based on the formation of blue-green colonies surrounded by Listeria species.11
an opaque, white halo. The addition of a chromogenic and
a phospholipid substrate in the medium facilitates the detec- Principles of the Procedure
tion and differentiation of L. monocytogenes and L. ivanovii BBL CHROMagar Listeria was originally developed by A.
from other Listeria species and organisms. An advantage BBL Rambach, CHROMagar, Paris, France. BD, under a licensing
CHROMagar Listeria has over recommended traditional media, agreement, has optimized this formulation utilizing proprietary
such as Modified Oxford and Oxford, is the ability to distinguish intellectual property used in the manufacturing of the BBL
L. monocytogenes and L. ivanovii from other Listeria species. CHROMagar Listeria prepared plated medium.
This facilitates the detection of L. monocytogenes/L. ivanovii in Specially selected Difco™ peptones supply nutrients. The ad-
the presence of other Listeria species and other bacterial flora dition of selective agents inhibits the growth of gram-negative
that may be present in a sample, thereby minimizing the risk of organisms, yeast and fungi. The chromogen is a chromogenic
not detecting L. monocytogenes or L. ivanovii. substrate that produces a blue-green colored compound when
BBL CHROMagar Listeria has been validated by the AOAC hydrolyzed by an enzyme specific to Listeria species. A specific
Research Institute under the Performance Tested Methods enzyme found in L. monocytogenes and L. ivanovii acts
Program.8 It was evaluated for the detection of Listeria mono- upon the phospholipid substrate in BBL CHROMagar Listeria
cytogenes in raw ground beef, smoked salmon, lettuce and Brie producing an opaque, white halo around the blue-green colonies.
cheese. The recovery of L. monocytogenes on CHROMagar The growth of a blue-green colony with well-defined edges
Listeria was compared to the FDA/BAM, USDA/FSIS, AOAC surrounded by an opaque, white halo is presumptive for
and ISO reference plated media using the recommended pre- L. monocytogenes or L. ivanovii on BBL CHROMagar Listeria.
142
Sample Collection and Handling RI approved Listeria identification biochemical test kits
Follow appropriate standard methods for details on sample are necessary for differentiation of L. monocytogenes and
collection and preparation according to sample type and geo- L. ivanovii.
graphic location.1-4 2. Incubation in CO2 may adversely affect the recovery of
Listeria species.
Procedure References
Consult appropriate references and follow applicable standard 1. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
methods. Inoculate the incubated enrichment broth sample tional, Gaithersburg, Md.
2. U.S. Department of Agriculture, Food Safety and Inspection Services, Office of Public Health and
onto a BBL CHROMagar Listeria plate and streak for isola- Science. 2008. Isolation and identification of Listeria monocytogenes from red meat, poultry, egg and
environmental samples. In Microbiology laboratory guidebook, Method 8.06, online. <http://www.
tion. Incubate plates aerobically at 35 ± 2°C for 24 hours. Do fsis.usda.gov/Science/Microbiological_Lab_Guidebook/index.asp>.
C
3. Horwitz (ed.). 2002. Listeria monocytogenes in milk and dairy products. In Official Methods of
not incubate in CO2. If negative, reincubate for an additional Analysis, AOAC International, 17th ed, AOAC International, Gaithersburg, Md.
24 hours to report final results. 4. International Organization for Standardization (ISO). 2004. Microbiology of food and animal feeding
stuffs-horizontal method for the detection of Listeria monocytogenes, ISO 11290-1. International
Organization for Standardization, Geneva, Switzerland.
5. Doyle and Beuchat. 2007. Food microbiology fundamentals and frontiers, 3rd ed. American Society
Expected Results for Microbiology, Washington, D.C.
6. Gombas, Chen, Clavero and Scott. 2003. J. Food Prot. 66: 559.
After proper incubation, read plates against a white background. 7. Reissbrodt. 2004. Int. J. Food Microbiol. 95:1.
8. AOAC Research Institute News. 2005. Inside laboratory management (September/October 2005),
L. monocytogenes/L. ivanovii produce blue-green colonies with AOAC International, Gaithersburg, Md.
opaque, white haloes on BBL CHROMagar Listeria. Listeria 9. Ottaviani, Ottaviani and Agosti. Quimper Froid Symposium Proceedings, P6 ADRIA Quimper, 16-18
June 1997.
species produce blue-green colonies without haloes. Gram- 10. Health Canada. 2001/2002. The compendium of analytical methods, Health Products and Food
Branch, Method MFHPB-30 and Supplement to the Method MFHPB-30.
negative organisms are inhibited. Gram-positive organisms, 11. Hegdea, Leon-Velardea, Stamb, Jaykusb and Odumerua. 2007. J. Micro. Methods. 68:82.
other than Listeria species, will either be inhibited or produce
white colonies. Availability
BBL™ CHROMagar™ Listeria
Limitations of the Procedure BAM
1. BBL CHROMagar Listeria cannot differentiate L. mono- United States and Canada
cytogenes from L. ivanovii based on colony color or halo Cat. No. 215085 Prepared Plates - Pkg. of 20*
formation. Supplemental tests, such as hemolysis, xylose, Mexico
Cat. No. 252743 Prepared Plates - Pkg. of 10*
rhamnose and CAMP or commercially available AOAC-
*Store at 2-8°C.
CHROMagar™ MRSA
Intended Use BBL CHROMagar MRSA is a selective and differential medium,
BBL™ CHROMagar™ MRSA* is a selective and differential which incorporates cefoxitin, for the detection of MRSA from
medium for the qualitative direct detection of nasal coloniza- anterior nares specimens.
tion by methicillin-resistant Staphylococcus aureus (MRSA) BBL CHROMagar MRSA was developed by A. Rambach and
to aid in the prevention and control of MRSA infections in BD. This product utilizes CHROMagar Staph aureus, which was
healthcare settings. The test is performed on anterior nares swab developed by A. Rambach and is sold by BD under a licensing
specimens from patients and healthcare workers to screen for agreement with CHROMagar, Paris, France.
MRSA colonization. BBL CHROMagar MRSA is not intended
to diagnose MRSA infection nor to guide or monitor treatment Principles of the Procedure
for infections. BBL CHROMagar MRSA medium permits the direct detection
*Patent Pending
and identification of MRSA through the incorporation of spe-
cific chromogenic substrates and cefoxitin. MRSA strains will
Summary and Explanation grow in the presence of cefoxitin3 and produce mauve-colored
MRSA is a major cause of nosocomial and life threatening
colonies resulting from hydrolysis of the chromogenic substrate.
infections. Infections with MRSA have been associated with a
Additional selective agents are incorporated for the suppression
significantly higher morbidity, mortality and costs than methicil-
of gram-negative organisms, yeast and some gram-positive
lin-susceptible S. aureus (MSSA).1 Selection of these organisms
cocci. Bacteria other than MRSA may utilize other chromogenic
has been greatest in the healthcare setting; however, MRSA has
substrates in the medium resulting in blue to blue/green colored
also become more prevalent in the community.2
colonies or if no chromogenic substrates are utilized, the colonies
To control the transmission of MRSA, the Society for Healthcare appear as white or colorless.
Epidemiology of America (SHEA) has recommended guidelines,
which include an active surveillance program to identify potential
reservoirs and a rigorous infection control program to control
the spread of MRSA.1
143
Staphylococcus aureus
ATCC™ 43300 exposure may result in reduced recovery and/or coloration
of isolates. Keep plates within the original sleeve wrapping
and cardboard box for the entire storage period.
2. At 48 hours occasional strains of coagulase-negative staphy-
lococci (such as, S. epidermidis, S. cohnii, S. intermedius,
S. haemolyticus, S. capitis, S. hominis and S. schleiferi),
Acinetobacter sp., Corynebacterium and yeast may produce
mauve-colored colonies requiring a confirmatory coagulase
test for confirmation of MRSA. This may also occur at a
much lower rate at 24 hours. In clinical studies, approxi-
mately 5% (6/120) of the mauve-colored colonies detected
at 24 hours were coagulase-negative staphylococci and/or
corynebacteria on the BBL CHROMagar MRSA medium.
If desired, a coagulase test may be performed at 24 hours on
mauve-colored colonies to increase specificity.
3. Surveillance testing determines the colonization status at a
given time and could vary depending on patient treatment
(e.g. decolonization regime), patient status (e.g. not actively
shedding MRSA) or exposure to high risk environments
(e.g. contact with MRSA carrier, prolonged hospitalization).
Monitoring colonization status should be done according to
Procedure
hospital policies.
As soon as possible after receipt in the laboratory, inoculate
4. Results from CHROMagar MRSA should be used as an
the specimen onto a BBL CHROMagar MRSA plate and
adjunct to nosocomial infection control efforts to identify
streak for isolation. Incubate plates aerobically at 35–37°C for
patients needing enhanced precautions. The test is not in-
24 ± 4 hours in an inverted position. If no mauve colonies are
tended to identify patients with staphylococcal infection.
recovered, reincubate for an additional 24 ± 4 hours. Do not
Results should not be used to guide or monitor treatment
incubate in an atmosphere supplemented with carbon dioxide.
for MRSA infections. This device can be used to identify
Avoid exposure to light during incubation as light may result
patients for isolation or removal from isolation to control
in reduced recovery and/or coloration of isolates. Exposure to
nosocomial transmission of MRSA.
light is permissible after colony color develops.
5. A CHROMagar MRSA negative result following a previous
positive test result may indicate treatment eradication success
Expected Results
or may occur due to intermittent shedding.
Read plates against a white background. Colonies of MRSA will
6. mecA-negative S. aureus may grow if the oxacillin or cefoxitin
appear mauve on the BBL CHROMagar MRSA medium. Other
MICs are at or near the resistant breakpoint.
organisms (non-MRSA) will be inhibited or produce colorless,
7. Incubation in 5% CO2 is not recommended and may result
white, blue or blue/green colonies. Refer to the table below for
in false negative cultures.
interpretation of results.
8. Use of phenylephrine hydrochloride, a component of some
Limitations of the Procedure nasal sprays, at a concentration of ≥10% shows an inhibi-
1. Minimize exposure (<4 hours) of BBL CHROMagar MRSA tory effect on organism growth that is unrelated to medium
to light both before and during incubation, as prolonged performance.
144
C
3. Clinical and Laboratory Standards Institute. 2008. Performance standards for antimicrobial susceptibil-
ity testing; 18th Informational Supplement, M100-S18. CLSI, Wayne, Pa.
CHROMagar™ MRSA II
Intended Use Availability
BBL™ CHROMagar™ MRSA II is a selective and differential BBL™ CHROMagar™ MRSA II
medium for the direct detection of methicillin-resistant Staphy- Europe only
lococcus aureus (MRSA) from clinical specimens. The test can be Cat. No. 257434 Prepared Plates – Pkg. of 20*
257435 Prepared Plates – Ctn. of 120*
performed on nares and alternative body sites. For more product *Store at 2-8°C.
information, contact your local BD office (Europe only).
CHROMagar™ O157
Intended Use and has been validated by the AOAC™ Research Institute under
BBL CHROMagar O157* is a selective medium for the isola-
™ ™ the Performance Tested MethodsSM Program for the analysis of
tion, differentiation and presumptive identification of Escherichia raw ground beef and unpasteurized apple cider.
coli O157:H7 from human clinical stool specimens. *U.S. Patent No. 6,165,743.
BBL™ CHROMagar™ O157 is a selective medium for the isola- Summary and Explanation
tion, differentiation and presumptive identification of Escherichia E. coli O157:H7 is the most frequently isolated pathogen from
coli O157:H7 from food, veterinary and environmental sources bloody stools.1-3 However, absence of bloody diarrhea does not
Escherichia coli O157 Enterobacter rule out the presence of E. coli O157:H7.4 This serotype causes
ATCC™ 43895 cloacae (blue) a broad range of illness from mild non-bloody diarrhea to severe
ATCC™ 13047
bloody diarrhea (hemolytic colitis), hemolytic uremic syndrome
and death.1-3 The isolation of E. coli O157:H7 exceeds that of
some other common enteric pathogens, especially Shigella in
many areas and age groups. Transmission most often occurs
through ingestion of raw or undercooked beef; other foods have
also been implicated.1,2 In addition, transmission may occur
person to person, as well as from recreational water sources.1,2
CHROMagar O157 is intended for the isolation, differentia-
tion and presumptive identification of E. coli O157:H7. Due to
the chromogenic substrates in the medium, colonies of E. coli
O157:H7 produce a mauve color, thus allowing presumptive
identification from the primary isolation plate and differentia-
tion from other organisms. In samples with low numbers of
E. coli O157:H7, enrichment methods may be helpful prior to
inoculating medium.
BBL CHROMagar O157 has been validated by the AOAC-
Escherichia coli Research Institute under the Performance Tested Methods Pro-
O157 (mauve) gram.5 BBL CHROMagar O157 was evaluated for the detection
ATCC™ 43895
145
of E. coli O157:H7 in raw ground beef and unpasteurized apple streak for isolation. If the specimen is cultured from a swab,
cider using seeded samples. The recovery of E. coli O157:H7 roll the swab over a small area of the surface at the edge, then
on BBL CHROMagar O157 was compared to the FDA/BAM, streak from this area with a loop. Incubate plates aerobically
USDA/FSIS and ISO reference plated media. The reference at 35 ± 2°C for 18-24 hours in an inverted position (agar-side
recommended enrichment and screening procedures were fol- up). Plates are not to be incubated beyond the 24-hour time
lowed for the reference media and BBL CHROMagar O157. period prior to reading. Interpretation of plate results must be
Immunomagnetic separation (IMS) was performed according to completed within 18-24 hours after inoculation of the BBL
the USDA and ISO methods. Of the 180 food samples tested, CHROMagar O157 plate.
45 were tested using FDA BAM and USDA FSIS methods, and
For food samples, consult appropriate references6-8 and follow
90 were tested using ISO methods. BBL CHROMagar O157
applicable standard methods. Inoculate incubated enrichment
produced a sensitivity of 100% and a specificity of 100% as com-
broth or screened food sample particle onto BBL CHROMagar
pared to the reference methods for both food matrices. No false
O157 and streak for isolation. Incubate plates aerobically at
negatives were found in testing the food matrices. No statistical
35 ± 2°C for 18-24 hours in an inverted position (agar-side up).
difference was found in recovery using the BBL CHROMagar
O157 method compared to the reference plated media based Expected Results
on Chi-square analysis. Known isolates, including 54 strains
After proper incubation, read plates against a white background.
of E. coli O157:H7 (three of which were non-motile strains)
Interpretation of plate results must be completed within 18-24
and 32 non-E. coli O157:H7 strains, were evaluated on BBL
hours after inoculation of the BBL CHROMagar O157 plate.
CHROMagar O157 with a sensitivity and specificity of 100%.
E. coli O157:H7 will produce mauve-colored colonies on BBL
The results of these studies demonstrate that BBL CHROMagar
CHROMagar O157 medium. All mauve colonies should be
O157 is an effective medium for the recovery and detection of
confirmed biochemically and/or serologically prior to report-
E. coli O157:H7 in raw ground beef and unpasteurized apple
ing as E. coli O157:H7.3,6-8 Gram-positive organisms should
cider using FDA BAM, USDA FSIS and ISO methods. be completely inhibited. Gram-negative organisms, other than
E. coli O157:H7, will either be inhibited or produce colorless,
Principles of the Procedure blue, green, blue-green (aqua) or natural color colonies.
CHROMagar O157 was originally developed by A. Rambach,
CHROMagar, Paris, France. BD, under a licensing agreement, Limitations of the Procedure
has optimized this formulation utilizing proprietary intellectual 1. BBL CHROMagar O157 does not detect enterohemorrhagic
property used in the manufacturing of BBL CHROMagar O157
or enteropathogenic serotypes of E. coli other than O157:
prepared plated medium.
H7, since they may differ biochemically. β-glucuronidase-
Specially selected Difco™ peptones supply the nutrients. The positive strains of E. coli O157:H7 will not be detected on
addition of potassium tellurite, cefixime and cefsulodin reduces BBL CHROMagar O157; however, such strains are rare.
the number of bacteria other than E. coli O157:H7 that grow on 2. BBL CHROMagar O157 does not differentiate between
this medium. The chromogen mix consists of artificial substrates toxin-producing and non-toxin-producing strains of E. coli
(chromogens), which release an insoluble colored compound O157:H7.
when hydrolyzed by a specific enzyme. E. coli O157:H7 utilizes 3. Organisms other than E. coli O157:H7, such as Proteus spp.
one of the chromogenic substrates producing mauve colonies. may grow on this medium; however, they generally produce
The growth of mauve colonies is considered presumptive for a different color. If unisolated mauve colonies are observed,
E. coli O157:H7 on BBL CHROMagar O157. Non-E. coli isolation can be achieved by subculturing to another BBL
O157:H7 bacteria may utilize other chromogenic substrates CHROMagar O157 plate. Rare strains of E. coli (biochemi-
resulting in blue to blue-green colored colonies or, if none of the cally similar to Shigella) have been found that produce false
chromogenic substrates are utilized, colonies may appear as their positive results on BBL CHROMagar O157.
natural color. This facilitates the detection and differentiation 4. Confirmatory tests are necessary for definitive identifica-
of E. coli O157:H7 from other organisms. tion.3,6-8
5. Incubation at lower than recommended temperatures may
Sample Collection and Handling delay detection of positive reactions. If the incubation tem-
For clinical specimens, refer to lab procedures for details on perature is below 35 ± 2°C, the plates should be incubated
specimen collection and handling procedures. a full 24 hours before reporting as negative.9
6. Plates are not to be incubated beyond the 24-hour time period
For agrifood or other industrial samples, follow appropriate
prior to reading.
standard methods for details on sample preparation and process-
7. For clinical specimens, internal cross reactivity testing has
ing according to sample type and geographic location.
demonstrated that Salmonella serotype Heidelberg exhib-
ited mauve colonies when plated on BBL CHROMagar
Procedure O157 medium. As recommended, all mauve colonies should
For clinical specimens, as soon as possible after receipt in the
be confirmed by biochemical or serological testing prior to
laboratory, inoculate onto a BBL CHROMagar O157 plate and
reporting results.
146
References Availability
1. Moe. 2002. Waterborne transmission of Infectious agents. In Hurst, Crawford, Knudsen, McInerney,
and Stetzenbach (eds.), Manual of environmental microbiology, 2nd ed. American Society for Micro-
BBL™ CHROMagar™ O157
biology, Washington, DC. CCAM
2. Doyle, Zhao, Meng and Zhao. 1997. In Doyle, Beuchat and Montville (eds.), Food microbiology
fundamentals and frontiers. American Society for Microbiology, Washington, DC. United States and Canada
3. Nataro, Bopp, Fields, Kaper, and Strockbine. 2007. Escherichia, Shigella and Salmonella. In Murray, Cat. No. 214984 Prepared Plates - Pkg. of 20*
Baron, Jorgensen, Landry, and Pfaller (eds.), Manual of clinical microbiology, 9th ed. American Society
for Microbiology, Washington, DC. Europe
4. Centers for Disease Control and Prevention. 2001. Diagnosis and management of foodborne illness, Cat. No. 254105 Prepared Plates - Pkg. of 20*
MMWR Jan 26, 2001/50 (RR02):1.
5. AOAC Research Institute News. 2006. Inside laboratory management (January/February 2006), Japan
AOAC International, Gaithersburg, Md.
6. U.S. Food and Drug Administration. 2002. Bacteriological analytical manual, online. Chapter 4A: Cat. No. 251361 Prepared Plates – Pkg. of 20*
Diarrheagenic Escherichia coli. AOAC International, Gaithersburg, Md. 251362 Prepared Plates – Ctn. of 100*
7. U.S. Department of Agriculture. 2008. Detection, isolation and identification of Escherichia coli O157:
H7 and O157:NM (nonmotile) from meat products. In Microbiology laboratory guidebook MLG Mexico
5.04.
8. International Organization for Standards. 2001. Microbiological methods, ISO 16654: Microbiology
of food and animal feeding stuffs - horizontal method for the detection of Escherichia coli O157, First
Cat. No. 252717 Prepared Plates - Pkg. of 10*
*Store at 2-8°C.
C
Edition, 2001-05-01.
9. Data on file, BD Diagnostics.
CHROMagar™ Orientation
Intended Use Due to the different antimicrobial susceptibility patterns of
BBL CHROMagar Orientation* medium is a nonselective
™ ™ the microorganisms involved, identification to the species
medium for the isolation, differentiation and enumeration of level is necessary for effective antimicrobial therapy. The most
urinary tract pathogens. BBL CHROMagar Orientation medium frequently isolated species or organism groups produce charac-
allows for the differentiation and identification of Escherichia teristic enzymes. Thus, it is possible to identify these organisms to
coli and Enterococcus without confirmatory testing. the species level with a limited number of substrate fermentation
*U.S. Patent Nos. 5,716,799 and 5,962,251 or utilization tests.1
Some of the organisms encountered in UTIs produce enzymes
Summary and Explanation
either for the metabolism of lactose or glucosides or both. Other
Escherichia coli, enterococci, the Klebsiella-Enterobacter-
organisms produce none of these enzymes. For example, E. coli
Serratia (KES) and the Proteus-Morganella-Providencia (PMP)
contains enzymes for lactose metabolism but is β-glucosidase
groups are frequently encountered organisms in urinary tract
negative. Some members of the family Enterobacteriaceae are
infections (UTI). Most UTIs are caused by E. coli alone, or in
β-glucosidase positive but do not contain enzymes necessary
combination with enterococci. Staphylococcus saprophyticus
for lactose fermentation; others may contain both types of
and Streptococcus agalactiae may be isolated from females,
enzymes or none of them. β‑glucosidases are also found in
although less frequently.
gram-positive cocci, such as S. agalactiae and enterococci.
Mixed Bacterial Colonies
Escherichia coli (rose) Enterococcus faecalis Escherichia coli Enterococcus feacalis
ATCC™ 25922 (blue-green) ATCC™ 25922 ATCC™ 29212
ATCC™ 29212
Proteus
mirabilis (beige)
ATCC™ 43071
147
Tryptophan deaminase (TDA) is an enzyme characteristically is possible by means of colony morphology, pigmentation and
found in the Proteus-Morganella-Providencia group. medium discoloration.
BBL™ CHROMagar™ Orientation medium was developed by
A. Rambach and is sold by BD under a licensing agreement with
Limitations of the Procedure
1. As this medium is nonselective, other UTI pathogens will
CHROMagar, Paris, France.
grow. Colonies that show their natural color and do not
react with the chromogenic substrates must be further
Principles of the Procedure
differentiated with appropriate biochemical or serological
Specially selected peptones supply the nutrients in BBL™
tests to confirm identification.
CHROMagar™ Orientation medium. The chromogen mix
2. E. coli colonies that are dark rose to pink but are pinpoint
consists of artificial substrates (chromogens) which release
to small in size, require additional confirmatory tests such
differently colored compounds upon degradation by specific
as spot indole (DMACA indole reagent).
microbial enzymes, thus assuring the differentiation of certain
3. Gram-negative organisms other than those belonging to
species or the detection of certain groups of organisms, with only
the KES group may produce large blue colonies and thus
a minimum of confirmatory tests. Proteus swarming is partially
require other biochemical tests for identification.
to completely inhibited.
4. In very rare cases, Listeria monocytogenes or other Listeria
species may be present in urine (e.g., after abortion due
Procedure
to these agents). Listeria will produce blue to blue-green
A dilution of the specimen on the plate (by using calibrated
colonies that are PYR-negative, mimicking Streptococcus
loops or other techniques commonly used for plating urine
agalactiae. Therefore, it may be useful to perform a Gram
specimens) is required to obtain isolated colonies with
stain of organisms producing small, blue to blue-green
typical colors and morphology. Incubate plates aerobically at
colonies on this medium that are PYR negative. The
35 ± 2°C for not less than 20-24 hours in an inverted position
presence of gram-positive bacilli may be indicative of
(agar-side up). Do not incubate in an atmosphere supplemented
Listeria species, but additional biochemical tests are
with carbon dioxide. Avoid exposure to light during incubation
necessary to confirm their identification.
as light may destroy the chromogens. Once the colony color
5. Very rarely, isolates of Aeromonas hydrophila may produce
develops, exposure to light is permissible.
rose colonies. They may be differentiated from E. coli with
the oxidase test (Aeromonas is positive; E. coli is negative).
Expected Results 6. This medium will not support the growth of fastidious
After incubation, the plates should show isolated colonies in the
organisms, such as Neisseria spp., Haemophilus spp. or
areas where the inoculum was diluted appropriately.
Mycoplasma spp.
Typical colony appearance on BBL™ CHROMagar™ Orientation 7. Use of this medium for nonclinical or clinical specimens other
medium is as follows: than urine has not been documented.
E. coli............................................ Dark rose to pink, transparent 8. Minimize exposure to light before and during incubation, as
colonies, with or without light may destroy the chromogens. Keep plates within the
halos in the surrounding original sleeve wrapping and cardboard box for the entire
medium
KES group..................................... Medium-blue to dark blue storage period.
colonies
PMP group.................................... Pale to beige colonies References
surrounded by brown halos* 1. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
Enterococcus................................ Blue-green small colonies American Society for Microbiology, Washington, DC.
2. Merlino, Siarakas, Robertson, Funnell, Gottlieb and Bradbury. 1996. J. Clin. Microbiol. 34: 1788.
S. agalactiae................................. Blue-green to light blue, 3. Hengstler, Hammann and Fahr. 1997. J. Clin. Microbiol. 35:2773.
pinpoint to small colonies, 4. Samra, Heifetz, Talmor, Bain and Bahar. 1998. J. Clin. Microbiol. 36: 990.
with or without halos
S. saprophyticus (most strains)...... Light pink to rose, small
opaque colonies with or Availability
without halos BBL™ CHROMagar™ Orientation
Other including yeasts..................Natural (cream) pigmentation United States and Canada
* About 50% of P. vulgaris strains produce blue colonies on a brownish medium. Cat. No. 254102 Prepared Plates – Pkg. of 20*
Key: KES = Klebsiella-Enterobacter-Serratia group; 215081 Prepared Plates – Ctn. of 100*
PMP = Proteus-Morganella-Providencia group.
Europe
Clinical studies have demonstrated that BBL™ CHROMagar™ Cat. No. 254107 Prepared Plates – Ctn. of 120*
Orientation medium has advantages over other differential Japan
media used in the isolation, differentiation and enumeration of Cat. No. 251781 Prepared Plates – Pkg. of 20*
UTI pathogens, such as CLED Agar or a combination of Blood 252086 Prepared Plates – Ctn. of 100*
CHROMagar™ Salmonella
Intended Use CHROMagar Salmonella is intended for the isolation and dif-
BBL™ CHROMagar™ Salmonella* is a selective and differential ferentiation of Salmonella species. The addition of chromogenic
medium for the isolation and presumptive identification of substrates in the medium facilitates detection of Salmonella
Salmonella species from other coliform and non-coliform bacte- species from other flora.
ria in clinical stool samples and a variety of food samples. CHROMagar Salmonella was originally developed by A.
BBL™ CHROMagar™ Salmonella, prepared plated medium, and Rambach, CHROMagar, Paris, France. BD, under a licensing
agreement, has optimized this formulation utilizing proprietary
Difco™ CHROMagar™ Salmonella, dehydrated culture medium,
have been validated by the AOAC™ Research Institute under intellectual property used in the manufacturing of the BBL C
the Performance Tested MethodsSM program for the analysis of CHROMagar Salmonella prepared plated medium using the
raw ground beef, raw chicken, raw fish, lettuce and shell eggs. Difco CHROMagar Salmonella dehydrated culture medium
ISO, USDA/FSIS and FDA/BAM methods were used for method formulation.
comparison testing.1-3 CHROMagar Salmonella was found to be CHROMagar Salmonella media (prepared plates and dehy-
equivalent to the plated media recommended in the ISO, FDA drated) have been validated by the AOAC Research Institute
and USDA methods. under the Performance Tested Methods Program for testing a
*U.S. Patent Nos. 5,098,832 and 5,194,374
variety of food types, including raw ground beef, raw chicken,
raw fish, lettuce and shell eggs.6 The prepared plates and plates
Summary and Explanation
made from the dehydrated culture medium were compared to
Salmonella is ubiquitous in animal populations and is generally
the USDA/FSIS and FDA/BAM reference methods. The prepared
isolated from the intestinal tract of animals and humans. It is
plates were also compared to the ISO reference media. BBL
one of the most prevalent organisms associated with foodborne
CHROMagar Salmonella prepared plates performed as well
illnesses, which is often linked to animal origin.4 Illnesses caused
as the reference media in all of the food samples with 100%
by Salmonella have been associated with poultry, beef, chocolate,
agreement for each of the three methods. The dehydrated
dairy and vegetable products.5
Identity Specifications
Difco™ CHROMagar™ Salmonella
Dehydrated Appearance: Free-flowing, homogeneous, very pale to light
yellow to tan or very pale pink.
Solution: 3.74% solution, soluble in purified water upon
boiling.
Prepared Appearance: Light to medium yellow to tan or very pale pink
with no significant precipitate.
Reaction of 3.74%
Solution at 25°C: pH 7.6 ± 0.2
Cultural Response
Difco™ CHROMagar™ Salmonella
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C
for 18-24 hours, up to 48 hours for Salmonella strains.
INOCULUM
ORGANISM ATCC™ CFU RECOVERY COLONY COLOR
Citrobacter freundii 8090 104-105 Good Blue to
blue-green
Escherichia coli 25922 104-105 Partial to Blue to
complete inhibition green-blue
Salmonella enterica
subsp. enterica
serotype Enteritidis 13076 103-104 Good Mauve
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028 103-104 Good Mauve
Staphylococcus Partial to
aureus 25923 104-105 complete inhibition Cream
149
medium also produced 100% agreement versus the FDA/BAM Directions for the Preparation
and USDA/FSIS reference media. The results of this study dem- of Antibiotic Stock Solutions
onstrate that BBL CHROMagar Salmonella prepared plates Novobiocin Stock Solution: Dissolve 0.2 g of novobiocin into
and Difco CHROMagar Salmonella dehydrated culture medium 20 mL of purified water and filter sterilize into a sterile tube/bottle.
are effective for the isolation and presumptive identification of Aseptically add 1 mL of stock solution per liter of medium.
Salmonella in raw chicken, raw ground beef, raw fish, lettuce
and shell eggs. Cefsulodin Stock Solution: Dissolve 0.3 g of cefsulodin into
50 mL of purified water and filter sterilize into a sterile tube/bottle.
In a separate study, Cox and Bailey compared BBL CHROMagar Aseptically add 1 mL of stock solution per liter of medium.
Salmonella to the USDA/FSIS reference methods for detection
of Salmonella in rinses of whole chicken carcasses.7 Based on Amphotericin B Stock Solution: Dissolve 0.44 g of amphotericin
480 samples, CHROMagar Salmonella produced a sensitivity B into 100 mL of purified water and filter sterilize into a sterile
of 96.7% when compared to the two standard media combined tube/bottle. Aseptically add 1 mL of stock solution per liter of
(Brilliant Green Sulfa Agar and Modified Lysine Iron Agar). The medium.
researchers concluded that CHROMagar Salmonella is a feasible
single plate method for the detection of Salmonella from whole
Sample Collection and Handling
For clinical specimens, refer to laboratory procedures for details
chicken carcass rinses.7
on specimen collection and handling.
Principles of the Procedure For food samples, follow appropriate standard methods for
Specially selected peptones supply the nutrients. Gram-positive details on sample collection and preparation according to sample
organisms are generally inhibited as a result of the selective medi- type and geographic location.
um base. The addition of an antifungal agent prevents the growth
of Candida species and other antimicrobial agents are used to Procedure
inhibit the growth of gram-negative, non-glucose fermenting For clinical specimens, as soon as possible after receipt in
bacteria and Proteus species, which could potentially overgrow the laboratory, inoculate the specimen onto a CHROMagar
Salmonella colonies. A chromogenic mixture is included in Salmonella plate and streak for isolation. If the specimen is
the medium. Due to metabolic differences in the presence of cultured from a swab, roll the swab gently over a small area of
selected chromogens, colonies of Salmonella species appear mauve the surface at the edge, then streak from this area with a loop.
(rose to purple) in color, whereas undesired bacteria are either Incubate plates aerobically at 35 ± 2°C in an inverted position
inhibited, or produce blue-green or colorless colonies. (agar-side up) for 24 hours. If negative at 24 hours, reincubate
for an additional 24 hours to report final results. Once the colony
Formula color develops, exposure to light is permissible. Typical colonies
Difco™ CHROMagar™ Salmonella of Salmonella should be subjected to confirmatory biochemical
Approximate Formula* Per Liter or serological testing.
Chromopeptone........................................................ 22.0 g
Chromogenic mix......................................................... 0.34 g For food samples, follow sample preparation methodology as
Agar.......................................................................... 15.0 g outlined in USDA/FSIS’s Microbiology Laboratory Guidebook:
*Adjusted and/or supplemented as required to meet performance criteria.
Isolation and Identification of Salmonella from Meat, Poultry,
Directions for Preparation from and Egg Products, FDA/BAM’s chapter on Salmonella, ISO
guidelines or the procedure guidelines appropriate to sample
Dehydrated Product
type and geographic location.
1. Suspend 37.4 g of the powder in 1 L of purified water. Mix
thoroughly. Inoculate the incubated enrichment broth sample onto a
2. Heat with frequent agitation and boil for 1 minute to CHROMagar Salmonella plate. Streak for isolation and incu-
completely dissolve the powder. bate plates aerobically at 35 ± 2°C in an inverted position (agar
3. DO NOT AUTOCLAVE. Cool to 45-50°C. side up) for 24 hours. If negative at 24 hours, reincubate for
4. Aseptically add the following: Sodium Novobiocin (0.01 g/L), an additional 24 hours to report final results. Typical colonies
Cefsulodin (0.006 g/L), and Amphotericin B (0.004 g/L). If of Salmonella growing on CHROMagar Salmonella should
stock solutions are prepared, add 1 mL of each stock solution be subjected to confirmatory testing as outlined in ISO, USDA/
per liter of medium. FSIS and FDA/BAM procedures.1-3
5. Mix well and dispense approximately 20 mL per Petri dish.
6. Immediately after plates have been poured and have solidified, Expected Results
protect from light. Store and incubate plates in the dark. After proper incubation, read plates against a white background.
7. Test samples of the finished product for performance using Salmonella Typhimurium and other Salmonella species will
stable, typical control cultures. appear as light mauve to mauve-colored colonies, with the
exception of Salmonella enterica subspecies arizonae and other
Salmonella species positive for lactose and beta-glucosidase.
150
151
BBL CHROMagar Staph aureus was compared to the AOAC Principles of the Procedure
and ISO reference plated medium, Baird-Parker Agar, using BBL CHROMagar Staph aureus was originally developed by
the recommended diluents at low, medium and high inoculum A. Rambach, CHROMagar, Paris, France. BD, under a licensing
levels of S. aureus. After 24 hours of incubation, enumeration agreement, has optimized this formulation utilizing proprietary
was performed on BBL CHROMagar Staph aureus and after intellectual property used in the manufacturing of the BBL
48 hours on Baird-Parker Agar. CHROMagar Staph aureus prepared plated medium.
Based on statistical analysis, no significant difference was found Specially selected Difco™ peptones supply nutrients. The ad-
between the reference methods and the BBL CHROMagar dition of selective agents inhibits the growth of gram-negative
Staph aureus method for any food type or contamination level, organisms, yeast and some gram-positive cocci. The chromogen
with the exception of a low-level smoked salmon sample. The low mix consists of artificial substrates (chromogens), which release
contamination level of smoked salmon demonstrated a statistical an insoluble colored compound when hydrolyzed by specific
difference in internal testing using the ISO method; i.e., the enzymes. This facilitates the detection and differentiation of
BBL CHROMagar Staph aureus method at 24 hours recovered S. aureus from other organisms. S. aureus utilizes one of the
more colonies (log10 2.04) than the ISO reference at 48 hours chromogenic substrates, producing mauve-colored colonies. The
(log10 1.64). The repeatability precision estimates of the BBL growth of mauve-colored colonies at 24 hours is considered posi-
CHROMagar Staph aureus method were satisfactory. The corre- tive for S. aureus on BBL CHROMagar Staph aureus. Bacteria
lation coefficients ranged from 92.6% to 99.4%, demonstrating other than S. aureus may utilize other chromogenic substrates
good correlation for all contamination levels in all food types. resulting in blue, blue-green, or if no chromogenic substrates
No false-positive colonies were recovered from the food matrices are utilized, natural colored colonies.
using BBL CHROMagar Staph aureus, and all mauve colonies
were confirmed as S. aureus with no discrepancies. Known Sample Collection and Handling
isolates, including 30 strains of S. aureus (several of which were For clinical specimens, refer to laboratory procedures for details
enterotoxin-producing strains) and 37 non-S. aureus isolates on specimen collection and handling.
were evaluated producing both a sensitivity and specificity of
For food samples, follow appropriate standard methods for
100% on BBL CHROMagar Staph aureus. The results of these
details on sample collection and preparation according to sample
studies demonstrate that BBL CHROMagar Staph aureus can be
type and geographic location.
used for the isolation, enumeration and presumptive identifica-
tion of S. aureus in cooked roast beef, smoked salmon and shell Procedure
eggs using AOAC and ISO methods. For clinical specimens, as soon as possible after receipt in the
laboratory, inoculate onto a BBL CHROMagar Staph aureus
Staphylococcus aureus
ATCC™ 25923 plate and streak for isolation. If the specimen is cultured from
a swab, roll the swab gently over a small area of the surface at
the edge, then streak from this area with a loop. Incubate plates
aerobically at 35 ± 2°C for 20-24 hours in an inverted position
(agar-side up).
For food samples, consult appropriate references and follow
applicable standard methods. Inoculate the homogenized food
samples onto BBL CHROMagar Staph aureus using the spread
plate technique. Incubate plates aerobically at 35-37°C for 20-28
hours in an inverted position (agar-side up).
Expected Results
After proper incubation, read plates against a white background.
S. aureus produces mauve to orange-mauve colored colonies on
the BBL CHROMagar medium. Most gram-positive organisms,
if not inhibited, will produce blue, blue-green or natural color
(colorless, white or cream) colonies. Gram-negative organisms
and yeasts are partially to completely inhibited.
152
be accomplished by coagulase, other biochemicals or Gram 3. Doyle and Beuchat (eds.). 2007. Food microbiology fundamentals and frontiers, 3rd ed. American
Society for Microbiology, Washington, DC.
stain. Resistant gram-negative bacilli, which typically appear 4. Bannerman and Peacock. 2007. Staphylococcus, Micrococcus, and other catalase-positive cocci. In
Murray, Baron, Jorgensen, Landry and Pfaller (eds.), Manual of clinical microbiology, 9th ed. American
as small blue colonies, may also breakthrough. Society for Microbiology, Washington, DC.
5. Bennett and Lancette. 1998. Staphylococcus aureus. In FDA bacteriological analytical manual, 8th
2. Incubation beyond 24 hours (clinical) and 28 hours (food) is ed. AOAC International, Gaithersburg, Md.
6. Hurst, Crawford, Garland, Lipson, and Mills (eds.). 2007. Manual of environmental microbiology,
not recommended due to an increase in potential false posi- 3rd ed., American Society for Microbiology, Washington, DC.
tives. If incubation time is exceeded, mauve-colored colonies 7. AOAC Research Institute News. 2006. Inside laboratory management (March/April 2006), AOAC
International, Gaithersburg, Md.
should be confirmed prior to reporting as S. aureus. 8. Data on file, BD Diagnostics.
Procedure
As soon as possible after receipt in the laboratory, inoculate the
specimen onto a reduced CDSA plate and streak for isolation.
As some strains of C. difficile may not grow well due to the
selective properties of the medium, it is advisable to include a
nonselective medium, such as CDC Anaerobe Blood Agar.
Media should be reduced prior to inoculation by placing under
anaerobic conditions for 6-24 hours prior to use.1 An efficient
and easy way to obtain suitable anaerobic conditions is through
the use of GasPak™ EZ anaerobic systems or other alternative
anaerobic systems.
Incubate immediately under anaerobic conditions or place in
a holding jar flushed with oxygen-free gas(es) until sufficient
plates are accumulated (but no longer than 3 hours).2 Incubation
should be at 35 ± 2°C for at least 48 hours.
153
In contrast to the earlier media, this formulation was developed Enterobacter 13048 103-104 Yellow colonies No zone
aerogenes with or without
as a general-purpose medium for fastidious organisms that also weak yellow zone
permitted differentiation of pathogenic staphylococci from other
Proteus vulgaris 8427 103-104 Negative No zone
bacteria.
Staphylococcus
aureus 13150 103-104 Yellow zone Opaque zone
Principles of the Procedure Staphylococcus
Coagulase Mannitol Agar aids in the differentiation of epidermidis 12228 103-104 Negative No zone
staphylococci by indicating the presence of coagulase and the *Recovery of all cultures should be good.
utilization of mannitol. Coagulase production is dependant on
the presence of mannitol, a protein factor in the brain heart
infusion and blood serum (plasma).5 During utilization of the yellow zones around these colonies. An opaque area of coagu-
mannitol, the pH of the medium drops, causing the bromcresol lated plasma forms around the colonies of organisms that also
purple indicator to change from purple to yellow and producing produce coagulase.
154
Columbia Agars
Columbia Agar Base • Columbia Blood Agar Base
Columbia Blood Agar Base EH • Columbia Agar
with 5% Sheep Blood • Columbia Agar with Fildes
Enrichment and Bacitracin
Intended Use Columbia Agar Base meets United States Pharmacopeia (USP),
Columbia Agar Base, without or with the addition of 5% European Pharmacopoeia (EP) and Japanese Pharmacopoeia
(or 10%) sheep blood, is a highly nutritious, general-purpose (JP)1-3 performance specifications, where applicable.
medium for the isolation and cultivation of nonfastidious and
fastidious microorganisms from a variety of clinical and non- Summary and Explanation
clinical materials. Ellner et al.,4 in 1966, reported the development of a blood
agar formulation, which has been designated as Columbia
Columbia Blood Agar Base EH (Enhanced Hemolysis) is used with
Agar. The base achieves the more rapid and luxuriant growth
blood in isolating and cultivating fastidious microorganisms.
obtained from casein hydrolysate media with the sharply defined
Columbia Agar with Fildes Enrichment and Bacitracin is hemolytic reactions, more typical colonial morphology and
used in qualitative procedures for isolation and cultivation of improved pigment production achieved with media containing
Haemophilus species from clinical specimens. infusion peptone.
155
Columbia Agar Base is utilized as the base for media containing since it contains NADase which destroys the NAD. For this
blood and for selective media formulations in which various reason, Haemophilus influenzae, which requires both the X
combinations of antimicrobial agents are used as additives. and V factors, will not grow on this medium. Fildes found that
supplementing nutrient agar with a digest of sheep blood sup-
Sheep blood allows detection of hemolytic reactions and supplies
plied both of these factors and the medium would support the
the X factor (heme) necessary for the growth of many bacterial
growth of H. influenzae.5,6 The inclusion of bacitracin makes
species but lacks V factor (nicotinamide adenine dinucleotide),
Cultural Response
Difco™ Columbia Blood Agar Base or Columbia Blood Agar Base EH
Prepare the medium per label directions without (plain) and with 5% sheep blood (SB) for Columbia Blood Agar Base and with 5% sheep blood for Columbia Blood
Agar Base EH. Inoculate and incubate at 35 ± 2°C with 5-10% CO2 for 18-48 hours.
ORGANISM ATCC™ INOCULUM CFU RECOVERY PLAIN RECOVERY WITH SB HEMOLYSIS
Escherichia coli 25922 30-300 Good Good Beta
Neisseria meningitidis 13090 30-300 Good Good Gamma (none)
Staphylococcus aureus 25923 30-300 Good Good Beta
Streptococcus pneumoniae 6305 30-300 Good Good Alpha
Streptococcus pyogenes 19615 30-300 Good Good Beta
Streptococcus Streptococcus
pneumoniae pyogenes
ATCC™ 6305 ATCC™ 19615 Continued
156
the enriched Columbia Agar medium selective for the isolation isolating Aeromonas sp. from stool samples of patients showing
of Haemophilus species from clinical specimens, especially from clinical symptoms of gastroenteritis.10
the upper respiratory tract.7 Columbia Agar Base is used to prepare Modified Butzler Agar,
Columbia Agar with 5% sheep blood is a general all-purpose which is a selective isolation medium for the detection of ther-
enriched primary isolation medium that allows growth of all motolerant Campylobacter in food and animal feed.11 Columbia
clinically significant anaerobes and facultative anaerobes.8,9 Agar Base is a component of Oxford Medium and Columbia
Columbia Agar supplemented with 5% sheep blood is recom- Blood Agar Base is a component of Modified Oxford Medium,
mended when processing clinical specimens for unusual organ- both of which are used to detect Listeria monocytogenes in
isms, such as Bartonella bacilliformis, the causative agent of food and milk samples.11-14 Columbia Agar is listed as one of
Oroya fever and Peruvian wart.8 Columbia Agar supplemented the recommended media for the isolation of Clostridia sp. from
with 5% sheep blood and 20 μg of ampicillin per mL is used in nonsterile pharmaceutical products.1 C
Identity Specifications Staphylococcus aureus Escherichia coli
ATCC™ 25923 ATCC™ 25922
BBL™ Columbia Agar Base
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution: 4.25% solution, soluble in purified water upon
boiling. Solution is medium, yellow to tan, hazy.
Prepared Appearance: Plain – Medium, yellow to tan, hazy.
With sheep blood – Cherry red, opaque, no
hemolysis.
Reaction of 4.25%
Solution at 25°C: pH 7.3 ± 0.2
BBL™ Columbia Agar (prepared)
Appearance: Light to dark yellow and hazy with small cream
particles in sediment; may appear as flocculation
and contain small suspended insolubles.
Reaction at 25°C: pH 7.3 ± 0.2
Cultural Response
BBL™ Columbia Agar Base
Prepare the medium per label directions without (plain) and with 5% sheep
blood (SB). Inoculate and incubate at 35 ± 2°C under appropriate atmospheric
conditions for 48 hours (incubate C. jejuni at 42 ± 2°C for 48-72 hours).
For Clostridium sporogenes (both strains), inoculate with fresh 24-48 hour
Reinforced Clostridial Medium cultures, in duplicate, and incubate one set at Enterococcus
faecalis
30-35°C and the other set at 35-37°C for 48 hours. ATCC™ 33186
ORGANISM ATCC™ INOCULUM CFU RECOVERY PLAIN RECOVERY WITH SB
Campylobacter jejuni 33291 10 3
N/A Good
Campylobacter jejuni 33292 103 N/A Good
Candida albicans 10231 103-104 N/A Good
Escherichia coli 25922 103-104 N/A Good
Listeria monocytogenes 19115 103-104 N/A Good
Pseudomonas aeruginosa 10145 10 -10
3 4
Good N/A
Shigella flexneri 12022 103-104 Good N/A
Staphylococcus aureus 25923 103-104 Good N/A
Streptococcus pneumoniae 6305 103-104 Good N/A
Clostridium sporogenes 11437 <100 Growth (at 30-35°C) N/A
Clostridium sporogenes 11437 <100 Growth (at 35-37°C) N/A
Clostridium sporogenes 19404 <100 Growth (at 30-35°C) N/A
Clostridium sporogenes 19404 <100 Growth (at 35-37°C) N/A
157
Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to com-
pletely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. For preparation of blood agar, cool the base to 45-50°C and
add 5% sterile, defibrinated blood. Mix well.
5. Test samples of the finished product for performance using
stable, typical control cultures.
158
Procedure References
This medium should be reduced at room temperature immediately 1. Ellner, Granato and May. 1973. Appl. Microbiol. 26:904.
2. Jousimies-Somer, Summanen and Finegold. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.),
prior to inoculation by placing under anaerobic conditions for Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
3. Gibbons and MacDonald. 1960. J. Bacteriol. 80:164.
18-24 hours.4 4. Dowell. 1975. In Balows (ed.), Clinical microbiology. How to start and when to stop. Charles C.
Thomas, Springfield, Ill.
Use standard procedures to obtain isolated colonies from speci- 5. Isenberg, Schoenknecht and von Graeventiz. 1979. Cumitech 9, Collection and processing of bacte-
riological specimens. Coord. ed., Rubin. American Society for Microbiology, Washington, D.C.
mens. Inoculate an enrichment broth, such as BBL™ Enriched 6. Martin. 1971. Appl. Microbiol. 22:1168.
7. Allen, Siders and Marler. 1985. In Lennette, Balows, Hausler and Shadomy (ed.), Manual of clinical
Thioglycollate Medium, at the same time as the primary plates microbiology, 4th ed. American Society for Microbiology, Washington, D.C.
to detect small numbers of anaerobes.
159
Availability Japan
Cat. No. 251974 Prepared Plates – Pkg. of 10*
BBL™ Columbia Anaerobe 5% Sheep Blood Agar
*Store at 2-8°C.
United States and Canada
Cat. No. 221928 Prepared Plates – Pkg. of 20*
221929 Prepared Plates – Ctn. of 100*
Columbia Broth
Intended Use User Quality Control
Columbia Broth is used for cultivating fastidious microor-
ganisms. Identity Specifications
Difco™ Columbia Broth
Summary and Explanation Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Columbia Broth is prepared according to the formulation Solution: 3.5% solution, soluble in purified water upon
warming. Solution is light amber, clear to very
described by Morello and Ellner.1 In their study Columbia slightly opalescent, may have a slight amount of
Broth, a medium developed for blood cultures, was superior fine precipitate.
to a commonly used general purpose broth for faster growth Prepared Appearance: Light amber, clear to very slightly opalescent,
of Staphylococcus aureus, Escherichia coli and streptococci may have a slight amount of fine precipitate.
Reaction of 3.5%
(viridans and enterococcus groups). Columbia Broth, in the Solution at 25°C: pH 7.5 ± 0.2
presence of CO2 and supplemented with SPS, is an excellent
blood culture medium.2 In the study by Morello and Ellner,1 Cultural Response
Difco™ Columbia Broth
the addition of sodium polyanetholsulfonate (SPS) in Columbia
Prepare the medium per label directions. Inoculate and incubate at
Broth was emphasized. SPS is an anticoagulant that inhibits 35 ± 2°C under appropriate conditions for 18-48 hours. Incubate
serum bactericidal activity against many bacteria, inhibits Bacteroides fragilis anaerobically.
phagocytosis, inactivates complement, and neutralizes lyso- Organism ATCC™ INOCULUM CFU RECOVERY
zymes and the aminoglycoside class of antibiotics.2 Bacteroides fragilis 25285 102-103 Good
Neisseria meningitidis 13090 102-103 Good
Principles of the Procedure
Pseudomonas aeruginosa 27853 10 -10
2 3
Good
Peptones and yeast extract provide nitrogen, carbon, vitamins
Staphylococcus aureus 25923 102-103 Good
and trace nutrients essential for growth. Dextrose is added
Streptococcus pyogenes 19615 102-103 Good
to the formula as a carbon energy source. The medium is
buffered with Tris. Corn starch is omitted to reduce opalescence.1
Cysteine is the reducing agent. Magnesium and iron are added
to facilitate organism growth. Directions for Preparation from
Dehydrated Product
Formula 1. Suspend 35 g of the powder in 1 L of purified water. Mix
Difco™ Columbia Broth thoroughly.
Approximate Formula* Per Liter 2. Heat with frequent agitation and boil for 1 minute to
Pancreatic Digest of Casein........................................ 10.0 g
Yeast Extract................................................................ 5.0 g
completely dissolve the powder.
Proteose Peptone No. 3................................................ 5.0 g 3. OPTIONAL: Sodium polyanetholesulfonate (SPS) may
Tryptic Digest of Beef Heart.......................................... 3.0 g be added at this time with agitation to ensure a uniform
L-Cysteine HCl............................................................ 0.1 g
solution. The culture medium should contain 0.025 to
Dextrose..................................................................... 2.5 g
Sodium Chloride......................................................... 5.0 g 0.05% SPS.
Magnesium Sulfate (anhydrous).................................. 0.1 g 4. Autoclave at 121˚C for 15 minutes.
Ferrous Sulfate............................................................ 0.02 g 5. Test samples of the finished product for performance using
Sodium Carbonate....................................................... 0.6 g
Tris (Hydroxymethyl) Aminomethane........................... 0.83 g stable, typical control cultures.
Tris (Hydroxymethyl) Aminomethane HCl...................... 2.86 g
*Adjusted and/or supplemented as required to meet performance criteria. Procedure
Process clinical specimens from different body sites as described
in Clinical Microbiology Procedures Handbook,2 Manual of
Clinical Microbiology3 or according to laboratory procedures.
Expected Results
Refer to appropriate references and procedures for results.
160
C
Columbia CNA Agar • Columbia CNA Agar, Modified
Columbia PNA Agar
Intended Use Ellner and his colleagues found that a medium consisting of 10 mg
Columbia CNA Agar, Columbia CNA Agar, Modified, and of colistin and 15 mg of nalidixic acid per liter in a Columbia Agar
Columbia PNA Agar, all supplemented with 5% sheep blood, Base enriched with 5% sheep blood would support the growth
are selective and differential media used for the isolation and of staphylococci, hemolytic streptococci and enterococci while
differentiation of gram-positive microorganisms from clinical inhibiting the growth of Proteus, Klebsiella and Pseudomonas
and nonclinical materials. species. In BBL™ Columbia CNA Agar with 5% Sheep Blood,
the concentration of nalidixic acid has been reduced to 10 mg/L
Summary and Explanation to increase the recovery of gram-positive cocci from clinical
Ellner et. al., in 1966, reported the development of a blood agar specimens. The concentration of nalidixic acid has been further
formulation, which has been designated as Columbia Agar.1 reduced in Columbia CNA Agar, Modified to 5 mg/L.
The Columbia Agar base, which achieves rapid and luxuriant In the Columbia PNA version of Ellner’s medium, polymyxin
growth and sharply defined hemolytic reactions, is utilized as B has been substituted for colistin (10 mg). Although the
the base for media containing blood and for selective formula- antimicrobial properties of the two agents are nearly the same,
tions in which various combinations of antimicrobial agents are some species of gram-negative bacteria are more sensitive to
used as additives. polymyxin B than colistin.2
Identity Specifications
BBL™ Columbia CNA Agar
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution: 4.25% solution, soluble in purified water upon
boiling. Solution is medium, tan to yellow, hazy.
Prepared Appearance: Tan to yellow, hazy.
Reaction of 4.25%
Solution at 25°C: pH 7.3 ± 0.2
Cultural Response
BBL™ Columbia CNA Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C
with 3-5% CO2 for 18-24 hours.
INOCULUM
ORGANISM ATCC™ CFU REcovery hemolysis
Proteus mirabilis 12453 104-105 Partial to –
complete inhibition
Staphylococcus
aureus 25923 103-104 Good Beta
Streptococcus
pneumoniae 6305 103-104 Good Alpha
Streptococcus Beta, slight greening
pyogenes 19615 103-104 Good may be present
161
162
Identity Specifications
Difco™ Cooke Rose Bengal Agar
Dehydrated Appearance: Pinkish-tan, free-flowing, homogeneous.
Solution: 3.6% solution, soluble in purified water upon
boiling. Solution is pinkish red, slightly opales-
cent.
Prepared Appearance: Deep pink, slightly opalescent.
Reaction of 3.6%
Solution at 25°C: pH 6.0 ± 0.2
Difco™ Antimicrobic Vial A
Desiccated Appearance: Yellow cake or powder.
Rehydrated Appearance: Yellow, clear solution.
Solution: Soluble in 10 mL purified water.
Cultural Response
Difco™ Cooke Rose Bengal Agar and Antimicrobic
Vial A
Prepare the medium with 35 µg per mL chlortetracycline (Antimicrobic
Vial A added aseptically) per label directions. Inoculate and incubate at
25-30°C for up to 72 hours.
Organism ATCC™ INOCULUM CFU RECOVERY Candida albicans
ATCC™ 26790
Aspergillus brasiliensis (niger) 16404 Undiluted Good
Candida albicans 26790 30-300 Good
Escherichia coli 25922 103 Inhibition
Saccharomyces cerevisae 9763 30-300 Good
163
Procedure
Refer to appropriate references for specific procedures on the Availability
isolation and cultivation of fungi. Difco™ Cooke Rose Bengal Agar
SMWW
Cat. No. 270310 Dehydrated – 500 g
Expected Results
Refer to appropriate references and procedures for results. Difco™ Antimicrobic Vial A
SMWW
Cat. No. 233331 Vial – 6 x 10 mL*
*Store at 2-8ºC.
stains or spore stains should be made to determine the shape Using a sterile inoculating loop or needle, transfer growth from
and location of spores. a fresh subculture medium, inoculating heavily in the area of
meat particles. Incubate the tubes at 35 ± 2°C under anaerobic
Formula conditions for up to 7 days. It is recommended that an indicator
Difco™ Cooked Meat Medium of anaerobiosis be used.
Approximate Formula* Per Liter
Beef Heart (from 454 g) ............................................ 98.0 g Expected Results
Proteose Peptone ...................................................... 20.0 g
Dextrose ..................................................................... 2.0 g In the cultivation of clostridia, saccharolytic organisms usually
Sodium Chloride ......................................................... 5.0 g produce acid and gas. Growth of proteolytic organisms is
*Adjusted and/or supplemented as required to meet performance criteria.
generally characterized by blackening and dissolution of the
meat particles.
Directions for Preparation from
Dehydrated Product References
1. Suspend 12.5 g of the particles in 100 mL purified water 1. Robertson. 1916. J. Pathol. Bacteriol. 20:327.
2. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC, International,
(1.25 g/10 mL). Gaithersburg, Md.
3. Holdeman, Cato and Moore. 1977. Anaerobe laboratory manual, 4th ed. Virginia Polytechnical Institute
2. Let stand until all particles are thoroughly wetted and form and State University, Blacksburg, Va.
an even suspension. 4. Willis. 1977. Anaerobic bacteriology: clinical and laboratory practice, 3rd ed. Butterworths, London,
England.
3. Autoclave at 121°C for 15 minutes. Reduce pressure slowly
and cool without agitation. Availability
4. If not used within 24 hours, reheat (100°C) prior to use to Difco™ Cooked Meat Medium
drive off absorbed oxygen. AOAC BAM CCAM COMPF
5. Test samples of the finished product for performance using Cat. No. 226730 Dehydrated – 500 g
stable, typical control cultures.
BBL™ Cooked Meat Medium
AOAC BAM CCAM COMPF
Procedure
Cat. No. 221507 Prepared Tubes, 8 mL (K Tubes) – Pkg. of 10
Liquid media for anaerobic incubation should be reduced prior 221508 Prepared Tubes, 8 mL (K Tubes) – Ctn. of 100
to inoculation by placing the tubes, with caps loosened, under
BBL™ Cooked Meat Medium with Glucose, Hemin
anaerobic conditions for 18-24 hours. An efficient and easy and Vitamin K1
way to obtain suitable anaerobic conditions is through the use BS12 CMPH2 MCM9
of the GasPak™ EZ anaerobic system or an alternative anaerobic Cat. No. 297809 Prepared Tubes, 10 mL (C Tubes) – Ctn. of 100
system. Alternatively, liquid media may be reduced immediately 295982 Prepared Tubes, 9 mL (K Tubes) – Pkg. of 10
prior to use by boiling with caps loosened and cooling with 299455 Prepared Tubes, 9 mL (K Tubes) – Ctn. of 100
tightened caps to room temperature before inoculation.
Organisms to be cultivated must first be isolated in pure
culture in an appropriate medium.
165
Section III
C Corn Meal Agar
Formula
BBL™ Corn Meal Agar
Approximate Formula* Per Liter
Corn Meal Infusion from (Solids).................................. 2.0 g
Agar.......................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.
Microscopic Photo of Chlamydospores
Cultural Response
BBL™ Corn Meal Agar
Prepare the medium per label directions.Test for chlamydospore production.
Using fresh cultures, streak two parallel lines approximately 1.5 cm long each
and 1.0 cm apart. Make an S-shape by lightly streaking back and forth across
the two parallel streak lines. Place a coverslip over the streak marks. Incubate
at 25 ± 2°C for 4 days and examine microscopically.
chlamydospore
ORGANISM ATCC™ REcovery production
Aspergillus brasiliensis (niger) 16404 Good N/A
Candida albicans 10231 Good Present
Candida albicans 60193 Good Present
Candida kefyr 8553 Good None
166
Procedure
Limitation of the Procedure C
Corn Meal Agar with Dextrose is not recommended for detecting
To prepare plated media from agar deeps, place the agar deeps the production of chlamydospores by Candida species.
in a boiling water bath until the medium becomes liquefied
(clear). Pour the molten medium into a sterile Petri dish and References
allow to solidify before use. Organisms to be cultivated for 1. Pollack and Benham. 1960. J. Lab. Clin. Med. 50:313.
2. Walker and Huppert. 1960. Tech. Bull. Reg. Med. Technol. 30:10.
identification must first be isolated in pure culture on an 3. McGinnis. 1980. Laboratory handbook of medical mycology. Academic Press, New York, N.Y.
4. Conant, Smith, Baker and Callaway. 1971. Manual of clinical mycology, 3rd ed. W.B. Saunders Co.,
appropriate medium. Philadelphia, Pa.
5. Haley and Callaway. 1978. Laboratory methods in medical mycology. HEW Publication No. (CDC)
Using an inoculating needle, streak the medium with growth 78-8361. Center for Disease Control, Atlanta, Ga.
6. Isenberg (ed.). 1992. Clinical microbiology procedures handbook, vol. 1. American Society for
from a pure culture and incubate at 25 ± 2°C. Examine at Microbiology, Washington, D.C.
7. Campbell and Stewart. 1980. The medical mycology handbook. John Wiley & Sons, New York,
intervals for up to 28 days for growth and pigmentation. N.Y.
Cultural Response
Difco™ Cystine Heart Agar
Prepare the medium per label directions without and with hemoglobin.
Incubate inoculated medium at 35 ± 2oC aerobically for 66-72 hours. Incubate
Neisseria meningitidis under increased CO2.
INOCULUM RECOVERY RECOVERY
Organism ATCC™ CFU w/o Hemoglobin w/Hemoglobin
Francisella tularensis
(BD 16223)* 102-103 N/A Good
Neisseria meningitidis 13090 102-103 Good Good
Staphylococcus aureus 25923 102-103 Good Good
Streptococcus
pneumoniae 6303 102-103 Good Good
*Minimally, one strain of F. tularensis should be used for performance testing. F. tularensis ATCC 29684 can be
substituted for BD Diagnostics strain 16223.
168
Prepared Appearance: Light amber, slightly opalescent, may have a Aspergillus niger 9642 102-103 Good
slight precipitate. Candida albicans 10231 10 -10
2 3
Good
Reaction of 4.9%
Penicillium rubrum 10520 102-103 Good
Solution at 25°C: pH 7.3 ± 0.2
Streptococcus albus 3004 102-103 Good
Procedure Availability
Refer to appropriate references for specific procedures for the Difco™ Czapek-Dox Broth
cultivation of fungi and bacteria capable of utilizing inorganic Cat. No. 233810 Dehydrated – 500 g
nitrogen. Difco™ Czapek Solution Agar
SMWW
Expected Results Cat. No. 233910 Dehydrated – 500 g
Refer to appropriate references and procedures for results.
References
1. Czapek. 1902-1903. Beitr. Chem. Physiol. Pathol. 1:540.
2. Dox. 1910. U.S. Dept. Agr. Bur. Anim. Ind. Bull. 120:70.
3. Thom and Raper. 1945. Manual of the aspergilli. Williams & Wilkins Co., Baltimore, Md.
4. Thom and Church. 1926. The aspergilli. Williams & Wilkins Co., Baltimore, Md.
5. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C.
DCLS Agar
Intended Use Principles of the Procedure
DCLS Agar (Desoxycholate Citrate Lactose Sucrose Agar) is This medium contains peptones and beef extract, which
a moderately selective culture medium for the isolation of supply essential nutrients for the support of bacterial growth.
Salmonella and Shigella from fecal specimens. The citrate and desoxycholate compounds serve as inhibitors
of gram-positive bacteria and coliforms. The incorporation
Summary and Explanation of two sugars permits the formation of red colonies by
DCLS Agar is a modification of Leifson’s Desoxycholate Agar, organisms that rapidly ferment either sucrose or lactose, or
a slightly selective and differential plating medium for enterics both; e.g., Proteus vulgaris, as well as typical coliforms.
in which the degree of inhibition is accurately controlled by This permits the more accurate selection of members of the
the substitution of pure chemicals for the largely undefined genera Shigella and Salmonella, which form colorless or nearly
composition of bile.1 DCLS Agar is only one of a number of colorless colonies on DCLS Agar.
modified desoxycholate-containing media and differs from the
rest by its inclusion of sucrose.2
DCLS Agar supports good growth of cultures of Shigella
and Salmonella, and inhibits the growth of coliforms and
Proteus. In addition to the human pathogens, S. pullorum and
S. gallinarum grow well.
170
Procedure
Inoculate and incubate plates, protected from light, at 35 ± 2°C
for 18-24 hours. If negative after 24 hours, reincubate an
additional 24 hours.
Identity Specifications
BBL™ DCLS Agar
Dehydrated Appearance: Fine, homogeneous powder.
Solution: 5.0% solution, soluble in purified water upon
boiling. Solution is medium to dark, red-orange
to orange-rose, clear to slightly hazy.
Prepared Appearance: Medium to dark, red-orange to orange-rose,
clear to slightly hazy.
Reaction of 5.0%
Solution at 25°C: pH 7.2 ± 0.2
Cultural Response
BBL™ DCLS Agar
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 24 hours.
INOCULUM colony
ORGANISM ATCC™ CFU REcovery color
Escherichia coli 25922 104-105 Good Pink to rose-red
Salmonella enterica
subsp. enterica Colorless to
serotype Typhimurium 14028 103-104 Good pale pink
Shigella flexneri 12022 103-104 Good Colorless to
pale pink
Enterococcus faecalis 29212 104-105 None –
171
172
Formulae
Difco™ D/E Neutralizing Agar
Approximate Formula* Per Liter
Pancreatic Digest of Casein.......................................... 5.0 g
Yeast Extract................................................................ 2.5 g
Dextrose.................................................................... 10.0 g
Sodium Thioglycollate.................................................. 1.0 g
Uninoculated Bacillus subtilis Escherichia coli
Tube ATCC™ 6633 ATCC™ 25922 Sodium Thiosulfate...................................................... 6.0 g
Sodium Bisulfite........................................................... 2.5 g
Polysorbate 80............................................................. 5.0 g
Lecithin........................................................................ 7.0 g
Bromcresol Purple........................................................ 0.02 g
Agar.......................................................................... 15.0 g
Difco™ D/E Neutralizing Broth
Consists of the same ingredients without the agar.
*Adjusted and/or supplemented as required to meet performance criteria.
174
175
Dehydrated Appearance: Light beige with slight green tint, free-flowing, Dehydrated Appearance: Fine, homogeneous, free of extraneous
homogeneous. material.
Solution: 4.2% solution, soluble in purified water upon Solution: 4.2% solution, soluble in purified water upon
boiling. Solution is green, very slightly to slightly boiling.Solution is medium to dark, blue,
opalescent with slight precipitate. trace hazy to hazy.
Prepared Appearance: Green, very slightly to slightly opalescent with Prepared Appearance: Medium to dark, blue, trace hazy to hazy.
slight precipitate. Reaction of 4.2%
Reaction of 4.2% Solution at 25°C: pH 7.3 ± 0.2
Solution at 25°C: pH 7.3 ± 0.2
Cultural Response
Cultural Response BBL™ DNase Test Agar or DNase Test Agar
Difco™ DNase Test Agar or DNase Test Agar with with Toluidine Blue
Methyl Green Prepare the medium per label directions. Inoculate with fresh cultures and
Prepare the medium per label directions. Inoculate by streaking with a line of incubate at 35 ± 2°C for 18-24 hours. For DNase Test Agar, flood the plates
undiluted culture across the medium and incubate at 35 ± 2°C for up to 48 with 1N HCl and examine for deoxyribonuclease activity. For DNase Test Agar
hours. For DNase Test Agar, flood the streak plates with 1N HCl and examine with Toluidine Blue, examine for deoxyribonuclease activity.
for clear zones around the streaks (positive reactions). For DNase Test Agar
recovery/ recovery/reaction
with Methyl Green, examine the streak plates for decolorized zones around reaction DNASE TEST AGAR
the streaks (positive reactions). ORGANISM ATCC™ DNASE TEST AGAR W/TOLUIDINE BLUE
Staphylococcus
epidermidis
ATCC™ 12228
Procedure
Inoculate by making a single streak line using inoculum from
an agar slant or plate. One plate may be inoculated with up
to eight isolates by spot inoculation (1/8 to 1/4 inch) or streak
inoculation (a single 1- to 2-inch line).
Incubate at 35 ± 2°C for 24-48 hours. Plates should be
incubated in an inverted position. Incubate tubes with loosened
caps.
Following incubation, flood DNase Test Agar plates with 1N
HCl reagent and observe for reaction. Reagent addition is not
required with DNase Test Agar with Methyl Green or with
DNase Test Agar with Toluidine Blue.
Expected Results
A clear area surrounding growth (band/spot inocula) on DNase
Test Agar after the addition of 1N HCl indicates a positive
reaction, DNase activity. A negative reaction is indicated by
no clearing and a cloudy precipitate around colonies and
throughout medium due to precipitated salts in the medium.
A positive reaction on DNase Test Agar with Methyl Green
is a distinct clear zone surrounding growth in an otherwise
green-colored medium. The color of the medium remains
unchanged if the test is negative.
On DNase Test Agar with Toluidine Blue, DNase activity is
indicated by pink to red zones surrounding growth. The color
of the medium remains unchanged if the test is negative.
177
DRBC Agar
Intended Use Formula
DRBC Agar is used for the enumeration of yeasts and molds. Difco™ DRBC Agar
Approximate Formula* Per Liter
Summary and Explanation Proteose Peptone No. 3................................................ 5.0 g
Dextrose.................................................................... 10.0 g
DRBC (Dichloran Rose Bengal Chloramphenicol) Agar is based
Monopotassium Phosphate.......................................... 1.0 g
on the Dichloran Rose Bengal Chlortetracycline Agar formula Magnesium Sulfate...................................................... 0.5 g
described by King, Hocking and Pitt.1 DRBC Agar conforms Dichloran..................................................................... 2.0 mg
with APHA guidelines for the mycological examination of Rose Bengal............................................................... 25.0 mg
foods, containing chloramphenicol rather than chlortetracycline Chloramphenicol.......................................................... 0.1 g
Agar.......................................................................... 15.0 g
as originally proposed.2 DRBC Agar is a selective medium that *Adjusted and/or supplemented as required to meet performance criteria.
supports good growth of yeasts and molds.
Directions for Preparation from
Principles of the Procedure Dehydrated Product
Peptone provides nitrogen, vitamins and minerals. Dextrose
is a carbohydrate source. Phosphate is a buffering agent. 1. Suspend 31.6 g of the powder in 1 L of purified water. Mix
Magnesium sulfate is a source of divalent cations and sulfate. thoroughly.
The antifungal agent, dichloran, is added to the medium to 2. Heat with frequent agitation and boil for 1 minute to
reduce colony diameters of spreading fungi. The pH of the completely dissolve the powder.
medium is reduced from 7.2 to 5.6 for improved inhibition 3. Autoclave at 121°C for 15 minutes.
of the spreading fungi.1 The presence of rose bengal in the 4. Test samples of the finished product for performance using
medium suppresses the growth of bacteria and restricts the stable, typical control cultures.
size and height of colonies of the more rapidly growing molds.
The concentration of rose bengal is reduced from 50 µg/mL Procedure2,3
to 25 µg/mL as found in Rose Bengal Chloramphenicol Agar 1. Inoculate 0.1 mL of appropriate decimal dilutions of the
for optimal performance with dichloran. Chloramphenicol is
sample in duplicate onto the surface of DRBC Agar plates.
included in this medium to inhibit the growth of bacteria pres-
ent in environmental and food samples. Inhibition of growth of The plates should be dried overnight at room temperature.
bacteria and restriction of spreading of more-rapidly growing Spread the inoculum over the entire surface of the plate using
molds aids in the isolation of slow-growing fungi by preventing a sterile, bent-glass rod.
their overgrowth by more-rapidly growing species. In addi- 2. Incubate plates upright at 22-25°C. Examine for growth of
tion, rose bengal is taken up by yeast and mold colonies, which yeasts and molds after 3, 4 and 5 days incubation.
allows these colonies to be easily recognized and enumerated.
Reduced recovery of yeasts may be encountered due to increased Expected Results
activity of rose bengal at pH 5.6.1 Agar is the solidifying agent. Colonies of molds and yeasts should be apparent within 5 days
of incubation. Colonies of yeast appear pink due to the uptake
of rose bengal. Report the results as colony-forming units per
gram or milliliter of sample.
178
Aspergillus niger
User Quality Control ATCC™ 1015
Identity Specifications
Difco™ DRBC Agar
Dehydrated Appearance: Pink, free-flowing, homogeneous.
Solution: 3.16% solution, soluble in purified water upon
boiling.Solution is reddish pink, very slightly to
slightly opalescent.
Prepared Appearance: Bright pink, very slightly to slightly opalescent.
Reaction of 3.16%
Solution at 25°C: pH 5.6 ± 0.2
Cultural Response
Difco™ DRBC Agar
Prepare the medium per label directions. Inoculate and incubate at
25 ± 2°C for up to 5 days. For A. niger, spot inoculate.
INOCULUM
D
Organism ATCC™ CFU RECOVERY
Aspergillus niger 1015 Undiluted Good
Candida albicans 10231 10 -10
2 3
Good
Escherichia coli 25922 103 None to poor
Micrococcus luteus 10240 103 None to poor
179
Identity Specifications
Difco™ Decarboxylase Base Moeller
Dehydrated Appearance: Light to medium tan, free-flowing, homogeneous.
Solution: 1.05% solution, soluble in purified water upon boiling.
Solution is yellowish-red, slightly opalescent.
Prepared Appearance: Yellowish-red, very slightly opalescent.
Reaction of 1.05%
Solution at 25°C: pH 6.0 ± 0.2
Difco™ Decarboxylase Medium Base
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 0.9% solution, soluble in purified water upon warming.
Solution is purple, clear. Escherichia coli E. coli Shigella S. flexneri
ATCC™ 25922 w/Lysine flexneri w/Lysine
Prepared Appearance: Purple, clear. ATCC™ 25922 ATCC™ 12022 ATCC™ 12022
Reaction of 0.9% Decarboxylase Base Moeller
Solution at 25°C: pH 6.8 ± 0.2
Difco Lysine Decarboxylase Broth
™
Cultural Response
Difco™ Decarboxylase Base Moeller
Prepare the medium per label directions with and without 1% L-lysine HCl.
Inoculate tubes, overlaying with sterile mineral oil, and incubate at 35 ± 2°C for
18-48 hours. Purple color indicates a positive decarboxylase reaction; a yellow
color is negative.
reaction REACTIOn
INOCULUM without WITH Uninoculated Salmonella Proteus vulgaris
ORGANISM ATCC™ CFU RECOVERY lysine LYSINE Tube Typhimurium ATCC™ 13315
ATCC™ 14028
Escherichia coli 25922 103 Good Yellow Purple
Decarboxylase Medium Base
Shigella flexneri 12022 103 Good Yellow Yellow
Prepare the medium per label directions. Inoculate tubes, overlaying with sterile
mineral oil, and incubate at 35 ± 2°C for 18-48 hours. Purple color indicates a positive
decarboxylase reaction; a yellow color is negative.
Escherichia coli Proteus vulgaris
ORGANISM ATCC™ INOCULUM CFU RECOVERY REACTION ATCC™ 25922 ATCC™ 13315
Escherichia coli 25922 103 Good + Lysine Decarboxylase Broth
180
Falkow obtained valid and reliable results with a lysine decar- To obtain the appropriate reactions, the inoculated tubes
boxylase medium he developed to differentiate and identify must be protected from air with a layer of sterile mineral oil.
Salmonella and Shigella.9 Although his modification of the Exposure to air may cause alkalinization at the surface of
Moeller formula was originally described as a lysine medium the medium, which could cause a decarboxylase-negative
only, further study by Falkow and then by Ewing, Davis and organism to appear positive.
Edwards,10 substantiated the use of the medium for ornithine
and arginine decarboxylase reactions as well. Formulae
Difco™ Decarboxylase Base Moeller
Ewing, Davis and Edwards10 compared the Falkow decar- Approximate Formula* Per Liter
boxylase medium base to the Moeller medium and reported Peptone....................................................................... 5.0 g
that, although the two methods compared favorably in most Beef Extract.................................................................. 5.0 g
Dextrose...................................................................... 0.5 g
cases, the Moeller medium was found to be more reliable for
Bromcresol Purple........................................................ 0.01 g
cultures of Klebsiella and Enterobacter. They concluded that Cresol Red................................................................... 5.0 mg
the Moeller method should be regarded as the standard or Pyridoxal...................................................................... 5.0 mg
reference method, although the Falkow fomula is suitable for BBL™ Moeller Decarboxylase Broth Base
determining decarboxylase reactions for most members of the Approximate Formula* Per Liter
Enterobacteriaceae except for Klebsiella and Enterobacter. Peptic Digest of Animal Tissue...................................... 5.0 g
The Moeller medium is also particularly useful in the iden- Beef Extract.................................................................. 5.0 g
tification of Aeromonas, Plesiomonas, Vibrio spp. and Dextrose...................................................................... 0.5 g
Bromcresol Purple........................................................ 0.01 g
nonfermentative gram-negative bacilli.11
Cresol Red................................................................... 5.0 mg
Decarboxylase tests are important in the differentiation and Pyridoxal...................................................................... 5.0 mg
identification of a wide variety of microorganisms and are
outlined in numerous standard methods.12-15
181
182
D
7th ed. American Society for Microbiology, Washington, D.C. BAM CCAM COMPF ISO SMD SMWW
8. Kiska and Gilligan. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical
microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
Cat. No. 287220 Dehydrated – 500 g
9. Falkow. 1958. Am. J. Clin. Pathol. 29:598.
10. Ewing, Davis and Edwards. 1960. Publ. Health Lab. 18:77. Difco™ Lysine Decarboxylase Broth
11. Baron, Peterson and Finegold. 1994. Bailey & Scott’s diagnostic microbiology, 9th ed. Mosby-Year
Book, Inc., St. Louis, Mo. BAM CCAM COMPF ISO SMD SMWW
12. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed. Cat. No. 211759 Dehydrated – 500 g
American Society for Microbiology, Washington, D.C.
13. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna- *Store at 2-8°C.
tional, Gaithersburg, Md.
14. Eaton, Rice and Baird (ed.) 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C.
15. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
16. MacFaddin. 2000. Biochemical tests for identification of medical bacteria, 3rd ed. Lippincott Williams
& Wilkins, Baltimore, Md.
Cultural Response
Difco™ Demi-Fraser Broth Base
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for
24-48 hours.
RECOVERY/
Organism ATCC™ INOCULUM CFU APPEARANCE
Enterococcus faecalis 29212 103-2×103 Partial to complete Uninoculated Listeria monocytogenes
inhibition Tube ATCC™ 19114
185
References Availability
1. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
American Society for Microbiology, Washington, D.C.
BBL™ Dermatophyte Test Medium Base
2. Forbes, Sahm and Weissfeld. 1994. Bailey & Scott’s diagnostic microbiology, 10th ed. Mosby, Inc., Cat. No. 212330 Dehydrated – 500 g
St. Louis, Mo.
3. Kane and Summerbell. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of BBL™ Dermatophyte Test Medium, Modified
clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
4. Kwon-Chung and Bennett. 1992. Medical mycology. Lea & Febiger, Philadelphia, Pa. with Chloramphenicol
5. Taplin, Zaias, Rebell and Blank. 1969. Arch. Dermatol. 99:203-209.
6. Larone. 1995. Medically important fungi: a guide to identification, 3rd ed. American Society for BS12 MCM9
Microbiology, Washington, D.C. United States and Canada
Cat. No. 299636 Prepared Plates – Pkg. of 10*
299701 Prepared Slants – Pkg. of 10*
Europe
Cat. No. 254429 Prepared Plates – Pkg. of 20*
*Store at 2-8°C.
Desoxycholate Agar
Intended Use Principles of the Procedure
Desoxycholate Agar is a slightly selective and differential Peptone provides nitrogen and carbon for general growth
plating medium used for isolating and differentiating gram- requirements. Lactose is the fermentable carbohydrate. Sodium
negative enteric bacilli. chloride and dipotassium phosphate maintain the osmotic
balance of the medium. Sodium desoxycholate, ferric citrate and
Summary and Explanation sodium citrate inhibit growth of gram-positive bacteria. Neutral
Desoxycholate Agar as formulated by Leifson1 demonstrated red is a pH indicator. Agar is the solidifying agent.
improved recovery of intestinal pathogens from specimens Differentiation of enteric bacilli is based on fermentation
containing normal intestinal flora. The medium was an of lactose. Bacteria that ferment lactose produce acid and, in
improvement over other media of the time because the the presence of neutral red, form red colonies. Bacteria that
chemicals, citrates and sodium desoxycholate, in specified do not ferment lactose form colorless colonies. The majority of
amounts, worked well as inhibitors. This medium has been used normal intestinal bacteria ferment lactose (red colonies), while
to screen for Salmonella spp. and Shigella spp. from clinical Salmonella and Shigella species do not ferment lactose (color-
specimens.2 less colonies).
Enterobacter aerogenes
User Quality Control ATCC™ 13048
Identity Specifications
Difco™ Desoxycholate Agar
Dehydrated Appearance: Pinkish beige, free-flowing, homogeneous.
Solution: 4.5% solution, soluble in purified water upon
boiling. Solution is reddish-orange, slightly
opalescent.
Prepared Appearance: Orange, slightly opalescent.
Reaction of 4.5%
Solution at 25°C: pH 7.3 ± 0.2
Cultural Response
Difco™ Desoxycholate Agar
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 18-24 hours.
Inoculum COLONY
Organism ATCC™ CFU RECOVERY COLOR
Enterococcus faecalis 29212 103-2×103 Marked –
inhibition
Escherichia coli 25922 30-300 Good Pink w/bile
precipitate
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028 30-300 Good Colorless
186
Formula Procedure
Difco™ Desoxycholate Agar For a complete discussion on the isolation of enteric bacilli, refer
Approximate Formula* Per Liter to appropriate procedures outlined in the references.2-4
Peptone..................................................................... 10.0 g
Lactose ..................................................................... 10.0 g
Sodium Desoxycholate................................................. 1.0 g Expected Results
Sodium Chloride.......................................................... 5.0 g Refer to appropriate references and procedures for results.2-4
Dipotassium Phosphate................................................ 2.0 g
Ferric Ammonium Citrate............................................. 1.0 g References
Sodium Citrate............................................................. 1.0 g 1. Leifson. 1935. J. Pathol. Bacteriol. 40:581.
Agar.......................................................................... 15.0 g 2. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
Neutral Red.................................................................. 0.03 g American Society for Microbiology, Washington, D.C.
3. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
*Adjusted and/or supplemented as required to meet performance criteria. 21st ed., online. American Public Health Association, Washington, D.C.
4. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
Directions for Preparation from
Dehydrated Product Availability
1. Suspend 45 g of the powder in 1 L of purified water. Mix Difco™ Desoxycholate Agar
thoroughly. COMPF SMWW
2. Heat with frequent agitation and boil for 1 minute to
completely dissolve the powder. Avoid overheating. DO
Cat. No. 227310 Dehydrated – 500 g D
Europe
NOT AUTOCLAVE. Cat. No. 254010 Prepared Plates – Pkg. of 20*
3. Test samples of the finished product for performance using Japan
stable, typical control cultures. Cat. No. 251550 Prepared Plates – Pkg. of 20*
251824 Prepared Plates – Ctn. of 200*
251507 Prepared RODAC™ Plates – Pkg. of 30*
*Store at 2-8°C.
187
Identity Specifications
Difco™ Desoxycholate Citrate Agar
Dehydrated Appearance: Pinkish-beige, free-flowing, homogeneous.
Solution: 7.0% solution, soluble in purified water upon
boiling. Solution is orange-red, very slightly to
slightly opalescent.
Prepared Appearance: Orange-red, slightly opalescent.
Reaction of 7.0%
Solution at 25°C: pH 7.5 ± 0.2
Cultural Response
Difco™ Desoxycholate Citrate Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C
for 18-24 hours.
Inoculum colony
Organism ATCC™ CFU RECOVERY color H2S
Enterococcus 29212 10 -2×10 Marked to
3 3
– –
faecalis complete
inhibition
Escherichia coli 25922 102-103 Partial to Pink –
complete with bile
inhibition precipitate
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028 102-103 Fair to good Colorless +
Shigella flexneri 12022 102-103 Fair Colorless –
Procedure
1. Inoculate specimen directly onto surface of medium.
2. Incubate plates at 35 ± 2°C for 18-24 hours. Plates can
be incubated for an additional 24 hours if no lactose
fermenters are observed.
Expected Results
Lactose nonfermenters produce transparent, colorless to light
pink or tan colored colonies with or without black centers.
Lactose fermenters produce a red colony with or without a bile
precipitate.
188
of these manuals.
Directions for Preparation from
Principles of the Procedure Dehydrated Product
Peptone provides nitrogen and carbon for general growth 1. Suspend 42.5 g of the powder in 1 L of purified water. Mix
requirements. Lactose is a fermentable carbohydrate. Sodium thoroughly.
chloride maintains the osmotic balance of the medium.
Cultural Response
Difco™ Desoxycholate Lactose Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C
for 18-24 hours.
Inoculum COLONY
Organism ATCC™ CFU RECOVERY COLOR
Bacillus subtilis 6633 ~103 Inhibition –
Enterobacter 13048 30-300 Good Pink, may
aerogenes have slight bile
precipitate
Enterococcus faecalis 29212 ~103 Inhibition –
Escherichia coli 25922 30-300 Good Pink w/bile
precipitate
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028 30-300 Good Colorless
189
Expected Results
Refer to appropriate references and procedures for results.2,3
190
Dextrose Agar and Dextrose Broth are specified in the OPTIONAL: To prepare blood agar, aseptically add 5%
Compendium of Methods for the Microbiological Examination sterile defibrinated blood to the medium at 45-50°C. Mix well
of Foods.3 and dispense as desired.
191
192
Identity Specifications
Difco™ Dextrose Tryptone Agar
Dehydrated Appearance: Light, greenish-beige, free-flowing, homoge-
neous.
Solution: 3.0% solution, soluble in purified water upon
boiling. Solution is purple, slightly opalescent.
Prepared Appearance: Purple, slightly opalescent without significant
precipitate.
Reaction of 3.0%
Solution at 25°C: pH 6.7 ± 0.2
Cultural Response
Difco™ Dextrose Tryptone Agar
Prepare the medium per label directions. Inoculate plates by the pour plate
method and incubate at 55 ± 2°C for 40-48 hours.
Organism ATCC™
INOCULUM
CFU RECOVERY
DEXTROSE
FERMENTATION D
Bacillus coagulans 7050 102-103 Good + (yellow)
Bacillus
stearothermophilus 7953 102-103 Good + (yellow)
193
Identity Specifications
Difco™ Differential Reinforced Clostridial Agar
Dehydrated Appearance: Light tan, free-flowing, homogeneous.
Solution: 4.25% solution, soluble in purified water upon
boiling. Solution is light to medium amber, clear
to slightly opalescent while hot; upon cooling,
solution becomes light red.
Prepared Appearance: Light pink, clear to slightly opalescent without
significant precipitate.
