Ne8499 Lab M Micro Manual 2016 Lo

MICROBIOLOGY
MANUAL 2016/17
A Neogen® Company
THE GATEWAY TO MICROBIOLOGY ™

ABOUT LAB M OUR BRANDS
Lab M specialises in microbiological culture media.

The company develops, manufactures and supplies its products
from its headquarters in Heywood, Greater Manchester, UK,
and has an established network of distributors around the Lab M´s Harlequin™ range of chromogenic media includes
world. Lab M’s customers include those in the food industry, products for the isolation of Listeria species, Salmonellae,
water, environmental, clinical, pharmaceutical and vaccine Escherichia coli and Cronobacter sakazakii.
manufacturers.
Lab M has an international reputation for the quality of its

dehydrated culture media, which is available in various formats
to support worklows and streamline eficiencies across different Lab M’s μPREP™ ready-to-reconstitute bagged dehydrated
microbiology laboratories. microbiological culture media are ready to use simply by
adding water. This new range takes up minimal storage
OUR HERITAGE space, offers quick, convenient reconstitution with no need
to autoclave.
Established in 1971, Lab M initially manufactured pre-poured
media for clinical laboratories – and its name, London
Analytical and Bacteriological Media, relects its origins.
During the 1970s, the company developed its own dehydrated The Pinnacle™ brand is a new line of ready-to-use plated
culture media, and by the early 1980s had added consumables culture medium, providing Lab M’s high quality DCM
and diagnostics to its products list. At that time Lab M was the prepared as ready-to-use plates under a stringent quality
only UK supplier offering such a range, and with a growing management system in a GMP environment.
reputation for quality it soon became an international supplier.
Lab M has since evolved and relocated to a new purpose built

head ofice and manufacturing facility in North West England,
and now offer a comprehensive range of microbial products Captivate™ antibody coated paramagnetic particles for
and solutions covering both traditional and rapid methods. the speciic immunomagnetic separation (IMS) of micro-
organisms. The range offers solutions for the concentration
PRODUCT QUALITY of a number of Shiga toxin-producing Escherichia coli
(STEC) serotypes.
The quality of Lab M’s products has a direct impact on the
service their customers provide. As a result of this Lab M
consider quality to be everybody’s responsibility.
Lab M is certiied in accordance with ISO 9001:2008 & ISO NutriTone™ - Lab M offer a range of peptones for
13485:2003 for the design, manufacture and supply of biotechnology applications. The NutriTone™ range is
microbiological culture media, antibiotic supplements and intended for use in mammalian cell culture. For a dedicated
diagnostic products. Lab M’s products for the clinical market brochure please contact a member of the Lab M team.
are supplied in compliance with the European IVD directive
and carry the CE mark. Lab M’s QC laboratory holds
BS EN ISO 17025 accreditation by UKAS for the physical
and microbiological performance testing of our ready-to-use
Pinnacle™ media range. OrganoTone™ is the brand name given to Lab M’s range
of media constituents. These include peptones for use in
These accreditations, alongside Lab M’s stringent quality microbial fermentation by vaccine manufacturers,
management system, ensure that only the highest standard carbohydrates, selective agents and agars.
of product is manufactured and distributed to our customers
around the world.
FOR MORE INFORMATION Tel: +44 (0) 161 820 3833 Fax: +44 (0) 161 820 5383
© 2016 Lab M Limited. All rights reserved Lab M, Harlequin™, µPREP™, Pinnacle™, Captivate™, ColourScreen™, Nutritone™, OrganoTone™ and The Gateway to Microbiology are trademarks of Lab M Limited.
1
CONTENTS
Introduction
ISO accreditation 03
Customised media service 05
Quality criteria 06
Quality control of culture media 07
Preservation of stock cultures 08
Microbiology methods 09
Dehydrated culture media selection guide 10
1. Dehydrated Culture Media 13
2. Harlequin™ Chromogenic Media 96
3. Harmonised Pharmacopoeia (USP/EP/JP) 102
4. Biomolecular Products 110
5. µPREP™ Ready-to-Reconstitute Media 115
6. Captivate™ Immunomagnetic Separation 116
7. Pinnacle™ Pre-Poured Plates 118
8. Lyophilised Media Supplements 123
9. OrganoTone™ Media Constituents 133
10. Sterile Additives and Ready Prepared Media 140
Indexes 143
The Microbiology Manual Version 10
2
3
4
Customised Media Service Lab M Culture Media:
The Process Outline
The Lab M Customised Media Service
Since its inception, Lab M has been committed to customer
needs by offering a wide range of quality media products
RAW MATERIALS
for global markets. Our continued programme of product
Agars, Peptones, Extracts, Dyes, Chemicals etc.
innovation and development has ensured that the Company Each component is individually tested for suitability.
has been responsive to market trends and changes in custom
and practice. The increasing importance of regulatory
compliance has also been a signiicant factor in this process
of innovation.
From time to time, however, Lab M has been asked to ‘design’

media to meet speciic customer applications. This has evolved
into a more proactive approach to resolving customer needs:
PRODUCTION
The Lab M Customised Media Service. Weighing, Milling, Blending
Aproduction batch is made from raw materials of speciied
What do we offer? batch number which have been pre-tested for compatibility.
The components are individually milled to ensure uniform
• Individually designed media, engineered to meet speciic particle size. Weighings are double checked before the
components are blended.
customer requirements.
• Close client collaboration.
• Development programmes that are both cost and time

eficient.
• A process approach to development, commencing with

feasibility tests, through product trials and into routine
QUALITY CONTROL
production.
Physical, Biological parameters. Comparison with previous
batch and competition
• Customised packaging and labelling.
Quality control irst checks that the batch is completely
blended, then a series of physical and biological tests
• Assurance of compliance with regulatory requirements. are performed to ensure the product meets the exacting
standards required by our customers. Comparisons with
• Total client conidentiality. previous and competitor’s batches are made. Results are
recorded and a reference sample stored.
• Post-development consultancy.
• Commitment to on-going production.
• Quality products and service.
This is a unique service that differentiates us from our

competitors and our Technical Support Group would be happy
BOTTLING
Into 500g sealed containers, or bulk containers at request
to discuss your needs on a conidential basis.
Automated equipment delivers pre-weighed amounts
into containers which are hermetically sealed. Each
container is immediately labelled with product details,
code and batch number.
CUSTOMERS
Lab M products are dispatched all over the world
to microbiologists in all types of laboratory. Strict batch
traceability in accordance with ISO9001 ensures we can recall
all products if necessary, safeguarding your products/process.
5
Lab M Culture Media – Dehydrated Culture Media
The quality criteria
Storage
Dehydrated culture media stored unopened under optimal conditions
have a shelf life of 2-5 years. In-house quality control by the user
will help determine the condition of product in opened containers.
Raw materials The best conditions for storing dehydrated media are in a cool, even
temperature away from any sources of moisture such as washing up
Peptones and Extracts – Clarity, pH, moisture, growth promoting
areas or laboratory autoclaves and away from strong light. Storage in a
properties with Gram positive and Gram negative organisms
refrigerator is generally not recommended (unless otherwise stated on
aerobically and anaerobically, freedom from toxicity. Compatibility
the product label) as there is the risk of condensation on the container
with other components, haemolysis patterns, antibiotic antagonists.
when it is brought out of the refrigerator. Storage instructions for
Agar – Clarity, pH, gel strength, melting point, setting point, heavy Lab M media products are stated on the product label and must be
metal content (particularly Ca++, Mg++) compatibility with other followed.
components. Clarity on re-melt.
TABLE 1 – Deterioration of SS Agar stored in various conditions
Bile Salts – Clarity, pH, thin layer chromatography, compatibility for 6 months.
with other components.
Dyes & Chemicals – pH, chemical parameters, growth promotion
inhibition, properties after incorporation into culture media. Storage conditions moisture gain %
Unopened bottle stored in cool,
dark, dry conditions 0
Production
Loose cap, stored in light on bench 1.1
All components from speciied batch numbers. All components
weighed accurately and checked. Loose cap, stored in light in autoclave room 4.4
Components milled to uniform particle size. Components blended for
speciied time, multiple samples taken to ensure thorough blending. The effect of the moisture gain on the performance of the agar can
be quite dramatic. A 1.1% gain in moisture on storage will lead to
a 53% reduction in the numbers of Salmonella isolated. Similarly
a 4.4% gain in moisture will result in a 78% reduction in isolation
rate. This demonstrates the importance of ensuring the container lid is
Quality control tightly closed and the pot stored in cool, dry, dark conditions. Barry,
A. L. and Fay, G. D. A review of some common sources of error in the
Physical – pH, clarity, gel strength, colour, heat stability, viscosity, Preparation of Agar Media. (1972). Am. J. Med. Tech. Vol. 38 No. 7.
redox. When a container is opened for the irst time the date should be noted
on the container. Dehydrated media should not be used if it shows any
Biological – Growth characteristics, productivity ratio, chemical sign of moisture gain i.e. becomes lumpy or discoloured. The lid on
reactions and colour changes, comparison with previous batch and the container should be replaced quickly after media has been taken
competition. out and closed tightly.
Preparing Culture Media Weighing Out

Using a top-pan balance with an accuracy of ±0.1 gram the powder
should be spooned onto a weighing boat or clean beaker. Do not tip
the media out of the container as this will cause excess dust which
may be irritating and will certainly need cleaning up. The components
Quality Assured of some formulations can be irritant so the wearing of a suitable face
Before each batch of Lab M Culture media is passed for sale it mask at this stage is advisable.
undergoes a rigorous quality control procedure to ensure it gives
maximum recovery and reproducibility. Reconstitution of media in
the user’s laboratory must be done with care to ensure the same high
standards of performance. Water
The following section outlines the correct procedures which will Puriication by distillation, deionisation or reverse osmosis is
ensure high quality reconstituted products, and suggests simple advisable. It is important that the equipment is properly maintained; the
quality control techniques that can be used to check the performance output of ion resins need to be electronically monitored and microbial
of prepared media. colonisation of the resin and tubing must be avoided. Storage vessels
for puriied water must also be monitored for microbial colonisation.
It is advisable to use only fresh puriied water with a conductivity
of less than 10 microsiemens. Stored water tends to become acidic
because it absorbs atmospheric CO2. Tap water is not recommended
because of the potential presence of heavy metal ions which can cause
inhibition and precipitation problems.
6
Selectivity and speciicity.
Quality Control of Quantitative assessment of selectivity requires inoculation of both
selective culture media and a reference medium with the speciied
Culture Media micro-organism, at an appropriate inoculum for testing. Selectivity,
as deined by the selectivity factor, has to reach the value given in
the corresponding standard or as set out in the technical speciication.
For semi-quantitative and qualitative methods, the growth of the non-
The routine quality control of culture media is an essential ‘good target strain(s) should be inhibited partly or completely.
laboratory practice’ necessary to maintain the standards and
performance of any bacteriological culture technique. Such practices
are a key requirement for many laboratory accreditation schemes such
Assessment of growth
as UKAS, and CLAS, INAB and ENAC etc. who accredit according Solid Culture Media
to ISO17025
The International Standards Organisation recently published BS EN Productivity Ratio (P.R.)
ISO 11133:2014, deining the requirements relating to the preparation, Productivity ratio is determined by assessing performance related to a
production, storage and performance testing of culture media that is control medium, which should be a nutritious agar such as Tryptone
intended for the microbiological analysis of food, feed and water. Soy Agar (LAB011). A controlled inoculum of approximately 100
This applies to laboratories preparing media from dehydrated culture colony forming units (cfu) must be used for both media and the P.R.
media (DCM) in-house, but also to media manufacturers. Lab M are is calculated by counting the colonies on the test and control media:
compliant to this new standard, applying the appropriate QC criteria P.R. = No. of colonies on test x dilution factor
where applicable. This is relected in the certiicates of analysis No. of colonies on control x dilution factor
provided by Lab M.
Lab M further supported this by gaining UKAS accreditation for their Or to express as a percentage:
quality control laboratory according to ISO 17025:2005 in 2015. Lab
M’s schedule of accreditation covers both the physical and microbial P.R. (%) = No. of colonies on test x 100
performance testing of the Pinnacle™ media range; pH, sterility, ill No. of colonies on control
volume, qualitative performance testing and quantitative performance There are many inoculation methods that may be used to determine the
testing. All methods are based on the new requirements of BS EN productivity ratio, including:
ISO 11133:2014.
• Spiral plate
• Miles-Misra and Modiied Miles-Misra technique
Example of a typical quality control • Pour plate
process • Surface inoculation e.g. serial dilution surface
incoulation using ‘L-shaped spreader’
A typical dehydrated culture medium (DCM) product will be subjected
Relative Growth Index (Ecometric Technique)
to a battery of tests. The manufacturer tests the product in its inal
prepared form (as a plate or broth) for all criteria. An end user needs The ecometric technique of Mossel is simple and gives numerical
only to perform a minimum QC assessment. readings that can form the basis of records suitable for trend analysis.
Both absolute growth index (AGI) and relative growth index (RGI)
can be obtained by this method.
A typical manufacturer’s testing regime is as follows:
This plating technique is less-frequently used, but still offers a robust
Determinants of physical quality. method of determining productivity. The ecometric technique is
ɶ Final pH. based on streaking an inoculum to extinction. The results obtained
ɶ Clarity and presence of optical artefacts.
can be compared with previous batches of the same medium or with
batches of the same medium from different manufacturers. The results
ɶ Gel stability and consistency.
can also be compared with results obtained using the same organisms
Determinants of microbiological quality. on non-selective media.
Tests to assess microbial contamination and microbiological
growth characteristics are required for each batch of end product. Liquid Culture Media
Microbial contamination.
The samples tested include at least one plate or tube from the beginning, Liquid media are challenged with 10-100cfu of the target organism.
and one plate or tube from the end of a pouring or dispensing process. Recovery of the organism is assessed qualitatively (visual) or semi-
Plates or tubes are incubated for at least 18 hours under the routine quantitatively (by sub-culture).
incubation conditions speciied for a particular media type. Target
limits for the percentage of contaminated plates or containers of liquid 1. Prepare an overnight culture of the test organism in Tryptone
medium should be established for each medium or speciied by the Soy Broth (USP/EP/JP) (LAB004).
manufacturer.
Microbiological growth. 2. Prepare a tenfold serial dilution (to 10-12) in Maximum
Each batch of complete culture medium, nutrient components or Recovery Diluent (LAB103).
supplements, are assessed for microbiological growth in terms of
productivity, selectivity and speciicity. Assessment may be by the 3. Add 1ml of each dilution to 9ml of test and control broths.
quantitative, semi-quantitative or qualitative methods described in Incubate at 37°C for 18 hours.
the standard, or by another generally accepted technique. Results are
interpreted by comparing the amount of growth on the test medium 4. Examine the broths and note the highest dilution showing
with that on a speciied reference medium. The growth of target strains growth (turbidity of the broth).
should be typical in appearance, size and morphology, while the
growth of non-target strains should be partly or completely inhibited. This method can be used in conjunction with the Miles-Misra
Test strains. technique to demonstrate recovery of known levels of CFU’s in broth
Micro-organism cultures from the WDCM reference collection are media.
documented in this standard, however it is stated that ‘the use of
equivalent strains from other culture collections is permitted’. Lab M
typically test media with QC organisms drawn from ATCC, NCTC
References:
and NCIMB approved sources. BS EN ISO 11133:2014 Microbiology of food, animal feed and
Productivity. water — Preparation, production, storage and performance testing of
Solid, semi-solid or liquid culture media are inoculated with an culture media
appropriate inoculum of the working culture of each of the deined
test micro-organisms. Productivity should reach a minimum limit as Mossel D.A.A. et al (1983) Quality Assurance of Selective Culture
deined in the corresponding standard or as detailed in the technical
speciication. Quantitative methods require determination of the
productivity ratio: a score of growth based on a comparison between
the test medium and the deined reference medium.
7
Preservation of
Stock Cultures
The following method has been used in our laboratory for several
years for long-term storage of microorganisms. For most strains a
freezer at -20°C will sufice, however it should be noted that storage
temperature may affect growth characteristics and viability.
Preservation of Bacteria using Protect ™

Microorganism Preservation System
Culture Preparation
1. Grow the organism on an appropriate non-selective solid
medium using the appropriate incubation temperature and
atmospheric conditions to achieve a heavy growth. Use several
plates with organisms forming small colonies.
2. Remove growth from plate using a sterile cotton swab and
emulsify in the Protect™ cryopreservation luid, making a
thick suspension.
3. Carefully cap the vial and invert it six times. Leave the vial
to stand for at least 30 seconds. . The vial may be tapped to
remove the bubbles from bead centres.
4. Withdraw as much liquid as possible using a sterile, ine-tip
pipette.
Cap the vial, label and place in freezer.
Culture Recovery
1. Remove Protect™ vial from the freezer or liquid nitrogen
container. Use a cryoblock, which has been stored in a freezer
for at least thirty minutes to extend the time available for
working with frozen vial.
2. Open vial. Remove one bead using sterile needle and recover
the culture by rubbing bead over suitable agar medium or
placing bead directly into broth. Removed beads must not be
returned to the vial.
Duplicate vials can be prepared from the same original culture. This
will assist should the vial become contaminated or should the pedigree
of the organism be questioned.
The Protect™ Microorganism Preservation System is available
to purchase from Lab M. Full details are available on the Lab M
website, www.labm.com.
Details of recommended QC strains of organisms are contained in the
individual entries for media.
8
Microbiology Methods Format and
There are numerous sources of information regarding microbiology
Abbreviation Guide
methods, just some of which are listed below:
Bacteriological Analytical Manual.
Product Name
Food and Drug Administration. Available online at www.fda.gov
(Alternative name or commonly used abbreviation)
(FDA’s Bacteriological Analytical Manual (BAM) presents the
agency’s preferred laboratory procedures for microbiological
analyses of foods and cosmetics. AOAC International published
previous editions of this manual in a loose-leaf notebook format, Product Code
and, more recently, on CD-ROM. This online BAM is now available Description:
to the public. Some changes have been made to methods since the A brief outline which may include any of the following information
previous version. A listing of chapters updated since the last hard-
on the medium:
copy version (Edition 8, Revision A /1998) can be found in About the
Bacteriological Analytical Manual) • History
• Mechanisms
British Standards Institute.
389 Chiswick High Road, London W4 4AL. www.bsigroup.co.uk • Applications
• Recognition By Regulatory/Advisory Bodies
Compendium of Methods for the Microbiological Examination of
Foods 4th edition. • Advantages
2002. Edited by Downes, F.P. and Ito, K. American Public Health
Association. Typical Formula: The product composition in grams per litre;
ISBN-10: 087553175X / ISBN-13: 978-0875531755 minor adjustments to the published formula may be made to
meet performance criteria.
European Pharmacopeia 8th Edition.
European Pharmacopoeia, 2014, 8th edition, European Directorate for
the Quality of Medicine, Council of Europe, 226 Avenue de Colmar Method for reconstitution
BP907, F-67029 Strasbourg Cedex 1, France Distilled water can be substituted for deionised water. “Allow to soak
times” are not critical. If agar media are to be dispensed prior to
Manual of Microbiological Methods for the Food and Drink sterilising, irst bring to the boil to dissolve the agar.
Industry, 5th edition Appearance: – of the inished cooled medium.
2007. Campden BRI, Station Road, Chipping Campden, pH: at 20˚C. For agars, pour a small quantity into a universal bottle,
Goucestershire, GL55 6LD. Guideline G43. ISBN 978-0-905942- allow to set and plunge the probe into the medium.
93-3.
Minimum Q.C. organisms – for use every time a new batch of
The Microbiology of Drinking Water prepared medium is reconstituted. This short form check should not be
2002. Methods for the Examination of Water and Associated confused with a full Q.C. of the medium. Where an organism should
Materials. Environment Agency. Available online from www. http:// show inhibition this could be complete or partial. Records should be
www.environment-agency.gov.uk/ kept of these results to help recognise changes in performance over a
period of time.
Practical Food Microbiology, 3rd edition Storage of Prepared Media – All prepared media should be stored
2002. Edited by Roberts, D. and Greenwood, M. Wiley-Blackwell. in the dark. If a medium is to be used beyond the suggested shelf life,
ISBN-10: 1405100753 / ISBN-13: 978-1405100755 appropriate quality control should be performed to demonstrate that
there has been no detectable fall off in performance.
Growth characteristics
Abbreviation key for colonial descriptions:
CV = convex CR = crenated
F = lat Rz = rhizoid
E = entire G = glossy
P.P. = pinpoint D = dull
( ) brackets are used to denote occasional variations.
References
A list of related publications and sources of information.
N.B. The typical formulae in this manual and on the product label are
adhered to wherever possible. However it is occasionally necessary to
make minor adjustments to meet performance criteria.
9
HAL003 Harlequin™ Tryptone Bile Glucuronide Agar (TBGA)
Culture Media Selection LAB126
LAB196
Lactose Broth
Lauryl Tryptose Broth
Guide LAB045
LAB005
MacConkey Agar No. 3
MacConkey Broth (Purple)
LAB077 ColourScreen™ MLSTB-MT (ISO) Modiied Lauryl Sulphate
Tryptose Broth with MUG & Tryptophan
Anaerobes PIN002 Pinnacle™ TBGA (Tryptone Bile Glucuronide Agar, TBX) Prepared
LAB072 Tryptone Bile Agar

LAB160 Brazier’s CCEY Agar LAB031 Violet Red Bile Agar (VRBA)
HP012 Columbia Agar (USP/EP/JP) LAB088 Violet Red Bile Glucose Agar (VRBGA)
LAB001 Columbia Agar Base LAB573 VRBA with MUG
LAB215 Columbia II Agar Base
LAB220 DRCM (ISO) - Differential Reinforced Clostridial Medium
LAB090 Fastidious Anaerobe Agar (F.A.A.) Dairy
LAB071 Fastidious Anaerobe Broth (F.A.B.) LAB081 CSEB - Cronobacter sakazakii Enrichment Broth
HP001 Fluid Thioglycollate Medium (USP/EP/JP) LAB060 Endo Agar Base
LAB425 Fluid Thioglycollate Medium (Clear) HAL012 Harlequin™ CSIM (ISO)
LAB222 Iron Sulphite Agar HAL013 Harlequin™ CSA-DFI - Cronobacter sakazakii Agar DFI
LAB109 Perfringens Agar (O.P.S.P.) Formulation
LAB194 Perfringens Agar (TSC) HAL012 Harlequin™ CSIM (ISO)
LAB023 Reinforced Clostridial Agar HAL010 Harlequin™ Listeria Chromogenic Agar
HP011 Reinforced Medium for Clostridia (USP/EP/JP) LAB126 Lactose Broth
LAB064 Thioglycollate Medium (Brewer) LAB196 Lauryl Tryptose Broth
LAB092 M17 Agar
Bacillus LAB019 Milk Agar
LAB115 Milk Plate Count Agar
LAB020 Dextrose Tryptone Agar LAB077 ColourScreen™ MLSTB-MT (ISO) Modiied Lauryl Sulphate
LAB193 PEMBA (Bacillus Cereus Medium) Tryptose Broth with MUG & Tryptophan
LAB073 PREP Bacillus cereus Medium LAB093 MRS Agar
LAB094 MRS Broth
Biomolecular PIN003 Pinnacle™ CSIM (ISO) Prepared
HAL004 Harlequin™ LB Agar LAB098 Potato Dextrose Agar

LAB087 Sugar Free Agar
LAB168 LB Agar LAB063 Tryptone Glucose Extract Agar
LAB174 LB Agar (Lennox) LAB031 Violet Red Bile Agar (VRBA)
LAB169 LB Broth LAB088 Violet Red Bile Glucose Agar (VRBGA)
LAB173 LB Broth (Lennox) LAB573 VRBA with MUG
LAB191 Luria Bertani (Hi-Salt) Broth LAB038 Wort Agar
LAB182 NZCYM Broth LAB099 Wort Broth (Modiied)
LAB181 NZY Broth (NZYM) LAB018 Yeast Extract Agar
LAB183 Terriic Broth LAB119 Yeast Extract Dextrose Chloramphenicol Agar
LAB175 YPD Broth
LAB176 YPD with Agar
LAB180 2xYT Agar Diagnostic Medical Microbiology
LAB179 2xYT Broth
LAB195 BCYE Legionella Isolation Medium
LAB121 Bromocresol Purple Lactose Agar
Blood Agar Bases LAB006 CLED (Bevis)-double indicator
LAB028 Blood Agar Base LAB041 CLED (Mackey & Sandys)-single indicator
LAB015 Blood Agar Base No. 2 LAB090 Fastidious Anaerobe Agar
LAB001 Columbia Agar Base LAB067 GC Agar Base
LAB215 Columbia II Agar Base LAB195 GVPC Legionella Isolation Medium (BCYE basal agar)
LAB525 Eugon Agar LAB027 Hoyle’s Medium (Modiied)
LAB090 Fastidious Anaerobe Agar (F.A.A.) LAB035 TYC Medium
PIN007 Pinnacle™ Blood Agar No. 2 (25ml ill) Prepared
Refer to other sections for our full range of Medical Products.
PIN006 Pinnacle™ Columbia Agar Base (25ml ill) Prepared
LAB011 Tryptone Soy Agar Diluents/ Isotonic solutions

HP017 Buffered Sodium Chloride-Peptone Solution pH 7.0 (USP/EP/JP)
Blood Culture Media LAB103 Maximum Recovery Diluent
LAB049 Brain Heart Infusion Broth LAB100Z Ringer’s Solution (1/4 Strength) - Tablets
LAB071 Fastidious Anaerobe Broth (F.A.B.)
LAB004 Tryptone Soy Broth (USP/EP/JP)
LAB205 Tryptone Soy Broth (without dextrose) Enteric Pathogens
LAB167 Aeromonas Agar
Brewing LAB013 Bismuth Sulphite Agar
LAB201 Lysine Agar LAB034 Brilliant Green Agar
LAB198 Raka-Ray No.3 Agar LAB046 Buffered Peptone Water - pre-enrichment broth
LAB199 Raka-Ray No.3 (Increased gel strength) LAB204 Buffered Peptone Water (ISO)
LAB079 W.L. Nutrient Agar LAB112 Campylobacter Agar (Blood Free - Improved)
LAB038 Wort Agar LAB135 Campylobacter Enrinchment Broth
LAB200 Yeast & Mould Agar CAP003 Captivate™ O26
CAP009 Captivate™ O45
Clostridia CAP005 Captivate™ O103
LAB160 Brazier’s CCEY Agar CAP004 Captivate™ O111
HP012 Columbia Agar (USP/EP/JP) CAP008 Captivate™ O121
LAB001 Columbia Agar Base CAP006 Captivate™ O145
LAB215 Columbia II Agar Base CAP001 Captivate™ O157
LAB220 DRCM (ISO) - Differential Reinforced Clostridial Medium LAB161 Sorbitol MacConkey Agar (SMAC)
LAB222 Iron Sulphite Agar LAB003 DCLS
LAB109 Perfringens Agar (O.P.S.P) LAB029 Desoxycholate Citrate Agar (DCA)
LAB194 Perfringens Agar (TSC) LAB065 Desoxycholate Citrate Agar (Hynes)
HP011 Reinforced Medium for Clostridia (USP/EP/JP) LAB537 Diassalm
LAB023 Reinforced Clostridial Medium Agar HAL008 Harlequin™ E. coli / Coliform Medium
HAL009 Harlequin™ mLGA
Coliform/Enterobacteriaceae HAL001 Harlequin™ Salmonella ABC Medium
LAB051 Brilliant Green Bile 2% Broth HAL006 Harlequin™ Sorbitol MacConkey Agar (SMAC-BCIG)
LAB091 E. E. Broth (Enterobacteriacae Enrichment Broth) HAL003 Harlequin™ TBGA
LAB060 Endo Agar LAB110 Hektoen Enteric Agar
LAB061 Eosin Methylene Blue Agar LAB116 MLCB Agar
HAL008 Harlequin™ E. coli/Coliform Medium LAB150 MSRV
HAL009 Harlequin™ mLGA LAB030 MacConkey Agar (with salt)
10
LAB002 MacConkey Agar (without salt) LAB202 Mueller Kaufmann Tetrathionate Novobiocin Broth
HP006 MacConkey Agar (USP/EP/JP) (MKTTn)
LAB216 MacConkey Agar No.2 LAB116 MLCB Agar
HP005 MacConkey Broth (USP/EP/JP) LAB165 O157 Broth (MTSB)
LAB202 Mueller Kaufmann Tetrathionate Novobiocin Broth (MKTTn) LAB147 Orange Serum Agar
LAB165 O157 Broth (MTSB) LAB148 Palcam Agar
PIN004 Pinnacle™mLGA Prepared
LAB144 Palcam Broth
PIN002 Pinnacle™ TBGA (TBX) Prepared
LAB193 PEMBA - Bacillus Cereus Medium
PIN005 Pinnacle™ Salmonella ABC Medium Prepared
LAB194 Perfringens Agar (TSC)
LAB086 Rappapport Vassiliadis Medium LAB109 Perfringens Agar (O.P.S.P.)
HP007 Rappapport Vassiliadis Medium (USP/EP/JP) PIN003 Pinnacle™ CSIM (ISO) Prepared
LAB209 Rhamnose MacConkey (VTEC O26) Agar PIN001 Pinnacle™ LCA Listeria Chromogenic Agar (ISO) Prepared
LAB052 S.S. Agar (Salmonella Shigella Agar) PIN002 Pinnacle™ TBGA (TBX) Prepared
LAB044 Selenite Broth PIN005 Pinnacle™ Salmonella ABC Medium Prepared
LAB055 Selenite Cystine Broth LAB149 Plate Count Agar

LAB096 TCBS Cholera Medium LAB010 Plate Count Agar A.P.H.A.
LAB097 Tetrathionate Broth Base LAB098 Potato Dextrose Agar
MPB001 μPREP™ BPW (ISO) LAB073 PREP Agar
LAB032 XLD Agar LAB108 Pseudomonas Agar
HP008 Xylose Lysine Deoxycholate Agar (USP/EP/JP) LAB086 Rappaport Vassiliadis Medium (Broth)
LAB120 Yersinia CIN Agar LAB023 Reinforced Clostridial Agar
LAB221 XLT4 Agar – Xylose Lysine Tergitol 4 Agar LAB209 Rhamnose MacConkey (VTEC O26) Agar
LAB055 Selenite Cystine Broth
Enterococci / Streptococci LAB161 Sorbitol MacConkey Agar
LAB028 Blood Agar Base LAB087 Sugar Free Agar
LAB015 Blood Agar Base No. 2 LAB097 Tetrathionate Broth Base A.P.H.A.
LAB207 Bile Aesculin Agar MPB001 μPREP™ BPW (ISO)
LAB001 Columbia Agar Base MPB004 μPREP™ Half Fraser Broth ISO (+FAC)
LAB215 Columbia II Agar Base LAB155 UVM Broth
LAB106 Kanamycin Aesculin Azide Agar LAB031 VRBA
LAB107 Kanamycin Aesculin Azide Broth LAB573 VRBA with MUG
LAB216 MacConkey Agar No.2 LAB088 VRBGA
LAB092 M17 Agar LAB079 W.L. Agar
PIN007 Pinnacle™ Blood Agar No. 2 (25ml ill) LAB038 Wort Agar
PIN006 Pinnacle™ Columbia Agar Base (25ml ill) LAB099 Wort Broth (Modiied)
LAB166 Slanetz and Bartley (m Enterococcus Medium) LAB032 XLD Agar
LAB035 TYC Medium LAB221 XLT4 Agar – Xylose Lysine Tergitol 4 Agar
LAB075 Todd Hewitt Broth
Harmonised Pharmacopoeia (USP/EP/JP)
Food Microbiology HP017 Buffered Sodium Chloride-Peptone Solution pH 7.0
(USP/ EP/JP)
LAB167 Aeromonas Agar HP016 Casein Soya Bean Digest Agar (USP/EP/JP)
LAB085 Baird-Parker Medium HP002 Casein Soya Bean Digest Broth (USP/EP/JP)
LAB285 Baird Parker Medium Base (ISO) HP010 Cetrimide Agar (USP/EP/JP)
LAB207 Bile Aesculin Agar HP012 Columbia Agar (USP/EP/JP)
LAB034 Brilliant Green Agar (Modiied) HP003 Enterobacteria Enrichment Broth – Mossel (USP/EP/JP)
LAB582 Buffered Listeria Enrichment BrothPLUS HP001 Fluid Thioglycollate Medium (USP/EP/JP)
LAB046 Buffered Peptone Water HP006 MacConkey Agar (USP/EP/JP)
LAB204 Buffered Peptone Water (ISO) HP005 MacConkey Broth (USP/EP/JP)
LAB112 Campylobacter (Blood Free - Improved) HP009 Mannitol Salt Agar (USP/EP/JP)
LAB135 Campylobacter Enrichment Broth HP015 Potato Dextrose Agar (USP/EP/JP)
CAP003 Captivate™ O26 HP007 Rappaport Vassiliadis Salmonella Enrichment Broth
CAP009 Captivate™ O45 (USP/EP/JP)
CAP010 Captivate™ O91 HP011 Reinforced Medium for Clostridia (USP/EP/JP)
CAP005 Captivate™ O103 HP014 Sabouraud Dextrose Agar (USP/EP/JP)
CAP007 Captivate™ O104 HP013 Sabouraud Dextrose Broth (USP/EP/JP)
CAP004 Captivate™ O111 HP004 Violet Red Bile Glucose Agar (USP/EP/JP)
CAP008 Captivate™ O121 HP008 Xylose Lysine Deoxycholate Agar (USP/EP/JP)
LAB081 CSEB - Cronobacter sakazakii Enrichment Broth Identiication Media
LAB218 DG18 Agar - Dichloran (18%) Glycerol Agar
LAB537 Diassalm LAB059 Kligler Iron Agar
LAB217 DRBC Agar - Dichloran Rose Bengal Chloramphenicol Agar LAB126 Lactose Broth
LAB220 DRCM (ISO) - Differential Reinforced Clostridial Medium LAB054 Lysine Iron Agar
LAB171 EC Medium (E. coli Medium) LAB104 Peptone Water
LAB164 Fraser Broth LAB069 Simmons Citrate Agar
LAB212 Fraser BrothPLUS LAB053 Triple Sugar Iron Agar
LAB211 Half Fraser BrothPLUS LAB129 Tryptone Water
HAL013 Harlequin™ CSA-DFI - Cronobacter sakazakii Agar DFI Formulation LAB130 Urea Agar
HAL012 Harlequin™ CSIM (ISO) LAB131 Urea Broth
HAL008 Harlequin™ E.coli/Coliform Medium
HAL010 Harlequin™ Listeria Chromogenic Agar Lactic Acid Bacteria
HAL001 Harlequin™ Salmonella ABC Medium LAB092 M17 Agar
HAL006 Harlequin™ Sorbitol MacConkey Agar (SMAC-BCIG) LAB093 MRS Agar - de Man, Rogosa and Sharpe Agar
HAL003 Harlequin™ Tryptone Bile Glucuronide Agar (TBGA) LAB223 MRS Agar (ISO)
LAB106 Kanamycin Aesculin Azide Agar LAB094 MRS Broth
LAB107 Kanamycin Aesculin Azide Broth LAB147 Orange Serum Agar
LAB222 Iron Sulphite Agar LAB198 Raka-Ray No.3 Agar
LAB196 Lauryl Tryptose Broth LAB199 Raka-Ray No.3 (Increased gel strength)
LAB138 Listeria Enrichment Broth
LAB139 Listeria Enrichment Broth (Buffered)
LAB589 LEE Broth - Listeria Express Enrichment Broth Listeria
LAB122 Listeria Isolation Medium (Oxford)
LAB206 Listeria Isolation Media LAB139 Buffered Listeria Broth
LAB172 Listeria Monocytogenes Blood Agar (LMBA) LAB582 Buffered Listeria Enrichment BrothPLUS
LAB216 MacConkey Agar No.2 LAB212 Fraser BrothPLUS
LAB077 ColourScreen™ MLSTB-MT (ISO) Modiied Lauryl Sulphate Tryptose LAB164 Fraser Broth Base
Broth with MUG & Tryptophan LAB211 Half Fraser BrothPLUS
LAB219 Modiied Giolitti and Cantoni Broth (ISO) HAL010 Harlequin™ Listeria Chromogenic Agar
LAB093 MRS Agar LAB138 Listeria Enrichment Broth
LAB223 MRS Agar (ISO) LAB589 LEE Broth - Listeria Express Enrichment Broth
LAB094 MRS Broth LAB122 Listeria Isolation Medium (Oxford)
LAB150 MSRV
11
LAB206 Listeria Isolation Media LAB195 BCYE Legionella Isolation Medium
LAB172 Listeria Monocytogenes Blood Agar (LMBA) LAB046 Buffered Peptone Water
LAB148 PALCAM Agar Base LAB001 Columbia Agar Base
LAB144 PALCAM Broth LAB537 Diagnostic Semi Solid Salmonella Agar (Diassalm)
MPB004 μPREP™ Half Fraser Broth ISO (+FAC) LAB220 DRCM (ISO) - Differential Reinforced Clostridial Medium
LAB155 UVM Broth Base LAB171 EC Medium
PIN001 Pinnacle™ LCA (Listeria Chromogenic Agar) LAB060 Endo Agar Base
LAB061 Eosin Methylene Blue Agar (Levine)
LAB195 GVPC Legionella Isolation Medium (BCYE basal agar)
Neutralising HAL009 Harlequin™ mLGA
LAB188 D/E Neutralising Agar HAL006 Harlequin™ SMAC-BCIG
LAB187 D/E Neutralising Broth LAB110 Hektoen Enteric Medium
LAB186 D/E Neutralising Broth Base LAB106 Kanamycin Aesculin Azide Agar
LAB185 Letheen Agar (AOAC) LAB126 Lactose Broth
LAB184 Letheen Broth (AOAC) LAB196 Lauryl Tryptose Broth
LAB189 Microbial Content Test Agar LAB054 Lysine Iron Agar
LAB005 MacConkey Broth (Purple)
Nutrient Media for general use LAB002 MacConkey Agar (Without Salt)
LAB030 MacConkey Agar (With Salt)
LAB048 Brain Heart Infusion Agar LAB216 MacConkey Agar No.2
LAB049 Brain Heart Infusion Broth LAB045 MacConkey Agar No 3
LAB525 Eugon Agar LAB103 Maximal Recovery Diluent
LAB526 Eugon Broth LAB082 Membrane Lauryl Sulphate Broth
LAB008 Nutrient Agar LAB080 Minerals Modiied Glutamate Broth
LAB068 Nutrient Broth ‘E’ LAB008 Nutrient Agar
LAB014 Nutrient Broth No. 2 LAB014 Nutrient Broth No. 2
LAB062 Tryptose Phosphate Broth LAB165 O157 Broth (MTSB)
LAB018 Yeast Extract Agar LAB109 Perfringens Agar (O.P.S.P)
LAB194 Perfringens Agar (TSC)
Sensitivity Testing LAB010 Plate Count Agar A.P.H.A
PIN009 Pinnacle™ BCYE Legionella Medium (ISO) Prepared
LAB039 Mueller Hinton Agar PIN008 Pinnacle™ GVPC Legionella Medium (ISO) Prepared
LAB114 Mueller Hinton Broth PIN004 Pinnacle™ mLGA Prepared
LAB012 Sensitivity Test Agar LAB108 Pseudomonas Agar Base

LAB170 Susceptibility Test (Iso) Agar LAB163 R2A Medium
LAB203 R2A Broth
Staphylococci LAB086 Rappaport-Vassiliadis Medium (RVS)
LAB100Z Ringer’s Solution (1/4 Strength) Tablets
LAB085 Baird-Parker Medium LAB044 Selenite Broth
LAB285 Baird Parker Medium Base (ISO)
LAB095 DN’ase Test Agar LAB166 Slanetz & Bartley Medium (Membrane Enterococcus Agar)
LAB161 Sorbitol MacConkey Aga
LAB007 Mannitol Salt Agar LAB096 T.C.B.S. Cholera Medium
HP009 Mannitol Salt Agar (USP/EP/JP)
LAB219 Modiied Giolitti and Cantoni Broth (ISO) LAB097 Tetrathionate Broth Base APHA
LAB053 Triple Sugar Iron Agar
LAB192 ORSIM - Oxacillin Resistant Staphylococci Isolation LAB129 Tryptone Water
Medium
LAB131 Urea Broth Base
LAB197 Water Plate Count Agar (ISO)
Streptococci / Enterococci LAB032 XLD Agar
LAB028 Blood Agar Base LAB221 XLT4 Agar – Xylose Lysine Tergitol 4 Agar
LAB015 Blood Agar Base No. 2 LAB018 Yeast Extract Agar
LAB207 Bile Aesculin Agar
LAB001 Columbia Agar Base Yeasts and Moulds
LAB215 Columbia II Agar Base
LAB106 Kanamycin Aesculin Azide Agar LAB117 DTM Dermatophyte Test Medium
LAB107 Kanamycin Aesculin Azide Broth LAB218 DG18 Agar - Dichloran (18%) Glycerol Agar
LAB216 MacConkey Agar No.2 LAB217 DRBC Agar - Dichloran Rose Bengal
LAB092 M17 Agar Chloramphenicol Agar
LAB166 Slanetz & Bartley Medium (Membrane Enterococcus Agar) LAB201 Lysine Agar
LAB035 TYC Medium LAB037 Malt Extract Agar
LAB075 Todd Hewitt Broth LAB159 Malt Extract Broth
LAB089 OGYE Agar
LAB098 Potato Dextrose Agar
Sterility Test Media HP015 Potato Dextrose Agar (USP/EP/JP)
HP002 Casein Soya Bean Digest Broth (USP/EP/JP) LAB036 Rose Bengal Chloramphenicol Agar
HP001 Fluid Thioglycollate (USP/EP/JP) LAB009 Sabouraud Dextrose Agar
LAB425 Fluid Thioglycollate Medium (Clear) HP014 Sabouraud Dextrose Agar (USP/EP/JP)
LAB159 Malt Extract Broth HP013 Sabouraud Dextrose Broth (USP/EP/JP)
LAB014 Nutrient Broth No. 2 LAB111 Sabouraud Maltose Agar
HP013 Sabouraud Dextrose Broth (USP/EP/JP) LAB079 W.L. Nutrient Agar
LAB011 Tryptone Soy Agar LAB038 Wort Agar
LAB004 Tryptone Soy Broth LAB099 Wort Broth (Modiied)
LAB205 Tryptone Soy Broth (without dextrose) LAB119 Yeast Extract Dextrose Chloramphenicol Agar
LAB200 Yeast & Mould Agar
LAB175 YPD Broth
LAB176 YPD Agar
Total Viable Counts
LAB019 Milk Agar
LAB115 Milk Plate Count Agar
LAB010 Plate Count Agar A.P.H.A.
LAB149 Plate Count Agar
LAB163 R2A Medium
LAB063 Tryptone Glucose Extract Agar A.P.H.A.
LAB011 Tryptone Soy Agar
LAB197 Water Plate Count Agar (ISO)
LAB018 Yeast Extract Agar
Water Testing
LAB224 Alkaline Saline Peptone Water (ISO)
LAB085 Baird Parker Medium Base
LAB207 Bile Aesculin Agar
LAB013 Bismuth Sulphite Agar
LAB048 Brain Heart Infusion Agar
LAB034 Brilliant Green Agar (Modiied)
LAB051 Brilliant Green Bile 2% Broth
12
An alternative method is to inoculate triple sugar iron tubes.
1. Dehydrated Culture • Aeromonas will typically produce an acid butt (yellow) and an
alkaline or unchanged slant (red).
Media • Pseudomonas spp. will remain unchanged in both the butt and
slant.
To fully identify colonies as Aeromonas spp. the above tests should be
Aeromonas Agar supported using a proprietary kit such as API 20NE or Microbact 24E
Bile Salt Irgasan Brilliant Green Agar (other products may be available).
LAB167 Interpretation
Organism Size Shape Colour
Description
Aeromonas Agar is a highly selective medium for the isolation of Aeromonas spp.* 0.5-3.0 CV.E.G Translucent pale green
Aeromonas spp. from food, clinical and environmental samples. Pseudomonas spp. 0.5-1.0 CV.E.G Translucent pale green
Based on the selective agents brilliant green and irgasan, this medium
S.aureus No growth
will not inhibit those strains of Aeromonas spp. sensitive to ampicillin
used in other media. E. coli No growth
Typical Formula g/litre * The selective nature of the medium may mean occasional strains do not
grow, or grow poorly.
Beef Extract 5.0
Meat Peptone 5.0
Xylose 10.0 Alkaline Saline Peptone Water (ISO)
Bile Salts No.3 8.5 Alkaline Peptone Water, Alkaline Saline Water
Sodium thiosulphate 5.44

Irgasan 0.005
LAB224
Brilliant green 0.005 Description
Neutral red 0.025 Alkaline Saline Peptone Water (ISO) is a medium for the enrichment
of Vibrio spp. from food and water samples according to ISO
Agar 11.5 21872:2007.
Grams per litre 45.5 Originally described by Shread, Donovan & Lee as an enrichment
broth for Aeromonas spp. and identiied by Cruickshank as an
Appearance: effective medium for the enrichment of Vibrio spp., Alkaline Saline
Peptone Water uses elevated pH and salt levels to provide a favourable
Powder: ine, free-lowing, homogeneous, buff environment for the enrichment of Vibrio spp.
Finished medium: clear, purple gel Peptone provides essential vitamins, minerals, amino acids &
nitrogen for growth requirements. Sodium chloride provides essential
pH: 7.0 ± 0.2
electrolytes for maintenance of the osmotic balance.
Method for reconstitution
Typical Formula g/litre
Weigh 45.5 grams of powder and disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix and sterilise by bringing Peptone 20.0
to the boil. Cool to 47°C and mix well before dispensing into Petri
dishes. Dry the agar surface prior to use. Sodium chloride 20.0
Grams per litre 40.0
Inoculation:
Faecal specimens: Inoculate surface of medium directly, spreading for
single colonies. Appearance:
Samples requiring enrichment: Inoculate alkaline peptone water and Powder: ine, free-lowing, homogenous, buff
incubate at 37°C for 18-24 hr. Subculture onto Aeromonas Agar, Finished medium: clear, very pale straw coloured liquid
surface spreading for single colonies.
pH: 8.6 ± 0.2
Incubation: Incubate plates aerobically at 37°C for 18-24 hr. Examine
for typical colonies and conirm as Aeromonas spp. Hazard classiication: NR – Not regulated
o
Storage: Dehydrated culture media: 10-25 C away from direct Method for reconstitution
sunlight. Disperse 40 grams of powder in 1 litre of deionised water. Allow to
Prepared media: 7 days at 2-8°C in the dark (may be extended if soak for 10 minutes, swirl to mix, and dispense into inal containers.
moisture tight packaging used). Sterilise by autoclaving at 121°C for 15 minutes.
Storage: Dehydrated culture media: 10-25°C.

