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Rev 08 - The Oxoid Manual 1998 PDF
Rev 08 - The Oxoid Manual 1998 PDF
The
OXOID MANUAL
8th Edition 1998
Compiled by E. Y. Bridson
(former Technical Director of Oxoid)
Price: £10
The
OXOID MANUAL
8th Edition 1998
Compiled by E. Y. Bridson
(former Technical Director of Oxoid)
Denmark
Oxoid A/S
Tempovej 42-44
2750 Ballerup
Denmark
Tel: 45 44 97 97 35
Fax: 45 44 97 97 45
chloride. To this day this simple formula is the basic The OXOID Quality Policy
one for culture media.
It is the policy of OXOID, Basingstoke to manufacture
In the following year (1882) Heuppe described the
and sell OXOID products which are fit for the
labour saving convenience of substituting commercial
purpose for which they are intended and are safe in
meat extract for Koch's watery extract of fresh meat.
use.
By 1902 an American text book of bacteriology was
recommending the use of LEMCO for this purpose. OXOID Ltd (Basingstoke) is registered with the BSI to
This is quite possibly the first record of exporting BS EN ISO 9001 (Reg No. FM 09914) with extended
culture media ingredients by the company. scope to include BS EN 46001: 1997. This standard
endorses our quality management system for
It will be seen that the business of manufacturing
products manufactured at the Basingstoke site and
dehydrated culture media was a natural consequence
includes: Dehydrated Culture Media, Selective
of the converging commercial activities of Oxoid and
Supplements, Sterile Reagents, Biochemical Reagents,
the development of the science of microbiology. The
Laboratory Preparations, Signal Blood Culture System
early history explains why OXOID is one of the very
bottles, Susceptibility Discs in cartridges, Diagnostic
few producers of culture media that actually
Reagents, Salmonella Rapid Test and Listeria Rapid
manufactures its own extracts and hydrolysates. Test.
Ready Prepared Media and Lab Ready Media are
manufactured by G. M. Procter and are covered
under BS EN ISO 9002 Reg No. FM 27644.
The essential elements of the Oxoid Quality
management System include:
- product lot testing according to a defined protocol
- documented complaints and technical enquiries
procedure
- policy for raw material procurement
- good manufacturing practice combined with in-
process control
- comprehensive training for staff at all levels
- conformance to statutory Health and Safety and
Environmental requirements
The Oxoid Quality Policy functions through all
procedures described above and maintains the
company's high reputation for the performance of its
products.
Light Quanticult
All prepared culture media should be stored away Store at 2±88C.
from light and exposure to direct sunlight avoided at Shelf life 6±10 months.
all times. Diagnostic Reagents (DR products)
Humidity Store at 2±88C, do not freeze.
Sealed glass and plastic containers are unaffected by Shelf life 9 months to 2 years.
normal laboratory humidity. Opened containers of Diagnostic Discs (DD range)
dehydrated powders will be affected by high Store at ±208C but keep working stock at 2±88C.
humidity. Hot, steamy media preparation rooms are Shelf life 1 to 2 years.
unsuitable environments to store containers of culture
DRYSPOT
media; particularly containers which are frequently
Store at room temperature 15±258C.
opened and closed. An adjacent cooler room or an
adequate storage cupboard are preferable storage Shelf life 2 years.
areas. Susceptibility Discs
Store at ±208C but keep working stock at 2±88C.
Temperature and time
Shelf life 1 to 3 years.
The temperature storage conditions of culture media
and their components vary widely. The following Dehydrated Culture Media (CM, L products)
product groupings will help to differentiate the Sealed, unopened containers should be stored at room
various requirements. temperature 15±208C.
Prepared Agar and Broth Media (PM, R products) Opened containers should have the cap carefully and
Store at 2±88C. do not allow the products to freeze. securely replaced. It is important that opened
Shelf life 3 months to 2 years. containers are stored in a dry atmosphere at room
temperature.
Biochemical Reagents (BR products)
Store at 2±88C. Shelf life 1 to 5 years.
Shelf life 1 to 5 years. Prepared Plates Of Culture Media
Gas Generating Kits Poured plates of agar media are especially vulnerable
Store at 2±258C. in a dry place. Do not store these kits to infection, dehydration and chemical degradation.
at a higher temperature for long periods. Aseptic preparation and storage are essential to
Shelf life 3 years. protect plates from microbial infection.
20 months 20 months 20 months Water losses on storage can be minimised by
Anaerogen2 Campygen2 CO2Gen2 impermeable wrapping and/or storage at 2±88C.
Selective and Sterile Reagents (SR products, Chemical degradation e.g. oxidation or antimicrobial
Selective supplements) loss, can be retarded by protection from light, heat
Store at 2±88C. and dehydration.
except Therefore storage of prepared plates at 2±88C (unless
Horse Serum SR35 otherwise stated) in the absence of light and protected
store at ±20 to +88C. against moisture loss will minimise agar media
Nitrocefin SR112 deterioration from these defects.
Reconstitution fluid SR112A
store at ±20 to +88C. It is important, however, to monitor the storage of
Penase SR129 prepared plates by quality control tests so that any
store at ±208C. deterioration can be detected and the storage period
Shelf life 1 week to 2 years. accurately determined. Simple weighing tests of fresh
and stored plates will determine the rate of moisture
Culti Loops loss. Greater than 5% loss of weight will indicate a
Store at 2±88C. or frozen for Campylobacter sp. significant loss of water.
Shelf life 6±10 months (except
Campylobacter jejuni ± 4±6 months).
Toxin Detection Kits
Store at 2±88C.
Shelf life 1 year.
TABLE 1
Classification of micro-organisms on the basis of hazard and laboratory containment level
Class Description Laboratory
1 Unlikely to cause human disease. Level 1
2 May cause human disease; might be a hazard to laboratory workers; unlikely Level 2
to spread in the community; laboratory exposure rarely causes infections;
effective prophylaxis and therapy available.
3 May cause serious human disease; may be serious hazard to Level 3
laboratory workers; may spread in the community; effective
prophylaxis and therapy available.
4 Causes severe human disease; serious threat to laboratory workers; Level 4
high risk of spread in the community; no effective prophylaxis and therapy.
Based on the classification of the UK Advisory Committee on Dangerous Pathogens6.
Classes (also known as Groups) 2, 3 and 4 include known pathogens.
Class 4 contains only viruses.
TABLE 2
Summary of laboratory design features for laboratory containment levels
Containment Level
1 2 3 4
Laboratory isolated ± ± + +
and sealable for decontamination
Directional ventilation (inward) ± D + +
Filtered air exhaust ± ± + +
Double door entry ± ± O +
Airlock with shower ± ± ± +
Autoclave on site + + + +
in workroom ± ± O +
double ended ± ± ± +
Microbiological safety cabinets
Class I or II available ± + + +
in workroom ± ± + +
Class III ± ± O +
Based on WHO Laboratory Biosafety Manual5
Key: ± not required + essential D desirable O optional
metal, leading to mechanical failure) to the space gases. They should not be used for micro-organisms
between the tube and the bucket. Instructions for use or other living material.
of centrifuges and action to be taken if a centrifuge
tube breaks, usually indicated by a sudden change in SPILLAGE AND BREAKAGE
sound and/or visible imbalance of the machine, Spillage of cultures and chemicals and breakage of
should be posted adjacent to each machine.
vessels containing them must be reported
Physical hazards associated with centrifuges are immediately to the supervisor or local safety office. If
discussed in detail by Kennedy11. the spillage is considerable the room should be
vacated pending decontamination by qualified staff
Water Baths (see below).
The water in water baths may become contaminated
from the outsides of culture tubes or the leakage of Instructions for dealing with small-scale spillages and
their contents. These baths, even those operated at breakages should be posted in each laboratory, and
temperatures >608C should be emptied when not in should include the following:
use or a deposit may form in which micro-organisms ± wear heavy-duty gloves
can grow. A disinfectant that does not attack metals
may be added to the water in baths that are in ± cover the spillage/breakage with absorbent
continuous use (hypochlorites should not be used; see material, e.g. large paper towels
below). ± pour disinfectant (see Table 3) over the paper
towels and leave for at least 15 minutes
Homogenisers and Shakers
Bench-mounted models may generate aerosols and ± scoop up the paper towels with a dust pan or stiff
should be covered, (e.g. by clear plastic boxes) when cardboard and place them along with the dust pan
in use. These covers should be disinfected after use. or cardboard, along with any broken glass into a
Hand-held homogenisers should be held in a wad of laboratory discard container
cotton wool in case they break. Homogenisers and
± pick up any residual broken glass with forceps and
containers from shakers should be opened in add it to the discard container
microbiological safety cabinets.
± cover the area again with paper towels and pour
Pipetting on more disinfectant. Leave for 30 minutes before
Pipetting by mouth, even water, should be banned. any further cleaning up
Pipetting devices should be provided. Pipettes should
not be blown out vigorously, otherwise bubbles and ± autoclave the discard container.
aerosols may be formed.
PRECAUTIONS AGAINST BLOOD-BORNE
Microbiological Safety Cabinets INFECTIONS
These should conform to national standards and
should be tested regularly by independent engineers In addition to the precautions listed above personnel
to ensure that their performance is in accordance with who handle blood specimens or blood-stained
the requirements of that standard. These cabinets are material should wear high quality disposable gloves
designed to protect the user from the inhalation of and also plastic disposable aprons over their normal
infectious aerosols and air-borne particles. They give protective clothing. Guidelines for the safe handling
no protection against spillages of cultures or against in laboratories of materials that may contain hepatitis
chemicals. Class II and Class III cabinets also protect and/or the human immunodeficiency virus have
the test or product from external air-borne been published1,7,8,12,13.
contamination.
PRECAUTIONS WITH CELL AND TISSUE
Microbiological safety cabinets should be used only CULTURE
by experienced personnel who have received proper
instructions about their limitations. They must not be Separate accommodations should be provided to
used as fume cupboards or for work with flammable minimise contamination of cultures.
or toxic substances. Some cells and tissue cultures may contain
They should be decontaminated at regular intervals adventitious and unidentified micro-organisms or
by qualified staff who follow manufacturers', or other viruses from which the operator must be protected.
recognised procedures1,5,10. All work with cells and cell lines should therefore be
conducted in Class II microbiological safety cabinets.
Laminar Outflow (clean air) Cabinets Laminar outflow cabinets (see above) must NOT be
These are NOT microbiological safety cabinets. They used.
are designed to protect the work from external air-
borne contamination and do not protect the worker, STERILISATION, DISINFECTION AND
whose face and respiratory tract receive air that has DECONTAMINATION
passed over the workpiece. (See Cell and Tissue
culture, below). These terms are not interchangeable. In microbiology:
Sterilisation ± implies the complete destruction of all
Fume Cupboards
micro-organisms.
Fume cupboards are designed to protect workers and
the environment from toxic chemical fumes and
TABLE 3
Properties of some disinfectants
Fungi Bacteria Myco- Spores Viruses Protein Materials Hard Deter- Skin Eyes Lungs
G+ G± bacteria Lipid Non Natural Man- water gent
lipid made
Phenolics +++ +++ +++ ++ ± + v + ++ ++ + C + + ±
Hypochlorites + +++ +++ ++ ++ + + +++ + + + C + + +
Alcohols ± +++ +++ +++ ± + v + + + + ± ± + ±
Formaldehyde +++ +++ +++ +++ +++a + + + + + + ± + + +
Glutaraldehyde +++ +++ +++ +++ +++b + + NA + + + ± + + +
Iodophors +++ +++ +++ +++ + + + +++ + + + A + + ±
QAC + +++ ++ ± ± ± ± +++ +++ +++ +++ A(C) + + ±
+++ Good: ++ Fair: + Slight: ± Nil: V Depends on virus: a Above 408C: b Above 208C: C Catonic: A Anionic
From Collins, C.H. (1993) Laboratory Acquired Infections. 3rd.edn. by permission of the publishers Butterworth-Heinemann, Oxford
Equipment to be serviced must also be 10 Collins, C.H., Lyne, P.M. and Grange, J.M. (1995) Collins and
decontaminated in this way and clearly labelled to Lyne's Microbiological Methods. 7th edn. Oxford: Butterworth-
indicate that this has been done and that it should not Heinemann.
be used until after servicing. 11 Kennedy, D.A. (1991) In Safety in Clinical and Biomedical
Laboratories ed. Collins C.H. London: Chapman and Hall.
The working surfaces and inner walls of
12 Advisory Committee on Dangerous Pathogens. HIV ± the
microbiological safety cabinets should be swabbed
Causative Agent of AIDS and Related Conditions. London: HMSO.
with a suitable disinfectant, and the cabinets be
13 World Health Organization (1991) Biosafety Guidelines for
fumigated with formaldehyde, as indicated above
Diagnostic and Research Laboratories Working with HIV. Geneva:
before filters are changed or maintenance carried out.
WHO.
Rooms rarely need disinfection unless a major 14 Collins, C.H. and Kennedy, D.A. (1993) The Treatment and
accident has released massive aerosols. Formerly this Disposal of Clinical Waste. Leeds: H & H Scientific.
was done by formaldehyde fumigation, but this is 15 Collins, C.H. (1994) Lett. Appl. Microbiol. 19, 61±62.
now regarded as hazardous and uncertain. Spraying
or washing with disinfectant/detergent mixtures is
safer and more effective. TABLE 4
Infected and potentially infected waste from
microbiological laboratories
DISPOSAL OF INFECTED WASTE
Disposables other than sharps
Infected laboratory waste is included in the ± Specimens or their remains (in their containers)
definitions of clinical waste and must ultimately be submitted for tests containing blood, faeces,
incinerated. Table 4 lists the materials that should be sputum, urine, secretions, exudates, transudates,
regarded as infectious in microbiological and clinical other normal or morbid fluids but not tissues.
laboratories. As these are likely to be the most heavily ± All cultures made from these specimens, directly
infected of all such waste and may have to travel or indirectly.
along the public highway, often for long distances. It ± All other stocks of micro-organisms that are no
is prudent to autoclave it first1,14,15. longer required.
± Used diagnostic kits (which may contain glass,
plastics, chemicals and biologicals).
CONCLUSIONS ± Used disposable transfer loops, rods, plastic
Every microbiological laboratory should have written Pasteur pipettes.
safety policy and instructions that describe in full the ± Disposable cuvettes and containers used in
safety precautions deemed necessary by the Director chemical analyses.
and Safety Officer. ± Biologicals, standards and quality control
All members of the staff should be aware of the materials.
authorised procedures for containing and destroying ± Food samples submitted for examination in
micro-organisms. outbreaks of food poisoning.
± Paper towels and tissues used to wipe benches
A schedule of regular microbiological safety cleaning and equipment and to dry hands.
should be maintained for all working surfaces and ± Disposable gloves and gowns.
adjacent areas.
Sharps
References ± Hypodermic needles (syringes attached if custom
1 Collins, C.H. (1993) Laboratory Acquired Infections. 3rd edn. so requires).
Oxford: Butterworth-Heinemann. ± Disposable knives, scalpels, blades, scissors,
2 Collins, C.H. and Kennedy, D.A. (1987) J. Appl. Bact. 62, 385± forceps, probes.
402. ± Glass Pasteur pipettes; slides and cover glasses.
3 European Commission (1990/93) Council Directive on the ± Broken glass, ampoules and vials.
Protection of Workers from Risks Relating to Biological Agents at Tissues and animal carcasses
Work. 90/679/EEC as modified 93/88/EEC.
4 Control of Substances Hazardous to Health Regulations (1994). Bedding from animal cages
Biological Agents: Approved Code of Practice. London: Health and Adapted from Collins and Kennedy14 by permission
Safety Executive. of the authors and publisher.
5 World Health Organization (1993) Laboratory Biosafety Manual.
2nd edn. Geneva: WHO.
6 Advisory Committee on Dangerous Pathogens (1990)
Categorization of Pathogens on the Basis of Hazard and Categories of
Containment. London: HMSO.
7 National Research Council (1989) Biosafety in the Laboratory.
Washington DC: National Academy Press.
8 Health Services Advisory Committee (1991) Safe working and the
Prevention of Infection in Clinical Laboratories. London: HMSO.
9 Centers for Disease Control (1993) Biosafety in Microbiological and
Biomedical Laboratories. 3rd edn. HHS Publication No (CDC) 93±
8395. Washington: US Government Printing Office.
content and soya peptone with its high energy Phosphates, acetates, citrates, zwitterion compounds
carbohydrate content are popular examples of non- and specific amino-acids are examples of buffering
meat peptones. agents that may be added to culture media.
A detailed description of these products is given in A side effect of such compounds is their ability to
Section 3.1 `Peptones-Hydrolysates'. chelate (or bind) divalent cations (Ca++ and Mg++).
Polyphosphate salts, sometimes present in sodium
The nutrient components of culture media are
phosphate, are compounds which can bind essential
carefully selected to recover the required spectrum of
cations so firmly that they are made inaccessible to
organisms in the sample e.g. coliforms or anaerobes.
the micro-organisms.
General purpose media such as blood agar in its
various forms will often contain mixtures of peptones The effect of these binding or chelating agents will be
to ensure that peptides of sufficient variety are seen in diminished growth or failure to grow at all,
available for the great majority of organisms likely to unless care has been taken to supplement the essential
be present. However, more demanding organisms cations in the formulation. Opacity forming in a
will require supplemental growth factors to be added medium, after heating or on standing at 508C for
and examples of such requirements can be seen in several hours, is commonly caused by phosphate
media for Legionella species. interaction with metals. Such phosphate precipitates
can very effectively bind Fe and lower the available
Most of the components used for the nutrition of
amount of this essential metal in the medium.
micro-organisms are undefined and require extensive
testing with careful selection to ensure a reasonable 5 Indicator Substances
degree of uniformity. Would it not be better to use The addition of coloured indicator substances is a
wholly defined peptides and amino-acids to produce very effective way of detecting fermentation of
a totally defined medium? Whilst such media would specific carbohydrates in a culture medium. Such
improve uniformity, experience has shown that they compounds should change colour distinctly and
lack good performance as general purpose media. rapidly at critical pH values.
They would also be very expensive compared with Most of the compounds used e.g. phenol red, bromo-
undefined media. The use of totally defined culture cresol purple, fuchsin, etc., are toxic and it is essential
media is an understandable goal of most to use low concentrations of pre-screened batches/
microbiologists but defined media have yet to prove lots. Known sensitive strains of micro-organisms are
themselves equal in performance to currently used used in the screening tests.
complex mixtures of meat and plant protein
hydrolysates. 6 Selective Agents
Chemicals or antimicrobials are added to culture
2 Energy media to make them selective for certain micro-
The most common substance added to culture media organisms. The selective agents are chosen and added
as a source of energy to increase the rate of growth of at specific concentrations to suppress the growth of
organisms is glucose. Other carbohydrates may be unwanted organisms in a polymicrobial sample. It is,
used as required. of course, essential to have established that the
Carbohydrates added to media at 5±10 grammes per selective agents, at the appropriate concentration, will
litre are usually present as biochemical substrates to allow uninhibited growth of the desired organisms.
detect the production of specific enzymes in the Common chemical selective agents are: bile salts, dye-
identification of organisms. It is usual to add pH stuffs, selenite, tetrathionate, tellurite and azide.
indicators to such formulations. Antimicrobial agents are commonly used in mixtures
3 Essential Metals and Minerals when suppressing polymicrobial contaminating flora.
The inorganic essential components of culture media Antimicrobials are more specific in their selective
are many and can be divided on a semi-quantitative action than the chemical agents shown above.
basis: However, the critical weighing and heat-lability of
most antimicrobials demand special care and post-
Typical macro-components (gm/litre):
sterilisation addition.
Na, K, Cl , P, S, Ca, Mg, Fe.
The wide variety of organisms and their almost
Typical micro-components (mgm-microgm/litre):
infinite ability to adapt to changing conditions makes
Zn, Mn, Br, B, Cu, Co, Mo, V, Sr, etc.
a truly selective medium unlikely. Selective media can
As previously mentioned, a formulation may not be said to suppress most of the unwanted organisms
have specific metals and minerals listed in its and allow most of the desired organisms to grow. The
formulation. In such cases it is assumed that all the final formulation is usually a compromise which
factors required are present in the hydrolysates, achieves the best of these criteria.
buffers and agar components.
7 Gelling Agents
4 Buffering Agents Although gelatin is still used for a few specific media
It is important that the pH of a culture medium is and carrageenans, alginates, silica gel and
poised around the optimum necessary for growth of polyacrylamides are sometimes used as gelling
the desired micro-organisms. The use of buffer agents, the outstanding gel-forming substance used in
compounds at specific pK values is especially culture media is agar.
necessary when fermentable carbohydrates are added
as energy sources.
Hesse, a worker in Robert Koch's laboratory, is Simple conversion of the published formula into a
credited with its first use in culture media, although mixture of dehydrated components is seldom
Frau Hesse gave him the idea from its use in table- achieved. Usually the peptone/hydrolysate base has
jellies in hot climates. to be adapted and variations in concentration of other
components may be required. Laboratory mixes of the
Its inertness to microbial action, the unique setting
medium are prepared as R&D trials and after testing
and melting temperatures (388C and 848C
in the laboratory are sent to the originator for
respectively) the high gel strength which allows low
comment. Opportunity may also be taken to get the
concentrations of agar to be used, its clarity and low
views of other experts in this field. Special strains of
toxicity have contributed to its wide popularity with
organisms may be required to check the finer points
microbiologists. Its ability to retain its gel structure at
of performance.
608C makes agar of special value to culture media
which have to be incubated at this temperature to Subject to good report, a trial batch will be
isolate thermophilic organisms. manufactured and this will be used for larger trials
and wider-scale testing. During these trials QC testing
Agar is obtained from agarophyte sea-weeds mainly
and performance criteria will be established and the
Gelidium, Gracilaria and Pterocladia species. It is
specifications of the components will be determined.
extracted as an aqueous solution at greater than
Bought-in components will have buying specifications
1008C, decolourised, filtered, dried and milled to a
and in-house components will have manufacturing
powder.
specifications and standard-operating-processes
Agar is not an inert gelling agent; it contributes produced. Stability trials will begin if there is
nutrients and/or toxic agents to culture media, confidence that the final formulation has been
depending on the chemical processing carried out by achieved.
the suppliers.
The reports on the larger and wider-spread trials are
Microbiological agar is specially processed to yield a studied and if the results are satisfactory preparation
low toxicity, high clarity, low mineral and high will be made to manufacture a full production batch/
diffusion gel. lot. All the components of the medium, including
Other Components special protein hydrolysates which may have to be
There are many other substances added to culture specially manufactured, are assembled and a
media for specific purposes e.g. growth factors for laboratory mix tested to see that it meets the
fastidious organisms, eH-reducing compounds for performance specification. Finally the components are
anaerobic organisms (thioglycollate and cysteine), milled, mixed and blended to produce a finely
whole blood to detect haemolytic enzymes and divided, homogeneous powder which is held in large
encourage the growth of organisms which are containers for further testing before release.
vulnerable to oxidation products. All this work, plus literature, labels and product
Development and Manufacture of Culture Media inserts is carried out under the aegis of R&D/
The development of dehydrated culture media is a Marketing. Subsequent production lots are
process leading to the large-scale manufacture of a manufactured under surveillance which includes
reproducible, stable product. The initial development GMP monitoring and end-product testing by the
of the formulation is usually carried out by Quality Department.
microbiologists who wish to create a novel medium No product can be released without clearance from
with specific characteristics or who wish to improve The Quality Department.
the performance of an existing product. Such work is
usually written up in microbiological journals, having
first been judged by some form of peer review and
proved to be of special value by other workers in the
field.
PREPARATION OF DEHYDRATED MEDIA above the level of the water may not be sterilised in
the autoclave and may be a source of
Dehydrated media are hygroscopic and are sensitive contamination.
to moisture, heat and light. They are adversely
affected by drastic changes in temperature e.g. hot/ Agar-free media will usually dissolve with gentle
cold cycling temperatures which may occur between agitation.
day and night laboratory temperatures in winter. Media containing agar should be heated to dissolve
Storage conditions are usually indicated on the the agar before autoclaving. Bring the medium to the
product label and should be followed. boil without scorching or burning. Those media
which should not be autoclaved will be ready to pour
1 Write on the label the date of receipt in the into dishes or other containers after this amount of
laboratory. heating. Most culture media will require final
2 Store as directed on the label; usually below 258C sterilisation in an autoclave at 1218C for 15 minutes.
in a dry area, away from direct sunlight, The pH of the dehydrated medium has been adjusted
autoclaves, drying ovens or other heat sources. so that the final pH of the prepared medium
Where indicated store at 2±88C. conforms with the label specification when the
3 Check expiry date on the label, some media have medium has been cooled to 258C. Do not adjust the
significantly shorter shelf-lives than others. pH before sterilisation.
4 Use stock in lot/batch number order. Do not open
a new bottle until the previous bottle has been
STERILISATION OF CULTURE MEDIA
emptied. Note on the label the date the container is Although sterilisation of culture media is best carried
first opened. After use, make sure the container is out in a steam autoclave at temperatures around
tightly closed and return it to the designated 1218C it has to be recognised that damage is caused to
storage area. the medium by the heating process.
5 Order the medium in an appropriate size of Heat-treatment of complex culture media which
container and in a quantity which accords to contain peptides, sugars, minerals and metals results
normal use requirements. A medium in a large in nutrient destruction, either by direct thermal
container which has been opened many times will degradation or by reaction between the medium
deteriorate on storage. Discard the medium if the components. Toxic products caused by chemo-
powder is not free flowing, if the colour has oxidation can also be formed during heat-treatment.
changed or if it appears abnormal in any way.
It is important, therefore, to optimise the heating
process so that a medium is sterile after heating but
RECONSTITUTION OF DEHYDRATED MEDIA minimal damage is caused to the ingredients of the
Complete instructions for the preparation of culture medium. As a general rule it is accepted that short-
media are given on the label of each bottle. As a duration, high-temperature processes are more lethal
general rule it is wise to prepare one week's to organisms and less chemically damaging than are
requirement only. longer, lower temperature processes.
1 Use water prepared by distillation, deionisation or A general instruction for sterilising culture media in
reverse osmosis. Toxic metal ions such as copper volumes up to one litre at 1218C for 15 minutes is
must be absent. Check the pH of the water, if given on each label. Autoclaves vary in performance,
below 5.5, heat to drive off CO2 and re-check. The however, and thermocouple tests using different
conductivity of the water should ideally be below volumes of media should be carried out to determine
15 micro siemens (mS). Rinse glassware before use. the `heat-up' and `cool-down' times. It will be
essential to do this when volumes of media greater
2 Prepare the medium in a vessel about twice the
than two litres are prepared. In order to avoid
final volume of the medium to allow adequate
overheating large volume units of media, the `heat-
mixing. Follow the instructions given on the label
of each product. up' and `cool-down' periods are normally integrated
into the 1218C holding time.
3 Open the culture medium container away from
Sterilisation Cycle
draughts and moisture. Avoid inhaling the powder
The sterilisation cycle can be divided into its four
and prolonged skin contact. Weigh the powder
stages:
quickly, accurately and without creating `clouds of
dust'. Reclose the container as soon as possible. 1 Chamber heat-up time
2 Heat penetration time of the medium container
4 Pour half the required volume of water in the
3 Holding time at the prescribed temperature
vessel, then the weighed quantity of medium and
4 Cool-down time for the chamber to reach 808C.
agitate briskly for a few minutes. Pour the rest of
the water down the sides of the vessel to wash any
adherent medium back into solution. This is an
important step because dry culture media powder
Table of Faults and Possible Causes in Media Heat-labile supplements should be added to the
Sterilisation medium after it has cooled to 508C. Allow the sterile
supplement to come to room temperature before
Fault adding it to the agar medium. Cold liquids may cause
Wrong pH value. agar to gel or form transparent flakes which can
Possible Causes easily be seen in blood-enriched agar. Mix all
pH test carried out above 258C. supplements into the medium gently and thoroughly,
then distribute into the final containers as quickly as
Overheating through prolonged sterilisation, possible.
remelting or overlong period at 508C.
Blood used for the preparation of blood agar should
Incomplete solution of medium.
be as fresh as possible and should have been stored at
Poor quality water or containers. 2±88C (blood must not be frozen). Warm the blood in
Dehydrated medium stored incorrectly or beyond an incubator to about 35±378C before addition to
the stated shelf-life. sterile molten agar base, which has been cooled to 40±
458C. Adequate mixing in a large head-space vessel is
Fault essential to ensure aeration of the blood. Poorly
Turbidity, precipitation. oxygenated blood plates are purplish in colour
whereas properly aerated blood agar is cherry-red.
Possible Causes
Defibrinated blood is recommended for use rather
Poor quality water or containers.
than blood containing an anticoagulant.
Overheating or prolonged storage at 508C.
pH value incorrect. STORAGE OF PREPARED MEDIA
Incomplete solution. The recommended shelf-life of prepared culture
media varies considerably. Screw-capped bottles of
Fault nutrient broth and agar can be stored for 6 months at
Darkening. low ambient temperatures (12±168C). It is important
Possible Causes to store all media away from light.
Overheating, incomplete solution or pH drift. Agar plates should be stored at 2±88C in sealed
containers to avoid loss of moisture.
Fault
Soft gel. DO NOT FREEZE.
Possible Causes Fresh media are better than stored media therefore
Agar not in solution, poor mixing, prolonged avoid long storage times. Some very labile beta-
storage at 508C. lactam selective agents have very short active lives
and media containing such substances should be used
Overheating at low pH values. within a few days of preparation.
Error in weighing or overdilution with inoculum or
It is good laboratory practice to establish shelf-lives
media supplements.
for all prepared media and date-stamp the containers
Fault or holders accordingly.
Poor bacterial growth. Loss of moisture from agar plates is a common cause
of poor bacteriological performance. Do not pre-
Possible Causes
Prolonged and excessive heating, incomplete incubate all plates overnight as a sterility check. Only
obviously wet plates require pre-inoculation drying.
solution.
Ensure that all plates are incubated in a humid
Inhibitory substances in water or containers. environment.
Darkening and pH drift. Examine prepared media before inoculation. Look for
PREPARATION OF STERILISED MEDIA evidence of contamination, uneven filling or bubbles
on surface of agar, colour changes, haemolysis and
Liquid media which are sterilised in their final signs of dehydration such as shrinking, cracking and
containers should be cooled down to room loss of volume. Discard any defective plates or tubes.
temperature as rapidly as possible. Screw caps should
then be tightened. PRECAUTIONS IN THE USE AND DISPOSAL
Containers of agar media which have been sterilised OF PREPARED MEDIA
should be placed in a 508C water bath and the It should be recognised that inoculation of culture
medium dispensed as soon as it reaches this media with bacteria, deliberately or accidentally,
temperature, or within a maximum of 3 hours in the leads to very great numbers of organisms being
bath. The medium should be mixed thoroughly, produced. High concentrations of any organisms are
without bubble formation and aseptically dispensed potentially hazardous and must be disposed of safely
into sterile containers. Do not expose dishes of agar by approved methods.
media to sunlight; it causes excessive condensation on
the lids and may cause the formation of inhibitory All infected specimens and inoculated culture media
substances by photo-oxidation. should be handled only by qualified personnel who
have been trained in microbiological procedures. Such
staff should ensure that all specimens and cultures drains. The same precaution applies to any biological
under their care are properly handled and finally solution which contains sodium azide as a
autoclaved before disposal. Any apparatus used and preservative.
contaminated must be safely disinfected or sterilised; Products containing Barbitone
this is particularly important when such apparatus These products are labelled POISON/TOXIC.
must be serviced or passed out of the laboratory.
They are subject to the Misuse of Drugs Act 1973. The
The environment in which microbiological cultures supply is controlled and in the United Kingdom such
are handled must also be taken into account. Most products are available to bona fide laboratories only
countries have categories of organisms which are (orders must be signed by the head of department) or
divided into those which may be handled in the to establishments authorised to possess such
general microbiological laboratory, those which products. Certain establishments are exempt under
require special laboratory conditions and for the most the regulations e.g. NHS hospital laboratories and
dangerous organisms a totally contained and highly other government departments but they will be asked
protected environment. It may be a criminal offence to confirm this status.
not to observe these rules and regulations. When
using culture media always label or identify the Exports to some countries may require an import
container with the specimen details before licence for the country of destination.
inoculation. Cycloheximide
Inoculate the medium using aseptic techniques and This compound, prepared in Supplement vials,
incubate under the appropriate conditions. reaches a concentration which is considered to be
toxic and is labelled accordingly. However, when
Examine the medium after incubation for evidence of diluted out into the culture medium its concentration
microbial growth and carry out the appropriate falls below the minimum level considered to be
isolation and identification procedures. hazardous. It is important when reconstituting vials
containing toxic levels of cycloheximide to ensure that
PRECAUTIONS ± DEHYDRATED MEDIA the vial solution does not touch the skin and to
Most of the products supplied by OXOID Limited prevent the creation of aerosols which would allow
have no known risks except those usually associated the compound to be inhaled. Wear personal
with fine powders. However, to prevent the risk of protective equipment in accordance with the
inhaling fine dust it is recommended that approved information in the Material Safety Data Sheet. The
masks should be worn whilst handling dehydrated following First Aid procedures should be taken in
media. cases of accident with any of the products described
above:
Material Safety Data Sheets are available for
individual products.
FIRST AID PROCEDURES
Dehydrated culture media supplied as powders,
Skin Contact
granules or tablets should not be eaten. Powders
Remove all contaminated clothing; wash the affected
should not be inhaled because irritation of the upper
respiratory tract may occur. To avoid mild skin rashes areas thoroughly with soap and water. If any
symptoms occur obtain medical advice.
prevent prolonged contact with the powder and
ensure excessive dust is not produced. Eye Contact
Irrigate thoroughly with water. Obtain medical
Powdered products, if spilt, can be collected and
advice.
disposed of in the normal way. Any residue should
be washed away with ample cold water. Ingestion
Wash out mouth thoroughly with water. Give one
HAZARDOUS PRODUCTS pint of water to drink immediately. Obtain medical
advice.
There are a number of substances which contain toxic
substances. These must be used in accordance with Inhalation
the product specific Material Safety Data Sheet. All Remove person from area of exposure. Rest and keep
relevant Risk and Safety Phrases are on the product warm. Obtain medical advice.
label.
Spillage
Media containing Sodium Azide Large quantities Wear protective overalls, gloves, eye
These products contain less than 1% sodium azide protection and face mask. Collect the material into a
and have low toxicity. Some persons, however, have suitable container and seal. Dispose according to local
enhanced sensitivity to azide and therefore could regulations. Wash away the residue with plenty of
react to accidental exposure to the product. water.
Precautions must be taken to prevent ingestion or Small quantities Wash away with large volumes of
inhalation of the dust. Sodium azide reacts with many
running water, using protective gloves.
metals, especially copper, to produce explosive metal
azides.
If Local or National legislation permits and disposal
to sink is employed, use sufficient water to prevent
the powder remaining in contact with pipework and
Quality control tests should be carried out by the end- 3 Growth performance: test the growth support
user laboratory to ensure that the performance properties of the product by inoculating the
characteristics of the medium are within specification medium with appropriate stock cultures and/or
and that the methodology of medium preparation is fresh isolates. Use a standard inoculation
satisfactory. procedure and examine the quantitative and
qualitative results obtained. If testing new lots/
Each lot/batch of prepared medium should be
subjected to a minimal testing programme which will batches of media, inoculate old and new lots in one
test and compare the performance of the two lots
ensure that it is acceptable and will demonstrate a
side by side.
typical bacterial performance.
4 Stability: periodically perform the above
1 pH value: check that the pH of the prepared procedures on stored prepared media in order to
medium, when tested in final form at ambient determine whether the storage conditions will give
temperature (258C) lies within the range given on optimal results.
the product label. The medium should be
discarded if the pH value lies outside the specified NOTE: If a medium does not perform to expectations
range. and all the manufacturer's recommendations have
been followed, then the following steps should be
2 Sterility: a representative sample of each lot/batch taken:
of medium should be incubated for 2±5 days at 35±
378C. As a general rule, for a lot of 100 or less units 1 Record the nature of the problem and the method
a 3±5% sample should be tested. For a larger lot, 10 of preparation of the medium.
random plates or tubes are taken. There should be 2 Note the lot/batch number and the date it was
no evidence of microbial growth after incubation. received.
Discard all sterility samples when the tests have 3 Call the Technical Support department of the
been completed. company.
Satisfactory samples, collected without extraneous raised polymorphs/low sugar ± indicates bacterial
contamination and before antimicrobial therapy infection
should be transferred to the laboratory with minimal raised lymphocytes/normal sugar ± indicates viral
delay. If transportation is required then appropriate infection
transport media should be used to protect delicate raised lymphocytes/high protein/low sugar ±
organisms. Where quantitative results are important indicates mycobacterial infection.
e.g. urine cytology and bacteriology, or where If a fibrin clot is present then particular attention
commensal overgrowth should be prevented, should be paid to Mycobacterium tuberculosis.
refrigeration of samples at 2±88C is essential.
Cell counts are of little validity when clots are
All samples should be clearly labelled and sent in present.
leak-proof, satisfactory containers. Sealed, transparent
plastic bags, containing the sample container and the Centrifuge a portion of the CSF and make three films
request form attached to but not inside the plastic of sufficiently small area so that the whole may be
bag, is the most acceptable method of sending examined under the microscope. Stain one film by
pathological samples to the laboratory. Gram's stain, one by Leishmann or Giemsa stain and
one by an acid-fast bacilli stain. In purulent samples
BLOOD CULTURES Haemophilus influenzae may be difficult to see under
A full description of the Oxoid Signal Blood Culture Gram's stain. Carbol-thionin or a similar nucleic-acid
System and the Isolator Blood Culture System is stain may be helpful to see the bacteria in such
given on pages 8±1 to 8±10. circumstances.
Examination of blood for infectious agents is one of Inoculate a portion of the centrifuged sample (taking
the most important and often most urgent suitable aseptic precautions) on blood agar (incubate
examinations requested. All the various systems of aerobically and anaerobically), `chocolate' Columbia
blood culturing require blood samples to be collected Agar (incubate in a 5% CO2 atmosphere) examine
with scrupulous care to avoid extravenous after 18±24 hours incubation at 358C. Carry out
contamination. The blood/broth medium should be antimicrobial susceptibility tests on any organisms
subcultured to appropriate media either at fixed time isolated. Select appropriate antimicrobials for blood/
intervals or whenever changes in appearance of the brain infections. Culture for Mycobacterium tuberculosis
medium are noted e.g. turbidity, darkening, lysis etc. if the examination results indicate tuberculosis.
Subculture after 24 hours incubation, regardless of Direct tests to identify common bacterial antigens in
appearance, is recommended to detect early evidence CSF are available.
of bacteraemia. All subcultures must be made with
great care to avoid contaminating the blood/broth Associated pathogens
medium. Haemophilus influenzae
Neisseria meningitidis
Associated pathogens Streptococcus pneumoniae
Staphylococci (coagulase positive and negative) Mycobacterium tuberculosis
Streptococci (alpha/beta/non-haemolytic strains) Listeria monocytogenes
November 1998 2-9
Culture Media
Enterotoxigenic Escherichia coli (ETEC): inoculate Inoculate all swabs on Thayer-Martin Medium
MacConkey Agar and MacConkey Sorbitol Agar (CM367 + SR90 + SR91 or SR101) or on Modified
CM813. Look for non-sorbitol fermenting colonies New York City Medium (CM367 + SR105+ SR95 or
indicative of Escherichia coli O157:H7; confirm identity SR104). Incubate in a 5% CO2 atmosphere at 358C for
with serological tests. Look for Staphylococcus aureus 24±48 hours. Sabouraud Dextrose Agar or Dermasel
also on MacConkey Agar in case the disease is Agar can be inoculated if Candida are suspected.
staphylococcal enterocolitis.
PUERPERAL INFECTIONS
Campylobacter: inoculate Campylobacter Selective High vaginal swabs from such conditions should be
media made with one of the various selective examined carefully for Clostridium perfringens. Non-
supplements available. sporing, square-ended Gram-positive rods which
appear to be capsulated may be seen in the Gram
Vibrios: V. cholerae or V. parahaemolyticus may be
suspected. Inoculate alkaline peptone water and stained film and should reported to the physician
immediately.
TCBS Agar CM333.
Inoculate blood agar plates and incubate aerobically
Yersinia: Y. enterocolitica may be isolated on Yersinia
and anaerobically at 358C for 18±24 hours.
Selective Agar Base (CM653 + SR109). Inoculate the
medium and incubate for 18±24 hours at 328C.
Clostridium perfringens: inoculate blood agar and EXAMINATION OF FOOD AND
incubate anaerobically (and aerobically as a control). DAIRY PRODUCTS
Inoculate two tubes of Cooked Meat Broth and heat
one at 808C for 30 minutes to detect heat-resistant There is no general agreement on methods for the
spores. Subculture to blood agar and incubate laboratory examination of foods and dairy products.
anaerobically and aerobically. The standard reference books used are:
Aeromonas: A. hydrophila and A. sobria are associated Compendium of Methods for the Microbiological
with enteritis of children and adults. Inoculate Examination of Foods by the American Public Health
Aeromonas Medium Base (Ryan) CM833 + SR136 or Association. Washington D.C. 1976.
Blood Agar containing 20mgm per litre of Ampicillin. Bacteriological Analytical Manual 8th Edn. Revision A by
Incubate 18±24 hours at 358C. the Association of Official Analytical Chemists.
Clostridium difficile: when this organism is isolated Washington D.C. 1978.
from antimicrobial-associated-colitis it is considered Microorganisms in Foods Vols. 1 & 2. by the
to be a pathogen. It can be found fairly commonly in International Commission on Microbiological
infant stools where it is usually non-toxigenic. Specifications for Foods. Toronto University Press.
Inoculate alcohol-treated faeces on Clostridium 1988 with Revision.
Difficile Agar Base (CM601 + SR96) and incubate
anaerobically at 358C for 18±24 hours. In Europe, the Codex Alimentarius Commission is
considering standard methods, aided by published
Associated pathogens standards from the International Organization for
Bacillus cereus Standardization (ISO).
Plesiomonas shigelloides
Clostridium botulinum The bacteriological examination of food and dairy
products falls into one or more of the following four
Commensal organisms categories:
Coliform bacilli, Proteus species, Pseudomonas species,
Bacteroides species and many Clostridium species. 1 Total Viable Count: this is an attempt to measure
the total number of bacteria, yeasts and moulds in
SEXUALLY TRANSMITTED DISEASE SWABS a product by inoculating dilutions of suspensions
STD samples may come from the eye, throat, rectum, of the sample into various culture media and
cervix, vagina or urethra. incubating them for fixed periods at temperatures
varying from 228C to 558C. The resulting colony
Eye swabs: look for Neisseria gonorrhoeae and counts are then calculated as organisms per gram
Chlamydia trachomatis as previously described. of product. The results obtained are compared with
Throat swabs: look specifically for N. gonorrhoeae. expected figures and the product is passed or
failed. It is not an accurate process and fairly gross
Vaginal/cervical swabs: examine a Gram's stained changes in numbers are looked for which indicate
smear for N. gonorrhoeae and a `wet' slide preparation unsatisfactory raw materials, processing or storage
for Trichomonas vaginalis and for `clue cells' diagnostic conditions.
for Gardnerella vaginalis. Yeast cells may be seen in 2 Indicator Organism Count: specific organisms are
either preparation. To isolate G. vaginalis inoculate sought, most often coliforms (lactose-fermenters) or
Columbia Blood Agar Base containing 10% human, Enterobacteriaceae (glucose-fermenters) using
rabbit or horse blood plus G. vaginalis Selective selective media. See section on Violet Red Bile Agar
Supplement (SR119). Incubate at 358C in a 7% CO2 and Violet Red Bile Glucose Agar. These organisms
atmosphere for 48 hours. indicate the standard of hygiene used in the
Urethral swabs: as well as N. gonorrhoeae, include C. manufacture of the food products.
trachomatis in the smear examination using Giemsa 3 Detection of Specific Spoilage organisms:
stain or a specific immunofluorescent reagent. spoilage organisms are usually associated with
taints and off-flavours in stored products. They are assessment and control. The hazards are
the major factor in determining the shelf-lives of determined, the critical control points of those
food products and are now considered to be of hazards are identified and procedures to monitor
more relevance than total viable counts. Moulds the critical control points are established. An
and psychrotrophic Gram-negative rods are HACCP audit is an essential stage in the
specifically sought, using selective culture media implementation of this process. [ICMSF (1989)
and low temperature incubation. ``Micro-organisms in Foods, 4. Application of
hazard analysis critical control point (HACCP)
4 Detection of Food Poisoning Organisms: Hazard
system to ensure microbiological safety and
analysis critical control point technique (HACCP)
quality''. Blackwell Scientific Publications, Oxford.]
is a systematic approach to hazard identification,
Plate Count Colony count for indicating the Nutrient Gelatin (CM135a) CM635
efficiency of water treatment Tryptone Glucose Extract Agar CM127
processes, or suitability of water for Tryptone Soya Agar CM131
food preparation. Nutrient gelatin is Yeast Extract Agar CM19
used for the plate count of Plate Count Agar CM325
psychrophilic organisms e.g. Standard Plate Count Agar (APHA) CM463
Pseudomonas species and for testing Milk Plate Count Agar with
gelatinase activity antibiotic free skim milk CM681
Milk Agar CM21
PPCT Selective Supplement SR159
Salmonellae and Pre-enrichment procedures prior to Buffered Peptone Water CM509
Shigellae isolation Mannitol Selenite Broth Base CM399
Enrichment procedures prior to Muller-Kauffmann Tetrathionate
isolation Broth Base CM343
Selenite Broth Base CM395
Sodium Biselenite L121
Tetrathionate Broth Base CM29
Isolation and identification of Rappaport-Vassiliadis (RV) ±
Salmonella and Shigella species Enrichment Broth CM669
Rappaport Vassiliadis Soya (RVS)
Peptone Broth CM866
Selenite Cystine Broth Base CM699
Tetrathionate Broth USA CM671
Bismuth Sulphate Agar CM201
Brilliant Green Agar CM263
Brilliant Green Agar (Modified)
(Edel-Kampelmacher Medium) CM329
Sulphamandelate Supplement SR87
DCLS Agar CM393
Desoxycholate Citrate Agar (Hynes) CM227
Hektoen Enteric Agar CM419
XLD Medium CM469
MLCB Agar CM783
Identification of salmonella and Kligler Iron Agar CM33
shigella. Lysine Iron Agar CM381
Triple Sugar Iron Agar CM277
Salmonella Rapid Test Elective Medium CM857
Rapid Test FT201
Latex Test FT203
Staphylococci Isolation and differentiation of Baird-Parker Agar Base CM275
pathogenic staphylococci Egg Yolk Tellurite Emulsion SR54
Egg Yolk Emulsion SR47
Giolitti-Cantoni Broth CM523
Staphylococcus Medium No.110 CM145
Staph/Strep. Supplement SR70
Vogel-Johnson Agar CM641
Mannitol Salt Agar
(Chapman Medium) CM85
Selective enumeration of the total Baird-Parker Agar Base CM275
staphylococci count from foodstuffs Egg Yolk Emulsion SR47
RPF Supplement SR122
Differentiation of staphylococci:
(a) DNase production DNase Agar CM321
(b) Phosphatase production Blood Agar Base CM55
(c) Coagulase production Staphylase Test
A test kit for the identification of
Staphylococcus aureus: DR595
Staphytect DR650
Plant Hygiene and Diluent or rinse in bacteriological Ringer Solution Tablets BR52
Food Sample examination of food products, plant
Dilution and apparatus
Solvent diluent solution for calcium 'Calgon' Ringer Tablets BR49
alginate swabs
Chlorine-neutralising Ringer Thiosulphate Ringer Tablets BR48
Solution to counteract the
bactericidal effect of hypochlorites
or other chlorine sources
Microbial Limit Testing ± Coliforms Brilliant Green Bile (2%) Broth CM31
Buffered Peptone Water CM509
Membrane Endo Agar LES MM551
MacConkey Broth (purple) CM5a/CM6a
Tergitol-7 Agar CM793
Violet Red Bile Agar CM107
Violet Red Bile Glucose Agar CM485
Differentiation of coliforms
(a) Methyl Red and
Voges-Proskauer Tests MRVP Medium CM43
Enterococci Detection and isolation of faecal Azide Blood Agar Base CM259
streptococci from sewage and water Azide Dextrose Broth (Rothe Broth) CM868
MacConkey Agar No.2 CM109
Slanetz and Bartley Medium
(Enterococcus Agar) CM377
Kanamycin Aesculin
Azide Agar Base CM591
K-F Streptococcus Agar CM701
Salmonellae and Pre-enrichment procedures prior to Mannitol Selenite Broth Base CM399
Shigellae isolation Selenite Broth Base CM395
Enrichment procedures prior to isolation Selenite Cystine Broth Base CM699
Sodium Biselenite L121
Tetrathionate Broth Base CM29
Tetrathionate Broth USA CM671
Rappaport Vassiliadis (RV)
Enrichment Broth CM669
Rappaport Vassiliadis Soya (RVS)
Peptone Broth CM866
Diluents used in For preparation of 1/4 strength Ringer Ringer Solution (1/4 strength Ringer
water and sewage Solution, used as a diluent or rinse Solution tablets) BR52
bacteriology
Preparation of a sodium 'Calgon' Ringer Tablets BR49
hexametaphosphate Ringer Solution,
used as a solvent diluent solution in
conjunction with calcium alginate
swabs
Organism Differentiation
Corynebacterium species MRS Broth CM359
Tomato Juice Agar CM113
Organism Isolation/Cultivation
Legionella species Blood Agar Base Media: see above
Charcoal Agar CM119 + SR50
Isolation/Differentiation
`Chocolate' Columbia Agar Base CM331 + SR50
Legionella CYE Agar Base CM655 with BCYE
GC Agar Base CM367 with Haemoglobin
Growth Supplement SR110
Powder Soluble L53 and Growth Supplements
+ BMPA-a Supplement SR111
Yeast Autolysate SR105, Vitox SR90
or + MWY Supplement SR118
+ GC supplement SR56
or + GVPC Supplement SR152
or + VCNT supplement SR91
Legionella Latex Test DR800
or + VCN supplement SR101
L. Pneumophila serogroup 1 DR801
or + LCAT supplement SR95
L. Pneumophila serogroup 2±14 DR802
or + VCAT supplement SR104
Legionella species test reagent DR803
Stuart Transport Medium (Modified) CM111
Organism
Differentiation
Listeria species
Identification beta-lactamase Sticks BR66
Isolation/Cultivation Identification oxidase Sticks BR64
Blood Agar Base Media: see above Broad-Spectrum beta-lactamase Mixture SR113
Brain Heart Infusion Agar CM375 Nitrocefin reagent SR112
Buffered Listeria Enrichment Broth CM897
Organism
Listeria Selective Enrichment Supplement SR141
Pasteurella/Yersinia species
Fraser Broth CM895
Fraser Supplement SR156 Isolation/Cultivation
Half-Fraser Supplement SR166 Blood Agar Base Media: see above
Listeria Selective Agar Base (Oxford) CM856 Cary Blair Transport Medium CM519
Listeria Selective Supplement SR140 Desoxycholate Citrate Agar (Hynes) CM227
Listeria Enrichment Broth CM862 MacConkey Agar CM7b
Listeria Selective Enrichment Supplement SR141 SS Agar CM99
Listeria Selective Enrichment Supplement Yersinia Selective Agar Base CM653 + supplement
(modified) with 10mg/l Acriflavine SR149 SR109
Listeria Enrichment Broth Base (UVM formulation) Differentiation
CM863 Kligler Iron Agar CM33
Listeria Primary Selective Enrichment Supplement Triple Sugar Iron Agar CM277
(UVM ) SR142 Urea Agar Base/Broth Base CM53/CM71 + SR20
Listeria Secondary Selective Enrichment
Supplement (UVM II) SR143 Organism
Listeria Rapid Test FT401 Pseudomonas species
PALCAM Agar Base CM877 Isolation/Cultivation
PALCAM Selective Supplement SR150 Blood Agar Base CM55
Organism Pseudomonas Agar Base CM559 + CFC supplement
Mycobacterium species SR103
or + CN supplement SR102
Isolation/Differentiation
Acid Egg Medium PM1a Differentiation
Lowenstein-Jensen Medium PM1 or PM2 Oxidase Sticks BR64
Pyruvic Acid Egg Medium PM2a MacConkey Agar CM7b
Modified Acid Egg Medium PM95 Organism
Modified Pyruvic Acid Egg Medium PM96 Salmonella and Shigella species
Simplified Pyruvate Loewenstein-Jensen Medium Enrichment Media
PM98 Buffered Peptone Water CM509
Standard Reference Acid Egg Medium PM99 EE Broth CM317
Standard Reference Pyruvic Acid Egg Medium Lactose Broth CM137
PM100 Mannitol Selenite Broth Base CM399
Simplified Lowenstein-Jensen Medium PM97 Maximum Recovery Diluent CM733
Organism Mueller-Kauffmann Tetrathionate Broth CM343
Mycoplasma species Rappaport Vassiliadis (RV) Broth CM669
Rappaport Vassiliadis Soya Peptone (RVS) Broth
Isolation/Differentiation CM866
Blood Agar Base Media: see above Selenite Broth Base CM395 + L121
Mycoplasma Agar Base CM401 + supplement SR59 Selenite Cystine Broth Base CM699
or SR60 Tetrathionate Broth Base CM29
Mycoplasma Broth Base CM403 + supplement SR59 Tetrathionate Broth (USA) CM671
or SR60
Isolation Media
Organism Bismuth Sulphite Agar (Modified) CM201
Neisseria species Brilliant Green Agar CM263
Brilliant Green Agar (Modified) CM329 + Brain Heart Infusion Agar/Broth CM375/CM225
Sulphamandelate supplement SR87 China Blue Lactose Agar CM209
DCLS Agar CM393 Dextrose Broth CM175
Desoxycholate Citrate Agar CM35 Dextrose Tryptone Agar/Broth CM75/CM73
Desoxycholate Citrate Agar (Hynes) CM227 Edwards Medium (Modified) CM27
Endo Agar Base CM479 + BR50 GBS Agar Base (Islam) CM755 + SR35
Hektoen Enteric Agar CM419 Kanamycin Aesculin Azide Agar Base CM591 +
MacConkey Agar CM115 supplement SR92
M-Endo Agar LES MM551 + BR50 Kanamycin Aesculin Azide Broth Base CM771
MLCB Agar CM783 + supplement SR92
SS Agar CM99 KF Streptococcus Agar CM701
SS Agar (Modified) CM533 MacConkey Agar No.2 CM109
XLD Medium CM469 MacConkey Broth CM5
M17 Agar CM785
Differentiation
Nutrient Broth CM67
Kligler Iron Agar CM33
Slanetz and Bartley Medium CM377
Lysine Decarboxylase Broth CM308
Stuart Transport Medium (Modified) CM111
Lysine Iron Agar CM381
Todd-Hewitt Broth CM189
MR-VP Medium CM43
Tryptone Soya Agar/Broth CM131/CM129
Simmons Citrate Agar CM155
Triple Sugar Iron Agar CM277 Differentiation
Tryptone Water CM87 Azide Blood Agar CM259
Urea Agar Base/Broth Base CM53/CM71 + SR20 Bacitracin Discs DD2
Salmonella Rapid Test: Edwards Medium (Modified) CM27
Elective Medium CM857 KAA Agar Base/Broth Base CM591/CM771 +
Rapid Test FT201 supplement SR92
Latex Test FT203
Nutrient Gelatin CM135a
Organism
Optochin Discs DD1
Staphylococcus species
Streptococcal Grouping Kit DR585
Isolation/Cultivation
Organism
Baird-Parker Medium CM275 + SR47/SR30 or SR54
Vibrionaceae:
or + RPF supplement SR122
Aeromonas species
Blood Agar Base Media: see above
Plesiomonas species
China Blue Lactose Agar CM209
Vibrio species
Columbia Agar Base CM331 + Staph/Strep
supplement SR70 Isolation/Cultivation
Giolitti-Cantoni Broth CM523 Aeromonas Medium Base (Ryan) CM833 +
MacConkey Agar CM7b supplement SR136
Mannitol Salt Agar CM85 Blood Agar Base Media: see above
Nutrient Broth CM67 Cary Blair Transport Medium CM519
Salt Meat Broth CM94 Cholera Medium TCBS CM333
Staphylococcus Medium No.110 CM145 DCLS Agar CM393
Vogel-Johnson Medium CM641 Peptone Water CM9
Tryptone Soya Agar/Broth CM131/CM129
Differentiation
Baird-Parker Medium CM275 + SR47/SR30 or SR54 Differentiation
or + RPF supplement SR122 Cholera Medium TCBS CM333
Beta-Lactamase Sticks BR66 Identification Oxidase Sticks BR64
DNase Agar CM321 MR-VP Medium CM43
Egg Yolk Emulsion SR47 Nutrient Gelatin CM135a
Egg Yolk Tellurite Emulsion SR54 0129 Discs DD14/DD15
Mannitol Salt Agar CM85 Peptone Water CM9
Nitrocefin SR112 Simmons Citrate Agar CM155
Nutrient Gelatin CM135a Tryptone Water CM87
Staphylococcus Medium No.110 CM145 Organism
Staphylase Test DR595 Yeasts and Moulds
Staphytect DR650
Isolation/Cultivation
Organism `Actidione' Agar PM118
Streptococcus and Enterococcus species Brain Heart Infusion Agar CM375
Isolation/Cultivation Corn Meal Agar CM103
Amies Transport Medium CM425 Czapek Dox Agar (Modified) CM97
Azide Blood Agar Base CM259 Dermasel Agar CM539 + SR75
Azide Dextrose Broth CM868 Dichloran-Glycerol (DG18) Agar Base CM729 +
Blood Agar Base Media: see above + Streptococcus SR78
supplement SR126 DRBC Agar Base CM727 + SR78
2-30 November 1998
Culture Media
4 Lev M. Keudell KC. and Milford AF. Succinate as a growth to remove dissolved oxygen. They should be allowed
factor for Bacteroides melaninogenicus. J. Bact. 1971:108:175±8. to cool without agitation and then inoculated.
5 Neilson PA. Role of reduced sulphur compounds in nutrition of
Proprionobacterium acnes. J. Clin. Micr. 1983:17:276±9. Storage conditions and Shelf life
6 Shanson DC. and Singh J. Effect of adding cysteine to brain- Anaerobe Basal Broth CM957 should be stored tightly
heart infusion broth on the isolation of Bacteroides fragilis from capped in the original container at 108C±258C. When
experimental blood cultures. J. Clin. Path. 1981:34:221±3. stored as directed, the medium will remain stable
until the expiry date printed on the bottle.
The assay of antibiotics is a highly skilled process The reservoirs may be holes cut into the agar and
which requires very close attention to the details filled with known concentrations of antibiotic, or
specified in the official publications and these must be metal cylinders placed on the surface of the agar and
consulted. The general principles are, however, similarly filled, or paper discs charged with antibiotic.
straightforward and can be divided into (1) diffusion Large square plates (250 x 250mm) or flat sheets of
plate assay and (2) turbidometric assay. glass are commonly used and a 4mm depth of base
Diffusion plate assays depend upon the diffusion of agar is poured and allowed to set. Then a thin (1mm)
antibiotics from reservoirs on or in the agar layer. layer of seeded agar is poured over the base layer and
Measurement of the amount of diffusion is made by allowed to set. The holes are punched into these
comparing the sizes of zones of growth inhibition, layers or the various reservoirs placed on the seed
using standard strains of susceptible organisms and layer. It is essential that the layers of agar are even in
known amounts of reference antibiotics. depth and care must be taken to pour them on
properly levelled benches. Up to 36 separate
The results depend on critical rates of diffusion of the
reservoirs can be placed in dishes of this size.
antibiotic, critical growth rates of the standard
organisms and critical minimal inhibitory coefficient
levels of each organism.
Reference
1 Hanus F. J., Sands J. G. and Bennett E. O. (1967) Applied
Microbiology 15(1) 31±34.
Description References
A selective medium for the detection and isolation of 1 Edwards S. J. (1933) J. Comp. Path. Therap. 46(4) 211±217.
streptococci from faeces, sewage, and other specimens 2 Snydar M. L. and Lichstein H. C. (1940) J. Infect. Dis. 67(2) 113±
containing a mixed flora. Azide Blood Agar Base is 115.
similar to the medium used by Edwards1 for the 3 Lichstein H. C. and Snyder M. L. (1941) J. Bact. 42(5) 653±664.
isolation of mastitis streptococci. The sodium azide 4 American Public Health Association (1978) Standard Methods for
has a bacteriostatic effect on most Gram-negative the Examination of Dairy Products. 14th Edn. APHA Inc. New York.
organisms but permits growth of Gram-positive 5 Packer R. A. (1943) J. Bact. 46. 343±349.
organisms such as streptococci and some strains of 6 Mossel D. A. A., Diepen H. M. J. van and De Bruin A. S. (1957) J.
staphylococci. Proteus species are slightly more Appl. Bact. 20(2) 265±272.
resistant than other Enterobacteriaceae but swarming 7 Wood R. L. (1965) Amer. J. Vet. Res. 26. 1303±1308.
is prevented (Snyder and Lichstein2, Lichstein and 8 Dale C. N. (1940) J. Bact. 40. 228±231.
Snyder3). 9 Bohm K. H. (1971) Zbl. Bakt. I. Orig. 218. 330±334.
At the above concentration and pH, sodium azide
exerts no appreciable effect on haemolysis so that the AZIDE DEXTROSE BROTH
medium, with added blood, may be used for the (ROTHE)
simultaneous determination of haemolytic reactions.
Code: CM868
Azide blood agar is recommended by the American
Public Health Association4 for the isolation of For the detection of enterococci in water.
streptococci from cheese. The plates, inoculated with
dilutions of emulsified cheese, are incubated at 358C Formula gm/litre
and representative colonies subcultured for Peptone 20.0
subsequent identification. Glucose 5.0
There are variations in formula of Azide Blood Agar Sodium chloride 5.0
Base which have been recommended for different Di-potassium hydrogen phosphate 2.7
purposes: Potassium dihydrogen phosphate 2.7
Sodium azide 0.2
1 Packer5 increased the sodium azide concentration to Final pH 6.8 + 0.2
0.9g per litre and added 0.002g per litre crystal violet.
The pH was also adjusted to 6.8 + 0.1. This is a more Directions
selective medium for faecal streptococci in foods6. Add 35.6g to one litre of distilled water for single
strength broth or 71.2g for double strength broth.
2 Packer5 and Wood7 used the above formulation Heat gently to dissolve. Dispense into final containers
with 5% blood and the crystal violet increased to
and sterilise by autoclaving at 1218C for 15 minutes.
0.01g per litre, for the isolation of Erysipelothrix
rhusiopathiae and Streptococcus pneumoniae. Description
3 Dale8 and Bohm9 recommended the addition of Azide Dextrose Broth (Rothe) is used for the detection
phenol (1.0 to 2.5g per litre) to Packer's formulation of enterococci in water and sewage1.
to isolate E. rhusiopathiae. The presence of enterococci serves as an indicator of
Storage conditions and Shelf life faecal contamination. Enterococci are better indicators
Store the dehydrated medium below 258C and use than E. coli of sewage pollution in chlorinated waters
before the expiry date on the label. because they have a greater resistance to chlorine.
Store prepared blood agar plates of medium at 2±88C. Mallmann and Seligmann2 recommended Azide
Dextrose Broth for the quantitative determination of
Quality Control enterococci in water, sewage, foods and other
Positive control: materials suspected of contamination with sewage.
Enterococcus faecalis ATCC1 29212
Staphylococcus aureus ATCC1 25923 A blend of peptone and glucose render Azide
Dextrose Broth highly nutritious, and sodium
Negative control:
Proteus vulgaris ATCC1 13315 chloride maintains osmotic equilibrium. The use of
sodium azide as an inhibitor of Gram-negative
Escherichia coli ATCC1 25922
organisms has been reported by several workers2,3,4,
Precautions and the concentration selected provides optimum
Proteus and Escherichia species may not always be protection for the enterococci while largely
inhibited on the Edward's formulation. suppressing the Gram-negative flora. The phosphate
Always use a light inoculum for best selective results. buffer system controls pH.
Anaerobic incubation will enhance haemolytic reactions. Technique
Inoculate 10ml of medium with 1ml of the test
Haemolytic reactions will not be typical on Packer's sample. Inoculate a further three tubes with 0.1ml,
modification of Azide Blood Agar Base. Streptococcus 0.01ml and 0.001ml sample respectively. For samples
lactis will not grow on Packer's modification with 5% of 10ml or more, use double strength broth. Incubate
sheep blood. all tubes at 358C and examine for turbidity after 24
Read the section on Hazard Precautions 2.7 for azide- and 48 hours. For a more detailed description please
containing media. consult `Standard Methods for the Examination of
Water and Wastewater'.
2-40 November 1998
Culture Media
The presence of enterococci in the sample is indicated add 25ml of sterile Egg Yolk Emulsion SR47. Mix well
by turbidity in the broth. Positive cultures should be and pour into sterile petri dishes.
inoculated into Ethyl Violet Azide Broth (Litsky) Description
CM869 to confirm the presence of enterococci. Bacillus Cereus Selective Agar CM617, is based on the
Storage conditions and Shelf life highly specific diagnostic and selective PEMBA
Store the dehydrated medium below 258C and use medium, developed by Holbrook and Anderson1 for
before the expiry date on the label. the isolation and enumeration of Bacillus cereus in
foods. It meets the requirements for a medium that is
Store the prepared medium at 2±88C. sufficiently selective to be able to detect small numbers
Quality Control of B. cereus cells and spores in the presence of large
Positive control numbers of other food contaminants. The medium is
Enterococcus faecalis ATCC1 29212 also sufficiently diagnostic that colonies of B. cereus are
Negative control readily identified and confirmed by microscopic
Escherichia coli ATCC1 25922 examination.