Reaction of 4.25%
Solution at 25°C: pH 7.1 ± 0.2
Cultural Response
Difco™ Differential Reinforced Clostridial Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C
in an anaerobic atmosphere for 72 hours.
INOCULUM Black
Organism ATCC™ CFU Recovery Colonies
Clostridium bifermentans 638 102-103 Good +
Clostridium perfringens 12924 102-103 Good +
Clostridium septicum 12464 102-103 Good +
Procedure References
1. Gibbs and Freame. 1965. J. Appl. Microbiol. 28:95.
1. Prepare serial 10-fold dilutions of the sample in 1/4 strength 2. Miller, Gerrett and Prickett. 1939. Food Res. 4:447.
Ringer’s solution or 0.1% peptone water.
2. Depending on the amount of the initial sample, transfer Availability
1 mL or 0.1 mL of the appropriate dilution, prepared in Difco™ Differential Reinforced Clostridial Agar
step 1, to the bottom of a molten (45-50°C) DRCA tube. Cat. No. 264120 Dehydrated – 500 g
Prepare a duplicate tube using the same procedure.
3. Tighten the caps on the tubes.
194
Dubos Media
Dubos Broth Base • Dubos Medium Albumin
Dubos Oleic Agar Base • Dubos Oleic Albumin
Complex • Dubos Broth, Enriched
Intended Use Mycobacteria grow more rapidly in broth media. Primary culture
Dubos Broth Base is used with Dubos Medium Albumin for of all specimens in broth media is recommended.10 Polysorbate
rapidly cultivating pure cultures of Mycobacterium tuberculosis. 80 in the medium acts as a surfactant, dispersing the bacilli,
which increases growth.
Dubos Oleic Agar Base is used with Dubos Oleic Albumin
Complex and penicillin for isolating and determining the Dubos Broth, Enriched is a modified medium based on the
susceptibility of M. tuberculosis. formulation of Dubos et al.4 This formulation differs from the
original in that it has a strong buffering system and an acid
Dubos Broth, Enriched is a prepared medium used for the
cultivation of pure cultures of M. tuberculosis.
pH.11 The particular value of Dubos Broth, Enriched is that
it provides dispersed growth, free of excessive clumps, which
D
can be used to prepare a relatively uniform suspension of
Summary and Explanation
mycobacteria for use in bacterial studies. It is also used as a
Mycobacterial infections, particularly tuberculosis, are a
subculture and enrichment medium for the rapid cultivation of
worldwide health problem. Almost three million people
M. tuberculosis and other mycobacterial species from treated
worldwide die of tuberculosis each year.1 During the mid
clinical specimens and from direct inoculation of specimens that
1980s, the number of tuberculosis (TB) cases in the U.S.
may yield pure cultures; e.g., cerebrospinal fluid.12
began increasing. Prior to this time, the number of cases
in the U.S. had been decreasing, reaching a low in 1984.2
Principles of the Procedure
Non-tuberculous mycobacterial infections have also increased
Peptone and asparagine are sources of nitrogen. Disodium
since the mid 1980s.3
phosphate and monopotassium phosphate are sources of
Dubos Broth is prepared according to the Dubos, Fenner phosphates and, along with calcium chloride, help maintain
and Pierce4 modification of the medium originally described the pH of the medium. Magnesium sulfate, ferric ammonium
by Dubos and Davis5 and Dubos and Middlebrook.6 sulfate, zinc sulfate and copper sulfate are sources of trace
Dubos and Middlebrook6 described Dubos Oleic Medium metals and sulfates. Polysorbate 80, an oleic acid ester,
Albumin as suitable for primary isolation and cultivation of supplies essential fatty acids for the replication of mycobac-
the tubercle bacillus and for studying colony morphology. teria. Bovine albumin acts as a protective agent by binding
In comparative studies, Dubos Oleic Albumin Agar Medium was free fatty acids that may be toxic to mycobacteria. The
superior to other media studied for primary isolation.7,8 albumin is heat-treated to inactivate lipase, which may
release fatty acids from the polysorbate 80. Phosphate
There are two types of solid culture media for the primary buffers maintain the pH of the medium. Agar is the solidifying
isolation of mycobacteria, those that have coagulated egg as agent.
a base and those that have agar. Lowenstein formulations are
examples of media that contain egg; Middlebrook and Dubos Formulae
formulations contain agar. Difco™ Dubos Broth Base
Agar based media are not liquefied by contaminating Approximate Formula* Per Liter
Pancreatic Digest of Casein.......................................... 0.5 g
proteolytic organisms but overgrowth may occur. These Asparagine................................................................... 2.0 g
media are recommended for specimens from nonsterile sites.9 Polysorbate 80............................................................. 0.2 g
The medium is clear so colonies of mycobacteria can be viewed Monopotassium Phosphate.......................................... 1.0 g
Disodium Phosphate (anhydrous)................................. 2.5 g
through a stereo microscope even if contaminating organisms Ferric Ammonium Citrate........................................... 50.0 mg
are present. Colonies can be observed in 10-12 days. Magnesium Sulfate.................................................... 10.0 mg
Calcium Chloride......................................................... 0.5 mg
Drugs may be added to Dubos media in exact concentrations Zinc Sulfate.................................................................. 0.1 mg
because the medium is solidified with agar rather than by Copper Sulfate............................................................. 0.1 mg
inspissation. Also, there is less drug inactivation when egg Difco™ Dubos Medium Albumin
ingredients are not present. A 5% solution of albumin fraction V from bovine plasma and
7.5% dextrose in normal saline (0.85%).
195
196
Procedure References
The test procedures are recommended by the Centers for 1. Musser. 1995. Clin. Microbiol. Rev. 8:496.
2. Klietmann. 1995. Clin. Microbiol. Newsl. 17:65.
Disease Control and Prevention (CDC) for primary isolation 3. Metchock, Nolte and Wallace. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual
of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
from specimens containing mycobacteria.13 N-acetyl-L-cysteine- 4. Dubos, Fenner and Pierce. 1950. Am. Rev. Tuberc. 61:66.
5. Dubos and Davis. 1946. J. Exp. Med. 83:409.
sodium hydroxide (NALC-NaOH) solution is recommended 6. Dubos and Middlebrook. 1947. Am. Rev. Tuberc. 56:334.
7. Roberts, Wallace and Erlich. 1950. Am. Rev. Tuberc. 61:563.
as a gentle, but effective digesting and decontaminating agent. 8. Byham. 1950. Am. J. Clin. Pathol. 20:678.
These reagents are provided in the BBL™ MycoPrep™ Specimen 9. Isenberg (ed.). 1994. Clinical microbiology procedures handbook, suppl. 1. American Society for
Microbiology, Washington, D.C.
Digestion/Decontamination Kit. For detailed decontamination 10. Tenover, Crawford, Huebner, Geiter, Horsburgh and Good. 1993. J. Clin. Microbiol. 31:767.
11. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol.
and culturing instructions, consult an appropriate text.3,9,12,13,15 1 Williams & Wilkins, Baltimore, Md.
12. Cernoch, Enns, Saubolle and Wallace. 1994. Cumitech 16A, Laboratory diagnosis of the mycobacte-
Specimens that are less likely to be contaminated with other rioses. Coord. Ed. Weissfeld, American Society for Microbiology, Washington, D.C.
13. Kent and Kubica. 1985. Public health mycobacteriology: a guide for the level III laboratory. USDHHS,
microorganisms (cerebrospinal fluid, pleural fluid, tissue Centers for Disease Control, Atlanta, Ga.
14. U.S. Public Health Service, Centers for Disease Control and Prevention, and National Institutes of
biopsy, etc.) may be inoculated directly into the medium. Consult Health. 2007. Biosafety in microbiological and biomedical laboratories, 5th ed. HHS Publication No.
(CDC) 93-8395. U.S. Government Printing Office, Washington, D.C.
appropriate texts for recommended procedures.3,9,12,13,15 15. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby, Inc.,
St. Louis, Mo.
Incubate the tubes at 35 ± 2°C in a CO2-enriched atmosphere.
Keep the tube caps loosened for at least one week to permit Availability
circulation of CO2, but tighten the caps thereafter to prevent Difco™ Dubos Broth Base
dehydration. Loosen briefly once a week to replenish CO2. Cat. No. 238510 Dehydrated – 500 g
Six to eight weeks of incubation may be necessary for evidence Difco™ Dubos Medium Albumin
of growth of many mycobacteria. AOAC
Cat. No. 230910 Tube, 20 mL – Pkg. of 12*
Expected Results
Growth of mycobacterial colonies on the agar medium or
Difco™ Dubos Oleic Agar Base
Cat. No. 237310 Dehydrated – 500 g E
in broth media, as indicated by turbidity compared to an
uninoculated control. Difco™ Dubos Oleic Albumin Complex
Cat. No. 237510 Tube, 20 mL – Pkg. of 12*
Limitations of the Procedure BBL™ Dubos Broth, Enriched
1. Negative culture results do not rule-out active infection Cat. No. 295697 Prepared Tubes – Pkg. of 10*
by mycobacteria. Some factors that are responsible for *Store at 2-8°C.
197
198
199
EC Medium
Intended Use Formula
EC Medium is a culture medium for the detection of coliform Difco™ EC Medium
bacteria at 35°C and of Escherichia coli at an elevated tempera- Approximate Formula* Per Liter
ture (44.5 or 45.5°C). Tryptose..................................................................... 20.0 g
Lactose........................................................................ 5.0 g
Bile Salts No. 3............................................................. 1.5 g
Summary and Explanation Dipotassium Phosphate................................................ 4.0 g
EC Medium was devised by Hajna and Perry1 and is used for Monopotassium Phosphate.......................................... 1.5 g
Sodium Chloride.......................................................... 5.0 g
the examination of water, milk, shellfish and other material for *Adjusted and/or supplemented as required to meet performance criteria.
evidence of fecal pollution. Tennant et al. reported on the use of
this medium for the estimation of E. coli densities in seawater Directions for Preparation from
and shellfish.2 Fishbein and Surkiewicz used the EC confirma- Dehydrated Product
tion test for recovery of E. coli from frozen foods and nut meats 1. Dissolve 37 g of the powder in 1 L of purified water. Mix
and reported that the test worked optimally when conducted at thoroughly.
45.5°C with incubation being limited to 24 hours.3 2. Warm slightly to completely dissolve the powder.
EC Medium is recommended for use in the fecal coliform 3. Dispense into tubes containing inverted fermentation vials.
Most Probable Number (MPN) procedure for the examination 4. Autoclave at 121°C for 15 minutes.
of water, wastewater and foods.4,5 The procedure employing 5. Test samples of the finished product for performance using
EC Medium provides information regarding the source of the stable, typical control cultures.
coliform group (fecal or nonfecal) when used as a confirmatory
test.6 It should not be used for the direct isolation of coliforms Procedure
since prior enrichment in a presumptive medium for optimal Refer to the various compendia for the specific procedures
recovery of fecal coliforms is required. employing EC Medium.4-8
Cultural Response
Difco™ EC Medium
Prepare the medium per label directions. Inoculate and incubate tubes with fermentation
vials at 44.5 ± 0.2°C for 24 ± 2 hours.
INOCULUM
ORGANISM ATCC™ CFU RECOVERY GAS
Enterococcus faecalis 19433 103 Inhibition –
Escherichia coli 25922 103 Good + Uninoculated Escherichia coli
Tube ATCC™ 25922
Escherichia coli 8739 103 Good +
200
References Availability
1. Hajna and Perry. 1943. Am. J. Public Health 33:550.
2. Tennant, Reid, Rockwell and Bynoe. 1961. Can. J. Microbiol. 1:733.
Difco™ EC Medium
3. Fishbein and Surkiewicz. 1964. Appl. Microbiol. 12:127. AOAC BAM CCAM COMPF EPA ISO SMD SMWW
4. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C. Cat No. 231420 Dehydrated – 100 g
5. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods, 231430 Dehydrated – 500 g
4th ed. American Public Health Association, Washington, D.C.
6. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy product, online. American
231410 Dehydrated – 10 kg
Public Health Association, Washington, D.C.
7. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
tional, Gaithersburg, Md.
8. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
International, Gaithersburg, Md.
9. Ray. 1986. J. Food. Prot. 49:651.
Cultural Response
Difco™ EC Medium with MUG
Prepare the medium per label directions. Inoculate tubes in duplicate with fresh 18-24 hour cultures. Incubate
the first set at 35 ± 2°C for 24 ± 2 hours and the second set at 44.5 ± 0.2°C for 24 ± 2 hours. Read fluorescence
under a long-wave UV light.
RECOVERY at RECOVERY at
Organism ATCC™ 35°C/Gas 44.5°C/Gas Fluorescence
Enterobacter aerogenes 13048 Good/± Inhibition to good/– –
Enterococcus faecalis 19433 Inhibition/– Inhibition to good/– –
Escherichia coli 25922 Good/+ Good/+ + Escherichia coli
ATCC™ 25922
201
EC Medium, Modified
Novobiocin Antimicrobic Supplement
Intended Use Principles of the Procedure
EC Medium, Modified is used with Novobiocin Antimicrobic Peptone supports good growth of E. coli O157:H7 and is rich
Supplement in the detection of Escherichia coli O157:H7 in in peptides and nitrogen. Lactose is an additional source of
meat and poultry products. carbon for organisms, such as E. coli, that can ferment this
sugar. Dipotassium phosphate and monopotassium phosphate
Summary and Explanation are buffers that facilitate recovery of injured cells. Sodium
EC Medium, Modified and Novobiocin Antimicrobic Supple- chloride provides a suitable ionic environment for growth of
ment are based on the formula for modified EC broth with microorganisms.
novobiocin (mEC+n) as described by Okrend and Rose.1 In Selectivity of the medium is achieved by the incorporation of Bile
modifying the EC Medium formula, Okrend and Rose reduced Salts No. 3 into the base medium and by the addition of sodium
the Bile Salts No. 3 from 1.5 g per liter to 1.12 g per liter and novobiocin to the complete medium. These agents suppress
added 20 mg per liter of sodium novobiocin. Okrend et al. the growth of nuisance organisms commonly found in foods.
reported that mEC+n was useful in the enrichment and detection The sodium novobiocin is provided in the freeze-dried state
of E. coli O157:H7 from meats and poultry products.2-4 as Novobiocin Antimicrobic Supplement. This supplement is
rehydrated before use with sterile purified water.
202
203
Formulae
Difco™ EE Broth Mossel Enrichment
Approximate Formula* Per Liter
Pancreatic Digest of Gelatin....................................... 10.0 g
Dextrose...................................................................... 5.0 g
Disodium Phosphate.................................................... 8.0 g
Monopotassium Phosphate.......................................... 2.0 g
Brilliant Green............................................................ 15.0 mg
Oxgall........................................................................ 20.0 g
BBL™ EE Broth Mossel Enrichment
Approximate Formula* Per Liter
Pancreatic Digest of Gelatin....................................... 10.0 g
Dextrose...................................................................... 5.0 g
Oxgall........................................................................ 20.0 g
Disodium Phosphate.................................................... 8.0 g
Monopotassium Phosphate.......................................... 2.0 g
Brilliant Green............................................................ 15.0 mg
*Adjusted and/or supplemented as required to meet performance criteria.
Uninoculated Escherichia coli
Tube ATCC™ 25922
204
Prepare the medium per label directions. Inoculate 9 mL tubes and incu- BBL™ EE Broth Mossel Enrichment (prepared)
bate at 35 ± 2°C for 18-24 hours and 48 hours, if necessary. Appearance: Medium to dark green and clear to trace hazy.
ORGANISM ATCC
™
INOCULUM CFU RECOVERY ACID Reaction at 25°C: pH 7.2 ± 0.2
Enterobacter
aerogenes 13048 30-100 Good + (yellow) Cultural Response
Escherichia coli 25922 30-100 Good + (yellow)
BBL™ EE Broth Mossel Enrichment
Prepare the medium per label directions. Inoculate 10 mL tubes and
Shigella boydii 12030 30-100 Good – incubate at 35 ± 2°C for 18-24 hours and 48 hours, if necessary.
Staphylococcus Marked to
E
ORGANISM ATCC™ INOCULUM CFU RECOVERY ACID
aureus 25923 30-100 complete inhibition –
Escherichia coli 25922 10 -10
3 4
Good + (yellow)
Inoculate 100 mL bottles and incubate at 30-35°C for 18-24 hours and Pseudomonas
48 hours, if necessary. Inoculate a 20 mL tube with Escherichia coli ATCC aeruginosa 10145 103-104 Good –
8739 and incubate at 35-37°C for 18-48 hours.
Salmonella enterica
INOCULUM INCUBATION INCUBATION subsp. enterica
ORGANISM ATCC™ CFU TEMP TIME (HOURS) RECOVERY serotype Typhimurium 14028 103-104 Good + (yellow)
Escherichia coli 8739 <100 30-35°C 24 Growth Shigella sonnei 9290 103-104 Good – to reduced
Escherichia coli 8739 <100 35-37°C 18-48 Growth (yellow green)
Pseudomonas
Inoculate 100 mL bottles and incubate at 30-35°C for 18-24 hours and
aeruginosa 9027 <100 30-35°C 24 Growth
48 hours, if necessary. Inoculate a 20 mL tube with Escherichia coli
Staphylococcus ATCC 8739 and incubate at 35-37°C for 18-48 hours.
aureus 6538 >100 30-35°C 48 No growth
INOCULUM INCUBATION INCUBATION
ORGANISM ATCC™ CFU TEMP TIME (HOURS) RECOVERY
Escherichia coli 8739 <100 30-35°C 24 Growth
Escherichia coli 8739 <100 35-37°C 18-48 Growth
Pseudomonas
aeruginosa 9027 <100 30-35°C 24 Growth
Staphylococcus
aureus 6538 >100 30-35°C 48 No growth
205
References Availability
1. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The national
formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc., Rockville, Difco™ EE Broth Mossel Enrichment
Md.
COMPF EP ISO JP USP
2. European Directorate for the Quality of Medicines and Healthcare. 2008. The European pharma-
copoeia, 6th ed., Supp. 1, 4-1-2008, online. European Directorate for the Quality of Medicines Cat. No. 256620 Dehydrated – 500 g†
and Healthcare, Council of Europe, 226 Avenue de Colmar BP907-, F-67029 Strasbourg Cedex 1,
France. BBL™ EE Broth Mossel Enrichment
3. Japanese Ministry of Health, Labour and Welfare. 2006. The Japanese pharmacopoeia, 15th ed.,
online. Japanese Ministry of Health, Labour and Welfare. COMPF EP ISO JP USP
4. Mossel, Vissar and Cornelisen. 1963. J. Appl. Bacteriol. 26:444.
5. Hartman and Minnich. 1981. J. Food Prot. 44:385. Cat. No. 297005 Dehydrated – 500 g†
6. International Organization for Standardization. 2004 Microbiology of food and animal feeding stuffs 292627 Prepared Bottles, 90 mL (wide mouth) –
– horizontal methods for the detection and enumeration of Enterobacteriaceae – Part 1: Detection and Pkg. of 10†
enumeration by MPN technique with pre-enrichment. ISO 21528-1, 1st ed., 2004-08-15. International
Organization for Standardization, Geneva, Switzerland. † QC testing performed according to USP/EP/JP performance specifications.
7. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
mEI Agar
Intended Use ments than fecal coliforms.2 In 1986, the USEPA recommended
mEI Agar is a selective culture medium used for the chromo- that both Escherichia coli and enterococci be used as bacterial
genic detection and enumeration of enterococci in water by the water quality indicators to monitor recreational waters.3
single-step membrane filtration technique. It conforms with U.S. A two-step membrane filter (MF) method4 was developed by
Environmental Protection Agency (USEPA) Approved Method Levin et al. to measure enterococci in fresh and marine recre-
1600: Enterococci in Water by Membrane Filtration Using ational waters. Using mE agar, the method required a 48-hour
membrane-Enterococcus Indoxyl-β-D-Glucoside Agar (mEI). incubation and a transfer of the membrane to another substrate
medium, Esculin Iron Agar, to differentiate enterococci.
Summary and Explanation
Enterococci are found in the feces of humans and other warm- In 1997, the USEPA improved on the mE agar formulation
blooded animals. Although some strains are ubiquitous and by reducing the triphenyltetrazolium chloride component
are not related to fecal pollution, the presence of enterococci and adding the chromogen, indoxyl-β-D-glucoside. The new
in water is an indication of fecal pollution and the possible medium, mEI Agar,1,5 was developed as a single-step procedure
presence of enteric pathogens.1 In epidemiological studies that does not require the transfer of the membrane filter
conducted by the USEPA, it was found that the presence to another substrate. Observation of a blue halo around
of enterococci had a higher correlation with swimming- colonies in 24 hours is confirmatory for the presence of
associated gastroenteritis in fresh and marine water environ- enterococci. A wide range of sample volumes or dilutions can
Identity Specifications
Difco™ mEI Agar
Dehydrated Appearance: Light to medium beige, free-flowing, homoge-
neous.
Solution: 7.2% solution, soluble in purified water upon
boiling. Solution is medium to dark amber, very
slightly to slightly opalescent.
Prepared Appearance: Light to medium amber, clear to very slightly
opalescent.
Reaction of 7.2%
Solution at 25°C: pH 7.1 ± 0.2
Cultural Response
Difco™ mEI Agar
Prepare the medium per label directions. Inoculate and incubate at 41± 0.5°C
for 24 ± 2 hours. Count all colonies with blue halos.
INOCULUM
ORGANISM ATCC™ CFU RECOVERY appearance
Enterococcus faecalis 19433 20-80 Good Blue halo
Enterococcus faecium 19434 20-80 Good Blue halo
Escherichia coli 25922 20-80 Marked to –
complete inhibition
206
be tested by this single-step MF procedure for the detection and 7. Test samples of the finished product for performance using
enumeration of enterococci in potable, fresh, estuarine, marine stable, typical control cultures.
and shellfish-growing waters.
BD mEI Agar conforms to the 1986 revisions to the bacte-
Procedure
1. Collect and prepare water samples in accordance to
riological ambient water quality criteria, that included the
recommended guidelines.7,8
indicator bacteria E. coli and enterococci, which provide
2. Test sample volumes following the membrane filtration pro-
better correlation with swimming-associated gastrointestinal
cedure described in Standard Methods for the Examination
illness. In response to this health risk, the USEPA established
of Water and Wastewater.7 Select sample volumes to produce
the Beaches Environmental Assessment Closure and Health
20-60 colonies on the membrane filter.
(Beach) Program. This method is published for use in the Beach
3. After sample has been filtered, aseptically remove membrane
Program.5
filter from filter base and roll it onto mEI Agar to avoid the
The USEPA published false-positive rate is 6.0% and false- formation of bubbles between the membrane and the agar
negative rate is 6.5%.5 Colonies having a blue halo can be surface.
verified as enterococci by appropriate biochemical procedures 4. Invert inoculated plates and incubate for 24 ± 2 hours at
in instances where required in evidence gathering or for 41 ± 0.5°C.
performing quality control for the initial use of the test.5 5. After incubation, count and record the number of colo-
nies with a blue halo using an illuminated lens with a
Principles of the Procedure 2-5× magnification.
mEI Agar contains peptone that supplies nitrogen and carbon 6. Calculate and report the number of enterococci colonies per
compounds. Sodium chloride maintains osmotic equilibrium. 100 mL of sample.
Esculin is hydrolyzed by enterococci to form esculetin and
dextrose. Cycloheximide inhibits fungi. Sodium azide acts as a Expected Results E
selective agent to inhibit gram-negative bacteria. Yeast extract Colonies with a blue halo regardless of color may be presump-
provides trace elements, vitamins and amino acids. The tively identified as enterococci. Refer to the USEPA Microbiology
addition of the chromogen indoxyl-β-D-glucoside results Methods Manual, Part II, Section C, 3.5 for general counting
in the production of an insoluble indigo blue complex by rules.9
β-D-glucosidase-positive enterococci, which diffuses into the
surrounding medium, forming a blue halo around the colony.6 Limitations of the Procedure
Agar is incorporated into the medium as the solidifying agent. 1. Choose a water sample size that will result in 20-60 colonies
per filter.
Formula 2. Minimize the exposure of mEI Agar to light before and during
Difco™ mEI Agar incubation, as light may destroy the chromogen.
Approximate Formula* Per Liter 3. Overheating may cause darkening of the medium.2
Peptone..................................................................... 10.0 g
Sodium Chloride........................................................ 15.0 g
Esculin......................................................................... 1.0 g References
Cycloheximide.............................................................. 0.05 g 1. U.S. Environmental Protection Agency. 1997. Method 1600: Membrane filter test method for entero-
cocci in water. Publication EPA-821-R-97-004a. Office of Water, USEPA, Washington, D.C.
Sodium Azide............................................................... 0.15 g 2. U.S. Environmental Protection Agency. 2000. Improved enumeration methods for the recreational
Yeast Extract.............................................................. 30.0 g water quality indicators: enterococci and Escherichia coli. Publication EPA/821/R-97/004. Office of
Indoxyl-β-D-glucoside................................................... 0.75 g Science and Technology, USEPA, Washington, D.C.
3. U.S. Environmental Protection Agency. 1986. Bacteriological ambient water quality criteria:
Agar.......................................................................... 15.0 g availability. Fed. Reg. 51(45):8012.
*Adjusted and/or supplemented as required to meet performance criteria. 4. Levin, Fischer and Cabelli. 1975. Appl. Microbiol. 30:66.
5. U.S. Environmental Protection Agency. 2002. Method 1600: Enterococci in water by membrane filtra-
tion using membrane-enterococcus indoxyl-β-D-glucoside agar (mEI). Publication EPA-821-R-02-022.
Directions for Preparation from USEPA Office of Water, Office of Science and Technology, USEPA, Washington, DC.
6. Messer and Dufour. 1998. Appl. Environ. Microbiol. 64:678.
Dehydrated Product 7. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st (ed.), online. American Public Health Association, Washington, D.C.
1. Suspend 72 g of the powder in 1 L of purified water. 8. ASTM International. 2002. Annual book of ASTM standards. Water and environmental
technology. ASTM International, West Conshohocken, Pa.
Mix thoroughly. 9. Bordner, Winter and Scarpino (ed.). 1978. Microbiological methods for monitoring the
environment: water and wastes. Publication EPA-600/8-78-017. Environmental Monitoring and
2. Heat with frequent agitation and boil for 1 minute to Support Laboratory, Office of Research and Development, U.S. Environmental Protection Agency,
completely dissolve the powder. Cincinnati, Ohio.
EVA Broth
Intended Use User Quality Control
EVA (Ethyl Violet Azide) Broth is used for detecting and
confirming enterococci in water and other specimens as an Identity Specifications
indication of fecal contamination. Difco™ EVA Broth
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Summary and Explanation Solution: 3.58% solution, soluble in purified water. Solution
The presence of enterococci in water and other specimens is light amber, clear to very slightly opalescent.
indicates fecal contamination. Mallmann and Seligmann 1 Prepared Appearance: Light amber, clear to very slightly opalescent.
compared various enrichment media for detecting fecal strep- Reaction of 3.58%
tococci and found that Azide Dextrose Broth presumptively Solution at 25°C: pH 7.0 ± 0.2
identified the streptococci. However, because gram-positive
bacteria other than enterococci grow in that medium, con- Cultural Response
firmation is necessary. Litsky et al.2 studied various dyes and Difco™ EVA Broth
Prepare the medium per label directions. Inoculate and incubate at
selective agents and formulated a medium using ethyl violet
35 ± 2°C for 18-48 hours.
and sodium azide as selective agents. The medium known as
Ethyl Violet Azide (EVA) Broth is specific for enterococci. In Organism ATCC™ INOCULUM CFU RECOVERY
conjunction with Azide Dextrose Broth, EVA Broth is used Enterococcus faecalis 19433 102-103 Good
to confirm the presence of enterococci. Enterococcus faecalis 29212 10 -10
2 3
Good
Escherichia coli 25922 103 Inhibition
Principles of the Procedure
EVA Broth contains peptones as sources of carbon, nitrogen,
vitamins and minerals. Dextrose is the carbohydrate. Sodium
azide and ethyl violet inhibit gram-positive bacilli and
gram-positive cocci other than enterococci. Monopotassium
and dipotassium phosphates buffer the medium. Sodium
chloride provides osmotic balance.
Formula
Difco™ EVA Broth
Approximate Formula* Per Liter
Proteose Peptone No. 3................................................ 8.0 g
Pancreatic Digest of Casein........................................ 12.0 g
Dextrose...................................................................... 5.0 g
Dipotassium Phosphate................................................ 2.7 g
Monopotassium Phosphate.......................................... 2.7 g
Sodium Chloride.......................................................... 5.0 g
Sodium Azide............................................................... 0.4 g
Ethyl Violet................................................................... 0.83 mg
*Adjusted and/or supplemented as required to meet performance criteria.
Uninoculated Enterococcus faecalis
Tube ATCC™ 29212
Directions for Preparation from
Dehydrated Product
1. Dissolve 35.8 g of the powder in 1 L of purified water. Mix
thoroughly. References
1. Mallmann and Seligmann. 1950. Am. J. Pub. Health 40:286.
2. Autoclave at 121°C for 15 minutes. 2. Litsky, Mallmann and Fifield. 1953. Am. J. Pub. Health 43:873.
Expected Results
Growth of enterococci.
208
209
5. Jousimies-Somer, Summanen and Finegold. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.),
Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C. Availability
6. Holdeman, Cato and Moore (ed.). 1977. Anaerobe laboratory manual, 4th ed. Virginia Polytechnic
Institute and State University Anaerobe Laboratory, Blacksburg, Va.
BBL™ Egg Yolk Agar, Modified
7. Baron, Peterson and Finegold. 1994. Bailey & Scott’s diagnostic microbiology, 9th ed. Mosby-Year BS12 CMPH MCM9
Book, Inc., St. Louis, Mo.
8. Martin. 1971. Appl. Microbiol. 22:1168. Cat. No. 297873 Prepared Plates – Pkg. of 10*
*Store at 2-8°C.
Elliker Broth
Intended Use User Quality Control
Elliker Broth, also known as Lactobacilli Broth, is used for
cultivating streptococci and lactobacilli, particularly in dairy Identity Specifications
procedures. Difco™ Elliker Broth
Dehydrated Appearance: Light to medium beige, free-flowing, homoge-
neous.
Summary and Explanation Solution: 4.85% solution, soluble in purified water upon
Testing for lactic acid bacteria in dairy products may be useful boiling. Solution is light to medium amber,
for various reasons.1 These include determining the cause of clear.
acid defects in dairy products, evaluating lactic starter cultures Prepared Appearance: Light to medium amber, clear.
and controlling the quality of cured cheese, cultured milks Reaction of 4.85%
and uncultured products.1 Lactic acid bacteria found in dairy Solution at 25°C: pH 6.8 ± 0.2
products are primarily Streptococcus, Lactococcus, Leuconostoc
Cultural Response
and Lactobacillus.1
Difco™ Elliker Broth
Elliker Broth is prepared according to the formulation of Elliker, Prepare the medium per label directions. Inoculate and incubate at
Anderson and Hannesson,2 and modified by McLaughlin.3 This 35 ± 2°C for 18-48 hours except Streptococcus cremoris which is
incubated at 30 ± 2°C for 18-48 hours.
slightly acidic medium contains nutrients to support the growth
Organism ATCC™ INOCULUM CFU RECOVERY
of streptococci and lactobacilli.
Lactobacillus casei 7469 102-103 Good
A modification of Elliker Broth, Lactic (Elliker) Agar is Lactobacillus delbrueckii
recommended for general purpose enumeration of lactic acid subsp. lactis 8000 102-103 Good
bacteria.1 Lactobacillus sp. 11506 102-103 Fair
Streptococcus cremoris 9596 102-103 Good
Principles of the Procedure
Peptone and gelatin provide the nitrogen and amino acids
in Elliker Broth. Yeast extract is the vitamin source in this 3. Autoclave at 121°C for 15 minutes.
formula. Dextrose, lactose and saccharose are the fermentable 4. Test samples of the finished product for performance using
carbohydrates. Sodium chloride maintains the osmotic balance stable, typical control cultures.
of the medium, and ascorbic acid is added to create a proper
environment for organism growth. Sodium acetate is a Procedure
selective agent against gram-negative bacteria. For a complete discussion on the isolation and identification
of streptococci and lactobacilli, refer to standard methods in
Formula food testing.1,4-6
Difco™ Elliker Broth
Approximate Formula* Per Liter Expected Results
Pancreatic Digest of Casein........................................ 20.0 g
Yeast Extract................................................................ 5.0 g
Refer to appropriate references and procedures for results.
Gelatin......................................................................... 2.5 g
Dextrose...................................................................... 5.0 g References
Lactose........................................................................ 5.0 g 1. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy products. 17th ed.
Saccharose................................................................... 5.0 g American Public Health Association, Washington, D.C.
2. Elliker, Anderson and Hannesson. 1956. J. Dairy Sci. 39:1611.
Sodium Chloride.......................................................... 4.0 g 3. McLaughlin. 1946. J. Bacteriol. 51:560.
Sodium Acetate........................................................... 1.5 g 4. Marshall (ed.). 1993. Standard methods for the examination of dairy products, 16th ed. American
Ascorbic Acid............................................................... 0.5 g Public Health Association, Washington, D.C.
5. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
*Adjusted and/or supplemented as required to meet performance criteria.
tional, Gaithersburg, Md.
6. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
Directions for Preparation from 4th. ed. American Public Health Association, Washington, D.C.
Endo Agar
Intended Use User Quality Control
Endo Agar is a differential and slightly selective culture
medium for the detection of coliform and other enteric micro- Identity Specifications
organisms. BBL™ Endo Agar
Dehydrated Appearance: Fine, homogeneous powder that may contain a
large amount of minute to small dark particles.
Summary and Explanation
Solution: 4.15% solution, soluble in purified water upon
The majority of the enteric plating media developed in the early boiling. Solution is light to medium, pink rose to
years of the 20th century utilized either mixtures of bile salts tan rose trace orange, moderately hazy to hazy.
or individual salts as selective agents to achieve inhibition of May contain a moderate amount of small dark
red particles and a large amount of minute dark
gram-positive species. In 1904, Endo reported the develop- red particles.
ment of a culture medium for the differentiation of lactose Prepared Appearance: Light to medium, pink rose to tan rose trace
fermenters from the nonfermenters in which no bile salts orange, moderately hazy to hazy. May contain a
were used.1 Inhibition of gram-positive microorganisms was moderate amount of small dark red particles and
a large amount of minute dark red particles.
achieved by the sodium sulfite and basic fuchsin contained in the
Reaction of 4.15%
formulation. Endo’s Fuchsin Sulphite Infusion Agar was the Solution at 25°C: pH 7.5 ± 0.2
original name for this medium,2 which is known today as
Endo Agar. It was developed initially in order to facilitate the Cultural Response
isolation and identification of the typhoid bacillus. BBL™ Endo Agar
Prepare the medium per label directions. Inoculate and incubate at
The original formula has been modified extensively since
its introduction. The meat infusions have been replaced by
35 ± 2°C for 48 hours. E
INOCULUM colony
a peptic digest of animal tissue. The dye composition and ORGANISM ATCC™ CFU Recovery color
concentration also have been adjusted. Enterococcus faecalis 29212 104-105 Poor to fair Pink to
rose-red
Over the years, Endo Agar has been an important medium Escherichia coli 25922 103-104 Good Rose-red,
in the microbiological examination of potable water and green metallic
wastewater, dairy products and foods; however, the current sheen
compendia of standard methods for the examination of these Klebsiella pneumoniae 33495 103-104 Good Pink to rose-
red mucoid
materials recommend alternative media formulations.3-5
Salmonella enterica
subsp. enterica Colorless
Principles of the Procedure serotype Typhimurium 14028 103-104 Good to pale pink
The selectivity of Endo Agar is due to the sodium sulfite/basic
fuchsin combination, which results in the suppression of
gram-positive microorganisms. It is classified as only slightly Directions for Preparation from
selective since other media contain more potent inhibitors Dehydrated Product
of the gram-positive microorganisms. Coliforms ferment the 1. Suspend 41.5 g of the powder in 1 L of purified water. Mix
lactose, produce pink to rose-red colonies and similar coloration thoroughly.
of the medium. The colonies of organisms that do not ferment 2. Heat with frequent agitation and boil for 1 minute to
lactose are colorless to faint against the pink background of completely dissolve the powder.
the medium. 3. Autoclave at 121°C for 15 minutes.
4. Cool to 45-50°C. Resuspend precipitate by gentle mixing
Formula before use. Endo Agar should be prepared as needed.
BBL™ Endo Agar 5. Test samples of the finished product for performance using
Approximate Formula* Per Liter stable, typical control cultures.
Dipotassium Phosphate................................................ 3.5 g
Peptic Digest of Animal Tissue.................................... 10.0 g
Agar.......................................................................... 15.0 g Procedure
Lactose...................................................................... 10.0 g Use standard procedures to obtain isolated colonies from
Sodium Sulfite.............................................................. 2.5 g
Basic Fuchsin................................................................ 0.5 g
specimens. A nonselective medium should also be streaked to
*Adjusted and/or supplemented as required to meet performance criteria. increase the chance of recovery when the population of gram-
negative organisms is low and to provide an indication of other
organisms present in the specimen. Incubate plates, protected
from light, at 35 ± 2°C for 18-24 hours. If negative after
24 hours, reincubate an additional 24 hours.
211
212
Availability
Difco™ m Endo Agar LES
COMPF SMD SMWW
Cat. No. 273610 Dehydrated – 100 g
273620 Dehydrated – 500 g
213
Procedure
1. Place a membrane filter absorbent pad inside a sterile
60 mm Petri dish.
2. Add 1.8-2.0 mL m Endo Broth MF to each pad.
3. Filter the water sample through a membrane filter.
4. Place filter top side up on the pad using a rolling motion to
avoid entrapping air bubbles.
5. Invert the dish and incubate for 22-24 hours at 35 ± 0.5°C.
6. Observe and count all colonies that are red and have a
metallic sheen.
Expected Results
All colonies that are red and have the characteristic metallic Availability
sheen are considered coliforms. The sheen may cover the Difco™ m Endo Broth MF™
entire colony, may only be in the center or may appear only COMPF SMD SMWW
around the edges. Cat. No. 274920 Dehydrated – 100 g
274930 Dehydrated – 500 g
References
1. Fifield and Schaufus. 1958. J. Am. Water Works Assoc. 50:193.
2. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C.
3. Kim and Feng. 2001. In Downes and Ito (ed.), Compendium of methods for the microbiological
examination of foods, 4th ed. American Public Health Association, Washington, D.C.
214
Cultural Response
Difco™ Enteric Fermentation Base
Prepare the medium per label directions, without and with 1% dextrose. Inoculate with
fresh cultures and incubate at 35 ± 2°C for 18-24 hours. Acid production is indicated by a
change in color from light amber to dark pink or red. Check for gas production in at least
3% of the volume of the fermentation vial.
Uninoculated Escherichia Escherichia
Plain w/ Dextrose Tube coli coli
Organism ATCC™ RECOVERY Acid/Gas Acid/Gas ATCC™ 25922 ATCC™ 25922
with Dextrose Plain
Escherichia coli 25922 Good –/– +/+
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028 Good –/– +/+
Shigella flexneri 12022 Good –/– +/–
215
Carbohydrate
Final
Concentration
Add before
Autoclaving
Add After
Autoclaving Expected Results
Adonitol 0.5% X – A positive result for gas includes production in at least 3% of the
Arabinose 0.5% – X volume of the fermentation tube. A positive reaction for acid is
Cellobiose 0.5% – X a change in color from light amber to dark pink or red.
Dextrose (Glucose) 1% X –
Dulcitol 0.5% X –
Limitation of the Procedure
Glycerol* 0.5% X –
Negative tubes remain colorless and should be observed regularly
for a total of 30 days.
Inositol 0.5% X –
Lactose 1% – X
References
Mannitol 1% X – 1. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th edition. Mosby,
Inc., St. Louis, Mo.
Salicin 0.5% X – 2. Murray, Baron, Jorgensen, Landry and Pfaller. (ed.). 2007. Manual of clinical microbiology, 9th ed.
Sucrose 1% – X American Society for Microbiology, Washington, D.C.
3. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology,
Xylose 0.5% – X 9th ed. Williams & Wilkins, Baltimore, Md.
4. Ewing. 1986. Edwards and Ewing’s identification of Enterobacteriaceae, 4th edition. Elsevier
*Medium containing glycerol should be autoclaved for 10 minutes at 15 lbs pressure (121°C). Science Publishing Co., Inc., New York, N.Y.
5. Edwards and Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co.,
7. Dispense 9 mL amounts into test tubes containing inverted Minneapolis, Minn.
6. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
vials (Durham tubes). American Society for Microbiology. Washington, D.C.
216
Identity Specifications
BBL™ Enterococcosel™ Agar
Dehydrated Appearance: Medium fine, homogeneous, may contain some
tan specks.
Solution: 5.6% solution, soluble in purified water upon
boiling. Solution is medium, tan with a trace blue
cast, clear to moderately hazy.
Prepared Appearance: Medium, tan with a trace blue cast, clear to mod-
erately hazy.
Reaction of 5.6%
Solution at 25°C: pH 7.1 ± 0.2
BBL Enterococcosel™ Broth
™
Cultural Response
BBL™ Enterococcosel™ Agar or Enterococcosel™ Broth
Prepare the medium per label directions. For agar, inoculate as described
E
below. For broth, inoculate with fresh cultures. Incubate at 35 ± 2°C for
48 hours (agar) or 24 hours (broth).
ORGANISM ATCC™ inoculum CFU recovery AGAR recovery BROTH
Enterococcus faecalis 29212 103-104 Good, blackening Good, blackening
Escherichia coli 25922 104-105 Complete inhibition Partial to complete
inhibition, no blackening
Streptococcus pyogenes 19615 104-105 Complete inhibition Partial to complete
inhibition, no blackening
Formulae 2. For agar, heat with frequent agitation and boil for 1 minute
BBL Enterococcosel Agar
™ ™
to completely dissolve the powder. For broth, heat if neces-
Approximate Formula* Per Liter sary to completely dissolve the powder.
Pancreatic Digest of Casein........................................ 17.0 g 3. Autoclave at 121°C for 15 minutes.
Peptic Digest of Animal Tissue...................................... 3.0 g
Yeast Extract................................................................ 5.0 g 4. Test samples of the finished product for performance using
Oxgall........................................................................ 10.0 g stable, typical control cultures.
Sodium Chloride.......................................................... 5.0 g
Esculin......................................................................... 1.0 g
Ferric Ammonium Citrate............................................. 0.5 g
Procedure
Sodium Azide............................................................... 0.25 g Agar
Sodium Citrate............................................................. 1.0 g Use standard procedures to obtain isolated colonies from
Agar.......................................................................... 13.5 g
specimens. Incubate plates 24-48 hours at 35 ± 2°C in and
BBL™ Enterococcosel™ Broth aerobic atmosphere.
Consists of the same ingredients without the agar.
*Adjusted and/or supplemented as required to meet performance criteria. Broth
Colonies, from a primary isolation plate, suspected of being
Directions for Preparation from enterococci or group D streptococci can be emulsified in 2 mL
Dehydrated Product of Enterococcosel Broth and incubated at 35 ± 2°C in an aerobic
1. Suspend the powder in 1 L of purified water: atmosphere.
BBL™ Enterococcosel™ Agar – 56 g;
BBL™ Enterococcosel™ Broth – 43 g.
Mix thoroughly.
217
care facilities.6 Guidelines include stool and rectal swab Incubate the plates in an inverted position (agar-side up) for
culture surveys of asymptomatic patients who may be carrying 24-48 hours at 35 ± 2°C in an aerobic atmosphere.
VRE.
Expected Results
Principles of the Procedure Examine plates after 24 and 48 hours for the presence of trans-
Enterococcosel Agar, a Bile Esculin Agar with Azide, is used for lucent to light gray, pinpoint colonies exhibiting black halos
the rapid, selective detection and enumeration of enterococci. (discoloration of the agar) in areas of heavy growth. Perform
a Gram stain, catalase test and PYR test. Gram-positive cocci
Enterococci hydrolyze the glucoside esculin to esculetin and
which are either catalase negative or weakly positive and PYR
dextrose. Esculetin reacts with an iron salt to form a dark
positive may be presumptively identified as VRE pending
brown or black complex.7 Ferric citrate is incorporated into
confirmatory tests.1
the medium as an indicator of esculin hydrolysis and resulting
esculetin formation. Oxgall is used to inhibit gram-positive
bacteria other than enterococci. Sodium azide is inhibitory for
References
1. Barton and Doern. 1995. Diagn. Microbiol. Infect. Dis. 23:119.
gram-negative organisms. 2.
3.
Jett, Huycke and Gilmore. 1994. Clin. Microbiol. Rev. 7:462.
Moellering. 1992. Clin. Infect. Dis. 14:1173.
4. Emori and Gaynes. 1993. Clin. Microbiol. Rev. 6:428.
Vancomycin at 8 µg/mL is used to detect resistance to vanco- 5. Landry, Kaiser and Wenzel. 1989. Am. J. Infect. Control. 17:323.
6. Subcommittee on Prevention and Control of Antimicrobial-Resistant Microorganisms in Hospitals.
mycin.1,8 1994. Preventing the spread of vancomycin resistance: a report from the hospital infection control
practices advisory committee. Fed. Regist. 59:25758.
7. MacFaddin. 2000. Biochemical tests for identification of medical bacteria, 3rd ed. Lippincott,
Procedure Williams & Wilkins, Baltimore, Md.
8. Clinical and Laboratory Standards Institute. 2006. Approved standard: M2-A9. Performance standards
Allow the contents of a rectal swab (or a cotton-tipped swab for antimicrobial disk susceptibility tests, 9th ed. CLSI, Wayne, Pa.
sample obtained from a stool specimen) to elute in 1 mL of
Trypticase™ Soy Broth.1 Using a new swab, absorb eluent, Availability
rotate swab firmly several times against the upper inside wall
of the tube to express excess fluid, roll the swab over a small
BBL™ Enterococcosel™ Agar with Vancomycin, 8 µg/mL
BS12 CLSI
E
area of the surface at the edge of the plate and streak from this Cat. No. 292234 Prepared Plates – Pkg. of 10*
inoculated area. *Store at 2-8°C.
m Enterococcus Agar
Intended Use brane filtration method has the advantages of being simpler
m Enterococcus Agar, also referred to as m Azide Agar, is used to perform, not requiring confirmation and permitting a direct
for isolating and enumerating enterococci in water and other count of enterococci in 48 hours. Burkwell and Hartman4
materials by membrane filtration or pour plate technique. added 0.2% sodium carbonate and 0.05% polysorbate 80 to
m Enterococcus Agar to increase the sensitivity for the direct
Summary and Explanation plating method.
The enterococcus group is a subgroup of the fecal strepto-
cocci that includes E. faecalis, E. faecium, E. gallinarum, and Principles of the Procedure
E. avium.1 Enterococci are differentiated from other strepto- Peptone provides nitrogen, minerals and amino acids. Yeast
cocci by their ability to grow in 6.5% sodium chloride, at pH extract is the vitamin source and dextrose supplies carbon.
9.6 and at 10°C and 45°C.1 The enterococcal portion of the Dipotassium phosphate acts as a buffer for the medium.
fecal streptococcus group is a valuable bacterial indicator for
Enterococcus faecalis
determining the extent of fecal contamination of recreational ATCC™ 19433
surface waters.1 m Enterococcus Agar is used in standard meth-
ods for the detection of fecal streptococcus and enterococcus
groups using the membrane filtration technique.1
m Enterococcus Agar was developed by Slanetz et al.2 for
the enumeration of enterococci by the membrane filtration
technique. A modification of m Enterococcus Agar, adding
triphenyltetrazolium chloride (TTC), was described by Slanetz
and Bartley3. This modified medium proved to be a superior
membrane filtration medium for the enumeration of entero-
cocci. Increased recovery and larger colonies were obtained
by incubating the inoculated membranes on the agar surface
instead of on pads saturated with liquid medium. The mem-
219
the enterococci are the fourth leading cause of bacteremia through the use of disodium phosphate. Streptomycin at
in the United States.3 The case/fatality rates for enterococcal 2000 µg/mL and gentamicin at 500 µg/mL are used to detect
bacteremia range from 12 to 68% with death due to sepsis in high level aminoglycoside resistance.9 Vancomycin at 6 µg/mL
4 to 50% of the cases.4 is used to detect resistance to vancomycin.9 The Food, Drug &
Cosmetic (FD&C) dyes are inert and added for easy visual
Treatment of enterococcal infections with either penicil-
identification of the antimicrobials.
lin or vancomycin alone fails to kill enterococci resulting in
relapse of infection.5 Enterococci for years were known to
have low intrinsic resistance to a variety of β-lactam as well as
Procedure
1. Prepare the inoculum by suspending several well-isolated
aminoglycoside antibiotics.6 The addition of an aminoglycoside
colonies of the enterococcal isolate from an 18-24 hour
to which the isolate has demonstrated susceptibility results
plate culture into a tube of Trypticase™ Soy Broth and adjust
in both in vitro and in vivo synergism producing a bacteri-
the turbidity to be equivalent to a 0.5 McFarland turbidity
cidal effect.7 This synergistic effect is thought to be due to the
standard.
penicillin or vancomycin damaging the integrity of the cell
2. Spot inoculate each quadrant of the plate with 10 µL of the
wall, thus allowing the aminoglycoside to penetrate and inhibit
adjusted suspension.
bacterial protein synthesis. 8 The emergence of high level
3. Allow the inoculum spots to absorb into the agar surfaces.
resistance to streptomycin (≥ 2000 µg/mL), gentamicin
4. Incubate plates at 35 ± 2°C aerobically for a full 24 hours.
(≥ 500 µg/mL) and vancomycin (≥ 6 µg/mL) results in the failure
If negative at 24 hours, reincubate streptomycin tests an
of the penicillin- or vancomycin-aminoglycoside combinations
additional 24 hours.
to eradicate the infecting organisms. Therefore, testing for
high level resistance to streptomycin, gentamicin and vanco-
Expected Results
mycin is important. The use of a Brain Heart Infusion Agar
Following a full 24 hours of incubation, observe plates for
(BHIA) containing streptomycin (2000 µg/mL), gentamicin
(500 µg/mL) or vancomycin (6 µg/mL) is recommended by
growth. Growth on Quadrant I (BHIA Control) indicates E
viable test organisms in the inoculum broth suspension and
the Clinical and Laboratory Standards Institute (CLSI) for
the test is valid. If there is no growth, the test is invalid and
testing high level resistance.9
must be repeated.
Principles of the Procedure Growth on:
Brain Heart Infusion Agar is a general-purpose medium Quadrant II – Red (BHIA with gentamicin) and/or
suitable for the cultivation of a wide variety of microorganisms Quadrant III – Yellow (BHIA with vancomycin) and/or
and is recommended for agar screen susceptibility testing of Quadrant IV – Blue (BHIA with streptomycin) indicates that
enterococci.9 the antimicrobial would not be synergistic in combination
The meat infusion solids and peptones are sources of organic therapy.
nitrogen, carbon, sulfur, vitamins, and trace substances. No growth indicates synergy may be predicted. See the CLSI
Dextrose is the carbohydrate source. The medium is buffered standard and supplemental tables for details of interpretation
and additional procedures.
Enterococcus faecalis
ATCC™ 51299
References
1. Jett, Huycke and Gilmore. 1994. Clin. Microbiol. Rev. 7:462.
2. Moellering. 1992. Clin. Infect. Dis. 14:1173.
3. Emori and Gaynes. 1993. Clin. Microbiol. Rev. 6:428.
4. Landry, Kaiser and Wenzel. 1989. Am. J. Infect. Control 17:323.
5. Moellering, Korzeniowski, Sande and Wennersten. 1979. J. Infect. Dis. 140:203.
6. Murray. 1990. Clin. Microbiol. Rev. 3:46.
7. Mandell. 1984. Ann. Intern. Med. 100:904.
8. Moellering and Weinberg. 1971. J. Clin. Invest. 50:2580.
9. Clinical and Laboratory Standards Institute. 2006. Approved standard: M7-A7. Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically, 7th ed. CLSI, Wayne, Pa.
Availability
BBL™ Enterococcus Screen Agar Quad Plate
with Streptomycin/Gentamicin/Vancomycin
BS12 CLSI CMPH2
Cat. No. 222201 Prepared Plates (QUAD) – Pkg. of 10*
*Store at 2-8°C.
221
Identity Specifications
BBL™ Eosin Methylene Blue Agar, Levine
Dehydrated Appearance: Fine, homogeneous, may contain up to a large
amount of minute to small dark red purple
particles.
Solution: 3.74% solution, soluble in purified water upon
boiling. Solution is medium to dark, green
orange brown, hazy.
Prepared Appearance: Medium to dark, green orange brown, hazy.
Reaction of 3.74%
Solution at 25°C: pH 7.1 ± 0.2
BBL EMB Agar, Levine, without Lactose
™
Cultural Response
BBL™ Eosin Methylene Blue Agar, Levine or EMB
Agar, Levine, without Lactose
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 24 hours.
ORGANISM ATCC™ INOCULUM CFU REcovery
Enterococcus faecalis 29212 104-105 Partial inhibition
Escherichia coli 25922 103-104 Good
Klebsiella pneumoniae 33495 103-104 Good
Salmonella enterica subsp. enterica serotype Typhi 19430 10 -10
3 4
Good
Salmonella enterica subsp. enterica serotype Typhimurium 14028 103-104 Good
Shigella dysenteriae 9361 103-104 Good
Shigella flexneri 12022 103-104 Good
222
positive bacteria to a limited degree. These dyes also play a Expected Results
role in differentiating between lactose fermenters and lactose Typical colonial morphology on Eosin Methylene Blue Agar,
nonfermenters due to the presence or absence of dye uptake in Levine is as follows:
the bacterial colonies. Coliforms, as lactose-fermenting organ- Escherichia coli.........................Large, blue-black, green metallic
isms, are visualized as blue-black colonies, whereas colonies sheen
of Salmonella and Shigella, as lactose nonfermenters, appear Enterobacter/Klebsiella.............Large, mucoid, blue-black
colorless, transparent or amber. Proteus....................................Large, colorless
Some gram-positive bacteria, such as fecal streptococci, Salmonella...............................Large, colorless
staphylococci and yeasts, will grow on this medium and Shigella....................................Large, colorless
usually form pinpoint colonies. A number of nonpathogenic,
Pseudomonas...........................Irregular, colorless
lactose-nonfermenting gram-negative bacteria will grow on this
Gram-positive bacteria.............No growth to slight growth
medium and must be distinguished from the pathogenic strains
by additional biochemical tests.
Results obtained with Levine EMB Agar without Lactose are
Formulae dependent upon the substituted carbohydrate.
BBL™ Eosin Methylene Blue Agar, Levine
Approximate Formula* Per Liter References
Pancreatic Digest of Gelatin....................................... 10.0 g 1. Holt-Harris and Teague. 1916. J. Infect. Dis. 18:596.
2. Levine. 1918. J. Infect. Dis. 23:43.
Lactose...................................................................... 10.0 g 3. Endo. 1904. Zentralbl. Bakteriol., Abt. 1, Orig. 35:109.
Dipotassium Phosphate................................................ 2.0 g 4. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy products, 17th ed.
Eosin Y........................................................................ 0.4 g American Public Health Association, Washington, D.C.
5. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
Methylene Blue.......................................................... 65.0 mg 4th ed. American Public Health Association, Washington, D.C.
Agar.......................................................................... 15.0 g 6. Baron, Spilman and Carey. 1959. Abstr. G7, p. 29. Bacteriol. Proc. 59th Gen. Meet. Soc. Am. Bacte-
223
Procedure References
Use standard procedures to obtain isolated colonies from 1. Endo. 1904. Zentralbl. Bakteriol., Abt. I Orig. 35:109.
2. Holt-Harris and Teague. 1916. J. Infect. Dis. 18:596.
specimens. A nonselective medium should also be streaked 3. Levine. 1918. J. Inf. Dis. 23:43.
Esculin Agar E
Intended Use Procedure
Esculin Agar is a differential medium for demonstrating esculin Organisms to be tested must first be isolated in pure culture on
hydrolysis by various microorganisms. an appropriate solid medium. Using a sterile inoculating loop
or needle, inoculate esculin agar with several isolated colonies.
Summary and Explanation Incubate tubes at 35°C with caps loosened for up to 48 hours.
Esculin hydrolysis is recommended in the differentiation and
identification of a variety of organisms.1-3 If the test organism Expected Results
does not hydrolyze esculin, the medium remains unchanged Blackening of the agar medium in the area of growth indicates
and the esculin will fluoresce when subjected to long-wave UV esculin hydrolysis.
light at 360 nm. When hydrolyzed, the medium turns black and
fluorescence is lost.1 References
1. Shigei. 1992. In Isenberg (ed.), Clinical microbiology procedures handbook, vol. 1. American Society
for Microbiology, Washington, D.C.
Principles of the Procedure 2. Murray, Baron, Pfaller, Tenover and Yolken (ed.). 1999. Manual of clinical microbiology, 7th ed.
American Society for Microbiology, Washington, D.C.
Animal tissue peptones and infusions from heart muscle provide 3. Koneman, Allen, Janda, Schreckenberger and Winn. 1997. Color atlas and textbook of diagnostic
microbiology, 5th ed. Lippincott-Raven Publishers, Philadelphia, Pa.
amino acids or other nitrogenous substances that support bacterial
growth. Sodium chloride maintains osmotic equilibrium.
Availability
Esculin is a glycoside incorporated as a differential agent BBL™ Esculin Agar
to facilitate the identification of various organisms, including Cat. No. 295951 Prepared Slants – Pkg. of 10*
Enterobacteriaceae, enterococci and anaerobes. Hydrolysis of *Store at 2-8°C.
225
Eugon Agar
Intended Use Principles of the Procedure
Eugon Agar is a general-purpose medium used for cultivating a Peptones provide the nitrogen, vitamins and amino acids in
wide variety of microorganisms. Eugon Agar. The high concentration of dextrose is the energy
source for rapid growth of bacteria. L-Cystine and sodium
Summary and Explanation sulfite are added to stimulate growth. Sodium chloride maintains
Eugon Agar is prepared according to the formula described the osmotic balance of the media. The high carbohydrate
by Pelczar and Vera.1 Eugon Agar and Eugon Broth were content along with high sulfur (cystine) content improves
developed to obtain eugonic (luxuriant) growth of fastidious growth with chromogenicity.2 Agar is the solidifying agent
microorganisms.2 Eugon Agar can be used with or without in Eugon Agar.
enrichment. Enriched with blood, Eugon Agar supports the
growth of pathogenic fungi including Nocardia, Histoplasma Formula
and Blastomyces. With the addition of Supplement B, excellent Difco™ Eugon Agar
growth of Neisseria, Francisella and Brucella is achieved. The Approximate Formula* Per Liter
unenriched medium supports rapid growth of lactobacilli Proteose Peptone No. 3................................................ 7.5 g
Pancreatic Digest of Casein.......................................... 7.5 g
associated with cured meat products, dairy products and other Soy Peptone................................................................. 5.0 g
foods. Dextrose...................................................................... 5.5 g
L-Cystine...................................................................... 0.7 g
Niven3 reported the use of Eugon Agar for the detection of Sodium Chloride.......................................................... 4.0 g
lactic acid in cured meats, and recommended it for investigating Sodium Sulfite.............................................................. 0.2 g
Agar.......................................................................... 15.0 g
spoilage in meats. Harrison and Hansen4 employed the medium
*Adjusted and/or supplemented as required to meet performance criteria.
for plate counts of the intestinal flora of turkeys. Frank5 showed
its usefulness in germinating anaerobic spores pasteurized Directions for Preparation from
at 104°C.
Dehydrated Product
Eugon Agar is included in the Compendium of Methods for the 1. Suspend 45.4 g of the powder in 1 L of purified water. Mix
Microbiological Examination of Foods.6 thoroughly.
2. Heat with frequent agitation and boil for 1 minute to com-
pletely dissolve the powder.
User Quality Control 3. Autoclave at 121°C for 15 minutes.
Identity Specifications 4. When an enrichment is being prepared, cool to 50-55˚C prior
Difco™ Eugon Agar to adding the desired enrichment.
Dehydrated Appearance: Beige, free-flowing, homogeneous. 5. Test samples of the finished product for performance using
Solution: 4.54% solution, soluble in purified water upon stable, typical control cultures.
boiling. Solution is light amber, very slightly to
slightly opalescent, cystine precipitate may be
visible.
Procedure
Prepared Appearance: Light amber, slightly opalescent, cystine precipi- For a complete discussion on bacteria and fungi from clinical
tate may be visible. specimens, refer to the appropriate procedures outlined in the
Reaction of 4.54% references.7,8 For the examination of bacteria and fungi in food
Solution at 25°C: pH 7.0 ± 0.2
refer to standard methods.6,9
Cultural Response
Difco™ Eugon Agar Expected Results
Prepare the medium (unsupplemented) per label directions. For Candida Refer to appropriate references and procedures for results.
albicans and Aspergillus brasiliensis inoculate using fresh broth cultures and
incubate at 30 ± 2°C for 18-48 hours. For all other cultures inoculate and Limitations of the Procedure
incubate at 35 ± 2°C for 18-48 hours.
1. Eugon Agar is not recommended as a blood agar base for
Organism ATCC™ INOCULUM CFU RECOVERY hemolytic reactions because of its high sugar content.
Aspergillus brasiliensis (niger) 16404 Fresh Fair to good 2. It is suggested that Eugon Agar be prepared as required.
Candida albicans 26790 Fresh Good Do not melt and resolidify media containing enrichments.
Lactobacillus fermentum 9338 30-300 Good
Shigella flexneri 12022 30-300 Good
Streptococcus pyogenes 19615 30-300 Good
226
References Availability
1. Pelczar and Vera. 1949. Milk Plant Monthly 38:30.
2. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol.
Difco™ Eugon Agar
1. Williams & Wilkins, Baltimore, Md. COMPF
3. Niven. 1949. J. Bacteriol. 58:633.
4. Harrison and Hansen. 1950. J. Bacteriol. 59:197. Cat. No. 258910 Dehydrated – 500 g
5. Frank. 1955. J. Bacteriol. 70:269.
6. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods, Difco™ Supplement B
4th ed. American Public Health Association, Washington, D.C.
7. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed. Cat. No. 227610 Lyophilized – 6 × 10 mL with Reconstituting Fluid*
American Society for Microbiology, Washington, D.C. 227620 Lyophilized – 100 mL with Reconstituting Fluid*
8. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C. *Store at 2-8°C.
9. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
tional, Gaithersburg, Md.
to support bacterial growth. L-cystine is an essential amino acid ORGANISM ATCC™ INOCULUM CFU RECOVERY
that improves growth. Dextrose is incorporated as a source Aspergillus brasiliensis (niger) 16404 30-300 Fair to good
of energy and sodium chloride provides osmotic equilibrium. Candida albicans 26790 30-300 Good
Sodium sulfite along with the cystine content improves growth Lactobacillus fermentum 9338 30-300 Good
with chromogenicity. Shigella flexneri 12022 30-300 Good
Streptococcus pyogenes 19615 30-300 Good
Formula
Bacto™ Eugon Broth
Approximate Formula* Per Liter Procedure
Proteose Peptone No. 3................................................ 7.5 g
Pancreatic Digest of Casein.......................................... 7.5 g Organisms to be cultivated must first be isolated in pure
Soy Peptone................................................................. 5.0 g culture on an appropriate solid medium.
Dextrose...................................................................... 5.5 g
L-Cystine...................................................................... 0.7 g Using a sterile inoculating loop or needle, transfer fresh growth
Sodium Chloride.......................................................... 4.0 g from the subculture medium to the tubed medium.
Sodium Sulfite.............................................................. 0.2 g
*Adjusted and/or supplemented as required to meet performance criteria. Incubate under conditions appropriate for the organism being
cultivated. Broth cultures should be held at least 1 week
Directions for Preparation from before discarding as negative.
Dehydrated Product
1. Suspend 30.4 g of the powder in 1 L of purified water. Mix
thoroughly.
2. Heat with frequent agitation and boil for 1 minute to com-
pletely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. When an enriched medium is being prepared, cool to
50-55°C prior to adding the desired enrichment.
5. Test samples of the finished product for performance using
stable, typical control cultures.
227
228
Solution:
free-flowing, fine crystalline powder.
1.0% solution, soluble in 0.2N NaOH. Solution is F
deep red, clear to very slightly opalescent.
G
Procedure Expected Results
Difco™ m FC Agar Colonies of fecal coliforms will be various shades of blue.
1. Prepare the agar medium from the dehydrated base according Non-fecal coliforms are gray to cream-colored.
to the label directions and with the addition of the Rosolic
Acid solution. Limitation of the Procedure
2. Pour molten agar, previously cooled to 45-50°C into special A few non-fecal coliform colonies may be observed on m FC
tight-fitting plastic dishes and allow to harden. media due to the selective action of the elevated temperature
3. Roll the membrane filter used to collect the water sample and the addition of the Rosolic Acid. It may be useful to
onto the surface of the agar, so as to avoid the formation of elevate the temperature to 45 ± 0.2°C to eliminate Klebsiella
air bubbles between the filter and the agar surface. strains from the fecal coliform group.6
4. Place the dishes in plastic bags and incubate, by immersion,
in a water bath at 44.5 ± 0.2°C for 24 ± 2 hours.
References
1. Geldreich, Huff and Best. 1965. J. Am. Water Works Assoc. 57:208.
2. Eaton, Rice and Baird (ed). 2005. Standard methods for the examination of water and wastewater,
Difco m FC Broth
™
21st ed., online. American Public Health Association, Washington, D.C.
3. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
1. Prepare the broth medium from the dehydrated base International. Gaithersburg, Md.
4. U.S. Environmental Protection Agency. 1992. Manual for the certification of laboratories analyzing
according to the label directions and with the addition of the drinking water. EPA-814B-92-002. Office of Ground Water and Technical Support Division, USEPA,
Cincinnati, Ohio.
Rosolic Acid solution. 5. Bordner, Winter and Scarpino (ed.). 1978. Microbiological methods for monitoring the environment:
2. Add 2 mL of the cooled broth to sterile absorbent pads in water and wastes. Publication EPA-600/8-78-017. Environmental Monitoring and Support Laboratory,
Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, Ohio.
special tight-fitting plastic dishes. 6. Eaton, Clesceri and Greenberg (ed.). 1995. Standard methods for the examination of water and
wastewater, 19th ed. American Public Health Association, Washington, D.C.
3. Roll the membrane filter used to collect the water sample onto
the moistened absorbent pad, so as to avoid the formation
of air bubbles between the filter and the pad.
4. Place the dishes in plastic bags and incubate, by immersion,
in a water bath at 44.5 ± 0.2°C for 24 ± 2 hours.
229
m FC Basal Medium
Intended Use In another study, Ciebin et al.4 formulated DC Medium using
m FC Basal Medium is used with MUG or BCIG for cultivating FC Basal Medium supplemented with lactose, BCIG and
and enumerating fecal coliforms by the membrane filter tech- cefsulodin. It is a differential coliform medium for the
nique at elevated temperatures. enumeration of coliforms and E. coli in potable water using
membrane filtration. Ciebin et al. compared DC Medium to
Summary and Explanation LES Endo Medium and FC-BCIG Medium. They found DC
Ciebin et al.1 described a modification of m FC Medium, Medium superior to LES Endo Medium in recovering coliforms
called FC Basal Medium, in which the chromogenic substrate and equivalent to FC-BCIG Medium in recovering E. coli.
5-bromo-6-chloro-3-indolyl-β-D-glucuronide (BCIG) is added
for quantitative recovery of Escherichia coli from untreated Principles of the Procedure
water samples to show fecal contamination using membrane m FC Basal Medium contains peptones as sources of carbon,
filter methods. nitrogen, vitamins and minerals. Yeast extract supplies B-complex
Standard method procedures use media with the fluorogenic vitamins that stimulate bacterial growth. Bile Salts No. 3 inhibits
substrate, 4-methylumbelliferyl-β-D-glucuronide (MUG) to the growth of gram-positive microorganisms. Agar is the solidify-
enumerate E. coli by membrane filter methods.2 Disadvantages ing agent.
of using MUG include the requirement of ultraviolet light,
possible diffusion of fluorescence from the colony to the sur- Formula
rounding medium and background fluorescence of membrane Difco™ m FC Basal Medium
filters.3 Using BCIG in place of MUG to detect β-glucuronidase Approximate Formula* Per Liter
activity gives visible blue colonies and an indigo-blue complex Tryptose..................................................................... 10.0 g
Proteose Peptone No. 3................................................ 5.0 g
that remains within the colony. Ciebin et al.1 found FC-BCIG Yeast Extract................................................................ 3.0 g
Medium comparable to standard MUG-based media for Bile Salts No. 3............................................................. 1.5 g
detection of β-glucuronidase activity of E. coli. Sodium Chloride.......................................................... 5.0 g
Agar.......................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.
Escherichia coli
User Quality Control ATCC™ 25922
Identity Specifications
Difco™ m FC Basal Medium
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 3.95% solution, soluble in purified water upon boiling. Solution
is light amber, very slightly to slightly opalescent, may have slight
precipitate.
Prepared Appearance: Light amber, slightly opalescent.
Reaction of 3.95%
Solution at 25°C: pH 7.4 ± 0.2
Cultural Response
Difco™ m FC Basal Medium
Prepare the medium per label directions (with the addition of 0.01% MUG). Using the membrane
filter technique, inoculate and incubate at 44.5 ± 0.5°C for 24 ± 2 hours.
Organism ATCC™ INOCULUM cfu RECOVERY FLUORESCENCE
Enterobacter aerogenes 13048 30-200 Inhibition –
Enterococcus faecalis 19433 3×102-103 Marked to –
complete inhibition
Escherichia coli 25922 30-200 Good + (blue-white)
230
FLN Agar
Intended Use Procedure
FLN (Fluorescence Lactose Nitrate) Agar was developed to Specimens must first be isolated in pure culture on an F
provide a screening procedure for partial identification of
nonfermentative gram-negative bacilli.
appropriate medium. The isolate should be Gram-stained and
examined to confirm that morphology is appropriate for the
G
gram-negative bacilli.
Summary and Explanation Using a sterile inoculating needle, streak the slant surface and
Pickett and Pedersen observed that over 50% of 183 strains of
stab the butt with several colonies from the subculture medium.
nonfermentative bacilli from clinical specimens consisted of only
Incubate the tubes, with caps loosened, at 35°C for 18-24
3 (of 17) species.1 These species were Pseudomonas aeruginosa,
hours.
Pseudomonas maltophilia (now Stenotrophomonas maltophilia),
and Achromobacter anitratus (now Acinetobacter baumannii). If the isolate fails to grow, reincubate at 25-30°C for up to
This observation led to the development of media and tests to 1 week; examine daily for growth and pigment production. If
differentiate and partially identify the species. pigmentation fails to develop after the initial 24 hours of incuba-
tion, reincubate the cultures at 22°C for 1 or more days.
FLN Agar provides a means to test for fluorescence, lactose
oxidation and denitrification. Further differentiation can be
Expected Results
obtained by testing for oxidase, lysine decarboxylation and
Examine FLN Agar under UV light for fluorescin, a greenish-
fructose oxidation.1
yellow fluorescent pigment in the colonies and surrounding
medium. Observe butt of slant for presence of gas bubbles
Principles of the Procedure as evidence of denitrification. Yellow color on slant indicates
To demonstrate the ability of isolates to produce fluorescin
oxidation of lactose.
pigment, to reduce nitrate to nitrite to nitrogen gas and to oxidize
lactose, medium B of King (Pseudomonas Agar P/Tech Agar)2
References
was modified.1 The ability of isolates to produce fluorescin and 1. Pickett, and Pedersen. 1968. Appl. Microbiol. 16:1631.
2. King, Ward, and Raney. 1954. J. Lab. Clin. Med. 44:301.
to reduce nitrate or nitrite to nitrogen gas are two important 3. Koneman, Allen, Janda, Schreckenberger and Winn. 1997. Color atlas and textbook of diagnostic
characteristics in differentiating certain Pseudomonas spp. microbiology, 5th ed. Lippincott-Raven Publishers, Philadelphia, Pa.
Fildes Enrichment
Intended Use Summary and Explanation
Fildes Enrichment may be used to enrich a variety of media for Fildes Enrichment is a peptic digest of sheep blood used to
the cultivation of various microorganisms. enhance the growth of fastidious organisms. It may be added to
Tryptic/Trypticase™ Soy Broth or Agar in a final concentration
User Quality Control of 5% for cultivation of Haemophilus influenzae.1,2
Identity Specifications Stokes and Willis both recommended that 5% Fildes Enrichment
BBL Fildes Enrichment
™ and 20% human serum be added to Nutrient Agar for Nagler
Appearance: Dark brown and hazy with fine dark brown plates for C. perfringens and C. bifermentans.3,4
sediment.
Principles of the Procedure
Cultural Response Fildes Enrichment is a rich source of growth factors stimula-
BBL™ Fildes Enrichment tory to various microorganisms, including the X (hemin) and
Prepare medium with added Fildes Enrichment. Inoculate with fresh
cultures and incubate at 35 ± 2°C for 2 days with CO2. V (nicotinamide adenine dinucleotide, NAD) factors necessary
for the growth of H. influenzae.
ORGANISM ATCC™ recovery
Haemophilus influenzae 10211 Good
Formula
Neisseria meningitidis 700344 Good
Fildes Enrichment is prepared by the action of the enzyme
pepsin on defibrinated sheep blood.
232
Fletcher’s Media
Fletcher Medium Base • Fletcher’s Medium
Fletcher’s Medium with 5-FU
Intended Use Formula
Fletcher’s Medium is an enriched, semisolid medium used for Difco™ Fletcher Medium Base
the cultivation of Leptospira. Approximate Formula* Per 920 mL
Peptone....................................................................... 0.3 g
Fletcher’s Medium with 5-FU contains 5-fluorouracil for Beef Extract.................................................................. 0.2 g
selective recovery and cultivation of Leptospira from clinical Sodium Chloride.......................................................... 0.5 g
Agar............................................................................ 1.5 g
specimens. *Adjusted and/or supplemented as required to meet performance criteria.
Leptospira may also be cultured from liver and kidney tissues. BBL™ Fletcher’s Medium
Cat. No. 297242 Prepared Tubes (K Tubes), 5 mL – Pkg. of 10*
Aseptically macerate tissue specimens and inoculate using
1:1, 1:10 and 1:100 dilutions. Consult appropriate texts for BBL™ Fletcher’s Medium with 5-FU
detailed information about the processing and inoculation of SMWW
tissues and other specimens.1,2 Cat. No. 297243 Prepared Tubes (K Tubes), 5 mL – Pkg. of 10*
*Store at 2-8°C.
Incubate tubes in the dark at 25-30°C for up to 6 weeks.
Flo Agar
(See Pseudomonas Agars)
Fluid A • Fluid D
Intended Use Fluid D contains polysorbate 80, which acts as a surfactant to
Fluid A (peptone water) is used for diluting or rinsing when break down the lecithin or oils present.
performing sterility testing. Fluid D (peptone water with poly-
sorbate 80) is used for diluting or rinsing samples containing Principles of the Procedure
lecithin or oil when performing sterility testing. Fluid A and Fluid D contain peptic digest of animal tissue, which
provides a source of nitrogen for bacteria. Polysorbate 80 in
Meets United States Pharmacopeia (USP) performance speci-
Fluid D is a surfactant.
fications.
Formulae
Summary and Explanation Difco™ Fluid A
Pharmaceuticals, biologicals, medical devices or any material Approximate Formula* Per Liter
claiming to be sterile must be tested for sterility according to Peptic Digest of Animal Tissue...................................... 1.0 g
the procedures described in the compendia.1,2 Sterility testing Difco™ Fluid D
is performed using the membrane filtration or direct testing Approximate Formula* Per Liter
methods, depending upon sample type and size. Peptic Digest of Animal Tissue...................................... 1.0 g
Polysorbate 80............................................................. 1.0 mL
Fluid A and Fluid D are used for diluting or rinsing when perform- *Adjusted and/or supplemented as required to meet performance criteria.
ing sterility testing. These fluids aid in the complete rinsing of the
membrane filter apparatus and are not toxic to microorganisms.
234
Procedure References
Fluid A and Fluid D are provided as prepared, ready-to-use 1. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The
national formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc.,
diluents in a variety of bottle sizes and closures. Consult Rockville, Md.
2. Council of Europe. 2008. European pharmacopeia, 6th ed. Council of Europe, Strasbourg, France.
appropriate references for detailed information and recom-
mended procedures.1,2 Availability
Difco™ Fluid A
Expected Results EP USP
Consult appropriate references for further information.1,2 Cat. No. 292391 Prepared Bottles, 100 mL
(combo cap) – Pkg. of 10
290821 Prepared Bottles, 100 mL (serum) – Pkg. of 10
User Quality Control 290652 Prepared Bottles, 300 mL
(septum screw cap) – Pkg. of 10
Identity Specifications 299111 Prepared Bottles, 500 mL – Pkg. of 10
Difco™ Fluid A 292478 Prepared Bottles, 600 mL
Appearance: Nearly colorless, clear solution. (septum screw cap) – Pkg. of 10
Reaction of Solution at 25°C: pH 7.1 ± 0.2 Europe
Cat. No. 254979 Prepared Bottles, 300 mL – Pkg. of 10
Difco™ Fluid D 257096 Prepared Bottles, 650 mL – Pkg. of 4
Appearance: Nearly colorless, clear solution. 257332 Prepared Bottles, 100 mL (infusion) – Pkg. of 25
Reaction of Solution at 25°C: pH 7.1 ± 0.2 257324 Prepared Bottles, 400 mL – Pkg. of 10
257330 Prepared Bottles, 700 mL – Pkg. of 4
Toxicity Test 257263 Prepared Bottles, 300 mL
(double bagged) – Pkg. of 10
Difco™ Fluid A or Fluid D
257262 Prepared Bottles, 300 mL
Perform a toxicity test by inoculating duplicate tubes of Tryptic Soy (double bagged) – Pkg. of 4
Broth with the test organisms and adding 1 mL of Fluid A or Fluid D to
one tube of each set. Incubate tubes at 20-25°C for up to 5 days. Good Difco™ Fluid D
recovery in the tubes containing Fluid A or Fluid D indicates that the EP USP
solution does not have antibacterial or antifungal properties and is
Cat. No. 299447 Prepared Bottles, 100 mL
suitable for use in appropriate procedures.
(septum screw cap) – Pkg. of 10
ORGANISM ATCC™ INOCULUM CFU RECOVERY 290831 Prepared Bottles, 100 mL (serum) – Pkg. of 10
290662 Prepared Bottles, 300 mL
Bacillus subtilis
Candida albicans
6633
10231
10-102
10-102
Good
Good
(septum screw cap) – Pkg. of 10
299110 Prepared Bottles, 500 mL – Pkg. of 10
F
Kocuria rhizophila 9341 10-102 Good
210049 Prepared Bottles, 600 mL
(septum screw cap) – Pkg. of 10
G
Europe
Cat. No. 257299 Prepared Bottles, 500 mL – Pkg. of 10
254978 Prepared Bottles, 300 mL
(double bagged) – Pkg. of 10
236
Expected Results 3. The use of altered or deficient media may cause mutants
1. Prepare a standard concentration response curve by plotting having different nutritional requirements that will not give
the response readings against the amount of standard in each a satisfactory response.
tube, disk or cup. 4. For successful results of these procedures, all conditions of
2. Determine the amount of vitamin at each level of assay the assay must be followed precisely.
solution by interpolation from the standard curve.
3. Calculate the concentration of vitamin in the sample from Reference
1. Horwitz (ed.). 2007. Official methods of analysis of AOAC international, 18th ed., online. AOAC
the average of these values. Use only those values that do not International, Gaithersburg, Md.
vary more than ±10% from the average. Use the results only
if two-thirds of the values do not vary more than ±10%. Availability
Difco™ Folic AOAC Medium
Limitations of the Procedure AOAC
1. The test organism used for inoculating an assay medium must Cat. No. 212169 Dehydrated – 100 g*
be cultured and maintained on media recommended for this *Store at 2-8˚C.
purpose.
2. Aseptic technique should be used throughout the assay
procedure.
237
Prepare the folic acid stock solution required for the standard
User Quality Control
curve as follows:
Identity Specifications 1. Dissolve 50 mg dried Folic Acid USP Reference Standard or
Difco™ Folic Acid Assay Medium
Dehydrated Appearance: Off-white to very light beige, free-flowing,
equivalent in about 30 mL of 0.01N NaOH and 300 mL
homogeneous. purified water.
Solution: 3.75% (single strength) or 7.5% (double 2. Adjust to pH 7.5 ± 0.5 with diluted HCl solution. Add purified
strength) solution, soluble in purified water upon water to give a volume of 500 mL.
boiling for 2-3 minutes. Single strength solution
3. Add 2 mL of the solution from step 2 to 50 mL purified
is light amber, may have a slight precipitate.
water. Adjust the pH to 7.5 ± 0.5 with HCl solution. Dilute
Prepared Appearance: Very light amber, clear, may have a very slight
precipitate. to 100 mL with purified water to give a stock solution con-
Reaction of 3.75% taining 2 µg folic acid per mL. Prepare the stock solution
Solution at 25°C: pH 6.8 ± 0.2 fresh daily.
Cultural Response Prepare the standard solution for the assay by diluting 1 mL of
Difco™ Folic Acid Assay Medium this stock solution in 1 liter with purified water. This solution
Prepare the medium per label directions. The medium supports contains 2 ng folic acid per mL. Use 0.0, 0.5, 1, 2, 3, 4 and
the growth of Enterococcus hirae ATCC™ 8043 when prepared in
single strength and supplemented with folic acid. The medium should
5 mL per assay tube.
produce a standard curve when tested using a folic acid reference Following incubation, place the tubes in the refrigerator for
standard at 0.0 to 10.0 ng per 10 mL. Incubate tubes with caps
loosened at 35-37°C for 18-24 hours. Read the percent transmittance 15-30 minutes to stop growth. The growth can be measured
using a spectrophotometer at 660 nm. by a turbidimetric method and the curve constructed from the
values obtained. The most effective assay range is between the
levels of 2 and 10 ng folic acid per 10 mL tube.
Directions for Preparation from
Dehydrated Product Expected Results
1. Suspend 7.5 g of the powder in 100 mL of purified water. 1. Prepare a standard concentration response curve by plotting
2. Heat with frequent agitation and boil for 2-3 minutes. the response readings against the amount of standard in each
3. Dispense in 5 mL amounts into tubes, evenly dispersing the tube, disk or cup.
precipitate. 2. Determine the amount of vitamin at each level of assay
4. Add standard or test samples. solution by interpolation from the standard curve.
5. Adjust the tube volume to 10 mL with purified water. 3. Calculate the concentration of vitamin in the sample from
6. Autoclave at 121°C for 10 minutes. the average of these values. Use only those values that do not
vary more than ±10% from the average. Use the results only
Procedure if two-thirds of the values do not vary more than ±10%.
Prepare stock cultures of E. hirae ATCC 8043 by stab inocula-
tion of Lactobacilli Agar AOAC. Incubate at 35-37°C for 24-48 Limitations of the Procedure
hours. Store tubes in the refrigerator. Make transfers at monthly 1. The test organism used for inoculating an assay medium must
intervals. Prepare the inoculum for assay by subculturing a stock be cultured and maintained on media recommended for this
culture of E. hirae ATCC 8043 into a tube containing 10 mL purpose.
of Lactobacilli Broth AOAC. After incubation at 35-37°C for 2. Aseptic technique should be used throughout the assay
18-24 hours, centrifuge the cells under aseptic conditions and procedure.
decant the supernatant. Wash the cells three times with 10 mL of 3. The use of altered or deficient media may cause mutants
sterile 0.85% saline. After the third wash, dilute the cell suspen- having different nutritional requirements that will not give
sion 1:100 with sterile 0.85% saline. Use one drop of this latter a satisfactory response.
suspension to inoculate each of the assay tubes. 4. For successful results of these procedures, all conditions of
the assay must be followed precisely.
It is essential that a standard curve be set up for each separate
assay. Autoclaving and incubation conditions that influence
Reference
the standard curve readings cannot always be duplicated. 1. Capps, Hobbs and Fox. 1948. J. Bacteriol. 55:869.
The standard curve is obtained by using folic acid at levels of
0.0, 2, 4, 6, 8 and 10 ng per 10 mL assay tube. Turbidimetric Availability
readings should be made after incubation at 35-37°C for Difco™ Folic Acid Assay Medium
18-24 hours. Refrigerate tubes for 15-30 minutes to stop growth Cat. No. 231810 Dehydrated –100 g*
before reading. *Store at 2-8˚C.
238
239
240
241
Cultural Response
Difco™ Fraser Broth Base and Fraser Broth Supplement
Prepare the medium per label directions. Add Fraser Broth Supplement. Inoculate and incubate
at 35 ± 2°C and read for growth and blackening at 18-24 and 42-48 hours.
INOCULUM ESCULIN Uninoculated Listeria monocytogenes
ORGANISM ATCC™ CFU RECOVERY REACTION Tube ATCC™ 19114
Limitations of the Procedure 9. Kramer and Jones. 1969. J. Appl. Bacteriol. 32:381.
10. Ryser and Donnelly. 2001. In Downes and Ito (ed.), Compendium of methods for the microbiological
1. Since Listeria species other than L. monocytogenes can grow examination of foods, 4th ed. American Public Health Association, Washington, D.C.
11. Bille, Rocourt and Swaminathan. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual
on these media, an identification of Listeria monocytogenes of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
must be confirmed by biochemical and serological
testing.11 Availability
2. Poor growth and a weak esculin reaction may be seen after Difco™ Fraser Broth Base
40 hours incubation for some enterococci. CCAM COMPF ISO USDA
Cat. No. 211767 Dehydrated – 500 g
References 211766 Dehydrated – 2 kg
1. Murray, Webb and Swann. 1926. J. Pathol. Bacteriol. 29:407.
2. Monk, Clavero, Beuchat, Doyle and Brackett. 1994. J. Food Prot. 57:969.
Difco™ Fraser Broth Supplement
3. Wehr. 1987. J. Assoc. Off. Anal. Chem. 70:769. CCAM COMPF ISO USDA
4. Bremer and Osborne. 1995. J. Food Prot. 58:604.
5. Grau and Vanderlinde. 1992. J. Food Prot. 55:4. Cat. No. 211742 Vial – 6 × 10 mL*
6. Patel, Hwang, Beuchat, Doyle and Brackett. 1995. J. Food Prot. 58:244. *Store at 2-8°C.
7. Fraser and Sperber. 1988. J. Food Prot. 51:762.
8. Lee and McClain. 1994. Laboratory Communication No. 57 (revised February 8, 1994). Food Safety
and Inspection Service, Microbiology Division, USDA, Bethesda, Md.
Summary and Explanation In 1964, Thayer and Martin6 formulated a selective medium
In 1945, Johnston1 described a medium that could success- incorporating the antibiotics polymyxin B and ristocetin into
fully produce colonies of N. gonorrhoeae in 24 rather than 48 GC Agar with added hemoglobin and Supplement B. Thayer
hours. The accelerated growth rates were primarily due to the and Martin7 improved their medium by replacing the two
decreased agar content (solidity) of the medium. GC Medium original antibiotics with a new antimicrobial solution of co-
Base was introduced in 1947 with reduced agar content. While listin, vancomycin and nystatin (VCN). In 1971, Martin and
investigating the growth rate of some gonococcal strains, a Lester8 improved the new Thayer-Martin medium by incorpo-
medium containing the growth factors glutamine and cocar- rating an additional antibiotic, trimethoprim lactate (T), into
boxylase was found to improve recovery.2,3 From this discovery, the formulation (VCNT). This improved medium is called
Supplement B, a yeast concentrate, was developed. In a compara- Modified Thayer-Martin (MTM) Medium.9 VCN and VCNT
tive study4 of 12 different media, an enriched chocolate agar are used in the preparation of Thayer-Martin and Modified
prepared with GC Medium Base, Hemoglobin and Supplement Thayer-Martin agars, respectively.