Minimum Q.C. organisms: Aeromonas hydrophila Final medium: 7 days 15-20°C
WDCM 00063
E. coli WDCM 00013 Inoculation & Incubation – as per ISO 21872
(inhibited)
Primary enrichment
Add test sample to 9ml prepared medium.
Conirmation
Typical colonies (translucent, pale green colonies 0.5-3.0mm For large quantities, it may be necessary to pre-heat the prepared
diameter) should be conirmed as presumptive Aeromonas spp. by medium to 37°C before inoculation with the test portion.
performing an oxidase test and inoculating into Hugh & Leifsons O/F Incubate the initial suspension as follows:
medium.
ISO 21872-1
• Aeromonas spp. will give a positive oxidase reaction and • 37°C for 6 hours ± 1 hour for deep-frozen products
demonstrate both oxidative and fermentative metabolism. • 41.5°C for 6 hours ± 1 hour for fresh, dried or salted products
• Pseudomonas spp. will also be oxidase positive, but do not ISO 21872-2
possess fermentative metabolism.
• 37°C for 6 hours ± 1 hour
13 Dehydrated Culture Media

LAB224 Minimum Q.C. organisms: B. cereus WDCM 00001
E. coli (inhibition) WDCM 00013
Secondary enrichment
Transfer 1ml of primary enrichment culture (after incubation) into Storage of Prepared Medium: – up to 7 days at 2-8˚C in the dark.
10ml prepared medium. Inoculation: Surface, spreading or streaking for single colonies.
Incubate the initial suspension as follows: Incubation: 30˚C aerobically for 24-48 hours.
ISO 21872-1
Growth Characteristics
• 41.5°C for 18 hours ± 1 hour
colony size shape &
ISO 21872-2 organism (mm) surface colour
• 37°C for 18 hours ± 1 hour B. cereus 3.0-4.0 F.CR.D. Pink, white halo
Sub-culture B. subtilis 2.0-3.0 F.CR.D. Yellow

B. licheniformis 2.0 F.Rz.D. Yellow
Using a loop, sub-culture both the primary enrichment and secondary
E. coli no growth
enrichments onto LAB096 Cholera TCBS Medium
S. aureus 1.0 CV.E.G. Yellow (white halo)
Minimum Q.C. organisms:
V.cholerae WDCM 00136
References
V.parahaemolyticus WDCM 00037
E.coli WDCM 00013 Inal, T.: Vergleictiende Untersuchungen über die Selektivmedien
zum qualitativen und quantitativen Nachweis von Vacillus cereus in
Lebensmitteln.
References
Mitteilung I. : Fleischwritsch, 51: 1629-1632 (1971). IV. Mitteilung:
Cruickshank R. (1968) Medical Microbiology. 11th ed. Livingstone Fleischwritsch, 52: 1160-1162 (1972).
Ltd, London, UK. Mossel, D.A.A., Koopman, M.J. and Jongerius, E. (1967).
ISO/TS 21872-1:2007 Microbiology of food and animal feeding stuffs Enumeration of Bacillus cereus in foods. Appl. Microbiol. 15: 650-
653. Thatcher, F.S., Clarke, D.S. (1978) Micro-organisms in foods.
– Horizontal method for the detection of potentially enteropathogenic Volume 1 second edition. University of Toronto.
Vibrio spp. – Part 1: Detection of Vibrio parahaemolyticus and Vibrio
BS5763 Part 1L:1994. ISO7932:(1993) 3/100
cholerae.
ISO/TS 21872-2:2007 Microbiology of food and animal feeding
stuffs – Horizontal method for the detection of potentially
enteropathogenic Vibrio spp. – Part 1: Detection of species other than
Baird-Parker Medium Base
Vibrio parahaemolyticus and Vibrio cholerae.
LAB085
Shread, P., Donovan, T.J. and Lee, J.V. (1991). Soc. Gen. Microbiol.
Q. 8. 184. Description
Originally introduced in 1962, this medium was developed by
Baird-Parker to overcome the problems of recovering damaged
Bacillus Cereus Medium Staphylococcus aureus from foodstuffs.
Baird-Parker medium is highly selective by nature, due to the
Phenol Red Egg Yolk Polymyxin Agar (P.R.E.P.) presence of potassium tellurite and lithium chloride. Tellurite inhibits
Mannitol Egg Yolk Polymyxin Agar most coliforms and is also reduced to telluride by S. aureus, giving the
typical black colonies. Glycine and sodium pyruvate are both used as
growth factors by staphylococci while the pyruvate also neutralises
LAB073 any toxic peroxides that may be formed.
Description Unlike some commercially available preparations, Lab M Baird-
Introduced by Mossel and his co-workers in 1967 for the enumeration Parker Medium can be used with either Egg Yolk Tellurite (X085) or
of Bacillus cereus in foods, this formula was shown to be the most Rabbit Plasma Fibrinogen (X086).
effective for this purpose by Inal in 1972. Two reactions on this When Baird-Parker medium is used with Egg Yolk Tellurite X085,
medium differentiate B. cereus from other members of the Bacillus presumptive S. aureus appear as black colonies demonstrating
group, these are mannitol fermentation and lecithinase production. lecithinase activity (an opaque zone around the colony) and lipase
Mannitol fermentation on this medium produces a yellow colour, activity (a zone of clearing encircling the opaque zone). Suspected S.
B. cereus is mannitol negative and produces red colonies. The aureus colonies should be conirmed with RPF for coagulase or latex
lecithinase production of B. cereus is indicated by a white precipitate agglutination test.
around the colonies. Polymyxin is added to suppress coliforms but
some Proteus spp and Gram positive cocci may grow through. Rabbit plasma ibrinogen (RPF X086) is a more speciic alternative to
egg yolk tellurite and allows the direct detection of coagulase-positive
Typical Formula g/litre S. aureus. Typical S. aureus appear as black colonies surrounded
by a zone of precipitation (demonstrating coagulase activity). This
Beef Extract 1.0 is recognised as the gold standard method for the identiication of
S.aureus. RPF overcomes any issues with atypical colony forms and
Balanced Peptone No. 1 10.0 its use means further conirmatory tests are not necessary.
D-Mannitol 10.0
Sodium chloride 10.0
Tryptone 10.0
Phenol red 0.025
Beef Extract 7.5
Agar No. 1 15.0
Yeast Extract 1.0
Method for reconstitution Lithium chloride 5.0
Weigh 46 grams of powder, disperse in 900ml of deionised water. Glycine 12.0
Allow to soak for 10 minutes, swirl to mix then sterilise by autoclaving
at 121˚C for 15 minutes. Cool to 47˚C and aseptically add 100ml of Sodium pyruvate 10.0
X073 egg yolk emulsion and 2 vials of X074 Polymyxin. Agar No. 2 20.0
Appearance: Pink, opaque gel.
pH: 7.2 ± 0.2
Dehydrated Culture Media 14

Method for reconstitution References
For Baird-Parker Medium LAB085 with Egg Yolk Tellurite Baird-Parker, A.C. (1962). An improved diagnostic and selective
X085 medium for isolating coagulase positive staphylococci. J. Appl. Bact.
25(1): 12-19.
Allow to soak for 10 minutes, swirl to mix and sterilise at 121oC for Baird-Parker, A.C. and Davenport, E. (1965). The effect of Recovery
15 minutes. Cool to 47°C and add 5% (50mL) X085. Mix well before medium on the isolation of S. aureus after heat treatment and after
aseptically pouring into sterile Petri dishes. Dry the agar surface prior storage of frozen or dried cells. J. Appl. Bact 28: 390-402.
to use. Ten Broeke, R. (1976). The Staphylococcus medium of Baird-Parker
in practical use. The occurrence of coagulase-positive, egg yolk
For Baird-Parker Medium LAB085 with Rabbit Plasma nonclearing staphylococci. Antonie van Leeuwenhoek 33: 220-236.
Fibrinogen (RPF) Supplement X086 Smith, B.A. and Baird-Parker, A.C. (1964). The use of sulphamethazine
Weigh 6.55 grams of powder and disperse in 90mL of deionised water. for inhibiting Proteus spp. on Baird-Parker’s isolation medium for
Allow to soak for 10 minutes, swirl to mix and sterilise at 121oC for Staphylococcus aureus. J. Appl. Bact 27(1): 78-82.
15 minutes. Cool to 47°C and add 1 vial of reconstituted X086. Mix Beckers N J. et al (1984). Canad. J. Microbiol. 30: 470-474.
well before aseptically pouring into sterile Petri dishes. Dry the agar Sawhney D. (1986) J. Appl Bact. 61:149-155.
surface prior to use.
Appearance:
Powder: ine, free-lowing, homogeneous, buff Baird-Parker Medium Base (ISO)
Final medium: opaque cream/pale fawn gel (with X085)
translucent, pale straw gel (with X086) LAB285
pH: 6.8 ± 0.2
Description
For the isolation of coagulase-positive staphylococci. Formulated
Minimum Q.C. organisms: to ISO 6888-1 and compliant to ISO 6888-2 and ISO 6888-3.
Staphylococcus aureus WDCM 00034 Originally introduced in 1962, this medium was developed by
Staphylococcus saprophyticus WDCM 00159 Baird-Parker to overcome the problems of recovering damaged
Escherichia coli WDCM 00013 Staphylococcus aureus from foodstuffs. This version of the medium
is formulated according to ISO 6888-1:1999+A1:2003 and is in
Storage of Prepared Medium: Dehydrated culture media: 10-25oC compliance with ISO 6888-2:2003+A1:2003 and ISO 6888-3:2003.
Poured plates: LAB085+X085 upto 3 days at 2-8°C in the dark; Baird-Parker medium is highly selective by nature, due to the
LAB085+X086 use on day of preparation. presence of potassium tellurite and lithium chloride. Tellurite inhibits
Inoculation: Surface inoculation. most coliforms and is also reduced to telluride by S. aureus, giving the
typical black colonies. Glycine and sodium pyruvate are both used as
Incubation: growth factors by staphylococci while the pyruvate also neutralises
LAB085+X085: 37oC aerobically for 48 hours. any toxic peroxides that may be formed.
LAB085+X086: 37o C aerobically for 24-48 hours.
As with Lab M’s traditional Baird-Parker Medium, LAB085, and
Growth characteristics(with X085) unlike some commercially available preparations, the new ISO
formulated Baird-Parker medium can be used with either Egg Yolk
colony size shape &
Tellurite (X085) or Rabbit Plasma Fibrinogen (X086).
organism (mm) surface colour other
When Baird-Parker medium is used with Egg Yolk Tellurite X085,
S. aureus 1.0-3.0 CV.E.G. Black Narrow opaque
presumptive S. aureus appear as black colonies demonstrating
margin surrounded
lecithinase activity (an opaque zone around the colony) and lipase
by a zone of
activity (a zone of clearing encircling the opaque zone). Suspected S.
clearing
aureus colonies should be conirmed with RPF for coagulase or latex
S. saprophyticus 0.5-2.0 CV.E.G. Black (poor growth) agglutination test.
Other Rabbit plasma ibrinogen (RPF X086) is a more speciic alternative to
Coagulase 0.5-1.0 CV.E.G. Black (no growth) egg yolk tellurite and allows the direct detection of coagulase-positive
negative S. aureus. Typical S. aureus appear as black colonies surrounded
staphylococci by a zone of precipitation (demonstrating coagulase activity). This
Proteus spp. 0.5-2.0 F.Rz.G Brown (no growth)
is recognised as the gold standard method for the identiication of
Black S.aureus. RPF overcomes any issues with atypical colony forms and
its use means further conirmatory tests are not necessary.
Bacillus spp. 0.5-1.0 F.Rz.D. Brown (no growth)
Entero- no growth
bacteriaceae Pancreatic digest of casein 10.0
Yeast extract 1.0
Growth characteristics(with X086) Meat extract 5.0
colony size shape &
organism (mm) surface colour other Sodium pyruvate 10.0
Coagulase 1.0-3.0 CV.E.G. White Narrow opaque L-Glycine 12.0
positive . Grey zone of coagulase
S. aureus . Black activity
Lithium chloride 5.0
Coagulase 0.5-2.0 CV.E.G. White (poor growth) Agar 20.5
negative . Grey
staphylococci Black Method for reconstitution
Proteus spp. 0.5-2.0 F.Rz.G Brown (no growth) For Baird-Parker Medium LAB285 with Egg Yolk Tellurite X085
Black
Bacillus spp. 0.5-1.0 F.Rz.D. Brown (no growth)
Allow to soak for 10 minutes, swirl to mix and sterilise at 121°C for
Entero- no growth 15 minutes. Cool to 48°C and add 5% (50mL) X085. Mix well before
bacteriaceae aseptically pouring into sterile Petri dishes. Dry the agar surface prior
to use. Sulphamezathine may be added at 0.05g/L to suppress the
swarming of Proteus spp.
For Baird-Parker Medium LAB285 with Rabbit Plasma Fibrinogen
(RPF) Supplement X086
Weigh 6.35 grams of powder and disperse in 90mL of deionised water.
Allow to soak for 10 minutes, swirl to mix and sterilise at 121°C for
15 minutes. Cool to 48°C and add 1 vial of reconstituted X086. Mix
well before aseptically pouring into sterile Petri dishes. Dry the agar

LAB285 BCYE Legionella Isolation Medium
Appearance: LAB195
Powder: ine, free-lowing, homogeneous, buff
Description
Final medium: opaque cream yellow gel (with X085) clear, straw gel BCYE (Buffered Charcoal Yeast Extract) Legionella Isolation
(with X086) Medium (LAB195) is a base medium used for the isolation of
Legionella from clinical and environmental samples. This medium
pH: 7.2 ± 0.2 is based on the charcoal yeast extract formulation of Feeley et al.1&2
The performance of this medium is further enhanced by the additions
Minimum Q.C. organisms: of ACES (N-2-acetamido-2-aminoethane - sulphonic acid) buffer
Staphylococcus aureus WDCM 00034 and α-ketoglutarate as deined by Edelstein 3. This medium is also
Staphylococcus saprophyticus WDCM 00159 detailed in internationally recognized methodology 4 for the isolation
Escherichia coli WDCM 00013 of Legionella spp. from water.
Specimens or samples are often heavily contaminated with other
Storage: bacteria and consequentially a range of selective supplements have
been developed to aid isolation. Lab M provide the GVPC supplement
Dehydrated culture media: 10-25°C
(X195) which is most effective for the isolation of L. pneumophila.
Poured plates: LAB285+X085 upto 3 days at 2-8°C in the dark; It is recommended that this supplement is used in conjunction with
LAB285+X086 use on day of preparation. heat and acid sample treatments, to further reduce the growth of non-
Inoculation: Legionella bacteria.
LAB285+X085: surface inoculation as per user’s validated methods. This product contains the ACES buffer and ferric pyrophosphate in
LAB285+X086: surface inoculation or pour plate as per user’s the base medium. This negates the need for complex freeze dried
validated methods. supplements. A complementary growth supplement is provided (X196)
which contains the L-cysteine and α-ketoglutarate. In addition, an
Incubation: α-ketoglutarate supplement (X197) is also available for the preparation
of conirmatory media for suspected Legionella colonies.
LAB285+X085: 37°C aerobically for 48 hours.
LAB285+X086: 37°C aerobically for 24-48 hours. Principle of isolation
Interpretation: Water samples are concentrated either by membrane iltration or
LAB285+X085: Presumptive S. aureus colonies appear as black centrifugation (turbid samples may also be centrifuged). To reduce the
colonies demonstrating lecithinase activity and lipase activity. All growth of unwanted bacteria, separate portions of the concentrated
black colonies (suspected S. aureus) should be conirmed with a sample may be subjected to heat and acid treatments. Treated and
coagulase test (RPF) or a latex agglutination kit. untreated portions are then inoculated onto Legionella selective
media.
LAB285+X086: Typical S. aureus appear as black colonies surrounded
by a zone of coagulase activity. Typical Formula g/litre
References Yeast Extract 10.0
ISO 6888-1:1999+A1:2003 Microbiology of food and animal feeding
stuffs - Horizontal method for the enumeration of coagulase-positive Charcoal 2.0
staphylococci (Staphylococcus aureus and other species) - Part 1: Ferric Pyrophosphate 0.25
Technique using Baird-Parker agar medium (includes amendment
A1:2003). ACES Buffer 10.0
ISO 6888-2:1999+A1:2003 Microbiology of food and animal feeding
stuffs - Horizontal method for the enumeration of coagulase-positive Potassium Carbonate 2.28
staphylococci (Staphylococcus aureus and other species) - Part 1: Agar 14.0
Technique using rabbit plasma ibrinogen agar medium (includes
amendment A1:2003).
ISO 6888-3:2003 Microbiology of food and animal feeding stuffs Supplements
- Horizontal method for the enumeration of coagulase-positive
staphylococci (Staphylococcus aureus and other species) - Part 3: BCYE Growth Supplement (X196)
Detection & MPN technique for low numbers.
Typical Formula
Baird-Parker, A.C. (1962). An improved diagnostic and selective
medium for isolating coagulase-positive staphylococci. J. Appl. Bact. L-Cysteine 400mg
25(1):12-19.
α-ketoglutarate 1000mg
Smith, B.A. and Baird-Parker, A.C. (1964). The use of sulphamezathine
for inhibiting Proteus spp. on Baird-Parker’s isolation medium for
Staphylococcus aureus. J. Appl. Bact. 27(1):78-82
Presumptive ID (X197)
Typical Formula
Add one vial per 500mL of sterilised medium as appropriate.

Method for reconstitution Storage:
Maintenance (BCYE) Dehydrated culture media: 10-25ºC.
Weigh 38.5 grams of powder and disperse in 1 litre of deionised Poured plates: 7 days at 2-8°C in the dark.
water. Soak for 10 minutes, swirl to mix and sterilize by autoclaving Inoculation: Surface inoculation as per user’s validated methods.
at 110ºC for 10 minutes. Cool to 47ºC and aseptically add 2 vials of
reconstituted growth supplement X196. Mix well and pour into Petri Incubation: Incubate at 37°C for 18-24 hours.
dishes.
Presumptive Identiication (BCYE no L-Cysteine) Interpretation
Weigh 38.5 grams of powder and disperse in 1 litre of deionised colony size
water. Soak for 10 minutes, swirl to mix and sterilize by autoclaving organism (mm) observations
at 110ºC for 10 minutes. Cool to 47ºC and aseptically add 2 vials of
Enterococcus spp. 0.1 - 0.3 Blackening of media around colony
reconstituted growth supplement X197. Mix well and pour into Petri
dishes. Enterobacter spp. 1-2 Blackening of media around colony
pH: 6.9 ± 0.1 Pseudomonas aeruginosa 0.3 - 0.6
Inoculation: Escherichia coli 1.5 - 2.5
Surface inoculation, streak for single colonies.
Incubation: Stahpylococcus aureus 0.5
Incubate at 36 + 1ºC in a humid atmosphere under aerobic conditions Streptococcus pyogenes Inhibited
for up to 10 days.
Interpretation:
Typical morphology should be regarded as presumptive Legionella. Bismuth Sulphite Agar
Presumptive isolates should be conirmed using a serological method,
(Wilson and Blair Medium)
e.g. Microgen M45 Latex.
LAB013A + LAB013B
Legionella spp. - Growth Description
A modiication of Wilson and Blair’s original medium for the isolation
References of Salmonella typhi and other Salmonella from clinical samples,
Feeley, J.C., Gibson, R.J. et al. (1979). Journal of Clinical sewage and other materials. The presence of bismuth sulphite and
Microbiology 10: 437-441 brilliant green make this medium highly selective. As the medium
contains neither lactose nor sucrose it can be used to detect lactose
Pesculle, A.H., Feeley, J.C. et al. (1980). Journal of Infectious Disease and sucrose fermenting Salmonella.
141: 727-732
Edelstein, P.H. (1982). Journal of Clinical Microbiology 14: 298-303 Typical Formula g/litre
International Standard. ISO 11731:1998(E). Water Quality – Detection Bismuth Sulphite Agar Base ‘A’ LAB013A
& Enumeration of Legionella.
Beef Extract 6.0
Balanced Peptone No. 1 10.0
Bile Aesculin Agar Ferric citrate BPC 0.4
Brilliant Green 0.01
LAB207
Agar No. 2 20.0
Description Bismuth Chemical Mixture ‘B’ LAB13B
For the isolation and presumptive identiication of Enterococci / Bismuth ammonium citrate 3.0
Group D Streptococci. The aesculin produced by organisms positive
for aesculin hydrolysis reacts with ferric citrate to form a dark brown Sodium sulphite 5.0
or black complex. Bile salts inhibit Gram-positive organisms other
than Enterococci or Group D Streptococci. This medium can also be Disodium phosphate 5.0
used for presumptive differentiation of the Klebsiella-Enterobacter- Glucose 5.0
Serratia group from other Enterobacteriaceae.
Typical Formula g/litre Method for reconstitution

Agar Base ‘A’: Weigh 36.4 grams of powder and mix with 1 litre
Peptone 8.0 of deionised water. Sterilise for 15 minutes at 121˚C. Cool to 50˚C
Bile salts 20.0 approx. and add 100ml of Chemical Mixture ‘B’. Mix well and
pour thin plates. Store at 4˚C for 3 days to mature, before use.
Ferric citrate 0.5 Chemical Mixture ‘B’: Suspend 18 grams of powder in 100ml of
Aesculin 1.0 deionised water. Bring to boil over a tripod and gauze, and cool
quickly in cold water. Add to 1 litre of Agar Base ‘A’ prepared as
Agar 15.0 above.
Appearance: Pale green, opaque gel.
Weigh 44.5 grams of powder and disperse in 1 litre of deionised pH: 7.6 ± 0.2
water. Allow to soak for 10 minutes, swirl to mix and sterilise by
autoclaving for 15 minutes at 121ºC. Cool to 47°C, mix well and Minimum Q.C. organisms: Salmonella sp. WDCM 00031
dispense into Petri dishes.. E. coli (inhibition) WDCM 00013
Appearance: Storage of Prepared Medium: Plates – store 3 days before use.
Use within 7 days. Store at 2-8˚C in the dark.
Powder: ine, free-lowing, homogeneous, buff.
Inoculation: Surface, streak out to single colonies.
Finished medium: Buff/pale brown gel.
Incubation: 37˚C for 24 hours aerobically.
pH: 7.1 ± 0.2
Hazard classiication
Xi – Irritant

Enterococcus faecalis WDCM 00087
Enterobacter aerogenes WDCM 00175
Streptococcus pyogenes

Growth characteristics Growth characteristics
colony size shape & colony size shape &
organism (mm) surface colour other organism (mm) surface colour other
S. typhi 1.5-2.0 CV.E.G. Black Metallic sheen S. aureus 0.5-1.5 CV.E.G. White- haemolytic
black deposit in Golden
medium. (H2S-ve S. pyogenes P.P.-1.0 CV.E.G. Grey beta haemolytic
strains green) alpha haemolytic
Other non-haemolytic
Salmonella spp. 1.0-2.5 CV.E.G. Black/ Metallic sheen S. pneumoniae P.P.-1.0 F.E.G. Grey alpha haemolytic
Green especially in draughtsman
heavy growth,
single colonies N. meningitidis P.P.-1.5 CV.E.G. Grey mucoid
may give rabbit
eye appearance E. coli 1.5-2.5 CV.E.G. Grey haemolytic
Ps. aeruginosa 0.5-3.0 F.CR.D. Grey many colonial
E. coli P.P.-1.0 CV.E.G. Green
forms
Klebsiella spp. P.P-2.0 CV.E.G. Green green pigment
Citrobacter spp. 1.0-2.5 CV.E.G. Green (black centre)
B. fragilis 0.5-1.5 CV.E.G. Grey mucoid
Proteus spp. 1.0-2.5 CV.E.G. Green / (black centre)
Brown
References
References Cruikshank, R. (1972). Medical Microbiology. 11th edn. Livingstone,
London
Wilson, W.J. and Blair, E.M. M’V (1926). A combination of bismuth
and sodium sulphites affording an enrichment and selective medium
for the typhoid-paratyphoid groups of bacteria. J. Pathol. Bacteriol.,
29: 310-311.
International Journal of Food Microbiology (1987) 5:3:200-202. Blood Agar Base No. 2
I.C.M.S.F. (1978) Micro organisms in Foods I. Their signiicance
and enumeration. 2nd edition Univ of Toronto Press. Speck M.L.
Compendium of methods for microbiological examination of foods. LAB015
(1984) 2nd edition. American Public Health Association, Washington.
3/102 Description
A very rich agar base which, with the addition of blood, is capable
of growing delicate clinical pathogens. The medium gives colonial
appearances, haemolysis patterns and pigment production of
Blood Agar Base diagnostic value. When the blood is ‘chocolated’ the medium gives
good recovery of Haemophilus spp. The medium can be made
LAB028 selective for various groups by the addition of appropriate antibiotic
mixtures eg:
Description Streptococci – Colistin/Oxolinic acid (X013)
An inexpensive general purpose agar base which, with the addition Gardnerella spp. – Colistin/Oxolinic acid (X011)
of 5% sterile blood, can be used to cultivate a wide range of micro C. perfringens – Neomycin (X015) (X016)
organisms of clinical signiicance. Typical haemolysis patterns are Staphylococci/streptococci – Colistin/Naladixic acid (X012)
obtained with this medium.
Tryptose 15.0
Beef Extract 10.0
Soy Peptone 2.5
Yeast Extract 5.0
Sodium chloride 5.0
Sodium chloride 5.0
Agar No. 2 12.0
Agar No. 2 12.0
Weigh 37 grams of powder, disperse in 1 litre of deionised water. Allow Method for reconstitution
to soak for 10 minutes, swirl to mix then sterilise by autoclaving for
15 minutes at 121˚C. Cool to 47˚C and add 5-7% sterile deibrinated Weigh 39.5 grams of powder, disperse in 1 litre of deionised water.
blood. Mix by swirling the lask and pour into Petri dishes. Soak for 10 minutes, swirl to mix then sterilise for 15 minutes at
121˚C. Cool to 47˚C then aseptically add 5-7% sterile, deibrinated
Appearance: Dependent upon blood additive. horse or sheep blood. Mix well before pouring.
pH: 7.4 ± 0.2 Appearance: Dependent upon blood additive.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: S. aureus. WDCM 00034
S. pyogenes ATCC 19615 Minimum Q.C. organisms: S. aureus WDCM 00034
S. pyogenes ATCC 19615
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
dark.
Inoculation: Surface, streaking to single colonies. dark.
Incubation: 37˚C aerobically, anaerobically or microaerobically for Inoculation: Surface, streaking out to single colonies.
24 hours.
Incubation: 37˚C aerobically or microaerobically for 24 hours,
anaerobically for 24 and 48 hours.

Growth characteristics References
Roseburg. T., Epps, L.J. and Clarke, A.R. (1944). A study of the
colony size shape & isolation, cultivation and pathogenicity of Actinomyces israeli
organism (mm) surface colour other recovered from the human mouth and from actinomycosis in man. J.
S. aureus 1.5-2.0 CV.E.G. White/ (haemolytic) inf. Dis., 74: 131-149.
Golden
Howell, E. (1948) Eficiency of methods of isolation of Histoplasma
S. pyogenes 1.0-1.5 CV.E.G. Grey beta haemolytic) capsulatum. Pbl. Hlth. Rep. 63: 173-178. 3/108
(alpha or non
haemolytic)
S. pneumoniae 0.5-1.0 F.E.G. Grey (draughtsman Brain Heart Infusion Broth
(alpha haemolytic)
(mucoid)
(require CO2) LAB049
N. meningitidis 0.5-1.0 CV.E.G. Grey (May require
CO2) Description
A rich isotonic infusion medium with tryptose (a mixture of meat
E. coli 2.0-3.0 CV.E.G. Grey (haemolytic) and milk peptones) providing a wide range of substrates. A low
Ps. aeruginosa 1.0-3.0 F.CR.D. Grey (green pigment concentration of glucose is used to stimulate early growth. The
(haemolytic) medium is lightly buffered to prevent the early death of some species
due to acid production. Organisms which produce signiicant amounts
B. fragilis 1.0-1.5 CV.E.G. Grey non haemolytic of acid may well overwhelm the buffering system and auto-sterilise.
The medium is suitable for use as a blood culture medium or as an
enrichment broth for fastidious organisms.

Brain Heart Infusion Agar Brain-Heart Infusion Mixture 17.5
LAB048 Tryptose 10.0

Glucose 2.0
Description
Sodium chloride 5.0
A general purpose nutritious agar base. This medium was irst used for
the isolation of dental pathogens. With the addition of 7% deibrinated Disodium hydrogen phosphate 2.5
blood the medium will support the growth of a wide range of
fastidious organisms, the phosphate buffer will help neutralise the Method for reconstitution
acids produced from the utilisation of glucose and thus maintain Weigh 37 grams of powder then disperse in 1 litre of deionised water.
viability. The medium is not recommended for the determination of Allow to stand for 10 minutes then dissolve with gentle heat before
haemolytic reactions because of the glucose content. dispensing into tubes or bottles. Sterilise at 121˚C for 15 minutes.
Overheating will cause caramelisation and darkening of the medium.
Appearance: Straw colour, clear liquid.
Brain-Heart Infusion Mixture 17.5
Tryptose 10.0 pH: 7.4 ± 0.2
Glucose 2.0 Minimum Q.C. organisms: S. aureus WDCM 00034
Sodium chloride 5.0 E. coli WDCM 00013
Disodium phosphate 2.5 Storage of Prepared Medium: Capped container – up to 3 months
at 15-20˚C in the dark.
Agar No. 2 12.0
Inoculation: (as a blood culture medium). Using a minimum volume
Method for reconstitution of 50ml of medium add the blood to a dilution of from 1:10 to 1:20.
Weigh 49 grams of powder, disperse in 1 litre of deionised water. Allow Use in conjunction with an anaerobic culture medium e.g. Fastidious
to stand for 10 minutes then swirl to mix. Sterilise by autoclaving at Anaerobe Broth LAB071.
121˚C for 15 minutes. Cool to 47˚C then pour into Petri dishes. Incubation: 37˚C aerobically for 7 to 15 days.
Appearance: Pale Straw colour, clear gel. Interpretation: Observe daily, subculture after 1, 2, 3, 7 and 15 days
or immediately on showing signs of growth.
pH: 7.4 ± 0.2
References
Minimum Q.C. organisms: S. aureus WDCM 00034 Rosenow. E.C. (1919). Studies on selective localisation; focal
E. coli WDCM 00013 infection with special reference to oral sepsis. J. Dent. Res. 1:205-
267.
dark. Capped container – up to 3 months at 15-20˚C in the dark.
Inoculation: Surface, streaking out to single colonies. Brazier’s CCEY Agar
Incubation: Time and temperature to suit specimen/organisms.
LAB160
Growth characteristics (with horse blood)
Description
colony size shape & Brazier’s CCEY agar is the formulation currently used by the
organism (mm) surface colour Anaerobe Reference Unit for the isolation of C.dificile, resulting
S. aureus 1.0-1.5 CV.E.G. White/ from work initiated by Ken Phillips and Paul Levett, and completed
Golden by Jon Brazier.
other Staphylococci 0.5-1.5 CV.E.G. White/ Based upon the market leading anaerobe medium, Fastidious Anaerobe
Yellow Agar, it incorporates additional ingredients to improve the isolation
S. pyogenes 0.5-1.0 CV.E.G. White and differentiation of C.dificile from clinical specimens.
E. faecalis 1.0-1.25 CV.E.G. Grey/ Cholic acid is present to promote spore germination following alcohol
Green shock treatment, and p-hydroxyphenylacetic acid to enhance the
S. pneumoniae 0.5-1.0 F.E.G. Grey/ production of p-cresol, a distinctive metabolite of C.dificile.
Green Selectivity is achieved by addition of supplement X093 (cefoxitin
E. coli 2.0-3.0 CV.E.G. Grey cycloserine), whilst egg yolk emulsion X073 is added to help
differentiate C.dificile from lecithinase positive clostridia. Finally,
the addition of lysed horse blood optimises the recognition of colony
luorescence when cultures are examined using UV light.

Typical Formula g/litre Typical Formula g/litre
Peptone Mix 23.0 Beef Extract 5.0
Sodium chloride 5.0 Balanced Peptone No. 1 10.0
Soluble Starch 1.0 Yeast Extract 3.0
Agar No. 2 12.0 Disodium hydrogen phosphate 1.0
Sodium bicarbonate 0.4 Sodium dihydrogen phosphate 0.6
Glucose 1.0 Lactose 10.0
Sodium pyruvate 1.0 Sucrose 10.0
Cysteine HCl 0.5 Phenol red 0.09
Haemin 0.01 Brilliant green 0.0047
Vitamin K 0.001 Agar No. 2 12.0
L-arginine 1.0
Soluble pyrophosphate 0.25 Method for reconstitution
Weigh 52 grams of powder and disperse in 1 litre of deionised water.
Sodium succinate 0.5 Allow to soak for ten minutes and then bring to the boil with frequent
Cholic acid 1.0 swirling to dissolve the solids and cool to 47˚C in a water bath. Pour
plates and dry the surface before inoculation. DO NOT remelt or
p-Hydroxyphenylacetic acid 1.0 autoclave: overheating causes precipitation of the medium. Store
plates away from light.
Appearance: Red/Brown, clear gel.
Weigh 48 grams of powder and add to 1 litre of deionised water. Allow
to soak for 10 minutes, swirl to mix, and sterilise by autoclaving at pH: 6.9 ± 0.2
121˚C for 15 minutes. Cool to 47˚C and aseptically add the following:
2 vials of X093, 40ml of Egg Yolk Emulsion X073 and 10ml lysed Minimum Q.C. organisms: Salmonella sp. WDCM 00031
horse blood. Mix well and pour into Petri dishes. E. coli (inhibition) WDCM 00013
Appearance: Tan opaque gel. Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
pH: 7.0 ± 0.2 dark.
Inoculation: Surface streaking for single colonies, a heavy inoculum
Minimum Q.C. organisms: C. dificile can be used.
E. coli (inhibition) WDCM 00013 Incubation: 37˚C for 18-24 hours aerobically.
Storage of prepared medium: Plates – up to 7 days at 2-8˚C in Growth characteristics

the dark. colony size shape &
Inoculation: Surface streak untreated or alcohol shocked specimens organism (mm) surface colour other
for single colonies. Salmonella spp. 1-1.5 CV.E.G. Pink (red zone in
Incubation: 37˚C for 24-48hrs under anaerobic conditions colonies medium)
Characteristics of C.dificile: Gray opaque lat colonies, raised S. typhi 1.0 CV.E.G. Pink/Red (may not grow)
elevation, 2-3mm diameter, generally circular but tending to elongate E. coli no growth (0.5-1.0 yellow
in the direction of spreading, ground glass appearance and a rough, colony)
imbriate edge. Lecithinase negative. Incubation longer than 48hrs Proteus spp no growth
may result in a lighter gray or white centre to the colony. Phenolic
odour due to the production of p-cresol. Colonies luoresce yellow- Enterococcus no growth
green under UV light. Conirm by latex agglutination. spp.
S. sonnei no growth
References
Brazier J.S. (1993) Rôle of the Laboratory in Investigations of References
Clostridium dificile Diarrhoea. Clinical Infectious Diseases 16 (4)
Edel, W. and Kamplemacher, E.H. (1968). Comparative studies on
228-33.
Salmonella isolation in eight European laboratories. Bull. Wld. Hlth.
Org. 39: 487-491.
Edel, W. and Kamplemacher, E.H. (1969). Salmonella infections in
Brilliant Green Agar (modiied) nine European laboratories using a standard technique. Bull Wld.
Hlth. Org. 41: 297-306.
(Phenol Red Brilliant Green Agar, BPLS)
American Public Health Association (1966). Recommended Methods
for the Microbiological Examination of Foods, 2nd end. (ed. J.M.
LAB034 Sharf) A.P.H.A. Washington.
Association of Oficial Analytical Chemists (AOAC) (1978)
Description Bacteriological Analytical Manual, 5th edn., Washington D.C.
First introduced by Kristensen et al in 1925 as a selective medium Pharmacopoeia of culture media for food microbiology. (1987). Int.
for the isolation of salmonellae (except S. typhi). The medium was J. Food Microbiol. 513: 245-247. 3/112
modiied by the Netherlands Institute for Public Health, Utrecht.
The modiication was to increase the selectivity of the medium by
increasing the dye concentration. This formulation is quoted by the
International Standards Organisation, standard European Community
Methods, the American Public Health Association and the Association
of Oficial Analytical Chemists. The medium is suitable for subcultures
from selective enrichment media. However because this medium is
highly selective, small numbers of salmonellae may be missed. This
medium is deinitely not recommended for S. typhi and Shigella spp.
Less inhibitory media such as X.L.D. and Hektoen Enteric Agar will
be useful in detecting salmonellae and shigellae inhibited by Brilliant
Green Agar.

Brilliant Green Bile 2% Broth Bromocresol Purple Lactose Agar
(Drigalski agar)
LAB051
Description LAB121
A modiication of MacConkey’s medium, formulated in 1926 by Description
Dunham and Schoenlein, for the recovery of coliform bacteria in A non-selective differential medium for the isolation and enumeration
foodstuffs and water. The brilliant green and bile inhibit most Gram of Enterobacteriaceae from urine, water and food products. Lactose
positive organisms thus overcoming the problem of some Clostridium fermenting organisms produce yellow colonies, non lactose fermenters
spp. fermenting lactose and giving false positive results. produce purple colonies.
Balanced Peptone No. 1 10.0 Peptone mixture 7.4
Lactose 10.0 Lactose 8.5
Ox Bile 20.0 Bromocresol purple 0.025
Brilliant green 0.0133 Agar No. 1 12.0
Method for reconstitution Method for reconstitution

Weigh 40 grams of powder, disperse in 1 litre of deionised water. Weigh 28 grams of powder and disperse in 1 litre of deionised
Allow to soak for 10 minutes, swirl to mix then warm to dissolve. water. Allow to soak for 10 minutes, swirl to mix then sterilise by
Dispense into tubes or bottles with inverted Durham tubes. Sterilise autoclaving at 121˚C for 15 minutes. Allow to cool to 47˚C then pour
by autoclaving at 115˚C for 15 minutes. into Petri dishes.
Appearance: Green, clear. Appearance: Purple, clear agar.
pH: 7.4 ± 0.2 pH: 6.8 ± 0.2
Minimum Q.C. organisms: Salmonella sp. WDCM 00031 Minimum Q.C. organisms: E. coli. WDCM 00013
E. coli WDCM 00013 S. aureus WDCM 00034
Storage of Prepared Medium: Capped containers – up to 1 month
at 2-8˚C in the dark. Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
dark.
Inoculation: Serial 1:10 dilutions of homogenised sample are
inoculated into the broth in the proportion of 1ml sample to 9ml broth. Inoculation: Surface, plating either over entire surface for colony
Ensure the Durham tube is free from gas bubbles before commencing count or streak out to single colonies.
inoculation. B.G.B. broth can be used at double strength if required Incubation: 37˚C aerobically for 18-24 hours.
but cannot be sterilised by autoclaving, pasteurisation must be used
instead. Growth Characteristics
Incubation: E. coli and thermotrophs 44˚C for 18 hours aerobically. colony size shape &
Mesopholic coliforms 32˚C for 24-48 hours aerobically. organism (mm) surface colour other
Psychrotrophic coliforms 4˚C for 10 days aerobically.
Interpretation: Turbidity, colour changes (to yellow or yellowish E. coli 1.5-2.0 CV.E.G. Yellow (N.L.F. – purple)
green) and production of gas are all presumptive evidence of the Proteus spp. 1.0-1.5 CV.E.G. Purple
growth of organisms of the coli-aerogenes group. Conirmation by Salmonella spp. 1.0-2.0 CV.E.G. Purple
indole production in Tryptone Water LAB129 (44˚C for E. coli).
S. aureus 0.5 CV.E.G. Cream (purple if N.L.F.)
References
E. faecalis 0.5 CV.E.G. Yellow
Pharmacopoeia of Culture Media for Food Microbiology (1987). Int.
J. Food Microbiol. 5:3:206-207.
American Public Health Association, American Water Works References
Association and Water Pollution Control Federation, (1975), Drigalski, C. (1902). Uber ein Verfahren zum Nachweis der
Standard Methods for the Examination of Water and Wastewater, 14th Typhusbacillen. Z. Hyg. Infekt. 39:283-300.
ed., Washington D.C.
Association of Oficial Analytical Chemists (AOAC). Bacteriological
Analytical Manual, 5th ed., Washington, D.C. Association of Oficial
Analytical Chemists. 1978.
Hausler, W. J. (ED) (1972). Standard Methods for the Examination of
Dairy Products. 13th ed., Washington. D.C. American Public Health
Association.
Shane, M.S. (1947). Studies on false conirmed test using B.G.B. and
comparison studies on Lauryl Sulfate Tryptose Broth as presumptive
medium. J. Am. Water Works Assoc., 39: (4), 337.

Buffered Listeria Enrichment Broth Minimum Q.C. organisms: E. coli WDCM 00013
LAB139 pH: 7.2 ± 0.2

Storage of Prepared Medium: Capped containers – up to 3
Description months at 15-20˚C in the dark.
A medium for the selective enrichment of food and environmental Inoculation: Add 25 grams of sample to 225ml of Buffered
samples for Listeria spp, LAB139 is a buffered version of the ‘FDA’ Peptone Water and homogenise.
broth LAB138. The extra buffering capacity maintains the pH of the
enrichment culture during incubation, ensuring optimum conditions Incubation: Aerobically at 37˚C for 18-24 hours.
for the recovery of Listeria spp. Subculture: 10ml aliquots in 100ml of Selenite Cystine Broth
LAB055 and 0.1ml into 10ml Rappaport Vassiliadis Medium
Typical Formula g/litre LAB086.
Tryptone 17.0 References

Edel W. and Kampelmacher E.H. (1973). Bull. Wld Hlth Org. 48:
Soy peptone 3.0
167-174.
Sodium chloride 5.0 Poemla P.K. and Silliker J.H. (1976) Salmonella in Compendium
Dipotassium hydrogen phosphate 2.5 of Methods for microbiological examination of foods. Am. Pub.
Health Ass., Washington.
Glucose 2.5
Yeast Extract 6.0
Potassium dihydrogen phosphate 1.35
Buffered Peptone Water (ISO)
Disodium hydrogen phosphate 9.6 LAB204
Method for reconstitution Description
Weigh 47 grams of powder and add to 1 litre of deionised water. Formulated to ISO 6579, this pre-enrichment medium is designed
Allow to soak for 10 minutes then swirl to mix and sterilise by to help sublethally damaged salmonellae recover before introducing
autoclaving at 121˚C for 15 minutes. Cool to 47˚C and add 2 vials of them into a selective medium. This nutrient medium is free from
X139 reconstituted in 50% alcohol. Aseptically dispense into sterile inhibitors and is well buffered to maintain pH 7.0 for the incubation
tubes or bottles. period. Sublethal injury to salmonellae occurs in many food processes
and this pre-enrichment step greatly increases recovery of these
Appearance: Yellow, clear. organisms.
pH: 7.2 ± 0.2 Typical Formula g/litre
Minimum Q.C. organisms: L. monocytogenes WDCM 00021 Enzymatic digest of casein 10.0
E. coli (inhibition) WDCM 00013 Sodium chloride 5.0
Storage of Prepared Medium: Capped containers – up to 14 days at Disodium hydrogen phosphate (anhydrous) 3.6*
2-8˚C in the dark.
Inoculation: Add 25 grams of sample to 225mls of Buffered Listeria
Enrichment Broth and homogenise. *Equivalent to 9.0g of disodium hydrogen phosphate dodecahydrate
Incubation: 30˚C aerobically for up to 48 hours.

Subculture: After 24 and 48 hours onto Listeria Isolation Medium – Method for reconstitution
LAB122. Weigh 20.1 grams of powder and disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then distribute into tubes or
bottles. Sterilise by autoclaving for 15 minutes at 121ºC.
Buffered Peptone Water Appearance:

LAB046
Finished medium: pale straw, clear liquid
Description pH: 7.0 ± 0.2
A pre-enrichment medium designed to help sublethally damaged Hazard classiication
salmonellae recover before introducing them into a selective medium. NR – Not regulated
This nutrient medium is free from inhibitors and is well buffered to
maintain the pH at 7.2 for the incubation period. Sublethal injury to
salmonellae occurs in many food processes and this pre-enrichment
Staphylococcus aureus WDCM 00034
step greatly increases recovery of these organisms.
Salmonella typhimurium WDCM 00031
Listeria monocytogenes WDCM 00021
Peptone 10.0 Storage:
Dehydrated culture media: 10-25°C away from direct sunlight
Sodium chloride 5.0
Prepared media: capped containers – up to 3 months at 15-20°C in
Disodium hydrogen phosphate 3.7 the dark.
Potassium dihydrogen phosphate 1.5 Inoculation: Add 25 grams of sample to 225ml of Buffered Peptone
Water and homogenise.
Method for reconstitution Incubation: Aerobically at 37°C for 18-24 hours.
Weigh 20 grams of powder and disperse in 1 litre of deionised water. References
Mix to dissolve then distribute into tubes or bottles. Sterilise by
BS EN ISO 6579:2002 Microbiology of food and animal feeding
autoclaving at 121˚C for 15 minutes.
stuffs – Horizontal method for the detection of Salmonella spp.
Appearance: Pale straw, clear liquid. (Incorporating Corrigendum No. 1)

Campylobacter Blood LAB112
Free Selective Medium References
(Modiied CCDA-Improved)
Bolton F.J. Hutchinson D.N., Parker G. Reassessment of Selective
Agars and Filtration Techniques for Isolation of Campylobacter
LAB112 Species from Feces. Eur.J. Clin. Microbiol. Infects. Dis. (1988) 7
p 155-160.
Description Bolton F. J. (1988) Personal Communication.
A blood free medium which will support the growth of most enteric
campylobacters. The selective cocktail X112 (or X212) makes the Bolton F.J. Hutchinson D.N., Parker G. Isolation of Campylobacter:
medium selective for C. jejuni. and C. coli when incubated at 37˚C. What are we missing? J.Clin.Path. (1987) 40 p 702-703.
With this product incubation at 42˚C is no longer necessary and higher Goosens H., De. Boeck M., Coignau H., Vlaes L., Van Den Borre C.,
recovery rates have been reported at 37˚C than at 42˚C. Butzler J.P. Modiied Selective Medium for Isolation of
The supplement X112 (or X212) consists of cefoperazone and Campylobacter spp from Feces: Comparison with Preston Medium, a
amphotericin and is superior to the selective cocktails of Skirrow, Blood Free Medium, and a Filtration System. J.Clin. Micro. (1986)
Butzler and Blazer-Wang all of which contain antibiotics shown to 24 p 840-843.
be inhibitors to C. coli. The colonial morphologies of Campylobacter Gun-Munro J., Rennie R.P., Thornley J.H. Richardson H.L.,
spp. on this medium are distinctive. Hodge D., Lynch J. Laboratory and Clinical Evaluation of Isolation
Media for Campylobacter jejuni J. Clin Micro. (1987). 25 p
Typical Formula g/litre 2274-2277.
Herbert G.A., Hollis D.G., Weaver R.E., Karmali M.A., Simor A.E.,
Peptone blend 25.0 Roscoe M., Fleming P.C., Smith, S.S. Lane J. Evaluation of a
Bacteriological Charcoal 4.0 Blood-Free, Charcoal-Based, Selective Medium for the Isolation of
Campylobacter organisms from Faeces. J. Clin. Micro. (1986) 23
Sodium chloride 3.0 p 456-459.
Sodium desoxycholate 1.0
Ferrous sulphate 0.25
Campylobacter Enrichment Broth
Sodium pyruvate 0.25
(Bolton Formulation)
Agar No. 2 12.0
LAB135
Description
Weigh 45.5 grams of powder, disperse in 1 litre of deionised water
and allow to soak for 10 minutes. Swirl to mix, then sterilise by A selective enrichment broth for the isolation of Campylobacter
autoclaving at 121˚C for 15 minutes. Cool to 47˚C then add 2 vials of spp. from food, environmental samples and faeces. The use of a
X112 supplement, mix well and pour into Petri dishes. Continuously selective enrichment broth enhances the recovery of sub-lethally
mix whilst pouring to prevent the charcoal settling. damaged organisms due to processing of foods, or if small numbers of
campylobacters are present in heavily contaminated specimens. This
Appearance: Black agar. broth has been shown to give appreciably better results than Preston
Broth.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: C. jejuni
E. coli (inhibition) WDCM 00013 Meat Peptone 10.0
Candida albicans (inhibition) Lactalbumin Hydrolsates 5.0
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the Yeast Extract 5.0
dark.
Sodium chloride 5.0
Inoculation: C. jejuni, C. coli. surface streaking to single colonies.
Haemin 10.0mg
Incubation: 37˚C for 48 hours in an atmosphere of 5% oxygen,
10% carbon dioxide and 85% nitrogen. C. cinaedi and C. fennelliae Sodium pyruvate 0.5
require up to 7 days.
α – ketoglutaric acid 1.0
Sodium metabisulphite 0.5
Sodium carbonate 0.6
colony size shape &
C. jejuni 2.0-3.0 F.E.G. Grey/
White Eflorescent Weigh 27.6 grams of powder, disperse in 1 litre of deionised water
(spreading moist) and allow to soak for 10 minutes. Swirl to mix and autoclave at 121˚C
for 15 minutes. Cool to 47˚C, add 2 vials of selective supplement
C. coli 1.0-2.5 CV.E.G. Creamy Moist
Grey
X132 reconstituted with 5ml of 50% alcohol and 50ml of saponin
lysed horse blood, mix well and dispense into sterile containers.
Appearance: Translucent, wine-red with a ine black suspension.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: Campylobacter jejuni

Storage of Prepared Medium: Capped containers: 7 days at 2-8˚C

in the dark.
Inoculation: Food homogenate is added to broth in a ratio of 1:4
(w/v) in screw cap containers leaving a head space of 1.5 cm. For
faeces 1ml of a 10% suspension in Buffered Peptone Water LAB046
is added to 5ml of broth.
Incubation: Aerobically at 37˚C for 2-4 hours, followed by a further
16-44 hours at 42˚C.
Subculture: Onto Campylobacter Blood Free Selective Medium
LAB112.

References C.E.M.O. Agar Base
Bolton, F.J. Personal Communication.
(Contagious Equine Metritis Organism)
Hunt J.M., Abeyta C., and Tran T. (1998) Chapter 7 Campylobacter in
FDA Bacteriological Analytical Manual 8th Edition.
LAB078
Description
Cary-Blair Medium This medium is a selective isolation medium for Taylorella equigenitalis
the causative organism of contagious equine metritis. The medium is a
LAB505 sugar free base with a mixture of high grade casein and soy peptones
as nutrients and with L-cystine and sodium sulphite as supplements
Description and reducing agents. The medium is made selective with the addition
of amphotericin (5 mg/L) and trimethoprim (10 mg/L). Streptomycin
Cary-Blair medium is a transport medium for the collection and
(200 mg/L) can also be used but sensitive variants of T. equigenitalis
shipment of clinical specimens based on the formulation of Cary
have been described.
and Blair. The low nutrient content of the medium and the inclusion
of phosphate buffer prevents bacterial overgrowth by E. coli,
Citrobacter freundii and Klebsiella aerogenes, which can occur in Typical Formula g/litre
other transport medium containing sodium glycerophosphate. The Tryptone 15.0
low oxidation-reduction potential of the medium ensures bacterial
survival over long periods. Soy Peptone 5.0
Cary and Blair reported recovery of cholera vibrios up to 22 days, Sodium chloride 5.0
Salmonella and Shigella after 49 days and Yersinia pestis up to 75
days storage at 28°C. Survival of Vibrio parahaemolyticus has been Agar No. 2 12.0
reported after a 35-day period at 70-80°F.
L-Cystine 0.3
The medium may be prepared as a pre-reduced anaerobic sterilised
medium (PRAS) by the Holdeman and Moore method. Sodium sulphite 0.2

Disodium hydrogen phosphate 1.1 Weigh 37.5 grams of powder, disperse in 1 litre of deionised water.
Sodium thioglycollate 1.5 at 121˚C for 15 minutes. Allow to cool to 80˚C, add 50ml of sterile
Sodium chloride 5.0 horse blood and allow to ‘chocolate’. Further cool to 47˚C before
adding antibiotic selective agents. Mix well and pour into Petri dishes.
Calcium chloride 0.09
Appearance: Chocolated Blood Agar.
Agar 5.6
pH: 7.3 ± 0.2
Minimum Q.C. organisms: H. equigenitalis
Allow the mixture to soak for 10 minutes, swirl to mix then bring to
the boil, with mixing, to dissolve the agar. Distribute into bijou bottles Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
and sterilise by immersing in free-steam for 15 minutes. Allow the dark.
medium to cool and tighten the screw caps to prevent water loss. Inoculation: Surface, streaking out for single colonies.
For transport of fastidious anaerobic bacteria prepare the medium as Incubation: 37˚C in 10% CO2 for 2-3 days.
directed and ill into long narrow screw capped tubes, or to the neck
of the Bijou bottle.
Appearance: Colourless soft gel. colony size shape &
pH: 8.4 ± 0.2
T. equigenitalis 0.1-1.0 CV.E.G. Cream colony size
variation is
Minimum Q.C. organisms: Shigella sonnei ATCC 25931
common
Vibrio furnissi NCTC 11218
References
Storage of Prepared Medium: Store away from light at 2-8°C or at
room temperature (22-25°C) for up to 19 months. Atherton, J.G. (1978). Inhibition of the C.E.M. organism in mixed
cultures. Vet. Rec. 432.
Inoculation: Use sterile, cotton-tipped swab on wooden sticks to
collect the specimen. Push the swab down one third of the depth of Mackintosh, M.E. (1981). Bacteriological techniques in the diagno-
the medium and cut the stick. Screw the cap irmly on the bottle. sis of equine genital infections. Vet. Rec. 108, 52-55. Atherton, J.G.
Label the bottle and send to the testing laboratory without delay. Personal Communication.
Fleming, M.P. Tribe. G. W. (1977). Vet. Rec. 101, 1470.
References
Cary, S.G. and Blair, E.B. (1964). J. Bact. 88. 96-98.
Cary, S.G., Matthew, M.S., Fusillo, M.H., and Harkins, C. (1965).
Survival of Shigella and Salmonella in a new transport medium for
shipment of clinical samples. Am. J. Clin, Path. 43. 294-296.
Crookes, E.M. and Stuart, R.D. (1959) J. Pathol. Bacteriol. 78. 283-
288.
Stuart, R.D. (1959) Public Health Reports 74. 431-438.
Neumann D.A., Benenson, M.W., Hubster, E. and Tuan, N.T.N.
(1971). Am. J. Clin. Path. 57.
Wren, M.W.D. J. Med. Microbiol. 10. 195-201.
Holdeman, L.V. and Moore, W.E.C (1975) Anaerobe Laboratory
Manual, Virginia Polytechnic Institute Anaerobe Laboratory, 3rd Ed.

Cetrimide Agar C.L.E.D. Medium
(USP/EP/JP) (Mackey and Sandys)
(Cystine Lactose Electrolyte Deicient-Single Indicator)
HP010
Description LAB041
A medium recommended by the Harmonised European Pharmacopoeia Description
for the isolation and identiication of Pseudomonas aeruginosa, in non-
sterile pharmaceutical samples. Conforms to USP/EP/JP performance A medium for urine culture irst described by Mackey and Sandys in
speciication. Gelatin is a source of nitrogen whilst glycerol acts as a 1960. The absence of electrolytes inhibits the swarming of Proteus
carbon source. Cetrimide is a quarternary ammonium compound that spp. Cystine is added for the beneit of those organisms which have a
inhibits the growth of a wide range of Gram-positive and some Gram- speciic cystine requirement. Differentiation of lactose and non lactose
negative micro-organisms. Magnesium chloride and dipotassium fermenters is achieved using bromothymol blue as pH indicator. This
sulphate improve the production of pyoverdin and pyocyanin pigments medium supports the growth of fastidious organisms that do not
that combine to give Pseudomonas aeruginosa characteristic green require blood.
colonies. According to the Harmonised European Pharmacopoeia,
subculture is carried out onto the medium after enrichment in Casein Typical Formula g/litre
Soya Bean Digest Broth. Balanced Peptone No. 1 4.0
Typical Formula g/litre Beef Extract 3.0
Pancreatic digest of gelatin 20.0 Tryptone 4.0
Magnesium chloride 1.4 Lactose 10.0
Dipotassium sulphate 10.0 L-Cystine 0.128
Cetrimide 0.3 Bromothymol blue indicator 0.02
Agar 13.6 Agar No. 1 15.0

Disperse 45.3 grams of powder in 1 litre of deionised water. Allow to Weigh 36 grams of powder, disperse in 1 litre of deionised water.
soak for 10 minutes, add 10mL of glycerol, swirl to mix and boil to Allow to soak for 10 minutes, swirl to mix then sterilise by autoclaving
dissolve. Sterilise by autoclaving atg121oC for 15 minutes. Cool to for 15 minutes at 121˚C. Cool to 47˚C mix and distribute into Petri
47°C and mix well before dispensing into sterile Petri dishes. Dry the dishes.
agar surface prior to use.
Appearance: Green/blue clear gel.
Appearance: pH: 7.3 ± 0.2
Minimum Q.C. organisms: E. coli
Finished medium: translucent, pale straw gel WDCM 00013
pH: 7.2 ± 0.2 S. aureus
Minimum Q.C. organisms: WDCM 00034
Pseudomonas aeruginosa ATCC 9027
Escherichia coli ATCC 8739 Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
dark.
Inoculation: Surface inoculation either spreading for single colonies
Hazard classiication: NR – Not regulated
or spread evenly over entire surface for colony counts.
Storage: Incubation: 37˚C aerobically for 18-24 hours.
Dehydrated culture media: 10-25°C away from direct sunlight.
Prepared media: 7 days at 2-8°C in the dark. Growth Characteristics
colony size shape &
Inoculation: According to the European Pharmacopoeia 8.0
subculture is performed from enrichment in casein soya bean digest organism (mm) surface colour other
broth onto the agar surface. E. coli 2.0-3.0 CV.E.G. Yellow (blue if non
lactose fermenters)
Incubation:
Proteus spp. 2.0-3.0 CV.E.G. Blue
Incubate at 30-35°C for 18-72 hours.
Salmonella spp. 2.0-3.0 CV.E.G. Blue (yellow if
Growth Characteristics lactose +ve)
Shape & Staph. aureus 1.0-1.5 CV.E.G. Yellow (blue if non-
Organism surface Colour Other lactose
fermenting)
P. aeruginosa CV.E.G. Yellow/green Fluorescent under
UV light other Staphylococcus (yellow if
spp. 0.5-1.5 CV.E.G. Blue-white lactose
fermenting)
References Enterococcus
spp. 0.5 CV.E.G. Yellow
European Pharmacopoeia 8th Edition
References
Mackey, J.P. and Sandys, G.H. (1966). Diagnosis of urinary infections.
Brit.Med.J. 1: 1173.
Guttman, D and Naylor, G.R.E. (1967). Dip-slide: an aid to quantitative
urine culture in general practice. Brit.Med. J. 3: 343-345.