Precautions The role of B. cereus in food poisoning, particularly
This product contains less than 1% azide and has low from the consumption of contaminated rice, is now
toxicity. However, when handling the powder, wear well documented.2,3,4 The organism has also been
gloves, mask and eye protection. When washing implicated in eye infections5,6 and a wide range of other
azide products down sinks, use sufficient water to conditions including abscess formation, meningitis,
prevent accumulation of azide in the plumbing. septicaemia and wound infection. B. cereus is
recognised as a significant pathogen in post-operative
References and post-traumatic wounds of orthopaedic patients.7
1 Greenberg A. E. et al (ed). (1985) Standard Methods for the Amongst veterinarians, B. cereus is a known cause of
Examination of Water and Wastewater, 16th ed. APHA, disease, especially mastitis, in ewes and heifers.8
Washington, D.C.
In the formulation of Bacillus cereus Selective Agar a
2 Mallmann W. L. and Seligmann E. B. (1950) Am. J. Public Health
peptone level of 0.1% and the addition of sodium
40. 286.
pyruvate improve egg yolk precipitation and enhance
3 Edwards S. J. (1933) J. Comp. Path. Therap. 46. 211.
sporulation. Bromothymol blue is added as a pH
4 Hartman G. (1937) Milchw. Forsch. 18. 166.
indicator to detect mannitol utilisation. The medium is
made selective by addition of Bacillus cereus Selective
BACILLUS CEREUS SELECTIVE Supplement SR99, which gives a final concentration of
AGAR BASE 100 IU of polymyxin B per ml of medium. Polymyxin B
as a selective agent for the isolation of B. cereus has
Code: CM617 been previously suggested by Donovan9 and found to
be satisfactory by Mossel.10 It is recommended that,
A selective and diagnostic medium for the isolation and where large numbers of moulds are expected in the
enumeration of Bacillus cereus. inoculum, filter-sterilised cycloheximide is added to
the medium at a final concentration of 40mg/ml.
Formula gm/litre
Peptone 1.0 The primary diagnostic features of the medium are
Mannitol 10.0 the colonial appearance, precipitation of hydrolysed
Sodium chloride 2.0 lecithin and the failure of B. cereus to utilise mannitol.
Magnesium sulphate 0.1 The typical colonies of B. cereus are crenated, about
Disodium hydrogen phosphate 2.5 5mm in diameter and have a distinctive turquoise to
Potassium dihydrogen phosphate 0.25 peacock blue colour surrounded by a good egg yolk
Bromothymol blue 0.12 precipitate of the same colour.
Sodium pyruvate 10.0 These features distinguish B. cereus from other Bacillus
Agar 14.0 species except B. thuringiensis. Other egg yolk-reacting
pH 7.2 + 0.2 organisms which can grow on the medium, including
Staphylococcus aureus, Serratia marcescens and Proteus
BACILLUS CEREUS SELECTIVE vulgaris are distinguished from B. cereus by colony
SUPPLEMENT form and colour. These organisms also produce an
egg yolk-clearing reaction in contrast to egg yolk
Code: SR99 precipitate produced by B. cereus.
Vial contents (each vial is sufficient for 500ml of Microscope examination for presence of lipid globules
medium) in the vegetative cells is recommended as a rapid and
Polymyxin B 50,000 IU confirmatory test for B. cereus and replaces the need for
Directions biochemical testing. Holbrook and Anderson1 have
Suspend 20.5g in 475ml of distilled water and bring confirmed that only B. cereus of the Bacillus species are
gently to the boil to dissolve completely. Sterilise by capable of possessing lipid globules in their vegetative
autoclaving at 1218C for 15 minutes. Cool to 508C and cells when grown on the selective medium. One further
aseptically add the contents of one vial of Oxoid advantage of this test is that strains of B. cereus that
Bacillus cereus Selective Supplement SR99 react only weakly or not at all with egg yolk can be
reconstituted with 2ml of sterile distilled water, then detected and confirmed.
Pour Plate Method 15 Beckers N. J., Leusden F. M., Bindscheidler O. and Guerraz D.
1 Prepare the RPF Agar as directed and hold at 488C. (1984) Canad. J. Microbiol. 30. 470±474.
2 Process the food sample in a stomacher or Waring 16 Sawhney D. (1986) J. Appl. Bact. 61. 149±155.
blender using the recommended sample size and 17 Holbrook R., Anderson J. M. and Baird-Parker A. C. (1965) J.
diluent. Appl. Bact. 32. 187±191.
Dry the plates before use but take care to avoid Salmonella typhi
overdrying. Correctly prepared plates should have a Appearance
smooth, cream-like opacity with a pale straw colour. Black `rabbit-eye' colonies with a black zone and
There should be no sedimentation of the indicator. metallic sheen surrounding the colony after 18
hours. Uniformly black after 48 hours incubation.
DO NOT OVERHEAT ± DO NOT AUTOCLAVE Other Salmonella species
Description Appearance
Bismuth Sulphite Agar is a modification of the Variable colony appearance after 18 hours, they
original Wilson and Blair1 selective medium for the may be black, green or clear and mucoid.
isolation and preliminary identification of Salmonella Uniformly black colonies are seen after 48 hours,
typhi and other salmonellae from pathological often with widespread staining of the medium and
material, sewage, water supplies, food and other a pronounced metallic sheen.
products suspected of containing these pathogens. Other organisms, e.g. coliform bacteria, Serratia,
In this medium freshly precipitated bismuth sulphite Proteus species
acts together with brilliant green as a selective agent Appearance
by suppressing the growth of coliforms, whilst Usually inhibited but occasional strains give dull
permitting the growth of salmonellae. Sulphur green or brown colonies with no metallic sheen or
compounds provide a substrate for hydrogen staining of the surrounding medium.
Storage conditions and Shelf life 8 Harvey R. W. S. and Price T. M. (1974) Public Health Laboratory
Store the dehydrated medium below 258C and use Service Monograph Series No.8. Isolation of Salmonellas. HMSO
before the expiry date on the label. Note the following London.
comments: 9 Hajna A. A. (1951) Pub. Hlth. Rep. 9. 48±51.
Due to its contents of reactive and hygroscopic
substances, dehydrated Bismuth Sulphite Agar BLOOD AGAR BASE
quickly deteriorates when exposed to the atmosphere. Code: CM55
This is usually indicated by aggregation into a solid
non-friable mass, and by the development of a brown A non-selective general purpose medium which may be
coloration. Medium reconstituted from such material enriched with blood or serum.
is brown, does not become green on storage, and is
characterised by loss of differential and selective Formula gm/litre
properties. For this reason the powder should be `Lab-Lemco' powder 10.0
stored in a cool, dry place and after use the container Peptone Neutralised 10.0
should be properly closed. Sodium chloride 5.0
Prepared medium Agar 15.0
It is recommended that the medium should be used pH 7.3 + 0.2
on the day of preparation. Directions
Quality Control Suspend 40g in 1 litre of distilled water. Bring to the
Salmonella typhi should be used only in a Class II boil to dissolve completely. Sterilise by autoclaving at
laboratory, not for routine testing or in food 1218C for 15 minutes.
laboratories. For blood agar, cool the Base to 508C and add 7% of
Positive Control: Defibrinated Horse Blood SR50. Mix with gentle
Salmonella enteritidis ATCC1 13076 rotation and pour into petri dishes or other
S. typhi-murium ATCC1 14028 containers.
Negative control: Description
Escherichia coli ATCC1 25922 Oxoid Blood Agar Base is a non-selective general
Citrobacter freundii ATCC1 8090 purpose medium widely employed for the growth of
pathogenic and non-pathogenic bacteria:
Precautions
Prepared plates of medium should not be stored for (i) Without additions, the medium may be
longer than two days at 2±88C; after which time the employed as a nutrient agar (a richer medium
dye oxidises to give a green medium that can be than Nutrient Agar CM3), or as a medium for the
inhibitory to some salmonellae. short-term maintenance of stock cultures.
Shigella species are usually completely inhibited. (ii) With added serum or other enrichments, the
medium becomes suitable for the cultivation of
Salmonella sendai, S. cholera-suis, S. berta, S. gallinarum many fastidious organisms. Serum and other
and S. abortus-equi are markedly inhibited9. thermolabile enrichments should be added to the
It is important that the spreading technique yields sterilised medium cooled to 45±508C.
well separated colonies. The typical colonial (iii) With added blood, the medium is not only
characteristics will not develop if the growth is too enriched, but becomes suitable for the
heavy or confluent; S. typhi colonies will appear light determination of the typical haemolytic reactions
green in these circumstances. Therefore, when in which are important diagnostic criteria for
doubt, almost any growth on the medium should be streptococci, staphylococci, and other organisms.
subject to further tests. For blood agar, 7% of sterile blood should be
added to the sterilised medium cooled to
References 45±508C.
1 Wilson W. J. and Blair E. M. McV (1927) J. Hyg. Camb. 26. 374.
Blood Agar Base was used during investigations on
2 Cook G. T. (1952) J. Path. Bact. 64. 559. irradiated Escherichia coli and other bacteria1,2. It was
3 McCoy J. M. and Spain G. E. (1969) in Isolation Methods for the most suitable medium for investigating the
Microbiologists, p. 20. Ed. by Shapton D. A. and Gould G. W. phages of Clostridium perfringens3 and as the basis of a
Academic Press London.
selective medium for Cl. perfringens4. It was used with
4 Hobbs B. C., King G. C. G. and Allison V. D. (1945) Monthly added phenolphthalein phosphate for the detection of
Bulletin of the Ministry of Health and Emergency Public Health Lab. phosphatase-producing staphylococci5 and with
Service 4. 40.
added salt and agar for the assessment of surface
5 Anon (1981) Int. Standard ISO 6579±1981. Geneva. Internat. contamination on equipment and pig carcasses6. It
Organization for Standardization. was used for determining the salinity range of growth
6 ICMSF (1978) Micro-organisms in Food 1. 2nd Edn. University of
of marine flavobacteria7.
Toronto Press, Ontario.
7 Speck M. L. (1984) Compendium of methods for the micro-biological Storage conditions and Shelf life
examination of foods. 2nd Edn. American Public Health Association. Store the dehydrated medium below 258C and use
before the expiry date on the label.
Store the prepared plates of medium at 2±88C.
2-48 November 1998
Culture Media
Examination of food for salmonellae (enumeration)4 7 Harvey R. W. S., Price T. H. and Hall L. M. (1973) J. Hyg. Camb.
This is carried out by adding equal volumes of 71. 481±486.
decimal dilutions of the homogenised sample to tubes
of double strength Selenite Broth. After incubation, a
loopful from each tube is plated on Bismuth Sulphite BRILLIANT GREEN AGAR
Agar and Brilliant Green Agar. Colonies with
salmonellae characteristics are identified and the most (MODIFIED)
probable number of salmonellae per gram of sample Code: CM329
is calculated from the three highest sample dilutions
which yield salmonellae on subculture. A selective and diagnostic agar for salmonellae (other
Examination of dairy products for salmonellae 3 than S. typhi) from food and feeds.
Milk and liquid milk products, dried milk, cheese,
eggs and egg products ± Brilliant Green Agar is Formula gm/litre
employed, with and without an enrichment phase, in `Lab-Lemco' powder 5.0
conjunction with other selective media for enteric Peptone 10.0
bacteria. Yeast extract 3.0
Disodium hydrogen phosphate 1.0
Colonial Characteristics Sodium dihydrogen phosphate 0.6
Non-lactose/sucrose-fermenting organisms Lactose 10.0
Red-pink-white opaque coloured colonies surrounded Sucrose 10.0
by brilliant red zones in the agar ± most probably Phenol red 0.09
salmonella (but not S. typhi). Brilliant green 0.0047
Proteus and Pseudomonas species Agar 12.0
These may grow as small red colonies. pH 6.9 + 0.2
Lactose/sucrose-fermenting organisms (normally Directions
inhibited) Suspend 52 grams in 1 litre of distilled water. Heat
Yellow to greenish-yellow coloured colonies gently with occasional agitation and bring just to the
surrounded by intense yellow-green zones in the agar boil to dissolve the medium completely. DO NOT
± E. coli or Klebsiella/Enterobacter group. AUTOCLAVE. Cool to 508C, mix well and pour
plates.
Storage conditions and Shelf life
Store the dehydrated medium below 258C and use
before the expiry date on the label.
SULPHAMANDELATE SUPPLEMENT
Store the prepared plates of medium at 2±88C.
Code: SR87
Quality Control
Positive control: Vial contents (each vial is sufficient for 500ml of
Salmonella typhimurium ATCC1 14028 medium)
Negative control: Sodium sulphacetamide 500mg
Escherichia coli ATCC1 25922 Sodium mandelate 125mg
Proteus vulgaris ATCC1 13315 Directions
Precautions To one vial add 5ml of sterile distilled water and mix
Lactose-fermenting salmonella (S. arizona) may be gently to dissolve the contents completely. Avoid
present in foods7. frothing. Add the solution to 500ml of sterile Oxoid
Brilliant Green Agar (Modified) CM329 cooled to
Salmonella typhi and Shigella species may not grow on 50±558C. Mix gently and pour into sterile petri dishes.
this medium, use the cited alternative media.
Description
Proteus, Citrobacter and Pseudomonas species may Brilliant Green Agar (Modified) was developed from
mimic enteric pathogens by producing small red a formula supplied by the Rijks Instituut voor de
colonies. Volksgezondheid (National Institute for Public
Health), Utrecht1,2.
References
1 Kristensen M., Lester V. and Jurgens A. (1925) Brit. J. Exp. Pathol. The medium has been widely assessed in Europe and
6. 291±297. is now used in the ISO standards3,4,5.
2 Kauffman F. (1935) Seit. F. Hyg. 177. 26±34.
The advantages claimed for the medium are the
3 American Public Health Association (1976) Compendium of greater inhibition of Escherichia coli and Proteus
Methods for the Microbiological Examination of Foods. APHA Inc. species than other formulations: the restriction of
Washington D.C.
growth of Pseudomonas species, whose colonies may
4 American Public Health Association (1978) Standard Methods for resemble salmonellae on Brilliant Green Agar and
the Examination of Dairy Products. 14th Edn. APHA Inc. cause confusion or much extra work to confirm their
Washington D.C.
identity: the absence of inhibitory properties towards
5 Association of Official Analytical Chemists (1978) Bacteriological small numbers of salmonellae6.
Analytical Manual 5th Edn. AOAC. Washington D.C.
6 Osborn W. W. and Stokes J. L. (1955) Appl. Microbiol. 3. 295±301.
Precautions Directions
Do not autoclave double-strength broth. Suspend 19.5g in 500ml of distilled water. Boil to
dissolve the medium completely. Sterilise by
Gram-positive sporing organisms may produce gas if
autoclaving at 1218C for 15 minutes.
the bile/brilliant green inhibition is attenuated by
food material. Or
Differential reactions and characteristics for species of the genera Campylobacter, Arcobacter and
Helicobacter pylori. (Adapted from Penner1)
Growth Susceptibility
Species Cata- Nitrate H2S Hipp- Urease 258C 378C 428C Nali- Cephol- G+C
lase (TSI) urate dixic othin content
acid (mol%)
+, Postive reaction; ±, negative reaction; ND, no test reactions; R, resistant; S, susceptible; I, intermediate
results found; (+), most strains positive but a low zones of inhibitions.
percentage negative: (±), most strains negative but a
Susceptibility to antibiotics was determined with
some positive or weakly positive; d, different
30mg disks.
*IU/L
CCDA ± Modified Charcoal Cefoperazone Desoxycholate Agar (Blood-free medium)
Gun-Monro et al.2 carried out a laboratory and Table 3. Suppression of faecal flora from 70
clinical evaluation of the various selective isolation simulated positive faecal samples.
media for thermophilic campylobacters. Summary
tables of their findings for the above antibiotic No. (%) of plates with 75% reduction
supplements are shown. of faecal flora compared with control
Medium number (%) of strains
Table 1. Recovery of 70 C. jejuni strains on five
isolated after
selective media.
incubation for:
Medium Colony Count P valueb 24h 48h
Blood agar control 7.95 + 0.36 Skirrow 46 (66) 38 (54)
Skirrow 7.82 + 0.48 Nsc Butzler 56 (80) 47 (67)
Butzler 7.77 + 0.51 <0.05 Blaser-Wang 28 (40) 15 (21)
Blaser-Wang 7.70 + 0.56 <0.05 Preston 58 (83) 50 (71)
Preston 7.76 + 0.52 <0.05 Modified 64 (91) 59 (84)
Modified CCDA 7.91 + 0.36 NS CCDA
The general conclusions reached by Gun-Munro et al.
Table 2. Isolation of C. jejuni from 70 simulated were confirmed by Griffiths and Ribeiro3.
positive faeces samples.
Enrichment broth cultures ± the value of enrichment
Medium 24h 48h media for campylobacters is controversial1 but in food
Blood agar control 69 (99) 70 (100) and environmental studies enrichment may be
Skirrow 39 (56) 67 (96) essential4. Enrichment at 428C4,5 and cold enrichment
Butzler 38 (54) 60 (86) at 48C6 have been reported. Where enrichment
increases competitive flora, the use of membrane
Blaser-Wang 17 (24) 31 (41)
filters on the surface of the agar can help select
Preston 32 (46) 64 (91) campylobacters7.
Modified CCDA 61 (87) 69 (99)
A comprehensive review of selective media for
a Campylobacter and Arcobacter species has been
Log10 mean colony counts + standard deviation. published in a special issue of International Journal of
b
significance determined by Student's t test for Food Microbiology.
unpaired samples.
c
NS, Not significant. Reference
Corry J.E.L., Post D.E., Colin P. and Laisney M.J. (1995). Int. J. Food
Microbiol. 26. 43±76.
Add the contents of one vial to 500ml of sterile blood CAMPYLOBACTER SELECTIVE
agar, cooled to 50±558C, prepared from Oxoid SUPPLEMENT (BLASER-WANG)
Columbia Agar CM331 or Blood Agar Base No.2
CM271, with 5±7% lysed defibrinated horse blood. Code: SR98 (Blaser-Wang)
Mix gently and pour into sterile petri dishes. Vial contents (each vial is sufficient for 500ml of
Description medium)
Campylobacter Selective Supplement SR69 is based Vancomycin 5mg
on the formulation of Skirrow1. Polymyxin B 1,250 IU
Trimethoprim 2.5mg
The antibiotic supplement is designed to be used at Amphotericin B 1mg
428C for optimum selective effect. Cephalothin 7.5mg
Reference Directions
1 Skirrow M. B. (1977) BMJ 2. 9±11. To rehydrate the contents of the vial, add aseptically
2ml of sterile distilled water and turn end-over-end to
dissolve. Avoid frothing the solution.
CAMPYLOBACTER SELECTIVE
SUPPLEMENT (BUTZLER) Add the contents of one vial to 500ml of a sterile
nutrient medium cooled to 50±558C prepared from
Code: SR85 (Butzler) Columbia Agar Base CM331 or Blood Agar Base No.2
Vial contents (each vial is sufficient for 500ml of CM271, with 10% sheep blood or 5±7% laked horse
medium) blood SR48. Mix gently and pour into sterile petri
Bacitracin 12,500 IU dishes.
Cycloheximide 25mg Description
Colistin sulphate 5,000 IU Campylobacter Selective Supplement (Blaser-Wang)
Cephazolin sodium 7.5mg SR98 is based on the formulation of Skirrow1, but
Novobiocin 2.5mg with the addition of amphotericin B and cephalothin2.
Directions The inclusion of amphotericin B inhibits the growth of
To rehydrate the vial aseptically add 3ml of 50/50 Candida albicans and cephalothin improves the
ethanol/water and turn end-over-end to dissolve. selectivity of the supplement.
Avoid frothing of the solution.
References
Add the contents of one vial to 500ml of sterile blood 1 Skirrow M.B. (1977) BMJ 2. 9±11.
agar cooled to 50±558C prepared from Oxoid 2 Blaser M.J., Hardesty H.L., Powers B. and Wang W.L.L. (1980) J.
Columbia Agar CM331, with 5±7% defibrinated horse Clin. Micro. 11. 309±313.
or sheep blood. Mix gently and pour into sterile petri
dishes.
CAMPYLOBACTER GROWTH SUPPLEMENT
Description
Campylobacter Selective Supplement SR85 is based Code: SR84
on the formulation of Lauwers, De Boeck and FBP Supplement for the enhanced growth and
Butzler1. aerotolerance of campylobacter.
Cephalothin (15mg/ml) has been replaced in the Vial contents (each vial is sufficient for 500ml of
Oxoid Supplement by cephazolin (15mg/ml) as this medium)
has been found to be more inhibitory to Pseudomonas Sodium pyruvate 0.125g
species1. Sodium metabisulphite 0.125g
This supplement differs from Skirrow Supplement Ferrous sulphate (hydrated salt) 0.125g
SR69 by its selective action at 378C. It therefore Directions
overcomes the need to use incubators at 428C and it To rehydrate the contents of the vial, aseptically add
selectively isolates those strains of Campylobacter 2ml of sterile distilled water and invert to dissolve.
species that fail to grow at 428C2 (e.g. C. fetus sub.sp. Avoid frothing of solution.
fetus).
Add the contents of one vial to 500ml of a sterile
References nutrient medium cooled to 50±558C prepared from
1 Lauwers S., De Boeck M. and Butzler J.P. (1978) Lancet, 1. 604± Oxoid Columbia Agar CM331, Blood Agar Base No.2
605. CM271, or Campylobacter Agar Base CM689, with
2 Butzler J.P., Dekeyser P., Detrain M. and Dehaen F. (1973) J. 5±7% lysed defibrinated horse or sheep blood, and the
Pediat. 82. 493. rehydrated contents of one vial of Campylobacter
Antibiotic Supplement SR69, SR85 or SR98. Mix
gently and pour aseptically into sterile petri dishes.
Description
Campylobacter species, which require less oxygen (i.e.
microaerophilic) are inhibited at the normal
atmospheric oxygen level. The optimum level of
oxygen required for growth has been reported to be
6%1. This exacting level, together with a carbon
2-64 November 1998
Culture Media
productive for C. upsaliensis in this application than 4 MAFF Validated Methods for the Analysis of Foodstuffs:
CAT medium. Modified CCDA medium has been Method for the detection of thermotolerant Campylobacter in
confirmed as suitable for isolation of Campylobacter Foods (v30) J. Assoc. Publ. Analysts (1993) 29. 253±262.
spp. from non-clinical samples following enrichment 5. Rice, B.E., Chinta Lamichhane, Joseph, S.W. and Rollins, D.M.
in Exeter broth7. (1996) Clin. Diag. Lab. Immunol. 3, 669±677.
6. Hald, B. and Madsen, M. (1997) J. Clin. Microbiol. 35, 3351±3352.
The use of Campylobacter Blood-Free Medium is
7. Humphrey, T.J., Martin, K.W. and Mason, M.J. (1997) PHLS
specified by the U.K. Ministry of Agriculture,
Microbiology Digest 13 (2), 86±88.
Fisheries and Food (MAFF) in a validated method for
isolation of Campylobacter from foods4.
CEFOPERAZONE, AMPHOTERICIN
Technique B, TEICOPLANIN SUPPLEMENT
1 Prepare Campylobacter Blood-Free Selective Agar (CAT)
as described in the directions.
2 Emulsify approximately 0.5g of the specimen in Code: SR174
5ml of sterile 0.1% peptone water to form an A selective supplement for the isolation of thermophilic
approximate 1:10 dilution. Campylobacter spp. and improved recovery of
3 Inoculate onto the selective medium with cotton Campylobacter upsaliensis from faeces.
tipped swabs so that single isolated colonies are Vial contents (each vial is sufficient for 500ml of
formed. medium)
4 Incubate the plates in an atmosphere consisting of
CAT Supplement gm/litre
approximately 5±6% oxygen, 10% carbon dioxide
Cefoperazone 8.0
and 84±85% nitrogen for 48 hours at 378C. This can
Teicoplanin 4.0
best be achieved by using the Oxoid Gas
Amphotericin B 10.0
Generating Kit for Campylobacters (BR56) in
conjunction with the Oxoid Anaerobic Jar and an Directions
active catalyst (BR42). For jars of smaller capacity Aseptically add 4ml of sterile distilled water to the
(2.5 litres) use the Oxoid Gas Generating Kit for vial. Mix gently to resuspend the supplement.
Campylobacters (BR60). Alternatively use
Prepare 500ml of sterile Blood-Free Campylobacter
CampyGen CN023A or CN035A which does not
Agar Base CM739 as directed. Cool to 508C and
require the use of a catalyst or the addition of
aseptically add one vial of SR174E reconstituted as
water.
directed above.
The colonial morphology of campylobacters can be
used as a guideline for identification to species level. Mix well and pour the resulting CAT medium into
C. jejuni strains produce grey, moist flat spreading sterile petri dishes. Incubate cultures at 378C for
colonies. Some strains may have a green hue or a dry 48±72 hours in a microaerobic atmosphere.
appearance, with or without a metallic sheen. C. coli
Description
strains tend to be creamy-grey in colour, moist,
Because of the sensitivity of C. upsaliensis to a wide
slightly raised and often produce discrete colonies.
range of antibiotics, isolation of the organism from
Colonies tend to swarm when initially isolated from faeces using selective media has hitherto been
clinical specimens. difficult. The recommended isolation method uses a
Storage conditions and Shelf life membrane filter culture technique on non-selective
Store the dehydrated medium below 258C and use agar. This does not give good recovery from faeces
before the expiry date on the label. containing less than 105 CFU/g5 however, and is a
technically demanding method which is relatively
Store the selective supplement in the dark at 2±88C slow to perform.
and use before the expiry date on the label.
CAT Supplement SR174 is based on the formulation
The prepared medium may be stored for up to 2 described by Aspinall et al7. When added to Blood-
weeks at 2±88C. Free Campylobacter Agar Base CM739 which
Quality Control contains charcoal, it gives good isolation of
Incubation at 378C for 48 hours. thermophilic Campylobacter spp., and makes the
isolation of C. upsaliensis possible on a selective
Positive control: medium because CAT Supplement contains reduced
Campylobacter jejuni ATCC1 29428 levels of cefoperazone compared to other
Negative control: Campylobacter supplements. This inhibits most
Escherichia coli ATCC1 25922 Enterobacteriaceae, but not enterococci. Teicoplanin is
included to inhibit enterococci. Amphotericin B is
References added as an antifungal agent.
1 Bolton, F.J., Hutchinson, D.N. and Coates, D. (1984) J. Clin.
Microbiol. 19, 169±171. Further work confirmed the effectiveness of CAT
2 Hutchinson, D.N. and Bolton, F.J. (1984) J. Clin. Path. 34, 956± medium as an alternative to membrane filtration
957. culture for selective isolation of thermophilic
3 Bolton, F.J., Hutchinson, D.N. and Parker, G. (1988) Eur. J. Clin. campylobacters including C. upsaliensis8.
Microbiol. Infect. Dis. 7. 155±160. Atabay, Corry and On9 isolated a previously
Negative control: To one vial add 2ml of sterile distilled water and
Uninoculated medium. dissolve the contents completely. Add this solution to
500ml of sterile, molten Charcoal Agar CM119, cooled
Precautions to 508C, together with 10% v/v defibrinated horse
The medium should not be incubated to check blood SR50. Mix well before pouring into sterile petri
sterility, prior to use. This should be carried out on dishes.
separate quality control samples.
For Haemophilus influenzae, omit the selective agents
The medium can maintain the viability of fastidious and convert to `chocolate' agar.
organisms for transport purposes but it should not be
used as a storage or enrichment medium. Transport Medium for B. pertussis
The vial contents may be added to 500ml of half-
The results obtained from the medium are dependent strength Charcoal Agar + 10% v/v defibrinated horse
on the quality of the specimen material. Commensal blood SR50 for use as a transport medium for B.
anaerobic organisms may overgrow in the medium pertussis.
and cause misleading results.
Description
References Charcoal Agar CM119 was developed by Oxoid to
1 Cary S. G. and Blair E. B. (1964) J. Bact. 88. 96±98. provide a non-blood containing medium for the
2 Crookes E. M. and Stuart R. D. (1959) J. Pathol. Bacteriol. 78. 283± cultivation of Bordetella pertussis and Haemophilus
288. influenzae. Proom1 showed that nicotinic acid was an
3 Stuart R. D. (1959) Public Health Reports 74. 431±438. essential growth factor for the bordetellae. Ensminger
4 Cary S. G., Fusillo M. H. and Harkins C. (1965) Am. J. Clin. Path. et al.2 used a charcoal medium for the growth of B.
43. 294±295. pertussis in vaccine production and found that the
5 DeWitt W. E., Gangarosa E.J., Huq I. and Zarifi A. (1971) Amer. medium could replace Bordet-Genou. Mishulow et
J. Trop. Med. Hyg. 20. 685±688. al.3 used charcoal agar for B. pertussis cultivation.
6 Neumann D. A., Benenson M. W., Hubster E. and Tuan N. T. N.
H. influenzae is cultivated on the medium containing
(1971) Am. J. Clin. Path. 57.
10% `chocolated' blood but no antibiotics. The
7 Patton C. M., Mitchell S. W., Potter M. E. and Kauffmann A. F.
inoculated plate is incubated for 2 to 3 days at 378C.
(1981) J. Clin. Microbiol. 13. 326±328.
The colonies are usually small, transparent and
8 Luechtefeid M.W., Wang W. L. L., Blaser M. J. and Reller L. B.
droplet-like, but some transformation to the `rough'
(1981) J. Clin. Microbiol. 13. 438±439.
type colony may occur. Species differentiation is
9 Wren M. W. D., Baldwin A. W. F., Eldon C. P. and Sanderson P.
performed by examination of the need for X and V
J. (1977) J. Med. Microbiol. 10. 49±61.
growth factors, on Blood Agar Base CM55.
10 Wren M. W. D. J. Med. Microbiol. 10. 195±201.
11 Holdeman L. V. and Moore W. E. C. (1975) Anaerobe Laboratory The greatest problem in the isolation of Bordetella
Manual, Virginia Polytechnic Institute Anaerobe Laboratory, 3rd Ed. species from naso-pharyngeal secretions is the
12 Wells J. G. and Morris G. K. (1981) J. Clin. Microbiol. 13. 789±791. suppression of unwanted flora during the long
13 Morris G. K and Heck J. (1978) J. Clin. Microbiol. 13. 438±440. incubation period on very nutritious media.
Fleming's first in vitro demonstration of penicillin was
to show that it could help isolate B. pertussis on
media4. Lacey5 confirmed this but found that the
CHARCOAL AGAR penicillin-resistant flora still caused problems. He
Code: CM119 supplemented penicillin with 2mg/ml 4,4' diamidino-
diphenylamine dihydrochloride (M & B 938) thereby
A medium for the cultivation and isolation of Bordetella increasing the selectivity of this medium.
pertussis and Haemophilus influenzae.
Broome et al.6 found methicillin to be superior to
Formula gm/litre penicillin in suppressing unwanted naso-pharyngeal
`Lab-Lemco' powder 10.0 flora but the earlier publication of Sutcliffe and
Peptone 10.0 Abbott7 where cephalexin (40mg/ml) was shown to be
Starch 10.0 superior to penicillin, has proved to be the most
Charcoal bacteriological 4.0 significant advance.
Sodium chloride 5.0
Nicotinic acid 0.001 The benefits of cephalexin as a selective agent for B.
Agar 12.0 pertussis have been confirmed8,9,10,11. The ability to
pH 7.4 + 0.2 recover stressed cells and the much longer shelf life
(6±8 weeks) are added benefits to its superiority at
Directions suppressing unwanted naso-pharyngeal growth.
Suspend 51g in 1 litre of distilled water. Bring to the
boil to dissolve completely. Sterilise by autoclaving at Regan and Lowe8 showed that half-strength Oxoid
1218C for 15 minutes. Cool to 508C, add 10% of Charcoal Agar, supplemented with 40mg/ml
defibrinated blood and mix gently. The medium is cephalexin SR82 v/v lysed, defibrinated horse blood
made selective for the isolation of Bordetella pertussis was an excellent enrichment and transport medium.
and B. parapertussis by the addition of Bordetella The efficacy of this transport medium has been
Selective Supplement SR82. confirmed by other workers12.
A random sample of sufficient quantity to be cultures grown in Clausen Medium at 30±328C. The
representative of the whole bulk, should be examined. Rh. rubra inoculum is prepared from a 48 hour culture
grown in the same broth at 20±258C.
Two methods of detecting non-sterile units may be
used in the microbial-contamination test. Dithionite-Thioglycollate Broth was formulated by
Clausen as a highly nutritious medium containing
Membrane Filter Method
reducing agents and essential metals for the recovery
The test substance is dissolved or suspended in 200ml
of anaerobic spore-bearing organisms. It also contains
of 0.1% w/v sterile (pH 7.0±7.2) CM9 and
lecithin and Tween 80 to overcome the effects of
immediately filtered through one or more membrane
cationic agents which may show powerful
filters (average pore diameter 0.45m or less).
bacteriostatic effects in vitro.
Each filter is then washed three times, by passing
The broth should be prepared as directed and
100ml volumes of peptone solution through the
transferred to tubes or bottles in sufficient volumes (at
membrane.
least 15ml) for the standard microbial-contamination
After filtration the membranes are transferred to test and rapidly cooled to 208C after sterilisation.
tubes of media, containing at least 15ml of Clausen
Storage conditions and Shelf life
Medium and tubes of Tryptone Soya Broth (soybean-
Store the dehydrated medium below 258C and use
casein digest medium) CM129. If only one filter is
before the expiry date on the label.
used, this is divided into two and the two halves
placed in separate tubes. Prepared medium: The medium should be stored in a
cool place (not at 48C) away from the light, for a
Tubes of Clausen Medium are incubated for at least
maximum period of one month.
14 days at 30±328C.
Quality Control
Tubes of Tryptone Soya Broth (soybean-casein digest
Positive control:
medium) are incubated for at least 14 days at
see Control section
20±258C.
Dilution Method Negative control:
From each sample 1.0ml of material or suspension is Uninoculated medium.
transferred to each of at least 10 tubes containing a Precautions
minimum quantity of 15ml of Clausen Medium. The medium is yellowish in colour and almost clear.
One half of the number of tubes is incubated at It turns pale-pink under aerobic conditions. The
30±328C for at least 14 days and the other half upper third only of the medium should be pink by
at 20±258C for the same time. the time it is to be used.
If the medium becomes turbid on incubation, Bibliography
subcultures must be taken as soon as possible. Clausen O. G., Aasgaard N. B. and Solberg O. (1973) Ann.
Subcultures must also be taken after normal Microbiol. (Inst. Pasteur) 124 B. 205.
incubation and observed for a further period of 14 Christensen E. A., Kristensen H. and Jensen K. M. (1969) Arch.
days. Pharm. Chem. 76. 625.
Clausen O. G. (1973) `A study of the growth-promoting properties of
Assessment Of The Results fluid and solid microbial-contamination test media on small numbers
The standard microbial contamination test is passed if of micro-organisms.' `Pharmaceutica Acta Helvetiae''48. 541±548.
growth is not observed in any of the tubes. If growth Clausen O. G. (1973) `An examination of the Bactericidal and
is observed, the test may be repeated with twice the Fungicidal Effects of Cetylpyridinium Chloride, separately and in
number of samples. The test is then passed if no combinations embodying EDTA and Benzyl Alcohol'. Die. Pharm.
growth is observed in any of these tubes. Growth is Ind. 35. Nr. 12 869±874.
diagnosed by the appearance of turbidity in fluid or Mohamed A. and Abdou F. (1974) `Comparative Study of Seven
semi-fluid media, by the formation of colonies on Media for Sterility Testing'. Jnl. of Pharma. Sci. Vol. 63. No.1 Jan.
solid media, or by microscopy of culture samples. Mohamed A. and Abdou F. (1974) `Sterilitatstest III
Controls Vergleichsuntersuchungen von 3 Medien zum Nachweis von
Both methods of testing must be controlled for Bakterien'. Pharm. Ind. 36. Nr. 5. 337±334.
microbial inhibitors by adding a small inoculum of
organisms (approximately 10 colony-forming
bacteria) either to the tubes prepared in Method II or
to peptone diluent, prior to filtration, in Method I.
If no growth occurs in the tubes containing the test
organisms then the test must be repeated with the
growth-inhibitory effect inactivated.
The test organisms recommended are:
Staphylococcus epidermidis
Clostridium sporogenes
Rhodotorula rubra
They are maintained on agar slants or deep agar stabs
and the test inoculum is prepared from 24 hour
found this medium was inhibitory compared with Technique for Alcohol Shock Treatment
growth on blood agar. They recommended the use of 1 Mix equal parts of industrial methylated spirit or
a fructose containing nutrient medium plus egg yolk, absolute alcohol and the faecal specimen.
with D-cycloserine and cefoxitin as selective agents 2 Homogenise using a vortex mixer.
for the isolation of Cl. difficile.
3 Leave at room temperature for 1 hour.
The combination of Oxoid Clostridium difficile Agar 4 Inoculate on to Clostridium Difficile Selective Agar
Base CM601 plus the Selective Supplement SR96 is and incubate anaerobically.
based on the formulation proposed by George et al.10
Storage conditions and Shelf life
The selective agents D-cycloserine (500mg/ml) and Store the dehydrated medium below 258C and use
cefoxitin (16mg/ml) inhibit growth of the majority of before the expiry date on the label.
Enterobacteriaceae, as well as Strep. faecalis,
staphylococci, Gram-negative non-sporing anaerobic Store the prepared medium at 2±88C no longer than
bacilli and Clostridia species. (except Cl. difficile) which 5±7 days.
may be found in large numbers in faecal samples. Quality Control
Levett11, noting reports12,13 that some strains of Positive control:
Clostridium difficile had low minimum inhibitory Clostridium difficile NCTC 11204
concentrations to both cycloserine and cefoxitin, Negative control:
reduced the antibiotic concentrations to 125mg per ml Staphylococcus aureus ATCC1 25923
cycloserine and 4mg per ml cefoxitin and combined
Escherichia coli ATCC1 25922
this with alcohol shock14 to compensate for the
reduction in selectivity. Cl. difficile was isolated from Precautions
all of the 33 faecal specimens plated on to CCFA Colonies of Cl. difficile from faecal cultures are smaller
Medium containing cycloserine and cefoxitin at 250mg when egg yolk is used in place of horse blood.
per ml and 8mg per ml respectively, but from only 25/
The Oxoid formula does not contain the neutral red
33 specimens plated on to medium containing 500mg
indicator proposed by George et al.10 because it is
per ml cycloserine and 16mg per ml cefoxitin. designed for use with horse blood. On this medium
The specimen should be treated with alcohol before the typical colour of the colony of Cl. difficile will not
inoculation (see technique). appear however there will be a fluorescent reaction.
It can be expected that medium containing the lower Typical Gram stain morphology of Cl. difficile may not
concentration of antibiotics will yield a greater be evident in colonies picked from this medium
growth of contaminating organisms if antibiotics are because of the antibiotics present. Subculture to blood
used alone, but Levett reported that there was no agar to obtain characteristic morphology10.
difference in the growth of contaminating organisms
on plates containing either concentration of antibiotics
following alcohol shock treatment of the specimen. CLOSTRIDIUM DIFFICILE
Phillips and Rogers15 have described a simple MOXALACTAM NORFLOXACIN
modification to the medium in which the ability of Cl. (CDMN) SELECTIVE SUPPLEMENT
difficile to produce p-cresol from p-hydroxyphenyl
Code: CR173
acetic acid is used for the rapid presumptive
identification by gas chromatographic detection of the Vial contents (each vial is sufficient for 500ml of
p-cresol. medium).
Addition of 7% horse blood to the agar base increases Mg Mg/litre
the recovery of Cl. difficile and produces larger Cysteine hydrochloride 250.0 500.0
colonies compared with Egg Yolk Emulsion used by
Norfloxacin 6.0 12.0
George et al.10
Moxalactam 16.0 32.0
Technique
1 Lightly inoculate the medium with the faecal Directions
sample spreading part of the original inoculum in Aseptically add 2ml of sterile distilled water to a vial
order to obtain well separated colonies. and mix gently to dissolve the supplement
completely. Avoid frothing. Add to 500ml of
2 Incubate plates at 358C for 18±24 hours in a Clostridium difficile Agar Base, CM601, prepared as
conventional anaerobic gas jar. The use of the directed and cooled to 508C. Add 7% v/v of
Oxoid Anaerobic Jar HP11 with an H2/CO2 Gas Defibrinated Horse Blood SR50. Mix well and pour
Generating Kit is strongly recommended. into petri dishes.
Alternatively use Anaerogen AN025A or AN035A.
Anaerogen does not require the addition of water Description
or a catalyst. Cl. difficile CDMN medium is an alternative selective
3 Colonies of Cl. difficile after 48 hours incubation are medium based on a formula described by Aspinall et
4±6mm diameter irregular, raised opaque, grey- al16 for the isolation of Cl. difficile from faeces. It has
white. been found to be significantly more productive than
CCFA medium. Inclusion of cysteine hydrochloride
speeds the growth rate of Cl. difficile. CDMN medium
agar. Streak an overnight (or older) culture of Staph./Strep. supplemented plates should be
Streptococcus agalactiae known to produce the CAMP incubated aerobically at 358C for 18 hours. Incubation
factor at right angles to the first inoculation taking in carbon dioxide-enriched air will cause inhibition of
care that the lines do not touch. Incubate staphylococcal growth15.
anaerobically at 35±378C for 18±24 hours. Strep. supplemented plates may be incubated
A positive reverse CAMP test is indicated by the aerobically or anaerobically at 358C for 18 hours.
formation of an ``arrowhead'' of haemolysis between
Prepared plates of both supplemented media should
the lines of the C. perfringens and Strep. agalactiae
be used within 18 hours of preparation for maximum
growth. selectivity. Gardnerella supplemented plates should
Storage conditions and Shelf life be incubated at 358C for 48 hours in an atmosphere
Store the dehydrated medium below 258C and use containing 7% carbon dioxide.
before the expiry date on the label.
Carry out confirmatory tests on all colonies from
Store the prepared plates of medium at 2±88C. horse blood medium and on beta-haemolytic colonies
from human or rabbit blood medium.
Quality Control
Columbia Agar Incubate plates of Clostridium E-Y Agar anaerobically
Positive control: at 358C for 18 hours, look for lecithinase activity
Staph. aureus ATCC1 25923 (pearly layer) and for proteolysis. Lecithinase activity
Strept. pyogenes ATCC1 19615 is inhibited in the presence of specific anti-toxin.
Negative control: References
Uninoculated plate 1 Ellner P.D., Stoessel C.J., Drakeford E. and Vasi F. (1966) Tech.
Brucella Medium Bull. Reg. Med. Techn. 36. No. 3, reprinted in Amer. J. Clin. Path.
Positive control: (1966) 45. 502±504.
*Brucella abortus ATCC1 4315 2 Farrel I.D. and Robinson L. (1972) J. Appl. Bact. 35. 625±630.
3 Hunter D. and Kearns M. (1977) Brit. Vet. J. 133. 486±489.
Negative control: 4 Skirrow M. B. (1977) B.M.J. (ii) 9±11.
Esch. coli ATCC1 25922 5 DeKeyser P., Goussuin-Detrain M., Butzler J.P. and Sternon J.
Campylobacter Media (1972) J. Infect. Dis. 125. 390±392.
Positive control: 6 Butzler J.P., De Keyser P., Detrain M. and Dehaen F. (1973) J.
C. jejuni ATCC1 29428 Pediat. 32. 493.
7 Blaser M.J., Hardesty H.L, Powers B. and Wang W. L. L. (1980)
Negative control: J. Clin. Microbiol. 11. 309±313.
Esch. coli ATCC1 25922 8 Blaser M.J., Berkowitz I.D., La Force F.M., Dravens J., Reller L.B.
and Wang W.L.L. (1979) Ann. Int. Med. 91. 179±185.
Staph Medium Strep
Positive control: 9 Blaser M.J., Cravens J., Powers B.U., La Force F.M. and Wang
Staph. aureus ATCC1 25923 W.L.L. (1979) Amer. J. Med. 67. 715±718.
Strept. pyogenes ATCC1 19615 10 George H.A., Hoffman P.S., Krieg M.R. and Smibert R.M. (1979)
Canad. J. Microbiol. 25. 8±16.
Negative control: 11 Hoffman P.S., George H.A., Krieg H.R. and Smibert R.M. (1979)
Esch. coli ATCC1 25922 Canad. J. Microbiol. 25. 8±16.
12 Westblom T.U., Madan E. and Midkiff B.R. (1991) J. Clin.
Streptococcus Selective Medium
Microbiol. 29. 819±821.
Positive control:
13 Lowbury E.J.L. and Lilly H.A. (1955) J. Path. Bact. 70. 105±108.
Strept. pyogenes ATCC1 19615
14 Hansen M.V. and Elliott L.P. (1980) J. Clin. Microbiol. 12. 617±
Negative control: 619.
Staph. aureus ATCC1 25923 15 Morton C.E.G. and Holt H.A. (1989) Med. Lab. Sci. 46. 72±73.
Directions Incubation
Suspend 10g in 100ml of distilled water (or 1g Aerobic organisms: incubate up to 7 days at 358C
amounts in 10ml volumes of water in tubes). Allow to with loosened caps, examine daily for turbidity, gas
stand for 15 minutes until the meat particles are or changes in the meat particles.
thoroughly wetted. Sterilise by autoclaving at 1218C Anaerobic organisms: use freshly reduced medium
for 15 minutes. Do not cool the bottles rapidly and incubate up to 21 days at 358C, examine daily for
because ebullition will expel the meat particles from changes in the medium; make films and subculture at
the containers. intervals.
Description Maintenance of stock cultures: hold at room
Cooked Meat Medium prepared from heart tissue is a temperature after the initial incubation at 358C.
well established medium for the cultivation of Subculture every 4±6 months.
anaerobic and aerobic organisms1.
Storage conditions and Shelf life
It has the ability to initiate growth of bacteria from Store the dehydrated medium below 258C and use
very small inocula and to maintain the viability of before the expiry date on the label.
cultures over long periods of time. Mixed cultures of
bacteria survive in Cooked Meat Medium without Store the prepared medium at room temperature, in
displacing the slower growing organisms. The the dark with tightened caps, up to 6 months.
products of growth do not rapidly destroy the Quality Control
inoculated organisms and therefore it is an excellent Positive proteolysis:
medium for the storage of aerobic and anaerobic Clostridium histolyticum ATCC1 19401
bacteria.
Positive saccharolysis:
The addition of glucose in the formulation allows
Clostridium perfringens ATCC1 13124
rapid, heavy growth of anaerobic bacteria in a short
time and leads to a more rapid identification of Negative control:
important anaerobes. The improved growth also Uninoculated medium
enhances GLC identification of anaerobic bacteria.
Precautions
The improved clarity of the supernatant broth permits The excellent recovery properties of Cooked Meat
earlier detection of growth especially when combined Medium mean that mixed cultures commonly result
with the increased growth of most organisms. Slower from sample inoculation.
growing isolates will yield detectable growth in 45
hours incubation. Blackening of the medium will not take place if the
pH is acid.
Technique
Anaerobic Culture. It is preferable to use freshly Carbohydrate fermentation may inhibit proteolysis.
reconstituted and sterile medium which is inoculated
Reference
as soon as it has cooled to approximately 358C. Tubes
1 Robertson M. (1916) J. Path. Bact. 20. 327±349.
which are not used on the day of preparation should
be placed in a boiling water bath or steamer for about
15 minutes to remove dissolved oxygen. They should
be allowed to cool without agitation and then
inoculated. CORN MEAL AGAR
Inoculation should be made near the bottom of the Code: CM103
tube in the meat particles. A recommended medium for chlamydospore production
Clostridia may be divided into two main groups by by Candida albicans and for the maintenance of fungal
their action on the medium. stock cultures.
(i) Saccharolytic Organisms Formula gm/litre
There is rapid production of acid and gas but no Corn Meal Extract 2.0
digestion of the meat. Cultures may have a slightly (from 50 grams whole maize)
sour smell, with reddened protein. Agar 15.0
pH 6.0 + 0.2
(ii) Proteolytic Organisms
Proteolysis causes decomposition of the meat with Directions
the formation of foul-smelling sulphur compounds Suspend 17g in 1 litre of distilled water. Bring to the
and blackening. However, some saccharolytic boil to dissolve completely. Sterilise by autoclaving at
strains also produce H2S which will cause 1218C for 15 minutes.
blackening but to a lesser degree. Description
Aerobic Culture Corn Meal Agar is a well established mycological
The tube of medium is incubated with the cap loose medium which is a suitable substrate for
and no seal is required. Aerobes grow at the top chlamydospore production by Candida albicans and
whilst more anaerobic species grow deeper in the the maintenance of fungal stock cultures.
medium. When grown on this medium, microscopic
examination of Candida albicans shows the
characteristic chlamydospore production which is an
2-82 November 1998
Culture Media
accepted criterion for the identification of this species. Corn Meal Agar with `Tween 80' (or other wetting
Prospero and Reyes1 investigated the use of corn meal agents) will allow C. stellatoides and C. tropicalis to
agar, soil extract agar, and purified polysaccharide produce chlamydospores.
medium for the morphological identification of C. Some Candida strains lose their ability to produce
albicans. Out of 290 yeast colonies isolated on chlamydospores after repeated subculturing.
Sabouraud agar, corn meal agar stimulated the
production of chlamydospores in 149 colonies (51%), References
soil extract agar in 103 (36%) and purified 1 Prospero Magdalene T. and Reyes A. C. (1955) Acta Mel.
polysaccharide medium in 94 (32%). Phillipina 12(2). 69±74.
The addition of `Tween 80' (e.g. 1%) to Corn Meal 2 Rosenthal S. A. and Furnari D. (1958) J. Invest. Derm. 31. 251±
Agar greatly enhances the development of 253.
chlamydospores on the medium2,3,4,5,6. 3 Kelly J. P. and Funigiello (1959) J. Lab. Clin. Med. 53. 807±809.
7 4 Walker L. and Huppert M. (1959) Am. J. Clin. Path. 31. 551±558.
Mackenzie found that all 163 isolates of Candida
5 Walker L., Huppert M. and Woods A. (1960) Am. J. Clin. Path.
albicans obtained from laboratories in the United
33. 190±194.
Kingdom produced chlamydospores on Oxoid Corn
6 Gordon M.A. and Little G. N. (1962±63) sabouraudia 2. 171±175.
Meal Agar but Dawson8 using only 27 isolates of 7 Mackenzie D. W. R. (1962) J. Clin. Path. 15(6). 563±565.
Candida albicans, found that Oxoid Czapek Dox Agar
8 Dawson Christine O. (1962) sabouraudia 1(4). 214±219.
and rice infusion agar were slightly superior for
9 Conant N. F., Smith D. T., Baker R. D., Callaway J. L. and
chlamydospore production. Martin D. S. (1971) Manual of Clinical Mycology. 3rd Edn. W. B.
Corn meal agar is a nutritionally impoverished Saunders, Philadelphia, USA.
medium and so may be employed for the 10 Washington J. A. (1981) Laboratory Procedures in Clinical
maintenance of stock cultures of fungi, especially the Microbiology. Springer-Verlag. New York, USA.
black-pigmented varieties.
The addition of glucose (0.2g% w/v) to Corn Meal
Agar will enhance the chromogenesis of some species
of Trichophyton e.g. T. rubrum9. CROSSLEY MILK MEDIUM
Technique Code: CM213
A single petri dish containing Corn Meal Agar may This medium is suitable for use where Litmus Milk was
be used to identify four or five different colonies of previously specified.
Candida grown on Sabouraud Dextrose Agar CM41.
Using a straight wire, pick a colony off the surface of Formula gm/litre
the latter medium and make a deep cut in the Corn Skim milk powder 100.0
Meal Agar (i.e. a horizontal furrow). Repeat for each Peptone 10.0
colony. Place a flamed sterile coverslip over the line of Bromocresol purple 0.1
inoculum. After incubation for 24 to 48 hours at 228C, pH 6.8 + 0.2
the streaks are examined microscopically, through the Directions
cover slip, using a low power objective. Along such Cream 110g of the powder with a little distilled water
streaks, C. albicans produces mycelium-bearing ball- and gradually dilute to 1 litre with continuous
like clusters of budding cells and the characteristic mixing. Tube in 10ml quantities and autoclave at
thick-walled round chlamydospores9. 1218C for 5 minutes.
The addition of 0.001g% w/v Trypan blue to Corn
Meal Agar provides a contrasting background for the Description
observation of characteristic morphological features of A simple medium originally described by Crossley1
yeast cultures10. for the routine examination of canned food samples
for anaerobic bacteria.
Storage conditions and Shelf life
Store the dehydrated medium below 258C and use This medium was evolved as the result of
before the expiry date on the label. comparative trials carried out by Crossley with
several standard media. It is capable of giving rapid
Store the prepared plates of medium at 2±88C. growth without the use of special anaerobic
Quality Control apparatus, yet the bacteria detected may be
Chlamydospore Production provisionally identified by their reactions upon the
Positive control: medium.
Candida albicans ATCC1 10231 Crossley milk medium is recommended, in the
Negative control: second edition of Tanner's `The Microbiology of
Candida krusei ATCC1 6258 Foods'2, for the examination of meat, meat products,
and canned foods for sporing anaerobes.
Precautions
Glucose supplemented Corn Meal Agar should not be
used for chlamydospore production.
The Special peptone, used in DCLS Agar, includes the DERMASEL SELECTIVE SUPPLEMENT
nucleic acid factors, vitamins and carbon compounds
of meat extract, as well as a rich variety of Code: SR75
polypeptides. It has improved the growth of shigellae Vial contents ( each vial is sufficient for 500ml of
and salmonellae, but it should be noted that Sh. sonnei medium)
may exhibit a translucent, pink colony which should Cycloheximide 200mg
not be confused with the red Esch. coli colony. Chloramphenicol 25mg
The selectivity of DCLS Agar is similar to Directions
Desoxycholate Citrate Agar and it will grow Vibrio Suspend 44.5g in 1 litre of distilled water and heat
species, as well as salmonellae and shigellae, whilst gently to dissolve completely. Add the contents of 1
inhibiting the growth of Esch. coli. vial of Antibiotic Supplement SR75, reconstituted
DCLS Agar may be inoculated directly from the with 3mls of ethanol, to each 500ml of medium to
specimen, or inoculated after enrichment through give a level of cycloheximide 0.4g/l and
Selenite Broth CM395 and L121, Muller-Kauffmann chloramphenicol 0.05g/l. Mix gently and sterilise by
Tetrathionate Broth CM343 or Tetrathionate Broth autoclaving at 1218C for 10 minutes. Avoid
CM29. The plates should be incubated overnight overheating at any time.
(18±24 hours) at 358C and examined for the presence Description
of pale, translucent or colourless colonies. Sub- Oxoid Dermasel Agar CM539 is used for the primary
cultures can be made into confirmatory media such as isolation and identification of dermatophyte fungi
Kligler Iron Agar CM33 or Triple Sugar Iron Agar from hair, nails or skin scrapings.
CM277 or picked for transfer to nutrient broth for
Emmons1 suggested that media for growth of
subsequent motility tests and serological
dermatophytes should have a pH of 6.8±7.0 rather
agglutinations.
than pH 5.6 as is often recommended. A near neutral
Storage conditions and Shelf life pH is better for the growth of some fungi and the acid
Store the dehydrated medium below 258C and use pH used to suppress bacterial contaminants can be
before the expiry date on the label. replaced by antibiotics.
Store the prepared agar plates at 2±88C. The addition to the medium of Oxoid Antibiotic
Quality Control Supplement SR75 to give a level of cycloheximide
Positive control: 0.4g/l and chloramphenicol 0.05g/l renders the
Lactose/sucrose fermenters medium selective for dermatophytes, inhibiting the
Proteus vulgaris ATCC1 13315 growth of saprophytic fungi, yeasts and bacterial skin
Non-lactose/sucrose fermenters flora2.
Salmonella typhimurium ATCC1 14028 The chloramphenicol and cycloheximide supplement
SR75 reduces the potential risk to health from these
Negative control:
antibiotics. To include them in the powder mix could
Staphylococcus aureus ATCC1 25923
allow them to be scattered as dust whilst weighing
Precautions the medium. It also ensures a fixed, accurate dose of
Boil the medium for the minimal period of time to get antibiotic that has been protected from degradation
the agar into solution. Overheating reduces the agar on storage.
gel strength and increases the degree of inhibition. It The addition of cycloheximide and an anti-bacterial
is therefore important not to hold the molten medium agent has been reported to improve considerably the
at 508C for more than the short time required to isolation of dermatophytes, especially when the
distribute it into dishes. inoculum, such as horse hair was heavily
contaminated3,4. The presence of staphylococci, which
Reference
may grow in the absence of the antibiotic has been
1 Leifson E. (1935) J. Path. Bact. 40. 581±599.
shown to prevent the in-vitro growth of Trichophyton
rubrum5.
The presence of cycloheximide in the medium inhibits
DERMASEL AGAR BASE the growth of Trichosporon cutaneum, Candida
parasilosis, Candida krusei, Aspergillus, Penicillium,
Code: CM539 Fusarium and Cephalosporium species which have been
A selective medium for dermatophyte fungi associated with diseased nails6,7.
recommended for the examination of hair, skin The incorporation of griseofulvin at a level of 20mg/
scrapings, nails, etc. ml into one of paired tubes of selective media has
Formula gm/litre been recommended as an additional aid in the
Mycological peptone 10.0 diagnosis of dermatophytosis8. The absence of
Glucose 20.0 growth, on the medium containing griseofulvin
Agar 14.5 provides presumptive identification of a
dermatophyte fungus.
Dermatophyte fungi cultured on Oxoid Dermasel
Agar show characteristic colonial morphology with
fermenters of enteric origin form colourless colonies. See Desoxycholate Citrate Agar (Hynes) CM227 for
Non-lactose fermenters which are not of enteric origin the description of colonies but note that DCA CM35
are generally inhibited by the sodium desoxycholate provides an opaque background against which one
in the medium. Identify suspect colonies in the usual may more easily discern the clearing produced by
manner. alkali-producing pathogens.
Storage conditions and Shelf life The use of a less selective medium for direct sampling
Store the dehydrated medium below 258C and use of faeces and a more selective medium for post-
before the expiry date on the label. enrichment sampling, would be advantageous.
Store the prepared agar plates at 2±88C. Similarly, the less inhibitory medium is often
preferable when shigellae are being sought as well as
Quality Control salmonellae2.
Positive control:
Lactose Fermenters Storage conditions and Shelf life
Escherichia coli ATCC1 25922 Store the dehydrated medium below 258C and use
before the expiry date on the label.
Klebsiella oxytoca NCTC 8167
Store the prepared agar plates at 2±88C.
Non-Lactose Fermenters
Shigella sonnei ATCC1 25931 Quality Control
Positive controls:
Negative control: Salmonella typhimurium ATCC1 14028
Staphylococcus aureus ATCC1 25923 Shigella sonnei ATCC1 25931
Precautions Negative control:
As with all desoxycholate media, this medium is heat Enterococcus faecalis ATCC1 29212
sensitive. Observe the precautions stated under
Directions. Precautions
Observe the precautions about overheating shown
Reference under Directions.
1 American Public Health Association (1978) `Standard Methods for
The medium is best used freshly prepared.
the Examination of Dairy Products' 14th ed,. APHA Inc., New York,
Stock cultures of Shigella species may become
pp. 58±59.
predominantly in the R-phase when subcultured
away from DCA media. Such cultures are difficult to
use for control purposes without first heavily
DESOXYCHOLATE CITRATE AGAR streaking the cultures on DCA plates and picking off
Code: CM35 the few S-phase colonies i.e. the macro-colonies on the
agar surface, for further subculture.
A modification of Leifson's medium for the isolation of
intestinal pathogens. When making biochemical tests on colonies picked
from the surface of DCA plates, purity subcultures
Formula gm/litre should be carried out because the colony may be
`Lab-Lemco' powder 5.0 contaminated with Escherichia coli present as micro-
Peptone 5.0 colonies.
Lactose 10.0
Sodium citrate 5.0 References
Sodium thiosulphate 5.0 1 Leifson E. (1935) J. Path. Bact. 40. 581±599.
Ferric citrate 1.0 2 Fricker C.R. (1987) J. Appl. Bact. 63. 99±116.
Sodium desoxycholate 2.5
Neutral red 0.025
Agar 15.0 DESOXYCHOLATE CITRATE AGAR
pH 7.0 + 0.2
(HYNES)
Directions
Suspend 48.5g in 1 litre of distilled water. With Code: CM227
frequent agitation bring to the boil over a gauze and A selective medium for the isolation of Salmonella and
flame to dissolve completely. Mix well and pour Shigella organisms.
plates immediately. Dry the agar surface before use.
Formula gm/litre
THIS MEDIUM IS HEAT SENSITIVE. AVOID `Lab-Lemco' Powder 5.0
EXCESSIVE OR PROLONGED HEATING DURING Peptone 5.0
RECONSTITUTION. DO NOT AUTOCLAVE, OR Lactose 10.0
REMELT. Sodium citrate 8.5
Description Sodium thiosulphate 5.4
An Oxoid modification of Leifson medium1, for the Ferric ammonium citrate 1.0
isolation and maximum recovery of intestinal Sodium desoxycholate 5.0
pathogens. It is less selective and inhibiting than Neutral red 0.02
Desoxycholate Citrate Agar (Hynes) but colonial Agar 12.0
characteristics are identical on the two media. pH 7.3 + 0.2
and at 558C. Organisms which produce acid from growth. The inclusion of the buffers (disodium
dextrose, such as Bacillus stearothermophilus and other phosphate and sodium acetate) helped prevent
`flat-sour' organisms, are detected by the colour excessive movements of pH values which could result
change of the medium from purple to yellow. from utilisation of glucose or amino-acids. Such pH
movements would interfere with haemolytic
Storage conditions and Shelf life
reactions1 and the MIC values of pH-susceptible
Store the dehydrated medium below 258C and use
antimicrobials2.
before the expiry date on the label.
Long before the mechanisms of folate antagonism had
Store the prepared broth below 258C.
been discovered, the addition of the bases adenine,
Quality Control guanine, uracil and xanthine were shown to improve
Positive control: the performance of the medium as an antimicrobial
Bacillus stearothermophilus NCIB 8919 test medium.
Negative control: Aneurine, added as a general purpose vitamin,
Uninoculated medium improved the growth of several organisms especially
staphylococci.
References
1 American Public Health Association (1976) Compendium of The agar used in the formulation has been specially
Methods for the Microbiological Examination of Foods. APHA processed to allow unimpeded diffusion of
Washington DC. antimicrobials from discs3.
2 Baumgartner J. G. and Hersom A. C. (1956) `Canned Foods' 4th
DSTA CM261 is now primarily used for susceptibility
ed., Churchill Ltd. London, pp. 229±230 and 247.
tests and its role in diagnostic microbiology i.e. the
primary isolation of organisms from clinical samples,
has diminished.
DIAGNOSTIC SENSITIVITY TEST An essential requirement for satisfactory
antimicrobial susceptibility media is that the reactive
AGAR (DST AGAR) levels of thymidine and thymine must be sufficiently
Code: CM261 reduced to avoid antagonism of trimethoprim and
sulphonamides4.
A susceptibility test agar for antimicrobial testing.
DSTA meets this requirement and in the presence of
Formula gm/litre lysed horse blood (or defibrinated horse blood if the
Proteose peptone 10.0 plates are stored long enough to allow some lysis of
Veal infusion solids 10.0 the erythrocytes) the level of thymidine will be further
Glucose 2.0 reduced. This is caused by the action of the enzyme
Sodium chloride 3.0 thymidine phosphorylase which is released from lysed
Disodium phosphate 2.0 horse erythrocytes5. Thymidine is an essential growth
Sodium acetate 1.0 factor for thymidine-dependent organisms and they
Adenine sulphate 0.01 will not grow in its absence or they will grow poorly
Guanine hydrochloride 0.01 in media containing reduced levels6. It is important
Uracil 0.01 that users of DSTA are aware of this limitation of
Xanthine 0.01 thymidine which now exists in the medium and the
Aneurine 0.00002 effect it will have on a small proportion of organisms.
Agar 12.0
pH 7.4 + 0.2 Details of the function of the medium and the
methodology used for antimicrobial susceptibility
Directions tests are discussed in the Section `Susceptibility
Add 40g to 1 litre of distilled water. Bring to the boil Testing'.
to dissolve completely. Sterilise by autoclaving at
1218C for 15 minutes. Storage conditions and Shelf life
Store the dehydrated medium below 258C and use
For blood agar, cool the base to 508C and add 7% of before the expiry date on the label.