B proved superior for isolating N. gonorrhoeae. Difco VX Martin and Lewis10 further improved the selectivity of MTM
Supplement and BBL™ IsoVitaleX™ Enrichment are chemically by increasing the concentration of vancomycin from 3.0 µg/mL
defined supplements developed to replace the yeast concentrate to 4.0 µg/mL for greater inhibition of gram-positive bacteria and
additive. replacing nystatin with anisomycin (VCA/VCAT) for greater
Difco Supplement B is a yeast concentrate for use in supple- inhibition of yeasts; this is known as Martin-Lewis (ML) Agar
menting media for growth of microorganisms with exacting Medium. Transgrow Medium bottles and Gono-Pak and
nutritional requirements. It is recommended for use in the JEMBEC™* plates are chocolate agar-based transport medium
preparation of chocolate agar described by Christensen and systems that can incorporate these formulations.11
Schoenlein.5 *JEMBEC is a trademark of Miles Scientific.
Difco Supplement VX and BBL™ IsoVitaleX™ Enrichment are Principles of the Procedure
lyophilized concentrates. These supplements are recommended Peptones provide nitrogen, vitamins and amino acids. Corn
for enriching GC Agar media, Proteose No. 3 Agar and the starch absorbs any toxic metabolites that are produced; dibasic
selective agars for the isolation of pathogenic Neisseria. and monobasic potassium phosphates buffer the medium.
Sodium chloride maintains osmotic balance. Agar is the solidify-
Hemoglobin, an autoclavable preparation of beef blood, provides
hemin, which is required by Haemophilus species and enhances ing agent. F
growth of Neisseria species. Chocolate Agar is prepared from GC agar medium base with G
Uninoculated Neisseria gonorrhoeae the addition of 2% Hemoglobin. Hemoglobin provides hemin
Plate ATCC™ 43069
(X factor) required for growth of Haemophilus and enhanced
growth of Neisseria.
The growth rate of Neisseria and Haemophilus is improved
with the addition of 1% nutritive enrichment, providing the
growth factors glutamine and cocarboxylase. Supplement B
contains yeast concentrate, glutamine, coenzyme (V factor),
cocarboxylase, hematin (X factor) and growth factors. Supple-
ment VX is a defined lyophilized concentrate of essential growth
factors; i.e., vitamins, amino acids, coenzymes, dextrose and
other factors to improve the growth of Haemophilus and
Neisseria species. IsoVitaleX Enrichment provides V factor
(nicotinamide adenine dinucleotide, NAD) for Haemophilus
species and vitamins, amino acids, coenzymes, dextrose, ferric
ion and other factors which improve the growth of pathogenic
Neisseria.
Antimicrobial agents are used as inhibitors in the selective media,
Thayer-Martin, Modified Thayer-Martin and Martin Lewis agars
and Transgrow Medium. Colistin inhibits gram-negative bacteria,
Haemophilus vancomycin inhibits gram-positive contaminants, nystatin
parainfluenzae
ATCC™ 7901
suppresses the growth of yeasts, anisomycin provides improved
inhibition of Candida albicans, an organism that has been
shown to inhibit N. gonorrhoeae,12,13 and trimethoprim lactate
suppresses the swarming of Proteus species.
243
244
245
246
247
8. Apply the discs by means of an antimicrobial disc dispenser, Disregard faint growth of tiny colonies that can be detected
using aseptic precautions. Most antimicrobial agents with difficulty near the edge of the obvious zone of inhibition.
produce larger zones of inhibition when tested against 3. Refer to the Zone Diameter Interpretive Standards in the
N. gonorrhoeae compared with other organisms; therefore, CLSI publication for interpretation of results obtained
no more than 9 discs per 150 mm plate are recommended. with clinical isolates of N. gonorrhoeae.3 Results may be
After discs have been placed on the agar, tamp them with reported as resistant, intermediate or susceptible depending
a sterile needle or forceps to make complete contact with on the zone diameters obtained. Organisms testing positive
the medium surface. This step is not necessary if the discs for β-lactamase production should be considered resistant to
are deposited using the Sensi-Disc™ Self Tamping 12-Place penicillin regardless of the zone diameters obtained.
Dispenser (tampers will not descend from holes lacking
Consult references for additional information.4-6
cartridges).
9. Within 15 minutes after the discs are applied, invert the References
plates and incubate for 20-25 hours at 35°C in an aerobic 1. Centers for Disease Control. 1987. Antibiotic-resistant strains of Neisseria gonorrhoeae: policy
guidelines for detection, management, and control. Morbid. Mortal. Weekly Rep. 36(Suppl.):1S.
atmosphere enriched with 5-7% carbon dioxide. 2. Clinical and Laboratory Standards Institute. 2006. Approved standard: M2–M9. Performance standards
for antimicrobial disk susceptibility tests, 9th ed. CLSI, Wayne, Pa.
3. Clinical and Laboratory Standards Institute. 2008. M100–S18 (M2), Disk diffusion supplemental
Expected Results tables. CLSI, Wayne, Pa.
4. Neumann, Sahm, Thornsberry and McGowan. 1991. Cumitech 6A, New developments in antimi-
1. Examine the plates after 20-24 hours of incubation. A conflu- crobial agent susceptibility testing: a practical guide. Coordinating ed., McGowan. American Society
for Microbiology, Washington D.C.
ent “lawn” of growth should be obtained. If only isolated 5. Koneman, Allen, Janda, Schreckenberger and Winn. 1997. Color atlas and textbook of diagnostic
colonies grow, the inoculum was too light and the test should microbiology, 5th ed. Lippincott-Raven Publishers, Philadelphia, Pa.
6. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
be repeated. American Society for Microbiology, Washington. D.C.
GC-Lect™ Agar
Intended Use of the pathogenic Neisseria in specimens containing Capnocy-
BBL™ GC-Lect™ Agar is a selective plated medium providing tophaga species and other strains resistant to the inhibitors in
enhanced growth and recovery of Neisseria gonorrhoeae and MTM Agar; i.e., vancomycin-resistant contaminants, including
better inhibition of contaminating bacteria and fungi, including certain strains of Staphylococcus epidermidis. As with MTM,
Capnocytophaga species in oropharyngeal specimens. N. lactamica, which is resistant to colistin, is not inhibited by
GC-Lect Agar.
Summary and Explanation GC-Lect Agar contains a decreased concentration of vancomycin
A succession of media have been developed for the isolation for improved recovery of N. gonorrhoeae strains that are sensi-
of the pathogenic Neisseria from specimens containing mixed tive to this antibiotic.
flora (Thayer-Martin Selective Agar, Modified Thayer-Martin
[MTM] Agar, Martin-Lewis Agar).1-3 Each provides greater The JEMBEC™* system consists of GC-Lect Agar in a JEMBEC
inhibition of contaminating organisms than the preceding (John E. Martin Biological Environmental Chamber)-style plate,
formulation but each is, to varying degrees, inhibitory to a carbon dioxide tablet and resealable plastic bag.7
certain strains that it is designed to recover.4,5 A 100 mm dish (with pill pocket) is also available with GC-Lect
BD Diagnostics developed BBL GC II Agar Base as an improved
™ Agar as the medium.
*JEMBEC is a trademark of Miles Scientific.
base for Chocolate II Agar, which is utilized in these selective
media. The superior growth-promotion achieved for pathogenic
Neisseria also enabled growth of stains of Capnocytophaga
Principles of the Procedure
BBL™ GC-Lect™ Agar is based on BBL Chocolate II Agar that
on the selective medium when inoculated with oropharyngeal
contains the improved GC II Agar Base, achieved through careful
specimens.6
selection and pretesting of raw materials, bovine hemoglobin and
GC-Lect Agar was developed and patented by BD Diagnostics to BBL™ IsoVitaleX™ Enrichment. The GC II Agar Base contains
provide the additional inhibition required to prevent overgrowth nitrogenous nutrients in the form of casein and meat peptones,
248
phosphate buffer to maintain pH and corn starch, which 2. Cross-streak the “Z” pattern with sterile wire loop, preferably
neutralizes toxic fatty acids that may be present in the agar. in the clinic. If not done previously, cross-streaking should
X (hemin) and V (nicotinamide adenine dinucleotide) factors are be done in the laboratory.
provided by hemoglobin and IsoVitaleX Enrichment. IsoVitaleX 3. Place the culture as soon as possible in an aerobic environ-
Enrichment also provides vitamins, amino acids, coenzymes, ment enriched with carbon dioxide.
dextrose, ferric ion and other factors that improve the growth With the JEMBEC System: With sterile forceps remove a
of pathogenic Neisseria. 8 In addition, GC-Lect Agar permits the CO2-generating tablet from its foil wrapper and place it in the
growth of some vancomycin-sensitive gonococcal strains which specially designed well in the plate. Place inoculated plates in
are inhibited on standard MTM Agar.9 the polyethylene bag provided (one plate per bag). DO NOT
ADD WATER TO THE TABLET. Seal the bag by pressing
To improve the selectivity of GC-Lect Agar, a combination
down on the “zipper” at the end of the bag with fingers and
of five antimicrobial agents was developed to inhibit gram
slide along to the opposite end. Be sure that the bag is sealed
positive bacteria, including vancomycin-resistant S. epidermidis,
completely. After the bag is sealed, incubate in an inverted
gram-negative species, including Proteus and Capnocytophaga,
position (agar side up) at 35°C for 18-48 hours.11
as well as fungi, including Candida albicans.9
To transport the culture after incubation, place the sealed
In the JEMBEC system, a tablet consisting of a mixture of citric JEMBEC system in a suitable mailing or shipping container.
acid and sodium bicarbonate is placed in a well within the plate Care should be taken to protect the culture from extreme
and is activated by the moisture (humidity) produced by the heat or cold and to ensure delivery to the testing laboratory
culture medium within the sealed plastic bag. The CO2 levels gen- as soon as possible.
erated are sufficient for the growth of Neisseria gonorrhoeae.7 4. Incubate at 35 ± 2°C and examine after overnight incubation
and again after approximately 48 hours.
Procedure 5. Subculture for identification of N. gonorrhoeae should be
Streak the specimen as soon as possible after it is received in the made within 18-24 hours. If shipped after incubation, colo-
laboratory. If material is being cultured directly from a swab, nies should be subcultured before performing biochemical
proceed as follows.10 identification tests in order to ensure that adequate viability
1. Roll swab directly on the medium in a large “Z” to provide is achieved.
adequate exposure of swab to the medium for transfer of
organisms. Expected Results F
After a minimum of 18 hours of incubation, the plates should
show isolated colonies in streaked areas and confluent growth G
in areas of heavy inoculation. Some strains may require up to
72 hours of incubation before visible colonies appear.
Neisseria gonorrhoeae appears as small, grayish-white to
colorless mucoid colonies. N. meningitidis forms a colony
similar to N. gonorrhoeae, but larger and bluish-gray.
A presumptive identification may be made by performing a Gram
stain and an oxidase test.12 Biochemical tests and other identifica-
tion procedures should be performed to confirm findings.
250
buffers are incorporated to maintain the pH of the medium. directly into the broth. For stool specimens, use 1 g of feces or
Sodium citrate and sodium desoxycholate are added to inhibit 1 mL of liquid stool per tube. Consult appropriate references
gram-positive and some gram-negative bacteria. for information about the processing and inoculation of other
clinical specimens or food samples.6-9
Proteus, Pseudomonas and coliforms do not overgrow
Salmonella and Shigella in GN Broth during the first 6 hours Incubate the tubes with loosened caps at 35 ± 2°C and subculture
of incubation. onto selective and differential media after 6-8 hours of incubation
and again after 18-24 hours of incubation.10
Formulae
Difco™ GN Broth, Hajna Expected Results
Approximate Formula* Per Liter Growth in broth media is indicated by turbidity compared to an
Pancreatic Digest of Casein........................................ 12.0 g uninoculated control. Subculture onto appropriate selective and
Proteose Peptone No. 3................................................ 8.0 g
Dextrose...................................................................... 1.0 g differential media to isolate pathogens for identification.
D-Mannitol.................................................................. 2.0 g
Sodium Citrate............................................................. 5.0 g Limitation of the Procedure
Sodium Desoxycholate................................................. 0.5 g
Dipotassium Phosphate................................................ 4.0 g Enrichment broths should not be used as the sole isolation
Monopotassium Phosphate.......................................... 1.5 g medium. They are to be used in conjunction with selective
Sodium Chloride.......................................................... 5.0 g and nonselective plating media to increase the probability of
BBL™ GN Broth isolating pathogens, especially when they may be present in
Approximate Formula* Per Liter small numbers. Consult references for detailed information and
Pancreatic Digest of Casein........................................ 10.0 g
recommended procedures.6-9
Peptic Digest of Animal Tissue.................................... 10.0 g
Dextrose...................................................................... 1.0 g
D-Mannitol.................................................................. 2.0 g References
Sodium Citrate............................................................. 5.0 g 1. Hajna. 1955. Public Health Lab. 13:59.
Sodium Desoxycholate................................................. 0.5 g 2. Hajna. 1955. Public Health Lab. 13:83.
3. Croft and Miller. 1956. Am. J. Clin. Pathol. 26:411.
Dipotassium Phosphate................................................ 4.0 g 4. Taylor and Schelhart. 1967. Am. J. Clin. Pathol. 48:356.
Monopotassium Phosphate.......................................... 1.5 g 5. Taylor and Schelhart. 1968. Appl. Microbiol. 16:1383.
6. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
Sodium Chloride.......................................................... 5.0 g 4th ed. American Public Health Association, Washington, D.C.
*Adjusted and/or supplemented as required to meet performance criteria. 7. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
Gelatin
Intended Use innovation, a solid culture method, became the foundation for
Gelatin is used in preparing microbiological culture media. investigation of the propagation of bacteria.1 However, gelatin-
based media were soon replaced by media containing agar as
Summary and Explanation the solidifying agent.
Gelatin is a protein of uniform molecular constitution derived Gelatin is used in culture media for determining gelatinolysis
chiefly by the hydrolysis of collagen.1 Collagens are a class of (elaboration of gelatinases) by bacteria. Levine and Carpenter2
albuminoids found abundantly in bones, skin, tendon, cartilage and Levine and Shaw3 employed gelatin media in their studies
and similar animal tissues.1 of gelatin liquefaction. Garner and Tillett4 used culture media
Koch1 introduced gelatin into bacteriology when he invented the prepared with gelatin to study the fibrinolytic activity of
gelatin tube method in 1875 and the plate method in 1881. This hemolytic streptococci.
251
Cultural Response
Difco™ Gelatin
Prepare a 12% Gelatin solution in 0.8% Nutrient Broth. Dispense into tubes and
autoclave. Inoculate and incubate at 35 ± 2°C under appropriate atmospheric
conditions for 18-48 hours or for up to 2 weeks for the gelatinase test. To read
gelatinase, refrigerate until well-chilled and compare to uninoculated tubes. Tubes
positive for gelatinase will remain liquid.
Inoculum Uninoculated Bacillus subtilis
Organism ATCC™ CFU RECOVERY Gelatinase Tube ATCC™ 6633
Bacillus subtilis 6633 102-103 Good +
Clostridium sporogenes 11437 102-103 Good +
Escherichia coli 25922 102-103 Good –
Gelysate™ Peptone
Intended Use Summary and Explanation
Gelysate Peptone is used for cultures requiring low carbohydrates, Gelatin hydrolysate is high in proline residues.1 Gelysate Peptone
cystine and tryptophan levels in cell culture and bacterial is deficient in carbohydrates and is characterized by low cystine,
fermentation. methionine and tryptophan content. When used alone as a basic
nutrient, it is suitable for preparing media for organisms not
particularly fastidious in their nutritional requirements.
Consult standard methods manuals for media formulations
containing Gelysate Peptone.2,3
252
Cultural Response
Biochemical Reactions
BBL™ Gelysate™ Peptone
Prepare a sterile solution of Gelysate Peptone as directed below. Adjust final pH to 7.2-7.4. Inoclate and incubate at 35 ± 2°C for 18-48 hours.
TEST TEST SOLUTION ORGANISM ATCC™ inoculum cfu RESULT
Fermentable Carbohydrates 2% Escherichia coli 29552 ~107 Negative
Indole Production 0.1% Escherichia coli 29552 0.1 mL, undiluted Negative
Acetylmethylcarbinol Production 0.1% with 0.5% dextrose Enterobacter aerogenes 13048 0.1 mL, undiluted Positive
Hydrogen Sulfide Production 1% Citrobacter freundii 8454 0.1 mL, undiluted Positive
Growth Response
BBL™ Gelysate™ Peptone
Prepare a sterile solution of 10 g of Gelysate Peptone, 2.5 g of sodium chloride and 6.5 g of agar in 500 mL of purified water. Adjust final pH to 7.2-7.4. Inoculate
and incubate plates at 35 ± 2°C for 2-3 days (incubate S. pyogenes with 3-5% CO2).
ORGANISM ATCC™ INOCULUM CFU recovery
Enterococcus faecalis 29212 103-104 Good
Pseudomonas aeruginosa 27853 10 -10
3 4
Good
Staphylococcus aureus 6538P 103-104 Good
Streptococcus pyogenes 49117 104-105 Good
F
Principles of the Procedure Procedure
Gelysate Peptone is a pancreatic digest of gelatin. Gelatin is See appropriate references for specific procedures using Gelysate G
extracted from collagen, which is the fibrous protein in bone, Peptone.
cartilage and connective tissue. Gelysate Peptone provides
nitrogen, amino acids and vitamins in microbiological culture Expected Results
media. Refer to appropriate references and procedures for results.
extract supplies B-complex vitamins which stimulate bacterial Prepared Appearance: Medium amber, clear.
Procedure
For food samples, follow appropriate standard methods for
details on sample collection and preparation according to sample References
1. Giolitti and Cantoni. 1966. J. Appl. Bacteriol. 29:395.
type and geographic location.3,4 2. Mossel, Harrewijn and Elzebroek. 1973. UNICEF.
3. International Organization for Standardization. 2003. Microbiology of food and animal feeding stuffs
Consult appropriate references for details on test methods using – Horizontal method for the enumeration of coagulase-positive staphylococci – Part 3: Detection and
MPN technique for low numbers. ISO 6888-3, 2003-03-15. ISO, Geneva, Switzerland.
Giolitti-Cantoni Broth Base.3,4 4. Downes and Ito (ed.). 2001. Compendium of methods for the microbiogical examination of foods,
4th ed. American Public Health Association, Washington, D.C.
254
filter method. The revised formula contains the indicator dye, Prepared Appearance: Medium, green to blue-green, clear to moderately
hazy.
bromcresol green. It is a relatively more complex formula than Reaction of 7.3%
many of the other media exclusively used for the recovery of Solution at 25°C: pH 4.6 ± 0.2
yeasts and molds.
Cultural Response
Principles of the Procedure BBL™ M-Green Yeast and Mold Broth
Prepare the medium per label directions. Inoculate using the membrane filter
The formulation is rich in nutrients provided by peptones, yeast technique and incubate at 30-35°C for 2 days (up to 5 days, if necessary).
extract and dextrose, but bacterial growth is inhibited by the
ORGANISM ATCC™ INOCULUM CFU recovery
acid pH. Diastase is a mixture of amylolytic (starch-hydrolyz-
Aspergillus brasiliensis (niger) 16404 102-3×102 Good
ing) enzymes. The bromcresol green indicator facilitates the
Candida tropicalis 1369 30-300 Good
visualization and counting of fungal colonies. The colonies are
Penicillium roquefortii 10110 102-3×102 Good
green due to the diffusion of bromcresol green into the colonies
Saccharomyces cerevisiae 9763 30-300 Good
(alkaline reaction). End products from the colonies diffuse into
the medium, further reducing the pH and causing the dye to
turn yellow (acid reaction).
Procedure F
Formula
BBL™ M-Green Yeast and Mold Broth
1. Saturate a sterile membrane filter pad in a sterile Petri dish
with 2.0-2.5 mL of M-Green Yeast and Mold Broth. G
Approximate Formula* Per Liter 2. Roll a membrane filter, which has been used to filter the test
Yeast Extract................................................................ 9.0 g sample, onto the surface of the moistened pad so as to avoid
Dextrose (anhydrous)................................................. 50.0 g the trapping of air bubbles between the filter and the pad.
Pancreatic Digest of Casein.......................................... 5.0 g
Peptic Digest of Animal Tissue...................................... 5.0 g 3. Incubate the plates at 30-35°C for 48 hours and up to 5 days
Magnesium Sulfate...................................................... 2.1 g in an aerobic atmosphere with increased humidity.
Potassium Phosphate................................................... 2.0 g
Diastase....................................................................... 0.05 g
Thiamine...................................................................... 0.05 g Expected Results
Bromcresol Green....................................................... 26.0 mg After incubation, colonies appearing on the filter surface can be
*Adjusted and/or supplemented as required to meet performance criteria. counted. Mold colonies generally appear green and filamentous,
whereas yeast colonies are green and opaque.
Directions for Preparation from
Dehydrated Product Availability
1. Suspend 7.3 g of the powder in 100 mL of purified water. BBL™ M-Green Yeast and Mold Broth
Mix thoroughly. COMPF
2. Warm slightly if necessary to completely dissolve the Cat. No. 211286 Dehydrated – 100 g*
powder. 211287 Dehydrated – 500 g*
3. Autoclave at 121°C for 10 minutes. *Store at 2-8°C.
255
Procedure
As soon as possible, inoculate the specimen onto a Group A
Selective Strep Agar with 5% Sheep Blood (ssA) plate by firmly
rolling the swab over a third of the agar surface. Streak the
remainder of the plate with a sterilized or sterile disposable
inoculating loop to obtain isolated colonies. After streaking, stab
the agar two or three times in the area of heaviest inoculation.
256
257
HC Agar Base
Intended Use User Quality Control
HC Agar Base, when supplemented with Polysorbate 80, is used
for enumerating molds in cosmetic products. Identity Specifications
Difco™ HC Agar Base
Summary and Explanation Dehydrated Appearance: Very light to light beige, free-flowing, homoge-
neous.
Methods for isolating molds from cosmetic products require
Solution: 5.45% solution, soluble in purified water upon
incubation for 5 to 7 days using traditional agar media.1 In boiling. Solution is medium to dark amber,
1986, Mead and O’Neill2 described a new medium, HC Agar, slightly opalescent to opalescent, may have a
for enumerating molds in cosmetic products that decreased slight precipitate.
incubation time to 3 days at 27.5 ± 0.5°C. HC Agar Base, based Prepared Appearance: Medium amber with yellow tint, very slightly to
on the HC Agar formula of Mead and O’Neill, is supplemented slightly opalescent, no significant precipitate.
with Polysorbate 80 to prepare HC Agar. Reaction of 5.45%
Solution at 25°C: pH 7.0 ± 0.2
Principles of the Procedure
Cultural Response
HC Agar Base contains peptones as sources of carbon, nitro-
Difco™ HC Agar Base
gen, vitamins and minerals. Yeast extract supplies B-complex Prepare the medium per label directions. Inoculate and incubate at
vitamins which stimulate bacterial growth. Dextrose provides 27.5 ± 0.5°C for 65-72 hours.
a source of fermentable carbohydrate. Ammonium chloride Organism ATCC™ Inoculum CFU RECOVERY
and magnesium sulfate provide essential ions. Disodium and Aspergillus brasiliensis (niger) 16404 102-103 Good
monopotassium phosphates buffer the pH to near neutrality. Pseudomonas aeruginosa 10145 10 -2×10
3 3
None to poor
Sodium carbonate inactivates low levels of preservatives that are Serratia marcescens 13880 103-2×103 None to poor
active at a more acidic pH (e.g., benzoic acid). Chloramphenicol
inhibits bacteria, including Pseudomonas aeruginosa and
Serratia marcescens, that are potential contaminants of cosmetic Procedure
products. Polysorbate 80 neutralizes preservatives and sequesters 1. Process each specimen as appropriate for that specimen and
inoculate directly onto the surface of the medium.1 Inoculate
surfactants that may be present in residual amounts from the
duplicate plates.
product sample.2 Agar is the solidifying agent.
2. Incubate plates aerobically at 27.5 ± 0.5°C.
3. Examine plates for growth and recovery after 72 hours of
Formula
incubation.
Difco™ HC Agar Base
Approximate Formula* Per Liter
4. Count mold colonies from duplicate plates and record
Pancreatic Digest of Casein.......................................... 2.5 g average count as mold count per gram or milliliter of
Proteose Peptone......................................................... 2.5 g sample.
Yeast Extract................................................................ 5.0 g
Dextrose.................................................................... 20.0 g
Disodium Phosphate.................................................... 3.5 g Expected Results
Monopotassium Phosphate.......................................... 3.4 g Mold cultures should yield good growth and recovery. Bacteria
Ammonium Chloride.................................................... 1.4 g should be inhibited.
Magnesium Sulfate...................................................... 0.06 g
Chloramphenicol.......................................................... 0.1 g
Sodium Carbonate....................................................... 1.0 g Limitation of the Procedure
Agar.......................................................................... 15.0 g The 27.5 ± 0.5°C incubation temperature is critical for obtain-
*Adjusted and/or supplemented as required to meet performance criteria.
ing statistically significant mold counts after three days using
Directions for Preparation from this medium.
Dehydrated Product References
1. Suspend 54.5 g of the powder in 1 L of purified water. Mix 1. U.S. Food and Drug Administration. 2001. FDA bacteriological analytical manual, online. AOAC
thoroughly. International, Gaithersburg, Md.
2. Mead and O’Neill. 1986. J. Soc. Cosmet. Chem. 37:49.
2. Heat with frequent agitation and boil for 1 minute to
completely dissolve the powder. Availability
3. Add 20 mL of Polysorbate 80. Difco™ HC Agar Base
4. Autoclave at 121°C for 15 minutes. Cat. No. 268510 Dehydrated – 500 g
5. Test samples of the finished product for performance using
stable, typical control cultures.
258
m HPC Agar
Intended Use Formula
m HPC Agar is used for enumerating heterotrophic organisms in Difco™ m HPC Agar
treated potable water and other water samples with low counts Approximate Formula* Per Liter
by membrane filtration. Peptone..................................................................... 20.0 g
Gelatin....................................................................... 25.0 g
Agar.......................................................................... 15.0 g
Summary and Explanation *Adjusted and/or supplemented as required to meet performance criteria.
m HPC Agar was developed by Taylor and Geldreich in 1979
in their pursuit of a suitable standard methods medium to Directions for Preparation from
use with the membrane filter procedure.1 m HPC Agar is also Dehydrated Product
known as m-Heterotrophic Plate Count Agar and previously as 1. Suspend 6 g of the powder in 100 mL of purified water. Mix
membrane filter Standard Plate Count Agar, m-SPC Agar. The thoroughly.
formulation was evaluated by many investigators who reported 2. Heat with frequent agitation and boil for 1 minute to
it as a suitable alternative medium for standard plate counts.2-4 completely dissolve the powder.
It is recommended for the membrane filter method in the recent 3. Add 1 mL of glycerol.
editions of Standard Methods for the Examination of Water 4. Autoclave at 121°C for 5 minutes. Cool to 45-50°C.
and Wastewater.5 5. Dispense 5 mL portions into 50 × 9 mm Petri dishes.
6. Test samples of the finished product for performance using
The advantages of the membrane filter procedure over the
stable, typical control cultures.
standard plate count method have been described by many
investigators.6-8 The volume of inoculum is limited with both NOTE: Excessive heat may cause breakdown of the gelatin.
pour and spread plate techniques while the membrane filter See “Limitations of the Procedure.”
method enables the use of large samples, which is desirable for
water with low counts. Procedure
Water samples should be collected and handled as described in
Principles of the Procedure Standard Methods for the Examination of Water and Waste-
Peptone provides nitrogen and carbon as well as other nutri- water, Section 9060.5
ents. The original concentration of 5% gelatin was reduced to 1. The volume to be filtered will vary with the sample. Select a
2.5% to avoid problems associated with liquefying gelatin and maximum sample size to give 20-200 CFU per filter.
spreading colonies. 2. Filter the appropriate volume through a sterile 47 mm,
0.45 µm, gridded membrane filter, under partial vacuum.
H
Rinse funnel with three 20-30 mL portions of sterile dilution
-
water. Place filter on agar in Petri dish.
K
User Quality Control Water Sample
Identity Specifications
Difco™ m HPC Agar
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution: 6% solution, soluble in purified water upon boiling. (Add
1% glycerol after boiling.) With glycerol, solution is light
amber, slightly opalescent to opalescent, may have a slight
precipitate.
Prepared Appearance: Light amber, opalescent, may have a precipitate.
Reaction of 6% Solution
with Glycerol at 25°C: pH 7.1 ± 0.2
Cultural Response
Difco™ m HPC Agar
Prepare the medium per label directions. Inoculate using the membrane filtration
technique. Incubate at 35 ± 2°C for 40-48 hours.
Organism ATCC™ Inoculum CFU RECOVERY
Enterococcus faecalis 29212 20-200 Growth
Escherichia coli 25922 20-200 Growth
Pseudomonas aeruginosa 10145 20-200 Growth
259
less colonies per square. For 3-10 colonies per square, count
10 squares and obtain average count per square. For 10-20 Availability
colonies per square, count 5 squares and obtain average count Difco™ m HPC Agar
per square. Multiply average count per square by 100 and COMPF SMWW
divide by the sample volume to give colonies per milliliter. If Cat. No. 275220 Dehydrated – 500 g
there are more than 20 colonies per square, record count as Difco™ Glycerol
> 2,000 divided by the sample volume. Report averaged counts Cat. No. 228210 Bottle – 100 g
as estimated colony-forming units. Make estimated counts only 228220 Bottle – 500 g
References Availability
1. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
BBL™ Haemophilus Isolation Agar with Bacitracin
2. Chapin and Doern. 1983. J. Clin. Microbiol. 17:1163. Cat. No. 295914 Prepared Plates – Pkg. of 20*
3. Fildes. 1920. Br. J. Exper. Pathol. 1:129.
4. Fildes. 1921. Br. J. Exper. Pathol. 2:16. *Store at 2-8°C.
5. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergeys Manual of determinative bacteriology,
9th ed. Williams & Wilkins, Baltimore, Md.
6. Garrod and O’Grady. 1971. Antibiotics and chemotherapy, 3rd ed. Williams & Wilkins, Baltimore,
Md.
7. Kilian. 1991. In Balow, Hausler, Herrmann, Isenberg and Shadomy (ed.), Manual of clinical microbiol-
ogy, 5th ed. American Society for Microbiology, Washington, D.C.
Procedure
1. Prepare a Gram stain before starting susceptibility testing to
confirm culture purity and to confirm tentative identification
of Haemophilus.
2. Use several well-isolated colonies taken directly from an
overnight (preferably 20-24 hours) Chocolate Agar plate as
the source of the inoculum.
3. A rapid β-lactamase test should be utilized for rapid detection
of strains that are resistant to penicillin, ampicillin or
amoxicillin.
261
4. Prepare a suspension of the test organism in Mueller Hinton above. The endpoint should be taken as the area showing no
Broth, Mueller Hinton II Broth or 0.9% saline. This suspen- obvious visible growth that can be detected with the unaided
sion should be adjusted to the turbidity of the 0.5 McFarland eye. Disregard faint growth of tiny colonies which can be
standard using a photometric device. This suspension will detected with difficulty near the edge of the obvious zone of
contain approximately 1-4 × 108 CFU/mL. Care must be the inhibition.
exercised in preparing this suspension because higher 3. Consult the product literature or the CLSI Approved Standard
inoculum concentrations may lead to false-resistant results M21 for details on interpretation of results.
with some β-lactam antibiotics, particularly when
β-lactamase-producing strains of H. influenzae are tested.1 References
5. Alternative methods of inoculum preparation involving 1. Clinical and Laboratory Standards Institute. 2006. Approved standard: M2-A9. Performance standards
for antimicrobial disk susceptibility tests, 9th ed. CLSI, Wayne, Pa.
devices that permit direct standardization of inocula without 2. Bauer, Kirby, Sherris and Turck. 1966. Am. J. Clin. Pathol. 45:493.
3. Ryan, Schoenknecht, and Kirby. 1970. Hospital Practice 5:91.
adjustment of turbidity, such as the BBL ™ Prompt ™ 4. Barry, Garcia, and Thrupp. 1970. Am. J. Clin. Pathol. 53:149.
Inoculation System, have been found to be acceptable for 5. Neumann, Sahm, Thornsberry and McGowan. 1991. Cumitech 6A, New developments in antimi-
crobial agent susceptibility testing, A practical guide. Coord. ed., McGowan. American Society for
routine testing purposes.10 This system has also been found Microbiology, Washington, D.C.
6. Jorgensen, Redding, Maher and Howell. 1987. J. Clin. Microbiol. 25:2105-2113.
to be satisfactory for testing H. influenzae.11 7. Jorgensen, Howell and Maher. J. Clin. Microbiol. 28:985-988.
8. Ericsson and Sherris. 1971. Acta. Pathol. Microbiol. Scand. Sect. B, Suppl. 217:1.
Consult the product literature or the CLSI Approved Standard M21 9. Jorgensen, Turnidge and Washington. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.),
Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
for details on plate inoculation and use of antimicrobial discs. 10. Baker, Thornsberry and Hawkinson. 1983. J. Clin. Microbiol. 17:450.
11. Marsik, Evans, Fowler and Thompson. 1989. Abstr. C-67, p. 404. Abstr. 89th Annu. Meet. Am. Soc.
Microbiol. 1989.
Expected Results
1. Examine the plates after 16-18 hours of incubation. A Availability
confluent “lawn” of growth should be obtained. If only BBL™ Haemophilus Test Medium Agar (HTM Agar)
isolated colonies grow, the inoculum was too light and the BS12 CLSI CMPH2
rest should be repeated. United States and Canada
Cat. No. 221992 Prepared Plates – Pkg. of 10*
2. Measure the diameter of the zones of complete inhibition 221954 Prepared Plates (150 × 15 mm-style) – Pkg. of 8*
(as judged by the unaided eye), including the diameter of
Europe
the disc, to the nearest whole millimeter, using calipers, a Cat. No. 254058 Prepared Plates – Pkg. of 20*
ruler, or a template prepared for this purpose. The measuring Japan
device is held on the back of the plate, which is held over Cat. No. 251992 Prepared Plates – Pkg. of 10*
a black, non-reflecting background and illuminated from 252037 Prepared Plates – Pkg. of 20*
251954 Prepared Plates (150 × 15 mm-style) – Pkg. of 8*
*Store at 2-8°C.
262
Identity Specifications
Difco™ Heart Infusion Agar
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution: 4% solution, soluble in purified water upon
boiling. Solution is light to medium amber,
very slightly to slightly opalescent.
Prepared Appearance: Plain – Light to medium amber, slightly opal-
escent.
With 5% sheep blood – Cherry red, opaque.
Reaction of 4%
Solution at 25°C: pH 7.4 ± 0.2
Cultural Response
Difco™ Heart Infusion Agar
Prepare the medium per label directions without (plain) and with 5% sheep
blood (SB). Inoculate and incubate at 35 ± 2°C for 18-48 hours.
inoculum recovery recovery
ORGANISM ATCC™ CFU PLAIN with 5% SB HEMOLYSIS
Escherichia coli 25922 102-103 Good Good Beta
Staphylococcus
aureus 25923 102-103 Good Good Beta
Streptococcus
Streptococcus pyogenes
pneumoniae 6305 10 -10
2 3
Fair Good Alpha ATCC™ 19615
Streptococcus
pyogenes 19615 102-103 Fair Good Beta
Expected Results
Refer to appropriate references and procedures for results.
263
Cultural Response
Bacto™ Heart Infusion Broth
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-48
hours.
Organism ATCC™ inoculum cfu RECOVERY
Escherichia coli 25922 102-103 Good
Staphylococcus aureus 25923 10 -10
2 3
Good
Streptococcus pneumoniae 6305 102-103 Good
Uninoculated Escherichia coli
Streptococcus pyogenes 19615 102-103 Good Tube ATCC™ 25922
264
Identity Specifications H
Difco™ Hektoen Enteric Agar -
Dehydrated Appearance: Light beige, may have a slight green cast, free-
flowing, homogeneous.
K
Solution: 7.6% solution, soluble in purified water upon
boiling. Solution is brown with greenish cast,
slightly opalescent.
Prepared Appearance: Green with yellowish cast, slightly opalescent.
Reaction of 7.6%
Solution at 25°C: pH 7.5 ± 0.2
Cultural Response
Difco™ Hektoen Enteric Agar
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 18-24 hours.
INOCULUM COLONY
ORGANISM ATCC™ CFU RECOVERY color
Enterococcus Marked to
faecalis 29212 103 complete inhibition –
Escherichia coli 25922 10 -3×10 2
Partial
2
Salmon-orange,
inhibition may have bile
precipitate
Salmonella enterica
subsp. enterica Greenish blue,
serotype Typhimurium 14028 102-3×102 Good w/black centers
Shigella flexneri 12022 102-3×102 Good Greenish blue
265
Dehydrated Product
1. Suspend 76 g of the powder in 1 L of purified water. Mix Availability
thoroughly. Difco™ Hektoen Enteric Agar
AOAC BAM BS12 CCAM CMPH2 COMPF MCM9 SMD
2. Heat to boiling with frequent agitation to dissolve
Cat. No. 285340 Dehydrated – 500 g
completely. Do not overheat. DO NOT AUTOCLAVE. 285310 Dehydrated – 2 kg
3. Cool to 45-50°C and use immediately. 285320 Dehydrated – 10 kg
4. Test samples of the finished product for performance using BBL™ Hektoen Enteric Agar
stable, typical control cultures. AOAC BAM BS12 CCAM CMPH2 COMPF MCM9 SMD
United States and Canada
Procedure Cat. No. 221365 Prepared Plates – Pkg. of 20*
Use standard procedures to obtain isolated colonies from speci- 221366 Prepared Plates – Ctn. of 100*
mens. A nonselective medium should also be streaked to increase Europe
Cat. No. 254009 Prepared Plates – Pkg. of 20*
the chance of recovery when the population of gram-negative
254075 Prepared Plates – Ctn. of 120*
organisms is low and to provide an indication of other organisms
Mexico
present in the specimen. Cat. No. 224450 Prepared Plates – Pkg. of 10*
Incubate plates, protected from light, at 35 ± 2°C for 18-24 BBL™ Hektoen Enteric Agar//Salmonella Shigella Agar
hours. Cat. No. 297426 Prepared I Plate™ Dishes – Pkg. of 20*
BBL™ Hektoen Enteric Agar//XLD Agar
Expected Results Cat. No. 295646 Prepared I Plate™ Dishes – Pkg. of 20*
After incubation most plates will show an area of confluent *Store at 2-8°C.
growth. Because the streaking procedure is, in effect, a “dilution”
technique, diminishing numbers of microorganisms are deposited
on the streaked areas. Consequently, one or more of these areas
should exhibit isolated colonies of the organisms contained in
the specimen. Better isolation is obtained due to the inhibitory
action of the medium.
Procedure The following table shows the expected growth results for
The initial specimens should be inoculated onto BBL Chocolate various Haemophilus spp.
II Agar or another suitable medium and incubated for 18-24 Growth on Quadrants*/ Factor(s) Present
hours in a CO2-enriched atmosphere. Choose one or two I (X) II (V) III (XV) IV Hemolysis
well-isolated colonies that resemble Haemophilus species and H. influenzae – – + –
perform a Gram stain to confirm that the isolate is a gram- H. haemolyticus – – + +
negative rod or coccobacillus. To prepare inoculum, suspend H. parainfluenzae – + + –
several well-isolated colonies of the test organism from an 18-24 H. parahaemolyticus – + + +
hour plate culture into 5 mL of distilled water or Trypticase™ H. aphrophilus** + + + –
Soy Broth or other suitable medium and adjust the turbidity H. paraphophilus – + + –
to a 0.5 McFarland turbidity standard. Dilute 10-1. Inoculate
H. segnis – + + –
each quadrant with one loopful of diluted specimen and streak
H. ducreyi + – + –
to obtain isolated colonies. To prevent carry-over of growth
factors, sterilize the loop between inoculations of each quadrant.
* + = growth or hemolysis
H
-
– = no growth or no hemolysis
** X factor requirement occasionally may be observed on primary isolation but will be lost on
Haemophilus
influenzae
subculture. Gram staining, biochemical tests and/or additional identification procedures should be
performed to confirm findings.4-6 K
ATCC™ 10211
References
1. Davis. 1917. J. Infect. Dis. 21:392.
2. Thjotta and Avery. 1921. J. Exp. Med. 34:97.
3. Lwoff and Lwoff. 1937. Proc. Roy. Soc. (London) 122:352.
4. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
5. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby, Inc.,
St. Louis, Mo.
6. Koneman, Allen, Janda, Schreckenberger and Winn. 1997. Color atlas and textbook of diagnostic
microbiology, 5th ed. Lippincott-Raven Publishers, Philadelphia, Pa.
Availability
BBL™ Hemo (Haemophilus) Identification (ID)
QUAD Plate
MCM9
United States and Canada
Cat. No. 297890 Prepared Plates (QUAD) – Pkg. of 10*
Japan
Cat. No. 251142 Prepared Plates (QUAD) – Pkg. of 10*
*Store at 2-8°C.
267
H
Pancreatic Digest of Casein.......................................... 5.0 g Prepared Appearance: Light to medium amber, slightly opalescent.
Yeast Extract................................................................ 3.0 g
Difco ISP Medium 2
™
Reaction of 3.8%
Solution at 25°C: pH 7.2 ± 0.2 -
Approximate Formula* Per Liter Difco™ ISP Medium 4 K
Yeast Extract................................................................ 4.0 g Dehydrated Appearance: White to light beige, free-flowing, homoge-
Malt Extract............................................................... 10.0 g neous.
Dextrose...................................................................... 4.0 g
Agar.......................................................................... 20.0 g Solution: 3.7% solution, soluble in purified water upon
boiling. Solution is white to off-white, opaque
Difco™ ISP Medium 4 with precipitate.
Approximate Formula* Per Liter Prepared Appearance: White to off-white, opaque, may have a precipi-
Soluble Starch............................................................ 10.0 g tate.
Dipotassium Phosphate................................................ 1.0 g
Reaction of 3.7%
Magnesium Sulfate USP............................................... 1.0 g
Solution at 25°C: pH 7.2 ± 0.2
Sodium Chloride.......................................................... 1.0 g
Ammonium Sulfate...................................................... 2.0 g
Calcium Carbonate...................................................... 2.0 g Cultural Response
Ferrous Sulfate............................................................. 1.0 mg Difco™ ISP Medium 1, ISP Medium 2 or ISP Medium 4
Manganous Chloride.................................................... 1.0 mg Prepare the medium per label directions. Inoculate tubes of prepared
Zinc Sulfate.................................................................. 1.0 mg ISP Medium 1 with the test organisms and incubate at 30 ± 2°C for up
Agar.......................................................................... 20.0 g to 96 hours. Inoculate prepared ISP Medium 2 and ISP Medium 4 with
*Adjusted and/or supplemented as required to meet performance criteria. the test organisms by placing approximately 0.1 mL of inoculum near
the edge of the plate. Five parallel streaks across the plate are made
from this 0.1 mL of inoculum, followed by four perpendicular streaks. Incu-
bate inoculated plates at 30 ± 2°C for 48-96 hours.
Organism ATCC™ INOCULUM CFU RECOVERY
Streptomyces albus 3004 102-103 Good
Streptomyces lavendulae 8664 102-103 Good
269
Procedure Availability
For details on the use of these media for characterization of Difco™ ISP Medium 1
Streptomyces species, consult the reference.1 For a complete Cat. No. 276910 Dehydrated – 500 g
discussion on the isolation and maintenance of Streptomyces Difco™ ISP Medium 2
species refer to appropriate references.2,3 Cat. No. 277010 Dehydrated – 500 g
Difco™ ISP Medium 4
Expected Results Cat. No. 277210 Dehydrated – 500 g
Refer to appropriate references and procedures for results.
References
1. Shirling and Gottlieb. 1966. Int. J. Syst. Bacteriol. 16:313.
2. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
American Society for Microbiology, Washington, D.C.
3. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
270
for various lengths of time. Incubate tubes with loosened Consult appropriate references for an explanation of the
caps at 35 ± 2°C in an aerobic atmosphere. The caps of tubes reactions involved and expected results with specific microor-
inoculated with obligate anaerobes should be tightened during ganisms.3,4
incubation.
1. Indole Test
Limitations of the Procedure
Indole Nitrite Medium should not be used for detecting indole
The test for indole may be performed as soon as heavy growth
production by members of the Enterobacteriaceae. The tubed
has taken place, usually after 18 to 48 hours of incubation.
medium should be boiled for 2 minutes and cooled, without
The test may be performed by any suitable method, such
agitation, before use.
as with Kovacs’ reagent (add 0.5 mL, Cat. No. 261185) or
Ehrlich’s reagent employing p‑dimethylaminobenzaldehyde.2
References
Testing for indole may be made after 24 hours of incuba- 1. Smith, Rogers and Bettge. 1972. Appl. Microbiol. 43:423.
tion; if negative, the test should be repeated on another 2. MacFaddin. 2000. Biochemical tests for identification of medical bacteria, 3rd ed. Lippincott
Williams & Wilkins, Baltimore, Md.
culture incubated for 48 hours. 3. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby, Inc.,
St. Louis, Mo.
2. Nitrite Test 4. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
The test for nitrites may be performed at several intervals
during the incubation process if replicate tubes were
Availability
inoculated. The presence of nitrites may be detected by any
BBL™ Indole Nitrite Medium (Trypticase™ Nitrate Broth)
of several methods.2 Addition of approximately 5 drops each
BAM
of sulfanilic acid (Cat. No. 261197) and N, N-dimethyl- Cat. No. 211299 Dehydrated – 500 g
1-naphthylamine (Cat. No. 261198) reagents permits the 221655 Prepared Tubes – Pkg. of 10*
detection of nitrites. If prior tests are negative, a final test Difco™/BBL™ Indole Reagent
should be conducted at 48 hours of incubation. Cat. No. 261185 Droppers, 0.5 mL – Ctn. of 50
Difco™/BBL™ Nitrate A Reagent
Expected Results
Cat. No. 261197 Droppers, 0.5 mL – Ctn. of 50
1. Indole Test
The production of a pink to red color following addition of Difco™/BBL™ Nitrate B Reagent
the reagent is a positive test for indole formation. Cat. No. 261198 Droppers, 0.5 mL – Ctn. of 50
2. Nitrite Test Difco™/BBL™ Nitrate C Reagent
A pink to red color develops, after addition of the reagents, Cat. No. 261207 Droppers, 1 g – Ctn. of 50
if nitrite is present, and indicates that nitrate reduction *Store at 2-8°C.
271
Identity Specifications
BBL™ Inhibitory Mold Agar
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution: 3.6% solution, soluble in purified water upon
boiling. Solution is light to medium, yellow to tan,
slightly hazy to hazy.
Prepared Appearance: Light to medium, yellow to tan, slightly hazy to
hazy.
Reaction of 3.6%
Solution at 25°C: pH 6.7 ± 0.2
Cultural Response
BBL™ Inhibitory Mold Agar
Prepare the medium per label directions. Inoculate with fresh cultures
(undiluted or diluted as described below) and incubate at 25 ± 2°C for
7 days under appropriate atmospheric conditions.
ORGANISM ATCC™ INOCULUM CFU recovery
Candida albicans 10231 Undiluted Good
Escherichia coli 25922 104-105 Partial to complete
inhibition
Trichophyton
mentagrophytes 9533 Undiluted Good
Decant the supernatant and resuspend cells in 10 mL 0.85% 2. Determine the amount of vitamin at each level of assay
sterile saline. Wash the cells three times with 10 mL sterile 0.85% solution by interpolation from the standard curve.
saline. After the third wash, resuspend the cells in 10 mL 0.85% 3. Calculate the concentration of vitamin in the sample from
saline. Dilute 1 mL of the cell suspension in 1000 mL of sterile the average of these values. Use only those values that do not
0.85% saline. This diluted suspension is the inoculum. Use 1 vary more than ± 10% from the average. Use the results only
drop of inoculum suspension to inoculate each assay flask. if two-thirds of the values do not vary more than ± 10%.
The concentrations of inositol required for the preparation
of the standard curve may be prepared by dissolving 200 mg Limitations of the Procedure
inositol in 100 mL purified water. Mix thoroughly. Dilute 1. The test organism used for inoculating an assay medium
1 mL of this solution with 999 mL purified water to make a final must be grown and maintained on media recommended for
solution containing 2 µg inositol per mL. Use 0.0, 0.5, 1, 2, 3, 4 this purpose.
and 5 mL per flask. Prepare this stock solution fresh daily. 2. Aseptic technique should be used throughout the assay
procedure.
It is essential that a standard curve be constructed each time 3. The use of altered or deficient media may cause mutants
an assay is run. Autoclave and incubation conditions can having different nutritional requirements that will not give
impact the standard curve readings and cannot always be a satisfactory response.
duplicated. The standard curve is obtained by using inositol at
4. For successful results of these procedures, all conditions of
levels of 0.0, 1, 2, 4, 6, 8 and 10 µg per assay flask (10 mL).
the assay must be followed precisely.
Following inoculation, flasks are incubated at 25-30°C for
20-24 hours. Place flasks in the refrigerator for 15-30 minutes Reference
to stop growth. Growth is measured turbidimetrically using any 1. Atkin, Schultz, Williams and Frey. 1943. End. & Eng. Chem., Ann. Ed. 15:141.
suitable spectrophotometer.
Availability
Expected Results Difco™ Inositol Assay Medium
1. Prepare a standard concentration response curve by plotting Cat. No. 212222 Dehydrated – 100 g*
*Store at 2-8°C.
the response readings against the amount of standard in each
tube, disk or cup.
References Availability
1. Jordan and Harmon. 1928. J. Infect. Dis. 42:238.
2. Edwards and Ewing. 1955. Identification of Enterobacteriaceae. Burgess Publishing Company, Min-
BBL™ Jordan’s Tartrate Agar
neapolis, Minn. USDA
3. Ewing. 1986. Edwards and Ewing’s identification of Enterobacteriaceae, 4th ed. Elsevier Science
Publishing Co., Inc., New York, N.Y. Cat. No. 221889 Prepared Agar Deeps (K size tubes) – Pkg. of 10*
4. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed. *Store at 2-8°C.
American Society for Microbiology, Washington, D.C.
5. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology,
9th ed. Williams & Wilkins, Baltimore, Md.
KF Streptococcus Agar
TTC Solution 1%
Intended Use TTC (2, 3, 5-Triphenyl Tetrazolium Chloride) in aqueous 1%
KF Streptococcus Agar is used with TTC Solution 1% in solution may be added to various agar and fluid media to lend
isolating and enumerating fecal streptococci. color to cells and to colonies on agar or on membranes, thereby
facilitating the detection of growth.
Summary and Explanation
Kenner et al. developed KF (Kenner Fecal) Streptococcal Agar Principles of the Procedure
with TTC for use in detecting streptococci in surface waters by Peptone provides a source of nitrogen, amino acids and
direct plating or by the membrane filtration method.1 These carbon. Yeast extract is a source of trace elements, vitamins and
investigators compared the performance of their formulation amino acids. Maltose and lactose are fermentable carbohydrates
to other media used for enumerating fecal streptococci and and carbon sources. Sodium azide is a selective agent. Bromcresol
achieved greater recoveries with KF Streptococcal Agar. The purple is an indicator dye.
medium is recommended for use in determining counts of fecal
streptococci in water.
Cultural Response
Difco™ KF Streptococcus Agar
Prepare the medium per label directions. Inoculate using the pour plate
technique and incubate at 35 ± 2°C for 46-48 hours.
INOCULUM COLONY
Organism ATCC™ CFU RECOVERY Color
Enterobacter Marked to
aerogenes 13048 3×102-103 complete inhibition –
Enterococcus Red to
faecalis 19433 30-300 Good pink centers
Enterococcus Red to
faecalis 29212 30-300 Good pink centers
Escherichia Marked to
coli 25922 3×102-103 complete inhibition –
275
KF Streptococcus Broth
Intended Use Principles of the Procedure
KF Streptococcus Broth is used for isolating fecal streptococci. Peptone provides a source of nitrogen, amino acids and carbon.
Yeast extract is a source of trace elements, vitamins and amino
Summary and Explanation acids. Maltose and lactose are the fermentable carbohydrates
Kenner et al. developed KF (Kenner Fecal) Streptococcal Broth and carbon sources. Sodium azide is the selective agent.
for the detection and enumeration of enterococci in waters.1,2 Bromcresol purple is the indicator dye.
They found that this formulation was superior to other liquid The addition of 1% TTC (2,3,5-Triphenyl Tetrazolium Chlo-
media in the recovery of enterococci in Most Probable Number ride), in the membrane filter procedure, causes the enterococci
(MPN) test systems. The medium is not specific for presump- to have a deep red color as a result of tetrazolium reduction to
tive identification of group D streptococci. Other tests are an acid azo dye.
required.2-4
276
Enterococcus faecalis
User Quality Control ATCC™ 29212
Identity Specifications
Difco™ KF Streptococcus Broth
Dehydrated Appearance: Light greenish-beige, free-flowing, homogeneous.
Solution: 5.64% solution, soluble in purified water upon boiling. Solution
is reddish to light purple, clear to very slightly opalescent.
Prepared Appearance: Purple, clear to very slightly opalescent.
Reaction of 5.64%
Solution at 25°C: pH 7.2 ± 0.2
Cultural Response
Difco™ KF Streptococcus Broth
Prepare the medium per label directions. Supplement with TTC Solution 1%. Using
the membrane filter technique, inoculate and incubate at 35 ± 1°C in an atmosphere
saturated with water vapor for 46-48 hours.
Organism ATCC™ INOCULUM CFU RECOVERY COLONY COLOR
Enterobacter aerogenes 13048 3×102-103 Inhibition –
Enterococcus faecalis 19433 30-200 Good Red
Enterococcus faecalis 29212 30-200 Good Red
Escherichia coli 25922 3×10 -10
2 3
Inhibition –
277
Cultural Response
BBL™ Kligler Iron Agar
Prepare the medium per label directions. Stab inoculate with fresh cultures and
incubate at 35 ± 2°C for 24 hours. Uninoculated Echerichia coli Morganella Salmonella
Tube ATCC™ 25922 morganii Typhimurium
ORGANISM ATCC™ recovery SLANT BUTT H2S ATCC™ 8019 ATCC™ 14028
Escherichia coli 25922 Good Acid Acid with gas –
Morganella morganii 8019 Good Alkaline Acid with –
or without gas
Pseudomonas Alkaline
aeruginosa 27853 Good Alkaline without gas –
Salmonella enterica
subsp. enterica
serotype Typhi 19430 Good Alkaline Acid without gas +
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028 Good Alkaline Acid with gas +
Shigella flexneri 12022 Good Alkaline Acid without gas –
278
red pH indicator in response to the acid produced during the To enhance the alkaline condition in the slant, free exchange of
fermentation of these sugars. The dextrose concentration is only air must be permitted through the use of a loose closure. If the
10% of the lactose concentration. The combination of ferric tube is tightly closed, an acid reaction (caused solely by dextrose
ammonium citrate and sodium thiosulfate enables the detection fermentation) will also involve the slant.
of hydrogen sulfide production.
Lactose nonfermenters (e.g., Salmonella and Shigella) initially
Expected Results
After incubation, record the reaction in the slant and butt, noting
produce a yellow slant due to acid produced by the fermentation
gas formation and hydrogen sulfide production.
of the small amount of dextrose. When the dextrose supply is
exhausted in the aerobic environment of the slant, the reaction Typical reactions produced by members of the Enterobacte-
reverts to alkaline (red slant) due to oxidation of the acids. riaceae (majority of the species in the particular genus) are
The reversion does not occur in the anaerobic environment in presented in the following table.5
the butt, which remains acid (yellow butt). Lactose fermenters Slant Butt Gas H2S
produce yellow slants and butts because enough acid is produced Citrobacter Alkaline Acid + + or –
in the slant to maintain an acid pH under aerobic conditions. Edwardsiella Alkaline Acid + +
Organisms incapable of fermenting either carbohydrate produce Escherichia coli Acid Acid + –
red slants and butts.
Enterobacter Acid* Acid + –
Hydrogen sulfide production is evidenced by a black color either Morganella Alkaline Acid ± –
throughout the butt, or in a ring formation near the top of Proteus Alkaline or Acid Acid + +
the butt. Gas production (aerogenic reaction) is detected as Providencia Alkaline Acid ± –
individual bubbles or by splitting or displacement of the agar. Salmonella Alkaline Acid + +
Shigella Alkaline Acid – –
Formula *May revert to alkaline even though lactose fermented (E. aerogenes).
BBL™ Kligler Iron Agar
Approximate Formula* Per Liter References
Pancreatic Digest of Casein........................................ 10.0 g 1. Russell. 1911. J. Med. Res. 25:217.
2. Kligler. 1917. Am. J. Public Health. 7:1041.
Peptic Digest of Animal Tissue.................................... 10.0 g 3. Kligler. 1918. J. Exp. Med. 28:319.
Lactose...................................................................... 10.0 g 4. Bailey and Lacy. 1927. J. Bacteriol. 13:183.
Dextrose...................................................................... 1.0 g 5. Ewing. 1986. Edwards and Ewing’s identification of the Enterobacteriaceae, 4th ed. Elsevier Science
Publishing Co., Inc. New York, N.Y.
Sodium Chloride.......................................................... 5.0 g
Ferric Ammonium Citrate............................................. 0.5 g
Sodium Thiosulfate...................................................... 0.5 g Availability
Agar.......................................................................... 15.0 g BBL™ Kligler Iron Agar
Phenol Red................................................................ 25.0 mg
*Adjusted and/or supplemented as required to meet performance criteria. BAM CCAM CMPH2 COMPF ISO MCM9
Cat. No. 211317 Dehydrated – 500 g
H
Directions for Preparation from 220896 Prepared Slants – Pkg. of 10*
-
K
220897 Prepared Slants – Ctn. of 100*
Dehydrated Product *Store at 2-8°C.
1. Suspend 52 g of the powder in 1 L of purified water. Mix
thoroughly.
2. Heat with frequent agitation and boil for 1 minute to com-
pletely dissolve the powder.
3. Dispense and autoclave at 121°C for 15 minutes.
4. Cool in a slanted position such that deep butts are formed.
For best results, the medium should be used on the date of
preparation or melted and resolidified before use.
5. Test samples of the finished product for performance using
stable, typical control cultures.
Procedure
To inoculate, carefully touch the center of an isolated colony
on an enteric plated medium with a cool, sterile needle, stab
into the medium in the butt of the tube, and then streak back
and forth along the surface of the slant. Several colonies from
each primary plate should be studied separately, since mixed
infections may occur. Incubate tubes with loosened caps for
18-24 hours at 35 ± 2°C in an aerobic atmosphere.
279
Procedure
1. Transfer growth from a single colony or a loopful of liquid
suspension and inoculate the broth medium.
2. Incubate at 35 ± 2°C for 18-24 hours.
280
281
282
3 material.
2.5 Solution: 2.0% solution, soluble in purified water.
2 Solution is light to medium, yellow to tan,
slightly hazy to hazy.
1.5
Prepared Appearance: Light to medium, yellow to tan, slightly
1
hazy to hazy.
0.5 Reaction of 3.5%
0 Solution at 25°C: pH 6.6 – 7.1
0 1 2 3 4 5 6 7 8
Time in Hours
Classical LB Broth Select APS LB Broth Cultural Response
BBL™ Select APS™ LB Broth Base
Prepare the medium per label directions. Inoculate and incubate at 37°C,
250 rpm for 16 hours.
Principles of the Procedure
Soy hydrolysate provides nitrogen and carbon essential for bac- ORGANISM ATCC™ RECOVERY
terial metabolism. Yeast extract supplies vitamins, amino acids Escherichia coli 700790 Good
and trace elements which enhance bacterial growth and plasmid
yield. Sodium chloride provides sodium ions for transport and
osmotic balance.
283
284
285
Identity Specifications
Difco™ LPM Agar Base
Dehydrated Appearance: Light tan, homogeneous, may have a tendency
to clump.
Solution: 5.05% solution, soluble in purified water upon
boiling. Solution is light to medium amber, very
slightly to slightly opalescent.
Prepared Appearance: Light to medium amber, slightly opalescent.
Reaction of 5.05%
Solution at 25°C: pH 7.3 ± 0.2
BBL™ Listeria Selective Supplement
Lyophilized Appearance: White to pale yellow and dry.
Rehydrated Appearance: Clear to pale yellow to light tan yellow; clear.
Cultural Response
Difco™ LPM Agar Base
Prepare the medium per label directions with Listeria Selective Supplement.
Inoculate and incubate at 35 ± 2°C for 18-48 hours.
Organism ATCC™ INOCULUM CFU Growth w/MOXALACTAM
Bacillus subtilis 6633 103 Partial inhibition
Enterococcus faecalis 29212 10 -2×10
3 3
Marked to complete inhibition
Escherichia coli 25922 103-2×103 Marked to complete inhibition
Listeria monocytogenes 19114 102-103 Good (at 40-48 hours)
Staphylococcus aureus 25923 103 Marked to complete inhibition
286
preserve the viability and sensitivity of the test organism for Dehydrated Appearance: Tan, free-flowing, homogeneous.
its intended purpose; Solution: 3.8% solution, soluble in purified water upon boiling
2. Inoculum Media: To condition the test culture for immediate 2-3 minutes. Solution is medium amber, opalescent
when hot, clear after cooling, may have a slight
use; precipitate.
3. Assay Media: To permit quantitation of the vitamin under Prepared Appearance: Medium amber, clear, may have a slight precipi-
test. They contain all the factors necessary for optimal tate.
growth of the test organism except the single essential Reaction of 3.8%
vitamin to be determined. Solution at 25°C: pH 6.8 ± 0.2
287
288
Lactobacillus delbrueckii
User Quality Control subsp. lactis
ATCC™ 7830
Identity Specifications
Difco™ Lactobacilli MRS Agar
Dehydrated Appearance: Light tan, free-flowing, homogeneous.
Solution: 7.0% solution, soluble in purified water upon
boiling. Solution is medium amber, clear to
slightly opalescent.
Prepared Appearance: Medium amber, very slightly to slightly opales-
cent.
Reaction of 7.0%
Solution at 25°C: pH 6.5 ± 0.2
Difco Lactobacilli MRS Broth
™
Cultural Response
Difco™ Lactobacilli MRS Agar or Lactobacilli MRS Broth
Prepare the medium per label directions. Inoculate Lactobacilli MRS Agar and
incubate in a 5% CO2 atmosphere at 35° ± 2°C for 24- 72 hours. Inoculate
Lactobacilli MRS Broth and incubate at 35° ± 2°C for 18-24 hours.
Organism ATCC™ INOCULUM CFU RECOVERY
Lactobacillus delbrueckii
subsp. lactis 7830 102-103 Good
Lactobacillus fermentum 9338 10 -10
2 3
Good
Lactobacillus johnsonii 11506 102-103 Good
289
2. Heat with frequent agitation and boil for 1 minute to Expected Results
completely dissolve the powder. Lactobacilli appear as large, white colonies embedded in or on
3. Autoclave at 121°C for 15 minutes. Lactobacilli MRS Agar or as turbidity in Lactobacilli MRS Broth.
4. Test samples of the finished product for performance using Growth may be subcultured onto the appropriate media for use
stable, typical control cultures. in additional procedures. Refer to appropriate references for
recommendations on the culture of Lactobacillus spp.2,3
Procedure
Direct Counts Limitation of the Procedure
1. To obtain direct counts of lactobacilli, pour 15-20 mL Organisms other than lactobacilli may grow in these media.
sterile, molten (45-50°C) Lactobacilli MRS Agar into ster- Isolates must be confirmed as lactobacilli by appropriate bio-
ile Petri dishes containing 1 mL volumes of diluted test chemical testing.
sample.
2. Distribute the inoculum throughout the medium by rotating References
1. deMan, Rogosa and Sharpe. 1960. J. Appl. Bacteriol. 23:130.
the plate in one direction and then in the reverse direction. 2. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
3. Allow the medium to solidify on a flat surface for 5-10 American Society for Microbiology, Washington, D.C.
3. Downes and Ito(ed.). 2001. Compendium of methods for the microbiological examination of foods,
minutes. 4th ed. American Public Health Association, Washington, D.C.
Lactose Broth
Intended Use Principles of the Procedure
Lactose Broth is used for detection of the presence of coliform The peptone and beef extract provide essential nutrients for
organisms, as a pre-enrichment broth for salmonellae and in the bacterial metabolism. Lactose provides a source of fermentable
study of lactose fermentation of bacteria in general. carbohydrate for coliform organisms. Growth with the formation
of gas is a presumptive test for coliforms.
Summary and Explanation
Lactose Broth was formulated in accordance with recommen- Formula
dations of the American Public Health Association (APHA) Difco™ Lactose Broth
and the American Water Works Association for testing dairy Approximate Formula* Per Liter
products and water for the presence of coliform organisms.1,2 Beef Extract.................................................................. 3.0 g
Peptone....................................................................... 5.0 g
This medium was, but no longer is, listed as an alternative Lactose........................................................................ 5.0 g
to Lauryl Sulfate Broth in the presumptive portion of the *Adjusted and/or supplemented as required to meet performance criteria.
290
Cultural Response
Difco™ Lactose Broth
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for
18-48 hours. After incubation, add 1-2 drops of 1% phenol red solution to observe
acid production.
INOCULUM
ORGANISM ATCC™ CFU RECOVERY ACID GAS
Enterobacter aerogenes 13048 30-300 Good + +
Escherichia coli 25922 30-300 Good + +
Uninoculated Escherichia coli
Enterococcus faecalis 19433 30-300 Good + – Tube ATCC™ 25922
Salmonella enterica
subsp. enterica
serotype Typhi 6539 30-300 Good – –
Expected Results
Cat. No. 256668 Prepared Bottles, 90 mL – Pkg. of 10*
*Store at 2-8°C.
L
After incubation at 35 ± 2°C for 24 ± 2 hours, examine tubes
for turbidity and for gas production in the Durham tubes. If no
gas has formed and been trapped in the inverted tube, reincubate
and reexamine after 48 ± 3 hours.
Turbidity of the medium accompanied by formation of gas in any
amount in the Durham tubes within 48 ± 3 hours is a positive
presumptive test for the presence of coliforms in the sample. The
result should be confirmed by additional standard testing.
291
293
Directions for Preparation from NOTE: Refrigerated broth generally becomes cloudy or forms
Dehydrated Product precipitates but clears upon warming to room temperature.
Difco™ Lauryl Tryptose Broth However, clarity is not important because only gas production
1. Suspend 35.6 g of the powder in 1 L of purified water. Mix is significant.
thoroughly.
2. Warm slightly to completely dissolve the powder.
Procedure
Refer to the official test procedures for the detection of coliforms
3. Dispense required amounts into tubes containing inverted
in the compendia of methods for microbiological examination
fermentation vials (see table).
of foods, dairy products and waters.2-5
4. Autoclave at 121°C for 15 minutes. Cool the broth as quickly
as possible.
Expected Results
5. Test samples of the finished product for performance using
After incubation of the tubes with loosened caps at 35 ± 0.5°C
stable, typical control cultures.
for 24 hours, examine for turbidity and for gas production in
BBL™ Lauryl Sulfate Broth the Durham fermentation tubes. If no gas has formed and been
1. Suspend 35.6 g of the powder in 1 L of purified water. trapped in the inverted tube, reincubate and reexamine after
2. Dispense in test tubes, containing inverted Durham tubes, 48 hours.2-5
in 10 mL amounts for testing samples of 1 mL or less. For Turbidity of the medium accompanied by formation of gas in
testing 10 mL quantities of samples, dissolve 71.2 g of the any amount in the Durham tubes within 48 hours is a positive
powder in 1 L of purified water and distribute in 10 mL presumptive test for the presence of coliforms in the sample.2-5
amounts. The concentration of the medium should be The result should be confirmed by additional standard testing.
varied according to the size of the test samples (see table).
3. Autoclave at 121°C for 15 minutes. After autoclaving, cool References
the broth as quickly as possible. 1. Mallmann and Darby. 1941. Am. J. Public Health 31:127.
2. Downes and Ito. 2001. Compendium of methods for the microbiological examination of foods. 4th
4. Test samples of the finished product for performance using ed. American Public Health Association, Washington, D.C.
3. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy products, 17th ed.
stable, typical control cultures. American Public Health Association. Washington, D.C.
4. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
Preparation of Lauryl Tryptose (Sulfate) Broth4 21st ed., online. American Public Health Association, Washington, D.C.
5. Horwitz (ed.). 2007. Official methods of analysis of AOAC International. 18th ed., online. AOAC
amount of dehydrated International. Gaithersburg, Md.
medium in volume of medium
Inoculum mL the tube mL medium+inoculum mL required g/L
1 10 or more 11 or more 35.6 Availability
10 10 20 71.2 Difco™ Lauryl Tryptose Broth
AOAC BAM CCAM COMPF ISO SMD SMWW
10 20 30 53.4
Cat. No. 224140 Dehydrated – 100 g
20 10 30 106.8 224150 Dehydrated – 500 g
100 50 150 106.8 224120 Dehydrated – 2 kg
100 35 135 137.1 224130 Dehydrated – 10 kg
294
295
color.6 Glycine, vancomycin, and polymyxin B are included to L. pneumophila produces small to large, smooth, pale green
provide moderate inhibition of competing flora and, therefore, colonies which are slightly mucoid.
enhance the recovery of Legionella species.7
References
Procedure 1. Feely, Gibson, Gorman, Langsford, Rasheed, Mackel, and Baine. 1979. J. Clin. Microbiol. 10:437.
2. Feely, Gorman, Weave, Mackel and Smith. 1978. J. Clin. Microbiol. 8:320.
Use standard procedures to obtain isolated colonies from 3. Pasculle, Feely, Gibson, Cordes, Myerowitz, Patton, Gorman, Carmack, Ezzell and Dowling. 1980.
J. Infect. Dis. 191:727.
specimens. Incubate the plates in an inverted position (agar-side 4. Edelstein. 1981. J. Clin. Microbiol. 14:298.
5. Data on file, BD Diagnostic Systems.
up) at 35°C for a minimum of 3 days. Growth is usually visible 6. Vickers, Brown and Garrity. 1981. J. Clin. Microbiol. 13:380.
7. Wadowski and Yee. 1981. Appl. Environ. Microbiol. 42:768.
within 3-days, but may take up to 2 weeks to appear.
Cultural Response
Difco™ Leptospira Medium EMJH with Enrichment
Prepare the medium per label directions. Inoculate tubes with undiluted fresh cultures of
Leptospira and incubate at 30 ± 2°C for up to 7 days.
Organism ATCC™ INOCULUM RECOVERY Uninoculated Leptospira interrogans
Tube serotype australis
Leptospira interrogans ATCC™ 23605
serotype australis 23605 2-3 Loopfuls Good
Leptospira interrogans
serotype canicola 23470 2-3 Loopfuls Good
Leptospira kirschneri
serotype grippotyphosa 23604 2-3 Loopfuls Good
298
Procedure7 Availability
Blood and Cerebrospinal Fluid Difco™ Leptospira Medium Base EMJH
Freshly drawn blood is preferable; otherwise, use blood taken SMWW
with SPS, sodium oxalate or heparin. Cat. No. 279410 Dehydrated – 500 g
1. Inoculate four 5 mL tubes of Leptospira Medium EMJH with
1-2 drops of fluid per tube.
Difco™ Leptospira Enrichment EMJH
SMWW
L
2. Incubate in the dark at 28-30°C or at room temperature. Cat. No. 279510 Bottle – 6 x 100 mL*
*Store at 2-8°C.
299
Letheen Agar and Letheen Broth are specified for use by the
User Quality Control
American Society for Testing and Materials (ASTM) in the
Identity Specifications Standard Test Method for Preservatives in Water-Containing
Difco™ Letheen Agar Cosmetics.4
Dehydrated Appearance: Tan, moist appearance, with a few clumps.
Solution: 3.2% solution, soluble in purified water upon Principles of the Procedure
boiling. Solution is light to medium amber,
clear to slightly opalescent, may have a slight,
Letheen Agar contains beef extract and peptone which
fine precipitate (opalescent immediately after provide the carbon and nitrogen sources required for growth of
autoclaving). a wide variety of organisms. Dextrose is provided as a source
Prepared Appearance: Light to medium amber, slightly opalescent, may of fermentable carbohydrate. Agar is the solidifying agent.
have a slight precipitate.
Lecithin and polysorbate 80 are added to neutralize surface
Reaction of 3.2%
disinfectants.2,5,6 Lecithin is added to neutralize quaternary
Solution at 25°C: pH 7.0 ± 0.2
ammonium compounds and polysorbate 80 is incorporated
Difco Letheen Broth
™
to neutralize phenols, hexachlorophene, formalin and, with
Dehydrated Appearance: Tan, appears moist, with a tendency to clump.
lecithin, ethanol.7
Solution: 2.57% solution, soluble in purified water upon
boiling. Solution is light amber, clear to slightly Letheen Broth contains peptone and beef extract which provide
opalescent (opalescent when hot). May have a the carbon and nitrogen sources necessary for growth. Lecithin
very slight precipitate.
and polysorbate 80 are added as surface active disinfectant
Prepared Appearance: Light to medium amber, clear to slightly opales-
cent, may have a slight precipitate.
neutralizing agents.2,5,6 Sodium chloride is included to maintain
Reaction of 2.57%
osmotic balance.
Solution at 25°C: pH 7.0 ± 0.2
Formulae
Cultural Response Difco™ Letheen Agar
Difco™ Letheen Agar Approximate Formula* Per Liter
Prepare the medium per label directions. Inoculate and incubate at Beef Extract.................................................................. 3.0 g
35 ± 2°C for 40-48 hours. Pancreatic Digest of Casein.......................................... 5.0 g
Dextrose...................................................................... 1.0 g
Organism ATCC™ Inoculum CFU RECOVERY Agar.......................................................................... 15.0 g
Escherichia coli 11229 102-103 Good Polysorbate 80............................................................. 7.0 g
Lecithin........................................................................ 1.0 g
Staphylococcus aureus 6538 10 -10
2 3
Good
Difco™ Letheen Broth Difco™ Letheen Broth
Approximate Formula* Per Liter
Prepare the medium per label directions. Inoculate and incubate at
Beef Extract.................................................................. 5.0 g
35 ± 2°C for 40- 48 hours.
Proteose Peptone No. 3.............................................. 10.0 g
Organism ATCC™ Inoculum CFU RECOVERY Polysorbate 80 ............................................................ 5.0 g
Escherichia coli 11229 102-103 Good Lecithin........................................................................ 0.7 g
Sodium Chloride.......................................................... 5.0 g
Salmonella enterica *Adjusted and/or supplemented as required to meet performance criteria.
subsp. enterica
serotype Typhi 6539 102-103 Good
Directions for Preparation from
Staphylococcus aureus 6538 10 -10
2 3
Good
Dehydrated Product
1. Suspend the powder in 1 L of purified water:
effectively neutralizes quaternary ammonium compounds in the Difco™ Letheen Agar – 32 g;
testing of germicidal activity. Letheen Agar is a modification of Difco™ Letheen Broth – 25.7 g.
TGE agar with the addition of lecithin and polysorbate 80. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to
Letheen Broth was developed as a subculture medium for
completely dissolve the powder.
the neutralization of quaternary ammonium compounds in
3. Autoclave at 121°C for 15 minutes.
disinfectant testing. Quisno, Gibby and Foter2 found that
4. Test samples of the finished product for performance using
the addition of lecithin and polysorbate 80 to F.D.A. Broth
stable, typical control cultures.
resulted in a medium that neutralized high concentrations of
quaternary ammonium salts. The resulting medium, termed NOTE: The dehydrated Letheen Agar has a characteristic
“Letheen” (a combination of Lecithin and Tween), was “brown sugar” appearance and may seem moist. This does not
easy to prepare and clear in appearance, which aided in visual indicate deterioration.
inspection for growth. Letheen Broth is recommended in the
Official Methods of Analysis of AOAC International3 for Procedure
use with disinfectants containing cationic surface active Letheen Agar and Letheen Broth are used in a variety of proce-
materials. dures. Consult appropriate references for further information.3,4
300
4. Incubate the diluted samples from step 1 at 35 ± 2°C for Difco™ Letheen Broth, Modified
7 days. Subculture enriched samples onto Letheen Agar, BAM
Modified only if there is no growth on the primary Letheen Cat. No. 263010 Dehydrated – 500 g*
Agar, Modified plates. Europe
Cat. No. 257327 Prepared Bottles, 500 mL – Pkg. of 4
*Store at 2-8°C.
Lim Broth
Intended Use and nalidixic acid is a medium recommended to maximize the
Lim Broth is used for the selective enrichment of group B strep- likelihood of recovering group B streptococci upon plating on
tococci (Streptococcus agalactiae), especially from genital sheep blood agar.1 Lim Broth is prepared from Todd Hewitt
specimens. Broth by the addition of colistin and nalidixic acid, at the
recommended concentrations, plus yeast extract for enhanced
Summary and Explanation growth of group B streptococci.2
Since its emergence in the 1970s, neonatal group B streptococcal Group B streptococci have also been found in cases of sepsis
disease has become the major infectious cause of illness and death in nonparturient women and men, and in joint infections,
among newborns. Prior to 1994, an estimated 7600 episodes osteomyelitis, urinary tract infections and wound infections.
of invasive group B streptococcal disease, primarily sepsis They are associated with endocarditis, pneumonia and pyelo-
and meningitis, occurred in newborns each year in the United nephritis in immunosuppressed patients.7
States, with approximately 80% of those episodes representing
early-onset disease occurring within the first week of life.1 The Principles of the Procedure
disease is spread to newborns through vertical transmission from Todd Hewitt Broth base is a general-purpose medium primarily
a mother who carries B streptococci in her anorectum or genital used for the cultivation of β-hemolytic streptococci, especially
tract. Lim and colleagues combined the use of an enriched, for serologic studies.8
selective broth medium and slide coagglutination test to rapidly
screen such maternity patients.2-5 The peptones, dextrose and salts provide an excellent nutritional
base for the growth of streptococci. The added yeast extract is
The Centers for Disease Control and Prevention (CDC) has a rich source of B-complex vitamins. Dextrose stimulates
published guidelines for screening and use of intrapartum hemolysin production. Disodium phosphate and sodium
chemoprophylaxis for prevention of neonatal group B strep- carbonate provide buffering action to counteract the acidity
tococcal disease.6 The use of Todd Hewitt Broth with colistin
302
produced during the fermentation of the carbohydrate, thereby (β- or non-hemolytic, gram-positive and catalase-negative).
protecting the hemolysin from inactivation by the acid. Specific identification may be performed; e.g., using streptococcal
Nalidixic acid and colistin suppress growth of gram-negative grouping sera, the CAMP test or other procedures.
bacteria. Jones et al. reported detection of group B streptococci by slide
coagglutination after 5 hours of incubation when the concentra-
Procedure tion of organisms in the culture was 107 per mL or greater.4
Inoculate tubes and incubate with loosened caps at 35 ± 2°C in
an aerobic atmosphere with or without added carbon dioxide. References
If desired, perform a slide coagglutination test for group B 1. Federal Register. 1994. Prevention of group B streptococcal disease: a public health perspective. Fed.
Regist. 59:64764.
streptococci after 5 hours of incubation.4 2. Jones, Friedl, Kanarek, Williams and Lim. 1983. J. Clin. Microbiol. 18:558.
3. Lim, Kanarek and Peterson. 1982. Curr. Microbiol. 7:99.
4. Jones, Kanarek and Lim. 1984. J. Clin. Microbiol. 20:438.
If turbidity is observed after 18-24 hours, subculture from the 5. Jones, Kanarek, Angel and Lim. 1983. J. Clin. Microbiol. 18:526.
broth culture to a sheep blood agar plate; otherwise, incubate 6. Centers for Disease Control and Prevention. 2002. Morbid. Mortal. Weekly Rep. 51 (No. RR-
11):1.
an additional 24 hours before discarding.1 7. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby Inc.,
St. Louis, Mo.
8. Todd and Hewitt. 1932. J. Pathol. Bacteriol. 35:973.
Expected Results
Growth in broth medium is indicated by the presence of Availability
turbidity compared to an uninoculated control. BBL™ Lim Broth
BS12 CMPH2 MCM9
Subculture to a Trypticase™ Soy Agar with 5% Sheep Blood Cat. No. 292209 Prepared Tubes (K Tubes), 5 mL – Pkg of 10*
(TSA II) plate and incubate for 18-24 hours, or up to 48 hours if 296266 Prepared Tubes (K Tubes), 5 mL – Ctn. of 100*
necessary. Identify organisms suggestive of group B streptococci *Store at 2-8°C.
Buffered Listeria Enrichment Broth Base is used as an enrichment The most common contaminating bacteria found in food sources
broth for the cultivation of Listeria spp. from food according to potentially containing Listeria are: streptococci, especially the
the U.S Food and Drug Administration (FDA).1 enterococci, micrococci, Bacillus species, Escherichia coli,
Pseudomonas aeruginosa and Proteus vulgaris.9 Identification
Summary and Explanation of Listeria is based on successful isolation of the organism,
First described in 1926 by Murray, Webb and Swann,2 Listeria biochemical characterization and serological confirmation.
monocytogenes is a widespread problem in public health and
the food industries. This organism can cause human illness
Listeria Enrichment Broth is based on the formula developed L
by Lovett et al.10 in which Tryptic Soy Broth is supplemented
and death, particularly in immunocompromised individuals
with yeast extract for optimum growth of Listeria. Listeria
and pregnant women.3 The first reported foodborne outbreak
of listeriosis was in 1985.4 Since then, microbiological and Enrichment Broth, Modified is a modification of Listeria
epidemiological evidence from both sporadic and epidemic cases Enrichment Broth in which the concentration of one of the
of listeriosis has shown that the principal route of transmission selective agents, acriflavine, has been reduced from 15 mg to
is via the consumption of foodstuffs contaminated with Listeria 10 mg per liter. This modification reflects the lower concentra-
monocytogenes.5 tion once specified by the IDF for isolation of L. monocytogenes
from milk and milk products (IDF Standard No. 143A).11 More
Implicated vehicles of transmission include turkey frankfurt-
recently, the IDF standard has been replaced by ISO Standard
ers,6 coleslaw, pasteurized milk, Mexican-style cheese, paté,
11290 which utilizes Half Fraser Broth for enrichment.12
and pickled pork tongue. The organism has been isolated from
commercial dairy and other food processing plants, and is Buffered Listeria Enrichment Broth Base is a modification of
ubiquitous in nature, being present in a wide range of unpro- Listeria Enrichment Broth with added buffering strength. The
cessed foods and in soil, sewage, silage and river water.7 addition of selective agents is delayed until after four hours of
enrichment with this formula.1
303
304
Medium.
4. Incubate the agar plates at 37°C for 48 ± 2 hours. Availability
Difco™ Listeria Enrichment Broth
Buffered Listeria Enrichment Broth Base Cat. No. 222220 Dehydrated – 500 g
For food samples, the FDA1 selective enrichment method is as 222210 Dehydrated – 10 kg
follows: Difco™ Listeria Enrichment Broth, Modified
1. Add 25 mL liquid or 25 g solid test material to 225 mL Cat. No. 220530 Dehydrated – 500 g
245152 Dehydrated – 2 kg
Buffered Listeria Enrichment Broth Base without selective 220520 Dehydrated – 10 kg
agents and mix or blend thoroughly.
Difco™ Buffered Listeria Enrichment Broth Base
2. Incubate for 4 hours at 30°C.
BAM COMPF SMD
3. Add 0.455 mL acriflavine HCl solution, 1.8 mL nalidixic Cat. No. 290720 Dehydrated – 500 g
acid solution and 1.15 mL cycloheximide solution to 225 mL 214919 Dehydrated – 2 kg
Buffered Listeria Enrichment Broth Base and continue incu-
bating another 44 hours, for a total of 48 hours, at 30°C.
4. At 24 and 48 hours incubation, streak incubated broth onto
both Oxford Medium and LPM Agar (or PALCAM Agar) L
plates. Incubate Oxford Medium and PALCAM plates at
35°C for 24-48 hours, and LPM plates at 30°C for 24-48
hours.
Litmus Milk
Intended Use Summary and Explanation
Litmus Milk is used for the maintenance of lactic acid bacteria Litmus Milk has been used for many years for determining
and as a differential medium for determining the action of the metabolic activities of microorganisms in milk as an aid to
bacteria on milk. the identification of bacterial species. It is especially useful in
species differentiation within the genus Clostridium.
This medium is also of value in the maintenance and propaga-
tion of lactic bacteria.
305
Cultural Response
BBL™ Litmus Milk
Prepare the medium per label directions. Inoculate with fresh cultures diluted 1:10
and incubate at 35 ± 2°C for 7 days. Uninoculated Bacillus subtilis Clostridium Lactobacillus
Tube ATCC™ 6633 perfringens rhamnosus
ORGANISM ATCC™ RESULT ATCC™ 12924 ATCC™ 7469
306
Identity Specifications
Difco™ Liver Infusion Agar Precautions6
Dehydrated Appearance: Dark beige to light tan, free-flowing, homoge- 1. Biosafety Level 2 practices, containment equipment and
neous. facilities are recommended for activities with clinical speci-
Solution: 5.5% solution, soluble in purified water upon mens of human or animal origin containing or potentially
boiling. Solution is medium to dark amber, slightly containing pathogenic Brucella spp.
opalescent to opalescent.
2. Biosafety Level 3 practices, containment equipment and
Prepared Appearance: Medium to dark amber, slightly opalescent.
facilities are recommended for all manipulations of cultures
Reaction of 5.5%
Solution at 25°C: pH 6.9 ± 0.2 of the pathogenic Brucella spp. and for experimental animal
Difco™ Liver Infusion Broth studies.
Dehydrated Appearance: Tan, free-flowing, homogeneous.
Solution: 3.5% solution, soluble in purified water. Solution Directions for Preparation from
is medium to dark amber, clear to very slightly Dehydrated Product
opalescent with a few particles. 1. Suspend/dissolve the powder in 1 L of purified water:
Difco™ Liver Infusion Agar – 55 g;
L
Prepared Appearance: Medium to dark amber, clear to very slightly
opalescent with a few particles. Difco™ Liver Infusion Broth – 35 g.
Reaction of 3.5% Mix thoroughly.
Solution at 25°C: pH 6.9 ± 0.2
2. Heat the Liver Infusion Agar with frequent agitation and
Cultural Response boil for 1 minute to completely dissolve the powder.
Difco™ Liver Infusion Agar or Liver Infusion Broth 3. Autoclave at 121°C for 15 minutes.
Prepare the medium per label directions. Inoculate and incubate at 4. Test samples of the finished product for performance using
35 ± 2°C for 18-48 hours, or up to 72 hours if necessary. Incubate Clostridium stable, typical control cultures.
under anaerobic conditions. Incubate Brucella spp. and S. pneumoniae with
3-5% CO2. Procedure
Organism ATCC™ INOCULUM CFU RECOVERY For a complete discussion of the isolation and identification
Brucella abortus 11192* 30-300 Good of Brucella, anaerobic microorganisms and other fastidious
Brucella melitensis 4309* 30-300 Good pathogens, refer to the procedures described in Bailey & Scott’s
Brucella suis 4314* 30-300 Good Diagnostic Microbiology,4 Clinical Microbiology Procedures
Clostridium sporogenes 11437 30-300 Good Handbook7 and Manual of Clinical Microbiology.8
Streptococcus pneumoniae 6305 30-300 Good
*Minimally, one strain of Brucella should be used for performance testing. These ATCC strains
should be used if available.