C.L.E.D. Medium (Bevis modiication) Columbia Agar Base
(Cystine Lactose Electrolyte Deicient – Double Indicator)
LAB001
LAB006 Description
Description A general purpose nutritious agar base formulated by Ellner et al.
When further enriched by the addition of sterile blood, Columbia agar
Bevis modiied Mackey and Sandys original medium by introducing can be used for the isolation of most clinically signiicant pathogens.
a double indicator to improve the differentiation of lactose and non The blood can be ‘chocolated’ if required. The medium can be made
lactose fermenting coliforms, staphylococci and streptococci. The selective for various groups by the addition of appropriate antibiotic
swarming of Proteus spp. is inhibited. Lab M C.L.E.D. will grow mixtures eg:
many of the more demanding streptococci of Lanceield groups A, B,
C, G and F. This medium may not grow Pasteurella spp. or halophilic Streptococci – Colistin/Oxolinic acid (X013)
organisms. Gardnerella spp. – Colistin/Nalidixic acid (X011)
C. perfringens – Neomycin (X015) (X016)
Typical Formula g/litre Campylobacters - (X214)
Staphylococci/streptococci – Colistin/Naladixic acid (X012)
Beef Extract 3.0 Typical Formula g/litre
Tryptone 4.0 Columbia Peptone Mixture 23.0
Lactose 10.0 Corn Starch 1.0
L-Cystine 0.128 Sodium chloride 5.0
Bromothymol blue indicator 0.02 Agar No. 2 12.0
Andrade’s indicator 0.08 Method for reconstitution

Agar No. 1 15.0 Weigh 41 grams of powder, disperse in 1 litre of deionised water. Allow
to soak for 10 minutes, swirl to mix then sterilise by autoclaving for
15 minutes at 121˚C. Cool to 48˚C and add 5-7% sterile, deibrinated
Method for reconstitution horse or sheep blood. Mix well before pouring.
Weigh 36 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise by autoclaving Appearance: Cherry red if blood is fresh and well oxygenated.
for 15 minutes at 121˚C. Cool to 47˚C and mix before pouring.
Appearance: Green/blue, clear gel. S.pyogenes NCTC 8198
E.coli ATCC 8739
pH: 7.5 ± 0.2 S.aureus ATCC 6538
Minimum Q.C. organisms: E. coli WDCM 00013 pH: 7.3 ± 0.2
S. aureus WDCM 00034
dark.
dark. Inoculation: Surface plating, streaking out for single colonies.
Inoculation method: Surface inoculation, either streaking for single Incubation: 37˚C aerobically or microaerobically for 24 hours.
colonies or spread evenly over entire surface for colony counts. Anaerobically for 24 and 48 hours.
Incubation: 37˚C for 24 hours aerobically. Growth Characteristics
Growth Characteristics colony size shape &
colony size shape &
S. aureus 1.5-2.0 CV.E.G. White-
E. coli 2.0-3.0 CV.E.G. Yellow/ (Blue if Yellow Haemolytic
Orange non-lactose S. pyogenes 0.5-1.0 CV.E.G.(D) White α, β-haemolytic
fermenter)
dependent on
Proteus spp. 2.0-3.0 CV.E.G. Blue strain
Salmonella spp. 2.0-3.0 CV.E.G. Blue (Yellow-orange S. pneumoniae 0.5-1.5 F.E.G. Grey greenish
if lactose +ve) discolouration
in medium, mucoid
S. aureus 1.0-1.5 CV.E.G. Yellow/ (Blue if in H2/C02
Orange non-lactose
Neisseria
fermenting)
meningitidis 1.0-2.0 CV.E.G. trans/ (mucoid)
Other (Yellow if Grey
staphylococci 0.5-1.5 CV.E.G. Blue-White lactose E. coli 2.0-3.0 CV.E.G. Opaque/ (haemolytic)
fermenting) Grey
Enterococcus spp. 0.5 CV.E.G. Yellow- Ps. aeruginosa 0.5-4.0 F.CR.D. Opaque many colonial
Grey forms
Orange
(green pigment)
(haemolytic)
References (mucoid)
Bevis, T.D. (1968). A modiied electrolyte-deicient culture medium. C. perfringens 1.5-2.0 CV.CR.G. Grey usually target
J. Med. Lab. Tech., 25: 38-41. haemolysis
Mackey, J.P. and Sandys, G.H. (1966). Diagnosis of urinary (non haemolytic)
infections, Brit.Med. J., 1: 1173. B. fragilis 1.0-1.5 CV.E.G. Grey (mucoid)
Sandys, G.H. (1960). A new medium for preventing swarming of P. anaerobius P.P.-0.5 CV.E.G. White/
Proteus spp. with a description of a new medium suitable for use in Grey
routine laboratory practice. J. Med.Lab. Tech., 17: 224-233.

References CSEB - Cronobacter sakazakii
Ellner, P.D., Stoessel, C.J., Drakeford, E and Vasi, F. (1966). A new
culture medium for medical bacteriology. Amer. J. Clin Pathol., Enrichment Broth
45:502-504. Modiied Lauryl Sulphate Tryptose Broth Vancomycin Medium
Goldberg, R.L., and Washington, J.A., (1976). Comparison of isolation
of Haemophilus vaginalis (Corynebacterium vaginale) from Peptone- LAB081
Starch-Dextrose Agar and Columbia Colistin-Nalidixic Acid Agar. J.
Clin. Microbiol., 4:245-247.
Description
Thayer, D.D. and Martin, H. E. (1966). An improved medium for Cronobacter sakazakii (formerly Enterobacter sakazakii) is a member
the cultivation of N. gonorrhoeae and N. meningitidis. Publ. Hlth. of the Enterobacteriaceae family and has been associated with serious
Report, 81:559-562. outbreak infections in neonates (premature infants) which have been
fed on infant formula milk. Although rarely causing infections in
immunocompetent adults, C. sakazakii has been implicated in sepsis,
Columbia II Agar Base meningitis and necrotising enterocolitis with a high death rate in
neonates. This opportunistic pathogen is common in the environment
and its ability to survive desiccation presents a signiicant risk for
LAB215 post pasteurisation contamination and survival in spray dried milk
products.
Description
A modiication of the original Columbia Agar base formulation, Based on lauryl sulphate tryptose broth, Cronobacter sakazakii
Columbia II Agar Base provides a medium that is suitable for use Enrichment Broth (CSEB) has added sodium chloride for extra
with both deibrinated horse and deibrinated sheep blood. selectivity against competing organisms. The antibiotic vancomycin is
also added to inhibit Gram-positive organisms such as Staphylococccus
Originally described as a general purpose nutritious agar base by aureus which may be able to grow in this medium.
Ellner et al. Columbia Agar is more frequently used when enriched by
the addition of sterile blood. The medium is suitable for supporting Typical Formula g/litre
the growth of a variety of microorganisms including the majority of
clinically signiicant pathogens. Enzymatic digest of animal and plant tissue 20.0
Lactose 5.0
Columbia peptone mixture 25.1 Dipotassium hydrogen phosphate 2.75
Soluble starch 1.0 Potassium dihydrogen orthophosphate 2.75
Sodium chloride 5.0 Sodium lauryl sulphate 0.1
Agar 12.0 Grams per litre 64.6
Method for reconstitution Weigh 64.6 grams of powder and disperse in 1 litre of deionised water.
Disperse 43.1 grams of powder in 1 litre of deionised water. Allow Allow to soak for 10 minutes, swirl to mix and if required, heat gently
to soak for 10 minutes, swirl to mix, then sterilise by autoclaving at to dissolve. Dispense in 10ml volumes and sterilise by autoclaving for
121oC for 15 minutes. Cool to 47°C and add 5-7% sterile deibrinated 15 minutes at 121°C. Cool to 47°C.
horse or sheep blood. Mix well before dispensing into sterile Petri Prepare a solution of vancomycin in distilled water at a concentration
dishes. Dry the agar surface before use. of 1mg/ml. Add 0.1ml of the vancomycin solution to the sterile
broth to obtain a inal concentration of 0.1mg per 10ml (10mg/L) of
Appearance: CSEB.
Powder: ine, free-lowing, homogeneous, buff Appearance:
Finished medium: Opaque red gel (with blood) Powder: ine, free-lowing, homogeneous, buff
pH: 7.3 ± 0.2 Finished medium: clear, straw liquid
Hazard classiication pH: 6.8 ± 0.2
NR – Not regulated
Minimum Q.C. organisms: NR – Not regulated
S.pyogenes NCTC 8198
E.coli ATCC 8739 Minimum Q.C. organisms:
S.aureus ATCC 6538 Cronobacter sakazakii ATCC 12868
Cronobacter muytjensii ATCC 51329
Storage: Escherichia coli ATCC 25922 (inhibition)
Storage:
Prepared media: 7 days at 2-8°C
Inoculation: Surface plating, streaking out for single colonies.
Prepared media (with vancomycin): 1 day at 2-8°C in the dark..
Incubation: Incubate for 24-48 hours at 30°C or 3°C in an aerobic,
anaerobic, CO2 or microaerophilic environment as requried for growth Inoculation: Following pre-enrichment in Buffered Peptone Water,
of target organism. transfer 0.1mL of the obtained culture into 10ml LAB081 CSEB.
Incubation: Incubate at 44°C + 0.5°C for 24 hours + 2 hours.
References
Sub-culture & Interpretation: After incubation, tubes showing
Ellner, P.D., Stoessel, C.J., Drakeford, E. and Vasi, F. (1966). A new turbidity should be streaked onto HAL012 CSIM (ISO).
culturemedium for medical bacteriology. Amer J. Clin Pathol. 45.
502-504.

References Method for reconstitution
Bowen AB, Braden CR (2006). “Invasive Enterobacter sakazakii Weigh 45.5 grams of powder, disperse in 1 litre of deionised water.
disease in infants”. Emerging Infect Dis 12 (8): 1185–9. Allow to soak for 10 minutes, swirl to mix, then bring to the boil
Caubilla-Barron J & Forsythe S (2007). “Dry stress and survival time with frequent stirring. When the medium boils up into the neck of
of Enterobacter sakazakii and other Enterobacteriaceae in dehydrated the lask, quickly remove from the source of heat and allow the froth
infant formula”. Journal Food Protection 13: 467-472. to subside. Return to the heat and allow the foam to boil up into
the neck of the lask once more. Remove at once and cool to 47˚C
“Enterobacter sakazakii infections associated with the use of powdered approx. before pouring plates. Dry the surface before inoculation.
infant formula--Tennessee, 2001” (2002). MMWR Morb Mortal Wkly DO NOT REMELT OR AUTOCLAVE THIS MEDIUM.
Rep 51 (14): 297–300.
Appearance: Pale pink, translucent, a ine precipitate of
Farmer JJ III, Asbury MA, Hickman FW, Brenner DJ, the desoxycholate may be present which may clear if the pH is increased
Enterobacteriaceae Study Group (USA) (1980). “Enterobacter by the growth of organisms.
sakazakii: a new species of “Enterobacteriaceae” isolated from clinical
specimens”. Int J Syst Bacteriol 30: 569–84. pH: 7.0 ± 0.2
ISO/TS 22964:2006(E) Milk and milk products – Detection of Minimum Q.C. organisms: Salmonella sp. WDCM 00031
Enterobacter sakazakii. E. coli WDCM 00013
Iversen C, Lehner A, Mullane N, et al (2007). “The taxonomy
of Enterobacter sakazakii: proposal of a new genus Cronobacter Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
gen. nov. and descriptions of Cronobacter sakazakii comb. nov. dark.
Cronobacter sakazakii subsp. sakazakii, comb. nov., Cronobacter
sakazakii subsp. malonaticus subsp. nov., Cronobacter turicensis sp. Inoculation: Surface, streaking for single colonies.
nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. Incubation: 37˚C for 18-24 hours aerobically.
nov. and Cronobacter genomospecies 1”. BMC Evol Biol 7: 64.
Iversen C, Mullane N, Barbara McCardell, et al (2008). “Cronobacter Growth Characteristics
gen. nov., a new genus to accommodate the biogroups of Enterobacter colony size shape &
sakazakii, and proposal of Cronobacter sakazakii gen. nov. comb. organism (mm) surface colour other
nov., C. malonaticus sp. nov., C. turicensis sp. nov., C. muytjensii sp.
S. typhi 0.5. 1.5 CV.E.G. transp (black centre)
nov., C. dublinensis sp. nov., Cronobacter genomospecies 1, and of
Yellow
three subspecies, C. dublinensis sp. nov. subsp. dublinensis subsp.
nov., C. dublinensis sp. nov. subsp. lausannensis subsp. nov., and C. Other Salmonella
spp. 1.5-2.0 CV.E.G transp (black centre)
dublinensis sp. nov. subsp. lactaridi subsp. nov.”. IJSEM.
(Opaque) (clearing
Lai KK (2001). “Enterobacter sakazakii infections among neonates, Yellow around colony)
infants, children, and adults. Case reports and a review of the
literature”. Medicine (Baltimore) 80 (2): 113–22. S. sonnei 1.5-2.0 CV.E.G. transp (more opaque
(pinkish) centre)
E. coli P.P.-1.5 CV.E.D. Red/Pink ppt in medium
(uninhibited) (G)
D.C.A. Citrobacter spp. P.P.-2.0 CV.E.D. Red/Pink ppt in medium
(Desoxycholate Citrate Agar) (G) (black centre)
Proteus spp. 1.0-2.0 CV.E.G. Yellow (black/grey
LAB029 centre)
ishy odour
(clearing around
Description colony)
This is Leifson’s original formulation of this selective medium for
the isolation of Salmonella spp. and Shigella spp. from faeces and References
environmental samples. It has approximately half the quantity of
inhibitors used in the Hynes modiication. The medium uses sodium Hynes. M. (1942). The isolation of intestinal pathogens by selective
citrate and sodium desoxycholate as inhibitors. Sodium thiosulphate media. J. Path. Bact. 54. 193-207.
is the substrate for the enzyme thiosulphate reductase being broken Liefson. E. (1935). New culture media based on Sodium
down to form sulphite and hydrogen sulphide. The hydrogen sulphide desoxycholate for the isolation of intestinal pathogens and for the
reacts with the ferric ions to produce a black precipitate of ferrous enumeration of colon bacilli in milk and water. J. Path. Bact. 40:
sulphide. This gives a typical black centre to the colonies of most 581-589.
species of Salmonella.

D.C.A. Hynes
Beef Extract 5.0
(Desoxycholate Citrate Agar -Hyne’s modiication)
Lactose 10.0 LAB065
Sodium citrate 5.0 Description
Sodium thiosulphate 5.0 This modiication of Leifson’s D.C.A. medium was introduced in
1942. The medium was designed to be more inhibitory to commensal
Ferric citrate 1.0 lora whilst allowing for adequate growth of Salmonella spp and
Shigella spp. The citrate and desoxycholate levels are signiicantly
Sodium desoxycholate 2.5 increased. To keep the desoxycholate in solution the pH also had to be
Neutral Red 0.025 increased. The medium still uses lactose fermentation and hydrogen
sulphide production as differential indicators.
Agar No. 2 12.0

Beef Extract 5.0 Allow to soak for 10 minutes then heat gently with frequent mixing
and bring to the boil. Simmer for 1 minute to complete dissolution of
Balanced Peptone No. 1 5.0 the solids. Cool to 47˚C then distribute 20ml into 90mm Petri dishes.
Lactose 10.0 Dry the surface by partial exposure, before use. DO NOT REMELT
OR AUTOCLAVE THIS MEDIUM.
Appearance: Pale Pink, clear.
Sodium citrate 8.5
pH: 7.2 ± 0.2
Ferric citrate 1.0
Sodium desoxycholate 5.0 Minimum Q.C. organisms: Salmonella typhimurium
WDCM 00031
Neutral red 0.02 E. coli
Agar No. 2 12.0 WDCM 00013
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in

Method for reconstitution the dark.
Weigh 52 grams of powder, disperse in 1 litre of deionised water in Inoculation: Surface plating, streaking out to single colonies.
a two litre lask. Bring to the boil over a gauze, swirling frequently
Incubation: 37˚C aerobically for 24 hours
to prevent burning. Simmer for 30 seconds to dissolve. Cool to 47˚C
before pouring plates. Dry the surface before inoculation. DO NOT Growth Characteristics
REMELT OR AUTOCLAVE THIS MEDIUM.
colony size shape &
Appearance: Pink, clear, bile aggregates may appear on the surface organism (mm) surface colour other
on refrigeration.
S. typhi 0.5-1.0 CV.E.G. Trans.
pH: 7.4 ± 0.2 colourless
Other
Minimum Q.C. organisms: Salmonella sp. WDCM 00031 Salmonella spp. 1.5-2.0 CV.E.G. Slight
E. coli WDCM 00013 cloudy
colourless
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the S. sonnei 1.5-2.0 CV.E.G Trans. (More opaque
dark. Pinkish centre)
E. coli P.P.-1.5 CV.E.G.(D) Red (ppt around
Inoculation: Surface, streaking out for single colonies.
(inhibited) colonies)
Incubation: 37˚C aerobically for 24 hours.
Citrobacter P.P.-2.0 CV.E.D.(G) Red (ppt around
spp (inhibited) colonies)
colony size shape & Proteus spp. 1.0-2.0 CV.E.G. Yellow (Fishy odour)
S. sonnei 1.0-2.0 CV.E.G.(D) Colourless References
- pale pink Hynes, M. (1942). The isolation of intestinal pathogens by selective
media. J. Path. Bact. 54: 193-207.
Salmonella spp. 1.0-4.0 CV.E.G. Colourless (black centre)
Leifson, E. (1935). New culture media based on sodium desoxycholate
S. typhi 0.5-1.5 CV.E.G. Colourless (Black/grey for the isolation of colon bacilli in milk and water. J. Path. Bact. 40:
centre) 581-589.
E. coli P.P.-1.5 CV.CR.D. Red (No growth)
Proteus spp 0.5-2.0 CV.E.G. Colourless (Yellow)
ishy odour
D/E Neutralising Agar
References (Dey & Engley)
Hynes, M. (1942). The isolation of intestinal pathogens by selective
media. J. Path. Bact, 54: 193-207 LAB188
Description
D/E Neutralising Agar is used to neutralise and determine the
D.C.L.S. Agar bactericidal activity of antiseptics and disinfectants. Developed
(Desoxycholate Citrate Lactose Sucrose Agar) by Dey and Engley, this agar neutralises a broad spectrum of
antimicrobial chemicals, producing better results than those obtained
using alternatives such as Letheen Agar. Complete neutralisation is
LAB003 required to prevent false results arising from disinfectant carryover.
D/E Neutralising Agar is used as the plating medium when testing
Description disinfectants using D/E Neutralising Broth and D/E Neutralising
A modiication of Leifson’s D.C.A. medium which incorporates Broth Base. It can also be used to test disinfectants by a disc diffusion
sucrose as an additional fermentable substrate to differentiate lactose method. D/E Neutralising Agar contains thioglycollate to neutralise
negative sucrose positive coliforms from Salmonella spp. This mercurial compounds, sodium thiosulphate to neutralise iodine
medium is unsuitable for the isolation of Yersinia spp. which are and chlorine and sodium bisulphite to neutralise formaldehyde
sucrose positive. and gluteraldehyde. Lecithin is included to neutralise quaternary
ammonium compounds and Polysorbate 80 neutralises phenols,
Typical Formula g/litre hexachlorophene, formalin, and combined with lecithin, ethanol.
Bromocresol purple allows detection of growth via a colour change
Balanced Peptone No. 1 7.0 from purple to yellow when organisms ferment the glucose contained
Beef Extract 3.0 in the medium.
Lactose 5.0
Sucrose 5.0
Sodium citrate 10.5
Agar No. 2 12.0
Neutral Red 0.03

Typical Formula g/litre D/E Neutralising Broth
Glucose 10.0 (Dey & Engley)
Lecithin 7.0
LAB187
Description
Polysorbate 80 5.0
D/E Neutralising Broth is used to neutralise and determine the
Tryptone 5.0 bacteriocidal activity of antiseptics and disinfectants. Developed by
Dey and Engley, D/E Neutralising Broth neutralises a broad spectrum
Sodium bisulphite 2.5 of antimicrobial chemicals, producing better results than those
Yeast extract 2.5 obtained using alternatives such as Letheen Broth, Thioglycollate
Medium and Neutralising Buffer. Complete neutralisation is required
Sodium thioglycollate 1.0 to prevent false results arising from disinfectant carryover. When used
with D/E Neutralising Broth Base the action of the antimicrobial agent
Bromocresol purple 0.02 can be assessed, i.e. whether it is bacteriostatic or has bactericidal
Agar 15.0 properties. The procedure is based upon D/E Neutralising Broth Base
being deicient of all neutralising agents, therefore the potency of the
Method for reconstitution disinfectant is not diminished after addition to the medium. Whereas,
when disinfectant is added to the D/E Neutralising Broth, its activity
Weigh 54.0 grams of powder and disperse in 1 litre of deionised water. is neutralised allowing for the detection of any bacteria presence. D/E
Allow to soak for 10 minutes. Sterilise by autoclaving at 121°C for Neutralising Broth contains thioglycollate to neutralise mercurial
15 minutes. Cool to 47°C and pour into sterile Petri dishes and allow compounds, sodium thiosulphate to neutralise iodine and chlorine
to set. and sodium bisulphite to neutralise formaldehyde and gluteraldehyde.
Appearance: Purple opaque gel. Lecithin is included to neutralise quaternary ammonium compounds
and Polysorbate 80 neutralises phenols, hexachlorophene, formalin,
pH: 7.6 ± 0.2 and combined with lecithin, ethanol. Bromocresol purple allows
detection of growth via a colour change from purple to yellow when
Minimum Q.C. organisms: organisms ferment the glucose contained in the medium.
Bacillus subtilis WDCM 00070
Escherichia coli WDCM 00013 Typical Formula g/litre
Pseudomonas aeruginosa WDCM 00025
Glucose 10.0
Staphylococcus aureus WDCM 00034 Lecithin 7.0
Storage of Powder: Store at 2-8°C in the dark. Formulation is very
hygroscopic, keep container tightly closed after use. Polysorbate 80 5.0
Storage of Prepared Medium: Plates – up to 7 days at 2-8°C in the Tryptone 5.0
dark.
Inoculation: Consult appropriate references as this product is used Sodium bisulphite 2.5
in several procedures. Yeast extract 2.5
Incubation: 37°C aerobically for 24-48 hours.
Sodium thioglycollate 1.0
Interpretation: Count all colonies for total counts, count yellow
colonies for differential acid producer count. Non-acid producing Bromocresol purple 0.02
colonies are grey to colourless.
Roberts, D., Hooper, W. and Greenwood, M., (1995). Methods for the Weigh 39.0 grams of powder and disperse in 1 litre of deionised
examination of food for micro-organisms of public health signiicance, water. Allow the mixture to soak for 10 minutes, swirl to mix and
2nd edition, section 5.10, Practical Food Microbiology. Butler & dispense into inal containers. Sterilise by autoclaving at 121°C for
Tanner. ISBN 0 901144 36 3. 15 minutes.
Engley Jr., F.B. and Dey, B.P. (1970). A universal neutralising medium Appearance: Purple opaque liquid.
for antimicrobial chemicals. Presented at the Chemical Specialities
pH: 7.6 ± 0.2
Manufacturing Association (CSMA) Proceedings, 56th Mid Year
Meeting.
Dey, B.P. and Engley Jr., F.B. (1995). Comparison of Dey and Bacillus subtilis WDCM 00070
Engley
(D/E) Neutralising medium to Lethhen medium and Standards Escherichia coli WDCM 00013
Methods Medium for recovery of Staphylococcus aureus from Pseudomonas aeruginosa WDCM 00025
sanitised surfaces. J. Ind. Microbiol. 14:21-25. Salmonella typhimurium WDCM 00031
Curry, A.S., Graf, J.G. and McEwen Jr., G.N. (ed.) (1993) CFTA Staphylococcus aureus WDCM 00034
Microbiology Guidelines. The Cosmetic, Toiletry and Fragrance
Association, Washington, D.C. Storage of Powder: Store at 2-8°C in the dark. The formulation is
very hygroscopic therefore keep the container tightly closed after
use.
Storage of Prepared Medium: Capped containers – up to 3 months
at 15-20°C in the dark.
Inoculation: Consult appropriate references as this product is used
in several procedures.
Interpretation: Examine all tubes for increased turbidity, formation
of a pellicle or a colour change from purple to yellow, indicating
bacterial growth.

References References
Roberts D., Hooper, W. and Greenwood, M., (1995). Methods Roberts, D., Hooper, W. and Greenwood, M., (1995). Methods
for the examination of food for micro-organisms of public health for the examination of food for micro-organisms of public health
signiicance, 2nd edition, section 5.10, Practical Food Microbiology. signiicance, 2nd edition, section 5.10, Practical Food Microbiology.
Butler & Tanner. ISBN 0 901144 36 3. Butler & Tanner. ISBN 0 901144 36 3.
Engley Jr., F.B. and Dey, B.P. (1970). A universal neutralising Engley Jr., F.B. and Dey, B.P. (1970). A universal neutralising
medium for antimicrobial chemicals. Presented at the Chemical medium for antimicrobial chemicals. Presented at the Chemical
Specialities Manufacturing Association (CSMA) Proceedings, 56th Specialities Manufacturing Association (CSMA) Proceedings, 56th
Mid Year Meeting. Mid Year Meeting.
Dey, B.P. and Engley Jr., F.B. (1995). Comparison of Dey and Dey, B.P. and Engley Jr., F.B. (1995). Comparison of Dey and Engley
Engley (D/E) Neutralising medium to Lethhen medium and Standards
(D/E) Neutralising medium to Lethhen medium and Standards Methods Medium for recovery of Staphylococcus aureus from
Methods Medium for recovery of Staphylococcus aureus from sanitised surfaces. J. Ind. Microbiol. 14:21-25.
sanitised surfaces. J. Ind. Microbiol. 14:21-25. Curry, A.S., Graf, J.G. and McEwen Jr., G.N. (ed.) (1993) CFTA
Curry, A.S., Graf, J.G. and McEwen Jr., G.N. (ed.) (1993) CFTA Microbiology Guidelines. The Cosmetic, Toiletry and Fragrance
Microbiology Guidelines. The Cosmetic, Toiletry and Fragrance Association, Washington, D.C..
Association, Washington, D.C.
D/E Neutralising Broth Base Dermatophyte Test Medium (D.T.M.)

(Dey & Engley) LAB117
LAB186 Description
A modiication of the formulation of Taplin, Zaias, Rebell and Blank
Description for the detection of dermatophytic fungi. This medium helps in the
D.E. Neutralising Broth Base is a nutritious medium deicient of differentiation between saprophytic and environmental fungi.
all neutralising agents. Therefore when a test disinfectant is added
to the broth, the potency is undiminished. Developed for use with Typical Formula g/litre
Dey and Engley’s Neutralising Broth (LAB187), incorporating D.E. Balanced Peptone No. 1 10.0
Neutralising Broth Base into the test procedure allows the user to
differentiate between bacteriostatic and bactericidal activity, and Glucose 40.0
to detect viable organisms that remain after treatment. Its use is
recommended in disinfectant evaluation, environmental sampling and Agar No. 2 12.0
water-miscible cosmetics in accordance with Cosmetic, Toiletry and Phenol Red 0.2
Fragrance Association (CTFA) guidelines..
Glucose 10.0 Allow to soak for 10 minutes then bring to the boil with frequent
stirring. Dissolve 2 vials of Chloramphenicol X009 (or 1 vial X209)
Tryptone 5.0 in ethanol and add these to the agar, mix well and distribute into tubes
Yeast extract 2.5 or universal containers. Sterilise at 121˚C for 15 minutes, allow to
cool in the sloped position.
Bromocresol purple 0.02 Note: Do not exceed the times stated for sterilisation, overheated
Grams per litre 17.5 acidiied agar loses gel strength and the sugars are caramelised.
Appearance: Orange, clear gel.
Weigh 17.5 grams of powder and disperse in 1 litre of deionised pH: 5.5 ± 0.2
water. Allow to soak for 10 minutes, swirl to mix and dispense into
inal containers. Sterilise by autoclaving for 15 minutes at 121oC. Minimum Q.C. organisms: Aspergillus spp. NCIMB 50097
Trichophyton spp.
Appearance:
Powder: ine, free-lowing, homogeneous, buff Storage of Prepared Medium: Slopes – up to 1 month at 2-8˚C in
the dark.
Finished medium: clear, purple liquid
Inoculation: Surface plating or stab inoculation.
pH: 7.6 ± 0.2
Incubation: 22-25˚C aerobically for 10-14 days.
Bacillus subtilis WDCM 00070 Interpretation: Dermatophytes appear as luffy colonies, colour
Escherichia coli WDCM 00013 varies with species, the medium is reddened. Fungi other than
Pseudomonas aeruginosa WDCM 00025 dermatophytes cause the medium to become yellow due to acid
Salmonella typhimurium WDCM 00031 production. If incubation is prolonged the medium may become
reddened. Yeasts appear as white creamy colonies. Blastomyces,
Histoplasma and Coccidiodes may also turn the medium red, though
these are rarely encountered in lesions associated with ring worm.
Storage of Powder: 10-25oC away from direct sunlight.
Storage of Prepared Medium: in capped containers for up to 3 References
months at 15-20°C in the dark. Taplin, D., Zaias, N., Rebell, G., Blank, H. (1969). Isolation and
Inoculation: Consult appropriate references as this product is used in recognition of dermatophytes on a new medium. (DTM) Arch.
several procedures. Dermatol. 99: 203-209.
Interpretation: Examine all tubes for turbidity, indicating growth.

Dextrose Tryptone Agar DG18 Agar
Dichloran (18%) Glycerol Agar
LAB020
Description
LAB218
A medium for the enumeration of thermophilic spore bearers in foods. Description
The medium was designed to detect the thermophilic bacteria causing
‘lat sour’spoilage of canned foods. The medium also detects the Lab M’s Dichloran 18% Glycerol Agar (DG18 Agar) is a medium for
‘lat sour’ organism Bacillus stearothermophilus in sugar and other the enumeration of osmophilic yeasts and xerophilic moulds in food
sweetening agents used in the preparation of frozen dairy foods, and animal products.
cereals and other food products. DG18 Agar is used for the enumeration of viable osmophilic yeasts
and xerophilic moulds in food or animal feed products with a water
Typical Formula g/litre activity of less than or equal to 0.95 by a colony count technique.
This includes such foods as dry fruits, jams, cakes, dried meat, salted
Tryptone 10.0 ish, grains, cereals, lours, nuts, spices, condiments and some animal
Glucose 5.0 feeds. This medium is not suitable for the examination of dehydrated
products with a water activity of less than or equal to 0.60 and does not
Bromocresol purple 0.04 allow the enumeration of mould spores or the detection of halophilic
xerophilic fungi found in dried ish.
Agar No. 2 12.0
The reduction in water activity in this medium is achieved by the
Method for reconstitution addition of glycerol at approximately 18% and this is very important
Weigh 27 grams of powder, disperse in 1 litre of deionised water. as many yeast and moulds actually require a low water activity to
Allow to soak for 10 minutes, swirl to mix then bring to the boil to enhance growth and colony development. The medium also contains
dissolve agar before dispensing in 20ml amounts for poured plate the antifungal agent dichloran, which restricts the spreading of
technique. Sterilise by autoclaving at 121˚C for 15 minutes. mucoraceous fungi and restricts the colony size of other genera
making colony counting an easier task.
Appearance: Purple clear agar.
Additional selectivity against bacterial growth is achieved by the
pH: 6.9 ± 0.2 incorporation of the heat-stable antibiotic Chloramphenicol. Glucose
is incorporated as the fermentable carbohydrate source, with casein
Minimum Q.C. organisms: B. stearothermophilus enzymatic digest providing the essential vitamins, minerals, amino
acids, nitrogen and carbon.
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the Developed with reference to ISO 21527-2:2008, this medium is tested
dark. Capped container – up to 3 months at 15-20˚C in the dark. to the performance requirements of the standard.
Inoculation: Pour plate technique, pre-heat sample by steaming for Typical Formula g/litre
20 minutes if a spore count is required.
Casein enzymatic digest 5.0
Incubation: For Thermophiles – Aerobically for 48 hours at 55˚C.
For Mesophiles – Aerobically for 48-72 hours at 30-32˚C. D-Glucose 10.0
Interpretation: Count all colonies for total counts, count yellow Potassium dihydrogen phosphate 1.0
colonies for differential acid producer count. Non acid producing
colonies are grey to colourless. Magnesium sulphate 0.5
Dichloran 0.002
Chloramphenicol 0.1
organism colony shape & colour
size (mm) surface Agar 15.0
B. stearothermophilus 2.0 Rz.D Yellow zone
mauve centre
Bacillus spp. 1.5-3.0 Rz.D Mauve
(Yellow halo)
Disperse 31.6g of powder in 1 litre of distilled water. Allow to soak
S. aureus 0.5-1.5 CV.E.G. Yellow for 10 minutes and swirl to mix. Add 220g Glycerol and if necessary,
heat gently to dissolve. Sterilise by autoclaving for 15 minutes at
E. coli 1.0-1.5 CV.E.G. Yellow
121°C. Cool to 47°C and mix well before dispensing into Petri dishes.
Klebsiella spp. 1.5-2.5 CV.E.G. Yellow (mucoid) Dry the agar surface prior to use.
Enterococci 0.5 CV.E.G. Yellow Appearance:
Proteus spp. 2.0-3.0 RzD Yellow (spreads) Powder: ine, free-lowing, homogeneous, buff
Finished medium: clear, straw gel
References pH: 5.6 ± 0.2
Williams, O.B. (1963). Tryptone Medium for the Detection of Flat
Sour Spores. Food Research 1, (3): 217-221. Hazard classiication
T – Toxic
American Public Health Association. (1972). Standard Methods
for the Examination of Dairy Products. 13th Edn. Ed. W.J. Hausler Minimum Q.C. organisms:
A.P.H.A. Washington. Saccharomyces cerevisiae WDCM 00058
Tanner, F.W. (1946). The Microbiology of Food 2nd edn., Garrard Escherichia coli WDCM 00013 (inhibited)
Press, Champners.
Baumgartner, J.G. and Hersom, A.C. (1956). Canned Foods. 4th Edn. Storage:
Churchill, London. Dehydrated culture media: 10-25°C away from direct sunlight.
Prepared media: 7 days at 2-8°C in the dark.
Inoculation (as per ISO 21527-2:2008): Inoculate plates in duplicate
with 0.1ml of test sample. Spread the liquid over the agar surface using
a sterile spreader until the liquid is completely absorbed.
Incubation (as per ISO 21527-2:2008): Incubate aerobically with
lids uppermost at 25°C ± 1°C for 5-7 days.
Interpretation (as per ISO 21527-2:2008): Read plates between 2 –
5 days. Select dishes containing less than 150 colonies/propagules and
count these colonies/propagules.
If fast-growing moulds are a problem, count colonies/propagules after
2 days and again after 5-7 days of incubation.

Bell, C., Neaves, P., Williams, A.P. (2005). Food microbiology and Weigh 53.0 grams of powder, disperse in 1 litre of deionised water.
laboratory practice. Blackwell, Oxford. p324. Mix well, bring quickly to the boil. Allow to cool to 47˚C and add
Beuchat, L.R. Media for detecting and enumerating yeasts and moulds. 1 vial of Novobiocin supplement – X150 (10mg/vial). Mix well and
In Corry, J.E.L., Curtis, GDW., Baird, R.M., Editors. Handbook of pour plates. Nitrofurantoin may be used instead of Novobiocin to
Culture Media for Food Microbiology, p369-386. improve the isolation of S. enteritidis.
Beuchat LR, Frandberg E, Deak T, Alzamora SM, Chen J, Guerrero AS, Appearance:- Green transparent, soft gel
López-Malo A, Ohlsson I, Olsen M, Peinado JM, Schnurer J, de Siloniz
MI, Tornai-Lehoczki J. (2001). Performance of mycological media in pH: 5.5 ± 0.2
enumerating desiccated food spoilage yeasts: an interlaboratory study.
Int. J. Food Microbiol. Oct 22;70(1-2):89-96. Minimum QC organisms:
BS ISO 21257-2:2008 Microbiology of food and animal feeding stuffs
Escherichia coli WDCM 00013
– Horizontal method for the enumeration of yeasts and moulds. Part
2: Colony count technique in products with water activity less than or
equal to 0,95. Storage of Prepared Medium: Plates – up to 7 days: at 2-8˚C in the
dark.
Deak, T., Chen. J., Golden D.A., Tapia, M.S., Tornai-Lehoczki, J.,
Viljoen, B.C., Wyder, M.T. Beuchat, L.R. (2001). Comparison of Inoculation: 3 drops (0.1ml) of 8 to 20hr. incubated pre-enrichment
dichloran 18% glycerol (DG18) agar with general purpose mycological broth are inoculated in one spot in the centre of one plate of
media for enumerating food spoilage yeasts. Int. J. Food Microbiol. Diassalm.
67, 49-53.
Hocking, A.D., Pitt, J.I. (1980). Dichloran-glycerol based medium for Incubation: At 42 ± 0.5˚C or 37˚C for 18-24 hours. Keep the lid
the enumeration of xerophilic fungi form low moisture foods. Appl. uppermost at all times.
Environ. Microbiol. 39, 488-492. Interpretation
After incubation the plates are examined for a mobility zone with a
purple/black colour change. When the mobility zone is absent, but the
Diagnostic Semi-Solid Salmonella centre is blackened, non-motile salmonellae may be present. A loopfull
of the motile zone which is the farthest from the sample
Agar (Diassalm) inoculum (or the blackened centre if non-motile) is sub-cultured onto
brilliant green agar and XLD agar. Futher biochemical and serological
According to Van Netten and Van der Zee et al identiication are performed according to recognised procedure.
Direct latex agglutination may also be carried out from the edge of
LAB537 the mobility zone.
Description References
Diassalm, as developed by Van Netten et al (1991), is a semi-solid Blazevics, D.J. (1968) Appl. Microbiol. 16, 688
differential medium for the isolation of Salmonella spp. from food De Smedt J.M. et al 1987 J. Food Protection 50, 658
and water. It is an improved modiication of MSRV (De Smedt and
Bolderdijk 1988) and SR (Perales and Audicana 1989) with regard Perales, I and Audicana. Evaluation of semi-solid Rappaport medium
to the composition of the basal medium, selective system and the for detection of Salmonellae in meat products. J. Food Protection 52,
introduction of a differential system. Van Netten, P., Van de Moosdijk, A., Perales, I. and Mossel, D.A.A.
The original basal medium was a commercially available sulphide Letters in Applied Microbiology
mobility-indole medium (SIM BBL) (Blazevic 1968). Lab M have Van Netten, P., Van der Zee, H., and Van der Moosdijk, A., (1991).
substituted their raw materials into Blazevic’s formula to create The use of diagnostic selective semi-solid medium for the isolation of
a richer base for Diassalm. Selectivity is achieved by the use of Salmonella enteritidis from poultry. Proceedings of the 10th
malachite green oxalate, magnesium chloride and novobiocin. Symposium on the quality of poultry meat, Spelderholt Beckbergen,
The diagnostic properties of Diassalm are based on the use of two pp. 59-67.
indicator systems; saccharose combined with bromocresol purple; Van der Zee, H., and Van Netten, P., (1992). Diagnostic semi-solid
and ferro-iron in combination with thiosulphate. media based on Rappaport-Vassiliadis Broth for the detection of
The eficiency of Diassalm is due to the ability of salmonellae to Salmonella spp. and S. enteritidis in foods. Proceedings of the
move through the highly selective mobility medium in a Petri dish, International Symposium of Salmonella and Salmonellosis.
whilst the double diagnostic system allows visualisation of motile and Van der Zee, H., (1992). Detection of Salmonella spp. with the use of
non-motile suspected salmonellae due to blacking zones against the a standard method, diagnostic semi-solid agars and immunocapture
turquoise background. Diassalm can be seeded after pre-enrichment kit. Proceedings Third World Congress Foodborne infections and
or after 8hr enrichment in selective broth (De Smedt and Bolderdijk intoxications, Berlin.
1987).

Tryptone 20.0
Meat Peptone 6.1
Ferrous ammonium sulphate 0.2
Sucrose 7.5
Lactose 0.5
Bromocresol purple 0.08
Malachite green oxalate 0.037
Magnesium chloride anhyd. 11.0
Agar No.1 2.8

DN’ase Agar DRBC Agar
Dichloran Rose Bengal Chloramphenicol Agar
LAB095
Description LAB217
DN’ase agar provides a convenient means of identifying potentially
pathogenic staphylococci, based on the ability of coagulase-positive Description
species to split DNA. DN’ases produced by the organisms hydrolyse Lab M’s Dichloran Rose Bengal Chloramphenicol Agar (DRBC
the DNA molecule to a mixture of smaller mono and poly nucleotides. Agar) is a medium for the enumeration of yeasts and moulds in food
DiSalvo observed perfect correlation between coagulase activity and and animal products.
DN’ase production using S. aureus strains from clinical specimens. Developed with reference to ISO 21527-1:2008, this medium is tested
Other publications have also reported a close correlation. to the performance requirements of the standard.
Used for the enumeration of viable yeasts and moulds in products with
Typical Formula g/litre a water activity of greater than 0.95 such as eggs, meat, some dairy
Tryptone 20.0 products, fresh pastes, fruit and vegetables, DRBC Agar is designed
to suppress the colonial growth of ‘spreader’ moulds and in doing so
Deoxyribonucleic acid (DNA) 2.0 allow easier performance of the colony count technique on yeasts and
moulds.
Sodium chloride 5.0
The use of the anti-fungal agent, dichloran, restricts spreading of
Agar No. 2 12.0 mucoraceous fungi and restricts the colony size of other genera. Rose
bengal also assists in the reduction of colony sizes and is selective
Method for reconstitution against bacteria.
Weigh 39 grams of powder, disperse in 1 litre of deionised water. Additional selectivity against bacterial growth is achieved by the
Allow to soak for 10 minutes, swirl to mix then sterilise by autoclaving incorporation of the heat-stable antibiotic Chloramphenicol. Glucose
at 121˚C for 15 minutes. Allow to cool to 47˚C then pour into Petri is incorporated as the fermentable carbohydrate source, with an
dishes. enzymatic digest of animal & plant tissues providing the essential
vitamins, minerals, amino acids, nitrogen and carbon.
Appearance: Pale cream, clear.
pH: 7.3 ± 0.2
Enzymatic digest of animal & plant tissues 5.0
Minimum Q.C. organisms: S. aureus NCIMB 50080
S. epidermidis NCIMB 50082 D-Glucose 10.0
Storage of Prepared Medium: Plates – up to 7 days: at 2-8˚C in the
dark. Capped container – up to 1 month at 4˚C in the dark. Magnesium sulphate 0.5
Inoculation: Use a heavy inoculum on a small area. Four or more Dichloran 0.002
organisms can be tested on one 90mm Petri dish. Chloramphenicol 0.1
Incubation: 37˚C aerobically for 18-24 hours. Rose bengal 0.025
Interpretation: Agar 15.0
Having obtained good growth lood the plate with 1N hydrochloric
acid. This will precipitate the DNA in the medium. DN’ase producing Grams per litre 31.7
organisms will be surrounded by a clear area where the DNA has
been broken down into fractions which are not precipitated by the Method for reconstitution
Hydrochloric acid. Gram positive, catalase positive cocci that produce Disperse 31.7g of powder in 1 litre of distilled water. Allow to soak for
DN’ase can be provisionally classiied as S. aureus, and conirmed 10 minutes, swirl to mix and sterilise by autoclaving for 15 minutes at
by tube coagluase or thermostable DN’ase tests. DN.’ase is also 121°C. Cool to 47°C and mix well before dispensing into Petri dishes.
produced by some Gram negative bacilli such as Serratia marcescens, Dry the agar surface prior to use.
Pseudomonas aeruginosa. Some corynebacteria and streptococci may
also produce DN’ase. Appearance:
References Powder: ine, free-lowing, homogeneous, pink
Baird-Parker, A. C. 1965. The classiication of staphylococci and Finished medium: clear, pink gel
micrococci from world-wide sources. J. Gen. Microbiol. 38, 363-387.
Black, W. A., Hodgson, R. and McKechnie, A. 1971. pH: 5.6 ± 0.2
DiSalvo, J. W. 1958 Deoxyribunuclease and coagulase activity of Hazard classiication
micrococci. Med. Tech. Bull. U.S. Armed Forces Med. J. 9, 191. T – Toxic
Martin, W. J and Ewing, W. H. 1967. The deoxryibonuclease test as Storage:
applied to certain gram-negative bacteria. Can. J. Microbiol. 13, 616-
618. Dehydrated culture media: 10-25°C away from direct sunlight.
Messinova, O. V., Yusupova, D. V. and Shamsutdinov, N. S. 1963. Prepared media: 7 days at 2-8°C in the dark.
Deoxyribonuclease activity of Corynebacterium and its relation to Inoculation (as per ISO 21527-1:2008): Inoculate plates in duplicate
virulence. Fed. Proc. 22, T1033. with 0.1ml of test sample. Spread the liquid over the agar surface
using a sterile spreader until the liquid is completely absorbed.
Streitfeld, M. M., Hoffmann, E. M. and Janklow, H. M. 1962.
Evaluation of extracellular deoxyribonuclease activity in Incubation (as per ISO 21527-1:2008): Incubate aerobically with
Pseudomonas. J. Bacteriol. 84, 77. Wannamaker, L. W. 1964. lids uppermost at 25°C ± 1°C for 5 days.
Streptococcal deoxryribonuclease, pp. 140-165. J. W. Uhr (ed.). The Interpretation (as per ISO 21527-1:2008): Read plates between 2 –
Streptococcus, Rheumatic Fever, Glomerulophritis. Baltimore: 5 days. Select dishes containing less than 150 colonies/propagules and
Williams & Williams. count these colonies/propagules.
Weckman, B. G. and Catlin, B. W. 1957 Deoxryribonuclease activity If necessary, use a magniier to distinguish between cells of yeasts or
of micrococci from clinical sources. J. Bacteriol. 73, 747-753. moulds and bacteria from colonies.
Zierdt, C. H. and Golde, D. W. 1970. Deoxryribonuclease-positive
Staphylococcus epidermidis strains. Appl. Microbiol. 20(1), 54-57.

References Appearance:
Bacteriological Analytical Manual, 8th edition, Revision A, 1998. Powder: ine, free-lowing, homogeneous, buff
Chapter 18 Yeasts, Molds and Mycotoxins. Authors: Valerie Tournas,
Michael E. Stack, Philip B. Mislivec, Herbert A. Koch and Ruth Finished medium: clear, straw liquid
Bandler. Revised: 2000-APR-17 pH: 7.1 ± 0.2
Beuchat and Cousin (2001). In Downes and Ito (ed.). Compendium of Hazard classiication: NR – Not regulated
Methods for the Microbiological Examination of Foods, 4th edition.
American Public Health Association. Storage:
BS ISO 21257-1:2008 Microbiology of food and animal feeding stuffs Dehydrated culture media: 10-25°C away from direct sunlight.
– Horizontal method for the enumeration of yeasts and moulds. Part Prepared media: 7 days at 2-8°C in the dark.
1: Colony count technique in products with water activity greater than
0.95. Inoculation (as per BS EN 26461-1:1993): Before the test, the
sample of water should be heated in a water bath at 75 ± 5°C for 15
King Jr, A.D., Hocking, A.D. and Pitt, J.I. (1979). Dichloran-Rose minutes from the time it reaches that temperature.
Bengal Medium for Enumeration and Isolation of Molds from Foods.
J. Appl. Environ. Microbiol. 1979, 37, 959-964. Add 50ml of sample to 50ml double strength medium (x5).
Add 10ml of sample to 10ml double-strength medium (x5).
Add 1ml of sample to 25ml single-strength medium (x5).
If required add 1ml of a 1 in 10 dilution of the sample to 25ml single-
DRCM (ISO) strength medium (x5).
To qualitatively examine 100ml drinking/bottled water without
Differential Reinforced Clostridial Medium (ISO) performing MPN, add 100ml sample to 100ml double-strength
medium.
LAB220 If required, top up all bottles with single-strength medium to bring the
volume of liquid level with the neck of the bottle, and to ensure that
Description only a very small volume of air remains. Seal the bottles hermetically,
or incubate under anaerobic conditions.
Differential Reinforced Clostridial Medium ISO (DRCM) is a medium
for the detection and enumeration of the spores of sulphite-reducing Incubation (as per BS EN 26461-1:1993): Incubate aerobically with
anaerobes as described in BS EN 26461-1. lids uppermost at 37°C ± 1°C for 44 ± 4 hours.
Sulphite reducing anaerobes, in particular clostridia, can be indicators Large volumes of culture in hermetically sealed glass bottles may
of remote and intermittent pollution. Widespread in the environment, explode due to gas production. The addition of iron wire, heated
to redness and placed into the medium before inoculation, may aid
being found in human and animal faeces, soil and waste water, the anaerobiosis
spores are more resistant to physical and chemical factors than
vegetative cells and able to survive for long periods in water. The Interpretation (as per BS EN 26461-1:1993): Bottles in which
spores may also be resistant to chlorination at the levels commonly blackening is observed, as a result of the reduction of sulphite and the
used in water treatment. precipitation of iron (II) sulphide, shall be regarded as positive.
DRCM has been developed for use with the Most Probable Number References
(MPN) method to determine the MPN of anaerobes (Clostridia) per BS EN 26461-1:1993 / BS 6068-4.8:1993 / ISO 6461-1:1986. Water
volume of sample. The formulation includes peptone, yeast extract, quality – Detection and enumeration of he spores of sulite-reducing
meat extract, starch & L-cysteine for nutrition with glucose providing anaerobes (clostridia) – Part 1: Method by enrichment in a liquid
the energy source. Sodium acetate provides partial selectivity. medium.
Clostridia are able to reduce sulphite to sulphide – forming iron Freame, B. & Fitzpatrick, B.W.F. (1967). The use of Differential
sulphide. Iron (III) citrate is included in the formulation as an indicator Reinforced Clostridial Medium for the isolation and enumeration of
of sulphite reduction. Blackening in the medium indicates that iron Clostridia from foods. The Society for Applied Microbiology Technical
sulphide has been formed and therefore that sulphite reduction has Series n. 5: Isolation of Anaerobes, ed. Shapton, D.A. & Board, R.G.
occurred. Vol. 5. London Academic Press. 49-55.
Other bacteria are able to form sulphide, so vegetative cells must be Gibbs, M.B. (1973). The detection of Clostridium welchii in the
irst be removed from the test sample by an appropriate process e.g. Differential Reinforced Clostridial Medium technique. J. Appl. Bact.
heat treatment. 36. 23-33.
Peptone mix 10.0
Yeast extract 1.5
Starch 1.0
Hydrated sodium acetate 5.0
Glucose 1.0
L-Cysteine hydrochloride 0.5
Sodium sulphite 0.4
Iron (III) citrate 0.7

Disperse 30.1g of powder in 1 litre of distilled water. Allow to soak for
10 minutes, swirl to mix and dispense into inal containers. Sterilise by
autoclaving for 15 minutes at 121°C.
Medium should be used on day of preparation. If medium is stored,
tubes should be reheated to deoxygenate the medium. Tubes should
not be reheated more than once.

EC Medium E.E. Broth
(Escherichia coli Medium) (Enterobacteriaceae Enrichment Broth)
LAB171 LAB091
Description Description
EC Medium (Escherichia coli Medium) is a selective enrichment E.E. Broth is recommended as an enrichment medium when examining
broth designed for the isolation of coliforms, including E. coli, from food and feedstuffs for Enterobacteriaceae. It is a modiication of
water and food samples. It was the recommended medium of the LAB051 Brilliant Green Bile Broth, with an improved buffering
American Public Health Association (APHA) and the AOAC. capacity to encourage early growth and prevent autosterilization. E.E.
Broth uses glucose instead of lactose to make the medium a test for all
EC Medium is made selective for coliforms by the inclusion of Bile enterobacteria including non lactose fermenting organisms.
Salts No.3 in the dehydrated medium. The selective nature of this
medium ensures that the growth of non-coliform bacteria is minimised. Typical Formula g/litre
The medium is buffered by the addition of potassium phosphates
and osmotically balanced by sodium chloride. The medium is used Balanced Peptone No. 1 10.0
at 37°C for coliform organisms and 45.5°C is recommended for the
isolation E. coli. Dextrose 5.0
Disodium hydrogen phosphate 6.45
Tryptone 20.0
Bile Salts 20.0
Lactose 5.0
Brilliant green 0.0135
K2HPO4 4.0
KH2PO4 1.5 Method for reconstitution
Sodium chloride 5.0 Weigh 43.5 grams of powder and add to 1 litre of deionised water.
Swirl to dissolve, warm gently if necessary, then distribute into
Bile Salts No. 3 1.5 bottles or tubes and heat at 100˚C for 30 minutes only. Cool rapidly.
OVERHEATING THIS MEDIUM WILL ADVERSELY AFFECT
Method for reconstitution ITS PERFORMANCE.
Weigh 37.0 grams of powder and disperse in 1 litre of deionised water. Appearance: Green, clear.
Allow the mixture to soak for 10 minutes, swirl to mix. Dispense
into tubes of appropriate volume and, where applicable, add Durham pH: 7.2 ± 0.2
tubes. Sterilise by autoclaving at 121°C for 15 minutes.
Minimum Q.C. organisms: E. coli WDCM 00013
Appearance: Clear straw broth. B. subtilis WDCM 00070 (inhibition)
pH: 6.9 ± 0.2
Storage of Prepared Medium: capped containers – up to 3 months
Escherichia coli WDCM 00013 Inoculation: Add 1 part of sample suspension or dilution to 10 parts
Enterococcus faecalis WDCM 00087 (inhibition) of medium.
Bacillus subtilis WDCM 00070 (inhibition) Incubation: 44˚C for 18 hours for thermotrophs. 32˚C for 24-48
hours for mesotrophs. 4˚C for 10 days for psychrotrophs.
Storage of Prepared Medium: Capped containers – up to 3 months Interpretation: Turbidity and a colour change to yellow-green is
at 15-20°C in the dark. presumptive evidence of Enterobacteriaceae. Subculture onto
Inoculation: Coliforms: Follow the methods and procedures as stated conirmatory media e.g. LAB088 V.R.B.G.A. must be carried out.
in Standard Methods for the Examination of Water and Wastewater
and Compendium of Methods for the Microbiological Examination References
of Foods. Mossel, D. A. A., Visser, M. and Cornelissen, A. M. R. 1963. The
Incubation: 45.5°C for 18-24 hours aerobically for E. coli and 37°C examination of foods for Enterobacteriaceae using a test of the type
for 18-24 hours, aerobically for coliforms. generally adopted for the detection of salmonellae. J.Appl. Bacteriol.
26, 444-452.
Interpretation: Turbidity of broth and gas collection in the Durham
tube indicates the presumptive growth of organisms from the coli- Mossel, D. A. E., Harrewijn, G. A. and Nesselrooy-van Zadelhoff,
aerogenes group. All broths should be sub-cultured onto selective C. F. M. 1974. Standardisation of the selective inhibitory effect
media whether turbid or not. of surface active compounds used in media for the detection of
Enterobacteriaceae in food and water. Health Lab. Sci. 11, 260-267.
References Richard, N. 1982. Monitoring the quality of selective liquid media
American Public health Association, (1980). Standards Methods for by the oficial French dilution technique used for the bacteriological
the Examination of Water and Wastewater, 15th Edition, American examination of foods. In: Quality assurance and quality control of
Public Health Association, Inc., Washington, D.C. microbiological culture media, edited by J. E. L. Corry, G.I.T.-Verlag
Darmstadt, pp. 51-57.
American Public health Association, (1976). Compendium of
Methods for the Microbiological Examination of Foods, American
Public Health Association, Inc., Washington, D.C.
Association of Oficial Analytical chemists. (1995). Bacteriological
analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
Perry and Hajna, (1943). American Journal of Public Health, 33:550.
Perry and Hajna, (1944). American Journal of Public Health, 34:735.