Defibrinated Horse Blood SR50. Mix with gentle
rotation and pour into petri dishes (12ml for a 9cm Store the prepared agar plates at 2±88C.
dish) or other containers. RECONSTITUTION AND Quality Control
MIXING SHOULD BE PERFORMED IN A FLASK Positive control:
AT LEAST 2.5 TIMES THE VOLUME OF MEDIUM Streptococcus pneumoniae ATCC1 6303
TO ENSURE ADEQUATE AERATION OF THE
BLOOD. with blood
Neisseria meningitidis ATCC1 13090
Description Staphylococcus aureus ATCC1 25923
Diagnostic Sensitivity Test Agar was developed in
Oxoid as a dual purpose medium which would without blood
satisfy both diagnostic and susceptibility Enterococcus feacalis ATCC1 29212
requirements. Pseudomonas aeruginosa ATCC1 27853
The diagnostic role was supported by the nutritional Negative control:
amino-acid base with glucose to encourage early Uninoculated medium
For the bacteriological evaluation of processed foods Storage conditions and Shelf life
the entire Enterobacteriaceae group can be used as Store the dehydrated medium below 258C and use
indicator organisms10. This will overcome the before the expiry date on the label.
discrepancies that can arise when lactose-negative, Store the prepared broth at 2±88C.
anaerogenic lactose-positive or late lactose fermenting
Enterobacteria are present but are missed by the Quality Control
standard `coli-aerogenes' tests. To overcome these Positive control:
problems lactose media have been replaced by those Yersinia enterocolitica NCTC 10460
containing glucose. Mossel et al.1 cited several Escherichia coli ATCC1 25922
examples in the literature which referred to various Negative control:
foods contaminated with salmonellae, although Staphylococcus aureus ATCC1 25923
results for coliforms were negative. A later example
quoted by Mossel9 involved an outbreak of diarrhoea Precautions
caused by French mould-fermented soft cheese Avoid overheating the medium, especially the
contaminated by Escherichia coli serotype 0124. This double-strength broth.
organism is lactose-negative and therefore was not
detected in coliform tests but only recognised when References
the commodity was tested for Enterobacteriaceae 1 Mossel. D. A. A., Vissar M. and Cornellisen A. M. R. (1963) J.
since it fermented glucose rapidly. Appl. Bact. 26(3). 444±452.
2 Van Schothurst M., Mossel D. A. A., Kampelmacher E. H. and
EE Broth should be used as an enrichment broth in Drion E. F. (1966) Vet. Med. 13(3) 273±285.
conjunction with Violet Red Bile Glucose Agar CM485. 3 Taylor W. I. (1961) Appl. Microbiol. 9. 487±490.
When specific organisms, rather than Enterobacteriaceae 4 North W. R. (1961) Appl. Microbiol. 9. 188±195.
in general, are required subcultures must be made onto 5 Mossel D. A. A. and Harrewijn G. A. (1972) Alimenta 11. 29±30.
lactose differential media e.g. Desoxycholate Citrate 6 Mossel D. A. A. and Ratto M. A. (1970) Appl. Microbiol. 20. 273±
Agar CM35, Brilliant Green Agar CM329, or MacConkey 275.
Agar CM7 for the detection of lactose-negative or 7 Mossel D. A. A., Shennan Jean L. and Vega Clare (1973) J. Sci.
delayed organisms. Fd. Agric. 24. 499±508.
Sample size should not be less than 10 grams to yield 8 Mossel D. A. A. and Ratto M. A. (1973) J. Fd. Technol. 8. 97±103.
the organisms being sought. 9 Mossel D. A. A., Harrewijn G. A. and Nesselrooy-van Zadelhoff
C. F. M. (1974) Health Lab. Sci. 11. 260±267.
10 Richard N. (1982) in Quality Assurance and quality control of
Technique microbiological culture media. Ed. J.E.L. Corry. G.I.T. ± Verlag
1 Resuscitate debilitated cells by incubating 1:10 Darmstadt. pp 51±57.
dilutions of the food samples under investigation 11 Mossel D. A. A. (1973) Food R. A. Technical Circular no 526,
in Tryptone Soya Broth CM129 at 258C for 2±8 February 1973.
hours. The fluid layer should not be much deeper
than one centimetre. Shake the flask to disperse the
contents alternately in clockwise and anti-
clockwise directions for 30 seconds on three
successive occasions.
2 After the period of time necessary for resuscitation,
ten-fold volumes of EE Broth are added to the
resuscitated suspensions.
3 Shake to disperse as above. For large samples it is
desirable to add the resuscitation medium
containing the product under examination, to
equal volumes of double strength EE Broth.
4 Incubate at :
448C for 18 hours for thermotrophic bacteria
328C for 24/48 hours for mesophilic bacteria
48C for 10 days for psychrotrophic bacteria
of Non-specific Vaginitis (NSV). The symptoms of this 7 Carry out confirmatory tests on isolates that show
mild condition prior to the isolation of the aetiological a beta-haemolytic zone. Use an inoculating wire to
agent(s) are: stab through the agar overlay to reach the colonies
beneath.
1 The absence of recognised pathogens.
The following tests have been compiled from the
2 Foul smelling discharge.
literature and personal communication.
3 pH greater than 4.5.
4 Release of `fish' odour on the addition of Test or Test %
potassium hydroxide (10%) to the discharge. Substrate Result Positive
5 The presence of `clue' cells in prepared wet mounts Oxidase Negative 0
(these are epithelial cells with a characteristic Catalase Negative 0
stippled or granular appearance caused by Gram Haemolysis of:
variable bacilli adhering to the cell surface). Human blood Positive 967
Several media and techniques have been described for Rabbit blood Positive 96
the isolation of G. vaginalis. The Oxoid Gardnerella Horse blood Negative some strains
Vaginalis Selective Medium can be used for the Sheep blood Negative 07
surface inoculation technique or the double layer
Hippurate hydrolysis Positive 92
technique2.
Starch hydrolysis Positive 90
With added human blood or rabbit blood3, a beta-
Metronidazole
haemolytic reaction is exhibited by G. vaginalis. This
(50mg) Susceptible 90
can be used as a preliminary diagnosis feature1. The
addition of `Tween 80' (0.02% v/v) to the medium Trimethoprim
containing human blood has been found to give (5mg) Susceptible 100
enhanced beta-haemolytic zones4,5. Sulphonamide
(1000mg) Resistant 0
G. vaginalis is a Gram variable, small, pleomorphic
bacillus which forms 0.25±0.44mm diameter colonies Storage conditions and Shelf life
producing beta-haemolysis on medium containing Store the dehydrated medium below 258C and use
human blood. before the expiry date on the label.
Technique Store the prepared plates at 2±88C.
Surface Inoculation Method (Isolation) Quality Control
1 Prepare the selective medium from Columbia Positive control:
Blood Agar Base CM331, Gardnerella Vaginalis Gardnerella vaginalis ATCC1 14018
Selective Supplement SR119 and defibrinated Negative control:
Horse Blood SR51, according to the directions. To Escherichia coli ATCC1 25922
demonstrate the characteristic haemolysis with
human or rabbit blood, substitute human or rabbit References
for horse blood when preparing the medium. 1 Ison C. A., Dawson S. G., Hilton J., Csonka G. W. and Easmon
2 Using a swab inoculate the vaginal discharge on to C. S. F. (1982) J. Clin. Path. 35. 550±554.
the medium. 2 Spiegel C. A., Eschenbach D., Schoenknech F. and Holmes K. K.
3 Incubate, at 358C for 48 hours in an atmosphere (1980) N. Engl. J. Med. 303. 601±607.
containing 7% carbon dioxide6. 3 King E. A. (1964) `The Identification of Unusual Pathogenic Gram
negative Bacteria' Center for Disease Control, Atlanta GA (quoted in
4 Carry out confirmatory tests on all colonies from
Reference 7).
medium containing horse blood and on beta-
4 Taylor E. and Phillips I. (1983) J. Med. Microbiol. 16. 83±92.
haemolytic colonies from medium containing
5 Totton P. A., Amsel R., Hale J., Piot P. and Holmes K. K. (1972)
human blood or rabbit blood.
J. Clin. Microbiol. 15. 141±147.
Double Layer Method (Isolation and Presumptive 6 Bailey R. K., Voss J. L. and Smith R. F. (1979) J. Clin. Microbiol. 9.
identification) 65±71.
1 Prepare two lots of selective medium from 7 Greenwood J. R. and Picket M. J. (1979) J. Clin. Microbiol. 9. 200±
Columbia Blood Agar Base CM331, Gardnerella 204.
Vaginalis Selective Supplement SR119 and sterile
human blood according to the directions.
2 Use one lot to prepare base medium plates and
place the second lot in a water bath at 508C.
3 Using the swab inoculate the vaginal discharge on
to the surface of the prepared plates. Allow to dry
at room temperature for half an hour.
4 Overlay with 5ml of the selective medium at 508C.
5 Allow the overlay medium to set.
6 Incubate at 358C for 48 hours in an atmosphere
containing 7% carbon dioxide.
GC SELECTIVE MEDIA VCN, LCAT and VCAT) have been described that can
be added to culture media in order to suppress Gram-
Isolation techniques for Pathogenic Neisseria. positive and Gram-negative contaminants.
The choice of the selective supplement is dependent
NEISSERIA ISOLATION MEDIA AND upon the preference of the laboratory as well as
TECHNIQUES regional and strain differences of the organism.
Introduction
The probability of success in the isolation and
identification of pathogenic Neisseria from clinical
OXOID GC AGAR BASE
specimens is related to five factors: Code: CM367
1 The amount of care taken in obtaining good Oxoid GC Agar Base has been formulated to include
specimens, their transport to the laboratory and Special Peptone L72 which is a mixture of meat and
correct inoculation on culture medium. plant enzymatic digests. The presence of starch ensures
2 The provision of a culture medium capable of that toxic metabolites produced by neisseria are
growing small inocula of demanding strains. absorbed. Phosphate buffers are included to prevent
changes in pH due to amine production that would
3 The provision of optimal incubation temperature
affect the survival of the organism.
and gaseous environment.
4 The inclusion of selective agents in the medium Formula gm/litre
which are capable of preventing overgrowth of Special peptone 15.0
commensal organisms but which will not inhibit Corn starch 1.0
the growth of the species required. Sodium chloride 5.0
Dipotassium hydrogen phosphate 4.0
5 Confirmatory tests to identify the species.
Potassium dihydrogen phosphate 1.0
Importance of Accurate Diagnosis Agar 10.0
Diagnosis of gonorrhoea is achieved by microscopic pH 7.2 + 0.2
examination and cultivation of material from infected
sites. Presumptive gonococci have then to be
distinguished from other organisms of the Neisseria SOLUBLE HAEMOGLOBIN POWDER
group which have a similar morphology and staining Code: L53
characteristics.
A specially prepared powder which will form a solution
Sampling Sites of 2% w/v in water and remain stable after sterilisation.
The diagnosis is achieved by examining smears and
cultures from a number of sampling sites. These YEAST AUTOLYSATE GROWTH
include the urethra and rectum of males, the urethra, SUPPLEMENT
cervix and rectum of females and occasionally also
the ducts of Bartholin's glands. The oropharynx of Code: SR105
both sexes may also require sampling1,2. Repeated Yeast Autolysate Supplement is a sterile lyophilised
examination may be necessary before a diagnosis is concentrate of specially prepared yeast fractions with
achieved. The calcium alginate or cotton wool swabs glucose and sodium bicarbonate.
that are used must be of low toxicity for
bacteriological purposes. Vial contents (each vial is sufficient for 500ml of
medium)
As with meningococcal infection, gonorrhoea may
Yeast autolysate 5.0g
present as septicaemia with signs of rash, fever and/
Glucose 0.5g
or arthritis and the responsible organism may be
Sodium bicarbonate 0.075g
isolated from the blood, joints or (rarely) the
cerebrospinal fluid. Ocular infections can occur in
neonates and occasionally in adults. VITOX
Inoculation Code: SR90/SR90B
The plates are inoculated by rolling the culture swab
Vitox is a sterile lyophilised concentrate of essential
across a segment of a moist plate or preferably in a
growth factors. Many workers prefer such a chemically
large `Z' pattern so that an adequate area of the plate
defined growth supplement to yeast extracts for the
is inoculated. Streaking the plate with a sterile
supplementation of Thayer Martin Medium.
inoculating loop is carried out to ensure adequate
dispersion of the organisms.
Media
Media used for the cultivation of gonococci are agars
of high peptone and starch content enriched with
fresh horse blood (lysed) or soluble haemoglobin and
GC growth supplements (Yeast Autolysate
Supplement SR105 and Vitox SR90) which have been
shown to stimulate growth from small inocula.
Several combinations of selective antibiotics (VCNT,
MEDIUM CONSTITUENTS
Soluble
Haemoglobin Growth Selective
or Blood Supplement Supplement
4 Dissolve the contents of a vial of either VCN 4 Examine the plates after 24 hours incubation and if
Antibiotic Supplement SR101 or VCNT Antibiotic negative reincubate for a further 24 hours.
Supplement SR91 as directed on the vial label. 5 Presumptive gonococcus colonies are identified by
5 Aseptically add the Vitox solution to 240ml of the Gram stain, oxidase and sugar fermentation
sterile GC Agar Base cooled to 508C. reactions.
6 Aseptically add the reconstituted antibiotic See also Mycoplasma page 2-120.
supplement VCN or VCNT to the GC Agar Base- Typical reactions of N. gonorrhoeae
Vitox solution. Gram negative cocci, usually arranged in pairs with
7 Asceptically add the 250ml of sterile haemoglobin long axes parallel. Oxidase positive.
solution, cooled to 508C to the GC Agar Base-
Vitox-Antibiotic Supplement solution. Mix gently Fermentation Reactions of the Neisseria Group
to avoid trapping air bubbles in the agar and pour Glucose Maltose Lactose Sucrose
into sterile petri dishes. N. meningitidis + + ± ±
`Transgrow' Medium N. gonorrhoeae + ± ± ±
`Transgrow' Medium is prepared in the same manner Bran. catarrhalis ± ± ± ±
as described for Thayer Martin Medium variants, N. flava and
except that 5g of Agar is added to the 18g of GC Agar related types + +/± ± +/±
Base. The medium is dispensed as agar slants in glass
bottles. The extra glucose required is contained in the + = Acid production
growth supplements. +/± = Variation in reaction among different types
GC Medium derived from the New York City List of products available from Oxoid for the
Formulation with LCAT or VCAT antibiotic Growth and Identification of Neisseria Species
supplement GC Agar Base CM367
1 Suspend 18g of Oxoid GC Agar Base in 425ml of VCN Selective Supplement SR101
distilled water and bring gently to the boil to Agar No.1 L11
dissolve the agar. Sterilise by autoclaving at 1218C VCNT Selective Supplement SR91
for 15 minutes. Soluble Haemoglobin Powder L53
2 Lyse 50ml of Defibrinated Horse Blood SR50 with LCAT Selective Supplement SR95
0.5% (by vol.) saponin.
Sterile Yeast Autolysate Supplement SR105
3 Dissolve the contents of a vial of sterile Yeast
Autolysate Supplement SR105 in 15ml of sterile VCAT Selective Supplement SR104
distilled water. Vitox SR90
4 Dissolve the contents of either LCAT SR95 or GC Supplement SR56
VCAT SR104 in 10ml of sterile distilled water. (Yeast Fractions plus VCNT Antibiotics)
5 Aseptically add the lysed defibrinated horse blood, Defibrinated Horse Blood SR50
Yeast Autolysed Supplement and the Antibiotic Oxidase Detection Papers BR54
Supplement (LCAT or VCAT) to the sterile GC Laked Horse Blood SR48
Agar Base cooled to 508C. Mix gently to avoid
trapping air bubbles in the agar and pour into Storage conditions and Shelf life
sterile petri dishes. Store the dehydrated medium below 258C and use
This is a derivative of NYC Medium11,12,13 based on before the expiry date on the label.
Young's publication14 where the higher level of Store the prepared plates at 2±88C.
glucose recommended by the originators was reduced
to allow sugar fermentation test to be carried out15. Quality Control
Positive control:
Technique with antibiotics
1 Prepare the medium as directed and pour into Neisseria gonorrhoeae ATCC1 19424
petri dishes. Neisseria meningitidis ATCC1 13090
2 Inoculate by rolling the culture swabs across a without antibiotics
segment of the plate or preferably in a large `Z' Haemophilus influenzae ATCC1 35056
pattern so that an adequate area of the plate is
inoculated. Streaking of the plate is carried out Negative control:
with antibiotics
with a sterile loop to ensure adequate dispersion of
Proteus vulgaris ATCC1 13315
the organisms.
Staphylococcus aureus ATCC1 25923
3 Plates are incubated at 378C in a sealed jar with at
least 70% humidity and 5±10% carbon dioxide Please note that for the LCAT Selective Supplement
provided by the introduction of the gas into the SR95, Proteus vulgaris ATCC1 13315 as a negative
incubator or by the use of a candle jar. The above control is inoculum dependent.
incubation conditions can be conveniently achieved without antibiotics
by using the Oxoid Carbon Dioxide Gas Uninoculated medium
Generating Kit BR39 in the Oxoid Gas Jar HP11.
Technique Directions
1 Prepare the inoculum in Mueller-Hinton Broth Suspend 76g of the medium in 1 litre of distilled
(Oxoid CM405) or 0.9% saline, to match the water and soak for 10 minutes. Heat gently and allow
turbidity of 0.5 McFarland standard. to boil for a few seconds to dissolve the agar. DO
2 Using a swab saturated with the above inoculum NOT AUTOCLAVE. Cool to 508C and pour plates.
suspension, inoculate the surface of a Haemophilus Description
Test Medium Agar plate to give confluent growth. Hektoen Enteric Agar was developed by King &
3 Apply the antimicrobial discs on to the surface of Metzger1. The high peptone content offsets the
the Haemophilus Test Medium plate. inhibitory effect of bile salts on Shigella species in
4 Incubate the plates at 358C in 5±7% carbon dioxide particular. The additional carbohydrates (sucrose and
for 16±18 hours and measure the zones of salicin) give better differentiation than lactose alone
inhibition. and the lower toxicity of the double indicator
improves recovery. The increased lactose content
NB: In the absence of a CO2 incubator, the CO2 helps early recognition of slow lactose-fermenting
enriched atmosphere can best be achieved by using organisms. The thiosulphate and ferric citrate are
the Oxoid Gas Generating Kit BR39 in conjunction present to detect H2S-producing organisms.
with Oxoid Anaerobic Jar.
Taylor & Schelhaut2 found the medium to be of value
Storage conditions and Shelf life in the differentiation of pathogenic organisms and for
HTM Base should be stored tightly capped in the better growth of shigellae.
original container in a cool, dry place away from
bright light. When stored as directed the medium will Hoben et al.3 added novobiocin 15mg/litre to
remain stable until the expiry date printed on the improve the selectivity of the medium by inhibiting
label. Citrobacter and Proteus species.
HTM Supplement as supplied should be stored in the Hektoen Enteric Agar meets the requirements of the
dark below 08C. When stored as directed the APHA4.
supplement is stable until the expiry date stated on Technique
the label. Inoculate the medium with fresh faeces suspended in
Quality Control Ringers solution or inoculate directly with rectal
H. influenzae ATCC149766 Good growth swabs. Spread the inoculum to obtain well separated
colonies. Incubate for 18±24 hours at 378C. Further
References incubation will improve differentiation between
1 NCCLS Documents M2-A4 Vol. 10. No. 7. and M7-A2 Vol. 10.
shigellae and salmonellae.
No 8.
2 Jorgensen J.H., Redding J.S., Maher L.A. and Howell A.W. Organism characteristics:
(1987) J. Clin. Micro. 25, 2105±2113. Shigella Green, moist raised colonies.
3 Doern G.V., Jorgensen J.H., Thornsberry C. and Snapper H. Salmonella Blue-green colonies with
(1990). Eur. J. Clin. Microbiol. Infect. Dis. 9, 329±336. or without black centres.
4 Barry A.L., Jorgensen J.H. and Hardy D.J. (1991) J. Antimic. Coliforms (rapid Salmon-pink to orange
Chem. 27, 295±301. lactose/sucrose/ colonies surrounded by a zone
5 Evans G., Marsik F., Thompson L. and Fowler J. (1990) Abstracts salicin fermenters) of bile precipitation.
of ASM Meeting 1990 C-252. Storage conditions and Shelf life
Store the dehydrated medium below 258C and use
before the expiry date on the label.
HEKTOEN ENTERIC AGAR Store the prepared plates at 2±88C.
Code: CM419 Quality Control
A differential, selective medium for the isolation of Positive control:
Shigella and Salmonella species from enteric Salmonella typhimurium ATCC1 14028
pathological specimens. Shigella flexneri ATCC1 12022
Formula gm/litre Negative control:
Proteose peptone 12.0 Escherichia coli ATCC1 25922
Yeast extract 3.0 Enterococcus faecalis ATCC1 29212
Lactose 12.0
Precautions
Sucrose 12.0
Do not overheat the medium or hold it molten for
Salicin 2.0
Bile salts No.3 9.0 long periods.
Sodium chloride 5.0 Proteus species may resemble salmonellae or shigellae.
Sodium thiosulphate 5.0 Further testing must be carried out to confirm the
Ammonium ferric citrate 1.5 presumptive identification of organisms isolated on
Acid fuchsin 0.1 this medium.
Bromothymol blue 0.065
Agar 14.0 References
pH 7.5 + 0.2 1 King S. and Metzger W. I. (1968) Appl. Microbiol. 16. 577±561.
2 Taylor W. I. and Schelhaut D. (1971) Appl. Microbiol. 21. 32±37.
3 Hoben D. A., Ashton D. H. A. and Peterson A. C. (1973) Appl. 3 Incubate at 358C for three to five days under micro-
Microbiol. 21. 126±129. aerophilic conditions. Use Campylobacter Gas
4 American Public Health Association (1976) Compendium of Generating Kit BR56 or BR60 with an active
Methods for the Microbiological Examination of Foods. APHA Inc. catalyst BR42. Alternatively use CampyGen
Washington DC. CN025A or CN035A. CampyGen does not require
the addition of water or a catalyst.
4 Examine for the presence of discrete, translucent
and non-coalescent colonies. Note that colonies of
HELICOBACTER PYLORI H. pylori do not resemble those of Campylobacter
ISOLATION species.
5 Confirm the identity of the isolates with the
HELICOBACTER PYLORI SELECTIVE following tests:
SUPPLEMENT (DENT)
Gram negative, curved or spiral bacillus.
Code: SR147
Growth at 358C, no growth at 258C, variable growth
A selective supplement for the isolation of H. pylori at 428C. Urease positive, Catalase positive, Oxidase
from clinical specimens. positive, Hippurate negative.
Vial contents (each vial is sufficient for 500ml of Quality Control
medium) Positive control:
Vancomycin 5.0mg Helicobacter pylori NCTC 11916/ATCC1 43526
Trimethoprim lactate 2.5mg
Cefsulodin 2.5mg Negative control:
Amphotericin B 2.5mg Candida albicans ATCC1 10231
Directions
References
To one vial add 2ml of sterile distilled water and
1 Marshall B. K., Warren J. R., Blincow E. D., Phillips M.,
dissolve the powder completely. Avoid frothing. Add
Goodwin C. S., Murray R., Blackbourne S. J. and Waters T. E.
the contents aseptically to 500ml of sterile Columbia (1988) The Lancet, December 24/31, No 8626/8627.
Blood Agar Base CM331 at 508C. Add 35ml of Laked
2 Dent J. C. and McNulty C. A. M. (1988) Eur. J. Clin. Microbiol.
Horse Blood SR48 and mix well before pouring into
Infec. Dis. 7. 555±568.
sterile petri dishes.
3 Buck G. E. (1988) Laboratory Management, 26, No.9.
Description 4 Skirrow M. B. (1977) BMJ, 1. 9±11.
Helicobacter pylori is associated with a number of
gastric conditions, chiefly gastritis and peptic
ulcers1,2,3.
Helicobacter pylori Selective Supplement (Dent)
SR147 was developed from Dent's selective medium HOYLE MEDIUM BASE
described for the isolation of H. pylori from gastric Code: CM83
biopsies2. This is a modification of Skirrow's medium4
in which polymixin B is replaced by cefsulodin and A modification of Neill's medium for the isolation and
amphotericin B is added to inhibit Candida species. differentiation of Corynebacterium diphtheriae types.
When used routinely in the laboratory for 100 gastric Formula gm/litre
biopsies, Dent's medium achieved a higher isolation `Lab-Lemco' powder 10.0
rate for H. pylori and lower contamination by other Peptone 10.0
organisms when compared with Skirrow's medium Sodium chloride 5.0
and chocolate blood agar2. The provision of a good Agar 15.0
selective medium for H. pylori will help establish the pH 7.8 + 0.2
role of this organism in the aetiology of gastric Directions
disease. Suspend 40g in 1 litre of distilled water and bring to
Technique the boil to dissolve completely. Sterilise by
1 Prepare the medium as directed. The plates can be autoclaving at 1218C for 15 minutes. Cool to 558C and
stored at 48C for three weeks but it is essential that add 50ml of Laked Horse Blood SR48 and 10ml of
they are kept moist. This can be achieved simply 3.5% Potassium Tellurite solution SR30, mix, and
by keeping the plates in a plastic bag. pour plates.
2 Smear the specimen on to the medium. Description
Hoyle medium is the well known modification1 of
Note ± the recovery of H. pylori from gastric biopsies Neill's medium for the cultural isolation and
is improved by direct cultivation as soon as possible differentiation of Corynebacterium diphtheriae types.
after collection. If transportation is necessary, then Hoyle medium does not exert the inhibitory effect
place the biopsy against the neck of a small, sterile manifested by Neill's on some mitis types, but gives
glass bottle containing 0.1ml of sterile saline2. The very rapid growth with all types of Corynebacterium
biopsy should adhere to the glass but be protected diphtheriae, so that diagnosis is possible after 18 hours'
from dehydration by water vapour. incubation.
November 1998 2-109
Culture Media
Technique Precautions
This is a highly selective medium which is used in It should be noted that not all corynebacteria produce
parallel with non-selective media such as blood agar the typical colonies described above ± so in all cases it
(e.g. Blood Agar Base CM55 with 10% of horse is advisable to use Hoyle medium in conjunction with
blood). Unlike the non-selective media, Hoyle the additional media and tests mentioned above.
medium should be inoculated by rubbing the throat
swab (or other material) over the entire surface ± References
spreading with a platinum loop is not necessary. 1 Hoyle L. (1941) Lancet. i. 175±176.
2 Elek S. D. (1948) Brit. Med. J. 1. 493±496.
Normally incubation for 18 hours at 358C is sufficient
but, when a negative result is obtained, incubation for
up to 72 hours may be advisable. Gentian violet
stained films made from colonies picked straight off
the medium, are satisfactory for C. diphtheriae HIGH RESOLUTION (H.R.) MEDIUM
morphology. For the demonstration of the Code: CM845
characteristic morphology and staining reactions of
C. diphtheriae by Neisser's or Albert's method, it is A chemically defined medium for fluconazole
preferable to utilise colonies from Loeffler medium. susceptibility testing.
The toxigenicity of C. diphtheriae strains may be Formula gm/litre
determined by the Elek2 method. Dextrose 19.98
Colonial Characteristics Potassium dihydrogen phosphate 1.99
It is best to examine with a low-power microscope, Ammonium sulphate 4.99
the colonies being illuminated from above by L-Glutamine 0.58
daylight. Magnesium sulphate (anhydrous) 0.99
Sodium chloride 0.20
Type differentiation is good, and typical colonial Calcium chloride 0.20
appearances after 18 hours' incubation are as follows: L-Lysine monohydrochloride 0.073
C. diphtheriae type gravis ± grey colonies, 1.5±2.5mm Valine 0.047
diameter dull, matt surface. May be slightly L-Arginine monohydrochloride 0.042
umbonate and show indented margins. Colonies can L-Histidine 0.023
be pushed along the surface of the medium. Methionine (DL) 0.0189
Tryptophane 0.020
C. diphtheriae type mitis -- grey colonies, 1.5±2.0mm Inositol 0.00397
diameter with regular margins and shining surface. Boric acid 0.00099
Variation in size common. Calcium D-pantothenic acid 0.00079
C. diphtheriae type intermedius ± grey colonies, Nicotinic acid 0.00079
0.5±0.75mm diameter with shining surface. Colonies Pyridoxine hydrochloride 0.00079
are very uniform in size with darker centres. Aneurine hydrochloride 0.00079
Manganous sulphate 0.00079
C. hofmannii ± usually rounded, white or greyish, Zinc sulphate 0.0014
0.5±0.75mm diameter. Colonies may be up to 1mm 4-Aminobenzoic acid 0.000395
diameter, when they are black in more heavily Riboflavin BP/USP 0.000395
inoculated areas and white when well isolated. Ferric chloride 0.000395
C. xerosis ± black shining colonies of variable size. Cupric sulphate 0.00012
Biotin crystalline 0.000004
Streptococci ± minute black or brownish-black
Folic acid 0.000395
colonies.
L-Isoleucine 0.052
Other organisms may occasionally grow which Sodium molybdate 0.00047
resemble C. diphtheriae type intermedius but are larger, Potassium iodide 0.00020
while sporing anaerobes may produce brownish L-Leucine 0.052
mucoid colonies. Threonine 0.0476
Storage conditions and Shelf life
Store the dehydrated medium below 258C and use Directions
before the expiry date on the label. PART A
Prepare a 2% w/v solution of Agar Technical (Oxoid
Store the prepared plates at 2±88C. L13) and buffer to pH 7.5 with 0.1ml phosphate
Quality Control buffer. Sterilise at 1158C for 10 minutes.
Positive control: PART B
Corynebacterium diphtheriae biotype gravis ATCC1 Dissolve 29.34g of High Resolution Medium in 900ml
19409 of distilled water. Stir continuously and add 2g of
Corynebacterium diphtheriae biotype intermedius sodium bicarbonate (analar) and make up the volume
ATCC1 14779 to 1 litre with distilled water. Sterilise by filtration.
Negative control: The medium can be kept at 48C for 2 weeks.
Uninoculated medium. Aseptically mix equal volumes of molten High
Resolution Medium Part A (cooled to 608C) and High
Resolution Medium Part B. Mix thoroughly and pour 5 Inoculate the plates using a multipoint inoculator.
into sterile petri dishes. It is possible to inoculate 20 isolates, evenly spaced,
per 9cm petri dish.
Description
High Resolution Medium is a chemically defined 6 Incubate at 288C for 6 days.
medium, specifically developed for the in vitro testing 7 Check the control plates to ensure that all isolates
of fluconazole. The MIC values generated using the have grown adequately.
medium give sensible correlations with efficacy in 8 Determine the endpoint as for Candida.
vitro and with clinical outcome.
Candida albicans (ATCC1 76615) has an MIC value of
In a comparison of a disc diffusion method against a 1.56mg/ml against the antifungal agent fluconazole.
microdilution method correlation using HR medium
was good for a number of antibiotics including Storage conditions and Shelf life
nystatin and amphotericin B but generally better for High Resolution Medium should be stored tightly
new triazoles such as fluconazole than for the other capped in the original container at 10 to 258C away
antifungals.6 from bright light. When stored as directed the
medium will remain stable until the expiry date
printed on the label.
Technique
Preparation of the inocula Please note shelf life of 2 years.
The preparation and standardisation of the inoculum
varies with different fungi: References
1 Hoeprich P.D. and Finn P.D. (1972) J. Infect. Dis. 126, 353±361.
CANDIDA spp
2 Cook R.A., McIntyre K.A. and Galgiani J.N. (1990). Antimicrob.
1 Grow Candida spp overnight at 378C in Sabouraud Agents and Chemother. 34, 1542±1545.
dextrose broth.
3 Pfaller M.A. et al. Antimicrob. Agents Chemother. 34, 1648±1654.
2 Vortex mix and make a 1 in 100 dilution of each 4 Pfaller M.A. et al. (1992) Antimicrob. Agents and Chemother. 36,
culture in normal saline and estimate the cell 1805±1809.
numbers using a haemocytometer. 5 Pfizer Ltd, private communication (1990).
3 Appropriately dilute each culture in normal saline 6. Carillo-Munoz A.J., Tur C., Etervill D. et al (1995) Revista
to give the following cell densities: EspanÄola de Quimioterapia 8, 221±228.
Candida albicans 105/ml
Candida krusei 105/ml
Candida tropicalis 105/ml
Candida guillermondii 106/ml
Candida parapsilosis 106/ml IRON SULPHITE AGAR
Candida pseudotropicalis 106/ml Code: CM79
Inoculation of the plates A medium for the detection of thermophilic anaerobic
1 Surface inoculate each series of plates using a organisms.
multipoint inoculator which delivers 1ml of each
culture inoculum. It is possible to inoculate 36 Formula gm/litre
isolates per 9cm petri dish. Inoculate control plates Tryptone 10.0
at both the beginning and end of the inoculation Sodium sulphite 0.5
run. Iron (III) citrate 0.5
Agar 12.0
2 Incubate the plates at 288C for 48 hours. pH 7.1 + 0.2
Endpoint determination Directions
1 Check the control plates to ensure that all the Suspend 23.0g in 1 litre of distilled water and boil to
organisms have grown adequately. dissolve completely. Sterilise by autoclaving for 15
2 Read all plates against a standard background and minutes at 1218C. Mix well before pouring.
record, for each isolate, the lowest concentration of Description
fluconazole that completely suppresses visible (to This medium is a modification of Cameron Sulphite
the naked eye) growth. This is the MIC value. Agar developed by the National Canners Association
DERMATOPHYTE spp of America1.
1 Grow the dermatophytes on Sabouraud Dextrose It had been shown that the medium was improved by
Agar (CM41) at 288C for 5±10 days. reducing the concentration of sodium sulphite.
2 Scrape off the mycelium from the agar surface Beerens2 showed that some strains of Clostridium
using a scalpel and place in a bijou bottle sporogenes would not tolerate 0.1% sulphite. This was
containing 4gm of glass beads (approximately confirmed by Mossel et al.3 who consequently used
2mm diameter) plus 2ml of 0.85% saline. iron sulphite agar containing only 0.05% sulphite and
3 Vortex mix to evenly disperse. obtained no apparent inhibition.
4 Adjust the density of the suspension with 0.85% Technique
saline to give a 65% light transmission on an Iron Sulphite Agar is particularly suitable for the
absorptiometer. detection of thermophilic anaerobic organisms
causing sulphide spoilage in food. The medium
5 Poor growth-supporting ability for streptococci Certain supplements interfere with antimicrobial
and variable growth rates with gram-positive activity and tests must be made to measure their
organisms generally. effect.
Some mutant strains which are totally dependent on Nutrient Antimicrobial
thymine and thymidine for their growth will, Thymidine Trimethoprim
however, not grow in Oxoid `Iso-Sensitest Agar', Blood Sulphonamides,
which has these two compounds at very low levels, as trimethoprim,
they are the naturally occurring antagonists of aminoglycosides
trimethoprim. Care must be taken to recognise these CO2 Aminoglycosides,
strains8,9,10. erythromycin,
Oxoid `Iso-Sensitest Agar', CM471 was developed lincomycin, tetracyline,
from Oxoid `Sensitest' Agar CM409 which has been novobiocin
used successfully in several centres throughout the Cysteine and other Aminoglycosides
world11,12,13. -SH compounds
Vitox/Isovitalex Aminoglycosides,
The role of metal ions as antagonists to certain sulphonamides,
antibiotics has been described by many trimethoprim
workers14,15,16,17,18,19. A stabilised mineral content in
sensitivity test media is, therefore, important. `Iso-Sensitest Agar' with the addition of 10% of horse
Reller, Schoenknecht, Kenny & Sherris3 demonstrated blood has been recommended as a suitable medium
the contribution of cations provided in media by when testing susceptibility of Helicobacter pylori21.
ordinary agars. A considerable difference in mineral Although Mueller-Hinton agar performed nearly as
content between the agar and broth media was well, `Iso-Sensitest Agar' was preferred because its
shown. Only by using an agar, which is specially contents are better defined.
processed to remove the free anions and cations, can a
stabilised mineral content be obtained. References
1 Ericsson H. M. and Sherris J. C. (1971) Acta. Pathol. Microbiol.
Oxoid `Iso-Sensitest Agar' has stability in mineral Scand. Suppl. 217. 1±90.
content and the ability to produce optimum 2 Garrod L. P. and Waterworth P. M. (1971) J. Clin. Path. 24. 779±
antimicrobial zones of inhibition. 789.
The amino-nitrogen base of acid-hydrolysed casein 3 Reller L. B., Schoenknecht F. D., Kenny M. A. and Sherris J. C.
and special peptones has been supplemented with (1974) J. Infect. Dis. 130. 454±463.
defined growth factors. Careful preparation of the 4 Duncan I. B. R. (1974) Antimicrob. Agents & Chemotherapy 5. 9±15.
nutrients ensures that trimethoprim and 5 Yourassowsky E., Vanderlinden M. P. and Schoutens E. (1974) J.
sulphonamide antagonists are at very low levels. Clin. Path. 27. 897±901.
6 Neussil H. (1976) Chemotherapy Vol.2. 33±40.
THE ADDITION OF LYSED HORSE 7 Bridson E. Y. (1976) Arztl. Lab. 22. 373±376.
ERYTHROCYTES TO THE MEDIUM IS NOT 8 Tanner E. I. and Bullin C. H. (1974) J. Clin. Path. 27. 565±568.
REQUIRED WHEN CARRYING OUT 9 Thomas M. and Bond L. (1973) Med. Lab. Technol. 30. 277±279.
ANTIMICROBIAL SUSCEPTIBILITY TESTS WITH 10 Barker J., Healing D. and Hutchinson J. G. P. (1972) J. Clin. Path.
THESE COMPOUNDS. 25. 1086±1088.
11 Reynolds A. V., Hamilton-Miller J. M. T. and Brumfitt W. (1974)
B.M.J. (ii) 778.
SUPPLEMENTAL NUTRIENTS FOR 12 Stewart Sheila M., Anderson Isobel M. E. and Malcolm Margaret
NUTRITIONALLY DEPENDENT ORGANISMS G. G. (1975) J. Clin. Path. 28. 195±197.
13 Bell S. M. (1975) Pathology 7. Suppl. 1±48.
Some pathogenic organisms may be nutritionally 14 Garrod L. P. and Waterworth P. M. (1969) J. Clin. Path. 22. 534±
dependent because of intrinsic demands for special 538.
growth factors, e.g. streptococci, neisseria, 15 Gilbert D. N., Kutscher E., Ireland P., Barnett J. A. and Sandford
haemophilus, campylobacter etc. Other organisms J. P. (1971) J. Infect. Dis. 124. (suppl.), S37-S45.
may exhibit mutant characteristics, e.g. dwarf Staph. 16 Zimelis V. M. and Jackson G. G. (1973) J. Infect. Dis. 127. 663±
aureus, thymidine-dependent Esch. coli. 669.
Supplemental nutrients can be added to `Iso-Sensitest 17 Davis S. D., Ianetta A., Wedgewood R. J. (1971) J. Infect. Dis. 124.
Agar' to obtain or improve growth of these 610±612.
organisms20. 18 Brenner V. C. and Sherris J. C. (1972) Antimicrob. Agents
Chemother. 1. 116±122.
Nutrient Micro-organism
19 Traub W. H. (1970) Appl. Microbiol. 20. 98±102.
Laked Blood (5% v/v) Neisseria, Streptococcus
20 Acar J. F. (1980) Antibiotics in Laboratory Medicine, Lorian V. (Ed.)
Fildes Peptic Digest Haemophilus Williams and Wilkens, Baltimore, USA, 48±51.
of Blood (5% v/v) 21 Hartzen S.H., Andersen L.P., Bremmelgaard A. et al (1997)
Menadione (0.5mg/ml) Dwarf colonies of Antimicrob. Ag. and Chemother. 41. 2634±2639.
Thiamine (2mg/ml) Staph. aureus and coliform
organisms
Pyridoxine Symbiotic streptococci
hydrochloride (1mg/ml)
November 1998 2-113
Culture Media
Streptococcus avium (Group Q) has been included in 5 Thoroughly mix the medium and sample to give a
the `enterococci' group as it has very similar uniform dispersion of the organisms.
biochemical and antigenic characteristics to the Group 6 Solidify the agar as rapidly as possible after
D species and also occurs in warm-blooded animals. pouring.
The detection of enterococci may provide more 7 Incubate the plates in an inverted position at 358C
specific information about the source of pollution for 48 hours.
because certain enterococci are host specific. For 8 Count all red to pink surface and subsurface
example, a predominance of E. bovis and E. equinus colonies.
would indicate pollution due to animal excrement.
9 Calculate the numbers of enterococci and report as
The detection of E. bovis and E. equinus species has faecal streptococci per 100ml.
been found to be associated with pollution involving 10 Confirm the colonies as enterococci2. Use an
meat processing plants, dairy wastes and feedlot and
inoculating wire to stab through the agar to reach
farmland run-off.
the colonies.
The detection of these enterococcal species is Storage conditions and Shelf life
indicative of recent contamination as the organisms Store the dehydrated medium below 258C and use
survive for only a short period outside their natural before the expiry date on the label.
habitat. The coliform/enterococci ratios may also
provide information on possible sources of pollution2. Store the prepared plates at 2±88C.
Caution must be observed when assessing the quality Quality Control
of marine recreational waters, particularly in tropical Positive control:
climates, because a high incidence of false-positive Enterococcus faecalis ATCC1 29212
presumptive counts for enterococci may occur. Negative control:
Anaerobic incubation of tropical marine water Escherichia coli ATCC1 25922
samples is recommended3.
Precautions
Colonies of enterococci on the membrane filter or agar Observe the HAZARD precautions stated on page 2-7
plate are red or pink with a variation in diameter about sodium azide when disposing of the medium.
from 0.3 to 2mm. It is recommended that counting The pH of the medium should not fall below 7.0 as it
should be done with the aid of a low power (10 to 15 may become inhibitory towards enterococci1. KF
magnification) binocular wide field dissecting Streptococcus Agar is not specific for the presumptive
microscope or equivalent optical device. identification of Group D streptococci. Further tests
must be made to confirm the identity of the
Technique organisms isolated.
Membrane Filtration Technique
1 Prepare the KF Streptococcus Agar Medium as References
directed. 1 Kennor G.A., Clark H.F. and Kabler P.W. (1961) J. Appl.
2 Filter samples through a sterile membrane to give Microbiol. 9. 15±20.
20 to 100 colonies on the membrane surface. Use 2 American Public Health Association (1981) Standard Methods for
volumes of 100, 10, 1, 0.1 or 0.01ml, depending on the Examination of Water and Wastewater, 15th Edn. APHA Inc.
the degree of pollution present. Washington DC.
3 Fujioka R.S., Ueno A.A. and Narikawa O.T. (1984). Technical
3 Transfer the membrane filter directly to agar
medium in the petri dishes, avoiding the formation Report number 168, Water Resources Research Center, University of
of air bubbles. Hawaii at Manoa, Honolulu.
Description Description
Lactose broth is recommended for use in the Lauryl Tryptose Broth provides a selective medium
presumptive identification of coliform organisms in which is used for the detection of coliform organisms
milk, water and foods as specified by the American in water, dairy products and other foods. The APHA1
Public Health Association1,2,3. recommend that Lauryl Tryptose Broth should be
used for the Mean Probable Number Presumptive
Tubes of Lactose Broth are inoculated with dilutions
Test of coliforms in waters, effluent or sewage as a
of the samples and incubated at 358C. Examination
confirmatory test of lactose fermentation with gas
for gas formation is carried out after 24 and 48 hours
production for milk samples (APHA2) and for the
incubation. This presumptive evidence of coliform
detection of coliforms in foods (APHA3).
organisms must be confirmed by further tests.
Surface active agents have long been used as the
Storage conditions and Shelf life
Store the dehydrated medium below 258C and use inhibitory ingredients in selective media. MacConkey4
before the expiry date on the label. introduced bile salts for this purpose and later Albus
and Holm3 working with lactobacilli found that
Store the prepared medium at room temperature sodium ricinoleate exerted a selective action. The
(18±228C). development of synthetic wetting agents opened up
Quality Control new fields of investigation. Mallmann and Darby6
Positive control: after comparative tests with a large number of these
Escherichia coli ATCC1 25922 compounds, showed that sodium lauryl sulphate
Enterobacter aerogenes ATCC1 13048 gave the best results in selective media for the
coliform group.
Negative control:
Lauryl Tryptose Broth was designed to promote a
Uninoculated medium
rich growth and copious gas production from small
Precautions inocula of coliform organisms. Aerobic sporing
Ensure that the fermentation tubes are free from air bacteria are completely inhibited. The advantage in
bubbles before inoculation. using this product is that in addition to giving the
fermentation reaction typical of MacConkey Broth it
Large water samples may require double-strength
can also be directly tested for the presence of indole.
lactose broth to reduce the final volumes. Do not
overheat double-strength broth or inhibitory products Unlike MacConkey Broth, the medium contains no
indicator, but this can be added (if required) after
will be produced.
incubation.
References Technique
1 American Public Health Association (1978) Standard Methods for For details of the APHA standard methods please
the Examination of Dairy Products. 14th Edn. APHA Washington consult the references below.
DC.
Lauryl Tryptose Broth is recommended for the
2 American Public Health Association (1980) Standard Methods for
detection and enumeration of coliform organisms in
the Examination of Water and Wastewater. 15th Edn. APHA Inc.
water and milk products, especially in the control of
Washington DC.
ice-cream manufacture and in dairy hygiene. A
3 American Public Health Association (1976) Compendium of
suggested procedure (Dyett7) is as follows:
Methods for the Microbiological Examination of Foods. APHA Inc.
Washington DC. Inoculate samples of ice-cream into tubes of Lauryl
Tryptose Broth in the manner normally employed in
LAURYL TRYPTOSE BROTH the MacConkey test. Examine the tubes after
overnight incubation at 358C and, if no gas is visible,
(LAURYL SULPHATE BROTH) examine again at the end of 48 hours' incubation. For
every tube showing fermentation ('primary
Code: CM451 fermentation') two further tubes of Lauryl Tryptose
A medium for the detection of coliform organisms in Broth are inoculated from a tube of the primary
water and waste water, according to the formula of the fermenting broth, and these are incubated at 358C and
American Public Health Association. 448C respectively. It is advisable that the tube to be
incubated at 448C be warmed up in a water bath at
Formula gm/litre this temperature before inoculation.
Tryptose 20.0
Lactose 5.0 If the 448C incubated broth ferments after seven
Sodium chloride 5.0 hours, test for indole production with either Ehrlich
Dipotassium hydrogen phosphate 2.75 or Kovac's reagent. Due to the lauryl sulphate
Potassium dihydrogen phosphate 2.75 present, shaking the reagent culture mixture forms a
Sodium lauryl sulphate 0.1 persistent emulsion which interferes with the test.
pH 6.8 + 0.2 This may be avoided by shaking with ether, which
separates rapidly, and then adding Kovac's reagent to
Directions the layer without shaking. If fermentation has not
Dissolve 35.6g in 1 litre of distilled water and occurred after seven hours, leave the tube overnight
distribute into containers with fermentation tubes at 448C and test the following day. A positive indole
(Durham). Sterilise by autoclaving at 1218C for 15 reaction in a broth that has produced gas at 448C
minutes. indicates the presence of Escherichia coli. The tube at
November 1998 2-119
Culture Media
Precautions gm/litre
If stored at 2±88C the broth will become cloudy or ACES Buffer/Potassium hydroxide 10
form a precipitate. This should clear at room Ferric pyrophosphate 0.25
temperature but gas formation is the criterion of L-cysteine hydrochloride Nil
growth not turbidity. a-Ketoglutarate 1.0
References
1 American Public Health Association (1980) Standard Methods for LEGIONELLA BMPA SELECTIVE
the Examination of Water and Wastewater. 15th Edn. APHA Inc. SUPPLEMENT
Washington DC.
2 American Public Health Association (1978) Standard Methods for Code: SR111
the Examination of Dairy Products. 14th Edn. APHA Inc. Vial contents (each vial is sufficient for 100ml of
Washington DC. medium)
3 American Public Health Association (1976) Compendium of Cefamandole 400mg
Methods for the Microbiological Examination of Foods. APHA Inc. Polymyxcin B 8,000 IU
Washington DC. Anisomycin 8mg
4 MacConkey A. T. (1938) J. Hyg. 8. 322±334.
5 Albus W. R. and Holm G. E. (1926) J. Bact. 12. 13±18.
6 Mallmann W. L. and Darby C. W. (1941) Am. J. Pub. Hlth. 31.
127±134.
7 Dyett E. J. (1957) Lab. Prac. 6(6). 327±328.
8 ISO Standard 11866±2 Milk and Milk Products - Enumeration of
presumptive Escherichia coli - part 2: Most probable number technique
using 4-methylumbelliferyl-b-D-glucuronide.
2 Examine microscopically for Legionella by the 5 Spread 0.1ml on to plates of BCYE Medium using a
Fluorescent Antibody Method (FA) and for other sterile spreader.
bacteria by Gram's stain. 6 Incubate the plates at 358C and examine daily for
3 Inoculate specimens that are FA-positive but with up to seven days.
no other organisms present on to plates of BCYE Colonies suspected of being Legionella are
Medium. Both FA-positive and FA-negative subcultured to Tryptone Soya Agar containing 5%
specimens in which other organisms have been sheep blood and BCYE Agar.
detected by the Gram stain, are inoculated on to
the selective medium BMPAa. Isolates that grow on BCYE Agar but fail to grow on
4 Incubate the plates at 358C in a 90% relative TSA Blood Agar and have characteristic morphology,
humidity atmosphere. may be presumed to be Legionella. Confirmation must
be made by biochemical and serological tests12.
5 Growth usually appears in 2±3 days but continue
to examine daily for 14 days before discarding the As the media described are not completely selective
plates. for Legionella species, it is recommended8 that the
Environmental Samples following criteria are used for the examination of
plates:
1 Take 10ml of the concentrated sample and
centrifuge at 2,500 rpm for 20 minutes (using 1 the colonies must have characteristic colour, size
sealed buckets). and appearance when examined under a dissecting
2 Remove the supernatant to leave approximately microscope.
1ml of fluid. Resuspend the deposit. This 2 the isolates should not grow on blood agar.
constitutes the inoculum.
3 the organisms should show characteristic Gram
3 Spread 0.1ml on to plates of BCYE Medium with
morphology.
and without selective agents using a sterile
spreader. Legionella spp. cannot be identified solely on growth
4 Add 9ml of HCl-KCl buffer* (pH 2.2); shake gently characteristics on various media or by biochemical
and leave for 5 minutes. tests. Further studies with DNA homology, cellular
fatty acids and serotyping must be undertaken.
*HCl-KCl buffer:
3.9ml of 0.2 M HCl Colony morphology after incubation at 358C for 2±3
25ml of 0.2 M KCl days on CYE/BCYE media
Adjust the pH to 2.2 using 1M KOH. L. pneumophila: diameter 1±2mm (increase in size on
further incubation). White, glistening, circular,
Alternatively, heat 10ml of the sample concentrate smooth, raised with an entire edge.
in a 508C water bath for 30 minutes.
L. gormanii: diameter 1±2mm Buff-white or cream,
Important slight raised, mucoid.
ACID AND HEAT PRETREATMENT OF
Other legionellae: (L. micdadei, L. bozemanii, L. dumoffii,
SAMPLES MUST NOT BE COMBINED.
L. longbeachae and L. jordanis) ± indistinguishable from
L. pneumophila.
Nitrates reduced ± ± ± ± ± ± + +
to nitrites
D-mannitol ± ± ± ± ± + + ±
L-rhamnose + ± V V ± ± V ±
D-xylose ± + ± + + ± ± +
a-methyl D-mannoside + ± + + V NS NS NS
Acriflavine is activated by light and may form The most common clinical manifestation in both
compounds inhibitory for Listeria. adults and neonates is meningitis. Widely
disseminated infection, abscesses, sub-acute bacterial
References endocarditis and opportunistic infections in
1 Lovett J., Francis D.W. and Hunt J.M. (1987) J. Food Prot. 50. immunosuppressed patients occur less
188±192. frequently.
2 Curtis G.D.W., Nichols W.W. and Falla T.J. (1989) Lett. Appl.
Birds, fish and other animals are all susceptible to
Micro. 8. 169±172.
infection with Listeria. It is of particular importance in
domestic farm animals. In the Federal Republic of
LISTERIA SELECTIVE AGAR Germany reporting of listeriosis in animals is
compulsory and meat inspection law in the same
(OXFORD FORMULATION) country requires examination for Listeria because of
Code: CM856 its significance in meat hygiene.
A selective and diagnostic medium for the detection of Listeria monocytogenes is very widespread in the
Listeria monocytogenes, when prepared from Listeria environment. Isolation has been reported from
Selective Agar Base CM856 and Listeria Selective milk2,3, cheese4, sewage and riverwater5, and silage6.
Supplement SR140. Because Listeria is so widespread sources of infections
are numerous. Uncooked vegetable foods have been
Formula gm/litre
implicated; an episode associated with consumption
Columbia Blood Agar Base 39.0
of coleslaw7 was linked with cabbage from a farm
Aesculin 1.0
using sewage fertiliser. In outbreaks caused by dairy
Ferric ammonium citrate 0.5 products, cattle with mastitis may be the source of the
Lithium chloride 15.0
organism. Of great importance to veterinarians is the
pH 7.0 + 0.2
considerable increase amongst sheep of infection
manifesting as abortion or encephalitis due largely to
LISTERIA SELECTIVE SUPPLEMENT changing practices in silage manufacture8.
(OXFORD FORMULATION) The ability to isolate the organism has been impeded
Code: SR140 in the past by lack of an effective selective medium, as
L. monocytogenes can be easily and completely
Vial contents (each vial is sufficient for 500ml of overgrown by competing flora.
medium)
Cycloheximide 200mg Listeria Selective Medium (Oxford Formulation) is
Colistin sulphate 10mg based on the formulation described by Curtis et al9
Acriflavine 2.5mg and is recommended for the detection of Listeria
Cefotetan 1.0mg monocytogenes from clinical and food specimens.
Fosfomycin 5.0mg The medium utilises:
Directions (i) the selective inhibitory components lithium
Suspend 27.75 of the Listeria Selective Agar Base chloride, acriflavine, colistin sulphate, cefotetan,
(Oxford Formulation) CM856 in 500ml of distilled cycloheximide and fosfomycin, and
water. Bring gently to the boil to dissolve. Sterilise by
autoclaving at 1218C for 15 minutes. Cool to 508C and (ii) the indicator system aesculin and ferrous iron for
aseptically add the contents of one vial of Listeria the isolation or differentiation of
Selective Supplement (Oxford Formulation) SR140 L. monocytogenes.
reconstituted with 5ml of ethanol/sterile distilled L. monocytogenes hydrolyses aesculin, producing black
water (1:1). Mix well and pour into sterile petri zones around the colonies due to the formation of
dishes. black iron phenolic compounds derived from the
aglucon. Gram-negative bacteria are completely
Prepared plates may be stored for up to 10 days at inhibited. Most unwanted gram-positive species are
48C in the dark room. suppressed, but some strains of enterococci grow
Description poorly and exhibit a weak aesculin reaction, usually
Foodborne infection by Listeria monocytogenes has after 40 hours incubation. Some staphylococci may
prompted increased concern for detecting this grow as aesculin-negative colonies.
organism in foods, in the environment and in Typical L. monocytogenes colonies are almost always
pathological specimens from both human and animal visible after 24 hours, but incubation should be
subjects. continued for a further 24 hours to detect slow-
Most infections in adult humans are symptomless and growing strains.
result in intestinal, vaginal and cervical carriage. Techniques for isolation vary with the author and the
Infection during pregnancy may cause abortion, material under examination10,11. For all specimens
premature delivery and neonatal infection. The selective enrichment and cold enrichment have been
possibility of listeriosis should be considered in any shown to increase isolation rates significantly12,13,14.
woman with unexplained recurrent miscarriage, The efficacy of Listeria Selective Medium (Oxford
premature labour or foetal death. The organism Formulation) has been confirmed for various
should be sought in blood cultures and genital-tract foods15,16 following the methodology and using
swabs1.
November 1998 2-127
Culture Media
24 Curtis G.D.W., Nichols W.W. and Falla T.J. (1989) Letters in 3 Subculture from the Listeria Selective Enrichment
Appl. Microbiol. 8. 169±172. Broth onto Listeria Selective Agar plates (see Note)
after 1, 2 and 7 days by:
LISTERIA ENRICHMENT (i) Direct plating onto Listeria Selective Agar
BROTH BASE plates.
(ii) Adding 1ml of the Listeria Selective Enrichment
Code: CM862 Broth to 9ml of 0.5% KOH, vortex mixing, and
Formula gm/litre plating onto Listeria Selective Agar plates.
Tryptone soya broth 30.0 Note:
Yeast extract 6.0 Suitable Listeria Selective Media are:
pH 7.3 + 0.2 1 Listeria Selective Medium (Oxford formulation)
(Oxoid CM856 and Oxoid SR140).
LISTERIA SELECTIVE ENRICHMENT 2 Listeria Selective Medium (MOX) (Oxoid CM856
SUPPLEMENT and Oxoid SR140).
Code: SR141 3 PALCAM Medium (Oxoid CM877 and Oxoid
SR150).
Vial contents (each vial is sufficient for 500ml of Storage conditions and Shelf life
medium) Store the dehydrated medium below 258C and use
Nalidixic acid 20.0mg before the expiry date on the label.
Cycloheximide 25.0mg
Acriflavine hydrochloride 7.5mg Store the prepared medium at 2±88C.
Directions Quality Control
Suspend 18g in 500ml of distilled water. Sterilise by Positive control:
autoclaving at 1218C for 15 minutes. Cool to 508C and Listeria monocytogenes ATCC1 19117
aseptically add the contents of one vial of Listeria
Negative control:
Selective Enrichment Supplement SR141,
Staphylococcus aureus ATCC1 25923
reconstituted with 2ml of sterile distilled water. Mix
well and distribute into sterile containers in volumes Precautions
as required. Note the precautions stated under Listeria Selective
Listeria Selective Enrichment Supplement (modified Medium (Oxford) CM856 and SR140. Broth cultures
with 10 mg/litre of Acriflavine) Code: SR149. are more dangerous than colonies on agar plates.
Vial contents (each vial is sufficient to supplement Store prepared medium away from light. Acriflavine
2.25 litres of CM862). can photo-oxidise to form inhibitory compounds
against listeria.
Nalidixic acid 90.0mg
Cycloheximide 112.5mg Supplement SR141 used in this medium contains a
Acriflavine hydrochloride 22.5mg toxic concentration of cycloheximide. Note the
precautions to be taken under HAZARDS page 2±7.
Directions
Aseptically add 10ml of sterile distilled water to one References
vial and invert gently to dissolve. Aseptically add the 1 Lovett J., Francis D. W. and Hunt J. M. (1987) Journal of Food
vial contents to 2.25 litres of Listeria Enrichment Broth Protection 50. 188±192.
Base (CM862), cooled to 508C. 2 Agello G., Hayes P. and Feeley J. (1986) Abstracts of the Annual
Meeting, ASM, Washington DC p5.
Description
Listeria Selective Enrichment Medium is based on the
formulation described by Lovett et al.1 and is
recommended for the selective enrichment of Listeria LISTERIA ENRICHMENT BROTH
species from food. The enrichment procedure has BASE
been shown to recover an inoculum of less than
10cfu/ml from raw milk. (UVM FORMULATION)
In order to achieve a higher isolation rate it is Code: CM863
recommended that the enrichment broth is Formula gm/litre
subcultured onto Listeria Selective Agar plates after 1, Proteose peptone 5.0
2 and 7 days. Agello et al.2, have shown that Tryptone 5.0
extending the incubation period to 7 days allows `Lab-Lemco' powder 5.0
better recovery of environmentally stressed listeria Yeast extract 5.0
from milk and milk products. Sodium chloride 20.0
Technique Disodium hydrogen phosphate 12.0
1 Add 25g or 25ml samples to 225ml of Listeria Potassium dihydrogen phosphate 1.35
Selective Enrichment Broth. Homogenise if Aesculin 1.0
required. pH 7.4 + 0.2
2 Incubate at 308C for 7 days.
6 McLain D. and Lee W.H. (1989) FSIS Method for the isolation and blackening. Another possible advantage to the
identification of Listeria monocytogenes from processed meat and addition of ferric ammonium citrate is that it has been
poultry products. Laboratory Communications number 57. shown that ferric ions enhance the growth of L.
monocytogenes3.
Lithium chloride is included in the medium to inhibit
FRASER BROTH the growth of enterococci which can also hydrolyse
Code: CM895 aesculin.
A secondary selective diagnostic enrichment medium for Care must be taken when using Fraser Broth with
the isolation of Listeria spp. from food and DNA probe methodology because the high salt
environmental specimens. content of the medium may have an inhibitory effect
on detection4.
Formula gm/litre
Proteose peptone 5.0 Technique
Tryptone 5.0 1 Inoculate 10ml of Fraser Broth with 0.1ml of the
`Lab-Lemco' powder 5.0 primary enrichment broth (i.e. FDA or UVM I
Yeast extract 5.0 enrichment broth) which has been incubated for 20
Sodium chloride 20.0 to 24 hours.
Di-sodium hydrogen phosphate 12.0 2 Incubate at 358C for 26 + 2 hours in air.
Potassium dihydrogen phosphate 1.35 3 Compare each inoculated tube to an inoculated
Aesculin 1.0 control against a white background. Tubes that
Lithium chloride 3.0 darken or turn black should be subcultured on to
pH 7.2 + 0.2 Oxford Medium, Modified Oxford Medium (MOX)
or PALCAM Medium. Tubes that retain the
FRASER SUPPLEMENT original yellow colour should also be inoculated on
plating media and confirmed as free from Listeria
Code: SR156 spp. before discarding.
Vial contents (each vial is sufficient to supplement It should be emphasised that the incubation period
500ml of medium) should be controlled. Fraser Medium should be
Ferric ammonium citrate 0.25g incubated for 26 + 2 hours to ensure at least 24 hours
Nalidixic acid 10.0mg incubation period to permit the development of the
Acriflavine hydrochloride 12.5mg black colour.
Directions Storage conditions and Shelf life
Suspend 28.7g in 500ml of distilled water. Sterilise by Store the dehydrated medium below 258C and use
autoclaving at 1218C for 15 minutes. Cool to 508C and before the expiry date on the label.
aseptically add the contents of one vial of Fraser
Store the selective supplement in the dark at 28C to
Selective Supplement SR156 reconstituted with 5ml of
88C and use before the expiry date on the label.
ethanol/sterile water (1:1). Mix well and distribute
into sterile containers. The prepared medium may be stored for up to 2
weeks at 28C to 88C.
Description
Fraser Medium is a modification of the USDA-FSIS
(United States Department of Agriculture-Food Safety Quality Control
Inspection Service) UVM secondary enrichment broth Positive control:
and is based on the formula described by Fraser and Listeria monocytogenes ATCC1 19117
Sperber1. It contains ferric ammonium citrate and Negative control:
lithium chloride. Blackening of the medium is Enterococcus faecalis ATCC1 29212
presumptive evidence of the presence of Listeria.
Contrary to early indications, cultures which do not References
blacken cannot be assumed to be Listeria-free. All 1 Fraser J.A. and Sperber W.H. (1988) J. Food Protect. 51, No.10,
Fraser Broth enrichment cultures should be 762±765.
subcultured to plating medium. 2 McClain D. and Lee W.H. (1988) J. Assoc. Off. Anal. Chem. 71,
The medium is intended for the isolation of Listeria NO.3, 660±664.
spp. from food and environmental samples when 3 Cowart R.E. and Foster B.G. (1985) J. Infect. Dis. 151, 721±730.
used as the secondary enrichment medium in the 4 Partis L., Newton K., Marby J. and Wells R.J. (1994) Appl. Env.
USDA-FSIS methodology for Listeria isolation. Microbiol. 60, 1693±1694.
To prepare 2.5 litres of medium, suspend 172.5g in 2.5 1 Inoculate one loopful of the selective enrichment
litres of distilled water. Sterilise and cool as above broth onto the PALCAM Medium plates.
and add the contents of one vial of SR150B, 2 Incubate at 378C for 48 hours under micro-
reconstituted with 10ml of sterile distilled water. aerophilic conditions. The micro-aerophilic
condition can be best achieved by using Oxoid
The addition of 2.5% (v/v) Egg Yolk Emulsion (Oxoid Campylobacter Gas Generating Kit (BR56) in
code SR47) to the medium may aid the recovery of
conjunction with the Oxoid Anaerobic Jar and an
damaged Listeria.
active catalyst (BR42). For jars of smaller capacity
(2.5 litres) use the Oxoid Campylobacter Gas
Description Generating Kit (BR60). Alternatively use
PALCAM Medium is based on the formulation CampyGen CN025A or CN035A. CampyGen does
described by Van Netten et al1 and is recommended not require the addition of water or a catalyst.
for the isolation of Listeria monocytogenes from foods.
3 Examine for typical colonies of Listeria after 48
The heightened awareness and concern surrounding hours incubation.
the presence of Listeria monocytogenes in food has 4 Colonies identified as presumptive Listeria species
resulted in the development of many media for its must be confirmed by biochemical and serological
isolation2±9. However, Cassiday and Brackett10 testing8.
conclude that no single method currently available is
suitable for use with all types of food. After 48 hours incubation, typical Listeria spp. form
colonies that are approximately 2mm in diameter,
PALCAM Medium is highly selective due to the grey-green in colour with a black sunken centre and a
presence of Lithium chloride, Ceftazidime, Polymixin black halo against a cherry-red medium background.
B and Acriflavine hydrochloride. It allows the easier
differential diagnosis of Listeria monocytogenes by Occasional Enterococcus or Staphylococcus strains
utilising the double indicator system: develop on PALCAM Medium to form grey colonies
Formula gm/litre
Infusion from fresh liver 23.0
Peptone 10.0
Potassium phosphate 1.0
Extracted liver tissue 30.0
pH 6.8 + 0.2 LYSINE DECARBOXYLASE BROTH
Directions (TAYLOR MODIFICATION)
Suspend 64 grams in 1 litre of distilled water and Code: CM308 (Tablets)
soak for 15 minutes, with occasional stirring.