307
308
Procedure Availability
For a complete discussion of the isolation and identification Difco™ Liver Veal Agar
of anaerobic bacteria and other fastidious aerobic pathogens, BAM CCAM COMPF
refer to the procedures described in Clinical Microbiology Cat. No. 259100 Dehydrated – 500 g
Procedures Handbook7 and Manual of Clinical Microbiology.8
Expected Results
Refer to appropriate references and procedures for results.
Lowenstein Media
Lowenstein Medium Base • Lowenstein-Jensen
Medium • Lowenstein-Jensen Medium, Gruft
Lowenstein-Jensen Medium with Iron
Lowenstein-Jensen Medium with Pyruvic Acid
Lowenstein-Jensen Medium with 5% Sodium Chloride
Intended Use percentage of tubercle bacilli recovered from clinical specimens
Lowenstein Medium and Lowenstein-Jensen (LJ) Medium are compared to recovery on the standard LJ Medium.4
used for the isolation and cultivation of mycobacteria and as Wayne and Doubek differentiated rapidly-growing from
bases for selective, differential and enriched media for myco- slow-growing mycobacteria based on iron intake.5 The rapid-
bacteria. growing mycobacteria take up iron in the medium, producing
LJ Medium, tubed as deeps, is used for the semi-quantitative rusty-brown colonies and a tan discoloration in the medium.6
catalase test. M. chelonae and slow-growing species do not take up the iron.7
LJ Medium, Gruft, is a selective medium used for the isolation
and cultivation of mycobacteria.
Hughes 8 and Dixon and Cuthbert 9 reported that the addition
of pyruvic acid to egg-based media resulted in improved
L
recovery of tubercle bacilli compared to recovery on egg-based
LJ Medium with Iron is used to determine iron uptake for
media supplemented only with glycerol. Dixon and Cuthbert
differentiation and identification of mycobacteria.
recommended using pyruvic acid-egg medium in addition to
LJ Medium with Pyruvic Acid is an enrichment medium used media supplemented with glycerol for optimum recovery of
for enhanced growth of mycobacteria. tubercle bacilli from clinical specimens.9
LJ Medium with 5% sodium chloride is used to characterize Additionally, the medium is available with the addition of
certain strains of mycobacteria. 5% sodium chloride. Most rapid growers, the slowly growing
M. triviale and some strains of M. flavescens grow on NaCl-
Summary and Explanation containing media. The inability of M. chelonae subsp. chelo-
LJ Medium is an inspissated, egg-based medium developed from nae to grow helps differentiate it from other members of the
Jensen’s modification of Lowenstein’s formula.1,2 M. fortuitum complex (e.g., M. chelonae subsp. abscessus).6,10
Gruft modified LJ Medium by adding penicillin and nalidixic In the semi-quantitative catalase test, mycobacteria can be
acid for selective isolation of mycobacteria.3 Gruft also found differentiated into groups, based upon catalase activity. 6,11,12
that the addition of ribonucleic acid (RNA) increased the
309
Cultural Response
Difco™ Lowenstein Medium Base
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C under appropriate
atmospheric conditions for up to 21 days.
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Escherichia coli 25922 103-2×103 Partial inhibition
Mycobacterium tuberculosis H37Ra 25177 102-3×102 Good
Mycobacterium tuberculosis 27294 102-3×102 Good
Uninoculated Mycobacterium
Mycobacterium kansasii Group I 12478 102-3×102 Good Tube fortuitum
ATCC™ 6841
Mycobacterium scrofulaceum Group II 19981 102-3×102 Good
Mycobacterium intracellulare Group III 13950 102-3×102 Good
Mycobacterium fortuitum Group IV 6841 10 -3×10
2 2
Good
Pyruvic acid (2.5 mg/mL) enhances the growth of tubercle bacilli. Directions for Preparation from
The ability to tolerate 5% sodium chloride is a characteristic Dehydrated Product
of certain strains of mycobacteria (e.g., M. fortuitum and 1. Suspend 37.4 g of the powder in 600 mL of purified water
M. chelonae subsp. abscessus).10 containing 12 mL of glycerol. Do not add glycerol if bovine
Catalase is an intracellular, soluble enzyme capable of split- tubercle bacilli or other glycerophobic organisms are to be
ting hydrogen peroxide into water and oxygen. The oxygen cultivated. Mix thoroughly.
bubbles into the reaction mixture creating a column of bubbles. 2. Heat with frequent agitation just until the medium boils.
With a column height breakpoint of 45 mm, the mycobacteria 3. Autoclave at 121°C for 15 minutes. Cool to approximately
can be divided into groups: those producing less than 45 mm 50°C.
(M. tuberculosis, M. marinum, M. avium complex and 4. Meanwhile, prepare 1,000 mL of whole eggs collected asepti-
M. gastri); and those producing more than 45 mm (M. kansasii, cally and mixed thoroughly, without introducing air bubbles.
M. simiae, most scotochromogens, the nonphotochromogenic 5. Admix base and egg gently until mixture is uniform and
saprophytes and the rapid growers).6 without bubbles.
310
6. Distribute in suitable sterile containers such as screw-capped Stained smears may show acid-fast bacilli, which are reported
tubes. only as “acid-fast bacilli” unless definitive tests are performed.
7. Arrange tubes in slanted position, then coagulate and
Bottles may be examined by inverting the bottles on the stage
inspissate at 85°C for 45 minutes.
of a dissecting microscope. Read at 10-60× with transmitted
8. Test samples of the finished product for performance using
light. Scan rapidly at 10-20× for the presence of colonies. Higher
stable, typical control cultures.
magnification (30-60×) is helpful in observing colony morphol-
ogy; i.e., serpentine cord-like colonies.
Procedure
The test procedures are those recommended by the Centers Examine LJ Medium with Iron for rusty-brown colonies with a
for Disease Control and Prevention (CDC) for primary isola- tan discoloration in the surrounding medium, indicating uptake
tion from specimens containing mycobacteria.6 N-Acetyl-L- of the iron.
cysteine-sodium hydroxide (NALC-NaOH) solution is recom- The presence or absence of growth in the tube of medium
mended as a gentle but effective digesting and decontaminating containing 5% NaCl aids in the differentiation of mycobacterial
agent. These reagents are provided in the BBL™ MycoPrep™ isolates. The salt tolerance test is positive when numerous colo-
Mycobacterial Specimen Digestion/Decontamination Kit. For nies appear on the control medium and more than 50 colonies
detailed decontamination and culturing instructions, consult grow on the medium containing 5% NaCl.6,15 Colonies on the
an appropriate reference.6,7,12,14,15 control medium, but no visible growth on the test medium after a
Following inoculation, keep test containers shielded from total of 4 weeks of incubation constitutes a negative test.6,12,15
light and place them in a suitable system providing an aerobic In the semi-quantitate catalase test, mycobacteria fall into two
atmosphere enriched with carbon dioxide. Incubate at 35 ± 2°C. groups with M. tuberculosis falling into the group producing a
Slanted and bottled media should be incubated in a horizontal column of bubbles less than 45 mm in height.6
plane until the inoculum is absorbed. Tubes and bottles should
have screw caps loose for the first 3 weeks to permit the circula- Limitations of the Procedure
tion of carbon dioxide for the initiation of growth. Thereafter, 1. Negative culture results do not rule-out active infection
to prevent dehydration, tighten caps; loosened briefly once a by mycobacteria. Some factors that are responsible for
week. Stand tubes upright if space is a problem. unsuccessful cultures are:
• The specimen was not representative of the infectious
NOTE: Cultures from skin lesions suspected to be M. marinum
material; i.e., saliva instead of sputum.
or M. ulcerans should be incubated at 25-33°C for primary
• The mycobacteria were destroyed during digestion and
isolation; cultures suspected to contain M. avium or M. xenopi
decontamination of the specimen.
exhibit optimum growth at 40-42°C.6 Incubate a duplicate
• Gross contamination interfered with the growth of the
culture at 35-37°C.
mycobacteria.
For LJ Medium with Iron, specimens must first be isolated in • Proper aerobic conditions and increased CO2 tension
pure culture on an appropriate solid medium. Inoculate LJ were not provided during incubation.
Medium with Iron with one drop of a barely turbid suspension 2. Mycobacteria are strict aerobes and growth is stimulated
of the culture to be tested. by increased levels of CO2. Screw caps on tubes or bottles
should be handled as directed for exchange of CO2.
For the semi-quantitative catalase test, 1 mL of a 1:1 mixture of
10% polysorbate 80 and 30% hydrogen peroxide is added to
References
each inoculated tube after 2 weeks of incubation. The height of
the column of bubbles is recorded after 5 minutes as <45 mm
1.
2.
Lowenstein. 1931. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig.120:127.
Jensen. 1932. Zentralb. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. I Orig. 125:222.
L
3. Gruft. 1971. Health Lab. Sci. 8:79.
or >45 mm.6,7 4. Gruft. 1963. Am. Rev. Respir. Dis. 88:412.
5. Wayne and Doubek. 1968. Appl. Microbiol. 16:925.
6. Kent and Kubica. 1985. Public health mycobacteriology: a guide to the level III laboratory. USDHHS.
Expected Results Centers for Disease Control, Atlanta, Ga.
7. Metchock, Nolte and Wallace. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual
Cultures should be read within 5-7 days after inoculation and of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
8. Hughes. 1966. J. Clin. Pathol. 19:73.
once a week thereafter for up to 8 weeks. 9. Dixon and Cuthbert. 1967. Am. Rev. Respir. Dis. 96:119.
10. Silcox, Good and Floyd. 1981. J. Clin. Micobiol. 14:686.
11. Wayne. 1962. Am. Rev. Respir. Dis. 86:651.
Record Observations: 12. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby, Inc.,
St. Louis, Mo.
1. Number of days required for colonies to become macroscopi- 13. U.S. Public Health Service, Centers for Disease Control and Prevention, and National Institutes of
Health. 2007. Biosafety in microbiological and biomedical laboratories, 5th ed. HHS Publication No.
cally visible. Rapid growers have mature colonies within 7 (CDC) 93-8395. U.S. Government Printing Office, Washington, D.C.
14. Cernoch, Enns, Saubolle and Wallace. 1994. Cumitech 16A, Laboratory diagnosis of the mycobacte-
days; slow growers require more than 7 days for mature rioses. Coord. ed., Weissfeld. American Society for Microbiology, Washington, D.C.
colony forms. 15. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
American Society for Microbiology, Washington, D.C.
2. Pigment production
White, cream to buff = Nonchromogenic (NC)
Lemon, yellow, orange, red = Chromogenic (Ch)
311
313
Procedure
Using an inoculating needle, stab the butt twice then streak
Uninoculated Proteus Salmonella
Tube mirabilis Typhimurium the slant with growth from a pure culture. Incubate tubes
ATCC™ 25933 ATCC™ 14028
with loosened caps for 18-48 hours at 35 ± 2°C in an aerobic
atmosphere.
314
315
M9 Minimal Salts, 5×
Intended Use User Quality Control
M9 Minimal Salts, 5× is used in preparing M9 Minimal
Medium which is used for cultivating recombinant strains of Identity Specifications
Escherichia coli. Difco™ M9 Minimal Salts, 5×
Dehydrated Appearance: White, free-flowing, homogeneous.
Solution: 5.64% solution, soluble in purified water.
Summary and Explanation Solution is colorless, clear, no significant
M9 Minimal Salts, 5× is a 5× concentrate that is diluted to precipitate.
a 1× concentration and supplemented with an appropriate Prepared Appearance: Colorless, clear, no siginficant precipitate.
carbon and energy source, such as dextrose, to provide a Reaction of 5.64% Solution
minimal, chemically defined medium. The medium will support (5× concentrate) at 25°C: pH 6.8 ± 0.2
the growth of “wild-type” strains of E. coli. M9 Minimal
Salts is useful for maintaining positive selection pressure on Cultural Response
Difco™ M9 Minimal Salts, 5×
plasmids coding for the ability to produce essential substances
Prepare the medium and dilute to 1x. Supplement with glucose per label
such as amino acids or vitamins. M9 Minimal Medium is also directions. Inoculate and incubate at 35 ± 2°C for 18-48 hours.
used to maintain stocks of F´-containing bacteria for use with Organism ATCC™ INOCULUM CFU RECOVERY
M13. The medium can be supplemented with specific amino Escherichia coli (Strain B) 23226 30-300 Good to excellent
acids or other metabolites, allowing for selection of specific
Escherichia coli (JM103) 39403 30-300 Good to excellent
auxotrophs.
316
317
Bacto™ M Broth
Intended Use are fermentation energy sources. Mannose prevents fimbrial
Bacto™ M Broth is used for cultivating Salmonella in foods and agglutination. 1 Sodium chloride helps maintain osmotic
feeds by the accelerated enrichment serology (ES) procedure. equilibrium, while dipotassium phosphate acts as a buffer. The
inorganic salts stimulate bacterial growth. Polysorbate 80 is a
Summary and Explanation surfactant and dispersing agent.
M Broth, prepared according to the formula of Sperber and
Diebel,1 contains all the nutrients necessary for good growth Formula
and flagella development of Salmonella. Bacto™ M Broth
Approximate Formula* Per Liter
Fantasia, Sperber and Deibel2 compared the enrichment serology Yeast Extract................................................................ 5.0 g
(ES) procedure with the traditional procedure that was outlined Pancreatic Digest of Casein........................................ 12.5 g
in the Bacteriological Analytical Manual3 (BAM) and reported D-Mannose.................................................................. 2.0 g
Sodium Citrate............................................................. 5.0 g
excellent agreement between the two. They found the ES Sodium Chloride.......................................................... 5.0 g
procedure not only to be faster and less complicated but also as Dipotassium Phosphate................................................ 5.0 g
accurate and sensitive as the BAM procedure. Manganese Chloride.................................................... 0.14 g
Magnesium Sulfate...................................................... 0.8 g
M Broth also conforms to the testing standards recommended Ferrous Sulfate............................................................. 0.04 g
Polysorbate 80............................................................. 0.75 g
by the Compendium of Methods for the Microbiological *Adjusted and/or supplemented as required to meet performance criteria.
Examination of Foods4 (APHA) for the isolation and identifica-
tion of foodborne Salmonella. Directions for Preparation from
Monoclonal enzyme immunoassay (EIA) methods have been Dehydrated Product
described in Official Methods of Analysis of AOAC Interna- 1. Suspend 36.2 g of the powder in 1 L of purified water. Mix
tional5 using M Broth. These methods are screening procedures thoroughly.
for the presence of Salmonella and positive results must be 2. Heat with frequent agitation and boil for 1 minute to
confirmed by culture. completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
Principles of the Procedure 4. Test samples of the finished product for performance using
Yeast extract is a source of B-complex vitamins. Peptone stable, typical control cultures.
provides organic nitrogen. D-Mannose and sodium citrate
Cultural Response
Bacto™ M Broth
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for
18-24 hours.
Organism ATCC™ INOCULUM CFU RECOVERY
Salmonella enterica Uninoculated Salmonella Choleraesuis
subsp. enterica serotype Tube ATCC™ 12011
Choleraesuis var. Kunzendorf 12011 102-103 Good
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028 102-103 Good
318
MI Agar
Intended Use (MF) technology. However, standard MF technology for the
MI Agar* is a chromogenic/fluorogenic medium used to detect detection of TC and fecal coliforms requires the use of several
and enumerate Escherichia coli and total coliforms in drinking different types of media and two different incubation tempera-
water by the membrane filtration technique. It conforms tures.1
with the U.S. Environmental Protection Agency (USEPA) The newest technology developed by the USEPA for testing
Approved Method 1604: Total Coliforms and Escherichia coli in drinking water is a single membrane filtration technique where
Water by Membrane Filtration Using a Simultaneous Detection no membrane filter transfers are required.1-4 The medium is
Technique (MI Medium). named after the two enzyme substrates included in the formula-
* U.S. Patent No. 6,063,590. Manufactured under license. Commercialization of dehydrated culture
medium as prepared medium is prohibited. tion: a fluorogen, 4-Methylumbelliferyl-β-D-galactopyranoside
(MUGal) and a chromogen, Indoxyl-β-D-glucuronide (IBDG).
Summary and Explanation MI Agar can simultaneously detect and enumerate both TC and
Coliform bacteria are species that inhabit the intestines of warm- E. coli in water samples in 24 hours or less based on their specific
blooded animals or occur naturally in soil, vegetation and water. enzyme activities. MI Agar detects the presence of the bacterial
They are usually found in fecally-polluted water and are often enzymes β-galactosidase and β-glucuronidase produced by TC
associated with disease outbreaks. Although these bacteria are and E. coli, respectively.
not usually pathogenic themselves, their presence in drinking MI Agar is approved for use by certified drinking water
water indicates the possible presence of other pathogens. E. coli laboratories for microbial analysis of potable water. Other uses
is one species in this group of coliform bacteria. Since it is always include recreational, surface or marine water, bottled water,
found in feces, it is a more direct indicator of fecal contamination groundwater, well water, treatment plant effluents, water from
and the possible presence of enteric pathogens. drinking water distribution line, drinking water source water
Chromogens or fluorogens have been used for many years to and possibly foods.5
detect and identify total coliforms (TC) and E. coli. Some As referenced in USEPA method 1604, this method has a detec-
methods use liquid media in a multiple-tube-fermentation (MTF) tion limit of one E. coli and/or one total coliform per sample
test, a presence-absence (PA) format or other tube tests. Agar volume or dilution tested.5 The false-positive and false negative
media are also used for direct plating or membrane filtration rates for E. coli are both 4.3%.5 Specificity for E. coli is 95.7%
319
Identity Specifications
Difco™ MI Agar
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 3.65% solution, soluble in purified water upon
boiling. Solution is light to medium tan, very
slightly to slightly opalescent.
Prepared Appearance: Light tan, clear to very slightly opalescent. Upon
removal from 2-8°C storage, plates may exhibit a
crystal precipitate that disappears upon warming
to room temperature. This is a typical character-
istic of the medium and is acceptable.
Reaction of 3.65%
Solution at 25°C: pH 6.95 ± 0.2
Cultural Response
Difco™ MI Agar
Prepare the medium per label directions. Inoculate using the membrane
filtration technique and incubate at 35 ± 2°C for 20-24 hours. Count all
blue or indigo colonies under ambient light. Expose MI Agar plates to
long-wave ultraviolet light (366 nm) and count all fluorescent colonies.
INOCULUM COLONY COLOR/
ORGANISM ATCC™ CFU RECOVERY FLUORESCENCE
Escherichia coli
Enterobacter aerogenes 13048 20-80 Good Tan / blue-white ATCC™ 25922
and Coliform
Escherichia coli 25922 20-80 Good Blue / blue-green
Proteus mirabilis 43071 20-80 Good Tan / none
Pseudomonas Marked to
aeruginosa 27853 20-80 complete inhibition Tan / none
and for total coliforms is 93.1%.5 The single lab recovery of Formula
E. coli is 97.9% of the heterotrophic plate count (pour plate) and Difco™ MI Agar
115% of the R2A spread plate count.5 For Klebsiella pneumoniae Approximate Formula* Per Liter
and Enterobacter aerogenes, recoveries are 87.5% and 85.7% Proteose Peptone No. 3................................................ 5.0 g
Yeast Extract................................................................ 3.0 g
of the heterotrophic plate count and 89.3% and 85.8% of the D-Lactose..................................................................... 1.0 g
R2A spread plate method, respectively.5 MUGal (4-Methylumbelliferyl-β-D-galactopyranoside)... 0.1 g
Indoxyl-β-D-glucuronide (IBDG).................................... 0.32 g
Sodium Chloride.......................................................... 7.5 g
Principles of the Procedure Dipotassium Phosphate................................................ 3.3 g
MI Agar contains peptone as a source of nitrogen, carbon and Monopotassium Phosphate.......................................... 1.0 g
amino acids. Yeast extract provides trace elements, vitamins Sodium Lauryl Sulfate................................................... 0.2 g
and amino acids. Lactose is a fermentable carbohydrate and Sodium Desoxycholate................................................. 0.1 g
Agar.......................................................................... 15.0 g
carbon source. Sodium chloride maintains osmotic equilibrium. *Adjusted and/or supplemented as required to meet performance criteria.
Monopotassium and dipotassium phosphates offer buffering
capabilities. Sodium lauryl sulfate and sodium desoxycholate are Directions for Preparation from
selective against gram-positive bacteria. E. coli that produce the Dehydrated Product
enzyme β-D-glucuronidase cleave the chromogen indoxyl-β-D- 1. Suspend 36.5 g of the powder in 1 L of purified water. Mix
glucuronide (IBDG) to form a blue- or indigo-colored compound. thoroughly.
The β-galactosidase produced by total coliforms cleaves the 2. Heat with frequent agitation and boil for 1 minute to com-
fluorogen 4-methylumbelliferyl-β-D-galactopyranoside (MU- pletely dissolve the powder.
Gal), producing 4-methylumbelliferone, a fluorescent compound 3. Autoclave at 121°C for 15 minutes and cool in a 50°C water
when exposed to long-wave UV light (366 nm). Agar is the bath.
solidifying agent. Celfsulodin is added to inhibit gram-positive 4. Add 5 mL of a freshly prepared 1 mg/mL filter-sterilized
bacteria and some non-coliform gram-negative bacteria that solution of cefsulodin per liter of tempered agar medium
may cause false positives. (final concentration of 5 μg/mL).
5. Dispense 5 mL amounts into 9 × 50 mm or 15 × 60 mm
plates and allow to solidify.
6. Test samples of the finished product for performance using
stable, typical control cultures.
320
321
MIL Medium
Intended Use Ferric ammonium citrate is an H2S indicator. Bromcresol purple
MIL Medium is used for differentiating Enterobacteriaceae is a pH indicator. Agar is the solidifying agent.
based on motility, lysine decarboxylation, lysine deamination
and indole production. Formula
Difco™ MIL Medium
Summary and Explanation Approximate Formula* Per Liter
MIL (Motility-Indole-Lysine) Medium, prepared according to Peptone..................................................................... 10.0 g
Pancreatic Digest of Casein........................................ 10.0 g
the formula of Reller and Mirrett,1 is a single culture medium Yeast Extract................................................................ 3.0 g
that provides four differentiating biochemical reactions. When L-Lysine HCl............................................................... 10.0 g
used in conjunction with Triple Sugar Iron Agar (TSI) and Urea Dextrose...................................................................... 1.0 g
Ferric Ammonium Citrate............................................. 0.5 g
Agar, as many as nine reactions are provided. This combination Bromcresol Purple........................................................ 0.02 g
enables reliable initial identification of Enterobacteriaceae.2,3 Agar............................................................................ 2.0 g
Extensive testing of 890 enteric cultures by Reller and Mirrett1 *Adjusted and/or supplemented as required to meet performance criteria.
gave essentially the same results with MIL Medium as with the
standard motility, indole and lysine decarboxylase (Moeller) Directions for Preparation from
test media. Dehydrated Product
1. Suspend 36.5 g of the powder in 1 L of purified water. Mix
Principles of the Procedure thoroughly.
2. Heat with frequent agitation and boil for 1 minute to
Peptones provide the carbon and nitrogen sources required
completely dissolve the powder.
for good growth of a wide variety of organisms. Yeast extract 3. Autoclave at 121°C for 15 minutes.
provides vitamins and cofactors required for growth. Lysine 4. Test samples of the finished product for performance using
hydrochloride is present as a substrate to detect lysine decarbox- stable, typical control cultures.
ylase or lysine deaminase activity. Dextrose is an energy source.
Cultural Response
Difco™ MIL Medium
Prepare the medium per label directions. Stab inoculate using fresh cultures and incubate at
35 ± 2°C for 18-24 hours. After reading the lysine decarboxylase, motility and lysine
deaminase reactions, add Indole Reagent Kovacs to determine the indole reaction.
Lysine Lysine Indole
Organism ATCC™ Decarboxylase Motility Deaminase Production
Escherichia coli 25922 + + – +
Providencia alcalifaciens 9886 – + + – Uninoculated Escherichia coli Shigella flexneri
Tube ATCC™ 25922 ATCC™ 12022
Salmonella enterica
subsp. enterica All with Indole Reagent
serotype Enteritidis 13076 + + – –
Shigella flexneri 12022 – – – –
322
323
Cultural Response
Difco™ MIO Medium
Prepare the medium per label directions. Inoculate with fresh cultures using an inoculating
needle and incubate with caps loosened at 35 ± 2°C for 24-48 hours. Detect the presence
of indole by the addition of 3-4 drops of Kovacs’ Reagent.
ORGANISM ATCC™ Motility Indole Ornithine
Enterobacter aerogenes 13048 + – +
Escherichia coli 25922 + + +
Uninoculated Enterobacter Escherichia coli
Klebsiella pneumoniae Tube aerogenes ATCC™ 25922
subsp. pneumoniae 13883 – – – ATCC™ 13048
primary isolation plate or other pure culture. Incubate all tubes References
for 18-24 hours at 35 ± 2°C in an aerobic atmosphere. 1. Ederer and Clark. 1970. Appl. Microbiol. 2:849.
2. Oberhofer and Hajkowski. 1970. Am. J. Clin. Pathol. 54:720.
3. MacFaddin. 2000. Biochemical tests for identification of medical bacteria, 3rd ed. Lippincott Williams
& Wilkins, Baltimore, Md.
Expected Results 4. Ewing. 1986. Edwards and Ewing’s identification of Enterobacteriaceae, 4th ed. Elsevier Science
Publishing Co., New York, N.Y.
Read motility and decarboxylase activity prior to the addition 5. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual of determinative bacteriology,
of the reagent for the detection of indole production. 9th ed. Williams & Wilkins, Baltimore, Md.
6. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
1. Motility is indicated by growth extending from the line of
inoculation. Nonmotile organisms grow only along the line Availability
of inoculation. Difco™ MIO Medium
2. Decarboxylation of ornithine is indicated by the development BAM
of a turbid purple to a faded yellow-purple color. A negative Cat. No. 273520 Dehydrated – 500 g
reaction is indicated by a yellow color.
BBL™ Motility Indole Ornithine Medium
3. Indole production is indicated by the formation of a pink to
BAM
red color after the addition of three or four drops of Kovacs’ Cat. No. 221517 Prepared Deeps (K Tubes), 5 mL – Pkg. of 10*
reagent to the surface of the medium and gentle shaking. 221518 Prepared Deeps (K Tubes), 5 mL – Ctn. of 100*
A negative reaction is indicated by the development of a *Store at 2-8°C.
yellow color.
Refer to appropriate texts for typical reactions produced by
various members of the Enterobacteriaceae.4-6
Prepare the medium per label directions. Inoculate with fresh cultures Cultural Response
and incubate at 35 ± 2°C for 40-48 hours. BBL™ MR-VP Broth
Prepare the medium per label directions. Inoculate two sets of tubes
METHYL VOGES-
ORGANISM ATCC™ RECOVERY RED PROSKAUER (3 mL, Voges-Proskauer and 5 mL, Methyl Red) with fresh cultures and
incubate at 35 ± 2°C for 48 hours (3 mL) and 5 days (5 mL).
Enterbacter aerogenes 13048 Good – +
(yellow) (red) METHYL VOGES-
ORGANISM ATCC™ RECOVERY RED PROSKAUER
Escherichia coli 25922 Good + –
(red) (no change) Citrobacter freundii 8454 Good + –
(red) (no change)
Klebsiella pneumoniae – +
subsp. pneumoniae 23357 Good (yellow) (red) Enterbacter aerogenes 13048 Good – +
(yellow) (red)
Escherichia coli 25922 Good + –
(red) (no change)
Klebsiella pneumoniae – +
subsp. pneumoniae 33495 Good (yellow) (red)
325
2. Voges-Proskauer Test
A positive reaction is indicated by the development of a
distinct red color which occurs within 5 minutes.
Certain species within Enterobacteriaceae genera may react
differently or give variable results. Consult appropriate texts
for reactions of specific species.3-6
Expected Results
1. Methyl Red Test
a. Positive – red color at surface of the medium.
b. Negative – yellow color at surface of the medium.
326
MYP Agar
Antimicrobic Vial P
Intended Use Principles of the Procedure
MYP Agar is used with Egg Yolk Enrichment 50% and Antimi- MYP Agar contains beef extract and peptone as sources of
crobic Vial P for enumerating Bacillus cereus from foods. carbon, nitrogen, vitamins and minerals. D-Mannitol is the
carbohydrate source. Phenol red is the pH indicator. Agar
Summary and Explanation is the solidifying agent. Egg Yolk Enrichment 50% provides
Mossel et al.1 formulated Mannitol-Egg Yolk-Polymyxin (MYP) lecithin. Antimicrobic Vial P is polymyxin B which inhibits the
Agar to isolate and enumerate Bacillus cereus from foods. This growth of most other bacteria.
medium differentiates B. cereus from other bacteria based on Bacteria that ferment mannitol produce acid products and
its resistance to polymyxin, lack of mannitol fermentation and form colonies that are yellow. Bacteria that produce lecithinase
presence of lecithinase.2,3 B. cereus is commonly found in nature, hydrolyze the lecithin and a zone of white precipitate forms
on vegetables and in some processed foods.4 Under favorable around the colonies. B. cereus is typically mannitol-negative
circumstances the microorganism grows to sufficient numbers (pink-red colonies) and lecithinase-positive (zone of precipitate
and causes gastrointestinal illness.4 Outbreaks of foodborne ill- around the colonies).
ness have been associated with boiled and cooked rice, cooked
meats and cooked vegetables. 5
MYP Agar is a recommended medium for testing foods.4-6
Identity Specifications
Difco™ MYP Agar
Dehydrated Appearance: Pink, free-flowing, homogeneous.
Solution: 46 g soluble in 900 mL purified water upon
boiling. Solution is red, slightly opalescent.
Prepared Appearance: Red, very slightly to slightly opalescent without
significant precipitate.
Reaction of 46 g/900 mL
at 25°C: pH 7.2 ± 0.1
Difco™ Antimicrobic Vial P
Dehydrated Appearance: White cake or powder.
Cultural Response
Difco™ MYP Agar
Prepare the medium per label directions. Supplement with Egg Yolk
Enrichment 50% and Antimicrobic Vial P. Inoculate and incubate at
30 ± 2°C for 18-48 hours. Lecithinase reaction is read as a zone of
precipitate. Colonies that ferment mannitol are yellow.
INOCULUM Mannitol Lecithinase
Organism ATCC™ CFU RECOVERY Fermentation Reaction
Bacillus cereus 13061 30-300 Good – + Bacillus cereus
ATCC™ 13061
Bacillus subtilis 6633 30-300 Good + –
Pseudomonas
aeruginosa 27853 103-2×103 Inhibition – –
327
Formulae Procedure
Difco™ MYP Agar Consult appropriate references.4-6
Approximate Formula* Per 900 mL
Beef Extract.................................................................. 1.0 g Expected Results
Peptone..................................................................... 10.0 g
D-Mannitol................................................................ 10.0 g Consult appropriate references.4-6
Sodium Chloride........................................................ 10.0 g
Phenol Red................................................................ 25.0 mg References
Agar.......................................................................... 15.0 g 1. Mossel, Koopman and Jongerius. 1967. Appl. Microbiol. 15:650.
2. Donovan. 1958. J. Appl. Bacteriol. 21:100.
Difco™ Antimicrobic Vial P 3. Coliner. 1948. J. Bacteriol. 55:777.
4. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
Approximately 30,000 units polymyxin B per vial. tional, Gaithersburg, Md.
*Adjusted and/or supplemented as required to meet performance criteria. 5. Bennett and Belay. 2001. In Downes and Ito (ed.), Compendium of methods for the microbiological
examination of foods, 4th ed. American Public Health Association, Washington, D.C.
6. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
Directions for Preparation from International, Gaithersburg, Md.
Dehydrated Product
Difco™ MYP Agar Availability
Difco™ MYP Agar
1. Suspend 46 g of the powder in 900 mL of purified water.
AOAC BAM COMPF ISO USDA
Mix thoroughly.
Cat. No. 281010 Dehydrated – 500 g
2. Heat with frequent agitation and boil for 1 minute to com-
Europe
pletely dissolve the powder. Cat. No. 257004 Prepared Plates – Pkg. of 20*
3. Dispense 225 mL into 500 mL flasks. Japan
4. Autoclave at 121°C for 15 minutes. Cool to 45-50°C. Cat. No. 251264 Prepared Plates – Pkg. of 20*
5. Aseptically add 12.5 mL Egg Yolk Enrichment 50% and Difco™ Antimicrobic Vial P
4.1 mL Antimicrobic Vial P rehydrated with 5 mL sterile AOAC BAM COMPF ISO USDA
water (25,000 units of polymyxin B). Mix thoroughly. Cat. No. 232681 Vial – 6 × 10 mL*
6. Test samples of the finished product for performance using
Difco™ Egg Yolk Enrichment 50%
stable, typical control cultures.
AOAC BAM COMPF ISO USDA
Difco™ Antimicrobic Vial P (Polymyxin B) Cat. No. 233471 Tube – 12 × 10 mL*
233472 Bottle – 6 × 100 mL*
1. To rehydrate, aseptically add 5 mL sterile purified water
*Store at 2-8°C.
(to achieve the desired concentration for MYP Agar).
2. Rotate in an end-over-end motion to dissolve the contents
completely.
MacConkey Agars
MacConkey Agar • MacConkey Agar Base
MacConkey Agar without Crystal Violet
MacConkey Agar without Crystal Violet or Salt
MacConkey Agar without Salt
Intended Use of staphylococci and enterococci. The medium can be used also
MacConkey agars are slightly selective and differential plating to separate Mycobacterium fortuitum and M. chelonae from
media mainly used for the detection and isolation of gram-nega- other rapidly growing mycobacteria.
tive organisms from clinical,1-3 dairy,4 food,5-7 water,8 pharma- MacConkey Agar without Crystal Violet or Salt and MacConkey
ceutical,9-11 cosmetic,6,7 and other industrial sources. Agar without Salt are used for isolating and differentiating
MacConkey Agar is used for isolating and differentiating gram-negative bacilli while suppressing the swarming of most
lactose-fermenting from lactose-nonfermenting gram-negative Proteus species.
enteric bacilli. MacConkey Agar meets United States Pharmacopeia (USP),
MacConkey Agar Base is used with added carbohydrate in European Pharmacopoeia (EP) and Japanese Pharmacopoeia
differentiating coliforms based on fermentation reactions. (JP)9-11 performance specifications, where applicable.
MacConkey Agar without Crystal Violet is used for isolating and
differentiating enteric microorganisms while permitting growth
328
Identity Specifications
Difco™ MacConkey Agar Difco™ MacConkey Agar without Crystal Violet
Dehydrated Appearance: Pink to pinkish beige, free-flowing, homoge- Dehydrated Appearance: Pinkish beige, free-flowing, homogeneous.
neous. Solution: 5.2% solution, soluble in purified water upon
Solution: 5.0% solution, soluble in purified water upon boiling. Solution is reddish orange, clear to very
boiling. Solution is reddish-purple, slightly opal- slightly opalescent.
escent. Prepared Appearance: Reddish orange, clear to very slightly opales-
Prepared Appearance: Pinkish red, slightly opalescent. cent.
Reaction of 5.0% Reaction of 5.2%
Solution at 25°C: pH 7.1 ± 0.2 Solution at 25°C: pH 7.4 ± 0.2
Difco MacConkey Agar Base
™
Difco MacConkey Agar without Salt
™
Dehydrated Appearance: Pinkish beige, free-flowing, homogeneous. Dehydrated Appearance: Beige to pinkish beige, free-flowing, homoge-
Solution: 4.0% solution, soluble in purified water upon neous.
boiling. Solution is red, very slightly to slightly Solution: 4.7% solution, soluble in purified water upon
opalescent. boiling. Solution is reddish orange, slightly
Prepared Appearance: Red, slightly opalescent. opalescent.
Reaction of 4.0% Prepared Appearance: Reddish orange, slightly opalescent.
Solution at 25°C: pH 7.1 ± 0.2 Reaction of 4.7%
Solution at 25°C: pH 7.4 ± 0.2
Cultural Response
Difco™ MacConkey Agar Difco™ MacConkey Agar without Crystal Violet
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C Prepare the medium per label directions. Inoculate and incubate at
for 18-24 hours (incubate E. coli ATCC 25922 for 40-48 hours). For E. coli 35 ± 2°C for 18-48 hours.
ATCC 8739, inoculate in duplicate and incubate one plate at 30-35°C for
INOCULUM COLOny BILE
18-24 hours and the other plate at 35-37°C for 18-72 hours. ORGANISM ATCC™ CFU RECOVERY COLOr PPT.
INOCULUM Colony BILE Enterococcus faecalis 29212 30-300 Good Red –
ORGANISM ATCC™ CFU RECOVERY COLOR PPT.
Escherichia coli 25922 30-300 Good Pink to red –
Enterococcus faecalis 29212 103 Marked to – –
complete inhibition Proteus mirabilis 12453 30-300 Good Colorless –
Escherichia coli 25922 30-300 Good Pink to red + Salmonella enterica
subsp. enterica
Proteus mirabilis 12453 30-300 Good Colorless – serotype Typhimurium 14028 30-300 Good Colorless –
Salmonella enterica Staphylococcus aureus 25923 30-300 Good Pink to red –
subsp. enterica
serotype Typhimurium 14028 30-300 Good Colorless – Difco™ MacConkey Agar without Salt
Escherichia coli 8739 <100 Growth Pink + Prepare the medium per label directions. Inoculate and incubate at
(18-24 hours to red 35 ± 2°C for 18-48 hours.
at 30-35°C)
INOCULUM Colony BILE
Escherichia coli 8739 <100 Growth Pink + ORGANISM ATCC™ CFU RECOVERY COLOR PPT.
(18-72 hours to red
Enterococcus faecalis 33186 30-300 Good Red –
at 35-37°C)
Escherichia coli 25922 30-300 Good Pink to red –
Difco™ MacConkey Agar Base Proteus mirabilis 12453 30-300 Good Colorless, –
Prepare the medium per label directions without and with 1% added no swarming
lactose. Inoculate and incubate at 35 ± 2°C for 18-24 hours.
Salmonella enterica
INOCULUM Colony Color BILE subsp. enterica
ORGANISM ATCC™ CFU RECOVERY Plain w/Lactose PPT. serotype Typhimurium 14028 30-300 Good Colorless –
Enterococcus 29212 103 Marked to – – – Shigella flexneri 12022 30-300 Good Colorless –
faecalis complete
inhibition
Continued
Escherichia coli 25922 30-300 Good Colorless Pink +
to red (w/lactose)
Proteus
mirabilis 12453 30-300 Good Colorless Colorless –
Salmonella
enterica subsp.
enterica serotype
Typhimurium 14028 30-300 Good Colorless Colorless –
329
Identity Specifications
BBL™ MacConkey Agar BBL™ MacConkey Agar without Crystal Violet
Dehydrated Appearance: Fine, homogenous, may contain dark Dehydrated Appearance: Fine, homogeneous, free of extraneous
particles. material.
Solution: 5.0% solution, soluble in purified water Solution: 5.2% solution, soluble in purified water
upon boing. Solution is medium to dark, upon boiling. Solution is medium, red-
rose to brown-rose with or without a trace orange to red-rose, slightly hazy to hazy.
orange tint; clear to slightly hazy. Prepared Appearance: Medium, red-orange to red-rose, slightly
Prepared Appearance: Medium to dark, rose to brown-rose with or hazy to hazy.
without a trace orange tint; clear to slightly Reaction of 5.2%
hazy. Solution at 25°C: pH 7.4 ± 0.2
Reaction of 5.0%
BBL MacConkey Agar without Crystal Violet or Salt
™
Solution at 25°C: pH 7.1 ± 0.2
Dehydrated Appearance: Fine, homogeneous, free of extraneous
BBL™ MacConkey Agar (prepared) material.
Appearance: Medium-dark, rose-tan and trace hazy. Solution: 4.37% solution, soluble in purified water
Reaction at 25°C: pH 7.1 ± 0.2 upon boiling. Solution is medium, red-
orange to red-rose, slightly hazy to hazy.
Prepared Appearance: Medium, red-orange to red-rose, slightly
hazy to hazy.
Reaction of 4.37%
Solution at 25°C: pH 7.4 ± 0.2
Cultural Response
BBL™ MacConkey Agar BBL™ MacConkey Agar without Crystal Violet
Prepare the medium per label directions. Inoculate and incubate at Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 48 hours. For E. coli ATCC 8739, inoculate in duplicate and 35 ± 2°C for 18-24 hours and up to 48 hours if necessary (up to 11 days
incubate one plate at 30-35°C for 18-24 hours and the other plate at for M. fortuitum).
35-37°C for 18-72 hours.
INOCULUM Colony BILE
INOCULUM COLOny BILE ORGANISM ATCC™ CFU RECOVERY Color PPT.
ORGANISM ATCC™ CFU RECOVERY COLOr PPT.
Enterococcus faecalis 29212 103-104 Good Rose red –
Enterococcus faecalis 29212 104-105 Partial to – – Escherichia coli 25922 103-104 Good Pink to –
complete inhibition rose red
Escherichia coli 25922 103-104 Good Red to + Mycobacterium
rose-red fortuitum 6841 103-104 Good Rose red –
Proteus mirabilis 12453 103-104 Good Colorless – Salmonella enterica
Salmonella enterica subsp. enterica
subsp. enterica serotype Typhimurium 14028 103-104 Good Colorless –
serotype Typhimurium 14028 103-104 Good Colorless – Staphylococcus aureus 25923 103-104 Good Pink to –
Shigella flexneri 12022 103-104 Good Colorless – rose red
Escherichia coli 8739 <100 Growth Red to +
(18-24 hours rose-red
BBL™ MacConkey Agar without Crystal Violet or Salt
at 30-35°C) Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 18-24 hours and up to 48 hours if necessary (up to 11 days
Escherichia coli 8739 <100 Growth Red to + for M. fortuitum).
(18-72 hours rose-red
at 35-37°C) INOCULUM COLOny BILE
ORGANISM ATCC™ CFU RECOVERY COLOr PPT.
BBL™ MacConkey I Agar (prepared) Enterococcus faecalis 29212 103-104 Good Rose red –
Inoculate and incubate at 35 ± 2°C for 18-24 hours. Incubate E. coli Escherichia coli 25922 103-104 Good Pink to –
ATCC 8739 at 30-35°C for 18-72 hours. rose red
INOCULUM COLOny BILE Proteus mirabilis 12453 103-104 Good Colorless, –
ORGANISM ATCC™ CFU RECOVERY COLOr PPT.
no swarming
Enterococcus faecalis 29212 104-105 Partial to – – Salmonella enterica
complete inhibition subsp. enterica
Escherichia coli 25922 103-104 Good Red to + serotype Typhimurium 14028 103-104 Good Colorless –
rose-red
Pseudomonas Greenish
aeruginosa 10145 103-104 Good yellow –
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028 103-104 Good No reaction –
Shigella dysenteriae 9361 10 -10
3 4
Good No reaction –
Escherichia coli 8739 10-100 Growth Red to +
rose-red
330
331
BBL™ MacConkey Agar without Crystal Violet For pharmaceutical samples, refer to USP General Chapter <62>
Approximate Formula* Per Liter for details on the examination of nonsterile products and tests
Pancreatic Digest of Casein........................................ 10.0 g
Peptic Digest of Animal Tissue.................................... 10.0 g
for isolating E. coli using MacConkey Agar.9
Lactose...................................................................... 10.0 g
Bile Salts...................................................................... 5.0 g Procedure
Sodium Chloride.......................................................... 5.0 g Refer to appropriate standard references for details on test
Agar.......................................................................... 12.0 g
Neutral Red.................................................................. 0.05 g methods to obtain isolated colonies from specimens or samples
Difco™ MacConkey Agar without Salt
using MacConkey Agar.1-11 Incubate plates for 18-72 hours
Approximate Formula* Per Liter at 35 ± 2°C under appropriate atmospheric conditions, or as
Peptone..................................................................... 20.0 g instructed in the standard reference.1-11
Lactose...................................................................... 10.0 g
Bile Salts...................................................................... 5.0 g Expected Results
Agar.......................................................................... 12.0 g
Neutral Red................................................................ 75.0 mg Lactose-fermenting organisms grow as pink to brick-red colonies
BBL MacConkey Agar without Crystal Violet or Salt
™ with or without a zone of precipitated bile. Lactose-nonferment-
Approximate Formula* Per Liter ing organisms grow as colorless or clear colonies.
Pancreatic Digest of Gelatin....................................... 10.0 g
Yeast Extract.............................................................. 10.0 g
Swarming by Proteus spp. is reduced on MacConkey agars
Lactose...................................................................... 10.0 g without salt.
Oxgall.......................................................................... 5.0 g
Magnesium Sulfate...................................................... 0.2 g On MacConkey Agar without Crystal Violet and MacConkey
Agar.......................................................................... 12.0 g agars without salt, staphylococci produce pale pink to red
Neutral Red................................................................ 75.0 mg colonies and enterococci produce tiny red colonies; these
*Adjusted and/or supplemented as required to meet performance criteria.
organisms are inhibited on MacConkey Agar.
Directions for Preparation from On MacConkey Agar without Crystal Violet, potentially
Dehydrated Product pathogenic rapid growers of the M. fortuitum complex usually
1. Suspend the powder in 1 L of purified water: grow in 5-11 days, while the commonly saprophytic species
Difco™ MacConkey Agar – 50 g; are inhibited.3,13
BBL™ MacConkey Agar – 50 g; On MacConkey agars without salt, the swarming of Proteus
Difco™ MacConkey Agar Base – 40 g; is reduced.
Difco™ MacConkey Agar without Crystal Violet – 52 g;
BBL™ MacConkey Agar without Crystal Violet – 52 g; Limitations of the Procedure
BBL™ MacConkey Agar without Crystal Violet or Salt – 47.3 g; 1. Although MacConkey media are selective primarily for
Difco™ MacConkey Agar without Salt – 47 g. gram-negative enteric bacilli, biochemical and, if indicated,
Mix thoroughly. serological testing using pure cultures are recommended for
2. Heat with frequent agitation and boil for 1 minute to com- complete identification. Consult appropriate references for
pletely dissolve the powder. further information.1,3
3. Autoclave at 121°C for 15 minutes. 2. Incubation of MacConkey Agar plates under increased CO2
NOTE: If MacConkey Agar Base is to be used within 12 hours, has been reported to reduce the growth and recovery of a
omit autoclaving and gently boil medium for 5 minutes. Add 1% number of strains of gram-negative bacilli.14
carbohydrate before or after autoclaving, depending upon heat 3. Some strains of M. smegmatis from humans may grow on
lability. The surface of MacConkey agars without salt should MacConkey Agar without Crystal Violet, but these strains
be thoroughly air-dried prior to inoculation. can be differentiated from M. fortuitum complex by the
3-day arylsulfatase test.9
4. Test samples of the finished product for performance using
stable, typical control cultures. References
1. Murray, Baron, Jorgensen, Landry and Pfaller (eds.). 2007. Manual of clinical microbiology, 9th ed.
Sample Collection and Handling American Society for Microbiology, Washington, D.C.
2. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby Elsevier,
For clinical specimens, refer to laboratory procedures for details St. Louis, Mo.
3. Isenberg and Garcia (eds.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd
on specimen collection and handling.1-3 ed., American Society for Microbiology, Washington, D.C.
4. Wehr and Frank (eds.). 2004. Standard methods for the examination of dairy products, 17th ed.
For food or dairy samples, follow appropriate standard methods American Public Health Association, Washington, D.C.
5. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
for details on sample collection and preparation according to 4th ed. American Public Health Association, Washington. D.C.
6. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online (25 Sept 2008).
sample type and geographic location.4-7 AOAC International, Gaithersburg, Md.
7. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
International, Gaithersburg, Md.
For cosmetics, water, or other industrial samples, follow 8. Eaton, Rice and Baird (eds.). 2005. Standard methods for the examination of water and wastewater,
appropriate standard methods for details on sample collection 21st ed., online. American Public Health Association, Washington, D.C.
9. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The national
and preparation according to sample type and geographic formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc., Rockville, Md.
location.6-11
332
333
Cultural Response
BBL™ MacConkey II Agar
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 48 hours.
INOCULUM colony bile
ORGANISM ATCC™ CFU recovery COLOR ppt.
Enterococcus faecalis 29212 104-105 Partial to – –
complete
inhibition
Escherichia coli 25922 103-104 Good Pink to red +
Proteus mirabilis 12453 103-104 Good, Colorless –
inhibition of
swarming
Pseudomonas 10145 103-104 Good Colorless –
aeruginosa to blue to
green to pink
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028 103-104 Good Colorless –
334
335
Escherichia coli 25922 102-103 Good Pink-red + Escherichia coli 25922 103-104 Good Pink to red +
to rose-red
Escherichia coli
O157:H7 35150 102-103 Good Colorless – Escherichia coli
O157:H7 35150 103-104 Good Colorless –
Proteus mirabilis 12453 103-104 Good Colorless –
of the fecal flora ferment sorbitol and appear pink on this Formulae
medium. MacConkey Agar with Sorbitol, therefore, permits Difco™ MacConkey Sorbitol Agar
ready recognition of E. coli O157:H7 in stool cultures.1-3 Approximate Formula* Per Liter
Peptone..................................................................... 15.5 g
The addition of cefixime and tellurite significantly reduces the Proteose Peptone......................................................... 3.0 g
number of sorbitol nonfermenters that need to be screened D-Sorbitol.................................................................. 10.0 g
Bile Salts...................................................................... 1.5 g
during the attempted isolation of E. coli O157:H7.4,5
Sodium Chloride.......................................................... 5.0 g
Agar.......................................................................... 15.0 g
Principles of the Procedure Neutral Red.................................................................. 0.03 g
MacConkey Sorbitol Agar and MacConkey II Agar with Crystal Violet............................................................... 1.0 mg
Sorbitol, modified MacConkey agars using sorbitol instead BBL™ MacConkey II Agar with Sorbitol
of lactose, are only slightly selective, since the concentration Approximate Formula* Per Liter
Pancreatic Digest of Gelatin....................................... 17.0 g
of bile salts, which inhibits gram-positive microorganisms, is Pancreatic Digest of Casein.......................................... 1.5 g
low in comparison with other enteric plating media. Crystal Peptic Digest of Animal Tissue...................................... 1.5 g
violet also is included in the medium to inhibit the growth D-Sorbitol.................................................................. 10.0 g
Bile Salts...................................................................... 1.5 g
of gram-positive bacteria, especially enterococci and staphylo-
Sodium Chloride.......................................................... 5.0 g
cocci. MacConkey II Agar with Sorbitol is also formulated to Agar.......................................................................... 13.5 g
reduce swarming of Proteus species. Neutral Red.................................................................. 0.03 g
Crystal Violet............................................................... 1.0 mg
Differentiation of enteric microorganisms is achieved by the *Adjusted and/or supplemented as required to meet performance criteria.
MacConkey Broth
Intended Use replaces the original sodium taurocholate to inhibit growth of
MacConkey Broth is used for the detection of coliform organ- gram-positive organisms.
isms in milk and water. MacConkey Broth is used for cultivating gram-negative,
Meets United States Pharmacopeia (USP), European Pharma- lactose-fermenting bacilli and as a presumptive test for coliform
copoeia (EP) and Japanese Pharmacopoeia (JP)1-3 performance organisms. It has been used to analyze food,8 milk9,10 and water
specifications, where applicable. samples10-13 for coliforms. In addition, this medium has also been
used in the rapid detection of shiga-toxin producing E. coli in
Summary and Explanation fecal samples.14 MacConkey Broth is recommended in the USP
MacConkey Broth is a modification of the original bile salt as a test medium for E. coli in the microbiological examination
broth recommended by MacConkey4 that contained 0.5% of nonsterile products.1
sodium taurocholate and litmus as an indicator. In later publi-
cations,5,6 MacConkey suggested variations of this formulation Principles of the Procedure
using neutral red indicator instead of litmus. Childs and Allen7 Peptone provides amino acids and other growth factors. Lactose
demonstrated the inhibitory effect of neutral red and substituted is a carbon energy source for gram-negative lactose-fermenting
the less inhibitory bromcresol purple. Oxgall in the medium bacilli. Oxgall inhibits the growth of gram-positive organisms.
Bromcresol purple is the indicator.
337
Identity Specifications
Difco™ MacConkey Broth
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 3.5% solution, soluble in purified water. Solution is purple,
clear.
Prepared Appearance: Purple, clear.
Reaction of 3.5%
Solution at 25°C: pH 7.3 ± 0.1
BBL™ MacConkey Broth (prepared)
Appearance: Purple and clear.
Reaction at 25°C: pH 7.3 ± 0.2
Cultural Response
Difco™ MacConkey Broth
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-24
hours. For E. coli ATCC 8739 and S. aureus ATCC 6538, inoculate 100 mL bottles and
incubate at 43-44°C for 18-48 hours.
INOCULUM Uninoculated Escherichia coli
ORGANISM ATCC™ CFU RECOVERY ACID GAS Tube ATCC™ 25922
Enterococcus faecalis 29212 103 Marked to – –
complete inhibition
Escherichia coli 25922 30-300 Good + +
Salmonella enterica
subsp. enterica serotype
Choleraesuis var. Kunzendorf 12011 30-300 Good – –
Escherichia coli 8739 <100 Growth N/A N/A
(at 24 hours)
Staphylococcus aureus 6538 >100 No growth N/A N/A
(at 48 hours)
KEY: + = positive, yellow for acid, gas
– = negative, no change for no acid, no gas
Malonate Broth
Intended Use group utilizes malonate whereas the Escherichia group is unable
Malonate Broth is used for differentiating Enterobacter from to grow on the medium.
Escherichia based on malonate utilization. Malonate Broth is further described for differentiating Enterobac-
teriaceae in food and dairy products.2,3 More often, the medium
Summary and Explanation referenced is the modified Edwards and Ewing4 formulation that
Malonate Broth, prepared according to the formula described contains yeast extract and dextrose. The modification permits
by Leifson,1 is a liquid medium containing ammonium sulfate as growth of organisms that would otherwise fail on the unsupple-
the only source of nitrogen and malonate as the only source of mented Leifson medium.
carbon. Leifson was able to demonstrate that the Enterobacter
Cultural Response
Difco™ Malonate Broth
Prepare the medium per label directions. Inoculate with fresh cultures and incubate
at 35 ± 2°C for 18-48 hours.
Organism ATCC™ MEDIUM color
Enterobacter aerogenes 13048 Blue
Enterobacter cloacae 13047 Blue
Escherichia coli 25922 Green Uninoculated Enterobacter Escherichia coli
Tube aerogenes ATCC™ 25922
Klebsiella pneumoniae 13883 Blue ATCC™ 13048
Salmonella enterica
subsp. arizonae 13314 Blue
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028 Green
339
References
Directions for Preparation from 1. Leifson. 1933. J. Bacteriol. 26:329.
2. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
Dehydrated Product 4th ed. American Public Health Association, Washington, D.C.
3. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy products, 17th ed.
1. Suspend 8 g of the powder in 1 L of purified water. Mix American Public Health Association, Washington, D.C.
4. Edwards and Ewing. 1962. Enterobacteriaceae. U.S. Public Health Service Bulletin No. 734:19.
thoroughly. 5. Oberhofer. 1985. Manual of nonfermenting gram-negative bacteria. Churchill Livingstone, New York,
2. Heat with frequent agitation and boil for 1 minute to N.Y.
340
Malt Agar
Intended Use Malt Agar allows for optimal growth of molds and yeasts while
Malt Agar is used for isolating and cultivating yeasts and molds restricting bacterial growth.
from food and for cultivating yeast and mold stock cultures.
Formula
Summary and Explanation Difco™ and BBL™ Malt Agar
Malt media for yeasts and molds have been widely used for many Approximate Formula* Per Liter
Malt Extract............................................................... 30.0 g
years. In 1919, Reddish1 prepared a satisfactory substitute for Agar.......................................................................... 15.0 g
beer wort from malt extract. Thom and Church2 used Reddish’s *Adjusted and/or supplemented as required to meet performance criteria.
medium for their studies of the aspergilli. Malt Agar was also
employed by Fullmer and Grimes3 for their studies of the growth Directions for Preparation from
of yeasts on synthetic media. Malt Agar is included in Official Dehydrated Product
Methods of Analysis of AOAC International.4 1. Suspend 45 g of the powder in 1 L of purified water. Mix
thoroughly.
Principles of the Procedure 2. Heat with frequent agitation and boil for 1 minute to
Malt Agar contains malt extract which provides the carbon, completely dissolve the powder.
protein and nutrient sources required for the growth of
microorganisms. Agar is the solidifying agent. The acidic pH of
341
Procedure
See appropriate references for specific procedures.
Expected Results
Refer to appropriate references and procedures for results.
References
1. Reddish. 1919. Abstr. Bacteriol. 3:6.
2. Thom and Church. 1926. The aspergilli. Williams & Wilkins, Baltimore, Md.
3. Fulmer and Grimes. 1923. J. Bacteriol. 8:585.
4. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
Candida Saccharomyces International, Gaithersburg, Md.
albicans cerevisiae
ATCC™ 10231 ATCC™ 9763
Availability
Difco™ Malt Agar
AOAC BAM
Cat. No. 224200 Dehydrated – 500 g
224100 Dehydrated – 10 kg
BBL™ Malt Agar
AOAC BAM
Cat. No. 211401 Dehydrated – 500 g
342
343
344
Cultural Response
BBL™ Mannitol Salt Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C
for 42-48 hours. Incubate plates with Staphylococcus aureus ATCC 6538 Staphylococcus
and E. coli ATCC 8739 at 30-35°C for 18-72 hours. aureus
ATCC™ 25923
COLOR OF MEDIUM
ORGANISM ATCC™ INOCULUM CFU RECOVERY AROUND COLONY
Proteus mirabilis 12453 104 – 105 Partial to complete inhibition –
Staphylococcus aureus 25923 103 – 104 Good Yellow
Staphylococcus epidermidis 12228 103 – 104 Good Red
Staphylococcus aureus 6538 <100 Growth N/A
Escherichia coli 8739 >100 No growth N/A
345
Agar.......................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria. Availability
BBL™ Mannitol Salt Agar
Directions for Preparation from BAM BS12 CMPH2 EP JP MCM9 USP
Dehydrated Product Cat. No. 211407 Dehydrated – 500 g†
1. Suspend 111 g of the powder in 1 L of purified water. Mix 211410 Dehydrated – 5 lb (2.3 kg)†
293689 Dehydrated – 25 lb (11.3 kg)†
thoroughly.
2. Heat with frequent agitation and boil for 1 minute to com- United States and Canada
Cat. No. 221173 Prepared Plates – Pkg. of 20*†
pletely dissolve the powder. 221271 Prepared Plates – Ctn. of 100*†
3. Autoclave at 121°C for 15 minutes. Europe
4. Test samples of the finished product for performance using Cat. No. 254027 Prepared Plates – Pkg. of 20*†
stable, typical control cultures. 254079 Prepared Plates – Ctn. of 120*†
Japan
Cat. No. 251173 Prepared Plates – Pkg. of 20*
Sample Collection and Handling
*Store at 2-8°C.
For clinical specimens, refer to laboratory procedures for details †QC testing performed according to USP/EP/JP performance specifications.
on specimen collection and handling.7-11
For cosmetic and pharmaceutical samples, follow appropriate
standard methods for details on sample collection and prepara-
tion according to sample type and geographic location.1,13-15
Procedure
Refer to appropriate standard references for details on test
methods to obtain isolated colonies from specimens or samples
using Mannitol Salt Agar.1,6,7,11 Incubate plates at 35 ± 2°C in
an aerobic atmosphere for 24-48 hours, or as instructed in the
standard reference.1,6,7,11
346
Cultural Response
Difco™ Marine Agar 2216
Prepare the medium per label directions. Inoculate and incubate at
20-25°C for 40-72 hours.
Organism ATCC™ INOCULUM CFU RECOVERY
Vibrio fischeri 7744 102-103 Good
Vibrio harveyi 14126 10 -10
2 3
Good
347
Neisseria gonorrhoeae ...... Small grayish-white to colorless, BBL™ Martin-Lewis, Modified//Chocolate II Agar
mucoid Cat. No. 298513 Prepared Bi-Plate Dishes – Pkg. of 20*
298206 Prepared Bi-Plate Dishes – Ctn. of 100*
Neisseria meningitidis . ...... Medium to large, blue-gray, mu-
coid BBL™ Martin-Lewis Agar (Gono-Pak)
Cat. No. 221793 Prepared Plates – Pkg. of 20*
Colonies may be selected for Gram staining, subculturing or
other diagnostic procedures. BBL™ Martin-Lewis Agar (JEMBEC™)
Cat. No. 221804 Prepared Plates – Pkg. of 10*
299602 Prepared Plates (with white patient
label on bottom) – Pkg. of 10*
*Store at 2-8°C.
Low levels of peptone help protect organisms in the diluent. Escherichia coli 25922 103-104 No significant reduction
Sodium chloride maintains proper osmotic pressure. Staphylococcus aureus 25923 103-104 No significant reduction
350
Identity Specifications
Difco™ McClung Toabe Agar Base
Dehydrated Appearance: Very light beige, free-flowing, homogeneous.
Solution: 7.5% solution, soluble in purified water upon
boiling. Solution is light amber, slightly opales-
cent, with a precipitate.
Prepared Appearance: Plain – Light amber, opalescent with precipitate.
With Egg Yolk Enrichment – Light yellow,
opaque.
Reaction of 7.5%
Solution at 25°C: pH 7.6 ± 0.2
Difco Egg Yolk Enrichment 50%
™
Cultural Response
Difco™ McClung Toabe Agar Base with Egg Yolk
Enrichment 50%
Prepare the medium with Egg Yolk Enrichment 50% per label direc-
tions. Inoculate and incubate at 35 ± 2°C for 18-48 hours. Incubate the
clostridia anaerobically.
INOCULUM Lecithinase
Organism ATCC™ CFU RECOVERY Reaction
Clostridium perfringens 12919 102-103 Good Opaque halo
Clostridium perfringens 12924 102-103 Good Opaque halo
Staphylococcus aureus 25923 102-103 Good None
Staphylococcus epidermidis 14990 102-103 Good None
351
Principles of the Procedure 3. Dispense 90 mL amounts into flasks and autoclave at 121°C
McClung Toabe Agar Base contains peptone as a source for 15 minutes.
of carbon, nitrogen, vitamins and minerals. Dextrose is the 4. Cool to 50°C and aseptically add 10 mL of Egg Yolk Enrich-
carbohydrate source. Sodium chloride provides essential ions. ment 50% to each 90 mL of base. Mix thoroughly.
Magnesium sulfate provides divalent cations and sulfate. 5. Dispense into sterile Petri dishes in approximately 15 mL
amounts.
Disodium phosphate and monopotassium phosphate maintain
6. Test samples of the finished product for performance using
pH balance and provide a source of phosphates. Agar is the
solidifying agent. Egg Yolk Enrichment 50% provides egg yolk stable, typical control cultures.
lecithin. Lecithinase-producing clostridia, such as C. perfringens, Difco™ Egg Yolk Enrichment 50%
hydrolyze the lecithin and produce opaque halos. Shake gently to resuspend the precipitate.
Formulae Procedure
Difco™ McClung Toabe Agar Base See appropriate references for specific procedures.
Approximate Formula* Per Liter
Proteose Peptone....................................................... 40.0 g
Dextrose...................................................................... 2.0 g Expected Results
Disodium Phosphate.................................................... 5.0 g Refer to appropriate references and procedures for results.
Monopotassium Phosphate.......................................... 1.0 g
Sodium Chloride.......................................................... 2.0 g
Magnesium Sulfate...................................................... 0.1 g References
1. McClung and Toabe. 1947. J. Bacteriol. 53:139.
Agar.......................................................................... 25.0 g 2. Labbe. 2001. In Downes and Ito (ed.), Compendium of methods for the microbiological examination
of foods, 4th ed. American Public Health Association, Washington, D.C.
Difco Egg Yolk Enrichment 50%
™
3. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
Concentrated egg yolk emulsion. tional, Gaithersburg, Md.
*Adjusted and/or supplemented as required to meet performance criteria.
Availability
Directions for Preparation from Difco™ McClung Toabe Agar Base
Dehydrated Product Cat. No. 294110 Dehydrated – 500 g
Difco McClung Toabe Agar Base
™
Difco™ Egg Yolk Enrichment 50%
1. Suspend 75 g of the powder in 1 L of purified water. Mix Cat. No. 233471 Tube – 12 × 10 mL*
thoroughly. 233472 Bottle – 6 × 100 mL*
2. Heat with frequent agitation and boil for 1 minute to *Store at 2-8°C.
352
References
1. Lorian (ed.). 1986. Antibiotics in laboratory medicine, 2nd ed. Williams & Wilkins, Baltimore, Md.
2. McFarland. 1907. J. Am. Med. Assoc. 49:1176.
3. Baron, Peterson and Finegold. 1994. Bailey & Scott’s diagnostic microbiology, 9th ed. Mosby-Year
Book, Inc., St. Louis, Mo.
4. Clinical and Laboratory Standards Institute. 2006. Approved Standard: M7-A7. Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically, 7th ed. CLSI, Wayne, Pa.
5. Clinical and Laboratory Standards Institute. 2006. Approved Standard: M2-A9. Performance standards
for antimicrobial disk susceptibility tests, 9th ed. CLSI, Wayne, Pa.
6. Clinical and Laboratory Standards Institute. 2007. Approved Standard: M11-A7. Methods for antimi-
crobial susceptibility testing of anaerobic bacteria, 7th ed. CLSI, Wayne, Pa.
the viability and sensitivity of the test organism for its Dehydrated Appearance: Beige, free-flowing, homogeneous.
intended purpose; Solution: 3.7% solution, soluble in purified water. Solution
is light to medium amber, clear to very slightly
2. Inoculum Media: To condition the test culture for immediate
opalescent.
use;
Prepared Appearance: Light to medium amber, clear to very slightly
3. Assay Media: To permit quantitation of the vitamin under opalescent, without precipitate.
test. They contain all the factors necessary for optimal Reaction of 3.7%
growth of the test organism except the single essential Solution at 25°C: pH 6.7 ± 0.2
vitamin to be determined.
Cultural Response
Micro Assay Culture Agar is used for maintaining stock Difco™ Micro Assay Culture Agar or Micro
cultures of lactobacilli and other test microorganisms. This Inoculum Broth
medium is also used for general cultivation of lactobacilli. Prepare the medium per label directions. Inoculate with test organisms.
Incubate Micro Assay Culture Agar at 35 ± 2°C for 18-48 hours; incubate
Micro Inoculum Broth is used for cultivating lactobacilli and Micro Inoculum Broth at 35-37°C for 18-24 hours.
preparing the inoculum for microbiological assays. Organism ATCC™ INOCULUM CFU RECOVERY
Enterococcus hirae 8043 102-103 Good
Principles of the Procedure Lactobacillus rhamnosus 7469 102-103 Good
Peptone provides nitrogen and amino acids in both Micro Assay Lactobacillus delbrueckii
Culture Agar and Micro Inoculum Broth. Yeast extract is a subsp. lactis 7830 102-103 Good
vitamin source. Dextrose is a carbon source. Monopotassium Lactobacillus plantarum 8014 102-103 Good
phosphate is a buffering agent. Polysorbate 80 acts as an emulsi-
fier. Agar is the solidifying agent (Micro Assay Culture Agar).
353
354
355
Cultural Response
Difco™ Middlebrook 7H10 Agar with BBL™ Middlebrook
OADC Enrichment
Uninoculated Mycobacterium
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C under Tube fortuitum
approximately 3-5% CO2 for up to 21 days. ATCC™ 6841
Petragnani Medium. Dubos and Middlebrook were instrumental primary effect of albumin is that of protection of the tubercle
in the development of a number of formulations which contained bacilli against toxic agents and, therefore, it enhances their
oleic acid and albumin as key ingredients to aid in the growth of recovery on primary isolation; dextrose is an energy source;
the tubercle bacilli and to protect the organisms against a variety and catalase destroys toxic peroxides that may be present in the
of toxic agents.1 Subsequently, Middlebrook and Cohn improved medium. Partial inhibition of bacteria is achieved by the presence
the formulation of oleic acid-albumin agar and obtained faster, of the malachite green dye.
more luxuriant growth of Mycobacterium species on their
medium designated as 7H10.2,3 The oleic acid and bovine Formulae
albumin, along with sodium chloride, dextrose and catalase, are Difco™ Middlebrook 7H10 Agar
provided by the Middlebrook OADC Enrichment. Approximate Formula* Per 900 mL
Ammonium Sulfate...................................................... 0.5 g
It has been reported that the 7H10 medium tends to grow fewer Monopotassium Phosphate.......................................... 1.5 g
contaminants than the egg-based media commonly used for the Disodium Phosphate.................................................... 1.5 g
Sodium Citrate............................................................. 0.4 g
cultivation of mycobacteria.4 Magnesium Sulfate.................................................... 25.0 mg
Calcium Chloride......................................................... 0.5 mg
Prepared plates of the complete medium are deep-filled to Zinc Sulfate.................................................................. 1.0 mg
reduce the effects of drying during prolonged incubation. Copper Sulfate............................................................. 1.0 mg
L-Glutamic Acid (sodium salt)....................................... 0.5 g
Principles of the Procedure Ferric Ammonium Citrate............................................. 0.04 g
Pyridoxine Hydrochloride.............................................. 1.0 mg
Middlebrook and Cohn 7H10 Agar Base contains a variety of Biotin........................................................................... 0.5 mg
inorganic salts that provide substances essential for the growth Malachite Green...................................................... 250.0 µg
of mycobacteria. The sodium citrate, when converted to citric Agar.......................................................................... 15.0 g
acid, serves to hold certain inorganic cations in solution. BBL™ Middlebrook OADC Enrichment
Glycerol is an abundant source of carbon and energy. Approximate Formula* Per Liter
Sodium Chloride.......................................................... 8.5 g
Supplementation of the agar base is required in order to obtain Dextrose.................................................................... 20.0 g
mycobacterial growth. In the enriched medium, sodium chloride Bovine Albumin (Fraction V)....................................... 50.0 g
Catalase....................................................................... 0.03 g
maintains osmotic equilibrium; oleic acid, as well as other Oleic Acid.................................................................... 0.6 mL
long chain fatty acids, can be utilized by tubercle bacilli and *Adjusted and/or supplemented as required to meet performance criteria.
358
Difco™ Glycerol
BS12 CMPH2 EP MCM9
Cat. No. 228210 Bottle – 100 g
United States and Canada
228220 Bottle – 500 g
Cat. No. 221174 Prepared Plates (Deep Fill) – Pkg. of 20*
*Store at 2-8°C.
295964 Prepared I Plate™ Dishes (Middlebrook 7H10
Agar and Middlebrook 7H10 Agar) – Pkg. of 20*
220958 Prepared Slants, (A Tubes) – Pkg. of 10*
220959 Prepared Slants, (A Tubes) – Ctn. of 100*
297448 Prepared Slants, (C Tubes) – Pkg. of 10*
297396 Prepared Slants, (C Tubes) – Ctn. of 100*
297274 Prepared 1 oz. Transgrow-style Bottles –
Ctn. of 100*
Milk Agar
Intended Use Poor cleaning of the milking equipment may cause contamination
Milk Agar is recommended by the British Standards Institute 1 with micrococci, streptococci, coliforms or heat resistant
for the enumeration of microorganisms in liquid milk, ice cream, Bacillus strains, giving an increase of the bulk milk count of
dried milk and whey. >5 × 104 organisms/mL. Spoilage of pasteurized or raw milk
by proteolytic psychrotrophic bacteria can occur on prolonged
Summary and Explanation storage below 7°C.
Liquid milk is a highly perishable foodstuff with a shelf life Milk Agar conforms to the EEC Commission for the examination
of only 5-10 days after pasteurization. Contamination of raw of ice cream.2 Milk Agar is recommended for performing plate
milk may arise from either the soiled or diseased udder or count tests on milks, rinse waters and dairy products.3
inadequately cleaned milking or storage equipment. Bovine
mastitis or udder inflammation may cause contamination with Principles of Procedure
Staphylococcus aureus, Streptococcus agalactiae, Escherichia Peptone and yeast extract provide essential nutrients while
coli or, more rarely, Yersinia enterocolitica and Leptospira skim milk powder is a source of casein. Dextrose is the carbon
species. Excretion of these organisms can increase the bulk milk energy source. Agar is the solidifying agent.
count by 105 organisms/mL.
Proteolytic bacteria will be surrounded by a clear zone from
User Quality Control the conversion of casein into soluble nitrogenous compounds.1
359
360
361
References Availability
1. Gray. 1959. J. Hyg., Camb. 57:249.
2. Gray. 1964. J. Hyg., Camb. 62:495.
Difco™ Minerals Modified Glutamate Broth
3. P. H. L. S. Standing Committee on the Bacteriological Examination of Water Supplies. 1968. Cat. No. 218501 Dehydrated – 500 g
J. Hyg., Camb. 65:67.
4. Joint Committee of the P. H. L. S. and the Standing Committee of Analysts. 1980. J. Hyg., Camb.
85:35.
5. Abbiss, Wilson, Blood and Jarvis. 1981. J. Appl. Bact. 51:121.
6. Holbrook, Anderson and Baird-Parker. 1980. Food Technol. Aust. 32:78.
7. Departments of the Environment, Health & Social Security, and P.H.L.S. 1982. The bacteriological
examination of drinking water supplies. Report on Public Health and Medical Subjects No. 71.,
H.M.S.O., London, England.
363
References Availability
1. Lederberg. 1950. Methods in Med. Res. 3:5.
2. Davis. 1949. Proc. Natl. Acad. Sci. 35:1.
Difco™ Minimal Agar Davis
3. Nester, Schafer and Lederberg. 1963. Genetics 48:529. Cat. No. 254410 Dehydrated – 500 g
Difco™ Minimal Broth Davis without Dextrose
Cat. No. 275610 Dehydrated – 500 g
Availability
Difco™ Mitis Salivarius Agar
Cat. No. 229810 Dehydrated – 500 g
BBL™ Tellurite Solution 1%
Cat. No. 211917 Tube – 20 mL
364
Identity Specifications
Difco™ Mitis Salivarius Agar
Dehydrated Appearance: Bluish-beige, free-flowing, homogeneous.
Solution: 9.0% solution, soluble in purified water upon
boiling. Solution is deep royal blue, very slightly
opalescent.
Prepared Appearance: Deep royal blue, slightly opalescent.
Reaction of 9.0%
Solution at 25°C: pH 7.0 ± 0.2
BBL Tellurite Solution 1%
™
Cultural Response
Difco™ Mitis Salivarius Agar with BBL™ Tellurite
Solution 1%
Prepare the complete medium per label directions. Inoculate and
incubate under 5-10% CO2 at 35 ± 2°C for 18-48 hours.
INOCULUM COLONY
Organism ATCC™ CFU RECOVERY COLOR
Enterococcus faecalis 19433 102-103 Good Blue/black
Escherichia coli 25922 103 Partial to Brown, if any Streptococcus
salivarius
complete inhibition ATCC™ 9758
Staphylococcus aureus 25923 103 Partial to –
complete inhibition
Streptococcus mitis 9895 102-103 Good Blue
Streptococcus salivarius 9758 102-103 Good Blue “gum
drop” shape
Motility GI Medium
Intended Use Procedure
Motility GI Medium is used for detecting motility of microorgan- 1. If tubes are desired, dispense the molten medium to a depth
isms and for separating organisms in their motile phase. of 60-75 mm and cool in cold water up to the depth of
the medium. Cool flasks of medium to 50-55°C; pour into
Summary and Explanation sterile Petri dishes to a depth of 1/8 inch or more and allow
Motility GI Medium is prepared according to the formulation to solidify.
of Jordan, Caldwell and Reiter.1 It is a semisolid gelatin-heart 2. Inoculate with growth from an 18-24 hour pure culture. If
infusion medium that is adaptable to use in both tubes and plates tubes are used, inoculate by stab inoculation. If plates are
for motility studies. used, spot the inoculum on the surface or stab just below the
medium surface.
Principles of the Procedure 3. Incubate at a temperature and duration appropriate for the
Beef heart infusion, peptone and gelatin provide nitrogen, suspected organism being tested.
vitamins and amino acids. Agar is the solidifying agent. Motility 4. Examine tubes or plates for growth and signs of motility.
is evidenced by the presence of diffuse growth away from the line
or spot of inoculation. Nonmotile organisms grow only along Expected Results
the line of inoculation. Motility is evidenced by the presence of diffuse growth away
from the line or spot of inoculation. Nonmotile organisms grow
Formula only along the line of inoculation.
Difco™ Motility GI Medium
Approximate Formula* Per Liter Limitations of the Procedure
Beef Heart, Infusion from 500 g................................. 10.0 g
Tryptose..................................................................... 10.0 g
1. All weak or questionable motility test results should be
Sodium Chloride.......................................................... 5.0 g confirmed by flagella stain or by direct wet microscopy.2
Gelatin....................................................................... 53.4 g 2. Some flagellar proteins are not synthesized at higher
Agar............................................................................ 3.0 g
*Adjusted and/or supplemented as required to meet performance criteria.
temperatures.3
3. Some isolates of Yersinia enterocolitica demonstrate motility
Directions for Preparation from at 35°C while others may be nonmotile at 25°C.2 The motil-
Dehydrated Product ity of Proteus is also temperature dependent. This effect of
1. Suspend 81.4 g of the powder in 1 L of purified water. Mix temperature on motility needs to be taken into account when
thoroughly. deciding on a testing regimen.
2. Heat with frequent agitation and boil for 1 minute to 4. Due to the temperature dependency of motility in some
completely dissolve the powder. organisms, a negative test tube or plate should be incubated
3. Autoclave at 121°C for 15 minutes. an additional 5 days at a lower temperature of 22-25°C.3
4. Test samples of the finished product for performance using
stable, typical control cultures.
366
Cultural Response
Difco™ Motility GI Medium
Prepare the medium per label directions. Inoculate tubes of the medium with fresh cultures
by stabbing with an inoculating wire and incubate at 35 ± 2°C for 18-48 hours.
Organism ATCC™ RECOVERY Motility
Enterobacter aerogenes 13048 Good +
Uninoculated Escherichia coli Klebsiella
Escherichia coli 25922 Good + Tube ATCC™ 25922 pneumoniae
ATCC™ 13883
Klebsiella pneumoniae 13883 Good –
Proteus mirabilis 25933 Good +/–*
*Motility of Proteus is temperature dependent, being more pronounced at 20°C and possibly absent at 35°C.
References Availability
1. Jordan, Caldwell and Reiter. 1934. J. Bacteriol. 27:165.
2. D’Amato and Tomfohrde. 1981. J. Clin. Microbiol. 14:347.
Difco™ Motility GI Medium
3. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. Cat. No. 286910 Dehydrated – 500 g
1. Williams & Wilkins, Baltimore, Md.
Indole is produced in MILS Medium by organisms that Lysine decarboxylation (read at the bottom of the tube) is in-
possess the enzyme tryptophanase. Tryptophanase degrades dicated by a dark purple color compared with an uninoculated
the typtophan present in the casein peptone, yielding indole. control tube. In a negative test, the medium is yellow throughout
Indole can be detected in the medium by adding Kovacs’ the tube or has a narrow band of purple at the top of the medium.
reagent to the agar surface. The indole combines with the A negative test with organisms that do not ferment dextrose may
p-dimethylaminobenzaldehyde of Kovacs’ reagent and show no color change.
produces a red complex.
The test for lysine deaminase is read in the upper portion
MILS Medium is also used in the demonstration of hydrogen of the medium. In a positive test, the medium at or near the
sulfide production. Hydrogen sulfide, which is produced by surface is purple. In a negative test, the medium near the
some enteric organisms from sulfur compounds contained in surface is yellow or remains unchanged.
the medium, reacts with ferric ion, producing a characteristic
Hydrogen sulfide production is indicated by blackening in the
black precipitate.
medium.
Procedure After reading the other reactions, indole production may be
Using a sterile inoculating loop or needle, remove growth from detected by adding three drops of Kovacs’ reagent (Cat. No.
the subculture medium and stab the center of the motility 261185) to the surface of MILS Medium and shaking gently.
medium to the bottom of the tube. Incubate the tubes at 35°C The medium turns red if indole is present and remains unchanged
for 48 hours. if indole has not been produced.
Some flagellar proteins are not synthesized at higher temperatures.
References
If motility tests are negative after two days incubation, place 1. Ederer, Lund, Blazevic, Reller and Mirrett. 1972. J. Clin. Microbiol. 2:266.
2. Ewing. 1986. Edwards and Ewing’s identification of Enterobacteriaceae, 4th ed. Elsevier Science
the cultures at 21-25°C for up to 5 days to induce flagellar Publishing Co., Inc., New York, N.Y.
development. 3. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby, Inc.,
St. Louis, Mo.
4. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
368
369
Cultural Response
BBL™ Motility Test Medium
Prepare the medium per label directions. Stab inoculate with fresh cultures and incubate
at 35 ± 2°C for 2 days.
ORGANISM ATCC™ recovery Motility
Enterobacter aerogenes 13048 Good +
Escherichia coli 25922 Good +
Klebsiella pneumoniae 33495 Good –
Uninoculated Escherichia coli Klebsiella
Salmonella enterica subsp. enterica Tube ATCC™ 25922 pneumoniae
serotype Typhimurium 14028 Good + ATCC™ 13883
Procedure References
Inoculate tubes with a pure culture by stabbing the center of the 1. Tittsler and Sandholzer. 1936. J. Bacteriol. 31:575.
2. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology,
column of medium to greater than half the depth. Incubate tubes 9th ed. Williams & Wilkins, Baltimore, Md.
3. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
for 24-48 hours at 35 ± 2°C in an aerobic atmosphere. American Society for Microbiology, Washington, D.C.
4. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol.
1. Williams & Wilkins, Baltimore, Md.
Expected Results
After incubation, observe the tubes for growth in relation to Availability
the stab line. Nonmotile organisms grow only along the line of BBL™ Motility Test Medium
inoculation, while motile organisms spread out from the line of BAM CCAM COMPF USDA
inoculation and may even grow throughout the medium. Cat. No. 211436 Dehydrated – 500 g
221509 Prepared Tubes – Pkg. of 10
Negative tubes can be reincubated at 25 ± 2°C for an additional 221510 Prepared Tubes – Ctn. of 100
5 days, if desired.
Difco™ TTC Solution 1%
Consult appropriate texts for results with specific organisms. 2,3 Cat. No. 231121 Tube – 30 mL
264310 Bottle – 25 g
Limitation of the Procedure
Many organisms fail to grow deep in semisolid media; inoculat-
ing pour plates may be advantageous.4
370
Unsupplemented Mueller Hinton agar, although adequate for Directions for Preparation from
susceptibility testing of rapidly growing aerobic pathogens,
Dehydrated Product
is not adequate for more fastidious organisms such as
1. Suspend 38 g of the powder in 1 L of purified water. Mix
S. pneumoniae. The CLSI Document M2, Performance Standards
thoroughly.
for Antimicrobial Disk Susceptibility Tests, recommends Mueller
2. Heat with frequent agitation and boil for 1 minute to
Hinton agar supplemented with 5% defibrinated sheep blood.
completely dissolve the powder.
Details of quality control procedures and interpretive criteria
372
3. Autoclave at 121°C for 15 minutes. DO NOT OVERHEAT. diluted inoculum and rotate it firmly several times against the
OPTIONAL: Cool medium to 45-50°C and aseptically add upper inside wall of the tube to express excess fluid.
5% sterile defibrinated sheep blood. 6. Inoculate the entire agar surface of the plate three times,
4. Pour cooled Mueller Hinton agar into sterile Petri dishes on rotating the plate 60° between streakings to obtain even
a level, horizontal surface to give a uniform depth of about inoculation. As a final step, swab the rim of the agar bed.
4 mm (60-70 mL of medium for 150 mm plates and 25-30 mL 7. The lid may be left ajar for 3-5 minutes and the plate held
for 100 mm plates) and cool to room temperature.4 at room temperature for no longer than 15 minutes to
5. Check prepared medium to ensure the final pH is 7.3 ± 0.1 allow any surface moisture to be absorbed before applying
at 25°C. the antimicrobial agent-impregnated discs.
6. Test samples of the finished product for performance using 8. Apply the discs by means of an antimicrobial disc dis-
stable, typical control cultures. penser, using aseptic precautions. Deposit discs so that
the centers are at least 24 mm apart. It is preferable to
Procedure deposit penicillin and cephalosporin discs so that they are
A. Standard Method4 not less than 10 mm from the edge of the Petri dish, and
1. Perform a Gram stain before starting a susceptibility test their centers are at least 30 mm apart. Avoid placing such
to confirm culture purity and to determine appropriate test discs adjacent to one another. After discs have been placed
battery. on the agar, tamp them with a sterile needle or forceps to
2. Select at least three to five well-isolated similar colonies and make complete contact with the medium surface. This step
transfer with an inoculation needle or loop into 4-5 mL of is not necessary if the discs are deposited using the
suitable broth. Sensi-Disc™ 12-place self-tamping dispenser.
3. Incubate the broth at 35°C until it achieves or just exceeds 9. Within 15 minutes after the discs are applied, invert
the turbidity of the 0.5 McFarland barium sulfate standard the plates and place them in a 35°C incubator. With non-
(usually 2-6 hours). This results in a suspension containing fastidious organisms, plates should not be incubated under
approximately 1 to 2 × 108 CFU/mL (for E. coli ATCC an increased concentration of carbon dioxide.
25922). 10. Examine plates after 16-18 hours incubation. A full 24
4. Adjust the turbidity to be equivalent to the barium hours incubation is recommended for Staphylococcus
sulfate standard. For the diluent, use sterile broth or sterile aureus with oxacillin to detect methicillin-resistant
saline. The turbidity of the standard and the test inoculum S. aureus (MRSA) and for Enterococcus spp. when tested
should be compared by holding both tubes in front of with vancomycin to detect vancomycin-resistant strains.
a white background with finely drawn black lines or a Growth within the apparent zone of inhibition is indicative
photometric device can be used. of resistance.
5. Within 15 minutes after adjusting the turbidity of the A confluent “lawn” of growth should be obtained. If only
inoculum, immerse a sterile cotton swab into the properly isolated colonies grow, the inoculum was too light and the
373
test should be repeated. Measure the diameter of the zones Expected Results
of complete inhibition (as judged by the unaided eye), includ- Zone diameters measured around discs should be compared
ing the diameter of the disc, to the nearest whole millimeter, with those in the CLSI Document M100 (M2). Results obtained
using sliding calipers, a ruler, or a template prepared for this with specific organisms may then be reported as resistant,
purpose. The measuring device is held on the back of the intermediate or susceptible.
inverted plate over a black, non-reflecting background, and
With Mueller Hinton Agar with 5% Sheep Blood, the zone
illuminated from above. of growth inhibition should be measured, not the zone of
The endpoint should be taken as the area showing no inhibition of hemolysis. The zones are measured from the
obvious visible growth that can be detected with the unaided upper surface of the agar illuminated with reflected light, with
eye. Disregard faint growth of tiny colonies which can be the cover removed. Zone diameters for the agents specified
detected with difficulty near the edge of the obvious zone of under “Intended Use” should be compared with those in the
inhibition. Staphylococcus aureus when tested with oxacillin CLSI Document M100 (M2), which provides interpretive
discs is an exception, as are enterococci when tested with criteria.5 Results obtained may then be reported as resistant,
vancomycin. In these cases, transmitted light should be used intermediate or susceptible.
to detect a haze of growth around the disc which is shown Isolates of S. pneumoniae with oxacillin zone diameters of
by “occult resistant” MRSA strains17 or vancomycin-resistant ≥20 mm are susceptible (MIC ≤0.06 mg/mL) to penicillin. CLSI
enterococci.4 With Proteus species, if the zone of inhibition is Document M100 (M2) should be consulted for other antimi-
distinct enough to measure, disregard any swarming inside the crobial agents to which penicillin-susceptible isolates may also
zone. With trimethoprim and the sulfonamides, antagonists in be considered susceptible.4
the medium may allow some slight growth; therefore, disregard
NOTE: Informational supplements to CLSI Document M2,
slight growth (20% or less of the lawn of growth) and measure
containing revised tables of antimicrobial discs and interpretive
the more obvious margin to determine the zone diameter.
standards are published periodically. The latest tables should
B. Direct Method4 be consulted for current recommendations. The complete
The direct colony suspension method should be used when testing standard and informational supplements can be ordered from
S. pneumoniae. Observe aseptic techniques. the Clinical and Laboratory Standards Institute, 940 West
Valley Road, Suite 1400, Wayne, PA 19087-1898. Telephone:
1. Suspend growth from an overnight (16-18 hour) sheep blood
(610) 688-1100.
agar plate in saline or broth, such as Mueller Hinton broth.