Endo Agar Eosin Methylene Blue Agar (Levine)
LAB060 LAB061
This medium was developed in 1914 for the isolation of Salmonella This medium was introduced in 1916 by Holt-Harris and Teague to
typhi; other media have since proved superior for this purpose, but differentiate Escherichia spp. and Aerobacter spp. It was modiied by
Endo Agar has a role as a coliform medium. It is recommended by
Levine in 1918 who removed sucrose from the formula and increased
the American Public Health Association as a standard medium for the the lactose content. The distinctive metallic sheen produced by E.
enumeration of coliforms in water and dairy products. In this medium coli on this medium is due to acid production resulting in an amide
acetaldehyde is produced by coliforms and then ixed by the sulphite
bonding between the eosin and methylene blue, other coliforms do not
to produce a metallic sheen with the basic fuchsin dye. Most enteric produce enough acid to cause this reaction. Eosin inhibits most Gram
Gram negative organisms will grow well, whilst Gram positive positive organisms. The prepared medium is sensitive to light.
organisms are mostly inhibited.
Peptone 10.0
Lactose 10.0
Lactose 10.0
Dipotassium phosphate 2.0
Eosin Y 0.4
Sodium sulphite 2.5
Methylene Blue 0.065
Agar No. 1 15.0
Agar No. 2 15.0
Weigh 41 grams of powder, disperse in 1 litre of deionised water. Method for reconstitution
Add 4ml of a 10% w/v alcoholic solution of basic fuchsins (95% Weigh 37.5 grams of powder, disperse in 1 litre of deionised water.
ethyl alcohol). Bring to the boil with frequent swirling to dissolve the Allow to soak for 10 minutes, swirl to mix then sterilise by autoclaving
solids. Sterilise by autoclaving at 121˚C for 15 minutes. Cool to 47˚C at 121˚C for 15 minutes. Cool to 50˚C and agitate gently to ensure
in a water bath before pouring. The precipitate typically associated uniform distribution of the locculant precipitate (which is a feature of
with this medium should be dispersed by gentle swirling prior to this medium) before pouring into Petri dishes.
pouring the plates. STORE IN THE DARK.
This medium is light sensitive and should therefore be stored in the Appearance: Blue/purple with a light precipitate.
dark, preferably under refrigeration. The medium will become dark
red in colour if exposed to light. pH: 6.8 ± 0.2
Basic Fuchsin is a potential Carcinogen and care should be taken
when handling it to avoid inhalation of the powdered dye and
contamination of the skin.
Appearance: Pale pink/orange dark.
pH: 7.5 ± 0.2 Inoculation: Surface, streaking for single colonies.
Minimum Q.C. organisms: E. coli WDCM 00013 Growth Characteristics
colony shape &
organism size (mm) surface colour other
dark.
E. coli 2.0-3.0 CV.E.G. Blue Black (Metallic
Inoculation: Surface, streaking out for single colonies. sheen)
Incubation: 37˚C for 18-48 hours aerobically. Klebsiella spp. 3.0-4.0 CV.E.G. Brown
Blue (mucoid)
Growth Characteristics Salmonella spp. 2.0-3.0 CV.E.G. Colourless

colony size shape & S. aureus P.P. CV.E.G. Colourless
E. faecalis P.P. CV.E.G. Colourless
E. coli 1.0-2.0 CV.E.G. Deep Red (Metallic sheen)
K. aerogenes 1.0-2.5 CV.E.G. Red (mucoid) References

Proteus spp 2.0-3.0 CV.E.G. Pale Pink American Public Health Association, American Water Works
colourless Association and Water Pollution Control Federation, (1975).
Standard Methods for the Examination of Water and Wastewater, 14th
Gram positive no growth. Edn., Washington, D.C. American Public Health Association.
organisms
Girolami, R.L. and Stamm, J.M. (1976). Inhibitory effect of light on
growth supporting properties of Eosin Methylene Blue Agar. Appl.
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the Environ. Microbiol., 31:1 141-142.
dark.
Haesler, W. J. (ed) (1972). Standard Methods for the Examination of
Inoculation: Surface, streaking out for single colonies. Dairy Products, 13th edn., Washington, D.C., American Public Health
Incubation: 37˚C for 18-48 hours aerobically. Association.
References
Levine, M. (1918). Differentiation of E. coli and B. aerogenes on a
Endo, 1914, Centr. Bakt., Abt 1, Orig., 35: 109. simpliied Eosin-Methylene Blue agar. J. Infect. Dis., 23: 43-47.
American Public Health Association, 1975. Standard Methods for the
Examination of Water and Wastewater, 14th Edn. American Public
Health Association, Inc. Washington D.C.
American Public Health Association, 1972. Standard Methods for the
Examination of Dairy Products, 13th End., American Public Health
Association, Inc., Washington, D.C.

Eugon Agar Frank, H.A. (1955). The inluence of various media on spore count
determinations of a putrefactive anaerobe. J. Bacteriol. 70:269.
(Eugonic Agar) Vanderzant, C. and Splittstoesser, D.F. (ed.). (1992). Compendium of
methods for the microbiological examination of food, 3rd ed. American
LAB525 Public Health Association, Washington, D.C.
Isenberg, H.D. (ed.) (1992). Clinical microbiological procedures
Description handbook, American Society for Microbiology, Washington, D.C.
Eugon Agar is used for the cultivation of a wide variety of Murray, P.R. et al (ed) (1995). Manual of Clinical Microbiology, 6th
microorganisms, particularly in mass cultivation procedures. ed. American Society for Microbiology, Washington, D.C.
The medium is prepared according to the formulation of Vera and Association of Oficial Analytical Chemists. (1995). Bacteriological
was developed to obtain eugonic (luxuriant) growth of fastidious analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
microorganisms. The medium can be used with additions to enhance
its performance with certain microorganisms, e.g. Eugon Agar
supplemented with 5% sterile deibrinated blood will enable the
growth of pathogenic fungi such as Nocardia, Histoplasma and
Blastomyces.
Eugon Broth
(Eugonic Broth)
Niven reported Eugon Agar for the detection of lactic acid bacteria
in cured meats and recommended it for investigating spoilage in
meats. Harrison and Hansen employed the medium for plate counts LAB526
of the intestinal lora of turkeys and Frank showed its use for the
germination of anaerobic spores pasteurised at 104°C. Eugon Agar Description
is also speciied in the APHA Compendium of Methods for the This is the broth version of Eugon Agar (LAB525) for the cultivation
Microbiological Examination of Food. of a wide variety of microorganisms, particularly in mass cultivation
The high sugar content of this medium dictates that it not suitable as procedures. The medium is prepared according to the formulation
a base for haemolytic reactions. of Vera and was developed to obtain eugonic (luxuriant) growth of
fastidious microorganisms. The medium can be used with additions
Typical Formula g/litre to enhance its performance with certain microorganisms e.g. Eugon
Broth supplemented with 5% sterile deibrinated blood the medium
Tryptose 15.0 will support the growth of pathogenic fungi such as Nocardia,
Soy Peptone 5.0 Histoplasma and Blastomyces.
Dextrose 5.5 Typical Formula g/litre

L-Cystine 0.7 Tryptose 15.0
Sodium chloride 4.0 Soy Peptone 5.0
Sodium sulphite 0.2 Dextrose 5.5
Agar 15.0 L-Cystine 0.7
Method for reconstitution Sodium chloride 4.0

Weigh 45.4 grams of powder and disperse in 1 litre of deionised Sodium sulphite 0.2
water. Allow the mixture to soak for 10 minutes, swirl to mix and then
sterilise by autoclaving at 121°C for 15 minutes. Cool to 47°C before Method for reconstitution
the addition of supplements or pouring into sterile Petri dishes.
Appearance: Light amber clear gel, may contain a slight precipitate. Allow the mixture to soak for 10 minutes, swirl to mix and sterilise
by autoclaving at 121°C for 15 minutes. Cool before the addition of
pH: 7.0 ± 0.2 enrichments and aseptically dispense into appropriate containers.
Minimum QC organisms: Appearance: Light amber solution, may contain a slight precipitate.
Aspergillus niger ATCC 16404
pH: 7.0 ± 0.2
Candida albicans ATCC 10231
Lactobacillus fermentum ATCC 9388 Minimum QC organisms:
Streptococcus pyogenes NCTC 8198 Aspergillus niger ATCC 16404
Storage of Prepared Medium: Plates can be stored up to 7 days at Lactobacillus fermentum ATCC 9388
2-8°C in the dark. Streptococcus pyogenes NCTC 8198
Inoculation: For the examination of clinical specimens for bacteria
and fungi refer to the appropriate published references. For the Storage of Prepared Medium: Store the prepared medium at
examination of food for the examination of bacteria and fungi refer 2-8°C.
to standard methods.
Inoculation: For the examination of clinical specimens for bacteria
Incubation: 35°C ± 2°C for up 72 ± 4 hours for bacteria. 30°C ± 2°C and fungi refer to the appropriate published references.
for up 72 ± 4 hours for fungi.
Incubation: 35°C ± 2°C for up 72 ± 4 hours for bacteria. 30°C ± 2°C
Interpretation: Refer to appropriate references and procedures. for up 72 ± 4 hours for fungi.
References Interpretation: Refer to appropriate references and procedures.
Vera, H.D. (1947). The ability of peptones to support surface growth References
of lactobacilli. J. Bacteriol. 54:14.
Vera, H.D. (1947). The ability of peptones to support surface growth
MacFaddin, J.D. (1985). Media for the isolation-cultivation- of lactobacilli. J. Bacteriol. 54:14.
identiication-maintenance of medical bacteria. 301-303. vol. 1.
Williams & Wilkens, MD. MacFaddin, J.D. (1985). Media for the isolation-cultivation-
identiication-maintenance of medical bacteria. 301-303. vol. 1.
Niven (1949). J. Bacteriol. 58:633. Williams & Wilkens, MD.
Harrison, A.P.Jr. and Hansen, P.A. (1950). The bacterial lora of the Isenberg, H.D. (ed.) (1992). Clinical microbiological procedures
cecal feces of healthy turkeys. J. Bacteriol. 59. 197. handbook, American Society for Microbiology, Washington, D.C.
Murray, P.R. et al (ed) (1995). Manual of Clinical Microbiology, 6th
ed. American Society for Microbiology, Washington, D.C.

Fastidious Anaerobe Agar (F.A.A.) Growth Characteristics (48 hours)
colony shape &
LAB090 organism size (mm) surface colour other
Bacteroides 1.0 - 2.0 CV.E.G. Grey
Description fragilis
A primary isolation medium capable of growing most clinically Clostridium 1.0 - 2.0 CV.E.G Grey ‘Target’
signiicant anaerobes. Developed by Lab M, comparisons have Perfringens haemolysis
shown this medium to be superior to other formulations as a primary (non haemolytic)
isolation medium for fastidious organisms. The peptones included Fusobacterium 1.0 - 2.0 CV.E.G (D) trans- (grey)
have been chosen for maximum growth stimulation. Starch and necrophorum parent (haemolytic)
sodium bicarbonate act as de-toxiication agents whilst haemin Porphyromonas 1.0 - 2.0 CV.E.G Grey /
encourages pigment production in Porphyromonas melaninogenicus. asaccharolyticus Brown (clearing)
Speciic growth promoting agents are Cysteine for Fusobacterium Peptostreptococcus 0.5 - 2.0 CV.E.G White /
necrophorum, Propionibacterium acne and Bacteroides fragilis, anaerobius Grey
arginine for Eubacterium spp. soluble pyrophosphate for Porph. Actinomyces israeli 0.5 - 1.0 CV.E.G White (‘molar tooth’)
gingivalis and Porph. asaccharolytica. Pyruvate helps neutralise (smooth)
hydrogen peroxide and is also utilised by Veillionella spp. as an
energy source. Vitamin K and sodium succinate provide essential References
growth factors for some anaerobes as does the 0.1% glucose. The low
level of glucose prevents the production of high levels of acids and Brazier, J.S. (1986). Yellow luorescence of Fusobacteria Letters in
alcohols which would inhibit colonial development. Applied Microbiol. 2: 124-126.
Brazier, J.S. (1986). A note on ultra violet red luorescence of
Typical Formula g/litre anaerobic bacteria in vitro. J. Appl. Bact. 60: 121-126.
Peptone mix 23.0 Eley, A., Clarry, T., Bennett, K.W. (1989). Selective and differential
medium for isolation of Bacteriodes ureolyticus from clinical
Sodium chloride 5.0 specimens. European Journal of Clinical Microbiology, Infectious
Diseases. 8: 83-85.
Soluble starch 1.0
Wade W. Grifiths, M. (1987). Comparison of Media for cultivation of
Agar No. 2 12.0 subgingival bacteria. J. Dent. Res. 66: no. 4 abstract 334.
Sodium bicarbonate 0.4 Heginbotham M., Fitzgerald T.C., and Wade W.G. (1990).
Comparison of solid media for the culture of anaerobes. J. Clin. Path.
Glucose 1.0 43: 253-256.
Sodium pyruvate 1.0
Cysteine HCl monohydrate 0.5
Fastidious Anaerobe Broth (F.A.B.)
Haemin 0.01
Vitamin K 0.001
LAB071
L-Arginine 1.0 Description
F.A.B. was developed by Lab M working in conjunction with the
Soluble pyrophosphate 0.25 microbiology department of a University of Manchester teaching
Sodium succinate 0.5 hospital. The medium was designed to give optimum growth of
fastidious anaerobes and has found applications as a blood culture
medium and an enrichment broth for the isolation of anaerobes.
Method for reconstitution The medium is very rich in nutrients from the specially selected
Weigh 46 grams of powder and add to 1 litre of deionised water. Allow peptone mixture. Vitamin K. haemin and L-cysteine are all growth
to soak for 10 minutes, swirl to mix then sterilise by autoclaving at factors required by some anaerobes. L-cysteine together with sodium
121˚C for 15 minutes. Cool to 47˚C then aseptically add 5-10% of thioglycollate reduce the Eh of the medium and the agar content
sterile deibrinated horse blood, mix well and pour into Petri dishes. inhibits absorption of oxygen and convection currents. Resazurin is a
This medium can be made selective for various species of anaerobes redox indicator. Several published evaluations show F.A.B. to be the
by the addition of appropriate selective cocktails e.g. liquid medium of choice for fastidious anaerobes.
Gram negative anaerobes X090
Non-sporing anaerobes X291
Clostridium dificile X093 Peptone mixture 15.0
Appearance: Red due to addition of blood. The blood will darken Yeast Extract 10.0
(reduce) because of the presence of reducing agents.
pH: 7.2 ± 0.2 Sodium chloride 2.5
Minimum Q.C. organisms: B. fragilis ATCC 25285 Agar No. 1 0.75
P. anaerobious ATCC 27337 L-Cysteine HCl 0.5
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the Resazurin 0.001

dark. Sodium bicarbonate 0.4
Inoculation: Surface plating, streaking out to single colonies.
Haemin 0.005
Incubation: 37˚C anaerobically with 10% CO2 for 48 hours to 5
days. Vitamin K 0.0005

Method for reconstitution Fluid Thioglycollate Medium (Clear)
Weigh 29.7 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix. Boil to dissolve the agar LAB425
then dispense into screw cap containers. Sterilise by autoclaving
at 121˚C for 15 minutes. Tighten the caps as soon as possible after
Description
autoclaving.
A medium for sterility tests to promote growth in both Aerobic and
Appearance: Pale straw, clear, viscous. May have a narrow band of anaerobic organisms even from small inocula. In appropriate tubes or
red/purple at the surface due to action of oxygen on the resazurin. bottles the thioglycollate ensures adequate anaerobic conditions. The
If the medium is reddish this indicates too much oxygen has been low level of agar reduces oxygen diffusion into the medium.
absorbed, the medium should be reheated to deoxygenate. Do not
reheat more than once. Performance of this medium complies with the requirements
pH: 7.2 ± 0.2 described in the EP/USP/JP for Fluid Thioglycollate Medium.
Minimum Q.C. organisms: Bacteroides fragilis ATCC 25285 Typical Formula g/litre
Tryptone 15.0
at 15-20˚C in the dark. L-Cystine 0.5
Inoculation: If used as a blood culture medium a minimum dilution Glucose 5.5
of 1:10 should be used.
Yeast Extract 5.0
Incubation: 37˚C for 24-72 hours. Keep the container airtight.
Growth indicators: The broth may become turbid or individual Sodium chloride 2.5
colonies may form suspended in the medium.
References Resazurin 0.001
Gould, J.H., Duerden, B.I. (1983). Blood culture – current state and
future prospects. J. Clin. Pathol. 36: 963-977. Gelling agent 0.75
Ganguli, G.A., O’Hare, W., Hyde, W.A. (1984). Rapid Detection of Grams per litre 29.75
Bacteraemia by early subculture. J. Med. Microbiol. 17: 311-315.
Ganguli, L.A., Keaney, M.G.L., Hyde, W.A., Fraser, B.J. (1985). More Method for reconstitution
Rapid identiication of bacteraemia by manual rather than radiometric Weigh 29.75 grams, disperse in 1 litre of deionised water. Soak for 10
methods. J. Clin. Pathol. 38: 1146-1149. minutes, swirl to mix, then bring to the boil to dissolve and dispense
Junt, G.H., Price, E.H. (1982). Comparison of a home made into suitable airtight containers. Sterilise by autoclaving for 15
blood culture broth containing a papain digest of liver, with four minutes at 121°C. Store the medium between 2°C - 25°C in the dark.
commercially available media, for the isolation of anaerobes from If more than 30% of the medium turns pink (oxidised) the Eh may
simulated paediatoic blood cultures. J. Clin. Pathol. 35: 1142-1149. be restored (once only) by heating in a boiling water bath or by free-
Ganguli, L.A., Turton, L.J., Tillotson, G.S. (1982). Evaluation of lowing steam. Take care to cool quickly after heating and prevent the
Fastidious Anaerobe Broth as a blood culture medium. J. Clin. Pathol. introduction of non-sterile air into the container.
35: 458-461.
Appearance:
Tillotson, G.S. (1981). Evaluation of ten commercial blood culture
systems to isolate a pryridoxal dependent streptococcus. J. Clin. Powder: ine, free - lowing, homogeneous, buff
Pathol. 34: 930-934. Finished medium: Pale straw colour, clear. Surface may be pink due
to oxidation of Resazurin
pH: 7.1 ± 0.2

Clostridium sporogenes ATCC 19404
Staphylococcus aureus ATCC 6538
Storage:
Prepared media: Capped container – up to 3 months at 15-20°C in
the dark.
Incubation: 30-35°C aerobically for 14 days.
Interpretation: Turbidity, colonies in medium.

Fluid Thioglycollate Medium Fraser Broth
(USP/EP/JP) LAB164
HP001 Description
Description Developed as a modiication of UVM II medium, Fraser broth is a
A medium recommended by the Harmonised European secondary enrichment broth for the isolation of Listeria spp., and
Pharmacopoeia for sterility testing. Conforms to USP/EP/JP is similar to Palcam broth in that it contains aesculin to indicate
performance speciication. Casein and yeast extract provide a source the presence of a potential Listeria isolate. It also contains lithium
of nitrogen, essential vitamins and amino acids. The glucose provides chloride in an attempt to suppress the growth of enterococci in the
a carbon source and sodium chloride maintains osmotic balance. medium (as does Palcam). Fraser broth may also be used as a primary
L-Cystine and sodium thioglycollate act as reducing agents to create enrichment medium by incorporating 1/2 strength supplement into
an anaerobic environment and maintain a low Eh. This is aided by the broth base (X164 or X564).
the low level of agar which reduces the oxygen permeability through
the medium. Resazurin is an oxidation indicator which turns from Typical Formula g/litre
colourless to red/pink when oxidised. Sodium thioglycollate also
serves to inactivate mercurial compounds. If after sterilisation more Peptone mixture 15.0
than the upper one third of the medium has become red/pink it may be
Yeast extract 5.0
restored once by heating in a water bath or in free-lowing steam until
the colour disappears. Ensure the media is cooled quickly and prevent Aesculin 1.0
the introduction of non-sterile air into the containers.
Typical Formula g/litre Potassium dihydrogen phosphate 1.35
L-Cystine 0.5 Sodium chloride 20.0
Agar 0.75 Lithium chloride 3.0
Sodium chloride 2.5
Glucose anhydrous 5.0 Weigh 55 grams of power and add to 1 litre of deionised water (add
to 900ml if preparing 1/2 Fraser). Allow to soak for 10 minutes, swirl
Yeast extract 2.5 to mix and sterilise at 121˚C for 15 minutes. Cool to 47˚C and add 2
Pancreatic digest of casein 15.0 vials of Fraser supplement X165 (or 2 vials of 1/2 Fraser supplement
X164), mix well and aseptically dispense into sterile tubes or bottles.
Sodium thioglycollate 0.3 Appearance: Straw opalescent broth with precipitate (clears on
Resazurin 0.001 storage).
pH 7.2 ± 0.2
Disperse 26.55 grams of powder in 1 litre of deionised water. Allow to Minimum Q.C. organisms:
soak for 10 minutes, swirl to mix and bring to the boil. Distribute into Listeria monocytogenes WDCM 00021
suitable vessels and sterilise at 121°C for 15 minutes. E. coli (inhibition) WDCM 00013
Appearance:
Storage of Prepared Medium: Bottles – up to 14 days at 2-8˚C.
Powder: ine, free-lowing, homogeneous, buff Inoculation: 1/2 Fraser – Add 25g sample to 225ml of 1/2 Fraser broth
Finished medium: straw, clear to slight haze with red/pink layer at the and homogenise Fraser – Subculture 0.1ml of primary enrichment
broth (UVM I or 1/2 Fraser) into 10ml of Fraser broth.
top of the medium
Incubation: 1/2 Fraser – 30˚C aerobically for 24hrs.
pH: 7.1 ± 0.2
Fraser – 35˚C aerobically for 24hrs and 48hrs. Subculture onto
selective agars at 24 and 48hrs.
Staphylococcus aureus ATCC 6538 Interpretation
Bacillus subtilis ATCC 6633 Blackening of the broth indicates the presence of a potential Listeria
Pseudomonas aeruginosa ATCC 9027 and should be subcultured onto Listeria isolation medium (Oxford)
Candida albicans ATCC 10231 LAB122 or Palcam agar LAB148. All broths should be subcultured
Clostridium sporogenes ATCC 19404 before discarding, irrespective of colour change.
Aspergillus brasiliensis ATCC 16404
References
Hazard classiication: NR – Not regulated Fraser J.A., and Sperber W.H., (1988) Rapid detection of Listeria
spp in food and environmental samples by esculin hydrolysis. J.Food
Storage: Protection 51 (10) 762-765.
Dehydrated culture media: 10-25°C away from direct sunlight. McClain D., and Lee W.H. (1989) FSIS method for isolation of
Prepared media: 7 days at 2-8°C in the dark. L.monocytogenes from processed meat and poultry products.
Lab.Comm.No.57, Revised May 24, (1989). US Dept of Agric.FSIS,
Use: According to the sterility protocol deined in the Harmonised Microbiol. Div.
European Pharmacopoeia the samples are incubated in portions of the
medium at 30-35°C for 14 days. No growth of micro-organisms is
required for a sterility pass.
According to the growth promotion test deined in the Harmonised
European Pharmacopeia, Clostridium sporogenes, Pseudomonas
aeruginosa and Staphylococcus aureus are inoculated (with not more
than 100 CFU) and incubated for not more than 3 days. The media
is suitable if a clearly visible growth of the micro-organisms occurs.
Interpretation:
Growth is indicated by turbidity, refer to speciic guidelines as deined
in the Harmonised European Pharmacopoeia.
References

Fraser Broth PLUS (ISO) G.C. Agar Base
LAB212 LAB067
A secondary enrichment broth for the isolation of Listeria spp. A nutritious agar base described by Thayer and Martin for the
formulated according to ISO 11290. The selective components isolation of Neisseria gonorrhoeae. The rich peptone mixture is
acrilavine and nalidixic acid are blended into the base powder and enhanced by the use of corn starch to absorb toxic metabolites and
the ferric ammonium citrate (X211) is added to the tempered broth a buffering system is used to maintain neutral pH. The medium is
after sterilisation. made selective by the use of various antibiotic cocktails. Thayer and
Martin originally recommended the use of vancomycin, colistin and
Typical Formula g/litre nystatin V.C.N. but the addition of trimethoprim (X068) is useful in
preventing the swarming of proteus. More recently the emergence
Peptone mixture 15.00 of vancomycin sensitive gonococci has made the New York City
Yeast extract 5.00 selective agents (lincomycin, colistin, amphotericin, trimethoprim
X070, LCAT) the combination of choice. Enrichment of the base is
Aesculin 1.00 usually by the addition of lysed blood. Alternatively chocolated blood
or haemoglobin powder and Thayer and Martin’s mixture of vitamins,
Disodium hydrogen phosphate 9.60 amino acids and coenzymes can be used. The growth supplement
Potassium dihydrogen phosphate 1.35 X271 can be added to this medium to aid in the isolation of Neisseria
spp.
Special Peptone 15.0
Acrilavine 0.025
Corn Starch 1.0
Nalidixic acid 0.02
Sodium chloride 5.0
Method for reconstitution Dipotassium hydrogen phosphate 4.0
Allow to soak for 10 minutes, swirl to mix and sterilise by autoclaving Potassium dihydrogen phosphate 1.0
for 15 minutes at 121°C. Cool to 47°C and add 2 vials X211. Mix well Agar No. 2 10.0
and dispense into sterile containers..
Appearance: Method for reconstitution
Powder: ine, free-lowing, homogeneous, buff Weigh 36 grams of powder, disperse in 1 litre of deionised water. Allow
to soak for 10 minutes, swirl to mix then sterilise by autoclaving at
Finished medium: straw opalescent broth with yellow luorescence 121°C for 15 minutes. Cool to 48°C and add 50-70ml of lysed blood
(in inal medium). and 2 vials of X070 selective agent. Mix well and pour into Petri
dishes.
pH: 7.2 ± 0.2
Hazard classiication Appearance: Dependent on blood supplement used.
NR – Not regulated pH: 7.2 ± 0.2
Minimum Q.C. organisms: Minimum Q.C. organisms: N. gonorrhoeae NCTC 8375

Listeria monocytogenes WDCM 00021 E. coli (inhibition) WDCM 00013
Enterococcus faecalis WDCM 00087 Storage of Prepared Medium: Plates – up to 7 days at 2-8°C in the
dark.
Storage:
Incubation: 37°C microaerobically for 24-48 hours.
Final medium: 14 days at 2-8°C in the dark
Inoculation: Sub-culture 0.1mL of LAB211 into 10mL of LAB212 Growth Characteristics
Fraser BrothPLUS (ISO). colony size shape &
Incubation: 35-37°C aerobically for 24hrs and 48hrs. Sub-culture organism (mm) surface colour other
onto selective agar at 24 and 48 hours. N. gonorrhoeae 1.0-2.0 CV.E.G. Transparent variations in
Interpretation: Blackening of the broth indicates the presence of a colony size
potential Listeria spp. and should be sub-cultured onto a selective
Listeria isolation medium, e.g. Harlequin™ Listeria Chromogenic References
Agar (HAL010). All broths should be sub-cultured before discarding
irrespective of colour change. Young, H. 1978. Cultural diagnoses of gonorrhoea with modiied
New York City (MNYC) medium. Brit. Journ. Ven. Dis. 54: 36-40:
References Thayer, J. D. and Martin, J. E. 1966. Improved medium selective for
Fraser J.A. and Sperber W.H. (1988). Rapid detection of Listeria spp the cultivation of N. gonorrhoeae and N. Meningitidis: Public Health
in food and environmental samples by esculin hydrolysis. J. Food rep. 81: 559-562.
Protect. 51, No.10, 762-765.
McClain D. and Lee W.H. (1989). FSIS method for isolation of L.
monocytogenes from processed meat and poultry products. Lab.
Comm.No.57, Revised May 24, (1989). US Dept of Agric.FSIS,
Microbiol. Div.
ISO 11290-1:1997 (Microbiology of food and animal feeding stuffs
- Horizontal method for the enumeration of Listeria monocytogenes -
part 1, Incorporating Amendment 1.)

GVPC Legionella Isolation Medium pH: 6.9 ± 0.1
Inoculation:
(BCYE basal medium) The concentrated sample should be split into 3 portions. One portion
is used without any further treatment, the other 2 portions should be
LAB195 treated, one with heat and the other with acid.
Description Heat Treatment
BCYE (Buffered Charcoal Yeast Extract) Legionella Isolation Take 1ml of the concentrated sample and place in a water bath at 50ºC
Medium (LAB195) is a base medium used for the isolation of for 30 minutes.
Legionella from clinical and environmental samples. This medium Acid Treatment
is based on the charcoal yeast extract formulation of Feeley et al.1&2
Take 1-10ml of the concentrated sample and centrifuge at 6000g for
The performance of this medium is further enhanced by the additions
10 minutes. Decant the supernatant to leave half the original volume.
of ACES (N-2-acetamido-2-aminoethane - sulphonic acid) buffer
Vortex to re-suspend the pellet and make up to the original volume
and α-ketoglutarate as deined by Edelstein 3. This medium is also
using an HCl-KCl buffer. Leave to stand for 5 minutes.
detailed in internationally recognized methodology 4 for the isolation
of Legionella spp. from water. Inoculate the irst plate of GVPC supplemented media with 0.1mL
of the untreated portion and spread over the entire surface of the
Specimens or samples are often heavily contaminated with other
plate. Inoculate the second plate of GVPC supplemented media in the
bacteria and consequentially a range of selective supplements have
same way with 0.1ml of the heat treated portion as soon as possible
been developed to aid isolation. Lab M provide the GVPC supplement
after removal from the water bath. Inoculate the third plate of GVPC
(X195) which is most effective for the isolation of L. pneumophila.
supplemented media in the same way with 0.1mL of the acid treated
It is recommended that this supplement is used in conjunction with
portion immediately after acid treatment.
heat and acid sample treatments, to further reduce the growth of non-
Legionella bacteria. Incubation:
This product contains the ACES buffer and ferric pyrophosphate in Incubate at 36 + 1ºC in a humid atmosphere under aerobic conditions
the base medium. This negates the need for complex freeze dried for up to 10 days.
supplements. A complementary growth supplement is provided Interpretation:
(X196) which contains the L-cysteine and α-ketoglutarate.
The plates should be examined for growth on days 3, 5, 7 and 10.
Suspect colonies should be sub-cultured on to “maintenance”
Principle of isolation
supplemented BCYE medium and “presumptive ID” supplemented
Water samples are concentrated either by membrane iltration or
BCYE medium, incubate as before. Isolates that fail to grow on the
centrifugation (turbid samples may also be centrifuged). To reduce the
growth of unwanted bacteria, separate portions of the concentrated “presumptive ID” medium but grow on the maintenance medium
sample may be subjected to heat and acid treatments. Treated and and have typical morphology should be regarded as presumptive
untreated portions are then inoculated onto Legionella selective media. Legionella.
Presumptive isolates should be conirmed using a serological method,
e.g. Microgen M45 Latex.
Yeast Extract 10.0 Minimum Q.C. organisms: Legionella spp. - Growth
Staphylococcus epidermidis - Growth
Charcoal 2.0
Escherichia coli - Inhibited
Ferric 0.25
Pyrophosphate ACES Buffer 10.0 References
Feeley, J.C., Gibson, R.J. et al. (1979). Journal of Clinical
Potassium Carbonate 2.28 Microbiology 10: 437-441
Agar 14.0 Pesculle, A.H., Feeley, J.C. et al. (1980). Journal of Infectious Disease
141: 727-732
Supplements Edelstein, P.H. (1982). Journal of Clinical Microbiology 14: 298-303
International Standard. ISO 11731:1998(E). Water Quality – Detection
GVPC Selective Supplement (X195) & Enumeration of Legionella.
Typical Formula
Glycine 3000mg
Vancomycin 1mg
Half Fraser Broth PLUS (ISO)
Polymyxin B 79200IU LAB211
Cycloheximide 80mg Description
Primary enrichment broth for the isolation of Listeria spp. formulated
according to ISO 11290. The selective components acrilavine
BCYE Growth Supplement (X196) and nalidixic acid are blended into the base powder and the ferric
ammonium citrate (X211) is added to the tempered broth after
Typical Formula
sterilisation.
L-Cysteine 400mg
Half Fraser Broth Plus (ISO), LAB211 was developed to give
improved results for the isolation of Listeria spp. with both traditional
and ELISA methods. As with the sister product, Fraser Broth Plus
Method for reconstitution (ISO), LAB212 the selective components acrilavine and nalidixic
Selective Isolation (GVPC BCYE) acid are blended into the base powder and the Ferric Ammonium
Weigh 38.5 grams of powder and disperse in 1 litre of deionised Citrate (X211) is added to the tempered broth after autoclaving. This
water. Soak for 10 minutes, swirl to mix and sterilise by autoclaving format has been shown to give improved selectivity with pure cultures
at 110ºC for 10 minutes. Cool to 47ºC and aseptically add 2 vials of and food samples. Furthermore, more stable ELISA results are seen,
reconstituted growth supplement X196 and 2 vials of reconstituted resulting in fewer false positive results when the ferric ammonium
selective supplement X195. Mix well and pour into sterile Petri dishes. citrate is omitted from the complete media.
Developed as a modiication of UVM medium and made according
to ISO 11290, Fraser Broth is a secondary enrichment broth for the
isolation of Listeria spp., and is similar to Palcam Broth in that it
contains aesculin to indicate the presence of a potential Listeria isolate.

It also contains lithium chloride in an attempt to suppress the Typical Formula g/litre
growth of Enterococci in the medium (as does Palcam). Listeria
spp. hydrolyse the aesculin to form aesculetin, which reacts with the Meat Peptone 12.0
ferric ammonium citrate in X211 resulting in a black precipitate and a
visible positive reaction. However, Enterococci can also perform this Yeast Extract 3.0
reaction, so further plating is required onto an isolation medium such Lactose 12.0
as Harlequin™ Listeria Chromogenic Agar ISO (HAL010).
Note: Acrilavine and Nalidixic Acid in LAB211 are half-strength of Sucrose 12.0
Fraser BrothPLUS (ISO) LAB212. Salicin 2.0
Typical Formula g/litre Bile Salts No. 3 7.0
Peptone mixture 15.00 Sodium desoxycholate 2.4
Yeast extract 5.00 Sodium chloride 5.0
Aesculin 1.00 Sodium thiosulphate 5.0
Disodium hydrogen phosphate 9.60 Ammonium ferric citrate 1.5
Potassium dihydrogen phosphate 1.35 Acid fuchsin 0.1
Sodium chloride 20.00 Bromothymol blue 0.065
Lithium chloride 3.00 Agar No. 1 14.0
Acrilavine 0.0125 Method for reconstitution

Nalidixic acid 0.01 Weigh 76 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then heat gently and bring
Method for reconstitution to the boil. Cool to 47˚C and pour plates. DO NOT AUTOCLAVE OR
OVERHEAT THIS MEDIUM.
Allow to soak for 10 minutes, swirl to mix and sterilise by autoclaving Appearance: Green, clear.
for 15 minutes at 121°C. Cool to 47°C and add 2 vials X211. Mix well
and dispense into sterile containers. pH: 7.5 ± 0.2
Appearance: Minimum Q.C. organisms:

Powder: ine, free-lowing, homogeneous, buff Shigella sp.
Finished medium: straw opalescent broth with yellow luorescence E. coli (some inhibition) WDCM 00013
(in inal medium).
pH: 7.2 ± 0.2 dark.
Inoculation: Surface plating, streak out to single colonies.
Listeria monocytogenes WDCM 00021 Incubation: 37˚C aerobically for 18-24 hours.
Escherichia coli WDCM 00013 Growth Characteristics
colony size shape &
Storage: organism (mm) surface colour other
Dehydrated culture media: 10-25°C H2S +ve 2-3 CV.E.G. Green+

Final medium: 14 days at 2-8°C in the dark Salmonella Black
Inoculation: Add 25g of sample to 225mL LAB211 and homogenise. H2S -ve 2-3 CV.E.G. Green
Sub-culture 0.1mL of LAB211 into 10mL of LAB212 Fraser BrothPLUS Salmonella
(ISO). S. sonnei 2-2.5 CV.E.G. Green (Rough)
E. coli 0.5-2 CV.E.G. Salmon ppt. (Rough)
Incubation: 30°C aerobically for 24 hours. around (No growth)
Interpretation: Blackening of the broth indicates the presence of colonies
a potential Listeria spp. All broths should be sub-cultured before Citrobacter spp. 1.0-2.0 CV.E.G. Salmon (Rough)
discarding irrespective of colour change. Proteus spp. 1.0-2.0 CV.E.G. Green/ (No growth)
References Black (brownish centre)
Fraser J.A. and Sperber W.H. (1988). Rapid detection of Listeria spp centre
in food and environmental samples by esculin hydrolysis. J. Food
Protect. 51, No.10, 762-765. References
King, S. and Metzger, W.I. (1967). A new medium for the isolation of
McClain D. and Lee W.H. (1989). FSIS method for isolation of L. Salmonella and Shigella species. Bact. Proc. Am. Soc. Microbiol. 77.
monocytogenes from processed meat and poultry products. Lab.
Comm.No.57, Revised May 24, (1989). US Dept of Agric.FSIS, King, S. and Metzger, W.I. (1968). A new plating medium for
Microbiol. Div. the isolation of enteric pathogens. Hektoen Enteric Agar, Appl.
Microbiol., 16(4), 577.
ISO 11290-1:1997 (Microbiology of food and animal feeding stuffs
- Horizontal method for the enumeration of Listeria monocytogenes - King, S. and Metzger, W.I. (1968). A new plating medium for the
part 1, Incorporating Amendment 1.) isolation of enteric pathogens. II. Comparison of Hektoen Agar with
SS and EMB agar. Appl. Microbiol., 16(4), 579.
Speck, M.L. (ed.). (1976). Compendium of Methods for the
Hektoen Enteric Agar Microbiological Examination of Food. Washington, D.C.: American
Public Health Association.
LAB110
Description Hoyle’s Medium
A medium developed at the Hektoen Institute in Chicago for the
enhanced recovery of shigellae from clinical specimens. This medium (modiied)
has high levels of peptones and sugar which counteract some of the
toxic effects of bile salts used to make the medium selective. This LAB027
allows the shigellae to grow as well as the salmonellae. Salicin is
fermented by many coliforms including those that do not ferment Description
lactose and sucrose. The medium employs a double indicator system A highly selective culture medium for the isolation and differentiation
similar to that used in LAB006 C.L.E.D., (Bevis) and an H2S indicator of Corynebacterium diphtheriae types gravis, mitis and intermedius.
system similar to that used in LAB032 XLD. Although intended This product, based on Hoyle’s medium gives rapid growth of all
primarily for clinical use this medium is quoted in B.S. 4285 as types of C. diphtheriae, which results in most specimens giving
suitable for the examination of dairy products for salmonellae. adequate growth with overnight incubation.

Typical Formula g/litre Appearance:
Powder: ine, free-lowing, homogeneous, buff with brown lecks
Peptone 1.0 Finished medium: clear, light tan gel
Yeast Extract 4.0
pH: 7.1 ± 0.2
Sodium chloride 4.0
Sugars 1.0 NR – Not regulated
Agar 12.0 Method for reconstitution
Disperse 23 grams of powder in 1 litre of deionised water. Allow
Method of Reconstitution to soak for 10 minutes, swirl to mix, then sterilise by autoclaving
Weigh 37 grams of powder and disperse in 1 litre of deionised water. at 121°C for 15 minutes. Cool to 50°C and mix well before
Allow to soak for 10 minutes, and sterilise by autoclaving at 121˚C for dispensing.
15 minutes. Cool to 47˚C, add 50ml of lysed horse or sheep blood and
10ml of X027 potassium tellurite solution. Mix well before pouring. Storage
Dehydrated culture media: 10-25°C.
Appearance: Dark Red, clear gel Final medium: Use on day of preparation
pH: 7.8 ± 0.2 Inoculation
Use ‘deep shake’ or ‘Attenborough and Scarr overlay’ methods
Inoculation: Spread the entire surface with the swab or sample for inoculation.
under investigation. Hoyle’s medium is very selective and spreading Deep-Shake Culture Method
for single colonies using a wire loop is not necessary. Use of a non- Dispense the medium in 10ml volumes in tubes. Inoculate the
selective blood agar alongside Hoyle’s is recommended. sample when the medium is at approximately 50°C. Allow to set.
Incubation: 37˚C for 18-48 hrs, aerobically Attenborough and Scarr Method
Storage: Plates – up to 7 days at 2-8˚C This membrane ilter technique is quicker, of comparable
accuracy and permits the examination of larger samples.
Interpretation In this method, diluted samples of sugar or any other food are
iltered through membrane ilters. These ilters are then rolled up
colony size shape & and placed in tubes containing just suficient Iron Sulphite Agar
organism (mm) surface colour other (at 50°C) to cover them. The medium is allowed to set.
C. diphtheriae 0.5-2.0 CV.E.G. Grey Easily
var mitis (dark centre) emulsiied Incubation
Streptococcus pp-1.5 CV.E.G Black Enterococci may Incubate for 24-48 hours at 55°C for thermophilic organisms or
Spp. be larger 37°C for mesophilic organisms. May also be used for mesophillic
sulphite reducers if incubated at 37°C.
Minimum Q.C. Organisms C. diphtheriae var mitis Interpretation
(non-toxigenic) NCTC 13056 Deep-Shake Culture Method
E. coli WDCM 00013 (inhibition) Typical thermophilic species, e.g. Desulfotomaculum nigriicans
, produce distinct black spherical colonies in the depth of the
Reference: medium.
Hoyle L. (1941) A Tellurite Blood Agar Medium for the Rapid Attenborough and Scarr Method
Diagnosis of Diphtheria. Lancet 1 175-176 Count the number of black colonies on the membrane ilter.
Conirmation tests should be carried out to identify the organism
176. Elek S.D. (1948) The Recognition of Toxigenic Bacterial Strains growing in the medium.
in vitro. Brit. Med. J. 1 493-496. The blackening reaction is only presumptive evidence of
clostridial growth. Conirmation tests must be carried out for
identiication. There are many gram-negative bacteria that are
able to reduce sulite with iron sulide production in this medium,
but in these cases the enzymes are extra cellular and the entire
Iron Sulphite Agar medium becomes dark, rendering their enumeration impossible.
LAB222 Desulfotomaculum nigriicans ATCC 7946
E.coli WDCM 00013
Description C.sporogenes WDCM 00008
Iron Sulphite Agar is a medium for the detection of thermophilic
anaerobic organisms causing sulphide spoilage in food. References
This formulation is a modiication of Cameron Sulphite Agar, which Attenborough, S.J. & Scarr, P.M. (1957). J. Appl. Bact. 20. p460-
was developed by the National Canners Association of America (now 466.
the Grocery Manufacturers Association). Beerens, H. (1958) DSIR. Proc. 2nd Internat. Symp. Food.
Iron Sulphite Agar has a reduced concentration of sodium sulphite to Microbiol. 1957, HMSO, London, pp. 235-245.
allow improved detection of some strains of Clostridium sporogenes. Bufton, A.W.J. (1959). J. Appl. Bact. 22. p278-280.
Beerens, and later Mossel, demonstrated that some strains of Mossel, D.A.A., Golstein Brouwers, G.W.M.V. & de Bruin, A.S.
C. sporogenes would not tolerate sodium sulphite levels of 0.1%. (1959). J. Path. Bact. 78. 290-291.
Mossel further observed that reducing sulphite content to 0.05% Tanner, F.W. (1944). The Microbiology of Foods, 2nd edition,
improved detection of these strains. Garrard press, Illinois, p1127.
Tryptone provides nitrogen and other nutrients necessary to support
bacterial growth. The presence of sulphite reducing bacteria is
indicated by the formation of black colonies. These colonies form
when bacteria reduce sulphite to sulphide, which reacts with iron (III) Kanamycin Aesculin Azide Agar
citrate to yield a black precipitate. (K.A.A. Agar)
LAB106
Tryptone 10.0
Description
Sodium sulphite 0.5
A selective isolation and enumeration medium for enterococci
Iron (III) citrate 0.5 (Lanceield group D streptococci) in food. Sodium azide and
kanamycin provide the selective inhibition required whilst aesculin
Agar 12.0 and iron salts form an indicator system for the presumptive
Grams per litre 23.0 identiication of enterococci. Incubation at 42˚C will increase the
medium’s selectivity.

Typical Formula g/litre Minimum Q.C. organisms: E. faecalis WDCM 00087
Tryptone 20.0
Yeast Extract 5.0
Sodium chloride 5.0 at 15-20˚C in the dark.
Sodium citrate 1.0 Inoculation: Inoculate tubes with decimal dilutions of food
suspension.
Aesculin 1.0 Incubation: 37˚C or 42˚C aerobically for 18-24 hours.
Ferric ammonium citrate 0.5 Interpretation: Blackening of the medium suggests the presence of
enterococci/faecal streptococci.
Sodium azide 0.15
Kanamycin sulphate 0.02 References
Mossel, D.A.A., Bijken, P.H.G., Eelderink, I. and. van Spreekens.
Agar No. 1 10.0 K.A. (1978). Streptococci, edited by Skinner, F.A. and Quesnel, L.B.
SAB Symposium Series No. 7 Academic Press, London.
Weigh 43 grams of powder, disperse in 1 litre of deionised water. Allow
121˚C for 15 minutes. Cool to 47˚C, then dispense into Petri dishes.
Kligler Iron Agar
Appearance: Pale straw, clear. LAB059
pH: 7.0 ± 0.2 Description
A differential medium for the recognition of enteric pathogens by
Minimum Q.C. organisms: E. faecalis WDCM 00087 their ability to ferment glucose and/or lactose, and liberate sulphides.
E. coli (inhibition) WDCM 00013 Fermentation liberates acid, with or without gas, turning phenol
red indicator yellow. Fermentation of glucose only, is followed by
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the reversion in pH on the slope, from initial acidity to inal alkalinity (red
dark. colour), but not in the anaerobic conditions of the butt, which remains
acid (yellow). Fermentation of lactose as well as glucose, produces
Inoculation: Surface, spread 0.1ml to 0.5ml over entire surface of acidity in both slope and butt (yellow). Liberation of sulphide results
plate. in the formation of iron sulphide (blackening of either slope or butt).
Incubation: 37˚C or 42˚C aerobically for 18-24 hours.
Interpretation: Count all white/grey colonies, approx 2mm diameter,
surrounded by a black halo to give presumptive enterococcus/faecal Peptone 20.0
streptococcus count.
Lactose 10.0
References
Mossel, D.A.A., Bijken, P.H.G., Eelderink, I. and van Spreekens, Glucose 1.0
K.A. (1978). Streptococci, edited by Skinner, F. A. and Quesnel, Sodium chloride 5.0
L.B. SAB Symposium Series No. 7 Academic Press, London.
Ferric ammonium citrate 0.5
Kanamycin Aesculin Azide Broth Phenol red 0.025
(K.A.A. Broth) Agar No. 2 12.0
LAB107 Method of reconstitution

Weigh 49 grams of powder and mix with 1 litre of distilled water.
Description Bring to the boil with frequent stirring to dissolve completely.
Dispense into tubes and sterilise for 15 minutes at 121˚C. Cool in a
An enrichment and isolation medium for enterococci. The medium
slanted position such that slopes are formed over deep butts approx.
can be used with the M.P.N. technique to enumerate enterococci
3cm in depth.
in food. This broth is identical to LAB106 K.A.A. agar with the
omission of the agar. Appearance: Reddish brown agar.
pH: 7.4 ± 0.2
Tryptone 20.0 Minimum Q.C. Organisms Salmonella typhimurium
WDCM 00031
Yeast Extract 5.0
Pseudomonas aeruginosa
Sodium chloride 5.0 WDCM 00025
Sodium citrate 1.0
Inoculation
Aesculin 1.0 Subcultures for further identiication are picked from the centre of
isolated colonies on selective media and streaked across the slant and
stabbed deep into the butt of tubes of Kligler Iron Agar.
Sodium azide 0.15
Incubation: 37˚C aerobically for 18-24 hours.
Kanamycin sulphate 0.02
Interpretation
Method for reconstitution Organism Butt Slope Sulphide
Salmonella typhi Acid Alkaline +
Allow to soak for 10 minutes, warm gently to dissolve completely
then disperse into tubes or bottles. Sterilise by autoclaving at 121˚C S. paratyhi A+ B Acid Alkaline -
for 15 minutes.
Other Salmonella Acid/gas Alkaline +
Appearance: Light straw, clear. E. coli Acid/gas Acid -
pH: 7.0 ± 0.2 Proteus spp Acid/gas Alkaline +
Shigella sonnei Acid Alkaline -
S. lexneri Acid Alkaline -

Storage: Tightly capped containers - up to 3 months at 15-20˚C in Method for reconstitution
the dark. Weigh 35.6 grams of powder and disperse in 1 litre of deionised water.
Allow the mixture to soak for 10 minutes, swirl to mix and dispense
References: Kligler, I.J. (1917). A Simple Medium for the into tubes or bottles containing inverted Durham tubes. Sterilise by
Differentiation of Members of the Typhoid - Paratyphoid Group. Am. autoclaving at 121°C for 15 minutes.
J. Publ. Hlth, 7:1042-1044.
Bailey, S.F. and Lacey, G.R. (1927). A modiication of the Kligler Appearance: straw, clear liquid.
Lead Acetate Medium. J. Bact. 13:182-189. pH: 6.8 ± 0.2

Lactose Broth Escherichia coli WDCM 00013
Enterococcus faecalis WDCM 00087 (inhibition)
LAB126
Storage of Prepared Medium: Store the prepared medium at room
Description temperature (18-22°C), in the dark.
A medium used for the performance and conirmation of the Pre-
Inoculation: Inoculate the medium in accordance with standard
sumptive Test for members of the coliform group in water and dairy
methods or laboratory policy.
products.
Incubation: 35°C ± 2°C for 24 and 48 hours.
Formula g/litre Interpretation: After incubation at 35°C for 24 hours examine for
Beef Extract 3.0 turbidity and gas production. If no gas has formed incubate for a
further 24 hours and re-examine.
Gelatin Peptone 5.0
Turbidity in the medium accompanied by the formation of gas within
Lactose 5.0 48 hours is a presumptive result for the presence of coliforms. The
results should be conirmed by standard testing methods.
References
Weigh 13 grams of powder, disperse in 1 litre of deionised water, heat
American Public Health Association (1980) Standard Methods for the
to dissolve then distribute into bottles with Durham tubes. Sterilise by
Examination of Water and Wastewater. 15th Edn. APHA Inc.
autoclaving at 121˚C for 15 minutes. Washington DC.
Appearance: Straw coloured, clear. American Public Health Association (1978) Standard Methods for
the Examination of Dairy Products. 14th Edn. APHA Inc. Washington
pH: 6.9 ± 0.2
DC.
Minimum Q.C. organisms: E. coli WDCM 00013 American Public Health Association (1976) Standard Methods for the
Examination of Foods. 15th Edn. APHA Inc. Washington DC.
Storage of Prepared Medium: Capped containers – up to 3 months Mallmann, W.L. and Darby, C.W. (1941) Am. J. Pub. Hlth. 31. 127-
at 15-20˚C in the dark. 134.
Inoculation: See methods for standard techniques. ISO Standard 11866-2 Milk and Milk Products –Enumeration of
Incubation: 35˚C aerobically for 48 hours. presumptive Escherichia coli – part 2: Most probable number
technique using 4-methyl umbelliferyl-β-D-glucuronide.
Interpretation: Coliforms are presumptively identiied by their
ability to ferment lactose and produce gas within 48 hours at 35˚C.
References Letheen Agar

American Public Health Association. (1975). Standard Methods for (Tryptone Glucose Extract Agar with Lecithin
the examination of water and waste water, 892. Washington. United and Polysorbate 80)
States Pharmocopeia, XXI, 1985.
LAB185
Lauryl Tryptose Broth
Description
(Lauryl Sulphate Broth, LTB, LSB)
Letheen Agar is used for evaluating the bactericidal activity of
quaternary ammonium compounds, and is used with Letheen Broth
LAB196 to determine the suitability of preservatives for use in cosmetic
formulations, as speciied by the American Society for Testing and
Description Materials (ASTM), Standard Test Method for Preservatives in
Lauryl Tryptose Broth is a selective medium for the detection of Water-Containing Cosmetics. Letheen Agar is a modiication of
coliforms in water, dairy products and other foods. The American Tryptone Glucose Extract (TGE) Agar, and is formulated to neutralise
Public Health Authority (APHA) recommend Lauryl Tryptose quaternary ammonium compounds used in testing of germicidal
Broth for the Most Probable Number Presumptive Test of coliforms activity, the importance of which was irst described by Weber and
in waters, efluent or sewage and as a conirmation test of lactose Black in 1948. The addition of Polysorbate 80 means Letheen Agar
fermentation with gas production from milk samples and for the also neutralises phenols, hexachlorophene, formalin and ethanol
detection of coliforms in foods. (in the presence of lecithin). Letheen Agar also allows calculation
Lauryl Tryptose Broth is prepared according to the formulation of of colony forming units to be assessed, when used with a hygiene
Mallmann and Darby. Mallmann and Darby showed that tryptose at swabbing protocol and will ensure against disinfectant carry-over
a concentration of 2% increased the early logarithmic growth phase from the swabbing diluent/medium.
when compared to meat peptone. These researchers added phosphate
buffers and sodium chloride, which improved gas production by Typical Formula g/litre
“slow lactose fermenting” organisms. Sodium lauryl sulfate was Dextrose 1.0
incorporated as a selective agent for the inhibition of non-coliform
organisms. Tryptone 5.0
This medium can also be used with the addition of MUG Beef extract 3.0
(4-methylumbelliferyl-β-D-glucuronide) according to the ISO
Standard 11866-1 to give enhanced detection of Escherichia coli. Lecithin 1.0
Typical Formula g/litre Polysorbate 80 7.0

Agar 15.0
Tryptose 20.0
Lactose 5.0
Sodium chloride 5.0
Dipotassium hydrogen phosphate 2.75
Sodium lauryl sulphate 0.1