Distribute into 18mm diameter tubes to a depth of To detect lysine decarboxylase production by
50mm so that the bottom of the tube is filled with salmonellae and some other Enterobacteriaceae.
liver particles. Agitate frequently during distribution Formula gm/litre
to keep the liver tissue in suspension. Sterilise by Yeast extract 3.0
autoclaving for 20 minutes at 1158C. Inoculate when Glucose 1.0
cool and then aseptically seal with a layer of sterile L-lysine 5.0
2% Oxoid Agar No.3 solution. Bromocresol purple 0.016
pH 6.1 + 0.2
Formula gm/litre One further useful property of this agar is its ability to
Tryptone 5.0 detect streptococcal mutants which are unable to
Soya peptone 5.0 ferment lactose1. These mutant Lac- strains form
`Lab-Lemco' powder 5.0 much smaller colonies than the parent lactose
Yeast extract 2.5 fermenting strain.
Ascorbic acid 0.5
Magnesium sulphate 0.25 Technique
Di-sodium-glycerophosphate 19.0 Bacteriophage assay.
Agar 11.0 Microbiologists wishing to assay phage activity
pH 6.9 + 0.2 should consult the paper of Terzaghi and Sandine1 for
a comprehensive description of the method.
Directions
Suspend 48.25g in 950ml of distilled water and bring For the enumeration of Streptococcus thermophilus
gently to the boil. Sterilise by autoclaving at 1218C for in yogurt.
15 minutes. Cool to 508C and add 50ml of sterile 1 Mix or blend the yogurt sample to obtain a
lactose solution (10% w/v). uniform homogenicity.
3 Windle Taylor E. (1958) `The examination of Waters and Water useful for the recognition of enterococci, in the
Supplies' 7th ed., Churchill Ltd., London. presence of coliforms and non-lactose fermenters from
4 Hoogendijk J. L. (1962) Antonie van Leeuwenhoek J. Microbiol. water, sewage, food products, etc.
Serol. 28(3) 315±320.
On this medium enterococci appear as small intensely
5 Wilson G. S. and Miles A. A. (1964) `Topley and Wilson's
red colonies with a pale periphery about 1mm in
Principles of Bacteriology and Immunity' 5th Ed., Edward Arnold
diameter. These organisms are frequently sought as
Ltd., London. vol.2.
an index of faecal pollution. Non-lactose fermenters
6 Stewart D. J. (1962) Nature 195(4845), 1023.
are colourless. Bile tolerant micrococci, such as
staphylococci and non-faecal streptococci, are
completely inhibited.
MACCONKEY AGAR
McGeachie & Kennedy1 employed Oxoid MacConkey
(WITHOUT SALT) Agar No.2 in a simplified method for counting the
Code: CM7b bacteria in urine. Using a bacteriological loop
(delivering a known volume) they streaked well
A differential medium on which swarming of Proteus mixed uncentrifuged urine directly on to a blood agar
species is suppressed. Recommended for urine and a MacConkey Agar plate ± and spread the urine
examination. in a 1cm wide strip across one edge of the plate using
Formula gm/litre 20 strokes. With a second sterile loop they spread a
Peptone 20.0 1cm wide portion to form a second strip at right
Lactose 10.0 angles to the first. This was repeated to give a square
Bile salts 5.0 pattern of four 1cm wide strips around the edge of
Neutral red 0.075 the plate. After incubation, growth was noted as +, +
Agar 12.0 +, + + +, or + + + + depending on whether 1, 2, 3 or 4
pH 7.4 + 0.2 sides of the square showed colonies. The approximate
estimate obtained agreed well with a more
Directions complicated pour-plate method and the simplified
Suspend 47g in 1 litre of distilled water. Bring to the method was recommended for routine use.
boil to dissolve completely. Sterilise by autoclaving at
Storage conditions and Shelf life
1218C for 15 minutes. Mix well before pouring. Dry Store the dehydrated medium below 258C and use
the surface of the gel before inoculation.
before the expiry date on the label.
Description
Store the prepared plates at 2±88C.
This medium has the same formulation as
MacConkey Agar CM7 except that it does not contain Quality Control
added salt and therefore provides a `low electrolyte Positive control:
medium' on which most Proteus species do not Enterococcus faecalis ATCC1 29212
spread. For this reason the medium has found Negative control:
particular favour for use in the examination of urine
Uninoculated medium
so that overgrowth of other organisms is prevented.
Reference
1 McGeachie J. and Kennedy A. C. (1963) J. Clin. Path. 16. 32±38.
a simple, inexpensive, rapid and reliable means of 6 Pai C. H., Gordon R., Sims H. V. and Bryant L. E. (1984) Ann.
screening Esch. coli O157. Intern. Med. 101. 738±742.
7 Waters J. R. (1985) Can. Dis. Weekly Rep. 11. 123±124.
Esch. coli O157 has recently been recognised as a cause
8 Doyle M. P. and Schoeni S. L. (1984) Appl. and Envir. Microbiol.
of haemorrhagic colitis, an illness characterised by
48. 855±856.
bloody diarrhoea and severe abdominal pain. There is
9 Karmali M. A. (1988) Culture 9. 2.
mounting evidence linking Esch. coli O157 and
10 Lior H. and Borcryk A. (1987) Lancet. i. 333.
haemorrhagic colitis with haemolytic uraemic
syndrome (HUS)3,4,5,6,7.
Technique
1 Make up the agar according to the directions and
pour into petri dishes. If necessary dry the surface
of the agar. CEFIXIME-TELLURITE
2 Inoculate the plates with a suspension of the food, SUPPLEMENT
faeces, etc. to produce separated colonies.
3 Incubate at 358C for 24 hours. Doyle and Schoeni8 Code: SR172
have reported that 35±378C is the optimal A freeze-dried supplement for use with Sorbitol
temperature for growth of Esch. coli O157. At 44 to MacConkey Agar, CM813, for the selective isolation of
45.58C this Esch. coli serotype does not grow well E. coli O157:H7.
even after 48 hours incubation.
Vial contents Milligrams Mg/litre
Delay in reading plates beyond 24 hours should be
avoided because the colour intensity of sorbitol- Potassium tellurite 1.25 2.5
fermenting colonies fades, reducing the contrast with Cefixime 0.025 0.05
non-fermenting colonies. Directions
Other Gram negative organisms including Aseptically add 2ml of sterile distilled water to 1 vial
Pseudomonas, Proteus and Klebsiella species are able to of Cefixime-Tellurite Supplement SR172E. Mix gently
grow on Sorbitol MacConkey Agar but may generally to dissolve the contents completely.
be differentiated by the appearance of their colonies. Add the vial contents to 500ml of Sorbitol
A diagnostic reagent Escherichia coli O157 latex test MacConkey Agar prepared as directed and cooled to
DR620 is available so that instant confirmatory tests 508C. Mix well and pour the medium into petri
can be made from suspicious colonies. dishes.
Colonial Morphology Description
Esch. coli O157 will form colourless but otherwise Chapman and co-workers1, added cefixime and
typical Esch. coli colonies. potassium tellurite to Sorbitol MacConkey Agar to
Storage conditions and Shelf life improve the selectivity of the medium. The level of
Store the dehydrated medium below 258C and use potassium tellurite selects serogroup O157 from other
before the expiry date on the label. E. coli serogroups and inhibits Providencia spp. and
Aeromonas spp. Cefixime is inhibitory to Proteus spp.
Store the prepared plates at 2±88C.
The use of cefixime and tellurite in Sorbitol
Quality Control
MacConkey Agar for isolation of E. coli O157:H7 is
Positive control:
described in the FDA Bacteriological Analytical
Escherichia coli O157
Manual2.
Negative control:
Escherichia coli ATCC1 25922 Storage conditions and Shelf life
Cefixime-Tellurite Supplement SR172 should be
Precautions stored in the dark at temperatures below 08C.
Although the great majority of Esch. coli O157 strains
have a typical appearance on Sorbitol MacConkey Prepared medium may be stored for up to 2 weeks in
Agar, some strains are atypical9. plastic bags.
Sorbitol MacConkey Agar cannot be used solely to Quality Control
detect VTEC strains of Esch. coli as some non-toxic Positive control:
strains will not ferment sorbitol10. E. coli O157 NCTC 12079
Negative control:
References E. coli ATCC1 25922
1 Rappaport F. and Henig E. (1952) J. Clin. Path. 5. 361.
2 March S. B. and Ratnam S. (1986) J. Clin. Microbiol. 23. 869±872. References
3 Centers for Disease Control 1985 ± United States, 1984, Morbid 1 Zadik P.M., Chapman P.A. and Siddons C.A. (1993) J. Med.
Mortal Weekly Rep., 34. 20±21. Microbiol. 39. 155±158.
4 Karmali M.A., Petric M., Lim C., Fleming P. C., Arbus G. S. and 2 Food and Drug Administration (1995) Bacteriological Analytical
Lior H. (1985) J. Infect. Dis. 151. 775±782. Manual. 8th Edition. AOAC International. Gaitherburg. MD.
5 Karmali M.A., Steele B.T., Petric M. and Lim C. (1983) Lancet i: Chapter 4, 20±23.
619±620.
358C for 48 hours. Choice of volumes for inoculation MALT EXTRACT AGAR
will depend on the bacteriological grade of the water
being tested; for `medium' waters the Public Health Code: CM59
Laboratory Service Water Committee (1961) A medium for the detection, isolation and enumeration
recommend one 50ml, five 10ml and five 1ml of yeasts and moulds. Bacteria may be suppressed by the
quantities of water ± 50ml and 10ml amounts being addition of lactic acid.
added to their own volume of double-strength
MacConkey Broth while the 1ml amounts are each Formula gm/litre
added to 5ml of single-strength MacConkey Broth. Malt extract 30.0
Acid formation is indicated by a yellow colouration of Mycological peptone 5.0
the broth, and gas formation is indicated by an Agar 15.0
amount of gas at least sufficient to fill the concavity at pH 5.4 + 0.2
the top of the Durham tube. From the number of Directions
tubes showing the presence of acid and gas, the most Suspend 50 grams in 1 litre of distilled water and boil
probable number of (presumed) coliform bacteria to dissolve. Sterilise by autoclaving at 1158C for 10
present in 100ml of the original water may be minutes.
estimated by reference to probability tables; these
tables based on McCrady's computations, are If it is desired to adjust the reaction to pH 3.5, cool to
included in Report No.71: `The Bacteriological 558C and add 10% Lactic Acid SR21 to the Malt
Examination of Water Supplies'2 and in many other Extract Agar. Once acidified with lactic acid, the
publications dealing with this subject. For the medium should not be re-heated.
differential coliform test, each MacConkey tube Description
showing acid and gas is then subcultured into a fresh This medium, similar to the one described by
tube of MacConkey Broth and incubated at 448C. Galloway & Burgess1 is recommended for the
Formation of gas within 48 hours is practically detection, isolation and enumeration of yeasts and
specific for Escherichia coli and indicative of faecal moulds. For mycological counts it may be desirable to
pollution of the original water sample. prepare the more acid medium in order to suppress
MacConkey Broth (Purple) is also suitable for the bacterial growth.
bacteriological examination of milk, as described by Also see Wort Agar.
Davis4. This method, which is basically similar to that
used for the examination of water, consisting in the
inoculation of suitable dilutions of the milk into tubes
of this medium followed by incubation and
inspection, was originally recommended by the Dept.
of Health, London5. MALT EXTRACT BROTH
MUG Reagent BR71 ± The addition of Code: CM57
4-methylumbelliferyl-b-D-glucuronide (MUG) BR71 A liquid medium recommended for the cultivation of
to this medium will enhance the detection of moulds and yeasts, especially during tests for sterility.
Escherichia coli. See MUG Reagent BR71 under
Biochemical Reagents for further details. Formula gm/litre
Malt extract 17.0
Storage conditions and Shelf life
Mycological peptone 3.0
Store the dehydrated medium below 258C and use
pH 5.4 + 0.2
before the expiry date on the label.
Directions
Quality Control
Add 20g to 1 litre of distilled water. Mix well,
Positive control:
distribute into final containers and sterilise by
Escherichia coli (Acid + Gas) ATCC1 25922
autoclaving at 1158C for 10 minutes. This liquid
Negative control: medium is recommended for the cultivation of
Staphylococcus aureus ATCC1 25923 moulds and yeasts, during tests for sterility, etc.
Storage conditions and Shelf life
References Store the dehydrated medium below 258C and use
1 Childs Eileen and Allen L. A. (1953) J. Hyg. Camb. 51(4). 468±477.
before the expiry date on the label.
2 Departments of the Environment, Health, Social Security and
Public Health Laboratory Service (1982) The Bacteriological Store the prepared medium at 2±88C.
Examination of Drinking Water Supplies. Report No. 71. HMSO Quality Control
London. Positive control:
3 World Health Organization (1963) `International Standards for Aspergillus niger ATCC1 9642
Drinking Water' 2nd ed., WHO, Geneva. Candida albicans ATCC110231
4 Davis J. G. (1959) `Milk Testing' 2nd ed., Dairy Industries Ltd.,
London. Negative control:
5 Dept. of Health (1937) Memo. 139/Foods, HMSO, London. Bacillus cereus (at pH 3.5) ATCC1 10876
Precautions
Avoid overheating as the acid pH will soften the agar
in the presence of heat.
November 1998 2-143
Culture Media
colony size and hence facilitates carrying out the 2 American Public Health Association (1980) Standard Methods for
colony count. the Examination of Water and Wastewater. 15th Edn, APHA Inc,
Washington DC.
Technique 3 McCarthy J.A., Thomas H.A.J., Delaney J.E. (1958) `Evaluation of
The water sample is filtered through a sterile the Reliability of Coliform Density Tests'. AJPH, 48. 16±28.
membrane filter6. 4 Calabrese J.P. and Bissonnette G.M. (1990) Appl. Env. Microbiol.
For the first stage of enrichment, place a sterile 56. 3558±3564.
5 Noble R.E. (1960) `Reliability of MPN Indexes for Coliform
incubating pad in the upper half of a sterile petri dish
and pipette onto this 2ml of Lauryl Tryptose Broth organisms'. JAWWA, 52. 803.
CM451. Aseptically place the filter membrane on to 6 Departments of the Environment, Health & Social Security and
PHLS (1982) The Bacteriological Examination of Drinking Water
the incubating pad and incubate, without inverting
the dish, for 1±1.5 hours at 358C in an atmosphere Supplies. Report on Public Health and Medical Subjects No.71.
having 100% humidity. Place petri dishes of M-Endo HMSO. London.
Agar LES in the incubator for the entire period so that
they will be at the correct temperature when required
for the second stage of enrichment. The first stage
enrichment culture is removed from the incubator
and the filter membrane is stripped aseptically from MEMBRANE LAURYL
the incubating pad and transferred to the surface of SULPHATE BROTH
the petri dish of M-Endo Agar LES. It is important
that complete contact is made between the membrane Code: MM615
and the agar surface. The plate is inverted and A replacement medium for Membrane Enriched Teepol
incubated for 22±24 hours at 358C. Broth for the enumeration of coliform organisms and
Alternatively, the membrane filter incubating pad can Escherichia coli in water.
be placed inside the lid of the petri dish of M-Endo Formula gm/litre
Agar LES and 2ml of Lauryl Tryptose Broth CM451 Peptone 39.0
pipetted onto the pad. The filter membrane is placed Yeast extract 6.0
face upwards on the pad and incubated for 1±1.5 Lactose 30.0
hours at 358C. Phenol red 0.2
To carry out the second stage of enrichment, the first- Sodium lauryl sulphate 1.0
stage enrichment is removed from the incubator and pH 7.4 + 0.2
the filter membrane is stripped from the pad and Directions
placed face upwards on the surface of the M-Endo Dissolve 76.2 grams in 1 litre of distilled water.
Agar LES medium. The incubating pad is left in the Distribute into final containers, e.g. 100ml screw cap
lid and the plates are incubated in the inverted bottles. Sterilise by steaming for 30 minutes on three
position for 24 hours at 358C. consecutive days or by autoclaving at 1218C for 15
If preferred the second stage only may be used. The minutes.
prepared membrane filter is placed directly on the Description
agar surface and incubated as described. In formulating Membrane Enriched Teepol Broth,
All the organisms which produce a colony with a Burman1 substituted Teepol in place of bile salts in
golden-green metallic sheen within 24 hours the membrane filtration test medium used to detect
incubation may be considered as presumptive coliform organisms in water. The use of Teepol in
coliforms. place of bile salts had been previously recommended
by Jameson and Emberley2 and its value was
Calculation of Coliform Density confirmed by other workers (Jebb3 and Windle-
Report the coliform density in terms of total coliform/ Taylor4,5). It is essential to use one standard grade of
100ml. Compute the count using those membrane Teepol and Teepol 610 (BDH Ltd.) has been
filters with 20±80 coliform colonies and not more than recommended.
200 of all types per membrane.
Burman6 showed that resuscitation media are not
Total Coliform Coliform colonies x 100 required with Membrane Enriched Teepol Broth if a
=
colonies/100ml ml of sample filtered preliminary incubation is carried out at a lower
Storage conditions and Shelf life temperature. Thus non-chlorinated organisms benefit
Store the dehydrated medium below 258C and use from 4 hours incubation at 308C, but chlorinated
before the expiry date on the label. organisms require 6 hours incubation at 258C.
Store the prepared medium in the dark and at 2±88C. Membrane Lauryl Sulphate Broth is similar to
Membrane Enriched Teepol Broth except that the
Precautions
selective agent Teepol 610 has been replaced by
Use care when handling basic fuchsin to avoid
sodium lauryl sulphate.
inhaling the powder and staining the skin.
In 1976 the production of Teepol 610 ceased and
References studies were carried out to identify a suitable
1 McCarthy J.A., Delaney J.E., Grasso R.J. (1961) `Measuring alternative selective agent that could be incorporated
Coliforms in Water', Water and Sewage Works, 108. 238±243. into the basal medium. As a result of this work it was
This latter medium was incorporated in another large Papadakis10 investigated the isolation of Esch. coli
trial carried out by the PHLS6 in which three from sea-water and found Minerals Modified
glutamate media were compared with Teepol Broth Glutamate Medium to be better than MacConkey
(Jameson & Emberly7) and MacConkey Broth. The Broth formulations. However, to avoid high salt
results showed that Gray's improved formate lactose concentrations in the broth he recommended 1ml only
glutamate medium was superior to the other of sea-water to be added to 10ml of single-strength
glutamate media on trial. MMG medium. Higher volumes of sea-water must be
diluted out 1/10 with MMG medium.
The report carried criticism of the mineral content of
the medium and it was considered that it could be Technique
improved by modifying the amounts of minerals. The technique known as the Multiple Tube Method,
Dilution Method or the Most Probable Number
A co-operative investigation was carried out between
the Metropolitan Water Board Laboratories and (MPN) method is used with Minerals Modified
Glutamate Medium. A trial comparing membrane
Oxoid Laboratories which resulted in a Minerals
filtration and multiple tube methods showed
Modified Glutamate Medium CM289.
glutamate medium to is unsatisfactory for use with
The Oxoid Minerals Modified Glutamate Medium membranes for enumerating coliform organisms in
was used in further PHLS6 trials and the results with water11.
the Oxoid medium confirmed the superior
With waters expected to be of good quality, the
performance of glutamate media reported previously
(PHLS6). medium should be inoculated with one 50ml volume
and five 10ml volumes. With waters of more doubtful
The superior performance of Minerals Modified quality, five 1ml volumes should be used in addition
Glutamate Medium over MacConkey Broth is due to the 50ml and 10ml volumes. Dilutions of the 1ml
mainly to improved detection of Escherichia coli. The volumes may be required for polluted water and the
table (adapated from PHLS8) illustrates the results 50ml volume may be omitted.
obtained in the trial.
The larger volumes of water (10ml and 50ml) are
The table shows that for chlorinated water, incubation added to equal volumes of double-strength medium,
for >18 hours is required for glutamate media to whereas the 1ml volumes (or dilutions of them) are
demonstrate their superiority. added to 5ml of single-strength medium.
The medium and method are fully described in Her The tubes are incubated at 358C and examined after
Majesty's Stationery Office Report 711. 18±24 hours. All those tubes showing acid (yellow
More recently further trials showed Minerals colour in the medium) and gas in the inverted inner
Modified Glutamate Medium to be the medium of (Durham) tube should be regarded as `presumptive
choice for the detection of Esch. coli in chlorinated positive' tubes, including those in which gas appears
waters, especially where the numbers of organisms after tapping the tube. The tube may only have a
concerned were small. bubble of gas after tapping. The remaining tubes
should be re-incubated and examined after another 24
It was also found better than Lauryl Tryptose Lactose hours. Any further tubes becoming `positive' should
Broth for detection of small numbers of Esch. coli in be treated as `presumptive positives'.
other water, although the latter medium gave quicker
results (18±24 hours compared to the 48 hours Each `presumptive positive' tube should be sub-
required by Minerals Modified Glutamate Medium). cultured to a tube of Brilliant Green Bile (2%) Broth
CM31 and incubated for 24 hours at 448C.
Unchlorinated samples
MacConkey Broth 17 37 100 625 806 1060 467 528 582
Minerals Modified
Glutamate Medium 2 20 97 557 858 1175 503 707 764
Chlorinated samples
MacConkey Broth 4 19 49 125 216 315 77 121 128
Minerals Modified
Glutamate Medium 0 1 37 59 223 395 39 144 203
The concentration of Mg++ appears to be critical for Pick all colonies presumed to be Salmonella spp. and
maximum growth of salmonellae on MLCB Agar. van confirm by biochemical and serological testing.
Schothorst et al.2 showed that Oxoid MLCB Agar did Storage conditions and Shelf life
not inhibit any of the Salmonellae species Store the dehydrated medium below 258C and use
investigated. before the expiry date on the label.
Salmonellae serotypes that have a high incidence of Store the prepared plates of medium at 2±88C.
H2S-negative strains e.g. S. sendai, S. berta, S. pullorum
and S. senftenberg may produce atypical pale colonies. Quality Control
MLCB Agar is not suitable for S. typhi and S. paratyphi Positive control:
A. because of the inhibitory concentration of brilliant Salmonella virchow NCTC 5742
green. Negative control:
The medium may be inoculated directly with the Escherichia coli ATCC1 25922
specimen or from an enrichment culture. Selectivity is
Precautions
relatively weak and its performance may be adversely
The identity of colonies presumed from their
effected by heavily contaminated specimens. Because
appearance to be Salmonella spp. must be confirmed
of these limitations MLCB Agar should not be used
by biochemical and serological testing. In common
alone.
with other enteric media care must be taken to ensure
MLCB Agar is specified as a plating medium the purity of colonies taken for further testing as
following enrichment in Modified Semi-Solid organisms that are inhibited from developing into
Rappaport Vassiliadis (MSRV) for isolation of colonies remain viable and may accidentally be
Salmonella spp. from human faeces3. picked on sub-culture.
van Schothorst et al.2 reported MLCB Agar to be
excellent for the isolation of H2S-positive salmonellae Reference
1 Takao Inoue et al. (1968) Proceedings of the Japanese Society of
after enrichment in Rappaport-Vassiliadis (RV)
Veterinary Science. Number 169. Jap. J. Vet. Sci. 30.
Enrichment Broth CM669. They found that the
2 van Schothorst M., Renaud A. and van Beek C. (1987) Food
selectivity of MLCB Agar was substantially increased
Microbiol. 4. 11±18.
after RV broth enrichment. They suggested Brilliant
3 Aspinall S.T., Hindle M.A. and Hutchinson D.N. (1992) Eur. J.
Green Agar and MLCB Agar should be used when
Clin. Microbiol. Inf. Dis. 11. 936±939.
examining heavily contaminated samples.
Salmonellae grow as large purple-black colonies due
to hydrogen sulphide production. Mannitol is utilised
by the organism and the resultant pH fall initiates
lysine decarboxylation which controls further MODIFIED LAURYL SULPHATE
downward pH movement and promotes blackening.
TRYPTOSE BROTH WITH MUG
MLCB Agar does not depend on lactose fermentation
and is therefore recommended when investigating Code: CM967
lactose-fermenting salmonellae (Salm. arizona). A modified Lauryl Tryptose Broth, incorporating
Atypical Salmonella strains that produce little or no 4-methylumbelliferyl-b-D-glucuronide (MUG) allowing
hydrogen sulphide grow as mauve-grey colonies and the enumeration of presumptive Escherichia coli as well
may develop a central black `bulls eye'. as other coliforms, using the Most Probable Number
(MPN) method.
To assist the detection of these atypical strains
Brilliant Green Agar (modified) CM329 or Bismuth Formula gm/litre
Sulphite Agar CM201 should also be used. Tryptose 20.0
Lactose 5.0
Gram-positive and most Gram-negative organisms Dipotassium hydrogen phosphate 2.75
are inhibited although some strains of Citrobacter spp. Potassium dihydrogen phosphate 2.75
may grow sufficiently well to mimic the appearance Sodium chloride 5.0
of Salmonella spp. and some Proteus spp. may swarm. Sodium lauryl sulphate 0.1
Most contaminating organisms that are able to grow 4-methylumbelliferyl-b-D-glucuronide
develop as small colourless colonies. (MUG) 0.1
Tryptophan 1.0
Technique
pH 6.8 + 0.2
Dry the surface of the agar before use.
Directions
Inoculate the medium heavily with the specimen or
Dissolve 36.7g of Modified Lauryl Sulphate Tryptose
enrichment culture and incubate for 18±24 hours at Broth with MUG CM967 in 1 litre of distilled water.
358C.
Dispense into final containers containing Durham
Examine for typical large purple-black colonies of H2S
tubes.
positive salmonella. Search carefully for H2S negative
strains that atypically grow as large mauve-grey Sterilise by autoclaving at 1218C for 15 minutes.
colonies with a cratered centre. A proportion may 500g of this medium makes 13.3 litres of single
show a black `bulls eye'. strength medium.
Description References
Oxoid Modified Lauryl Sulphate Tryptose Broth with 1 IDF-170L (1994) Milie & Milie products. Enumeration of
MUG CM967 is formulated to allow use of the MPN presumptive Escherichia coli.
technique for coliforms and also the enumeration of 2 ISO-11866±2: (1997) (E).
presumptive E. coli by means of a culture technique
involving a liquid medium containing MUG.
The formulation contains 4-methylumbelliferyl-b-D- MODIFIED SEMI-SOLID
glucuronide (MUG), which is cleaved by the enzyme RAPPAPORT VASSILIADIS (MSRV)
b-glucuronide to release 4-methylumbelliferone, a MEDIUM BASE
blue-green fluorophore exhibiting blue-green
fluorescence visible when viewed under long wave Code: CM910
ultra-violet (366nm). The inclusion of tryptophan acts A semi-solid medium for the detection of motile
as a substrate for indole production. Both reactions
Salmonella spp. from food and environmental samples.
are characteristic of E. coli and can therefore be used
to identify presumptive E. coli. Formula gm/litre
Coliform organisms will ferment lactose to produce Tryptose 4.59
gas. This production of gas can be taken as positive Casein hydrolysate 4.59
for the presence of coliforms. Sodium chloride 7.34
Potassium dihydrogen phosphate 1.47
Magnesium chloride (anhydrous) 10.93
Technique Malachite green oxalate 0.037
Prepare a sufficient number of dilutions of original Agar 2.7
sample to ensure tubes for the final dilution will yield pH 5.2 + 0.2
a negative result. Inoculate each dilution into tubes of
Modified Lauryl Sulphate Tryptose Broth with MUG
containing inverted Durham tubes. For the MPN MSRV SELECTIVE SUPPLEMENT
technique, inoculate each dilution in triplicate. Code: SR161
Incubate the tubes at 308C for 24±48 hours. Examine Vial contents (each vial is sufficient to supplement
the tubes for growth turbidity, gas production, 500ml of MSRV medium base)
fluorescence and formation of indole. Read as follows: Novobiocin 10mg
1 Tubes showing fluorescence gas and formation of Directions
indole indicate presumptive E. coli. Suspend 15.8g of MSRV Medium Base in 500ml of
2 Tubes showing gas formation indicate coliforms. distilled water. Bring to the boil with frequent
The MPN index can be determined from the numbers agitation. DO NOT AUTOCLAVE. Cool to 508C and
of positive tubes of selected dilutions by means of an aseptically add the contents of 1 vial of MSRV
MPB table, and a calculation of the MPN of Selective Supplement reconstituted with 2ml of sterile
presumptive E. coli or coliforms per gram or per distilled water. Mix well and pour into sterile petri
millilitre of the original sample carried out. dishes. Air dry at room temperature for at least one
hour. (Plates may be air-dried overnight prior to
storage at 28C to 88C.)
Storage conditions and Shelf life
Modified Lauryl Sulphate Tryptose Broth with MUG Description
CM967 should be stored tightly capped in the original Modified Semi-solid Rappaport Vassiliadis (MSRV)
container at 108C±258C. When stored as directed the Medium is based on the formulation described by De
medium will remain stable until the expiry date Smedt et al which has been shown to detect more
printed on the bottle. Salmonella-positive samples than the traditional
enrichment procedures1,2. Further collaborative
studies have confirmed these findings3,4.
Quality Control
Positive control: Motility enrichment on MSRV Medium has been
Escherichia coli ATCC1 25922 designed as a simple, sensitive method for the
isolation of salmonellae from food and environmental
Negative control: samples. The efficiency of the medium is based on the
Staphylococcus aureus ATCC1 25923 ability of salmonellae to migrate through the selective
Enterobacter aerogenes ATCC1 13048* medium ahead of competing motile organisms, thus
* also MUG-ve. producing opaque halos of growth.
Further tests can be carried out directly from the
Precautions migrated culture with the inoculum being taken from
Modified Lauryl Sulphate Tryptose Broth with MUG the edge of the growth. The Oxoid Salmonella Latex
CM967 should only be used for in vitro diagnostic Test (FT203) is recommended for serological
purposes. confirmation of Salmonella species.
Do not use beyond the stated expiry date, or if the The medium is not suitable for the detection of non-
product is caked, discoloured or shows any signs of motile strains of Salmonella (incidence <0.1%)5.
deterioration.
(Figures obtained from records of the Department of
MRS Agar with sorbic acid has been described3,4. This 3 Reuter G. (1985) Intern. J. Food Microbiol. 2. 55±68.
is MRS medium with its pH reduced to 5.7 and 0.14% 4 ISO/TC 34/SC 6/WG 15, No.3 and 5 (1984) Enumeration of
w/v sorbic acid added (=0.2% w/v potassium Lactobacteriaceae in meat and meat products.
sorbate). 5 Lankaputhra W.E.V., Shah N.P. and Britz M.L. (1996) Food
Australia 48. 113±118.
An evaluation of media for selective enumeration of
Lactobacillus acidophilus and Bifidobacterium species
showed that minor adjustments to the basic formula
of MRS Agar can readily be made to optimise its
performance for determining the content of
L. acidophilus and Bifidobacterium spp. in the presence MRS BROTH (DE MAN, ROGOSA,
of other lactic acid bacteria which are present in SHARPE)
yoghurt5.
Code: CM359
The lactobacilli are micro-aerophilic and generally
require layer plates for aerobic cultivation on solid A non-selective medium for profuse growth of `lactic
media. Submerged or surface colonies may be acid bacteria'.
compact or feathery, and are small, opaque and Formula gm/litre
white. Peptone 10.0
Technique `Lab-Lemco' powder 8.0
Products to be examined for lactobacilli content are Yeast extract 4.0
macerated or diluted in a diluent such as quarter- Glucose 20.0
strength Ringer solution, and further dilutions are Sorbitan mono-oleate 1ml
made in MRS Broth. Dipotassium hydrogen phosphate 2.0
Sodium acetate 3H2O 5.0
1ml volumes of the diluted samples are added to Triammonium citrate 2.0
sterile petri dishes, and molten MRS Agar (458C) is Magnesium sulphate 7H2O 0.2
poured into the dish and mixed thoroughly. Manganese sulphate 4H2O 0.05
When the medium has set, another layer of pH 6.2 + 0.2
uninoculated MRS Agar is poured over the surface to Directions
produce a layer-plate. Add 52 grams to 1 litre of distilled water at 608C. Mix
Plates are incubated as described below. It is until completely dissolved. Dispense into final
important that adequate moisture vapour is present containers and sterilise by autoclaving at 1218C for 15
in the atmosphere above the agar because drying of minutes.
the plates during incubation will concentrate the Description
selective factors on the surface and make the medium MRS Broth may be used for tests in the identification
inhibitory. The presence of carbon dioxide stimulates of lactobacilli, such as temperature dependence,
growth and plates should be incubated in an growth in 4% NaCl, growth in 0.4% Teepol, etc. as
atmosphere of 5% CO2. MRS medium is selective for recommended by Sharpe, Fryer and Smith1.
lactobacilli but some growth of leuconostocs and
pediococci may occur. Storage conditions and Shelf life
Store the dehydrated medium below 258C and use
Incubation method before the expiry date on the label.
428C thermophilic: 2 days
358C mesophilic: 2 days Store the prepared medium at 2±88C.
308C + 228C mesophilic-psychrotrophic: 2+1 days Quality Control
258C psychrotrophic: 3 days Positive control:
Incubation carried out under anaerobic or micro- Lactobacillus gasseri ATCC1 19992
aerophilic conditions Negative control:
Select isolated colonies on the agar medium and stain Staphylococcus aureus ATCC1 25923
a smear from each to identify the presumptive
Lactobacillus colonies; pick these off into MRS Broth. Reference
An advantage of this broth is that any other micro- 1 Sharpe M. Elisabeth, Fryer T. F. and Smith D. G. (1966)
organisms, originally lying dormant in the selective `Identification of the Lactic Acid Bacteria' in `Identification Method for
agar, are not given the opportunity to multiply, as Microbiologists Part A' (Gibbs B. M. and Skinner F. A. eds.) London
may occur in a non-selective broth. Incubate the and New York, Academic Press. Pages 65±79.
broths at temperatures and times similar to those
used for the MRS Agar; they can then be examined
microscopically and further sub-cultured to MRS
Agar for subsequent confirmation and identification
of species.
References
1 de Man J. C., Rogosa M. and Sharpe M. Elisabeth (1960) Appl.
Bact. 23. 130±135.
2 Briggs M. (1953) J. Dairy Res. 20. 36±40.
MRVP MEDIUM Incubate not less than 48 hours at 358C for the MR
test but more usually 3±5 days at 308C. A heavy
Code: CM43 inoculum and 18±24 hours incubation at 358C may
A medium recommended for the Methyl-red and Voges- give a rapid result10. A rapid VP test may be carried
Proskauer tests for the differentiation of the coli- out from a heavy inoculum and incubation in a water
aerogenes group. bath at 358C for 4±5 hours. Some organisms (Hafnia
alvei) require incubation at 258C to give a positive VP
Formula gm/litre test.
Peptone 5.0
Glucose 5.0 After incubation, test one portion of the broth with 5
drops of 0.4% w/v methyl-red solution and read the
Phosphate buffer 5.0
colour on the surface of the medium immediately.
pH 7.5 + 0.2
The second portion of the broth is used for the VP
Directions
reaction by one of the following methods:
Add 15g to 1 litre of distilled water. Mix well,
distribute into final containers and sterilise by 1 Add 3ml of 5% w/v alcoholic a-naphthol solution
autoclaving at 1218C for 15 minutes. and 3 ml of 40% w/v KOH solution (Barritt's
method12).
Description
This glucose-phosphate medium is recommended for 2 Add a trace amount of creatine (2 drops of a 0.3%
the Methyl-red and Voges-Proskauer tests, for the w/v solution) and 5ml of 40% KOH solution
differentiation of the coli-aerogenes group1. (O'Meara's method 13).
Smith2 noted the low acid production of Enterobacter A bright pink or eosin red colour will appear after
aerogenes cultures as compared with those of gentle shaking for 30 seconds. A pink colour is
Escherichia coli. Clark & Lubs3 employed methyl-red positive; no colour is negative.
as a hydrogen-ion concentration indicator in order to Barry & Feeney14 obtained rapid results by adding
differentiate glucose phosphate peptone water creatine to Barritt's reagents.
cultures of members of the coli-typhoid group. This
Storage conditions and Shelf life
test, now known as the Methyl-red test, distinguishes
Store the dehydrated medium below 258C and use
those organisms able to form large amounts of acid
before the expiry date on the label.
from glucose so that the pH falls below 4.4 and those
organisms which cannot produce a low pH level. Store the prepared medium at 2±68C.
The difference in pH value is visualised by adding Quality Control
methyl-red to the culture, (<pH 4.4 red: pH 5.0±5.8 Positive MR:
orange: >pH 6.0 yellow). Escherichia coli ATCC1 25922
7 Durham H. E. (1900±1901) J. Exper. Med. 5. 353±388. 3 Differences in the characteristics of the agar used in
8 Levine M. (1916a) J. Bact. 1. 87. the medium, especially diffusion properties15.
9 Levine M. (1916b) J. Bact. 1. 153±164. In the light of such criticisms the NCCLS called
10 Barry A. L., Bernsohn K. L., Adams A. B. and Thrupp L. D. interested manufacturers together to discuss the
(1970) Appl. Microbiol. 20. 866±870. standardisation and stabilisation of Mueller-Hinton
11 Cowan S. T. & Steel K. J. (1966) Manual for the Identification of Agar. Control methods were established whereby
Medical Bacteria. Cambridge University Press. pp. 32±33. critical antimicrobial/organism combinations had to
12 Barritt M. M. (1936) J. Path. Bact. 42. 441±454. yield consistent zones of inhibition within 2mm of the
13 O'Meara R. A. Q. (1931) J. Path. Bact. 34. 401- specified diameters in the standards6.
14 Barry A. L. and Feeney K. L. (1967) Appl. Microbiol. 15. 1138±
1141. The result of this cooperative effort is that Mueller-
15 Vaughn R., Mitchell N. B. and Levine M. (1939) J. Amer. Water Hinton Agar is now a standard medium and declares
Works Assoc. 31. 993- on the label that it conforms to the NCCLS standard
M6-A.
MUELLER-HINTON AGAR `This lot meets the NCCLS standard M6-A for
dehydrated Mueller-Hinton agar.'
Code: CM337
For further details of antimicrobial susceptibility
An antimicrobial susceptibility testing medium which testing see Section 6.
may be used in internationally recognised standard
procedures. Mueller-Hinton Agar supplemented with yeast, NAD
and haematin is used specifically for the susceptibility
Formula* gm/litre testing of Haemophilus influenzae16. For further details
Beef, dehydrated infusion from 300.0 see Haemophilus Test Medium (HTM), CM989.
Casein hydrolysate 17.5
Starch 1.5 Mueller-Hinton Agar and Broth are used as the basis
Agar 17.0 of solid and liquid media containing cefoperazone,
pH 7.4 + 0.2 trimethoprim, piperacillin and cycloheximide for
selective isolation of Arcobacter spp. from meats16.
* modified to meet performance standards.
Storage conditions and Shelf life
Directions Store the dehydrated medium below 258C and use
Add 38 grams to 1 litre of distilled water. Bring to the before the expiry date on the label.
boil to dissolve the medium completely. Sterilise by
autoclaving at 1218C for 15 minutes. Store the prepared plates at 2±88C.
5 National Committee for Clinical Laboratory Standards (1990) used in microtitre plates in an agar dilution method
Performance Standards for Antimicrobial Disk Susceptibility Tests. for determining the MIC for Helicobacter pylori of a
4th Edn. Approved Standard M2-A4. NCCLS. Villanova Pa. number of antibiotics. The method does not require
6 Pollock H. M. et al. (1986) J. Clin. Microbiol. 24. 1±6. prolonged incubation in carbon dioxide-enriched air
7 WHO (1961) Standardization of Methods for Conducting Microbic and results are available in 48 hours compared to 3±4
Sensitivity Tests. Geneva. Tech. Rep. Ser. No.210. days for agar diffusion testing on solid medium4.
8 WHO (1977) Expert Committee on Biological Standardization.
For further details of antimicrobial susceptibility
Geneva. Tech. Rep. Ser. No.610.
testing see Section 6.
9 Barry A. L. and Effinger L. J. (1974) Amer. J. Clin. Path. 62. 113±
117. Storage conditions and Shelf life
10 Reller L. B., Schoenknecht F. D., Kenny M. A. and Sherris J. C. Store the dehydrated medium below 258C and use
(1974) J. Infect. Dis. 130. 454±463. before the expiry date on the label.
11 D'Amato R. F., Thornsberry C., Baker N. and Kirven L. A. (1975) Store the prepared medium at 2±88C.
Antimicrob. Agents Chemotherap. 7. 596±600.
12 D'Amato R. F. and Thornsberry C. (1979) Curr. Microbiol. 2. Quality Control
135±138. Positive control:
13 Ferone R., Bushby S. R. M., Burchall J. J., Moore W. D. and Escherichia coli ATCC1 25922
Smith D. (1975) Antimicrob. Agents Chemotherap. 7. 91±98. Pseudomonas aeruginosa ATCC1 27853
14 Ferguson R. W. and Weissfeld A. S. (1984) J. Clin. Microbiol. 19. Staphylococcus aureus ATCC1 25923
85±86. Enterococcus faecalis ATCC1 29212
15 Bridson E. Y. and Brecker A. (1970) in `Methods in Microbiology'
Negative control:
Eds. Norris and Ribbons. Vol.3A Academic Press London. pp. 257± Uninoculated medium
266.
16 Jorgensen J. H., Redding J. S., Maher L. A. and Howell A. W. Precautions
(1987) J. Clin. Microbiol. 25. 2105±2113. Monitor the performance of the broth routinely using
17 De Boer E., Tilburg J. J. H. C., Woodward D. L., Lior H. and the standard QC organisms. If the broth does not
Johnson W. M. (1996) Left. Appl. Microbiol. 23. 64±66. yield the expected MIC values, modify the volumes of
Mg++ and Ca++ solutions until the MIC values
approximate to those in Table 3 in reference1.
MUELLER-HINTON BROTH If the thymidine content is lowered, after the addition
Code: CM405 of lysed horse blood or thymidine phosphorylase, the
MIC values may be lower.
An antimicrobial susceptibility testing medium which
may be used in internationally recognised standard References
procedures. 1 National Committee for Clinical Laboratory Standards (1985)
Methods for Dilution Antimicrobial Susceptibility Tests for bacteria
Formula* gm/litre
that grow Aerobically. Approved Standard M7-A. NCCLS. Villanova,
Beef, dehydrated infusion from 300.0
Pa.
Casein hydrolysate 17.5
2 Thornsberry C., Gavan T. L. and Gerlach E. H. (1977) Cumitech
Starch 1.5
6. American Society for Microbiology. Washington DC.
pH 7.3 + 0.1
3 Swenson J. M. and Thornsberry C. (1978) Curr. Microbiol. l. 189±
* modified to meet performance standards. 193.
4 Kobayashi, Hasegawa M., Saika T. et al (1997) J. Antimicrob.
Directions Chemother. 40. 713±716.
Dissolve 21g in 1 litre of distilled water. Sterilise by
autoclaving at 1218C for 15 minutes.
Description
Oxoid Mueller-Hinton Broth has been produced in MULLER-KAUFFMANN
parallel with Oxoid Mueller-Hinton Agar CM337. TETRATHIONATE BROTH BASE
Where studies on antibiotic susceptibilities are being
made both in broth and agar, it will be found to be of Code: CM343
particular value to have media of identical nutrient An improved enrichment medium for the isolation of
formulation. salmonellae and the suppression of Proteus species.
Mueller-Hinton Broth is recommended for broth Formula gm/litre
dilution MIC studies1. Tryptone 7.0
Oxoid Mueller-Hinton Broth will require Soya peptone 2.3
supplementation with the divalent cations Mg++ and Sodium chloride 2.3
Ca++ after sterilisation2. Calcium carbonate 25.0
Sodium thiosulphate 40.7
Lysed horse blood or thymidine phosphorylase may Ox bile 4.75
be added to the broth to improve the MIC endpoints
of sulphonamides and trimethoprim3. Directions
Suspend 82 grams in 1 litre of distilled water and
Mueller-Hinton Broth containing horse serum and bring to the boil. Cool below 458C and add, just prior
agar added to create a semi-solid agar medium was to use, 19ml of iodine solution and 9.5ml of a 0.1%
November 1998 2-157
Culture Media
brilliant green solution. Mix well and fill out into Quality Control
sterile tubes or flasks. Positive control:
Salmonella typhimurium ATCC1 14028
Iodine Solution
Iodine 20 grams Negative control:
Potassium iodide 25 grams Escherichia coli ATCC1 25922
Distilled water to 100ml
Precautions
Dissolve the potassium iodide in approximately 5ml Do not autoclave the base broth.
of distilled water, add the iodide and gently warm the
solution to completely dissolve it. Make up the Add the iodine solution and brilliant green just prior
volume to 100ml with distilled water. to use.
Brilliant Green Solution The medium is not suitable for the growth of S. typhi,
Brilliant Green (BDH or Chroma) 0.1 grams S. sendai, S. pullorum and S. gallinarum.
Distilled water 100ml References
1 Muller L. (1923) C. R. Soc. Biol. (Paris) 89. 434±443.
Add the brilliant green to the distilled water and
shake to dissolve the dye. Heat the solution to 1008C 2 Kauffmann F. (1930) Z. f. Hyg. 113. 148±157.
for 30 minutes and shake from time to time whilst 3 Kauffmann F. (1935) Z. f. Hyg. 117. 26±32.
4 Jeffries L. (1959) J. Clin. Path. 12. 568±570.
cooling, to ensure that the dye has completely
dissolved. Store in a brown glass bottle or away from 5 Edel W. and Kampelmacher E. H. (1969) Bull. Wld Hlth Org. 41.
light. 297±306.
Description
Muller1 developed this medium in 1923. It was later
modified by Kauffmann2,3 with the addition of
brilliant green and ox bile to suppress commensal MYCOPLASMA AGAR BASE
organisms and thus improve the isolation of
salmonellae. Code: CM401
The brilliant green dye used in the medium has been A basic medium which, after enrichment with
shown to be critical and Chroma or BDH brands supplements, will support the growth of Mycoplasma
should be used. It is essential that the dye is added as species.
directed because heating the brilliant green or
Formula gm/litre
attempting to incorporate it in the basal medium
Bacteriological peptone 10.0
seriously impairs its selective action.
`Lab-Lemco' powder 10.0
The addition of novobiocin at 40mg per litre of broth Sodium chloride 5.0
was described by Jeffries4 to suppress the growth of Mineral supplement 0.5
Proteus species. Agar 10.0
pH 7.8 + 0.2
Muller-Kauffmann Tetrathionate Broth should not be
used if Salmonella typhi is suspected. Directions
Add 35.5g to 1 litre of distilled water. Boil to dissolve
Muller-Kauffmann Tetrathionate Broth was used in a
large-scale investigation between nine laboratories in the agar and distribute in 80ml volumes. Sterilise by
autoclaving at 1218C for 15 minutes. Cool to 508C,
eight different countries5.
and add the sterile supplement.
Incubation of Muller-Kauffmann Broth at 438C was
shown to be essential in this trial and the technique
used for enrichment of the salmonellae is as follows:
Add approximately 10 grams of sample to 100ml of
Muller-Kauffmann Broth. Shake vigorously and
immediately place the flasks of medium in a 458C MYCOPLASMA SUPPLEMENT-G
water-bath for 15 minutes. Remove the flasks from Code: SR59
the water-bath, without drying them, and place in an
incubator or another water-bath at 438C. POISON ± CONTAINS THALLIUM SALT
Sub-culture the broth after 18±24 hours and again Vial contents (each vial is sufficient for 80ml of
after 48 hours. Take one loopful of broth from the medium)
edge of the surface of the fluid and inoculate either Horse serum 20ml
two Oxoid Brilliant Green Agar (Modified) CM329 Yeast extract (25% w/v) 10ml
plates (9cm diameter) without recharging the loop Thallous acetate 25mg
between plates, or one large plate (14cm diameter). Penicillin 20,000 IU
Incubate the plates at 358C for 18±24 hours. Directions
The sterile supplement is prepared by aseptically
Storage conditions and Shelf life
adding 20ml of sterile distilled water to the vial and
Store the dehydrated medium below 258C and use
mixing gently. Add this to 80ml of sterilised Oxoid
before the expiry date on the label.
Mycoplasma Agar or Broth Base CM401/CM403,
Store the prepared medium at 2±88C. previously cooled to 508C.
2-158 November 1998
Culture Media
Precautions Reference
Thallous acetate is toxic, observe the precautions 1 Lapage S. P., Shelton J. E. and Mitchell T. G. (1970) in `Methods
stated under Hazards on page 2±7. in Microbiology' Eds. Norris J. R. and Ribbons D. W. Vol.3A.
Academic Press. London. p.116.
Sub-culture the broth to agar as soon as the indicator
begins to change colour before the pH change
destroys the organism.
Quality Control
Positive gelatinase:
Serratia liquefaciens ATCC1 27592
Negative gelatinase:
Escherichia coli ATCC1 25922
Precautions
Do not shake the gelatin tubes whilst they are warm
because growth and liquefaction of gelatin frequently
occurs on the surface layer only7.
In routine diagnostic work report gelatin liquefaction
or not. The type or shape of liquefaction is of less
importance2.
References
1 American Public Health Association (1946) Standard Methods for
the Examination of Water and Sewage. 9th Edn. APHA Inc.
Washington DC.
2 American Society for Microbiology (1981) Manual of Methods for
General Bacteriology. ASM. Washington DC.
3 Cowan S. T. and Steel K. J. (1966) Manual for the Identification of
Medical Bacteria. Cambridge University Press. Cambridge. pp. 19.
27±28, 116 and 156.
4 Wilson G. S. and Miles A. A (1964) Topley and Wilson's Principles
of Bacteriology and Immunity. 5th Edn. Vol.1. Edward Arnold.
London. pp. 493±494.
5 Windle Taylor. E. (1958) `The Examination of Waters and Water
Supplies' 7th ed., Churchill Ltd., London, pp. 414 and 422.
6 Stone R. V. (1935) Proc. Soc. Exper. Biol. Med. 185±187.
7 Frobisher M. (1957) Fundamentals of Microbiology. 6th Edn. W. B.
Saunders. Philadelphia. p. 39.
Extract Agar more selective than `Mycophil' Agar by 4 Koburger J. A. and Mace F. E. (1967) Proc. W. Va. Acad. Sci. 39.
inhibiting the growth of lactobacilli, most of which 102±106.
grow at the acid pH of the latter medium. 5 Mossel D. A. A. (1951) Antonie Van Leeuwenhoek 17. 146.
6 Sharf J. M. (1960) Ann. Inst. Pasteur, Lille II. 117.
The choice of a suitable medium for enumeration of
7 Mossel D. A. A., Vega Clara L. and Put H. M. C. (1975) J. Appl.
yeasts and moulds is greatly dependent on the nature
Bact. 39. 15±22.
of the foodstuffs under investigation and the
8 Dijkmann K.E., Koopmans M. and Mossel D.A.A. (1979) J. Appl.
organisms that occur on them7. Oxytetracycline-
Bact. 47. ix.
Glucose-Yeast Extract Agar remains bacteriostatic
when inoculated with not greater than 1ml of a 10-1
dilution of foods and subsequently incubated for not
greater than 5 days at 258C as is the customary
practice in food mycology2.
For isolation of psychotrophic yeasts from chilled
proteinaceous foods a combination of oxytetracycline
and gentamicin is effective8.
Very proteinaceous foods and the higher incubation
temperatures around 358C required for some
organisms will inactivate oxytetracycline allowing
growth of Gram positive and Gram negative rods. For
such applications Rose-Bengal Chloramphenicol Agar
CM549 may be substituted or Dichloran-Glycerol
(DG18) Agar Base CM729.
Technique
Transfer 1ml aliquots of a series of suitable dilutions
of the sample to empty 9cm diameter petri dishes.
Two dishes are used for each of the dilutions. Add
approximately 15ml of medium prepared as
described. Mix gently, turning the plates three times
clockwise and three times counter-clockwise.
Incubate for 5 days at 228C +2 with the petri dishes
upside down, checking for formation of aerial mycelia
after 2 days.
Count the numbers of colonies in plates containing
50±100 colonies after 5 days, or in any countable
plates when aerial mycelia threaten to obscure further
readings after 2 days. The counts obtained for each
dilution should be similar on both plates.
Calculate the number of yeasts or moulds per 1g or
1ml by multiplying the number of colonies by the
dilution factor.
Storage conditions and Shelf life
Store the dehydrated medium below 258C and use
before the expiry date on the label.
Store the prepared medium at 2±88C.
Quality Control
Positive control:
Aspergillus niger ATCC1 9642
Saccharomyces cerevisiae ATCC1 9763
Negative control:
Escherichia coli ATCC1 25922
Precautions:
The lactic-acid bacteria are inhibited on this medium.
References
1 Mossel D. A. A., Harrewijn G. A. and Elzebroek J. M. (1973)
UNICEF.
2 Mossel D. A. A., Kleynen-Semmeling A. M. C., Vincentie H. M.,
Beerens H. and Catsaras M. (1970) J. Appl. Bact. 33. 454±457.
3 Mossel D. A. A., Visser M. and Mengerink W. H. J. (1962) Lab.
Prac. II, 109±112.
Egg yolk free TSC Agar is used with the techniques Basically the APHA method consists of the
described above. Cl. perfringens colonies are black but preparation of pour-plates using diluted samples, and
in the absence of egg yolk no lecithinase activity can counting colonies after incubation. Incubation is for
be detected. 48 hours at 328C or at 358C for the Standard Plate
Tests for confirmation are described in a study Count. For the enumeration of micro-organisms with
other temperature requirements, plates may also be
initiated by the International Commission on
incubated for 7±10 days at 5±78C: for 3±5 days at
Microbiological Specifications for Foods6 involving
208C; for 2±3 days at 458C; or for 48 hours at 558C.
nitrate reduction, lactose fermentation, gelatin
See the APHA1 publication for details.
liquefaction and the absence of motility. All black
colonies growing on TSC or SFP Agars should be Storage conditions and Shelf life
tested. Store the dehydrated medium below 258C and use
Storage conditions and Shelf life before the expiry date on the label.
Store the dehydrated medium below 258C and use Store the prepared plates at 2±88C.
before the expiry date on the label.
Quality Control
Store the prepared medium at 2±88C. Compare with previous lot/batch using pasteurised and
raw milk samples, incubated at 32±358C for 48 hours.
Quality Control
Positive control: Precautions
Clostridium perfringens ATCC1 13124 Make sure that the procedures and media used in
milk product testing comply with the National
Negative control:
Regulations required for each country.
Clostridium sordellii ATCC1 9714
Precautions Reference
Black colonies appearing on these two media may be 1 American Public Health Association (1978) Standard Methods for
organisms other than Cl. perfringens. the Examination of Dairy Products. 14th Edn. APHA Inc.
Washington DC.
References
1 Shahidi S. A. and Ferguson A. R. (1971) Appl. Microbiol. 21. 500±
506.
2 Harmon S. M., Kauttar D. A. and Peeler J. T. (1971) Appl.
Micobiol. 22. 688±692.
3 Harmon S. M., Kautter D. A. and Peeler J. T. (1971) Appl. STANDARD PLATE COUNT AGAR
Microbiol. 21. 922±927.
4 Hauschild A. H. W. and Hilsheimar R. (1973) Appl. Microbiol. 27.
(APHA)
78±82. Code: CM463
5 Hauschild A. H. W. and Hilsheimar R. (1973) Appl. Microbiol. 27.
A standard medium corresponding to the APHA
521±526.
formulation for milk, water, food and dairy products.
6 Hauschild A. H. W., Gilbert R. J., Harmon S. M., O'Keefe M. F.
and Vahlfeld R. (1977) Can. J. Microbiol. 23. 884±892. Formula gm/litre
Yeast extract 2.5
Pancreatic digest of casein 5.0
PLATE COUNT AGAR Glucose 1.0
Agar 15.0
TRYPTONE GLUCOSE YEAST AGAR pH 7.0 + 0.2
Code: CM325 Directions
A medium for the enumeration of viable organisms in Suspend 23.5g in 1 litre of distilled water. Bring to the
milk and dairy products. boil to dissolve completely. Dispense into bottles and
sterilise by autoclaving at 1218C for 15 minutes.
Formula gm/litre
Tryptone 5.0 Description
Yeast extract 2.5 Standard Plate Count Agar was developed by
Glucose 1.0 Buchbinder et al.1 who wished to use an agar without
Agar 9.0 milk solids in the formulation and investigated the
pH 7.0 + 0.2 control tests necessary to give standard results in
dairy products with statistically valid counts.
Directions
Add 17.5g to 1 litre of distilled water. Dissolve by It is prepared from selected ingredients and tested by
bringing to the boil with frequent stirring, mix and the APHA protocol2.
distribute into final containers. Sterilise by Standard Plate Count Agar meets the prescribed
autoclaving at 1218C for 15 minutes. standards of the APHA3, and AOAC4.
Description Storage conditions and Shelf life
Plate Count Agar is equivalent to the medium Store the dehydrated medium below 258C and use
recommended by the APHA1 for the plate count of before the expiry date on the label.
micro-organisms in milk and other dairy products.
Store the prepared plates at 2±88C.
3 Swab a large inoculum over half the area of the foods may carry a wide range of pseudomonads and
plate. the colonies on C-F-C Medium, incubated at lower
4 Using a sterile loop, streak out the inoculum over temperatures, may be Ps. fluorescens or Ps. putida as
the remainder of the plate to obtain isolated well as Ps. aeruginosa. Aeromonas species will also
colonies. appear as pink/brown colonies, particularly from fish
products.
5 Incubate at 358C and examine after 24 and 48
hours, using both white and ultraviolet light. References
Food, Water and Environmental Samples for 1 King E.O., Ward M.K. and Raney D.E. (1954) J. Lab. & Clin. Med.
Pseudomonads 44. 301±307.
1 Prepare Pseudomonas C-F-C Medium as directed. 2 Goto S. and Enomoto S. (1970) Jap. J. Microbiol. 14. 65±72.
2 Pour plates and dry the surface. 3 Lowbury E.J. and Collins A.G. (1955) J. Clin. Path. 8. 47±48.
4 Mead G.C. and Adams B.W. (1977) Br. Poult. Sci. 18. 661±667.
3 Prepare food samples by diluting 1 in 5 or 1 in 10
5 Geftic S.G., Heymann H. and Adair F.W. (1970) App. &
with 1% (w/v) sterile Peptone Water, CM9, and
Environmental Microbiol. 37. 505±510.
homogenise in a `Stomacher' or a laboratory
6 Stanbridge L.H. and Board R.G. (1994) Lett. Appl. Microbiol. 18.
blender.
327±328.
4 Pipette 0.5 or 1ml of the homogenate on to the
plate and spread over the surface with a sterile
glass spreader. Inoculate water and swab samples
directly on the surface of the medium.
5 Incubate at 258C and examine after 24 and 48
hours, using both white and ultraviolet light.
Colonial Appearance
Growth on C-N or C-F-C Medium is usually limited
to Pseudomonas spp. but some members of the family
Enterobacteriaceae may also be present. The presence
of blue-green or brown pigmentation, or fluorescence
may be taken as presumptive evidence of
Pseudomonas spp. but further tests must be carried out
to confirm the identity of the organism.
Stanbridge and Board6 modified C-F-C Medium to
differentiate pseudomonads from Enterobacteriaceae
developing on beef steaks packaged in modified
atmospheres. Arginine 1% w/v and phenol red
0.002% w/v were added to the medium.
Pseudomonads produce ammonia from the arginine
and colonies may be distinguished by a pink
coloration.
Storage conditions and Shelf life
Store the dehydrated medium below 258C and use
before the expiry date on the label.
Store the prepared medium at 2±88C.
Quality Control
C-N formulation
Positive control:
Pseudomonas aeruginosa ATCC1 27853
Negative control:
Proteus vulgaris ATCC1 13315
C-F-C formulation
Positive control:
Burkholderia cepacia NCTC 10661
Negative control:
Staphylococcus aureus ATCC1 25923
Precautions
Fresh media should be prepared as required. Molten
agar should not be kept longer than 4 hours. Medium
should not be stored and remelted. If swarming
colonies of Proteus species are a problem in food
samples then the incubation temperature can be
lowered to 208C for a period of 3±5 days. Chilled
numbers and incubation time when compared with Because of the diversity of environmental conditions
unmodified Universal Beer Agar. required for growth of lactic acid bacteria a semi-
anaerobic atmosphere may be needed. This is
These investigations provided the basis for the
achieved using Oxoid Gas Generating Kit BR56 in the
formula of Raka-Ray 3 Medium in which sorbitan
Oxoid Anaerobic Jar. Alternatively use CampyGen
mono-oleate is included as a stimulant for lactic acid
CN025A or CN035A. CampyGen does not require the
bacteria in general4. Fructose is present as the
addition of water or a catalyst.
essential carbohydrate source for Lactobacillus
fructivorans5 while maltose is present to detect Storage conditions and Shelf life
lactobacilli which cannot utilise glucose6. Store the dehydrated medium below 258C and use
before the expiry date on the label.
Detailed changes to the published Raka-Ray 3
formula are common, arising from attempts to further Store the prepared medium at 2±88C.
improve the performance for particular organisms Quality Control
and strains. Pediococci appear to have a universal Positive control:
ability to utilise glucose7. The value of partial Lactobacillus fermentans ATCC1 9338
substitution by glucose of the fructose content has
been noted. Negative control:
Escherichia coli ATCC1 25922
Selectivity is achieved by the addition of 3gm/litre of
2-phenylethanol to inhibit Gram-negative organisms Precautions
and 7mg of cycloheximide to inhibit yeasts8. Although the concentration of cycloheximide in the
In a review of the performance of various media, Van medium is below toxic levels, precautions should be
Keer et al.5 found that Raka-Ray 3 yielded the highest observed as detailed under HAZARDS page 2±7.
colony count and allowed the enumeration of the
greatest number of strains within 48 hours from a References
1 Saha R. B., Sondag R. J. and Middlekauff J. E. (1974) Proceedings
total of 30 strains of Lactobacillus taken from different
of the American Society of Brewing Chemists, 9th Congress, 1974.
origins and incubated under semi-anaerobic
2 Methods of Analysis of the ASBC (1976) 7th Edition. The Society,
conditions.
St. Paul. Mn. USA.
Hsu and Taparowsky9, when comparing Raka-Ray 3 3 European Brewing Convention, EBC Analytica Microbiologica:
and MRS Agar found the Raka-Ray formulation to be Part II J. Inst. Brewing (1981) 87. 303±321.
superior for Pediococcus cerevisiae although it was not 4 Mauld B. and Seidel H. (1971) Brauwissenschaft 24. 105.
as efficient for L. gayonii. In another study Hug, 5 Van Keer C., Van Melkebeke L., Vertriest W., Hoozee G. and
Schlienger and Pfenniger10 compared Raka-Ray 3 Van Schoonenberghe E. (1983) J. Inst. Brewing 89. 361±363.
with a number of other lactobacillus media including 6 Lawrence D. R. and Leedham P. A. (1979) J. Inst. Brewing 85.
MRS and sucrose agars and concluded that Raka-Ray 119.
3 and MRS were the best. 7 Coster E. and White H.R. (1951) J. Gen. Microbiol. 37. 15.
Technique 8 S. Shaw ± Personal communication.
Surface Inoculation 9 Hsu W. P. and Taparowsky J. A. (1977) Brewers Digest 52. 48.
0.1ml of the sample is spread on agar plates. Incubate 10 Hug H., Schlienger E. and Pfenniger H. (1978) Braueri-Rundschau
89. 145.
at 25±308C under anaerobic conditions using the
Oxoid Gas Generating Kit BR38 with the Oxoid
Anaerobic Jar. Alternatively, the specimen can be
filtered and the membrane placed on the agar surface RAPPAPORT-VASSILIADIS (RV)
for incubation. ENRICHMENT BROTH
Overlay Technique Code: CM669
Aseptically dispense 4ml volumes of Raka-Ray Agar
into small test tubes and keep molten in a water bath A selective enrichment broth for the isolation of
at 558C. salmonellae.
Mix 1ml of the test sample with 4ml of molten agar Formula (Classical) gm/litre
and immediately pour the contents into a petri dish Soya peptone 5.0
containing 15±20ml of solid Raka-Ray Agar to give Sodium chloride 8.0
well distributed colonies. Incubate under anaerobic Potassium dihydrogen phosphate 1.6
conditions at 25±308C in an Oxoid Anaerobic Jar with Magnesium chloride 6H2O 40.0
a Gas Generating Kit BR38. Alternatively use Malachite green 0.04
AnaeroGen AN025A or AN035A. AnaeroGen does pH 5.2 + 0.2
not require the addition of water or a catalyst. THIS MEDIUM IS VERY HYGROSCOPIC AND
Because the agar layer is very thin, individual MUST BE PROTECTED FROM MOISTURE.
colonies can be picked easily for further examination. The quantities given for the formula as classically
Incubation Conditions described made 1110ml of medium. They have been
An incubation period of 4 days is generally sufficient published this way in the Oxoid literature to coincide
but slower growing organisms may require up to 7 with the scientific literature.
days. The directions for reconstituting Oxoid Rappaport-
Vassiliadis (RV) Enrichment Broth CM669 follow
2-174 November 1998
Culture Media
11 McGibbon L., Quail E. and Fricker C.R. (1984) Inter. J. Food also highlighted the importance of the concentration
Microbiol. 1. 171±177. of magnesium chloride in the final medium.
12 Fricker C.R. (1987) J. Appl. Bact. 63. 99±116.
Technique
Food and environmental specimens.
1 Prepare Buffered Peptone Water (Oxoid CM509) as
instructed on the label in volumes of 225ml.