Adjust the turbidity to be equivalent to the 0.5 McFarland Refer to other texts for additional information on antimicrobial
barium sulfate standard. For the diluent, use sterile broth susceptibility testing.19,20 Protocols developed by the CLSI and
or sterile saline. The turbidity of the standard and the test used by manufacturers to evaluate the performance of Mueller
inoculum should be compared by holding both tubes in front Hinton Agar in comparison to a reference medium are published
of a white background with finely drawn black lines or a in CLSI document M6-A.21
photometric device can be used.
NOTE: Alternative methods of inoculum preparation involv- Limitations of the Procedure
ing devices that permit direct standardization of inocula 1. Numerous factors can affect results: inoculum size; rate of
without adjustment of turbidity, such as the BBL™ Prompt™ growth; medium formulation and pH, length of incubation
Inoculation System, have been found to be acceptable for and incubation environment; disc content and drug diffusion
routine testing purposes.18 rate; and measurement of endpoints. Therefore, strict adher-
ence to protocol is required to ensure reliable results.22
2. Within 15 minutes of adjusting the turbidity of the inoculum,
2. When Mueller Hinton agar is supplemented with blood,
dip a sterile swab into the properly diluted inoculum and
the zone of inhibition for oxacillin and methicillin may be
rotate it firmly several times against the upper inside wall of
2-3 mm smaller than those obtained with unsupplemented
the tube to express excess fluid.
agar.23 Conversely, sheep blood may markedly increase the
3. Inoculate onto Mueller Hinton Agar with 5% Sheep Blood
zone diameters of some cephalosporins when they are tested
by streaking the entire agar surface of the plate three times,
against enterococci.24 Sheep blood may cause indistinct zones
rotating the plate 60° between streakings to obtain even
or a film of growth within the zones of inhibition around
inoculation. As a final step, swab the rim of the agar bed.
sulfonamide and trimethoprim discs.23
4. Replace the lid of the plate and hold the plate at room tem-
3. Mueller Hinton agar deeper than 4 mm may cause false-
perature for at least 3 minutes, but no longer than 15 minutes, resistant results, and agar less than 4 mm deep may be
to allow surface moisture to be absorbed before applying the associated with a false-susceptibility report.23
drug-impregnated discs. Use no more than nine discs per 4. A pH outside the range of 7.3 ± 0.1 may adversely affect
150 mm plate, or four discs per 100 mm plate. susceptibility test results. If the pH is too low, aminoglyco-
5. Incubate for 20-24 hours at 35°C in an atmosphere of 5% sides and macrolides will appear to lose potency; others may
CO2. appear to have excessive activity.23 The opposite effects are
possible if the pH is too high.23
374
21. Clinical and Laboratory Standards Institute. 2006. Approved standard: M6-A2. Protocols for evaluating BS12 CCAM CLSI CMPH2 MCM9
dehydrated Mueller-Hinton agar, 2nd ed. CLSI, Wayne, Pa.
22. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed. United States and Canada
American Society for Microbiology, Washington, D.C. Cat. No. 221176 Prepared Plates – Pkg. of 20*
23. Wood and Washington. 1995. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical
microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
221993 Prepared Plates (150 × 15 mm) – Pkg. of 8*
24. Buschelman, Jones and Bale. 1994. J. Clin. Microbiol. 32:565. 221801 Prepared Plates (150 × 15 mm) – Box of 24*
Europe
Availability Cat. No. 254030 Prepared Plates – Pkg. of 20*
254080 Prepared Plates – Ctn. of 120*
Difco™ Mueller Hinton Agar
255080 Prepared Plates (150 × 15 mm-style) – Pkg. of 20*
BAM BS12 CCAM CLSI CMPH2 ISO MCM9 254517 Prepared Plates (square 120 × 120 mm-style) –
Cat. No. 225250 Dehydrated – 500 g Pkg. of 20*
225220 Dehydrated – 2 kg Japan
225230 Dehydrated – 10 kg Cat. No. 251176 Prepared Plates – Pkg. of 20*
BBL™ Mueller Hinton II Agar 252129 Prepared Plates – Ctn. of 200*
251801 Prepared Plates (150 × 15 mm-style) – Pkg. of 24*
BAM BS12 CCAM CLSI CMPH2 ISO MCM9
*Store at 2-8°C.
Cat. No. 211438 Dehydrated – 500 g
211441 Dehydrated – 5 lb (2.3 kg)
212257 Dehydrated – 25 lb (11.3 kg)
375
Procedure
Use standard procedures to obtain isolated colonies from Availability
specimens. Incubate plates at 35 ± 2°C for 18-24 hours and BBL™ Mueller Hinton Chocolate Agar
United States and Canada
up to 72 hours, if necessary, in an aerobic atmosphere enriched
Cat. No. 221860 Prepared Plates – Pkg. of 20*
with 5-10% CO2.5 221869 Prepared Plates (150 × 15 mm-style plates) –
Pkg. of 8*
Expected Results 221802 Prepared Plates (150 × 15 mm-style plates) –
Box of 24*
After a minimum of 18 hours of incubation, the plates should
Japan
show isolated colonies in streaked areas and confluent growth Cat. No. 251860 Prepared Plates – Pkg. of 20*
in areas of heavy inoculation. 251802 Prepared Plates (150 × 15 mm-style) – Pkg. of 24*
*Store at 2-8°C.
The growth of Haemophilus appears as small (1 mm), moist,
pearly colonies with a characteristic “mousy” odor.
Procedure
Inoculate the medium with a pure culture, streaking to obtain
isolated colonies. Place a sterile blank disc on the inoculated
plate. Incubate plates at 35 ± 2°C in an aerobic atmosphere for
18-24 hours.
After incubation, add a few drops of Kovacs’ Reagent to the
sterile blank disc. Read the disc for the indole reaction within 1
minute after the addition of the reagent.
376
377
378
379
380
381
382
383
References Availability
1. Lowenstein. 1931. Zentralbl. Bakteriol. Parasitenkd. Infectionskr. Hyg. Abt. I Orig. 120:127.
2. Lowenstein. 1933. Ann. Inst. Pasteur. 50:161.
BBL™ Mycobactosel™ L-J Medium
3. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed. CMPH2 MCM9
American Society for Microbiology, Washington, D.C.
4. Jensen. 1932. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. I. Orig. 125:222. Cat. No. 221413 Prepared Slants (A Tubes) – Pkg. of 10*
5. Petran and Vera. 1971. Health Lab. Sci. 8:225. 221414 Prepared Slants (A Tubes) – Ctn. of 100*
6. U.S. Public Health Service, Centers for Disease Control and Prevention, and National Institutes of
Health. 2007. Biosafety in microbiological and biomedical laboratories, 5th ed. HHS Publication No. *Store at 2-8°C.
(CDC) 93-8395. U.S. Government Printing Office, Washington, D.C.
7. Kent and Kubica. 1985. Public health mycobacteriology: a guide for the level III laboratory. USDHHS.
Centers for Disease Control, Atlanta, Ga.
8. Cernoch, Enns, Saubolle and Wallace. 1994. Cumitech 16A, Laboratory diagnosis of the mycobacte-
rioses. Coord. ed., Weissfeld. American Society for Microbiology, Washington, D.C.
9. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby, Inc.,
St. Louis, Mo.
10. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
American Society for Microbiology, Washington, D.C.
Mycological Media
Mycological Agar • Mycophil™ Agar
Mycophil™ Agar with Low pH
Intended Use Formulae
Mycological media are used for the cultivation and maintenance of Difco™ Mycological Agar
fungi, for the demonstration of chromogenesis and for obtaining Approximate Formula* Per Liter
yeast and mold counts. Soy Peptone............................................................... 10.0 g
Dextrose.................................................................... 10.0 g
Agar.......................................................................... 15.0 g
Summary and Explanation BBL™ Mycophil™ Agar
Many different culture media have been developed for the Approximate Formula* Per Liter
growth of fungi. In comparison with media for the majority Papaic Digest of Soybean Meal................................... 10.0 g
of bacterial strains, fungal media are of simple composition, Dextrose.................................................................... 10.0 g
Agar.......................................................................... 16.0 g
usually consisting of a peptone, dextrose and agar. Selectivity
is achieved by lowering the pH, incorporating dyes or adding BBL™ Mycophil™ Agar with Low pH
antimicrobial agents. Approximate Formula* Per Liter
Papaic Digest of Soybean Meal................................... 10.0 g
Mycological Agar and Mycophil Agar are nonselective media Dextrose.................................................................... 10.0 g
Agar.......................................................................... 18.0 g
of value in general work with yeasts and molds rather than *Adjusted and/or supplemented as required to meet performance criteria.
for isolation from materials possessing mixed flora. It is often
desirable to use these media in parallel with selective media as Directions for Preparation from
some of the selective agents are inhibitory for certain fungi. Dehydrated Product
Mycophil Agar with Low pH has had its base adjusted to Difco™ Mycological Agar
approximately pH 4.7, which obviates the need for pH adjustment 1. Suspend 35 g of the powder in 1 L of purified water. Mix
with lactic or tartaric acids in the laboratory. It also differs from thoroughly.
Mycophil Agar in that an additional 2 g/L of agar has been 2. Heat with frequent agitation and boil for 1 minute to
incorporated so that the medium may be sterilized and remelted completely dissolve the powder.
without losing its ability to solidify. 3. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using
Wetzler et al. employed Mycophil Agar with Low pH for
stable, typical control cultures.
enumeration of yeasts and molds in poultry processing plants.1
The formulation also has been recommended for isolation of BBL™ Mycophil™ Agar
yeasts and most filamentous fungi from clinical material.2 1. Suspend 36 g of the powder in 1 L of purified water. Mix
thoroughly.
Principles of the Procedure 2. Heat with frequent agitation and boil for 1 minute to
The peptone and dextrose ingredients supply sufficient completely dissolve the powder.
nutrients for the metabolism of fungal species. 3. Autoclave at 118°C for 15 minutes.
4. For yeast and mold counts, adjust the pH to 4.0 by adding
15 mL of sterile 10% lactic acid to each L of sterile melted
medium prior to plating.
5. Test samples of the finished product for performance using
stable, typical control cultures.
384
Prepare the medium per label directions. Inoculate and incubate at BBL™ Mycophil™ Agar with Low pH
30 ± 2°C (incubate Penicillium at 20-25°C) for 18-72 hours. Dehydrated Appearance: Fine, homogeneous, free of extraneous material,
may contain tan specks.
ORGANISM ATCC
™
INOCULUM CFU RECOVERY
Solution: 3.8% solution, soluble in purified water upon
Aspergillus brasiliensis (niger) 16404 30-300 Good boiling. Solution is light to medium, yellow to
Candida albicans 10231 30-300 Good tan, clear to slightly hazy.
Penicillium abeanum 22346 30-300 Good Prepared Appearance: Light to medium, yellow to tan, clear to slightly
Saccharomyces cerevisiae 9080 30-300 Good hazy.
Reaction of 3.8%
Staphylococcus aureus 25923 103-104 Good
Solution at 25°C: pH 4.7 ± 0.2
Cultural Response
BBL™ Mycophil™ Agar with Low pH BBL™ Mycophil™ Agar or Mycophil™ Agar with
1. Suspend 38 g of the powder in 1 L of purified water. Mix Low pH
Prepare the medium per label directions. Inoculate with fresh cultures and
thoroughly. incubate at 25 ± 2°C for 7 days.
2. Heat with frequent agitation and boil for about 30 seconds
recovery
to completely dissolve the powder. Recovery MYCOPHIL™
MYCOPHIL™ AGAR WITH
3. Autoclave at 118°C for 15 minutes or at 121°C for 10 ORGANISM ATCC™ Agar LOW PH
minutes. Aspergillus brasiliensis (niger) 16404 Good Good
4. The medium should be cooled and used at once. If the Candida albicans 60193 Good Good
medium is allowed to solidify after autoclaving, it may be Nocardia asteroides 19247 Good N/A
remelted once. DO NOT OVERHEAT. Penicillium roquefortii 9295 Good N/A
5. Test samples of the finished product for performance using Penicillium roquefortii 10110 N/A Good
stable, typical control cultures. Saccharomyces cerevisiae 9763 N/A Good
Trichophyton mentagrophytes 9533 Good N/A
Procedure
Inoculate plated media with test specimens or materials so as
to obtain isolated colonies. Consult appropriate references for in- typical color and morphology. Yeast and mold colonies can
formation about the processing and inoculation of specimens.3,4 be counted to determine the level of contamination in the test
For isolation of fungi from potentially contaminated specimens, sample. Biochemical tests and serological procedures should be
also inoculate a selective medium. Incubate plates at 25-30°C in performed to confirm findings.5
an inverted position (agar side up) with increased humidity. For
isolation of fungi causing systemic mycoses, two sets of media References
should be inoculated, with one set incubated at 25-30°C and 1. Wetzler, Musick, Johnson and MacKenzie. 1962. Am. J. Public Health 52:460.
2. Von Riesen and Jensen. 1958. Am. J. Med. Technol. 24:123.
a duplicate set at 35 ± 2°C. All cultures should be examined at 3. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
American Society for Microbiology, Washington, D.C.
least weekly for fungal growth and should be held for 4-6 weeks 4. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
before being reported as negative. 5. Larone. 1995. Medically important fungi: a guide to identification, 3rd ed. American Society for
Microbiology, Washington, D.C.
Expected Results
After sufficient incubation, the plates should show isolated
colonies in streaked areas and confluent growth in areas of
heavy inoculation. Examine plates for fungal colonies exhibiting
385
Mycoplasma Media
(See PPLO Media)
Mycosel™ Agar
Intended Use Summary and Explanation
Mycosel Agar is a highly selective medium containing cyclohexi- Mycosel Agar was developed by using the ingredients of
mide and chloramphenicol. It is recommended for the isolation Mycophil™ Agar as a nutritive base to which cycloheximide
of pathogenic fungi from materials having a large amount of and chloramphenicol were added as selective agents. It is widely
flora of other fungi and bacteria.1,2 BBL™ prepared plates of used for the isolation of fungi from a variety of sources, and is
Mycosel Agar are deep-filled to reduce the effects of drying recommended for the recovery of dermatophytes.3
during prolonged incubation.
Identity Specifications
BBL™ Mycosel™ Agar
Dehydrated Appearance: Fine, homogeneous, free of extraneous
material.
Solution: 3.6% solution, soluble in purified water upon
boiling. Solution is light to medium, yellow
to tan, clear to moderately hazy.
Prepared Appearance: Light to medium, yellow to tan, clear to
moderately hazy.
Reaction of 3.6%
Solution at 25°C: pH 6.9 ± 0.2
Cultural Response
BBL™ Mycosel™ Agar
Prepare the medium per label directions. Inoculate with fresh cultures and
incubate at 25 ± 2°C for 7 days.
ORGANISM ATCC™ recovery
Aspergillus brasiliensis (niger) 16404 Partial to
complete inhibition
Aureobasidium pullulans 9348 Partial to
complete inhibition
Blastomyces dermatitidis 56218 Good
Candida albicans 10231 Good
Escherichia coli 25922 Partial to
complete inhibition
Microsporum audouinii 9079 Good
Penicillium roquefortii 9295 Partial to
complete inhibition
Phialophora verrucosa 10223 Good
Staphylococcus aureus 25923 Complete inhibition
Streptomyces rimosus 10970 Partial to
complete inhibition
Trichophyton mentagrophytes 9533 Good
386
387
388
Procedure
As soon as possible after the specimen is received in the Availability
laboratory, inoculate the specimen onto a Neomycin Blood BBL™ Neomycin Blood Agar
Unites States and Canada
Agar plate by firmly rolling swab over a third of the agar Cat. No. 221792 Prepared Plates – Ctn. of 100*
surface. Streak the remainder of the plate with a sterilized
Europe
inoculating loop to obtain isolated colonies. Without re-sterilizing Cat. No. 254444 Prepared Plates – Pkg. of 20*
*Store at 2-8°C.
Bacto™ Neopeptone
Intended Use activity. Neopeptone has been cited as a component of culture
Bacto Neopeptone is used in preparing microbiological culture media used for cultivation of human pathogens, notably,
media. Bordetella pertussis and group A streptococci.
Neopeptone has also been reported to provide nutrients
Summary and Explanation for support of spirochetes and protozoa. Wyss et al.3 used
Neopeptone is recommended for use in media for detection neopeptone as a component of a medium for cultivation of
of fungi.1 Apodaca and McKerrow2 used neopeptone for the Treponema maltophilum sp. nov., a fastidious oral anaerobe.
cultivation of Trichophyton rubrum for study of its proteolytic
389
Cultural Response
Biochemical Reactions
Bacto™ Neopeptone
Prepare a sterile solution of Bacto Neopeptone as directed below. Adjust final pH to 7.2-7.4. Inoculate and incubate at 35 ± 2°C for 18-48 hours.
TEST TEST SOLUTION ORGANISM ATCC™ INOCULUM CFU RESULT
Fermentable Carbohydrates 2% Escherichia coli 25922 ~107 Negative
Indole Production 0.1% Escherichia coli 29552 0.1 mL, undiluted Positive
Acetylmethylcarbinol 0.1% with Enterobacter aerogenes 13048 0.1 mL, undiluted Positive
Production 0.5% dextrose
Hydrogen Sulfide 1% Salmonella enterica subsp. enterica 14028 0.1 mL, undiluted Positive
Production serotype Typhimurium
Growth Response
Bacto™ Neopeptone
Prepare a sterile solution with 2% Bacto Neopeptone, 0.5% sodium chloride and 1.5% agar. Adjust final pH to 7.2-7.4. Inoculate and incubate plates at
35 ± 2°C for 18-48 hours.
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Escherichia coli 25922 30-300 Good
Staphylococcus aureus 25923 30-300 Good
Neutralizing Buffer
Intended Use Principles of the Procedure
Neutralizing Buffer is recommended for detection of microor-
ganisms found on dairy and food equipment disinfected with
Monopotassium phosphate provides the buffering capability.
Sodium thiosulfate inactivates the effect of chlorine compounds.
N
chlorine or quaternary ammonium compounds. The aryl sulfonate complex neutralizes the effects of quaternary
ammonium compounds.
Summary and Explanation
Neutralizing Buffer has the ability to inactivate the bacteri- Formula
cidal and bacteriostatic effect of chlorine as well as quaternary Difco™ Neutralizing Buffer
ammonium compounds. Neutralizing Buffer is recommended Approximate Formula* Per Liter
for use in the microbiological examination of surfaces in Monopotassium Phosphate........................................ 42.5 mg
Sodium Thiosulfate...................................................... 0.16 g
standard methods for the examination of dairy products Aryl Sulfonate Complex................................................ 5.0 g
and foods.1,2 Neutralizing Buffer is also recommended for the *Adjusted and/or supplemented as required to meet performance criteria.
391
392
393
Nitrate Broth
Intended Use gram-negative bacilli. The end product of reduction depends
Nitrate Broth is recommended as an aid in the identification of upon the bacterial species.2
aerobic and facultative anaerobic gram-negative microorganisms Nitrate Broth is a basal medium containing potassium nitrate.
by means of the nitrate reduction test. The microorganism under evaluation is inoculated into the
medium and after incubation, nitrate reduction may be
Summary and Explanation determined. An inverted Durham fermentation tube in the
Microorganisms may be differentiated according to their prepared tubed medium serves to trap nitrogen gas produced
metabolism of certain substrates. The ability to reduce nitrate through denitrification. The medium is evaluated for nitrate
to nitrite is characteristic of the family Enterobacteriaceae.1 reduction by the addition of two reagents, Nitrate A Reagent
Nonfermenters and other miscellaneous gram-negative bacilli (0.8% sulfanilic acid in 5N acetic acid) and Nitrate B Reagent
vary in their ability to reduce nitrates. Some members of this (0.6% N, N-dimethyl-alpha-naphthylamine in 5N acetic acid),
group are capable of denitrification, which is a reduction which detect the presence of a catabolic end product, and by the
of nitrate to nitrogen gas. The production of gas from nitrate addition of Nitrate C Reagent, zinc dust, which detects the
is an important differential test for glucose-nonfermenting absence of remaining nitrate in the medium.2
Cultural Response
Difco™ Nitrate Broth
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-24
hours. Test for nitrate reduction using Nitrate A Reagent, Nitrate B Reagent and Nitrate
C Reagent following label directions.
INOCULUM NITRATE
ORGANISM ATCC™ CFU RECOVERY REDUCTION
Acinetobacter calcoaceticus 19606 102-103 Good –
Enterobacter aerogenes 13048 102-103 Good +
Uninoculated Acinetobacter Escherichia coli
Escherichia coli 25922 102-103 Good + Tube calcoaceticus ATCC™ 25922
ATCC™ 19606
Pseudomonas aeruginosa 27853 102-103 Good +
394
Expected Results
If growth is apparent after 24-48 hours of incubation, examine
for presence of gas in the Durham tube. If gas is present
and the test organism is a nonfermenter, the test is positive
for denitrification (nitrate was reduced to nitrogen gas). If
the organism is a fermenter, gas may or may not be present.
Add 10 drops of Nitrate A Reagent and 10 drops of Nitrate B
Reagent to the tube. Development of a red color within 2 minutes
denotes a positive test for nitrate. If there is no color develop-
ment, add a small amount (approximately 20 mg on the tip of
395
396
N
214320 Dehydrated – 10 kg
Cat. No. 297222 Prepared Pour Tubes, 20 mL – Pkg. of 10*
*Store at 2-8°C.
BBL™ Xanthine Agar
Cat. No. 297224 Prepared Pour Tubes, 20 mL – Pkg. of 10*
Nocardia ID QUAD
Intended Use Quadrant III contains Tyrosine Agar (tyrosine and nutrient agar)
The Nocardia ID QUAD plate is a four-sectored plate for testing the ability of isolates to decompose tyrosine.
containing four different media used for differentiation and Quadrant IV contains Xanthine Agar (xanthine and nutrient
identification of Nocardia species and other aerobic actinomy- agar) for testing the ability of isolates to decompose xanthine.
cetes isolated from clinical specimens.
Principles of the Procedure
Summary and Explanation The four biochemical media contained in the Nocardia ID
The most frequently encountered aerobic actinomycetes, QUAD plates offer a convenient means of conducting four
members of the order Actinomycetales, include the genera tests used in the differentiation and identification of Nocardia
Nocardia, Streptomyces, Actinomadura, Nocardiopsis, Rhodo- species following observation of staining reactions and micro-
coccus and Dermatophilus. The testing algorithm that permits scopic characteristics.1,2
identification of most aerobic actinomycetes consists of direct
microscopic techniques and a minimum number of biochemical Decomposition of casein in Quadrant I may be detected by
reactions.1 observing clear zones in the white, opaque skim milk around
the inoculum. Growth without clearing around the inoculum is
Quadrant I contains Casein Agar (skim milk and agar) for considered to be a negative test result.
determining the ability of isolates to hydrolyze casein.
The ability of isolates to break down and utilize starch may be
Quadrant II contains Starch Agar (potato starch and nutrient detected in Quadrant II. Starch hydrolysis may be detected by
agar) for testing the ability of isolates to utilize starch. colorless zones surrounding colonies after the plate is flooded
397
with Gram’s iodine. Blue or purple zones surrounding colonies reaction. N. brasiliensis decomposes casein and gives a positive
indicate a negative test. reaction. N. asteroides shows a negative reaction or no clearing
around the inoculum.
The decomposition of tyrosine can be detected in Quadrant III.
A clear halo around a colony is a positive test. Growth without To determine starch utilization, flood Quadrant II with
the presence of clear halos or growth with the production of Gram’s or Lugol’s iodine and observe the plate for colorless
melanin-like pigment is a negative test. zones around the inoculum, which indicates a positive reac-
tion, such as that obtained with S. rimosus. N. asteroides and
The ability of isolates to decompose xanthine may be detected
N. brasiliensis give a negative reaction with no clear zones; blue
in Quadrant IV. A clear halo around a colony is a positive test.
or purple zones surround colonies.
Growth without the presence of clear halos or growth with the
production of a melanin-like pigment is a negative test. The decomposition of tyrosine in Quadrant III is indicated by
clear halos around colonies. There are no clear halos around
Procedure colonies in a negative test. N. brasiliensis decomposes tyrosine,
Inoculate each sector with a pure culture of the isolate. Use whereas N. asteroides does not.
a small sterile spatula to obtain approximately 1 mm of the
The ability of isolates to decompose xanthine in Quadrant
colony from a pure culture. Using the spatula, cut a small
IV is shown by a clear halo around colonies, such as that
groove through the agar to the bottom of the plate, depositing
obtained with S. rimosus. The absence of clear halos or the pro-
the inoculum near the bottom of the groove. Alternatively, the
duction of a melanin-like pigment indicates a negative test. Both
tip of a sterile wooden applicator stick can be used to make a
N. asteroides and N. brasiliensis give a negative reaction.
well through the agar to the bottom of the plate, depositing the
inoculum at the bottom of the well.
References
Incubate the plates at 30°C in an inverted position (agar side 1. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.,
American Society for Microbiology, Washington, D.C.
up) under aerobic conditions and observe every 3-4 days for 2. Koneman, Allen, Janda, Schrechenberger and Winn. 1997. Color atlas and textbook of diagnostic
microbiology, 5th ed. Lippencott-Raven Publishers, Philadelphia, Pa.
14-21 days.1
Availability
Expected Results BBL™ Nocardia ID QUAD
Examine plates for growth periodically for 14-21 days of BS12 CMPH2 MCM9
incubation. Cat. No. 298309 Prepared Plates (QUAD) – Pkg. of 10*
Examine Quadrant I for the presence of a clear halo in the white *Store at 2-8°C.
Nutrient Agar
Intended Use User Quality Control
Nutrient Agar is used for the cultivation of bacteria and for the
enumeration of organisms in water, sewage, feces and other Identity Specifications
materials. Difco™ Nutrient Agar
Dehydrated Appearance: Tan, free-flowing, homogeneous.
Summary and Explanation Solution: 2.3% solution, soluble in purified water upon
boiling. Solution is light to medium amber, clear
Early in the 20th century, the American Public Health Associa- to slightly opalescent.
tion published the formula for a general purpose medium for Prepared Appearance: Light amber, very slightly to slightly opalescent.
the growth of a wide variety of nonfastidious microorganisms.1 Reaction of 2.3%
This was in recognition of the need for a standardized medium Solution at 25°C: pH 6.8 ± 0.2
for the use in the examination of water and wastewater, dairy
products and various foods. This relatively simple formula- Cultural Response
tion has stood the test of time, and with the name of Nutrient Difco™ Nutrient Agar
Prepare the medium per label directions. Inoculate and incubate at
Agar, is still specified in current compendia of methods for 35 ± 2°C for 18-48 hours.
the microbiological examination of a broad spectrum of
ORGANISM ATCC™ INOCULUM CFU RECOVERY
materials.2-5 Additionally, it is used in the laboratory for the
Enterococcus faecalis 19433 102-103 Good
cultivation and maintenance of nonfastidious species.
Escherichia coli 25922 102-103 Good
Pseudomonas aeruginosa 27853 102-103 Good
398
Principles of the Procedure Tubed slants are used primarily for the cultivation and main-
Nutrient Agar consists of peptone, beef extract and agar. This tenance of pure cultures. They should be inoculated with an
relatively simple formulation provides the nutrients necessary inoculating loop and incubated under the same conditions as
for the replication of a large number of microorganisms that the plated medium.
are not excessively fastidious. The beef extract contains water-
soluble substances including carbohydrates, vitamins, organic Expected Results N
nitrogen compounds and salts. Peptones are the principle sources Examine plates for growth.
of organic nitrogen, particularly amino acids and long-chained Growth from tubes inoculated with pure cultures may be used
peptides. Agar is the solidifying agent. for biochemical and/or serological testing.
Formula References
Difco™ Nutrient Agar 1. American Public Health Association. 1917. Standard methods of water analysis, 3rd ed. American
Public Health Association, New York, N.Y.
Approximate Formula* Per Liter 2. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
Beef Extract.................................................................. 3.0 g tional, Gaithersburg, Md.
Peptone....................................................................... 5.0 g 3. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C.
Agar.......................................................................... 15.0 g 4. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
*Adjusted and/or supplemented as required to meet performance criteria. International, Gaithersburg, Md.
5. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
Directions for Preparation from
Dehydrated Product Availability
1. Suspend 23 g of the powder in 1 L of purified water. Mix Difco™ Nutrient Agar
thoroughly. AOAC BAM CCAM COMPF ISO SMWW USDA
2. Heat with frequent agitation and boil for 1 minute to Cat. No. 212000 Dehydrated – 100 g
completely dissolve the powder. 213000 Dehydrated – 500 g
211665 Dehydrated – 2 kg
3. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using BBL™ Nutrient Agar
AOAC BAM CCAM COMPF ISO SMWW USDA
stable, typical control cultures.
United States and Canada
Cat. No. 297801 Prepared Plates – Pkg. of 10*
Procedure 220968 Prepared Pour Tubes – Pkg. of 10
Liquefy the agar if prepared tubes are used, cool to 45-50°C 220971 Prepared Slants – Ctn. of 100
and pour into Petri dishes. Allow to solidify for at least 30 Mexico
minutes. Use standard procedures to obtain isolated colonies Cat. No. 257500 Prepared Plates – Pkg. of 10*
*Store at 2-8°C.
from specimens. Incubate plates at 35 ± 2°C for 18-24 hours
and 42-48 hours, if necessary.
399
Identity Specifications
Difco™ Nutrient Agar 1.5%
Dehydrated Appearance: Beige to light tan, free-flowing, homogeneous.
Solution: 3.1% solution, soluble in purified water upon
boiling. Solution is light to medium amber, very
slightly to slightly opalescent.
Prepared Appearance: Light to medium amber, very slightly to slightly
opalescent.
Reaction of 3.1%
Solution at 25°C: pH 7.3 ± 0.2
Cultural Response
Difco™ Nutrient Agar 1.5%
Prepare the medium per label directions without (plain) and with
5% sheep blood (SB). Inoculate and incubate the plates at 35 ± 2°C
for 40-48 hours under appropriate atmospheric conditions.
INOCULUM recovery RECOVERY
ORGANISM ATCC™ CFU PLAIN with SB HEMOLYSIS
Escherichia coli 25922 102-103 Good Good Beta
Neisseria
meningitidis 13090 102-103 Good Good Gamma Streptococcus Streptococcus
pneumoniae pyogenes
Staphylococcus ATCC™ 6305 ATCC™ 19615
aureus 25923 102-103 Good Good Beta
Streptococcus
pneumoniae 6305 102-103 Good Good Alpha
Streptococcus
pyogenes 19615 102-103 Good Good Beta
Procedure
Availability
For a complete discussion of the isolation and identification of
Difco™ Nutrient Agar 1.5%
aerobic and anaerobic microorganisms, refer to appropriate
COMPF
references. Cat. No. 269100 Dehydrated – 500 g
400
Identity Specifications
Difco™ Nutrient Agar with MUG
N
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution: 2.31% solution, soluble in purified water upon
boiling. Solution is light amber, clear to very
slightly opalescent.
Prepared Appearance: Light amber, clear to slightly opalescent.
Reaction of 2.31%
Solution at 25°C: pH 6.8 ± 0.2
Cultural Response
Difco™ Nutrient Agar with MUG
Prepare the medium per label directions. After incubation on m Endo
Agar LES using the membrane filter technique, aseptically transfer the
membrane to Nutrient Agar with MUG. Incubate 4-24 hours at 35 ± 2°C.
Examine for fluorescence under long-wave (approximately 366 nm) UV
light.
Organism ATCC™ INOCULUM CFU Fluorescence
Enterobacter aerogenes 13048 30-300 –
Escherichia coli 25922 30-300 +
Nutrient Broth
Intended Use Formula
Nutrient Broth is used for the cultivation of many species of Difco™ Nutrient Broth
nonfastidious microorganisms. Approximate Formula* Per Liter
Beef Extract.................................................................. 3.0 g
Peptone....................................................................... 5.0 g
Summary and Explanation *Adjusted and/or supplemented as required to meet performance criteria.
Nutrient Broth has the formula originally designed for use
in the Standard Methods for Examination of Water and Waste- Directions for Preparation from
water. It is not a recommended bacteriological medium in later Dehydrated Product
editions of this publication. It is one of several nonselective media 1. Dissolve 8 g of the powder in 1 L of purified water.
useful in routine cultivation of microorganisms.1-3 2. Autoclave at 121°C for 15 minutes.
3. Test samples of the finished product for performance using
Principles of the Procedure stable, typical control cultures.
This relatively simple formulation supports the growth of
nonfastidious microorganisms due to its content of peptone Procedure
and beef extract. Inoculate tubes of the broth medium with the test samples.
Incubate tubes for 18-24 hours at 35 ± 2°C in an aerobic
User Quality Control atmosphere.
Identity Specifications Expected Results
Difco™ Nutrient Broth
After incubation, growth is evidenced by the appearance of
Dehydrated Appearance: Medium tan, free-flowing, homogeneous.
turbidity in the broth. Aliquots of the broth can be used for
Solution: 0.8% solution, soluble in purified water. Solu-
tion is light to medium amber, clear. subculturing to solid media for purification and identification
Prepared Appearance: Light to medium amber, clear. purposes.
Reaction of 0.8%
Solution at 25°C: pH 6.8 ± 0.2 References
1. Marshall (ed.). 1993. Standard methods for the examination of dairy products, 16th ed. American
Public Health Association, Washington, D.C.
Cultural Response 2. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
tional, Gaithersburg, Md.
Difco™ Nutrient Broth 3. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
Prepare the medium per label directions. Inoculate and incubate at 4th ed. American Public Health Association, Washington, D.C.
35 ± 2°C for 18-24 hours.
ORGANISM ATCC™ INOCULUM CFU RECOVERY Availability
Escherichia coli 25922 102-103 Good Difco™ Nutrient Broth
Staphylococcus aureus 25923 102-103 Good AOAC BAM CCAM COMPF SMD
Cat. No. 233000 Dehydrated – 100 g
234000 Dehydrated – 500 g
231000 Dehydrated – 2 kg
232000 Dehydrated – 10 kg
BBL™ Nutrient Broth
BAM CCAM COMPF SMD
Cat. No. 221669 Prepared Tubes, 5 mL (K Tubes) – Pkg. of 10
Nutrient Gelatin
Intended Use of the Enterobacteriaceae and nonfermenting gram-negative
Nutrient Gelatin is used for the detection of gelatin liquefaction bacteria. The use of Nutrient Gelatin for determining gelatin
by microbial species. liquefaction patterns is considered to be the “standard” method
for taxonomic studies, since the rate of liquefaction is impor-
Summary and Explanation tant in the characterization of groups within the Enterobac-
Nutrient Gelatin is made in accordance with the formula teriaceae family as well as other groups of microorganisms.2,3
formerly used in the examination of water, sewage, and other Edwards and Ewing consider gelatin liquefaction to be an
materials of sanitary importance.1 Gelatin liquefaction is one essential test for differentiation of enteric bacilli.4
of the characteristics used in the classification of members
402
Nutrient Gelatin is used chiefly for identification of pure Incubate tubes, including an uninoculated control, at 35 ± 2°C
cultures of bacteria that are not particularly fastidious in for 24-48 hours and up to 14 days.
regard to nutritional requirements.
Expected Results
Principles of the Procedure At various intervals during the incubation process, examine the
The peptone and beef extract supply sufficient nutrients for tubes for growth (turbidity) and liquefaction. Use uninoculated N
the growth of nonfastidious bacterial species. The gelatin is the control tubes for comparison. At each interval, tighten caps and
substrate for the determination of the ability of an organism to transfer the tubes to a refrigerator or ice bath for a sufficient
produce gelatinases, which are proteolytic-like enzymes active time period to determine whether liquefaction has or has not
in the liquefaction of gelatin. occurred. It is important that the tubes not be shaken during
the transfer from incubator to refrigerator. When reading
Formula results, invert the chilled tubes to test for solidification or
Difco™ Nutrient Gelatin liquefaction.3
Approximate Formula* Per Liter
Beef Extract.................................................................. 3.0 g Consult appropriate texts for results with specific organisms.3-6
Peptone....................................................................... 5.0 g
Gelatin..................................................................... 120.0 g Limitations of the Procedure
*Adjusted and/or supplemented as required to meet performance criteria.
1. This medium is not recommended for determination
Directions for Preparation from of gelatin liquefaction by fastidious species and obligate
anaerobes.
Dehydrated Product
2. Gelatin is liquid at temperatures above 20°C. If tubes are
1. Suspend 128 g of the powder in 1 L of purified water.
incubated at 35°C, they must be refrigerated in order to
2. Warm to 50°C to completely dissolve the powder.
read for liquefaction. Include an uninoculated tube in the
3. Autoclave at 121°C for 15 minutes.
test procedure for comparison.
4. Test samples of the finished product for performance using
3. Growth and liquefaction frequently occur only at the
stable, typical control cultures.
surface of the tube. To prevent a false-negative interpretation,
Procedure handle tubes carefully when warm so that liquified gelatin
Using a heavy inoculum (growth from an 18-24 hour pure remains at the surface of the tube.3
culture), stab the tubes of Nutrient Gelatin with an inoculating
needle directly down the center of the medium to a depth of
References
1. American Public Health Association. 1960. Standard methods for the examination of water and
approximately one-half an inch from the bottom of the tube. sewage, 9th ed. American Public Health Association, New York, N.Y.
2. MacFaddin. 2000. Biochemical tests for identification of medical bacteria, 3rd ed. Lippincott Williams
& Wilkins, Baltimore, Md.
3. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
User Quality Control American Society for Microbiology, Washington, D.C.
4. Ewing. 1986. Edwards and Ewing’s identification of Enterobacteriaceae, 4th ed. Elsevier Science
Publishing Co., Inc., New York, N.Y.
Identity Specifications 5. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology,
9th ed. Williams & Wilkins, Baltimore, Md.
Difco™ Nutrient Gelatin 6. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
Dehydrated Appearance: Tan, fine granular, free-flowing.
Solution: 12.8% solution, soluble in purified water upon
warming in a 50-55°C water bath. Solution Availability
is light to medium amber, clear to slightly Difco™ Nutrient Gelatin
opalescent, may have a slight precipitate.
USDA
Prepared Appearance: Medium amber, clear to slightly opalescent, Cat. No. 211100 Dehydrated – 500 g
may have a slight precipitate.
Reaction of 12.8% BBL™ Nutrient Gelatin
Solution at 25°C: pH 6.8 ± 0.2 USDA
Cat. No. 220974 Prepared Tubes, 8 mL (Deeps) – Pkg. of 10
Cultural Response
Difco™ Nutrient Gelatin
Prepare the medium per label directions. Stab inoculate using a heavy
inoculum of fresh cultures and incubate at 35 ± 2°C for 1-7 days.
ORGANISM ATCC™ RECOVERY GELATINASE
Escherichia coli 25922 Good –
Staphylococcus aureus 25923 Good +
403
Cultural Response
Difco™ OF Basal Medium
Prepare the medium per label directions without (plain) and with 1% dex-
trose. Inoculate tubes in duplicate with fresh cultures using an inoculating
needle and add an overlay of mineral oil to one set of tubes. Incubate at
35 ± 2°C for 18-48 hours.
PLAIN PLAIN WITH DEXTROSE WITH DEXTROSE
ORGANISM ATCC™ OPEN CLOSED OPEN CLOSED
Uninoculated Acinetobacter Escherichia Pseudomonas
Acinetobacter Tube calcoaceticus coli aeruginosa
ATCC™ 19606 ATCC™ 25922 ATCC™ 27853
calcoaceticus 19606 K K A K
Tubes above are closed, with Dextrose
Enterobacter
aerogenes 13048 K K A, G A, G
Escherichia coli 25922 K K A, G A, G
Pseudomonas
aeruginosa 27853 K K A K
Shigella flexneri 12022 K K A A
K = alkaline reaction, green medium
A = acid reaction, yellow medium
G = gas production
404
OFPBL Agar
(See PC Agar)
405
Oatmeal Agar
Intended Use Formula
Oatmeal Agar is used for cultivating fungi, particularly for Difco™ Oatmeal Agar
macrospore formation. Approximate Formula* Per Liter
Oatmeal..................................................................... 60.0 g
Summary and Explanation
Agar.......................................................................... 12.5 g
*Adjusted and/or supplemented as required to meet performance criteria.
Fungi are extremely successful organisms, as evidenced by their
ubiquity in nature. Of the estimated 250,000 species, fewer than Directions for Preparation from
150 are known primary pathogens of humans.1 Dehydrated Product
Identification and classification of fungi is primarily based on the 1. Suspend 72.5 g of the powder in 1 L of purified water. Mix
morphologic differences in their reproductive structures.2 Fungi thoroughly.
reproduce by producing spores.2 Large, multi-celled spores are 2. Heat with frequent agitation and boil for 1 minute to
called macroconidia, macroaleuriospores or macrospores and completely dissolve the powder.
are produced by aerial sporulation.2 3. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using
The detection of fungi is a great concern in the pharmaceutical,
stable, typical control cultures.
food and cosmetic industry.
407
Directions for Preparation from Difco™ Orange Serum Broth Concentrate 10×
Dehydrated Product Orange Serum Broth Concentrate 10× diluted to single-strength
BBL™ Orange Serum Agar is used for small samples to initiate growth.
1. Suspend 45 g of the powder in 1 L of purified water. Mix
thoroughly. Expected Results
BBL™ Orange Serum Agar
2. Heat with frequent agitation and boil for 1 minute to
completely dissolve the powder. Record colony morphology for each type of growth.
3. Dispense in quantities under 50 mL and autoclave at 121°C Difco™ Orange Serum Broth Concentrate 10×
for 10 minutes. For larger quantities, increase the autoclave Turbidity indicates growth.
time. Avoid overheating with consequent darkening and poor
solidification. Limitations of the Procedure
4. Test samples of the finished product for performance using 1. Orange Serum Agar is not a differential medium. Perform
stable, typical control cultures. microscopic examination and biochemical tests to identify
Difco™ Orange Serum Broth Concentrate 10× isolates to genus and species if necessary.
1. To prepare the single-strength medium, aseptically add 2. If Orange Serum Agar is divided into aliquots and
100 mL of Orange Serum Broth Concentrate 10× to allowed to solidify, remelt only once. Repeated heating may
900 mL sterile purified water and mix thoroughly. produce a softer medium.
2. Aseptically dispense 10 mL amounts into sterile test tubes.
References
1. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
Procedure 4th ed. American Public Health Association, Washington, D.C.
2. Hays. 1951. Proc. Fla. State Hortic. Soc. 54:135.
BBL™ Orange Serum Agar 3. Murdock, Folinazzo and Troy. 1952. Food Technol. 6:181.
4. Stevens. 1954. Food Technol. 8:88.
1. For the plate count method, prepare serial 10-fold dilutions 5. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol.
1. Williams & Wilkins, Baltimore, Md.
of the test material.
2. Add 1 mL of test sample to a sterile Petri dish.
3. Add 18-20 mL of molten agar (cooled to 45-50°C) and swirl
plate gently to mix well.
4. Allow to solidify before incubating at 30°C for 48 hours.
Plates can be held up to 5 days.
408
Procedure
1. Prepare the inoculum by suspending several well-isolated
colonies of the S. aureus test isolate from an 18- to 24-hour
plate culture into a tube of suitable broth medium, such
as Trypticase™ Soy Broth and adjust the turbidity to a 0.5
McFarland turbidity standard, or use the BBL™ Prompt™
inoculation system.
2. Spot inoculate with 10 µL of test suspension using micro-
pipette.
3. Alternatively, saturate a cotton swab with the test suspen-
sion and gently press out excess fluid against the inner wall
of the tube. Streak plate by drawing swab over an approxi-
mately 1 inch (2.54 cm) area.
409
4. Include a Trypticase Soy Agar with 5% Sheep Blood (TSA the heterogeneous resistant population, is improved with
II) plate as a nonselective growth control. the larger inoculum obtained by using a micropipette and
5. The test and control plates may be divided into several inoculating the plate with 10 µL.9
wedge-shaped sectors by marking the bottom of the plate. 3. Any isolate that grows on this medium should be tested
Several isolates may be tested on each plate. However, use quantitatively by broth or agar dilution to confirm oxacillin
and incubate each plate only once. DO NOT REUSE AND resistance and also resistance to other antimicrobial agents
REINCUBATE a BBL Oxacillin Screen Agar plate. that are characteristic of MRSA, such as chloramphenicol,
6. Incubate plates at 30-35°C for a full 24 hours. Do not clindamycin, erythromycin, gentamicin and tetracycline.
exceed 35°C. 4. The use of Oxacillin Screen Agar for the detection of
methicillin/oxacillin resistant coagulase-negative staphylo-
Expected Results cocci is not recommended.7
Following incubation, observe plates for growth. Any growth,
even one colony, indicates that the isolate is methicillin (oxacillin) References
resistant. No growth indicates that the organism is susceptible 1. Chain, Florey and Jennings. 1949. In Florey, Chain, Heatley, Jennings, Sanders, Abraham and Florey
(ed.), Antibiotics, vol. II. Oxford University Press, London.
to PRPs (methicillin, nafcillin and oxacillin). Isolates that grow 2. Barrett, McGehee and Finland. 1968. N. Engl. J. Med. 279:444.
3. Swenson, Patel and Jorgensen. 2007. In Murray, Baron, Jorgensen, Landry and Pfaller (ed.), Manual
on Oxacillin Screen Agar should be reported as resistant to all of clinical microbiology, 9th ed. American Society for Microbiology, Washington, D.C.
4. Leitch and Boonlayangoor. 1994. In Isenberg (ed.), Clinical microbiology procedures manual, vol. 1
β-lactam antimicrobial agents, including β-lactam/ β-lactamase (suppl. 1). American Society for Microbiology, Washington, D.C.
5. Harberberger, Kallen, Driscoll and Wallace. 1998. Lab. Med. 29:302.
inhibitor combinations and cephalosporins. 6. Clinical and Laboratory Standards Institute. 2006. Approved standard: M2-A9. Performance standards
for antimicrobial disk susceptibility tests, 9th ed. CLSI, Wayne, Pa.
7. Clinical and Laboratory Standards Institute. 2008. Disk diffusion supplemental tables, M100-S18
Limitations of the Procedure (M2). CLSI, Wayne, Pa.
8. Clinical and Laboratory Standards Institute. 2008. MIC testing supplemental tables: M100-S18 (M7).
1. Occasionally, S. aureus isolates with borderline resistant CLSI, Wayne, Pa.
9. Data on file, BD Diagnostics.
MICs may not grow within 24 hours. It is recommended
that any equivocal results demonstrated on the screening
plate be confirmed with a standard MIC test.
Availability
BBL™ Oxacillin Screen Agar
2. In-house studies have shown that there is a difference
BS12 CLSI CMPH2 MCM9
in inoculum size between inoculating with 10 µL of the
United States and Canada
test suspension using a micropipette and inoculating the Cat. No. 221952 Prepared Plates – Pkg. of 10*
plate with a swab. The likelihood of the emergence of the Europe
resistant sub-population is greater in a large population Cat. No. 254570 Prepared Plates – Pkg. of 10*
of bacterial cells. Detection of resistance, especially with *Store at 2-8°C.
410
Identity Specifications
Difco™ Oxford Medium Base
Dehydrated Appearance: Tan, free-flowing, homogeneous (may contain
small dark particles).
Solution: 5.75% solution, soluble in purified water upon
boiling. Solution is medium amber, slightly to
moderately opalescent with a blue ring at the
surface of the liquid.
Prepared Appearance: Light to medium amber, very slightly to slightly
opalescent. O
Reaction of 5.75%
Solution at 25°C: pH 7.2 ± 0.2 P
Difco™ Modified Oxford Antimicrobic Supplement
Appearance: White cake may be broken; colorless solution with
a pale yellow tint upon rehydration.
Cultural Response
Difco™ Oxford Medium or Modified Oxford Medium
Prepare the medium with corresponding supplement. Inoculate and incubate
at 35 ± 2°C for 18-48 hours.
Inoculum Recovery on Recovery on Modified
Organism ATCC™ CFU Oxford Medium Oxford Medium
Enterococcus 29212 103-2×103 Marked to Marked to
faecalis complete inhibition complete inhibition
Escherichia coli 25922 103-2×103 Marked to Marked to
complete inhibition complete inhibition
Listeria 19114 102-103 Good at Good at
monocytogenes 40-48 hours, 40-48 hours,
black colonies black colonies
Identification of Listeria is based on successful isolation of most widely recognized antimicrobial agent combinations are
the organism, biochemical characterization and serological the Oxford Medium formulation11 and the Modified Oxford
confirmation. Medium formulation.2 The Oxford Medium formulation
contains cycloheximide, colistin sulfate, acriflavine, cefotetan and
Oxford Medium Base is prepared according to the formulation
fosfomycin. The Modified Oxford Medium formulation contains
of Curtis et al.11 who originally described the medium and its use
moxalactam and colistin methane sulfonate or colistin sulfate
in the selective isolation of Listeria from mixed cultures.
(available as Modified Oxford Antimicrobic Supplement).
Principles of the Procedure Modified Oxford Medium is recommended for isolating and
Peptones and beef heart digest provide nitrogen, carbon, amino identifying Listeria monocytogenes from processed meat and
acids and vitamins. Agar is the solidifying agent. Sodium poultry products.2 Oxford Medium is recommended for isolating
chloride maintains the osmotic balance. Listeria from enrichment broth cultures.13
Ferric ammonium citrate aids in the differentiation of Listeria
spp. Since all Listeria spp. hydrolyze esculin, the addition
Formulae
Difco™ Oxford Medium Base
of ferric ions to the medium will detect the reaction. A black-
Approximate Formula* Per Liter
ening of the colony and surrounding medium in cultures Pancreatic Digest of Casein.......................................... 8.9 g
containing esculin-hydrolyzing bacteria results from the Proteose Peptone No. 3................................................ 4.4 g
formation of 6,7-dihydroxycoumarin which reacts with the Yeast Extract................................................................ 4.4 g
Tryptic Digest of Beef Heart.......................................... 2.7 g
ferric ions.12 Starch.......................................................................... 0.9 g
Sodium Chloride.......................................................... 4.4 g
Selectivity is provided by the presence of lithium chloride in the
Esculin......................................................................... 1.0 g
formula. The high salt tolerance of Listeria is used as a means Ferric Ammonium Citrate............................................. 0.5 g
to markedly inhibit growth of enterococci. Lithium Chloride........................................................ 15.0 g
Agar.......................................................................... 15.3 g
Selectivity is increased by adding various antimicrobial agents
to the base. Incorporating these agents into Oxford Medium
Base will completely inhibit gram-negative organisms and most
gram-positive organisms after 24 hours of incubation. The
411
M-PA-C Agar
Intended Use Many of the membrane filter media used for the recovery of
M-PA-C Agar is used for the selective recovery and enumeration P. aeruginosa lacked specificity and were of limited value when
of Pseudomonas aeruginosa from water. large heterogeneous microbial flora were present in the water
samples. Levin and Cabelli devised M-PA Agar as a selective
Summary and Explanation membrane filter medium for P. aeruginosa.1 This formulation
A variety of methods have been used for the enumeration of incorporated four antimicrobics, kanamycin, nalidixic acid,
P. aeruginosa from water samples, some of which have sulfapyridine and cycloheximide, which render the medium
been more widely accepted than others. The most-probable- moderately selective. This original formulation was modified
number (MPN) procedures result in satisfactory recovery by raising the pH2 and altering the content or concentration
levels of P. aeruginosa, but are not usable for the testing of of ingredients.3 The resulting medium was designated M-PA-B
large-volume water samples and lack precision. These two Agar.
deficiencies are eliminated in membrane filter (MF) techniques.
412
References
Directions for Preparation from 1. Levin and Cabelli. 1972. Appl. Microbiol. 24:864.
Dehydrated Product 2.
3.
Carson, Peterson, Favero, Doto, Collins and Levin. 1975. Appl. Microbiol. 30:935.
Dutka and Kwan. 1977. Appl. Environ. Microbiol. 33:240.
1. Suspend 35 g of the powder in 1 L of purified water. Mix 4.
5.
Brodsky and Ciebin. 1978. Appl. Environ. Microbiol. 36:26.
Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
thoroughly. 21st ed., online. American Public Health Association, Washington, D.C.
6. Estevez. 1984. Lab. Med. 15:258.
2. Heat with frequent agitation and boil for 1 minute to
completely dissolve the powder. DO NOT AUTOCLAVE. Availability
3. Cool to 45-50°C and pour into sterile 50-mm Petri dishes. BBL™ M-PA-C Agar
Use the medium within 1 week after preparation. SMWW
4. Test samples of the finished product for performance using Cat. No. 298153 Dehydrated – 500 g
stable, typical control cultures.
Procedure
Following filtration of the water sample through a sterile
47 mm, 0.45 µm gridded filter, place the membrane filter on
the surface of a plate of M-PA-C Agar taking care to avoid
the entrapment of bubbles between the agar and filter surface.
Incubate for 72 hours at 41.5 ± 0.5°C in an aerobic atmo-
sphere. Consult the standard method for additional information
regarding the M-PA-C membrane filter technique.5
413
Identity Specifications
Difco™ PALCAM Medium Base
Dehydrated Appearance: Pink, free-flowing, homogeneous.
Solution: 6.8% solution, soluble in purified water upon
boiling. Solution is dark red, slightly opalescent.
Prepared Appearance: Medium red, very slightly to slightly opalescent
with slight precipitate.
Reaction of 6.8%
Solution at 25°C: pH 7.2 ± 0.2
Difco™ PALCAM Antimicrobic Supplement
Lyophilized Appearance: White, free-flowing, homogeneous powder.
Rehydrated Appearance: Colorless solution.
Cultural Response
Difco™ PALCAM Medium Base
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C
for 48 hours in a microaerophilic environment.
INOCULUM ESCULIN
ORGANISM ATCC™ CFU RECOVERY REACTION
Enterococcus faecalis 29212 103-2 × 103 Inhibition –
Escherichia coli 25922 10 -2 × 10 Inhibition
3 3
–
Listeria monocytogenes 19114 100-300 Good +
Staphylococcus aureus 25923 103-2 × 103 Inhibition –
414
Formulae Procedure
Difco™ PALCAM Medium Base Consult appropriate references2-8 and follow applicable standard
Approximate Formula* Per Liter methods. Inoculate incubated enrichment broth or screened food
Columbia Blood Agar Base......................................... 39.0 g sample particle onto PALCAM Medium and streak for isolation.
Pancreatic Digest of Casein..................... 10.0 g
Proteose Peptone No. 3............................ 5.0 g Incubate plates at 35°C for 24-48 hours under aerobic or micro-
Yeast Extract............................................. 5.0 g aerophilic conditions in an inverted position (agar side up).
Beef Heart, Infusion from 500 g................ 3.0 g
Corn Starch.............................................. 1.0 g
Sodium Chloride....................................... 5.0 g Expected Results
Agar....................................................... 15.0 g On PALCAM Medium, colonies of Listeria appear gray-green
Mannitol.................................................................... 10.0 g with a black precipitate. Confirmation of the presence of
Dextrose...................................................................... 0.5 g
Listeria is made following subculture onto appropriate media O
Esculin......................................................................... 1.0 g
Ferric Ammonium Citrate............................................. 0.5 g
Lithium Chloride........................................................ 15.0 g
and biochemical/serological identification.2-8 Colonies of man- P
nitol-fermenting organisms such as staphylococci, which may
Phenol Red.................................................................. 0.08 g
Acriflavine HCl............................................................. 5.0 mg
grow on this medium, appear yellow with a yellow halo.
Polymyxin B Sulfate...................................................... 0.01 g
Agar ............................................................................ 2.0 g References
1. Van Netten, Perales, Van de Moosalijk, Curtis, and Mossel. 1989. Int. J. Food Microbiol. 8:299.
Difco™ PALCAM Antimicrobic Supplement 2. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. Chapter 10:
Formula Per 10 mL Vial Detection and enumeration of Listeria monocytogenes in foods (January 2003). AOAC International,
Gaithersburg, Md.
Ceftazidime............................................................... 40.0 mg 3. Downes and Ito (eds.). 2001. Compendium of methods for the microbiological examination of foods,
*Adjusted and/or supplemented as required to meet performance criteria. 4th ed. American Public Health Association, Washington, D.C.
4. Pagotto, Daley, Farber, and Warburton. 2001. Isolation of Listeria monocytogenes from all food and
environmental samples. Health Products and Food Branch Ottawa, MFHPB-30. Published on the
Directions for Preparation from Food Directorate (Health Canada’s) website at <www.hc-sc.gc.ca/food-aliment>.
5. Pagotto, Daley and Farber. 2002. Enumeration of Listeria monocytogenes in foods. Health Products
Dehydrated Product and Food Branch Ottawa, MFLP-74. Published on the Food Directorate (Health Canada’s) website
at <www.hc-sc.gc.ca/food-aliment>.
Difco™ PALCAM Medium Base 6. International Organization for Standardization. 1996. Microbiology of food and animal feeding stuffs
– Horizontal method for the detection and enumeration of Listeria monocytogenes; Part 1: Detection
1. Suspend 68 g of the powder in 1 L of purified water. Mix method. ISO 11290-1. International Organization for Standardization, Geneva, Switzerland.
7. International Organization for Standardization. 2004. Microbiology of food and animal feeding stuffs
thoroughly. – Horizontal method for the detection and enumeration of Listeria monocytogenes; Part 1: Detection
method. Amendment 1: Modification of the isolation media and the haemolysis test, and inclusion of
2. Heat with frequent agitation and boil for 1 minute to com- precision data. ISO 11290-1, Amendment 1. International Organization for Standardization, Geneva,
pletely dissolve the powder. Switzerland.
8. Henning, Flowers, Reiser, and Ryser. 2004. Pathogens in milk and milk products. In Wehr and Frank
3. Autoclave at 121°C for 15 minutes. Cool to 45-50°C. (eds.), Standard methods for the examination of dairy products, 17th ed. American Public Health
Association, Washington, D.C.
4. Aseptically add 2 mL rehydrated PALCAM Antimicrobic
Supplement. Mix well. Availability
5. Test samples of the finished product for performance using Difco™ PALCAM Medium Base
stable, typical control cultures. BAM CCAM COMPF ISO SMD
6. Store the prepared medium at 2-8°C. Cat. No. 263620 Dehydrated – 500 g
263610 Dehydrated – 2 kg
Difco™ PALCAM Antimicrobic Supplement
1. Aseptically add 10 mL sterile purified water to the vial. Difco™ PALCAM Antimicrobic Supplement
BAM CCAM COMPF ISO SMD
2. Shake to dissolve the contents.
Cat. No. 263710 Vial – 3 × 10 mL*
3. Upon rehydration, Difco PALCAM Antimicrobic Supplement
Europe
is stable for 1 month when stored at 2-8°C. Cat. No. 254539 Prepared Plates (complete) – Pkg. of 20*
Sample Collection and Handling *Store at 2-8°C.
isolates, such as Pseudomonas aeruginosa, Escherichia coli and PC Agar contains the pH indicator phenol red to facilitate
Staphylococcus aureus, overgrow the slower-growing colonies detection of B. cepacia. Alkaline end products from the
of B. cepacia and mask its presence. metabolism of pyruvate raise the pH of the medium, causing
the color of the indicator to change from light orange to pink
Gillian et al. developed PC Agar for improved recovery of
or pink-red in the area of growth. In areas of heavy growth of
B. cepacia.2 Crystal violet, bile salts and two antimicrobial
B. cepacia, the pink color intensifies.
agents are used as selective agents. Phenol red facilitates
detection of B. cepacia by a color change in the medium. They OFPBL Agar contains the pH indicator bromthymol blue to
reported isolating B. cepacia on PC Agar from respiratory facilitate detection of B. cepacia. Acid end products from the
secretions of 35 CF patients, but isolated the organism from metabolism of lactose lower the pH of the medium resulting
only 21 patients on MacConkey Agar.2 in a yellow color change. B. cepacia colonies will also have a
yellow color.
Welch et al. developed a differential but less selective medium
for the recovery of B. cepacia.4,5 This medium, OFPBL Agar,
is OF (oxidation-fermentation) basal medium supplemented
Procedure
Use standard procedures to obtain isolated colonies from
with polymyxin B, bacitracin, lactose and agar. The indicator,
specimens. Incubate the plates in an inverted position (agar-
bromthymol blue, aids in the detection of B. cepacia isolates
side up) at 30-35°C for a minimum of 4 days to allow
through a color change in the medium. These investigators
sufficient time for colony development and for the color of the
reported isolating B. cepacia on OFPBL Agar from 58 CF
indicator change.6,7
patients, while only isolating this organism from 19 patients on
MacConkey Agar.4
Expected Results
Typical colonies of B. cepacia on PC Agar are grayish-white with
Principles of the Procedure
a pink-red zone in the surrounding medium.2 Typical colonies of
These media provide a variety of enzymatic digests of
B. cepacia on OFPBL Agar are yellow with yellow zones in the
proteinaceous substrates, inorganic salts and other nutrients to
surrounding medium.
satisfy the nutritional requirements of these organisms.
Selective agents are incorporated to improve the recovery Limitation of the Procedure
of B. cepacia by inhibiting common contaminants. PC agar Organisms other than B. cepacia may also grow on PC Agar
incorporates crystal violet to inhibit gram-positive cocci, and produce alkaline end products that cause the medium to
especially enterococci and staphylococci, bile salts to inhibit become pink. Other organisms, e.g., B. gladioli, may also grow
most gram-positive cocci other than enterococci, and ticarcillin on OFPBL Agar and resemble B. cepacia (yellow colonies).
and polymyxin B to inhibit gram-negative bacilli. OFPBL Agar Therefore, these media should not be used as the sole method
incorporates polymyxin B to inhibit gram-negative flora, while of identification of B. cepacia.8
bacitracin inhibits the gram-positive organisms and Neisseria.4
416
References Availability
1. Gilligan and Whittier. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical BBL™ PC Agar
microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
2. Gilligan, Gage, Bradshaw, Schidlow and DeCicco. 1985. J. Clin. Microbiol. 22:5. BS12 CMPH2 MCM9
3. Gilligan and Schidlow. 1984. Clin. Microbiol. Newsl. 6:42.
4. Welch, Muszynski, Pai, Marcon, Hribar, Gilligan, Matsen, Ahlin, Hilman and Chartrand. 1987. Cat. No. 297755 Prepared Plates – Pkg. of 20*
J. Clin. Microbiol. 25:1730.
5. Carson, Tablan, Cusick, Jarvis, Favero and Bland. 1988. J. Clin. Microbiol. 26:2096. BBL™ OFPBL Agar
6. MacDonald, Gilligan, Welch, Reller and Menegus. 1994. In Consensus conference: microbiology and
infectious disease in cystic fibrosis, vol. 5:1. Cystic Fibrosis Foundation, Washington, D.C. BS12 MCM9
7. Gilligan. 1996. Clin. Microbiol. Newsl. 18:83. United States and Canada
8. Christenson, Welch, Mukwaya, Muszynski, Weaver and Brenner. 1989. J. Clin. Microbiol. 27:270.
Cat. No. 299970 Prepared Plates – Pkg. of 20*
Europe
Cat. No. 254481 Prepared Plates – Pkg. of 20*
*Store at 2-8°C.
O
P
PPLO Media (Mycoplasma Media)
PPLO Agar (Mycoplasma Agar Base)
PPLO Broth (Mycoplasma Broth Base)
Mycoplasma Broth Base (Frey) • Mycoplasma
Supplement • Mycoplasma Enrichment w/o Penicillin
Intended Use Principles of the Procedure
PPLO (Mycoplasma) agars and broths, when supplemented Meat digests, peptones, beef extract and yeast extract provide
with nutritive enrichments, are used for isolating and cultivating the nitrogen, vitamins, amino acids and carbon in these media.
Mycoplasma. Mycoplasma Broth Base (Frey) is used for the Sodium chloride maintains the osmotic balance of these
cultivation of avian mycoplasmas. formulations. Agar, the solidifying agent, is used in PPLO
(Mycoplasma) Agar at a concentration slightly reduced from
Summary and Explanation usual to ensure formation of the largest possible colonies
Members of the class Mollicutes, Mycoplasma was first recog-
because the organisms grow into the agar with only slight
nized from a case of pleuropneumonia in a cow.1 The organism
was designated “pleuropneumonia-like organism,” or PPLO.1 surface growth.13
Although some species are normal human respiratory tract The base media are supplemented with Mycoplasma Supple-
flora, M. pneumoniae is a major cause of respiratory disease ment or Mycoplasma Enrichment w/o Penicillin because
(primary atypical pneumonia, sometimes called “walking
Mycoplasma spp. are fastidious in their growth require-
pneumonia”).1 M. hominis, M. genitalium and Ureaplasma
urealyticum are important colonizers (and possible pathogens) ments.14
of the human genital tract.1 Mycoplasma Supplement contains fresh yeast extract and
PPLO (Mycoplasma) Agar was described by Morton, Smith horse serum. Yeast extract provides the preformed nucleic acid
and Leberman.2 It was used in a study of the growth require- precursors that are required by Mycoplasma spp.14 Horse
ments of Mycoplasma,3 along with the identification and serum supplies cholesterol, a growth stimulant.14
cultivation of this organism.4-6
Mycoplasma Enrichment without Penicillin is a selective
PPLO (Mycoplasma) Broth (without crystal violet) is prepared enrichment containing the inhibitor thallium acetate, to
according to the formula described by Morton and Lecci.3 which a penicillin of choice (penicillin G or a broad-spectrum
Crystal violet is omitted from this formula due to its inhibitory
semisynthetic penicillin) can be added at the time of use to make
action on some Mycoplasma. It has been used for the cultivation
of Mycoplasma for research studies.7,8 it selective against gram-positive and gram-negative bacteria.
417
Pancreatic Digest of Casein.......................................... 7.5 g Prepare the medium per label directions. Inoculate and incubate at
Papaic Digest of Soybean Meal..................................... 2.5 g 35 ± 2°C under 3-5% CO2 for 7 days. Subculture to Mycoplasma Agar
Yeast Extract................................................................ 5.0 g plates and incubate aerobically at 35 ± 2°C for 7 days. Examine plates
Sodium Chloride.......................................................... 5.0 g microscopically for growth.
Potassium Chloride...................................................... 0.4 g Organism ATCC™ INOCULUM CFU recovery
Magnesium Sulfate...................................................... 0.2 g
Disodium Phosphate.................................................... 1.6 g Escherichia coli 25922 102-103 Growth in a dilution
containing 103 CFU/mL
Monopotassium Phosphate.......................................... 0.1 g
Mycoplasma gallisepticum 19610 Undiluted Good
Mycoplasma synoviae 25204 Undiluted Good
418
Difco™ Mycoplasma Supplement 4. For recovery of M. synoviae, add 0.01% (w/v) nicotinamide
Approximate Formula* Per 30 mL Vial adenine dinucleotide (NAD) and 0.01% (w/v) L-cysteine
Yeast Extract................................................................ 0.09 g
Horse Serum.............................................................. 22.8 mL
HCl. Inactivated swine serum is preferred in place of horse
serum.
BBL™ Mycoplasma Enrichment without Penicillin
5. Test samples of the finished product for performance using
Approximate Formula* Per 30 mL Vial
Horse Serum.............................................................. 20.0 mL stable, typical control cultures.
Yeast Extract (fresh autolysate)................................... 10.0 mL
Difco™ Mycoplasma Supplement
Thallium Acetate........................................................ 50.0 mg
*Adjusted and/or supplemented as required to meet performance criteria.
BBL™ Mycoplasma Enrichment without Penicillin
1. Rehydrate with 30 mL of sterile purified water.
Directions for Preparation from
Dehydrated Product
2. Rotate gently to dissolve.
3. Add 30 mL (the contents of one vial) to 70 mL of sterile
O
Difco™ PPLO Agar medium base. P
Difco™ PPLO Broth 4. Dispense in plates or tubes as desired.
1. PPLO Agar: Suspend 35 g of the powder in 700 mL
of purified water. Mix thoroughly. Heat with frequent Procedure
agitation and boil for 1 minute to completely dissolve the Agar
powder. Inoculate the surface of plates containing the complete medium
PPLO Broth: Dissolve 21 g of the powder in 700 mL of by adding drops of liquid inoculum or by a swab-inoculation
purified water. Mix thoroughly. technique. Incubate plates at 35 ± 2°C for up to 21 days in a
2. Autoclave at 121°C for 15 minutes. Cool medium to 50- moist atmosphere containing 5-10% carbon dioxide or anaero-
60°C. bically if the presence of M. buccale, M. faucium, M. orale or
3. Aseptically add 300 mL Difco Mycoplasma Supplement to M. salivarium is suspected.13
the medium. Mix well.
Broth
4. Add selective agents if desired (i.e., thallium acetate or
penicillin). Test material, either solid or liquid, should be directly inoculated
5. Test samples of the finished product for performance using into the broth medium. For preparation of stock organism sus-
stable, typical control cultures. pensions, a block of agar culture can be added to the broth.
BBL™ Mycoplasma Agar Base Following incubation at 35 ± 2°C in a moist aerobic atmosphere
BBL™ Mycoplasma Broth Base containing 5-10% carbon dioxide or anaerobically, if appropri-
1. Mycoplasma Agar Base: Suspend 34 g of the powder in 1 L of ate,13 for various lengths of time, subculture aliquots of the broth
purified water. Mix thoroughly. Heat with frequent agitation to PPLO (Mycoplasma) Agar plates for visualization of typical
and boil for 1 minute to completely dissolve the powder. colonies. The broth usually does not become turbid enough to
Mycoplasma Broth Base: Suspend 20 g of the powder in confirm the presence of growth.
1 L of purified water. Mix thoroughly. Warm slightly to For a complete discussion of the isolation and identification of
completely dissolve the powder. Mycoplasma spp. from clinical specimens, refer to appropriate
2. Autoclave at 121°C for 15 minutes. procedures outlined in the references.13-15
3. Cool to 50°C and add enrichment. Recommended
enrichments include addition of 20 mL of horse serum and Expected Results
5 mL of specially prepared yeast extract11 to each 75 mL of Agar
cooled medium. PPLO colonies are round with a dense center and a less dense
4. For a selective medium inhibitory to bacteria, add 30 mL of periphery, giving a “fried egg” appearance on PPLO (Mycoplasma)
BBL Mycoplasma Enrichment without Penicillin to 70 mL Agar. Vacuoles, large bodies characteristic of Mycoplasma spp.,
of molten agar medium (50°C) or 70 mL of broth medium are seen in the periphery. Colonies vary in diameter from 10 to
and add sterile penicillin G to a final concentration of 500 500 microns (0.01-0.5 mm) and penetrate into the medium.
units/mL.
Broth
5. Test samples of the finished product for performance using
stable, typical control cultures. After subculture to plates of PPLO (Mycoplasma) Agar, positive
broth cultures produce colonies exhibiting the typical morphology;
BBL™ Mycoplasma Broth Base (Frey) i.e., “fried egg” appearance.
1. Dissolve 22.5 g of the powder in 1 L of purified water. Mix
thoroughly. Limitation of the Procedure
2. Autoclave at 121°C for 15 minutes. Thallium acetate can partially inhibit some mycoplasmas.13
3. Cool to 50°C and add 100 mL of sterile inactivated horse
serum. Mix thoroughly.
419
References Availability
1. Baron, Peterson and Finegold. 1994. Bailey & Scott’s diagnostic microbiology, 9th ed. Mosby-Year
Book, Inc. St. Louis, Mo.
Difco™ PPLO Agar (Mycoplasma Agar)
2. Morton, Smith and Leberman. 1951. Am. J. Syphilis Gonorrh. 35:361. Cat. No. 241210 Dehydrated – 500 g
3. Morton and Lecce. 1953. J. Bacteriol. 66:646.
4. Chanock, James, Fox, Turner, Mufso and Hayflick. 1962. Soc. Exp. Biol. Med. 110:884. BBL™ Mycoplasma Agar Base (PPLO Agar Base)
5. Craven, Wenzel, Calhoun, Hendley, Hamory and Gwaltney. 1976. J. Clin. Microbiol. 4:225.
6. Gregory and Cundy. 1970. Appl. Microbiol. 19:268. Cat. No. 211456 Dehydrated – 500 g
7. Adler and Da Massa. 1967. Appl. Microbiol. 15:245.
8. Leland, Lapworth, Jones and French. 1982. J. Clin. Microbiol. 16:709. Difco™ PPLO Broth (Mycoplasma Broth)
9. Frey, Hanson and Anderson. 1968. Am. J. Vet. Res. 29:2163.
10. Hayflick. 1965. Tex. Rep. Biol. Med. 23:285. Cat. No. 255420 Dehydrated – 500 g
11. Chanock, Hayflick and Barile. 1962. Proc. Nat. Acad. Science 48:41. 255410 Dehydrated – 10 kg
12. Hayflick. 1968. Personal communication.
13. Kenny. 1985. In Lennette, Balows, Hausler and Shadomy (ed.). Manual of clinical microbiology, 4th
ed. American Society for Microbiology, Washington, D.C.
BBL™ Mycoplasma Broth Base (PPLO Broth Base)
14. Waites and Taylor-Robinson. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.). Manual of Cat. No. 211458 Dehydrated – 500 g
clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
15. Isenberg (ed.). 1992. Clinical microbiology procedures handbook, vol. 1. American Society for
Microbiology, Washington, D.C.
BBL™ Mycoplasma Broth Base (Frey)
Cat. No. 212346 Dehydrated – 500 g
212347 Dehydrated – 5 lb (2.3 kg)
Difco™ Mycoplasma Supplement
Cat. No. 283610 Vial – 6 x 30 mL*
BBL™ Mycoplasma Enrichment w/o Penicillin
Cat. No. 212292 Vial – 10 x 30 mL*
*Store at 2-8°C.
420
Directions for Preparation from The standard curve is obtained by using calcium pantothenate
Dehydrated Product solution at levels of 0.0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08
1. Suspend 7.3 g of the powder in 100 mL of purified water. and 0.1 µg per assay tube (10 mL). Turbidimetric determinations
2. Heat with frequent agitation and boil for 2-3 minutes to are made after 18-24 hours incubation at 35-37°C. Construct
completely dissolve the powder. a standard curve and determine the concentration of the
3. Dispense in 5 mL amounts into tubes, evenly dispersing the unknown by interpolation from the standard curve.
precipitate. The concentration of pantothenic acid required for the prepa-
4. Add standard or test samples. ration of the standard curve may be prepared by dissolving
5. Adjust tube volume to 10 mL with purified water. 50 mg dried calcium pantothenate in a solution containing
6. Autoclave at 121°C for 10 minutes. approximately 500 mL purified water, 10 mL 0.2N acetic acid
and 100 mL 0.2N sodium acetate. Dilute to 1,150 mL with
O
Procedure additional water to make the calcium pantothenate concentra- P
Prepare stock cultures of L. plantarum ATCC 8014 in tripli- tion 43.47 µg per mL; one mL equals 40 µg pantothenic acid.
cate by stab inoculation of Lactobacilli Agar AOAC. Incubate
cultures for 18-24 hours at 35-37°C. Store the tubes at 2-8°C. This solution is diluted by adding 25 mL to a solution containing
Prepare a fresh stock culture every week. Do not use a culture 500 mL purified water, 10 mL 0.2N acetic acid and 100 mL
older than 1 week for this assay. 0.2N sodium acetate. Dilute to 1 liter with purified water to
make a stock solution containing 1.0 µg pantothenic acid per
Inoculum mL. The standard solution is made by diluting 2 mL of the stock
Subculture from a stock culture of Lactobacillus plantarum solution to 100 mL with purified water. This solution contains
ATCC 8014 to 10 mL of sterile single-strength Pantothenate 0.02 µg pantothenic acid per mL. Use 0.0, 0.5, 1.0, 1.5, 2.0, 2.5,
Assay Medium supplemented with 0.02 µg pantothenate. 3.0, 4.0 and 5.0 mL per assay tube. Prepare the stock solution
Incubate for 18-24 hours at 35-37°C. Centrifuge the cells fresh daily.
under aseptic conditions and decant the supernatant. Wash
the cells three times with 10 mL sterile 0.85% saline. After Expected Results
the third wash, resuspend the cells with sterile 0.85% saline 1. Prepare a standard concentration response curve by plotting
and adjust to a turbidity of 40-45% transmittance when read the response readings against the amount of standard in each
on a spectrophotometer at 660 nm. Aseptically inoculate each tube, disk or cup.
assay tube with one drop of the cell suspension. 2. Determine the amount of vitamin at each level of assay
Standard Curve solution by interpolation from the standard curve.
3. Calculate the concentration of vitamin in the sample from
It is essential that a standard curve be constructed each time an
the average of these values. Use only those values that do not
assay is run. Autoclave and incubation conditions can influence
vary more than ±10% from the average. Use the results only
the standard curve readings and cannot always be duplicated.
if two-thirds of the values do not vary more than ±10%.
421
Dehydrated Appearance: White to 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
Solution: 3.65% (single strength) or 7.3% (double other chemicals must be used. Glassware must be heated to
strength) solution,soluble in purified water 250°C for at least 1 hour to burn off any organic residues that
upon boiling for 2-3 minutes. Single-strength
solution is very light amber, clear, may have a
might be present. Take precautions to keep sterilization and
slight precipitate. cooling conditions uniform throughout the assay.
Prepared Appearance: (Single strength) light amber, clear, may have
a very slight precipitate. Directions for Preparation from
Reaction of 3.65% Dehydrated Product
Solution at 25°C: pH 6.7 ± 0.1
1. Suspend 7.3 g of the powder in 100 mL of purified water.
Cultural Response 2. Heat with frequent agitation and boil for 2-3 minutes to
Difco™ Pantothenate Medium AOAC completely dissolve the powder.
Prepare the medium per label directions. The medium supports the 3. Dispense 5 mL amounts into tubes, evenly dispersing the
growth of Lactobacillus plantarum ATCC™ 8014 when prepared in single precipitate.
strength and supplemented with pantothenic acid. The medium should 4. Add standard or test samples.
produce a standard curve when tested with a pantothenic acid reference
standard at 0.0 to 0.05 µg per 10 mL. Incubate tubes with caps loosened 5. Adjust the tube volume to 10 mL.
at 35-37°C for 18-24 hours. Read the percent transmittance using a 6. Autoclave at 121°C for 10 minutes.
spectrophotometer at 660 nm.
422
Bacto™ Peptone
Intended Use Researchers uncovered estrogenic activity associated with Bacto
Bacto Peptone is used as an organic nitrogen source in micro- Peptone when including the peptone in medium for culture of
biological culture media for cultivation of a variety of bacteria yeast. The estrone contained in Bacto Peptone was converted
and fungi. to estrodiol by Saccharomyces cerevisiae. These findings suggest
that adding estrogens to a medium containing Bacto Peptone
Summary and Explanation for studies of estrodiol production by yeast may confound
Bacto Peptone was first introduced in 1914 and became the results.6,7
standard peptone for the preparation of bacteriological culture Several media containing peptone are specified in standard
media. Bacto Peptone is used as an organic nitrogen source in methods for multiple applications.8-15
microbiological culture media for cultivation of a variety of
bacteria and fungi. For example, Iwanaga et al.1 utilized Bacto Principles of the Procedure
Peptone for production of cholera toxin by Vibrio cholerae O1 Bacto Peptone is an enzymatic digest of animal protein. Bacto
El Tor. Benkerroum et al.2 reported using Bacto Peptone in a Peptone contains nitrogen in a form that is readily available for
selective medium developed for isolating Leuconostoc sp. from bacterial growth. Bacto Peptone has a high peptone and amino
food samples. Bacto Peptone was used in a culture medium for acid content, with only a negligible quantity of proteoses and
two anaerobic, extremely thermophilic Archaea, Thermococcus more complex nitrogenous constituents.
celer and Pyrococcus woesei, by Blamey et al.3
Bacto Peptone has also been utilized as a nitrogen source Typical Analysis
in cell culture media formulations. Taylor et al.4 used Bacto Refer to Product Tables in the Reference Guide section of this
Peptone to supplement serum-free medium for several mammalian manual.
cell lines and reported that the solubility of Bacto Peptone is very
good at 10 g/100 mL water. Sakoda and Fukusho5 also utilized Directions for Preparation from
Bacto Peptone in serum-free culture medium for maintaining Dehydrated Product
porcine kidney epithelial cells. Bacto Peptone is also useful as a Refer to the final concentration of Bacto Peptone in the formula
supplement in cell culture with serum. of the medium being prepared. Add appropriate product as
required.
424
Cultural Response
O
Biochemical Reactions
Bacto™ Peptone
P
Prepare a sterile solution of Bacto Peptone as directed below. Adjust final pH to 7.2-7.4. Inoculate and incubate at 35 ± 2°C for 18-48 hours.
TEST TEST SOLUTION ORGANISM ATCC™ INOCULUM CFU RESULT
Fermentable Carbohydrates 2% Escherichia coli 25922 ~10
7
Negative
Indole Production 0.1% Escherichia coli 29552 0.1 mL, undiluted Positive
Acetylmethylcarbinol 0.1% with Enterobacter aerogenes 13048 0.1 mL, undiluted Positive
Production 0.5% dextrose
Hydrogen Sulfide 1% Salmonella enterica subsp. enterica 14028 0.1 mL, undiluted Positive
Production serotype Typhimurium
Growth Response
Bacto™ Peptone
Prepare a sterile solution with 2% Bacto Peptone, 0.5% sodium chloride and 1.5% agar. Adjust final pH to 7.2-7.4. Inoculate and incubate plates at 35 ± 2°C for
18-48 hours.
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Escherichia coli 25922 30-300 Good
Staphylococcus aureus 25923 30-300 Good
Procedure Availability
See appropriate references for specific procedures using Bacto Bacto™ Peptone
Peptone. AOAC BAM COMPF EP EPA SMD SMWW USDA USP
Cat. No. 211677 Dehydrated – 500 g
Expected Results
211820 Dehydrated – 2 kg
211830 Dehydrated – 10 kg
Refer to appropriate references and procedures for results.
References
1. Iwanaga, Yamamoto, Higa, Ichinose, Nakasone and Tanabe. 1986. Microbiol. Immunol. 30:1075.
2. Benkerroum, Misbah, Sandine and Elaraki. 1993. Appl. Environ. Microbiol. 59:607.
3. Blamey, Chiong, Lopez and Smith. 1999. J. Microbiol. Methods. 38:169.
4. Taylor, Dworkin, Pumper and Evans. 1972. Exp. Cell Res. 74:275.
5. Sakoda and Fukusho. 1998. In Vitro Cell. Dev. Biol. Anim. 34:53.
6. Feldman and Krishnan. 1995. Environ. Health Perspect. 103 Suppl 7:129.
7. Miller, Bottema, Stathis, Tokes and Feldman. 1986. Endocrinology. 119:1362.
8. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
9. Horowitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
International, Gaithersburg, Md.
10. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C.
11. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy products, 17th ed.
American Public Health Association, Washington, D.C.
12. U.S. Environmental Protection Agency. 2000. Improved enumeration methods for the recreational
water quality indicators: Enterococci and Escherichia coli. EPA-821/R-97/004. Office of Water,
Washington, D.C.
13. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
tional, Gaithersburg, Md.
14. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The national
formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc., Rockville,
Md.
15. U.S. Department of Agriculture. Microbiology laboratory guidebook, online. Food Safety and Inspec-
tion Service, USDA, Washington, D.C.
425
Procedure
1. Obtain a pure culture of a test organism. Pick the center of
a single colony with an inoculating needle.
2. Inoculate a tube of Peptone Iron Agar by the stab method.
Stab the needle to within 1/4 to 1/2 inch of the bottom. With-
draw the needle following the initial line of inoculation.
3. Incubate tubes at 35 ± 2°C for 18-48 hours.
4. Read tubes for growth and hydrogen sulfide production.
Uninoculated Escherichia coli Salmonella
Tube ATCC™ 25922 Enteritidis
ATCC™ 13076
Expected Results
Any blackening of the medium along the line of inoculation or
throughout the butt indicates hydrogen sulfide production.
For a complete discussion of the identification of coliform bacteria,
refer to appropriate references.4-6
426
References Availability
1. Levine, Vaughn, Epstein and Anderson. 1932. Proc. Soc. Exp. Biol. Med. 29:1022.
2. Levine, Epstein and Vaughn. 1934. Am. J. Public Health 24:505.
Difco™ Peptone Iron Agar
3. Tittsler and Sandholzer. 1937. Am. J. Public Health 27:1240. Cat. No. 289100 Dehydrated – 500 g
4. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
American Society for Microbiology, Washington, D.C.
5. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
6. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
Peptone Water
O
Intended Use
Peptone Water is used for cultivating nonfastidious organisms,
Formula
Difco™ Peptone Water
P
for studying carbohydrate fermentation patterns and for Approximate Formula* Per Liter
performing the indole test. Peptone..................................................................... 10.0 g
Sodium Chloride.......................................................... 5.0 g
*Adjusted and/or supplemented as required to meet performance criteria.
Summary and Explanation
The formulation of Peptone Water makes it useful for cultivating Directions for Preparation from
nonfastidious organisms.1 This nonselective medium has been Dehydrated Product
used as a basal medium for biochemical tests such as carbohy- 1. Dissolve 15 g of the powder in 1 L of purified water.
drate fermentation patterns and production of indole.1,2 2. Warm slightly with frequent agitation to completely
dissolve the powder.
Principles of the Procedure 3. Autoclave at 121°C for 15 minutes.
Peptone Water contains peptone as a source of carbon, nitrogen, 4. Test samples of the finished product for performance using
vitamins and minerals. Sodium chloride maintains the osmotic stable, typical control cultures.
balance of the medium.
For Determining Carbohydrate Fermentation Patterns
1. Add 1.8 mL 1% phenol red solution to 1 liter rehydrated
User Quality Control
Peptone Water. Mix thoroughly.
Identity Specifications 2. Dispense into test tubes containing inverted Durham vials.
Difco™ Peptone Water 3. Autoclave at 121°C for 15 minutes.
Dehydrated Appearance: Cream-white to light tan, free-flowing, homoge- 4. Aseptically add sufficient sterile carbohydrate solution
neous.
to yield a 1% final concentration. Rotate each tube to
Solution: 1.5% solution, soluble in purified water upon
warming with frequent agitation. Solution is light thoroughly distribute the carbohydrate.
amber, clear to very slightly opalescent.