Method for reconstitution Minimum Q.C. organisms:
Weigh 32.0 grams of powder and disperse in 1 litre of deionised Escherichia coli ATCC 11229
water. Allow the mixture to soak for 10 minutes, swirl to mix, and Staphylococcus aureus ATCC 6538
then sterilise by autoclaving at 121°C for 15 minutes. Cool to 47°C
and pour into sterile Petri dishes and allow the medium to set.
Storage of Powder: Store at 2-8°C in the dark. Formulation is very
Appearance: Straw, opalescent gel. hygroscopic, keep container tightly closed after use.
pH: 7.0 ± 0.2 Storage of Prepared Medium: Capped containers – up to 3 months
Minimum Q.C. organisms: Inoculation: There are a variety of methods which use Letheen Broth
Escherichia coli ATCC 11229 and the appropriate references should be consulted. For example:
Phenol co-eficient testing – Subculture from disinfectant dilutions
into 10ml volumes of Letheen Broth
Storage of Dehydrated Medium: Store at 2-8°C in the dark.
Formulation is very hygroscopic, keep container tightly closed after Hygiene swabbing – Swab measured area or speciic equipment and
use. place in 10ml volume of Letheen Broth. Area to be swabbed and
volume of medium may vary depending upon swabbing protocol
Storage of Prepared Medium: Plates – up to 7 days at 2-8°C in the used.
dark. Incubation: 37°C aerobically for 24-48 hours.
Inoculation: Preservative testing - From the dilutions of product Interpretation: Examine all tubes for turbidity or as stipulated in the
in Letheen Broth, subculture to Letheen Agar using a pour plate method.
technique, or surface inoculation.
References
Hygiene swabbing – Subculture from the swab diluent using a pour
plate or surface inoculation to allow calculation of the colony forming American Society for Testing Materials, (1998). Standard Test Method
units (cfu) for the area swabbed. for Preservatives in Water-Containing Cosmetics. E640-78.
Annual Book of ASTM Standards, Philadelphia, PA.
Incubation: 37°C aerobically for 24-48 hours. Association of Analytical Chemists, (1995). Oficial methods of
Interpretation: Count all colonies and calculate the number of cfu analysis, 16th edition, section 6. Association of Oficial Analytical
per ml of sample allowing for dilution factors, or the cfu of the area Chemists, Washington, D.C.
swabbed (typically 25cm2). Roberts, D., Hooper, W. and Greenwood, M. (1995). Methods
for the examination of food for micro-organisms of public health
References signiicance, 2nd edition, section 5.10, Practical Food Microbiology.
Weber, G.R. and Black, L.A. (1948). Relative eficiencies of Butler & Tanner. ISBN 0 901144 36 3.
quaternary inhibitors. Soap and Sanit. Chem. 24: 134-139.
American Society for Testing Materials. (1998). Standard Test Method
for Preservatives in Water-Containing Cosmetics. E640-78.
Annual Book of ASTM Standards, Philadelphia, PA. Listeria Enrichment Broth
Association of Analytical Chemists. (1995). Oficial methods of
analysis, 16th edition, section 6.Association of Oficial Analytical LAB138
Chemists, Washington, D.C.
Description
Roberts, D., Hooper, W., and Greenwood, M. (1995). Methods
for the examination of food for micro-organisms of public health A medium for the selective enrichment of food and environmental
signiicance, 2nd edition, section 5.10, Practical Food Microbiology. samples for Listeria spp. irst described in 1987 by J. Lovett. The
Butler & Tanner. ISBN 0 901144 36 3. medium offers more rapid enrichment than the low temperature
enrichment techniques. The medium is incubated at 30˚C and utilises
acrilavine, nalidixic acid and cycloheximide as selective agents.
Letheen Broth Typical Formula g/litre
LAB184 Tryptone 17.0

Soy Peptone 3.0
Description
Letheen Broth is primarily used for the assessing the bactericidal Sodium chloride 5.0
activity of quaternary ammonium compounds, and for determining Dipotassium hydrogen phosphate 2.5
the phenol co-eficient of cationic surfactants as recommended by the
Oficial Methods of Analysis of the Association of Oficial Analytical Glucose 2.5
Chemists (AOAC). It is also used in hygiene swabbing protocols
Yeast Extract 6.0
where it is necessary to neutralise quaternary ammonium compounds.
A modiication of FDA Broth, Letheen Broth contains lecithin to
neutralise quaternary ammonium compounds and Polysorbate 80 to Method for reconstitution
neutralise phenols, hexachlorophene, formalin and (with lecithin) Weigh 36 grams of powder and add to 1 litre of deionised water. Allow
ethanol. Letheen Broth is easily prepared and has a clear appearance to soak for 10 minutes then swirl to mix and sterilise by autoclaving at
aiding in visual inspection for growth. The American Society for 121˚C for 15 minutes. Cool to 47˚C and add 2 vials of reconstituted
Testing Materials (ASTM) speciies the use of Letheen Broth in X139. Aseptically dispense into sterile tubes or bottles.
the Standard Test Method for Preservatives in Water Containing
Cosmetics. Appearance: Yellow, clear.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: L. monocytogenes WDCM 00021
Peptone 10.0 E. coli (inhibition) WDCM 00013
Beef extract 5.0
Storage of Prepared Medium: Capped containers – up to 14 days
Sodium chloride 5.0 at 2-8˚C in the dark.
Lecithin 0.7 Inoculation: Add 25 grams of sample to 225mls of Listeria
Enrichment Broth and homogenise.
Polysorbate 80 5.0
Incubation: 30˚C aerobically for up to 48 hours.
Method for reconstitution Subculture: After 24 and 48 hours onto Listeria Isolation Medium –
LAB122.
Weigh 25.7 grams of powder and disperse in 1 litre of deionised
water. Allow the mixture to soak for 10 minutes, swirl to mix, and References
then sterilise by autoclaving at 121°C for 15 minutes. Lovett, J. Frances, D.W. Hunt, J.M. J. Food Protect. 50: 188-192.
Bolton, F. J. Personal Communication Public Health Laboratory,
Appearance: Straw, clear liquid. Preston U.K.
pH: 7.0 ± 0.2

LEE Broth Listeria Isolation Medium (Oxford)
Listeria Express Enrichment Broth
LAB122
LAB589 Description
Description A selective identiication medium for the isolation of Listeria
monocytogenes from food and clinical material. Columbia agar is the
Listeria Express Enrichment Broth (LEE Broth) is a selective nutrient base to which selective inhibitors have been added. Lithium
enrichment broth for the detection of Listeria. Developed to give chloride is used to inhibit enterococci and acrilavine to inhibit some
improved growth rates of Listeria over traditional selective enrichment Gram negative and Gram positive species. Further selective agents
media, LEE Broth enhances the expression of target antigens for may be added after autoclaving to increase the selectivity; these
most commercially available immunological test kits/methods whilst are colistin, fosfomycin, cefotetan and cyclohexamide. Aesculin is
maintaining adequate suppression of potential non-target organisms. included in the formula as a differential indicator. L. monocytogenes
Selective components are blended into the powder, removing the will hydrolyse aesculin to aesculutin which reacts with the iron salt to
requirement for supplementation. give a black precipitate around the colonies.
Compared with more traditional methods, immunological tests, such Lab M’s formulation has been used to successfully isolate Listeria
as ELISA, require relatively high levels of target organisms to achieve from such diverse products as chicken giblets and dairy cheeses. The
a reliable positive result. LEE Broth has been speciically designed to advisability of using this medium at two levels of selectivity has been
stimulate growth from low numbers to the required high levels within recognised.
a 24 hour period. Therefore, LEE Broth offers an excellent choice for
laboratories employing immunological test methods for detection of
Listeria in food products. Typical Formula g/litre
Lab M LEE Broth can be used as an enrichment broth prior to plating Columbia Agar Base 41.0
on selective media such as Harlequin™ Listeria Chromogenic Agar
ISO (HAL010 / PIN001); as secondary selective enrichment medium Aesculin 1.0
following primary enrichment in, for example, Half Fraser Broth Ferric ammonium citrate 0.5
(LAB211 or LAB164); or as the enrichment step of a rapid method
e.g. ELISA, lateral low device, PCR. Lithium chloride 15.0

Peptone 13.0 Weigh 57.5 grams of powder. Add to 1 litre of deionised water. Allow
Growth enhancers 8.0 121˚C for 15 minutes. Allow to cool to 47˚C, add 2 vials of selective
supplement X123 mix well and pour plates.
Buffer 22.2
Selective mix 3.0 Appearance: Pale yellow, slightly opaque gel.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: L. monocytogenes WDCM 00021
Appearance: E. coli (inhibition) WDCM 00013
Finished medium: clear, dark straw liquid with yellow luorescence Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
dark.
pH: 7.2 ± 0.1
Inoculation: Surface, streak out to single colonies. This medium is
Hazard classiication: highly selective, a heavy inoculum can be used.
Xi – Irritant Incubation: 30˚C aerobically for 24-48 hours.
Method for reconstitution:
Disperse 46.2g of powder in 1 litre of distilled water. Allow to soak
colony size shape &
for 10 minutes, swirl to mix and dispense into suitable containers.
Sterilise by autoclaving for 15 minutes at 121°C. Allow to cool to
ambient temperature prior to use. L. monocytogenes 0.5-1.0 CV.E.G. Grey/ Black/brown
Green around colonies
diffusion
Incubation:
Enterococci p.p. - 0.5 CV.E.G. Black Usually no growth
Incubate at 30°C ± 1°C for 24 ± 1 hour (for optimum performance
check at 24 hours precisely). Alternative incubation temperatures may
be used if lagella are not the target antigen. References
Garayzabal, J.F.F., Rodriguez, L.D., Boland, J.A.V., Cancelo,
Interpretation: J.L.B., Fernandez, G.S. (1986). Listeria monocytogenes dans le lait
Low cell numbers may express high levels of detection targets pasteurise. Can. J. Microbiol. 32: 149-150.
therefore further tests (plating, ELISA etc) should be conducted on all Donnelly. C.W., Gregory J. Baigent (1986). Method for low
samples regardless of presence/absence of turbidity. cytometric detection of Listeria monocytogenes in milk. Appl. &
Environ. Microbiol.Oct. 689-695.
Storage: Bolton, C.F.J. Preston P.M.L. Personal communication. Lovett, J.
Dehydrated culture media: 10-25°C away from direct sunlight. Francis, D.W. Hunt. J.M. (1987). Listeria monocytogenes in raw
Prepared media: 1 month at 2-8°C in the dark. milk: Detection, Incidence and Pathogenicity. Journ. Food Protect.
Vol. 50. No. 3: 188-192.
Minimum Q.C. organisms: Van Netten, P., Van de Van, A., Perales, I., Mossel, P.A.A. (1988). A
Listeria monocytogenes WDCM 00020 selective and diagnostic medium for use in enumeration of Listeria
Listeria ivanovii WDCM 00018 spp. in foods. International Journal of Food Microbiology 6:187-198.

Listeria Isolation Media LMBA contains lithium chloride in concentrations that inhibit the
growth of enterococci yet allow good haemolysis by L. monocytogenes.
(Oxford Formulation)
LMBA is supplemented by polymyxin plus ceftazidine (X072) and
nalidixic acid (X072N) to suppress competing lora such as members
LAB206 of the bacillus group and staphylococci.
Description The addition of donated sheep blood (deibrinated with sodium
citrate) to LMBA allows differentiation between haemolytic and
A selective identiication medium for the isolation of Listeria non-haemolytic stains of Listeria. The use of sheep blood is standard
monocytogenes from food and clinical material. Columbia agar is the methodology for Listeria testing. However, ingredients in selective
nutrient base to which selective inhibitors have been added. Lithium agars can result in partial lyses or darkening of the blood supplement.
chloride is used to inhibit Enterococci, whilst acrilavine inhibits some The use of citrated sheep blood prevents this and allows differentiation
Gram-negative and Gram-positive species. Further selective agents of L. monocytogenes from other haemolytic Listeria species e.g.
may be added after sterilisation to increase the selectivity. L. seeligeri and L. ivanovii, due to its distinctive haemolytic pattern.
L. seeligeri is rarely isolated from foods and produces very weak
Typical Formula g/litre haemolysis, whilst L. ivanovii produces wide zones of haemolysis
Columbia agar base 41.0 compared to the narrow zone of L. monocytogenes and is an animal
rather than human pathogen.
Aesculin 1.0 LMBA is a cost effective method by which to speciically isolate and
Ferric ammonium citrate 0.5 enumerate L. monocytogenes
Lithium chloride 15.0 Typical Formula g/litre

Tryptone 15.0
Weigh 57.5 grams of powder and disperse in 1 litre of deionised water. Soy peptone 5.0
Allow to soak for 10 minutes, swirl to mix and sterilise by autoclaving Sodium chloride 5.0
for 15 minutes at 121°C. Cool to 47°C, and add 2 vials of X123. Mix
well before dispensing into sterile Petri dishes. Lithium chloride 10.0
Appearance: Magnesium sulphate (3/4H2O) 3.8
Powder: Fine, free-lowing, homogeneous, buff. Agar 15.0
Final medium: Straw-yellow gel.
pH: 7.0 ± 0.2 Method for Reconstitution
Weigh 53.8 grams of powder and disperse into 1 litre of deionised
Hazard classiication water. Allow the mixture to soak for 10 minutes, swirl to mix then
Xn - Harmful sterilise at 121°C for 15 minutes. Cool to 47°C, and aseptically add
citrated sheep blood to 5%, 2 vials of X072 supplement and 2 vials
Minimum Q.C. organisms: of X072N supplement. Mix well, and pour into sterile Petri dishes
Listeria monocytogenes WDCM 00021 and allow to set.
Enterococcus faecalis WDCM 00087 Appearance: Opaque and blood red.
pH: 7.0 ± 0.2
Storage:
Dehydrated culture media: 10-25°C Minimum Q.C. organisms:
Poured plates: 7 days at 2-8°C in the dark Listeria monocytogenes WDCM 00021
Inoculation: Surface inoculation as per user’s validated methods.
Listeria innocua WDCM 00018
Incubation: Incubate for 24-48 hours at 30, 35 or 37°C according to
user’s validated methods. Storage of Prepared Medium: Plates can be stored up to 7 days at
References 2-8°C in the dark.
ISO 11290-1:1996 Microbiology of food and animal feeding stuffs Inoculation: Surface plating, streaking out to single colonies, from
– Horizontal method for the detection and enumeration of Listeria the enrichment broth. For enumeration, 0.1ml of neat or 10-1 dilution
monocytogenes – Part 1: Detection method. of the food sample is spread over the entire surface of the plate. Use
multiple plates if the volume required is greater than 0.1 ml.
Incubation: Aerobically at 37°C for 24-48 hours.
Listeria Monocytogenes Blood Agar Interpretation:
(LMBA) Organism Shape and Surface Colour & Haemolysis
L. monocytogenes C.V.E.G. Cream, narrow zone of
LAB172 β haemolysis
L. ivanovii C.V.E.G. Cream, wide zone of
Description β haemolysis
Listeria monocytogenes Blood Agar (LMBA) has been developed for L. innocua C.V.E.G. Cream, no haemolysis
the speciic detection and enumeration of L. monocytogenes in food
samples. This medium has been shown to improve the isolation rate L. seeligeri C.V.E.G. Cream, very weak
of L. monocytogenes in ready to eat foods by up to 22%. β haemolysis
L. monocytogenes is the only important human pathogen among the Enterococcus faecalis No growth
species of Listeria currently recognised. It is distinguished from Escherichia coli No growth
L. innocua on LMBA using colonial appearance and haemolysis.
Studies have shown that the most commonly isolated species of
listeria, other that L. monocytogenes from food and processing References
environments is L. innocua. Johansson, T (1998). Enhanced detection and enumeration of Listeria
L. monocytogenes can be found in all main categories of products e.g. monocytogenes from foodstuffs and food processing environments.
dairy, meat and poultry. The symptoms of infection with this organism International Journal of Food Microbiology, 40; 77-85.
include fever, generalised aches and pains, sore throat, diarrhoea Jay, J.M. (1996). Prevalence of Listeria spp. in meat and poultry
and abdominal pains. In severe cases pneumonia, septicaemia and products. Food Control, 7; 209-214.
meningitis may develop. Pregnant women are particularly susceptible Kozak, J., Balmer, T., Byrne, R. and Fisher, K. (1996). Prevalence of
to listeriosis, due to immune suppression. L. monocytogenes can Listeria monocytogenes in foods: incidence in dairy products. Food
cross the placenta causing abortion, still birth, or meningitis of the Control, 7; 215-222.
new born.

Lysine Agar Lysine Iron Agar
LAB201 LAB054
Originally described by Morris and Eddy, this complex synthetic This is a differential medium for the detection of salmonellae and
medium is designed for the isolation and enumeration of wild other enteric pathogens, by means of lactose fermentation, lysine
yeasts in pitching yeast. Lysine is utilised by wild yeasts, but not decarboxylase activity and hydrogen sulphide production. Salmonella
by Saccharomyces cerevisiae, S. carlsbergensis and S. pastorianus. strains (including Salmonella arizona) which ferment lactose and
Lab M Lysine Agar is made to the Morris and Eddy published produce black colonies on Bismuth Sulphite Agar (LAB013) can
formulation. be recognised by the alkaline reaction (purple colour) produced
throughout the medium, together with blackening due to sulphide
Typical Formula g/litre production. Enteric organisms that do not decarboxylate lysine yield
an alkaline slant over an acid butt (yellow). Thus no distinction
Dextrose 4.0 between Shigella and E. coli is possible and Triple Sugar Iron Agar
(LAB053) is recommended in parallel. Proteus and Providencia
Lysine Agar Chemical 4.0
cultures characteristically produce a distinctive red slant over an acid
Agar 1.0 butt since these organisms deaminate lysine but without sulphide
production. Salmonella arizona strains which produce pink to red
colonies on bile salt media are often overlooked in outbreaks of food
Method for reconstitution: poisoning, however the use of Bismuth Sulphite Agar with subculture
Disperse 6.6g of powder in 100ml of distilled water and add 1 vial into Lysine Iron Agar allows determination of their presence.
(1ml) of X034, Poptassium lactate. Allow to soak for 10 minutes,
swirl to mix and bring to the boil with frequent agitation to prevent
superheating. Cool to 50oC and add 0.1ml of X036, 10% Lactic Acid. Balanced Peptone No. 1 5.0
Mx well and dispense into sterile petri dishes.
Yeast Extract 3.0
Appearance: Glucose 1.0
Powder: ine, free-lowing, homogeneous, buff L-Lysine 10.0
Finished medium: colourless clear gel
Ferric Ammonium Citrate 0.5
pH: 4.8 ± 0.2 (complete medium)
Hazard classiication:
Bromocresol Purple 0.02
Agar No. 2 12.0
Storage:
Dehydrated culture media: 10-25°C Method for reconstitution
Final medium: 7 days at 2-8°C in the dark Weigh 31.5 grams of powder and disperse in 1 litre of deionised water.
Allow the mixture to soak for 10 minutes, swirl to mix and bring to
Minimum Q.C. organisms: the boil, with frequent stirring to dissolve completely. Dispense into
Pichia fermentans NCYC 850 tubes and sterilise by autoclaving at 121°C for 15 minutes. Cool in a
Saccharomyces pastorianus NCYC 185 slanted position such that slopes are formed over deep butts approx.
3cm in depth.
Appearance: Clear purple gel.

pH: 6.7 ± 0.2
Storage of Prepared Medium: Tightly capped containers – up to 3
months at 15-20°C in the dark.
Inoculation: Subcultures for further identiication are picked from the
centre of isolated colonies on selective media and streaked across
the slant and stabbed into the butt of tubes of Lysine Iron Agar.
Organism Butt Slant Sulphide Production
Salmonella Alkaline Alkaline +

Enterobacter aerogenes)
Citrobacter Acid Alkaline +

Escherichia coli Acid (NC) Alkaline -
Shigella Acid Alkaline -
Proteus Acid ‘red’ -
References
Edwards, P.R. and Fife, M.A. (1961). Lysine iron agar in the detection
of Arizona cultures. Appl. Microbiol. 9:478-480.
Edwards, P.R. and Ewing, W.H. (1964). Identiication of
Enterobacteriaceae. Burgess Publishing Co. Minn.

M17 Agar MacConkey Agar
(With Salt)
LAB092
Description LAB030
A medium for the enumeration of Lactococci in dairy products. Description
The medium can also be used to investigate the bacteriophage
susceptibilities of these organisms. Another application is for the A selective medium for the isolation of bile tolerant organisms from
enumeration of Streptococcus thermophilus in yoghurts. faeces, urine, sewage and foodstuffs. Bile-tolerant Gram positive
organisms as well as Gram negative organisms will grow on this
medium. This formula is recommended by W.H.O. and other bodies
for the examination of water and milk. Some strains of Proteus spp.
will spread on this medium making interpretation dificult, for this
Balanced Peptone 5.0 reason LAB002 MacConkey Agar (without salt) may be preferred as
it is less prone to this phenomenon.
Soy Peptone 5.0
Yeast Extract 2.5 Typical Formula g/litre
Beef Extract 5.0 Peptone 20.0
Sodium glycerophosphate 19.0 Bile Salts 5.0
Magnesium sulphate 0.25 Sodium chloride 5.0
Ascorbic acid 0.5 Neutral red 0.05
Agar No. 2 15.0 Agar No. 2 12.0

Weigh 57.2 grams of powder, disperse in 1 litre of deionised water. Weigh 52 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then bring to boil to dissolve Allow to soak for 10 minutes, swirl to mix then sterilise by autoclaving
agar before dispensing in 15ml aliquots. Sterilise by autoclaving at at 121˚C for 15 minutes. Cool to 47˚C and mix well before pouring
115˚C for 20 minutes. into Petri dishes.
Appearance: Pale straw, translucent agar. Appearance: Pink/red, clear

pH: 7.1 ± 0.2 pH: 7.4 ± 0.2
Minimum Q.C. organisms: Lactococcus lactis WDCM 00016 Minimum Q.C. organisms: E. coli WDCM 00013
Storage of Prepared Medium: Capped containers – up to 1 month Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
at 15-20˚C in the dark. dark.
Inoculation: Pour plate technique. Inoculation: Surface plating, streaking out to single colonies.
Incubation: 30˚C for 48-72 hours for mesophilic streptococci, 37˚C Incubation: 37˚C aerobically for 24 hours.
for 48 hours for Streptococcus thermophilus.
Interpretation: Count all colonies. Streptococci form colonies of Growth Characteristics
1-2mm in diameter. approx. size shape &
References
Proteus spp. 1.5-2.5 CV.E.G. Yellow
Terzaghi, B.E. Sandine, W.E.. (1975). Improved medium for lactic (spreading)
Streptococci and their Bacteriophages. Appl. Microbiol. 29 No. 6 pp
Salmonella spp. 1.5-2.5 CV.E.G. Colourless
807-813.
S. aureus 0.5-2.0 CV.E.G. White/ (dependent on
Pink lactose
fermentation
Orange and pigment
Opaque production)
Enterococcus
spp. P.P.-0.5 CV.E.G. Pink/Deep
Red
Opaque
References
Environment Agency: The Microbiology of Drinking Water (2002).
Methods for the Examination of Water and Associated Materials.
World Health Organisation (1971). International Standards for
Drinking Water. 3rd Edn. W.H.O., Geneva.
Taylor, E.W. (1958). The Examination of Water and Water Sup-
plies. 7th Edn. Churchill, London.
Cruikshank, R. (1973). A Guide to the Laboratory Diagnosis and
Control of Infection. Medical Microbiology. 12th Edn. Churchill.

MacConkey Agar MacConkey Agar No.2
(without salt)
LAB216
LAB002
A medium irst introduced by MacConkey in 1905 for the isolation MacConkey Agar No.2 is a modiication of MacConkey Agar which
and differentiation of lactose and non lactose fermenting enteric contains bile salts No. 2 for the recognition of enterococci. This is
bacteria. The medium has since been modiied to improve the especially useful when looking for enterococci in the presence of
recovery of staphylococci and enterococci, it is used for culturing a coliforms and non-lactose fermenters from water, sewage and food
wide range of clinical material and has applications in food, water and products. Enterococci are frequently sought as an index of faecal
dairy bacteriology. pollution and appear on this medium as small, intensely coloured
red-purple colonies. Non-lactose fermenters appear colourless, whilst
Typical Formula g/litre bile tolerant Gram-positive organisms, such as staphylococci and non-
faecal streptococci, are completely inihibited.
Mixed Peptones 20.0
Lactose 10.0 Typical Formula g/litre
Bile 5.0 Peptone 20.0
Neutral red 0.05 Lactose 10.0
Agar No. 2 13.5 Bile salts No.2 1.5
Sodium chloride 5.0
Weigh 48.5 grams of powder, disperse in 1 litre of deionised water. Neutral red 0.05
Allow to soak for 10 minutes, swirl to mix then sterilise by autoclaving Crystal violet 0.001
for 15 minutes at 121˚C. Cool to 47˚C and mix well before pouring
plates. Prior to inoculation, dry the surface of the agar by partial Agar 15.0
exposure at 37˚C.
Appearance: Pink/red, clear. Method for reconstitution
pH: 7.4 ± 0.2 Allow to soak for 10 minutes, swirl to mix and sterilise by autoclaving
for 15 minutes at 121°C. Cool to 47°C and mix well before dispensing
Minimum Q.C. organisms: E. coli WDCM 00013 into Petri dishes. Dry the agar surface prior to use.
Appearance:
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the Powder: ine, free-lowing, homogeneous, buff
dark.
Finished medium: clear, red-purple gel
Inoculation: Surface inoculation, streaking for single colonies.
Incubation: 37˚C aerobically for 24 hours. pH: 7.2 ± 0.2
colony size shape &
organism (mm) surface colour other Minimum Q.C. organisms:
E. coli 2.0-3.0 CV.E.G. Red (non lactose Enterococcus faecalis WDCM 00087
fermenting Escherichia coli WDCM 00013
yellow) Salmonella typhimurium WDCM 00031
Staphylococcus aureus WDCM 00034 (inhibited)
Proteus spp. 2.0-3.0 CV.E.G. Yellow ishy odour
Storage:
Salmonella spp. 2.0-3.0 CV.E.G. Yellow
S. aureus 0.5-1.0 CV.E.G. Pink- (lactose-negative) Dehydrated culture media: 10-25°C away from direct sunlight.
Orange Prepared media: 7 days at 2-8°C in the dark.
Enterococcus spp. 0.5 CV.E.G. Pink- Inoculation: Surface inoculation as per user’s validated methods.
Deep Red
Incubation: Incubate at 37°C + 1°C for 18 - 48 hours.
References Interpretation: After incubation the plate should be assessed for
MacConkey, A.T. (1905) Lactose-fermenting bacteria in faeces. typical colonies.
J.Hyg. (Camb), 5: 333-379.
MacConkey, A. T. (1908) Bile salt media and their advantages in Interpretation
some bacteriological examinations, J.Hyg. (Camb.), 8: 322-341. colony size shape &
Environment Agency: The Microbiology of Drinking Water (2002). organism (mm) surface colour
Methods for the Examination of Water and Associated Materials. Enterococcus faecalis 0.5mm Convex, entire, Red-purple
World Health Organisation (1971), International Standards for glossy
Drinking Water, 3rd Edn. W.H.O., Geneva. Taylor, E.W. (1958). Escherichia coli 3 - 4mm Convex, entire, Red-purple
The Examination of Water Supplies, 7th Edn. Churchill, London glossy
Salmonella spp. 2 – 3mm Convex, entire, Translucent
glossy
Staphylococcus aureus No growth

MacConkey Agar No. 3 . MacConkey Broth
(Purple)
LAB045
Description
LAB005
A modiication recommended by the W.H.O. and the American Public Description
Health Association for the isolation of Enterobacteriaceae from This medium is used in the detection and enumeration of faecal
waters and sewage. The medium has been made more selective than coliforms (37˚C) and E. coli (44˚C). The replacement of neutral red
MacConkey’s original formula by the use of crystal violet as well as used in the original formulation by bromocresol purple makes the
bile salts. Gram positive organisms will not grow on this medium. colour change caused by acid producing organisms easier to read.
Peptone 20.0 Peptone 20.0
Bile Salts No. 3 1.5 Bile Salts 5.0
Sodium chloride 5.0 Bromocresol purple 0.01
Neutral red 0.03
Crystal violet 0.001 Weigh 35 grams of powder, disperse in 1 litre of deionised water. Mix
Agar No. 2 15.0 well and dispense into tubes or bottles with inverted Durham tubes.
Sterilise by autoclaving for 15 minutes at 121˚C. Prepare double
strength broth (70g/l) if 50ml or 10ml amounts of inoculum are to
Method for reconstitution be added to equal volumes of broth. Prepare single strength broth
Weigh 51.5 grams of powder and add to 1 litre of deionised water. (35g/l) if 1ml or 0.1ml amounts of inoculum are to be added to 10ml
Allow to soak for 10 minutes, swirl to mix then sterilise for 15 minutes of broth.
at 121˚C. Cool to 47˚C and pour into Petri dishes. Dry the surface
before inoculation. Appearance: Purple, clear.
Appearance: Pale red slight violet tinge. pH: 7.3 ± 0.2
pH: 7.1 ± 0.2 Minimum Q.C. organisms: E. coli WDCM 00013

B. subtilis WDCM 00070
Ent. faecalis (inhibition) Storage of Prepared Medium: Capped containers – up to 1 month
WDCM 00087 at 15-20˚C in the dark.
Incubation: 37˚C aerobically for coliforms, 44˚C aerobically for
Storage of prepared medium: Plates – up to 7 days at 2-8˚C in the E. coli. Use Durham tubes to detect gas production for E. coli.
dark.
Growth Indicators: Turbidity, gas production. Lactose-fermenting
Inoculation: Surface, streaking for single colonies.
organisms cause a colour change from purple to yellow.
References
Growth Characteristics Ministry of Health (1937). Bacteriological Tests for Graded Milk,
colony size shape & Memo 139/Foods. H.M.S.O., London.
organism (mm) surface colour other Minister of Health, Public Health Laboratory Service Water Committee
E. coli 3.0-4.0 CV.E.G. Red (red ppt around (1969). The Bacteriological Examination of Water Supplies, 4th Edn.
(D) colony) report No. 71. H.M.S.O., London.
Proteus spp. 3.0-4.0 CV.E.G. Pale- (ishy odour) World Health Organisation (1971). International Standards for
yellow Drinking Water, 3rd Edn, W.H.O., Geneva.
Staph. aureus No growth
Enterococcus
spp. No growth
References
American Public Health Association (1950). Diagnostic Procedures
and Reagents. 3rd edn. A.P.H.A., New York.
American Public Health Association (1946). Standard Methods for
the examination of Water and Sewage. 9th edn. A.P.H.A., New York

Malt Extract Agar Method for reconstitution
Weigh 20g of powder and disperse in 1 litre of deionised water.
LAB037 Allow to soak for 10 minutes, swirl to dissolve and dispense into inal
containers. Sterilise by autoclaving at 115˚C for 10 minutes.
Description Appearance: Pale brown/straw, clear broth.
An acidic medium which will support the growth of most yeasts
and moulds whilst inhibiting most bacteria. It was irst described by pH 5.4 ± 0.2
Thom and Church in 1926 in a study of Aspergillus spp. claiming
the high carbohydrate content ensured rapid growth. Selectivity Inoculation: Inoculate samples direct into tubes of broth according
can be increased by further lowering the pH with the addition, after to the particular method being employed.
sterilisation, of X037 Lactic Acid. It should be noted that excess
heating of this medium together with its low pH can easily result in
Incubation: 25˚C (or 37˚C) for up to 7 days aerobically, depending
hydrolysis of the agar gel producing soft plates. upon protocol used. Subculture turbid tubes onto solid media for
identiication of growth.
Minimum QC organism: Candida albicans WDCM 00054
Malt Extract 30.0 Aspergillus niger
Mycological Peptone 5.0
References
Agar No. 2 15.0 Galloway, L.D. and Burgess, R. (1952) Applied Mycology and
Bacteriology, 3rd ed, Leonard Hill, London pp 54 & 57.
Weigh 50 grams of powder, disperse in 1 litre of deionised water,
allow to soak for 10 minutes, swirl to mix then sterilise at 115˚C for
10 minutes. If the addition of X037 Lactic Acid is required this should Mannitol Salt Agar
be done after sterilisation. One 5ml vial of X037 will lower the pH of
250ml of medium to 3.5-4.0. Cool to 47˚C before making additions LAB007
and pouring plates.
Description
Appearance: Pale brown/straw, clear. Mannitol Salt Agar is a medium for Staphylococcus aureus which
pH: 5.4 ± 0.2 (if X037 is added pH 3.5-4.0) is selective because the high sodium chloride level inhibits most
other species with the exception of halophilic Vibrios. The majority
Minimum Q.C. organisms: Candida spp. WDCM 00054 of S. aureus ferment mannitol producing yellow colonies, occasional
strains of coagulase-negative staphylococci may also ferment
mannitol. It is necessary to conirm the identity of presumptive S.
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the aureus colonies by other means e.g. coagulase, protein A, DN’ase,
dark. Capped container – up to 1 month at 15-20˚C in the dark. thermonuclease or latex agglutination. This medium performs as per
Inoculation: Pour plate technique or surface streaking for single the requirements of the Harmonised Pharmacopoeia (USP/EP/JP).
colonies.
Incubation: 25˚C aerobically for 5 days. Typical Formula g/litre
Beef Extract 1.0
colony size shape & Balanced Peptone No. 1 10.0
organism (mm) surface colour other Sodium chloride 75.0
Candida albicans 4 CV.E.D. White
D-Mannitol 10.0
Candida krusei 10 F.CR.D. White
Agar No. 2 12.0
Penicillium
notatum 25 Green (white/yellow - Phenol Red 0.025
velvet strain dependent)
Aspergillus niger 25 White (yellow/black
border, centre) Method for reconstitution
black
centre Weigh 108 grams of powder and disperse in 1 litre of deionised
water. Allow to soak for 10 minutes, swirl to mix and sterilise by
autoclaving for 15 minutes at 121oC. Cool to 47°C before pouring
References
into Petri dishes.
Galloway, L.D. and Burgess, R. (1952). Applied Mycology and
Bacteriology, Leonard Hill, London. Thom and Church, 1926. Appearance:
The Aspergilli.
Finished medium: clear, red gel
Malt Extract Broth pH: 7.4 ± 0.2
LAB159 Minimum Q.C. organisms: S. aureus ATCC 6538

E. coli ATCC 8739 (inhibition)
Description
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
A liquid medium of low pH for the growth of yeasts and moulds,
the dark.
typically employed as part of sterility testing protocols for various
products. The high carbohydrate content of the medium ensures rapid Inoculation Method: Surface plating, streak out for single colonies.
growth of yeasts and moulds. Incubation: In accordance with Harmonized Pharmacopoeia (USP/
EP/JP), incubate aerobically at 30-35oC for 18-72 hours. The product
Typical Formula g/litre may also be used for other applications at 37oC for 48 hours.
Malt Extract 17.0
Mycological Peptone 3.0

Growth Characteristics Membrane Lauryl Sulphate Broth
colony size shape &
organism (mm) surface colour other LAB082
S. aureus 1.5-2.0 CV.E.G. Bright
Yellow
Description
Other
This medium superseded Membrane Enriched Teepol broth when Shell
Staphylococci 1.0-1.5 CV.E.G. White or (some ferment Chemicals withdrew Teepol 610 from sale. Sodium lauryl sulphate
Yellow mannitol) was found to be an adequate reproducible substitute and this medium
is recommended for the enumeration of coliform and organisms in
Enterobacteriaceae no growth water and sewage.
References Typical Formula g/litre

American Public Health Association (1966). Recommended Methods Peptone 39.0
for Microbiological Examination of Foods, 2nd Edn. (ed. J.M. Sharf)
A.P.H.A. Washington. Yeast Extract 6.0
Davis, J.G., (1959). Milk Testing 2nd edn, Dairy Industries, London. Lactose 30.0
Phenol red 0.2
Sodium lauryl sulphate 1.0
Maximum Recovery Diluent
(Peptone/Saline diluent) Method for reconstitution
LAB103 Distribute into screw cap containers and sterilise by autoclaving at
115˚C for 10 minutes.
Description
An osmotically controlled solution which is an alternative to, and Appearance: Red, clear solution.
a replacement for, 1/4 strength Ringer’s Tablets (LAB100Z). The pH: 7.4 ± 0.2
presence of a low level of peptone lessens the physiological shock
normally experienced by bacterial cells when they are introduced to
a diluent such as Ringer’s Solution. The level of peptone is such that Minimum Q.C. organisms: E. coli WDCM 00013
multiplication of the organisms is not possible in the time in which
the sample will be present in the diluent (1-2 hours). This formula is Storage of Prepared Medium: Capped containers – up to 3 months
recommended by ISO 6887: BS5763. at 15-20˚C in the dark.
Inoculation: E. coli and coliform counts should be made on separate
Typical Formula g/litre samples of water. The volumes should be chosen so as the number
Peptone 1.0 of colonies on the membrane lies between 10 and 100. With waters
expected to contain less than 1 coliform per ml, a sample of 100ml
Sodium chloride 8.5 should be iltered. The membrane ilter should be placed face upwards
on a pad soaked in Membrane Lauryl Sulphate Broth, after iltration.
Grams per litre 9.5
These membranes should be incubated in a container which does not
allow evaporation to occur. Water tight metal containers placed in
Method for reconstitution an accurate water bath are required for incubation of membranes at
Weigh 9.5 grams of powder and disperse in 1 litre of deionised water. 44˚C.
Allow to soak for 10 minutes, swirl to mix and if required, heat gently Incubation: E. coli 4 hours at 30˚C 14 hours at 44˚C; Coliforms 4
to dissolve. Distribute into inal containers. Sterilise by autoclaving hours at 30˚C 14 hours at 35˚C.
for 15 minutes at 121oC.
Interpretation: No colonies:- assume a nil count. Small colonies of
Appearance: an intermediate colour:- return to incubation for a full period.
Powder: ine, free-lowing, homogeneous, buff E. coli: Yellow-coloured colonies from membranes incubated at 44˚C
Finished medium: clear, colourless liquid should be subcultured to Lactose Broth LAB126 and Tryptone Water,
pH: 7.0 ± 0.2 LAB129 to conirm gas and indole production respectively, after 24
hours incubation at 44˚C.
N.B. The absence of any buffer results in the product taking on the pH Coliform organisms: Yellow colonies from membranes incubated
of any sample added. at 35˚C or 37˚C should be subcultured into Lactose Broth LAB126.
After 48 hours incubation at 37˚C a result should be obtained
Minimum Q.C. organisms: E. coli WDCM 00031 regarding the production of gas.
Full details of the methodology can be found in The Bacteriological
Examination of Water Supplies 71, 1969.
at 15-20˚C in the dark. References
References Burnham, N.P. (1967). Proc. Soc. Wat. Treat, Exam. 16:40.
Straka, R.P. and Stokes, J.L. (1957). Rapid destruction of bacteria in Environment Agency: The Microbiology of Drinking Water (2002).
commonly used diluents and its elimination. Appl. Microbiol. 5: 21- Methods for the Examination of Water and Associated Materials.
25. Windle Taylor, E. (1961) Glutamic acid medium, 40th Ann Rep. Div.
Water Exam. Met. Water Board London pp 18-22.
ISO 6887-1:1999 Microbiology of food and animal feeding stuffs -
Preparation of test samples, initial suspension and decimal dilutions
for microbiological examination – Part 1: General rules for the
preparation of the initial suspension and decimal dilutions.

Microbial Content Test Agar (MCA) Milk Agar
(Tryptone Soy Agar with Lecithin and Polysorbate 80 (TSALT)) LAB019
(Casein Soy Peptone Agar with Lecithin and Polysorbate 80)
Description
LAB189 An approved formulation for the enumeration of micro-organisms in
milk, rinse waters and dairy products. With the addition of a further
Description 5 g/l Agar No. 1 the medium is suitable for the preparation of Roll-
The use of Microbial Content Test Agar (MCA) is recommended Tubes using established mechanical equipment. Also see Milk Plate
for the detection of microorganisms on surfaces sanitised with Count Agar LAB115. Milk Agar can also be used with the P-INC
quaternary ammonium compounds, phenolic compounds and supplement (X019, X219) for accelerated shelf-life determination of
formalin. The medium is a modiication of Tryptone Soy Agar dairy products.
with added neutralising compounds lecithin and Polysorbate 80. It
is recommended for determining the hygiene status of containers, Typical Formula g/litre
equipment and work areas treated with disinfectants or other
sanitisers. The addition of Lecithin and Polysorbate 80 in the formula Yeast Extract 3.0
inactivates some preservatives that may inhibit bacterial growth, Peptone 5.0
reducing “preservative carryover”. The formulation is recommended
for Aerobic Plate Count (Microbial Limit Test) for water miscible Antibiotic Free Skim Milk Powder 1.0
cosmetic products containing preservatives. Lecithin is included to
Agar No. 1 15.0
neutralise quaternary ammonium compounds and Polysorbate 80 is
incorporated to neutralise phenols, hexachlorophene, formalin and
with lecithin, ethanol. Method for reconstitution
Typical Formula g/litre Allow to soak for 15 minutes, swirl to mix then sterilise for 15 minutes
at 121˚C. Cool to 45˚C before mixing with sample dilutions.
Tryptone 15.0
Appearance: White, opalescent gel.
Soy Peptone 5.0
pH: 7.2 ± 0.2
Sodium Chloride 5.0
Polysorbate 80 5.0 Minimum Q.C. organisms: E. coli WDCM 00013
Lecithin 0.7
Agar No.2 15.0 Storage of Prepared Medium: Capped containers – up to 3 months
Method for Reconstitution Inoculation: Pour plate technique.
Incubation: Aerobically at 30˚C for 72 hours.
Allow the mixture to soak for 10 minutes, swirl to mix and sterilise References
by autoclaving at 121°C for 15 minutes. Cool to 47°C and pour into Ministry of Health (1937). Bacteriological Tests for Graded
sterile Petri dishes and allow the medium to set. Milk. Memo 139/Foods H.M.S.O., London. British Standard 4285:
Appearance: Straw opalescent gel. Methods of Microbiological Examination for Diary Purposes.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: Milk Plate Count Agar

Escherichia coli ATCC 11229
Staphylococcus aureus ATCC 6538 LAB115
Storage of Powdered Medium: Store at 2-8°C in the dark. Description
Formulation is very hygroscopic, keep container tightly closed after
use. A medium recommended by the British Standards Institute and the
International Organisation for Standardisation for the enumeration of
Storage of Prepared Medium: Plates – up to 7 days at 2-8°C in the viable bacteria in milk and other dairy products.
dark.
Inoculation: Consult the appropriate references as this product is Typical Formula g/litre
used in several procedures.
Tryptone 5.0
Interpretation: Count all colonies and calculate the number of Yeast Extract 2.5
colony forming units, (cfu) per ml of sample allowing for dilution Dextrose 1.0
factors.
Antibiotic Free Skim Milk Powder 1.0
References
Orth, D.S. (1993) Handbook of Cosmetic Microbiology. Marcel Agar No. 1 10.0
Dekker, Inc., New York, NY.
Brummer, B. (1976). Inluence of possible disinfectant transfer on Method for reconstitution
Staphylococcus aureus plate counts after contact sampling. App. Weigh 19.5 grams of powder, disperse in 1 litre of deionised water.
Environ. Microbiol. 32:80-84. Allow to soak for 10 minutes, swirl to mix then bring to the boil to
dissolve the agar. Allow to cool to 47˚C and dispense into suitable
containers. Sterilise by autoclaving at 121˚C for 15 minutes.
Appearance: Pale cream, opalescent gel.
pH: 6.9 ± 0.2

Storage of Prepared Medium: Capped container – up to 3 months

Inoculation: Pour plate technique.
Interpretation: Count all colonies. Calculate back to determine
viable organisms per ml

References References
British Standards Institute. (1984). BS 4285 Section 1.2. International Gray, R.D. (1964). An improved formate lactose glutamate medium
Organisation for Standardisation Draft International Standard. (1982) for the detection of Escherichia coli and other coliform organisms in
ISO/DIS 6610. D.I.N. 10192. water. J. Hyg. Camb. 62: 495-508.
PHLS Water Sub-Committee. (1958). A comparison between
MacConkey broth and Glutamic acid media for the detection of
Minerals Modiied Glutamate Medium coliform organisms in water. J. Hyg. Camb. 56: 377-388.
PHLS Standing Committee on Bacteriological Examination of Water
LAB080A & LAB080B Supplies. (1968). Comparison of MacConkey Broth, Teepol Broth and
Glutamic Acid Media for the enumeration of Coliform organisms in
Description water. J. Hyg. Camb. 66: 67-87.
This medium was developed for use with the Most Probable Numbers Environment Agency: The Microbiology of Drinking Water (2002).
Technique (M.P.N.) for the enumeration of coliforms in water Methods for the Examination of Water and Associated Materials.
supplies. The medium is an improved version of the chemically
deined glutamic acid medium described by Gray in 1964. The product
is supplied in two parts because it has been shown that separating the
sodium glutamate from the base improves its stability.
M.L.C.B. Agar
(Mannitol Lysine Crystal Violet Brilliant Green Agar)
LAB080A (double strength)
LAB116
Lactose 20.0 Description
A medium for the selective isolation of Salmonella spp. (with the
Sodium formate 0.5 exception of S. typhi and S. paratyphi A) from food and faeces.
L-Cystine 0.04 Salmonella colonies are recognised by distinctive colonial appearance
and H2S production and like the Bismuth Sulphite Agar of Wilson &
L(-) Aspartic acid 0.048 Blair, this medium will detect lactose and sucrose fermenting strains.
Some problems may occur with H2S negative strains, eg S. pullorum,
L(+) Arginine 0.04
S. senftenberg, S. sendai and S. berta. This medium should not be
Thiamine 0.002 used to detect S. typhi and S. paratyphi A, as these strains are more
susceptible to the brilliant green dye.
Nicotinic acid 0.002
Pantothenic acid 0.002 Typical Formula g/litre
Magnesium sulphate (MgSO4.7H20) 0.2 Yeast Extract 5.0
Ferric ammonium citrate 0.02 Tryptone 5.0
Calcium chloride (CaCl2.2H2O) 0.02 Meat Peptones 7.0
Dipotassium hydrogen phosphate 1.8 Sodium chloride 4.0
Bromocresol purple 0.02 Mannitol 3.0
LAB080B L-Lysine HCL 5.0

Glutamic acid (sodium salt) 12.7 Sodium thiosulphate 4.0
Double strength: Dissolve 22.7 grams of base medium (LAB080A) Brilliant green 0.012
together with 12.7 grams of sodium glutamate (LAB080B) in 1 litre Crystal violet 0.01
of deionised water containing 5 grams of ammonium chloride eg
BDH cat no. 27149. Dispense 10ml and 50ml volumes into tubes with Agar No. 2 15.0
inverted Durham tube.
Single strength: Dissolve in 11.35 grams of base medium (LAB080A) Method for reconstitution
together with 6.35 grams of sodium glutamate (LAB080A) in 1 litre Weigh 49 grams of powder, disperse in 1 litre of deionised water.
of distilled water containing 2.5 grams ammonium chloride. Dispense Allow to soak for 10 minutes, swirl to mix then bring to the boil with
5ml volumes into tubes with inverted Durham tubes. frequent agitation to completely dissolve the powder. Cool to 47˚C
Sterilise by autoclaving for 10 minutes at 115˚C, alternatively heat to and pour plates. DO NOT AUTOCLAVE OR OVERHEAT.
100˚C for 30 minutes on three successive days. Appearance: Pale purple, translucent gel.
Appearance: Purple, clear solution. pH: 6.8 ± 0.2
pH: 6.7 ± 0.2
Minimum Q.C. organisms: Salmonella spp. WDCM 00031
Minimum Q.C. organisms: E. coli WDCM 00013 E. coli (inhibition) WDCM 00013

Inoculation: Use the Most Probable Number technique. With 10ml
and 50ml of sample add to equal volumes of double strength medium.
With 1ml volumes of sample add to 5ml of single strength medium.
Ensure the Durham tube is free of bubbles.
Incubation: 37˚C for 18-24 hours aerobically.
Interpretation Tubes showing the production of acid (medium turns
yellow) and gas in the Durham’s tube are considered presumptive
positive. Each presumptive positive tube should be subcultured to
Brilliant Green Bile Broth LAB051 with Durham tube and incubated
at 44˚C for 24 hours and examined for gas production. A tube of
Tryptone Water LAB129 should also be inoculated and incubated at
44˚C for 24 hours for the production of indole. The production at
44˚C of gas from lactose and the formation of indole are evidence
of E. coli.

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the Method for reconstitution
dark. Disperse 36.7g of powder in 1 litre of distilled water. Allow to soak
Inoculation: Surface plating, streaking for single colonies. for 10 minutes, swirl to mix and heat gently to dissolve. Dispense
Inoculation can be carried out directly, or from enrichment broths. 10 ml volumes into test tubes (e.g. 16mm x 160mm tubes of non-
Because of the low selectivity of this medium the inoculum should autoluorescent glass) containing inverted Durham’s tubes. Sterilise
not be heavy, and it is recommended that this medium should be used at 121ºC for 15 minutes. This medium may also be used at double-
in conjunction with other more selective media. strength (73.4g/l).
Incubation: 37˚C aerobically for 24 hours. Appearance:
Growth Characteristics Powder: ine, free-lowing, homogeneous, buff
colony size shape & Finished medium: clear, straw liquid
organism (mm) surface colour
pH: 6.8 ± 0.2
Salmonella spp. 2.0-3.0 CV.E.G. Black Hazard classiication
Salmonella spp. 2.0-3.0 CV.E.G. Pale NR – Not regulated
(H2S negative)
Proteus spp. 1.0-2.0 CV.E.G. Grey brown Escherichia coli WDCM 00013
Shigella spp. mainly
inhibited
Citrobacter spp. 0.5-2.5 CV.E.G. Mainly
Storage:
inhibited Dehydrated culture media: 10-25°C away from direct sunlight.
pale may Prepared media: 1 month at 2-8°C in the dark.
have black Inoculation: Add 1ml of test sample per tube of single-strength
centre
medium. For double-strength medium add 10ml of test sample per
E. coli mainly inhibited tube. Repeat for each further dilution.
Gram positive Incubation: Incubate all inoculated tubes at 30°C for 24 hours (± 2
organisms mainly inhibited hours). Incubation may be extended for up to 48 hours (± 2 hours) if
gas formation and/or turbidity not observed at 24 hours (± 2 hours).
After incubation, add 0.5ml sodium hydroxide solution to all tubes.
Examine for luorescence under a long-wave (360-366nm) UV lamp.
References Add 0.5ml indole (Kovac’s) reagent to all tubes showing luorescence.
Inove et al. 66th meeting of the Japanese Vet. Medicine Society. Mix & observe after 1 minute. The presence of indole (positive tube)
is indicated by the formation of a red colour in the alcoholic phase.
Interpretation: Presumptive E. coli are indicated by those tubes
ColourScreen™ MLSTB-MT (ISO) showing luorescence and a positive indole reaction.
Presumptive coliforms are indicated by those tubes which show gas
Modiied Lauryl Sulphate Tryptose Broth with MUG & formation.
Tryptophan (ISO)
Counts should then be performed according to Most Probable Number
(MPN technique).
LAB077
References
Description Health Protection Agency National Standard Method. Enumeration
Modiied Lauryl Sulphate Tryptose Broth with MUG & Tryptophan of coliforms and presumptive Escherichia coli by the Most Probable
(ISO) is used for the presumptive enumeration of Escherichia coli Number (MPN) technique. Reference number D5i2.4; issued May
from milk and milk products using the Most Probable Number (MPN) 2005.
technique according to ISO 11866-1:2005. ISO 11866-1:2005 Milk and milk products – Enumeration of
presumptive Escherichia coli – Part 1: Most probable number
The original Lauryl Tryptose Broth described by Mallmann and Darby technique using 4-methylumbelliferyl-β-D-glucuronide (MUG).
(1941) has been modiied to incorporate 4-Methylumbelliferyl-β-D-
glucuronide (MUG) and tryptophan allowing presumptive E. coli to Mallmann, W.L. and Darby, C.W. (1941). Am. J. Pub. Hlth. 31: 127-
be enumerated from presumptive coliforms. The addition of MUG to 134.
the medium allows the positive discrimination of E. coli strains. As the
majority of E. coli produce the β-glucuronidase enzyme, they are able
to hydrolyse MUG, releasing a luorogenic compound. Tryptophan Modiied Giolitti and Cantoni Broth (ISO)
acts as a substrate for the indole test. Tubes which luoresce under UV
are conirmed for E. coli by a positive indole reaction when indole
(Kovac’s) reagent is added to the tube. Tubes showing gas formation
LAB219
are identiied as being positive for presumptive coliforms.
Description
Phosphate buffers and sodium chloride improve gas production by Modiied Giolitti and Cantoni Broth (ISO) is used for the detection
slow lactose fermenting organisms whilst sodium lauryl sulphate acts and enumeration of coagulase-positive staphylococci from food
as a selective agent for the inhibition of non-coliform organisms. and animal feeding stuffs using the Most Probable Number (MPN)
Modiied Lauryl Sulphate Tryptose Broth with MUG & Tryptophan technique according to ISO 6888-3:2003.
(ISO) is described in ISO 11866-1:2005 and is used in the HPA Originally described by Giolitti and Cantoni as a medium for the
National Standard Method D5 for Enumeration of coliforms and enrichment of staphylococci from foodstuffs, Mossel later applied the
presumptive Escherichia coli by the Most Probable Number (MPN) medium to use with samples from dried milk and infant food.
technique.
Optimised for use in samples where staphylococci may be stressed
Typical Formula g/litre and/or in low numbers, growth of the target organisms is promoted
by sodium pyruvate, Glycine and the high concentration of mannitol.
Tryptose 20.0 Selectivity is achieved via lithium chloride, which inhibits Gram-
negative bacilli, and potassium tellurite, which inhibits Gram-positive
Lactose 5.0 organisms other than staphylococci. Further selectivity is achieved
Dipotassium hydrogen phosphate 2.75 by use of anaerobiosis either by pouring a plug of agar/parafin or
by incubation in a jar or incubator under anaerobic conditions.
Potassium dihydrogen phosphate 2.75 Anaerobiosis particularly inhibits the growth of Micrococcus spp.
Sodium chloride 5.0 The presence of coagulase-positive staphylococci is indicated by
the reduction of tellurite, resulting in a blackening of the broth or a
Sodium lauryl sulphate 0.1 black precipitate. Coagulase-positive staphylococci are principally
Staphylococcus aureus but may also include the species Staphylococcus
4-Methylumbelliferyl-β-D-glucuronide (MUG) 0.1
intermedius and Staphylococcus hyicus.
Tryptophan 1.0

Typical Formula g/litre Sub-culture:
24 hours: tubes suspected as positive for coagulase-positive
Peptone 1.0 staphylococci after 24 hours should be conirmed by sub-culture on
Yeast extract 5.0 to either Baird-Parker Medium (LAB285+X085) or Rabbit Plasma
Fibrinogen Agar (LAB285+X086). Suspected positive tubes should
Lithium chloride 5.0 then be reincubated for full 48 hours.
Mannitol 20.0 48 hours: tubes suspected as positive at 48 hours should be conirmed
by sub-culture on to either Baird-Parker Medium (LAB285+X085) or
Sodium chloride 5.0 Rabbit Plasma Fibrinogen Agar (LAB285+X086).
Glycine 1.2 Tubes showing a presumptive negative result after 48 hours incubation
should also be sub-cultured on to either Baird-Parker Medium or RPF
Sodium pyruvate 3.0 Agar.
References
Method for reconstitution Giolitti, G. and Cantoni, C. (1966). A medium for the isolation of
For single-strength media: disperse 54.2g of powder in 1 litre of staphylococci from foodstuffs. J. Appl. Bacteriol. 29:395-398.
distilled water. Allow to soak for 10 minutes, swirl to mix and add
1g polyoxyethelene sorbitan mono-oleate (Polysorbate 80). Swirl Mossel, D.A.A., Harrewijn, G.A. and Elzebroek, J.M. (1973).
to disperse and heat gently to dissolve. Dispense the medium in UNICEF.
appropriate quantities into tubes of suitable dimensions e.g. 9ml in ISO 6888-3:2003 Microbiology of food and animal feeding stuffs
16mm x 160mm tubes. Sterilise at 121ºC for 15 minutes. - Horizontal method for the enumeration of coagulase-positive
For double-Strength media: disperse 108.4g of powder in 1 litre of staphylococci (Staphylococcus aureus and other species) - Part 3:
distilled water. Allow to soak for 10 minutes, swirl to mix and add Detection & MPN technique for low numbers.
2g polyoxyethelene sorbitan mono-oleate (Polysorbate 80). Swirl
to disperse and heat gently to dissolve. Dispense the medium in
appropriate quantities into tubes of suitable dimensions e.g. 10ml in
20mm x 200mm tubes. Sterilise at 121ºC for 15 minutes.
M.R.S. Agar
If product is to be used on day of preparation, allow to cool to 44-47ºC (de Man, Rogosa and Sharpe Agar)
and use immediately.
If the medium is not used as above then the medium must be re-heated
LAB093
to 100ºC for 15 minutes to expel any dissolved oxygen and cooled to Description
44-47ºC.
MRS Agar is a medium for the cultivation and enumeration of
Prior to use add X043 1% Potassium Tellurite to give a inal Lactobacillus spp.
concentration of 0.1g/L, e.g. add 0.1ml X043 to 9ml of single strength
base or add 0.2ml X043 to 10ml of double strength base. DO NOT Originally developed in 1960 by de Man, Rogosa & Sharpe, the
REHEAT MEDIA CONTAINING POTASSIUM TELLURITE. medium is suitable for most lactic acid bacteria and is intended as a
substitute for Tomato Juice Agar.
Appearance: When acidiied to pH 5.4 M.R.S. Agar can be used to enumerate
Powder: ine, free-lowing, homogeneous, buff Lactobacillus bulgaricus in yoghurts.
Finished medium: clear, straw liquid (will be darker if prepared at Nutrition is provided by a mixture of carefully selected peptones,
double-strength) glucose, beef & yeast extracts whilst Polysorbate 80, magnesium
and manganese sulphates act as growth stimulants. Selectivity
pH: 6.9 ± 0.2 against streptococci & moulds is provided by ammonium citrate and
sodium acetate. Used at low pH, ammonium citrate allows growth
Hazard classiication of lactobacilli whilst inhibiting a number of other organism groups.
Occasionally, sterilisation of this medium at 121˚C for 15 minutes, in
some autoclaves, may cause the pH to fall outside of the speciied pH
Minimum Q.C. organisms: limits 6.4 +/- 0.2. In these rare cases, adjustment of the medium using
Staphylococcus aureus WDCM 00034 acetic acid or sodium hydroxide is recommended.
Storage:
Mixed Peptones 10.0
Prepared media: use immediately after preparation. The sterilised base Yeast Extract 5.0
medium can be stored at 2-8°C for 1 week but prior to use heat the Beef Extract 10.0
base medium at 100ºC for 15 minutes to expel any dissolved oxygen,
cool to 44-47ºC and aseptically add X043 as per directions for use. Glucose 20.0
Inoculation: Dipotassium phosphate 2.0
Detection method: add 1g/1ml of sample to 9ml/9g single-strength
medium or 10g/10ml of sample to 10ml/10g double-strength broth. Sodium acetate 5.0
Enumeration method: add 1ml of sample to each of three tubes of Triammonium citrate 2.0
single-strength medium or 10ml of sample to each of three tubes of
double-strength medium. Repeat for any subsequent dilutions. Magnesium sulphate 0.2
Incubation: Incubate anaerobically (either by agar/parafin plug in Manganese sulphate 0.05
each tube or under anaerobic conditions in a gas jar or anaerobic
workstation) at 37°C for 24 to 48 hours (± 2 hours). Polysorbate 80 1.08
Interpretation: Formation of a black precipitate or the blackening of Agar No. 1 15.0
the broth indicates the presence of coagulase-positive staphylococci.