2 Prepare RVS Broth CM866 as instructed.
RAPPAPORT-VASSILIADIS SOYA 3 Add 25g or 25ml of the test sample to 225ml of
(RVS) PEPTONE BROTH Buffered Peptone Water and incubate at 378C for
16±20 hours. Transfer 0.1ml of the pre-enrichment
Code: CM866 peptone water culture to 10ml of RVS Broth and
A selective enrichment broth for the isolation of incubate at 428 +1.08C for 24 hours.
salmonellae. 4 Sub-culture the enrichment broth by streaking onto
Formula gm/litre plates of MLCB Agar CM783 and Brilliant Green
Soya peptone 4.5 Agar (Modified) CM329. Incubate at 358C for 18±24
Sodium chloride 7.2 hours. Colonies suspected as salmonellae should be
Potassium dihydrogen phosphate 1.26 confirmed by biochemical or serological methods.
Di-potassium hydrogen phosphate 0.18 Faecal specimens ± no pre-enrichment needed.
Magnesium chloride (anhydrous) 13.58 Add one or two 3mm loopfuls of liquid faeces (or an
Malachite green 0.036 emulsion of faeces in saline) to 10ml of RVS Broth
pH 5.2 + 0.2 CM866 pre-warmed to 428C. Incubate at 428C +1.08C
for 24 hours, and then streak onto selective agars of
Directions choice.
Suspend 26.75 grams in 1 litre of distilled water and
heat gently to dissolve. Dispense 10ml volumes into Storage conditions and Shelf life
screw-capped bottles or tubes and sterilise by Store the dehydrated medium below 258C and use
autoclaving at 1158C for 15 minutes. before the expiry date on the label.
Description Store the prepared medium at 2±88C.
Rappaport-Vassiliadis Soya (RVS) Peptone Broth is Quality Control
recommended as a selective enrichment medium for Positive control:
the isolation of Salmonellae from food and Salmonella typhimurium ATCC114028
environmental specimens.
Negative control:
RVS Broth CM866 shares with the original Escherichia coli ATCC125922
formulation1, the ability to exploit the full
characteristics of Salmonella species when compared Precautions
with other Enterobacteriaceae. These are: RVS Broth should not be used if Salm. typhi is
suspected.
1 The ability to survive at relatively high osmotic
pressure. In order to achieve optimum recovery it is
2 To multiply at relatively low pH values. recommended that the enrichment broth is incubated
at 428C + 1.08C.
3 To be relatively more resistant to malachite green.
4 To have relatively less demanding nutritional References
requirements. 1 Rappaport F., Konforti N. and Navon B. (1956) J. Clin. Path 9.
RVS Broth is based on the revised formulation 261±266.
described by van Schothorst et al2, and is 2 van Schothorst M., Renauld A. and van Beek C. (1987) Food
recommended as the selective enrichment medium for Microbiology 4. 11±18.
the isolation of salmonellae from food and 3 van Schothorst M. and Renauld A. (1983) J. Appl. Bact. 54. 209±
environmental specimens. It can also be used to 215.
isolate salmonellae from human faeces without the 4 Peterz M., Wiberg C. and Norberg P. (1989) J. Appl. Bact. 66.
need for pre-enrichment. 523±528.
7 Barnes Ella M. and Goldberg H. S. (1962) J. Appl. Bact. 25. Preparation of Differential Reinforced Clostridial
94±106. Medium
8 Goldberg H. S., Barnes Ella M. and Charles A. B. (1964) J. Bact. Make separate solutions of 4% sodium sulphite
87. 737±742. (anhydrous) and 7% ferric citrate in distilled water.
9 Williams Smith H. (1961) J. Appl. Bact. 24. 235±241. Heat the ferric citrate solution to dissolve. Sterilise
10 Sneath P. H. A. (1962) Nature 195. 643±646. both solutions by filtration. The solutions may be
11 Gregory P. H., Lacey M. E., Festenstein G. N. and Skinner F. A. stored at 48C for up to 14 days.
(1963) J. Gen. Microbiol. 33. 147±174.
On the day of use mix equal volumes of the two
12 Barnes Ella M. and Ingram M. (1956) J. Appl. Bact. 19. 117±128.
solutions. Add 0.5ml of the mixture to each 25ml
13 Ingram M. and Barnes Ella M. (1956) Lab. Practice 5. 145.
volume of single-strength freshly steamed and cooled
14 Miller N. J., Garrett O. W. and Prickett P. S. (1939) Food Res. 4.
Reinforced Clostridial Medium. To each 10ml and
447±451.
50ml volume of double-strength medium add 0.4ml
15 Mossel D. A. A., De Bruin A. S., Diepen H. M. J., van Vendrig C.
and 2ml respectively of the mixed solutions.
M. A. and Zoutwelle G. (1956) J. Appl. Bact. 19. 142±154.
All cultures showing blackening must be sub-cultured
for confirmatory tests.
Weenk, Fitzmaurice and Mossel6 modified
REINFORCED CLOSTRIDIAL Differential Reinforced Clostridial Medium by
MEDIUM (RCM) increasing the iron content to 1 gram/litre of ferric
ammonium citrate and accurately adjusting the
Code: CM149 sulphite concentration to 0.05% disodium sulphite
A semi-solid medium for the enumeration and heptahydrate. The time required for sulphite-reducing
cultivation of clostridia and other anaerobes occurring clostridium colonies to blacken was significantly
in food and pathological specimens. It is the basal shorter than that when using iron sulphite agar. The
medium for Differential Reinforced Clostridial Medium. modified medium to a great extent suppressed the
formation of black colonies by hydrogen sulphide-
Formula gm/litre positive Bacillus spp. Resistance to metronidazole and
Yeast extract 3.0 growth on aerobically incubated tryptone soya agar
`Lab-Lemco' powder 10.0 are reliable criteria for recognising Bacillus spp.
Peptone 10.0 colonies that develop.
Soluble starch 1.0
Glucose 5.0 Storage conditions and Shelf life
Cysteine hydrochloride 0.5 Store the dehydrated medium below 258C and use
Sodium chloride 5.0 before the expiry date on the label.
Sodium acetate 3.0 Store the prepared medium at 2±88C.
Agar 0.5
pH 6.8 + 0.2 Quality Control
Positive control:
Directions Clostridium perfringens ATCC1 13124
Suspend 38g in 1 litre of distilled water. Bring to the
boil to dissolve completely. Sterilise by autoclaving at Negative control:
1218C for 15 minutes. Uninoculated medium
Description Precautions
A semi-solid medium for the enumeration and Further identification tests must be carried out on
cultivation of anaerobes. Recommended for the organisms isolated from this medium.
isolation and cultivation of anaerobic organisms
occurring in a variety of habitats, including food and References
pathological specimens. 1 Hirsch A. and Grinstead E. (1954) J. Dairy Res. 21. 101±110.
2 Mundt J. O. and Jones V.W. (1952) Bact. Proc. p. 106.
Reinforced Clostridial Medium (RCM) was designed 3 Gibbs B.M. and Hirsch A. (1956) J. Appl. Bact. 19. 129±141.
by Hirsch & Grinstead1 for the cultivation and 4 Gibbs B.M. and Freame B. (1965) J. Appl. Bact. 28. 95±111.
enumeration of clostridia. They showed that the 5 The Microbiology of Water 1994 Part 1 ± Drinking Water.
medium was more fertile and enabled growth to be Report on Public Health and Medical Subjects Number 71:
initiated from small inocula more readily than five Methods for the Examination of Waters and Associated
other media tested. In a further comparison, the Materials. HMSO London.
highest viable count obtainable was the criterion 6 Weenk G., Fitzmaurice E. and Mossel D.A.A. (1991) J. Appl. Bact.
used, and again, RCM proved superior. Compared 70. 135±143.
with the spleen infusion medium of Mundt & Jones2,
RCM gave somewhat higher counts (Gibbs &
Hirsch3).
Reinforced Clostridial Medium can be made
differential for sulphite-reducing clostridia by the
addition of sodium sulphite and ferric citrate4.
Differential Reinforced Clostridial Medium is
recommended for detection of sulphite-reducing
clostridia and Cl. perfringens in drinking water5.
November 1998 2-179
Culture Media
Note
ROSE-BENGAL PHOTO-OXIDISES TO FORM
TOXIC COMPOUNDS. STORE PLATES OF THE
MEDIUM IN THE DARK AND AVOID EXPOSURE
TO LIGHT8.
Quality Control
Positive control:
Saccharomyces cerevisiae ATCC1 9763
Mucor racemosus ATCC1 42647
SABOURAUD DEXTROSE AGAR pale pink whilst other Candida species and other fungi
form deeper pink or red colonies. The test is adequate
Code: CM41 for screening purposes but other diagnostic criteria
An acid pH medium for the isolation of dermatophytes, should also be utilised for the identification of Candida
other fungi and yeasts. albicans10,11,12,13.
12 Sinski J. T. (1960) J. Invest Dermat. 35. 131±133. Storage conditions and Shelf life
13 Ridley M. F. (1960) Australian J. Dermat. 5. 209±213. Store the dehydrated medium below 258C and use
14 McDonough E. S., Georg L. K., Ajello L. and Brinkman S. (1960) before the expiry date on the label.
Mycopath. Mycol. Appl. 13. 113±116.
Store the prepared medium below 258C.
Quality Control
Positive control:
Candida albicans ATCC1 102131
Aspergillus niger ATCC1 9642
SABOURAUD LIQUID MEDIUM Negative control:
Code: CM147 Uninoculated medium
A liquid medium recommended for sterility testing and References
for the determination of the fungistatic activity of 1 Pharmacopoeia of the United States: 1995 Sterility Testing.
pharmaceutical products. 2 Food and Drug Administration (1992) Bacteriological Analytical
Formula gm/litre Manual 7th Ed. F.D.A. Washington D.C.
Pancreatic digest of casein 5.0 3 Reeder J.C., Ganguli L.A., Drucker D.B., Keaney M.G.L. and
Peptic digest of fresh meat 5.0 Gibbs A.C.C. (1989) Microbios. 60. 71±77.
Glucose 20.0
pH 5.7 + 0.2
Directions
Dissolve 30g in 1 litre of distilled water. Mix well,
distribute into final containers and sterilise by
autoclaving at 1218C for 15 minutes.
SABOURAUD MALTOSE AGAR
Code: CM41a
Description
Sabouraud Liquid Medium is a mycological sterility An acid medium for the isolation of dermatophytes,
test medium conforming to the medium described in other fungi and yeasts.
the USP1 and FDA Bacteriological Analytical Manual2
Formula gm/litre
for the determination of the fungistatic activity of
pharmaceutical and cosmetic products in order to Mycological peptone 10.0
Maltose 40.0
avoid false sterility tests. The medium may also be
Agar 15.0
used for the cultivation of moulds, yeasts, and
acidophilic bacteria. pH 5.6 + 0.2
Directions
In clinical microbiology, the use of Sabouraud Liquid
Suspend 65g in 1 litre of distilled water. Bring to the
Medium has been shown to increase the isolation rate
boil to dissolve completely. Sterilise by autoclaving at
of Candida albicans in blood culture3.
1218C for 15 minutes.
Technique
Description
The USP recommends that the fungistatic activity of
This medium differs from Sabouraud Dextrose Agar,
pharmaceutical products be determined as follows:
only in the carbohydrate incorporated. Sabouraud
1 Test Culture Maltose Agar may be used, with or without
A 1 in 1,000 dilution of a 24 to 28 hour culture of antibiotics, where a maltose medium is preferred.
Candida albicans in Sabouraud Liquid Medium and
Sabouraud Maltose Agar may be modified to form a
inoculate with the Test Culture.
selective indicator medium for the isolation of Candida
2 Tests albicans by the addition of Tergitol-7, bromocresol
Add specified amounts of the product to be tested purple, potassium tellurite and triphenyltetrazolium
to volumes of Sabouraud Liquid Medium and chloride (Chapman1).
inoculate with the Test Culture.
The storage conditions, quality control tests and
3 Controls precautions to be observed are exactly those
Inoculate tubes of Sabouraud Liquid Medium only, described under Sabouraud Dextrose Agar CM41.
with the Test Culture.
4 Incubate at 228C to 258C for at least 10 days. Reference
1 Chapman G. H. (1952) Trans. New York Acad. Sci., Series II 14(6).
5 If growth in the test series is comparable to that in
254.
the control tubes, then the product is not
fungistatic ± therefore use the amount of product
and medium specified for all routine sterility tests
on the product.
If the product is fungistatic when tested as above, add
a suitable sterile inactivating reagent, or, use a larger
ratio of medium to product in order to determine the
ratio of product to medium in which growth of the
test organism is not affected.
November 1998 2-183
Culture Media
Agar Base No.2 when tested with the same 2 Mata L. J., Carrillo C. and Villatoro E. (1969) Appl. Microbiol. 17.
organisms. 596±599.
3 Hibbert H. R. and Spencer R. (1970) J. Hyg. Camb. 68. 131±135.
Technique
4 Mossel D. A. A., Beerens H., Tahon-Castel Baron G. and
The sample suspension is diluted as necessary in
Potspeel B. (1965) Ann. Inst. Pasteur de Lille 16. 147±156.
order to obtain separated and countable colonies. A
5 Kari C., Nagy Z., Kovacs P. and Hernadi F. (1971) J. Gen. Micro.
calibrated loopful is then spread on the surface of a
68. 349±356.
previously dried Schaedler Anaerobe Agar plate.
6 de Waart J. and Pouw H. (1970) Zbl. I. Abt. 0rig. 214. 551±552.
Conditions of incubation will vary according to the
7 de Waart J. (1973) Personal Communication.
type of culture under test. Pure cultures may grow on
the base medium and this is also used for general
aerobic and anaerobic counts.
In the enumeration of Enterococcus faecalis
(facultatively anaerobic) as an indicator organism in SCHAEDLER ANAEROBE BROTH
dehydrated or frozen foods and water, and for the
detection of Clostridium, the medium can be used as Code: CM497
follows: A broth version of Schaedler Anaerobe Agar (CM437)
Food sample (e.g. pre-cooked frozen food) for the general growth of anaerobes, for use in blood
suspensions are plated out by the surface spread cultures and antibiotic MIC studies of these organisms.
technique and an aerobic viable count may be carried Formula gm/litre
out at 258C and 358C. For pre-cooked meat products, Tryptone Soya Broth (Oxoid CM129) 10.0
an anaerobic viable count and a selective plate Special peptone 5.0
examination for Cl. perfringens should also be Yeast extract 5.0
performed. Glucose 5.0
Addition of Selective Agents Cysteine HCl 0.4
To 1,000ml of base agar, the following selective agents Haemin 0.01
may be added: Tris buffer 0.75
pH 7.6 + 0.2
1 Medium for anaerobic lactobacilli and anaerobic
streptococci. Directions
NaCl 10.0g Add 26.5g to 1 litre of distilled water and mix to
Neomycin 0.002g dissolve completely. Sterilise by autoclaving at 1218C
for 15 minutes.
Incubate anaerobically at 358C.
Description
2 Medium for bacteroides and clostridia. Schaedler Anaerobe Broth CM497 is a clear medium
Placenta powder 2.0g which can support the growth of those anaerobic
(Nutritional Biochemicals Corp, Cleveland) bacteria commonly associated with human and
Neomycin 0.002g veterinary disease. It is identical to the formula of
Incubate anaerobically at 358C. Schaedler Anaerobe Agar CM437, except that the agar
has been omitted.
3 Medium for flavobacteria.
7ml of 0.5% tyrothricin in ethanol. Used as a fluid medium, under the appropriate
atmosphere, Schaedler Anaerobe Broth CM497
Incubate aerobically at 358C.
showed enhanced growth with a number of
Storage conditions and Shelf life demanding anaerobic organisms when compared
Store the dehydrated medium below 258C and use with seven other commonly used broth media1.
before the expiry date on the label. Schaedler Anaerobe Broth CM497 can also be used to
Prepared plates may be stored at 2±88C if suitably determine antibiotic MIC levels of anaerobic
protected. organisms. The extreme variations of growth rates
usually prevent the existing linear regression plots of
Quality Control
MIC versus zone diameter being used. The use of
Positive control:
tube methods overcomes this problem1.
Staphylococcus aureus ATCC1 25923
Clostridium perfringens ATCC1 13124 Fass, Prior and Rotilie2 described a simple tube
Bacteroides fragilis ATCC1 23745 method that does not require special atmospheres or
special equipment to carry out the test. By adding a
Negative control: 6mm solid glass bead to the tube of broth prior to
Uninoculated medium. autoclaving, growth of most organisms could be
Precautions detected after one day's incubation, by slowly
Note the comment on strict anaerobic conditions for rotating the tube and observing the swirl of
obligate anaerobe isolation without blood. organisms. The addition of 0.0001 of w/v resazurin to
the medium was used to determine whether
References oxidation had occurred in stored media. For
1 Schaedler R. W., Dubos R. and Castello R. (1965) J. Exp. Med. anaerobic cocci, heat-inactivated horse serum was
122. 59±66. added to a final concentration of 1% v/v before use3.
The addition of menadione (0.1g/litre), sodium Although no further reports have been received
polyanethol-sulphonate (SPS, 0.3g/litre) and carbon sodium biselenite is now considered to be very toxic
dioxide (3% v/v) to Schaedler Broth enables it to be and should be handled with great care.
used as a blood culture medium and for the
cultivation of especially fastidious Bacteroides species. SODIUM BISELENITE (SODIUM HYDROGEN
Storage conditions and Shelf life SELENITE)
Store the dehydrated medium below 258C and use Code: L121
before the expiry date on the label.
Store prepared broth in the dark at low ambient Directions
Dissolve 4g in 1 litre of distilled water and use this
temperature <158C.
solution to reconstitute the base medium or tablets.
Quality Control
Toxic by inhalation and if swallowed. Danger of
Positive control:
cumulative effects.
Clostridium perfringens ATCC1 13124
Bacteroides fragilis ATCC1 25285 Description
Prevotella loescheii ATCC1 15930 (with menadione Klett1 first demonstrated the selective inhibitory
addition) effects of selenite and Guth2 used it to isolate
Salmonella typhi. It was twenty years later before
Negative control:
Leifson3 fully investigated selenite and promoted
Uninoculated. wide use of the medium.
Precautions Selenium toxicity to certain micro-organisms is not
As with all anaerobic broth media, it is important to fully understood but it is suggested that it reacts with
avoid chemo-oxidation (overheating) and photo- sulphur and sulphydral groups in critical cell
oxidation (storage in the light) because such oxidative components4,5.
effects will cause inhibition of growth.
Proteus and Pseudomonas species appear to be resistant
References to its effects4. Lactose is added as a fermentable
1 Stalons D. R., Thornsberry C. and Dowel V. R. (1974) Appl. carbohydrate to prevent a rise in pH value during
Microbiol. 27. 1098±1104. incubation because any increase in pH will reduce the
2 Fass R. J., Prior R. B. and Rotilie C. A. (1975) Antimicrob. Agents selective activity of selenite. The fact that Proteus and
Chemother. 8. 444±452. Pseudomonas species do not ferment lactose may
3 Rotilie C. A., Fass R. J., Prior R. B. and Perkins R. L. (1975) explain why they escape inhibition.
Antimicrob. Agents Chemother. 7. 311±315.
There have been many modifications and alterations
to the original medium described by Leifson,
including mannitol to replace lactose (Mannitol
Selenite Broth CM399), addition of cystine (Selenite
SELENITE BROTH BASE Cystine Broth CM699), brilliant green, sodium
(LACTOSE) taurocholate, sulphapyridine and streptomycin. The
performance of these modifications has been
Code: CM395 investigated but with no overall agreement6.
An enriched medium for the isolation of Salmonella Technique
from faeces and food products. For routine purposes Selenite Broth cultures should
Formula gm/litre be incubated at 358C for 18 to 24 hours and then sub-
Peptone 5.0 cultured on any combination of greater and lesser
Lactose 4.0 inhibitory selective agars for Enterobacteriaceae. The
Sodium phosphate 10.0 development of Escherichia coli and Proteus species is
pH 7.1 + 0.2 not indefinitely retarded in selenite media. Where the
initial proportion of these organisms is high, it is often
Directions advantageous to sub-culture on to the solid media
Dissolve 4 grams of sodium biselenite L121 in 1 litre after 6 hours as well as after 18 hours.
of distilled water and then add 19 grams of CM395.
Warm to dissolve, mix well and fill out into If a high proportion of debris is present, in the sample
containers to a depth of 5cm. Sterilise in a boiling of material being examined, the selective powers of
water bath, or in free flowing steam, for 10 minutes. the selenite may be nullified. This is well established
DO NOT AUTOCLAVE. in the examination of faeces and egg powder. It is
common practice to emulsify the specimen in sterile
To minimise any possible risk of teratogenicity to saline, allow the gross particles to settle, and inoculate
laboratory workers, the sodium biselenite must be the medium with the supernatant. An alternative
added as a solution to this medium. method is as follows: Add 2 to 3 grams of solid
Robertson7 reported miscarriages and possible specimen to 15ml of saline in a wide-necked 1oz.
teratogenic effects on pregnant laboratory assistants bottle, emulsify, separate the debris by slowly
which may have been caused by ingested sodium pressing a plug of cotton-wool down through the
biselenite. Oxoid therefore removed this substance suspension. Withdraw approximately 1ml of the
from the powdered medium. supernatant and inoculate 10ml of Selenite Broth.
Description Reference
Sensitest Agar CM409 was developed in the Oxoid 1 Bell S. M. (1975) Supplement to Pathology (Journal of the Royal
laboratories as an antimicrobial susceptibility testing College of Pathology of Australia) Vol.7 No.4. pp. 1±48.
medium that did not require the addition of lysed
horse blood to overcome sulphonamide and
trimethoprim antagonists.
Using hydrolysed casein as the major source of SIMMONS CITRATE AGAR
amino-nitrogen in the medium, it was possible to Code: CM155
lower the peptone content to the minimum necessary
to supply essential peptides and other growth factors. An agar medium for the differentiation of
Careful control of the hydrolysis of the peptones Enterobacteriaceae based on the utilisation of citrate as
ensures that antagonists to critical antibiotics do not the sole source of carbon.
arise. Formula gm/litre
Bell1 in a monograph on antimicrobial susceptibility Magnesium sulphate 0.2
testing chose Oxoid Sensitest Agar as the preferred Ammonium dihydrogen phosphate 0.2
medium, from those tested, on the following criteria: Sodium ammonium phosphate 0.8
Sodium citrate, tribasic 2.0
1 Ability to support the growth of common Gram Sodium chloride 5.0
positive and Gram negative organisms under the Bromothymol blue 0.08
conditions of test. Agar 15.0
2 Ability to yield reproducible results. pH 7.0 + 0.2
3 Did not require the addition of lysed horse blood Directions
when a heavy inoculum method was employed. Suspend 23g in 1 litre of distilled water. Bring to the
4 Ability to demonstrate standard zones of inhibition boil to dissolve completely. Sterilise by autoclaving at
with reference organisms and antibiotics. 1218C for 15 minutes.
In the final development of the CDS method Bell Description
selected Sensitest Agar because of its superiority in Simmons Citrate Agar is recommended (Ewing and
sulphonamide testing, easier reconstitution of the Edwards1) for the differentiation of the family
dehydrated powder and stability of the powdered Enterobacteriaceae based on whether or not citrate is
medium on storage. utilised as the sole source of carbon.
Whilst the addition of 5% horse blood to the medium The medium is virtually a solidified form of Koser
is required for demanding strains, e.g. Strept. pyogenes citrate medium which, in its original form, suffered
and Strept. pneumoniae, there is no significant from the disadvantage that false appearance of
difference in zone sizes from the addition of blood. growth occurred when large inocula were employed.
The agar used in the medium, has been specially The addition of bromothymol blue indicator to the
processed to yield a gel that does not impede the medium was a distinct improvement. See Koser
diffusion of antimicrobials. Citrate Medium CM65.
For further details of antimicrobial susceptibility Simmons Citrate Agar complies with the
testing see Section 6. recommendations of the APHA2.
Storage conditions and Shelf life Technique
Store the dehydrated medium below 258C and use The medium may be used either as slopes in test
before the expiry date on the label. tubes or as a plate medium in petri dishes. In both
cases the surface of the medium is lightly inoculated
Store the prepared plates of agar at 2±88C. by streaking and, where slopes are used, the butt of
Quality Control medium is inoculated by stabbing. Incubation for 48
Positive control: hours at 358 is recommended.
Escherichia coli ATCC1 25922 Positive growth (i.e. citrate utilisation) produces an
Pseudomonas aeruginosa ATCC1 27853 alkaline reaction and changes the colour of the
Staphylococcus aureus ATCC1 25923 medium from green to bright blue, whilst in a
Enterococcus faecalis ATCC1 29212 negative test (i.e. no citrate utilisation) the colour of
Negative control: the medium remains unchanged.
Uninoculated medium Escherichia coli including serotypes from epidemic
Precautions infantile enteritis, as well as Shigella, Yersinia and
As with other susceptibility testing media, Sensitest Edwardsiella species do not grow on the medium.
Agar should be used for rapidly growing aerobic Serratia and the majority of the Enterobacter,
organisms only. It should not be modified by the Citrobacter, Klebsiella, Proteus and Providence species,
addition of carbohydrates or incubated in a CO2 except Morganella morganii and Klebsiella
enriched atmosphere. rhinoscleromatis utilise citrate and produce the
characteristic blue coloration3.
If the medium is used for Bell's CDS method then the
specified discs and technique must be used. Refer to Simmons Citrate Agar may be used to differentiate
the monograph cited in the References. citrate-positive Salmonella enteritidis and members of
Salmonella subgenus II, III and IV from the citrate- chemical substitution results in ferrous sulphide being
negative Salmonella typhi, Salmonella paratyphi A, formed along the line of inoculation.
Salmonella pullorum and Salmonella gallinarum. The presence of fermentable sugars may suppress the
Storage conditions and Shelf life enzyme mechanism which forms hydrogen sulphide,
Store the dehydrated medium below 258C and use as a result of the acid products formed (Bulmash and
before the expiry date on the label. Fulton2) and therefore sugars are not included in the
medium. Oxoid SIM Medium can be used in
Store the prepared medium at 2±88C.
conjunction with Triple Sugar Iron Agar CM277 to
Quality Control assess the ability of the culture to ferment lactose,
Positive control: sucrose and glucose.
Klebsiella pneumoniae ATCC1 13883
The production of indole from tryptophan is one of
Negative control: the diagnostic tests used in identifying enteric
Escherichia coli ATCC1 25922 bacteria. For example, unless it is an unusual form, a
Salmonella culture never produces indole from
Precautions tryptophan in amounts detectable in usual tests.
It is important not to carry over any nutrients into the
citrate medium because this will result in false Tryptone is incorporated into the medium since it is a
positive tests. Dilute the inoculum in saline before tryptophan-rich peptone, and after incubation, indole
inoculating the citrate medium to avoid a carry-over can be identified by a red dye complex reaction with
of other carbon sources4. one of several reagents, e.g. Kovac's Reagent which
consists of amyl alcohol, para-
References dimethylaminobenzaldehyde and concentrated
1 Ewing W. H. and Edwards P. R. (1960) Bull. Bact. Nomen. and hydrochloric acid3.
Taxon. 10. 1±12.
The presence of glucose in the medium is avoided as
2 American Public Health Association (1981) Standard Methods for
recommended4. False negative reactions have been
the Examination of Water and Wastewater. 15th Edn. APHA Inc.
recorded when fermentation has occurred5.
Washington DC.
3 Kauffman F. (1954) `Enterobacteriaceae' 2nd ed., Munksgaard, The use of only 0.35% agar in the medium results in
Copenhagen. the production of a semi-solid medium, ideal for the
4 Matsen J. M. and Sherris J. C. (1969) Appl. Microbiol. 18. 452±454. examination of motility. Non-motile organisms will
grow along the line of inoculation only, whereas
motile species will grow away from it.
SIM MEDIUM SIM Medium CM435 is therefore designed to
Code: CM435 determine three characteristics: hydrogen sulphide
production, indole production and motility.
A medium for the differentiation of enteric bacteria on
the basis of sulphide production, indole production and Technique
motility. The medium should be dispensed in tubes or bottles
Formula gm/litre and when cool, inoculated once with a pure culture,
Tryptone 20.0 by inserting a straight wire to about one third of the
Peptone 6.1 depth of the medium. If papers are used for the
Ferrous ammonium sulphate 0.2 detection of indole, then these are wedged between
Sodium thiosulphate 0.2 the cotton wool plug or cap, and side of the container.
Agar 3.5 The inoculated medium is incubated at 358C for 18
pH 7.3 + 0.2 hours or longer, if necessary, and examined for
motility, hydrogen sulphide production and finally
Directions
indole production from tryptophan.
Suspend 30g in 1 litre of distilled water and boil to
dissolve the medium completely. Dispense into final To Test for Indole Production:
containers and sterilise by autoclaving for 15 minutes 1 Add 0.2ml of Kovac's Reagent to the tube and
at 1218C. allow to stand for 10 minutes. A dark red colour in
the reagent constitutes a positive indole test. No
Description change in the original colour of the reagent
A motility-indole medium has been found to be constitutes a negative test.
helpful in the identification of the Enterobacteriaceae; or
e.g. in the differentiation of Klebsiella from Enterobacter
2 Suspend a strip of filter paper, soaked in a solution
and Serratia species1. For convenience, these two of saturated oxalic acid and dried, over the
important tests have been combined with sulphide- medium4. Indole formed by positive organisms is
production in one tube.
volatile and causes the test paper to turn pink.
The production of hydrogen sulphide is a useful
diagnostic test in the identification of enteric bacteria Colonial Appearances
and is helpful in the differentiation between Non-motile organisms grow only along the line of
Salmonella and Shigella. The sulphate-reducing inoculation, whereas motile species show either a
bacteria will produce hydrogen sulphide and further diffuse even growth spreading from the inoculum,
turbidity of the whole medium, or more rarely, The medium is very selective for enterococci and,
localised outgrowths which are usually fan-shaped or when it is incubated at elevated temperatures
occasionally nodular. (44±458C), all red or maroon colonies may be accepted
as presumptive enterococci4,5.
Hydrogen sulphide production is shown by
blackening of the line of inoculation. Burkwall and Hartman showed that the addition of
0.5ml of `Tween 80' and 20ml of a 10% solution of
Storage conditions and Shelf life
sodium carbonate or bicarbonate to each litre of
Store the dehydrated medium below 258C and use
medium was of value when examining frozen foods
before the expiry date on the label.
for enterococci; the original article should be
Store the prepared medium at 2±88C. consulted for procedural details2.
Quality Control
Organism Motility H2S Indole Technique
Escherichia coli ATCC1 25922 V ± + The Department of Health6 in their `Report 71'
Proteus vulgaris ATCC1 13315 + + + recommend the use of Slanetz and Bartley medium
Shigella sonnei ATCC1 25931 ± ± ± for the enumeration of enterococci in water supplies.
The water is filtered through a membrane filter which
Precautions is then placed on the surface of a well dried plate of
To avoid delay in initiating growth always sub- the medium. Plates are incubated at 358C for 4 hours
culture from solid media. The reactions given by SIM and then at 44±458C for 44 hours. Membranes are
Medium are not sufficient to speciate organisms. examined, with a hand lens in a good light, and all
Additional biochemical and serological tests are red or maroon colonies counted as enterococci.
required for confirmation.
Food samples can be examined for enterococci by the
References method suggested by the Nordic Committee of Food
1 Blazevic D. J. (1968) Appl. Microbiol. 16. 668. Analysis3. Samples are homogenised and so diluted
2 Bulmash J. M. and Fulton M. (1964) J. Bact. 88. 1813. with physiological saline that only 15±150 colonies
3 Harrigan W. F. and McCance M. E. (1966) `Laboratory Methods in
grow on each petri dish. Homogenates or dilutions
Microbiology' Academic Press. 53. are spread evenly over the agar surface with a glass
4 Wilson G. S. and Miles A. A. (1964) Topley and Wilson's rod and allowed to soak in. Dishes are inverted and
`Principles of Bacteriology and Immunity' 5th ed., Arnold, 1. 490. incubated at 358C for 48 hours, after which typical
5 Giles R. R. (1956) J. Clin. Path. 9. 368±371.
colonies (pink or dark red, with a narrow whitish
border) are counted.
Storage conditions and Shelf life
Store the dehydrated medium below 258C and use
SLANETZ AND BARTLEY MEDIUM before the expiry date on the label.
Code: CM377 Store the prepared medium at 2±88C away from light.
A medium for the detection of enterococci. Quality Control
Positive control:
Formula gm/litre Enterococcus faecalis ATCC1 29212
Tryptose 20.0
Yeast extract 5.0 Negative control:
Glucose 2.0 Escherichia coli ATCC1 25922
Disodium hydrogen phosphate 4.0 Precautions
Sodium azide 0.4 Count all red, maroon or pink colonies as
Tetrazolium chloride 0.1 presumptive enterococci. Not all species reduce TTC
Agar 10.0 therefore pale colonies should not be ignored.
pH 7.2 + 0.2 Although incubation at 358C yields a higher count, it
allows the growth of organisms which do not
Directions conform to the definition of enterococci. Incubation at
Suspend 42 grams in 1 litre of distilled water and 44±458C has a selective effect and produces fewer
bring to the boil to dissolve the agar completely. false-positives. However, the preliminary incubation
EXCESSIVE HEATING MUST BE AVOIDED. at 358C encourages the recovery of stressed
Dispense into petri dishes and allow to solidify. It organisms.
should not be remelted. The medium may be used Although the selective properties of this medium are
with membrane filters or by spreading dilutions of very good it is advisable to regard the colony count as
the sample over the surface of the agar with a glass a presumptive or unconfirmed count. Further
rod. identification may be required depending on the
scope of the examination.
Description
Slanetz & Bartley1 originally devised this medium to References
detect and enumerate enterococci by the technique of 1 Slanetz L. W. and Bartley C. H. (1957) J. Bact. 74. 591±595.
membrane filtration, but it has also proved useful as a 2 Burkwall M. K. and Hartman P. A. (1964) Appl. Microbiol. 12.
direct plating medium2,3. 18±23.
3 Nordic Committee on Food Analysis (1968) Leaflet 68.
4 Taylor E. W. and Burman N. P. (1964) J. Appl. Bact. 27. 294±303. Quality Control
5 Mead G. C. (1966) Proc. Soc. Wat. Treat. Exam. 15. 207±221. Positive control:
6 Department of Health and Social Security. Report 71 (1982) The Salmonella enteritidis ATCC1 13076
Bacteriological Examination of Drinking Water Supplies. HMSO. Shigella sonnei ATCC1 25931
London.
Negative control:
Enterococcus faecalis ATCC1 29212
Precautions
This medium is highly selective and R-strains of
shigellae will not grow on it. It is not recommended
SALMONELLA SHIGELLA for the primary isolation of shigellae1,2.
AGAR (SS AGAR) References
Code: CM99 1 Leifson E. (1935) J. Path. Bact. 40. 581-
2 Taylor W. I. and Harris B. (1965) Am. J. Clin. Path. 44. 476-
A differential selective medium for the isolation of
Salmonella and some Shigella species from clinical
specimens, foods etc.
Formula gm/litre
`Lab-Lemco' powder 5.0
Peptone 5.0
Lactose 10.0 SALMONELLA SHIGELLA AGAR
Bile salts 8.5 (SS AGAR MODIFIED)
Sodium citrate 10.0
Sodium thiosulphate 8.5 Code: CM533
Ferric citrate 1.0
An improved formulation which gives better growth of
Brilliant green 0.00033
shigellae and better colony characteristics for
Neutral red 0.025
salmonellae.
Agar 15.0
pH 7.0 + 0.2 Formula gm/litre
`Lab-Lemco' powder 5.0
Directions
Peptone 5.0
Suspend 63g in 1 litre of distilled water. Bring to the
boil with frequent agitation and allow to simmer Lactose 10.0
Bile salts 5.5
gently to dissolve the agar. DO NOT AUTOCLAVE.
Sodium citrate 10.0
Cool to about 508C, mix and pour into sterile petri
dishes. Sodium thiosulphate 8.5
Ferric citrate 1.0
Description Brilliant green 0.00033
SS Agar is a differential, selective medium for the Neutral red 0.025
isolation of Shigella and Salmonella species from Agar 12.0
pathological specimens, suspected foodstuffs, etc. pH 7.3 + 0.2
Gram-positive and coliform organisms are inhibited
by the action of the selective inhibitory components Directions
brilliant green, bile salts, thiosulphate and citrate. Suspend 57g in 1 litre of distilled water. Bring to the
boil with frequent agitation, and allow to simmer
Thiosulphate in combination with iron also acts as an gently to dissolve the agar. DO NOT AUTOCLAVE.
indicator for sulphide production, which is indicated Cool to about 508C and pour into petri dishes.
by blackening in the centres of the colonies.
Technique Description
Inoculate the medium heavily with the specimen, Although widely used, SS Agar has been criticised
spreading a portion of the original inoculum in order because of excessive inhibition of Shigella species.
to obtain well separated colonies on some part of the Investigation has shown that modification to the
plate. Incubate for 18 to 24 hours at 358C; non-lactose formulation by alterations to the bile salt mixture,
fermenters form colourless colonies, whilst occasional peptone and pH value considerably improve its
resistant coliforms or other lactose fermenters performance in the growth of shigellae without too
produce pink or red colonies. much increased growth of commensal organisms.
In parallel with the SS Agar plate, inoculate a tube of Salmonella colonies are also larger with improved
Selenite Broth CM395 enrichment medium, incubate blackening at the centre.
for 12 hours at 358C, and sub-culture on to another SS
Agar plate. The change in formulation has reduced the number of
gm/litre from 63g to 57g.
Storage conditions and Shelf life
Store the dehydrated medium below 258C and use
Technique
before the expiry date on the label.
Inoculate the medium heavily with the specimen,
Store the prepared medium at 2±88C. spreading a portion of the original inoculum in order
to obtain well separated colonies on some part of the Stone3 suggested that gelatinase activity was
plate. Incubate for 18 to 24 hours at 358C; non-lactose indicative of food poisoning strains but Chapman et
fermenters form colourless colonies. Occasional al.4 reported that typical food poisoning staphylococci
resistant coliforms and other lactose fermenters should also produce an orange pigment, be
produce pink or red colonies. haemolytic, be coagulase-positive, and ferment
mannitol. Chapman5 showed that incubation at 308C
In addition to the SS Agar (Modified) plate, inoculate
produced deeper pigmentation and no interference
a tube of Selenite Broth Enrichment Medium, CM395.
with the Stone reaction or with acid production from
Incubate it for 12 hours at 358C, and sub-culture on to
mannitol ± both of the latter being about as intense as
another SS Agar (Modified) plate.
at 358C.
Colonial Characteristics Smuckler & Appleman6 made Staphylococcus
Non-Lactose Fermenting Organisms Medium No.110 selective, for the determination of
Salmonella species Transparent colonies usually coagulase-positive staphylococci in meat pies
with black centres containing large numbers of Bacillus species, by the
Shigella species Transparent colonies addition of sodium azide 0.75mM (4.875 grams per
litre).
Proteus species Transparent colonies with
Citrobacter species grey-black centres Staphylococcus Medium No.110 is formulated
according to the APHA7 and AOAC8 specifications.
Late-lactose fermenting organisms will develop Carter9 modified the medium by adding egg yolk (5%
colonies with pink centres after 48 hours incubation. v/v SR47) so that the characteristic egg yolk reactions
Storage conditions and Shelf life of staphylococci can be seen.
As for SS Agar CM99. Technique
Quality Control Streak or smear the Staphylococcus Medium No.110
As for SS Agar CM99. plate with the specimen and incubate for 43 hours at
358C or for 48 hours at 308C. Pigmented colonies are a
deep orange colour, whilst non-pigmented colonies
are white.
Acid production from mannitol is best demonstrated
STAPHYLOCOCCUS MEDIUM by adding a drop of 0.04% bromothymol blue
indicator to the sites of the individual colonies; yellow
NO.110 indicates acid production.
Code: CM145 Gelatin hydrolysis may be demonstrated by adding a
A selective medium for the isolation and differentiation drop of a saturated aqueous solution of ammonium
of pathogenic staphylococci based on salt tolerance, sulphate or, preferably, of a 20% aqueous solution of
pigmentation, mannitol fermentation and gelatin sulphosalicylic acid to an individual colony ('Stone
liquefaction. reaction'). A positive `Stone reaction' is denoted by
the presence of a clear zone round gelatinase-
Formula gm/litre producing colonies after 10 minutes' contact with the
Yeast extract 2.5 reagent.
Tryptone 10.0
Lactose 2.0 The above reactions may be conveniently performed
Mannitol 10.0 using short sleeves, 5mm long and 10mm diameter,
Sodium chloride 75.0 cut from polythene tubing. The sleeves act as
Dipotassium hydrogen phosphate 5.0 receptacles for the reagents when placed over discrete
Gelatin 30.0 colonies, and may be stored in 70% alcohol prior to
Agar 15.0 use.
pH 7.1 + 0.2 Coagulase tests should not be carried out without first
Directions sub-culturing in Nutrient Broth No.2 CM67 or on
Suspend 150g in 1 litre of distilled water. Bring to the Blood Agar Base CM55.
boil to dissolve completely. Sterilise by autoclaving at Storage conditions and Shelf life
1218C for 15 minutes. Disperse the precipitate by Store the dehydrated medium below 258C and use
gentle agitation before pouring. before the expiry date on the label.
Description Store the prepared medium at 2±88C.
Staphylococcus Medium No.110 is a selective medium
for isolation and differentiation of pathogenic Quality Control
staphylococci (Chapman12) on a basis of salt Positive control:
tolerance, pigmentation, mannitol fermentation, and Staphylococcus aureus ATCC1 25923
gelatin liquefaction. Pathogenic staphylococci Negative control:
(coagulase-positive) are able to grow on the high-salt Escherichia coli ATCC1 25922
mannitol medium to form orange colonies which give
positive reactions for acid production and gelatin Precautions
liquefaction. Enterococcus faecalis may grow on this medium as tiny
colonies with slight mannitol fermentation.
7 Incubate the plates as described for the Direct (as appropriate) with 0.1% (w/v) sterile Peptone
Plating Method, strain, and count the number of Water CM9, and homogenise in a stomacher or a
pink indole positive colonies. laboratory blender.
If required the unstained plates may be placed in the Pipette 0.5ml or 1.0ml (as appropriate) of the
refrigerator overnight and the indole test carried out homogenate on to the plate and spread over the
the following morning. surface with a sterile glass spreader. Incubate plates
Indole Reagent for 4 hours at 308C then 18 hours at 448C.
5% p-dimethylaminobenzaldehyde in 1N Multiply the number of blue/green colonies by the
hydrochloric acid. dilution factor and express the result as the number of
Storage conditions and Shelf life E. coli per gram of food.
Store the dehydrated medium below 258C and use
before the expiry date on the label. Description
TBX Medium is based on Tryptone Bile Agar CM595.
Store the prepared plates at 2±88C. Tryptone Bile Agar was originally formulated to
Quality Control improve on earlier methods used to detect E. coli in
Stain colonies on the membrane filter with indole foods2 in terms of speed, reliability, better recovery
reagent. from frozen samples and the detection of poor lactose
fermenters.
Positive control:
Escherichia coli ATCC1 25922 TBX Medium builds on these advantages through the
addition of a chromogenic agent ± X-glucuronide ±
Negative control: which detects glucuronidase activity. This is the same
Enterobacter aerogenes ATCC1 13048 enzyme detected by MUG reagent3, and has been
shown to be highly specific for E. coli4. However,
References approximately 3±4% of E. coli are glucuronidase
1 Anderson J. M. and Baird-Parker A. C. (1975) J. Appl. Bact. 39.
negative, notably E. coli O157 strains5.
111±117.
2 Delaney J. E., McCarthy J. A. and Grasso R. J. (1962) Wat. Sewage Unlike MUG, where the flurophore leaches out of the
Works. 109. 289. cell into the surrounding agar, the released
3 Ewing W. H. (1972) COC Atlanta, US Dept. of Health, Education & chromophore in TBX Medium is insoluble and
Welfare. accumulates within the cell. This ensures that
4 International Commission on Microbiological Specifications for coloured target colonies are easy to identify.
Foods (1979) Can. J. Microbiol. 25. 1321±1327. Most E. coli strains can be differentiated from other
5 Holbrook R., Anderson J. M. and Baird-Parker A. C. (1980) Food coliforms by the presence of the enzyme
Technol. in Aust. 32. 78±83. glucuronidase. The chromogen in TBX Medium is 5-
6 Clarke P. H. and Cowen S. T. (1952) J. Gen. Microbiol. 6. 187±197. bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-
7 Vracko R. and Sherris J. C. (1963) Amer. J. Clin. Path. 39. 429±432. glucuronide), and is targeted by this enzyme. E. coli
8 Sharpe A. N. and Jackson A. K. (1972) Appl. Microbiol. 24. 175± cells are able to absorb this complex intact and
178. intracellular glucuronidase splits the bond between
the chromophore and the glucuronide. The released
chromophore is coloured and builds up within the
cells, causing E. coli colonies to be coloured blue/
green.
TRYPTONE BILE
X-GLUCURONIDE MEDIUM (TBX) Storage conditions and Shelf life
TBX Medium CM945 should be stored tightly capped
Code: CM945 in the original container at 108C±258C. When stored
A selective, chromogenic medium for the detection and as directed, the medium will remain stable until the
enumeration of Escherichia coli in food. expiry date printed on the bottle.
Formula Quality Control
gm/litre Positive control:
Tryptone 20.0 Escherichia coli ATCC1 25922 ± blue/green colonies
Bile Salts No. 3 1.5
Agar 15.0 Negative control:
X-glucuronide 0.075 Klebsiella pneumoniae ATCC1 11228 ± colourless
pH 7.2 + 0.2 colonies
Directions
Suspend 36.6g of TBX Medium CM945 in 1 litre of Precautions
distilled water. Sterilise by autoclaving at 1218C for 15 TBX Medium CM945 should only be used for in vitro
minutes. Cool to 508C and pour the medium into diagnostic purposes.
sterile petri dishes. Do not use beyond the stated expiry date, or if the
Dry the surface of the medium in the prepared plates. product is caked, discoloured or shows any sign of
Prepare the food sample by diluting 1 in 5 or 1 in 10 deterioration.
Directions
TRYPTONE GLUCOSE Add 40g to 1 litre of distilled water. Bring to the boil
EXTRACT AGAR to dissolve completely. Sterilise by autoclaving at
1218C for 15 minutes.
Code: CM127
Description
For the plate count of water, dairy products and for the A general purpose agar medium, containing two
detection of thermophilic organisms. peptones, which will support the growth of a wide
Formula gm/litre variety of organisms. It is suitable for the cultivation
`Lab-Lemco' powder 3.0 both of aerobes and anaerobes, the latter being grown
Tryptone 5.0 either in deep cultures or by incubation under
Glucose 1.0 anaerobic conditions. The medium may also be used
Agar 15.0 as a blood agar base ± for this purpose 7% of sterile
pH 7.0 + 0.2 blood should be added to the sterile molten medium
which has been cooled to approximately 458C.
Directions Tryptone Soya Agar can also be used for the
Suspend 24g in 1 litre of distilled water. Bring to the preparation of `chocolate' agar.
boil to dissolve completely. Sterilise by autoclaving at
1218C for 15 minutes. Mix well before pouring. When Since Tryptone Soya Agar contains no added
the dilution of the original specimen is greater than 1 carbohydrate it may be used, with added blood, in
in 10, add 10ml of sterile 10% solution of Skim Milk the determination of haemolysis.
Powder L31 per litre. Horse blood agar plates prepared with Oxoid
Description Tryptone Soya Agar are used for the colicine typing
This medium, of American origin, is recommended of Shigella sonnei1,2,3,4.
for the plate count of water and dairy products1,2. It The Oxoid medium has also been used as a
can also be used for the detection of thermophilic replacement for yeastrel-milk agar plates in the
bacteria in dairy products. The Standard Methods Lisboa test5 and for bacterial counts on eviscerated
Committee of the American Public Health Association poultry6.
recommended that sterile milk should be added to
this medium only when the dilution of the original When supplemented with 0.7g lecithin and 5g
specimen was greater than 1 in 10. The addition of Polysorbate (Tween 80) per litre of Tryptone Soya
10ml of a sterile 10% solution of Skim Milk Powder Agar, the medium can be used as Microbial Content
L31 per litre is suitable for this purpose. It is essential Test Agar for testing quaternary ammonium
that this medium is not overheated during compounds7.
sterilisation. Tryptone Soya Agar is recommended as a reference
See also Plate Count Agar CM325. medium when testing selective media, to measure the
degree of inhibition8.
Storage conditions and Shelf life
Store the dehydrated medium below 258C and use A medium for isolation of Bacteroides gracilis is
before the expiry date on the label. prepared from Tryptone Soya Agar by adding
formate, fumarate and nitrate. The medium is made
Store the prepared medium at 2±88C. selective using nalidixic acid and teicoplanin9.
Quality Control Enchanced haemolysis agar (EHA) used to improve
Compare with previous lot/batch of medium using detection of Listeria monocytogenes when present
samples of pasteurised and unpasteurised milk. amongst other listeriae has been modified to optimise
its performance by substituting Tryptone Soya Agar
References for Columbia Agar in the original formulation10.
1 American Public Health Association (1980) Standard Methods for
the Examination of Water and Wastewater. 15th Edn. APHA Inc. Storage conditions and Shelf life
Washington DC. Store the dehydrated medium below 258C and use
2 American Public Health Association (1972) Standard Methods for before the expiry date on the label.
the Examination of Dairy Products. 13th Edn. APHA Inc. Store the prepared plates of medium at 2±88C.
Washington DC.
Carry out microscopy and other identification tests on sodium azide is recommended for the isolation of
the isolated colonies. pathogenic streptococci from cheese and other dairy
products2,3.
Storage conditions and Shelf life
Store the dehydrated medium below 258C and use Tryptose Phosphate Broth with added agar is also
before the expiry date on the label. recommended by the American Public Health
Association for the examination of throat swabs and
Store the prepared plates of medium at 2±88C.
blood for Streptococcus pneumoniae and as a growth
Quality Control medium for pneumococci prior to the bile solubility
Positive control: test.
Staphylococcus aureus ATCC1 25923
Tryptose Phosphate Agar Broth may also be
Streptococcus pyogenes ATCC1 19615
employed for the emulsification of cheese prior to the
Negative control: plate isolation method for Brucella species2.
Uninoculated medium.
The small proportion of agar (0.1±0.2%) necessary for
the above methods may be most conveniently added
References to the Oxoid broth before sterilisation, in the form of
1 Casman E. P. (1942) J. Bact. 43. 33±37.
Oxoid Agar Tablets CM49 (one tablet per 100ml).
2 Casman E. P. (1947) Amer. J. Clin. Path. 17. 281±282.
3 American Public Health Association (1970) Diagnostic Procedures Technique
and Reagents. 5th Edn. APHA Inc. New York. Examination of throat swabs for S. pneumoniae4
1 Prepare Tryptose Phosphate Broth in the usual
manner but add a small amount of agar by
dissolving 10 Agar Tablets CM49 per litre of broth
before autoclaving.
TRYPTOSE PHOSPHATE 2 Place a pharyngeal or sputum swab in 3ml of
Tryptose Phosphate (Agar) Broth and incubate for
BROTH 2 hours at 358C. If sufficient pneumococci are
Code: CM283 present, type directly or preferably employ the
culture for mouse inoculation.
A buffered glucose broth for the cultivation of fastidious
bacteria. Examination of cheese for Lancefield Group A
streptococci2
Formula gm/litre 1 Heat 25ml of Tryptose Phosphate (Agar) Broth
Tryptose 20.0 (prepared as above) to 458C.
Glucose 2.0
Sodium chloride 5.0 2 Immediately emulsify 5 grams of cheese in the
Disodium hydrogen phosphate 2.5 broth, using a heavy sterile glass rod. Plate suitable
pH 7.3 + 0.2 dilutions of the emulsion on Tryptose Agar plates
containing 1.5% agar and 0.04% of sodium azide.
Directions
Alternatively, add sterile aqueous sodium azide to the
Dissolve 29.5g in 1 litre of warm distilled water and
emulsion to give a final concentration of 2.5%,
distribute into final containers. Sterilise by
incubate for 12±14 hours at 358C, shake, plate on
autoclaving at 1218C for 15 minutes.
Tryptose Agar plates containing 1.5% agar and 0.04%
For the cultivation of anaerobes, if the reconstituted of sodium azide.
medium has been stored prior to use, remove
The sodium azide suppresses the growth of most
dissolved oxygen by placing the tubes in a boiling
bacteria except streptococci and some lactobacilli.
water bath for 15 minutes and cool without agitation
before inoculation. Blood Culture
1 Add up to 10ml of blood to 150ml of Tryptose
Description
Phosphate Broth in a 300ml flask or bottle.
A buffered dextrose broth for use as an adjuvant to
tissue culture media and for the cultivation of 2 Incubate and sub-culture on to other media in the
fastidious bacteria. Ginsberg et al.1 maintained tissue usual manner, according to the exact purpose of
cultures of HeLa cells for at least 10 days in a mixture the investigation.
of Tryptose Phosphate Broth 15±25%, Scheren Storage conditions and Shelf life
maintenance solution 67.5±77.5% and chicken serum Store the dehydrated medium below 258C and use
7.5%. The cells increased 3±5 fold in number during before the expiry date on the label.
this period, smaller quantities of ARD, AD and type 1
Store the prepared medium at 2±88C.
poliomyelitis virus could be detected and more ARD
virus could be propagated in HeLa cells in the Quality Control
Tryptose Phosphate. Positive control:
Streptococcus pneumoniae ATCC1 6303
Broth supplemented medium.
Neisseria meningitidis ATCC1 13090
Tryptose Phosphate Broth is recommended for the
cultivation of streptococci, pneumococci, Negative control:
meningococci and other fastidious organisms. Uninoculated medium
Tryptose Phosphate Broth with added agar and
2-210 November 1998
Culture Media
References
1 Ginsberg H. S. et al. (1955) Proc. Soc. Exper. Biol. Med. 89. 66±71.
2 American Public Health Association (1953) `Standard Methods for
the Examination of Dairy Products' 10th ed., APHA Inc., New York,
pp. 179, 180, 181.
3 Newman R. W. (1950) J. Milk Food Tech. 13. 226±233.
4 American Public Health Association (1953) `Diagnostic Procedures
and Reagents' 4th ed., APHA Inc., New York, p. 141.
40% Urea Solution is supplied, as a sterile solution in 2 Rapid growth ensued and it was possible to
ampoules, for the convenient preparation of this discern a clear-cut positive reaction within two to
medium. five hours at 358C.
Storage conditions and Shelf Life 3 It was easier to detect any contamination during
Store the dehydrated medium below 258C and use storage.
before the expiry date on the label. Technique
Store the prepared medium at 2±88C. For the examination of faeces, specimens are cultured
in enrichment and selective media in the usual
Quality Control manner. Discrete colonies are then picked off the
Positive control: surface of the solid selective media.
Proteus vulgaris ATCC113315
Inoculate tubes of Urea Broth with single colonies of
Negative control: non-lactose-fermenting organisms and incubate for 2
Escherichia coli ATCC125922 to 6 hours at 358C. (Maslen states that the cultures
should be incubated in a water bath in order to obtain
Precautions
the highest proportion of positive reactions within 5
The alkaline reaction produced in this medium after
hours.) Regard all organisms producing a pink
prolonged incubation may not be caused by urease
coloration in the medium (i.e. due to the alkalinity
activity. Check using medium without urea.
caused by urea hydrolysis) as not belonging to the
Do not heat or reheat the medium because urea Salmonella or Shigella groups, and discard.
decomposes very easily.
Inoculate all cultures showing no colour change (i.e.
For the detection of urease-positive Proteae the no urea hydrolysis) into `sugar' peptone waters
reaction must be read within the first 2±5 hours of (Andrade Peptone Water CM61) plus the appropriate
incubation. carbohydrate and on to a Blood Agar Base CM55
slope.
Reference
1 Christensen W. B. (1946) J. Bact. 52. 461±466. Incubate the new cultures, together with the Urea
Broth, until the next morning. No further examination
is necessary if the urea tube now shows an alkaline
UREA BROTH BASE reaction (pink colour), otherwise continue the
Code: CM71 diagnostic tests ± including slide agglutinations from
the Blood Agar Base culture, if necessary.
A liquid version of Christensen's medium for the
differentiation of urease-producing Enterobacteriaceae. Storage conditions and Shelf Life
Store the dehydrated medium below 258C and use
Formula gm/litre before the expiry date on the label.
Peptone 1.0
Store the prepared medium at 2±88C.
Glucose 1.0
Disodium phosphate 1.2 Quality Control
Potassium dihydrogen phosphate 0.8 Positive control:
Sodium chloride 5.0 Proteus vulgaris ATCC113315
Phenol red 0.004
Negative control:
pH 6.8 + 0.2
Escherichia coli ATCC125922
Directions
Add 0.9g to 95ml of distilled water. Sterilise by Precautions
autoclaving at 1158C for 20 minutes. Cool to 558C and It is preferable that the medium be used on the day of
aseptically introduce 5ml of sterile 40% Urea Solution preparation. If not, examine the tubes carefully to
SR20. Mix well and distribute 10ml amounts into ensure sterility.
sterile containers. After overnight incubation other members of the
Description Enterobacteriaceae may show alkaline reactions.
This is a liquid modification of Christensen medium1. Reference
The modification is suitable for the differentiation of 1 Maslen L. G. C. (1952) Brit. Med. J. 2. 545±546.
urease-producing organisms from members of the
Salmonella and Shigella groups, during the routine
examination of rectal swabs and faeces. Maslen noted
that in the routine examination of faeces for Salmonella
and Shigella organisms many non-lactose-fermenting
colonies isolated were later found to belong to the
urease-positive Proteae. He evolved this medium as a
means whereby the latter organisms could be rapidly
detected and eliminated ± thus saving a considerable
amount of time and media. Maslen claimed that the
advantages of the fluid medium were:
1 A Pasteur pipette could be used to inoculate other
diagnostic media.
VIOLET RED BILE AGARS and plant hygiene. However, when testing, for
example, raw vegetables, Violet Red Bile Agar may
A guide to the choice of an appropriate medium. remain a more practical choice for the assessment of
their hygienic status because certain non-lactose-
Media containing bile and the dye violet red used in
fermenting but glucose-utilising organisms, e.g.
the examination of foods are based on the medium
Pseudomonas species predominate amongst the
developed by MacConkey for detection of bile-
naturally-occurring associated flora and may easily
tolerant Gram-negative bacteria. Violet Red Bile Agar
overgrow the indicator organisms on VRBGA.
CM107 (VRBA) contains lactose as a fermentable
carbohydrate which enables the medium to be used If there is any concern that non-lactose-fermenting
for the detection of lactose-fermenting bacteria and pathogens may also be present, then consideration
differentiation of the group of bacterial genera known should be given to performing additional specific
as coliforms or coli-aerogenes bacteria from non- tests for these organisms, if necessary by a consulting
lactose-fermenting organisms. Violet Red Bile Glucose laboratory.
Agar CM485 (VRBGA) differs from VRBA only by Further information about the significance of
substitution of glucose for lactose. By definition all Enterobacteriaceae present may be obtained by
members of the Enterobacteriaceae ferment glucose incubating VRBGA at different temperatures. When
and grow as purple-red `positive' colonies. testing for enteric pathogens is not feasible,
VRBA was used routinely during the years 1925 to incubation at elevated temperatures, e.g. 42±448C for
1935 to monitor the efficacy of milk pasteurisation detecting populations of thermotrophic
and milking parlour hygiene. It is still considered to Enterobacteriaceae may be helpful because these
be a useful medium for testing the hygienic status of organisms and major enteric pathogens thrive at
water and milk. All Gram-negative bacteria capable similar temperatures. A test for thermotrophic
of growth on bile-containing media and which Enterobacteriaceae should not be regarded as a
ferment lactose are included in the coliform count. substitute for a search for specific enteric pathogens.
Lactose-fermenting colonies must not be assumed to Some non-Enterobacteriaceae such as Aeromonas
be E. coli and if required colony identification should species may also grow on VRBA and VRBGA. It is
be carried out. important to have a general understanding of the
Extension of the use of VRBA into food examination microflora likely to be encountered in any specific
revealed weaknesses in using the ill-defined `coli- sample. This will assist in the selection of the most
aerogenes' group of organisms for assessments of appropriate medium and also guide the
processing and general hygiene. Lack of microbiologist in deciding the necessity for colony
standardisation of methodology and differences in identification.
interpretation of colony morphology of coli-aerogenes Because of the multiplicity of genera designated `coli-
organisms sometimes led to very considerable aerogenes' and Enterobacteriaceae that grow on
differences in accuracy and precision of results. This VRBA and VRBGA, variety may be expected in their
situation is brought about because generally all colony appearance and size. These differences may be
Gram-negative bacteria capable of growth on bile- further influenced by parameters such as the numbers
containing media and which ferment lactose are of colony-forming units present, their distance from
included in the coliform count. This mixture of each other and by incubation temperature. Although
organisms may vary because of a number of generally colonies may be up to 2mm in diameter,
influences including the type of sample under they may be considerably smaller, sometimes less
investigation, the culture medium used, incubation than 0.5mm. In practice, all purple-red colonies on
temperature and criteria chosen for reading the Violet Red Bile Agar CM107 should be regarded as
results. This last issue might erroneously exclude presumptive coli-aerogenes and all purple-red
relevant organisms because of an unusual colony colonies on Violet Red Bile Glucose Agar CM485 as
appearance; such as colour, size and presence or Enterobacteriaceae. If required, further tests may be
absence of bile precipitation surrounding colonies. carried out to confirm their identity.
Substitution of glucose for lactose in the same Mossel1 has carried out much of the work on the role
selective medium resulted in Violet Red Bile Glucose
of Violet Red Bile Agar and Violet Red Bile Glucose
Agar CM485. All Enterobacteriaceae on this medium
Agar in food microbiology and his paper is a useful
produce `purple-red' colonies and use of the clearly
introduction to the subject. Further detailed
delineated family of Enterobacteriaceae eliminates the
discussion and methodology for examining foods for
inaccuracies in methods and interpretation inherent in
the presence of Enterobacteriaceae and coli-aerogenes
the ill-defined `coli-aerogenes' group. However, there
(coliform) bacteria is given by Mossel et al2.
is the possibility of falsely reassuring results in
situations where lactose-negative organisms Neither VRBA or VRBGA are intended to be used for
predominate and where these may include non- detection of enteropathogenic E. coli. If it is necessary
lactose-fermenting pathogens, e.g. Salmonella species. to determine whether any of the E. coli colonies
In summary, generally little is to be gained by present are of pathogenic strains then it will be
continued use of `coli-aerogenes' bacteria instead of necessary first to establish the serogroup and
Enterobacteriaceae as index or indicator organisms subsequently submit the isolate to tests of
but much may be lost. Violet Red Bile Glucose Agar is pathogenicity.
becoming the preferred medium for use in many
investigations into raw materials, processed foods
2-214 November 1998
Culture Media
VIOLET RED BILE GLUCOSE AGAR examined and Mossel drafted a specification as
follows:
Code: CM485
1 Approved media have to be clear and yield
A glucose-containing selective medium for the detection colonies of satisfactory size. They have to give
and enumeration of Enterobacteriaceae in food products. reproducible counts of typical colonies of
Enterobacteriaceae.
Formula gm/litre
Yeast extract 3.0 2 When challenged for intrinsic toxicity by an
Peptone 7.0 anaerobic metabolic test13 using a strain of Yersinia
Sodium chloride 5.0 enterocolitica (Serotype 03) as a sensitive indicator,
Bile Salts No.3 1.5 media must promote adequate growth, acid
Glucose 10.0 formation and, where required, adequate gas
Neutral red 0.03 formation.
Crystal violet 0.002 3 Media have to satisfy the confirmation rate of
Agar 12.0 typical colonies, i.e. the number of colonies
pH 7.4 + 0.2 confirmed as Enterobacteriaceae divided by the
Directions number of colonies tested.
Suspend 38.5g in 1 litre of distilled water. Bring to the Violet Red Bile Glucose Agar CM485 has been
boil. Continue to boil for 2 minutes or for the developed to satisfy all of these criteria and complies
minimum time necessary to dissolve completely and with the recommendations of ISO14.
ensure that there are no remaining flecks of unmelted
agar. No further sterilisation is necessary or desirable. Technique
Mix well and dispense into tubes or dishes. Prepare a series of dilutions of the samples so that at
Description least one will be included that will yield 100±200
Results from tests that may be applied to water to colonies from a 1ml aliquot. Transfer 1ml aliquots of
detect coli-aerogenes organisms as possible indicators each dilution to 9cm petri dishes using 2 plates for
of faecal contamination possess far less significance each dilution. Add 15ml of medium, cool to 478C.
when applied to raw foods. In the examination of Gently swirl the plates 3 times clockwise and 3 times
foodstuffs, detection of a more defined group of anti-clockwise. After the medium has solidified
organisms, the Enterobacteriaceae, that ferment overlay with 10ml of the same medium and leave to
glucose to produce acid and/or gas has been solidify. Invert the dishes and incubate at >428C for
recommended1,2. In addition to coliforms this group 18 hours, 328C for 24±48 hours or 48C for 10 days
depending on the groups of Enterobacteriaceae to be
includes salmonellae and shigellae, which do not
recovered15.
ferment lactose, and enterotoxicogenic Esch. coli. It
also contains organisms, such as Klebsiella and The agar overlay ensures anaerobic conditions which
Citrobacter, which are more resistant to heat than suppress the growth of non-fermentative Gram
coliforms and are therefore better indicators of failure negative bacteria. It also encourages the fermentation
of processes that use minimal heat. of glucose which favours the formation of clearly
visible purple colonies, surrounded by a purple halo.