Prepared Appearance: Light amber, clear to slightly opalescent. Procedure
Reaction of 1.5% For Determining Carbohydrate Fermentation Patterns
Solution at 25°C: pH 7.2 ± 0.2
1. Inoculate tubes with test organisms.
2. Incubate tubes at 35 ± 2°C for 18-48 hours.
Cultural Response
Difco™ Peptone Water 3. Observe for color change.
Growth/Indole Reaction For Performing the Indole Test
Prepare the medium per label directions. Inoculate with a fresh culture and 1. Inoculate tubes with test organisms.
incubate at 35 ± 2°C for 18-48 hours. Indole reaction is read using the BBL™
DrySlide™ Indole test slide (Cat. No. 231748). 2. Incubate tubes at 35 ± 2°C for 24 or 48 hours.
3. Using an inoculation loop, spread a loopful of culture over
Indole
Organism ATCC™ RECOVERY Reaction the reaction area of a BBL™ DrySlide™ Indole slide.
Escherichia coli 25922 Good Positive 4. Examine the reaction area for appearance of a pink color
within 30 seconds.
Carbohydrate Fermentation
Prepare the medium per label directions with the addition of phenol red and
dextrose. Inoculate and incubate at 35 ± 2°C for 18-48 hours. Expected Results
For Determining Carbohydrate Fermentation Patterns
INOCULUM Acid
Organism ATCC™ CFU RECOVERY Production Acid is produced when carbohydrates are fermented. This is
Escherichia coli 25922 102-103 Good Positive indicated by a yellow color in the medium. Gas production is
Staphylococcus aureus 25923 102-103 Good Positive indicated by the presence of gas bubbles in the fermentation
tube.
427
Petragnani Medium
Intended Use Slanted media should be incubated in a horizontal plane until
Petragnani Medium is used in qualitative procedures for the inoculum is absorbed. Tubes should have screw caps loose
the isolation and cultivation of mycobacteria from clinical for the first 3 weeks to permit circulation of carbon dioxide for
specimens. the initiation of growth. Thereafter, to prevent dehydration,
tighten caps; loosen briefly once a week. Stand tubes upright if
Summary and Explanation space is a problem.
Petragnani Medium was described by Norton et al. in their paper Note: Cultures from skin lesions suspected to contain
regarding culture methods for tubercle bacilli.1,2 M. marinum or M. ulcerans should be incubated at 25-33°C
Petragnani Medium is glycerolated egg medium made with a for primary isolation; cultures suspected to contain M. avium
milk base containing malachite green. Somewhat more inhibi- or M. xenopi exhibit optimum growth at 40-42°C.3 Incubate a
tory than ATS and Lowenstein-Jensen media because of higher duplicate culture at 35-37°C.
dye content, Petragnani Medium is particularly recommended
for old specimens and for use in parallel with other media for Expected Results
isolation of tubercle bacilli.2,3 Cultures should be read within 5-7 days after incubation and
once a week thereafter for up to 8 weeks.
Principles of the Procedure Record Observations.3
The casein peptone, potato flour and skim milk contain amino
acids, proteins and carbohydrates necessary for the growth of 1. Number of days required for colonies to become macroscopi-
mycobacteria. The glycerol is a source of energy. Asparagine cally visible. Rapid growers have mature colonies within 7
promotes the initiation of growth and increases the growth rate. days. Slow growers require more than 7 days for mature
Egg yolk is a source of lipids for mycobacterial metabolism. colony forms.
Partial inhibition of bacteria is achieved by the presence of the 2. Pigment production
malachite green dye. White, cream or buff = Nonchromogenic (NC)
Lemon, yellow, orange, red = Chromogenic (Ch)
Precaution
Laboratory procedures involving mycobacteria require special Stained smears may show acid-fast bacilli, which are reported
equipment and techniques to minimize biohazards.4 only as “acid-fast bacilli” unless definitive tests are performed.
Procedure References
1. Norton, Thomas and Broom. 1932. Am. Rev. Tuberc. 25:378.
The test procedures are those recommended by the Centers 2. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. 1.
Williams & Wilkins, Baltimore, Md.
for Disease Control and Prevention (CDC) for primary isola- 3. Cernoch, Enns, Saubolle and Wallace. 1994. Cumitech 16A, Laboratory diagnosis of the mycobacte-
rioses. Coord. ed., Weissfeld. American Society for Microbiology, Washington, D.C.
tion from specimens containing mycobacteria.3 N-Acetyl-L- 4. U.S. Public Health Service, Centers for Disease Control and Prevention, and National Institutes of
cysteine-sodium hydroxide (NALC-NaOH) solution is recom- Health, 2007. Biosafety in microbiological and biomedical laboratories, 5th ed. HHS Publication No.
(CDC) 93-8395. U.S. Government Printing Office, Washington, D.C.
mended as a gentle but effective digesting and decontaminating 5. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby, Inc.,
St. Louis, Mo.
agent. These reagents are provided in the BBL™ MycoPrep™ 6. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
Mycobacterial Specimen Digestion/Decontamination Kit. For
detailed decontamination and culturing instructions, consult an
Availability
appropriate reference.3,5,6
BBL™ Petragnani Medium
Media may be inoculated up to the expiration date and Cat. No. 221389 Prepared Slants (C Tubes) – Pkg. of 10*
incubated for up to 8 weeks. Following inoculation, keep *Store at 2-8°C.
429
Prepare the medium per label directions. Inoculate slant tubes with fresh Cultural Response
cultures by stabbing the butt and streaking the slant surface. Incubate at BBL™ Phenol Red Agar Base
35 ± 2°C for 18-48 hours. Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 12-18 hours.
ORGANISM ATCC™ RECOVERY acid GAS
ORGANISM ATCC™ INOCULUM CFU REcovery
Escherichia coli 25922 Good + +
Salmonella enterica Escherichia coli 25922 102-103 Good
subsp. enterica Pseudomonas aeruginosa 10145 102-103 Good
serotype Typhimurium 14028 Good + +
Staphylococcus aureus 25923 Good + –
Streptococcus mitis 9895 Good – –
430
Cultural Response
BBL™ Phenol Red Broth Base or Phenol Red Broth with Dextrose or Lactose or Mannitol or Sucrose
Prepare the medium per label directions. Inoculate with fresh cultures and incubate at 35 ± 2°C for 42-48 hours.
ORGANISM ATCC™ BASE DEXTROSE LACTOSE MANNITOL SUCROSE
Escherichia coli 25922 K AG AG AG
Enterococcus faecalis 33186 A A A
Proteus vulgaris 8427 K A K AG
Pseudomonas aeruginosa 10145 K K
Salmonella Typhimurium* 14028 K K K
Shigella flexneri 9199 K A K A K
Staphylococcus aureus 25923 A
*S. enterica subsp. enterica serotype Typhimurium
KEY: A = growth with acid (yellow color)
K = growth with alkaline reaction (red color)
G = gas formation
For quality control organisms for prepared tubes of Phenol Red Broth with the various carbohydrates, consult the BBL™ Quality Control and
Product Information Manual for Plated and Tubed Media.5
431
Formulae References
BBL™ Phenol Red Broth Base 1. MacFaddin. 2000. Biochemical tests for identification of medical bacteria, 3rd ed., Lippincott Williams
& Wilkins, Baltimore, Md.
Approximate Formula* Per Liter 2. Forbes, Sahm and Weissfeld. 2007. Diagnostic microbiology, 12th ed. Mosby, Inc., St. Louis, Mo.
Pancreatic Digest of Casein........................................ 10.0 g 3. Vera. 1950. Am. J. Public Health, 40:1267.
4. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Intera-
Sodium Chloride.......................................................... 5.0 g tional, Gaithersburg, Md.
Phenol Red................................................................ 18.0 mg 5. Becton, Dickinson and Co. 2007. BBL™ quality control and product information manual for plated
*Adjusted and/or supplemented as required to meet performance criteria. and tubed media, BD Diagnostics, Sparks, Md.
6. Ewing. 1986. Edwards and Ewing’s identification of Enterobacteriaceae, 4th ed. Elsvier Science
Publishing Co., New York, N.Y.
BBL™ Phenol Red Carbohydrate Broths 7. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology,
Contain the above ingredients with, per liter, 5.0 g of the speci- 9th ed. Williams & Wilkins, Baltimore, Md.
8. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
fied carbohydrate. American Society for Microbiology, Washington, D.C.
Phenylalanine Agar
Ferric Chloride Reagent
Intended Use Principles of the Procedure
Phenylalanine Agar is used for the differentiation of enteric The phenylalanine serves as the substrate for enzymes which
bacilli on the basis of their ability to produce phenylpyruvic are able to deaminate it to form phenylpyruvic acid. The addition
acid by oxidative deamination. Ferric Chloride Reagent is used of 3-5 drops of a 10% aqueous ferric chloride solution (or a
to visualize the phenylalanine deamination reaction. 12% aqueous ferric chloride solution acidified with 2.5 mL
of concentrated HCl per 100 mL of reagent) to the cultures O
Summary and Explanation
Henrickson initially demonstrated that Proteus species were
following incubation results in the appearance of a light to deep
green color (positive reaction) or no color change (negative reac-
P
able to transform phenylalanine to phenylpyruvic acid.1 Singer tion). In a positive reaction, any phenylpyruvic acid present will
and Volcani,2 Hamida and LeMinor3 and others studied the react with the ferric salt in the reagent to give a green color.
reaction and emphasized its usefulness in the taxonomy of the
Enterobacteriaceae. Formulae
Difco™ Phenylalanine Agar
Buttiaux et al. developed a culture medium containing
Approximate Formula* Per Liter
phenylalanine in their study of the characteristic biochemi- DL-Phenylalanine.......................................................... 2.0 g
cal properties of the Proteus and Providencia genera.4 This Yeast Extract................................................................ 3.0 g
medium was designed to differentiate members of the Proteeae Sodium Chloride.......................................................... 5.0 g
Dipotassium Phosphate................................................ 1.0 g
from other members of the Enterobacteriaceae by the ability of Agar.......................................................................... 12.0 g
organisms in the genera within the Proteeae to deaminate phe-
BBL™ Phenylalanine Agar
nylalanine to phenylpyruvic acid by enzymatic activity.5 Proteus, Approximate Formula* Per Liter
Providencia and Morganella species possess this capability. This DL-Phenylalanine.......................................................... 2.0 g
formula conforms to the modified formula of Ewing et al.6 Yeast Extract................................................................ 3.0 g
Sodium Chloride.......................................................... 5.0 g
Ferric Chloride Reagent is used to determine if a specific Sodium Phosphate....................................................... 1.0 g
microorganism is capable of producing phenylpyruvic acid Agar.......................................................................... 12.0 g
from phenylalanine.5 Difco™/BBL™ Ferric Chloride Reagent Droppers
Contain 0.5 mL of 10% ferric chloride in aqueous solution.
*Adjusted and/or supplemented as required to meet performance criteria.
433
Availability
Difco™ Phenylalanine Agar
BAM
Cat. No. 274520 Dehydrated – 500 g
BBL™ Phenylalanine Agar
BAM
Cat. No. 211537 Dehydrated – 500 g
Difco™/BBL™ Ferric Chloride Reagent (10%)
Cat. No. 261190 Droppers, 0.5 mL – Ctn. of 50
*Store at 2-8°C.
Staphylococcus aureus
User Quality Control ATCC™ 25923
Identity Specifications
BBL™ Phenylethyl Alcohol Agar
Dehydrated Appearance: Slightly moist and softly clumped, resembling
“brown sugar” in consistency and appear-
ance.
Solution: 4.25% solution, soluble in purified water upon
boiling. Solution is light to medium, yellow to
tan, clear to slightly hazy.
Prepared Appearance: Light to medium, yellow to tan, clear to slightly
hazy. O
Reaction of 4.25%
Solution at 25°C: pH 7.3 ± 0.2 P
Cultural Response
BBL™ Phenylethyl Alcohol Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C
with 3-5% CO2 for 18-24 hours.
INOCULUM
ORGANISM ATCC™ CFU recovery
Proteus mirabilis 12453 104-105 Partial to complete
inhibition
Staphylococcus aureus 25923 103-104 Good
Streptococcus pneumoniae 6305 103-104 Good, alpha hemolysis
Streptococcus pyogenes 19615 103-104 Good, beta hemolysis
436
3. Japanese Ministry of Health, Labour and Welfare. 2006. The Japanese pharmacopoeia, 15th ed.,
online. Japanese Ministry of Health, Labour and Welfare. Availability
4. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C.
BBL™ Phosphate Buffer, pH 7.2
5. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy products, 17th ed. AOAC BAM COMPF EP JP SMD SMWW USDA USP
American Public Health Association, Washington, D.C.
6. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods, Cat. No. 211544 Dehydrated – 500 g†
4th ed. American Public Health Association, Washington, D.C. 214973 Prepared Bottles (Working Solution), 500 mL
7. U.S. Food and Drug Administration. Bacteriological analytical manual, online. AOAC International,
Gaithersburg, Md.
(septum screw cap) – Pkg. of 10†
8. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC 257385 Prepared Bottles (Stock Solution), 100 mL
International, Gaithersburg, Md. (septum screw cap) – Ctn. of 25†
9. U.S. Department of Agriculture. Microbiology laboratory guidebook, online. Food Safety and Inspec-
tion Service, USDA, Washington, D.C. † QC testing performed according to USP/EP/JP performance specifications.
437
Identity Specifications
BBL™ Phytone™ Peptone
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution: 2.0% solution, soluble in purified water. Solution is clear to slightly hazy.
Reaction of 2.0%
Solution at 25°C: pH 6.5-7.5
Difco Select Phytone™ UF
™
Cultural Response
Biochemical Reactions
BBL™ Phytone™ Peptone
Prepare a sterile solution of Phytone Peptone as directed below. Adjust final pH to 7.2-7.4. Inoculate and incubate at 35 ± 2°C for 18-48 hours.
TEST TEST SOLUTION ORGANISM ATCC™ INOCULUM CFU RESULT
Fermentable Carbohydrates 2% Escherichia coli 29552 ~10
7
Positive
Indole Production 0.1% Escherichia coli 29552 0.1 mL, undiluted Positive
Acetylmethylcarbinol 0.1% with Enterobacter aerogenes 13048 0.1 mL, undiluted Positive
Production 0.5% dextrose
Hydrogen Sulfide
Production 1% Citrobacter freundii 8454 0.1 mL, undiluted Positive
Growth Response
BBL™ Phytone™ Peptone
Prepare a sterile solution of peptone agar without (plain) and with 5% sheep blood (SB) using 10 g of Phytone Peptone, 2.5 g of sodium chloride
and 6.5 g of agar in 500 mL of purified water. Adjust final pH to 7.2-7.4. Inoculate and incubate plates at 35 ± 2°C for 2-3 days (incubate streptococci with
3-5% CO2).
ORGANISM ATCC™ INOCULUM CFU recovery PLAIN recovery WITH SB
Staphylococcus aureus 6538P 103-104 Good N/A
Streptococcus pneumoniae 6305 10 -10
3 4
N/A Good, alpha hemolysis
Streptococcus pyogenes 49117 104-105 Good Good, beta hemolysis
Continued
438
Identity Specifications
Difco™ Select Soytone
Dehydrated Appearance: Tan, free-flowing, homogeneous powder.
Solution: 2.0% solution, soluble in purified water. Solution is clear to moderately hazy.
Reaction of 2.0%
Solution at 25°C: pH 6.5-7.5
Bacto™ Soytone
Dehydrated Appearance: Light to medium tan, free-flowing, homogeneous.
Solution: 2.0% solution, soluble in purified water. Solution is light to medium amber, clear to very slightly hazy. A small amount of precipitate is
acceptable.
Reaction of 2.0% O
Solution at 25°C: pH 6.5-7.5
P
Cultural Response
Biochemical Reactions
Bacto™ Soytone
Prepare sterile solutions of Bacto Soytone as directed below. Adjust final pH to 7.2-7.4. Inoculate and incubate at 35 ± 2°C for 18-48 hours.
TEST TEST SOLUTION ORGANISM ATCC™ INOCULUM CFU RESULT
Fermentable Carbohydrates 2% Escherichia coli 25922 ~107 Slight positive
Indole Production 0.1% Escherichia coli 29552 0.1 mL, undiluted Positive
Acetylmethylcarbinol 0.1% with Enterobacter aerogenes 13048 0.1 mL, undiluted Positive
Production 0.5% dextrose
Hydrogen Sulfide 1% Salmonella enterica 14028 0.1 mL, undiluted Positive
Production subsp. enterica
serotype Typhimurium
Growth Response
Bacto™ Soytone
Prepare a sterile solution with 2% Bacto Soytone, 0.5% sodium chloride and 1.5% agar. Adjust final pH to 7.2-7.4. Inoculate and incubate plates at
35 ± 2°C for 18-48 hours.
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Staphylococcus aureus 6538P 10 -10
3 4
Good
Streptococcus pneumoniae 6305 103-104 Good
Streptococcus pyogenes 49117 104-105 Good
439
Cultural Response
Availability
BBL™ Phytone™ Yeast Extract Agar
BBL™ Phytone™ Yeast Extract Agar
Cat. No. 211546 Dehydrated – 500 g
Prepare the medium per label directions. Inoculate with fresh cultures as
described below and incubate at 25 ± 2°C for 7 days.
ORGANISM ATCC™ INOCULUM CFU recovery
Aspergillus brasiliensis (niger) 16404 Undiluted Good
Candida albicans 10231 Undiluted Good
Penicillium roquefortii 9295 Undiluted Good
Staphylococcus aureus 25923 104-105 Complete inhibition
Trichophyton verrucosum 38485 Undiluted Good
440
Formula
Difco™ Plate Count Agar or BBL™ Standard Methods Agar
Approximate Formula* Per Liter
Pancreatic Digest of Casein.......................................... 5.0 g
Yeast Extract................................................................ 2.5 g
Dextrose...................................................................... 1.0 g
Agar.......................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.
441
References
1. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater, Availability
21st ed., online. American Public Health Association, Washington, D.C.
2. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
Difco™ m Plate Count Broth
4th ed. American Public Health Association, Washington, D.C. Cat. No. 275120 Dehydrated – 500 g
Polypeptone™ Peptone
Intended Use Principles of the Procedure
Polypeptone Peptone is used as a component in microbiological Polypeptone Peptone is a mixture of peptones made up of equal
culture media. parts of pancreatic digest of casein and peptic digest of animal
tissue. Polypeptone Peptone includes the high content of amino
Summary and Explanation acids and small polypeptides characteristic of pancreatic digest
Researchers have found that Polypeptone Peptone meets of casein and the larger polypeptides characteristic of peptic
the nutritional requirements of various bacteria, fungi and digest of animal tissue. Polypeptone Peptone provides nitrogen,
mammalian cells, where a single source of casein meat amino acids and vitamins in microbiological culture media.
peptone has been unsatisfactory. Polypeptone Peptone has been
utilized in culture media for the production of trypsin inhibitor Typical Analysis
by Cephalosporium sp.,1 in the production of bacterial cellulose Refer to Product Tables in the Reference Guide section of this
by Acetobacter sp. A92 and in the production of succinic manual.
acid from whey by Anaerobiospirillum succiniciproducens.3
In addition, Polypeptone Peptone has been used in the mass Directions for Preparation from
production of luciferase-bacterial magnetic particles by Dehydrated Product
recombinant Magnetospirillum magneticum AMB-14 and the Refer to the final concentration of Polypeptone Peptone in
production of a novel tumor-killing factor by human the formula of the medium being prepared. Add product as
macrophage-monocyte hybridomas.5 required.
Media formulations containing Polypeptone Peptone are speci-
fied in standard methods for various applications.6-11
Procedure
See appropriate references for specific procedures using
Polypeptone Peptone.
443
444
Cultural Response
Difco™ Potato Dextrose Agar 2. Heat with frequent agitation and boil for 1 minute to com-
Prepare the medium per label directions. Inoculate and incubate at 25- pletely dissolve the powder.
30°C for 18-48 hours (up to 7 days for T. mentagrophytes). For Aspergillus 3. Autoclave at 121°C for 15 minutes.
brasiliensis, incubate at 20-25°C for 5 days.
4. To alter the reaction of the agar medium to pH 3.5, cool the
ORGANISM ATCC™ INOCULUM CFU RECOVERY
base to 45-50°C and aseptically add an appropriate amount
Candida albicans 10231 103-104 Good of sterile 10% tartaric acid to each liter of medium. Mix well.
Saccharomyces Do not reheat the medium.
cerevisiae 9763 103-104 Good
5. Test samples of the finished product for performance using
Trichophyton
mentagrophytes 9533 Undiluted Good stable, typical control cultures.
Aspergillus
brasiliensis (niger) 16404 <100 Growth Sample Collection and Handling
For clinical specimens, refer to laboratory procedures for details
Difco™ Potato Dextrose Broth
on specimen collection and handling.8,9
Prepare the medium per label directions. Inoculate and incubate at
25 ± 2°C for 40-48 hours. For food, dairy and cosmetic samples, follow appropriate stan-
ORGANISM ATCC™ INOCULUM CFU RECOVERY dard methods for details on sample collection and preparation
Aspergillus according to sample type and geographic location.4-7
brasiliensis (niger) 16404 30-300 Good
Candida albicans 10231 30-300 Good
For pharmaceutical samples, refer to the USP for details on
Lactobacillus casei 7469 30-300 Fair to good
sample collection and preparation for testing of nonsterile
products.1
Saccharomyces
cerevisiae 9763 30-300 Good
Procedure
For clinical specimens, refer to appropriate standard references
for details on testing protocol to obtain isolated colonies from
Directions for Preparation from specimens using Potato Dextrose Agar.8,9
Dehydrated Product
1. Suspend the powder in 1 L of purified water: For food, dairy and cosmetic samples, refer to appropriate
Difco™ Potato Dextrose Agar – 39 g; standard references for details on test methods using Potato
Difco™ Potato Dextrose Broth – 24 g. Dextrose Agar.4-7
Mix thoroughly. For pharmaceutical samples, refer to USP General Chapter
<61> for details on the examination of nonsterile products and
Microbial Enumeration Tests using Potato Dextrose Agar.1
445
Candida albicans
ATCC™ 10231
Aspergillus brasiliensis
ATCC™ 16404
Growth from tubes inoculated with pure cultures may be used
for biochemical and/or serological testing.
For broth, observe cultures for surface growth and pellicle
formation.
Streak the specimen onto prepared media with a sterile inoculat- Availability
ing loop to obtain isolated colonies. When used for determining Difco™ Potato Dextrose Agar
yeast and mold counts, the medium should be adjusted to a pH AOAC BAM BS12 CCAM CMPH2 COMPF EP
of approximately 3.5 with sterile tartaric acid and used in the JP MCM9 SMD USP
standard pour plate technique. Incubate the plates at 25-30°C Cat. No. 213300 Dehydrated – 100 g†
213400 Dehydrated – 500 g†
with increased humidity for up to 7 days. 213200 Dehydrated – 2 kg†
Tubed slants are used primarily for the cultivation and main- BBL™ Potato Dextrose Agar
tenance of pure cultures. They should be inoculated with an AOAC BAM BS12 CCAM CMPH2 COMPF EP
inoculating loop and incubated under the same conditions as JP MCM9 SMD USP
the plated medium. Cat. No. 221002 Prepared Pour Tubes, 20 mL – Pkg. of 10
297241 Prepared Slants – Pkg. of 10
For isolation of fungi from potentially contaminated specimens, 299906 Prepared Bottles,500 mL
a selective medium should be inoculated along with the nonselec- (septum screw cap) – Pkg. of 10†
tive medium. For isolation of fungi causing systemic mycoses, United States and Canada
Cat. No. 296272 Prepared Plates (Deep Fill) – Pkg. of 20*
two sets of media should be inoculated, with one set incubated 297945 Prepared Plates (Deep Fill) – Ctn. of 100*
at 25-30°C and a duplicate set at 35 ± 2°C. All cultures should
Japan
be examined at least weekly for fungal growth and should be Cat. No. 251545 Prepared Plates – Ctn. of 100*
held for 4-6 weeks before being reported as negative. 251821 Prepared Plates (Deep Fill) – Ctn. of 100*
251544 Prepared Plates (150 × 15 mm-style) –
Inoculation of Potato Dextrose Broth with pure cultures of yeasts Pkg. of 24*
can assist in their identification. Mexico
Cat. No. 252632 Prepared Bottles, 140 mL – Pkg. of 12
Expected Results NOTE: None of the prepared media contain tartaric acid.
After sufficient incubation, the plates which were streak in- Difco™ Potato Dextrose Broth
oculated should show isolated colonies in streaked areas and Cat. No. 254920 Dehydrated – 500 g
confluent growth in areas of heavy inoculation. The colonies in * Store at 2-8°C.
† QC testing performed according to USP/EP/JP performance specifications.
pour plates should be counted and the results expressed as yeast
and mold counts per gram or milliliter of material, taking into
account the applicable dilution factor.
446
447
Presence-Absence Broth
Intended Use or absence of lactose fermentation.1 This test is based on the
Presence-Absence Broth is used for detecting coliforms in treated principle that coliforms and other pollution indicator organisms
water. should not be present in a 100 mL water sample.2-8
Comparative studies with the membrane filter procedure
Summary and Explanation indicate that the P-A test may maximize coliform detection
The Presence-Absence (P-A) test is a presumptive detection test in samples containing many organisms that could overgrow
for coliforms in water. The test is a simple modification of the coliform colonies and cause problems in detection.1 The P-A test
multiple-tube procedure.1 One test sample, 100 mL, is inoculated is described in standard methods for water testing1 and by U.S.
into a single culture bottle to obtain qualitative information Environmental Protection Agency.9
on the presence or absence of coliforms based on the presence
448
O
Solution at 25°C: pH 6.8 ± 0.2
Cultural Response
Difco™ Presence-Absence Broth
P
Prepare Presence-Absence Broth in triple strength solution (9.15%). Sterilize in 50 mL
quantities in milk dilution bottles with capacity greater than 150 mL. Add 100 mL of
drinking water after medium is sterilized and cooled to room temperature. Inoculate
bottles with the test organisms. Incubate bottles at 35 ± 0.5°C for 18-48 hours. Presence-Absence Broth
References Availability
1. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C.
Difco™ Presence-Absence Broth
2. Weiss and Hunter. 1939. J. Am. Water Works Assoc. 31:707. CCAM EPA SMWW
3. Clark. 1968. Can. J. Microbiol. 14:13.
4. Clark. 1969. Can. J. Microbiol. 15:771. Cat. No. 219200 Dehydrated – 500 g
5. Clark and Vlassoff. 1973. Health Lab. Sci. 10:163. 219100 Dehydrated – 2 kg
6. Clark and Pagel. 1977. Can. J. Microbiol. 23:465.
7. Clark. 1980. Can. J. Microbiol. 26:827.
8. Clark, Burger and Sabatinos. 1982. Can. J. Microbiol. 28:1002.
9. Federal Register. 1989. National primary drinking water regulations; total coliforms (including fecal
coliforms and E. coli). Fed. Regist. 54:27544.
450
Dehydrated Appearance: Tan, free-flowing, homogeneous. Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 1.0%, 2.0% and 10.0% solutions, soluble in Solution: 1.0%, 2.0% and 10.0% solutions, soluble in
purified water. 1.0% solution is very light amber, purified water. 1.0% solution is very light amber,
clear to very slightly opalescent, may have a clear to very slightly opalescent, may have a
slight precipitate. 2.0% solution is light amber, slight precipitate. 2.0% solution is light amber,
clear to slightly opalescent, may have a slight clear to slightly opalescent, may have a slight
precipitate. 10.0% solution is light to medium precipitate. 10.0% solution is medium amber,
amber, clear to slightly opalescent, may have a slightly opalescent to opalescent, may have a
slight precipitate. slight precipitate.
Reaction of 1.0% Reaction of 1.0%
Solution at 25°C: pH 6.5-7.5 Solution at 25°C: pH 6.6-7.6
Bacto™ Proteose Peptone No. 2
Dehydrated Appearance: Tan, free-flowing, granules.
Solution: 1.0%, 2.0% and 10.0% solutions, soluble in
purified water. 1.0% solution is light to medium
amber, clear. 2.0% solution is medium amber,
clear. 10.0% solution is medium to dark amber,
slightly opalescent to opalescent, may have a
slight precipitate.
Reaction of 1.0%
Solution at 25°C: pH 7.2-7.6 Continued
451
Cultural Response
Biochemical Reactions
Bacto™ Proteose Peptone, BiTek™ Proteose Peptone, Bacto™ Proteose Peptone No. 2,
Bacto™ Proteose Peptone No. 3 or Bacto™ Proteose Peptone No. 4
Prepare a sterile solution as directed below. Adjust final pH to 7.2-7.4. Inoculate and incubate at 35 ± 2°C for 18-48 hours.
TEST TEST SOLUTION ORGANISM ATCC™ INOCULUM CFU RESULT
Fermentable Carbohydrates 2% Escherichia coli 25922 ~107 Negative
Indole Production 0.1% Escherichia coli 29552 0.1 mL, undiluted Positive
Acetylmethylcarbinol 0.1% Enterobacter aerogenes 13048 0.1 mL, undiluted Positive
Production with 0.5% dextrose
Hydrogen Sulfide 1% Salmonella enterica subsp. enterica 14028 0.1 mL, undiluted Positive
Production serotype Typhimurium
Growth Response
Bacto™ Proteose Peptone, BiTek™ Proteose Peptone Bacto™ Proteose Peptone No. 2
or Bacto™ Proteose Peptone No. 4 Prepare a sterile solution with 2% Bacto Proteose Peptone No. 2, 0.5%
1. Prepare a sterile solution with 2% Bacto Proteose Peptone, BiTek sodium chloride and 1.5% agar. Adjust final pH to 7.2-7.4. Inoculate and
Proteose Peptone or Bacto Proteose Peptone No. 4, 0.5% sodium chloride incubate plates at 35 ± 2°C for 18-48 hours under appropriate atmospheric
and 1.5% agar. Adjust final pH to 7.2-7.4. Inoculate and incubate plates at conditions.
35 ± 2°C for 18-48 hours under appropriate atmospheric conditions.
ORGANISM ATCC™ INOCULUM CFU RECOVERY
ORGANISM ATCC™ INOCULUM CFU RECOVERY Escherichia coli 25922 30-300 Good
Neisseria meningitidis 13090 30-300 Good* Staphylococcus aureus 25923 30-300 Good
Staphylococcus aureus 25923 30-300 Good
Bacto™ Proteose Peptone No. 3
Streptococcus pneumoniae 6303 30-300 Good Prepare a sterile solution with 2% Bacto Proteose Peptone No. 3, 0.5%
*Fair to good for BiTek Proteose Peptone. sodium chloride and 1.5% agar. Adjust final pH to 7.2-7.4. Inoculate and
2. For Bacto Proteose Peptone and Bacto Proteose Peptone No. 4 only, incubate plates at 35 ± 2°C for 18-48 hours under appropriate atmospheric
prepare KL Virulence Agar from individual ingredients using 2 g of Bacto conditions.
Proteose Peptone or Bacto Proteose Peptone No. 4. Sterilize, cool to ORGANISM ATCC™ INOCULUM CFU RECOVERY
55-60°C and add KL Virulence Enrichment and Tellurite Solution. Dispense
into Petri dishes. Inoculate with a loopful of surface growth and incubate Staphylococcus aureus 25923 30-300 Good
at 35 ± 2°C for 72 hours. Examine at 24, 48 and 72 hours for growth Streptococcus pneumoniae 6303 30-300 Good
and blackening. Streptococcus pyogenes 19615 30-300 Good
ORGANISM ATCC™ RESULT
Corynebacterium diphtheriae
biotype intermedius 8032 Growth
Corynebacterium diphtheriae
biotype gravis 8028 Growth
Corynebacterium diphtheriae
biotype mitis 8024 Growth
References Availability
1. Kirkbride, Berthelsen and Clark. 1931. J. Immunol. 21:1.
2. Hazen and Heller. 1931 J. Bacteriol. 23:195.
Bacto™ Proteose Peptone
3. Nelson. 1927. J. Infect. Dis. 41:9. EPA SMD SMWW USDA
4. Mollby and Holme. 1976 J. Gen. Microbiol. 96:137.
5. Kirkbride and Wheeler. 1926. J. Immunol. 11:477. Cat. No. 211684 Dehydrated – 500 g
6. Kneeland and Dawes. 1932. J. Exp. Med. 55:735. 212010 Dehydrated – 10 kg
7. Hanks and Rettger. 1931. J. Immunol. 22:283.
8. Berg, Nord and Wadstrom. 1978. Appl. Environ. Microbiol. 35:269. BiTek™ Proteose Peptone
9. Mamo and Gessesse. 1999. J. Ind. Microbiol. Biotechnol. 22:622.
10. Hezayen, Rehm, Eberhardt and Steinbuchel. 2000. Appl. Microbiol. Biotechnol. 54:319. Cat. No. 253310 Dehydrated – 10 kg
11. Jan, Jones, Emery and Al-Rubeai. 1994. Cytotechnol. 16:17.
12. Shukla, Kaul and Mehlotra. 1989. Indian J. Exp. Biol. 27:785. Bacto™ Proteose Peptone No. 2
13. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy products, 17th ed.
American Public Health Association, Washington, D.C. Cat. No. 212120 Dehydrated – 500 g
14. U.S. Department of Agriculture. Microbiology laboratory guidebook, online. Food Safety and Inspec- 212110 Dehydrated – 10 kg
tion Service, USDA, Washington, D.C.
15. U.S. Environmental Protection Agency. 2000. Improved enumeration methods for the recreational
water quality indicators: Enterococci and Escherichia coli. EPA-821/R-97/004. Office of Water, USEPA, Bacto™ Proteose Peptone No. 3
Washington, D.C. BAM EPA SMWW USDA
16. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C. Cat. No. 211693 Dehydrated – 500 g
17. Bunney and Thomas. 1936. J. Immunol. 31:95. 212220 Dehydrated – 2 kg
18. Ifediba and Vanderberg. 1980. J. Parasitol. 66:236.
19. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna- 212230 Dehydrated – 10 kg
tional, Gaithersburg, Md.
Bacto™ Proteose Peptone No. 4
Cat. No. 211715 Dehydrated – 10 kg
452
Procedure
For a complete discussion of the isolation and identification
of Haemophilus or Neisseria spp., refer to the procedures
outlined in the references.4-6
453
Pseudomonas Agars
Pseudomonas Agar F • Flo Agar
Pseudomonas Agar P • Tech Agar
Intended Use Pseudomonas Agar P (Tech Agar) contains enzymatic digest of
Pseudomonas Agar F, also known as Flo Agar, is used for the gelatin to provide amino acids and other essential nitrogenous
enhancement of fluorescin production and Pseudomonas Agar substances. The gelatin peptone is low in phosphorous to
P, also known as Tech Agar, is used for the enhancement of minimize the inhibitory action on pyocyanin production.4
pyocyanin production by Pseudomonas. Magnesium, potassium and sulfate ions promote pyocyanin
production.4
Summary and Explanation Both media contain glycerol, which acts as a source of energy
Pseudomonas aeruginosa is widely distributed in soil, water and enhances pigment production.
and foods. It is frequently isolated from infusion fluids,
disinfectants and cosmetics. The organism causes disease in Formulae
humans; e.g., ocular infections, burn wound infections and Difco™ Pseudomonas Agar F
respiratory tract infections.1 Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 10.0 g
Most strains of P. aeruginosa produce pyocyanin, a blue, Proteose Peptone No. 3.............................................. 10.0 g
water- and chloroform-soluble, nonfluorescent pigment that Dipotassium Phosphate................................................ 1.5 g
diffuses into the surrounding medium.2 P. aeruginosa is the only Magnesium Sulfate...................................................... 1.5 g
Agar.......................................................................... 15.0 g
Pseudomonas species known to produce this pigment. (However,
certain strains are apyocyanogenic.) Difco™ Pseudomonas Agar P
Approximate Formula* Per Liter
Some strains of P. aeruginosa produce other pigments, such as Pancreatic Digest of Gelatin....................................... 20.0 g
the brown-black pyomelanin, the red pyorubin or the yellow Magnesium Chloride.................................................... 1.4 g
Potassium Sulfate....................................................... 10.0 g
pyoverdin. Pyoverdin is a water soluble fluorescent pigment Agar.......................................................................... 15.0 g
often produced by P. aeruginosa and other pseudomonads *Adjusted and/or supplemented as required to meet performance criteria.
isolated from humans.2 The presence of these pigments can,
however, mask the production of pyocyanin.2 Directions for Preparation from
Pseudomonas Agar F (Flo Agar) and Pseudomonas Agar P
Dehydrated Product
1. Suspend the powder in 1 L of purified water containing 10 g
(Tech Agar) are modifications of two media (Medium A and
of glycerol:
Medium B) that King et al. developed to enhance pigment
Difco™ Pseudomonas Agar F – 38 g;
production for improved differentiation of pseudomonads.3
Difco™ Pseudomonas Agar P – 46.4 g.
Principles of the Procedure Mix thoroughly.
The 1:1 ratio of casein to meat peptone in Pseudomonas 2. Heat with frequent agitation and boil for 1 minute to com-
Agar F (Flo Agar) is conducive to fluorescin production by pletely dissolve the powder.
Pseudomonas. These peptones contain phosphorus, which 3. Autoclave at 121°C for 15 minutes.
is stimulatory to fluorescin production.4 The addition of 4. Test samples of the finished product for performance using
dipotassium phosphate increases the phosphorus content of stable, typical control cultures.
the medium, thereby enhancing production of the fluorescent
pigment. Magnesium sulfate provides essential ions for fluorescin
Procedure
Specimens must first be isolated in pure culture on an
production.4
appropriate medium. The isolate should be Gram-stained and
examined to confirm that morphology is appropriate for
Pseudomonas.
454
Pseudomonas aeruginosa
User Quality Control ATCC™ 9027
Pseudomonas Pseudomonas
Identity Specifications Agar F Agar P
Difco™ Pseudomonas Agar F
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 3.8% solution, soluble in purified water with 1%
glycerol upon boiling. Solution is light to medium
amber, very slightly to slightly opalescent.
Prepared Appearance: Light to medium amber, slightly opalescent.
Reaction of 3.8%Solution
with 1% glycerol at 25°C: pH 7.0 ± 0.2
Difco™ Pseudomonas Agar P
O
Dehydrated Appearance: Light beige, free-flowing, homogeneous. P
Solution: 4.64% solution, soluble in purified water with 1%
glycerol upon boiling. Solution is light amber, very
slightly to slightly opalescent.
Prepared Appearance: Light to medium amber, slightly opalescent.
Reaction of 4.64% Solution
with 1% glycerol at 25°C: pH 7.0 ± 0.2
Cultural Response
Difco™ Pseudomonas Agar F or Pseudomonas Agar P
Prepare the medium per label directions. For Pseudomonas Agar F
inoculate as described below and incubate at 35 ± 2°C for 18-24 hours.
For Pseudomonas Agar P inoculate with fresh cultures and incubate at
35 ± 2°C for 18-24 hours.
INOCULUM CFU PIGMENT PRODUCTION PIGMENT PRODUCTION
ORGANISM ATCC™ PSEUDOMONAS AGAR F RECOVERY PSEUDOMONAS AGAR F PSEUDOMONAS AGAR P
Pseudomonas aeruginosa 9027 30-300 Good Greenish yellow Blue
Pseudomonas aeruginosa 27853 30-300 Good Greenish yellow Blue to green
Pseudomonas cepacia 25609 30-300 Good No pigment No pigment
Using a sterile inoculating loop or needle, streak plates or slants 2. The formation of nonpigmented colonies does not
with several colonies from the subculture medium. Incubate completely rule-out a Pseudomonas aeruginosa isolate.
plates or tubes, with caps loosened, at 35 ± 2°C for 18-24 3. A pyocyanin-producing Pseudomonas strain will usually
hours. If the isolate fails to grow or grows slowly, reincubate also produce fluorescin. It must, therefore, be differentiated
at 25-30°C for 1-2 days and observe for growth and pigment from other simple fluorescent pseudomonads by other means.
production.5 Temperature can be a determining factor as most other
fluorescent strains will not grow at 35°C. Rather, they grow
Expected Results at 25-30°C.4
Examine Pseudomonas Agar F (Flo Agar) under long wavelength
UV light (366 nm) for fluorescin, a greenish-yellow fluorescent References
1. Kiska and Gilligan. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical
pigment in the colonies and surrounding medium. Examine microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
Pseudomonas Agar P (Tech Agar) for pyocyanin, a blue to 2. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby, Inc.,
St. Louis, Mo.
blue-green pigment seen in the colonies and surrounding 3. King, Ward and Raney. 1954. J. Lab. Clin. Microbiol. 44:301.
4. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol.
medium. Confirm the presence of pyocyanin by adding 1. Williams & Wilkins, Baltimore, Md.
5. Sewell. 1987. In Wentworth (ed.), Diagnostic procedures for bacterial infections, 7th ed. American
several drops of chloroform and observe for a blue color in Public Health Association, Washington, D.C.
the chloroform. (Pyocyanin is more soluble in chloroform
than in water.) Availability
Difco™ Pseudomonas Agar F
Limitations of the Procedure BAM CCAM
1. Occasionally, a Pseudomonas culture is encountered that will Cat. No. 244820 Dehydrated – 500 g
produce small amounts of pigment in the medium. When this ™
BBL Flo Agar
happens, a yellow-green color will appear on Pseudomonas BAM CCAM
Agar F (Flo Agar) or a blue-green color on Pseudomonas Agar Cat. No. 296003 Prepared Slants – Pkg. of 10
P (Tech Agar). If a blue-green color occurs on Pseudomonas
Agar P (Tech Agar), confirmation of the presence of pyocya-
nin can be made by extraction with chloroform (CHCl3).4
455
Formula
Difco™ Pseudomonas Isolation Agar
Approximate Formula* Per Liter
Peptone..................................................................... 20.0 g
Magnesium Chloride.................................................... 1.4 g
Potassium Sulfate....................................................... 10.0 g
Irgasan™. ................................................................... 25.0 mg
Agar.......................................................................... 13.6 g
*Adjusted and/or supplemented as required to meet performance criteria.
Procedure References
Inoculate the medium using the streak plate method to obtain 1. Kiska and Gilligan. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical
microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
isolated colonies. Incubate for 18-48 hours at 35 ± 2°C. 2. King, Ward and Raney. 1954. J. Lab. Clin. Med. 44:301.
3. Furia and Schenkel. January, 1968. Soap and Chemical Specialties.
4. Gaby and Free. 1931. J. Bacteriol. 22:349.
Expected Results 5. Isenberg and Garcia (ed.). 2004 (update, 2007) Clinical microbiology procedures handbook, 2nd ed.
American Society for Microbiology, Washington, D.C.
Examine for the presence of good growth. Pseudomonas aeru-
ginosa colonies may be greenish after incubation for 18 hours Availability
and turn blue to blue-green as incubation continues up to 24-48 Difco™ Pseudomonas Isolation Agar
hours, with diffusion of the pigment into the medium. Cat. No. 292710 Dehydrated – 500 g
O
Europe
Limitations of the Procedure Cat. No. 257002 Prepared Plates – Pkg. of 20*
1. Some strains of Pseudomonas aeruginosa may fail to
produce pyocyanin.1,4
Difco Glycerol
™
P
Cat. No. 228210 Bottle – 100 g
2. Non-Pseudomonas aeruginosa strains that are not 228220 Bottle – 500 g
*Store at 2-8°C.
completely inhibited on this medium may be encountered
and must be differentiated from Pseudomonas aeruginosa.
Consult appropriate references.1,5
Pseudosel™ Agar
(See Cetrimide Agar Base)
Procedure
Carbohydrates should be added to the basal medium either as
sterile solutions or as BBL Taxo Carbohydrate Discs (broth).
Inoculate tubes of carbohydrate agar with an inoculating
needle to within 1/4 inch from the bottom of the tube. In-
oculate tubes of broth containing an appropriate carbohydrate
using a light inoculum from an 18- to 24-hour pure culture.
Incubate tubes for 24-72 hours or up to 30 days at 35 ± 2°C
either in an aerobic or anaerobic atmosphere depending on the
organism being tested.
Uninoculated Typical positive Typical negative
Tube reaction with acid reaction with
and gas growth Expected Results
Purple Broth Base Examine the tubes daily for growth. A yellow color (acid) is a
positive reaction for fermentation of the carbohydrate incor-
458
porated into the medium. Bubbles in the inverted fermentation BBL™ Purple Broth with Carbohydrates and
vials are an indication of gas production. Durham Tube
Cat. No. 295863 Prepared Tubes (K Tubes) with Arabinose – Pkg. of 10*
Consult appropriate texts for expected reactions of specific 295864 Prepared Tubes (K Tubes) with Cellobiose – Pkg. of 10*
organisms with specific carbohydrates.1-4 296013 Prepared Tubes (K Tubes) with Dextrose – Pkg. of 10*
295865 Prepared Tubes (K Tubes) with Dulcitol – Pkg. of 10*
297734 Prepared Tubes (K Tubes) with Fructose – Pkg. of 10*
References 296014 Prepared Tubes (K Tubes) with Galactose –Pkg. of 10*
1. Ewing. 1986. Edwards and Ewing’s identification of Enterobacteriaceae, 4th ed. Elsevier Science 295866 Prepared Tubes (K Tubes) with Inositol – Pkg. of 10*
Publishing Co, Inc., New York, N.Y.
2. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby, Inc.,
296015 Prepared Tubes (K Tubes) with Lactose – Pkg. of 10*
St. Louis, Mo. 295999 Prepared Tubes (K Tubes) with Maltose – Pkg. of 10*
3. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology, 297018 Prepared Tubes (K Tubes) with Mannitol – Pkg. of 10*
9th ed. Williams & Wilkins, Baltimore, Md.
4. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
295867
297203
Prepared Tubes (K Tubes) with Raffinose – Pkg. of 10*
Prepared Tubes (K Tubes) with Rhamnose – Pkg. of 10* O
Availability
297019
297020
Prepared Tubes (K Tubes) with Salicin – Pkg. of 10*
Prepared Tubes (K Tubes) with Sorbitol – Pkg. of 10* P
296016 Prepared Tubes (K Tubes) with Sucrose – Pkg. of 10*
Difco™ Purple Agar Base 295870 Prepared Tubes (K Tubes) with Trehalose – Pkg. of 10*
Cat. No. 222810 Dehydrated – 500 g 295871 Prepared Tubes (K Tubes) with Xylose – Pkg. of 10*
*Store at 2-8°C.
BBL™ Purple Broth Base
AOAC BAM CCAM COMPF ISO SMD USDA
Cat. No. 211558 Dehydrated – 500 g
296012 Prepared Tubes (K Tubes) with Durham Tube –
Pkg. of 10*
Pyridoxine Y Medium
Intended Use Formula
Pyridoxine Y Medium is used for determining pyridoxine Difco™ Pyridoxine Y Medium
concentration by the microbiological assay technique. Approximate Formula* Per Liter
Dextrose.................................................................... 40.0 g
L-Asparagine................................................................ 4.0 g
Summary and Explanation Ammonium Sulfate...................................................... 4.0 g
Vitamin assay media are prepared for use in the microbiologi- Monopotassium Phosphate.......................................... 3.0 g
cal assay of vitamins. Three types of media are used for this Magnesium Sulfate...................................................... 1.0 g
Calcium Chloride......................................................... 0.49 g
purpose: DL-Methionine........................................................... 40.0 mg
1. Maintenance Media: For carrying the stock culture to DL-Tryptophan........................................................... 40.0 mg
preserve the viability and sensitivity of the test organism for DL-Isoleucine.............................................................. 40.0 mg
DL-Valine................................................................... 40.0 mg
its intended purpose; L-Histidine Hydrochloride........................................... 20.0 mg
2. Inoculum Media: To condition the test culture for immediate Riboflavin................................................................... 20.0 mg
use; Biotin Salt.................................................................... 8.0 mg
Inositol......................................................................... 5.0 mg
3. Assay Media: To permit quantitation of the vitamin under Ferrous Sulfate......................................................... 500.0 µg
test. They contain all the factors necessary for optimum Thiamine Hydrochloride........................................... 400.0 µg
growth of the test organism except the single essential Calcium Pantothenate.............................................. 400.0 µg
Nicotinic Acid........................................................... 400.0 µg
vitamin to be determined. Boric Acid................................................................ 200.0 µg
Potassium Iodide...................................................... 200.0 µg
Pyridoxine Y Medium is patterned after the formulation of Ammonium Molybdate.............................................. 40.0 µg
Campling and Nixon,1 and modified by Hurley2 and Parrish, Loy Manganese Sulfate.................................................... 80.0 µg
and Kline.3 This medium is used in the microbiological assay Copper Sulfate........................................................... 90.0 µg
of pyridoxine using Saccharomyces cerevisiae ATCC™ 9080 as Zinc Sulfate................................................................ 80.0 µg
*Adjusted and/or supplemented as required to meet performance criteria.
the test organism.
Precautions
Principles of the Procedure Great care must be taken to avoid contamination of media
Pyridoxine Y Medium is free from pyridoxine, but contains or glassware in microbiological assay procedures. Extremely
all other nutrients and vitamins essential for the growth of small amounts of foreign material may be sufficient to give
S. cerevisiae ATCC 9080. The addition of pyridoxine in erroneous results. Scrupulously clean glassware free from
specified increasing concentrations gives a growth response that detergents and other chemicals must be used. Glassware
can be measured turbidimetrically or titrimetrically. must be heated to 250°C for at least 1 hour to burn off any
organic residues that might be present. Take precautions to keep
sterilizing and cooling conditions uniform throughout assay.
459
460
R2A Agar
Intended Use balance the pH and provide phosphate. Magnesium sulfate is
R2A Agar is used for enumerating heterotrophic organisms in a source of divalent cations and sulfate. Agar is the solidifying
treated potable water. agent.
Limitations of the Procedure 4. Means, Hanami, Ridgway and Olson. 1981. J. Am. Water Works Assoc. 53:585.
5. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
1. R2A Agar is intended for use only with treated potable water 21st ed., online. American Public Health Association, Washington, D.C.
6. Kim and Feng. 2001. In Downes and Ito (ed.), Compendium of methods for the microbiological
since it is recommended for compromised bacteria. examination of foods, 4th ed. American Public Health Association, Washington, D.C.
7. Van Soestberger and Lee. 1969. Appl. Microbiol. 18:1092.
2. Use of the pour plate method is discouraged because 8. Klein and Wu. 1974. Appl. Microbiol. 27:429.
of cycloheximide dissolved in a small quantity of acetone per 3. Autoclave at 121°C for 15 minutes. Avoid overheating, which
liter of medium before autoclaving. Yeasts and gram-negative will cause a softer medium.
bacteria are suppressed, facilitating enumeration of the lactic 4. Test samples of the finished product for performance using
bacterial flora. stable, typical control cultures.
463
Principles of the Procedure indicated by a change of the phenol red indicator from red to
Rapid Fermentation Medium produces rapid results in detect- yellow. The control cultures should produce results as shown
ing acid production from dextrose, maltose, sucrose and in the table. If the results with the control cultures do not agree
other carbohydrates. Although much of the literature refers to with those in the table, review the procedure, check the control
fermentation patterns for N. gonorrhoeae, it has been shown cultures by Gram-staining and performing the oxidase test and
that this species metabolizes dextrose by strictly aerobic repeat the fermentation test if necessary. The control tube (no
mechanisms; i.e., by a combination of the Entner-Doudoroff carbohydrate) should be negative (red).
and pentose phosphate pathways. The utilization of dextrose Control Organisms ATCC™ Production of Acid from:
by N. gonorrhoeae, as indicated by an acid change in the pH Neisseria gonorrhoeae 43070 Dextrose only
indicator present in the medium, is due to the production of Neisseria meningitidis 13090 Dextrose and Maltose
acetic acid and small amounts of lactic acid.4 The negative Neisseria lactamica 23970 Dextrose, Maltose and Lactose
carbohydrate test is the result of the deamination of the Neisseria sicca 29193 Dextrose, Maltose and Sucrose
peptone in the absence of any utilizable carbohydrate.
If no positive carbohydrate reactions are observed within
Procedure 4 hours, the tubes may be incubated overnight or longer to allow
1. Remove a full loopful of fresh colony growth from the a positive reaction to develop.
surface of a Chocolate II Agar plate or slant. A large
inoculum must be used in order to obtain a rapid reaction. References
2. Deposit the inoculum below the surface of the medium and 1. Flynn and Waitkins. 1972. J. Clin. Pathol. 25:525.
2. Morse and Bartenstein. 1976. J. Clin. Microbiol. 3:8.
mix well. 3. Knapp and Koumans. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical
microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
3. Repeat for each tube to be inoculated. Inoculate a control 4. Morse, Stein and Hines. 1974. J. Bacteriol. 120:702.
tube (no carbohydrate) with Neisseria sp.
4. Incubate all tubes at 35 ± 2°C in an aerobic atmosphere Availability
without carbon dioxide. Observe periodically after 4 hours BBL™ Rapid Fermentation Medium and Rapid
for reactions noted below. Continue incubation overnight if Fermentation Medium with Carbohydrates
necessary. A few strains may require incubation for up to Cat. No. 221890 Prepared Tubes (K Tubes) – Pkg. of 10*
221891 Prepared Tubes, Dextrose – Pkg. of 10*
48-72 hours. 221893 Prepared Tubes, Lactose – Pkg. of 10*
221894 Prepared Tubes, Maltose – Pkg. of 10*
Expected Results 221895 Prepared Tubes, Sucrose – Pkg. of 10*
*Store at 2-8°C.
After incubation, compare the reactions produced by the
unknown isolates with those produced by known control
organisms. Negative reactions are red. A positive reaction is
464
Identity Specifications
Difco™ Rappaport-Vassiliadis Medium, Semisolid
Modification
Dehydrated Appearance: Pale green, free-flowing, homogeneous.
Solution: 3.16% solution, soluble in purified water upon boil-
ing. Solution is blue, clear to slightly opalescent.
Prepared Appearance: Blue, slightly opalescent, semisolid.
Reaction of 3.16%
Solution at 25°C: pH 5.2 ± 0.2
Cultural Response
Difco™ Rappaport-Vassiliadis Medium, Semisolid
Modification
Prepare the medium per label directions. Inoculate and incubate at
42 ± 0.5°C for 18-24 hours.
INOCULUM halo/
Organism ATCC™ CFU RECOVERY Motility
Citrobacter freundii 8090 103-2×103 Marked –
inhibition
Proteus mirabilis 9240 103-2×103 None –
Pseudomonas aeruginosa 27853 103-2×103 None –
Salmonella enterica
subsp. enterica
serotype Enteritidis 13076 102-103 Good +
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028 102-103 Good +
Salmonella senftenberg
(NCTC 10384) 102-103 Good +
467
468
469
References 14. United Stated Department of Agriculture. 2008. Microbiology laboratory guidebook, online. MLG
4.04, Isolation and identification of Salmonella from meat, poultry and egg products. USDA, Food
1. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The national Safety and Inspection Service, Office of Public Health Science, Athens, Ga.
formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc., Rockville, Md. 15. International Organization for Standardization. 2002. Microbiology of food and animal feeding
2. European Directorate for the Quality of Medicines and Healthcare. 2008. The European pharmacopoeia, stuffs – horizontal method for the detection of Salmonella spp. ISO 6579, 2002-07-15. International
6th ed., Supp. 1, 4-1-2008, online. European Directorate for the Quality of Medicines and Healthcare, Organization for Standardization, Geneva, Switzerland.
Council of Europe, 226 Avenue de Colmar BP907-, F-67029 Strasbourg Cedex 1, France.
3. Japanese Ministry of Health, Labour and Welfare. 2006. The Japanese pharmacopoeia, 15th ed.,
online. Japanese Ministry of Health, Labour and Welfare.
4. Rappaport, Konforti and Navon. 1956. J. Clin. Pathol. 9:261.
Availability
5. Rappaport and Konforti. 1959. Appl. Microbiol. 7:63. Difco™ RVS Soy Broth
6. Vassiliadis, Paternaki, Papaiconomon, Papadakis and Trichopoulos. 1976. Ann. Microbiol. Inst.
Pasteur. 127B:195. CCAM EP ISO JP USDA USP
7. Van Schothorst and Renaud. 1983. J. Appl. Bacteriol. 54:209.
8. McGibbon, Quail, and Fricker. 1984. Int. J. Food Microbiol. 1:171. Cat. No. 214943 Dehydrated – 500 g†
9. Fricker and Girdwood. 1985. J. Appl. Bacteriol. 58:343.
10. Fricker, Quail, McGibbon, and Girdwood. 1985. J. Hyg. Cambridge. 95:337. BBL™ RVS Soy Broth
11. Quail, McGibbon and Fricker. 1986. J. Hyg. Cambridge. 96:425.
12. Peterz, Wiberg and Norberg. 1989. J. Appl. Bacteriol. 66:523. CCAM EP ISO JP USDA USP
13. International Organization for Standardization. 2001. Milk and milk products – Detection of Salmo- Cat. No. 215199 Prepared Tubes, 10 mL – Pkg. of 10†
nella spp. ISO 6785, IDF 93, 2001-05-15. International Organization for Standardization, Geneva,
Switzerland. † QC testing performed according to USP/EP/JP performance specifications.
Identity Specifications
BBL™ Regan-Lowe Charcoal Agar Base
Dehydrated Appearance: Fine, homogeneous, free of extraneous
material.
Solution: 5.1% solution, soluble in purified water upon
boiling. Solution is charcoal black, homoge-
neous, opaque.
Prepared Appearance: Charcoal black, homogeneous, opaque.
Reaction of 5.1%
Solution at 25°C: pH 7.4 ± 0.2
Cultural Response
BBL™ Regan-Lowe Charcoal Agar Base
Prepare the medium per label directions. Inoculate with fresh broth
cultures diluted 1:10 and incubate at 35 ± 2°C for 7 days.
ORGANISM ATCC™ recovery
Bordetella pertussis 9797 Good
Bordetella parapertussis 15311 Good
470
Agar.......................................................................... 12.0 g
*Adjusted and/or supplemented as required to meet performance criteria. References Q
Directions for Preparation from
1. Regan and Lowe. 1977. J. Clin. Microbiol. 6:303.
2. Sneed. 1992. In Isenberg (ed.), Clinical microbiology procedure handbook, vol. 1. American Society
for Microbiology, Washington, D.C.
R
Dehydrated Product 3. Marcon. 1995. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology,
6th ed. American Society for Microbiology, Washington, D.C.
4. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
1. Suspend 51 g of the powder in 1 L of purified water. Mix American Society for Microbiology, Washington, D.C.
thoroughly.
2. Heat with frequent agitation and boil for 1 minute to Availability
completely dissolve the powder. BBL™ Regan-Lowe Charcoal Agar Base
3. Autoclave at 121°C for 15 minutes. DO NOT OVERHEAT. Cat. No. 298123 Dehydrated – 500 g
4. For preparation of blood plates, add 10% sterile, defibrinated BBL™ Regan-Lowe Charcoal Agar
horse blood to sterile agar which has been previously melted BS12 CMPH2 MCM9
and cooled to 45-50°C. Cat. No. 297883 Prepared Plates – Pkg. of 10*
5. For selective isolation of B. pertussis and B. parapertussis, 297855 Prepared Tubes (Deeps), 10 mL – Pkg. of 10*
add 40 µg of cephalexin per mL. BBL™ Regan-Lowe Charcoal Agar without Cephalexin
6. Test samples of the finished product for performance using Cat. No. 298326 Prepared Plates – Pkg. of 10*
stable, typical control cultures. *Store at 2-8°C.
471
472
CCAM EP JP USP Cat. No. 215192 Prepared Bottles, 100 mL (septum screw cap) –
Pkg. of 10†
Cat. No. 218081 Dehydrated – 500 g†
Europe
CCAM EP
Cat. No. 254548 Prepared Plates – Pkg. of 20*
* Store at 2-8°C.
† QC testing performed according to USP/EP/JP performance specifications.
473
Directions for Preparation from standard curve is obtained by using Riboflavin USP Reference
Dehydrated Product Standard or equivalent at levels of 0.0, 0.025, 0.05, 0.075, 0.1,
1. Suspend 4.8 g of the powder in 100 mL of purified water. 0.15, 0.2 and 0.3 µg riboflavin per assay tube (10 mL).
2. Heat with frequent agitation and boil for 2-3 minutes to The concentration of riboflavin required for the preparation
completely dissolve the powder. of the standard curve may be prepared by dissolving 0.1 g of
3. Dispense 5 mL amounts into tubes, evenly dispersing the Riboflavin USP Reference Standard or equivalent in 1,000 mL
precipitate. of purified water by heating, giving a stock solution of 100 µg
4. Add standard or test samples. per mL. Dilute the stock solution by adding 1 mL to 999 mL
5. Adjust the volume to 10 mL with purified water. purified water. Use 0.0, 0.25, 0.5, 0.75, 1, 1.5, 2 and 3 mL of
6. Autoclave at 121°C for 10 minutes. the diluted stock solution per tube. Prepare the stock solution
fresh daily.
Procedure
Follow applicable assay procedures.2 Levels of riboflavin used Expected Results
in the determination of the standard curve should be prepared 1. Prepare a standard concentration response curve by
according to this reference or according to the following plotting the response readings against the amount of standard
procedure. in each tube, disk or cup.
Stock Cultures 2. Determine the amount of vitamin at each level of assay solu-
tion by interpolation from the standard curve.
Stock cultures of L. rhamnosus ATCC 7469 are prepared
3. Calculate the concentration of vitamin in the sample from
by stab inoculation into 10 mL of Lactobacilli Agar AOAC.
the average of these values. Use only those values that do not
After 24-48 hours incubation at 35-37°C, the stock cultures are
vary more than ±10% from the average and use the results
kept in the refrigerator. Transfers are made at monthly intervals
only if two-thirds of the values do not vary by more than
in triplicate.
±10%.
Inoculum
Inoculum for assay is prepared by subculturing a stock culture Limitations of the Procedure
of L. rhamnosus ATCC 7469 into 10 mL of Lactobacilli 1. The test organism used for inoculating an assay medium must
Broth AOAC or Micro Inoculum Broth. Following incubation be cultured and maintained on media recommended for this
for 16-24 hours at 35-37°C, the culture is centrifuged under purpose.
aseptic conditions and the supernatant liquid decanted. After 2. Aseptic technique should be used throughout the assay
washing 3 times with 10 mL sterile 0.85% saline, the cells are procedure.
resuspended in 10 mL sterile 0.85% saline. The cell suspen- 3. The use of altered or deficient media may cause mutants
sion is then diluted with sterile 0.85% saline, to a turbidity of having different nutritional requirements that will not give
35-40% transmittance when read on the spectrophotometer a satisfactory response.
at 660 nm. One drop of this latter suspension is then used to 4. For successful results of these procedures, all conditions of
inoculate each of the assay tubes. the assay must be followed precisely.
5. Maintain pH below 7.0 to prevent loss of riboflavin.
Riboflavin Assay Medium may be used for both turbidimetric
and titrimetric determinations. Turbidimetric readings should
References
be made after 18-24 hours incubation at 35-37°C, whereas 1. Snell and Strong. 1939. Ind. Eng. Chem. 11:346.
titrimetric determinations are best made after 72 hours incuba- 2. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
International, Gaithersburg, Md.
tion at 35-37°C. Using Riboflavin Assay Medium, the most
effective assay range is between 0.025 and 0.15 µg riboflavin. Availability
Standard Curve Difco™ Riboflavin Assay Medium
It is essential that a standard curve be constructed each time AOAC
an assay is run. Conditions of autoclaving and temperature Cat. No. 232510 Dehydrated – 100 g*
*Store at 2-8°C.
of incubation, which influence the standard curve readings,
cannot be duplicated exactly from assay to assay. The
474
477
Identity Specifications
Difco™ Rose Bengal Agar Base
Dehydrated Appearance: Beige to faint pink, free-flowing, homo-
geneous.
Solution: 3.2% solution, soluble in purified water
upon boiling. Solution is reddish pink,
very slightly to slightly opalescent.
Prepared Appearance: Bright pink, very slightly to slightly opal-
escent.
Reaction of 3.2%
Solution at 25°C: pH 7.2 ± 0.2
Difco Rose Bengal Antimicrobic Supplement C
™
Cultural Response
Difco™ Rose Bengal Agar Base with Antimicrobic
Supplement C
Prepare the medium per label directions. Inoculate using the pour plate
technique (for Aspergillus niger, inoculate the surface of an agar slant)
and incubate aerobically at 25-30°C for up to 7 days.
Candida albicans
INOCULUM Colony ATCC™ 10231
Organism ATCC™ CFU Recovery Color
Aspergillus niger 1015 Fresh Good White to black
Candida albicans 10231 102-3×102 Good Pink
Escherichia coli 25922 103-2×103 Marked to –
complete inhibition
Micrococcus luteus 10240 103-2×103 Marked to –
complete inhibition
478
Availability
Expected Results Difco™ Rose Bengal Agar Base
Colonies of yeast appear pink due to the uptake of rose bengal. SMWW
Count plates containing 15-150 colonies and report the counts Cat. No. 218312 Dehydrated – 500 g
as colony-forming units (CFU) per gram or mL of sample.
Difco™ Rose Bengal Antimicrobic Supplement C
SMWW
Limitations of the Procedure Cat. No. 214904 Vial – 10 × 3 mL*
1. Although this medium is selective primarily for fungi,
microscopic examination is recommended for presumptive Difco™ Hycheck™ Hygiene Contact Slides
Cat. No. 290006 Rose Bengal Chloramphenicol Agar//
identification. Biochemical testing using pure cultures is
Tryptic Soy Agar – Pkg. of 10 slides*
required for complete identification. 290007 Rose Bengal Chloramphenicol Agar//
2. Due to the selective properties of this medium and the Tryptic Soy Agar with 0.01% TTC –
type of specimen being cultured, some strains of fungi may
be encountered that fail to grow or grow poorly on the
*Store at 2-8°C.
Pkg. of 10 slides*
S
complete medium; similarly, some strains of bacteria may
be encountered that are not inhibited or only partially
inhibited.
3. Care should be taken not to expose this medium to light,
since photodegradation of rose bengal yields compounds
that are toxic to fungi.13
SF Medium • SF Broth
Intended Use Principles of the Procedure
SF (Streptococcus Faecalis) Medium (Broth) is used for the Peptone and dextrose supply the nutrients required for the
differentiation of Enterococcus species from the Streptococcus growth of enterococci. Sodium chloride maintains the osmotic
bovis group and other streptococci. balance of the medium. Sodium azide exhibits a bacteriostatic
effect on gram-negative bacteria through its inhibitory action
Summary and Explanation on enzymes in the electron transport system. Bromcresol purple
The formulation of SF Medium was developed by Hajna and serves as a pH indicator.
Perry1 as a result of their comparative study of presumptive
and confirmatory media for the detection of coliforms and Formula
fecal streptococci. It was recommended for use in the ex- Difco™ SF Medium
amination of waters and other materials for the presence of Approximate Formula* Per Liter
fecal streptococci as an indicator of pollution. The use of SF Tryptone.................................................................... 20.0 g
Dextrose...................................................................... 5.0 g
Medium in sanitary bacteriology has been replaced by more Dipotassium Phosphate................................................ 4.0 g
selective media recommended in current compendia of methods Monopotassium Phosphate.......................................... 1.5 g
for the examination of waters and foods.2-4 Sodium Chloride.......................................................... 5.0 g
Sodium Azide............................................................... 0.5 g
For diagnostic microbiology purposes, the medium is useful in Bromcresol Purple...................................................... 32.0 mg
*Adjusted and/or supplemented as required to meet performance criteria.
differentiation of enterococci from streptococci. Pure cultures
of streptococci are inoculated into SF Medium in order to
determine if the respective culture is Enterococcus sp. Entero-
cocci ferment dextrose and grow in the presence of the inhibitor
sodium azide.
479
480
Identity Specifications
Difco™ SFP Agar Base
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution: 47 g, soluble in 900 mL purified water
upon boiling. Solution is medium to dark
amber, slightly opalescent.
Prepared Appearance (Final): Canary yellow, opaque.
Reaction of 47g/900 mL
Solution at 25°C: pH 7.6 ± 0.2
Difco Egg Yolk Enrichment 50%
™
S
ATCC™ 12924
Prepare the base layer and cover layer per label directions, inoculating the base
layer. Incubate at 35 ± 2°C under anaerobic conditions for 18-48 hours.
INOCULUM
Organism ATCC™ CFU RECOVERY COLONY COLOR
Clostridium perfringens 12919 30-300 Good Black with a zone of precipitation (halo)
Clostridium perfringens 12924 30-300 Good Black with a zone of precipitation (halo)
in food samples plays a role in the epidemiological investigation Difco™ Egg Yolk Enrichment 50%
of outbreaks of foodborne illness.2 Concentrated egg yolk emulsion.
Difco™ Antimicrobic Vial K
SFP Agar (with added kanamycin and polymyxin B) is compa-
25 mg Kanamycin per 10 mL vial.
rable to Tryptose Sulfite Cycloserine (TSC) Agar, which uses
cycloserine as the inhibitory component.2,4,5 Difco™ Antimicrobic Vial P
30,000 units Polymyxin B per 10 mL vial.
*Adjusted and/or supplemented as required to meet performance criteria.
Principles of the Procedure
SFP Agar Base contains peptones as sources of carbon, nitrogen, Directions for Preparation from
vitamins and minerals. Yeast extract supplies B-complex Dehydrated Product
vitamins, which stimulate bacterial growth. Ferric ammonium Difco™ SFP Agar Base
citrate and sodium sulfite are H2S indicators. Clostridia
Base Layer:
reduce sulfite to sulfide, which reacts with iron to form a black
1. Suspend 47 g of the powder in 900 mL of purified water.
iron sulfide precipitate. Antimicrobic Vial P contains polymyxin
Mix thoroughly.
B and Antimicrobic Vial K contains kanamycin; both are
2. Heat with frequent agitation and boil for 1 minute to
inhibitors to organisms other than Clostridium spp. Egg Yolk completely dissolve the powder.
Enrichment 50% provides egg yolk lecithin, which some 3. Autoclave at 121°C for 15 minutes. Cool to 50°C.
clostridia hydrolyze. Agar is the solidifying agent. 4. Add 100 mL Egg Yolk Enrichment 50%, 10 mL of
rehydrated Antimicrobic Vial P (30,000 units polymyxin
Formulae B sulfate) and 4.8 mL rehydrated Antimicrobic Vial K
Difco™ SFP Agar Base
(12 mg kanamycin). Mix thoroughly.
Approximate Formula* Per Liter
Yeast Extract................................................................ 5.0 g Cover Layer:
Proteose Peptone No. 3................................................ 7.5 g
Pancreatic Digest of Casein.......................................... 7.5 g
1. Suspend 47 g of the powder in 1 L of purified water.
Soytone....................................................................... 5.0 g 2. Prepare as above, except omit Egg Yolk Enrichment 50%.
Ferric Ammonium Citrate............................................. 1.0 g 3. Test samples of the finished product for performance using
Sodium Bisulfite........................................................... 1.0 g
Agar.......................................................................... 20.0 g
stable, typical control cultures.
481
SIM Medium
Intended Use Principles of the Procedure
SIM Medium is used to differentiate enteric bacilli on the The ingredients in SIM Medium enable the determination of
basis of sulfide production, indole formation and motility. three activities by which enteric bacteria can be differentiated.
Sodium thiosulfate and ferrous ammonium sulfate are indica-
Summary and Explanation tors of hydrogen sulfide production. The ferrous ammonium
Hydrogen sulfide production, indole formation and motility sulfate reacts with H2S gas to produce ferrous sulfide, a black
are distinguishing characteristics which aid in the identification precipitate.1 The casein peptone is rich in tryptophan, which is
of the Enterobacteriaceae, especially Salmonella and Shigella. attacked by certain microorganisms resulting in the production
SIM Medium, therefore, is useful in the process of identification of indole. The indole is detected by the addition of chemical
of enteric pathogens. reagents following the incubation period. Motility detection
Cultural Response
BBL™ SIM Medium
Prepare the medium per label directions. Stab inoculate using heavy inocula of fresh cultures
and incubate at 35 ± 2°C for 18-24 hours. Uninoculated Escherichia coli Salmonella
Tube ATCC™ 25922 Typhimurium
ORGANISM ATCC™ recovery MOTILITY H2S INDOLE with indole ATCC™ 13311
reagent with indole
Escherichia coli 25922 Good + – + reagent
Salmonella enterica
subsp. enterica
serotype Typhimurium 13311 Good + + –
Shigella sonnei 9290 Good – – –
482
is possible due to the semisolid nature of the medium. Growth Expected Results
radiating out from the central stab line indicates that the test Following incubation, observe for motility (diffuse growth
organism is motile. outward from the stab line or turbidity throughout the
medium) and for H2S production (blackening along the stab line).
Formula To detect indole production, add three or four drops of Kovacs’
BBL™ SIM Medium reagent2 and observe for a red color (positive reaction).
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 20.0 g Consult appropriate references for activities of specific micro-
Peptic Digest of Animal Tissue...................................... 6.1 g organisms.2-4
Ferrous Ammonium Sulfate.......................................... 0.2 g
Sodium Thiosulfate...................................................... 0.2 g
Agar............................................................................ 3.5 g References
*Adjusted and/or supplemented as required to meet performance criteria. 1. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol.
1. Williams & Wilkins, Baltimore, Md.
2. Ewing. 1986. Edwards and Ewing’s identification of Enterobacteriaceae, 4th ed. Elsevier Science
Directions for Preparation from Publishing Co., Inc., New York, N.Y.
3. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology,
Dehydrated Product 9th ed. Williams & Wilkins, Baltimore, Md.
4. Farmer. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology,
1. Suspend 30 g of the powder in 1 L of purified water. Mix 7th ed. American Society for Microbiology, Washington, D.C.
thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely Availability
dissolve the powder. BBL™ SIM Medium
BAM
3. Dispense and autoclave at 121°C for 15 minutes.
Cat. No. 211578 Dehydrated – 500 g
4. Test samples of the finished product for performance using 221010 Prepared Tubes – Pkg. of 10
stable, typical control cultures. 221011 Prepared Tubes – Ctn. of 100
Procedure S
Loosen caps, boil and cool before use. Using growth from
a pure culture, stab an inoculating needle two-thirds of the
distance to the bottom in the center of the tube. Incubate tubes
with loosened caps for 18-24 hours at 35 ± 2°C in an aerobic
atmosphere.
SOB Medium
Intended Use Summary and Explanation
SOB Medium is used for cultivating recombinant strains of SOB Medium was developed by Hanahan1 as a nutritionally
Escherichia coli. rich growth medium for preparation and transformation of
competent cells. Transformation requires making perforations
User Quality Control in the bacterium (i.e., making the cells “competent”) to allow
the introduction of foreign DNA into the cell. To survive this
Identity Specifications
process, competent cells need a rich, isotonic environment.
Difco™ SOB Medium
Dehydrated Appearance: Light beige, free-flowing, homogeneous. SOC Medium, used in the final stage of transformation, may
Solution: 2.8% solution, soluble in purified water. Solution be prepared by aseptically adding 20 mL of a filter-sterilized
is light to medium amber, clear.
20% solution of glucose (dextrose) to the sterile SOB Medium.
Prepared Appearance: Light to medium amber, clear.
This addition provides a readily available source of carbon and
Reaction of 2.8%
Solution at 25°C: pH 7.0 ± 0.2
energy in a form E. coli can use in mending the perforations
and for replication.2
Cultural Response
Difco™ SOB Medium Principles of the Procedure
Prepare the medium per label directions. Inoculate and incubate at Peptone and yeast extract provide sources of nitrogen and
35 ± 2°C for 18-24 hours. growth factors which allow the bacteria to recover from the
Organism ATCC™ INOCULUM CFU RECOVERY stress of transformation and grow well. Sodium chloride
Escherichia coli (DH-5) 53868 102-3×102 Good and potassium chloride provide essential ions. Magnesium
sulfate is a source of magnesium ions required in a variety of
enzymatic reactions, including DNA replication.
483
Formula Procedure
Difco™ SOB Medium Consult appropriate references for recommended test proce-
Approximate Formula* Per Liter dures.2
Tryptone.................................................................... 20.0 g
Yeast Extract................................................................ 5.0 g
Sodium Chloride.......................................................... 0.5 g Expected Results
Magnesium Sulfate (anhydrous)................................... 2.4 g Growth is evident in the form of turbidity.
Potassium Chloride.................................................. 186.0 mg
*Adjusted and/or supplemented as required to meet performance criteria.
References
1. Hanahan. 1983. J. Mol. Biol. 166:557.
Directions for Preparation from 2. Sambrook, Fritsch and Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.
Dehydrated Product
1. Suspend 28 g of the powder in 1 L of purified water. Mix Availability
thoroughly. Difco™ SOB Medium
2. Heat with frequent agitation and boil for 1 minute to Cat. No. 244310 Dehydrated – 500 g
completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. If desired, SOC Medium may be prepared by adding
20 mL filter-sterilized 20% glucose solution after cooling the
medium to 45-50°C.
5. Test samples of the finished product for performance using
stable, typical control cultures.
SPS Agar
Intended Use Formula
SPS Agar is used for detecting and enumerating Clostridium Difco™ SPS Agar
perfringens in food. Approximate Formula* Per Liter
Tryptone ................................................................... 15.0 g
Yeast Extract.............................................................. 10.0 g
Summary and Explanation Ferric Citrate................................................................ 0.5 g
In the 1950s, Mossel1 and Mossel et al.2 proposed media for Sodium Sulfite.............................................................. 0.5 g
enumerating anaerobic sulfite-reducing clostridia in foods. Sodium Thioglycollate.................................................. 0.1 g
Polysorbate 80............................................................. 0.05 g
Angelotti et al.3 modified the formula as Sulfite Polymyxin Sulfadiazine................................................................. 0.12 g
Sulfadiazine (SPS) Agar and used it to quantitate C. perfringens Polymyxin B Sulfate...................................................... 0.01 g
in foods. Agar.......................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.
Identity Specifications
Difco™ SPS Agar
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution: 4.1% solution, soluble in purified water upon
boiling. Solution is light to medium amber, very
slightly to slightly opalescent.
Prepared Appearance: Light to medium amber, slightly opalescent.
Reaction of 4.1%
Solution at 25°C: pH 7.0 ± 0.2
Cultural Response
Difco™ SPS Agar
Prepare the medium per label directions. Inoculate using the pour plate
technique and incubate anaerobically at 35 ± 2°C for 24-48 hours.
INOCULUM COLONY
Organism ATCC™ CFU RECOVERY COLOR
Clostridium perfringens 12919 102-103 Good Black
Clostridium sporogenes 11437 102-103 None to fair Black
Escherichia coli 25922 102-103 Marked to –
complete inhibition
Salmonella enterica Staphylococcus
aureus
subsp. enterica Marked to
S
ATCC™ 25923
serotype Typhimurium 14028 102-103 complete inhibition –
Staphylococcus aureus 25923 102-103 Fair to good White
References
1. Mossel. 1959. J. Sci. Food Agric. 19:662.
2. Mossel, DeBruin, van Diepen, Vendrig and Zoutewelle. 1956. J. Appl. Microbiol. 19:142.
3. Angelotti, Hall, Foster and Lewis. 1962. Appl. Microbiol. 10:193.
4. Labbe. 2001. In Downes and Ito (ed.), Compendium of methods for the microbiological examination
of foods, 4th ed. American Public Health Association, Washington, D.C.
5. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
tional, Gaithersburg, Md.
Formulae 2. Heat with frequent agitation and boil for 1 minute to com-
Difco SS Agar
™ pletely dissolve the powder. DO NOT AUTOCLAVE.
Approximate Formula* Per Liter 3. Cool the medium to approximately 45-50°C and pour into
Beef Extract................................................................. 5.0 g Petri dishes.
Proteose Peptone........................................................ 5.0 g
Lactose..................................................................... 10.0 g 4. Allow the plates to dry for approximately 2 hours with the
Bile Salts No. 3............................................................ 8.5 g covers partially removed.
Sodium Citrate............................................................ 8.5 g 5. Test samples of the finished product for performance using
Sodium Thiosulfate.................................................... 8.5 g
Ferric Citrate............................................................... 1.0 g stable, typical control cultures.
Agar......................................................................... 13.5 g
Brilliant Green............................................................. 0.33 mg Procedure
Neutral Red............................................................... 25.0 mg Use standard procedures to obtain isolated colonies from speci-
BBL™ Salmonella Shigella Agar mens. A nonselective medium should also be streaked to increase
Approximate Formula* Per Liter the chance of recovery when the population of gram-negative
Beef Extract................................................................. 5.0 g
Pancreatic Digest of Casein......................................... 2.5 g organisms is low and to provide an indication of other organisms
Peptic Digest of Animal Tissue..................................... 2.5 g present in the specimen. Incubate plates, protected from light, at
Lactose..................................................................... 10.0 g 35 ± 2°C for 18-24 hours. If negative after 24 hours, reincubate
Bile Salts..................................................................... 8.5 g
Sodium Citrate............................................................ 8.5 g an additional 24 hours.
Sodium Thiosulfate..................................................... 8.5 g
Ferric Citrate............................................................... 1.0 g Expected Results
Agar......................................................................... 13.5 g Typical colonial morphology on Salmonella Shigella Agar is
Brilliant Green............................................................. 0.33 mg
Neutral Red............................................................... 25.0 mg as follows:
*Adjusted and/or supplemented as required to meet performance criteria. Escherichia coli................... Slight growth, pink or red
Enterobacter/Klebsiella....... Slight growth, pink
Directions for Preparation from Proteus.............................. Colorless, usually with black center
Salmonella......................... Colorless, usually with black center
Dehydrated Product Shigella.............................. Colorless
1. Suspend 60 g of the powder in 1 L of purified water. Mix Pseudomonas..................... Irregular, slight growth
thoroughly. Gram-positive bacteria....... No growth
486
Shigella flexneri
ATCC™ 12022
Salmonella Typhimurium
ATCC™ 14028 References
1. Leifson. 1935. J. Pathol. Bacteriol. 40:581.
2. Taylor and Harris. 1965. Am. J. Clin. Pathol. 44:476.
3. Pollock and Dahlgren. 1974. Appl. Microbiol. 27:197.
Availability
Difco™ SS Agar
BS12 CMPH2 COMPF MCM9
Cat. No. 274500 Dehydrated – 500 g
212118 Dehydrated – 2 kg
274300 Dehydrated – 10 kg
BBL™ Salmonella Shigella Agar
BS12 CMPH2 COMPF MCM9
Cat. No. 211596 Dehydrated – 100 g
211597 Dehydrated – 500 g
211600 Dehydrated – 5 lb (2.3 kg)
293306 Dehydrated – 25 lb (11.3 kg)
United States and Canada
Cat. No. 221181 Prepared Plates – Pkg. of 20*
221279 Prepared Plates – Ctn. of 100*
Europe
Cat. No. 254047 Prepared Plates – Pkg. of 20*
254085 Prepared Plates – Ctn. of 120*
Japan
Cat. No. 251181 Prepared Plates – Pkg. of 20*
487
Expected Results
After 18-48 hours of incubation, the plates should show Availability
isolated colonies in streaked areas and confluent growth in BBL™ SXT Blood Agar
areas of heavy inoculation. Group A or B streptococci may be BS12 cmph2 mcm9
presumptively identified as small, translucent to opaque, white Cat. No. 221809 Prepared Plates – Pkg. of 20*
221810 Prepared Plates – Ctn. of 100*
to gray colonies surrounded by zones of beta hemolysis. Gram *Store at 2-8°C.
stains, biochemical tests, susceptibility to bacitracin, utilizing
Taxo™ A (0.04 unit) discs, and serological procedures should
be performed to confirm findings.
488
Identity Specifications
Difco™ Sabouraud Brain Heart Infusion Agar Base
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 5.9% solution, soluble in purified water upon
boiling. Solution is medium amber, very slightly to
slightly opalescent.
Prepared Appearance: Medium amber, very slightly to slightly opales-
cent.
Reaction of 5.9%
Solution at 25°C: pH 7.0 ± 0.2
Cultural Response
Difco™ Sabouraud Brain Heart Infusion Agar Base
Prepare the medium per label directions without (plain) and with 10% sheep
blood (SB). Inoculate and incubate at 30 ± 2°C for 18-48 hours and up to
7 days for Trichophyton.
INOCULUM RECOVERY
ORGANISM ATCC™ CFU plain AND WITH SB
Aspergillus brasiliensis (niger) 16404 102-103 Good
Candida albicans 10231 102-103 Good
Escherichia coli 25922 103-2×103 Marked to Saccharomyces
complete inhibition cerevisiae
S
ATCC™ 9763
Saccharomyces cerevisiae 9763 102-103 Good
Staphylococcus aureus 25923 103-2×103 Marked to
complete inhibition
Trichophyton
mentagrophytes 9533 102-103 Good
Formula side up) with increased humidity. For isolation of fungi causing
Difco™ Sabouraud Brain Heart Infusion Agar Base systemic mycoses, two sets of media should be inoculated, with
Approximate Formula* Per Liter one set incubated at 25-30°C and a duplicate set at 35 ± 2°C.
Brain Heart Digest........................................................ 9.25 g
Proteose Peptone......................................................... 5.0 g All cultures should be examined at least weekly for fungal
Enzymatic Digest of Casein.......................................... 5.0 g growth and should be held for 4-6 weeks before being reported
Dextrose.................................................................... 21.0 g
as negative.
Sodium Chloride.......................................................... 2.5 g
Disodium Phosphate.................................................... 1.25 g
Agar.......................................................................... 15.0 g Expected Results
*Adjusted and/or supplemented as required to meet performance criteria.
After sufficient incubation, the plates should show isolated
colonies in streaked areas and confluent growth in areas of
Directions for Preparation from heavy inoculation.
Dehydrated Product
1. Suspend 59 g of the powder in 1 L of purified water. Mix Examine the plates for fungal colonies exhibiting typical color
thoroughly. and morphology. Biochemical tests and serological procedures
2. Heat with frequent agitation and boil for 1 minute to should be performed to confirm findings.
completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes. Cool to 50-55°C. Limitation of the Procedure
4. Aseptically add 1 mL chloramphenicol solution (100 mg/mL) Some fungi may be inhibited by the antibiotics in selective
and, if desired, 10% sterile sheep blood. formulations.3,4
5. Test samples of the finished product for performance using
stable, typical control cultures.
References
1. Gorman. 1967. Am. J. Med. Technol. 33: 151.
2. Merz and Roberts. 1995. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical
microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
Procedure 3. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
American Society for Microbiology, Washington, D.C.
Use standard procedures to obtain isolated colonies from 4. Kwon-Chung and Bennett. 1992. Medical mycology. Lea & Febiger, Philadelphia, Pa.
specimens.
For isolation of fungi from potentially contaminated specimens,
both a nonselective and a selective medium should be inoculated.
Incubate the plates at 25-30°C in an inverted position (agar
489
Prepared Appearance: Light amber, clear. Prepare the medium per label directions. Inoculate tubes and incubate at
Reaction of 3.0% 30± 2°C for 18-48 hours or up to 7 days if necessary. For (*) culture inocu-
Solution at 25°C: pH 5.6 ± 0.2 late a 125 mL bottle and incubate at 30-35°C for 48 hours. For (**) culture
inoculate a 125 mL bottle and incubate at 20-25°C for 3 days.
Difco Fluid Sabouraud Medium
™
Dehydrated Appearance: Off-white, free-flowing, homogeneous. ORGANISM ATCC™ INOCULUM CFU RECOVERY
Solution: 3.0% solution, soluble in purified water. Solution Aspergillus brasiliensis (niger) 16404 30-300 Good
S
is light amber, clear to very slightly opalescent. Candida albicans 10231 30-300 Good
Prepared Appearance: Light amber, clear to very slightly opalescent. Lactobacillus casei 9595 30-300 Good
Reaction of 3.0% Saccharomyces cerevisiae 9763 30-300 Good
Solution at 25°C: pH 5.7 ± 0.2
Candida albicans* 10231 <100 Growth
Difco™ Sabouraud Maltose Agar
Candida albicans** 10231 <100 Growth
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 6.5% solution, soluble in purified water upon Difco™ Fluid Sabouraud Medium
boiling. Solution is light amber, slightly opales- Prepare the medium per label directions. Inoculate and incubate at 30 ± 2°C
cent, may have a slight precipitate. for 18-72 hours.