Weigh 70 grams of powder and add 1 litre of deionised water. Allow Disperse 70 grams of powder in 1 litre of deionised water. Allow to
to soak for 10 minutes, swirl to mix then sterilise by autoclaving at soak for 10 minutes, swirl to mix, then sterilise by autoclaving at
121°C for 15 minutes. Cool to 47°C and pour into sterile Petri dishes. 121°C for 15 minutes. Cool to 47°C and mix well before dispensing
If acidiied medium is required, adjust pH prior to pouring. Dry the into sterile Petri dishes. Dry the agar surface before use.
agar surface before use.
Appearance:
Appearance: Powder: ine, slightly cohesive, light tan powder with some lumps
Powder: ine, slightly cohesive, light tan powder with some lumps
Finished medium: clear, tan gel
Finished medium: Light amber, clear gel
pH: 6.4 ± 0.2 pH: 5.7 ± 0.1
Hazard classiication: NR – Not regulated Hazard classiication
Lactobacillus casei subsp. rhamnosus WDCM 00101 Minimum Q.C. organisms:
Lactobacillus plantarum ATCC 8014 Lactobacillus sakei subsp. sakei WDCM 00015
Lactobacillus delbrueckii subsp. lactis ATCC 4797 Lactococcus lactis subsp. lactis WDCM 00016
Storage of Prepared Medium: Escherichia coli WDCM 00013 (inhibited)
Dehydrated culture media: 10-25˚C. Storage:
Final medium: Plates - 7 days at 2-8˚C. Dehydrated culture media: 10-25°C away from direct sunlight.
Capped containers – up to 1 month at 15-20˚C in the dark. Prepared media: 7 days at 2-8°C.
Inoculation: Surface, spread to cover surface, or use pour plate Inoculation: Using the pour plate method, inoculate 1ml of the test
technique. sample (serial dilutions should be used) in to the Petri dish, before
Incubation: 25˚C microaerobically for 2-5 days. pouring over the molten agar. Mix carefully & allow to solidify.
Interpretation: Count all colonies exhibiting typical morphology. Overlays may be used if required.
Surface inoculations may also be used
References
Incubation: Incubate microaerobically at 30°C for 72 hours ± 3
de Man, J.C.,. Rogosa, M and Sharpe, M.E. (1960). A medium for hours.
the cultivation of lactobacilli. J. Appl. Bacteriol. 23: 130-135.
Interpretation: Count all colonies.
Some Leuconostoc spp. may form large, slimy colonies which
may hinder the development of other colonies, thus causing an
MRS Agar (ISO) underestimation of the number of lactic acid bacteria.
de Man, Rogosa & Sharpe Agar at pH 5.7 according to ISO Due to the possible development of microorganisms other than
15214:1998 lactic acid bacteria, it may be necessary in some cases to conirm the
colonies obtained using simple techniques such as Gram stain or test
for catalase.
LAB223
References
Description de Man, J.C., Rogosa, M and Sharpe, M.E. (1960). A medium for the
MRS Agar (ISO) is a medium for the enumeration of mesophilic cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-135.
lactic acid bacteria according to ISO 15214:1998.
This medium was originally developed in 1960 by de Man, Rogosa ISO 15214:1998 Microbiology of food and animal feeding stuffs
& Sharpe for the cultivation and enumeration of Lactobacillus spp. – Horizontal method for the enumeration of mesophilic lactic acid
from various sources and is intended as a substitute for Tomato Juice bacteria – Colony count technique at 30°C.
Agar. This original formulation has been adapted and adjusted to pH
5.7 according to ISO 15214:1998.
Nutrition is provided by enzymatic digest of casein, glucose, meat
& yeast extracts whilst polyoxyethylenesorbitan monooleate,
magnesium and manganese sulphates act as growth stimulants.
Selectivity against streptococci & moulds is provided by ammonium
citrate and sodium acetate. Used at low pH, ammonium citrate allows
growth of lactobacilli whilst inhibiting a number of other organism
groups.
Although MRS Agar (ISO) is optimised to be selective for
Lactobacilli, some growth of Leuconostoc spp. and Pediococci may
also occur.
Occasionally, sterilisation of this medium at 121oC for 15 minutes, in
some autoclaves, may cause the pH to fall outside of the speciied pH
limits 5.7 +/- 0.1. In these rare cases, adjustment of the medium using
acetic acid or sodium hydroxide is recommended.

Enzymatic digest of casein 10.0
Meat extract 10.0
Yeast extract 4.0
Triammonium citrate 2.0
Sodium acetate 5.0
Magnesium sulphate heptahydrate 0.2
Manganese sulphate tetrahydrate 0.05
Dipotassium hydrogen phosphate 2.0
Glucose 20.0
Polyoxyethylenesorbitan monooleate 1.08
Agar 15.5

M.R.S. Broth MSRV
(de Man, Rogosa and Sharpe Broth) (Semi-solid Rappaport Medium)
LAB094 LAB150
MRS Broth is a medium for the cultivation and enumeration of MSRV was developed in 1986 by De Smedt, Bolderdijk and Rappold
Lactobacillus spp. This product has the same formulation as LAB093 as a rapid means of Salmonella detection. The medium, based upon
MRS Agar with the omission of agar. Rappaport Vassiliadis broth, is inoculated directly from the pre-
Originally developed in 1960 by de Man, Rogosa & Sharpe, the enrichment medium, in the centre of the plate. Motile organisms
medium can be used for conirmatory tests on organisms isolated spread from the centre in the semi-solid agar, but non-salmonellas are
on MRS Agar. The medium can also be used for enumeration by the inhibited by the selective agents.
Miles and Misra technique. After overnight incubation the use of polyvalent salmonella antisera
Nutrition is provided by a mixture of carefully selected peptones, or a latex kit can conirm the presence of a Salmonella. Alternatively,
glucose, beef & yeast extracts whilst Polysorbate 80, magnesium and a paper disc wetted with polyvalent H antiserum can be placed 1/3
manganese sulphates act as growth stimulants. Selectivity against of the way from the edge of the dish, and will signal the presence of
streptococci & moulds is provided by ammonium citrate and sodium a Salmonella by inhibiting the mobility of the organism around the
acetate. disc.
Occasionally, sterilisation of this medium at 121˚C for 15 minutes, in Using this medium De Smedt and Bolderdijk have reported the
some autoclaves, may cause the pH to fall outside of the speciied pH possibility of detecting Salmonella in 24hrs (1987)
limits 6.4 +/- 0.2. In these rare cases, adjustment of the medium using
acetic acid or sodium hydroxide is recommended. Typical Formula g/litre
Typical Formula g/litre Tryptone 2.3
Mixed Peptones 10.0 Meat Peptone 2.3
Yeast Extract 5.0 Acid Hydrolysed Casein 4.7
Beef Extract 10.0 Sodium chloride 7.3
Glucose 20.0 Potassium dihydrogen phosphate 1.5
Potassium phosphate 2.0 Magnesium chloride 10.9
Sodium acetate 5.0 Malachite green 0.037
Magnesium sulphate 0.2 Agar No. 1 2.5
Manganese sulphate 0.05 Method for reconstitution

Polysorbate 80 1.08 Weigh 31.5 grams of powder and disperse in 1 litre of deionised water.
Soak for 10 minutes, swirl to mix and bring to the boil. Cool to 47˚C.
Ammonium citrate 2.0 and add 2 vials of X150 novobiocin supplement (10mg/vial). Mix
well before dispensing.
Weigh 55 grams of powder and add 1 litre of deionised water. Appearance: Turquoise/blue, clear, soft gel.
Allow to soak for 10 minutes, swirl to mix then warm to completely pH: 5.2 ± 0.2
dissolove solids. Dispense into suitable tubes or bottles then sterilise
by autoclaving at 121°C for 15 minutes. Minimum Q.C organisms: Salmonella typhimurium
WDCM 00031
Appearance: E. coli (inhibition) WDCM 00013
Powder: ine, slightly cohesive, light tan powder with some lumps
Finished medium: Light amber, clear Storage of prepared medium: Plates – up to 7 days at 4˚C.
pH: 6.4 ± 0.2 Inoculation: From pre-enrichment broth (6-24hrs) adding 0.1ml to
the centre of the plate.
Incubation: 37˚C. or 42 ± 0.5˚C. for 18-24 hours. Keep lid uppermost
Minimum Q.C. organisms: at all times.
Lactobacillus casei subsp. rhamnosus WDCM 00101 Interpretation: A spreading growth indicates a Salmonella may
Lactobacillus plantarum ATCC 8014 be present, substantiated if a disc with polyvalent H antiserum has
Lactobacillus delbrueckii subsp. lactis ATCC 4797 been added and is inhibiting the zone. This should be conirmed
by subculturing from the edge of the mobility zone onto XLD and
Storage of Prepared Medium: brilliant green agar and performing biochemical and serological tests.
Direct latex agglutination may be carried out from the edge of the
Dehydrated culture media: 10-25°C. mobility zone.
Final medium: capped containers – up to 3 months at 15-20°C in the
dark. References
Inoculation: Either with suspect colonies from M.R.S. agar or with De Smedt, J.M. and Bolderdijk, R.F. (1987): ‘One Day Detection of
serial dilutions of test material. Salmonella from Foods and Environmental Samples by Mobility
Enrichment’. Fifth International Symposium on Rapid Methods and
Incubation: 25˚C microaerobically for 2-5 days. Automation in Microbiology and Immunology, Florence (1987).
Brixia Academic Press.
Interpretation: For enumeration purposes count tubes showing signs
of growth as positive. De Smedt, J.M. and Bolderdijk, R.F., Rappold H. and Lautenschlaeger,
D. Rapid Salmonella Detection in Foods in Mobility Enrichment on
References a Modiied Semi-Solid Rappaport- Vassiliadis Medium. Journal of
de Man, J.C., Rogosa, M. and Sharpe, M.E. (1960). A medium for Food Protection 49 510-514. (1986).
the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-135. De Smedt, J.M. and Bolderdijk, R.F. Dynamics of Salmonella Isolation
with Modiied Semi-Solid Rappaport-Vassiliadis Medium.
Journal of Food Protection 50 658-661. (1987).

De Smedt, J.M. and Bolderdijk, R.F. Collaborative Study of the
International Ofice of Cocoa. Chocolate and Sugar Confectionery on
Mueller Hinton Broth
the Use of Mobility Enrichment for Salmonella Detection in Cocoa
and Chocolate. Journal of Food Protection 53 659-664. (1990). LAB114
Goossens, H., Wauters, G., De Boeck, M., Janssens, M., and Butzler, Description
J.P. Semi-solid selective mobility enrichment medium for isolation This medium is the broth version of Mueller Hinton Agar. It is an
of Salmonella from faecal specimens J. Clin. Microbiol 19 940-941. antagonist free medium for use in the tube dilution technique for the
(1984). determination of antibiotic M.I.C. values. The medium is carefully
standardised to meet N.C.C.L.S. standards for antimicrobial
susceptibility tests on bacteria which grow aerobically.
Mueller Hinton Agar Typical Formula g/litre
LAB039 Beef Extract 2.0
Acid Hydrolysed Casein 17.5
Description
Starch 1.5
A medium for antimicrobial sensitivity testing by the disc diffusion
method. This medium, used in the technique of Bauer and Calcium ions 50 mg/litre
Kirby, has been adopted by the National Committee for Clinical
Laboratory Standards (NCCLS) in the USA as the deinitive method Magnesium ions 20 mg/litre
for susceptibility testing. The medium has a very low thymine
and thymidine content, making it suitable for trimethoprim and Method for reconstitution
sulphonamide testing, controlled to ensure correct zone sizes Weigh 21 grams powder, disperse in 1 litre distilled water. Allow
with aminoglycoside and tetracyline antibiotics. The medium was to soak for 10 minutes, swirl to mix then heat gently to dissolve.
originally formulated as a heat labile protein free medium for the Distribute into tubes or bottles, and sterilise at 121˚C for 10 minutes.
isolation of pathogenic Neisseriaceae.
Appearance: Pale straw, cloudy.
pH: 7.4 ± 0.2
Beef Extract 2.0
Acid Hydrolysed Casein 17.5 Minimum Q.C. organisms: S. aureus WDCM 00034
E. coli WDCM 00013
Starch 1.5 (M.I.C. values)
Agar No. 1 17.0
Calcium ions 50-100mg/litre at 15-20˚C in the dark.
Magnesium ions 20-35mg/litre Inoculation: Standard inocula are required. As described by
NCCLS.
Method for reconstitution Incubation: As recommended by methodology for particular
Weigh 38 grams of powder, disperse in 1 litre of deionised water. organisms and antibiotics by NCCLS.
Allow to soak for 10 minutes, swirl to mix then sterilise at 121˚C for
15 minutes. Cool to 47˚C, mix well and pour plates. References
MacFaddin, J. (1985). Media for isolation cultivation, identiication
Appearance: Straw coloured, clear gel. maintenance of medical bacteria. Williams & Williams, Baltimore.
pH: 7.3 ± 0.1 N.C.C.L.S.-M7-A. (1985). Methods for dilution antimicrobiol
susceptibility tests for bacteria that grow aerobically. Approved
Minimum Q.C. organisms: E. coli WDCM 00013 Standard.
S. aureus (antibiotic sensitivity
zones) WDCM 00034

dark.
Inoculation: Surface, inoculum as described by N.C.C.L.S.
Incubation: As recommended by methodology for particular
organisms and antibiotics by NCCLS.
References
Mueller, J.H. and Hinton, J. (1941). Protein-free medium for primary
isolation of gonococcus and meningococcus. Proc. Soc. Exp. Biol.
and Med., 48: 330-333.
Goodale, W.I., Gould, G. and Schwab, L. (1943). Laboratory
Identiication of sulphonamide resistant gonococcic infection. J.Am.
Med. Ass., 123: 547-549.
American Public Health Association. (1950). Diagnostic Procedures
and Reagents. 3rd edn., A.P.H.A., New York.
NCCLS. (1986). Performance standards for antimicrobial susceptibility
testing – second informational supplement.

Mueller-Kauffmann Tetrathionate Nutrient Agar
novobiocin Broth (MKTTn) LAB008
LAB202 Description
Description A general purpose medium for the cultivation of organisms that are
not demanding in their nutritional requirements e.g. organisms that
A selective enrichment medium for the isolation of salmonellae from
can be isolated from air, water, dust etc. Nutrient Agar is suitable
food and animal feeds. The recent addition of novobiocin is to inhibit
for teaching and demonstration purposes, it is isotonic and can be
the growth of Proteus spp.
enriched with biological luids such as sterile blood and egg yolk.
Meat Extract 4.3 Peptone 5.0
Enzymatic digest of casein 8.6 Beef Extract 3.0
Sodium chloride 2.6 Sodium chloride 8.0
Calcium carbonate 38.7 Agar No. 2 12.0
Sodium thiosulphate (anhydrous)* 30.45
Ox bile 4.78
Brilliant green 0.0096 Allow to soak for 10 minutes, swirl to mix then sterilise by autoclaving
for 15 minutes at 121˚C. Cool to 47˚C, mix well then pour plates.
*Equivalent to 47.8g of sodium thiosulphate pentahydrate.
Appearance: Buff, opalescent gel.
Method for reconstitution pH: 7.3 ± 0.2
water. Allow to soak for 10 minutes, swirl to mix and bring to the boil. Minimum Q.C. organisms: S. aureus WDCM 00034
Cool to below 45°C, prior to use add 20 ml of iodine-iodide solution E. coli WDCM 00013
and 4 vials of X150 Novobiocin. Mix well and distribute into sterile
containers. Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
Iodine-iodide solution dark. Capped containers – up to 3 months at 15-20˚C in the dark.
Dissolve 25g of potassium iodide in 10 ml of water. Add 20g iodine Inoculation: Surface streaking for single colonies.
and dilute to 100ml with sterile deionised water. Incubation: Temperature and time to suit organisms. Usually
Appearance: aerobic.
Powder: ine, free-lowing, homogeneous, buff Growth Characteristics
Finished medium: Green turbid solution that precipitates on standing colony size shape &
pH: 8.2 ± 0.2 (base medium)
Hazard classiication Yellow
NR – Not regulated other
Staphylococcus 0.5-2.0 CV.E.G. White-
Minimum Q.C. organisms: spp. Yellow
Salmonella Typhimurium WDCM 00031 Strep. pyogenes P.P.-0.5 CV.E.G. Transp.
E. coli 1.5-2.5 CV.E.G. Grey
Storage: Proteus spp. spreading - Grey ishy odour
Klebsiella spp. 2.0-4.0 CV.E.G. Grey mucoid
Base medium (without supplements): 2 weeks at 10-25°C
Complete medium: use on day of preparation Bacillus spp. 2.0-6.0 various Grey may spread
Inoculation and Incubation: Following pre-enrichment in non- Ps. aeruginosa 2.0-4.0 F.CR.D. Grey-Green odour if
selective liquid media (see ISO 6579:2002), transfer 1ml of the broth pigmented
to 10ml of MKTTn and 0.1ml to 10ml of RVS broth (LAB086).
Incubate LAB202 MKTTn at 37°C ± 1°C for 24h ± 3h and LAB086
RVS broth at 41.5 ± 1°C, for 24h ± 3h. Subculture these selective
broths onto XLD agar (LAB032) and a second isolation medium of
your choice and incubate for 24h ± 3h. Salmonella should be conirmed
by appropriate biochemical and serological techniques.
References
BS EN ISO 6579:2002 Microbiology of food and animal feeding
stuffs – Horizontal method for the detection of Salmonella spp.

Nutrient Broth “E” O157 Broth MTSB
(Modiied Tryptone Soy Broth)
LAB068
Description
LAB165
An inexpensive broth for the growth of nutritionally non-demanding Description
organisms. Ideal for teaching purposes.
Modiied tryptone soy broth has emerged as the medium of choice
for the enrichment of E. coli O157:H7 in red meats . As concern
1,2
Typical Formula g/litre regarding this organism has grown due to the severity of the disease
Beef Extract 1.0 syndromes caused, and the increase in foodborne infection , so too
3
has the need to optimise methods for its eficient isolation. Symptoms
Yeast Extract 2.0 start with severe stomach cramps and watery, bloody diarrhoea, and a
percentage of individuals infected will develop Haemolytic Uraemic
Peptone 5.0 Syndrome (HUS) leading to acute renal failure . In a comparison of 4
4
Sodium chloride 5.0 different selective broth media, MTSB was the most productive and
selective for the isolation of E. coli O157:H7. MTSB is made selective
for O157:H7 by including bile salts in the dehydrated medium, and the
addition of novobiocin supplement (X150).
Weigh 13 grams of powder, add to1 litre of deionised water. Heat to
dissolve then dispense into bottles or tubes. Sterilise by autoclaving Typical Formula g/litre
at 121˚C for 15 minutes.
Tryptone 17.0
Appearance: Straw coloured, clear.
Sodium chloride 5.0
pH: 7.4 ± 0.2
K2HPO4 4.0
Minimum Q.C. organisms: S. aureus WDCM 00034 Dextrose 2.5
E. coli WDCM 00031
Soy Peptone 3.0
Storage of Prepared Medium: Capped containers – up to 3 months Bile Salts No.3 1.5
Inoculation and incubation: To suit chosen organism.
Growth indicator: Turbidity.
Weigh 33 grams of powder and add to 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix and autoclave at 121˚C for
15 minutes. Cool to 47˚C and add 2 vials of Novobiocin supplement
Nutrient Broth No. 2 X150. Mix well and distribute aseptically into sterile containers.
Appearance: Clear straw broth
LAB014 pH: 7.2 ± 0.2
Description Minimum QC organisms: E. coli O157:H7
A general purpose broth which can be used for sterility testing for (non-toxigenic) WDCM 00014
aerobic organisms. This broth can also be used as the suspending E. coli WDCM 00013
medium for cooked meat granules for the cultivation of anaerobic (inhibition)
organisms.
Inoculation: Add 25g sample to 225ml of supplemented MTSB and
Typical Formula g/litre homogenise for 2 minutes.
Beef Extract 10.0 Incubation: 42˚C aerobically for 24hrs. Subculture onto CT-SMAC
Peptone 10.0 (LAB161 plus X161) or SMAC-BCIG (HA006) and examine for
non-sorbitol fermenting colonies and/or glucuronidase negative
Sodium chloride 5.0 organisms. Some workers recommend the use of an immunomagnetic
1
separation step after 6hrs incubation.

Weigh 25 grams of powder, disperse in 1 litre of deionised water. Interpretation: Turbidity in the broth indicates growth. All broths
Allow to soak for 10 minutes, swirl to mix then dispense into tubes or should be subcultured to selective media whether turbid or not.
bottles, and sterilise for 15 minutes at 121˚C.
References
Appearance: Pale straw, clear. 1) Bolton, E.J., Crozier, L., Williamson, J.K. (1995) Optimisation of
methods for the isolation of Escherichia coli O157 from beefburgers.
pH: 7.3 ± 0.2 PHLS Microbiology Digest 12 (2) 67-70.
Minimum Q.C. organisms: S. aureus WDCM 00034 2) Willshaw, G.A., Smith, H.R., Roberts, D., Thirlwell, J., Cheasty, T.,
E. coli WDCM 00013 Rowe, B. (1993) Examination of raw beef products for the presence
of verocytotoxin producing Escherichia coli, particularly those of
serogroup O157. J.Appl.Bacteriol. 75 420-426.
at 15-20˚C in the dark. 3) Sharp, J.C.M., Coia, J.E., Curnow, J., Reilly, W.J. (1994)
Escherichia coli O157 infections in Scotland. J.Med.Microbiol 40
Incubation: 37˚C for 18-24 hours aerobically. 3-9.
References 4) Doyle, M.P. (1991) Escherichia coli O157:H7 and its signiicancein
foods. Int.J.Food Microbiol. 12 289-302.
British Pharmacopoeia. (1973). H.M.S.O., London. Cruikshank, R.
(1972). Medical Microbiology. 11th edn. Livingstone, London.

Orange Serum Agar ORSIM
(Oxacillin Resistant Staphylococci Isolation Medium)
LAB147
Description
LAB192
A medium developed for the investigation of organisms involved
in the spoilage of citrus products including fruit juices and citrus Description
concentrates. The low pH of these products restricts the growth of The over-prescription of therapeutic antibiotics in recent years is
organisms to those capable of tolerating an acid environment such thought to be a contributing factor in the rising numbers of multi-
as yeasts and moulds and bacteria belonging to the genera Bacillus, resistant bacteria being encountered. One such bacterium, MRSA
Lactobacillus, Leuconostoc, Streptococcus and Clostridium. By (multi resistant Staphylococcus aureus), is particularly prevalent
having a low pH and incorporating orange extract, Orange Serum within the hospital environment, and is well recognised as a pathogen
Agar is the ideal isolation medium. amongst immuno-compromised patients. In this situation, early
detection is vital to ensure the individual’s survival. This media is an
Typical Formula g/litre improved version of the highly regarded Mannitol Salt Agar (LAB007)
and incorporates an enhanced indicator system using aniline blue
Tryptone 10.0 and mannitol fermentation. This combination produces intense blue
colonies as presumptive MRSA, which are unmistakable amongst
Yeast Extract 3.0 mixed cultures and easily visualised against the media background.
Orange Extract 5.0 To complement this, ORSIM possesses a reined selectivity, derived
from a reduction in the salt level to 55g/L, and the introduction of
Glucose 4.0 Lithium chloride at 5g/L. This chemical mixture still provides the
required inhibition towards competing organisms, whilst ensuring
Di-potassium phosphate 3.0
optimal recovery of MRSA, even at low numbers. To complete the
Agar No.2 17.0 medium, the selective supplement X192 is included. This contains
two antibiotics, oxacillin to inhibit multi sensitive Staphylococcus
aureus (the cause of false positives), and polymyxin B to suppress
Method for reconstitution other halophillic bacteria such as Proteus spp.
Allow to soak for 10 minutes. Bring to the boil, swirling frequently. Typical Formula g/litre
Sterilise by autoclaving for 15 minutes at 115°C. Cool to 47°C, mix
well and dispense into Petri dishes. Peptone 11.8
Appearance: Yeast Extract 9.0
Powder: ine, free-lowing, homogeneous, buff Mannitol 10.0
Finished medium: Amber, slightly opalescent gel Sodium chloride 55.0
pH: 5.5 ± 0.2 Lithium chloride 5.0
Minimum Q.C. organisms Lactobacillus plantarum Aniline blue 0.2
Aspergillus niger Agar 12.5
Storage of Prepared Medium: Plates – up to 7 days at 2-8°C. in the Method for Reconstitution
dark. Capped containers – 10-25°C. in the dark.
Weigh 103.5 grams of powder and disperse into 1 litre of deionised
Inoculation: Pour plate technique. water. Allow the mixture to soak for 10 minutes, swirl to mix then
Incubation: 3 days at 30°C. for bacteria, 5 days at 30˚C for yeasts sterilise at 121°C for 15 minutes. Cool to 47°C, and aseptically add
and moulds. 2 vials of X192 supplement. Mix well, and pour into sterile Petri
Interpretation: Count bacterial colonies and yeasts/moulds dishes.
separately. Calculate the colony forming units (CFU) per ml of the
sample, allowing for dilution factors. Appearance: Straw/grey gel.
pH: 7.2 ± 0.2
References
Hays, G.L. (1951) The isolation, cultivation and identiication of Minimum Q.C. organisms:
organisms which have caused spoilage in frozen concentrated orange Staphylococcus aureus (MRSA Strain) NCTC 11940
juice. Proc. Florida State Hort. Soc. E.coli NCTC 25922 (inhibition).
Hays, G.L. and Reister, D.W. (1952) The control of ‘off-odour’
spoilage in frozen concentrated orange juice. Food Tech 6 p386. Storage of Prepared Medium: Plates can be stored up to 7 days at
Murdock, D.I., Folinazzo, J.F., and Troy, V.S. (1952) Evaluation of 2-8°C in the dark.
plating media for citrus concentrates. Food Tech. 6 p181. Inoculation: Take a swab sample from a suspected infection and
apply the swab end directly to the surface of a supplemented plate of
ORSIM and streak out for single colonies.
Incubation: Aerobically at 37°C for 24 and 48 hours.
Interpretation: After incubation for 24 hours, examine the plate
for intense blue colonies and conirm using either coagulase/latex
agglutination and Penicillin binding protein 2’ test (PBP2’). Once
conirmed, all positive plates should be discarded safely.
*Typical strains of MRSA will be detected within 24 hours on this
medium. However, some strains may require longer incubation, so all
negative plates should be re-incubated for a further 24 hours*.
References
Orth, D.S. (1993) Handbook of Cosmetic Microbiology. Marcel
Dekker, Inc., New York, NY.
Brummer, B. (1976). Inluence of possible disinfectant transfer on
Staphylococcus aureus plate counts after contact sampling. App.
Environ. Microbiol. 32:80-84.

Oxytetracycline Glucose Yeast Extract PALCAM Agar Base
Agar Base (Polymyxin, Acrilavine, Lithium chloride, Ceftazidine,
(O.G.Y.E.) Aesculin, Mannitol)
LAB089 LAB148
A selective medium for the enumeration of yeasts and moulds in food, Palcam Agar was developed by Van Netten et al in 1989 as an
introduced by Mossel in 1970. Unlike many selective media for yeasts improved selective differential medium for the isolation of Listeria
OGYE has a neutral pH and it has been shown to give better recovery monocytogenes from food, clinical and environmental specimens.
rates than those media with a low pH. Oxytetracycline is used to inhibit Improved selectivity is achieved by the combination of antibiotic
bacteria, certain high protein foods may reduce the effectiveness of supplements and microaerobic incubation, whilst the double indicator
this antibiotic as a selective agent. Rose Bengal Chloramphenicol system of aesculin hydrolysis and mannitol fermentation aids
Agar (LAB036) is recommended in these instances. differentiation of Listeria spp from enterococci and staphylococci
which can be confused with Listeria spp on other types of culture
Typical Formula g/litre media.
Yeast Extract 5.0
Dextrose 20.0
Columbia Peptone Mix 23.0
Biotin 0.001
Sodium chloride 5.0
Agar No. 2 12.0
Corn Starch 1.0
Method for reconstitution Yeast Extract 3.0
Weigh 37 grams of powder, disperse in 1 litre of deionised water. Glucose 0.5
at 115°C for 10 minutes. Cool to 47°C and aseptically add 2 vials Mannitol 10.0
of X089 Oxytetracycline selective supplement. Mix thoroughly and
pour into Petri dishes. DO NOT REHEAT THIS MEDIA ONCE Aesculin 0.8
PREPARED. Lithium chloride 15.0
Appearance: Ferric ammonium citrate 0.5
Powder: ine, free-lowing, homogeneous, buff Phenol red 0.08
Finished medium: Clear, straw gel
pH: 7.0 ± 0.2 Agar No. 2 12.0
Hazard classiication: NR – Not regulated Method for Reconstitution

Minimum Q.C. organisms: Aspergillus sp. Soak for 10 minutes, swirl to mix then sterilise by autoclaving at 121˚C
Saccharomyces cerevisiae WDCM 00058 for 15 minutes. Cool to 47˚C and add 2 vials of P.A.C. Supplement –
X144. Mix thoroughly and pour into Petri dishes.
Storage of Prepared Medium: Dehydrated culture media: 10-25oC
Prepared media: Plates - up to 7 days at 2-8oC, in the dark Appearance: Red, translucent
Capped containers – up to 1 month at 2-8oC, in the dark pH: 7.2 ± 0.2
Inoculation: Surface spreading or pour plate.
Minimum Q.C. organisms L. monocytogenes
Incubation: 25˚C aerobically for 5 days. WDCM 00020
colony size shape & Storage of Prepared Medium: Plates – up to 7 days at 4˚C in the
organism (mm) surface colour dark.
Inoculation: 0.1ml of sample selectively enriched in Palcam Broth
Candida spp. 3.0-4.0 CV.E.D. Cream (or other enrichment medium) spread over surface of plate.
Incubation: 30˚C aerobically or microaerobically for 24-48 hours.
Candida krusei 7.0-8.0 F.CR.D. White
S. cerevisiae 3.0-4.0 CV.E.D. White Growth Characteristics

colony size shape &
Pen. notatum 1.5 Green/blue centre organism (mm) surface colour other
Pen. lavescens 1.5-2.0 Yellow centre L. monocytogenes 1.5-2.0mm F.E.D. Grey/green Black halo,
(draughtsman
colonies).
References
Other
Mossel, D.A.A. et al. (1970). O.G.Y.E. for Selective Enumeration of Listeria spp. 0.5-2.0mm F.E.D. Grey/green Black halo,
Moulds and Yeast in Foods and Clinical Material. J. Appl. Bact. 35: (draughtsman
454-457.
colonies).
Banks, J.G., Board, R.G. (1987). Some factors inluencing the recovery
of yeasts and moulds from chilled foods. Int. J. Food Microbiol. 4: Enterococci Inhibited - - (Small yellow
197-206. colonies
with yellow/
green
halo).
Staphylococci Inhibited - - (Small white/
yellow colonies
with yellow/
green halo)
Bacillus spp. Inhibited - - -
References
Van Netten, P., Perales, I., Curtis, G.D.W., Mossel, D.A.A. (1989)
Liquid and solid selective differential media for the enumeration of
L. monocytogenes Int. J. Food Micro. 8 (4) 299-316.

PALCAM Broth PEMBA
(Bacillus Cereus Medium)
L-PALCAM Broth (Liquid, Polymyxin, Acrilavine, Lithium
chloride, Ceftazidime, Aesculin, Mannitol) LAB193
LAB144 Description
This medium is based on the highly speciic and sensitive PEMBA
Description medium. It is used for the isolation and enumeration of Bacillus cereus.
This formulation speciically enhances egg yolk precipitation and
Developed by Van Netten et al (1989) L-Palcam is a selective sporulation of Bacillus cereus. The bromothymol blue pH indicator
differential medium for the enrichment of Listeria spp. in food, gives clear visualisation of alkaline mannitol non-fermenting colonies
environmental and clinical samples. It is unique amongst Listeria and egg yolk precipitation indicative of B. cereus. The selectivity
enrichment media in that it contains an indicator system (aesculin) is provided by the polymyxin B supplement (X193) and provides
which will signal the presence of a possible Listeria by a browning/ excellent results for the majority of sample types.
blackening of the broth; the result being the indication of a potential
problem up to 48 hours before growth on plating media can be Microscopic examination of presumptive B. cereus colony can conirm
observed. identity by presence of lipid globules in vegetative cells.
Columbia Peptone Mix 23.0 Peptone 1.0
Yeast Extract 5.0 Mannitol 10.0
Peptonised Milk 5.0 Sodium chloride 2.0
Sodium chloride 5.0 Magnesium sulphate 0.1
Mannitol 5.0 Disodium hydrogen phosphate 2.5
Aesculin 0.8 Bromothymol blue 0.12
Ferric ammonium citrate 0.5 Sodium pyruvate 10.0
Phenol red 0.08 Agar 15.0
Lithium chloride 10.0 Method for Reconstitution

Weigh 41g of powder and disperse in 950ml of deionised water,
Method for Reconstitution allow the mixture to soak for 10 minutes, swirl to mix and sterilize by
Weigh 54.4 grams of powder and disperse in 1 litre of deionised autoclaving for 15 minutes at 121ºC for 15 minutes. Cool to 47ºC and
water. Soak for 10 minutes, swirl to mix and autoclave at 121˚C for add two vials of X193 and 50ml of Egg Yolk Emulsion (X073) mix
15 minutes. Cool to 47˚C and add two vials of X144. Mix well and well and pour the plates. Dry the agar surface before inoculation.
dispense into sterile tubes or bottles.
Appearance: Yellow and opaque.
Appearance: Clear red broth pH: 7.2 ± 0.2
pH: 7.2 ± 0.2
Minimum Q.C. organisms: Bacillus cereus WDCM 00001.
L. monocytogenes WDCM 00020 Escherichia coli WDCM 00013 (inhibition).
Storage of Prepared Medium: up to 7 days at 2-8ºC in the dark.
Storage of prepared medium: Capped containers – up to 7 days at Inoculation: Surface spreading or streaking for single colonies.
4˚C.
Incubation: 30ºC aerobically for 24-48 hours.
Inoculation: Sample or pre-enriched sample added to the broth in the
ratio 1 : 10.
Incubation: 30˚C for 24 hours and 48 hours.
colony size shape &
Subculture: Onto Palcam Agar – LAB148. If low numbers of Listeria organism (mm) surface colour
are present the medium may not produce the brown black colour. All
B. cereus 3.0-4.0 F.CR.D Blue white halo
tubes should be subcultured onto selective agar before a sample is
scored as negative. B. subtilis 2.0-3.0 F.CR.D Yellow
B. licheniformis 2.0 F.CR.D Yellow
References E. coli no growth -
Van Netten, P., Perales, I., Curtis, G.D.W., Mossel, D.A.A. (1989)
S. aueus 1.0 CV.E.G. Yellow white halo
Liquid and solid selective differential media for the enumeration of L.
monocytogenes Int. J. Food Micro. 8 (4) 299-316.
References
Holbrook, R. & Anderson, J.M. (1980). Can. J. Microbiol., 26(7)
753-759.
Donovan, K.O. (1958). J. Appl. Bacteriol., 21(1) 100-103.
Mossel, D.A.A., Koopman, M.J. & Jongerius. E. (1967). J. Appl.
Bacteriol. 15(3) 650-653.

Peptone Water Method for reconstitution
LAB104 Allow to soak for 10 minutes, swirl to mix and sterilise by autoclaving
for 15 minutes at 121oC. Allow to cool to 47°C before adding 2 vials
each of selective supplements X109 and X110. Mix well before
Description
dispensing into Petri dishes.
A general purpose growth medium that can be used as a base for
carbohydrate fermentation studies. The medium has a high level of Appearance:
tryptone making it suitable for use in the indole test. Powder: ine, free-lowing, homogeneous, buff
Typical Formula g/litre Finished medium: pale straw, clear gel
Peptone 5.0 pH: 7.3 ± 0.2
Tryptone 5.0 Minimum Q.C. organisms: C. perfringens WDCM 00007

E. coli WDCM 00013
Sodium chloride 5.0
(inhibition)
Weigh 15 grams of powder, and disperse in 1 litre of deionised water. Storage of prepared medium:
Allow to dissolve then distribute into inal containers. Sterilise by Dehydrated culture media: 10-25oC away from direct sunlight.
autoclaving at 121°C for 15 minutes. If sterile additions are to be Prepared media: Use on day of preparation.
made to this medium e.g. carbohydrates, the volume of water for
Inoculation: Pour plates, 1ml sample plus 9ml medium. When set
reconstitution must be reduced accordingly. A pH indicator may be
overlay with sterile medium.
added to detect acid production from carbohydrate utilisation.
Incubation: Incubate at 37oC anaerobically for 24 hours.
Appearance:
Interpretation: Count large black colonies, presumptively identiied
Powder: ine, free-lowing, homogeneous, buff as C. perfringens.
Finished medium: clear, colourless
References
pH: 7.2 ± 0.2
Bowen AB, Braden CR (2006). “Invasive Enterobacter sakazakii
Hazard classiication: NR – Not regulated disease in infants”. Emerging Infect Dis 12 (8): 1185–9.
Handford, P.M. (1974). A new medium for the detection and
Minimum Q.C. organisms: enumeration of C. perfringens in foods. J. Appl. Bact. 37: 559-570.
Escherichia coli WDCM 00013 (indole positive)
Hauschild A.H.W. and Hilsheimer R. (1973). Evaluation and
Salmonella typhimurium WDCM 00031 (indole negative) Modiications of Media for Enumeration of Clostridium perfringens.
Applied Microbiology 27 p78-82.
Storage of Prepared Medium:
Shahidi, S.A. and Ferguson, A.R. (1971). A new quantitative and
Dehydrated culture media: 10-25°C.
conirmatory medium for C. perfringens in food. Appl. Microbiol.
Final medium: capped containers – up to 3 months at 15-20°C in the 21:500-506.
dark..
Marshall, R.S., Steenberger, J.F. and McClung, L.S. (1965). A rapid
Inoculation: A light inoculum from a pure culture. technique for the enumeration of C. perfringens. Appl. Microbiol. 13:
Incubation: According to organism. 559.
References Pharmacopoeia of culture media for food microbiology. (1987). Int. J.
Food Microbiol. 5:3:240-241.
Bergey’s Manual of Systematic Bacteriology, Vol. 1, (1984).
Williams and Wilkins, Baltimore/London.
MacFadden, J.F. (1983). Biochemical Tests for the Identiication
of Medical Bacteria, 2nd edn. Williams and Wilkins, Baltimore/ Perfringens Agar TSC
London.
(Tryptose Sulphite Cycloserine (TSC) Agar)
LAB194
Perfringens Agar OPSP
Description
LAB109 Perfringens Agar Base is a nutrient medium to which egg yolk
emulsion (X073) and cycloserine (X194) are added for the preparation
Description of Tryptose Sulphite Cycloserine (TSC) Agar. Sodium metabisulphite
Oleandomycin, Polymixin, Sulphadiazine, Perfringens (OPSP) agar, and ferric ammonium citrate are used as an indicator of sulphite
has been used as a standard medium for Clostridium perfringens reduction by Clostridium perfringens. The reduction of sulphite by
for many years. This medium was developed by Handford in 1974 Cl. perfringens produces black colonies and the egg yolk emulsion
to overcome some of the problems associated with enumerating incorporated into the media detects the lecithinase activity of this
Clostridium perfringens in foods. The medium is buffered and utilises bacteria. However not all strains produce lecithinase and therefore
sodium metabisulphite and liver extract as sources of H2S with ferric black lecithinase positive and black lecithinase negative colonies
ammonium citrate as the indicator. should be considered as presumptive Cl. perfringens.
The medium is made selective with the addition of X109 Sulphadiazine Typical Formula g/litre
and X110 Oleandomycin / Polymyxin supplements. Some strains
of C. perfringens may demonstrate sensitivity to the sulphadiazine Tryptose 15.0
antibiotic (X109) in such cases use of LAB194 TSC Perfringens Agar
Base (with X194 D-Cycloserine) should be considered. Soy Peptone 5.0
Beef extract 5.0
Yeast extract 5.0
Tryptone 15.0
Yeast Extract 5.0
Soy Peptone 5.0
Agar 14.0
Liver Extract 7.0
Tris buffer 1.5
Agar No. 2 10.0

Method for reconstitution Minimum Q.C. organisms: S. aureus WDCM 00032
Weigh 46.0 grams of powder and disperse in 1 litre of deionised E. coli WDCM 00013
water. Allow the mixture to soak for 10 minutes, swirl to mix and
sterilise by autoclaving at 121°C for 10 minutes. Allow the medium
to cool to 47°C and supplement with 2 vials of X194 (cycloserine) Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
and 50ml of egg yolk emulsion (X073), mix well and pour into sterile dark.
Petri dishes. The egg yolk emulsion is omitted for preparation of Egg Inoculation: Surface, or pour plate.
Yolk Free TSC Agar and Egg Yolk Free TSC Agar should be used for Incubation: 30˚C aerobically for 48 hours for aerobic mesotroph
an overlay medium. count. 6˚C aerobically for 10 days for aerobic psychrotroph count.
55˚C aerobically for 48 hours for aerobic thermotroph count.
Appearance: Straw, clear gel or pale yellow opaque gel.
Interpretation: Count all colonies or use spiral plating colony count
pH: 7.6 ± 0.2 equipment.
Storage of Prepared Medium: Plates can be stored up to 7 days at
2-8°C in the dark. References
Reasoner, D.J., Geldreich, E.E. (1985) A New Medium for the
Minimum Q.C. organisms: Enumeration and Subculture of Bacteria from potable water. App &
Clostridium perfringens WDCM 00007 Env. Microbiol. Jan. 1985 p.1-7.
Escherichia coli WDCM 00013 (inhibition) American Public Health Association (1985) Standard Methods for the
Enumeration of Water and Wastewater. 16th Edition. American Public
Inoculation: For a spread plate inoculate the agar plate with 0.1ml Health Association Inc. Washington D.C.
aliquots of an appropriate serial dilution of the homogenised test Environment Agency: The Microbiology of Drinking Water (2002).
sample and overlay if required. For a pour plate mix 1ml aliquots of Methods for the Examination of Water and Associated Materials.
an appropriate serial dilution of the homogenised test sample with
approximately 20 ml of TSC plus egg yolk emulsion. For full details
refer to appropriate references and standard method protocols.
Plate Count Agar (A.P.H.A.)
Incubation: 35°C ± 2°C anaerobically for 18-24 hours.
(Standard Methods Agar, Tryptone Glucose Yeast Agar)
Interpretation: Count all black colonies with or without a halo as
presumptive C. perfringens. Further conirmation should be carried LAB010
out according to standard method protocols e.g. nitrate reduction,
lactose fermentation, gelatin liquefaction and absence of motility. Description
Formulated to A.P.H.A. speciications, this medium is used for
References establishing total viable counts for aerobes in food, dairy and water
Shahidi, S.A. and Furguson, A.R. (1971). Appl. Microbiol. 21. 500- bacteriology. The product uses agar of very high gel strength in
506. order that it can be used in pour-plate as well as surface inoculation
techniques. The product can be remelted prior to use although it should
Harmon, S.M., Kauttar, D.A. and Peeler, J.T. (1971). Appl. not be held for a prolonged period in the molten stage.
Microbiol. 22. 688-692.
Hauschild, A. H. W. and Hilsheimar R. (1973). Appl. Microbiol. 27. Typical Formula g/litre
78-82.
Tryptone 5.0
Hauschild A.H.W. and Hilsheimar, R. (1973). Appl. Microbiol. 27.
521-526. Yeast Extract 2.5
Hauschild, A.H.W. et al (1977). Can. J. Microbiol. 23. 884-892. Glucose 1.0
Labbe, R. G. and Harmon, S.M. (1992). Compendium of methods for Agar No. 1 15.0
the microbiological examination of foods, 3rd ed 623-635. American
Public Health Association, Washington, D.C.
Rhodehamel, E.J. and Harmon, S.M. (1995). Bacteriological Weigh 23.5 grams of powder, disperse in 1 litre of deionised water.
Analytical Manual 8th ed. 16.01-16.06 AOAC International, Bring to the boil with frequent stirring to dissolve. Dispense into tubes
Gaithersberg, MD. and sterilise by autoclaving at 121˚C for 15 minutes. Cool to 44-46˚C
Andrews, W. (1995) Oficial methods of analysis AOAC International for not more than 3 hours prior to use.
16th ed. 1-119. AOAC International, Arlington, VA. ROLL-TUBES. Add an additional 10g/litre Agar No. 1 prior to
reconstitution of the medium.
Appearance: Pale straw coloured, clear gel.
Plate Count Agar
pH: 7.0 ± 0.2
LAB149
Minimum Q.C. organisms: S. aureus WDCM 00032
Description E. coli WDCM 00013
A medium designed for use with the spiral plating system and other
surface inoculation techniques. The formula is suitable for the Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
determination of total viable counts in food products by surface count dark. Capped containers – up to 3 months at 15-20˚C in the dark.
and pour plate methods. Inoculation method: Pour plate technique or surface inoculation.
Incubation: 30˚C aerobically for 48 hours for aerobic mesotroph
Typical Formula g/litre count. 6˚C aerobically for 10 days for aerobic psychrotroph count.
Tryptone 5.0 55˚C aerobically for 48 hours for aerobic thermotroph count.
Interpretation: Count all colonies and calculate the number of
Yeast Extract 2.5 organisms (or ‘colony forming units’ c.f.u.) per ml of sample allowing
Glucose 1.0 for dilution factors.
Agar No. 2 12.0 References
Method for reconstitution the Examination of Dairy Products. 13th edn. (ed. Hausler, W.J.)
Weigh 20.5 grams of powder, disperse in 1 litre of deionised water. A.P.H.A., Washington.
Allow to soak for 10 minutes, swirl to mix then sterilise by autoclaving American Public Health Association (1966). Recommended Methods
at 121˚C for 15 minutes. Cool to 47˚C then pour into Petri dishes. for the Microbiological Examination of Foods, 2nd edn. (ed. Sharf,
Appearance: Pale straw colour, clear. J.M.) A.P.H.A., Washington.
pH: 7.0 ± 0.2 the Examination of Water and Waste Water, 14th edn. (ed.. Franson,
M.A) A.P.H.A., Washington.

Potato Dextrose Agar Pseudomonas Agar Base
(C.F.C./C.N. Agar)
LAB098
Description LAB108
Potato Dextrose Agar is recommended by the American Public Health Description
Association for the enumeration of yeasts and moulds in examination
of dairy products, soft drinks, dried and frozen foods and other types of The base medium is a modiication of King’s medium A which
product. Depending on whether the medium is to be used as a selective uses magnesium and potassium salts to enhance production of the
or non-selective agar it can be used with or without acidiication. pigments pyocyanin (green) and luorescein (detected by U.V./blue
light). The medium is made selective for Pseudomonas aeruginosa by
Typical Formula g/litre the addition of X107 C.N. supplement. Alternatively the medium can
be made selective for Pseudomonas species generally by the addition
Potato Extract 4.0 of X108 C.F.C. supplement. This medium can be made selective for
Dextrose 20.0 the isolation of Burkholderia cepacia by the addition of X140.
Agar No. 1 15.0 Typical Formula g/litre

Acid Hydrolysed Casein 10.0
Weigh 39 grams of powder, disperse in 1 litre of deionised water, then Gelatin Peptone 16.0
sterilise at 121˚C for 15 minutes. Mix well before pouring into sterile Potassium sulphate 10.0
Petri dishes. In certain cases it may be desirable to lower the pH of the
medium to 3.5 in order to suppress bacterial growth. This can be done Magnesium chloride 1.4
by adding 10ml of sterile 10% Lactic Acid X037, to one litre of Potato
Dextrose Agar LAB098. This addition must be after autoclaving and Agar No. 2 11.0
cooling to 47˚C. Once the pH has been lowered the medium may not
be heated again without resultant loss of gel strength caused by agar Method for reconstitution
hydrolysis. Weigh 48.4 grams of powder and disperse in 1 litre of deionised
water. Add 10ml of glycerol. Sterilise by autoclaving at 121˚C for 15
Appearance: Translucent white agar. minutes. Allow the medium to cool to 47˚C then add the contents of
pH: 5.6 ± 0.2 (3.5-4.0 if X037 is added) 2 vials of either X107 C.N. supplement or X108 C.F.C. supplement.
Mix well and pour into Petri dishes.
Appearance: Pale straw, opaque.
Aspergillus brasiliensis WDCM 00053
Saccharomyces cerevisiae WDCM 00058 pH: 7.1 ± 0.2
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the Minimum Q.C. organisms: P. aeruginosa WDCM 00025
dark. Capped containers – up to 1 month at 15-20˚C in the dark. E. coli (inhibition) WDCM 00013
Inoculum: Pour plate technique.
Incubation: 21˚C aerobically for 5 days. Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
dark.
Growth Characteristics Inoculation: Surface, spread 0.1 to 0.5ml of sample over entire
surface.
colony size shape &
organism (mm) surface colour Incubation: 25-30˚C aerobically for 48 hours.
Candida spp. 2.0 C.V.E.D. White Interpretation: Count all colonies as Pseudomonas species.
Colonies that exhibit the pyocyanin and luorescein pigments count
Candida krusei 2.0 F.Rz.D. Grey/White
as P. aeruginosa.
Asp. niger 4.0 Black spores Yellow
centre obverse Growth Characteristics
White surround
colony size shape &
Pen. notatum 4.0 Green spores Green organism (mm) surface colour luorescence
centre
White surround obverse
Ps. aeruginosa 2.0-3.0 CV.Cr.D. Green/Blue yes
P. luorescens 2.0-3.0 CV.Cr.D. Yellow yes
References P. fragi 1.0-3.0 CV.Cr.D. Grey no
Association of Oficial Analytical Chemists (AOAC). Bacteriological
Analytical Manual, 5th ed. (1978). Washington D.C. Hausler,
W.J. (ed.). References
Standard Methods for the Examination of Dairy Prod. 14th edn., Burton, M.O., Campbell, J.J.R. and Eagles, B.A. (1948). The mineral
Washington D.C.: American Public Health Association, (1976). requirement for pyocyanin production. Can. J. res. Sect. C. Bot. Sci.
26:15.
King, E.O., Ward, M.K. and Raney, D.E. (1954). Two simple media
for the demonstration of pyocyanin and luorescein. J. Lab. Clin.
Med. 44: 301.
Goto, S. and Enomoto, S. (1970). Jap. J. Microbiol. 14: 65-72.
Mead, G.C. and Adams, B.W. (1977). Br. Poult. Sci. 18: 661-667.