The difficulties of measuring the total
Enterobacteriaceae content of foodstuffs have been Characteristic appearance of colonies
studied by Mossel et al.3, who showed that the Round, purple 1±2mm diameter surrounded by
addition of glucose to an existing medium for the purple haloes. Although colony size is generally
detection of coliforms improves the performance. 1±2mm, size can be affected by a number of
They added 10g per litre of glucose to crystal violet influences and all red colonies should be counted.
neutral red bile lactose agar (Violet Red Bile Agar Confirmation of the identity of these colonies must be
CM107), and named the modified formulation made by further tests.
MacConkey Glucose Agar.
Storage conditions and Shelf Life
Further work by Mossel et al.4,5 showed that the Store the dehydrated medium below 258C and use
lactose could be omitted resulting in the formulation before the expiry date on the label.
of Violet Red Bile Glucose Agar CM485. The Store the prepared medium at 2±88C and use as
continued inclusion of lactose would not provide test freshly as possible.
results leading to more accurate identification.
Exclusion of lactose renders the medium more
Quality Control
economical to make as less weight is required per
Positive control:
litre.
Escherichia coli ATCC1 25922
Media that contain bile salts have an intrinsic toxicity
for Enterobacteriaceae, even for cells that have not Negative control:
been under stress6,7,8,9,10,11. Staphylococcus aureus ATCC1 25923
The selective activity of this medium diminishes after from heavily contaminated foods and clinical
24 hours incubation and organisms previously specimens. It corresponds to the specification of the
suppressed may exhibit growth. United States Pharmacopoeia1 in terms of its formula.
Medium for the poured plate procedure should be Vogel and Johnson2 modified the Tellurite Glycine
freshly prepared, cooled to 478C and used within 3 Agar formula of Zebovitz et al.3 by doubling the
hours. mannitol concentration to 1% (w/v) and adding
Phenol Red as a pH indicator. The enhanced
References fermentation reaction which occurs as a result of the
1 WHO Technical Report Series N.598 (1976) Geneva, p. 51. increase in mannitol content is clearly indicated by
2 Mossel D. A. A. (1958) Zbl. Bakt. I. Ref. 166. 421±432. the development of yellow zones surrounding the
3 Mossel D. A. A., Mengerink W. H. J. and Scholts H. H. (1962) J. colonies.
Bacteriol. 84. 381.
Staph. aureus is able to reduce tellurite to metallic
4 Mossel D. A. A, Eelderink I., Koopmans M. and van Rossem F.
tellurium resulting in growth as black colonies.
(1978) Lab. Practice 27. No.12. 1049±1050.
5 Mossel D. A. A., Eelderink I., Koopmans M. and van Rossem F. During the first 24 hours of incubation contaminating
(1979) J. Food Protect. 42. 470±475. organisms are almost completely inhibited by
6 Mossel D. A. A. (1978) Food Technol. Austral. 30. 212±219. tellurite, lithium chloride and the high glycine
7 Kroninger D. L. and Banwart G. J. (1978) J. Food Sci. 43. 1328± concentration. Virtually all the organisms that grow
1329. in this time are coagulase positive.
8 Bridson E. Y. (1978±79) in `Van Monster tot Resultaat' Nederland
Organisms that grow as black colonies surrounded by
Society for Microbiology. Wageningen, pp. 58±67.
a yellow zone after incubation at 35±378C for 24 hours
9 Burman N. P. (1955) Proc. Soc. Water Treatm. Exam. 4. 10±20.
may be presumed to be Staph. aureus.
10 Mossel D. A. A. and Harrewijn G. A. (1972) Alimenta 11. 29±30.
11 Mossel D. A. A., Harrewijn G. A. and Nesselrooy-van Zadelhoff Prolonged incubation may result in the growth of
C. F. M. (1974) Health Labor. Sci. 11. 260±267. black coagulase-negative colonies and if these
12 Mossel D. A. A. (1971) Miscell. Papers Agricult. University organisms also ferment mannitol they may be falsely
Wageningen, The Netherlands 9. 29±39. identified from their appearance as Staph. aureus. In
13 Mossel D. A. A., Eelderink I. and Sutherland J. P. (1977) Zbl. these circumstances further tests of identity should be
Bakt. I. Orig. A238. 66±79. carried out before concluding that the organism is
14 International Organization for Standardization: Meat and meat Staph. aureus.
products - detection and enumeration of Enterobacteriaceae. Techniques
ISO/DIS 5552. 1977.
15 Mossel D. A. A., van der Zee H., Hardon A. P. and van Netten Food samples
P. (1986) J. Appl. Bact. 60. 289±295. 1 Dry the surface of the plates.
2 With a glass spatula spread from 0.1 to 1.0ml of
diluted food (macerated in 0.1% Peptone Water)
over the surface of each well dried plate.
VOGEL-JOHNSON AGAR 3 Incubate at 35±378C and examine after 24 and 48
Code: CM641 hours.
Clinical specimens
A selective medium for the isolation of Staphylococcus 1 Dry the surface of the prepared plates.
aureus from clinical specimens and food.
2 Inoculate directly with the specimen.
Formula gm/litre 3 Incubate at 35±378C and examine after 24 and 48
Tryptone 10.0 hours.
Yeast extract 5.0
Mannitol 10.0 Colonial appearance
Dipotassium phosphate 5.0 Staphylococcus aureus appear as black, convex shiny
Lithium chloride 5.0 colonies surrounded by a yellow zone.
Glycine 10.0 Storage conditions and Shelf life
Phenol red 0.025 Store the dehydrated medium below 258C and use
Agar 16.0 before the expiry date on the label.
pH 7.1 + 0.2
Store the prepared medium at 2±88C.
Directions
Suspend 61 grams in 1 litre of distilled water and Quality Control
bring gently to the boil to dissolve completely. Positive control:
Sterilise by autoclaving at 1218C for 15 minutes. Cool Staphylococcus aureus ATCC1 25923
to 508C and add 5.7ml of sterile 3.5% potassium Negative control:
tellurite solution SR30 (equivalent to 20ml of 1% Escherichia coli ATCC1 25922
potassium tellurite).
Precautions
Description All presumptive Staph. aureus colonies should be
Vogel-Johnson Agar, by selecting and identifying confirmed with further tests. Do not heat the medium
coagulase positive and mannitol-fermenting strains, after the addition of potassium tellurite.
permits the early detection of Staphylococcus aureus
References
1 United States Pharmacopoeia XXI (1985) Microbial. Limit Tests.
Rockville. Md.
2 Vogel R. A. and Johnson M. J. (1961) Pub. Hlth Lab. 18. 131.
3 Zeboritz E., Evans J. B. and Niven C. F. (1955) J. Bact. 70. 687.
References
Formula gm/litre
1 Wilkins T. D. and Chalgren S. (1976) Antimicrob. Agents
Tryptone 10.0
Chemother. 10. 926±928.
Gelatin peptone 10.0
2 Rogosa M. (1964) J. Bacteriol. 87. 162±170.
Yeast extract 5.0
3 Hoffman P. S., George H. A., Kreig N. R. and Smibert R. A.
Glucose 1.0
(1979) Can. J. Microbiol. 25. 8±16.
Sodium chloride 5.0
4 Quinto G. and Sebald M. (1964) Am. J. Med. Technol. 30. 381±384.
L-Arginine 1.0
5 Gibbons R. J. and MacDonald J. B. (1960) J. Bacterio. 80. 164±170.
Sodium pyruvate 1.0
6 Sutter V. L., Barry A. L., Wilkins T. D. and Zabransky R. J.
Menadione 0.0005
(1979) Anti-Microb. Agents Chemother. 16. 495±502.
Haemin 0.005
7 Brown W. J. and Waatti P. E. (1980) Antimicrob. Agents
Agar 10.0
Chemother. 17. 629±635.
pH 7.1 + 0.2
8 Castel O., Grollier G., Agius G. et al (1990) Eur. J. Clin. Microbiol.
Inf. Dis. 9. 667±671.
Directions
Suspend 43 grams in 1 litre of distilled water. Bring to
the boil to dissolve completely. Sterilise by N-S ANAEROBE SELECTIVE SUPPLEMENT
autoclaving at 1218C for 15 minutes.
Code: SR107
Recognising the need for a standard medium for
antimicrobial susceptibility testing of anaerobic For the selective isolation of non-sporing anaerobes.
bacteria, Wilkins and Chalgren1 developed a new Vial contents (each vial is sufficient for 500ml of
medium which would not require the addition of medium)
blood. Their formulation included yeast extract to Haemin 2.5mg
supply vitamins and other growth factors such as Menadione 0.25mg
purines and pyrimidines, that are necessary for good Sodium pyruvate 500mg
growth of Peptostreptococcus anaerobius and Bacteroides Nalidixic acid 5mg
melaninogenicus. Arginine was added to ensure
sufficient amino acid was available for the growth of
Eubacterium lentum. Pyruvate was added as an energy G-N ANAEROBE SELECTIVE SUPPLEMENT
source, for asaccharolytic cocci such as Veillonella2. It
Code: SR108
also acts similarly to catalase and degrades traces of
hydrogen peroxide, which may be produced by the For the selective isolation of Gram-negative anaerobes.
action of molecular oxygen on medium constituents
Vial contents (each vial is sufficient for 500ml of
and interfere with the metabolism of anaerobes3.
medium)
Haemin was found to be essential for the growth of
Haemin 2.5mg
Bacteroides species4 and menadione for
Menadione 0.25mg
B. melaninogenicus5.
Sodium succinate 1,250mg
Peptones derived from the single protein sources Nalidixic acid 5.0mg
casein and gelatin, were used to improve Vancomycin 1.25mg
standardisation of the medium. Wilkins and
Chalgren1 considered that this medium consistently
Directions
grew anaerobes as well or better than media such as
To prepare Non-Selective Medium for all anaerobic
Brucella Agar or Schaedler Blood Agar. A
organisms
collaborative study in ten laboratories showed that it
Suspend 21.5g of Wilkins-Chalgren Anaerobe Agar
could be used in an agar dilution method for
CM619 in 475ml of distilled water. Bring to the boil to
susceptibility testing of anaerobic bacteria and
dissolve completely. Sterilise by autoclaving at 1218C
recommended a procedure as a reference method6.
for 15 minutes. Cool to 508C and aseptically add 25ml
The value of such a procedure was further confirmed defibrinated blood SR50/SR51. Mix gently, and pour
by Brown and Waatti7, who found that the incidence into sterile petri dishes (see plate 1).
of resistance of anaerobic bacteria to frequently used
To prepare Selective Medium for Non-Sporing
antibiotics had increased. They considered it essential
Anaerobes
that diagnostic laboratories should have the capability
Suspend 21.5g of Wilkins-Chalgren Anaerobe Agar
of carrying out susceptibility tests on anaerobic
CM619 in 475ml of distilled water containing 0.5ml
bacteria.
`Tween 80'. Bring to the boil to dissolve completely by the action of molecular oxygen on media
and sterilise by autoclaving at 1218C for 15 minutes. constituents. Hydrogen peroxide is known to affect
Cool to 50±558C and aseptically add the contents of 1 the metabolism of anaerobes7.
vial of N-S Anaerobe Supplement SR107 rehydrated
Downes et al.8 showed that NAT Medium was
with 10ml of sterile distilled water, together with superior to kanamycin agar (KA) and neomycin agar
25ml of defibrinated blood SR50/SR51. Mix gently
(NA) in the recovery of all non-clostridial anaerobes.
and pour into sterile petri dishes (see plate 2).
The major superiority was in the recovery of
To prepare Selective Medium for Gram-Negative anaerobic, Gram-positive cocci.
Anaerobes Selective Medium for Gram-Negative Anaerobes
Suspend 21.5g of Wilkins-Chalgren Anaerobe Agar
This medium is described9 as NAV Medium and is
CM619 in 475ml of distilled water. Bring to the boil to
recommended for the isolation of Gram-negative
dissolve completely and sterilise by autoclaving at anaerobes from clinical specimens.
1218C for 15 minutes. Cool to 50±558C and aseptically
add the contents of 1 vial of G-N Anaerobe NAV Medium is a modification of NAT Medium1 in
Supplement SR108 rehydrated with 10ml of sterile which `Tween 80' and sodium pyruvate have been
distilled water, together with 25ml defibrinated blood replaced by sodium succinate. Vancomycin has been
SR50/SR51. Mix gently and pour into sterile petri added, thus making the medium totally selective for
dishes (see plate 3). Gram-negative anaerobes.
Wilkins-Chalgren Anaerobe Agar is recommended G-N Anaerobe Supplement SR108 contains nalidixic
but other media may be used satisfactorily, e.g. acid and vancomycin as selective agents; haemin,
Columbia Agar Base CM331 and Blood Agar Base menadione and sodium succinate as growth factors.
No.2 CM271. Sufficient haemin and menadione are Haemin was found to be essential for the growth of
contained in N-S and G-N Supplements to provide Bacteroides species5 and menadione for B.
adequate levels in these media when used as directed. melaninogenicus6. Some Gram-negative anaerobes
require succinate as a source of energy10.
Note
Use N-S Supplement with Wilkins-Chalgren Medium The recovery of Gram-negative anaerobes on NAV
for NAT medium. Use G-N Supplement with Medium has been shown8 to be superior to that on
Wilkins-Chalgren Medium for NAV Medium1. media containing neomycin and kanamycin as
selective agents.
Discussion
Selective Medium for Non-Sporing Anaerobes In order to isolate the maximum non-sporing
This medium is referred to in the published literature1 anaerobic bacteria from clinical specimens the
as NAT Medium and is recommended for the following scheme must be followed.
isolation of non-sporing anaerobes from clinical
specimens. Specimen
Inoculate on to each of the following media and
The recovery of non-sporing anaerobes from clinical
incubate anaerobically for 48 hours.
material may sometimes prove difficult in specimens
containing mixtures of aerobic and anaerobic bacteria. Plate 1 Plate 2 Plate 3
A medium which contains nalidixic acid as the Wilkins-Chalgren CM619 CM619
selective agent was described by Wren1 for isolating Anaerobe Agar + `Tween 80' + 5% (v/v)
these organisms. It was shown to be virtually non- CM619 + 5% (v/v) +5%(v/v) defibrinated
inhibitory to most non-sporing anaerobes whilst defibrinated defibrinated blood + SR108
retaining good selectivity for these organisms when blood blood + SR107
present in mixed cultures. The medium is particularly All bacteria capable (NAT Medium) (NAV Medium)
useful for the recovery of non-sporing Gram-positive of growing under Non-Sporing Gram Gram-negative
anaerobic conditions +ve and Gram -ve anaerobic bacteria
anaerobes since the presence of `Tween 80' stimulates anaerobic bacteria
their growth2.
A non-selective plate is included for attempted
Another advantage of this medium is the earlier isolation of any strain, in particular B. corrodens which
colonial pigmentation of the Bacteroides is sensitive to the selective agents.
melaninogenicus group due to the slow lysis of the
blood by `Tween 80' during incubation. It is also a Technique
less inhibitory medium than aminoglycoside- 1 Prepare supplies of Plate 1 (CM619 + blood) Plate 2
containing media for non-sporing anaerobes in (CM619 + blood + Tween 80 + SR107) and Plate 3
general. (CM619 + blood + SR108) as described in the
The NS Anaerobe Supplement SR107 for non-sporing section marked Directions.
anaerobes contains nalidixic acid as the selective 2 Inoculate the specimens on to plates of each
agent, together with haemin, menadione and sodium medium. Best results are obtained if freshly
pyruvate as an additional energy source1,4. prepared plates are used but plates may be stored
Haemin was found to be essential for the growth of at 48C for up to 3 days.
Bacteroides species5 and menadione for B. 3 Incubate the plates anaerobically at 358C for 48
melaninogenicus.6 Pyruvate, in addition to being an hours. The Oxoid Anaerobic System with a Gas
energy source, acts similarly to catalase and degrades Generating Kit BR38 is recommended.
traces of hydrogen peroxide which may be produced Alternatively use Anaerogen AN025A or AN035A.
Storage conditions and Shelf life the determination of osmophilic yeasts occurring in
Store the dehydrated medium below 258C and use materials used in the manufacture of soft drinks.
before the expiry date on the label. Technique
Store the prepared medium at 2±88C. For the microbiological examination of butter, make
suitable dilutions in quarter-strength Ringer solution
Quality Control
(prepared with Ringer Solution Tablets BR52).
w/o cycloheximide
Transfer 1ml of each dilution to a separate petri dish;
Positive control:
add 15ml of melted Wort Agar, cooled to 458C to
Saccharomyces cerevisiae ATCC1 9763
488C; mix by rotary movements in a horizontal plane;
Negative control: allow the poured-plates to set (protected from the
Uninoculated medium light) at room temperature for 30±50 minutes.
Incubate in an inverted position, e.g. for 5 days at
with cycloheximide 258C, and count the number of yeasts and mould
Positive control: colonies which develop.
Escherichia coli ATCC1 25922
For the examination of sugar products for osmophilic
Negative control: yeasts, dissolve dehydrated Wort Agar in a syrup
S. cerevisiae ATCC1 9763 containing 35 parts w/w of sucrose and 10 parts w/w
Precautions of glucose, and autoclave for 20 minutes at 1108C.
When handling cycloheximide observe the Inoculate and mix as above. Scarr recommends
precautions to be taken under HAZARDS page 2±7. incubation at 278c for 3±4 days for Schizosaccharomyces
species and for 5 days for less common osmophilic
References yeasts. Colonies on the medium are well defined and
1 Green S. R. and Gray P. P. (1950) Wallerstein Lab. Comm. 13. 357. normally opaque.
2 Hall Jean F. (1971) J. Inst. Brewing 77. 513±516. Storage conditions and Shelf life
Store the dehydrated medium below 258C and use
before the expiry date on the label.
}
bacteria. Shigella
Rapid xylose fermentation is almost universal Providencia Red colonies
amongst enteric bacteria, except for members of the H2S-negative Salmonella
(e.g. S. paratyphi A)
}
Shigella, Providencia and Edwardsiella genera1. Xylose is
thus included in the medium so that Shigella spp. may Escherichia
be identified by a negative reaction. Enterobacter
Salmonella spp. are differentiated from non-pathogenic Klebsiella Yellow, opaque
xylose fermenters by the incorporation of lysine in the Citrobacter colonies
medium. Salmonellae exhaust the xylose and Proteus
decarboxylate the lysine, thus altering the pH to Serratia
alkaline and mimicking the Shigella reaction. Note
However, the presence of Salmonella and Edwardsiella False positive, red colonies may occur with some
spp. is differentiated from that of shigellae by a Proteus and Pseudomonas species.
hydrogen sulphide indicator.
Storage conditions and Shelf life
The high acid level produced by fermentation of Store the dehydrated medium below 258C and use
lactose and sucrose, prevents lysine-positive coliforms before the expiry date on the label.
from reverting the pH to an alkaline value, and non-
pathogenic hydrogen sulphide producers do not Store the prepared medium at 2±88C.
decarboxylate lysine. The acid level also prevents Quality Control
blackening by these micro-organisms until after the 18 Positive control:
to 24 hour examination for pathogens. Salmonella typhimurium ATCC1 14028
Sodium desoxycholate is incorporated as an inhibitor Negative control:
in the medium. The concentration used allows for the Escherichia coli ATCC1 25922
inhibition of coliforms without decreasing the ability
to support shigellae and salmonellae.
References
1 Taylor W.I. (1965) Am. J. Clin. Path. 44. 471±475.
2 McCarthy M.D. (1966) N.Z. J. Med. Lab. Technol. 20. 127±131.
3 Isenberg H.D., Kominos S. and Sigeal M. (1969) Appl. Microbiol.
18. 656±659.
4 Taylor W. I. and Harris B. (1965) Am. J. Clin. Path. 44. 476±479.
5 Taylor W. I. and Harris B. (1967) Am. J. Clin. Path. 48. 350±355.
6 Taylor W. I. and Schelhart D. (1967) Am. J. Clin. Path. 48. 356±
362.
7 Taylor W.I. and Schelhart D. (1966) Appl. Microbiol. 16. 1387±
1392.
8 Rollender M.A., Beckford O., Belsky R.D. and Kostroff B. (1969)
Am. J. Clin. Path. 51. 284±286.
9 Taylor W. I. and Schelhart D. (1969) Appl. Microbiol. 18. 393±395.
10 Dunn C. and Martin W.J. (1971) Appl. Microbiol. 22. 17±22.
11 Chadwick P., Delisle G.H. and Byer M. (1974) Can. J. Microbiol.
20. 1653±1664.
12 Aspinall S.T., Hindle M.A. and Hutchinson D.N. (1992) Eur. J.
Clin. Microbiol. Inf. Dis. 11. 936±939.
13 Weissman J.B., Gangarosa E.J., Schmerler A., Marier R.L. and
Lewis J.N. (1975) Lancet I. 1898, 88±90.
Cultures are initially incubated for 3 days under YERSINIA SELECTIVE SUPPLEMENT
anaerobic conditions and then for a further 2 days
aerobically. Development of mould colonies is Code: SR109
impeded during the anaerobic phase of incubation. Vial contents (each vial is sufficient for 500ml of
Dimorphic moulds e.g. (mucor spp) may form yeast- medium).
like colonies during anaerobic incubation.
Cefsulodin 7.5mg
Technique Irgasan 2.0mg
Cultures on plates or membrane filters are incubated for Novobiocin 1.25mg
3 days at 258C under strictly anaerobic conditions.
Continue incubation of the cultures under aerobic Reconstitute the antibiotic supplement SR109 by
conditions for a further 2 days. Yeast colonies may be aseptically adding 2ml of a sterile 50:50 solution of
very small immediately following anaerobic incubation ethanol and water to one vial and mix gently to
but will increase in size in air. Mould growth may dissolve the contents completely.
become completely unrestricted after 3 days in air.
Directions
Storage conditions and Shelf life Suspend 29.0g in 500ml of distilled water and bring
Yeast and Mould Agar should be stored tightly gently to the boil to dissolve completely. Sterilise by
capped in the original container at 108C to 258C. autoclaving at 1218C for 15 minutes. Allow to cool to
When stored as directed the medium will remain 508C and aseptically add the contents of one vial of
stable until the expiry date printed on the label. Yersinia Selective Supplement SR109 reconstituted as
The prepared medium may be stored for up to 2 directed. Mix gently and pour into sterile petri dishes.
weeks at 2±88C. Description
Quality Control Yersinia Selective Medium (CIN Medium) is based on
Yeast and Mould Agar may be tested for performance the formulation of Schiemann1,2 and is recommended
using stable, typical control cultures. for the isolation and enumeration of Yersinia
enterocolitica from clinical specimens and food.
Precautions
For in vitro diagnostic use. Y. enterocolitica is becoming increasingly recognised as
a cause of diarrhoeal disease of man. Infection by the
Do not use beyond the stated expiry date or if the organisms results in diarrhoea, malaise, nausea and
product is caked, discoloured or shows any sign of fever, plus constant abdominal pain over a period of
deterioration. 1±2 days. The organism has also been shown as a
cause of polyarthritis, mesenteric adenitis and
References septicaemia. It is likely that human infections are
1 Wickerham L.J. (1951) U.S. Dept. Agric. Tech. Bull. No 1029, 1±19. directly or indirectly derived from animal sources and
2 Wickerham L.J. and Rettger L.F (1939) J. Tropical Med. Hyg. 42. may be contracted through the ingestion of
174±179. contaminated food. Initially serotypes 0:3 and 0:9
3 De Jong J. and Put H.M.C. (1980) Biology and Activities of Yeasts. were implicated in human infections but since then
Society for Applied Bacteriology Symposium series No. 9. Skinner other serotypes, mainly 0:5 and 0:8 have also been
F.A., Passmore S.M. and Davenport R.R. (eds). Academic Press, involved3. It is important to note that incidence of
London. Pages 289±292. disease caused by the various serotypes of
Y. enteroccolitica is currently reported to vary
considerably with geographical location. It is expected
that with provision of a selective medium, a higher
YERSINIA SELECTIVE AGAR isolation rate will result, and Y. enterocolitica will be
BASE recognised as more common and widespread than
previously suspected.
Code: CM653
Yersinia Selective Agar Base CM653 and the selective
A selective medium for Yersinia enterocolitica when supplement SR109 have been developed specifically
used with Yersinia Selective Supplement SR109 for the optimum growth and recovery of Y.
(Schiemann CIN Medium). enterocolitica after 18±24 hours incubation at 328C.
Formula gm/litre Schiemann2 modified his earlier formulation for CIN
Special peptone 20.0 Medium by replacing Bile Salts with sodium
Yeast extract 2.0 desoxycholate (0.5g/l) and by reducing the
Mannitol 20.0 concentration of novobiocin from 15 to 2.5mg/l in
Sodium pyruvate 2.0 order to eliminate the inhibition of some strains of
Sodium chloride 1.0 serotype 0:8.
Magnesium sulphate 0.01 The typical colonies of Y. enterocolitica will develop as
Sodium desoxycholate 0.5 a dark red `bullseye' surrounded by a transparent
Neutral red 0.03 border and will vary considerably among serotypes in
Crystal violet 0.001 colony size, smoothness and the ratio of the border to
Agar 12.5 centre diameter. Most other organisms that are
pH 7.4 + 0.2 capable of growing will produce larger colonies
(>2mm in diameter) with diffuse pinkish centres and
opaque outer zones. Serratia liquefaciens, Citrobacter
November 1998 2-227
Culture Media
References
1 United Sates Pharmacopoeia XXII 1990, Mack Publishing Co.
2 Clesceri L.S., Greenberg A.E. & Trussel R.R. eds. 1989. Standard
Methods for the Examination of Water and Wastewater. 17th ed.
A.P.H.A., A.W.W.A. & W.P.C.F.
3 Mehlman I.J. 1984. Appendix I. Culture media. In Bacteriological
Analytical Manual. A.O.A.C. Arlington, VA.
QUANTI-CULTPLUS(TM)
Each tube contains 10 packs (10 tests per pack).
Each kit is designed to deliver between 10±100 cfu/
0.1ml.
Total diluent volumes per vial is 1.2ml.
Note
The organisms used in QUANTI-CULTPLUS(TM) are
derived from original ATCC stock cultures according
to the number shown.
QUANTI-CULTPLUS(TM) are manufactured for Oxoid
Ltd by Chrisope Technologies Inc., an FDA approved
company.
FDA Registration No: 1625984. QUANTI-
CULTPLUS(TM) is a registered trademark of Chrisope
Technologies Inc, a division of remel.
1 INTRODUCTION
The first time the term `peptone' appeared was in
papers published in 1880 and 1882 by Nageli. He has
been credited as the first bacteriologist to discover
that chemo-organotrophic organisms grow best in
culture media containing a partially digested protein.
The problems associated with the production of
protein hydrolysates were quickly recognised and
their manufacture became the concern of commercial
suppliers. In fact protein hydrolysate was the first
complex culture medium ingredient to be supplied
commercially. This was the fore-runner of the large
range of commercial culture media now available.
November 1998 3-1
Peptones, Hydrolysates, Agars & Constituents
Oxoid (then the Medical Division of Oxo) started its specific amino acid linkage. Also, since proteins have
investigation into the manufacture of peptone in 1924. a very consistent primary structure, the mixture of
peptides produced after proteolytic digestion by a
The variety of peptones and extracts available reflects
specific enzyme is also consistent.
the differing demands of micro-organisms for amino
acids, peptides and other nutrients. Substrates used Enzymes commonly used are papain, pepsin and
by Oxoid for hydrolysis include: meat, casein, pancreatin, Figure A.
lactalbumin, milk, gelatin, soya and yeast cells. Pepsin will cut the chain anywhere there is a
phenylalanine or leucine bond3.
2 BASIC INFORMATION
Papain cuts adjacent to arginine, lysine, phenylalanine
Biochemistry of Proteins
and glycine4.
Proteins are macro-molecules and are fundamental to
the structure and function of all living organisms. Pancreatin has its action at arginine, lysine, tyrosine,
Chemically, proteins are made up of one or more tryptophan, phenylalanine and leucine bonds4.
chains of alpha-amino-carboxylic acids (amino acids), Raw materials may vary considerably in composition
consecutively linked covalently between the alpha- and the extent to which the protein components have
amino group of one moiety and the alpha carboxylic been denatured during any processing procedures,
group of the next with the elimination of water. This therefore the conditions of manufacture must be
linkage is termed the `peptide bond'. Chains of three carefully controlled to minimise the variations
or more amino acids are termed `polypeptides', whilst inherent in biological materials and so maintain
larger structures, with an arbitrarily determined quality.
lower molecular weight limit of 5,000 are the proteins.
An example of a Peptide and Amino Acid is shown in More defined protein sources, such as casein and
Figure A. gelatin will give more consistent mixtures of peptides
when treated with enzymes or acid.
Only 20 amino acids commonly occur in proteins.
They can occupy any position in the protein chain In practical terms, total breakdown of a protein to its
which can be at least 80 units long with molecular individual component amino acids is difficult even
weights of several millions. The chains are folded in a with a mixture of enzymes; the result, even with well
variety of complex forms and the structures may defined proteins such as casein, is a peptone
incorporate other macro-molecules such as containing a chemically undefined mix of peptides
carbohydrates and lipids. and amino acids.
Hydrolysis of Proteins Manufacture of Peptones
The hydrolysis of proteins, which breaks them down The manufacturing process is illustrated
to their constituent amino acids and peptides can be diagrammatically in Figure B and the syrup formed
achieved by the use of strong acids, strong bases or can be stored for long periods at room temperature
proteolytic enzymes such as pepsin, papain and because the high dissolved solid content inhibits
pancreatin (which contains trypsin)1. bacterial contamination. This syrup can be used in
fermentation processes without drying to a powder.
Hydrolysis with strong mineral acids, often at high
temperatures and pressures is much used in the food Quality Assurance
industry to produce food flavourings. The most It is essential that the quality of these products is
commonly used product in microbiology is based on maintained at the highest level and lot to lot variation
the hydrolysis of casein. In this process all peptide reduced to a minimum by closely following codes of
bonds are attacked and in theory, complete Good Manufacturing Practice (GMP)5. In order to
breakdown into component parts could be obtained. achieve this several types of analysis are carried out
However, because the reaction conditions are so and strict quality control specifications must be met
severe, some of the amino acids produced are for a lot to be accepted. A list of average analyses of
themselves destroyed by the process, notably hydrolysed products is shown on page 3±12.
tryptophan which is totally lost. Cystine, serine and To ensure that the product conforms to
threonine are partially broken down but asparagine predetermined specifications tests are carried out and
and glutamine are converted to their acidic forms. the following criteria are routinely monitored: clarity
Any vitamins present are largely destroyed. A series and colour, moisture content, pH value, ash residue,
of reactions may also take place between chloride, nitrogen content and microbiology.
carbohydrates and amino acids (such as the Maillard
reactions) which give rise to very dark products often Clarity and pH Value
toxic to the growth of micro-organisms2. For These tests are performed on an autoclaved 2%
microbiology the amount of hydrolysis is controlled solution of the final product and are controlled by
to produce a suitable nitrogen source for bacteria. comparison with reference materials.
Proteolytic enzymes act on proteins under less severe Moisture Content
conditions. They will function at much lower The level of moisture should be below 5% to ensure
temperatures and at normal pressures and are usually no chemical changes occur if the product is stored at
specific to the peptide bond they will attack. This high ambient temperatures.
means that the protein is not completely hydrolysed Ash Residue
to its constituent amino acids but into polypeptides of The ash residue consists mainly of inorganic material
varying lengths, depending on the frequency of the and is estimated after ignition.
3-2 November 1998
Peptones, Hydrolysates, Agars & Constituents
has its action at specific bonds, less hydrolysis is the maintain quality and reproducibility of existing
result. Proteose peptone is specially digested to processes. The peptone profiles above show the affect
contain higher molecular weight peptides and so has of hydrolysis time on the molecular profile (Figure E).
the lowest DH of all. From work by Adler-Nissen (ref Thus a range of peptones can be made with a wide
8) Figure D shows that during the course of a digest variety of chemical and bacteriological properties to
the DH achieved depends on a number of factors such different specifications.
as enzyme concentration or hydrolysing agent used. How the Test can Help the End User
Other variables that affect DH include type of Molecular profiles can give valuable information
substrate, temperature and pH. about the user's application and particularly shows
Size exclusion is perhaps one of the most useful how to improve yields or growth of organisms.
analyses of protein hydrolysates and assists in the Profiles can be run on peptones or complete
development of new products while helping to fermentation media before and after microbial
growth. By making a comparison of the profiles
before and after use, an indication of the efficiency of
the peptone for growing a particular organism is
obtained. These peptones can then be modified to
maximise organism growth or product yield. An
example of the peptone utilisation is shown in
Figure F.
3 MEAT PEPTONES
Oxoid manufactures a comprehensive range of Meat
Peptones derived from different animal tissues to suit
a range of nutritional requirements, using a number
of proteolytic enzymes and manufacturing processes.
Since the origin of these animal materials is
important, Oxoid only source from countries whose
disease status is acceptable and only use tissues from
selected portions of the animal. The tissues are
hydrolysed to produce straw coloured peptones
which are highly nutritious and clearly soluble in
water. The product reaches the consumer as an easily
handled, spray dried powder, although for some
applications the product can be used in syrup form.
The digest is water soluble and compatible with other L37 Typical analysis (% w/w)
culture media ingredients and may be sterilised by Total Nitrogen 15.2
filtration or autoclaving; thus it is suitable for use as Amino Nitrogen 2.9
an integral part of many culture media or as a Sodium chloride 1.0
valuable supplement. Being derived from liver this pH (2% solution) 6.3
product contains relatively high levels of iron. The
profile shows the characteristic even spread of
peptides obtained from papaic digests.
Typical Analysis (% w/w)
Total Nitrogen 11.0
Amino Nitrogen 3.6
Sodium chloride 1.6
pH (2% solution) 7.1
SPECIAL PEPTONE
Code: L72
A specially designed mixture of peptones, including
meat, plant and yeast digests designed to encourage
the growth of the most demanding organisms. It
contains a wide spectrum of peptide sizes together
with those minerals, vitamins, nucleotides and other
carbon compounds present in the individual
peptones.
Special Peptone is an ingredient of media where a
wide range of fastidious organisms are to be cultured
such as, Columbia Agar or Schaedler media and GC
Agar Base.
Typical analysis (% w/w)
Total Nitrogen 12.2
Amino Nitrogen 3.5
Sodium chloride 3.5
pH (2% solution) 7.2
November 1998 3-7
Peptones, Hydrolysates, Agars & Constituents
PEPTONISED MILK
Code: L32
This is a pancreatic digest of high grade skimmed
milk powder. It constitutes a source of nitrogen more
readily available than milk or milk powder and has a
high level of carbohydrate. As with milk powder, the
calcium level is relatively high.
The product may be used on its own or in conjunction
with other ingredients in media for isolation of
lactobacilli and bacteriological examination of dairy
products.
4 VEGETABLE PEPTONE It has a high tryptophan content and is therefore used
in media for testing the indole reaction.
SOYA PEPTONE Typical analysis (% w/w)
Code: L44 Total Nitrogen 5.3
Amino Nitrogen 1.8
Soya Peptone is obtained by the papain hydrolysis of Sodium chloride 1.6
soya flour and complies with the USP specification Tryptophan 0.53
(ref 9). In additon to its nitrogen constituents, this pH (2% solution) 6.5
peptone has a high carbohydrate content and is
suitable for many purposes. The presence of the
sugars stachyose, raffinose, sucrose and various
reducing sugars may be of importance in certain
applications. It is widely used in culture media and is
TRYPTONE T
Code: L43
This product was developed from Oxoid Tryptone
L42 by controlled enzymatic hydrolysis and modified
by the method of Meuller and Miller11. This produces
a lower level of calcium, magnesium and iron than in
Tryptone which makes it ideal for the production of
toxin by Clostridium tetani.
Typical analysis (% w/w)
TRYPTONE Total Nitrogen 11.7
Amino Nitrogen 3.5
Code: L42 Sodium chloride 3.5
Tryptone is a pancreatic digest of casein. It can be Calcium 280ppm
used in any formulation where a pancreatic or tryptic Magnesium 24ppm
digest of casein is specified and complies with the Iron 3ppm
specification for pancreatic digest of casein in the U.S. pH (2% solution) 7.0
Pharmacopoeia10. Casein is the main protein of milk LACTALBUMIN HYDROLYSATE
and is a rich source of amino acid nitrogen. The
profile shows a broad spread of peaks throughout the Code: L48
molecular weight range characteristic of a pancreatic After removal of casein from milk, lactalbumin is a
digest. This hydrolysate is often mentioned in protein extracted from the resulting whey. L48 is a
published works, either as a constituent of culture pancreatic digest of this protein and contains high
media for metabolic or growth studies, or for other levels of essential amino acids.
purposes where high performance and uniformity of
composition are of paramount importance. It has a It is most commonly used in media for tissue culture
high tryptophan content and is therefore used in and therefore production of vaccines of viral origin
media for testing the indole reaction. including foot and mouth disease, polio, dengue,
coxsackie B3 and many other viruses.
November 1998 3-9
Peptones, Hydrolysates, Agars & Constituents
Other uses include growth of lactobacilli, spore It will enhance the growth of many bacteria and is
growth of clostridia and in fermentation procedures therefore incorporated into a wide range of culture
for hormone production. media as a solid foundation material.
Typical analysis (% w/w) It is used in fermentation processes.
Total Nitrogen 12.5 Typical analysis (% w/w)
Amino Nitrogen 5.4 Total Nitrogen 13.3
Sodium chloride 0.2 Amino Nitrogen 2.5
pH (2% solution) 6.5 Sodium chloride 1.1
pH (2% solution) 7.2
6 EXTRACTS
LAB-LEMCO References
1 Haurowitz F. (1963)`The Chemistry & Function of Proteins' 2nd
Code: L29 Edition Academic Press.
Lab-Lemco is a meat extract made from specially 2 Einarsson H., Snygg B. G., Ericsson G. (1983) J. Agric. Food Chem.
selected raw materials, adjusted to neutrality and 31. 10.
dried to a fine powder. The product has considerable 3 Dixon M., Webb E. C. (1979) `Enzymes'. 3rd Edition Longman, GP
advantages over conventional meat extracts. Being a Limited, page 892.
refined and clarified extract it can be used with other 4 Dixon M., Webb E. C. (1979) `Enzymes', 3rd Edition Longman, GP
refined ingredients to make culture media which Limited, page 886.
require no filtration. Being only slightly hygroscopic 5 `Guide to Good Pharmaceutical Manufacturing Practice' (1983)
this product is very easy to handle. Its use eliminates Editor J. Sharp. Her Majesty's Stationery Office.
the troublesome procedures associated with handling 6 Bradstreet (1965) `The Kjeldahl Method for Organic Nitrogen'
conventional meat extracts which have a paste-like Academic Press, New York.
consistency. 7 Taylor (1957) Analyst 82. 488.
8 Adler-Nissen J. (1978) Ann. Nutr. Alim. 32. 205±216.
9 United States Pharmacopoeia (1985) 21st Revision p. 1396.
10 United States Pharmacopoeia (1985) 21st Revision pp. 1394±1396.
11 Meuller J. H. and Miller P. A. (1958) J. Bact. 67. 271±277.
Products not in our normal range, can be To prepare a liver infusion medium, add 50 grams of
manufactured on request provided a minimum of Liver Desiccated to 1 litre of distilled water and allow
100K is required. Customers interested in this service to infuse (with frequent agitation) for 1 hour at 508C.
are asked to state their specifications and discuss the Boil the mixture for a few minutes to coagulate
feasibility of manufacturing any such product with protein, strain through 60-mesh stainless steel gauze,
their Oxoid contact. add 10 grams of Peptone L34 and 5 grams of sodium
chloride. Adjust the reaction to pH 7.2, boil, strain
LIVER DESICCATED through gauze as above, and sterilise by autoclaving
at 1218C for 15 minutes.
Code: L26
Dehydrated whole liver, specially manufactured for
the preparation of infusion media. Liver Desiccated is
prepared by the dehydration of fresh ox livers under
carefully controlled conditions designed to ensure
maximum retention of nutritive properties, and is
equivalent to five times its weight of fresh liver.
Vegetable Peptone
Soya peptone Code L44 . . . Antibiotic production
Extracts
Yeast Extract Code L21 . . . . Increases yield
Lab-Lemco Code L29 . . . . Easy to use meat extract
Malt Extract Code L39 . Yeast and mould media
TABLE OF ANALYSIS
GENERAL ANALYSIS
TABLE OF ANALYSIS
AMINO ACID ANALYSIS
PURIFIED AGAR
Code: L28
An agar that has been extensively processed to give a
low electroendosmosis factor (mr) enabling the
product to be used in electrophoresis studies without
the high expense of using agarose preparations. It can
also be used for bacteriological culture media where
its special properties are required. An agar
recommended for immuno-electrophoresis and gel
diffusion studies.
Although this process yields bile salts, the product It can be seen from these variable factors that the
obtained may be a mixture of conjugates and free bile production of standard bile-containing media is a
acids, depending on the quality of the bile used in the difficult process which requires much skill and
process. experience.
LACTOSE BACTERIOLOGICAL
Code: L70
A special grade for inclusion in microbiological
media. Each batch is tested chromatographically to
ensure purity and correct identity.
SODIUM BISELENITE
(Sodium hydrogen selenite)
Code: L121
For use in Oxoid Selenite Broth Base CM395/CM396
and Mannitol Selenite Broth Base CM399.
CAP SECURELY AFTER USE.
Dissolve 4 grams in 1 litre of distilled water and use
this solution to reconstitute the base medium or
tablets.
Toxic by inhalation and if swallowed. Danger of
cumulative effects.
SODIUM CHLORIDE
Code: L5
See also Saline Tablets BR53.
This product is prepared from analytical grade salt to
avoid problems associated with additives.
SODIUM GLUTAMATE
Code: L124
For use with Minerals Modified Medium Base
(CM607).
concentration of the components in the final medium Oxoid Selective Media have the following features
and ± more serious still ± actual physical deterioration 1 Labile and hazardous components are presented in
may occur, particularly if storage is in warm nitrogen-flushed glass vials.
conditions. The effect of this on performance of 2 The stability of critical components is assured. A
selective media would be considerable. long shelf life (up to two years in most cases) is
Some selective agents are photo-sensitive and guaranteed.
although storage should be in the dark the problem is 3 Each vial contains a measured dose sufficient for a
probably of greater importance when the medium has standard volume of medium (usually 500ml). It is
been reconstituted ready for use. easy to reconstitute.
For best performance, preparation of the selective 4 The performance of the medium made with the
medium needs careful attention to the following selected basal medium and its compatible
points: supplement will be reproducible.
1 Use only completely clean equipment. Residues 5 Because the selective agents are not held for long
from incomplete cleaning can have a significant periods as solutions, but reconstituted immediately
effect. before use, they always have optimal activity.
Care must always be taken to prevent cross 6 A futher advantage of this concept is that some
contamination of one medium with another by flexibility of concentration is available. Although
dirty spatulae, balance pans, pipettes, etc. the vials are designed to be emptied into standard
volumes of medium, there is individual choice of
2 The choice of water is important. Glass distilled using less or more of the selective agent. In some
water is probably best, but de-ionised water is circumstances there may be advantages in
satisfactory. It should be remembered, however, changing the concentration of the selective agent in
that de-ionisers may harbour microbial flora, some order to yield optimum recovery of the desired
of which may produce inhibitory substances. organism and suppression of other flora.
Tap water should not be used.
3 Weighing and measurement must be precise The benefits of using the Oxoid range of
otherwise pH and concentrations of the Selective Media
constituents will be incorrect. These are evident from features described above, but
4 Finely powdered products can be scattered as dust they may be summed up as follows:
and some selective agents could become a health
1 More reliable media with greatly reduced
hazard if inhaled. Selective agents presented in
likelihood that labile ingredients will have
vials overcome this objection as the reconstitution
deteriorated.
is done by adding the sterile distilled water or
other solvent before the cap is removed. 2 The work of the media preparation staff is made
safer and easier.
5 The medium must be well mixed to ensure even
distribution, particularly of selective agents which 3 The microbiologist is free to concentrate on end
may be present in very small amounts. The product performance, confident of obtaining
addition of a solution of the selective agent to the reproducible results.
basal medium makes proper distribution more
certain.
6 Carry out the sterilisation procedure meticulously, SELECTIVE MICROBIOLOGY
taking care not to overheat. In most cases a better
medium will be obtained if the selective agent is Oxoid freeze-dried, selective and growth supplements
added aseptically after sterilisation. are used to prepare selective, differential or enriched
7 Selective agents, including antibiotics, may be culture media. They are designed to give optimum
labile especially in solution. Antibiotics are best performance when added to the appropriate Oxoid
kept dry until the time of use. Limited shelf life, base media shown in the directions.
unknown potency and cross-contamination are all All supplements should be stored at 2±88C.
special difficulties associated with antibiotics.
NOTE: before opening supplements containing
8 Media should be used as soon as possible after cycloheximide read the comments under HAZARDS
preparation, but if storage is needed this should be page 2-7.
in cool, moist and dark conditions to keep changes
in the medium to a minimum.
It was consideration of the special requirements of
selective media detailed above that led to the
conception of the Oxoid range of freeze-dried
supplements, which have been carefully standardised
for use with specified Oxoid dehydrated media. This
has not only greatly increased convenience for the
user, but has virtually eliminated health hazards to
the laboratory preparations staff.
Vial contents (each vial is sufficient for 500ml of A selective supplement for the isolation of Bordetella
medium) species when used with Bordet-Gengou Agar Base
Haemin 2.5mg CM267 or Charcoal Agar CM119. See Section 2.
Menadione 0.25mg Vial contents (each vial is sufficient for 500ml of
Sodium succinate 1.25g medium)
Nalidixic acid 5.0mg Cephalexin 20mg
Vancomycin 1.25mg
Directions
Directions To one vial add 2ml of sterile distilled water and
To one vial aseptically add 10ml of sterile distilled dissolve the powder completely. Add the contents
water and mix gently to dissolve completely. Avoid aseptically to 500ml of sterile, molten Charcoal Agar
frothing. Add the contents to 500ml of sterile Wilkins- CM119, cooled to 508C, together with 10%
Chalgren Medium, prepared from Wilkins-Chalgren defibrinated horse blood SR50. Mix well before
Anaerobe Agar CM619 plus 5% (v/v) defibrinated pouring into sterile petri dishes.
horse blood SR50 or SR51. Do not add `Tween 80'.
Mix gently and pour into sterile petri dishes. Bordet-Gengou Agar CM267 may be used instead of
Charcoal Agar. Add the reconstituted contents of one
ANAEROBE SELECTIVE SUPPLEMENT N-S vial to 500ml of Bordet-Gengou Agar cooled to 508C,
to which 75±100ml of fresh defibrinated horse blood
Code: SR107 SR50 has been added.
A selective supplement for the isolation of Non-Sporing The vial contents may also be added to half strength
anaerobes when used with Wilkins-Chalgren Agar Charcoal Agar plus 10% defibrinated horse blood
CM 619. See Section 2. (SR50), for use as a transport medium for Bordetella
pertussis.
Vial contents (each vial is sufficient for 500ml of
medium)
Haemin 2.5mg
BRUCELLA SELECTIVE SUPPLEMENT
Menadione 0.25mg Code: SR83
Sodium pyruvate 500.0mg
A selective supplement for the isolation of Brucella
Nalidixic acid 5.0mg
species when used with Blood Agar Base No.2
Directions CM271, Columbia Agar CM331 or Brucella Agar Bases
To one vial aseptically add 10ml of sterile distilled CM169 or CM691. See Section 2.
water and mix gently to disolve completely. Avoid
Vial contents (each vial is sufficient for 500ml of
frothing. Add the contents to 500ml of sterile Wilkins-
medium)
Chalgren Medium, prepared from Wilkins-Chalgren
Polymixin B 2,500 IU
Anaerobe Agar CM619 plus 0.1% (v/v) `Tween 80'
Bacitracin 12,500 IU
and 5% (v/v) defibrinated horse blood SR50 or SR51.
Cycloheximide 50mg
Mix gently and pour into sterile petri dishes.
Nalidixic acid 2.5mg
Nystatin 50,000 IU
Vancomycin 10mg
Directions Directions
To one vial add 2ml of sterile distilled water and mix To one vial add 5ml of ethanol/sterile distilled water
gently to dissolve the contents completely. Avoid (1:1) and mix gently to dissolve the contents
frothing. Aseptically add the vial contents to 500ml of completely. Avoid frothing. Aseptically add the vial
sterile Listeria Enrichment Broth Base (UVM contents to 500ml of sterilised Listeria Selective Agar
Formulation) CM863, below 508C. Mix well and pour Base (Oxford Formulation) CM856, cooled to 508C.
required volumes into sterile containers. Mix well and pour into sterile petri dishes.
Precautions
Colonies of some contaminating organisms growing STERILE REAGENTS
in close proximity to the coagulase positive colonies Sterile reagent products are offered in ready-to-use
may partially digest the coagulase halo reaction. form as enrichment solutions, essential growth
factors, enzyme substrates or biochemical indicators.
References
Each product has been processed with an appropriate
1 Beckers H. J., van Leusden F. M., Hogeboom W. M. and
sterilisation procedure, e.g. aseptic preparation,
Delfgon-van Asch E. H. M. (1980) (English summary) De Ware(n)-
filtration or irradiation, which will not affect the
Chemicals 10. 125±130.
performance of the product. All sterile reagents
2 Beckers H. J., van Leusden F. M., Bindshedler O. and Guerraz D.
should be stored at 2±88C away from light.
(1984) Can. J. Microbiol. 30. 470±474.
3 Baird-Parker A. C. (1962) J. Appl. Bacteriol. 25. 12±19.
EGG YOLK EMULSION
4 Hauschild A. H. W., Park C. E. and Hilsheimer R. (1979) Can. J.
Microbiol. 25. 1052±1057. Code: SR47
5 Sawhney D. (1986) J. Appl. Bact. 61. 149±155.
Description
6 Beckers H. J. (1985) Personal Communication.
A sterile stabilised emulsion of egg yolk for use in
7 van Schothorst M. (1985) Personal Communication.
culture media. It may be added directly to nutrient
media for the identification of Clostridium, Bacillus and
Staphylococcus species by their lipase activity.
units of beta-lactamase II. 1 unit of enzyme activity 5 Sabath L. D., Casey J. I., Ruch P. A., Stumpf L. L. and Finland
will hydrolyse 1 mmol of substrate per minute at pH M. (1971) J. Lab. Clin. Med. 78. 457±463.
7.0 and at 258C; beta-lactamase I is assayed using 6 Code of Federal Regulations, Title 21, Part 436, Sec.436.20 U.S.
benzyl penicillin in the presence of EDTA, and beta- Govt. Printing Office, Washington, D.C.
lactamase II using cephalosporin C in the presence of
Zn2+.
NITROCEFIN (GLAXO RESEARCH 87/312)
Code: SR112, SR112A
Definition of Units of Enzyme Activity
The scientific literature describes a number of For the rapid chromogenic detection of beta-lactamase
methods which are used to measure and define a unit activity.
of penicillinase of beta-lactamase activity2. Note that 1
Reagents
IU of activity = 600 Levy units of activity.
SR112
Application Vial of lyophilised Nitrocefin, containing 1mg
There are four major uses of this preparation of Nitrocefin.
enzymes.
SR112A
1 Inactivation of beta-lactam antibiotics in blood or Rehydration fluid. The vial contains 1.9ml of
other tissue samples prior to routine phosphate buffer (0.1M, pH 7.0) and 0.1ml of
microbiological examination2,3,4. dimethylsulphoxide.
2 Inactivation of beta-lactam antibiotics in blood and
other tissue samples prior to the microbiological Directions
estimate of aminoglycosides or other non-lactam Reconstitute the contents of one vial of lyophilised
antibiotics4,5. Nitrocefin SR112 by adding the entire contents (2ml)
of one vial of rehydration fluid SR112A. This yields a
3 The inactivation of beta-lactam antibiotic working Nitrocefin solution of 500mg/ml, (approx
preparations to enable sterility testing to be carried 10-3 M) suitable for most applications.
out before the administration of such preparations
to patients undergoing therapy with immuno- Precautions
suppressants, or who have a naturally low level of Nitrocefin, particularly in solution, is very light
immunity6. sensitive. The solution may be stored at ±208C for up
4 Assessment of the susceptibility of new beta-lactam to two weeks. INGESTION OR INHALATION, OR
antibiotics to inactivation by lactamase. CONTACT WITH THE SKIN AND EYES SHOULD
BE AVOIDED.
Methods
1 Blood Culture Procedures General Introduction and Intended Uses
Nitrocefin is the chromogenic cephalosporin
Inject 5ml of sterile distilled water into a vial of
developed by Glaxo Research Limited. (Coded 87/
enzyme mixture and mix gently. Add 1ml of this
312; 3-(2,4 dinitrostyrl) ± (6R,7R±7±(2-
solution aseptically to the blood culture bottle,
thienylacetamido)±ceph±3±em±4±carboxylic acid, E-
preferably before or immediately after inoculation
isomer)1.
with the blood sample (5±10ml).
2 Microbiological Assay of Non-Lactam Antibiotics This compound exhibits a rapid distinctive colour
change from yellow (max at pH 7.0 = 390nm) to red
1ml of the beta-lactamase enzyme solution should
(max at pH 7.0 = 486nm) as the amide bond in the
be added aseptically to 1ml of blood sample or beta-lactam ring is hydrolysed by a beta-lactamase
serum. This should be incubated at 308C for a
(E.C 3.5.2.6); it is sensitive to hydrolysis by all known
period of time depending on the beta-lactam
lactamases produced by Gram-positive and Gram-
antibiotic present. A minimum time would be 5
negative bacteria. This characteristic reaction forms
minutes and a maximum 60 minutes. After
the basis of a number of methods suitable for
incubation, the blood or serum samples should be
diagnostic use.
applied to wells in previously seeded antibiotic
assay plates in the normal manner. Apart from its use in giving rapid indication of beta-
Stability of Reagents lactamase potential, the reagent has been found
Solutions of the enzyme will remain active for several extremely useful for the detection of beta-lactamase
days when stored at 48C or several weeks when patterns from bacterial cell extracts on iso-electric
stored at minus 208C. focusing2,3,4 and has been used in inhibition studies in
development work on beta-lactamase resistant
Repeated freezing and thawing should be avoided. antibiotics5.
However, it is not advisable to store the solution for
long periods because of the possibility of Description of Use
contamination. Demonstration of beta-lactamase activity in
bacterial cells.
References Nitrocefin degradation should be used to give a rapid
1 Davis R. B., Abraham E. P. and Melling J. (1974) Biochem. J. 143. indication of beta-lactam inactivating systems and the
115±127. result so obtained will, in most cases, predict the
2 Waterworth P. M. (1973) J. Clin. Path. 26. 596±598. outcome of susceptibility tests with beta-lactam anti-
3 Selwyn S. (1977) J. Antimicrob. Chemother. 3. 161±168. microbials. However, it should not entirely replace
4 Newson S. W. B. and Walshingham B. M. (1973) J. Med. conventional susceptibility testing as other factors
Microbiol. 6. 59±66.
also influence the results of such tests, and on are stained by applying Whatman No.54 paper
occasion intrinsic resistance to beta-lactam impregnated with the Nitrocefin solution2. Focused
antimicrobials has not been correlated with bands in the gel with beta-lactamase activity appear
production of beta-lactamase6. pink on a yellow background.
Nitrocefin degradation has been found to be highly Determination of beta-lactamase activity by
efficient in detecting beta-lactamase producing spectrophotometric assay
isolates of Neisseria gonorrhoeae7,8, Haemophilus The working solution of Nitrocefin (500mg/ml) is
influenzae7,9,10,11 and staphylococci10,11. diluted tenfold in buffer (0.1M phosphate; 1mM
Excellent results have also been obtained with certain EDTA, pH 7.0). Spectrophotometric assays for beta-
lactamase are carried out measuring changes in
anaerobic bacteria, notably with Bacteroides
wavelength at 486nm. The molar extinction coefficient
species13,14,15. It should be emphasised that the
efficacy of the Nitrocefin tests in predicting the beta- of Nitrocefin at this wavelength is 20,500.
lactam susceptibilities of other micro-organisms is at Test samples of the finished product for performance
present unproven. with control cultures.
Another chromogenic cephalosporin, PADAC References
(Hoechst-Roussel) was not as effective as Nitrocefin in
1 O'Callaghan C. H., Morris A., Kirby S. M. and Shingler A. H.
detecting staphylococcal beta-lactamase12.
(1972) Antimicrob. Ag. & Chemother. 1. 283±288.
Technique 2 Mathew M., Harris A. M., Marshall M. J. and Ross G. W. (1975)
Rehydrate the Nitrocefin as directed, and use this J. Gen. Microbiol. 88. 169±178.
solution in the following ways: 3 Sparks J. and Ross G. W. (1981) J. Med. Microbiol. 15. p. iv.
1 4 King A., Shannon K. and Phillips I. (1980) Antimicrob. Ag. &
1 Direct Plate Method
Chemother. 17. 165±169.
Add one drop of the Nitrocefin solution on to the 5 Guay R., Letarte R., Pechere J. C. and Roy B. (1980) IRCS Med.
surface of the colony. If the isolate is a high beta- Science 8. 209.
lactamase producer then the colony and the 6 Markowitz S. M. (1980) Antimicrob. Ag. & Chemother. 6. 80±83.
surrounding area will quickly turn red. 7 Shannon K. and Phillips I. (1980) J. Antimicrob. Chemother. 6.
To detect a weak beta-lactamase producer the plate 617±621.
should then be incubated for 30 minutes before 8 Sng E. H., Yeo K. L., Rajan V. S. and Lim A. L. (1980) Br. J.
being reported as negative. Vener. Dis. 56. 311±313.
2 Slide Method1 9 Bell S. M. and Plowman D. (1980) Lancet i. 279.
10 Montgomery K., Raymundo L. and Drew W. L. (1979) J. Clin.
Add one drop of the Nitrocefin solution on to a
Micro. 9. 205±207.
clean glass slide. Using a sterile loop, pick one
11 Lucas T. J. (1979) J. Clin. Pathol. 32. 1061±1065.
colony from the plate and emulsify into the
12 Anhalt J. P. and Nelson R. (1982) Antimicrob. Ag. & Chemother.
Nitrocefin drop. Report as positive if the colour
21. 993±994.
changes from yellow to red within 30 minutes 13 Gabay E. L., Sutter V. L. and Finegold S. M. (1981) J. Antimicrob.
(protect the slide from desiccation during the
Chemother. 8. 413±416.
waiting period).
14 Timewell R., Taylor E. and Phillips I. (1981) J. Antimicrob.
3 Broth Method1 Chemother. 7. 137±146.
Add four drops of Nitrocefin solution to 1ml of the 15 Bourault A. M. and Rosenblatt J. E. (1979) J. Clin. Micro. 9. 654±
grown culture. Report as positive if the colour 656.
changes to red within 30 minutes.
4 Broken Cell Method1 PENASE
Sonicate 1ml of the culture in order to break open Code: SR129
the cells. Add 4 drops of Nitrocefin solution.
Report as positive if the colour changes to red 569/H9 Lactamase active against a range of penicillins.
within 30 minutes. Materials Supplied
5 Paper Disc Spot Test10 Penase SR129 is a Bacillus cereus 569/H9 lactamase
A Whatman No.1 filter paper disc (diameter 7cm) (E.C.5.2.6) presented as a sterile freeze-dried powder
is placed in a petri dish and impregnated with containing buffer salts. Each vial contains 3,300 IU of
Nitrocefin solution (0±5ml). This impregnated activity (1 unit of enzyme activity will hydrolyse 1.0
paper is generally usable for one day, but should mmol of benzylpenicillin to benzylpenicilloic acid per
be kept away from light to avoid spontaneous minute pH 7.0 and at 258C). The preparation will
degradation. An isolated colony is applied to the successfully inactivate a range of penicillins1.
impregnated paper with a loop; a pink to red Definition of Units of Enzyme Activity
reaction developing within 15 minutes indicates The scientific literature describes a number of
beta-lactamase presence. methods which are used to measure and define a unit
Detection of beta-lactamase activity on gels of penicillinase activity2. Note that 1 IU of activity =
Methods for preparing extracts containing the beta- 600 Levy units of activity.
lactamase activities from bacterial cells and the
Application
technique for analytical iso-electric focusing have
The major use of this enzyme preparation is for the
been described by Matthew et al.2 The developed gels inactivation of susceptible beta-lactam antibiotic
4-16 November 1998
Selective Supplements, Sterile Reagents
a differential cell count, fix a dried smear in methyl approved protective cabinets. Staff should be
alcohol and stain with haematoxylin and eosin or specially trained, tested for adequate immunity and
with Lieshmann stain. medically examined at appropriate intervals.
Reconstituted solutions of Sputasol, if kept sterile, are
stable for at least 48 hours stored at 2±88C. ACTIDIONE1 AGAR
An investigation into the survival of respiratory Code: PM118
pathogens in specimens that had been stored for 48 Formula gm/litre
hours at 48C following homogenisation using Yeast extract 4.0
Sputasol, showed that the organisms remained viable Tryptone 5.0
and, when necessary, treated specimens could be Glucose 50.0
succesfully re-cultured8. Potassium dihydrogen phosphate 0.55
Potassium chloride 0.425
References Calcium chloride 0.125
1 Mulder J. (1938) Acta. Med. Scand. 94. 98.
Magnesium sulphate 0.125
2 May J. R. (1952) Lancet 20.12.52. 1206±1207. Ferric chloride 0.0025
3 Rawlins G. A. (1955) J. Med. Lab. Technol. 13. 133±143. Manganese sulphate 0.0025
4 Dixon J. M. S. and Miller D. C. (1965) Lancet ii, 1046±1048. Bromocresol green 0.022
5 Cleland W. W. (1964) Biochemistry 3. 480±482.
Cycloheximide 0.01
6 Hirsch S. R., Zastrow J. E. and Kory R. C. (1969) J. Lab. & Clin. Agar 15.0
Med. 74. 346±353. Distilled water to 1 litre
7 Shah R. R. and Dye W. E. (1965) Amer. Rev. Resp. Dis. 94. 454.
pH 5.5 + 0.2
8 Could F. K., Freeman R., Hudson S. et al (1996) J. Clin. Pathol.
49. 684±686. Description
Actidione1 (cycloheximide) at a concentration of
TTC SOLUTION (0.05%) 0.001% w/v permits the growth of bacteria but
Code: SR148 inhibits the growth of most yeasts and moulds except
dermatophytes. Media containing this antibiotic are
TTC Solution is supplied as 5ml of filter-sterilised invaluable for the enumeration and detection of
aqueous solution of tri-phenyltetrazolium chloride bacteria in specimens containing large numbers of
(TTC). It is used to supplement Tergitol-7 Agar yeasts and moulds. For example, the medium is used
CM793 for the enumeration of coliforms in food and for the estimation of bacterial contamination of
water supplies. pitching yeast. `Actidione' Agar with added penicillin
and streptomycin is also valuable as a selective
READY PREPARED MEDIA AND medium for the isolation of dermatophytes.
DIP SLIDES The Oxoid medium, based on that of Green and
Gray1, may be used for microbiological investigations
Coded PM, R and DS during brewing and baking. As an approximate
These products are supplied in a form which is ready guide, incubation may be at 258C or 308C for up to 14
for inoculation and incubation. days, according to the flora present. Green and Gray
employed their medium at two different reactions,
Storage conditions and Shelf life pH 5.5 and pH 6.5, the latter may be attained by
PM and R products should be stored at 2±88C. DS adding approximately 16ml of sterile 1.5% sodium
products can be stored at low room temperature. All carbonate to each litre of molten medium.
products should be used before their expiry date
shown on the label. Reference
Quality Control 1 Green S. R. and Gray P. P. (1950) Wallerstein Lab. Communication
These products have been prepared and tested to 13. 357.
high standards. Microbiological control tests, using SELENITE BROTH
appropriate organisms, are made and the product is
released only if it satisfies these tests. If the medium is Code: R39
stored as directed and used within the expiry date, Formula gm/litre
then further tests are not required. However, if it is to Peptone 5.0
be used for purposes other than those described Lactose 4.0
below, then it would be wise to make suitable control Sodium biselenite 4.0
tests to ensure growth of the expected organism. Sodium phosphate 10.0
Precautions Distilled water to 1 litre.
As with all culture media, these products should be pH 7.1 + 0.2
used by trained staff only, in suitable laboratories and Description
disposed of safely by autoclaving at 1218C for at least Selenite Broth in ready-to-use form is prepared from
20 minutes. Selenite Broth Base CM395 with Sodium Biselenite
Mycobacterium tuberculosis (incl. M. bovis) is classified L121.
as a hazardous organism, specimens and cultures It is a modification of the original Leifson1 selenite
must be processed in contained laboratories, using broth F, used as an enrichment broth for the isolation
References
1 Davey B. B. and Turner, Myfanwy (1961) J. Appl. Bact. 24(1).
78±82.
2 Hayes P. R. (1963) J. Gen. Microbiol. 30(1). 1±19.
3 Innes A. G. (1956) J. Appl. Bact. 19(1). 39±45.
4 Tanner F. W. (1944) `The Microbiology of Foods', 2nd ed., Garrard
Press, Illinois, pp. 474±479.