Prepared Appearance: Very light amber, slightly opalescent without ORGANISM ATCC™ INOCULUM CFU RECOVERY
significant precipitate.
Aspergillus brasiliensis (niger) 16404 <100 Good
Reaction of 6.5%
Solution at 25°C: pH 5.6 ± 0.2 Candida albicans 10231 <100 Good
Continued
491
Appearance: Light to medium tan yellow; clear to trace Candida albicans* 10231 <100 Growth
hazy. Candida albicans** 10231 <100 Growth
Reaction at 25°C: pH 5.6 ± 0.2
BBL™ Sabouraud Dextrose Agar (prepared)
Candida albicans Inoculate and incubate at 25 ± 2°C for 7 days. Incubate Candida albicans
ATCC™ 10231 ATCC™ 60193 and Trichophyton for 3 days. Incubate (*) cultures at
20-25°C; 5 days for Aspergillus and 2 days for Candida. Incubate (**)
culture at 30-35°C for 2 days.
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Candida albicans 60193 Undiluted Good
Microsporum audouinii 9079 Undiluted Good
Nocardia asteroides 19247 Undiluted Good
Penicillium roquefortii 9295 Undiluted Good
Trichophyton mentagrophytes 9533 Undiluted Good
Aspergillus brasiliensis (niger)* 16404 <100 Growth
Candida albicans* 10231 <100 Growth
Candida albicans** 10231 <100 Growth
M. lanosum and Trichophyton gypseum.17 Davidson and Dowd- a large number of bacteria and makes the medium particularly well
ing also used this medium in isolating T. gypseum from a case suited for cultivating fungi and acidophilic microorganisms.
of tinea barbae.18
Sabouraud Dextrose Broth is used for culturing yeasts and molds
Principles of the Procedure
Sabouraud dextrose media are peptone media supplemented with
in cosmetics.9 General Chapter <62> of the USP recommends
dextrose to support the growth of fungi. Sabouraud agar is also
the use of Sabouraud Dextrose Broth when isolating Candida
available with maltose substituted for the dextrose. Peptones
albicans from nonsterile pharmaceutical products.1
are sources of nitrogenous growth factors. The carbohydrate
Sabouraud Maltose Broth is a modification of Sabouraud Dextrose provides an energy source for the growth of microorganisms.
Broth in which maltose is substituted for dextrose. It is selective Gentamicin is an aminoglycoside antibiotic that inhibits the
due to its acid pH and is used for the detection of fungi. growth of gram-negative bacteria. Chloramphenicol is inhibitory
to a wide range of gram-negative and gram-positive bacteria,
Fluid Sabouraud Medium is employed in sterility test procedures
and cycloheximide is an antifungal agent that is primarily
for determining the presence of molds, yeasts and aciduric micro-
active against saprophytic fungi and does not inhibit yeasts or
organisms. The acid reaction of the final medium is inhibitive to
dermatophytes.19
492
493
For the Sterile Pack media, sample selected surfaces by firmly 17. Davidson, Dowding and Buller. 1932. Can. J. Res. 6:1.
18. Davidson and Dowding. 1932. Arch. Dermatol. Syphilol. 26:660.
pressing the agar medium against the test area. Hold the plate 19. Lorian (ed.). 2005. Antibiotics in laboratory medicine: making a difference, 5th ed. Lippincott Williams
& Wilkins, Baltimore, Md.
with thumb and second finger and use index finger to press plate 20. Quisno, Gibby and Foter. 1946. Am. J. Pharm. 118: 320.
21. Erlandson and Lawrence. 1953. Science. 118: 274
bottom firmly against surface. Pressure should be the same for 22. Brummer. 1976. Appl. Environ. Microbiol. 32: 80.
23. Association for the Advancement of Medical Instrumentation. 2006. Sterilization of health care products –
every sample. Do not move plate laterally as this spreads con- radiation – Part 2: Establishing the sterilization dose. ANSI/AAMI/ISO 11137-2:2006. Association
taminants over the agar surface making resolution of colonies for the Advancement of Medical Instrumentation, Arlington, Va.
24. Larone. 1995. Medically important fungi: a guide to identification, 3rd ed. American Society for
difficult. Slightly curved surfaces may be sampled with a rolling Microbiology, Washington, D.C.
25. ICMSF. 2006. Microorganisms in foods 7. Intern. Comm. on Microbiol. Spec. for Foods. Kluwer
motion. Academic/Plenum Publishers, New York, N.Y.
494
BBL™ Sabouraud Dextrose Agar with Lecithin BBL™ Sabouraud Dextrose Broth
and Polysorbate 80 BAM EP JP USP
Cat. No. 221233 Sterile Pack RODAC™ Plates – Pkg. of 10* Cat. No. 215193 Prepared Bottles, 100 mL – Pkg. of 10†
222244 Sterile Pack RODAC™ SL Plates – Pkg. of 10*
215224 Sterile Pack RODAC™ SL Plates – Ctn. of 100* Difco™ Fluid Sabouraud Medium
292653 Isolator Pack, Finger Dab™ Prepared Plates Cat. No. 264210 Dehydrated – 500 g
(100 × 15 mm-style) – Pkg. of 10*
292654 Isolator Pack, Finger Dab™ Prepared Plates Difco™ Sabouraud Maltose Agar
(150 × 15 mm-style) – Pkg. of 5* Cat. No. 211020 Dehydrated – 500 g
495
Procedure References
Consult appropriate references for information about the 1. Sabouraud. 1892. Ann. Dermatol. Syphil. 3:1061.
2. Ajello, Georg, Kaplan and Kaufman. 1963. CDC laboratory manual for medical mycology. PHS
processing and inoculation of specimens.2,3 Publication No. 994, U.S. Government Printing Office, Washington, D.C.
3. LaRocco. 2007. In Murray, Baron, Jorgensen, Landry and Pfaller (ed.), Manual of clinical microbiol-
ogy, 9th ed. American Society for Microbiology, Washington, D.C.
Prepared tubed slants primarily are intended for use with pure 4. Kwon-Chung and Bennett. 1992. Medical mycology. Lea & Febiger, Philadelphia, Pa.
5. Lorian (ed.). 1996. Antibiotics in laboratory medicine, 4th ed. Williams & Wilkins, Baltimore, Md.
cultures for maintenance or other purposes. 6. Larone. 1995. Medically important fungi: a guide to identification, 3rd ed. American Society for
Microbiology, Washington, D.C.
For isolating fungi from potentially contaminated specimens,
a selective medium should be inoculated along with the nonse- Availability
lective medium. Incubate the plates at 25-30°C in an inverted Difco™ Sabouraud Agar, Modified
position (agar side up) with increased humidity. For isolation SMWW
of fungi causing systemic mycoses, two sets of media should be Cat. No. 274720 Dehydrated – 500 g
inoculated, with one set incubated at 25-30°C and a duplicate 274710 Dehydrated – 2 kg
set at 35 ± 2°C. BBL™ Sabouraud Dextrose Agar, Emmons
All cultures should be examined at least weekly for fungal CMPH2 MCM9 SMWW
growth and should be held for 4-6 weeks before being reported Cat. No. 221849 Prepared Plates (Deep Fill) – Pkg. of 20*
221867 Prepared Plates (Deep Fill) – Ctn. of 100*
as negative. 221826 Prepared Slants (C Tubes) – Pkg. of 10
221827 Prepared Slants (C Tubes) – Ctn. of 100
Expected Results 296308 Mycoflask™ Bottles – Pkg. of 10
After sufficient incubation, the plates or tubes should show BBL™ Sabouraud Dextrose Agar, Emmons with
growth with or without isolated colonies. Transfer of growth Chloramphenicol
from tubes to plated media may be required in order to obtain MCM9
pure cultures of fungi. Cat. No. 297931 Prepared Plates (Deep Fill) – Pkg. of 10*
297474 Prepared Plates (Deep Fill) – Ctn. of 100*
Examine plates or tubes for fungal colonies exhibiting typical BBL™ Sabouraud Dextrose Agar, Emmons with
color and morphology.6 Biochemical tests and serological Chloramphenicol and Cycloheximide
procedures should be performed to confirm findings. Cat. No. 297932 Prepared Plates (Deep Fill) – Pkg. of 10*
BBL™ Sabouraud Dextrose Agar, Emmons with Gentamicin
Limitation of the Procedure Cat. No. 296348 Prepared Plates (Deep Fill) – Pkg. of 20*
Antimicrobial agents incorporated into a medium to inhibit *Store at 2-8°C.
bacteria may also inhibit certain pathogenic fungi.
References Availability
1. Aldridge, Jones, Gibson, Lanham, Meyer, Vannest and Charles. 1977. J. Clin. Microbiol. 6:406.
2. Koneman, Allen, Janda, Schreckenberger and Winn. 1997. Color atlas and textbook of diagnostic
BBL™ Saline, 0.45%
microbiology, 5th ed. Lippincott-Raven Publishers, Philadelphia, Pa. Cat. No. 299489 Prepared Tubes (K Size), 1 mL – Ctn. of 100
3. Clinical and Laboratory Standards Institute. 2006. Approved standard M2-A9, Performance standards
for antimicrobial disk susceptibility tests, 9th ed. CLSI, Wayne, Pa. BBL™ Saline, Normal
4. Clinical and Laboratory Standards Institute. 2006. Approved standard M7-A7, Methods for dilution
susceptibility tests for bacteria that grow aerobically, 7th ed. CLSI, Wayne, Pa. Cat. No. 297815 Prepared Tubes (K Size), 1 mL – Ctn. of 100
221818 Prepared Tubes (K Size), 5 mL – Pkg. of 10
221819 Prepared Tubes (K size), 5 mL – Ctn. of 100
295771 Prepared Tubes (C size), 5 mL – Ctn. of 100
297753 Prepared Tubes (D size), 10 mL – Ctn. of 100
Europe
Cat. No. 257255 Prepared Tubes, 10 mL – Pkg. of 50
Schaedler Media
Schaedler Agar • Schaedler Agar with Vitamin K1
and 5% Sheep Blood • Schaedler K-V Agar with
5% Sheep Blood • Schaedler Broth • Schaedler
Broth with Vitamin K1
Intended Use anaerobes from a variety of clinical and nonclinical specimens.
Schaedler Agar is a base for several media formulations used for It is especially useful for the recovery of the fastidious anaerobic
the recovery of anaerobic microorganisms. bacteria such as Bacteroides, Prevotella and Porphyromonas
species. Schaedler K-V Agar with 5% Sheep Blood, containing
Schaedler Agar with Vitamin K1 and 5% Sheep Blood is used
kanamycin and vancomycin, is especially useful in the selective
for the isolation and cultivation of fastidious aerobes and
isolation of Bacteroides and Prevotella species.
497
Schaedler Broth and Schaedler Broth with Vitamin K1 are media mycin inhibits gram-positive bacteria by interfering with cell
used for the cultivation of fastidious aerobic and anaerobic wall synthesis.8
microorganisms.
Using Schaedler media, fastidious aerobes and anaerobes
grow well; however, the type of organisms recovered is
Summary and Explanation dependent on the environment utilized in the incubation process
In 1965, Schaedler, Dubos and Costello1 reported on the
(aerobic, aerobic supplemented with carbon dioxide or anaerobic
bacterial flora of the gastrointestinal tract of mice. In these
conditions).
studies, several new media formulations were introduced.
The majority of these contained inhibitors of specific bacterial
Formulae
species or groups since the authors indicated the need for BBL™ Schaedler Agar
selective media when processing specimens which contain Approximate Formula* Per Liter
large numbers of a heterogeneous bacterial population. The Pancreatic Digest of Casein.......................................... 8.2 g
basal medium, without inhibitors, is the original version of the Peptic Digest of Animal Tissue...................................... 2.5 g
Papaic Digest of Soybean Meal..................................... 1.0 g
medium designated as Schaedler Agar. It was formulated to sup- Dextrose...................................................................... 5.8 g
port the growth of fastidious anaerobic microorganisms such as Yeast Extract................................................................ 5.0 g
lactobacilli, streptococci, clostridia and Bacteroides. Sodium Chloride.......................................................... 1.7 g
Dipotassium Phosphate................................................ 0.8 g
Mata and coworkers,2 studying the fecal microflora in healthy L-Cystine...................................................................... 0.4 g
persons in Central America, modified Schaedler Agar to Hemin.......................................................................... 0.01 g
Tris (hydroxymethyl) aminomethane............................. 3.0 g
produce a number of new formulations. The modifications Agar.......................................................................... 13.5 g
in the basal medium of Schaedler included adjustments in the
BBL™ Schaedler Broth
peptone content, since Trypticase™ Soy Broth was substituted for Consists of the same ingredients without the agar.
the Trypticase peptone component of the original formulation, *Adjusted and/or supplemented as required to meet performance criteria.
and an increase in the sodium chloride content. Additionally,
the dextrose concentration was reduced to avoid interference Directions for Preparation from
with hemolytic reactions and the yeast extract level lowered to Dehydrated Product
avoid darkening of the medium.3 1. Suspend the powder in 1 L of purified water:
The inclusion of vitamin K1, is an additional modification and BBL™ Schaedler Agar – 41.9 g;
was added since it is a growth requirement for some strains of BBL™ Schaedler Broth – 28.4 g.
Prevotella melaninogenica (Bacteriodes melaninogenicus)4 and Mix thoroughly.
is reported to enhance the growth of some strains of Bacteroides 2. If desired, add 1 mL of a 1% vitamin K1 solution in absolute
and gram-positive nonsporeformers.5 ethanol.
3. Heat with frequent agitation and boil for 1 minute to com-
The combination of kanamycin and vancomycin in Schaedler pletely dissolve the powder.
K-V Agar with 5% Sheep Blood is used for the selective isolation 4. Autoclave at 121°C for 15 minutes.
of gram-negative anaerobes.6 5. For the agar medium, cool to approximately 45°C and add
Schaedler Broth has the same formula as Schaedler Agar except 5% sterile defibrinated sheep blood when required.
that the agar is omitted. Stalons et al.7 found Schaedler Broth 6. Test samples of the finished product for performance using
to be the most effective medium of nine broth media tested for stable, typical control cultures.
the growth of obligately anaerobic bacteria when incubated
in an anaerobic atmosphere. The incorporation of vitamin K1 Procedure
broadens the spectrum of organisms that can be cultivated in Agars
Schaedler Broth. These media should be reduced immediately prior to inoculation
by placing them under anaerobic conditions for 18-24 hours.9
Principles of the Procedure Use standard procedures to obtain isolated colonies from
The combination of three peptones derived from both animal specimens. Inoculate an enrichment broth, such as Enriched
and vegetable sources, dextrose and yeast extract render the basic Thioglycollate Medium, at the same time as the primary plates
formulation highly nutritious by providing nitrogenous growth to detect small numbers of anaerobes.
factors, carbohydrates as energy sources and vitamins. The sheep
blood and hemin also are important in stimulating the growth of Incubate plates and tubes immediately after inoculation, with
fastidious microorganisms. As discussed above, the vitamin K1 plates in an inverted position (agar side up) under anaerobic
additive is crucial for the recovery of certain anaerobes. conditions at 35°C, or place the media in a holding jar flushed
with oxygen-free gas(es) until a sufficient number of plates
The addition of the antimicrobial agents kanamycin and and tubes is accumulated (no longer than 3 hours). Incubate
vancomycin in the agar medium renders the medium selective for at least 48 hours and, if no growth occurs, continue
for gram-negative microorganisms. The kanamycin inhibits incubation for up to 7 days. It is recommended that an indicator
protein synthesis in susceptible organisms, whereas the vanco- of anaerobiosis be used.
498
499
Procedure Availability
To culture a specimen from a swab, inoculate the medium by BBL™ Selective Streptococcus Agar
rolling the swab over a third of the agar surface, and streak United States and Canada
the remainder of the plate to obtain isolated colonies. Material Cat. No. 221934 Prepared Plates – Pkg. of 20*
221935 Prepared Plates – Ctn. of 100*
not being cultured from swabs may be streaked onto the
Japan
medium with a sterilized inoculating loop. Without resteril- Cat. No. 212498 Prepared Plates – Pkg. of 20*
*Store at 2-8°C.
Formula Availability
Difco™ Selenite Broth Difco™ Selenite Broth
Approximate Formula* Per Liter BS12 MCM9 SMWW
Pancreatic Digest of Casein.......................................... 5.0 g
Cat. No. 227540 Dehydrated – 500 g
Lactose........................................................................ 4.0 g
Sodium Selenite........................................................... 4.0 g BBL™ Selenite-F Broth
Sodium Phosphate..................................................... 10.0 g BS12 MCM9 SMWW
*Adjusted and/or supplemented as required to meet performance criteria.
Cat. No. 221020 Prepared Tubes (K Tubes), 8 mL – Pkg. of 10*
221021 Prepared Tubes (K Tubes), 8 mL – Ctn. of 100*
Directions for Preparation from *Store at 2-8°C.
Dehydrated Product
1. Suspend 23 g of the powder in 1 L of purified water.
2. Heat to boiling. Avoid overheating. DO NOT AUTO-
CLAVE.
3. Test samples of the finished product for performance using
stable, typical control cultures.
Formula Availability
Difco™ Selenite Cystine Broth Difco™ Selenite Cystine Broth
Approximate Formula* Per Liter AOAC BAM CCAM COMPF ISO SMD SMWW
Pancreatic Digest of Casein.......................................... 5.0 g Cat. No. 268740 Dehydrated – 500 g
Lactose........................................................................ 4.0 g 268710 Dehydrated – 2 kg
Sodium Phosphate..................................................... 10.0 g
Sodium Selenite........................................................... 4.0 g BBL™ Selenite Cystine Broth
L-Cystine...................................................................... 0.01 g AOAC BAM CCAM COMPF ISO SMD SMWW
*Adjusted and/or supplemented as required to meet performance criteria.
Cat. No. 297711 Prepared Tubes (A Tubes), 20 mL – Ctn. of 100*
*Store at 2-8°C.
502
References
1. Albers. 1947. U.S. Naval Med. Bull. 47:33.
2. Funke and Bernard. 2007. In Murray, Baron, Jorgensen, Landry and Pfaller (ed.). Manual of clinical
microbiology, 9th ed. American Society for Microbiology, Washington, D.C.
Availability
BBL™ Serum Tellurite Agar
BS12 CMPH2 MCM9
Cat. No. 221183 Prepared Plates – Pkg. of 20*
221024 Tubed Slants – Pkg. of 10*
*Store at 2-8°C.
503
505
For information on the niacin test, consult the BBL™ Quality References
Control and Product Information Manual for Plated and Tubed 1. Cohn, Waggoner and McClatchy. 1968. Am. Rev. Respir. Dis. 98:295.
2. Murray, Baron, Jogensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
Media and other appropriate references.2,8-11 BBL™ Taxo™ TB American Society for Microbiology, Washington, D.C.
3. Mitchison, Allen, Carrol, Dickinson and Aber. 1972. J. Med. Mycol. 5:165.
Niacin Test Reagents (strips and control) may be used instead 4. McClatchy, Waggoner, Kanes, Cernich and Bolton. 1976. Am. J. Clin. Pathol. 65:412.
5. Kilburn, Stottmeier and Kubica. 1968. Am. J. Clin. Pathol. 50:582.
of the test reagents. 6. Garrod and O’Grady. 1971. Antibiotics and chemotherapy, 3rd ed. Williams & Wilkins, Baltimore, Md.
7. U.S. Public Health Service, Centers for Disease Control and Prevention, and National Insitutes of
Health. 2007. Biosafety in microbiology and biomedical laboratories, 5th ed. HHS Publication No.
Expected Results (CDC) 93-8395. U.S. Government Printing Office, Washington, D.C.
8. Kent and Kubica. 1985. Public health mycobacteriology: a guide for the level III laboratory. USDHHS,
Cultures on Seven H11 Agar should be read within 5-7 days after Centers for Disease Control, Atlanta, Ga.
9. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
incubation and once a week thereafter for up to 8 weeks. American Society for Microbiology, Washington, D.C.
10. Cernoch, Enns, Saubolle and Wallace. 1994. Cumitech 16A, Laboratory diagnosis of the mycobacte-
Record Observations:8 rioses. Coord. ed., Weissfeld. American Society for Microbiology, Washington, D.C.
11. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby, Inc.,
St. Louis, Mo.
1. Number of days required for colonies to become macroscopi-
cally visible. Rapid growers have mature colonies within 7
days. Slow growers require more than 7 days for mature
Availability
Difco™ Mycobacteria 7H11 Agar
colony forms.
Cat. No. 283810 Dehydrated – 500 g
2. Pigment production
White, cream or buff = Nonchromogenic (NC) BBL™ Seven H11 Agar Base
Cat. No. 212203 Dehydrated – 500 g
Lemon, yellow, orange, red = Chromogenic (Ch)
BBL™ Seven H11 Agar
Stained smears may show acid-fast bacilli, which are reported
BS12 CMPH2 MCM9
only as “acid-fast bacilli” unless definitive tests are performed. Cat. No. 221870 Prepared Plates (Deep Fill) – Pkg. of 10*
Test all nonchromogenic mycobacteria on Seven H11 Agar 221391 Prepared Slants (A Tubes) – Pkg. of 10*
221392 Prepared Slants (A Tubes) – Ctn. of 100*
with Aspartic Acid and Sodium Pyruvate for niacin production; 296105 Prepared Slants (C Tubes) – Pkg. of 10*
only the rough nonchromogenic strains need to be tested for 297704 Prepared Slants (C Tubes) – Ctn. of 100*
niacin. A culture must have at least 50-100 colonies with Japan
growth 3-4 weeks old. M. tuberculosis and the more rare Cat. No. 252119 Prepared Plates (Deep Fill) – Pkg. of 20*
M. simiae are usually niacin positive. Most other mycobacteria BBL™ Selective Seven H11 Agar
are niacin negative. BS12 CMPH2 MCM9
Cat. No. 221868 Prepared Plates (Deep-fill) – Pkg. of 10*
Limitations of the Procedure 297315 Prepared Slants (A Tubes) – Pkg. of 10*
297639 Prepared Slants (A Tubes) – Ctn. of 100*
1. Negative culture results do not rule-out active infection by 297184 Prepared Slants (C Tubes) – Pkg. of 10*
mycobacteria. Some factors that are responsible for unsuc- 297654 Prepared Slants (C Tubes) – Ctn. of 100*
cessful cultures are: BBL™ Seven H11 Agar with Aspartic Acid and Sodium
• The specimen was not representative of the infectious Pyruvate
material; i.e., saliva instead of sputum. Cat. No. 221958 Prepared Slants (A Tubes) – Pkg. of 10*
• The mycobacteria were destroyed during digestion and BBL™ Middlebrook 7H11 Agar//Selective 7H11 Agar
decontamination of the specimen. BS12 CMPH2 MCM9
• Gross contamination interfered with the growth of the Cat. No. 297250 Prepared Bi-Plate Dishes – Pkg. of 20*
mycobacteria.
Difco™ Glycerol
• Proper aerobic conditions and increased CO2 tension
Cat. No. 228210 Bottle – 100 g
were not provided during incubation. 228220 Bottle – 500 g
2. Mycobacteria are strict aerobes and growth is stimulated
BBL™ Taxo™ TB Niacin Test Strips and Control
by increased levels of CO2. Screw caps on tubes or bottles Cat. No. 231741 Vial – 25 strips*
should be handled as directed for exchange of CO2. 231735 Cartridge, Control – 50 discs*
*Store at 2-8°C.
Shigella Broth
Intended Use Summary and Explanation
Shigella Broth is a selective enrichment broth for the isolation Shigella was first recognized as the etiologic agent of bacillary
of Shigella species from food. dysentery or shigellosis in the 1890s.1 Humans are the only
natural reservoir. No natural food products harbor endogenous
Shigella species, but a wide variety of foods may be contami-
nated.1
506
Prepare the medium per label directions. Inoculate and incubate under
Directions for Preparation from
anaerobic conditions at 40-44°C for 18-24 hours. Dehydrated Product
ORGANISM ATCC™ INOCULUM CFU RECOVERY
1. Dissolve 31.5 g of the powder in 1 L of purified water. Mix
Escherichia coli 25922 100 Good
thoroughly.
Shigella flexneri 12022 100 Good
2. Autoclave at 121°C for 15 minutes. Cool to 45-50°C.
Shigella sonnei 25931 100 Good
3. Prepare novobiocin solution by weighing 50 mg of novobiocin
into 1 L of purified water. Sterilize by filtration using a 0.45μ
filter.
Shigellosis can manifest itself as a waterborne or a foodborne 4. Add 2.5 mL of sterile novobiocin solution from Step 3 to
disease. It is usually spread among people by food handlers 225 mL of Shigella Broth. S
with poor personal hygiene. Foods most often incriminated in 5. Test samples of the finished product for performance using
the transmission of the disease have been potato salad, shellfish, stable, typical control cultures.
raw vegetables and Mexican food.2
The infectivity dose is extremely low. As few as ten S. dysente-
Procedure
For food samples, follow appropriate standard methods for
riae bacilli can cause clinical disease, whereas 100-200 bacilli
details on sample collection and preparation according to sample
are needed for S. sonnei or S. flexneri infection.1 One possible
type and geographic location.3-6
reason for this low-dose response may be that virulent Shigellae
can withstand the low pH of gastric juice.1 Consult appropriate standard references for details on test
methods using Shigella Broth.3-6
Shigella species are gram-negative, nonmotile, facultatively
anaerobic, non-sporeforming rods. They utilize glucose and
Expected Results
other carbohydrates, producing acid but not gas. They do not
Growth is evident by the appearance of turbidity.
decarboxylate lysine or ferment lactose. Shigella organisms
may be difficult to distinguish biochemically from E. coli. The
References
genus Shigella consists of four species: S. dysenteriae, S. flexneri, 1. Sureshbabu, Poothirikovil, Abuhammour, and Burny. 2008. Shigella infection. <http://emedicine.
S. boydii and S. sonnei. medscape.com/article/968773>.
2. Mehlman, Romero and Wentz. 1985. J. Assoc. Off. Anal. Chem. 68:552.
3. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
Common contaminating bacteria found in food sources could tional, Gaithersburg, Md.
4. Health Canada. The compendium of analytical methods, online. Food Directorate, Health Products
mask the presence of any Shigella that could be present in the and food Branch, Health Canada, Ottawa, Ontario Canada.
5. International Organization for Standardization. 2004 Microbiology of food and animal feeding
sample. Identification of Shigella is based on successful isolation stuffs – horizontal method for the detection of Shigella spp. ISO 21567, 2004-11-01. International
of the organism, biochemical characterization and serological Organization for Standardization, Geneva, Switzerland.
6. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
confirmation. 4th ed. American Public Health Association, Washington. D.C.
507
Cultural Response
BBL™ Simmons Citrate Agar
Prepare the medium per label directions. Inoculate with fresh cultures and incubate at 35 ± 2°C
for 4 days.
ORGANISM ATCC™ recovery reaction
Uninoculated Enterobacter Escherichia coli
Enterobacter aerogenes 13048 Good Alkaline (blue) Tube aerogenes ATCC™ 25922
ATCC™ 13048
Escherichia coli 25922 Partial to complete inhibition –
Klebsiella pneumoniae 33495 Good Alkaline (blue)
Shigella flexneri 9199 Complete inhibition –
508
Prepare the medium per label directions. Inoculate with a drop or loopful of
fresh culture and incubate at 35 ± 2°C for 1-7 days. Availability
ORGANISM ATCC™ GROWTH APPEARANCE Difco™ Skim Milk
Clostridium perfringens 12919 Good Stormy fermentation Cat. No. 232100 Dehydrated – 500 g
Escherichia coli 25922 Good Acid, curd BBL™ Skim Milk Medium
Lactobacillus rhamnosus 9595 Good Acid, curd Cat. No. 298240 Prepared Tubes (D Tubes) – Pkg. of 10
509
Skirrows Medium
(See Campylobacter Agars)
Cultural Response
Difco™ Snyder Test Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-72 hours.
INOCULUM Acid
Organism ATCC™ CFU RECOVERY Production Uninoculated Lactobacillus
Tube fermentum
Lactobacillus rhamnosus 9595 10 -10
2 3
Good + ATCC™ 9338
510
Before adding ferric chloride solution to tubes containing test Add the appropriate amount of the ferric chloride solution
organism or positive cultures, use the small, negative control to all tubes containing 0.8 mL of incubated test and positive
tubes to determine the amount of ferric chloride to add by means control cultures.
of the following procedure.
1. To small negative control tube #1 containing 0.8 mL of
Expected Results
A positive test for hippurate hydrolysis is indicated by production
incubated broth, add 0.2 mL of the 12% ferric chloride
of a brown flocculating, insoluble precipitate that persists on
solution and immediately shake gently.
shaking. The amount of the precipitate is related to the degree
2. Allow the tube to stand 10-15 minutes before reading the
of hippurate hydrolysis.
result.
3. If negative (initial precipitate clears within 15 minutes), the A negative test (hippurate not hydrolyzed) is indicated by the
ferric ion is in excess and the ferric chloride solution can be lack of precipitate formation or the formation of a precipitate
used in the testing of the test cultures. that dissolves on shaking.
4. If positive (initial precipitate does not clear within 15 Consult an appropriate text for additional differentiating
minutes), ferric ion is not in excess and must be titrated to characteristics.4
determine the optimal amount of solution required to be
in excess. References
1. Ayers and Rupp. 1922. J. Infect. Dis. 30:388.
If titration is necessary, rapidly add 0.2, 0.3, 0.4 and 0.5 mL 2. Facklam, Padula, Thacker, Wortham and Sconyers. 1974. Appl. Microbiol. 27:107.
amounts of ferric chloride solution to small negative control 3. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria,
vol. 1. Williams & Wilkins, Baltimore, Md.
tubes #2 through #5 each containing 1.0 mL of the incu- 4. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology,
9th ed. Williams & Wilkins, Baltimore, Md.
bated uninoculated broth. Immediately shake gently. Let stand
10-15 minutes with occasional shaking. The smallest amount of Availability
FeCl3 solution giving a clear solution indicates that ferric ion is in BBL™ Sodium Hippurate Broth
excess. Use this amount in the evaluation of the test cultures. Cat. No. 221618 Prepared Tubes (K Tubes) – Pkg. of 10
Soytone
(See Phytone™ Peptone)
the dark for 4-7 days. Examine plates for growth after 18-24 Aspergillus
hours incubation and up to 7 days. brasiliensis (niger) 16404 30-300 Good
Candida albicans 26790 30-300 Good
Expected Results Enterococcus faecalis 29212 30-300 Marked to
complete inhibition
Yeasts and molds should show growth in 4-7 days at room tem-
perature. Bacteria should be inhibited on the complete medium
Escherichia coli
25922 30-300 Marked to
complete inhibition
S
containing antibacterial agents. Trichophyton
mentagrophytes 9533 30-300 Poor to fair
References
1. Morey, Otten, Burge, Chatigny, Feeley, LaForce and Peterson. 1986. Applied Industrial Hygiene
1:R-19.
2. Guide regarding condition and attendance of houses. 1990. Finnish Medical Board. Helsinki, Finland.
Availability
Difco™ Special Yeast and Mold Medium
Cat. No. 210810 Dehydrated – 500 g
513
Identity Specifications
Difco™ Spirit Blue Agar
Dehydrated Appearance: Grayish-beige, free-flowing, homoge-
neous.
Solution: 3.5% solution, soluble in purified
water upon boiling. Solution is royal
blue, slightly opalescent.
Prepared Appearance: Plain – Royal blue, opalescent.
With 3% Lipase Reagent – Pale blue,
opalescent.
Reaction of 3.5%
Solution at 25°C: pH 6.8 ± 0.2
Difco Lipase Reagent
™
Cultural Response
Difco™ Spirit Blue Agar and Lipase Reagent
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C
for up to 72 hours.
inoculum Halo/
Organism ATCC´™ cfu RECOVERY Lipolysis Staphylococcus
epidermidis
Proteus mirabilis 25933 102-103 Good – ATCC™ 12228
Staphylococcus aureus 25923 102-103 Good +
Staphylococcus aureus 6538 102-103 Good +
Staphylococcus epidermidis 12228 10 -10 2 3
Good +
Lipase Reagent contains tributyrin, a true fat and the simplest Procedure
triglyceride occurring in natural fats and oils. It is a good Inoculate the organism onto the medium. Incubate plates at
substrate when testing for lipolytic microorganisms because some 35 ± 2°C for up to 72 hours, or at other temperatures and times
microorganisms that hydrolyze tributyrin will not hydrolyze according to standard methods.2
other triglycerides or fats containing longer chain fatty acids.2
Expected Results
Formulae Lipolytic microorganisms metabolize the lipid in the medium
Difco™ Spirit Blue Agar and form colonies with halos indicating lipolysis.
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 10.0 g
Yeast Extract ............................................................... 5.0 g
References
1. Starr. 1941. Science 93:333.
Agar.......................................................................... 20.0 g 2. Frank and Yousef. 2004. In Wehr and Frank (ed.), Standard methods for the examination of dairy
Spirit Blue.................................................................... 0.15 g products, 17th ed. American Public Health Association, Washington, D.C.
514
Spirolate Broth
Intended Use Formula
Spirolate Broth was developed for mass culture of the Reiter BBL™ Spirolate Broth
treponeme in a medium without agar. It can be used for cultivat- Approximate Formula* Per Liter
ing other spirochetes. Pancreatic Digest of Casein........................................ 15.0 g
Dextrose...................................................................... 5.0 g
Yeast Extract................................................................ 5.0 g
Summary and Explanation Sodium Chloride.......................................................... 2.5 g
In 1956, Omata and Disraely developed FM Medium as a Sodium Thioglycollate.................................................. 0.5 g
L-Cysteine HCl............................................................. 1.0 g
selective medium for the growth of oral fusobacteria.1 BBL™ *Adjusted and/or supplemented as required to meet performance criteria.
Spirolate Broth is a modification of that medium and is
recommended for bulk production of Reiter treponemes for Directions for Preparation from
use in antigen production or research studies. Supplementation Dehydrated Product
with fatty acids has a stimulatory effect on growth of the Reiter 1. Suspend 29 g of the powder in 1 L of purified water. Mix
treponeme.2 thoroughly. (Add equal quantities of palmitic, stearic,
oleic and linoleic acids to a total fatty acid concentration of
Principles of the Procedure 0.20-0.25 g/L, if desired.)
The casein peptone, dextrose and yeast extract supply nitrog- 2. Heat with frequent agitation and boil for 1 minute to
enous growth factors, carbon, minerals and vitamins required completely dissolve the powder.
for the metabolism of Reiter treponemes. Sodium chloride 3. Dispense in test tubes, filling them half full, using 15-20 mL in
aids in the maintenance of the osmotic equilibrium of the
medium. Sodium thioglycollate reduces the oxygen tension to a
6-inch tubes, preferably with screw caps. If larger containers
are used, the ratio of surface to volume should be similar to
S
level conducive to the growth of treponemes. L-Cysteine that for tubes.
hydrochloride is a reducing agent and is slightly inhibitory to 4. Autoclave at 121°C for 15 minutes. Close caps upon removal
fusobacteria.1 from the autoclave.
The addition of ether-soluble pure palmitic, stearic, oleic and 5. Cool and add sterile inactivated sheep, rabbit or bovine
linoleic acids, in equal amounts at a total fatty acid concentra- serum, 10% by volume, to each tube. Tighten caps.
tion of 0.20-0.25 mg/mL of medium, provides fatty acids which 6. Store at room temperature, not in the refrigerator, unless in
enhance the growth of Reiter treponemes.2 sealed containers.
7. Test samples of the finished product for performance using
stable, typical control cultures.
User Quality Control
Identity Specifications Procedure
BBL™ Spirolate Broth Inoculate containers of Spirolate Broth with 0.05-mL aliquots
Dehydrated Appearance: Fine, homogeneous, free of extraneous of a 7-day pure culture in Thioglycollate Medium without
material.
Indicator-135C supplemented with 15% inactivated sheep,
Solution: 2.9% solution, soluble in purified water upon
boiling. Solution is light to medium, tan to
rabbit or bovine serum. Incubate containers for a minimum of
yellow, clear to slightly hazy. 7 days at 35 ± 2°C in an anaerobic atmosphere (BD GasPak™
Prepared Appearance: Light to medium, tan to yellow, clear to EZ anaerobic system or equivalent).
slightly hazy.
Reaction of 2.9% Expected Results
Solution at 25°C: pH 7.1 ± 0.2 After obtaining sufficient growth, process the cultures according
to the particular method being utilized.
Cultural Response
BBL™ Spirolate Broth
Prepare the medium per label directions (with 10% inactivated rabbit
References
1. Omata and Disraely. 1956. J. Bacteriol. 72: 677.
serum). Inoculate with a fresh culture and incubate at 35 ± 2°C for 2. Power and Pelczar. 1959. J. Bacteriol. 77: 789.
7 days.
ORGANISM Recovery Availability
Reiter treponeme Good BBL™ Spirolate Broth
Cat. No. 211636 Dehydrated – 500 g
515
516
For use in the sampling of surfaces, remove the top of the plate. References
Apply the agar surface to a flat surface, pressing down gently 1. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
but firmly and making certain that the entire agar meniscus 2. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy products, 17th ed. American
Public Health Association, Washington, D.C.
touches the surface. Use a rolling uniform pressure on the back 3. McGowan. 1985. In Lennette, Balows, Hausler and Shadomy (ed.), Manual of clinical microbiology,
4th ed. American Society for Microbiology, Washington, D.C.
of the plate to effect contact. Lift the plate straight up from the 4. Quisno, Gibby and Foter. 1946. Am. J. Pharm. 118: 320.
surface, being careful not to allow it to slide along the surface. 5. Erlandson and Lawrence. 1953. Science 118: 274.
Replace the top of the plate. Incubate plates with the agar side
up at 32°C for 24-48 hours depending upon whether contamina- Availability
tion is heavy or light.1,2 BBL™ Standard Methods Agar with Lecithin and
Polysorbate 80
COMPF SMD
Expected Results Cat. No. 211643 Dehydrated – 500 g*
After incubation, count the colonies and record as either number 221939 Prepared Contact Plates – Pkg. of 20*
of colonies per RODAC plate or number of colonies per cm2.1,2 221032 Prepared Pour Tubes, 18 mL – Pkg. of 10*
Subculture those colonies which are of interest so that positive *Store at 2-8°C.
m Staphylococcus Broth
Intended Use Formula
m Staphylococcus Broth is used for isolating staphylococci by
the membrane filtration technique.
Difco™ m Staphylococcus Broth
Approximate Formula* Per Liter
S
Pancreatic Digest of Casein........................................ 10.0 g
Yeast Extract................................................................ 2.5 g
Summary and Explanation Lactose........................................................................ 2.0 g
Staphylococci, along with other bacteria, are indicators of recre- Mannitol.................................................................... 10.0 g
ational water quality.1 Indicators of health risk include normal Dipotassium Phosphate................................................ 5.0 g
skin flora that are likely to be shed, such as Pseudomonas, Sodium Chloride........................................................ 75.0 g
*Adjusted and/or supplemented as required to meet performance criteria.
Streptococcus and Staphylococcus.2 These organisms account
for a large percentage of swimming pool-associated illness.1
Directions for Preparation from
The coagulase-positive species, Staphylococcus aureus, is well Dehydrated Product
documented as a human opportunistic pathogen.3 Coagulase-neg- 1. Dissolve 104 g of the powder in 1 L of purified water.
ative Staphylococcus spp. are a major component of the normal 2. Warm slightly to completely dissolve the powder.
microflora of humans.3 Staphylococci are widespread in nature, 3. Autoclave at 121°C for 15 minutes.
though they are mainly found living on the skin, skin glands and
NOTE: For field studies where autoclaving is not practical, boil
mucous membranes of mammals and birds.3
the medium for 5 minutes.
Chapman4 added 7.5% NaCl to Phenol Red Mannitol Agar to
achieve a selective medium for staphylococci. While studying 4. Test samples of the finished product for performance using
this medium formulation, Chapman5 developed Staphylococcus stable, typical control cultures.
Medium 110. m Staphylococcus Broth is patterned after the
formula of Staphylococcus Medium 110. Procedure
1. Follow the membrane filtration procedure described in
m Staphylococcus Broth, with the addition of sodium azide, is Standard Methods for the Examination of Water and
used in a multiple-tube procedure to monitor swimming pool Wastewater.1
water for the presence of S. aureus.1 2. Use 2.0-2.5 mL of medium to saturate the paper pads on
which the inoculated membrane is placed.
Principles of the Procedure 3. Incubate at 35 ± 2°C for 40-48 hours.
Peptone provides the nitrogen, amino acids and minerals in
m Staphylococcus Broth. Yeast extract is the vitamin source Expected Results
in this formula. Lactose and mannitol are the carbohydrates Observe membranes for growth and pigment production. Test
for bacterial growth. Dipotassium phosphate is the buffering for mannitol fermentation by adding a drop of bromthymol
agent. The high concentration of sodium chloride permits this blue to the site from which a colony is removed; a yellow color
medium to be selective for staphylococci. indicates mannitol fermentation.
517
Cultural Response
Difco™ m Staphylococcus Broth
Prepare the medium per label directions. Use the membrane filtration technique with the test organisms. Inoculate and incubate at 35 ± 2°C under humid
conditions for 40-48 hours. Observe the membranes for recovery and pigment production. Detect mannitol fermentation by adding a drop of bromthymol
blue to the site where a colony was removed. A yellow color indicates a positive result for mannitol fermentation.
Mannitol Pigment
Organism ATCC™ INOCULUM CFU RECOVERY Fermentation Production
Escherichia coli 25922 20-200 Inhibition N/A –
Staphylococcus aureus 25923 20-200 Good + +
Staphylococcus epidermidis 12228 20-200 Good – –
518
Cultural Response
Difco™ Staphylococcus Medium 110
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-48 hours. To observe mannitol fermentation, remove a colony from the medium
and add a drop of 0.04% bromthymol blue to the area from which the colony was removed. Observe for the formation of a yellow color (positive reaction).
To observe the gelatinase reaction, flood the plate with 5 mL of saturated ammonium sulfate solution and incubate at 35 ± 2°C for 10 minutes. Observe for a zone
of clearing around the colonies (positive reaction).
Organism ATCC™ INOCULUM CFU RECOVERY Pigment* Gelatinase Mannitol
Escherichia coli 25922 102-3×102 Marked to complete inhibition – N/A N/A
Staphylococcus aureus 25923 102-3×102 Good + + +
Staphylococcus epidermidis 12228 102-3×102 Good – + –
*Pigment is seen as a yellow to orange color.
Starch Agar
(For Nocardia, see Nocardia Differentiation Media)
519
Starch Agar
Intended Use Formula
Starch Agar is used for cultivating microorganisms being tested Difco™ Starch Agar
for starch hydrolysis. Approximate Formula* Per Liter
Beef Extract.................................................................. 3.0 g
Soluble Starch............................................................ 10.0 g
Summary and Explanation Agar.......................................................................... 12.0 g
In 1915,1 Vedder formulated Starch Agar for cultivating *Adjusted and/or supplemented as required to meet performance criteria.
Neisseria. Since then, other media have been developed that are
superior to Starch Agar for the isolation of Neisseria spp., including Directions for Preparation from
enriched GC medium base. Starch Agar is used in differentiating Dehydrated Product
microorganisms based on the starch hydrolysis test. 1. Suspend 25 g of the powder in 1 L of purified water. Mix
thoroughly.
Principles of the Procedure 2. Heat with frequent agitation and boil for 1 minute to
Beef extract provides the nitrogen, vitamins, carbon and completely dissolve the powder.
amino acids in Starch Agar. Starch reacts with Gram Iodine to 3. Autoclave at 121°C for 15 minutes.
give a blue color. Organisms hydrolyzing starch through 4. Test samples of the finished product for performance using
amylase production will produce a clearing around the isolate stable, typical control cultures.
while the remaining medium is blue. Agar is the solidifying
agent.
Procedure
Starch Hydrolysis Test
User Quality Control
Flood the surface of a 48-hour culture on Starch Agar with
Identity Specifications Gram Iodine.
Difco™ Starch Agar
Dehydrated Appearance: Light beige, free-flowing, homogeneous. For a complete discussion of the collection, isolation and identi-
Solution: 2.5% solution, soluble in purified water upon fication of microorganisms, refer to appropriate references.2,3
boiling. Solution is light amber, slightly opales-
cent. Expected Results
Prepared Appearance: Light amber, slightly opalescent. Starch hydrolysis (+) is indicated by a colorless zone surrounding
Reaction of 2.5% colonies. A blue or purple zone indicates that starch has not
Solution at 25°C: pH 7.5 ± 0.2
been hydrolyzed (-).
Cultural Response
Difco™ Starch Agar References
1. Vedder. 1915. J. Infect. Dis. 16:385.
Prepare the medium per label directions. Inoculate with a single streak 2. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
of undiluted test organism and incubate at 35 ± 2°C for 40-48 hours. American Society for Microbiology, Washington, D.C.
3. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
Test for starch hydrolysis by removing growth from each streak to American Society for Microbiology, Washington, D.C.
expose the agar and flood plates with Gram Iodine.
Organism ATCC™ Recovery Starch hydrolysis
Availability
Bacillus subtilis 6633 Good + Difco™ Starch Agar
Escherichia coli 25922 Good – Cat. No. 272100 Dehydrated – 500 g
Staphylococcus aureus 25923 Good –
Streptococcus pyogenes 19615 Good –
520
521
Strep ID Quad
Intended Use Quadrant IV contains Blood Agar Base with 6.5% sodium
The Streptococcus Identification Quadrant plate is a four-sectored chloride for the determination of salt tolerance. Enterococcal
plate that is used with a battery of tests for the differentiation and streptococci may be easily differentiated from other streptococci
presumptive identification of streptococci. by their ability to grow in the presence of salt.6
522
Bile Esculin Agar enables the differentiation of group D from Expected Results
non-group D streptococci. The bile in the esculin medium in Interpret results in conjunction with hemolytic reactions of the
quadrant III inhibits most gram-positive organisms other than isolate.
group D streptococci. Esculin is incorporated as a differential
Quadrant I (bacitracin): Streptococci strains susceptible to
agent. Hydrolysis of esculin by group D species results in the
bacitracin are inhibited on quadrant I while bacitracin-resistant
production of esculetin and dextrose. The esculetin reacts with
strains exhibit growth. A bacitracin-susceptible strain may be
the iron salt in the medium, producing a dark brown to black
presumptively identified as a group A streptococcus.
complex that appears as zones around the group D colonies.
Quadrant II (CAMP test): A definite arrowhead or crescent-
Enterococcal species may be differentiated in quadrant IV by
shaped clearing at the junction of S. aureus and the isolate
the 6.5% NaCl tolerance test. Enterococci usually grow heavily;
indicates a positive reaction. The absence of clearing indicates
nonenterococcal species do not grow in the salt-supplemented
a non-group B streptococcus. Bacitracin-resistant, CAMP-positive,
medium. However, salt tolerant, nonenterococcal streptococci
beta-hemolytic streptococci may be identified presumptively as
do grow occasionally.1
group B streptococci. CAMP-positive group A streptococci
may be differentiated from group B streptococci by hemolysis,
Procedure
bacitracin susceptibility and hippurate hydrolysis. Group B
Subculture the organism to be tested onto a plate of Trypticase
streptococci generally have smaller hemolytic zones than group
Soy Agar enriched with sheep blood, or another suitable
A streptococci.
medium, streaking to obtain isolated colonies. Incubate for
18-24 hours in a CO2-enriched atmosphere. Using a sterile Quadrant III (Bile Esculin Agar): The presence of brownish-
inoculating loop, choose one or two isolated colonies and black to black pigmentation of the medium indicates that
perform a Gram stain, examining to confirm that the the isolate may be presumptively identified as a group D
morphology of the isolate is appropriate for streptococci.
Choose three or four well-isolated colonies and streak the
streptococcus. Any blackening of the medium is enough to report
as a positive esculin reaction. The test is negative if there is no S
blackening of the medium.
surface of quadrants I, III, and IV of the Strep ID Quad
plate. Quadrant IV (6.5% sodium chloride): Growth indicates a
positive reaction. No growth indicates a negative reaction. A
Inoculate quadrant II by streaking Staphylococcus aureus
bile esculin positive, salt-tolerant isolate may be presumptively
ATCC™ 33862 across the widest area of the quadrant. If a
identified as an enterococcus.
loop is used, do not use it parallel to the agar surface, since
the streak will be too wide and results will not be satisfac-
References
tory. Then, streak the unknown isolate perpendicular to the 1. Ruoff. 1995. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology,
S. aureus culture, leaving 2-3 mm space between the two 6th ed. American Society for Microbiology, Washington, D.C.
2. Rochaix. 1924. Comt. Rend. Soc. Biol. 90:771.
streak lines. Alongside the unknown isolate, streak a known 3. Meyer and Schonfeld. 1926. Zentralbl. Bakteriol. Parasitenk. Infectionskr. Hyg. Abt. Orig. 99:402.
4. Swan. 1954. J. Clin. Pathol. 7:160.
S. agalactiae strain as a positive control and S. pyogenes as a 5. Facklam and Moody. 1970. Appl. Microbiol. 20:245.
6. Facklam, Padula, Thacker, Wortham and Sconyers. 1974. Appl. Microbiol. 27:107.
negative control. This procedure should be practiced with known 7. Bernheimer, Linder and Avigard. 1979. Infect. Immun. 23:838.
cultures before using it to identify unknown isolates. 8. Darling. 1975. J. Clin. Microbiol. 1:171.
Sulfite Agar
Intended Use Clark and Tanner2 described the thermophilic organisms that
Sulfite Agar is used for detecting thermophilic, H2S-producing cause spoilage in canned foods as flat-sour spoilage organisms,
anaerobes, particularly in foods. thermophilic anaerobes and sulfide-spoilage organisms. They
used Sulfite Agar to study sulfide-spoilage organisms in sugar
Summary and Explanation and starch.
Sulfide spoilage of foods is due to three factors: high spore Both beet and cane sugar can carry spores of the thermo-
counts, the heat resistance of the spores and subjecting the philic bacteria that are spoilage agents.3 Desulfotomaculum
finished product to elevated temperatures. The last factor may nigrificans, first classified as Clostridium nigrificans, causes
occur if the processed food is not cooled adequately.1
spoilage in non-acid canned foods such as vegetables and
523
Cultural Response
Difco™ Sulfite Agar
Prepare the medium per label directions. Inoculate molten medium, solidify and
incubate aerobically at 55 ± 2°C for 18-48 hours.
INOCULUM Sulfite
Organism ATCC™ CFU RECOVERY Reduction
Bacillus
stearothermophilus 10149 30-100 Good – Uninoculated Bacillus Desulfotomaculum
Tube stearothermophilus nigrificans
Clostridium ATCC 10149
™
ATCC™ 19858
thermosaccharolyticum 7956 30-100 Good +
Desulfotomaculum
nigrificans 19858 30-100 Good +
524
525
526
527
528
TCBS Agar
Intended Use metabolism of vibrios. The alkaline pH of the medium enhances
Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS Agar) is the recovery of V. cholerae. Thymol blue and bromthymol blue
used for the selective isolation of cholera vibrios and Vibrio are included as indicators of pH changes.
parahaemolyticus from a variety of clinical and nonclinical
specimens.1,2 Formula
Difco™ TCBS Agar
Summary and Explanation Approximate Formula* Per Liter
Yeast Extract................................................................ 5.0 g
Vibrio species are most widely recognized for their role in human Proteose Peptone No. 3.............................................. 10.0 g
intestinal infections. Diarrheas caused by Vibro cholerae and Sodium Citrate........................................................... 10.0 g
V. parahaemolyticus are important worldwide.3 The isolation of Sodium Thiosulfate.................................................... 10.0 g
Oxgall.......................................................................... 8.0 g
Vibrio species has been enhanced by the development of media Saccharose................................................................. 20.0 g
which are highly selective for vibrios. Sodium Chloride........................................................ 10.0 g
Ferric Ammonium Citrate............................................. 1.0 g
TCBS is the primary plating medium universally used for the Bromthymol Blue......................................................... 0.04 g
selective isolation of vibrios that cause cholera, diarrhea and Thymol Blue................................................................. 0.04 g
food poisoning. It was developed by Kobayashi et al.4, who Agar.......................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.
modified the selective medium of Nakanishi.5 The combina-
tion of alkaline peptone water and TCBS Agar is used in many Directions for Preparation from
procedures for the isolation of V. cholerae and other Vibrio
Dehydrated Product
species from feces.1-3,6,7
1. Suspend 89 g of the powder in 1 L of purified water. Mix
TCBS Agar Deeps (pour tubes) are provided in a 20 mL fill so thoroughly.
that the medium may be liquefied and poured into a Petri dish. 2. Heat with frequent agitation and boil for 1 minute to
This provides a convenient source of medium with a longer completely dissolve the powder.
shelf-life than pre-poured plated media. 3. Cool to 45-50°C and use immediately. DO NOT AUTO-
CLAVE.
Principles of the Procedure 4. Test samples of the finished product for performance using
TCBS Agar is highly selective for the isolation of V. cholerae stable, typical control cultures.
and V. parahaemolyticus as well as other vibrios. Inhibition
of gram-positive bacteria is achieved by the incorporation of Procedure
oxgall, which is a naturally occurring substance containing To prepare plated media, place agar deeps with caps loosened in
a mixture of bile salts, and sodium cholate, a pure bile salt. a boiling water bath until the medium becomes liquefied. Pour
Sodium thiosulfate serves as a sulfur source and, in combination the molten medium into a sterile Petri dish. Allow the medium
with ferric citrate, detects hydrogen sulfide production. Saccharose to solidify. Store the plates, protected from light, in an inverted
(sucrose) is included as a fermentable carbohydrate for the position (agar side up) at 2-8°C until ready to use.
530
Identity Specifications
Difco™ TCBS Agar
Dehydrated Appearance: Light tan with greenish cast, free-flowing,
homogeneous.
Solution: 8.9% solution, soluble in purified water upon
boiling. Solution is forest green, very slightly
opalescent.
Prepared Appearance: Green, slightly opalescent.
Reaction of 8.9%
Solution at 25°C: pH 8.6 ± 0.2
Cultural Response
Difco™ TCBS Agar
Prepare the medium per label directions. Inoculate with fresh cultures
(E. coli grown in TSB; vibrios grown in BHI) and incubate at 35 ± 2°C for
18-24 hours.
ORGANISM ATCC™ RECOVERY COLONY COLOR
Escherichia coli 25922 None –
Vibrio alginolyticus 17749 Good Yellow
Vibrio cholerae El Tor 14033 Good Yellow
Vibrio parahemolyticus 17802 Good Blue green
531
m TEC Agar
Intended Use dipotassium phosphate offer buffering capabilities. Lactose is
m TEC Agar is used for isolating, differentiating and rapidly a fermentable carbohydrate and carbon source. Sodium lauryl
enumerating thermotolerant Escherichia coli from water by sulfate and sodium desoxycholate are selective against gram-
membrane filtration and an in situ urease test. positive bacteria. Bromcresol purple and bromphenol red are
indicator components. Agar is the solidifying agent.
Summary and Explanation
m TEC is an acronym for “membrane Thermotolerant E. coli.” Formula
Escherichia coli is widely used as an indicator of fecal pollution Difco™ m TEC Agar
in water, and there are many procedures for enumerating Approximate Formula* Per Liter
Proteose Peptone No. 3................................................ 5.0 g
E. coli based on its ability to grow at elevated temperatures Yeast Extract................................................................ 3.0 g
and produce indole from tryptophan.1,2 The determination Lactose...................................................................... 10.0 g
of indole production in conjunction with the most-probable- Sodium Chloride.......................................................... 7.5 g
Monopotassium Phosphate.......................................... 1.0 g
number procedure often requires the use of another medium Dipotassium Phosphate................................................ 3.3 g
and additional incubation time. Sodium Lauryl Sulfate................................................... 0.2 g
Sodium Desoxycholate................................................. 0.1 g
In 1981, Dufour et al. developed a simple, accurate, nonlethal Bromcresol Purple........................................................ 0.08 g
membrane filter technique for the rapid enumeration of E. coli.3 Bromphenol Red.......................................................... 0.08 g
This medium, m TEC Agar, quantifies E. coli within 24 hours Agar.......................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.
without requiring subculture and identification of isolates. The
authors reported that they were able to recover E. coli from Directions for Preparation from
marine, estuarine and fresh water samples.
Dehydrated Product
m TEC Agar and urea substrate are recommended for use in the 1. Suspend 45.3 g of the powder in 1 L of purified water. Mix
detection of E. coli when evaluating the microbiological quality thoroughly.
of recreational waters.4,5 2. Heat with frequent agitation and boil for 1 minute to com-
pletely dissolve the powder.
Principles of the Procedure 3. Autoclave at 121°C for 15 minutes. (Cool to 45-50°C and
m TEC Agar contains sufficient nutrients to support the growth dispense 4-5 mL amounts into 50 × 10 mm Petri dishes and
of E. coli. Peptone is a source of nitrogen, amino acids, carbon allow to solidify; store in the refrigerator.)
and amino acids. Yeast extract provides trace elements, 4. Test samples of the finished product for performance using
vitamins and amino acids. Monopotassium phosphate and stable, typical control cultures.
Escherichia coli
User Quality Control ATCC™ 8739
Identity Specifications
Difco™ m TEC Agar
Dehydrated Appearance: Green to grayish tan, free-flowing, homogeneous.
Solution: 4.53% solution, soluble in purified water upon boiling.
Solution is deep purple with red cast, slightly opalescent.
Prepared Appearance: Deep purple with red cast, slightly opalescent.
Reaction of 4.53%
Solution at 25°C: pH 7.3 ± 0.2
Cultural Response
Difco™ m TEC Agar
Prepare the medium per label directions. Inoculate using the membrane filter
technique and incubate the plates at 35 ± 2°C for 2 hours. Transfer plates and
incubate at 44.5 ± 0.5°C for 22 ± 2 hours. After incubation, remove filters and
place over pads saturated with approximately 2 mL of urease substrate. Count
yellow to yellow-brown colonies (urease negative) after 15-20 minutes.
Inoculum
Organism ATCC™ CFU Recovery colony COLOR
Escherichia coli 8739 20-80 Good Yellow to yellow-brown
532
Identity Specifications
Difco™ Modified mTEC Agar
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 4.56% solution, soluble in purified water upon
boiling. Solution is light to medium tan, very
slightly to slightly opalescent, without significant
precipitate.
Prepared Appearance: Light tan, clear to very slightly opalescent,
without significant precipitate. Upon removal
from 2-8°C storage, plates may exhibit a crystal
precipitate that disappears upon warming to
room temperature. This is a typical characteristic
of the medium and is acceptable.
Reaction of 4.56%
Solution at 25°C: pH 7.3 ± 0.2
Cultural Response
Difco™ Modified mTEC Agar
Prepare the medium per label directions. Inoculate using the membrane
filtration technique and incubate at 35°C for 2 hours. Transfer plates and
incubate at 44.5 ± 0.2°C for approximately 22-24 hours. Count all red
or magenta colonies.
INOCULUM COLONY
ORGANISM ATCC™ CFU RECOVERY COLOR
Enterococcus faecalis 19433 20-80 Marked to –
complete inhibition
Escherichia coli 13762 20-80 Good Red or magenta
Proteus mirabilis 25933 20-80 Good Tan
534
TSN Agar T
Intended Use Summary and Explanation
TSN (Trypticase™ Sulfite Neomycin) Agar is used for the selective TSN Agar was developed by Marshall et al. as a medium that
isolation of Clostridium perfringens. could achieve rapid enumeration of Clostridium perfringens.1
The formulation is a modification of Mossel’s medium for the
enumeration of sulfite-reducing clostridia in foods.2 The 46°C
temperature of incubation for TSN Agar permits specific and
quantitative results.
Cultural Response
BBL™ TSN Agar
Prepare the medium per label directions. Inoculate and incubate at 46 ± 1°C anaerobically for 18-24 hours.
ORGANISM ATCC™ INOCULUM CFU recovery appearance
Clostridium bifermentans 17836 Undiluted Partial to complete inhibition With or without blackening
Clostridium perfringens 3624 Undiluted Good Blackening
Salmonella enterica subsp.
enterica serotype Enteritidis 13076 104-105 Partial to complete inhibition No blackening
535
Principles of the Procedure 3. Dispense and autoclave at 118°C for 12 minutes. Do not
Neomycin and polymyxin are inhibitory for gram-negative overheat.
enteric bacilli. Neomycin at the concentration employed at least 4. Test samples of the finished product for performance using
partially inhibits C. bifermentans. The relatively high incubation stable, typical control cultures.
temperature of 46°C renders the medium highly specific for
C. perfringens. The colonies are black due to the formation of Procedure
ferric sulfide as a result of the reduction of the sulfite. Use on the day of preparation. Inoculate tubes or plates of the
medium by stabbing deep tubes or streaking plates with the test
Formula specimen. Incubate containers for 18-24 hours at 46 ± 0.1°C in
BBL™ TSN Agar an anaerobic atmosphere (BD GasPak™ EZ anaerobic system
Approximate Formula* Per Liter or equivalent).
Pancreatic Digest of Casein........................................ 15.0 g
Sodium Sulfite.............................................................. 1.0 g
Neomycin Sulfate......................................................... 0.05 g
Expected Results
Polymyxin Sulfate......................................................... 0.02 g C. perfringenes produces black colonies at 46°C. C. perfringens
Yeast Extract.............................................................. 10.0 g and C. bifermentans produce black colonies on TSN Agar at
Ferric Citrate................................................................ 0.5 g
37°C; however, C. bifermentans is inhibited at 46°C.1
Agar.......................................................................... 13.5 g
*Adjusted and/or supplemented as required to meet performance criteria.
References
Directions for Preparation from 1. Marshall, Steenbergen and McClung. 1965. Appl. Microbiol. 13:559.
2. Mossel. 1959. J. Sci. Food Agric. 10:662.
Dehydrated Product
1. Suspend 40 g of the powder in 1 L of purified water. Mix Availability
thoroughly. BBL™ TSN Agar
2. Heat with frequent agitation and boil for 1 minute to com- Cat. No. 211690 Dehydrated – 500 g
pletely dissolve the powder.
Cultural Response
Difco™ TT Broth Base, Hajna
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-24 hours. After incubation, plate the inoculated broth onto MacConkey
Agar and incubate at 35 ± 2°C for 18-24 hours.
Tech Agar
(See Pseudomonas Agars)
Identity Specifications
Difco™ Tellurite Glycine Agar
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 6.25% solution, soluble in purified water upon
boiling. Solution is amber, opalescent with
precipitate.
Prepared Appearance: Medium amber, opalescent with precipitate.
Reaction of 6.25%
Solution at 25°C: pH 7.2 ± 0.2
Cultural Response
Difco™ Tellurite Glycine Agar
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 18-48 hours.
Inoculum Colony
Organism ATCC™ CFU RECOVERY COLOR
Escherichia coli 25922 30-300 Marked to –
complete inhibition
Salmonella enterica
subsp. enterica Marked to
serotype Typhimurium 14028 30-300 complete inhibition –
Staphylococcus aureus 25923 30-300 Good Black
Staphylococcus epidermidis 12228 30-300 Partial inhibition Gray, if any
Expected Results
Coagulase-positive staphylococci produce black colonies within
24 hours of incubation at 35°C.
538
Terrific Broth
Intended Use E. coli. The yeast extract concentration is increased to allow for
Terrific Broth is used with glycerol in cultivating recombinant elevated cell yields. Potassium phosphates are added to provide
strains of Escherichia coli. potassium for cellular systems and prevent cell death due to a
drop in pH. Glycerol is added as a carbon and energy source.
Summary and Explanation
Terrific Broth is a highly enriched medium developed by Tartoff Formula
and Hobbs to improve yield in plasmid-bearing E. coli.1 Difco™ Terrific Broth
Recombinant strains have an extended growth phase in the Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 12.0 g
medium. The addition of extra peptone and yeast extract in Yeast Extract.............................................................. 24.0 g
the medium allows higher plasmid yield per volume. Glycerol Dipotassium Phosphate................................................ 9.4 g
is used as the carbohydrate source in this formulation. Unlike
glucose, glycerol is not fermented to acetic acid.
Monopotassium Phosphate.......................................... 2.2
*Adjusted and/or supplemented as required to meet performance criteria.
g
T
Directions for Preparation from
Principles of the Procedure
Peptone and yeast extract provide necessary nutrients and
Dehydrated Product
1. Dissolve 47.6 g of the powder in 1 L of purified water.
cofactors for excellent growth of recombinant strains of
2. Add 4 mL of glycerol to the medium.
3. Autoclave at 121°C for 15 minutes.
User Quality Control 4. Test samples of the finished product for performance using
stable, typical control cultures.
Identity Specifications
Difco™ Terrific Broth
Dehydrated Appearance: Light beige, free-flowing, homogeneous. Procedure
Solution: 4.76% solution, soluble in purified water. Consult appropriate references for recommended test proce-
Solution is light to medium amber, clear. dures.1,2
Prepared Appearance: Light to medium amber, clear.
Reaction of 4.76% Expected Results
Solution at 25°C: pH 7.2 ± 0.2 Growth is evident in the form of turbidity.
539
Cultural Response
Difco™ Tetrathionate Broth Base
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-24 hours. After incubation, subculture onto MacConkey Agar plates and
incubate plated media at 35 ± 2°C for 18-24 hours.
COLONIES ON
ORGANISM ATCC™ INOCULUM CFU RECOVERY MACCONKEY AGAR
Escherichia coli 25922 102-103 Little or no increase in Pink with bile
number of colonies precipitate
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028 102-103 Good Colorless
540
541
Expected Results
Typical colonial morphology on these media is as follows:
Neisseria gonorrhoeae...... Small, grayish-white to colorless,
mucoid
Neisseria meningitidis....... Medium to large, blue-gray, mucoid
542
T
Thermoacidurans Agar
Intended Use Summary and Explanation
Thermoacidurans Agar is used for isolating and cultivating Stern et al.1 described a medium for isolating B. coagulans
Bacillus coagulans (Bacillus thermoacidurans) from foods. (B. thermoacidurans), which causes “flat sour” spoilage in
tomato juice and other canned foods. Bacterial growth
User Quality Control results in a 0.3-0.5 drop in pH; the ends of the can remain flat.
B. coagulans is a soil microorganism that can be found in
Identity Specifications canned tomato products and dairy products. Conditions
Difco™ Thermoacidurans Agar
favorable to multiplication of the organism can result in
Dehydrated Appearance: Light tan, free-flowing, homogeneous.
spoilage of the food product.2
Solution: 3.9% solution, soluble in purified water upon
boiling. Solution is light amber, opalescent. Thermoacidurans Agar can also be used to isolate mesophilic
Prepared Appearance: Light amber, opalescent. spore-forming anaerobes (Clostridium spp.) from foods. These
Reaction of 3.9% microorganisms tolerate high heat, grow in the absence of oxygen
Solution at 25°C: pH 5.0 ± 0.2
and grow over the range of temperatures used in canned and
Cultural Response processed foods. They are of primary importance in spoilage of
Difco™ Thermoacidurans Agar low-acid foods packed in hermetically sealed containers.2
Prepare the medium per label directions. Inoculate and incubate at
55 ± 1°C for 18-48 hours. Principles of the Procedure
Organism ATCC™ Inoculum CFU RECOVERY Thermoacidurans Agar contains peptone to provide the
Bacillus coagulans 7050 102-103 Good carbon and nitrogen for general growth requirements. Yeast
extract supplies B-complex vitamins which stimulate bacterial
growth. Dextrose is the carbohydrate source. Agar is the
solidifying agent.
543
Formula Procedure
Difco™ Thermoacidurans Agar Consult appropriate references for recommended test proce-
Approximate Formula* Per Liter dures.1,2
Yeast Extract................................................................ 5.0 g
Proteose Peptone......................................................... 5.0 g
Dextrose ..................................................................... 5.0 g Expected Results
Dipotassium Phosphate................................................ 4.0 g Refer to appropriate references and procedures for results.
Agar.......................................................................... 20.0 g
*Adjusted and/or supplemented as required to meet performance criteria.
Limitation of the Procedure
Directions for Preparation from Microorganisms other than B. coagulans may grow on this
medium. Perform microscopic examination and biochemical
Dehydrated Product
tests to identify to genus and species if necessary.
1. Suspend 39 g of the powder in 1 L of purified water. Mix
thoroughly.
References
2. Heat with frequent agitation and boil for 1 minute to 1. Stern, Hegarty and Williams. 1942. Food Research 7:186.
completely dissolve the powder. 2. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
3. Autoclave at 121°C for 15 minutes. Avoid overheating which
could cause a softer medium. Availability
4. Test samples of the finished product for performance using Difco™ Thermoacidurans Agar
stable, typical control cultures. CCAM COMPF
Cat. No. 230310 Dehydrated – 500 g
544
T
Ferrous Sulfate............................................................. 0.08 g loosened at 35-37°C for 16-18 hours. Read the percent transmittance
Polysorbate 80............................................................. 2.0 g using a spectrophotometer at 660 nm.
*Adjusted and/or supplemented as required to meet performance criteria.
Difco™ Thiamine Assay Medium LV
Prepare the medium per label directions. The medium supports the
Precautions growth of Weissella viridescens ATCC™ 12706 when prepared in
Great care to avoid contamination of media or glassware must single strength and supplemented with thiamine. The medium should
produce a standard curve when tested using a thiamine hydrochloride
be taken in microbiological assay procedures. Extremely small reference standard at 0.0 to 25.0 ng per 10 mL. Incubate tubes with caps
amounts of foreign material may be sufficient to give erroneous loosened at 30 ± 2°C for 16-20 hours. Read the percent transmittance
results. Scrupulously clean glassware free from detergents and using a spectrophotometer at 660 nm.
other chemicals must be used. Glassware must be heated to
250°C for at least 1 hour to burn off any organic residues that
might be present. Take precautions to keep sterilization and Procedure
cooling conditions uniform throughout the assay. Thiamine Assay Medium
Prepare stock cultures of the test organism, Lactobacillus
Directions for Preparation from fermentum ATCC 9338, by stab inoculation on Lactobacilli
Dehydrated Product Agar AOAC or Micro Assay Culture Agar. After 24-48 hours
1. Suspend the powder in 100 mL of purified water: incubation at 35-37°C, keep the tubes in the refrigerator. Make
Difco™ Thiamine Assay Medium - 8.5 g; transfers in triplicate at monthly intervals.
Difco™ Thiamine Assay Medium LV - 8.4 g. Prepare the inoculum by subculturing a stock culture of
2. Heat with frequent agitation and boil for 2-3 minutes. the test organism in 10 mL of Lactobacilli Broth AOAC or
3. Dispense in 5 mL amounts into tubes, evenly dispensing the Micro Inoculum Broth. After 16-18 hours incubation at 35-
precipitate. 37°C, centrifuge the cells under aseptic conditions and decant
4. Add standard or test samples. the supernatant liquid. Wash the cells three times with 10 mL
5. Adjust the volume to 10 mL with purified water. sterile 0.85% NaCl. After the third wash, resuspend the cells
6. Autoclave at 121°C for 5 minutes. in 10 mL sterile 0.85% NaCl. Add 0.5 mL of this suspension
to 100 mL sterile 0.85% NaCl. Use one drop of the resulting
suspension to inoculate the assay tubes.
545
A standard curve should be run with each assay because condi- The solution for preparing the standard curve for Thiamine
tions of heating and incubation temperature that influence the Assay Medium LV may be prepared as follows:
standard curve readings cannot always be duplicated.
1. Dissolve 50 mg of thiamine hydrochloride in 500 mL
The tubes for the Thiamine Assay Medium standard curve purified water (100 µg/mL).
contain 0.0, 0.005, 0.01, 0.015, 0.02, 0.03, 0.04 and 0.05 µg 2. Add 1 mL of the solution in Step 1 to 99 mL purified water
of thiamine hydrochloride per 10 mL tube. The most effective (1 µg/mL).
assay range for Thiamine Assay Medium is between 0.005 and 3. Add 1 mL of the solution in Step 2 to 199 mL purified water
0.03 µg thiamine. to give a final concentration of 5 ng (0.005 µg) per mL.
Prepare the stock solution of thiamine required for the prepa- Following incubation of W. viridescens ATCC 12706 at
ration of the standard curve in Thiamine Assay Medium as 30 ± 2°C for 16-20 hours, the growth response is measured
follows: turbidimetrically.
1. Dissolve 0.1 g of thiamine hydrochloride in 1,000 mL of
purified water (100 µg/mL).
Expected Results
Thiamine Assay Medium and Thiamine Assay Medium LV
2. Add 1 mL of the solution in Step 1 to 99 mL purified water
(1 µg/mL). 1. Prepare a standard concentration response curve by plotting
3. Add 1 mL of the solution in Step 2 to 99 mL purified water the response readings against the amount of standard in each
to give a final concentration of 10 ng (0.010 µg/mL). Use 0.0, tube, disk or cup.
0.5, 1, 1.5, 2, 3, 4 and 5 mL of this final solution per tube. 2. Determine the amount of vitamin at each level of assay
Prepare fresh stock solution daily. solution by interpolation from the standard curve.
3. Calculate the concentration of vitamin in the sample from
After 20-24 hours incubation at 35-37°C, L. fermentum the average of these values. Use only those values that do
ATCC 9338 is capable of using the pyrimidine and thiazole not vary more than ±10% from the average and use the
moieties of the thiamine molecule. It is essential that the growth results only if two-thirds of the values do not vary more
response be measured turbidimetrically prior to this time. than ± 10%.
Incubate the tubes at 35-37°C for 16-18 hours, then place in the
refrigerator for 15-30 minutes to stop growth. The growth can Limitations of the Procedure
then be measured by any suitable nephelometric method. 1. The test organism used for inoculating an assay medium must
Thiamine Assay Medium LV be cultured and maintained on media recommended for this
Prepare stock cultures of the test organism, W. viridescens purpose.
ATCC 12706, by stab inoculation on APT Agar or Lactobacilli 2. Aseptic technique should be used throughout the microbio-
Agar AOAC. After 24-48 hours incubation at 30 ± 2°C, keep logical assay procedure.
the tubes in the refrigerator. Make transfers in triplicate at 3. The use of altered or deficient media may cause mutants
monthly intervals. having different nutritional requirements which will not give
a satisfactory response.
Prepare the inoculum by subculturing a stock culture of the test 4. For successful results, all conditions of the assay must be
organism to 10 mL APT Broth or Lactobacilli Broth AOAC. followed exactly.
After 16-20 hours incubation at 30 ± 2°C, centrifuge the cells
under aseptic conditions and decant the supernatant liquid. Wash References
the cells three times with 10 mL sterile 0.85% NaCl. After the 1. Sarett and Cheldelin. 1944. J. Biol. Chem. 155:153.
2. Deibel, Evans and Niven. 1957. Abstr. A68, p. 28. Bacteriol. Proc. 57th Gen. Meet. Soc. Am. Bacte-
third wash, resuspend the cells in 10 mL sterile 0.85% NaCl. Add riologists. 1957.
3. Evans and Niven. 1951. J. Bacteriol. 62:599.
1 mL of this cell suspension to 100 mL sterile 0.85% NaCl. Use 4. Diebel, Evans and Niven. 1955. Abstr. G56, p. 48. Bacteriol. Proc. 55th Gen. Meet. Soc. Am. Bacte-
one drop of this suspension to inoculate the assay tubes. riologists. 1955.
546
Thioglycollate Media
Fluid Thioglycollate Medium • NIH Thioglycollate
Broth • Sterility Test Broth • Thioglycollate Medium,
Brewer Modified • Fluid Thioglycollate Medium
with Beef Extract • Thioglycollate Medium without
Dextrose • Thioglycollate Medium (Fluid), without
Dextrose or (Eh) Indicator • Thioglycollate Medium
without Indicator (135C) • Fluid Thioglycollate
Medium, Enriched • Enriched Thioglycollate Medium
Thioglycollate Medium with Calcium Carbonate
Enriched Thioglycollate Medium with Calcium
Carbonate
Intended Use Thioglycollate Medium with Calcium Carbonate and Thiogly-
Fluid Thioglycollate Medium (FTM) is used for the steril- collate Medium, Enriched, with Calcium Carbonate are recom-
ity testing of biologics and for the cultivation of anaerobes, mended for the maintenance of stock cultures.
aerobes and microaerophiles. Fluid Thioglycollate Medium and NIH Thioglycollate Broth/
Sterility Test Broth meet United States Pharmacopeia (USP)
NIH Thioglycollate Broth and Sterility Test Broth (USP
Alternative Thioglycollate Medium) may be used for sterility performance specifications. T
testing instead of FTM.
Summary and Explanation
Thioglycollate Medium, Brewer Modified is used for the
Quastel and Stephenson1 found that the presence of a small
cultivation of obligate anaerobes, microaerophiles and faculta-
amount of a compound containing an –SH group (cysteine,
tive organisms.
thioglycollic acid, glutathione) permitted “aerobic” growth of
Fluid Thioglycollate Medium with Beef Extract is used in Clostridium sporogenes in tryptic digest broth.
cultivating microorganisms from normally sterile biological
Falk, Bucca and Simmons2 pointed out the advantages of
products.
using small quantities of agar (0.06-0.25%) in detecting
Thioglycollate Medium without Dextrose and Thioglycol- contaminants during sterility testing of biologicals. The value
late Medium (Fluid), without Dextrose or (Eh) Indicator are of combining a small amount of agar and a reducing substance
used as bases for fermentation studies of anaerobes, as well was demonstrated by Brewer.3 Brewer’s experiments revealed
as for detecting microorganisms in normally sterile materials, that in a liquid medium containing 0.05% agar, anaerobes
especially those containing mercurial preservatives. grew equally well in the presence or absence of sodium thio-
Thioglycollate Medium without Indicator (135C) is an enriched glycollate. Marshall, Gunnish and Luxen4 reported satisfactory
general-purpose medium for the recovery of a wide variety cultivation of anaerobes in Brewer’s Thioglycollate Medium
of microorganisms, particularly obligate anaerobes, from in the presence of a mercurial preservative. Nungester, Hood
clinical specimens and other materials. and Warren5 and Portwood6 confirmed the neutralization of
the bacteriostatic effect of mercurial compounds by sodium
Fluid Thioglycollate Medium, Enriched and Enriched thioglycollate. Incorporation of casein peptone was introduced
Thioglycollate Medium are general-purpose media used in by Vera.7 Malin and Finn8 reported the commonly used medium
qualitative procedures for the cultivation of fastidious, as well containing thioglycollate is inhibitory to some organisms in the
as nonfastidious microorganisms, including aerobic and anaerobic presence of a carbohydrate. In 1941, the National Institutes
bacteria, from a variety of clinical and nonclinical specimens. of Health specified the use of two thioglycollate media in
Enriched Thioglycollate Medium when supplemented with sterility testing, the Brewer Formula and the Linden Formula.9
sodium bicarbonate or a marble chip is used to prepare a The Linden Formula was later referred to as Modified Brewer
standardized inoculum by the growth method for antimicrobial Thioglycollate Medium in which meat infusion was replaced by
susceptibility testing of anaerobic bacteria. plant (soy) peptones.10
547
Identity Specifications
Difco™ Fluid Thioglycollate Medium Difco™ Thioglycollate Medium without Dextrose
Dehydrated Appearance: Light beige, free-flowing, homogeneous. Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 2.98% solution, soluble in purified water upon Solution: 2.4% solution, soluble in purified water upon
boiling. When hot, solution is light amber, boiling. When hot, solution is light amber, clear
clear. to very slightly opalescent.
Prepared Appearance: Light amber, slightly opalescent, 10% or less of Prepared Appearance: Light amber, slightly opalescent, 10% or less of
upper layer may be medium pink. After shaking upper layer is green.
solution becomes pink throughout. Reaction of 2.4%
Reaction of 2.98% Solution at 25°C: pH 7.2 ± 0.2
Solution at 25°C: pH 7.1 ± 0.2
Difco Thioglycollate Medium without Dextrose or
™
Reaction of 3.47%
Solution at 25°C: pH 7.2 ± 0.2
548
Cultural Response
Difco™ Fluid Thioglycollate Medium Difco™ Fluid Thioglycollate Medium with Beef Extract
Prepare the medium per label directions. Inoculate and incubate at 30-35 C °
Prepare the medium per label directions. Inoculate and incubate at
for 18-48 hours (up to 72 hours, if necessary). To test for growth promotion 35 ± 2°C for 18-48 hours.
according to the USP/EP, inoculate using organisms marked with (*) and
ORGANISM ATCC™ INOCULUM CFU RECOVERY
incubate aerobically at 30-35°C for up to 5 days.
Bacillus subtilis 6633 10-102 Good
INOCULUM USP/EP
ORGANISM ATCC™ CFU RECOVERY GROWTH Bacteroides vulgatus 8482 10-102 Good
Clostridium novyi 7659 10-102 Good N/A Candida albicans 10231 10-102 Good
Clostridium perfringens 13124 10-10 2
Good N/A Clostridium chauvoei 10092 10-10 2
Good
Staphylococcus aureus 25923 10-102 Good N/A Clostridium perfringens 13124 10-102 Good
Bacillus subtilis* 6633 10-102 N/A Growth Clostridium sporogenes 19404 10-102 Good
Bacteroides vulgatus* 8482 10-102 N/A Growth Kocuria rhizophila 9341 10-102 Good
Clostridium sporogenes* 11437 10-102 N/A Growth Mercurial Neutralization Test – To perform, add 1% Merthiolate™ to medium,
Clostridium sporogenes* 19404 10-102 N/A Growth inoculate (103 CFU) with Staphylococcus aureus ATCC 6538P and Strepto-
coccus pyogenes ATCC 19615, and incubate at 30-35°C for 18-48 hours.
Kocuria rhizophila* 9341 10-102 N/A Growth Recovery of organisms indicates that Merthiolate has been neutralized.
Pseudomonas aeruginosa* 9027 10-102 N/A Growth
Difco™ Thioglycollate Medium without Dextrose,
Staphylococcus aureus* 6538 10-102 N/A Growth Thioglycollate Medium without Indicator* or Thio-
Mercurial Neutralization Test – To perform, add 1% Merthiolate™* to glycollate Medium without Dextrose or Indicator*
medium, inoculate (103 CFU) with Staphylococcus aureus ATCC 6538P Prepare the medium per label directions. Inoculate and incubate at
and Streptococcus pyogenes ATCC 19615, and incubate at 30-35°C for 35 ± 2°C for 18-48 hours.
18-48 hours. Recovery of organisms indicates that Merthiolate has been
ORGANISM ATCC™ INOCULUM CFU RECOVERY
neutralized.
Bacteroides fragilis 25285 10-102 Poor to fair†
Difco™ NIH Thioglycollate Broth
Prepare the medium per label directions. Inoculate duplicate tubes and Bacteroides vulgatus 8482 10-102 Poor to fair†
incubate at 30-35°C for 18-48 hours (up to 72 hours, if necessary) under Clostridium novyi 7659 10-10 2
Good
anaerobic conditions (tight caps). Clostridium sporogenes 11437 10-102 Good
ORGANISM ATCC™ INOCULUM CFU RECOVERY Staphylococcus aureus 25923 10-102 Good
Bacteroides vulgatus 8482 10-102 Poor to good *Mercurial Neutralization Test – To perform, add 1% Merthiolate™ to
Clostridium sporogenes 11437 10-102 Poor to good medium, inoculate (103 CFU) with Staphylococcus aureus ATCC 6538P
and Streptococcus pyogenes ATCC 19615, and incubate at 30-35°C for
T
Clostridium sporogenes 19404 10-10 2
Poor to good 18-48 hours. Recovery of organisms indicates that Merthiolate has been
Mercurial Neutralization Test – To perform, add 1% Merthiolate™ to neutralized.
medium, inoculate (103 CFU) with Staphylococcus aureus ATCC 6538P † Poor to good for Thioglycollate Medium without Indicator.
and Streptococcus pyogenes ATCC 19615, and incubate at 30-35°C for
Continued
18-48 hours. Recovery of organisms indicates that Merthiolate has been
neutralized.
*Merthiolate is a trademark of Eli Lilly and Company.
Thioglycollate Medium without Indicator (135C) is the medium to be required by certain anaerobes for growth.19,20 The addi-
of choice for diagnostic work because the lack of indicator avoids tion of calcium carbonate enhances the maintenance of stock
possible toxicity to organisms.11 This medium supports a minimal cultures by neutralizing acids produced during growth.16 The
inoculum with early visibility of growth. Enriched Thioglycollate Medium (Broth) recommended by the
CLSI for inoculum preparation for susceptibility tests of anaer-
When used as an enrichment broth to support plated media,
obes consists of Enriched Thioglycollate Medium (Thioglycollate
thioglycollate media are often supplemented with hemin and
Medium without Indicator [135] with 1 µg/mL of Vitamin K1
vitamin K1.16 Fluid Thioglycollate Medium, Enriched is BBL™
and 5 µg/mL of hemin) supplemented with 1 mg/mL of sodium
Fluid Thioglycollate Medium supplemented with vitamin
bicarbonate or a marble chip to neutralize acids produced during
K1 and hemin. Enriched Thioglycollate Medium is BBL
growth of the test organisms.21
Thioglycollate Medium without Indicator-135C supple-
mented with vitamin K1 and hemin. Enriched broth media
are recommended for use in the isolation and cultivation of
Principles of the Procedure
Dextrose, peptone, L-cystine and yeast extract provide the
fastidious or slow growing, obligately anaerobic microorganisms
growth factors necessary for bacterial replication. Sodium
present in clinical materials.17,18 They are also recommended for
chloride provides essential ions. Sodium thioglycollate is a
the isolation and cultivation of a wide variety of aerobic and
reducing agent that prevents the accumulation of peroxides
facultatively anaerobic microorganisms. Enriched Thioglycollate
which are lethal to some microorganisms. The L-cystine is also
Medium is prepared with an anaerobic head space and is
a reducing agent, since it contains sulfhydryl groups which
provided in screw-capped tubes in accordance with CDC
inactivate heavy metal compounds and maintain a low redox
recommendations.17 Vitamin K1 and hemin have been shown
potential, thereby supporting anaerobiosis. Methylene blue is
549
Prepare the medium per label directions. Inoculate and incubate as indicated
Dehydrated Appearance: Fine, homogeneous, free of extraneous
below.
material.