R2A Broth pH: 7.2 ± 0.2
Appearance:
LAB203 Powder: ine, free-lowing, homogeneous, buff
Finished medium: clear, opalescent gel
Description
A medium of low nutritional content for use with membrane methods Method for reconstitution
for the enumeration of bacteria from water samples. Weigh 18 grams of powder and disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix and sterilise by autoclaving
Typical Formula g/litre for 15 minutes at 121oC. (If required, bring to the boil to dissolve the
agar, and pour into smaller volumes before sterilizing.) Cool to 44-
Yeast Extract 0.5 46°C for not more than 3 hours before use. Mix well before dispensing
into Petri dishes. Dry the agar surface prior to use.
Meat Peptone 0.5
Inoculation: Pour 15ml into a Petri dish containing 1ml of sample,
Casamino acids 0.5 mix well and allow to set. Pour a further 10ml as an overlay and again
Glucose 0.5 allow to set. Alternatively it may be used as a spread plate, inoculating
0.1ml onto the plate and spreading over the entire surface of the
Starch 0.5 medium. It can also be used with membrane ilters if required.
Dipotassium hydrogen Phosphate 0.3 Incubation: When plates have set, incubate at 22°C for 5-7 days or
30°C for 3 days. Other incubation temperatures between 20°C and
Magnesium sulphate 0.05 28°C may be used.
Sodium pyruvate 0.3 Interpretation: Count all colonies and report the number of bacteria
in the original sample as the heterotrophic plate count.
pH: 7.2 ± 0.2
Minimum QC organisms: Pseudomonas luorescens WDCM 00115
Hazard classiication: NR – Not regulated Aeromonas hydrophila WDCM 00063
Appearance:
Powder: ine, free-lowing, homogeneous, buff Storage:
Finished medium: clear, pale straw liquid Dehydrated culture media: 10-25oC away from direct sunlight.
Method for reconstitution Prepared media:
Disperse 3g of powder in 1 litre of distilled water. Allow to soak for Plates - 7 days at 2-8°C in the dark
10 minutes, swirl to mix and dispense into tubes or bottles. Sterilise Capped containers – 3 months at 15-20°C in the dark
by autoclaving at 121ºC for 15 minutes.
References
Minimum QC organisms: Aeromonas hydrophila WDCM 00063 Reasoner, D.J., Geldreich, E.E. (1985) A New Medium for the
Pseudomonas luorescens WDCM 00115 Enumeration and Subculture of Bacteria from potable water. App &
Escherichia coli WDCM 00013 Env. Microbiol. Jan. 1985 p. 1-7.
American Public Health Association (1985) Standard Methods for the
Storage: Enumeration of Water and Wastewater. 16th Edition. American Public
Dehydrated culture media: 10-25°C away from direct sunlight. Health Association Inc. Washington DC.
Prepared media: 1 month at 2-8°C in the dark. Environment Agency: The Microbiology of Drinking Water (2002).
R2A Medium Raka-Ray No.3 Agar

LAB163
LAB198
Description
Description
R2A medium was developed to determine the bacterial count in
potable waters during treatment and distribution, and has been shown Based on the formulation of Saha, Sondag and Middlekauff, Raka-
to give signiicantly higher counts than plate count agar (PCA) or Ray No.3 Agar is for the detection of lactic acid bacteria in beer and
similar high-nutrient media. The standard plate count (SPC) method for monitoring in-process beer quality. It is recommended for this
using PCA provides an enumeration of bacteria which grow best at, or application by the European Brewing Convention (EBC) and the
near, body temperature and this estimation at best may correlate to the American Society of Brewing Chemists (ASBC).
coliforms present in the sample. However, there will be a population Contamination of beer and the beer making process by members of
of heterotrophic bacteria which cannot grow at all under the conditions the lactobacilli family results in spoilage, primarily through their
of the SPC method or may grow so slowly that the colonies fail to production of metabolic products which are detrimental to the lavour
reach a size detectable to the eye in the 48-h incubation period. In of the inal product. Detection of these organisms is complicated by
order to enumerate this section of the bacterial population in water, their diverse nutritional and environmental requirements.
a medium of low nutritional content (R2A) and extended incubation A number of different formulations have been described for the
times are required. isolation of lactic acid bacteria in brewing products and processes.
Raka-Ray Agar was developed by the addition of various growth
Typical Formula g/litre promoting compounds to Universal Beer Agar. This work led to the
recognition that the addition of sorbitan mono-oleate, liver extract
Yeast Extract 0.5
and N-acetylglucosamine produced superior growth when compared
Meat Peptone 0.5 to the standard Universal Beer Agar formulation.
Casamino acids 0.5 Further investigations provided the basis for the inal formula of Raka-
Ray No. 3 Medium in which fructose is an essential carbohydrate
Glucose 0.5 source for Lactobacillus fructivorans. Maltose is present to allow the
growth of lactobacilli which cannot utilise glucose. The media can be
Starch 0.5 made selective against yeasts by the addition of 7mg/l cycloheximide
Dipotassium hydrogen Phosphate 0.3 (Actidione®) and against Gram-negative bacteria by the addition of
3g/l 2-phenylethanol.
Magnesium sulphate 0.05
Sodium pyruvate 0.3
Agar No.2 15.0

Typical Formula g/litre Raka-Ray No.3 Agar
Yeast extract 5.0 (Increased Gel Strength)
Tryptone 20.0
LAB199
Liver concentrate 1.0
Description
Maltose 10.0
Based on the formulation of Saha, Sondag and Middlekauff, Raka-
Fructose 5.0 Ray No.3 Agar is for the detection of lactic acid bacteria in beer and
for monitoring in-process beer quality. It is recommended for this
Dextrose 5.0 application by the European Brewing Convention (EBC) and the
Betaine HCl 2.0 American Society of Brewing Chemists (ASBC). LAB199 Raka-Ray
No.3 Agar Increased Gel Strength contains the same components
Diammonium hydrogen citrate 2.0 as LAB198 Raka-Ray No.3 Agar, however the agar level has been
increased to 27g/l.
Potassium aspartate 2.5
Contamination of beer and the beer making process by members of
Potassium glutamate 2.5 the lactobacilli family results in spoilage, primarily through their
production of metabolic products which are detrimental to the lavour
Magnesium sulphate 7H 2O 2.0 of the inal product. Detection of these organisms is complicated by
Manganese sulphate 4H 2O 0.66 their diverse nutritional and environmental requirements.
A number of different formulations have been described for the
Potassium phosphate 2.0
isolation of lactic acid bacteria in brewing products and processes.
N-acetyl glucosamine 0.5 Raka-Ray Agar was developed by the addition of various growth
promoting compounds to Universal Beer Agar. This work led to the
Agar 17.0 recognition that the addition of sorbitan mono-oleate, liver extract and
N-acetylglucosamine produced superior growth when compared to
Method for reconstitution the standard Universal Beer Agar formulation.
Disperse 77.1g of powder in 1 litre of distilled water. Add 10ml Sorbitan Further investigations provided the basis for the inal formula of Raka-
mono-oleate and 7mg cycloheximide. Allow to soak for 10 minutes, Ray No. 3 Medium in which fructose is an essential carbohydrate
swirl to mix then sterilise by autoclaving for 15 minutes at 121°C. source for Lactobacillus fructivorans. Maltose is present to allow the
Cool to 50°C. If required, aseptically add 3g of 2-phenylethanol. Mix growth of lactobacilli which cannot utilise glucose. The media can be
well and pour into sterile Petri dishes. made selective against yeasts by the addition of 7mg/l cycloheximide
(Actidione®) and against Gram-negative bacteria by the addition of
Appearance: 3g/l 2-phenylethanol.
Powder: ine, free-lowing, homogeneous, buff Due to customer feedback regarding the soft gel strength of the
Finished medium: clear to slightly opalescent amber coloured gel standard formulation, Lab M has developed this modiied version of
Raka-Ray Agar with a higher gel strength. The increased gel strength
pH: 5.4 ± 0.2 is especially useful when performing surface plating techniques.
Yeast extract 5.0
Lactobacillus fermentum ATCC 9338 Tryptone 20.0
Pediococcus acidilactici NCTC 6990 Liver concentrate 1.0
Escherichia coli ATCC 25922 (inhibited / suppressed)
Maltose 10.0
Inoculation:
Fructose 5.0
Surface technique
Spread 0.1ml of the sample over the surface of the agar. Alternatively, Dextrose 5.0
the sample may be iltered and the membrane placed on the surface
of the agar. Betaine HCl 2.0
Overlay technique Diammonium hydrogen citrate 2.0
Aseptically dispense 4ml volumes of Raka-Ray No.3 Agar into test Potassium aspartate 2.5
tubes and keep molten at 50°C. Mix 1ml of the test sample with
4ml of molten agar and immediately pour the contents into a Petri Potassium glutamate 2.5
dish containing 15-20ml Raka-Ray No.3 Agar. Mix to give isolated
colonies. As the agar layer is very thin, individual colonies can be Magnesium sulphate 7H 2O 2.0
picked for further examination.
Manganese sulphate 4H 2O 0.66
Incubation: Incubate anaerobically at 25-30°C for 7 days.
Potassium phosphate 2.0
Interpretation: Lactobacilli are visible after 48 hours incubation
and appear as smooth, cream-coloured, moist colonies approximately N-acetyl glucosamine 0.5
1mm in diameter.
Agar 27.0
Incubation for 4 days may be suficient, however slow-growing
organisms such as Pediococcus may require upto 7 days.
If the number of colonies on the plate exceeds 300, dilute the sample
1:10 in Maximum Recovery Diluent (LAB103) and retest. Disperse 77.1g of powder in 1 litre of distilled water. Add 10ml Sorbitan
mono-oleate and 7mg cycloheximide. Allow to soak for 10 minutes,
References swirl to mix then sterilise by autoclaving for 15 minutes at 121°C.
Coster, E., and White, H.R. (1951). J. Gen. Microbiol. 37:15. Cool to 50°C. If required, aseptically add 3g of 2-phenylethanol. Mix
well and pour into sterile Petri dishes.
European Brewing Convention, EBC Analytica Microbiologica: Part
II J. institute of Brewing (1981) 87. 303-321. Appearance:
Lawrence D. R. and Leedham P. A. (1979) Journal of the Institute of Powder: ine, free-lowing, homogeneous, buff
brewing 85. 119
Finished medium: clear to slightly opalescent amber coloured gel
Mauld B. and Seidel H. (1971) Brauwissenschaft 24, 105
Methods of Analysis of the American Society of Brewing Chemists pH: 5.4 ± 0.2
ASBC (1976) 7th edition, The Society St. Paul. Mn. USA. Hazard classiication
Saha R. B., Sondag R. J. AND Middlekauff J. E. (1974). Proceedings NR – Not regulated
of the American Society of Brewing Chemists, 9th Congress 1974.
Van Keer C., Van Melkebeke l., Vertrieste W., Hoozee g. and Van
Schoonenberghe E. (1983). Journal of the Institute of brewing 89.
361 – 363.

Minimum Q.C. organisms: Method for reconstitution
Lactobacillus fermentum ATCC 9338 Weigh 26.8 grams powder, disperse in 1 litre of deionised water, swirl
Pediococcus acidilactici NCTC 6990 to mix, when dissolved dispense in 10ml volumes in screw capped
bottles and sterilise by autoclaving at 115˚C for 15 minutes.
Escherichia coli ATCC 25922 (inhibited / suppressed)
Appearance: Clear, blue luid.
Inoculation:
pH: 5.2 ± 0.2
Surface technique
Spread 0.1ml of the sample over the surface of the agar. Alternatively, Minimum Q.C. organisms: E. coli (inhibited) WDCM 00013
the sample may be iltered and the membrane placed on the surface S. typhimurium WDCM 00031
of the agar.
Overlay technique Storage of Prepared Medium: Capped container – 6 months at
Aseptically dispense 4ml volumes of Raka-Ray No.3 Agar into test 2-8˚C
tubes and keep molten at 50°C. Mix 1ml of the test sample with Inoculation: From pre-enrichment broth in the proportions of 1-part
4ml of molten agar and immediately pour the contents into a Petri
dish containing 15-20ml Raka-Ray No.3 Agar. Mix to give isolated inoculum to 99 parts R.V. Broth. Sub-culture onto either XLD Agar,
colonies. As the agar layer is very thin, individual colonies can be M.L.C.B. Agar or other salmonella selective agars.
picked for further examination. Incubation: 41.5 ± 0.5˚C for 24 hours (incubator) or 42 ± 0.1˚C for
Incubation: Incubate anaerobically at 25-30°C for 7 days. 24hrs (water bath).
Interpretation: Lactobacilli are visible after 48 hours incubation References
and appear as smooth, cream-coloured, moist colonies approximately Vassiliadis, P., (1983) The Rappaport Vassiliadis (R.V.) Enrichment
1mm in diameter. Medium for the Isolation of salmonellas: An overview J. Appl.
Incubation for 4 days may be suficient, however slow-growing Bacteriol. 56 69-76.
organisms such as Pediococcus may require upto 7 days. Vassiliadis, P., Mavromatti, CH. Efstratiou, M. and Chronas, G.
If the number of colonies on the plate exceeds 300, dilute the sample (1985). A note on the stability of Rappaport-Vassiliadis Enrichment
1:10 in Maximum Recovery Diluent (LAB103) and retest. Medium J. Appl. Bacteriol. 59 143-145.
Bolton, F.G., Preston, P.H.L. Personal communication.
References
Int. J. Food Micro. Pharmacopoeia of culture media for Food
Coster, E., and White, H.R. (1951). J. Gen. Microbiol. 37:15. Microbiology.
European Brewing Convention, EBC Analytica Microbiologica: Part Peterz, M., Wiberg, C., and Norberg, P. 1989. The effect of incubation
II J. institute of Brewing (1981) 87. 303-321. temperature and magnesium chloride concentration on growth of
Lawrence D. R. and Leedham P. A. (1979) Journal of the Institute of Salmonella in home-made and in commercially available dehydrated
brewing 85. 119 Rappaport-Vassiliadis broths.
Mauld B. and Seidel H. (1971) Brauwissenschaft 24, 105
Methods of Analysis of the American Society of Brewing Chemists
ASBC (1976) 7th edition, The Society St. Paul. Mn. USA. Reinforced Clostridial Agar
Saha R. B., Sondag R. J. AND Middlekauff J. E. (1974). Proceedings
of the American Society of Brewing Chemists, 9th Congress 1974. LAB023
Van Keer C., Van Melkebeke l., Vertrieste W., Hoozee g. and Van
Schoonenberghe E. (1983). Journal of the Institute of brewing 89. Description
361 – 363. This is a solidiied version of R.C.M. (LAB022) and can be used for
the enumeration of anaerobes by pour plate, shake tube or membrane
iltration methods. When solidiied in tubes or bottles with minimal
head space it can be used for anaerobic culture without the need for
Rappaport Vassiliadis (R.V.) Medium anaerobic atmosphere.
LAB086 Typical Formula g/litre

Yeast Extract 3.0
Introduction
Rappaport Vassiliadis Broth (R10 modiication) was born out of a long Beef Extract 10.0
series of experiments carried out to determine the correct levels of
malachite green and magnesium chloride that would allow Salmonella Peptone 10.0
to multiply freely yet still inhibit the other enteric organisms. Glucose 5.0
This formulation has been shown to be superior to Mueller
Soluble Starch 1.0
Kauffmann and Selenite Broth for the isolation of Salmonella from
meat products. Sodium chloride 5.0
The development work carried out on the formulation shows that it Sodium acetate 3.0
is extremely eficient in detecting small numbers of Salmonella in
heavily contaminated products. This formulation is very hygroscopic L-Cysteine hydrochloride 0.5
and will produce a slight exothermic reaction when mixed with
water. Agar No. 2 12.0

Soy Peptone 4.5 Weigh 49.5 grams of powder, disperse in 1 litre of deionised water,
allow to soak for 10 minutes, swirl to mix then sterilise for 15 minutes
Sodium chloride 7.2 at 121˚C. Cool to 47˚C. and distribute into sterile dishes or tubes
containing decimal dilutions of the sample under test.
Dipotassium hydrogen phosphate 0.18 Appearance: Pale straw, translucent gel.
pH: 6.8 ± 0.2
Magnesium chloride anhydrous 13.58
Malachite green 0.033 Minimum Q.C. organisms: C. perfringens WDCM 00007

Storage of Prepared Medium: Capped container – up to 3 months
at 15-20˚C in the dark. Growth Characteristics
Inoculation: Pour plate technique or tube culture. Expected result Expected result on
Incubation: 30˚C for up to 72 hours. Anaerobic conditions for Organism in RCM Columbia Agar subculture
pour plate. Count as early as possible as prolonged incubation may Clostridia spp. Growth Growth, typical colonies
result in the medium being disrupted due to gas production.
Interpretation: Count all colonies as presumptive clostridia. References
References European Pharmacopoeia 8th Edition
Miller, N.J., Garrett, O.W. and Prickett, P.S. (1939). Anaerobic
technique – a modiied deep agar shake. Food Res. 4: 447-451.
Ingram, M. and Barnes, E.M. (1956). A simple modiication of Rhamnose MacConkey (VTEC O26)
the deep shake tube for counting anaerobic bacteria. Lab. Pract. Agar (RMAC)
5: 145.
LAB209
Reinforced Medium for Clostridia Description
(USP/EP/JP) This medium is selective for the isolation of the verocytotoxin
producing Escherichia coli O26. This strain has been associated with
HP011 haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS).
It is based on the MacConkey formulation No.3, where the fermentable
Description carbohydrate of lactose has been substituted for rhamnose. VTEC
A medium recommended by the Harmonised European O26 colonies are not able to ferment the rhamnose so will remain a
Pharmacopoeia for the selective enrichment of Clostridia from non- translucent colour on the medium. All other non-O26 VTEC colonies
sterile pharmaceutical samples. Conforms to USP/EP/JP performance present are able to ferment Rhamnose and will appear as pink to red
speciication. The medium is also commonly referred to as Reinforced colonies. Selectivity of the medium can be increased by adding X161
Clostridial Medium and abbreviated to RCM. Peptone, beef and yeast Ceixime Tellurite (CT) supplement.
extract provide a source of nitrogen, essential vitamins and amino acids.
Starch aids the detoxiication of harmful metabolites and glucose is a Typical Formula g/litre
fermentable carbohydrate. Sodium chloride provides osmotic balance
and sodium acetate acts as a buffer. L-Cysteine act as reducing agents Peptone 15.0
to create an anaerobic environment and maintain a low Eh. This is Rhamnose 20.0
aided by the low level of agar which reduces the oxygen permeability
through the medium. According to the Harmonised European Bile salts No.3 1.5
Pharmacopoeia, Reinforced Medium for Clostridia is used as a selective
enrichment broth, with subculture performed onto Columbia Agar. Sodium chloride 5.0
Neutral red 0.03
Crystal violet 0.001
Beef extract 10.0
Agar No.2 12.0
Peptone 10.0
Yeast extract 3.0
Soluble starch 1.0 Allow to soak for 10 minutes, swirl to mix and sterilise by autoclaving
for 15 minutes at 121°C. Cool to 47°C, and add 2 vials of reconstituted
Glucose monohydrate 5.0 X161. Mix well and dispense into sterile Petri dishes.
Cysteine hydrochloride 0.5
Appearance:
Sodium chloride 5.0 Powder: ine, free-lowing, homogeneous, buff
Sodium acetate 3.0 Finished medium: red/purple gel
Agar 0.5 pH: 7.1 ± 0.2

Method for reconstitution Hazard classiication
Disperse 38 grams of powder in 1 litre of deionised water. Allow to
soak for 10 minutes, swirl to mix and bring to the boil. Distribute into
suitable vessels and sterilise at 121°C for 15 minutes. Minimum Q.C. organisms:
Escherichia coli NCTC 8783
Appearance: Escherichia coli WDCM 00013
Powder: ine, free-lowing, homogeneous, buff Salmonella typhimurium WDCM 00031
Finished medium: straw, clear to slight haze Enterococcus faecalis WDCM 00087
pH: 6.8 ± 0.2
Storage:
Minimum Q.C. organisms: Dehydrated culture media: 10-25°C away from direct sunlight
Clostridium sporogenes ATCC 19404 Poured plates: 7 days at 2-8°C in the dark
Inoculation: Surface inoculation as per user’s validated method.
Hazard classiication: NR - Not regulated Incubation: Incubate at 37°C for 18-24 hours.
Storage: References
Dehydrated culture media: 10-25°C away from direct sunlight. Hiramatsu R, Matsumoto M, Miwa Y, Saito M, Yatsuyanagi J,
Prepared media: 7 days at 2-8°C in the dark. Uchimura M, Kobayashi K, Tanaka H, Horikawa K, Mori R,
Miyazaki Y. Characterization of enterohemorrhagic Escherichia coli
Inoculation: According to the Harmonised European Pharmacopoeia, O26 and development of its isolation media. Kansenshogaku Zasshi.
a sample is prepared with and without heat treatment and transferred 1999 May; 73(5):407-13.
to Reinforced Medium for Clostridia.
Hiramatsu, R., Matsumoto, M., Miwa, Y., Suzuki, Y., Saito, M., and
Incubation: Miyazaki, Y. Characterization of Shiga Toxin-Producing Escherichia
coli O26 Strains and Establishment of Selective Isolation Media for
Incubate at 30-35°C for 48-72 hours These Strains. J Clin Microbiol. 2002 March; 40(3): 922–925.

Ringer’s Solution (1/4 strength) Tablets Minimum Q.C. organisms:
Aspergillus niger WDCM 00053
Saccharomyces cerevisiae WDCM 00058
LAB100Z Escherichia coli WDCM 00013 (inhibition)
Description
Storage of Prepared Medium:
An osmotically controlled solution for the preparation of suspensions Dehydrated culture media: 10-25˚C
of food samples and for use as a diluent in dilution techniques for Prepared media: Plates - up to 7 days at 2-8˚C, in the dark
bacterial enumeration. The solution can also be used in the sampling Capped containers – up to 1 month at 2-8˚C, in the dark
of food production apparatus by the rinse and swab method.
Inoculation: Surface spreading.
Incubation: 25˚C aerobically for 24 hours to 5 days.
Sodium chloride 2.25 Growth Characteristics
colony size shape &
Potassium chloride 0.105
Calcium chloride 0.12
Rhizopus spp. 13.5 Fluffy White
Sodium bicarbonate 0.05
Aspergillus lavus 8 Flat & Yellow/ hyphae
hyphae Green
Method for reconstitution Candida spp. 1.5 CV.E.G. White
Dissolve 1 tablet in 500ml deionised water. When completely dissolved Sacchromyces
dispense into containers as required and sterilise by autoclaving for 15 spp. 1.0 CV.E.G. White
minutes at 121oC.
E. coli no growth
Staphylococcus no growth
Minimum QC organisms: Escherichia coli WDCM 00013 spp.
Appearance: Tablet: white tablet References

Finished medium: colourless, clear liquid Banks, J.G. Board, R.G. (1987). Some factors inluencing the recovery
of yeasts and moulds from chilled foods. Int. J. Food Microbiol. 4:
Storage: 197-206.
Dehydrated culture media: 10-25oC away from direct sunlight. Jarvis, B. (1973). Comparison of an improvised Rose-Bengal
Prepared media: 7 days at 2-8°C in the dark. Chlortetracycline Agar with other media for the selective isolation
and enumeration of moulds and yeasts in food. J. Appl. Bact. 36:
723-727.
Rose Bengal Chloramphenicol Overcast, W.W. and Weakley, D.L. (1969). An aureomycin rose-
bengal agar for the enumeration of yeast and mould in cottage cheese.
Agar Base J.Milk and Fd.Tech. 32: 442-445.
LAB036
Description
Sabouraud Dextrose Agar
A selective medium for the enumeration of moulds and yeasts in foods. LAB009
The original formulation of Jarvis (1973) used chlortetracycline,
this has been substituted by chloramphenicol because of superior Description
selectivity. The Rose Bengal dye is taken up by the growing colonies
making them easier to see and inhibiting their spreading. Rose Bengal Introduced by Sabouraud in 1910 as a selective medium for fungi
becomes increasingly toxic on exposure to light so it is important to and yeasts. The acidic pH (5.6) of this medium inhibits many species
store plates in the dark. of bacteria. The medium can be made more selective by the addition
of chloramphenicol supplement (X009) (X209). Diagnostic features,
Typical Formula g/litre such as sporing structures and pigmentation are well developed on
this medium. Because of its low pH this medium is very sensitive
Mycological Peptone 5.0 to overheating which will soften the agar and caramelise the
Dextrose 10.0 carbohydrate.
Dipotassium phosphate 1.0 Typical Formula g/litre

Magnesium sulphate 0.5 Balanced Peptone No. 1 10.0
Rose bengal 0.05 Dextrose 40.0
Agar No. 2 12.0 Agar No. 2 12.0

Weigh 28.5 grams of powder, disperse in 1 litre of deionised water. Weigh 62 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise for 15 minutes Allow to soak for 10 minutes, swirl to mix then sterilise by autoclaving
at 121°C. Allow to cool to 47°C then add 2 vials of X009 (or 1 vial of for 15 minutes at 121˚C. NOTE: The gel strength of the medium
X209) Chloramphenicol. X089 Oxytetracycline (2 vials per litre) may may diminish if recommended sterilising time or temperature is
also be used. Mix well, then pour into Petri dishes. exceeded.
This medium should be protected from light. Chloramphenicol may Cool to 47˚C, mix well then pour plates.
be added before autoclaving.
Appearance: Buff opalescent gel.
DO NOT REHEAT THIS MEDIA ONCE PREPARED.
pH: 5.6 ± 0.2
Appearance:
Powder: ine, free-lowing, homogeneous, pink Minimum Q.C. organisms: Candida sp. WDCM 00054
Finished medium: Pink gel E. coli (inhibition) WDCM 00013
pH: 7.2 ± 0.2

dark. Capped container – up to 3 months at 15-20˚C in the dark. Growth Characteristics
Inoculation method: Surface streaking for single colonies or stab Expected result Expected result on
method. Organism in SDB SDA subculture
Incubation: Aerobically, yeasts 37˚C for 48 hours; fungi 25-30˚C Candida albicans Growth Growth, white colonies
for up to 3 weeks.
References
Growth Characteristics European Pharmacopoeia 8th Edition
colony size shape &
C. albicans 0.5-2.0 CV.E.D. White Yeasty smell Sabouraud Maltose Agar
C. krusei 1.0-3.0 F.CR.D. Grey- Yeasty smell
White LAB111
T. rubrum 25 White- Reverse-
luffy shades of Description
red
This is a modiication of Sabouraud Dextrose Agar, substituting
M. canis 25 White- reverse- maltose for dextrose, recommended by the American Public Health
centre- yellow-
Association.
yellow
radial Typical Formula g/litre
Balanced Peptone 10.0
References
Sabouraud, R. (1910). Les Teignes Paris. Pagano. J., Levin, J.D. and Maltose 40.0
Trejo, W. (1957-8). Diagnostic medium for the differentiation of Agar No. 2 12.0
species of Candida. Antibiotics Annual, 137-143.
Weigh 62 grams of powder, disperse in 1 litre of deionised water,
Sabouraud Dextrose Broth allow to soak for 10 minutes, swirl to mix then autoclave at 121˚C for
15 minutes. Do not overheat or the agar gel will be softened and the
(USP/EP/JP) carbohydrate will be caramelised. This medium may be made selective
by the addition of 2 ampoules X009 Chloramphenicol selective
HP013 supplement which may be added either before or after autoclaving.
Description Cool to 47˚C and mix well before pouring plates.
A medium recommended by the Harmonised European Appearance: Cream/yellow, translucent.
Pharmacopoeia for the enrichment of Candida albicans from non-
sterile pharmaceutical samples. Conforms to USP/EP/JP performance pH: 5.6 ± 0.2
speciication. The medium is also used for the cultivation of fungal test
strains as described by the Harmonised European Pharmacopoeia. The Minimum Q.C. organisms: Candida albicans WDCM 00054
peptone digests and dextrose provide a nutritious base for luxuriant E. coli (inhibition) WDCM 00013
fungal growth and the acidic pH affords selectivity against bacteria.
Due to the high carbohydrate content and low pH this medium is highly Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
sensitive to overheating. According to the Harmonised European dark. Capped containers – up to 3 months at 15-20˚C in the dark.
Pharmacopoeia, Sabouraud Dextrose Broth is used as an enrichment
Inoculation: Surface streaking or stab inoculum.
broth, with subculture performed onto Sabouraud Dextrose Agar.
Incubation: Aerobic; yeasts 37˚C for 48 hours; other fungi 25˚C for
Typical Formula g/litre up to 3 weeks.
Dextrose 20.0 Growth Characteristics
Peptic digest of animal tissue 5.0 colony size shape &
Pancreatic digest of casein 5.0
C. albicans 0.5-2.0 CV.E.D. White Yeasty smell
Method for reconstitution C. krusii 1.0-3.0 F.CR.D. Grey- Yeasty smell
Disperse 30 grams of powder in 1 litre of deionised water. Allow to White
soak for 10 minutes then swirl to mix. Distribute into suitable vessels T. rubrum 25mm White- Reverse-
and sterilise at 121°C for 15 minutes. luffy shades of
Red
Appearance:
M. canis 25mm White- Reverse-
Powder: ine, free-lowing, homogeneous, buff centre yellow-
yellow orange
Finished medium: straw, clear to slight haze
radial
pH: 5.6 ± 0.2
References
Minimum Q.C. organisms: Sabouraud, R. (1910). Les Teignes. Paris. Pagano, J., Levin, J. D.
Candida albicans ATCC 10231 and Trejo, W. (1957-8) Diagnostic medium for the differentiation of
Aspergillus brasiliensis ATCC 16404 species of Candida. Antibiotics Annual. 137-143.
Hazard classiication: NR - Not regulated

Storage:
Inoculation: According to the Harmonised European Pharmacopoeia,
a quantity corresponding to 1g or 1mL of the sample is use to inoculate
100mL of Sabouraud Dextrose Broth.
Incubation:
Incubate at 30-35°C for 3-5 days

Selenite Broth Method for reconstitution
Dissolve 4 grams of Sodium biselenite (LAB044B) in 1 litre of
LAB044A & LAB044B deionised water. Add 19 grams of Selenite Cystine Broth Base and
heat to dissolve. Distribute into tubes or bottles, and sterilise for 10
minutes in a boiling water bath, or steamer. DO NOT AUTOCLAVE
Description
THIS MEDIUM.
A medium for the selective enrichment of salmonellae from faeces,
food and sewage. First described by Leifson in 1936 the medium is Appearance: Pale straw colour, clear with slight precipitate. (A brick
a peptone lactose broth, moderately buffered, which utilises sodium red precipitate indicates overheating).
biselenite as a selective agent. This medium can be incubated at pH: 7.0 ± 0.2
various temperatures from 35-43˚C to vary the selectivity. Subcultures
should be performed after no more than 24 hours incubation as there Minimum Q.C. organisms:
is an increasing loss of selectivity if incubation is prolonged. Salmonella typhimurium WDCM 00031
Selenite Broth Base LAB044A: Storage of Prepared Medium: Capped containers – up to 3 months
Peptone 5.0
Inoculation: Add sample to broth in the ratio of 1:10. Use a pre-
Lactose 4.0 enrichment broth if damaged organisms are to be recovered.
Sodium phosphate 10.0 Incubation: 37˚C for 24-48 hours aerobically. Subculture onto
Salmonella selective media.
Sodium biselenite LAB044B: References
Sodium hydrogen selenite 4.0 International Organisation for Standardization. Microbiology (1981).
General guidance on methods for the detection of Salmonella.
Method for reconstitution ISO, 6579-1981.
Dissolve 4 grams of sodium biselenite in 1 litre of cold deionised International Organization for Standardization. Milk and milk
waer. Add 19 grams of Selenite Broth Base and warm to dissolve. products (1985). Detection of Salmonella. ISO 6785-2985 (E).
Distribute into tubes or bottles and sterilise for 5-10 minutes in a ICMSF, (1978). Micro-organisms in foods. 1. Their signiicance
boiling water bath, or by free steaming. DO NOT AUTOCLAVE. and methods of enumeration, 2nd edn., University of Toronto Press,
Appearance: Pale orange/red with slight precipitate (overheating Toronto, Ont.
will cause excessive precipitate and loss of selectivity). Leifson, E. (1936). New selenite enrichment media for the isolation of
pH: 7.1 ± 0.2 (complete medium) typhoid and paratyphoid (Salmonella) bacilli. Am, J. Hyg. 24, 423-
432.
Minimum Q.C. organisms: North, W.R. and. Bartram, M.T. (1953). The eficiency of selenite
Salmonella typhimurium WDCM 00031 broth of different compositions in the isolation of Salmonella. Appl.
E. coli (inhibition) WDCM 00013 Microbiol. 1, 130, 134.
Speck, M.L. (1984). Compendium of methods for the microbiological
Storage of Prepared Medium: Capped containers – up to 3 months examination of foods, 2nd edn., American Public Health Association.
Inoculation: Approximately 0.5-1 gram of sample per 10ml tube.
Incubation: Up to 24 hours aerobically at 35-43˚C. Sensitivity Test Agar
Subculture: Onto two or more selective agars.
(S.T.A.)
References
Leifson, E. (1936). New selenite enrichment media for isolation of LAB012
typhoid and paratyphoid (Salmonella) bacilli. Amer. J.Hyg. 24: 423-
432. Description
MacFaddin, J.F. (1985). Media for the isolation, cultivation, A medium formulated for antibiotic susceptibility testing by the Joan
identiication of Medical Bacteria Vol 1. Williams and Wilkins, Stokes technique. S.T.A. is inhibitor-free, very rich and includes
Baltimore. various nucleotides to enable fastidious organisms to be tested. It is
necessary to add lysed or ‘chocolated’ blood for some organisms.
Selenite Cystine Broth Typical Formula g/litre

Peptone-Infusion Solids 21.5
LAB055A & LAB044B
Starch 0.6
Description Sodium chloride 5.0
This formulation is a result of the investigation of North and Bartram
in 1953. They examined the effect of varying concentrations of cystine Disodium citrate 1.0
and phosphate on the recovery of salmonellae in egg products using
Adenine sulphate 0.01
selenite broth. It was found that the addition of 10 micrograms/ml of
cystine to Leifson’s selenite broth enhanced recovery of salmonellae. Guanine hydrochloride 0.01
Typical Formula g/litre Uracil 0.01
Selenite Cystine Broth Base LAB055A Xanthine 0.01
Balanced Peptone No. 1 5.0 Aneurine hydrochloride 0.01

Lactose 4.0 Uridine 0.1
Sodium phosphate 10.0 Agar No. 2 12.0
L-Cystine 0.01
Sodium biselenite LAB044B Weigh 40 grams of powder, disperse in 1 litre of deionised water,
Sodium hydrogen selenite 4.0 allow to soak for 10 minutes, swirl to mix then sterilise by auto-
claving for 15 minutes at 121˚C. To prepare blood agar cool to
45˚C and add 7% lysed blood or 6% deibrinated blood according
to preference. Mix well then pour plates.

Appearance: Dependent upon the blood additive. References
pH: 7.4 ± 0.2 Simmons, J.S. (1926). A Culture medium for differentiating organisms
of typhoid - colon aerogenes groups and for isolation of certain fungi.
Minimum Q.C. organisms: S. aureus NCTC 6571 J.Inf. Dis. 39: 209-215.
E. coli NCTC 10418 Koser, S.A. (1923). Utilisation of the salts of organic Acids by the
(antibiotic sensitivity zones) Colon-aerogenes group. J. Bact. 8: 493-520.
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the MacFaddin, J.F. (1983). Biochemical Tests for Identiication of
dark. Medical Bacteria. Williams and Wilkins.
Inoculation method: Surface, according to technique.
Incubation: 37˚C, atmosphere to suit organisms metabollic
requirements. Slanetz and Bartley Medium
Interpretation: There are no deined zone sizes as in Mueller Hinton, (Membrane Enterococcus Agar)
but all antibiotics should give adequate zone sizes when compared to
controls using standard organisms, e.g. S. aureus NCTC 6571, E. coli
NCTC 10418, Ps. aeruginosa NCTC 10662.
LAB166
References Description
Stokes, E.J. (1968). Clinical Bacteriology 3rd edn. Arnold, London.
Committee of the A.C.P. (1965). Report on the Antibiotic Sensitivity This medium was originally described by Slanetz and Bartley for the
test trial organised by the bacteriology committee of the Association enumeration of enterococci from water samples using a membrane
of Clinical Pathologists. J.Clin. Pathol., 18: 1-5. iltration technique, but it may also be used as a spread plate for the
examination of other sample types. Enterococci reduce tetrazolium
Hanus, F.J. Sands, J.G. and Bennett, E.O. (1967). Antibiotic activity in chloride to the insoluble red dye formazan, producing colonies which
the presence of agar. Appl. Microbiol., 15: 31-34. are dark red or maroon on the surface of the membrane or agar. This
Bechtle, R.M. and Scherr, G.H. (1958). A new agar for in vitro reaction is not exclusive to enterococci, and the count at this stage
antimicrobial sensitivity testing. Antibiot. Chemother., 8: 599-606. should be considered presumptive. Colonies may be conirmed as
enterococci by demonstrating aesculin hydrolysis using Kanamycin
Aesculin Azide Agar LAB106.
Simmons Citrate Agar Typical Formula g/litre
LAB069 Tryptose 20.0
Description Yeast Extract 5.0

A medium devised by Simmons in 1926 to help in the differentiation Glucose 2.0
of enteric bacteria and in the isolation of fungi. Certain
Enterobacteriacae have the ability to utilize citrate as the sole source Dipotassium hydrogen phosphate 4.0
of carbon and utilize inorganic ammonium salts as the sole source of
nitrogen resulting in an increase in alkalinity. Bromothymol Blue is Sodium azide 0.4
used as a pH indicator.
2,3,5 Tetrazolium chloride 0.1
Typical Formula g/litre Agar 12.0
Ammonium dihydrogen phosphate 1.0 Weigh 43.5 grams of powder and mix with 1 litre of deionised water.
Dipotassium phosphate 1.0 Bring to the boil with frequent stirring to dissolve completely. Cool
to 47˚C and pour into sterile Petri dishes. DO NOT AUTOCLAVE,
Sodium citrate 2.0 OVERHEAT, OR LEAVE FOR GREATER THAN 4hr AT 47˚C.
Sodium chloride 5.0 Appearance: Rose coloured gel.
Bromothymol blue 0.08 pH: 7.2 ± 0.2
Agar No. 2 15.0
Mininmum QC organisms:
Method for reconstitution Escherichia coli WDCM 00013 (inhibition)
Allow to soak for 10 minutes, swirl to mix then heat to dissolve Storage: Plates – upto 7 days at 2-8˚C. Storage in bottles is not
the agar and solids. Dispense into tubes or bottles then sterilise by recommended as re-melting the medium will cause damage.
autoclaving at 121˚C for 15 minutes. Allow to set as slopes.
Inoculation
Appearance: Green, opalescent. Water: Filter 100ml of the water through a suitable membrane, and
pH: 6.9 ± 0.2 place this on the surface of a properly dried Slanetz and Bartley
plate.
Minimum Q.C. organisms: E. coli WDCM 00013 Other samples: Dilute as necessary and spread 0.5ml over the surface
C. freundii WDCM 00006 of the plate using a spreader, and allow to soak into the agar.
Storage of Prepared Medium: Capped containers – up to 3 months Incubation

at 15-20˚C in the dark. Water: at 37˚C for 48hr if testing potable waters or processed foods.
Inoculation: Streak on surface and stab into the butt. At 37˚C for 4hr then 44˚C for 44hr if testing untreated waters or raw
materials.
Incubation: 37˚C aerobically for 24-48 hours, with loose caps to
allow gaseous exchange. Interpretation
Interpretation: Utilisation of citrate and ammonium salts results in Count all red and maroon colonies as presumptive enterococci.
growth and a change in colour of the medium from green to blue. Conirmation of isolates can be achieved by demonstration of a
positive aesculin reaction on KAAA LAB106.
organism growth colour of medium
Reference:
Slanetz, L.W., and Bartley, C.H. (1957) J.Bact. 74 591-595.
(most) Salmonella spp yes blue Environment Agency: The Microbiology of Drinking Water (2002).
Shigella spp. no green
E. coli no green
C. freundii yes blue

Sorbitol MacConkey Agar S.S. Agar
(SMAC, CT-SMAC) (Salmonella Shigella Agar)
LAB161 LAB052
This is a selective differential medium for the isolation of Escherichia This medium is a modiication of Leifson’s DCA Medium irst
coli O157:H7, the primary serovar associated with haemorrhagic described in 1941 by Mayield and Goeber shortly before Hynes
colitis (HC) and haemolytic uraemic syndrome (HUS). Pathogenicity described his modiication of DCA. The selectivity of the medium
of the organism is linked to the production of verocytotoxins (VT1 was increased by the addition of extra bile salts, sodium citrate and
and VT2), but it should be noted that not all strains of O157:H7 the addition of brilliant green dye. There is also the extra thiosulphate
produce verocytotoxins, and that strains from other serovars can be giving good H2S production which reduces the ferrous ammonium
toxin producers (e.g. O26, O111, O113, O145). sulphide giving black centred colonies with H2S positive organisms.
O157:H7 has been associated epidemiologically with food poisoning The selectivity of this medium can be such that it was suggested
outbreaks involving beefburgers and cold cooked meats. The medium by Taylor et al in 1965 to be unsuitable for the isolation of Shigella
is a modiication of MacConkey Agar No. 3 with the substitution species. Greater understanding of the selection mechanisms involved
of the fermentable carbohydrate from lactose to sorbitol. O157:H7 enable us to adjust the reaction and allow the more delicate Shigella to
is sorbitol negative and produces translucent colonies whereas
grow without unduly impairing the medium’s selective properties.
most other E. coli strains are sorbitol positive and so produce pink/
red colonies. Selectivity of the medium can be increased by adding
Ceixime-Tellurite (C.T.) supplement X161. Typical Formula g/litre
Beef Extract 5.0
Peptone 20.0
Lactose 10.0
Sorbitol 10.0
Bile Salts No. 3 8.5
Bile salts no.3 1.5
Sodium citrate 8.5
Sodium chloride 5.0
Neutral red 0.03
Ferric citrate 1.0
Brilliant Green 0.00033
Agar 12.0
Neutral Red 0.025
Method for reconstitution Agar No. 2 13.5
Weigh 48.5 grams of powder and add to 1 litre of de-ionised water.
Allow to soak for 10 minutes, swirl to mix and sterilise by autoclaving Method for reconstitution
at 121˚C for 15 minutes. Cool to 47˚C, add 2 vials of CT supplement Weigh 60 grams of powder, disperse in 1 litre of deionised water,
X161, and pour plates. Dry the surface prior to inoculation. and allow to soak for 10 minutes. Swirl to mix, then bring to the boil,
Appearance: Pale red, slight violet tinge. and allow to cool to 47˚C. Mix well then pour plates. Dry the surface
before incubation. DO NOT AUTOCLAVE THIS MEDIUM.
pH: 7.1 ± 0.2
Appearance: Pale Pink, clear.
Minimum QC organisms: pH: 7.0 ± 0.2
E. coli O157:H7 (non-toxigenic) WDCM 000014 (translucent)
E. coli WDCM 00013 (Pink/red)
Ent. faecalis WDCM 00087 (inhibition) Minimum Q.C. organisms:
dark. Storage of Prepared Medium: Plates – up to 7 days at 4˚C in the
Inoculation: Surface streak for single colonies. dark.
Incubation: 37˚C aerobically for 18-24 hr. Inoculation method: Surface plating, streaking for single colonies.
Growth Characteristics:
Organism Size (mm) Shape Colour Growth Characteristics
E. coli O157:H7 2.5 – 4.0 CV.E.G Translucent colony size shape &
Other E. coli 2.5 – 4.0 CV.E.G Pink/red
E. coli 0.1-2.0 CV.E.D. Red Red ppt around
Sorbitol +ve colonies
organisms 2.5 – 5.0 Any Pink/red
(No growth)
K. aerogenes 0.1-3.0 CV.E.G. Pink-Red (No growth)
References
Proteus spp. 1.0-2.0 CV.E.G. Yellow (grey centre)
Law, D., Ganguli, L.A., Donohue-Rolfe, A., Acheson, D.W.K. (1992) (ishy odour)
J. Med. Micro. 36 198-202.
Salmonella spp. 2.0-3.0 CV.E.G. Yellow (black centre)
Hitchins, A.D., Hartman, P.A., and Todd, E.C.D. (1992) in
Shigella spp. 0.5-2.0 CV.E.G. Pink-
“Compendium of methods for the microbiological examination of Yellow
foods” Ch.24. Published by American Public Health Association.
Gram positive organisms – no growth.
Varnam, A.H., Evans, M.G., (1991) Foodborne Pathogens an
Illustrated Text. Published by Wolfe Publishing Ltd.
Riley, L.W. (1991) Ann. Rev. Micro. 41 383-408. References
Isenberg, H.D., Kominos, S., and Siegel, M. (1969). Isolation of
Riley, L.W. et al (1983) New Eng. J. Med 308 681-685. salmonellae and shigellae from an artiicial mixture of fecal bacteria.
Salmon, R.L., Farrel, I.D., Hutchinson, J.G.P. (1989) Epid. Inf. 103 Appl. Microbiol., 18: 4, 656-659.
249-254. Leifson, E. (1935). New culture media based on sodium desoxycholate
for the isolation of intestinal pathogens and for the enumeration of
colon bacilli in milk and water. J. Pathol. Bacteriol., 40: 581-589.
Taylor, W.I., Harris, B. (1965) Isolation of shigellae. II. Comparison
of plating media and enrichment broths. Am. J. Clin. Pathol. 44: 4,
476-479.

Sugar Free Agar This medium will support the growth of the majority of pathogens
requiring susceptibility testing, without the addition of supplements.
However, certain organisms such as some streptococci, staphylococci,
LAB087 Enterobacteriaceae and Neisseria may require the addition of
intrinsic growth factors e.g. lysed horse blood, thymidine, thiamine
Description and menadione. However these supplements can introduce errors as
A formula described by the International Dairy Federation for the they can affect the activity of certain antibiotics and consequently
enumeration of psychrotrophic and mesophilic Gram-negative rods their affects must assessed before use.
in butter and other dairy products. The Gram-negative rods are able
to deaminate proteins as a carbon source, whilst some enterococci are Typical Formula g/litre
inhibited by this formula. The medium conforms to the formulation of
the International Dairy Federation (I.D.F.). Peptone Mixture 16.0
Glucose 2.0
Starch 1.0
Gelatin Peptone 7.5
Sodium chloride 2.8
Tryptone 7.5
Na2HPO4 0.4
Sodium chloride 5.0
Sodium glycerophosphate 0.22
Agar No. 1 14.0
Sodium gluconate 0.1
Method for reconstitution Sodium acetate 1.0
Weigh 34 grams of powder and disperse in 1 litre of deionised water. Uridine 0.3
Allow to soak for 10 minutes, boil to dissolve and disperse into tubes
or lasks. Sterilise by autoclaving at 121˚C for 15 minutes. Deined Chemical Mixture 0.078
Appearance: Light straw, clear. Agar 12.0
pH: 7.6 ± 0.2
Method for Reconstitution
Minimum Q.C. organisms: E. coli WDCM 00013 Weigh 35.9 grams of powder and disperse in 1 litre of deionised water.
Swirl to mix and sterilise by autoclaving at 121°C for 15 minutes.
Cool to 47°C and if required add 5-7% sterile lysed horse blood. Pour
Storage of Prepared Medium: Capped container – up to 3 months into sterile petri dishes and allow to set. Cool to 47°C, mix well and
at 15-20˚C in the dark. dispense into petri dishes.
Inoculation: 0.2ml of butter fat in a pour plate technique.
Incubation: 30˚C for 2 days then 20˚C for a further two days – Appearance: Straw clear gel.
aerobically. pH: 7.3 ± 0.2
Interpretation: Count colonies.
References (as recommended by the British Society for Antimicrobial
International Dairy Federation (1964). International standard count Chemotherapy (BSAC))
of contaminating organisms in butter. International Standard FIL- Escherichia coli NCTC 10418
IDF30. Staphylococcus aureus NCTC 6571
Ritter, P. and Eschmann, K.H. (1966). Alimenta 5(2), 43-45. Pseudomonas aeruginosa NCTC 10662
Thomas, S. B. (1969). J. Appl. Bact. 32, 269-296. Enterococcus faecalis NCTC 29212
Mossel, D.A.A., Krol, B. and Moerman, P.C. (1972). Alimenta 11(2), Haemophilus inluenzae NCTC 11931
51-60. Streptococcus pneumoniae ATCC 49619
Storage: Capped containers - up to 3 months at 15-20°C in the dark.

Plates - up to 7 days at 2-8°C, in the dark.
Susceptibility Test (Iso) Agar Inoculation: Surface, inoculum as described by standard methods.
Incubation: As stipulated in the BSAC methodology.
LAB170
Ericsson, H.M., Sherris, J.C. (1971). Antibiotic Sensitivity Testing.
Susceptibility Testing ‘Iso’ Agar is a semi-deined medium for Report of an International Collaborative Study. Acta. Pathol.
antimicrobial susceptibility (sensitivity) testing (AST), in which Microbiol. Scand. Sect B Suppl.; 217: 1-90.
the undeined elements are maintained at minimum levels. The Amato, R.F., Thornberry, C., (1979). Calcium and Magnesium in
antimicrobial susceptibility test is utilised in epidemiological Mueller Hinton Agar and their inluence on disc diffusion susceptibility
studies and in determining the appropriate usage of antimicrobials results. Current Microbiol. 2: 135-138.
in the clinical environment. The response of clinical isolates to
antimicrobials, and the detection of microbial resistance, allows Hawkey, P.M., Birkenhead, D., Kerr, K.G., Newton, K.E., Hyde, W.A.
for precise and rapid treatment. The AST is performed to detailed (1993). Effect of divalent cations in bacteriological media on the
standards, the results of which must be reproducible; a major factor is susceptibility of Xanthomonas maltophila to imipenem with special
the medium on which it is performed. reference to zinc ions. J. Antimicrobial Chermother; 31: 181-183.
The presence of antagonists in the medium e.g. thymidine and metal Garrod, L.P., Waterworth, P.M. (1969). Effect of medium composition
ions, have a detrimental effect on results obtained. The addition on the apparent sensitivity of pseudomonas aeruginosa to gentimicin.
of thymidine for the growth of dependant strains antagonises the J. Clin. Pathol; 22: 534-538.
antimicrobial action of Trimethoprim and sulphonamides and results Duncan, I.B.R. (1974). Susceptibility of 1500 isolates of Pseudomonas
in false resistance results. Metal ions can exert known antagonistic aeruginosa to gentimicin, carbenecillin, colistin, and polymyxin B.
effects on a number of antibiotics. Therefore the anion and cation Antimicrobial Agents and Chermother; (Jan) 9-15.
content of the medium must be regulated to prevent adverse effects
on performance.
Susceptibility Testing ‘Iso’ Agar is produced having a stable mineral
content, the presence of minimum antagonistic elements, and a
constant isotonic pH (preventing the blocking or enhancement of
antimicrobials), thereby ensuring production of optimum zones of
microbial inhibition.