5 Willis A. T. (1960) J. Path. Bact. 80(2). 379±390.
6 Willis A. T. and Gowland G. (1965) Nature 187(4735). 432±433.
between 75 and 858C for 45 minutes. The high egg formula gave earlier or heavier growth in 25 cases
content is needed to provide a firm slope. and the original medium in 3 cases, a significant
advantage of the new medium (P<0.001); growth was
Acid Egg Medium No.1b: This is prepared as above,
equal in 14 cases and absent in the remainder.
but the 12.0ml glycerol are replaced with 7.0g of
sodium pyruvate. The Reference Laboratory's method for sputum was
used to compare Acid Egg Medium No.2b with the
Reference Laboratory method for sputum (Marks and
pyruvate version of the method's original `buffered
Thomas4)
egg medium'. This trial was conducted on unselected
Two ml of 4% NaOH are made up to 4ml with
sputum or in the case of scanty specimens, with routine specimens. The new formula gave earlier or
heavier growth in 102 cases and the original medium
sputum and sterile water. After incubation for 15 to
in 23 cases, which is again a significant advantage to
30 minutes as above, the treatment bottle is held at an
angle and 16ml of sterile water poured in from a 28ml the new medium (P<0.001); growth was equal in 62
cases. All the media in these two trials were given the
(universal) container. Centrifugation and decanting
same low content of malachite green (approximately
follow and the deposit is divided between two slopes
0.0125%) as the object was to isolate the effect of
and a film. The medium recommended for this
phosphate concentration.
method will be called Acid Egg Medium No.2; it is also
made in glycerol (a) and pyruvate (b) versions. The third trial compared 0.0125% and 0.025%
malachite green in Acid Egg Medium No.2b using
Acid Egg Medium No.2a contains 2.4g KH2PO4, 0.3g
unselected specimens. With the weaker dye, growth
MgSO4.7H20, 12.0ml glycerol and 600ml distilled
was earlier or heavier in 32 cases and with the
water. The ingredients are dissolved in the water
stronger dye in 7 cases, a significant advantage to the
autoclaved at 1108C for 15 minutes. To this solution
lower concentration of malachite green in this
the following are then added aseptically: 1000ml
medium (P<0.001); growth was equal in 26 cases.
whole egg (mixed and strained through gauze), 40ml
N HCl, 10ml malachite green (2% w/v) and 100 000 Discussion
IU of sodium penicillin. The medium is distributed Four procedures for preparing specimens for culture
and inspissated as for Acid Egg Medium No.1. In the are described above and these provide respectively
pyruvate version (2b) the 12.0ml glycerol are replaced inocula which are (1) highly alkaline, (2) moderately
by 7.0g sodium pyruvate. alkaline, (3) neutral or slightly acid (by over-shoot)
and (4) moderately acid. Lowenstein-Jensen Medium
Petroff's method and modifications
and its simplified versions are suitable for the inocula
In the classical method of Petroff and in its
in groups (3) and (4) which vary from neutral to
modifications, sputum is incubated with an equal
moderately acid, but the pH of this medium rises to
volume of 3% or 4% NaOH. The mixture is then
an unfavourable level with alkaline inocula. The
neutralised, centrifuged and the deposit inoculated.
media originally recommended for the methods
Some workers centrifuge first, with or without
providing alkaline inocula depended on phosphate
preliminary dilution, and neutralise the deposit. The
buffer for control of the pH.
medium recommended for this method is called
Simplified Lowenstein-Jensen (L-J) and it is made in Experiments preliminary to the present work showed
glycerol and pyruvate versions. that the concentrations of phosphate used were
detrimentally high. In the new media, phosphate has
Simplified L-J Medium This medium has the same
been partially replaced by HCl. Malachite green is
formula as Acid Egg Medium No.2 except that the
more antibacterial at lower pH levels and the present
hydrochloric acid is omitted and the volume of
work has confirmed that lower concentrations than
malachite green solution is increased to 20ml.
usual should be used in acid egg medium.
Sulphuric acid treatment
The Simplified L-J Medium described above is based on
Apart from sputum and the deposit of gastric
earlier work aimed at simplifying Lowenstein-Jensen
contents, most specimens are best treated with
Medium (Marks and Thomas4, Stonebrink (1961)6)
sulphuric acid as described by Marks5. However, the
and has not been subjected to further trials. Credit for
Reference Laboratory has now reduced the maximum
the introduction of pyruvate medium is due to
reagent concentration of 5% to 4% (v/v).
Stonebrink6. We advise that most types of specimen
Treatment is followed by dilution, centrifugation and should be cultured on both glycerol and pyruvate
decanting which leaves a moderately acid deposit for medium as tubercle bacilli vary in their preference.
inoculation. The medium recommended for this
method is Simplified L-J Medium. References
1 Zaher F. and Marks J. (1977) Tubercle 58. 143±145. Reproduced by
Results
permission of the Authors and the Editor and Publisher of Tubercle.
The preliminary experiments which led to the
2 Zaher F. (1977) Factors promoting the growth of tubercle bacilli in
improvements of medium formulation is cited
artificial culture. M.Sc. Thesis, University of Wales.
elsewhere (Zaher 1977). However, the results of three
3 Marks J. (1959) A simple method for the cultivation of tubercle bacilli.
final trials are presented here. The `simple method' of
Monthly Bulletin of the Ministry of Health and the PHLS, 18. 81.
sputum culture used to compare Acid Egg Medium
4 Marks J. and Thomas C. H. H. (1958) Notes on the cultivation of
No.1 with the `acid egg medium' originally devised
tubercle bacilli. Monthly Bulletin of the Ministry of Health and the
for this method of culture. Glycerol and pyruvate
PHLS, 17. 194.
versions of both media were used in parallel for 50
5 Marks J. (1972) Endiong the routine guinea-pig test. Tubercle, 53. 31.
specimens selected as likely positives. The new
6 Stonebrink B. (1961) A new medium for the cultivation of container. By dipping the slide into freshly voided
Mycobacterium tuberculosis. Selected Papers. The Royal Netherlands urine, reliable counts can be obtained. These show the
Tuberculosis Association, 2. 1±22. number of organisms present in the urine at the time
of voiding.
The method has the following advantages:
THE OXOID DIP SLIDE
1 The Dip Slide technique is the simplest, most rapid
Code: DS and most reliable method for determining the
Kass1 defined significant bacteriuria on the basis of presence or absence of urinary tract infection.
colony counts of carefully collected, freshly voided, 2 Counts on a Dip Slide have been shown to
total specimens of urine obtained from populations of correlate closely with counts made by careful
asymptomatic women. He found that, on the basis of quantitative methods in the laboratory6,8,9,10.
the counts obtained, it was possible to distinguish 3 Since the slide is dipped into freshly voided urine,
between infected and contaminated urine. When the the counts obtained reflect the number of
count was more than 105 organisms per ml in a single organisms present at the time the urine was
specimen, the probability was about 80 per cent that passed. It is well-known that, as a result of its
infection was present. If the observation was repeated growth supporting properties, urine held for some
in a further specimen, the probability of infection was time at room temperature before examination
greater than 90 per cent. cannot be relied upon to yield reliable colony
Since these original observations it has become counts.
apparent that when carefully collected, mid-stream 4 It is usually a simple matter for the practised eye,
urine samples are obtained from symptomatic when the Dip Slide is used, to distinguish between
patients, the colony counts required for the diagnosis urinary infection and contamination from other
of urinary tract infection are lower than those defined sources.
by Kass for asymptomatic populations2,3. A count of 5 The time required by the medical laboratory
more than 102 organisms per ml can now be regarded scientist to examine each specimen is minimal.
as diagnostic in a woman with acute symptoms of Screening of large numbers of specimens can be
urinary tract infection, while in men the diagnostic achieved almost at a glance.
count is 103 organisms per ml. In most circumstances,
6 The Dip Slide inoculation technique is sufficiently
however, the counts obtained will be greater than 105
simple that it can be carried out at home by the
organisms per ml.
patient.
OXOID DIP SLIDE REGRESSION SLOPE 7 The Dip Slide technique can be used equally for the
examination of urine from infants and children.
Since the original observations by Kass many cultural
and chemical methods have been devised to measure METHOD OF USE
the bacterial population of urine.
The object is to wet both sides of the Oxoid Dip Slide
All these methods depend on freshly voided mid- with a mid-stream specimen of urine. This is done by
stream urine promptly delivered to the bacteriology collecting a mid-stream specimen in a sterile container
laboratory for examination, either immediately or and then dipping the slide into the urine. After
after brief refrigerated storage. dipping, the slide is held vertically for a moment to
Mackey and Sandys4 devised the method of filling a allow any surplus urine to drain from its lower end,
spoon with a solid medium and using this as a dip- after which the slide is returned into its sterile
inoculum transport medium, thus avoiding erroneous container.
counts that may result from bacterial growth in the Arniel11,12 devised a `dip-stream' technique that is
urine between collection and examination in the particularly suitable for children but can equally be
laboratory. used with adults. The patient commences micturition
The `Dip Slide' was a logical development of this and, when the stream is well-established, the Dip Slide
method5,6. By use of a microscope slide coated on is held into it sufficiently to wet each side thoroughly.
each side with a different culture medium larger areas The slide is then allowed to drain briefly and is
of medium were available to obtain a semi- returned into its sterile container. The Dip Slide dip-
quantitative measure of the bacterial count. stream technique is completely reliable and
inexpensive6,7 and can be used for large-scale screening.
Oxoid investigated the dip slide and concluded that
though the basic principle was satisfactory, useful With both methods care must be taken to ensure that
changes could be made in the design. The difficulty of the agar surface is not touched and patients must be
handling microscope dip slides without carefully instructed about the technique they should
contaminating the media, the relatively thin layers of follow13. It is helpful if the vulva are held apart
the coating agar and the uneven draining of the urine during the collection of mid-stream urine, but
on to a paper disc in contact with the bottom of the cleansing is not important. If it is done for any reason,
slide, were disadvantages not evident with the dip antibacterial soaps and antiseptics must be avoided.
spoon. The patient's name and details of the date and time of
The principle is simple. An agar-coated slide is collection are noted on the label and the container
provided, attached to the cap of a screw-capped with the Dip Slide is either posted or taken to the
References
1 Whillans D. (1950) J. Clin. Path. 3. 57.
2 Mayer M. M. et al (1946) J. Exp. Med. 84. 535±548.
3 Bradstreet C. M. Patricia and Taylor C. E. D. (1962) Mon. Bull.
Min. Hlth Pub. Hlth Lab. Serv. 21. 96±104.
4 Fulton F. and Dumbell K. R. (1949) J. Gen. Microbiol. 3. 97.
colony with the impregnated tip of the stick, rotate OXIDASE IDENTIFICATION
the stick, picking up a small mass of cells.
STICKS
4 Place the inoculated tip of the stick between the lid
and the base of the inverted plate. Code: BR64
5 The reaction requires moisture. The inoculated A convenient and stable presentation of oxidase
tip of the stick should be placed in the moisture reagent for the detection of oxidase-positive bacteria.
condensate in the lid. If condensate is not
The enzyme cytochrome oxidase is produced by
available in the inverted lid add one or two drops
many organisms including Neisseria and Pseudomonas
of distilled water to the lid and moisten the tip of
species and the `Oxidase Test' is an important and
the stick.
commonly used reaction for the screening and
6 Examine the reagent impregnated tip of the stick presumptive identification of microbial cultures.
for up to five minutes and, if negative re-examine Unfortunately, the reagent used is unstable in
after fifteen minutes. solution and needs frequent preparation for reliable
Note: Some staphylococci may take up to 1 hour results to be obtained.
before reaction shows a colour change. Oxidase Identification Sticks utilise a dry reagent
7 A positive reaction is shown by the development of specially stabilised to give it a long life. They
a pink/red colour. No colour change is observed therefore overcome the necessity for daily preparation
with organisms that do not produce beta- of fresh reagent and are very convenient to use.
lactamase. To ensure correct reading the colour of Formula
the stick should be compared to an unused stick. The tip of each stick is impregnated with a solution of
Caution N,N-dimethyl-p-phenylenediamine oxalate, ascorbic
Organisms producing pigmented colonies i.e. Staph. acid and a-napthol. The other end is coloured red for
citreus, may give false positive results. It is identification and to ensure that the correct end is
recommended therefore that when pigmented held.
colonies are to be tested Nitrocefin in solution ± code
SR112, should be used. Description
The Oxidase test is an important differential
Quality Control procedure which should be performed on all Gram-
Reference strains should be tested to control the negative bacteria that are to be identified. The
product at appropriate intervals. Oxidase reaction, based upon the ability of certain
Positive control: bacteria to produce indophenol blue from the
Beta-lactamase producing staphylococcus oxidation of dimethyl-p-phenylenediamine and
a-napthol was introduced by Gordon and McLeod1 to
Negative controls: aid in the identification of gonococci. Its wider use
Non beta-lactamase producing staphylococcus originated with the test devised by Kovacs2, to
Storage Temperature distinguish Pseudomonas species from enteric bacteria.
Store below ±108C. Kovacs smeared bacterial growth on filter-paper
impregnated with 1% w/v aqueous tetramethyl-p-
References phenylenediamine dihydrochloride solution. Steel3
1 O'Callaghan C. H., Morris A., Kirby S. M. and Shingler A. H. found Kovacs' method too sensitive, with some
(1972) Antimicrob. Ag. & Chemother. 1. 283±288. staphylococci giving weak or delayed reactions. More
2 Shannon K. and Phillips I. (1980) J. Antimicrob. Chemother. 6. useful results were obtained by the method described
617±621. by Gaby and Hadley4 using N,N-dimethyl-p-
3 Sng E. H., Yeo K. L., Rajan V. S. and Lim A. L. (1980) Br. J. phenylenediamine oxalate where all staphylococci
Vener. Dis. 56.311±313. were oxidase negative.
4 Bell S. M. and Plowman D. (1980) Lancet i. 279. Barry and Bernsohn5 confirmed the observations of
5 Montgomery K., Raymundo L. and Drew W. L. (1979) J. Clin. Carpenter et al6 that dried crystals of the oxalate salt
Micro. (9) 205±207. dimethyl-p-phenylenediamine have a longer shelf life
6 Lucas T. J. (1979) C. Clin. Pathol. 32. 1061±1065. than the tetra-methyl-p-phenylenediamine
7 Gabay E. L., Sutter V. L. and Finegold S. M. (1981) J. Antimicrob. dihydrochloride. The loss of activity of the oxidase
Chemother. 8. 413±416. reagents is caused by autoxidation which may be
8 Timewell R., Taylor E. and Phillips I. (1981) J. Antimicrob. retarded by the addition of 0.1% ascorbic acid3. Filter
Chemother. 7. 137±146. paper strips impregnated with the oxidase reagents
9 Bourgault A. M. and Rosenblatt J. E. (1979) J. Clin. Micro. 9. 654± and their use have been described by Rogers7 and by
656. Barry and Bernsohn5. However, the Oxoid oxidase
10 Lee D. T. F. and Rosenblatt J. E. (1983) Diagn. Microbial. Infect. stick, impregnated with oxidase reagents described by
Dis. 1. 173±175. Gaby and Hadley4, is a much more convenient
11 Markowitz S. M. (1980) Antimicrob. Ag. & Chemother. 17. 80±83. technique to use. The colony under test is touched
with the impregnated end of the stick so that some
microbial mass is picked from the colony. The use of
the stick also overcomes the problems of iron
oxidation of the reagent associated with nichrome
wire loops.
References
1 Gordon J. and McLeod J. W. (1928) J. Path. Bact. 31. 185.
2 Kovacs W. (1956) Nature Lond. 178. 703.
3 Steel K. J. (1962) J. Appl. Bact. 25. 445±447.
4 Gaby W. L. and Hadley C. (1957) J. Bact. 74. 356±358.
5 Barry A. L. and Bernsohn K. L. (1969) Appl. Micro. 17. 933±934.
6 Carpenter C. M., Suhrland L. G. and Morrison M. (1947) Science
105. 649±650.
7 Rogers K. G. (1963) Lancet ii. 686.
Description
Wideman et al.1 reported that Peptostreptococcus ENTEROBACTERIACEAE
anaerobius may account for one-fifth to one-third of all
Gram-positive cocci encountered in clinical specimens ONPG DISCS
and confirmed that all strains of Peptostreptococcus Code: DD13
anaerobius are totally inhibited by sodium polyanethol
sulphonate (SPS) as described previously by Graves et For the rapid detection of beta-galactosidase activity in
al.2. It has been recommended1 that the SPS disc micro-organisms.
method should be used as a rapid and simple method Contents
for the presumptive identification of Peptostreptococcus 1 cartridge. Each contains 50 discs.
anaerobius. By this method all strains of
Peptostreptococcus anaerobius give inhibition zones of Disc Contents
12 to 30mm. Peptostreptococcus micros and Peptococcus Each disc is impregnated with phosphate buffered
prevotii may give zones that overlap the zone size O-nitrophenyl-b-D-galacto-pyranoside (ONPG).
range obtained with Peptostreptococcus anaerobius. Description
Lactose fermentation is a classical identification test
SPS Disc Sensitivity of Clinical Isolates1
for many organisms. It is normally demonstrated by
SPS Disc acid production after the disaccharide has been
Inhibition cleaved into galactose and glucose by the enzyme
Zone beta-galactosidase. However, it is essential for the
Organism Diameter lactose to be conveyed into the cell by a specific
(Range mm) galactoside-permease enzyme before such cleavage.
Peptostreptococcus anaerobius 18±27
The role of these two essential enzymes is important
P. micros 6±12a
in the classification of micro-organisms into:
P. parvulus No zone
P. productus No zone 1 Active lactose fermenters (taking 18±24 hours)
Peptococcus asaccharolyticus No zone possessing both permease and galactosidase P+G+.
P. magnus No zoneb 2 Delayed lactose fermenters (taking longer than 24
P. prevotii 6/16c hours) lacking permease but possessing
P. saccharolyticus No zone galactosidase: P± G+.
P. variabilis No zone
3 Non-lactose fermenters lacking both permease and
Peptococcus species No zone
galactosidase: P± G.
Acidaminococcus fermentans No zone
`Gaffkya anaerobia' No zone For the ONPG test1 a synthetic galactoside (ortho-
Streptococcus intermedius No zone nitrophenyl-b-D-galacto-pyranose) is substituted for
S. morbillorum No zone lactose. It is hydrolysed in the same way as lactose
Streptococcus species No zone but the ortho-nitrophenol is chromogenic and when
Veillonella alcalescens No zone cleaved off in alkaline solution it produces a yellow
V. parvula No zone solution:
a
Two isolates had zone diameters of 10mm and one Bacterium + ONPG hydrolysed O-nitrophenol
isolate had a 12mm zone diameter. (colourless) b-galactosidase (yellow)
b
One isolate had a 17mm zone of diminished growth. This test is independent of an induced or constitutive
c
One isolate had a zone of 16mm. permease enzyme and can be very rapid.
Technique The ONPG test can be used to:
1 Prepare Wilkins-Chalgren Anaerobe Agar CM619
plates as directed. 1 Differentiate late lactose fermenters (P± G+) from
non-lactose fermenters (P± G±).
2 Adjust the 18±24 hour culture of the test organism
to give 0.5 of the McFarland No.1 nephelometer (a) Citrobacter (+) and Salm. arizonae (+) from
standard. Salmonella (±).
3 Evenly inoculate the surface of a Wilkins-Chalgren (b) Escherichia coli (+) from Shigella sonnei (±).
Anaerobe Agar plate with the culture under test. 2 Aid in species differentiation.
November 1998 5-9
Biochemical Reagents, Diagnostic Discs
(a) Pseudomonas cepacia (+) and Ps. maltophila (+) microscopic morphology, Zinnemann and Turner3
from other Pseudomonas species. argued that it should be reclassified in the genus
(b) Neisseria lactamica (+) from other Neisseria (±). Corynebacterium and suggested the name
Corynebacterium vaginale.
Technique Taxonomic studies4,5 have led to the naming of a new
1 Place one disc into a sterile tube.
genus Gardnerella for inclusion of the organisms
2 Add 0.1ml of sterile 0.88% sodium chloride previously classified as H. vaginalis or C. vaginale with
(physiological saline). the type species G. vaginalis.
3 Pick the colony under test with a sterile loop and Bailey et al.6 have recommended that the
emulsify it in the tube containing the disc and susceptibility and resistance to metronidazole and
physiological saline. sulphonamide in conjunction with fermentation tests
4 Incubate at 358C. should be used as an aid in the separation of G.
5 Examine at hourly intervals for up to 6 hours to vaginalis from other possibly unrecognised biotypes of
detect active lactose fermenters. G. vaginalis or other vaginal bacteria that
presumptively resemble the organism. They
6 Organisms that are negative after 6 hours should
be incubated for up to 24 hours to detect the late recommended a content of 50mg metronidazole per
disc.
lactose fermenters.
Interpretation of Results Bacterial group or species
Colourless ONPG Negative Susceptibility to disc of
Yellow ONPG Positive sulphonamide metronidazole
(1mg) (50mg)
The reaction speed depends on the size of inoculum. G. vaginalis R S
Quality Control Bifodobacteria and G. vaginalis-
Use known positive and negative beta-galactosidase like organisms S S
producing organisms to monitor the disc reactions. Streptococci R R
Lactobacilli R R
Reference S: Susceptible R: Resistant
1 Lowe G. H. (1962) J. Med. Lab. Technol. 19. 21±25.
It has been shown7 that in the treatment of G. vaginalis
± associated vaginitis with metronidazole, the
GARDNERELLA VAGINALIS hydroxy metabolite may contribute a significant
antimicrobial effect, in view of its excellent activity in
METRONIDAZOLE DIAGNOSTIC DISCS vitro. The diagnosis and treatment of non-specific
(50MG) vaginitis has been reviewed8.
Code: DD8 Technique
Inoculate the isolation medium with the specimen
SULPHONAMIDE DIAGNOSTIC DISCS and place a 50mg metronidazole disc and a 1000mg
(1000MG) sulphonamide disc on an area of the plate where
heavy, but not confluent, growth can be expected.
Code: DD11
Smith and Dunkelberg1 incubated at 358C in air
An aid in the identification of Gardnerella vaginalis. containing approximately 8% carbon dioxide, but
more recently Ralph et al.9 in a study of MICs of a
Contents
number of antibiotics for G. vaginalis reported that
DD8. 1 cartridge. Each contains 50 discs.
susceptibility to metronidazole was significantly
DD11. 1 cartridge. Each contains 50 discs.
increased by incubation in an anaerobic atmosphere
Disc Contents and more reliable results may be expected by
DD8 Metronidazole 50mg incubation under these conditions.
DD11 Sulphonamide 1000mg
G. vaginalis is best isolated on Columbia Agar CM331
Description with Gardnerella vaginalis Supplement SR119. See
Smith and Dunkelberg1 reported that metronidazole, Section 2.
previously thought to inhibit obligately anaerobic
Quality Control
bacteria only was found in vitro to inhibit the growth
Use known strains of G. vaginalis to monitor the
of facultatively anaerobic strains of Gardnerella
performance of the discs.
vaginalis when using the agar diffusion method.
Small pleomorphic Gram negative rods associated References
with `non-specific' bacterial vaginitis were recognised 1 Smith R. F. and Dunkelberg W. E. (1977) Sex. Trans. Dis. 4.
by Gardner and Dukes2 as the aetiologic agent. They 20±21.
named the organism Haemophilus vaginalis. However, 2 Gardner H. L. and Dukes C. D. (1955) Am. J. Obstet. Gynecol. 60.
in obvious conflict with the accepted definition of the 962±976.
genus Haemophilus it does not have a requirement for 3 Zinnemann K. and Turner G. C. (1963) J. Pathol. Bacteriol. 85.
Haemin (X factor), Nicotinamide Adenine 213±219.
Dinucleotide (NAD or V factor) or any other co- 4 Piot P., Van Dyke E., Goodfellow M. and Falkow S. (1980) J.
enzyme-like growth factor. On the basis of Gen. Microbiol. 119. 373±396.
5 Greenwood J. R. and Pickett M. J. (1980) Int. J. Syst. Becteriol. 30. Growth of Bacterial Species with and without X
170±178. and V factors
6 Bailey R. K., Voss J. L. and Smith R. F. (1979) J. Clin. Microbiol. 9. No With
65±71. growth With X With V X + V
7 Ralph E. D. and Amatnieks Y. E. (1980) Sex. Trans. Dis. 7. factors factor factor factor
157±160. H. influenzae ± ± ± +
8 Clay J. (1981) J. Antimicrob. Chemotherapy 7. 501±504. H. aegyptius
9 Ralph E. D., Austin T. W., Pattison F. L. M. and Schieven B. C. (Koch-Weeks bacillus) ± ± ± +
(1979) Sex. Trans. Dis. 6. 199±202. H. parainfluenzae ± ± + +
H. ducreyi ± + ± +
B. pertussis* + + + +
HAEMOPHILUS SPECIES +Growth ±No growth
X FACTOR DISCS *Requires special media for initial isolation, e.g.
Bordet-Gengou medium, but laboratory strains show
Code: DD3 adaptation.
V FACTOR DISCS N.B. ±V and X + V FACTOR DISCS MUST BE
STORED AT ±208C to ±108C.
Code: DD4
Quality Control
X + V FACTOR DISCS Use known strains of Haemophilus species to monitor
the performance of the discs and the medium.
Code: DD5
Reference
Discs impregnated with growth factors for the 1 Kilian M. (1980) Haemophilus. in Manual of Clinical Microbiology.
differentiation of the Haemophilus group of bacteria. Eds. Lennette et al. Amer. Soc. for Microbiol. 3rd Edn. Washington.
Contents STREPTOCOCCUS PNEUMONIAE
1 cartridge. Each contains 50 discs.
Description OPTOCHIN DISCS
Paper discs impregnated with growth factors for the Code: DD1
differentiation of the Haemophilus group of bacteria.
Haemophilus and Bordetella species may be identified For the differentiation of Streptococcus pneumoniae.
according to whether basal media require the Contents
addition of `X' and `V' growth factors before growth 1 cartridge. Each contains 50 discs.
will occur. X factor (haemin) and V factor (coenzyme
I) may be directly incorporated into a basal medium Description
or, more conveniently, are impregnated into paper Bowers and Jeffries1 have shown that there is
discs or strips which are placed upon the surface of complete correlation between bile-solubility and full
an inoculated basal medium. Colonies of those species `Optochin' susceptibility for the differentiation of
which show growth only in the vicinity of a disc or Strept. pneumoniae from other streptococci. Oxoid
strip impregnated with the particular growth factor `Optochin' Discs are paper discs ready impregnated
are unable to synthesise it in optimal amounts1. with `Optochin' (ethylhydrocuprein hydrochloride)
which provide a convenient and reliable alternative to
Technique the bile-solubility test. Pneumococci are sensitive to
Evenly inoculate the surface of a Blood Agar Base `Optochin' so that a culture shows a zone of inhibition
plate (CM55 without blood) with the organism to be around the impregnated disc, whilst streptococci
tested and aseptically apply the Diagnostic Discs in either grow right up to the edge of the disc or,
the following positions around the periphery of the occasionally, show a very small zone of inhibition.
plate (approximately 1 or 2cm in from the edge of the
medium): Technique
Streak a pure culture of the organism to be tested
X Factor Disc 12 o'clock across one half of a Blood Agar plate (Blood Agar
V Factor Disc 4 o'clock Base CM55 with 7% of sterile blood) and apply an
X+V Factor Disc 8 o'clock `Optochin' Disc immediately before incubation. At the
Incubate overnight at 358C or for 48 hours if same time apply a second `Optochin' Disc to the other
necessary, and observe for growth or no growth in half of the plate, previously streaked with a known
the neighbourhood of a disc. If the organism requires pneumococcus, in order to provide a positive control.
X factor alone, it will grow only in the vicinities of the After incubation, pneumococci show a zone of
X and X + V factor discs; if it requires V factor alone, inhibition at least 5mm from the edge of the disc,
it will grow only in the vicinities of the V and the X + whilst streptococci are completely resistant or show a
V factor discs; if both X and V factors are required, it small zone of inhibition extending not more than
will grow only in the vicinity of the X + V factor disc. 2mm from the edge of the disc.
Oxoid `Optochin' Discs may also be placed on the
primary culture plate, before incubation, in order to
provide rapid indication of the presence of large
numbers of pneumococci.
are stored at low temperatures. Shelf-life studies of discs are applied. Similarly once the discs have
the discs in their packaging demonstrate that they been applied, plates should be placed in the
meet the stated storage life printed on the labels. incubator within a 15 minute interval, to prevent
pre-diffusion of the antimicrobial at room
Finally, samples are taken from every batch/lot
manufactured and the discs tested microbiologically temperature.
to confirm that the antimicrobial content lies within 3 Uniformity of agar depth ± plates should be
80±120% of the stated content on the label. poured on levelled surfaces, using dishes with flat
bottoms, to ensure a uniform depth of agar
Storage (3±4mm in depth).
Discs must be stored at ±208C if kept for long periods.
Storage at 2±88C is suitable for discs currently being 4 Application of discs ± it is essential that discs are
used or to be used very soon. Discs should be in intimate contact with a moist agar surface.
returned to the refrigerator as quickly as possible after Therefore either use dispensers which have a
use. The most common cause of moisture reaching the tamping action on the discs or press them
discs and causing destruction of labile antimicrobials separately after application. Do not overdry the
is condensation of warm laboratory air on cold discs agar surface before applying the discs. Do not
removed from the refrigerator. attempt to place too many discs on the agar
surface; six 6mm discs are ideal for a 90mm dish.
It is important to allow the cartridge blister pack to Some workers apply up to 12 discs onto a 150mm
reach room temperature before exposing the discs, a dish.
period of one hour is generally sufficient. Use discs in 5 Incubation period ± ensure uniform times of
order of expiry date, which is valid only for unopened incubation for the plates, 16±18 hours at 35±378C is
blisters stored under proper conditions. usually satisfactory.
6 Interpretation of zone sizes ± after incubation the
3 INOCULUM plates are removed and the zones of inhibition
noted and measured. (See Oxoid aura
One of the very critical factors for accuracy and
page 6-3.)
precision in disc diffusion tests is the inoculum
preparation. It is therefore important to use a The diameter of the zone (including the diameter of
technique which will always yield a uniform the disc) is measured to the nearest millimetre,
suspension of the correct number of organisms, using calipers. If the NCCLS Bauer-Kirby method
105±106 orgs/ml. is being used then the zone diameters can be
compared with the current NCCLS Standards
Various techniques are described in which which can be obtained from the National
suspensions of pre-grown organisms are prepared or Committee for Clinical Laboratory Standards,
small inocula are incubated for fixed periods of time. NCCLS Publications, Villanova, Pa. USA. This
It is important that more than one colony is sampled standard should not be used unless the test is
(4±10 cols) to ensure a representative sample of the carried out following the described methodology
organism has been taken. on Mueller-Hinton medium with the appropriate
Some form of standardisation of the final suspension discs.
is necessary and it should be noted that different Where other systems are used the zone size
organisms will display different opacities of solution breakpoints should have been determined using
to yield a dense but not confluent growth. MIC/zone comparative tests, following the
To complement the newly launched Oxoid aura particular methodology chosen.
System (see page 6-3) Oxoid have launched the Oxoid 7 Control cultures ± it is essential that each
Turbidometer. This instrument provides the inoculum laboratory maintains adequate control over AST
density standardisation necessary to ensure accurate methods by testing reference cultures at regular
reproducible results. intervals. Daily tests may be required if new media
and discs are constantly being used.
4 OTHER FACTORS INFLUENCING The following reference strains are used in the
NCCLS test method.
THE RESULTS Staphylococcus aureus ATCC1 29213
1 Temperature of incubation ± the incubators Enterococcus faecalis ATCC1 29212
should be checked for satisfactory performance
Escherichia coli ATCC1 25922
and their recording thermometers should show air
temperatures of 35±378C with fluctuations of not Pseudomonas aeruginosa ATCC1 27853
more than 28C. Agar plates should not be placed in Escherichia coli ATCC1 35218 for testing clavulanic
high stacks because the middle plates will take acid compounds.
longer to reach the incubator temperature and this Records of the results obtained with these reference
delay could cause overlarge zones. strains should be maintained in log books and any
2 Pre-incubation and pre-diffusion conditions ± a deviation of zone sizes from the range accepted
routine procedure should be established so that should be investigated.
inoculated plates have discs applied not later than
15 minutes after inoculation. This prevents a pre-
incubation of organisms before the antimicrobial
6-2 November 1998
Antimicrobial Susceptibility Testing
3
(Ed) Analytical Microbiology. Academic Press. New York. 1±86.
Bell S. M. (1975) Pathology. 7. l-48 (Suppl).
Antimicrobial Susceptibility
4 Bauer A. W., Kirby W. M. M., Sherris J. C. & Turck M. (1966) Testing Media
Amer. J. Clin. Path. 45. 493±496. Media for Antimicrobial Susceptibility Testing (coded
5 Pollock H. M. et al. (1986) J. Clin. Microbiol. 24. 1±6. CM) should be stored in their closed containers at an
6 Nat. Comm. Clin. Lab. Standards (1979) Proposed reference even temperature in a cool, dry place, away from
dilution procedure for AST of anaerobic bacteria. NCCLS PSM- direct light.
11. Villanova. Pa. USA.
7 WHO (1977) Tech. Rep. Ser. No. 610.
Product Pack Size Order Code
8 FDA (1978) Codes of Fed. Rebs. 21. Part 460.
Diagnostic SensitivityTest
Agar Base (DST Agar) 500g CM261B
Note HR Medium 100g CM845A
The list of antimicrobial discs manufactured, the Haemophilus Test Medium (HTM)
agents, generic and proprietary names, the symbols HTM Supplement 10 vials SR158E
and range of levels available are supplied in the HTM Base 500g CM898B
Oxoid Product List and the list is updated annually. `Iso-Sensitest' Agar 500g CM471B
`lso-Sensitest' Broth 500g CM473B
Mueller-Hinton Agar 500g CM337B
Antimicrobial Susceptibility Mueller-Hinton Broth 500g CM405B
Discs `Sensitest' Agar 500g
Wilkins-Chalgren Anaerobe Agar 500g
CM409B
CM619B
Discs in routine use should be stored at 2 to 88C.
Wilkins-Chalgren Anaerobe Broth 500g CM643B
Longer term storage should be at ±208C. After cold
storage allow discs to reach room temperature before
opening storage containers. Discs are presented in OXOID aura
cartridges for dispensing either individually with The importance of standardisation.
Oxoid ejectors or with the Oxoid Dispenser System.
There are 50 discs per cartridge, 5 cartridges per pack. Antimicrobial Susceptibility Testing by the disc
diffusion method is simple, flexible, informative and
Cartridge packs cost-effective1.
Each cartridge is individually sealed together with a
silica gel capsule in a foil-covered see-through blister. However, a recent survey demonstrated that, between
Designed to allow the microbiologist better control laboratories, there is ``considerable variation in the
and storage of the discs in use. medium used, the method of inoculum preparation and
application, disc concentrations, incubation conditions
THE OXOID DISC DISPENSER MKIII and the basis of interpretation of zone sizes''2.
An enhanced system for antimicrobial susceptibility It is generally accepted that there is a need for greater
testing incorporating the new ergonomically designed standardisation of methodology and interpretation of
Disc Dispenser. It has a simple one-handed operation results to ensure accurate, reproducible results, within
and is fully height-adjustable to cater for various and between laboratories.
depths of media. The cover and base of the dispenser The Oxoid aura database enables clinical isolates to be
are fully interlocked to prevent the ingress of compared to an appropriate standard for every
moisture: cartridges `click' positively into their correct antibiotic and concentration in the system. The
locations and a plastic skirt ensures that each agar comparison is performed at species level and
plate is precisely centred every time the dispenser is automatically generates interpretation of the result.
used.
Interpretations are based on the principles set forth by
Product Pack size Order Code the Swedish Reference Group of Antibiotics (SRGA)
Disc Dispenser Mk III (90mm) and its sub-committee on Methodology (SRGA-M)3.
for 90mm petri dishes The value and function of this system has been
Disc Dispenser Mk III (90mm) 1 ST6090 proven over many years.
for 6 cartridges
The Complete Antimicrobial Susceptibility Testing
Disc Dispenser Mk III (90mm) 1 ST8090
(AST) System.
for 8 cartridges
Disc Dispenser Mk III (100mm) Accurate, reproducible AST results depend on the
for 100mm petri dishes following:
Disc Dispenser Mk III (100mm) 1 ST6100 . High quality products used in a defined
for 6 cartridges methodology
Disc Dispenser Mk III (100mm) 1 ST8100
. Standardised inoculum density
for 8 cartridges
Disc Dispenser (150mm) 1 ST1215 . Precise zone size measurement (aura)
for 12 cartridges . Accurate and meaningful interpretation of results
(aura)
References
1 Bridson E. (1996) Antimicrobial Susceptibility Testing ± the disc
diffusion method. Oxoid, Basingstoke, UK. folio number 547.
2 Andrews J. M. et al (1996) Antimicrob. Chemother. 37: 187±204.
3 Report from the Swedish Reference Group of Antibiotics and its
Methodology Subcommittee (SRGA-M), 1991.
be created which would support micro-aerophilic THE OXOID ANAEROBIC JAR HP11
organisms (about 6% oxygen in the jar) or capnoeic
organisms (about 10% CO2). A 3.4 litre capacity Anaerobic Jar of advanced design
that gives great flexibility in use by coping equally
Anaerobic indicators will show whether the redox well with Gas Generating Envelopes or Gas
potential (Eh) in the jar has been reduced below Cylinders.
±50mV, which is the level required to change the
resazurin indicator from pink to white. Both Jar and Lid are of robust construction and used
with the Low Temperature Catalyst BR42 ensure
Collection and transport of anaerobic specimens unprecedented protection to operator and equipment.
It must be emphasised that in spite of every care
taken in cultivation, poorly collected and transported The Oxoid Anaerobic Jar has a number of novel
samples will yield poor or negative results. design features and for greater convenience in use it is
supplied with a corrosion-resistant plate carrier that
Samples for anaerobic investigation should be greatly reduces the time and effort needed to load the
collected with care and protected from air. Swabs will jar. A test tube carrier is available as an optional extra
not be suitable, unless they are specially treated8 or minimising the risk of spillage of broth cultures.
placed in Stuarts Transport Medium9. Generous
samples of pus or fluids do not need anaerobic Anaerobiosis is achieved rapidly, safely and
transport but they should not be unduly delayed efficiently using the Gas Generating Kit BR38 or
before examination10. hydrogen obtained from cylinders.
It was Dack who said 50 years ago that it should be The Oxoid Anaerobic Jar may also be used for the
possible to isolate and study anaerobes in a relatively isolation of microaerophilic and CO2-dependent
convenient, routine fashion using `modern organisms by using the Campylobacter Gas
apparatus'11. Much later and armed with the Generating Kit BR56 or the CO2 Gas Generating Kit
components described below, anaerobic bacteriology BR39.
is within reach of all microbiological laboratories. Catalytic activity may be checked both by the
pressure gauge for an immediate indication of
References efficiency and by the Anaerobic Indicator BR55 which
1 Halliwell B. (1984) Med. Lab. Sci. 41. 157±171. provides supporting evidence of the pressure
2 Dubos R. (1988) in Pasteur and Modern Science. Sci. Tech. Publ. changes. These checks ensure that the absence of
Madison USA. pp. 76±79. growth does not reflect poor anaerobic incubations.
3 Veillon A. (1893) C. R. Soc. Biol (Paris) 45. 807±809.
4 Veillon A. and Zuber A. (1897) C. R. Soc. Biol (Paris) 49. 253±
The Oxoid Anaerobic Jar HP11 is part of the complete
389.
Oxoid Anaerobic System consisting of:
5 Finegold S.M. et al. (1975) Ann. lnt. Med. 83. 375±389.
. 3.4 Litre Anaerobic Jar of advanced design.
6 Walden W.C. and Hentges D.I. (1975) Appl. Microbiol. 30. 781± . Gas Generating Kit which is superior in design to
785. any other on the market.
7 McCord J.M. Keele B.B. and Fridovich I. (1971) Proc. Natl. Acad.
. A new safe low temperature catalyst.
Sci. USA. 68. 1024±1027.
8 Smith L.L. and Ferguson J.R. (1977) Med. Lab. Sci. 34. 247±258. . An Anaerobic Indicator that is reliable and reacts
9 Yrios J.M. et al. (1975) J. Clin. Microbiol. 1. 196±200 faster than any other equivalent products.
10 Bartlett J.G., Sullivan-Sigler N., Louie T.J. and Gorbach S.L.
(1976) J. Clin. Microbiol. 3. 133±136. OXOID GAS GENERATING KIT BR38
11 Dack G.M. (1940) Bact. Rev. 4. 227±259.
The Oxoid Gas Generating Kit BR38 is a laminated
foil envelope presented with an inner container
ANAEROBIC SYSTEMS holding tablets of sodium borohydride, sodium
bicarbonate and tartaric acid. The addition of water to
For some time now Oxoid has been marketing two the envelope activates the system causing hydrogen
distinctly different systems used for growing bacteria and carbon dioxide to be produced.
which require special atmospheres including
anaerobic organisms, microaerophilic organisms and Gas production takes place smoothly and
CO2 dependent organisms. reproducibly because the porous membrane of the
inner container regulates the passage of water
The original Oxoid system consisted of an anaerobic inwards and gas outwards.
jar of advanced design (Code HP11) together with a
gas generating kit for anaerobes (BR38), a CO2 gas Each individual Gas Generating Kit when activated
generating kit (BR39) and gas generating kits for with water evolves sufficient hydrogen for the
Campylobacter (BR56 and BR60). catalytic removal of oxygen present in the jar and
leaves the final internal pressure approximating to
In the case of BR38, BR56 and BR60 water is added to that of the atmosphere.
the sachets to initiate a reaction and the production of
hydrogen and CO2. A low temperature catalyst Carbon dioxide is also evolved to give a final
(BR42) is required to be used in the anaerobic jar on concentration of 10% v/v in the atmosphere.
each occasion. Gas generation is completed within 30 minutes and
because the resultant solution is acid it does not
reabsorb the carbon dioxide so necessary for the
growth of fastidious anaerobes.
7-2 November 1998
Anaerobic Systems
Rate of Oxygen catalysis aureus that grew normally in 1±2% of CO2 but which
This graph shows the rapidity with which the oxygen on aerobic culture plates grew as minute
level in the gas jar is lowered using the Oxoid Gas unpigmented colonies that were coagulase and
Generating Kit BR38. catalase negative.
Other reports9,10,11 also concern dwarf variants of
differing phage types of Staph. aureus that grow
normally in a CO2 enriched atmosphere.
Barker et al12 have identified strains of Klebsiella
species that are CO2 dependent and Eykyn and
Phillips13 reported the isolation of a CO2 dependent
Escherichia coli from a urine specimen.
It can be important clinically that CO2 dependent
strains of such commonly occurring organisms are
recognised and routine incubation of all specimens in
CO2 is recommended.
Disposal
After opening the jar, the exhausted sachet should be
removed without spilling the contents. The solution
remaining in the sachet is mildly acidic and may be
poured away into a sink or flushed with running
water. The empty sachet can then be discarded with
normal laboratory litter.
References
GAS GENERATING KIT CARBON DIOXIDE 1 Griffin P. J. and Racker E. (1965) J. Bact. 71. 717±721.
2 Jones-Holmquest A. M., Wendle R. D., Hudd R. L. and Williams
SYSTEM BR39
R. P. (1973) Appl. Microbiol. 26. 466±469.
Description 3 Chapin C. W. (1918) J. Infect. Dis. 23. 342±344.
The Oxoid Carbon-Dioxide Generating Kit is a 4 Jones R. T. and Talley R. S. (1977) J. Clin. Micro. 5. 427±432.
reliable and convenient method for producing 5 Jones L. M. and Brinley Morgan W. J. (1958) Bull. Wld Hlth Org.
suitable conditions, in standard jars, for organisms 19. 200. 576.
requiring an enhanced CO2 atmosphere. 6 Mair N. S. (1955) Mon. Bull. Minist. Hlth. 14. 184.
Each sachet contains two tablets, both of which are 7 Hale J. H. and Brit. J. (1951) Exp. Path. 32. 307.
composed of tartaric acid and sodium bicarbonate. 8 Thomas M. E. M. and Cowlard J. H. (1955) J. Clin. Path. 8. 288.
When used as directed, they will together produce 9 Sherris J. C. (1952) J. Clin. Path. 5. 534.
350ml carbon dioxide, which in the Oxoid Anaerobic 10 Goudie J. G. and Goudie R. B. (1955) J. Clin. Path. 8. 284.
Jar will give a final carbon dioxide level of 11 Wise R.I. and Spink W. W. (1954) J. Clin. Invest. 33. 1611.
approximately 10% (v/v) within 1 hours. 12 Barker J., Brookes G. and Johnson T. (1978) B.M.J. 1. 300.
13 Eykyn S. and Phillips I. (1978) B.M.J. 1. 576.
The requirement of CO2, by gonococci is well
documented1,2 although strains vary widely in their
requirement for this gas.
GAS GENERATING KITS FOR
Chapin3 introduced the candle-jar producing CAMPYLOBACTER BR56 & BR6O
approximately 2.5% (v/v) CO2, but this is below the
optimum level for the growth of carbon dioxide Description
requiring gonococci, particularly if the number of The Oxoid Gas Generating Kits for Campylobacter
bacteria is small4. isolation, BR56 and BR60 constitute a reliable and
convenient method for producing suitable gaseous
The Carbon-Dioxide Gas Generating Kit may also be conditions, in standard jars, for organisms such as
used to provide the enhanced CO2 atmosphere Campylobacter species which require a reduced oxygen
required for growth of meningococci. atmosphere.
A 10% (v/v) CO2 atmosphere is required for isolation BR56 and BR60 are disposable gas generating kits that
of Brucella species5,6. Plates of Blood Agar Base No.2, produce hydrogen and carbon dioxide in sufficient
CM271, or Brucella Medium Base, CM169, quantity that after reaction with a palladium catalyst
supplemented with 5±10% (v/v) inactivated horse in an anaerobic jar will produce an optimal gaseous
serum and Brucella Selective Supplement, SR83, atmosphere for the growth of campylobacters and
should be incubated at 358C in a carbon dioxide other microaerophilic organisms. BR56 is designed for
enriched atmosphere for ten days and examined 3.0±3.5 litre jars and is suitable for the Oxoid
every two days. Anaerobic Jar HP11 and for many other jars currently
Hale7 recorded the isolation from an abscess of a in use in laboratories. When used as directed, each
dwarf colony Staphylococcus aureus which was sachet will produce about 1,000ml hydrogen and
dependent on CO2 for characteristic growth. 350ml carbon dioxide.
Thomas and Cowlard8 reported strains of Staph. BR60 is designed for 2.5±3.0 litre jars and is for use
7-4 November 1998
Anaerobic Systems
THE ATMOSPHERE
GENERATION SYSTEM
In 1993, Oxoid launched a new range of innovative
products under the title of Atmosphere Generation
System (AGS). These novel products are safer (no
hydrogen produced) and more convenient (no water
to add). They include a new jar of advanced design ±
Anaerojar ± in which it is not necessary to use a
catalyst.
This range has frequently been extended and now
consists of the following products:
Anaerobic Indicator Code BR055B Anaerobic
AnaeroGenTM (for 2.5 Code Atmosphere
litre jar) AN025A Generation
AnaeroGenTM (for 3.5 Code System
litre jar) AN035A
AnaeroGen Code
THE OXOID ANAEROBIC INDICATOR BR55 CompactTM (for use AN010C
The Oxoid Anaerobic Indicator BR55 consists of a with plastic pouches)
Code Atmosphere
cotton strip impregnated with a redox indicator CampyGenTM (for 2.5
solution enclosed in a laminated foil envelope. This CN025A Generation
litre jar)
Code for Micro-
formulation and a pure cotton strip gives a CampyGenTM (for 3.5
reproducible redox colour change in a shorter time CN035A aerophilic
litre jar)
than similar products that are available. Use of the Code organisms
CampyGen Compact
Oxoid Anaerobic Indicator will support the evidence (for use with plastic CN020C
of pressure changes which occur with active catalysts pouches)
and ensure that the absence of growth does not reflect Code Atmosphere
CO2GenTM (for 2.5
poor anaerobic incubation. CD025A Generation
litre jar)
How the Oxoid Anaerobic Indicator BR55 helps the Code for CO2
CO2Gen Compact (for
microbiologist CD020C dependent
use with plastic
organisms
Features pouches)
1 Changes from red to white. AnaeroJarTM Code
AG025A
2 Improved sensitivity to detect lower levels of A range of accessories for the Compact products and
oxygen than has previously been achievable. the AnaeroJar completes the Atmosphere Generation
Benefits System.
1 Indicates when true anaerobiosis has been
achieved. ANAEROGEN
2 Indicates better anaerobic conditions. Code: AN25 & AN35
References Description
1 Patent application 54354/7 developed by Don Whitley Scientific Where an AnaeroGen sachet is placed in a sealed jar,
Limited. the atmospheric oxygen in the jar is rapidly absorbed
2 United Kingdom Department of Health and Social Security. with the simultaneous generation of carbon dioxide.
February 1979. This novel method differs from those commonly used
in that the reaction proceeds with no evolution of
hydrogen, and therefore does not require a catalyst.
Furthermore, no addition of water is needed to
activate the reaction.
When used as directed, the AnaeroGen sachet will
reduce the oxygen level in the jar to below 1% within
30 minutes. The resulting carbon dioxide level will be
between 9% and 13%.
AnaeroGen was used in methodology for detecting
bifidobacteria in meat and meat products in an
investigation into the suitability of these organisms as
indicators of faecal contamination.
pouch sealed within one minute. Micrococcus luteus ATCC1 9341 no growth
The reaction of the ascorbic acid with oxygen is Disposal
exothermic. However, the temperature of the On removal from the pouch after incubation, the
AnaeroGen Compact paper sachet will not exceed AnaeroGen Compact paper sachet will retain a small
658C. amount of activity and become warm. The sachets
should be allowed to cool to room temperature prior
This temperature will only be maintained while
to sterilisation and disposal with the non-hazardous
anaerobic conditions are being achieved. Once the
laboratory waste.
oxygen in the pouch has been absorbed, the
temperature within the pouch will return to ambient
temperature.
ANAEROJAR
Storage Code: AG25
Store at 2±258C. Under these conditions, the Description
AnaeroGen Compact sachets will retain their activity The 2.5 litre Oxoid AnaeroJar is an important addition
until the expiry date declared on the outer box and on to the Oxoid range of Atmosphere Generation
the foil wrapped sachet. Products. The jar is designed for use with the 2.5 litre
Directions AnaeroGen/CampyGen sachet.
1 Place the inoculated media plates or identification Important features include:
panel in the plastic pouch provided. Disposable
. No catalyst required.
plastic petri dishes should be of the vented variety
to aid gas transfer between the interior and exterior . Polycarbonate base which is secured to the lid by 4
of the plates. clips.
2 Tear open an AnaeroGen Compact foil sachet at These clips are designed to allow venting in the
the tear-nick indicated. Remove the AnaeroGen unlikely event of a positive pressure build-up
Compact paper sachet from within. occurring i.e. by allowing lid to lift and reseal to
3 Immediately place the AnaeroGen Compact paper maintain correct conditions.
sachet in the plastic pouch. . A carrying handle for the safe transportation of the
N.B. The AnaeroGen Compact paper sachet will jar from bench to incubator.
become warm to the touch on exposure to air. . Vacuum Relief Screw to overcome any vacuum
4 Expel excess air from the plastic pouch. Seal the which may occasionally occur.
plastic pouch immediately with the AnaeroGen
Compact clip (AN005C). Operating Instructions
Note
N.B. the time taken between opening the foil sachet Before use check:
and sealing the plastic pouch should not exceed
1 minute. Extended exposure will result in loss of a. `O' ring is correctly seated
reactivity, and full anaerobic conditions may not be b. The vacuum relief screw is in the closed position.
achieved in the pouch. 1 Place inoculated plates into the plate carrier.
5 Incubate appropriately. Disposable plastic petri dishes should be of the
6 After the incubation period remove the plates or ID vented variety to aid gas transfer between interior
panel and examine for the presence of colonies or and exterior of the dishes.
biochemical reaction. If the plates require re- 2 When using the anaerobic system (i.e. AN25)
incubation then a fresh AnaeroGen Compact sachet prepare the Oxoid Anaeraobic Indicator (BR55) by
must be used following steps 2±5 described above. cutting and exposing 10mm of the fabric strip,
N.B. The plates may be initially inspected through insert into the smaller, upper clip on the dish
the transparent plastic pouch. If the bag is not carrier.
opened, a fresh AnaeroGen Compact sachet is not 3 Lower the carrier into the polycarbonate base.
required for re-incubation. 4 Tear open an AnaeroGen/CampyGen/CO2Gen
7 After incubation, the exhausted AnaeroGen sachet at the tear-nick indicated, and remove the
Compact paper sachet and plastic pouch should be paper sachet from within.
sterilised and discarded with the non-hazardous 5 Immediately place the paper sachet in the
laboratory waste. appropriate clip in the plate carrier within the jar
Control Testing (see Technical insert).
It is recommended that OXOID Anaerobic Indicator 6 Having inserted the sachet into the carrier
(BR055B) is also used in the plastic pouch as a visual immediately place the lid on the jar, making sure
check that anaerobic conditions have been achieved the `O' ring is in place. Secure the clips with fingers
and maintained. shown in figure1. Repeat this process with each of
The user should check their anaerobic technique the four clips to properly secure the lid.
periodically for its ability to provide adequate 7 Use carrying handle situated on the lid to transport
conditions for the growth of anaerobic bacteria. The jar to the incubator.
following strains are recommended: 8 The anaerobic indicator will change from pink to
Clostridium novyii ATCC1 9690 growth white giving a visual indication of anaerobiosis.
Disposal 4 Expel excess air from the plastic pouch. Seal the
On removal from the jar after incubation, the plastic pouch immediately with a sealing clip. The
CampyGen paper sachet may retain a small amount time taken between opening the foil sachet and
of reactivity and will warm up. The sachets should sealing the plastic pouch should not exceed 1
therefore be allowed to cool to room temperature on minute.
an inert surface prior to disposal with the laboratory 5 Incubate appropriately.
waste.
6 After the incubation period, remove the plates and
Reference examine for the presence of colonies. If the plates
1 Bolton F. J., Wareing D. R. A. and Sails A. D. (1997) Eur. J. Clin. require re-incubation, a fresh CampyGen Compact
Microbiol. Inf. Dis. 16. 839±842. sachet must be used following steps 2±5 described
above.
CAMPYGEN COMPACT N.B. The plates may be initially inspected through
Code: CN020C the transparent plastic pouch. If the bag is opened,
a fresh CampyGen Compact sachet is required for
Description re-incubation.
CampyGen Compact for 1 or 2 petri dishes, is a
simple system for generating microaerobic conditions. 7 After incubation, the exhausted CampyGen
The system consists of a plastic pouch and sealing clip Compact paper sachet and plastic pouch should be
and a paper gas generating sachet. The paper sachet sterilised and discarded with the non-hazardous
contains ascorbic acid which reacts on contact with air laboratory waste.
to produce the microaerobic conditions for the growth Control Testing
of microaerophilic organisms. The user should check their technique periodically for
their ability to provide adequate conditions for the
Components growth of microaerophilic bacteria. Campylobacter
20 CampyGen Compact paper sachets, jejuni (ATCC1 33291) may be used for this purpose.
individually wrapped in foil
1 product leaflet Disposal
On removal from the pouch after incubation, the
Materials Required but not Provided CampyGen Compact paper sachet will retain a small
Sealing Clips (AN005C) amount of activity and become warm. The sachets
Plastic Pouches (AG020C). should be allowed to cool to room temperature prior
to sterilisation and disposal with the non-hazardous
Precautions
laboratory waste.
This product is for in vitro use only.
The CampyGen Compact paper sachet will become
active on contact with air. It is essential that the CO2GEN
plastic pouch is sealed within one minute of exposing
the paper sachet to the air. Code: CD025A
The reaction of ascorbic acid with oxygen is Description
exothermic. However, the temperature of the CO2Gen is designed for the generation of a carbon
CampyGen Compact paper sachet will not exceed dioxide-rich atmosphere within a gas jar. The paper
658C. sachet contains ascorbic acid which reacts on contact
with air to produce a level of approximately 6%
Storage
carbon dioxide within a 2.5 litre gas jar such as the
Store at 2±258C. Under these conditions, the
Oxoid AnaeroJar (AG025A). The final concentration
CampyGen Compact sachets will retain their activity
of oxygen is 15%.
until the expiry date given on the outer box and on
the foil wrap of the sachets. Components
10 CO2Gen paper sachets, individually wrapped in
Directions foil
1 Place 2 inoculated plates in a plastic pouch. 1 product leaflet
Disposable plastic petri dishes should be of the
vented variety to aid gas transfer between the Materials Required but not Provided
interior and exterior of the plates. If only one plate 2.5 litre gas jar (Oxoid AnaeroJar AG025A).
is to be inoculated, an uninoculated plate should Precautions
also be placed in the plastic pouch to prevent This product is for in vitro use only.
further activity as the volume of O2 and CO2 is
critical. The CO2Gen paper sachet will become active on
contact with air. It is therefore essential that the paper
2 Tear open a CampyGen Compact foil sachet at the sachet is placed in the jar and the jar sealed within
tear-nick indicated. Remove the CampyGen one minute.
Compact paper sachet from within.
The reaction of the ascorbic acid with oxygen is
3 Immediately place the paper sachet in the plastic
exothermic. However, the temperature of the CO2Gen
pouch with the plates.
paper sachet will not exceed 658C.
N.B. The paper sachet will become warm to the
touch on exposure to air.
cultured8,17. Culturing 10ml instead of 5ml can seven days. Diagnostic Microbiology and Infectious Diseases 16,
increase the isolation rate by about 15%, from 20ml 31±34.
the rate was 35% greater than 5ml14. When a 18 Becton Dickinson Diagnostic Section, Between Towns Road,
commercial blood culture system is used the Cowley.
manufacturer's recommendations should be followed, 19 McGowan J.E., Metchock B.G. (1992) Determination of growth
but with some systems this could involve culturing an value thresholds for BACTEC PLUS aerobic blood culture vials.
inadequate amount of blood. With children, because Journal of Clinical Microbiology 30, 771±774.
the total blood volume is much less than in adults, it 20 Weinstein M.P., Mirrett S., Wilson M.L., Harrell L.J., Stratton
is not feasible to culture large volumes of blood. C.W., Barth-Reller L. Controlled evaluation of BACTEC plus 26 and
However, many colony counts in children tend to be Roche Septi-Chek aerobic blood culture bottles. Journal of Clinical
higher than in adults and satisfactory results can be Microbiology 29, 879±882.
obtained when 1±5ml of blood are cultured. 21 Hubbard M., Chong K., Eiess-Levy E. (1992) An evaluation of
Bactec 860 and 660 automated blood culture systems. Australian
Contributed by Mrs Alison Elizabeth Eyre FIBMS., MSc.
Microbiologist 13, 158.
First published in the Newsletter of the British Society of 22 Marcelis L., Verhaegen J., Vandeven J., Bosman A., Verbist L.
Microbial Technology. (1992) Evaluation of BACTEC high blood volume resin media.
Diagnostic Microbiology of Infectious Diseases 15, 385±391.
References
1 Durack D. (1989) Blood and Circulation in: Mechanisms of Microbial
Disease. Eds Schaechter M., Medoff G., Schlessinger D., Williams OXOID SIGNAL BLOOD CULTURE SYSTEM
and Wilkins, Baltimore, pp 710±722. The following section describes briefly the Oxoid
2 Sprung C.L. (1991) Definitions of sepsis ± have we reached a SIGNAL Blood Culture System, the principles of its
consensus? Critical Care Medicine 19, 849±851. function and the equipment required for its optimal
3 Weinstein M.P., Barth-Reller L., Murphy J.R., Lichtenstein K.A. performance. For full details of the usage of the
(1983). The clinical significance of positive blood cultures: a System the product insert should be consulted.
comprehensive analysis of 500 episodes of bacteraemia and fungemia
in adults. Review of Infectious Diseases 5, 35±53. Principle of the Test
4 Graves S., Sinikas V., Hellyar A. (1992) Positive blood cultures in a Blood samples are collected from patients, using strict
large city hospital. Australian Microbiologist 13, 159.
aseptic technique and sterile equipment. The samples
5 Mileski W.J. (1991) Sepsis. What is it and how to recognize it. are inoculated into the blood culture bottles and
Surgical Critical Care 71, 749±764.
mixed with the medium.
6 Bone R.C. (1991) The pathogenesis of sepsis. Annals of Internal The formulation of the medium encourages the
Medicine 115, 457±469. growth of aerobic, anaerobic and micro-aerophilic
7 Shanson D.C. (1989) Modern blood culture techniques and other organisms. The medium is also designed to create
methods for detecting microbes in the blood. In ± septicaemia and pressure in the sealed bottle when organisms are
endocarditis (Shanson, D.C. Editor) 76±102. Oxford University Press. growing.
8 Shanson D.C., Dryden M.S. (1988) Comparison of methods for
isolating Mycobacterium avium-intracellulare from blood of patients
The detection of positive pressure is by means of a
growth indicator device which is connected to the
with AIDS. Journal of Clinical Pathology 41, 687±690.
bottle after the blood sample is added. A positive
9 Washington J.A. (1989) Blood cultures: An overview. European
pressure in the bottle displaces a quantity of blood/
Journal of Clinical Microbiology 8, 803±806.
broth mixture into the chamber as a sign of microbial
10 Welby P.L., Zusag T.M., Storch G.A. (1992) Comparison of the
activity.2,3,4,5
BACTEC Peds plus pediatric blood culture vial with Roche pediatric
Septi-Chek for blood cultures from pediatric patients. Journal of A positive result is indicated when the blood/broth
Clinical Pathology 30, 1361±1362. mixture rises above the green locking sleeve of the
11 Campos J.M. (1898) Detection of blood stream infections in children. growth indicator device.
European Journal of Clinical Microbiology and Infectious Diseases 9,
Medium Composition
815±824.
Typical formulation (European Patent 0124193 Al)
12 Klein J.O. (1990) Bacteriology of neonatal sepsis. The Pediatric
Infectious Diseases Journal 9, 778. gm/litre
13 Ascher D.P., Shoupe B.A., Robb D.A. (1992) Comparison of Tryptone Soya Broth 10.0
standard and quantitative blood cultures in the evaluation of children Gelatin peptone 10.0
with suspected central venous line sepsis. Diagnostic Microbiology of Yeast extract 5.0
Infectious Diseases 15, 499±503. Meat extract 5.0
14 Ackerman V.P., Pritchard R.C. (1987) Blood culture techniques. A Sodium chloride 8.0
survey in Australian laboratories. Pathology 19, 265±273. Potassium nitrate 2.0
15 Bates D.W., Goldman L., Lee T.H. (1991) Contaminant blood Glucose 1.0
cultures and resource utilization. JAMA 3, 365±369. L-arginine 1.0
16 Capderila J.A., Planes A.M., Palomar M., Grasser I., Almirante Sodium pyruvate 1.0
B., Pahissa A., Crespo E., Martinez-Vazquez J.M. (1992) Value of Gelatin 1.0
differential quantitative blood cultures in the diagnosis of catheter- Sodium thioglycollate 0.5
related sepsis. European Journal of Clinical Microbiology and Cysteine HCl 0.4
Infectious Diseases 5, 403±407. Sodium bicarbonate 0.4
17 Wilson M.L., Mirrett S., Weinstein M.P., Reimer L.G., Barth- Phosphate buffer 0.3
Reller L. (1993) Recovery of clinically important microorganisms Sodium polyanethol sulphonate 0.3
from BacT/Alert blood culture system does not require testing for Dithiothreitol 0.2
November 1998 8-3
Blood Cultures
Quality Assurance 20 Statham G. B., Barratt A. I., Wilson J. A. and Gray J. (1987) 3rd
The following organisms are used by Oxoid as part of European Congress of Clinical Microbiology. Hague, Holland.
the quality assurance of the product. The total Abstract 445.
inoculum challenge for each test organism per bottle 21 Daley D., Tomlinson P., Monro R. (1987) Poster No. l'222. 8
is 10 to 50 colony forming units (CFU's). Australian Microbiologist.
22 Schmideder H. (1987) Poster Presentation, Symposium on
NTCC No. ATCC No.
``Rapid Methods and Automation in Microbiology and
Bacillus cereus 7464 10876
Immunology'', Florence.
Bacteroides fragilis 9343 25285
23 Rohner P. and Auckenthaler R. (1989) Eur. J. Clin. Microbiol.
Clostridium novyi 27606
Infec. Dis. 8. 150±153.
Escherichia coli 10418 10536
Fusobacterium nucleatum 10562 10953
Haemophilus influenzae 4560 19418 OXOID and OXOID SIGNAL are trademarks.
Klebsiella pneumoniae 11228 29665
Neisseria meningitidis 10025 13077 ISOLATOR* 1.5 TUBES
Peptostreptococcus anaerobius 11460 27337
Intended use
Pseudomonas aeruginosa 10662 25668
The ISOLATOR 1.5 Tube is intended for the collection
Staphylococcus aureus 6571 9144
of small volume, paediatric blood samples to be used
Staphylococcus epidermidis 14990
for isolation of micro-organisms. The blood sample is
Streptococcus pneumoniae 6303
transferred from the tube directly to conventional
Streptococcus mutans 10449 25175
agar growth media for the purpose of isolation and
Candida albicans (NCPF3179) 10231
identification of micro-organisms.
User Quality Assurance
Principles of the test
1 Examine the bottles of broth for turbidity and/or
The ISOLATOR 1.5 Tube contains agents which lyse
change of colour before adding any blood. Discard
leucocytes and erythrocytes in blood, and block
any bottles showing abnormal characteristics.
coagulation.