Solution: 3.0% solution, soluble in purified water upon INOCULUM INCUBATION
ORGANISM ATCC™ CFU TIME/TEMP RESULT
boiling. When hot, solution is pale to light,
tan to yellow, clear. Bacteroides fragilis 25285 ≤103 7 days/35 ± 2°C Satisfactory
Prepared Appearance: Pale to light, tan to yellow, moderately hazy Campylobacter jejuni
to hazy. subsp. jejuni 33291 ≤103 7 days/40-44°C Satisfactory
Reaction of 3.0% Saccaharomyces
Solution at 25°C: pH 7.0 ± 0.2 cerevisiae 9763 ≤103 7 days/25 ± 2°C Satisfactory
Staphylococcus
aureus 25923 ≤103 7 days/35 ± 2°C Satisfactory
Streptococcus
pyogenes 19615 ≤103 7 days/35 ± 2°C Satisfactory
550
an indicator of the level of oxidation/reduction in the medium; BBL™ Thioglycollate Medium, Brewer Modified
increased oxidation raises the Eh, causing the methylene blue Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 17.5 g
indicator to become green. Resazurin is an oxidation-reduction
Papaic Digest of Soybean Meal..................................... 2.5 g
indicator, being pink when oxidized and colorless when Dextrose.................................................................... 10.0 g
reduced. The small amount of agar assists in the maintenance Sodium Chloride.......................................................... 5.0 g
of a low redox potential by stabilizing the medium against Sodium Thioglycollate.................................................. 1.0 g
Dipotassium Phosphate................................................ 2.0 g
convection currents, thereby maintaining anaerobiosis in the Methylene Blue............................................................ 2.0 mg
lower depths of the medium. The USP lists 5.5g/L of dextrose Agar............................................................................ 0.5 g
in the formulations for Fluid Thioglycollate Medium and Difco™ Fluid Thioglycollate Medium with Beef Extract
Alternative Thioglycollate Medium; some of the following Approximate Formula* Per Liter
formulations include the anhydrous form of dextrose (5.0g/L). Beef Extract................................................................. 5.0 g
Yeast Extract................................................................ 5.0 g
Vitamin K1 is a growth requirement for some strains of Pancreatic Digest of Casein........................................ 15.0 g
Prevotella melaninogenica18 and is reported to enhance the Dextrose ..................................................................... 5.5 g
Sodium Chloride......................................................... 2.5 g
growth of some strains of Bacteroides species and gram- L-Cystine...................................................................... 0.5 g
positive nonsporeformers.22 Hemin is the source of the X Sodium Thioglycollate.................................................. 0.5 g
factor, which stimulates the growth of many microorganisms. Agar............................................................................ 0.75 g
Resazurin..................................................................... 1.0 mg
Calcium carbonate neutralizes acids produced during growth, Difco™ Thioglycollate Medium without Dextrose
which helps to maintain the viability of fastidious organisms; Approximate Formula* Per Liter
e.g., pneumococci, gram-negative cocci, Clostridium perfringens Pancreatic Digest of Casein........................................ 15.0 g
and other acid-sensitive bacteria. Yeast Extract................................................................ 5.0 g
Sodium Chloride.......................................................... 2.5 g
Dipotassium phosphate is a buffering agent. L-Cystine...................................................................... 0.25 g
Sodium Thioglycollate.................................................. 0.5 g
Agar............................................................................ 0.75 g
Formulae Methylene Blue............................................................ 2.0 mg
Difco™ Fluid Thioglycollate Medium Difco™ Thioglycollate Medium without Dextrose
Approximate Formula* Per Liter or Indicator
Pancreatic Digest of Casein........................................ 15.0 g
Approximate Formula* Per Liter
Yeast Extract................................................................ 5.0 g
Dextrose...................................................................... 5.5 g
Sodium Chloride.......................................................... 2.5 g
Pancreatic Digest of Casein........................................ 15.0
Yeast Extract................................................................ 5.0
Sodium Chloride.......................................................... 2.5
g
g
g
T
L-Cystine...................................................................... 0.5 g
L-Cystine...................................................................... 0.25 g
Sodium Thioglycollate.................................................. 0.5 g
Sodium Thioglycollate.................................................. 0.5 g
Agar............................................................................ 0.75 g
Agar............................................................................ 0.75 g
Resazurin..................................................................... 1.0 mg
BBL™ Thioglycollate Medium, Fluid, without Dextrose
BBL™ Fluid Thioglycollate Medium
or Eh Indicator
Approximate Formula* Per Liter
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 15.0 g
Pancreatic Digest of Casein........................................ 20.0 g
Yeast Extract................................................................ 5.0 g
Sodium Chloride.......................................................... 2.5 g
Dextrose (anhydrous)................................................... 5.0 g
L-Cystine...................................................................... 0.5 g
Sodium Chloride.......................................................... 2.5 g
Sodium Thioglycollate.................................................. 0.5 g
L-Cystine...................................................................... 0.5 g
Agar............................................................................ 0.75 g
Sodium Thioglycollate.................................................. 0.5 g
Agar............................................................................ 0.75 g Difco™ Thioglycollate Medium without Indicator
Resazurin..................................................................... 1.0 mg Approximate Formula* Per Liter
Difco™ NIH Thioglycollate Broth Pancreatic Digest of Casein........................................ 15.0 g
Yeast Extract................................................................ 5.0 g
Approximate Formula* Per Liter
Dextrose...................................................................... 5.0 g
Casitone.................................................................... 15.0 g
Sodium Chloride.......................................................... 2.5 g
Yeast Extract................................................................ 5.0 g
L-Cystine...................................................................... 0.25 g
Dextrose...................................................................... 5.5 g
Sodium Thioglycollate.................................................. 0.5 g
Sodium Chloride.......................................................... 2.5 g
Agar............................................................................ 0.75 g
L-Cystine...................................................................... 0.5 g
Sodium Thioglycollate.................................................. 0.5 g BBL™ Thioglycollate Medium without Indicator – 135C
BBL Sterility Test Broth
™ Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 17.0 g
Approximate Formula* Per Liter
Papaic Digest of Soybean Meal..................................... 3.0 g
Pancreatic Digest of Casein........................................ 15.0 g
Dextrose...................................................................... 6.0 g
Yeast Extract................................................................ 5.0 g
Sodium Chloride.......................................................... 2.5 g
Dextrose (anhydrous).................................................. 5.0 g
L-Cystine...................................................................... 0.25 g
Sodium Chloride.......................................................... 2.5 g
Sodium Thioglycollate.................................................. 0.5 g
L-Cystine...................................................................... 0.5 g
Agar............................................................................ 0.7 g
Sodium Thioglycollate.................................................. 0.5 g
Sodium Sulfite.............................................................. 0.1 g
*Adjusted and/or supplemented as required to meet performance criteria.
551
552
Difco™ NIH Thioglycollate Broth (USP Alternative BBL™ Thioglycollate Medium without Indicator – 135C
Thioglycollate Medium) BS12 CMPH2
USP Cat. No. 211720 Dehydrated – 500 g
Cat. No. 225710 Dehydrated – 500 g 221199 Prepared Tubes, 8 mL (K Tubes) – Pkg. of 10*
221200 Prepared Tubes, 8 mL (K Tubes) – Ctn. of 100*
BBL™ Sterility Test Broth (USP Alternative Thioglycollate 221797 Prepared Tubes, 10 mL (D Tubes) – Pkg. of 10*
Medium) 221798 Prepared Tubes, 10 mL (D Tubes) – Ctn. of 100*
USP 221047 Prepared Tubes, 20 mL (A Tubes) – Ctn. of 100*
Cat. No. 211651 Dehydrated – 500 g BBL™ Fluid Thioglycollate Medium, Enriched
BBL™ Thioglycollate Medium, Brewer Modified Cat. No. 297642 Prepared Tubes (K Tubes) – Ctn. of 100*
Cat. No. 211716 Dehydrated – 500 g BBL™ Enriched Thioglycollate Medium
Difco Fluid Thioglycollate Medium with Beef Extract
™
BS12 CLSI CMPH2 MCM9
Cat. No. 269720 Dehydrated – 500 g Cat. No. 221741 Prepared Tubes, 5 mL (K Tubes) – Pkg. of 10*
269710 Dehydrated – 10 kg 221742 Prepared Tubes, 5 mL (K Tubes) – Ctn. of 100*
221787 Prepared Tubes, 8 mL (K Tubes) – Pkg. of 10*
Difco™ Thioglycollate Medium without Dextrose 221788 Prepared Tubes, 8 mL (K Tubes) – Ctn. of 100*
Cat. No. 236310 Dehydrated – 500 g 297289 Prepared Tubes, 10 mL (D Tubes) – Pkg. of 10*
297292 Prepared Tubes, 10 mL (D Tubes) – Ctn. of 100*
Difco™ Thioglycollate Medium without Dextrose or
Indicator BBL™ Thioglycollate Medium with Calcium
Cat. No. 243210 Dehydrated – 500 g Carbonate Chip
Cat. No. 298518 Prepared Tubes (K Tubes) – Ctn. of 100
BBL™ Thioglycollate Medium, Fluid, without Dextrose
or Eh Indicator BBL™ Enriched Thioglycollate Medium with
Cat. No. 211727 Dehydrated – 500 g Calcium Carbonate
221398 Prepared Tubes (K Tubes) – Ctn. of 100* Cat. No. 297264 Prepared Tubes, 10 mL (D Tubes) – Ctn. of 100*
Difco™ Thioglycollate Medium without Indicator *Store at 2-8°C.
Cat. No. 243010 Dehydrated – 500 g
Thiol Broth
T
Intended Use Formula
Thiol Broth is used for cultivating organisms from body fluids Difco™ Thiol Broth
and other materials containing penicillin, streptomycin or Approximate Formula* Per Liter
sulfonamides. Proteose Peptone No.3............................................... 10.0 g
Pancreatic Digest of Casein.......................................... 4.35 g
Gelatin......................................................................... 1.0 g
Summary and Explanation Yeast Extract................................................................ 5.0 g
Szawatkowski1 and Shanson and Barnicoat2 reported Thiol Dextrose...................................................................... 0.2 g
Sodium Chloride.......................................................... 5.0 g
Broth to be superior in supporting the growth of Bacteroides L-Cystine, Disodium .................................................... 2.4 g
species in blood cultures. Thiol Broth was used to study the Sodium Thioglycollate.................................................. 1.0 g
optimum incubation period of blood culture broths.3 Media p-Aminobenzoic Acid................................................... 0.05 g
*Adjusted and/or supplemented as required to meet performance criteria.
containing thiol and thioglycollate are recommended for
recovery of nutritionally variant streptococci (NVS).4 Directions for Preparation from
Thiol Broth is cited in the first edition of Clinical Microbiology Dehydrated Product
Procedures Handbook5 as a medium specific for anaerobic 1. Suspend 29 g of the powder in 1 L of purified water. Mix
bacteria in blood cultures. thoroughly.
2. Heat with frequent agitation and boil for 1 minute to com-
Principles of the Procedure pletely dissolve the powder.
Peptones and yeast extract provide nitrogen, vitamins and 3. Autoclave at 121°C for 15 minutes.
amino acids in Thiol Broth. Dextrose is a carbon source. 4. Test samples of the finished product for performance using
Sodium chloride maintains osmotic balance. Para-aminobenzoic stable, typical control cultures.
acid is a preservative. Sodium thioglycollate and L-cystine are
rich in sulfhydryl (-SH) groups, which neutralize the bacterio- Procedure
static and bactericidal effects of penicillin, streptomycin and For a complete discussion on processing and interpretation of
sulfonamides. blood cultures and other specimens, refer to appropriate refer-
ences.5,6
553
Identity Specifications
Difco™ Tinsdale Agar Base
Dehydrated Appearance: Light beige, free flowing, homogeneous.
Solution: 4.5% solution, soluble in purified water upon
boiling. Solution is light to medium amber,
slightly opalescent to opalescent.
Prepared Appearance: Light to medium amber, slightly opalescent to
opalescent.
Reaction of 4.5%
Solution at 25°C: pH 7.4 ± 0.2
Difco Tinsdale Enrichment Desiccated
™
Cultural Response
Difco™ Tinsdale Agar Base with Tinsdale Enrichment
Desiccated
Prepare the medium per label directions. Inoculate to obtain discrete colonies
and stab several times using an inoculating needle; incubate at 35 ± 2°C
for 18-48 hours.
Organism ATCC™ Inoculum CFU RECOVERY Appearance
Corynebacterium diphtheriae
biotype gravis 8028 102-103 Good Brown with halos
Corynebacterium diphtheriae
biotype mitis 8024 102-103 Good Brown with halos
Klebsiella pneumoniae 13883 102-103 Marked to complete –
Streptococcus pyogenes 19615 102-103
inhibition
Poor to fair Brown to black without halos
T
555
Procedure Availability
Incubate throat swabs in loosely-capped tubes of Todd Hewitt Bacto™ Todd Hewitt Broth
Broth at 35 ± 2°C in an aerobic atmosphere with or without Cat. No. 249240 Dehydrated – 500 g
249210 Dehydrated – 2 kg
added carbon dioxide for 2-5 hours prior to use in fluorescent
249220 Dehydrated – 10 kg
antibody procedures for the identification of group A strep-
tococci. Incubation may be continued for approximately 24 BBL™ Todd Hewitt Broth
Cat. No. 297778 Prepared Tubes (K Tubes), 0.5 mL – Pkg. of 10
hours prior to streaking for isolation on blood agar plates. Pure
cultures of streptococci may be cultured in Todd Hewitt Broth
221713 Prepared Tubes (K Tubes), 5 mL – Pkg. of 10
221714 Prepared Tubes (K Tubes), 5 mL – Ctn. of 100
T
prior to the preparation of extracts for serological typing. BBL™ Todd Hewitt Broth with Gentamicin and
Consult appropriate references for specific serological test Nalidixic Acid
procedures.2,8 Cat. No. 299486 Prepared Tubes (K Tubes) – Ctn. of 100*
*Store at 2-8°C.
Incubate tubes of Todd Hewitt Broth with Gentamicin and
Nalidixic Acid in an aerobic atmosphere with or without added
carbon dioxide. If turbidity is observed, subculture from the
broth culture to a sheep blood agar plate; otherwise, incubate
an additional 24 hours before discarding.9
557
Dehydrated Appearance: Tan, free-flowing, homogeneous. Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 18-48 hours (72 hours if necessary).
Solution: 6.0% solution, soluble in purified water
upon boiling. Solution is medium to dark Organism ATCC™ Inoculum CFU RECOVERY
amber, slightly opalescent. Lactobacillus acidophilus 4356 10 -10
2 3
Good
Prepared Appearance: Medium to dark amber, slightly opales- Lactobacillus rhamnosus 9595 102-103 Good
cent.
Lactobacillus delbrueckii
Reaction of 6.0% subsp. lactis 4797 102-103 Good
Solution at 25°C: pH 5.0 ± 0.2
Difco™ Tomato Juice Broth Difco™ Tomato Juice Broth
Prepare the medium per label directions. Inoculate and incubate at
Dehydrated Appearance: Tan, free-flowing, homogeneous and may
35 ± 2°C for 18-72 hours.
contain dark particles.
Solution: 4.1% solution, soluble in purified water Organism ATCC™ Inoculum CFU RECOVERY
upon boiling. Solution is dark amber, Lactobacillus rhamnosus 9595 102-103 Good
clear.
Lactobacillus delbrueckii
Prepared Appearance: Dark amber, clear. subsp. lactis 4797 102-103 Good
Reaction of 4.1% Saccharomyces cerevisiae 9080 102-103 Good
Solution at 25°C: pH 6.7 ± 0.2
Saccharomyces cerevisiae 9763 102-103 Good
Uninoculated
Plate
Lactobacillus rhamnosus
ATCC™ 9595
Tomato Juice Agar Special is recommended for the direct plate
count of lactobacilli from saliva and for cultivation of other
acidophilic microorganisms. The acidic pH of Tomato Juice
Agar Special encourages growth of lactobacilli while inhibiting
growth of accompanying bacteria. The number of lactobacilli in
saliva is an index of a predisposition to dental caries as described
by Jay.5, 6 Many dentists use the direct count of lactobacilli for
the diagnosis of caries. This medium is more selective for
lactobacilli than Tomato Juice Agar.
Tomato Juice Broth is recommended for use in cultivating and
isolating yeasts, lactobacilli and other aciduric microorganisms
from clinical specimens and foods.
Availability
Directions for Preparation from
Difco™ Tomato Juice Agar
Dehydrated Product Cat. No. 211794 Dehydrated – 500 g*
Equilibrate the medium to room temperature before opening.
Difco™ Tomato Juice Agar Special
1. Suspend the powder in 1 L of purified water: Cat. No. 238910 Dehydrated – 500 g*
Difco™ Tomato Juice Agar – 51 g;
Difco™ Tomato Juice Broth
Difco™ Tomato Juice Agar Special – 60 g;
Difco™ Tomato Juice Broth – 41 g.
Cat. No. 251720 Dehydrated – 500 g*
251710 Dehydrated – 10 kg*
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Mix thoroughly. *Store at 2-8°C.
Transport Media
Transport Medium Amies • Transport Medium
(Stuart, Toshach and Patsula) • Cary and Blair
Transport Medium
Intended Use Toshach and Patsula improved this formulation, introducing
Transport Medium Amies and Transport Medium (Stuart, what is now known as Stuart’s Transport Medium.2 The
Toshach and Patsula) are used for collecting, transporting and ability of Stuart’s medium to maintain the viability of gonococci
preserving microbiological specimens. during transport3,4 led other researchers to explore its use with
a variety of specimens. This medium is currently recommended
Cary and Blair Transport Medium is used for collecting, trans-
for throat, vaginal and wound samples.
porting and preserving microbiological specimens, particularly
those containing Vibrio cholerae. In 1964, Cary and Blair modified Stuart’s medium by substituting
inorganic phosphates for glycerophosphate and raising the
Summary and Explanation pH to 8.4.5 The modified medium was effective in maintaining
Transport media are chemically defined, semisolid, nonnutritive, the viability of Salmonella and Shigella6,7 in fecal samples.
phosphate buffered media that provide a reduced environment. Due to its high pH, Cary and Blair Transport Medium is also
Transport media are formulated to maintain the viability of effective in maintaining the viability of Vibrio cultures for up
microorganisms without significant increase in growth. to four weeks.8 Cary and Blair Transport Medium is currently
recommended for fecal and rectal samples.
In 1948, Moffett, Young and Stuart described a medium for
transporting gonococcal specimens to the laboratory.1 Stuart,
559
Amies9 confirmed Cary and Blair’s observations that an 4. Autoclave at 121°C for 15 minutes.
inorganic salt buffer was superior to the glycerophosphate. 5. Retighten caps, if necessary. Invert vials just prior to solidifica-
He further modified the formulation by using a balanced salt tion to uniformly distribute the charcoal.
solution containing inorganic phosphate buffer, omitting the 6. Test samples of the finished product for performance using
methylene blue and adding charcoal. This modified medium stable, typical control cultures.
yielded a higher percentage of positive cultures than the transport BBL™ Transport Medium (Stuart, Toshach and Patsula)
medium of Stuart. Transport Medium Amies is recommended for
1. Suspend 14.1 g of the powder in 1 L of purified water. Mix
throat, vaginal and wound samples. Amies media are especially
thoroughly.
suited for specimens containing Neisseria gonorrhoeae.
2. Heat with frequent agitation and boil for 1 minute to
Principles of the Procedure completely dissolve the powder.
In the formulations, potassium chloride, calcium chloride, 3. Dispense in small screw-capped bottles or vials, filling them
magnesium chloride and sodium chloride provide essential almost to capacity. Leave only enough space to permit
ions that help maintain osmotic balance while controlling acceptance of a small swab without overflow when in use.
permeability of bacterial cells. Monopotassium phosphate and 6. Autoclave at 121°C for 10 minutes or steam for 1 hour. After
disodium phosphate provide buffering capabilities. Sodium autoclaving, tighten caps immediately.
thioglycollate suppresses oxidative changes and provides a 7. Test samples of the finished product for performance using
reduced environment. Sodium glycerophosphate is a buffer for stable, typical control cultures.
use with calcium chloride. Methylene blue is a colorimetric BBL™ Cary and Blair Transport Medium
pH indicator of the oxidation-reduction state. Charcoal 1. Suspend 12.6 g of the powder in 991 mL of purified water.
neutralizes fatty acids that are toxic to microorganisms. Agar Mix thoroughly.
makes the media semi-solid. 2. Heat with frequent agitation and boil for 1 minute to
completely dissolve the powder.
Formulae 3. Cool to 50°C and add 9 mL of 1% aqueous calcium chloride.
Difco™ Transport Medium Amies 4. Adjust the pH to approximately 8.4, if necessary.
Approximate Formula* Per Liter 5. Dispense in 7 mL amounts in 9 mL screw-capped test tubes.
Sodium Chloride.......................................................... 3.0 g
Potassium Chloride...................................................... 0.2 g 6. Steam for 15 minutes. Cool. Tighten caps.
Calcium Chloride......................................................... 0.1 g 7. Test samples of the finished product for performance using
Magnesium Chloride.................................................... 0.1 g stable, typical control cultures.
Monopotassium Phosphate.......................................... 0.2 g
Disodium Phosphate.................................................... 1.15 g
Sodium Thioglycollate.................................................. 1.0 g Procedure
Charcoal.................................................................... 10.0 g 1. Obtain specimen with sterile swab. Insert specimen swab(s)
Agar............................................................................ 4.0 g
into the upper third of the medium in the transport con-
BBL™ Transport Medium (Stuart, Toshach and Patsula) tainer.
Approximate Formula* Per Liter
2. Cut with sterile scissors or break-off the protruding portion of
Sodium Thioglycollate.................................................. 1.0 g
Sodium Glycerophosphate ..................................... 10.0 g the swab stick. Tightly screw the lid on the bottle or vial.
Calcium Chloride......................................................... 0.1 g 3. Label the bottle or vial and send to the laboratory with
Methylene Blue............................................................ 2.0 mg minimum delay. Specimens may be refrigerated until ready
Agar............................................................................ 3.0 g
for shipment.
BBL™ Cary and Blair Transport Medium
4. Submit to laboratory within 24 hours for culture and analysis.
Approximate Formula* Per Liter
Sodium Thioglycollate.................................................. 1.5 g
Disodium Phosphate.................................................... 1.1 g Expected Results
Sodium Chloride.......................................................... 5.0 g Survival of bacteria in a transport medium depends on many
Agar ........................................................................... 5.0 g
*Adjusted and/or supplemented as required to meet performance criteria.
factors including the type and concentration of bacteria in
the specimen, the formulation of the transport medium, the
Directions for Preparation from temperature and duration of transport and inoculation to
Dehydrated Product appropriate culture media within 24 hours.
Difco™ Transport Medium Amies Optimal growth and typical morphology can only be expected
1. Suspend 20 g of the powder in 1 L of purified water. Mix following direct inoculation and appropriate cultivation.
thoroughly.
2. Heat with frequent agitation and boil for 1 minute to
completely dissolve the powder.
3. Dispense into 6-8 mL screw-cap vials to within 5 mm of the
top. Cap tightly.
560
561
Trichophyton Agars 1 – 7
Intended Use isolate requires inositol, thiamine or both. Trichophyton Agar
Trichophyton Agars are differential media used in the presump- 5, equivalent to Trichophyton Agar 1 with added nicotinic acid
tive identification of Trichophyton species based on nutritional (2 mg/L), is used with medium 1 to determine the requirement
requirements. for nicotinic acid, and medium 7 is used with medium 6 to
determine the requirement for histidine.
Summary and Explanation
Members of the genus Trichophyton have specific nutritional Principles of the Procedure
requirements that are essential for definitive identification.1-3 Nutritional requirements are determined by inoculating a
Georg and Camp devised a set of chemically-defined media control medium and a medium enriched with a specific
for differentiation and identification of Trichophyton isolates vitamin or amino acid with Trichophyton isolates that have
based on specific vitamin and amino acid requirements.4 These been presumptively identified by gross colony characteristics
requirements are determined by comparing growth in a basal and microscopic morphology.1-6 Moderate to heavy growth in
medium (Trichophyton Agar 1 or 6) with the amount of growth the vitamin- or amino acid-enriched medium compared to little
obtained by providing a specific nutrient. Trichophyton Agar or no growth in the basal medium indicates that the isolate
2, 3 and 4 are used with medium 1 to determine whether an requires that nutrient.
Cultural Response
Difco™ Trichophyton Agars 1, 2 or 3
Prepare the medium per label directions. Inoculate with fresh cultures and incubate at
30 ± 2°C for up to 2 weeks.
RECOVERY RECOVERY
ORGANISM ATCC™ AGARS 1 & 2 AGAR 3
Trichophyton concentricum 9358 Good Good
Trichophyton schoenleinii 4822 Good Good
Trichophyton verrucosum 34470 None to poor Good Uninoculated Trichophyton
Tube schoenleinii
ATCC™ 4822
Difco™ Trichophyton Agar 4
Prepare the medium per label directions. Inoculate with fresh cultures and incubate at
30 ± 2°C for up to 2 weeks.
ORGANISM ATCC™ RECOVERY
Trichophyton rubrum 28188 Good
Trichophyton verrucosum 34470 Poor
Trichophyton violaceum 8376 Good
562
Formulae Procedure
Difco™ Trichtophyton Agar 1 Using a sterile inoculating loop or needle, remove a small amount
Approximate Formula* Per Liter of colony growth from the isolation medium and streak the agar
Vitamin Assay Casamino Acids..................................... 2.5 g surface. A small inoculum should be used to prevent carry-over
Dextrose.................................................................... 40.0 g
Monopotassium Phosphate.......................................... 1.8 g of essential nutrients from the isolation medium.
Magnesium Sulfate...................................................... 0.1 g
Agar.......................................................................... 15.0 g Incubate medium at room temperature for up to 2 weeks.
Difco™ Trichtophyton Agar 2
Approximate Formula* Per Liter
Expected Results
Vitamin Assay Casamino Acids..................................... 2.5 g Record the amount of growth using + to indicate a trace of
Dextrose.................................................................... 40.0 g submerged growth to 4+ to indicate maximum growth. Consult
Monopotassium Phosphate.......................................... 1.8 g appropriate texts for information needed for interpretation of
Magnesium Sulfate...................................................... 0.1 g
Agar.......................................................................... 15.0 g the results.1,2
Inositol....................................................................... 50.0 mg
Difco™ Trichtophyton Agar 3 Limitations of the Procedure
Approximate Formula* Per Liter 1. It is important that pure cultures from a medium that is
Vitamin Assay Casamino Acids..................................... 2.5 g not vitamin enriched, such as Sabouraud Dextrose Agar or
Dextrose.................................................................... 40.0 g another general-purpose fungal medium, be used for the
Monopotassium Phosphate.......................................... 1.8 g
Magnesium Sulfate...................................................... 0.1 g inoculum.
Agar.......................................................................... 15.0 g 2. If cultures are contaminated with bacteria, the cultures
Inositol....................................................................... 50.0 mg should be grown on a fungal medium containing antibiotics
Thiamine HCl........................................................... 200.0 µg
for several generations to eliminate the bacteria. Many
Difco™ Trichtophyton Agar 4
bacteria synthesize vitamins and may invalidate the test
Approximate Formula* Per Liter
Vitamin Assay Casamino Acids..................................... 2.5 g results.
Dextrose.................................................................... 40.0 g 3. When inoculating Trichophyton Agars, take care not to
Monopotassium Phosphate.......................................... 1.8 g carry-over growth substances from primary cultures to the
Magnesium Sulfate...................................................... 0.1 g
Agar.......................................................................... 15.0 g
tube media used in the differential tests. Inocula transferred
Thiamine HCl........................................................... 200.0 µg to the nutrition tubes should be very small.
Difco™ Trichtophyton Agar 6
References
T
Approximate Formula* Per Liter
1. Roberts. 1985. In Washington (ed.), Laboratory procedures in clinical microbiology, 2nd ed. Springer-
Ammonium Nitrate...................................................... 1.5 g Verlag, New York, N.Y.
Dextrose.................................................................... 40.0 g 2. Weitzman, Rosenthal and Silva-Hutner. 1988. Superficial and cutaneous infections caused by molds:
Monopotassium Phosphate.......................................... 1.8 g dermatomycoses. In Wentworth (ed.), Diagnostic procedures for mycotic and parasitic infections, 7th
ed. American Public Health Association, Washington, D.C.
Magnesium Sulfate...................................................... 0.1 g 3. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
Agar.......................................................................... 15.0 g American Society for Microbiology, Washington, D.C.
4. Georg and Camp. 1957. J. Bacteriol. 74:113.
Difco™ Trichtophyton Agar 7 5. Haley, Transdel and Coyle. 1980. Cumitech 11, Practical methods for culture and identification of
fungi in the clinical mycology laboratory. Coord. ed., Sherris. American Society for Microbiology,
Approximate Formula* Per Liter Washington, D.C.
Ammonium Nitrate...................................................... 1.5 g 6. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
Histidine HCl.............................................................. 30.0 mg American Society for Microbiology, Washington, D.C.
Dextrose.................................................................... 40.0 g
Monopotassium Phosphate.......................................... 1.8 g Availability
Magnesium Sulfate...................................................... 0.1 g
Agar.......................................................................... 15.0 g
Difco™ Trichophyton Agar 1
*Adjusted and/or supplemented as required to meet performance criteria. Cat. No. 287710 Dehydrated – 500 g
BBL™ Trichophyton Agar 1
Directions for Preparation from Cat. No. 296243 Prepared Slants (C Tubes) – Pkg of 10*
Dehydrated Product Difco™ Trichophyton Agar 2
Difco™ Trichophyton Agars 1, 2, 3, 4, 6 and 7 Cat. No. 287410 Dehydrated – 500 g
1. Suspend 59 g of the powder in 1 L of purified water. Mix
BBL™ Trichophyton Agar 2
thoroughly. Cat. No. 296244 Prepared Slants (C Tubes) – Pkg. of 10*
2. Heat with frequent agitation and boil for 1 minute to
Difco™ Trichophyton Agar 3
completely dissolve the powder.
Cat. No. 296510 Dehydrated – 500 g
3. Autoclave at 121°C for 12 minutes.
4. Test samples of the finished product for performance using BBL™ Trichophyton Agar 3
stable, typical control cultures. Cat. No. 296245 Prepared Slants (C Tubes) – Pkg. of 10*
Difco™ Trichophyton Agar 4
Cat. No. 219710 Dehydrated – 500 g
BBL™ Trichophyton Agar 4
Cat. No. 296246 Prepared Slants (C Tubes) – Pkg. of 10*
563
564
Availability
Difco™ Triple Sugar Iron Agar
AOAC BAM BS12 CCAM CMPH2 COMPF EP ISO MCM9
SMD SMWW USDA
Cat. No. 226540 Dehydrated – 500 g
Uninoculated Escherichia coli Salmonella Shigella flexneri BBL™ TSI Agar
Tube ATCC™ 25922 Enteritidis ATCC™ 12022
ATCC™ 13076 AOAC BAM BS12 CCAM CMPH2 COMPF EP ISO MCM9
SMD SMWW USDA
Cat. No. 221038 Prepared Slants – Pkg. of 10*
221039 Prepared Slants – Ctn. of 100*
*Store at 2-8°C.
566
Streptococcus Streptococcus
pneumoniae pyogenes
ATCC™ 6305 ATCC™ 19615
567
568
Identity Specifications
BBL™ Trypticase™ Soy Agar BBL™ Trypticase™ Soy Agar (prepared bottle)
Dehydrated Appearance: Fine, homogeneous, free of extraneous material. Appearance: Light to medium tan yellow, clear to trace hazy.
Solution: 4.0% solution, soluble in purified water upon Reaction at 25°C: pH 7.3 ± 0.2
boiling. Solution is light to medium, yellow to
tan, clear to slightly hazy.
BBL™ Trypticase™ Soy Agar (prepared plate)
Appearance: Light to medium tan yellow, hazy.
Prepared Appearance: Plain – Light to medium, yellow to tan, clear to
slightly hazy. Reaction at 25°C: pH 7.3 ± 0.2
With 5% sheep blood – Bright red, opaque. BBL Trypticase Soy Agar (prepared Sterile Pack
™ ™
Cultural Response
BBL™ Trypticase™ Soy Agar BBL™ Trypticase™ Soy Agar (prepared plate)
Prepare the medium per label directions, without (plain) and with 5% Inoculate and incubate at 35 ± 2°C for 48 hours (incubate S. pyogenes
sheep blood (SB). Inoculate and incubate at 35 ± 2°C for 48 hours (incubate with 3-5% CO2). Incubate (*) cultures at 30-35°C for up to 3 days (up to
S. pneumoniae and S. pyogenes with 3-5% CO2). Incubate (*) cultures at 5 days for A. brasiliensis and C. albicans).
30-35°C for up to 3 days (up to 5 days for A. brasiliensis and C. albicans).
ORGANISM ATCC™ INOCULUM CFU RECOVERY
INOCULUM RECOVERY
ORGANISM ATCC™ CFU PLAIN w/SB HEMOLYSIS
Shigella flexneri 12022 50-100 Good
569
570
Procedure
100 × 15 mm and 150 × 15 mm-Style Plates
1. If specimen is being cultured from a swab, roll the swab
directly on the medium surface.
2. Incubate all plates at 35-37°C for 48 hours, and 25°C for 7
days or as required.
3. When incubation has been completed, count the colonies.
RODAC™/Contact Plates
Selected surfaces are sampled by firmly pressing the agar
medium against the test area. Hold the plate with thumb and
second finger and use index finger to press plate bottom firmly
against surface. Pressure should be the same for every sample.
Do not move plate laterally; this spreads contaminants over
the agar surface making resolution of colonies difficult. Slightly
curved surfaces may be sampled with a rolling motion. Grid method:
Areas (walls, floors, etc.) to be assayed may be divided into 1. Subdivide surface (floor or wall) into 36 equal squares per
sections or grids and samples taken from specific points within 100 square feet of area by striking five equidistant dividing
the grid. lines from each of the two adjacent sides.
2. These dividing lines intersect at twenty-five points.
3. Number these intersections consecutively in a serpentine
configuration.
572
4. Use red numerals for odd numbers, black numerals for even Availability
numbers. Difco™ Tryptic Soy Agar with Lecithin and
5. Omit number 13 which falls in the center of the total area. Polysorbate 80 (Microbial Content Test Agar)
6. Sample odd points at one sampling period, even points at CCAM
the next sampling period. Cat. No. 255320 Dehydrated – 500 g*
255310 Dehydrated – 2 kg*
7. For areas greater than 100 square feet, extend grid to include
entire area. BBL™ Trypticase™ Soy Agar with Lecithin and
Polysorbate 80
8. For areas smaller than 25 square feet, divide the areas into
CCAM
twenty-five equal squares (sixteen intersections). Sample
Cat. No. 211764 Dehydrated – 500 g*
eight even-numbered or odd-numbered intersections at each 212263 Dehydrated – 5 lb (2.3 kg)*
sampling period. United States and Canada
9. For areas between 25 and 100 square feet, divide into 36 Cat. No. 221943 Prepared Plates (Double Bag) – Ctn. of 100*
equal squares as in #1. 221945 Contact Plates (Double Bag) – Pkg. of 20*
221288 Prepared RODAC™ Plates – Pkg. of 10*
10. Mark plates with intersection numbers. 221287 Prepared RODAC™ Plates – Ctn. of 100*
222242 Prepared RODAC™ SL Plates – Pkg. of 20*
Incubate exposed plates at 35-37°C for 48 hours, and 25°C for
222249 Prepared RODAC™ SL Plates – Ctn. of 100*
7 days or as required. 221961 Sterile Pack Contact Plates – Pkg. of 10*
222208 Sterile Pack Contact Plates – Ctn. of 100*
Expected Results 221238 Sterile Pack RODAC™ Plates – Pkg. of 10*
222207 Sterile Pack RODAC™ Plates – Ctn. of 100*
Because interpretations are relative, each laboratory should 222248 Sterile Pack RODAC™ SL Plates – Pkg. of 10*
establish its own values for what constitutes a clean area. 222247 Sterile Pack RODAC™ SL Plates – Ctn. of 100*
292335 Isolator Pack RODAC™ Plates – Ctn. of 100*
Count all developing colonies. Spreading colonies should be 222252 Isolator Pack RODAC™ SL Plates – Pkg. of 10*
counted as one but care should be taken to observe other distinct 222253 Isolator Pack RODAC™ SL Plates – Ctn. of 100*
colonies intermingled in the growth around the plate periphery or 292271 Sterile Pack Finger Dab™ Plates – Ctn. of 100*
292648 Isolator Pack Finger Dab™ Plates – Pkg. of 10*
along a hair line. These should also be counted as one colony, as 292649 Isolator Pack Finger Dab™ Plates – Ctn. of 100*
should bi-colored colonies and halo type spreaders. 292650 Isolator Pack Finger Dab™ Plates
(150 × 15 mm-style) – Pkg. of 5*
It is generally agreed that 200 colonies is the approximate Europe
maximum that can be counted on contact plates. Cat. No.
254038
254542
Contact Plates – Pkg. of 33*
Contact Plates – Pkg. of 220* T
Colony counts may be recorded by: 257383 Isolator Pack Plates – Pkg. of 10*
257384 Isolator Pack Plates – Ctn. of 100*
1. Simply keeping individual counts. 257379 Isolator Pack Plates (HF) – Ctn. of 100*
2. Number of viable particles per square foot (agar area is 3.97 257380 Isolator Pack RODAC™ Plates – Pkg. of 10*
square inches). 257381 Isolator Pack RODAC™ Plates – Ctn. of 100*
257378 Isolator Pack RODAC™ SL Plates – Pkg. of 10*
3. Means and standard deviations.
257382 Isolator Pack RODAC™ SL Plates – Ctn. of 100*
Subculture colonies of interest so that positive identification can BBL™ Trypticase™ Soy Agar with Penicillinase
be made by means of biochemical and/or serological testing. Cat. No. 221839 Sterile Pack Plates – Pkg. of 10*
221837 Sterile Pack Plates (150 × 15 mm-style) –
Limitation of the Procedure Pkg. of 5*
The effectiveness of preservative neutralization with this BBL™ Trypticase™ Soy Agar with Lecithin, Polysorbate 80
medium depends on both the type and concentration of the and Pencillinase
preservative(s). United States and Canada
Cat. No. 221987 Contact Plates – Pkg. of 10*
221234 Sterile Pack RODAC™ Plates – Pkg. of 10*
References 222246 Sterile Pack RODAC™ SL Plates – Pkg. of 10*
1. Vesley and Michaelson. 1964. Health Lab. Sci. 1:107.
2. Pryor and McDuff. 1969. Exec. Housekeeper, March. Europe
3. Dell, L. A. 1979. Pharm. Technol. 3:47. Cat. No. 257400 Sterile Pack RODAC™ Plates – Ctn. of 100*
4. Hickey, Beckelheimer and Parrow. 1993. In Marshall (ed.), Standard methods for the examination of
dairy products, 16th ed. American Public Health Association, Washington, D.C.
257421 Isolator Pack RODAC™ SL Plates – Pkg. of 10*
5. Orth. 1993. Handbook of cosmetic microbiology. Marcel Dekker, Inc., New York, N.Y. 257455 Sterile Pack Plates – Ctn. of 100*
6. Hall and Hartnett. 1964. Public Health Rep. 79:1021. 257403 Isolator Pack Plates – Ctn. of 100*
7. McGowan. 1985. In Lennette, Balows, Hausler and Shadomy (ed.), Manual of clinical microbiology, *Store at 2-8°C.
4th ed. American Society for Microbiology, Washington, D.C.
8. Bryan. 1995. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology,
6th ed. American Society for Microbiology, Washington, D.C.
9. Favero, Gabis and Vesley. 1984. In Speck (ed.), Compendium of methods for the microbiological
examination of foods, 2nd ed. American Public Health Association, Washington, D.C.
10. Quisno, Gibby and Foter. 1946. Am. J. Pharm. 118:320.
11. Erlandson and Lawrence. 1953. Science 118:274.
12. Sveum, Moberg, Rude and Frank. 1992. In Vanderzant and Splittstoesser (ed.), Compendium of
methods for the examination of foods, 3rd ed. American Public Health Association, Washington,
D.C.
13. Association for the Advancement of Medical Instrumentation. 1984. Process control guidelines for
gamma radiation sterilization of medical devices. AAMI, Arlington, Va.
573
574
Enterococcus Streptococcus
faecalis pyogenes
ATCC™ 29212 ATCC™ 19615
575
may be mistakenly reported as group A. If a hemolytic reaction Incubate plates at 35 ± 2°C for 18-72 hours. Since many
is obtained, the organisms should be tested with a Taxo A disc pathogens require carbon dioxide on primary isolation, plates
and grouped serologically or tested by the fluorescent method.6 may be incubated in an atmosphere containing approximately
Beta-hemolytic streptococci and Haemophilus hemolyticus 5% CO2.
may be differentiated by performing a Gram stain on a smear CAMP Test10
prepared from the colony.7
Non-hemoytic, bile-esculin negative streptococci or bacitra-
Defibrinated rabbit blood is also used for enriching agar-based cin-resistant beta-hemolytic streptococci may be tested by
media.8 Hemolytic reactions on Trypticase Soy Agar with 5% the CAMP test for presumptive identification as S. agalactiae
Rabbit Blood (TSA II) prepared plates are similar to those on (Lancefield group B). The inoculum may be taken from an
sheep blood. However, rabbit blood does not inhibit Haemophilus overnight broth culture or from colonies picked from a blood
haemolyticus, a bacterium inhibited by sheep blood that pro- agar plate. Make a single streak of Staphylococcus aureus
duces colonies indistinguishable from those of beta-hemolytic ATCC 33862 across the center of a blood agar plate. If a loop
streptococci. is used, do not use it parallel to the agar surface, since the
streak will be too wide and the results will not be satisfac-
Formulae tory. The streptococcal isolates to be tested are inoculated by
Difco™ Tryptic Soy Blood Agar Base No. 2 making a simple streak perpendicular to the S. aureus line
Approximate Formula* Per Liter coming as close as possible (2-3 mm), but not touching it.
Tryptone H................................................................. 15.0 g
Soytone....................................................................... 5.0 g Several streptococcal isolates may be tested on the same plate.
Sodium Chloride.......................................................... 5.0 g Perpendicular streptococcal streaks should be 5-8 mm apart.
Agar.......................................................................... 15.0 g Include a known S. agalactiae for a positive control and
Difco™ Tryptic Soy Blood Agar Base EH S. pyogenes as a negative control. The procedure should be
Approximate Formula* Per Liter practiced with known cultures before using it to identify
Tryptone H Plus.......................................................... 15.0 g
Soytone....................................................................... 5.0 g unknown isolates.
Sodium Chloride.......................................................... 5.0 g NOTE: Studies on the CAMP Test have shown that the
Agar.......................................................................... 15.0 g
reaction is most reliable early in the shelf life of some lots
BBL™ Trypticase™ Soy Agar, Modified (TSA II)
of the prepared plated medium. It is recommended that
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 14.5 g S. agalactiae ATCC 12386 be included along with patient
Papaic Digest of Soybean Meal..................................... 5.0 g isolates to verify satisfactory performance.
Sodium Chloride.......................................................... 5.0 g
Agar.......................................................................... 14.0 g Incubate plates in an aerobic atmosphere at 35 ± 2°C for
Growth Factors............................................................ 1.5 g 18-24 hours. Do not incubate anaerobically or in a CO2
*Adjusted and/or supplemented as required to meet performance criteria.
incubator. False-positive results may occur with group A strep-
tococci when incubation is in an anaerobic or CO2-enriched
Directions for Preparation from
atmosphere.10,11
Dehydrated Product
1. Suspend 40 g of the powder in 1 L of purified water. Mix Expected Results
thoroughly. Hemolytic streptococci may appear translucent or opaque,
2. Heat with frequent agitation and boil for 1 minute to grayish, small (1 mm), or large matt and mucoid (2-4 mm)
completely dissolve the powder. colonies, encircled by a zone of hemolysis. Gram stains should
3. Autoclave at 121°C for 15 minutes. DO NOT OVER- be made and examined to check the macroscopic findings. (Other
HEAT. organisms which may cause hemolysis include Listeria, various
4. For preparation of blood plates, add 5-10% sterile, corynebacteria, hemolytic staphylococci, Escherichia coli and
defibrinated blood to sterile agar which has been cooled to Pseudomonas.) In reporting, approximate quantitation of the
45-50°C. Mix well. number of colonies of hemolytic streptococci may be helpful
5. Test samples of the finished product for performance using to the clinician.
stable, typical control cultures.
• Pneumococci usually appear as very flat, smooth, translu-
Procedure cent, grayish and sometimes mucoid colonies surrounded
Use standard procedures to obtain isolated colonies from by a narrow zone of “green” (alpha) hemolysis.
specimens. After streaking, stab the agar several times to • Staphylococci appear as opaque, white to gold-yellow
deposit beta-hemolytic streptococci beneath the agar surface. colonies with or without zones of beta hemolysis.
Subsurface growth will display the most reliable hemolytic • Listeria produce small zones of beta hemolysis. They
reactions owing to the activity of both oxygen-stable and may be distinguished by their rod shape in stains, and
oxygen-labile streptolysins.9 by motility at room temperature.
576
T
3. Vera and Power. 1980. In Lennette, Balows, Hausler and Truant (ed.), Manual of clinical microbiology,
3rd ed. American Society for Microbiology, Washington, D.C.
4. Bernheimer, Linder and Avigad. 1979. Infect. Immun. 23:838.
BBL™ Trypticase™ Soy Agar with 5% Sheep Blood
5. Krumweide and Kuttner. 1938. J. Exp. Med. 67:429. (TSA II)//MacConkey II Agar
6. Vera. 1971. Health Lab Sci. 8:176.
7. Finegold and Martin. 1982. Bailey & Scott’s diagnostic microbiology, 6th ed. The C.V. Mosby BS12 CMPH2 MCM9
Company, St. Louis, Mo. United States and Canada
8. Nash and Krenz. 1991. In Balows, Hausler, Herrmann, Isenberg and Shadomy (ed.), Manual of clinical
microbiology, 5th ed. American Society for Microbiology, Washington, D.C. Cat. No. 221290 Prepared I Plate™ Dishes – Pkg. of 20*
9. Ruoff, Whiley and Beighton. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of 221291 Prepared I Plate™ Dishes – Ctn. of 100*
clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
10. Darling. 1975. J. Clin. Microbiol. 1:171. Europe
11. Facklam and Washington. 1991. In Balows, Hausler, Herrmann, Isenberg and Shadomy (ed.), Manual Cat. No. 251290 Prepared I Plate™ Dishes – Pkg. of 20*
of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C.
Japan
Cat. No. 251290 Prepared I Plate™ Dishes – Ctn. of 20*
Availability 251572 Prepared I Plate™ Dishes – Ctn. of 100*
Difco™ Tryptic Soy Blood Agar Base No. 2
BBL™ Trypticase™ Soy Agar with 5% Sheep Blood
BAM COMPF
(TSA II)//MacConkey II Agar with MUG
Cat. No. 227300 Dehydrated – 500 g
Cat. No. 221949 Prepared I Plate™ Dishes – Pkg. of 20*
227200 Dehydrated – 10 kg
BBL™ Trypticase™ Soy Agar with 5% Sheep Blood
Difco™ Tryptic Soy Blood Agar Base EH
(TSA II)//Chocolate II Agar//MacConkey II Agar
BAM COMPF
Cat. No. 299580 Prepared Y Plate™ Dishes – Ctn. of 100*
Cat. No. 228300 Dehydrated – 500 g
228200 Dehydrated – 10 kg BBL™ Trypticase™ Soy Agar with 5% Horse Blood (TSA II)
United States and Canada
BBL™ Trypticase™ Soy Agar, Modified (TSA II)
Cat. No. 221372 Prepared Plates – Pkg. of 20*
BAM COMPF
Europe
Cat. No. 212305 Dehydrated – 500 g Cat. No. 212099 Prepared Plates – Pkg. of 20*
297941 Prepared Pour Tubes, 20 mL – Ctn. of 100
BBL™ Trypticase™ Soy Agar with 5% Rabbit Blood (TSA II)
BBL™ Trypticase™ Soy Agar with 5% Sheep Blood (TSA II)
Cat. No. 221356 Prepared Plates – Pkg. of 20*
BAM BS12 CCAM CMPH2 MCM9 USDA
United States and Canada BBL™ Trypticase™ Soy Agar (TSA II) with Defibrinated
Cat. No. 221239 Prepared Plates – Pkg. of 20* Sheep Blood Slant
221261 Prepared Plates – Ctn. of 100* Cat. No. 220830 Prepared Slants – Pkg. of 10*
Europe 220831 Prepared Slants – Ctn. of 100*
Cat. No. 254053 Prepared Plates – Pkg. of 20* *Store at 2-8°C.
254087 Prepared Plates – Ctn. of 120*
577
578
amino acids and longer-chained peptides. Sodium chloride main- shaped and may be confused with viridans streptococci, which
tains osmotic equilibrium. Defibrinated sheep blood supplies will also grow on this medium. Gram staining, biochemi-
nutrients necessary to support the growth of fastidious organ- cal tests and serological procedures should be performed to
isms and to detect hemolytic reactions while also inhibiting the confirm findings.
growth of Haemophilus haemolyticus, a bacterium commonly
found in nose and throat specimens that is indistinguishable from References
1. Spellerberg and Brandt. 2007. In Murray, Baron, Jorgensen, Landry and Pfaller (ed.), Manual of
beta-hemolytic streptococci.1 Gentamicin is an aminoglycoside clinical microbiology, 9th ed. American Society for Microbiology, Washington, D.C.
antibiotic that inhibits the growth of gram-negative bacteria. 2. Dilworth, Stewart, Gwaltney, Hendley and Sande. 1975. J. Clin. Microbiol. 2:453.
3. Sondag, Morgens, Hoppe and Marr. 1977. J. Clin. Microbiol. 5:397.
Agar is the solidifying agent.
Availability
Procedure BBL™ Trypticase™ Soy Agar with 5% Sheep Blood
Use standard procedures to obtain isolated colonies from speci- (TSA II) with Gentamicin
mens. Incubate the plates in an inverted position (agar side up) Cat. No. 297457 Prepared Plates – Pkg of 20*
at 35°C in a CO2-enriched atmosphere for 18-48 hours. *Store at 2-8°C.
Expected Results
Staphylococci and gram-negative bacteria are inhibited. Circular,
flat, translucent colonies surrounded by zones of alpha hemolysis
may be presumptively identified as Streptococcus pneumoniae.
However, when the colonies are young, they may be dome-
579
Identity Specifications
Bacto™ Tryptic Soy Broth
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 3.0% solution, soluble in purified water upon
warming. Solution is light amber, clear.
Prepared Appearance: Light amber, clear.
Reaction of 3.0%
Solution at 25°C: pH 7.3 ± 0.2
Difco™ Tryptic Soy Broth (prepared bottles)
Appearance: Light to medium tan yellow, clear to trace hazy.
Reaction at 25°C: pH 7.3 ± 0.2
Bacto Tryptic Soy Broth without Dextrose
™
Cultural Response
Bacto™ Tryptic Soy Broth Difco™ Tryptic Soy Broth (prepared bottles)
Prepare the medium per label directions. Inoculate and incubate at Inoculate and incubate at 30-35°C for 18-24 hours (up to 3 days for
30-35°C for 18-72 hours (up to 5 days for A. brasiliensis and C. albicans). B. subtilis). For (*) cultures incubate at 20-25°C for up to 3 days (up to
Prepare duplicate cultures of A. brasiliensis, B. subtilis and C. albicans 5 days for A. brasiliensis).
and incubate at 20-25°C for up to 3 days (up to 5 days for A. brasiliensis
ORGANISM ATCC™ INOCULUM CFU RECOVERY
and C. albicans).
Aspergillus brasiliensis (niger)* 16404 10-100 Growth (20-25°C)
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Bacillus subtillis 6633 10-100 Growth (30-35°C)
Neisseria meningitidis 13090 10-100 Fair to good
Bacillus subtillis* 6633 10-100 Growth (20-25°C)
Staphylococcus epidermidis 12228 10-100 Good
Candida albicans* 10231 10-100 Growth (20-25°C)
Streptococcus pneumoniae 6305 10-100 Good
Escherichia coli 8739 10-100 Growth
Streptococcus pyogenes 19615 10-100 Good
Pseudomonas aeruginosa 9027 10-100 Growth
Aspergillus brasiliensis (niger) 16404 <100 Growth (30-35°C)
Salmonella enterica
Aspergillus brasiliensis (niger) 16404 <100 Growth (20-25°C) subsp. enterica serotype
Bacillus subtillis 6633 <100 Growth (30-35°C) Typhimurium 14028 10-100 Growth
Bacillus subtillis 6633 <100 Growth (20-25°C) Staphylococcus aureus 6538 10-100 Growth
Candida albicans 10231 <100 Growth (30-35°C)
Bacto™ Tryptic Soy Broth without Dextrose
Candida albicans 10231 <100 Growth (20-25°C) Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C
Escherichia coli 8739 <100 Growth for 18-48 hours.
Pseudomonas aeruginosa 9027 <100 Growth ORGANISM ATCC™ INOCULUM CFU RECOVERY
Salmonella enterica Neisseria meningitidis 13090 30-300 Fair to good
subsp. enterica serotype
Typhimurium 14028 <100 Growth Staphylococcus epidermidis 12228 30-300 Good
Staphylococcus aureus 6538 <100 Growth Streptococcus pneumoniae 6305 30-300 Good
Streptococcus pyogenes 19615 30-300 Good
Continued
Tryptic Soy Broth without Dextrose, a modification of TSB, is Principles of the Procedure
a basal medium to which carbohydrates may be added for use Enzymatic digests of casein and soybean provide amino acids
in fermentation studies. Phenol red and other indicators may and other complex nitrogenous substances. Dextrose is an energy
also be added. source. Sodium chloride maintains the osmotic equilibrium.
Dibasic potassium phosphate acts as a buffer to control pH.
580
Identity Specifications
BBL™ Trypticase™ Soy Broth
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution: 3.0% solution, soluble in purified water upon warming. Solution is light, tan to yellow, clear to slightly hazy.
Prepared Appearance: Light, tan to yellow, clear to slightly hazy.
Reaction of 3.0%
Solution at 25°C: pH 7.3 ± 0.2
BBL Trypticase Soy Broth (prepared bottles)
™ ™
Cultural Response
BBL™ Trypticase™ Soy Broth BBL™ Trypticase™ Soy Broth (prepared bottles)
Prepare the medium per label directions. Inoculate tubes and incubate at Inoculate and incubate at 35-37°C for 48 hours. Incubate (*) cultures at
30-35°C for up to 3 days (up to 5 days for A. brasiliensis and C. albicans). 30-35°C for up to 3 days. Incubate (**) cultures at 20-25°C for up to 3
Prepare duplicate cultures of A. brasiliensis, B. subtilis and C. albicans days (up to 5 days for A. brasiliensis and C. albicans).
and incubate at 20-25°C for up to 3 days (up to 5 days for A. brasiliensis
ORGANISM ATCC™ INOCULUM CFU RECOVERY
and C. albicans).
Escherichia coli 25922 <100 Growth
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Staphylococcus aureus 25923 <100 Growth
Aspergillus brasiliensis (niger) 16404 <100 Growth (30-35°C)
Aspergillus brasiliensis (niger)** 16404 <100 Growth (20-25°C)
Aspergillus brasiliensis (niger) 16404 <100 Growth (20-25°C)
Bacillus subtillis* 6633 <100 Growth (30-35°C)
Bacillus subtilis 6633 <100 Growth (30-35°C)
Bacillus subtillis** 6633 <100 Growth (20-25°C)
Bacillus subtilis 6633 <100 Growth (20-25°C)
Candida albicans** 10231 <100 Growth (20-25°C)
Candida albicans 10231 <100 Growth (30-35°C)
Pseudomonas aeruginosa* 9027 <100 Growth (30-35°C)
Candida albicans 10231 <100 Growth (20-25°C)
Staphylococcus aureus* 6538 <100 Growth (30-35°C)
Escherichia coli 8739 <100 Growth
Pseudomonas aeruginosa 9027 <100 Growth
Salmonella enterica
subsp. enterica serotype
Typhimurium 14028 <100 Growth T
Staphylococcus aureus 6538 <100 Growth
581
582
583
Procedure Availability
Using a sterile swab or inoculating loop, remove fresh growth BBL™ Trypticase™ Soy Broth with 20% Glycerol
from the plated or slanted medium and suspend in the broth Cat. No. 296346 Prepared Tubes (K Tubes), 1.5 mL – Pkg. of 10
297808 Prepared Tubes (K Tubes), 1.5 mL – Ctn. of 100
maintenance medium to achieve the desired concentration of
297352 Prepared Tubes (C Tubes), 10 mL – Ctn. of 100
viable cells. Freeze suspension immediately at -20°C or below.
Uninoculated Pasturized
User Quality Control Plate Milk
Identity Specifications
Difco™ Tryptone Glucose Extract Agar
Dehydrated Appearance: Light to medium tan, free-flowing, homo-
geneous.
Solution: 2.4% solution, soluble in purified water
upon boiling. Solution is light amber, clear
to slightly opalescent.
Prepared Appearance: Light amber, clear to slightly opalescent.
Reaction of 2.4%
Solution at 25°C: pH 7.0 ± 0.2
Difco m TGE Broth
™
Cultural Response
Difco™ Tryptone Glucose Extract Agar
Prepare the medium per label directions in parallel with a control (approved)
lot of medium. Inoculate with serial dilutions of pasteurized and raw
milk samples using the pour plate technique and incubate at 32 ± 1°C for
47-49 hours. Recovery of bacteria from the milk samples should be
comparable for both the test and control lots.
Difco™ m TGE Broth
Prepare the medium per label directions. Inoculate using the membrane
filter technique and incubate at 35 ± 2°C for 18-24 hours in a humid
atmosphere.
T
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Escherichia coli 25922 30-300 Good
Staphylococcus aureus 25923 30-300 Good
Procedure References
Consult the reference for information regarding the processing 1. Bowers and Hucker. 1935. Tech. Bull. 228. New York State Agr. Exp. Sta., Geneva, N.Y.
2. Yale. 1938. Am. J. Pub. Health 28:148.
and inoculation of bottled water samples.10 3. Proc. 36th Cong. Intern. Assoc. Ice Cream Manufacturers. 1936. 2:132.
4. Dennis and Weiser. 1937. J. Dairy Science 20:445.
Agar (Pour Plate) 5. Prickett. 1928. Tech. Bull. 147. New York State Agr. Exp. Sta., Geneva, N.Y.
6. Standard Methods of Milk Analysis, 6th ed. 1934.
Usually 1 mL samples of appropriate dilutions of the test sample 7. American Public Health Association. 1948. Standard methods for the examination of dairy products,
9th ed. American Public Health Association, New York, N.Y.
are pipetted into sterile Petri dishes and molten, cooled Tryptone 8. American Public Health Association. 1972. Standard methods for the examination of dairy products,
13th ed. American Public Health Association, Washington, D.C.
Glucose Extract Agar is added followed by gentle mixing to 9. American Public Health Association. 1980. Standard methods for the examination of water and
distribute the sample dilution throughout the agar. Incubate wastewater, 15th ed. American Public Health Association, Washington, D.C.
10. Kim and Feng. 2001. In Downes and Ito (ed.), Compendium of methods for the microbiological
hardened plates at 32 ± 1°C for 47-49 hours, or as specified in examination of foods, 4th ed. American Public Health Association, Washington, D.C.
Tryptone Water
Intended Use hydrolyzed and deaminated to produce indole, pyruvic acid
Tryptone Water is recommended for use in the detection and ammonia.2 Indole can then be detected by the addition
of Escherichia coli in food and water samples based on indole of either Kovacs’ or Ehrlich’s Reagent, which contain an
production. aldehyde group. The aldehyde group combines with indole to
produce a red color in the alcohol layer. Sodium chloride is added
Summary and Explanation to the medium to provide a suitable osmotic environment.
Tryptone Water is based on the Tryptone Water formula
described in ISO standard 7251.1 In this procedure, Tryptone Formula
Water is used with Lauryl Tryptose (or Sulfate) Broth and EC Difco™ Tryptone Water
Broth to determine the most probable number (MPN) of E. coli Approximate Formula* Per Liter
Tryptone.................................................................... 10.0 g
present in the sample. Gas production in both media and indole Sodium Chloride.......................................................... 5.0 g
production in Tryptone Water is used as the basis for this pre- *Adjusted and/or supplemented as required to meet performance criteria.
sumptive E. coli test.
Directions for Preparation from
Tryptone Water may also be used for differentiation of other
bacteria based on indole production.
Dehydrated Product
1. Dissolve 15 g of the powder in 1 L of purified water.
Principles of the Procedure 2. Autoclave at 121°C for 15 minutes.
Tryptone Water contains both tryptone (1%) and sodium chloride. 3. Test samples of the finished product for performance using
Due to its high tryptophan content, tryptone is suitable for stable, typical control cultures.
use in detecting indole production by bacteria. Tryptophan is
Procedure
Test For Enumeration of Presumptive E. coli
User Quality Control
1. Suspend one part sample in 9 parts diluent. Homogenize
Identity Specifications sample.
Difco™ Tryptone Water 2. Prepare serial 10-fold dilutions to 10-6 using 1 mL of homog-
Dehydrated Appearance: White to light beige, free flowing, homoge-
neous.
enate and 9 mL of diluent. Mix each dilution thoroughly.
Solution: 1.5% solution, soluble in purified water. 3. Transfer 10 mL of test sample or initial suspension to each
Solution is pale to medium amber, clear to of 3 tubes of double-strength Lauryl Tryptose Broth (LTB).
slightly opalescent. Repeat using 3 tubes of single-strength LTB. Mix well.
Prepared Appearance: Light to medium amber, clear to slightly 4. For each of the serial 10-fold dilutions, transfer 10 mL of test
opalescent.
sample to each of 3 tubes of double-strength LTB. Repeat
Reaction of 1.5%
Solution at 25°C: pH 7.3 ± 0.2 using 3 tubes of single-strength LTB. Mix well.
5. Incubate all tubes of LTB at 35-37°C for 24 ± 2 hours and
Cultural Response up to 48 hours, if necessary, observing tubes for gas forma-
Difco™ Tryptone Water tion.
Prepare the medium per label directions. Inoculate and incubate at 6. Inoculate one 3-mm loopful of broth from each tube in Step 5
35 ± 2°C for 18-24 hours. Add 0.5 mL Indole Reagent (Kovacs) to the showing gas formation to 10 mL of EC Broth warmed to
tubes to test for indole production. Formation of a red color denotes a
positive indole test.
45°C.
7. Incubate the EC Broth tubes in a water bath at 45°C for 24 ± 2
Inoculum Indole
Organism ATCC™ CFU RECOVERY Production hours (up to 48 hours if necessary), observing for gas forma-
Enterobacter cloacae 13047 102-3×102 Good – tion.
Escherichia coli 25922 10 -3×10
2 2
Good + 8. Inoculate one 3-mm loopful of broth from each tube in Step 7
showing gas formation to 5-10 mL of Tryptone Water warmed
to 45°C.
586
9. Incubate Tryptone Water tubes in a water bath at 45°C for Limitations of the Procedure
48 hours. 1. Detection of E. coli in meats using Tryptone Water is a
10. Add 0.5 mL of Indole Reagent to Tryptone Water tubes, mix presumptive test. If confirmatory testing is required, please
well and examine after 1 minute. consult appropriate references.
Indole Determination Using Pure Cultures 2. Indole testing is recommended as an aid in the differen-
tiation of microorganisms based on indole production.
1. Inoculate Tryptone Water using a light inoculum of an
For complete identification of the organism, further
18-24 hour pure culture.
biochemical evaluation is necessary.
2. Incubate the tubes at 35 ± 2°C with loosened caps for
18-24 hours.
References
3. Add 0.5 mL of Indole Reagent (Kovacs) directly to the tube 1. International Organization for Standardization. 1993. Microbiology – general guidance for enumera-
and agitate. Allow tubes to stand for 5-10 minutes. tion of presumptive E. coli – most probable number technique. ISO 7251, 1993-12-15, 2nd ed. ISO,
Geneva, Switzerland.
2. MacFaddin. 2000. Biochemical tests for identification of medical bacteria, 3rd ed. Lippincott
Williams & Wilkins, Baltimore, Md.
Expected Results
Test For Enumeration of Presumptive E. coli
Availability
For each dilution, record tubes as positive if a red ring forms at Difco™ Tryptone Water
the top of the medium indicating indole production. Determine ISO
the MPN (Most Probable Number) of E. coli present in the Cat. No. 264410 Dehydrated – 500 g
sample based on the number of tubes that are positive for both
Difco™/BBL™ Indole Reagent
gas and indole. Consult the appropriate 3-tube MPN table.1 Cat. No. 261185 Droppers – 50 × 0.5 mL
Indole Determination Using Pure Cultures
Examine tubes for the formation of a red ring at the top of the
tube indicating indole production.
Bacto™ Tryptose
Intended Use parasitizes vectors of Chagas’ disease, on its insect cell host.7 T
Bacto Tryptose is an enzymatic digest of protein used in preparing Spodoptera frugiperda, a cotton pest in Argentina8 and several
microbiological culture media. tick cell lines have also been grown using a TPB-supplemented
medium.9 Tryptose Phosphate Broth has been reported as a
Summary and Explanation suitable supplement for growth of baby hamster kidney cells10
Tryptose was originally developed as a peptone particularly and porcine kidney cells.11
adapted to growth requirements of Brucella. Tryptose is very Media formulations containing Bacto Tryptose are specified in
useful for cultivation of streptococci, pneumococci, menin-
standard methods for various applications.12-17
gococci and other fastidious organisms, and was found to be
superior to meat infusion peptone media previously used Principles of the Procedure
for these organisms.1,2 Mobley et al.3 reported that Tryptose Bacto Tryptose is a mixed enzymatic hydrolysate with
Broth was the preferred medium for strains of Bordetella distinctive nutritional properties. The digestive process of
bronchiseptica in studies of phosphatase activity. Bacto Tryptose results in assorted peptides of higher molecular
Tryptose has been reported as beneficial for cell culture applica- weight suitable for long chain amino acid requirements.
tions. Litwin4 found Tryptose to be suitable for supplementing a Bacto Tryptose provides nitrogen, amino acids and vitamins in
serum-free medium to grow human diploid fibroblasts. Vaughn microbiological culture media.
and Fan5 established that Tryptose provided free amino acids
necessary for growth of Spodoptera frugiperda and Lymantria Typical Analysis
dispar insect cell lines. Tryptose is often used as a biomass Refer to Product Tables in the Reference Guide section of this
enhancer for recombinant E. coli production. manual.
Tryptose is the major ingredient and only peptone in the Directions for Preparation from
formulation for Tryptose Phosphate Broth (TPB), an often-used
Dehydrated Product
medium for various culture applications. Hata and Kojima6 have
Refer to the final concentration of Bacto Tryptose in the formula
shown TPB to be a useful supplement in culturing the nematode,
of the medium being prepared. Add product as required.
Angiostrongylus cantonensis. TPB was also reported as a supple-
ment to a medium for cultivating a protozoan parasite, which
587
Cultural Response
Biochemical Reactions
Bacto™ Tryptose
Prepare a sterile solution of Bacto Tryptose as directed below. Adjust final pH to 7.2-7.4. Inoculate and incubate at 35 ± 2°C for 18-48 hours.
TEST TEST SOLUTION ORGANISM ATCC™ INOCULUM CFU RESULT
Fermentable Carbohydrates 2% Escherichia coli 25922 ~107 Negative
Indole Production 0.1% Escherichia coli 29552 0.1 mL, undiluted Positive
Acetylmethylcarbinol 0.1% with Enterobacter aerogenes 13048 0.1 mL, undiluted Positive
Production 0.5% dextrose
Hydrogen Sulfide 1% Salmonella enterica 14028 0.1 mL, undiluted Positive
Production subsp. enterica serotype Typhimurium
Growth Response
Bacto™ Tryptose
Prepare a sterile solution with 2% Bacto Tryptose, 0.5% sodium chloride and 1.5% agar. Adjust final pH to 7.2-7.4. Inoculate and incubate plates at
35 ± 2°C for 18-48 hours.
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Staphylococcus aureus 25923 30-300 Good
Streptococcus pneumoniae 6303 30-300 Good
Streptococcus pyogenes 19615 30-300 Good
Procedure 11. Sakoda and Fukusho. 1998. In Vitro Cell Dev. Biol. Anim. 34:53.
12. Horowitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed, online. AOAC
See appropriate references for specific procedures using Bacto International, Gaithersburg, Md.
13. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
Tryptose. tional, Gaithersburg, Md.
14. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
Expected Results 15. U.S. Environmental Protection Agency (USEPA). 2000. Improved enumeration methods for the
recreational water quality indicators: Enterococci and Escherichia coli. EPA-821/R-97/004. Office of
Refer to appropriate references and procedures for results. Water, USEPA, Washington, D.C.
16. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C.
17. U.S. Department of Agriculture. Microbiology laboratory guidebook, online. Food and Safety Inspec-
References tion Service, USDA, Washington, D.C.
1. Casman. 1942. J. Bacteriol. 43:33.
2. Casman. 1947. Am. J. Clin. Pathol. 17:281.
3. Mobley, Chengappa, Kadel and Stuart. 1984. Can. J. Comp. Med. 48:175.
Availability
4. Litwin. 1985. Dev. Biol. Stand. 60:25. Bacto™ Tryptose
5. Vaughn and Fan. 1997. In Vitro Cell. Dev. Biol. Anim. 33:479.
6. Hata and Kojima. 1990. Exp. Parasitol. 70:467. AOAC BAM COMPF EPA SMWW USDA
7. Reduth, Schaub, and Pudney. 1989. Parasitology 98:387.
8. Deutschmann and Jager. 1994. Enzyme Microb. Technol. 16:506.
Cat. No. 211713 Dehydrated – 500 g
9. Munderloh and Kurtti. 1989. Exp. Appl. Acarol. 7:219. 211709 Dehydrated – 10 kg
10. Prodafikas and Plavsic. 2000. Focus 22:35.
The high productivity of tryptose media in the isolation and Difco™ Tryptose Broth
cultivation of Brucella supports use of these formulas as 1. Dissolve 26 g of the powder in 1 L of purified water.
general-purpose media, especially when avoidance of animal 2. Autoclave at 121°C for 15 minutes.
tissue products is desired. Tryptose Agar with 5% bovine 3. Test samples of the finished product for performance using
serum, with or without antibiotics, remains a standard plating stable, typical control cultures.
medium for the isolation of brucellae.5 For isolation of
Brucella stains from contaminated milk, crystal violet (gentian
589
Procedure References
Methodologies for the multiple applications using tryptose media 1. Huddleson. 1943. Brucellosis in man and animals, rev. ed. The Commonwealth Fund, New York,
N.Y.
are outlined in the references. 2. Castañeda. 1947. Proc. Soc. Exp. Biol. Med. 64:114.
3. Huddleson. 1939. Brucellosis in man and animals. Oxford University Press, Oxford, England.
4. McCullough, Mills, Herbst, Roessler and Brewer. 1947. J. Bacteriol. 53:5.
Expected Results 5. Moyer and Holcomb. 1995. In Murray, Baron, Pfaller, Tenover, and Yolken (ed.), Manual of clinical
microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
Refer to appropriate references and procedures for results. 6. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol.
1. Williams & Wilkins, Baltimore, Md.
7. Atlas. 1995. Handbook of microbiology media for the examination of food. CRC Press, Boca Raton,
Fla.
Limitations of the Procedure 8. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods.
4th ed. American Public Health Association, Washington, D.C.
1. Tryptose media are general-purpose, non-selective media. 9. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
Although certain diagnostic tests may be performed directly tional, Gaithersburg, Md.
10. U.S. Public Health Service, Centers for Disease Control and Prevention, and National Institutes of
on the medium, biochemical and, if indicated, immunological Health. 2007. Biosafety in microbiological and biomedical laboratories, 5th ed. HHS Publication No.
(CDC) 93-8395. U.S. Government Printing Office, Washington, D.C.
testing using pure cultures are recommended for complete 11. Ruoff, Whiley and Beighton. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of
clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
identification.
2. When preparing blood agar, hemolytic reactions of some
Availability
strains of group D streptococci have been shown to be
Difco™ Tryptose Agar
affected by differences in animal blood.
BAM CCAM COMPF
3. Atmosphere of incubation has been shown to influence Cat. No. 264300 Dehydrated – 500 g
hemolytic reactions of beta-hemolytic streptococci.11 264100 Dehydrated – 2 kg
For optimal performance, incubate tryptose media supple- Difco™ Tryptose Broth
mented with blood under increased CO2 or anaerobic BAM CCAM COMPF
conditions. Cat. No. 262200 Dehydrated – 500 g
4. Dextrose has been shown to inhibit hemolysin production 262100 Dehydrated – 10 kg
by some organisms.
Identity Specifications
Difco™ Tryptose Blood Agar Base
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution: 3.3% solution, soluble in purified water upon
boiling. Solution is light amber, very slightly to
slightly opalescent.
Prepared Appearance: Plain – Light amber, slightly opalescent.
With 5% sheep blood – Cherry red, opaque.
Reaction of 3.3%
Solution at 25°C: pH 7.2 ± 0.2
Cultural Response
Difco™ Tryptose Blood Agar Base
Prepare the medium per label directions without (plain) and with 5%
sterile defibrinated sheep blood (SB). Inoculate and incubate at 35 ± 2°C
for 18-48 hours (blood plates under 5-10% CO2).
Inoculum RECOVERY RECOVERY Hemolysis
Organism ATCC™ CFU PLAIN with SB 18-48 HR
Escherichia coli 25922 102-103 Good Good Beta
Neisseria
meningitidis 13090 102-103 None to poor Good None Streptococcus Streptococcus
pneumoniae pyogenes
Staphylococcus ATCC™ 6305 ATCC™ 19615
aureus 25923 102-103 Good Good Beta
Streptococcus
pneumoniae 6305 102-103 Fair to good Good Alpha
Streptococcus
pyogenes 19615 102-103 Fair to good Good Beta
591
References Availability
1. Casman. 1942. J. Bacteriol. 43:33.
2. Casman. 1947. Am. J. Clin. Pathol. 17:281.
Difco™ Tryptose Blood Agar Base
3. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna- BAM
tional, Gaithersburg, Md.
4. Ruoff, Whiley and Beighton. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of Cat. No. 223220 Dehydrated – 500 g
clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C. 223210 Dehydrated – 2 kg
5. Isenberg. (ed.). 1992. Clinical microbiology procedures handbook, vol. 1. American Society for
Microbiology, Washington, D.C.
6. Baron, Peterson and Finegold. 1994. Bailey & Scott’s diagnostic microbiology, 9th ed. Mosby-Year
Book, Inc., St. Louis, Mo.
The addition of 0.1-0.2% agar to Tryptose Phosphate Broth Neisseria meningitidis 13090 10 -10
2 3
Good
facilitates anaerobic growth and aids in dispersion of reducing Staphylococcus epidermidis 12228 102-103 Good
substances and CO2 formed in the environment.3 The low agar Streptococcus pneumoniae 6305 102-103 Good
concentration provides suitable conditions for both aerobic Streptococcus pyogenes 19615 102-103 Good
growth in the upper zone and for microaerophilic and anaerobic
growth in the lower zone.
Formula
Bacto™ Tryptose Phosphate Broth
Approximate Formula* Per Liter
Tryptose..................................................................... 20.0 g
Dextrose...................................................................... 2.0 g
Sodium Chloride.......................................................... 5.0 g
Disodium Phosphate.................................................... 2.5 g
*Adjusted and/or supplemented as required to meet performance criteria.
Procedure
See appropriate references for specific procedures.
592
Tween™ 80 Water
Intended Use Procedure
Tween™* 80 Water may be used to restore and/or inoculate Follow those procedures or test methods requiring the use of
microdilution panels. water with 0.02% polysorbate 80.
*Tween is a trademark of ICI Americas.
Reference
Summary and Explanation 1. Thrupp. 1986. In Lorian (ed.), Antibiotics in laboratory medicine, 2nd ed. Williams & Wilkins,
Baltimore, Md.
Tween 80 (polysorbate 80) is a surface active agent that is
recommended for use at a 0.02% concentration in routine Availability
inoculum preparation and dispensing procedures in various BBL™ Tween™ 80 Water
microdilution systems.1 Cat. No. 297381 Prepared Tubes (D Tubes), 12.5 mL – Ctn. of 100
296184 Prepared Tubes (A Tubes), 25 mL – Ctn. of 100
Principles of the Procedure
Tween 80 Water is a 0.02% concentration of polysorbate
80 in purified water that is convenient for use in dispersing
microorganisms during inoculum preparation and for reconsti-
tuting antimicrobial agents in microdilution plates.
T
Tyrosine Agar
(See Nocardia Differentiation Media)
593
594
595
Procedure
See appropriate references for specific procedures.
Availability
Difco™ Universal Beer Agar
Cat. No. 285610 Dehydrated – 500 g
Expected Results
Mexico
Refer to appropriate references and procedures for results. Cat. No. 252644 Prepared Plates (60 × 15 mm-style) – Pkg. of 20*
252645 Prepared Flasks, 140 mL
*Store at 2-8°C.
Cultural Response
Difco™ Universal Preenrichment Broth
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-24
hours.
Organism ATCC™ Inoculum CFU RECOVERY
Listeria monocytogenes 19115 10-102 Good Uninoculated Salmonella
Tube Typhimurium
Salmonella enterica subsp. ATCC™ 14028
enterica serotype Enteritidis 13076 10-102 Good
Salmonella enterica subsp.
enterica serotype Typhimurium 14028 10-102 Good
596
Formula Procedure
Difco™ Universal Preenrichment Broth Procedures for the preenrichment of Salmonella and Listeria are
Approximate Formula* Per Liter provided in appropriate references.1,2
Pancreatic Digest of Casein.......................................... 5.0 g
Proteose Peptone......................................................... 5.0 g
Monopotassium Phosphate........................................ 15.0 g Expected Results
Disodium Phosphate ................................................... 7.0 g Salmonella and Listeria demonstrate good growth and
Sodium Chloride.......................................................... 5.0 g recovery following preenrichment in this broth.
Dextrose...................................................................... 0.5 g
Magnesium Sulfate...................................................... 0.25 g
Ferric Ammonium Citrate............................................. 0.1 g References
Sodium Pyruvate.......................................................... 0.2 g 1. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
*Adjusted and/or supplemented as required to meet performance criteria. 2. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
tional, Gaithersburg, Md.
3. Bailey and Cox. 1992. J. Food Prot. 55:256.
Directions for Preparation from 4. Bailey, Fletcher and Cox. 1990. J. Food Prot. 53:473.
5. Juven, Cox, Bailey, Thomson, Charles and Shutze. 1984. J. Food Prot. 47:299.
Dehydrated Product
1. Suspend 38 g of the powder in 1 L of purified water. Mix
Availability
thoroughly.
Difco™ Universal Preenrichment Broth
2. Autoclave at 121°C for 15 minutes.
CCAM
3. Test samples of the finished product for performance using Cat. No. 223510 Dehydrated – 500 g
stable, typical control cultures.
Urea Media
Urea Agar Base • Urea Agar Base Concentrate 10×
Urea Agar • Urea Broth • Urease Test Broth
Urease Broth Concentrate 10×
Intended Use subsp. morganii, Providencia rettgeri, and a few Providencia
Urea Agar and Urease Test Broth are used for the differentiation stuartii strains with the reclassification of the members of the
of organisms, especially the Enterobacteriaceae, on the basis of Proteeae.
urease production. Urease base is also supplied as a filter sterilized 10× concen-
U
Summary and Explanation
trated solution for use in preparing Urease Test Broth in the
-
Urea Agar was devised by Christensen for use as a solid
laboratory.
Z
medium for the differentiation of enteric bacilli.1 It differentiates Principles of the Procedure
between rapid urease-positive Proteeae organisms (Proteus spp., The urea medium of Rustigian and Stuart3 is particularly suited
Morganella morganii subsp. morganii, Providencia rettgeri, for the differentiation of Proteus species from other gram-
and some Providencia stuartii) and other urease-positive negative enteric bacilli capable of utilizing urea;1 the latter
organisms: Citrobacter, Enterobacter and Klebsiella and are unable to do so in Urease Test Broth because of limited
bacteria other than Enterobacteriaceae; i.e., some Bordetella nutrients and the high buffering capacity of the medium. To
and Brucella spp.2 provide a medium with greater utility, Urea Agar was devised
The base is also supplied as a filter-sterilized 10× concentrated by Christensen1 with peptone and dextrose included and
solution in tubes for use in preparing Urea Agar slants in the reduced buffer content to promote more rapid growth of many
laboratory. of the Enterobacteriaceae and permit a reduction in incubation
time. The complete Urea Agar contains 15.0 g/L of agar in
Urease Test Broth was developed by Rustigian and Stuart.3
addition to the ingredients in the base medium.
It may be used for the identification of bacteria on the basis
of urea utilization and it is particularly recommended for the When organisms utilize urea, ammonia is formed during
differentiation of members of the genus Proteus from those of incubation which makes the reaction of these media alkaline,
Salmonella and Shigella in the diagnosis of enteric infections.4 producing a red-pink color. Consequently, urease production
The medium is positive for Proteus, Morganella morganii may be detected by the change in the phenol red indicator.
597
Uninoculated Proteus vulgaris Escherichia coli Uninoculated Proteus vulgaris Escherichia coli
Tube ATCC™ 13315 ATCC™ 25922 Tube ATCC™ 13315 ATCC™ 25922
Urea Broth Urea Agar
BBL™ Urea Agar Base Concentrate 10× (Prepared Tubes) A negative reaction is no color change. The agar medium
1. To prepare Urea Agar medium, add 1.7 g of granulated agar remains pale yellow to buff; the broth remains yellowish-
to 100 mL of purified water. Heat with agitation and boil orange.
for 1 minute. For a listing of urease-positive organisms, consult appropriate
2. Dispense in 9 mL aliquots into tubes and autoclave at 121°C texts.2, 4-7
for 15 minutes.
3. Cool the agar to 45-50°C, and allow one tube of Limitations of the Procedure
concentrate to come to room temperature. Add 1 mL of Urea Agar Base
concentrate to each 9 mL of cooled agar solution and mix 1. The alkaline reaction produced in this medium after prolonged
thoroughly. incubation may not be caused by urease activity. False positive
4. Allow the tubes to cool in a slanted position so that slants reactions may occur due to the utilization of peptones
with deep butts are formed. (especially in slant agar by Pseudomonas aeruginosa, for
5. Test samples of the finished product for performance using example) or other proteins which raise the pH due to
stable, typical control cultures. protein hydrolysis and the release of excessive amino acid
Difco™ Urea Broth residues. To eliminate possible protein hydrolysis, perform a
1. Equilibrate the medium to room temperature before control test with the same test medium without urea.7
opening. The presence of urea in this medium renders it 2. Do not heat or reheat the medium because urea decomposes
inherently lumpy. This condition will not adversely affect a very easily.
properly stored medium. 3. Urea Agar detects rapid urease activity of only the urease-
2. Dissolve 38.7 g of the powder in 1 L of purified water. Mix positive Proteus species. For results to be valid for the
thoroughly to completely dissolve the powder. detection of Proteus, the results must be read within the first
3. Filter sterilize. DO NOT BOIL OR AUTOCLAVE THE 2-6 hours after incubation. Urease-positive Enterobacter,
MEDIUM. Citrobacter or Klebsiella, in contrast, hydrolyze urea much
4. Test samples of the finished product for performance using more slowly, showing only slight penetration of the alkaline
stable, typical control cultures. reaction into the butt of the medium in 6 hours and requiring
3-5 days to change the reaction of the entire butt.
BBL™ Urease Broth Concentrate 10× (Prepared Tubes)
1. To prepare medium, aseptically add 10 mL of the concentrate Urea Broth
to 90 mL of cold sterile purified water. Mix thoroughly. 1. To rule out false positives due to protein hydrolysis (as op-
2. Dispense aseptically in 1-3 mL amounts, in small sterile test posed to urea hydrolysis) that may occur in the medium after
tubes. prolonged incubation, perform a control test with the same
test medium without urea.7
Procedure 2. Do not heat or reheat the medium because urea decomposes
U
If Urea Agar Base Concentrate 10× or Urease Broth Con- very easily. -
centrate 10× is being used, prepare the complete medium as
described above. If crystals form in the concentrate, they will
3. The high buffering system in this medium masks ure-
ase activity in organisms that are delayed positive. This
Z
usually dissolve at room temperature, or in a few minutes in a medium is therefore recommended for the detection of
40°C water bath. urease activity in all Proteus spp., Providencia rettgeri and
urease-positive Providencia stuartii.1 M. morganii slowly
Using a heavy inoculum (2 loopfuls) of growth from an 18- to
hydrolyzes urea and may require approximately a 36 hour
24-hour pure culture (TSI Agar or other suitable medium),
incubation for a strong urease-positive reaction to occur.1 If
inoculate the broth or agar (streaking back and forth over the
in doubt as to a result, compare with an uninoculated tube
entire slant surface). Do not stab the butt since it serves as a
or incubate for an additional 24 hours.
color control. For broth, shake tubes gently to suspend the
4. Variations in the size of the inoculum can affect the time
bacteria. Incubate tubes with loosened caps at 35 ± 2°C in an
required to reach positive (alkaline, pH 8.1) results.
incubator or water bath. Observe reactions after 2, 4, 6, 18,
24 and 48 hours. For agar, continue to check every day for
References
a total of 6 days; even longer incubation periods may be 1. Christensen. 1946. J. Bacteriol. 52:461.
necessary. 2. MacFaddin. 2000. Biochemical tests for identification of medical bacteria, 3rd ed. Lippincott Williams
& Wilkins, Baltimore, Md.
3. Rustigian and Stuart. 1941. Proc. Soc. Exp. Biol. Med. 47:108.
4. Ewing. 1985. Edwards and Ewing’s identification of Enterobacteriaceae, 4th ed. Elsevier Science
Expected Results Publishing Co, Inc., New York, N.Y.
5. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology,
The production of urease is indicated by an intense pink-red 9th ed. Williams & Wilkins, Baltimore, Md.
(red-violet) color on the slant or throughout the broth. The 6. Farmer. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology,
7th ed. American Society for Microbiology, Washington, D.C.
color may penetrate into the agar (butt); the extent of the color 7. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. 1.
Williams & Wilkins, Baltimore, Md.
indicates the rate of urea hydrolysis.5
599
V Agar
Intended Use References
V Agar is an enriched medium used in qualitative procedures for 1.
2.
Ellner, Stoessel, Drakeford and Vasi. 1966. Am. J. Clin. Pathol. 45:502.
Greenwood, Pickett, Martin and Mack. 1977. Health Lab Sci. 14:102.
the isolation and differentiation of Gardnerella vaginalis from 3. Greenwood and Pickett. 1980. Int. J. Syst. Bacteriol. 30:170.
4. Piot, Van Dyck, Goodfellow and Falkow. 1980. J. Gen. Microbiol. 119:373.
clinical specimens. 5. Greenwood and Pickett. 1979. J. Clin. Microbiol. 9:200.
6. Funke and Bernard. 2007. In Murray, Baron, Jorgensen, Landry and Pfaller (ed), Manual of clinical
microbiology, 9th ed. American Society for Microbiology, Washington, D.C.
Summary and Explanation
In 1966, Ellner et al. developed an improved blood agar base Availability
formulation, which has been designated as Columbia Agar.1 BBL™ V Agar
CMPH2 MCM9
Greenwood et al., in 1977, described a modification of Columbia
United States and Canada
Agar in which the peptone concentration was increased and Cat. No. 221874 Prepared Plates – Pkg. of 10*
human blood was used.2 This enriched medium was designed 221875 Prepared Plates – Ctn. of 100*
for the isolation and differentiation of G. vaginalis by means of Mexico
beta hemolysis of human blood.3,4 Greenwood et al. reported that Cat. No. 221874 Prepared Plates – Pkg. of 10*
96% of G. vaginalis isolated produced beta hemolysis of human *Store at 2-8°C.
Procedure
Use standard procedures to obtain isolated colonies from speci-
mens. Since G. vaginalis requires carbon dioxide on primary
isolation, plates should be incubated in an aerobic atmosphere
containing approximately 3-10% CO2 at 35 ± 2°C for 48
hours.6
Expected Results
Typical colonies of G. vaginalis appear small and white, yield
gram-variable diphtheroid-like forms and exhibit distinctive
diffuse beta hemolysis after 48 hours of incubation in an
aerobic atmosphere supplemented with carbon dioxide.
600