2 If further user quality control is required, it is
recommended that 3 aerobes and 1 anaerobe from The specific agents used in the tube are:
the above list be used. Purified Saponin, an effective and rapid cell lysing
agent, non-toxic to micro-organisms.
References
1 Finegold S. M. and Martin W. J. (1982) Diagnostic Microbiology Polypropylene Glycol to block the foaming
6th Edn. Published C. V Mosby Co. St Louis. p.42. tendency of Saponin.
2 European Patent No. EP 0124 193A1. Sodium Polyanetholsulphonate (SPS) which acts as
3 Hinder S. M., Sawhney D. and Swaine D. 2nd European an anticoagulant, neutralises the bactericidal
Congress of Clinical Microbiology 1985, Abstract 12/2. properties of blood and inhibits phagocytosis.
4 King A., Bone G. and Phillips I. 2nd European Congress of Reagents
Clinical Microbiology 1985, Abstract 12/4. Each ISOLATOR 1.5 Tube contains the following
5 King A., Bone G. and Phillips I. (1986) J. Clin. Pathol. 39. 661± reagents in aqueous solution (content prior to
665. sterilisation).
6 Sawhney D., Hinder S., Swaine D. and Bridson E. Y. (1986) J.
Clin. Pathol. 39. 1259±1263.
Polypropylene Glycol 8 millimetres/litre
7 Van Haebler T. and Miles A. A. (1938) J. Path. Bact. 46. 245±252.
Sodium
8 Lowrance B. L. and Traub W. H. (1969) Appl. Microbiol. 17. 839±
Polyanetholsulphonate 9.6 grams/litre
842.
Purified Saponin 40 grams/litre
9 Rosner R. (1972) Amer. J. Clin. Path. 57. 220±227. The internal components of the tube are sterile.
10 Traub W. H. (1969) Experientia 25. 206±207.
Precautions
11 Traub W. H. and Lowrance B. L. (1969) Experientia 24. 1184-
Used tubes, syringes and needles contain human
1185.
body fluids. All materials should be handled as if
12 Eng J. and Holten E. (1977) J. Clin. Microbiol. 6. 1±3.
they are capable of transmitting disease. Handle with
13 Wilkins T. D. and West S. E. H. (1976) J. Clin. Microbiol. 3. 393±
appropriate care. Autoclave all used materials before
396.
discarding.
14 A Weinstein M. P., Mirrett S. and Reller L. B. (1988) J. Clin.
Microbiol. 5. 962±964. Tube reagents can cause transient eye irritation. In the
15 A Weinstein M. P., Reller L. B., Mirrett S. and Reimer L. G. event of contact with the eyes, flush with copious
(1987) 27th ICAAC Meeting. Abstract 198. amounts of water and seek medical advice.
16 Weinstein M. P., Mirrett S., Reimer L. G. and Reller L. B. (1989)
Care must be exercised to avoid injury when needles
J. Clin. Microbiol. 3. 427±430.
are used.
17 Weinstein M. P., Mirrett S., Reimer L. G. and Reller L. B. (1988)
Poster Presentation, 28th ICAAC Meeting, Los Angeles. The ISOLATOR 1.5 Tube is not intended for the
18 Rene P. and Lavallee J. (1987) 27th ICAAC Meeting Abstract transportation of specimens through the mail.
199. FOR IN VITRO DIAGNOSTIC USE ONLY
19 Clayton P., Mitchell C. J. and Swan R. A. (1987) 3rd European
Congress of Clinical Microbiology. Hague, Holland. Abstract 451. Storage Instructions
The tubes can be stored between 28C and 408C; room
temperature (258C) is recommended. Turbidity within
November 1998 8-5
Blood Cultures
the solution in the tube is normal. Tubes in use should Alternative needle/syringe method for specimen
be at 208C to 308C to ensure proper mixing of the collection
blood with the reagents at the time of collection. 1 Assemble a sterile needle onto a 3ml syringe or use
sterile needle/syringe combinations. Loosen but do
Specimen Collection and Preparation
not remove the needle shield.
1 Open a needle cartridge. Twist to break the
tamper-evident seal. Remove cap, exposing the 2 Use 10% PVP iodine solution for disinfecting the
rear end of the needle and threaded hub. Do not stopper of the tube.
remove front needle cover. 3 Prepare the venipuncture site as previously
2 Assemble needle and holder. Thread needle into described.
holder until firmly seated. Take care not to touch 4 Remove needle shield and perform venipuncture.
the needle valve to the holder. Collect 1.6ml of blood.
3 Use an appropriate disinfectant (e.g. 10% PVP 5 Add 1.5ml of the blood to the ISOLATOR 1.5 Tube
iodine solution) for disinfecting the stopper of the by puncturing the stopper with the needle. Do not
ISOLATOR 1.5 Tube. Do not allow the iodine force the blood into the tube. This may cause the
solution to pool on the stopper. Pooling could top to pop off the tube.
result in the introduction of disinfectant into the 6 After removal of the needle from the tube,
tube; this may interfere with the recovery of micro- immediately mix the blood with the reagents in the
organisms. tube by gently inverting four or five times.
4 Allow the disinfectant to dry completely. Insert the 7 Replace the protective cover onto the needle and
stopper of the ISOLATOR 1.5 Tube into the holder. discard in a suitable manner.
Advance the tube straight onto the needle but no
further than the guideline on the holder. Specimen Processing
1 Specimens should be processed as soon as they are
Blood Drawing Procedure received in the laboratory. Immediate processing of
1 Apply a tourniquet and select a venipuncture site. the ISOLATOR 1.5 Tube results in faster isolation,
Loosen the tourniquet, double cleanse and disinfect minimises antimicrobial effects of blood, maximises
the site with an appropriate agent (e.g. alcohol and the opportunity for polymicrobial isolation and
PVP iodine). Allow the disinfectant to dry for at may provide valuable quantitative information.
least one minute. Always collect the ISOLATOR
specimen before collecting the other specimens to Specimens may be held in ISOLATOR 1.5 Tubes for
avoid contaminating the blood culture. up to 16 hours at room temperature without adverse
effect on the recovery of micro-organisms. Specimens
2 Reapply the tourniquet. Remove the needle cover. obtained from patients on antimicrobial
Perform venipuncture with the patient's arm or chemotherapy should be processed immediately.
other venipuncture site in a downward position. Colony counts will not reflect the colony forming
During venipuncture hold the tube/needle units per millilitre of blood if the specimen is held in
assembly so that the needle is elevated relative to the tube for more than 4 hours.
the bottom of the tube.
Do not refrigerate specimens collected in ISOLATOR
During the collection procedure, do not permit
1.5 Tubes. The recovery of cold-sensitive organisms
contents of the tube to contact the stopper in order
such as Neisseria gonorrhoeae may be dramatically
to avoid the possibility of backflow of reagents
decreased.
from the tube with the attendant possibility of
adverse patient reaction. 2 Vigorously mix the contents of the tube. A Vortex-
Push the evacuated tube to the end of the tube type mixer (highest setting for 5±10 seconds) is
holder or until blood flow is visible. When blood recommended.
flows into the tube, remove the tourniquet. 3 Disinfect the stopper with an appropriate
3 Immediately remove the tube when fill is complete disinfectant. Allow to dry for one minute.
and flow has ceased (approximately 1.5ml). 4 Using a 3ml syringe enter the upright tube at an
4 When sampling is completed, remove the needle/ angle so that the needle emerges from the bottom
holder assembly with the last tube. Apply and hold of the stopper between the wall of the tube and the
a dry sterile compress to the venipuncture site. side of the stopper. Tilt (do not invert) the tube to a
Elevate the arm. horizontal position and collect the blood. Be sure to
remove the blood that may have accumulated in
5 Remove the tube from the needle/holder assembly. the base of the stopper.
Immediately mix the collection tube to prevent
coagulation and to initiate red blood cell lysis by Expel any air in the syringe into the tube and
gently inverting the tube four or five times. remove the needle/syringe. Discard the tube.
Incomplete mixing will result in blood clotting in 5 Divide the lysate evenly among the primary
the tube. isolation media, using a maximum of 0.35ml per
6 Handle and discard used needle in a suitable plate. Suggested culture media and growth
manner. conditions are shown in Table 1.
7 Label the specimen appropriately. 6 Keeping the lids of the plates as low as possible,
position the needle over the medium (don't touch
the agar with the needle).
7 Dispense up to 0.35ml of inoculum in a straight
8-6 November 1998
Blood Cultures
line across the surface of the plate, avoiding the ISOLATOR* 10 TUBES
edge of the agar. Discard the needle and syringe
appropriately. Intended use
The ISOLATOR 10 Tube is intended for the collection
8 Raising the plate cover only far enough to admit a and concentration of micro-organisms from blood and
long sterile disposable or wire loop, cross-streak other body fluids. The tube is used by clinical
the inoculum starting at the top and proceeding to laboratories to concentrate micro-organisms before
the bottom of the inoculum line (do not streak to transfer to conventional agar media for isolation and
edge of the agar). Rotate the plate 90 degrees and identification.
streak parallel to the original inoculum line. Rotate
plate 45 degrees and streak a third time to ensure Principles of the test
maximum distribution of the inoculum. Do not The ISOLATOR 10 Tube contains agents which lyse
sterilise the loop between plates. leucocytes and erythrocytes in blood, and block
coagulation.
9 After plating and streaking, either appropriately
discard the disposable loop, or sterilise the The specific agents used in the tube are:
inoculating loop. Purified Saponin, an effective and rapid cell lysing
10 Plates should be placed under appropriate agent, non-toxic to micro-organisms.
incubation conditions as soon as possible after Polypropylene Glycol to block the foaming
inoculation to optimise the isolation of fastidious tendency of Saponin.
and anaerobic micro-organisms.
Sodium Polyanetholsulphonate (SPS) which acts as
11 Incubate aerobic plates upright for the first 24 an anticoagulant, neutralises the bactericidal
hours, and anaerobic plates upright for the first 48 properties of blood and inhibits phagocytosis.
hours. Thereafter incubate all plates inverted.
12 Examine all plates daily until discarded. Even Reagents
when growth appears early, the plates should be Each ISOLATOR 10 Tube contains the following
reincubated to check for a second organism which reagents in aqueous solution (content prior to
may grow later. Plates should be examined with sterilisation).
lids in place whenever possible. If a lid must be Polypropylene Glycol 8 millimetres/litre
removed due to condensation or to better visualise Sodium
colonies, do not remove the lid completely; raise it Polyanetholsulphonate 15.3 grams/litre
only high enough to examine the area in question. Purified Saponin 28 grams/litre
Table 1.
The internal components of the tube are sterile.
Suggested culture media and growth conditions.
No Medium Incubation conditions Discard Precautions
Used tubes, syringes and needles contain human
1 Blood Agar Anaerobic, 358C±378C 6 days
body fluids. All materials should be handled as if
1±4 Chocolate Agar 5% CO2, 358C±378C 4 days
they are capable of transmitting disease. Handle with
1 Sab Dext Agar Aerobic, 228C±308C 8 days
appropriate care. Autoclave all used materials before
Plates should be pre-dried at least overnight at room discarding.
temperature. This enhances absorption of the
Tube reagents can cause transient eye irritation. In the
inoculum and reduces condensation on the plate lid.
event of contact with the eyes, flush with copious
Interpretation of Results amounts of water and seek medical advice.
1 If a colony appears only within the area inoculated,
Care must be exercised to avoid injury when needles
it should be considered a significant positive
are used.
culture regardless of genus or species. While
colony counts in paediatric blood cultures are The ISOLATOR 10 Tube is not intended for the
generally higher than those found in adults, it is transportation of specimens through the mail.
not uncommon for the counts to be low (<10 cfu/ FOR IN VITRO DIAGNOSTIC USE ONLY
ml) during episodes of bacteraemia associated with
upper respiratory tract infections or occurring after Storage Instructions
antimicrobial therapy. The tubes can be stored between 48C and 408C; room
2 If colonies appear on both the inoculated area and temperature (258C) is recommended. Turbidity within
outside the inoculated area, consider the colony the solution in the tube is normal. Tubes in use should
within the inoculated area as a positive culture and be at 208C to 308C to ensure proper mixing of the
the one outside as a contaminant. blood with the reagents at the time of collection.
3 If a colony appears only outside the inoculated Specimen Collection and Preparation
area, it may be considered a plate contaminant. 1 Open a needle cartridge. Twist to break the
tamper-evident seal. Remove cap, exposing the
Clinical Significance
rear end of the needle and threaded hub. Do not
The clinical significance of a micro-organism isolated
remove front needle cover.
from a patient's blood should be determined by the
Physician, taking into consideration the patient's 2 Assemble needle and holder. Thread needle into
history, clinical status, repetitive cultures and other holder until firmly seated. Take care not to touch
pertinent laboratory findings. the needle valve to the holder.
3 Use an appropriate disinfectant (e.g. 10% PVP force the blood into the tube. This may cause the
iodine solution) for disinfecting the stopper of the top to pop off the tube.
ISOLATOR 10 Tube. Do not allow the iodine 6 After removal of the needle from the tube,
solution to pool on the stopper. Pooling could immediately mix the the blood with the reagents in
result in the introduction of disinfectant into the the tube by gently inverting four or five times.
tube; this may interfere with the recovery of micro-
7 Replace the protective cover onto the needle and
organisms.
discard in a suitable manner.
4 Allow the disinfectant to dry completely. Insert the
stopper of the ISOLATOR 10 Tube into the holder. Specimen Processing
Advance the tube straight onto the needle but no 1 Specimens should be processed as soon as they are
further than the guideline on the holder. received in the laboratory. Immediate processing of
Blood Drawing Procedure the ISOLATOR 10 Tube results in faster isolation,
1 Apply a tourniquet and select a venipuncture site. minimises antimicrobial effects of blood, maximises
Loosen the tourniquet, double cleanse and disinfect the opportunity for polymicrobial isolation and
the site with an appropriate agent (e.g. alcohol and may provide valuable quantitative information.
PVP iodine). Allow the disinfectant to dry for at Specimens may be held in ISOLATOR 10 Tubes for
least one minute. Always collect the ISOLATOR up to 16 hours at room temperature without adverse
specimen before collecting the other specimens to effect on the recovery of micro-organisms. Specimens
avoid contaminating the blood culture. obtained from patients on antimicrobial
2 Reapply the tourniquet. Remove the needle cover. chemotherapy should be processed immediately.
Perform venipuncture with the patient's arm or Colony counts will not reflect the colony forming
other venipuncture site in a downward position. units per millilitre of blood if the specimen is held in
During venipuncture hold the tube/needle the tube for more than 4 hours.
assembly so that the needle is elevated relative to Do not refrigerate specimens collected in ISOLATOR
the bottom of the tube. 10 Tubes. The recovery of cold-sensitive organisms
During the collection procedure, do not permit such as Neisseria gonorrhoeae may be dramatically
contents of the tube to contact the stopper in order decreased.
to avoid the possibility of backflow of reagents 2 Place the tube into an adaptor in a fixed angle rotor
from the tube with the attendant possibility of (35 degree) in a suitable centrifuge. Use only the
adverse patient reaction. unique adaptors intended for the ISOLATOR 10
Push the evacuated tube to the end of the tube Tubes. Centrifuge at 3000xg for 30 minutes. The
holder or until blood flow is visible. When blood centrifuge brake should not be used; this could
flows into the tube, remove the tourniquet. disturb the concentrate and decrease recovery of
3 Immediately remove the tube when fill is complete micro-organisms. Be careful when removing the
and flow has ceased (approximately 10ml). tube from the centrifuge to avoid mixing the
supernatant fluid and the microbial concentrate.
4 When sampling is completed, remove the needle/
holder assembly with the last tube. Apply and hold 3 The proper orientation of the adaptor is critical for
a dry sterile compress to the venipuncture site. the centrifugation of ISOLATOR 10 Tubes without
Elevate the arm. breakage or leakage. Putting the adaptor in upside
down or spinning it empty may cause it to deform.
5 Remove the tube from the needle/holder assembly.
It is therefore important to properly orientate the
Immediately mix the collection tube to prevent
adaptor in the rotor and to remove adaptors that
coagulation and to initiate red blood cell lysis by
are not being used before centrifugation. Periodic
gently inverting the tube four or five times.
application of a light lubricant inside the adaptor
Incomplete mixing will result in blood clotting in
will make tube insertion and removal easier.
the tube.
4 Following centrifugation, carefully remove each
6 Handle and discard used needle in a suitable
ISOLATOR 10 Tube from its adaptor and place in
manner.
the ISOSTAT rack. A slight clockwise twist will
7 Label the specimen appropriately. facilitate insertion of the tube into the rack. Be sure
Alternative needle/syringe method for specimen that tubes are firmly seated and vertically aligned,
collection to avoid breakage while applying the cap.
1 Assemble a sterile needle onto a 20ml syringe or 5 Disinfect the stopper with an appropriate
use sterile needle/syringe combinations. Loosen disinfectant. Do not allow the disinfectant to pool
but do not remove the needle shield. in the stopper cavity. Allow to dry for one minute.
2 Use 10% PVP iodine solution for disinfecting the 6 Place the rack on the base of the ISOSTAT Press.
stopper of the tube.
7 Remove an ISOSTAT Cap by pushing the base of
3 Prepare the venipuncture site as previously the cap out through the sterile pack. To avoid
described. contamination, handle by the sides only. Do not
4 Remove needle shield and perform venipuncture. touch the top of the cap or the tip of the internal
Collect 11ml of blood. spike.
5 Add 10ml of the blood to the ISOLATOR 10 Tube Place a cap over the stopper of each ISOLATOR 10
by puncturing the stopper with the needle. Do not Tube. If more than one tube is being processed,
position caps on all tubes in the rack before collapse it and to provide a vacuum for concentrate
proceeding to the next step. withdrawal. Do this before inserting the stem of
8 Position a tube with its cap under the press head. the pipette into the tube.
Gently pull the handle of the press down as far as Carefully insert the stem of the concentrate pipette
possible and hold down for five seconds. The spike into the ISOLATOR 10 Tube through the
will penetrate the stopper and the cap will be membrane in the ISOSTAT cap while maintaining
firmly seated on top of the tube. Return the handle pressure on the bulb.
to the upright position. Insert the pipette into the tube so that the tip
If more than one tube is being processed, rotate the reaches the bottom. It may be necessary to
rack to position the next tube. Press the cap onto manipulate both pipette and tube to properly
this tube, and continue until caps have been orientate the pipette tip.
pressed onto each tube in the rack. Carefully move Gradually release pressure on the bulb and allow
the rack of tubes from the press to the work area. the concentrate to be drawn into the pipette. A
9 Open the heat seal at the top of a pack of ISOSTAT slow controlled release of the bulb is necessary to
supernatant pipettes, then pull apart the zippered achieve maximum recovery of concentrate.
seal. Remove a supernatant pipette from the pack. 16 Immediately remove the pipette and use it to
To avoid contamination, handle pipettes by the distribute the concentrate evenly onto the selected
bulb only. Do not touch the pipette stem. agar media. Keeping the lids of the plates as low as
The pack may be reclosed by pressing the edges of possible, dispense the concentrate in a straight line
the zippered seal together. across the surface of the agar. Keep the inoculum
10 Squeeze the bulb of the ISOSTAT supernatant away from the edge of the plate. For suggested
pipette to collapse it and to provide a vacuum for culture media and growth conditions see Table 2.
supernatant withdrawal. Do this before inserting 17 Using the tip of the concentrate pipette, streak
the stem of the pipette into the ISOLATOR 10 through the concentrate, making about 15 to 20
Tube. passes perpendicular to the original inoculum line.
Do not squeeze the pipette bulb after insertion of Streak lines should be kept away from the edges of
the pipette stem into the tube. Bubbling may the plate.
disturb the microbial concentrate and result in the 18 Discard used pipettes and ISOLATOR 10 Tubes
decreased recovery of micro-organisms. If this into an appropriate receptacle for contaminated
occurs the ISOLATOR 10 Tube should be waste.
centrifuged again. The ISOSTAT cap must be 19 Plates should be placed under appropriate
removed prior to centrifugation. incubation conditions as soon as possible after
11 Carefully insert the stem of the supernatant pipette inoculation to optimise the isolation of fastidious
into the ISOLATOR 10 Tube through the and anaerobic micro-organisms.
membrane of the ISOSTAT cap while maintaining 20 Incubate aerobic plates upright for the first 24
pressure on the bulb. hours, and anaerobic plates upright for the first 48
Insert the pipette into the tube as far as possible; hours. Thereafter incubate all plates inverted.
the base of the bulb must rest on the cap. 21 Examine all plates daily until discarded. Even
Release the bulb and allow the supernatant fluid to when growth appears early, the plates should be
be drawn into the pipette. Repeat this procedure reincubated to check for a second organism which
with the remaining tubes in the rack, using a new may grow later. Plates should be examined with
pipette for each tube. Confirm that air has entered lids in place whenever possible. If a lid must be
the pipettes indicating that all the supernatant fluid removed due to condensation or to better visualise
has been withdrawn. colonies, do not remove the lid completely; raise it
12 When the supernatant fluid has been withdrawn only high enough to examine the area in question.
from all ISOLATOR 10 Tubes, remove and discard Table 1.
the pipettes into an appropriate receptacle for Suggested culture media and growth conditions.
contaminated waste.
13 Open the heat seal at the top of the pack of No Medium Incubation conditions Discard
ISOSTAT concentrate pipettes, then pull apart the 1 Blood Agar Anaerobic, 358C±378C 6 days
zippered seal. Remove a concentrate pipette from 2 Chocolate Agar 5% CO2, 358C±378C 4 days
the pack. 1 Sab Dext Agar Aerobic, 228C±308C 8 days
To avoid contamination, handle pipettes by the Plates should be pre-dried at least overnight at room
bulb only. Do not touch the pipette stem. The pack temperature. This enhances absorption of the
may be reclosed by pressing the edges of the inoculum and reduces condensation on the plate lid.
zippered seal together.
14 Remove the first ISOLATOR 10 Tube from the rack Interpretation of Results
and vigorously mix the contents for 5±10 seconds 1 If a colony appears only within the area inoculated,
in order to achieve a homogeneous emulsion. A it should be considered a significant positive
vortex-type mixer (highest setting) is culture regardless of genus or species. In adults,
recommended. bacteraemia at a level of one colony forming unit
15 Squeeze the bulb of the concentrate pipette to or less per millilitre of blood is common. Thereafter
All of these products are based on particle New products are regularly being introduced into the
agglutination with simple visual reading. The tests Diagnostic Reagent range to cover an ever wider
can be performed easily on single or batched samples range of analytes.
and the provision of appropriate controls ensures the
reliability of results. CAMPYLOBACTER TEST KIT
The tests are rapid and offer the benefit of minimal Code: DR150
sample preparation with actual assay times of The Oxoid Campylobacter Test Kit is a latex
between only 20 seconds for the Staphytect kit up to agglutination test for the identification of
eight minutes for the VDRL kit. enteropathogenic Campylobacter spp. from solid culture
Three particle types are used for the assays; stabilised media.
sheep red blood cells, carbon particles and blue Introduction
coloured latex particles. All of these formats give Campylobacters are helical, or curved, Gram-
highly visible reactions when viewed against the negative, oxidase positive rod-shaped bacteria.1 They
supplied, high quality white disposable reaction have been isolated from the environment as well as
cards. Most of the tests are based upon antibody/ from humans and animals. The adoption of
antigen reactions, which are made visible by the Campylobacter culture in laboratory routines for
presence of the coloured particles. investigating enteritis has shown Campylobacter spp.
to be the leading cause of diarrhoeal disease. Infection
Culture Confirmation Tests for the Identification of has been associated with the consumption of
Plate Isolates contaminated water and foods, particularly poultry
As an example, the test reagent in the E. coli 0157 kit and unpasteurised milk.
(DR620) consists of a suspension of sub-micron blue
latex particles which have been sensitised with rabbit Current methods for the isolation and culture of
antibodies that react specifically with the Campylobacters have been recently reviewed.2
lipopolysaccharide found on E. coli strains of the The Dryspot Campylobacter test reagent consists of
serotype 0157. When a portion of a colony of E. coli blue latex particles sensitised with rabbit antibody
0157 is emulsified with the latex reagent, the antibody reactive with selected Campylobacter cell surface
present on the latex surface binds the LPS on the cells antigens. The control reagent consists of blue latex
and also any antigen fragments present. This binding particles sensitised with rabbit antibody not reactive
causes considerable cross-linking of the latex particles with Campylobacters.
to produce large clumps. The clumps, when of a
sufficient size become visible to the naked eye during The latex reagents are dried onto reaction cards.
the rocking of the card. In addition to the presence of When a Campylobacter extract is mixed with the test
the clumps the latex particles are removed from the reagent, agglutination occurs due to cross-linking of
liquid phase causing a noticeable (and often latex-bound antibody and Campylobacter antigens. If
complete) clearing of the smooth blue background. In the extract does not contain recognised
a negative reaction (when the organism does not Campylobacter antigens agglutination will not occur
carry the 0157 antigen) the latex particles do not bind and the result will be negative.
to the bacteria and therefore no cross-linking occurs The Oxoid Dryspot Campylobacter Test includes
and the blue suspension remains smooth. antigen extraction reagents and a positive control
Other culture confirmation test kits currently antigen preparation.
available are listed below. Components of the Kit
DR151M Dryspot Campylobacter Reagent Cards
Direct Tests for Detection of Specific Antibody Test areas: Blue latex particles sensitised with
The Helicobacter pylori latex kit (DR700) is an example rabbit antibody reactive with selected
of a test that may be used to test a specimen directly. Campylobacter cell surface antigens.
It utilises antigen-sensitised blue latex particles to
detect circulating antibodies present in patients who Control areas: Blue latex particles sensitised with
are infected with the H. pylori bacterium. Specific rabbit antibody not reactive with Campylobacters.
antibodies if present will cross-link the latex particles 10 foil-sealed plastic trays each containing five
causing visible agglutination. reaction cards and a desiccant pouch. Each card
Other direct test kits currently available are listed has a test and a control area. 50 tests in total.
below.
DR152M Extraction Reagent 1 2 Konowalchuk J., Speirs J. and Stavric S. (1977) Infect. Immune 18.
A solution of acetic acid (1.2m) 775±779.
3 Scotland S., Day N. and Rowe B. (1980) FEMS Microbiol. Lett. 7.
DR153M Extraction Reagent 2
15±17.
A neutralising reagent of Tris buffer containing
4 Centers for Disease Control (1982) Morbid Mortal Weekly 31. 580±
0.09% sodium azide as a preservative.
585.
DR154M Positive Control Reagent 5 Karmali M., Steel B., Petric M. and Lim C. (1983) Lancet i. 619±
Consists of a neutralised acid extract of appropriate 620.
Campylobacter organisms in buffer containing 6 Johnson W., Lior H. and Bezanson (1983) Lancet i. 76.
0.09% sodium azide as a preservative. 7 March S. and Tarnam (1986) J. Clin. Microbiol. 23. 869±872.
8 Krishnan C., Fitzgerald V., Dakin S. and Behme R. (1987) J. Clin.
DR699M Paddle Pastettes
Microbiol. 25. 1043±1047.
DR155M Storage Bag
Instructions for use. INFECTIOUS MONONUCLEOSIS KIT
Materials Required but not Provided Code: DR680
Timer
The Oxoid Infectious Mononucleosis Kit is a simple,
Sterile Loop (5ml calibrated) two-minute latex agglutination test for the detection of
12 x 75mm test tubes the heterophile antibody associated with infectious
mononucleosis in serum and plasma.
A suitable laboratory disinfectant.
Infectious mononucleosis (glandular fever) is an acute
For full procedure please see product insert.
infectious disease caused by the Epstein-Barr virus
and primarily affects lymphoid tissue. It is
References
1 Cowan S. T. and Steel K. J. (1965) Characters of Gram-negative
characterised by the appearance of enlarged and often
tender lymph nodes, enlarged spleen, and abnormal
bacteria. In Manual for the identification of medical bacteria. Barrow
lymphocytes in blood. Patients usually, but not
G. I. and Feltham R. K. A. (ed.) Third Edition. Cambridge
University Press. Cambridge, U.K.
always, develop a transient heterophile antibody
response.
2 Corry J. E. L., Post D. E., Colin P. et al. (1995) Culture media for
the isolation of campylobacters. Int. J. Food Microbiol. 26. The detection of heterophile antibodies to infectious
43±76. mononucleosis by the agglutination of sheep red
blood cells was first reported by Paul and Bunnell1.
Subsequent work by Davidsohn,2,3 Lee3 and Beer4
E. COLI 0157 LATEX TEST showed the need for differential absorption of sera to
Code: DR620 remove non-infectious mononucleosis heterophile
antibodies. Fletcher and Woolfolk5 showed that
A latex agglutination test for the identification of E. coli
antigens obtained from the membranes of bovine
Serogroup 0157. erythrocytes were more effective in combining with
Certain strains of Escherichia coli have recently been the infectious mononucleosis heterophile antibodies
implicated in some cases of haemorrhagic colitis (HC) than those antigens obtained from either sheep or
and haemolytic uraemic syndrome (HUS). horse erythrocytes6,7.
It has been shown that these strains produce a vero- References
cytotoxin (VT). The E. coli serotype most frequently 1 Paul J. R. and Bunnell W. N. The presence of heterophile
isolated from HC and HUS cases is 0157:H7. Isolation antibodies in infectious mononucleosis. Am. J. Med. Sci. 1932;
of this serotype from a diarrhoeal stool, especially 183. 90±104.
with blood, is indicative of a verocytotoxin-producing 2 Davidsohn I. Serologic diagnosis of infectious mononucleosis.
strain1,2,3,4,5,6,7,8. JAMA 1937: 108. 289±295.
3 Davidsohn I. and Slaby R. Horse agglutinins in infectious
The Oxoid E. coli 0157 Latex Test will demonstrate by
mononucleosis. Am. J. Clin. Path. 1968: 49. 3±11.
slide agglutination E. coli strains possessing the 0157 4 Beer P. The heterophile antibodies in infectious mononucleosis
antigen. The test is best used in conjunction with
and after injection of serum. J. Clin. Invest. 1935: 15. 591±599.
Sorbitol MacConkey Agar (Oxoid CM813). E. coli
5 Fletcher M. A and Woolfolk B. J. Immunochemical studies of
0157:H7 strains do not ferment sorbitol and therefore
infectious mononucleosis. Isolation and Characterisation of
give colourless colonies on this medium. The majority
heterophile antigens from hemoglobin-free stroma. J. Immunol.
of E. coli isolates do ferment sorbitol and give
1971: 107. 842±853.
characteristic pink colonies.
6 Data on file at Oxoid Limited.
Sorbitol MacConkey Agar should be used as the 7 Henle G. E Horwitz C. A Hum. Pathol. 1974: 5. 551±565.
primary screen. Non-sorbitol-fermenting colonies can
Paddle Pastettes is a registered trademark of Alpha
then be tested with the latex reagents, to determine if
Laboratories.
the isolate belongs to the 0157 serogroup and
therefore a potential VT-producing strain.
References
1 Borczyk A., Lior H. and Crebin B. (1957) Int. J. Food Microbiol. 4.
347±349.
The TPHA test will detect these antibodies. The VDRL TEST KIT
Oxoid TPHA test is a sensitive passive
haemagglutination test specifically for the Code: DR525
detection of antibodies to Treponema pallidum. A test kit for the detection and quantitative assessment
Components of the Kit (DR530) of reagin antibodies in syphilis screening.
DR531 Test Cell Suspension The Oxoid VDRL Test Kit is a macroscopic non-
2 bottles each containing 8.5ml of antigen coated treponemal flocculation test for use in the detection
formolised tanned fowl erythrocytes. The dropper and quantification of reagin antibodies. A
bottle will dispense 75ml drops. Each kit contains presumptive diagnosis of syphilis can be made when
sufficient suspension for 200 tests. these antibodies are detected in serum. The Card Test
DR532 Control Cell Suspension uses a modified form of the VDRL antigen1
2 bottles each containing 8.5ml of uncoated containing micro-particulate carbon to improve the
formolised tanned fowl erythrocytes. The dropper reading of results.
bottle will dispense 75ml drops. Each kit contains The test can be performed using unheated serum or
sufficient suspension for 200 tests. plasma. The VDRL Carbon Antigen Suspension can
DR533 Diluent Buffer also be used on both single and multi-channel Auto-
2 bottles each containing 20ml of buffer. Analyser equipment2,3,4,5,6.
DR534 Positive Control Serum VDRL ANTIGEN SUSPENSION
1 bottle containing 2ml of pre-diluted (1/20) serum,
positive for antibodies to T. pallidum. The serum Code: DR526
should cause agglutination in the screening test Oxoid VDRL Carbon Antigen Suspension is used for
and remain positive to a serum dilution of 1/2560 the detection of reagin antibodies, which indicate a
plus or minus one doubling dilution in the serological diagnosis of syphilis. It is a modified form
quantitative test. of VDRL antigen utilising micro-particulate carbon to
DR535 Negative Control Serum enhance the visual reading of results. The product is
1 bottle containing 2ml of pre-diluted (1/20) serum suitable for use on both single and multi-channel
negative for antibodies to T. pallidum. Anto-Analyser equipment and for a manual slide
test2,3,4,5,6.
The human sera used in the manufacture of the
controls have been shown to be negative for
VDRL CARBON ANTIGEN SUSPENSION
HBsAG (Hepatitis B surface antigen), Hepatitis C
and HIV 1 and 2 antibodies by FDA approved Code: DR520
tests.
Oxoid VDRL Carbon Antigen Suspension is used for
Instruction Leaflet the detection of reagin antibodies, which indicate a
The following materials are required but not serological diagnosis of syphilis. It is a modified form
provided. of the VDRL antigen utilising micro-particulate
U-well microtitration plate. carbon to enhance the visual reading of results. The
product is suitable for use on both single and multi-
Micropipettes and tips to deliver 25 and 100ml channel Auto-Analyser equipment and for a manual
volumes. slide test2,3,4,5,6.
Suitable laboratory disinfectant.
References
For full procedure please see product insert. 1 Portnoy J., Brewer J. H. and Harris A. D. (1962) US Public Health
Report 77. 645.
References 2 McGrew B. E., Stout G. W. and Falcone V. H. (1968) Am. J. Med.
1 Garner M. F., Backhouse J. L., Daskalopoulos G. and Walsh J. L. Tech. 34. 634.
(1973) J. Clin Path. 26. 258±260. 3 McGrew B. E., Ducross M. J. F., Stout G. W. and Falcone V. H.
2 Rathlev T. (1965) W.H.O. VDT/RES/77 65. (1968) Am. J. Clin. Pathol. 59. 52.
3 Rathlev T. (1976) Brit. J. Vener. Dis. 43. 181. 4 Norins L. C. (1968) Automation in Analytical Chemistry,
4 Tomizawa T., Kasamatsu S. and Yamaya S.-I. (1969) Jap. J. Med. Technicon Symposium 1967, 1 157 New York Mediad.
Sci. Biol. 22. 341±350. 5 Stevens R. W. and Stroebel E. (1970) Am. J. Clin. Pathol. 53. 32.
5 Sequeria P. J. L. and Eldridge A. E. (1973) Brit. J. Vener. Dis. 49. 6 Stout G. W. McGrew B. E. and Falcone, V. H. (1968) J. Conf.
242±248. Public Health Lab. Directors 2. 67.
6 Cox P. M., Logan L. C. and Norins L. C. (1969) Appl. Microbiol.
18. 485±489.
7 Johnston N. A. (1972). Brit. J. Vener. Dis. 48. 474±478.
8 Uete T., Fukazawa S., Ogi K. and Takeuchi Y. (1971) Brit. J.
Vener. Dis. 47. 73±76.
9 Young H., Henrichsen C. and Robertson D. H. H. (1974) Brit. J.
Vener. Dis. 50. 341±346.
10 Coffey E. M., Bradford L. L., Naritomi L. S. and Wood R. M.
(1972) Appl. Microbiol. 24. 26±30.
11 Dyckman J. D., Storms S. and Huber T. W. (1980) J. Clin.
Microbiol. 12. 629±630.
RAPID FOOD TESTS Sample Window. This contains blue latex labelled
with antibody. The extract rehydrates the complex
and the specific antigen reacts, if present, with the
(AOAC and AFNOR approved) antibody.
OXOID LISTERIA RAPID TEST The complex moves through the pad by capillary
action to a test strip containing an immobilised line of
Code: FT401 antibody midway along the Result Window.
Intended Use A further reaction between antigen/latex complex
The Oxoid Listeria Rapid Test is designed for the and the fixed antibody results in a blue line in the
detection of Listeria species in foods and Result Window.
environmental samples within 43 hours. The test
protocol allows for the availability of the result two If no flagella antigen is present the Result Window
working days after the sample is received in the will remain clear.
testing laboratory. The procedure uses two carefully The Clearview Listeria device also provides an
selected enrichment steps for the maximum recovery integral control feature. The appearance of a blue line
and growth of Listeria followed by an immunoassay in the Control Window shows the test has been
in the ClearviewTM format. This simple system gives a carried out correctly.
clear visual result 20 minutes after the addition of the
heated and cooled sample to the test device with no Components of the Kit
further manipulations being required. Oxoid SR166M Half Fraser Supplement: 50 vials
Half Fraser Supplement is used in conjunction with
Introduction Fraser Broth (CM895), it is modified by the
Listeria is a genus of Gram positive, non-sporing addition of only half the level of selective agents
bacilli with a DNA G+C content of 36±38%. They normally found in Fraser Supplement. Each vial is
have up to 6 peritrichous flagella and are motile when sufficient for 225ml of broth.
grown at 308C or below. They are aerobic and Clearview Listeria Test Units: 50
facultatively anaerobic, catalase positive and oxidase
negative. The genus comprises six species, L. Positive Clearview Control: 3 vials
monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. Non-viable Listeria monocytogenes suspension.
seeligeri and L. grayi subsp. Grayi and L. Grayi subsp. Instruction leaflet
murrayi. All Listeria, except L. grayi, variously share 4
flagella antigens A, B, C, and D, of which flagella Materials Required but not Provided:
antigen B is common1. Oxoid Fraser Broth, (FB) CM895
Pathogenic and non-pathogenic Listeria are Oxoid Buffered Listeria Enrichment Broth,
ubiquitous in nature and can be isolated from soil, (B.L.E.B.) CM 897
vegetables and natural waters as well as from healthy B.L.E.B. Selective Enrichment Supplement, SR141E
animals and man. They are able to grow over a
Incubator at 30 + 28C
temperature range of 1±458C. Consequently, L.
monocytogenes is a food poisoning risk to susceptible Water bath at 80 + 28C
individuals if present in foods that are subsequently Glass test tubes of 5±8ml capacity
stored at these temperatures for sufficient time for the
organism to grow to infectious levels before ingestion. Sterile water/ethanol mixture 1:1, (v/v).
Clinical symptoms include flu-like illness, For full procedure please see product insert.
spontaneous abortion, still birth, meningitis,
pneumonitis, septicaemia and endocarditis. Listeria References
monocytogenes infections mainly occur in neonates, 1 Seeliger H.P.R. and Jones D. Genus Listeria Pirie. In: Sneath
pregnant women, the elderly and P.H.A., Mair H.S., Sharp N.E. and Holt J.G. (Eds): Bergey's
immunocompromised individuals. Manual of Systematic Bateriology. William & Wilkin Co. Baltimore,
Test Principle 1986.
1 Enrichment Broth System 2 Parry S.H., Briggs T., Blades J.A., Garni M & Piron J. (1993) A
Culture of the test sample is in two sequential rapid Clearview Immunoassay for detection of Listeria. 7th.
enrichments, taking 42 hours. Any Listeria organisms International Congress on Rapid Methods and Automation in
present in the food or environmental sample are Microbiology and Immunology.
selectively enriched using growth conditions which 3 Holbrook R., Briggs T.A., Anderson J.A., & Sheard P.N. (1993)
are optimal for flagella expression. Detection of Listeria species in foods in 43 hours using enrichment
and the Listeria ClearviewTM Immunoassay. 7th. International
2 Antigen Extraction Congress on Rapid Methods and Automation in Microbiology and
The second enrichment media is heated at 808C for 20 Immunology.
minutes to extract the flagella antigen. 4 Holbrook R., Briggs T., Anderson J., Blades J. & Sheard P.N.
3 ClearviewTM Listeria Device (1994) A 43 hour Test for Detecting Listeria in Foods using the
The Clearview Listeria device contains specific Unipath Clearview Immunoassay. 81st Annual Meeting of the
monoclonal antibodies to the B flagella antigen2 that International Association of Milk, Food and Environmental
is common to the Listeria species indicated earlier. Sanitarians, Inc., San Antonio.
References
1 Compendium of Methods for the Microbiological Examination of
Foods, 2nd Ed. (1984), American Public Health Association Inc,
Ed. M.L. Speck.
2 F.D.A. Bacteriological Analytical Manual, 6th Ed. (1984). Published
by Association Official Analytical Chemists.
TEST PRINCIPLE
Clostridium difficile toxin A is extracted from faecal
samples as described in ``Sample Preparation''.
The Test Unit contains monoclonal antibodies to C.
difficile toxin A.
The extracted antigen is added to the pad in the
Sample Window. The pad contains blue latex labelled
with antibody. The extract rehydrates the complex
and, if present, the specific antigen reacts with the
antibody.
The antibody-antigen-latex complex travels through
the test strip by capillary action to an area midway
along the Result Window. This area contains an
immobilised line of monoclonal antibody to C. difficile
toxin A.
A further reaction between the antigen-antibody-latex
complex and the fixed antibody is shown by the
formation of a blue line in the Result Window.
If no C. difficile toxin A is present no line will appear
in the Result Window.
11
INDEX
PRODUCT INDEX C
`Calgon' Ringers Solution . . . . . . . . . . . . . . . . . . . . . . . 5-6
A CampyGen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-9
CampyGen Compact . . . . . . . . . . . . . . . . . . . . . . . . . 7-10
Acid Egg Medium. . . . . . . . . . . . . . . . . . . . . . . . . .
. 4-20
`Actidione' Agar . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 4-18 Campylobacter Agar Base . . . . . . . . . . . . . . . . . . . . 2-63, 66
Aeromonas Medium (Ryan) . . . . . . . . . . . . . . . . . . . .
. 2-32 Campylobacter Agar Base (Karmali) . . . . . . . . . . . . . . . . 2-65
Agars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 3-14 Campylobacter Blood-Free Selective Agar Base . . . . . . . . . 2-67
Agar Bacteriological. . . . . . . . . . . . . . . . . . . . . . . . .
. 3-15 Campylobacter Growth Supplement. . . . . . . . . . . . . . 2-64, 4-4
Agar Purified. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 3-15 Campylobacter Selective Media. . . . . . . . . . . . . . . . . . . 2-61
Agar Tablets (Agar No.3) . . . . . . . . . . . . . . . . . . . . .
. 3-15 Campylobacter Selective Supplement (Blaser-Wang) . . . . 2-64, 4-4
Agar Technical (Agar No.3) . . . . . . . . . . . . . . . . . . . .
. 3-15 Campylobacter Selective Supplement (Butzler) . . . . . . . 2-64, 4-4
Amies Transport Medium . . . . . . . . . . . . . . . . . . . . .
. 2-33 Campylobacter Selective Supplement (Karmali) . . . . . . . 2-65, 4-5
Ampicillin Selective Supplement . . . . . . . . . . . . . . . . 2-32, 4-3 Campylobacter Selective Supplement (Skirrow) . . . . . . . 2-63, 4-4
Anaerobe Basal Agar . . . . . . . . . . . . . . . . . . . . . . . . . 2-34 Campylobacter Selective Supplement (Preston) . . . . . . . 2-66, 4-4
Anaerobe Basal Broth. . . . . . . . . . . . . . . . . . . . . . . . . 2-35 Campylobacter Test Kit . . . . . . . . . . . . . . . . . . . . . . . . 9-1
Anaerobic gas generating kits . . . . . . . . . . . . . . . . . . . . . 7-2 Cary-Blair Medium . . . . . . . . . . . . . . . . . . . . . . . . . . 2-69
Anaerobic Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . 7-6 Casein Hydrolysate . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9
Anaerobic Jar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-2, 3 CAT Supplement . . . . . . . . . . . . . . . . . . . . . . . . . 2-68, 4-5
Anaerobic Selective Medium . . . . . . . . . . . . . . . . . . . . 2-219 Catalyst - Anaerobic Jar . . . . . . . . . . . . . . . . . . . . . . . . 7-5
Anaerobe Selective Supplements . . . . . . . . . . . . . . . 2-219, 4-3 CCDA Selective Supplement . . . . . . . . . . . . . . . . . . 2-67, 4-5
AnaeroGen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-6 CDMN Selective Supplement . . . . . . . . . . . . . . . . . . 2-79, 4-6
AnaeroGen Compact . . . . . . . . . . . . . . . . . . . . . . . . . . 7-7 CFC Pseudomonas Supplement . . . . . . . . . . . . . . . 2-171, 4-5
AnaeroJar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-8 C. Difficile Toxin A Test . . . . . . . . . . . . . . . . . . . . . . . 10-2
An-Ident Discs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-8 Charcoal Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 2-50, 2-70
Antibiotic Assay Media . . . . . . . . . . . . . . . . . . . . . . . 2-35 China Blue Lactose Agar . . . . . . . . . . . . . . . . . . . . . . . 2-71
Antibiotic Medium No.1 . . . . . . . . . . . . . . . . . . . . . . . 2-37 Chloramphenicol Selective Supplement . . . . . . . 2-92, 94, 180, 4-6
Antibiotic Medium No.2 . . . . . . . . . . . . . . . . . . . . . . . 2-38 Cholera Medium TCBS . . . . . . . . . . . . . . . . . . . . . . . . 2-72
Antibiotic Medium No.3 . . . . . . . . . . . . . . . . . . . . . . . 2-38 Chromogenic E. Coli/Coliform Medium . . . . . . . . . . . . . 2-73
Antimicrobial Susceptibility Testing . . . . . . . . . . . . . . . . . 6-1 Chromogenic Urinary Tract Infection (UTI) Medium . . . . . . 2-74
Antimicrobial Susceptibility Testing - aura . . . . . . . . . . . . . 6-3 Clausen Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-75
Arcobacter Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-39 CLED Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-77
Assay Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-35 CLED Medium (with Andrade indicator) . . . . . . . . . . . . . 2-77
Atmosphere Generation System . . . . . . . . . . . . . . . . . . . 7-6 Clostridium difficile Agar Base . . . . . . . . . . . . . . . . . . . 2-78
aura - Complete Susceptibility System . . . . . . . . . . . . . . . . 6-3 Clostridium difficile Selective Supplement . . . . . . . . . . 2-78, 4-6
Azide Blood Agar Base . . . . . . . . . . . . . . . . . . . . . . . . 2-39 Clostridium perfringens Selective Media . . . . . . . . . . . . . 2-166
Azide Dextrose Broth (Rothe) . . . . . . . . . . . . . . . . . . . . 2-40 COBA Streptococcus Selective Medium . . . . . . . . . . . . . . 2-195
CO2Gen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-10
B CO2Gen Compact . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-11
Columbia Blood Agar Base . . . . . . . . . . . . . . . . . . . . . 2-80
Bacillus cereus Selective Agar Base. . . . . . . . . . . . . . . . . 2-41
Bacillus cereus Selective Supplement . . . . . . . . . . . . . 2-41, 4-3 Cooked Meat Medium . . . . . . . . . . . . . . . . . . . . . . . . 2-81
Bacitracin Discs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12 Columbia CNA Agar . . . . . . . . . . . . . . . . . . . . . . . . . 2-194
Complement Test Diluent . . . . . . . . . . . . . . . . . . . . . . . 5-2
Baird-Parker Agar Base. . . . . . . . . . . . . . . . . . . . . . . . 2-43
CN Pseudomonas Supplement . . . . . . . . . . . . . . . . 2-171, 4-5
Barbitone Complement Fixation Test Diluent . . . . . . . . . . . 5-1
Basic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . 2-97, 145, 5-2 Corn Meal Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-82
BCET-RPLA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-1 Corynebacterium diphtheriae Selective Medium . . . . . . . . 2-201
Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10 Crossley Milk Medium . . . . . . . . . . . . . . . . . . . . . . . . 2-83
Beta Lactamase Broad Spectrum Mixture . . . . . . . . . . . . . 4-14 CT Supplement . . . . . . . . . . . . . . . . . . . . . . . . . 2-141, 4-6
Culti-Loops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-229
Beta Lactamase Detection Papers . . . . . . . . . . . . . . . . . . . 5-3
Czapek Dox Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-84
Beta Lactamase Touch Sticks . . . . . . . . . . . . . . . . . . . . . 5-4
Biggy Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-45 Czapek Dox Liquid Medium (Modified) . . . . . . . . . . . . . 2-85
Bile Aesculin Agar . . . . . . . . . . . . . . . . . .
Bile Salts . . . . . . . . . . . . . . . . . . . . . . . .
.
.
.
.
.
.
. . . . . 2-46
. . . 3-16, 17 D
Bile Salts No.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17 DCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-88
Biochemical Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1 DCA Hynes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-88
Biological extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1 DCLS Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-85
Bismuth Sulphite Agar . . . . . . . . . . . . . . . . . . . . . . . . 2-47 Dermasel Agar Base. . . . . . . . . . . . . . . . . . . . . . . . . . 2-86
Blaser-Wang (Campylobacter) Selective Medium . . . . . . 2-64, 4-4 Dermatophyte Selective Medium . . . . . . . . . . . . . . . . . . 2-86
Blood Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-48 Dermasel Selective Supplement. . . . . . . . . . . . . . . . . 2-86, 4-6
Blood Agar Base No.2 . . . . . . . . . . . . . . . . . . . . . . . . 2-49 Desoxycholate Agar. . . . . . . . . . . . . . . . . . . . . . . . . . 2-87
Blood Agar Base (Sheep) . . . . . . . . . . . . . . . . . . . . . . . 2-50 Dextrose Bacteriological . . . . . . . . . . . . . . . . . . . . . . . 3-17
Blood Culture Systems . . . . . . . . . . . . . . . . . . . . . . . . . 8-1 Dextrose Tryptone Agar . . . . . . . . . . . . . . . . . . . . . . . 2-89
Blood Culture System `Isolator' . . . . . . . . . . . . . . . . . . 8-5, 10 Dextrose Tryptone Broth . . . . . . . . . . . . . . . . . . . . . . . 2-90
Blood Culture System `SIGNAL' . . . . . . . . . . . . . . . . . . . 8-3 Diagnostic Discs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-8
Blood Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-13 Diagnostic Kits and Reagents . . . . . . . . . . . . . . . . . . . . . 9-1
Bordetella pertussis Selective Media . . . . . . . . . . . . . . . . 2-50 Diagnostic Sensitivity Test Agar (DSTA) . . . . . . . . . . . . . 2-91
Bordetella Selective Supplement . . . . . . . . . . . . . . . . 2-51, 4-3 Dichloran-Glycerol (DG 18) Agar Base . . . . . . . . . . . . . . 2-92
Brain Heart Infusion . . . . . . . . . . . . . . . . . . . . . . . . . 2-51 Dichloran Rose Bengal Chloramphenicol Agar. . . . . . . . . . 2-94
Brain Heart Infusion Agar . . . . . . . . . . . . . . . . . . . . . . 2-52 Dip Slides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-23
Brewers Thioglycollate Medium . . . . . . . . . . . . . . . . . . 2-199 Disc dispensers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3
Brilliant Green Agar. . . . . . . . . . . . . . . . . . . . . . . . . . 2-53 DNase Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-93
Brilliant Green Agar (Modified). . . . . . . . . . . . . . . . . . . 2-54 DRBC Agar Base. . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-94
Brilliant Green Bile (2%) Broth . . . . . . . . . . . . . . . . . . . 2-56 Dryspot E. Coli O157 Latex Test . . . . . . . . . . . . . . . . . . . 9-8
Brucella Selective Media . . . . . . . . . . . . . . . . . . . . . . . 2-57 Dryspot IM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-8
BMPA (Legionella) Selective Medium . . . . . . . . . . . . . . . 2-120 Dryspot Staphytect Plus . . . . . . . . . . . . . . . . . . . . . . . . 9-8
Buffered charcoal yeast extract agar (BCYE) . . . . . . . . . . . 2-120 Dryspot Streptococcal Grouping Kit . . . . . . . . . . . . . . . . . 9-9
Buffered Listeria Enrichment Broth . . . . . . . . . . . . . . . . 2-126 DST Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-91
Buffered Peptone Water . . . . . . . . . . . . . . . . . . . . . . . 2-59 Dulbecco A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2
321 DNase Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 2-93 833 Aeromonas Medium Base (Ryan). . . . . . . . . . . . . . 2-32
325 Plate Count Agar . . . . . . . . . . . . . . . . . . . . . . . 2-169 845 HR Medium . . . . . . . . . . . . . . . . . . . . . . . . . . 2-110
327 Antibiotic Medium No 1 . . . . . . . . . . . . . . . . . . . 2-37 854 Sheep Blood Agar Base . . . . . . . . . . . . . . . . . . . . 2-50
329 Brilliant Green Agar (Modified). . . . . . . . . . . . . . . 2-54 856 Listeria Selective Agar Base (Oxford) . . . . . . . . . . . 2-127
331 Columbia Blood Agar Base . . . . . . . . . . . . . . . . . 2-80 857 Salmonella Rapid Test Elective Medium . . . . . . . . . 9-11
333 TCBS Cholera Medium . . . . . . . . . . . . . . . . . . . . 2-72 862 Listeria Enrichment Broth Base . . . . . . . . . . . . . . . 2-129
335 Antibiotic Medium No 2 . . . . . . . . . . . . . . . . . . . 2-38 863 Listeria Enrichment Broth Base (UVM) . . . . . . . . . . 2-129
337 Mueller Hinton Agar . . . . . . . . . . . . . . . . . . . . . 2-156 866 Rappaport Vassiliadis Soya Peptone (RVS) Broth . . . . 2-176
343 Muller Kauffman Tetrathionate Broth Base . . . . . . . . 2-157 868 Azide Dextrose Broth (Rothe) . . . . . . . . . . . . . . . . 2-40
353 Clausen Medium . . . . . . . . . . . . . . . . . . . . . . . 2-75 877 PALCAM Agar Base . . . . . . . . . . . . . . . . . . . . . 2-132
359 MRS Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-154 888 Bile Aesculin Agar . . . . . . . . . . . . . . . . . . . . . . 2-46
361 MRS Agar. . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-153 895 Fraser Broth . . . . . . . . . . . . . . . . . . . . . . 2-98, 2-131
367 GC Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . 2-102 897 Buffered Listeria Enrichment Broth . . . . . . . . . . . . 2-126
375 Brain Heart Infusion Agar . . . . . . . . . . . . . . . . . . 2-52 898 HTM Base. . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-107
377 Slanetz & Bartley Medium . . . . . . . . . . . . . . . . . . 2-191 906 R2A Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-173
381 Lysine Iron Agar. . . . . . . . . . . . . . . . . . . . . . . . 2-134 910 Modified Semi-Solid Rappaport Vassiliadis (MSRV)
391 Thioglycollate Broth USP (alternative) . . . . . . . . . . . 2-199 Medium Base. . . . . . . . . . . . . . . . . . . . . . . . 2-152
393 D.C.L.S. Agar . . . . . . . . . . . . . . . . . . . . . . . . . 2-85 920 Yeast and Mould Agar . . . . . . . . . . . . . . . . . . . . 2-226
395 Selenite Broth Base (Lactose) . . . . . . . . . . . . . . . . 2-186 935 Campylobacter Agar Base (Karmali) . . . . . . . . . . . . 2-65
399 Selenite Broth Base (Mannitol) . . . . . . . . . . . . . . . 2-187 945 TBX Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-206
401 Mycoplasma Agar. . . . . . . . . . . . . . . . . . . . . . . 2-158 949 Chromogenic UTI . . . . . . . . . . . . . . . . . . . . . . . 2-74
403 Mycoplasma Broth . . . . . . . . . . . . . . . . . . . . . . 2-160 956 Chromogenic E.coli/Coliform Medium . . . . . . . . . . 2-73
405 Mueller Hinton Broth . . . . . . . . . . . . . . . . . . . . . 2-157 957 Anaerobe Basal Broth. . . . . . . . . . . . . . . . . . . . . 2-35
409 Sensitest Agar . . . . . . . . . . . . . . . . . . . . . . . . . 2-188 965 Arcobacter Broth . . . . . . . . . . . . . . . . . . . . . . . 2-39
419 Hektoen Enteric Agar. . . . . . . . . . . . . . . . . . . . . 2-108 966 S.P.R.I.N.T. Salmonella Enrichment Broth . . . . . . . . 9-11
423 CLED Medium with Andrade indicator. . . . . . . . . . 2-77 967 Modified Lauryl Tryptose Broth with MUG and
425 Amies Transport Medium . . . . . . . . . . . . . . . . . . 2-33 added Tryptophan . . . . . . . . . . . . . . . . . . . . 2-151
435 SIM Medium . . . . . . . . . . . . . . . . . . . . . . . . . . 2-190 972 Anaerobe Basal Agar . . . . . . . . . . . . . . . . . . . . . 2-34
437 Schaedler Anaerobe Agar . . . . . . . . . . . . . . . . . . 2-184
451 Lauryl Tryptose Broth . . . . . . . . . . . . . . . . . . . . 2-119 L
463 Standard Plate Count Agar (APHA) . . . . . . . . . . . . 2-175 L5 Sodium Chloride Bacteriological . . . . . . . . . . . . . . 3-18
469 XLD Medium . . . . . . . . . . . . . . . . . . . . . . . . . 2-224 L8 Gelatin Bacteriological . . . . . . . . . . . . . . . . . . . . 3-17
471 Iso-Sensitest Agar . . . . . . . . . . . . . . . . . . . . . . . 2-112 L11 Agar Bacteriological (Agar No.1) . . . . . . . . . . . . . . 3-15
473 Iso-Sensitest Broth. . . . . . . . . . . . . . . . . . . . . . . 2-114 L13 Agar Technical (Agar No.3) . . . . . . . . . . . . . . . . . 3-15
479 Endo Agar Base . . . . . . . . . . . . . . . . . . . . . . . . 2-97 L21 Yeast Extract Powder . . . . . . . . . . . . . . . . . . . . . 3-10
485 Violet Red Bile Glucose Agar . . . . . . . . . . . . . . . . 2-216 L26 Liver Desiccated Bacteriological . . . . . . . . . . . . . . 3-11
487 Tinsdale Agar Base . . . . . . . . . . . . . . . . . . . . . . 2-201 L27 Liver Digest Neutralised . . . . . . . . . . . . . . . . . . . . 3-5
497 Schaedler Anaerobe Broth . . . . . . . . . . . . . . . . . . 2-185 L28 Purified Agar. . . . . . . . . . . . . . . . . . . . . . . . . . 3-15
501 WL Nutrient Broth . . . . . . . . . . . . . . . . . . . . . . 2-222 L29 Lab-Lemco Powder (for Beef Extract) . . . . . . . . . . . 3-10
509 Buffered Peptone Water . . . . . . . . . . . . . . . . . . . 2-59 L31 Skim Milk Powder . . . . . . . . . . . . . . . . . . . . . . 3-18
519 Cary Blair Medium . . . . . . . . . . . . . . . . . . . . . . 2-69 L32 Peptonised Milk . . . . . . . . . . . . . . . . . . . . . . . . . 3-8
523 Giolitti-Cantoni Broth. . . . . . . . . . . . . . . . . . . . . 2-106 L34 Peptone Bacteriological Neutralised . . . . . . . . . . . . . 3-6
533 SS Agar (Modified) . . . . . . . . . . . . . . . . . . . . . . 2-192 L37 Peptone Bacteriological. . . . . . . . . . . . . . . . . . . . . 3-6
539 Dermasel Agar Base. . . . . . . . . . . . . . . . . . . . . . 2-86 L39 Malt Extract Desiccated . . . . . . . . . . . . . . . . . . . 3-10
543 Perfringens Agar Base (OPSP). . . . . . . . . . . . . . . . 2-166 L40 Peptone Mycological . . . . . . . . . . . . . . . . . . . . . . 3-7
545 Oxytetracycline-Glucose-Yeast Extract Agar . . . . . . . 2-164 L41 Casein Hydrolysate (Acid). . . . . . . . . . . . . . . . . . . 3-9
549 Rose Bengal Chloramphenicol Agar . . . . . . . . . . . . 2-180 L42 Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9
559 Pseudomonas Agar Base . . . . . . . . . . . . . . . . . . . 2-171 L43 Tryptone T . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9
587 Perfringens Agar Bas (TSC/SFP) . . . . . . . . . . . . . . 2-167 L44 Soya Peptone Neutralised . . . . . . . . . . . . . . . . . . . 3-8
589 BIGGY Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 2-45 L47 Tryptose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7
591 Kanamycin Aesculin Azide Agar Base. . . . . . . . . . . 2-114 L48 Lactalbumin Hydrolysate . . . . . . . . . . . . . . . . . . . 3-9
595 Tryptone Bile Agar . . . . . . . . . . . . . . . . . . . . . . 2-204 L49 Peptone P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7
601 Clostridium difficile Agar Base . . . . . . . . . . . . . . . 2-78 L53 Haemaglobin Powder (Soluble) . . . . . . . . . . . 2-102, 3-17
607 Minerals Modified Glutamate Medium . . . . . . . . . . 2-148 L55 Bile Salts . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16, 17
617 Bacillus cereus Agar Base . . . . . . . . . . . . . . . . . . 2-41 L56 Bile Salts No.3 . . . . . . . . . . . . . . . . . . . . . . . . . 3-17
619 Wilkins Chalgren Anaerobe Agar . . . . . . . . . . . . . 2-219 L70 Lactose Bacteriological . . . . . . . . . . . . . . . . . . . . 3-17
627 Rogosa Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 2-180 L71 Glucose Bacteriological . . . . . . . . . . . . . . . . . . . . 3-17
641 Vogel Johnson Agar. . . . . . . . . . . . . . . . . . . . . . 2-217 L72 Special Peptone . . . . . . . . . . . . . . . . . . . . . . . . . 3-7
643 Wilkins Chalgren Anerobe Broth . . . . . . . . . . . . . . 2-221 L85 Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . 3-8
651 Universal Beer Agar. . . . . . . . . . . . . . . . . . . . . . 2-212 L120 Sodium Thioglycollate . . . . . . . . . . . . . . . . . . . . 3-18
653 Yersinia Selective Agar Base. . . . . . . . . . . . . . . . . 2-227 L121 Sodium Biselenite . . . . . . . . . . . . . . . . . . . . . . . 3-18
655 Legionella CYE Agar Base . . . . . . . . . . . . . . . . . . 2-120 L124 Sodium Glutamate . . . . . . . . . . . . . . . . . . . . . . 3-18
657 Orange Serum Agar. . . . . . . . . . . . . . . . . . . . . . 2-164
669 Rappaport Vassiliadis (RV) Enrichment Broth . . . . . . 2-174 SR
671 Tetrathionate Broth Base . . . . . . . . . . . . . . . . . . . 2-197 20 Urea 40% . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14
681 Plate Count Agar with antibiotic-free skim milk . . . . . 2-170 21 Lactic Acid 10% . . . . . . . . . . . . . . . . . . . . . . . . 4-14
689 Campylobacter Agar Base . . . . . . . . . . . . . . . . . . 2-63 30 Potassium Tellurite 3.5% . . . . . . . . . . . . . . . . . . . 4-14
699 Selenite Cystine Broth Base . . . . . . . . . . . . . . . . . 2-188 32 Tomato Juice . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14
701 KF Streptococcus Agar . . . . . . . . . . . . . . . . . . . . 2-115 35 Horse Serum . . . . . . . . . . . . . . . . . . . . . . . . . . 5-13
727 Dichloran Rose Bengal Chloramphenicol Agar. . . . . . 2-94 37 Potassium Lactate 50% . . . . . . . . . . . . . . . . . . . . 4-14
729 Dichloran-Glycerol (DG18) Agar Base . . . . . . . . . . . 2-92 46 Fildes Extract. . . . . . . . . . . . . . . . . . . . . . . . . . 4-14
733 Maximum Recovery Diluent . . . . . . . . . . . . . . . . 2-144 47 Egg Yolk Emulsion . . . . . . . . . . . . . . . . . . . . . . 4-13
739 Campylobacter Blood-Free Selective Agar Base . . . . . 2-69 48 Laked Horse Blood . . . . . . . . . . . . . . . . . . . . . . 5-13
755 GBS Agar Base (Islam) . . . . . . . . . . . . . . . . . . . . 2-101 49 Horse Blood Oxalated . . . . . . . . . . . . . . . . . . . . 5-13
777 Raka Ray Agar. . . . . . . . . . . . . . . . . . . . . . . . . 2-173 50 Horse Blood Defibrinated . . . . . . . . . . . . . . . . . . 5-13
783 MLCB Agar. . . . . . . . . . . . . . . . . . . . . . . . . . . 2-150 51 Sheep Blood Defibrinated . . . . . . . . . . . . . . . . . . 5-13
785 M17 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-136 52 Sheep Blood (formalised) . . . . . . . . . . . . . . . . . . 5-14
793 Tergitol 7 Agar. . . . . . . . . . . . . . . . . . . . . . . . . 2-197 53 Sheep Blood in Alsevers Solution. . . . . . . . . . . . . . 5-14
813 Sorbitol MacConkey Agar . . . . . . . . . . . . . . . . . . 2-145 54 Egg Yolk Tellurite Emulsion. . . . . . . . . . . . . . . . . 4-14
817 M17 Broth. . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-137 56 GC Selective Supplement . . . . . . . . . . . . . . . 2-103, 4-7