T.C.B.S. Cholera Medium Tetrathionate Broth Base A.P.H.A.
(Thiosulphate Citrate Bile Salts Sucrose Agar)
LAB097
LAB096
Description
Description A selective enrichment broth for the growth of Salmonella typhi
T.C.B.S. is designed for the selective isolation of Vibrio species, and other Salmonella spp. from faeces, foods etc. It conforms to
particularly V. cholerae. The formulation was developed by the formulation recommended by the American Public Health
Kobayashi, Enomoto, Sakazaki and Kuwahara and inhibits most Association for use in the examination of dairy products and foods
of the Enterobacteriaceae for at least 24 hours. Therefore heavy for salmonellae. Organisms which reduce tetrathionate, such as
inoculation of the medium is possible. salmonellae, proliferate in the medium, whilst most enteric organisms
are inhibited. Certain members of the Proteus group will also reduce
tetrathionate thereby impairing the performance of the medium
Typical Formula g/litre in some cases. To overcome this, Novobiocin may be added to the
Yeast Extract 5.5 medium at a level of 40 microgram/ml before addition of the iodine.
Gram-positive organisms are inhibited by the inclusion of bile salts.
Peptone Mix 10.0
Sodium thiosulphate 10.0 Typical Formula g/litre
Sodium citrate 10.0 Balanced Peptone No. 1 5.0
Bile salts 9.0 Bile Salts 1.0
Sucrose 17.0 Calcium carbonate 10.0
Sodium chloride 10.0 Sodium thiosulphate 30.0
Ferric citrate 1.0 Method for reconstitution

Bromothymol blue 0.04 Weigh 46 grams of powder and add to 1 litre of deionised water.
Bring to the boil with frequent swirling to fully dissolve the medium.
Thymol blue 0.04 Cool to 45˚C and add 20ml of iodine solution prepared as indicated
Agar No. 1 15.0 below. Mix well before dispensing into bottles and continue swirling
whilst dispensing to avoid the calcium carbonate sedimenting. For the
best results the medium should be used the same day as prepared.
Method of reconstitution
Iodine solution: Dissolve 5g of potassium iodide and 6g of iodine
Weigh 88 grams of powder and add to 1 litre of deionised water. crystals in 20ml of distilled water.
Allow to soak for 10 minutes, swirl to mix then bring to the boil.
Cool to 47˚C and pour into Petri dishes. DO NOT AUTOCLAVE OR Appearance: Turbid white.
OVERHEAT THIS MEDIUM.
pH: 8.4 ± 0.2
Appearance: Dark green clear agar.
pH: 8.6 ± 0.2 Minimum Q.C. organisms: Salmonella sp. WDCM 00031
Minimum Q.C. organisms: V. cholerae WDCM 00136
E. coli (inhibition) WDCM 00013 Storage of Prepared Medium: Capped containers – up to 7 days at
4˚C in the dark (without iodine solution).
Storage of Prepared Medium: Plates – up to 7 days at 4˚C in the Inoculation: Add 1 part of sample suspension or inoculated pre-
dark. Capped containers – up to 1 month at 15-20˚C in the dark. enrichment medium to 9 parts of Tetrathionate Broth.
Inoculation: Surface plating with a heavy inoculum, streak out to Incubation: 12-24 hours at 37˚C.
single colonies. Subculture: Onto LAB034 Brilliant Green Agar and either LAB032
Incubation: 37˚C aerobically for 18-24 hours. XLD or LAB110 Hektoen Enteric or other Salmonella selective
media.
Growth Characteristics References
colony size shape & Standard methods for the Examination of Dairy products, 10th
organism (mm) surface colour other Edition. APHA, (1953).
Vibrio. cholerae 2.0-3.0 CV.E.G. Yellow may revert to
green at R.T.
V. parahaemolyticus 3.0-5.0 CV.E.G. Blue or
Green
Enterococci 1.0 CV.E.G. Yellow
Proteus spp. 1.0 F.CR.G. Green/
Yellow
References
Kobayashi, T., Enomoto, S., Sakazaki, R. and Kuwahara, S. (1963).
Jap. Bacteriol 18: 10-11, 387-391.

Thioglycollate Medium (Brewer) Typical Formula g/litre
LAB064 Infusion from fat-free minced meat 10.0

Tryptone 20.0
Description
Dextrose 2.0
This is the original formula introduced by Brewer in 1940 as a clear
medium for the cultivation of anaerobes. It has found applications Sodium bicarbonate 2.0
as a sterility test medium and as a blood culture medium although it
has been superseded by Fluid Thioglycollate LAB025 and Fastidious Sodium chloride 2.0
Anaerobe Broth LAB071 for these purposes. Disodium phosphate anhydrous 0.4
The agar makes the medium viscous slowing down the permeation
of oxygen and any convection currents. Sodium thioglycollate acts as
a reducing agent and also neutralises the bacteriostatic properties of Method for Reconstitution
mercurial compounds. Methylene blue is a redox indicator which is Weight 36.4grams of powder and disperse in 1 litre of deionised water.
colourless at low Eh but turns green on exposure to oxygen. Allow to soak for 10 minutes, swirl to mix and warm to dissolve.
Dispense into 10ml volumes in screw capped containers and sterilise
Typical Formula g/litre by autoclaving at 121˚C for 15 minutes.
Beef Extract 1.0 Appearance: Pale straw, clear broth.
Yeast Extract 2.0 pH: 7.8 ± 0.2

Inoculation: Pick a well isolated colony for subculture into Todd
Hewitt Broth.
Dextrose 5.0 Incubation: 37˚C for 18-48hrs, aerobically.
Sodium chloride 5.0 Storage: Capped containers - up to 3 months at 15-20oC in the dark.
Minimum Q.C. Organisms Streptococcus pyogenes
Methylene blue 0.002 ATCC 19615.
Agar No. 1 1.0
Reference:
Method for reconstitution Todd, E.W., and Hewitt, L.F., (1932) A New Culture Medium for the
Production of Antigenic Streptococcal Haemolysin. J. Path. Bact. 35
Weigh 20 grams of powder, disperse in 1 litre of deionised water. (1) 973-974.
Allow to soak for 10 minutes then bring to the boil with gentle
agitation to dissolve the solids. Distribute into screw top containers Updyke, E.,L., and Nickle, M.I. (1954) A Dehydrated Medium for the
leaving minimal headspace. Sterilise by autoclaving at 121˚C for 15 Preparation of Type Speciic Extracts of Group A Streptococci. Appl.
minutes. Tighten caps as soon as possible after autoclaving. Microbiol. 2 117-118.
Appearance: Straw coloured, translucent, viscous liquid which may
have a green surface due to contact with oxygen. If the medium has
a diffuse green tinge it should not be used until the oxygen has been Triple Sugar Iron Agar
driven off by holding in a boiling water bath for 5 minutes. Do not
reheat more than once. LAB053
pH: 7.2 ± 0.2
Description
Minimum Q.C. organisms: C. perfringens WDCM 00007 This is a modiication of the Krumwiede and Kohn medium of 1917
which differentiates some of the Enterobacteriaceae on the basis of
Storage of Prepared Medium: Capped container – up to 3 months four reactions; fermentation of lactose, glucose and sucrose and H2S
at 15-20˚C in the dark. production. This medium should be used in conjunction with a urease
Inoculation: Ensure adequate dispersal of the inoculum in the broth. test to eliminate Proteus spp. when screening for Salmonella spp.
Incubation: 37˚C for 24-72 hours.
Growth Indicators: A diffuse turbidity or individual colonies.
Beef Extract 3.0
References
Yeast Extract 3.0
Brewer, J.H. (1940). Clear liquid mediums for the culture of anaerobes.
J. Amer. Med. Ass. 115: 598-600. Balanced Peptone No. 1 20.0
Sodium chloride 5.0
Todd Hewitt Broth Lactose 10.0

Sucrose 10.0
LAB075 Glucose 1.0
Description Ferric citrate 0.3
A nutritious broth medium formulated by Todd and Hewitt for the
production of antigenic streptococcal haemolysin. Todd Hewitt Broth Sodium thiosulphate 0.3
is also used to cultivate streptococci prior to serological grouping. Phenol red 0.025
The use of a fermentable sugar in the formulation leads to the
production of acid which would normally inactivate the haemolysin. Agar No. 2 12.0
This is prevented by the inclusion of buffers to maintain the pH of
the medium thus preserving the haemolysin, as well as promoting the Method for reconstitution
growth of pneumococci. Weigh 65 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then bring to the boil with
frequent swirling to dissolve the solids. Distribute into tubes and
sterilise at 121˚C for 15 minutes. Allow to set as a slope ensuring that
the slant is over a butt approximately 3cm deep.
Appearance: Reddish-orange gel.
pH: 7.4 ± 0.2

Minimum Q.C. organisms: E. coli WDCM 00013 Method for reconstitution
Storage of Prepared Medium: Capped containers – up to 3 at 121˚C for 15 minutes. Cool to 47˚C and pour into Petri dishes.
months at 15-20˚C in the dark.
Inoculation: A heavy inoculum is streaked over the surface of the Appearance: Straw coloured, clear gel.
slope and stabbed into the butt. pH: 7.2 ± 0.2
Interpretation
Slant/butt Colour Utilisation Storage of Prepared Medium: Plates – up to 4 days at 2-8˚C in the
Alkaline/acid Red/yellow Glucose only fermented dark. Capped container – up to 1 month at 2-8˚C in the dark.
Peptones utilised
Inoculation: 1ml of a 1:10 dilution of homogenised sample onto a
Acid/acid Yellow/yellow Glucose fermented Lactose membrane. The recommended membranes are 85mm in diameter
+ or sucrose fermented with a 0.45 micron pore size manufactured from cellulose esters.
Alkaline/alkaline Red/Red Neither glucose, lactose, Incubation: 4 hours at 37˚C on Nutrient Agar LAB008 or Minerals
nor sucrose fermented Modiied Glutamate Medium LAB080A and 80B plus agar – then 18-
Peptones utilised 24 hours on Tryptone Bile Agar at 44˚C.
Organism Butt Slant Sulphide Indole reagent: 5% p-dimethylaminobenzaldehyde in N-HC1
production (Vracko & Sherris 1963).
Indole reaction: Pipette 1-2ml of reagent into Petri dish lid, remove
S. sonnei Acid or -
membrane with forceps and place on reagent. Allow to stand for 5
S. lexneri Alk. minutes for reaction to develop, then dry in sunlight to ‘ix’ the colour.
Salmonella typhi Acid NC + Count all pink colonies as E. coli.
S. typhimurium Acid
S. enteritidis Gas NC + Growth characteristics
colony size shape & indole reaction on
E. coli
organism (mm) surface membrane
Enterobacter Acid Acid -
aerogenes Gas E. coli 1.0-3.0 CV.E.G. positive –
pink colour
Proteus mirabilis Acid Acid +
Gas other
Enterobacteriaceae 0.5-2.0 CV.E.G. negative – no colour
NC = No Change Pseudomonas spp. no growth
Staphylococcus
References spp. no growth
American Public Health Association (1963). Diagnostic Procedures Bacillus spp. no growth
and Reagents, 4th edn., A.P.H.A., New York.
American Public Health Association (1966). Recommended Methods References
for the Microbiological Examination of Foods. 2nd edn., A.P.H.A., Anderson, J.M., Baird-Parker, A.C. (1975). A rapid and direct method
New York. for enumerating Eschericia coli biotype I in food. J. Appl. Bact. 39:
Edwards, P.R. and Ewing, W.H. (1962). Identiication of 111-117.
Enterobacteriaceae. Burgess Publishing Co., Minneapolis. Delaney, J.E., McCarthy, J.A. & Grasso, R.J. (1962). Measurement
of E. coli type I by the membrane ilter technique Wat. Sewage Wks.
109, 289.
Tryptone Bile Agar Baird, R.M., Corry, J.E.L., Curtis, G.D.U. (1988). Pharmacopoeia
of culture media for food microbiology. Int. J. Food Microbiol. 276-
277.
LAB072
Description
First introduced by Delaney, McCarthy and Grasso in 1962 as a
method for detecting faecal coliforms in water supplies based on the
production of indole on a bile medium at 44˚C. The idea was applied
to foodstuffs by Anderson and Baird-Parker in 1975. The inoculum
is placed onto the membrane on a resuscitation agar and incubated
at 37˚C for 4 hours. The membrane is then transferred to a Tryptone
Bile Agar plate and incubated at 44˚C: after incubation the membrane
is looded with indole reagent. Indole positive colonies produce a red
colour on the membrane and are easily counted.

Tryptone 20.0
Bile Salts No. 3 1.5
Agar No. 2 15.0

Tryptone Glucose Extract Agar Minimum Q.C. organisms:
LAB063 Pseudomonas aeruginosa ATCC 9027
Bacillus subtilis ATCC 6633
Description Candida albicans ATCC 10231
A plate count agar suggested by the American Public Health
Association (A.P.H.A.) for estimation of total viable counts in
food and dairy products. This medium is also recommended by the Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
Association of Oficial Analytical Chemists (A.O.A.C.). dark. Capped containers – up to 3 months at 15-20˚C in the dark.
Inoculation: Surface plating.
Incubation: Time and temperature to suit organisms, usually
Beef Extract 3.0 aerobic.
Tryptone 5.0
Glucose 1.0 colony size shape &
Agar No. 1 15.0 organism (mm) surface colour other
Method for reconstitution Yellow
Weigh 24 grams of powder, disperse in 1 litre of deionised water. Ps. aeruginosa 0.5-3.0 F.CR.D. Grey- (marked strain
Allow to soak for 10 minutes, swirl to mix then boil to dissolve before Green variation)
distributing into tubes or bottles. Sterilise at 121˚C for 15 minutes.
Appearance: Pale straw colour, clear. References
pH: 7.0 ± 0.2 Harmonised Pharmacopeia 8.0, volume 1.
Blair, J.E. and Carr, M. (1953). The bacteriophage typing of
Minimum Q.C. organisms: S. aureus WDCM 00032 staphylococci. J. Infect. Dis. 93: 1-13.
E. coli WDCM 00013 Examination of Dairy Products. A.P.H.A., New York.

dark. Capped containers – up to 3 months at 15-20˚C in the dark.
Inoculation: Pour plate technique.
Tryptone Soy Broth
Incubation: 30˚C aerobically for 48 hours for aerobic mesophile (Soybean Casein Digest Medium
count. 6˚C aerobically for 10 days for aerobic psychrotroph count.
55˚C aerobically for 48 hours for aerobic thermophile count. LAB004
References Description
Association of Oficial Analytical Chemists (AOAC). (1995). A general purpose nutritious broth capable of growing a wide range
Bacteriological Analytical Manual, 8th ed.: Association of Oficial of bacteria and fungi. This medium is formulated to and performs to
Analytical Chemists. the requirements of the Harmonised Pharmacopeia for the sterility
Hausler, W.J. (Ed.) (1976). Standard Methods for the Examination of testing of a wide range of pharmaceutical products. The medium is
Dairy Products, 14th edn.: American Public Health Association. also widely used for blood cultures although the high carbohydrate
level may cause rapid growth and subsequent death of acid-producing
Speck, M.J. (Ed.) (1976). Compendium of Methods for the organisms.
Microbiological Examination of Foods.: American Public Health
Association. Typical Formula g/litre
Tryptone (Casein Digest USP) 17.0
Tryptone Soy Agar Soy Peptone 3.0
(Soybean Casein Digest Medium) Sodium chloride 5.0
LAB011
Dextrose 2.5
Description
A general purpose agar which will support the growth of a wide Method for reconstitution
range of micro organisms. The performance of this product conforms Weigh 30 grams of powder, disperse in 1 litre of deionised water.
with that laid down by the Harmonised Pharmacopeia (USP/EP/ Swirl to mix and warm if necessary to dissolve. Dispense into
JP) for sterility testing. The medium can be used for phage typing, tubes or lasks and sterilise at 121˚C for 15 minutes. Do not exceed
colicine typing and for testing the X and V factor requirements of temperature.
Haemophilus spp.
Appearance: Straw coloured, clear.
Tryptone (Casein Digest USP) 15.0 pH: 7.3 ± 0.2
Soy Peptone 5.0 Minimum Q.C. organisms:

Sodium chloride 5.0
Bacillus subtilis ATCC 6633
Agar No. 2 12.0 Aspergillus brasiliensis ATCC16404
Method for reconstitution Storage of Prepared Medium: Capped containers – up to 3 months

Weigh 37 grams of powder, disperse in 1 litre of deionised water, at 15-20˚C in the dark.
allow to soak for 10 minutes, swirl to mix then sterilise for 15 minutes Incubation: 20-25˚C aerobically for 14 days, for sterility tests. 37˚C
at 121˚C. Cool to 47˚C, mix well and then pour plates. aerobically for 14 days for blood cultures.
Appearance: Pale straw coloured, clear gel. Growth indicators: Turbidity or precipitate.
pH: 7.3 ± 0.2 References

Harmonised Pharmacopeia 8.0, volume 1.

Tryptone Soy Broth Storage of Prepared Medium: Capped containers – up to 3 months
(without dextrose) Inoculation: From pure culture.
Incubation: 37˚C for 24-48 hours.
LAB205
Interpretation: Indole positive organisms will give a distinct colour
Description change when either Kovac’s or Ehrlich’s indole reagent is added.
A basal media that can be supplemented with carbohydrates and References
indicators for fermentation studies.
American Public Health Association. (1955). American Water Works
Association 10th edn. 391-392.
MacFaddin, J. (1983). Biochemical tests for the identiication of
Tryptone 17.0 medical bacteria. 2nd edn. Williams & Wilkins, Baltimore.
Soy Peptone 3.0
Sodium chloride 5.0
Tryptose Phosphate Broth
LAB062
Weigh 27.5 grams of powder and disperse in 1 litre of deionised water. Description
Allow to soak for 10 minutes, swirl to mix and warm to dissolve if This is a versatile, nutritionally rich buffered glucose broth. The
necessary. Distribute into tubes or bottles and sterilise by autoclaving medium is a general purpose broth that has been used as a blood
for 15 minutes at 121°C. Do not exceed stated temperature. culture medium, and with the addition of sodium azide 0.025% as a
selective medium for streptococci in dairy products.
Appearance:
Powder: ine, free-lowing, homogeneous, buff Typical Formula g/litre
Finished medium: straw, clear liquid Tryptose 20.0
pH: 7.3 ± 0.2 Dextrose 2.0
Hazard classiication Sodium chloride 5.0
Disodium phosphate 2.5
Escherichia coli WDCM 00013 Method for reconstitution
Staphylococcus aureus WDCM 00032 Weigh 29.5 grams of powder and disperse in 1 litre of deionised water.
Bacillus subtilis WDCM 00003 Allow to soak for 10 minutes, heat to dissolve solids then distribute
Candida albicans WDCM 00054 into inal containers. Sterilise by autoclaving at 121oC for 15 minutes.
Cool to 47°C.
Aspergillus niger WDCM 00053
Appearance:
Storage: Powder: ine, free-lowing, homogeneous, buff
Dehydrated culture media: 10-25°C away from direct sunlight Finished medium: clear, pale straw liquid
Prepared media: capped containers – up to 3 months at 15-20°C in Minimum Q.C. organisms: E. coli WDCM 00013
the dark.
Incubation:
Strep. pyogenes NCTC 8198
Aerobically at 20-25°C for 14 days, for sterility tests.
Aerobically at 37°C for 14 days for blood cultures. pH: 7.3 ± 0.2
Tryptone Water Inoculation: For blood culture work dilute sample at least 1:10 in
broth.
LAB129 Incubation: Dependent on application.
A substrate for the testing of an organism’s ability to produce indole American Public Health Association (1948). Standard Method for the
from tryptophan. The indole test is frequently used in the classiication Examination of Dairy Products, 10th edn. A.P.H.A., New York.
of coliform organisms. This product is preferable to peptone water American Public Health Association, (1950). Diagnostic Procedures
LAB104 because it has a higher content of tryptophan. and Reagents, 3rd edn., A.P.H.A., New York.

Tryptone 10.0
Sodium chloride 5.0

Heat to dissolve then distribute into screw cap bottles. Sterilise by
autoclaving at 121˚C for 15 minutes.
Appearance: Colourless, clear.
pH: 7.5 ± 0.2

T.Y.C. Medium Gold, O.G., Jordon, H.V., Van Houte, J. (1973). A selective medium
for Streptococcus mutans. Archives of Oral Biology 19: 1357-1364.
(Tryptone Yeast Cystine) Ikeda, T., Sandham, H.J. (1972). A high-sucrose medium for the
identiication of Streptococcus mutans. Archives of Oral Biol. 4: 781-
LAB035 783.
Description Wade, W.G., Alldred, M. J., Walker, D.M. (1986). J. Med. Microbiol.
22: 319-323. An improved medium for isolation of Streptococcus
A medium designed by J. D. de Stoppelaar in 1967 to differentiate
mutans.
Streptococcus sanguis (frequently found in dental plaque) from
Streptococcus mutans (implicated in dental caries). The medium uses
a high sucrose content to promote the formation of speciic glucans by
S. mutans thus forming distinctive colonies. It can be made selective
by the addition of 0.2 units per ml of Bacitracin.
Urea Agar Base
(Christensen)
LAB130
Tryptone 15.0
Yeast Extract 5.0 Description
This is a modiication of Christensen’s urea base for the detection of
L-Cystine 0.2 rapid urease production by Proteus spp. Other enterobacteria will
Sodium sulphite 0.1 split the urea, but this will be delayed. This delay is achieved by the
incorporation of glucose and the introduction of a buffering system
Sodium chloride 1.0 into the medium. The indicator for ammonia production is phenol
red.
Disodium phosphate anhydrous 0.8
Sodium bicarbonate 2.0 Typical Formula g/litre
Sodium acetate anhydrous 12.0 Peptone 1.0
Sucrose 50.0 Glucose 1.0
Agar No. 2 12.0 Sodium chloride 5.0
Disodium phosphate 1.2
Weigh 98 grams of powder, disperse in 1 litre of deionised water. Potassium dihydrogen phosphate 0.8
Allow to soak for 10 minutes, swirl to mix, then sterilise for 15
minutes at 121˚C. Cool to 47˚C, mix and pour plates. Phenol red 0.012
Agar No. 1 12.0
Appearance: White, translucent gel.
pH: 7.3 ± 0.2 Method for reconstitution
Weigh 2.1 grams of powder, disperse in 95ml of deionised water.
Minimum Q.C. organisms: S. mutans NCTC 10832 Allow to soak for 10 minutes, swirl to mix, then sterilise at 121˚C for
15 minutes. Allow to cool to 47˚C, add aseptically 5ml sterile urea
Storage of Prepared Medium: Plates – up to 7 days at 4˚C in the solution X130/X135. Distribute into sterile bottles and slopes, allow
dark. to set in the sloped position.
Inoculation: Surface, streaking out for single colonies. Appearance: Yellow/pale pink, translucent.
Incubation: 37˚C for 4-5 days in an atmosphere of 90% H2, 10%
CO2. pH: 6.8 ± 0.2
Growth Characteristics Minimum Q.C. organisms:

Proteus mirabilis ATCC 29906/WDCM 00023
colony size shape & E. coli WDCM 00013
Strep. mutans 1.0-3.0 ‘heaped’ Yellow Crumbles when Storage of Prepared Medium: Capped container – up to 1 month at
Type A colony touched with wire 2-8˚C in the dark.
granular Inoculation: Pure culture using straight wire for stab/streak
surface technique.
irregular
edge Incubation: 37˚C for 4-6 hours or overnight, aerobically.
Type B 1.0-3.0 As Type A Grey white soft Interpretation: Production of red colour in under 6 hours is positive
consistency, for rapid urease production.
white precip in agar
(glistening drop) Organism Growth Characteristics
S. sanguis 1.0-3.0 Convex White very rubbery
Proteus spp. Red colour 4-6 hours
glossy (glistening drop)
crenated Klebsiella spp. Red colour 18-24 hours
Staphylococcus spp. Red colour 24-48 Hours
References
de Stoppelaar, J.D. van de Houte, J. and de Moor, C.E. (1967). The
presence of dextran forming bacteria resembling Streptococcus bovis References
and Streptococcus sanguis in dental plaque. Arch. Oral Biol. 12: Christensen, W.B. (1946). Urea decomposition as a means of
1199-1201. differentiating Proteus and paracolon cultures from each other and
de Stoppelaar, J.D. (1971). Streptococcus mutans, Streptococcus from Salmonella and Shigella types. J. Bacteriol. 52: 461-466.
sanguis and dental caries. Thesis, Rijksuniversiteit, Utrecht.
Emilson, C.G., Bratthall, D. (1976). Growth of Streptococcus mutans
on various selective media. Journal of Clinical Microbiology 4: 95-
98.

Urea Broth Base UVM Base
(Christensen) LAB 155
LAB155
LAB131 Description
Description UVM (University of Vermont Medium) Base is a two stage selective
This is a liquid version of Christensen’s medium (LAB130) introduced enrichment broth for the isolation of Listeria from meat products
and environmental swabs. The original method has been modiied to
by Maslen in 1952. This modiication allows inoculation by Pasteur
replace the second stage broth (UVM II) with Fraser broth LAB164
pipette, and it is easier to detect contamination in a luid rather than (McClain & Lee 1989).
in a slope. Maslen also claimed that it is easier to detect positive
results.
Peptone 1.0 Meat Peptone 5.0
Glucose 1.0 Beef Extract 5.0
Disodium phosphate 1.2 Yeast Extract 5.0
Potassium dihydrogen phosphate 0.8 Sodium chloride 20.0
Sodium chloride 5.0 Disodium hydrogen phosphate 9.6
Phenol red 0.004 Potassium dihydrogen phosphate 1.35
Aesculin 1.0
Weigh 0.9 grams of powder, add to 95ml of deionised water. Swirl
pH: 7.4 ± 0.2
to mix then sterilise by autoclaving at 121˚C for 15 minutes. Allow
to cool to 47˚C then add aseptically 5ml of X130/X135 sterile urea Appearance: Straw opalescent broth
solution. Distribute into sterile screw cap bijou bottles.
Appearance: Yellow, clear. Weigh 52 grams of powder and add to 1 litre of deionised water. Allow
to soak for 10 minutes, swirl to mix and sterilise at 121˚C for 15
pH: 6.8 ± 0.2 minutes. Cool to 47˚C and add 2 vials of UVM I supplement (X155)
as required. Mix well and distribute into sterile tubes or bottles.
Proteus mirabilis ATCC 29906/WDCM 00023 Inoculation: Add 25g sample to 225ml of and homogenise.
E. coli WDCM 00013 Incubation: 30˚C aerobically for 24 hrs. Subculture onto selective
agars. .
Storage of Prepared Medium: Capped container – up to 1 month at
2-8˚C in the dark. Minimum QC organism:
Inoculation: Fluid culture by pasteur pipette or straight wire from
Listeria monocytoges WDCM 00020
pure growth.
Incubation: 37˚C for 4-6 hours – preferably in a water bath for most
rapid growth, aerobically. References
McClain D., and Lee W.H. (1989) FSIS method for isolation of
Interpretation: The production of a red colour in under six hours is L.monocytogenes from processed meat and poultry products.
a positive result for rapid urease. Lab.Comm.No.57, Revised May 24, 1989. US Dept of Agric.FSIS,
Microbiol. Div.
Organism Growth Characteristics
Warburton D.W. et al (1991) A Canadian comparative study of
Proteus spp. Red colour 4-6 hours modiied versions of the FDA and USDA methods for the detection of
L.monocytogenes. J.Food Protection 54 (9) 669-676.
Some strains Klebsiella Red colour 18-24 hours
Escherichia ,, ,,
Staphylococcus, ,, ,,
References
Maslen L.G.C. (1952). Routine use of liquid urea medium for
identifying Salmonella and Shigella organisms. J. Brit. Med. 2: 545-
546.

Violet Red Bile Agar Violet Red Bile Agar with MUG
(V.R.B.A.) (V.R.B.A. with MUG)
LAB031 LAB573
A medium for the enumeration of coliform organisms in food and Violet Red Bile Agar with MUG (Methylumbelliferyl-β-D-
dairy products. The selectivity of the medium is due to the presence glucuronide) is a medium for the simultaneous enumeration of
of bile salts and crystal violet. Lactose fermenters produce red/purple coliform organisms and Escherichia coli in food and dairy products.
colonies often surrounded by a halo of the same colour. Non lactose The selectivity of the medium is due to the presence of bile salts and
fermenters produce pale colonies. Selectivity can be increased by crystal violet. Lactose fermenters produce red/purple colonies often
incubation at 42-44°C. surrounded by a halo of bile precipitate. Escherichia coli produce
red/purple luorescent colonies due to the fermentation of lactose and
Typical Formula g/litre production of the enzyme glucuronidase, which hydrolyses MUG to
yield the luorescent compound methylumbelliferone, detectable by
Yeast Extract 3.0 long-wave UV light. Non-lactose fermenters produce pale colonies.
Balanced Peptone No. 1 7.0 Standard Methods procedures specify VRBA with MUG for detecting
E. coli in food and dairy products by luorescence.
Sodium chloride 5.0
Bile Salts No. 3 1.5 Typical Formula g/litre
Lactose 10.0 Yeast extract 3.0
Neutral red 0.03 Balanced peptone No.1 7.0
Crystal violet 0.002 Sodium chloride 5.0

Agar No. 2 12.0 Bile Salts No. 3 1.5
Lactose 10.0
Neutral red 0.03
Disperse 38.5g of powder in 1 litre of distilled water. Dissolve by
bringing to the boil with frequent swirling of the lask to prevent Crystal violet 0.002
overheating. DO NOT AUTOCLAVE. Cool to 45°C and distribute into
bottles or tubes. If held molten in a water bath, use within 3 hours. Agar No. 2 12.0
Appearance: MUG, 4 methylumbelliferyl-ß-D-glucuronide 0.1
Finished medium: light, purple-violet clear gel Method for reconstitution
pH: 7.4 ± 0.2 Allow the mixture to soak for 10 minutes, swirl to mix and then
sterilise the medium, with frequent mixing, by bringing to the boil.
Minimum Q.C. organisms: E. coli WDCM 00013 DO NOT AUTOCLAVE. Cool to 47°C and distribute into bottles or
Enterococcus faecalis (inhibition) tubes. If held molten in a water bath, use within 3 hours.
WDCM 00087
Appearance: Light purple-violet clear gel.
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the pH: 7.4 ± 0.2
dark. Capped containers – up to 1 month at 15-20˚C in the dark.
Inoculation: Pour plate (with or without overlay) or surface spread. Minimum Q.C. organisms:
Incubation: 37˚C for 18-24 hours for ‘coliforms’; 4˚C for 10 days Enterobacter aerogenes WDCM 000175
for psychrotrophs; 32˚C for 24-48 hours for mesotrophs; 42˚C for 18 Staphylococcus aureus WDCM 00034
hours for thermotrophs.
Interpretation: Count all red/purple colonies > 0.5mm in diameter. Storage of Prepared Medium: Plates - up to 7 days at 2 - 8°C in the
Calculate the number of coliforms in original sample. dark. Capped containers up to 1 month at 15-20°C in the dark.
References Inoculation: Pour plate method (with or without overlay) or surface
American Public Health Association (1972), Standard Methods for spread.
the Examination of Dairy Products. 13th edn. (ed. W.H. Hausler), Incubation: 37°C for 18-24 hours for ‘coliforms’; 4°C for 10 days
A.P.H.A., Washington. for psychrotrophs; 32°C for 24-48 hours for mesotrophs; 42°C for 18
American Public Health Association (1966). Recommended Methods hours for thermotrophs.
for the Microbiological Examination of Foods. 2nd edn. (ed. J.M. Interpretation: Examine plates for growth and luorescence. Count
Sharf) A.P.H.A., Washington. all red/purple colonies > 0.5mm in diameter as coliforms and all
Davis, J.G. (1951). Milk Testing Dairy Industries, London. luorescent colonies as presumptive E. coli. Calculate the number of
coliforms and E. coli in the original sample.
Mossel, D.A.A., Eelderink, I. and Sutherland, J.P. (1977).
Development and use of single, ‘polytropic’ diagnostic tubes for the References
approximate taxonomic grouping of bacteria, isolated from foods, Christen, G.L., Davidson, P.M., McAllistair, J.S. and Roth, L.A.
water and medicinal preparations. Zbl. Bakt. Hyg. I., Orig., A 278, (1993). Coliform and other indicator bacteria. 247-269. R.T. Marshall
66-79. (ed.) Standard methods for the microbiological examination of dairy
Mossel, D.A.A., Eelderink, I., Koopmans, M. and van Rossem, products, 16th ed. American Public Health Association, Washington,
F. (1979). Inluence of carbon source, bile salts and incubation D.C.
temperature on the recovery of Enterobacteriaceae from foods using Hitchens, A.D., Hartman, P.A. and Todd, E.C.D. (1992) Coliforms-
MacConkey type agars. J. Food Protec. 42, 470-475. Escherichia coli and its toxins. C. Vanderzant and D. F. Splittstoesser
Mossel, D.A.A., van der Zee, H., Hardon, A.P. and van Netten, (ed.). Compendium of methods for the microbiological examination
P. (1986). The enumeration of thermotropic types amongst the of foods, 3rd ed. American Public Health Association, Washington,
Enterobacteriaceae colonizing perishable foods. J. Appl. Bacteriol. D.C.
60, 289-295. Hitchens, A.D., Peng, P., Watkins, W.D., Rippey, S.R. and Chandler,
L.A. (1995). Escherichia coli and the coliform bacteria. 4.01-4.29.
Bacteriological Analytical Manual, 8th ed. AOAC International,
Gaithersburg, MD.

Feng, P.C.S. and Hartman, P.A. (1982). Fluorogenic assays for
immediate conirmation of Escherichia coli. Appl. Environ. Microbiol.
Water Plate Count Agar (ISO)
43:1320-1329.
LAB197
Chang, G.W., Brill, J. and Lum, R. (1989). Proportion of β-D-
glucuronidase negative Escherichia coli in human fecal samples. Description
Appl. Environ. Microbiol. 55:335-339.
A nutritious non-selective medium which conforms to ISO
Hansen, W. and Yourassowsky, E. (1984). Detection of β-D- 6222:1999(E) Water quality - Enumeration of culturable micro-
glucuronidase in lactose fermenting members of the family organisms - Colony count by inoculation in a nutrient agar culture
Enterobacteriaceae and its presence in bacterial urine cultures. medium. The estimation of overall numbers of microorganisms can
J. Clinical Microbiol. 20:1177-1179. be used for the assessment and surveillance of water quality. Colony
Kilian, M. and Bulow, P. (1976). Rapid diagnosis of Enterobacteriaceae. counts are useful for assessment of ground water integrity and the
Acta. Pathol. Microbiol. Scand. Sect. B. 84:245-251. eficiency of water treatment processes. They also give an indication
Mates, A. and Shaffer, M. (1989). Membrane iltration differentiation of the cleanliness and integrity of the distribution system. They can
of E. coli from coliforms in the examination of water. J. Appl. be used to assess the suitability of a water supply for the preparation
Microbiol. 67:343-346. of food and drink, thus avoiding contamination of the product with
spoilage organisms. The main value of colony counts lies in the
Damare, J.M., Campbell, D.F. and Johnston, R.W. (1985). Simpliied detection of changes in water supply quality from those expected,
direct plating method for enhanced recovery of Escherichia coli in based on frequent long term monitoring. A sudden increase in the
food. Journal of Food Science. 50:1736-1746. numbers can be a warning of pollution and can call for immediate
remedial action
Violet Red Bile Glucose Agar Typical Formula g/litre
(V.R.B.G.A.) Tryptone 6.0

Yeast Extract 3.0
LAB088
Agar 15.0
Description
A modiication of Violet Red Bile Agar LAB031 introduced by Mossel Method for reconstitution
in 1978. V.R.B.A. LAB031 contains lactose which is fermented by Weigh 24.0 grams of powder and disperse in 1 litre of deionised water.
members of the coli/aerogenes group, this medium gives a ‘coliform’ Allow the mixture to soak for 10 minutes, swirl to mix then sterilise at
count. V.R.B.G.A. LAB088 has substituted lactose with glucose. 121°C for 15 minutes. Cool to 47°C before use.
Glucose is fermented by all members of the Enterobacteriaceae thus
V.R.B.G.A. gives a presumptive Enterobacteriaceae count. Bile salts Appearance: Pale straw coloured, clear gel.
and crystal violet are used to inhibit Gram positive and non-enteric
pH: 7.2 ± 0.2
organisms. The overlay procedure ensures anaerobic conditions and
suppresses the growth of non-fermentative Gram negative bacteria.
Typical Formula g/litre Escherichia coli WDCM 00013
Yeast Extract 3.0
Storage of Prepared Medium: Plates can be stored up to 7 days at
Balanced Peptone No. 1 7.0 2-8°C in the dark.
Sodium chloride 5.0 Inoculation: Pour plate technique or surface inoculation.
Bile Salts No. 3 1.5 Incubation: Aerobically at 36°C ± 2°C for 44 ± 4 hours and 22°C ±
2°C for 68 ± 4 hours.
Glucose 10.0
Interpretation: Count all colonies and calculate the number of
Neutral red 0.03 organisms (or ‘colony forming units’ c.f.u.) per ml of sample allowing
for dilution factors.
Agar No. 2 12.0 References
ISO 6222 (1999) Water quality - Enumeration of culturable
Method for reconstitution microorganisms - Colony count by inoculation in a nutrient agar
medium.
Allow to soak for 10 minutes then swirl to mix. Bring to the boil with
frequent swirling to prevent overheating. Further sterilisation is not
required. Cool to 45˚C, mix well and dispense into tubes or bottles. If
held molten in a water bath, use within 3 hours.
Appearance: Light purple-violet, clear.
pH: 7.4 ± 0.2

E. coli WDCM 00013

dark. Capped containers – up to 1 month at 15-20˚C in the dark.
Inoculation: Pour plate method with overlay.
Interpretation: Count all red/purple colonies > 0.5mm in diameter.
Calculate the number of Enterobacteriaceae in original sample.
References
Pharmacopoeia of Culture Medium for Food Microbiology (1987).
Int. J. Food Microbiol. 5: 3: 280-81.
Mossel, D.A.A., Mengerink, W.H.J. and Scholts, H.H. (1962). Use
of a modiied MacConkey agar medium for the selective growth and
enumeration of Enterobacteriacaea. J. Bacteriol. 84: 381.

W. L. Nutrient Agar Wort Agar
(Wallerstein Laboratory)
LAB038
LAB079
Description
Description A medium for the enumeration of yeasts and moulds in butter. The
This medium was developed by Green and Gray in 1950 for the medium can be modiied to enable it to isolate osmophilic yeasts from
isolation and enumeration of yeasts, moulds and bacteria in the soft drinks and sugar products by the addition of high concentrations
brewing process. The medium has a pH of 5.5 which is optimum for of sucrose and glucose.
Brewers yeast and will allow the growth of a wide range of organisms
including Enterobacteriaceae, Flavobacterium, Lactobacillus and Typical Formula g/litre
Pediococcus spp. as well as yeasts and moulds. If a process involving
bakers or distillers yeast is under examination the pH should be Malt Extract 15.0
adjusted to 6.5. The medium may be adapted to detect bacteria only
by the addition of 0.004 g/litre of Actidione to suppress the yeasts. Yeast Extract 7.0
Sugars 9.0
Tartaric acid 0.3
Yeast Extract 4.0
Peptone 1.0
Tryptone 5.0
Dextrose 50.0
Ammonium chloride 1.0
Agar 14.0
Calcium chloride 0.125 Method for reconstitution
Magnesium sulphate 0.125 water. Add 2.35ml of glycerol. Allow to soak for 10 minutes, swirl to
Ferric chloride 0.0025 mix then sterilise for 15 minutes at 121°C. Use 60 grams per litre if
required for inoculation by plate streaking with a wire loop. Do not
Manganese sulphate 0.0025 exceed time or temperature of sterilisation. If osmophilic modiication
is required add 48.3 grams of powder to 1 litre of a solution containing
Bromocresol green 0.022
35% w/v sucrose and 10% w/v glucose then sterilise at 108˚C (5 p.s.i.)
Agar No. 2 15.0 for 20 minutes.
Appearance: Light Brown, translucent.
pH: 5.0 ± 0.2
Allow to soak for 10 minutes, swirl to mix and sterilise by autoclaving Minimum Q.C. organisms: S. cerevisiae. WDCM 00058
for 15 minutes at 121o C. If adjustment of pH to 6.5 is required used
1% sodium bicarbonate. Cool to 47°C, mix well and dispense into
Petri dishes. Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
dark. Capped container – up to 1 month at 15-20˚C in the dark.
Appearance:
Inoculation: Pour plate or surface spread.
Finished medium: Green-blue, clear gel Incubation: 25˚C aerobically for 5 days.
pH: 5.5 ± 0.2 Growth Characteristics

colony size shape &
Minimum Q.C. organisms: S. cerevisiae. ATCC 9763 organism (mm) surface colour other
Candida spp. 4.0 CV.E.G White
Storage: Fungi Varies with
Dehydrated culture media: 10-25o C species
Final medium: up to 7 days (plates) or up to 1 month (capped S. cerevisiae 2.0-3.0 Varies with Cream
containers) at 2-8°C in the dark strain
Inoculation: Surface plating or pour plate.
Incubation: 30˚C aerobically for 48 hours (bacteria); 20˚C References
aerobically for 48 hours (yeasts). Paritt, E.H. (1933). The inluence of media upon the yeast and mould
Interpretation: Count all colonies. Calculate organisms per ml in count of butter. J. Dairy Sci. 16: 141-147.
original sample. Scarr, M.P. (1959). Selective media used in the microbiological
examination of sugar products. J. Sci. Fd. Agric. 10: 678-681.
References
Green, S.R. and Gray, P.P. (1950). Differential Procedure Applicable
to Investigation in Brewing. Wallerstein Lab. Comm. 13,357.
Hall, J.F. (1971). Detection of Wild Yeasts in the Brewery. J. Inst.
Brewing, 77: 513-516.

Wort Broth Method for reconstitution
(modiied) Weigh 53.5 grams of powder, disperse in 1 litre of deionised water.

Allow to soak for 10 minutes, swirl to mix. Bring rapidly to the boil
with frequent stirring, and transfer immediately to a 47˚C water bath.
LAB099 Pour into plates as soon as the medium has cooled. Protracted boiling
or prolonged holding at elevated temperature induces precipitation.
Description
Appearance: Light rose, clear gel.
A broth for the enumeration of yeasts and moulds, in butter. The
medium can be modiied for the isolation of osmophilic yeasts from pH: 7.4 ± 0.2
soft drinks and sugar products by the addition of high concentrations
of sucrose and glucose. Minimum Q.C. organisms: S.typhimurium WDCM 00031
Malt Extract 15.0 Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
dark.
Peptone 0.78
Maltose 12.75 Incubation: 37˚C for 18-24 hours aerobically.
Dextrin 2.75
colony size shape &
Ammonium chloride 1.0 organism (mm) surface colour other
Salmonella spp. 1.0-2.5 CV.E.G. Trans. (clearing of acid
Method for reconstitution black ppt of coliforms)
centre
Weigh 33.3 grams of powder and disperse in 1 litre of deionised water,
add 2.35ml of glycerol. Allow to soak for 10 minutes, swirl to mix then Shigella sonnei 1.5-2.5 CV.E.G. Pink
sterilise by autoclaving at 121˚C for 15 minutes. If osmophilic version E. coli 0.5-1.5 CV.E.G.(D) Yellow inhibited (ppt
is required disperse 33.3 grams of powder in 1 litre of a solution of around colony)
35% w/v sucrose and 10% w/v glucose then sterilise at 108˚C (5 p.s.i.)
Citrobacter spp. 1.0-1.5 CV.E.G.(D) Yellow (black centre)
for 20 minutes.
Proteus spp 1.0-2.5 CV.E.G. Trans. Pink ishy odour
Appearance: Light Brown, translucent. (black
pH: 4.8 ± 0.2 centre)
Minimum Q.C. organisms: S. cerevisiae. WDCM 00058 References

Taylor, W.I. (1965). Isolation of shigellae. I. Xylose Lysine Agars:
Storage of Prepared Medium: Capped container – up to 1 month at New media for the isolation of enteric pathogens. Am. J. Clin.
15-20˚C in the dark. Pathol., 44: 471-475.
Incubation: 25˚C aerobically for 5 days. Taylor, W.I., and Harris, B. (1965). Isolation of shigellae. II.
Comparison of plating media and enrichment broths. Am. J. Clin.
Pathol., 44(4), 476-479.
Taylor, W.I., and Harris, B. (1967). Isolation of shigellae. III.
X.L.D. Agar Comparison of new and traditional media with stool specimens. Am.
J. Clin. Pathol., 48: 350-355.
(Xylose Lysine Decarboxylase Agar)
Taylor, W.I., and Schelhart, D. (1967). Isolation of shigellae. IV.
LAB032 Comparison of plating media with stools. Am. J. Clin. Pathol., 48:
356-362.
Description
This medium was introduced by Taylor in 1965 to improve the
recovery and recognition of Shigella spp, and has proved to be an
excellent medium for Salmonella spp. The medium is low in nutrients
and relies on a small amount of sodium desoxycholate for selectivity.
The indicator system is novel and complex. Most enteric organisms
except Shigella, will ferment xylose to produce acid. However the
salmonellae will also decarboxylate the lysine to keep the pH neutral.
At near neutral pH Salmononella can produce H2S from the reduction
of thiosulphate producing black or black centred colonies. Citrobacter
spp. can also decarboxylate lysine, however, the acid produced by
fermentation of both lactose and sucrose will keep the pH too acid for
H2S to be produced.

Xylose 3.75
L-Lysine 5.0
Lactose 7.5
Sucrose 7.5
Sodium chloride 5.0
Yeast Extract 3.0
Phenol red 0.08
Agar No. 2 13.0

XLT4 Agar Minimum Q.C. organisms:
Salmonella Typhimurium WDCM 0031
Xylose Lysine Tergitol-4 Agar
Salmonella Enteritidis WDCM 00030
LAB221 Enterococcus faecalis WDCM 00087
Description Storage:
XLT4 Agar is a selective differential isolation medium for the speciic Dehydrated culture media: 10-25°C.
detection of Salmonella spp. from environmental, food and clinical Final medium: 7 days at 2-8°C in the dark
samples. Due to its highly selective nature, XLT4 Agar is particularly Inoculation: Surface inoculation, streaking out / spreading to achieve
effective when used with samples where overgrowth of contaminating single colonies.
lora is expected, for example, faecally-contaminated agricultural
samples. Incubation: Incubate plates at 37°C. Examine plates for growth at 24
hours and 48 hours.
Developed to perform as per Miller & Tate in 1990, this medium
Interpretation: Typical Salmonella (lactose-negative, H2S positive)
was found to improve the recovery of non-typhi Salmonella from
appear as colourless or red colonies with a black centre, giving the
chicken and farm environmental samples. Dusch & Altwegg further
traditional “ish-eye” appearance. All isolates with this appearance
established the application of XLT4 Agar to salmonellae detection
should be regarded as presumptive salmonellae.
in clinical samples, with the notable exceptions of Salmonella
Typhi and Salmonella Paratyphi. The presence of peptone and yeast Lactose-positive, H2S positive salmonellae, e.g. Salmonella Arizonae
extract provides suficient nutrients to allow the optimal growth of will appear yellow-red with black centre.
Salmonella spp. Lactose-negative, H2S negative salmonellae, e.g. Salmonella New
Selectivity is provided by the anionic surfactant Niaproof ® 4 (formerly Brunswick will appear yellow-red without a black centre.
known as Tergitol-4 / sodium tetradecylsulfate). This compound acts Some strains of Salmonella Poona may demonstrate sensitivity to
as an effective selective agent which is active against Gram-positive Niaproof ® 4.
and many Gram-negative organisms, including Proteus spp.
Differentiation is based on fermentation of the sugars xylose, Interpretation
lactose and sucrose in addition to the decarboxylation of lysine. The
inclusion of the pH indicator, phenol red, provides visual evidence of colony size shape &
a pH decrease (yellow) or increase (red) in the medium. Ammonium organism growth (mm) surface H2S colour
iron (III) citrate is present to distinguish hydrogen-sulphide (H2S) Salmonella Good 1.2 - 2.5 CV, E, G + Clear/red,
Typhimurium >50% recovery black centre
producing from non-H2S producing organisms.
Salmonella Good 1.5 - 2.5 CV, E, G + Clear/red,
Most enteric organisms, except Shigella, will ferment xylose to produce Enteritidis >50% recovery black centre
acid. However the salmonellae will also decarboxylate the lysine to Citrobacter Good 1.5 - 2.5 CV/DR, E, D +/- Yellow
keep the pH neutral to alkali, thus maintaining red colouration. At freundii
near-neutral pH Salmonella can produce H2S from the reduction of Escherichia Suppressed
ammonium iron (III) citrate and thiosulphate ions producing black or coli
black-centred colonies. Non H2S-producing salmonellae will be red Proteus Inhibited
without a black centre. mirabilis
Other Enterobacteriaceae (non-salmonellae) which are not inhibited Enterococcus Inhibited
by Niaproof-4, will ferment xylose, lactose and/or sucrose but will faecalis
not decarboxylate lysine. This fermentation activity causes a decrease Staphylococcus Inhibited
in pH, resulting in a colour change within the colonies from red to aureus
yellow. KEY CV = Convex D = Dull DR = Draughtsman
E = Entire G = Glossy
References
Proteose peptone 12.00 Dusch, H. and Altwegg, M. (1995). Evaluation of ive new plating
Yeast extract 1.5 media for the isolation of Salmonella species. Journal of Clinical
Microbiology. 33. No.4. 802-804.
L-lysine 5.0 Miller, R.G. and Tate, C.R. (1990). A highly selective plating medium
Xylose 3.5 for the isolation of Salmonella. The Maryland Poultryman, April:
2-7.
Lactose 7.0
Miller, R.G., Tate, C.R., Mallinson, E.T. and Scherrer, J.A. (1991).
Sucrose 7.5 Xylose-Lysine-Tergitol 4: An improved selective agar for the isolation
of Salmonella. Poultry Science 70. 2429-2432.
Ammonium iron (III) citrate 0.8
Miller, R.G., Tate, C.R., Mallinson, E.T. and Scherrer, J.A. (1992).
Sodium thiosulphate 5.5 Erratum. Xylose-Lysine-Tergitol 4: An improved selective agar for
the isolation of Salmonella. Poultry Science 71. 398.
Sodium chloride 5.0
Tate, C.R., Miller, R.G. and Mallinson, E.T. (1992). Evaluation of two
Phenol red 0.08 isolation and non-isolation methods for detecting naturally occurring
salmonellae from broiler lock environmental drag-swab samples. J.
Agar 13.0 Food Prot. 55. 964-967.

Add 4.6ml of Niaproof-4 supplement (Sigma Niaproof ® 4, product
code N1404). Allow to soak for 10 minutes, swirl to mix. Heat the
medium with frequent agitation and boil for 1 minute. DO NOT
OVERHEAT OR AUTOCLAVE THIS MEDIUM. Cool to 48-50°C,
mix well and dispense into sterile Petri dishes.
Appearance:
Powder: ine, free-lowing, homogeneous, buff (may have a slight
pink colouration)
Finished medium: clear, red gel
pH: 7.4 ± 0.2

Yeast Extract Agar Minimum Q.C. organisms:
Aspergillus sp. WDCM 00053
(Yeastrel Milk Agar) S. cerevisiae WDCM 00058
LAB018
Description dark.
A nutrient agar corresponding to the Standard Formulation for the Incubation: 25˚C for 5 days, aerobically.
plate count of micro-organisms in water and dairy products. This Inoculation: Pour plate technique.
medium is also useful for teaching and demonstration purposes
using non-fastidious organisms. Interpretation: Count all colonies.
Typical Formula g/litre References

Engel, G. (1982). Verglich verschieden Nährböden zum quantitativen
Yeast Extract 3.0 Nachweis von Hefen und Schimmelpilzen in Milch und
Milchprodukten. Milchwiss. 37: 727-730.
International Organisation for Standardization (ISO): Milk and
Agar No. 1 15.0 milk products – enumeration of yeasts and moulds – colony count
technique at 25˚C – standard method ISO/DIS 6611.
Method for reconstitution International Milchwirtschaftsverband: Milch und Milchprodukten –
Weigh 23 grams of powder, disperse in 1 litre of deionised water. Zählung von Hefen und Schimmelpilzen (Kolonieählung bel 25 C). –
Free steam or boil to dissolve. Mix well, and dispense into containers. International IMV Standard 94: (1980) in Milchwiss. 36: 220-222.
Sterilise for 15 minutes at 121˚C. Normenausschul Lebensmittel und landwirtshaft. Produkte
To prepare Yeastrel Milk Agar add 10ml of fresh milk before in DIN Deutsches Institut für Normung e.V. Mikrobiologische
autoclaving. Milchuntersuchung. Bestimmung der Anzahl von Hefen und
Schimmelpilzen. Reference method DIN 10186.
Appearance: Pale straw, clear gel.
British Standards Institute. B.S. 4285. Section 3.6: (1986).
pH: 7.2 ± 0.2

S. aureus WDCM 00032 Yeast & Mould Agar
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the LAB200
dark. Capped container – up to 3 months at 15-20˚C in the dark.
Description
Inoculation: Pour plate technique or surface spreading.
A medium recommended for the isolation and maintenance of yeasts
Incubation: 30˚C aerobically for 48 hours for aerobic mesophile and moulds. This medium can also be used for the detection of wild
count. 6˚C aerobically for 10 days for aerobic psychrotroph count. yeasts in beer.
55˚C aerobically for 48 hours for aerobic thermophile count.
References
Environment Agency: The Microbiology of Drinking Water (2002). Yeast extract 3.0
Malt extract 3.0
British Standard 4285: Methods of Microbiological Examination for
Dairy Purposes. Peptone 5.0
Dextrose 10.0
Agar 20.0
Yeast Extract Dextrose
Chloramphenicol Agar Method for reconstitution
LAB119 Allow to soak for 10 minutes, swirl to mix and sterilise by autoclaving
for 15 minutes at 121°C. Cool to 47°C, mix well and dispense into
sterile Petri dishes.
Description
A selective medium for the enumeration of yeasts and moulds in milk Increased selectivity of the medium can be achieved by the addition of
and other dairy products. The medium is said to have superior storage 12-15ml of sterile lactic acid (X037) to reduce the pH to 4.0. For the
properties to O.G.Y.E. and also has the advantage of incorporating an detection of wild yeasts, supplement the medium to the desired level
autoclavable supplement. with copper sulphate prior to sterilisation.
Appearance:
Yeast Extract 5.0 Finished medium: clear straw gel
Dextrose 20.0
pH: 6.2 ± 0.2
Agar No. 1 15.0
Weigh 40 grams of powder, disperse in 1 litre of deionised water. Minimum Q.C. organisms:
Allow to soak for 10 minutes, swirl to mix then bring to boil. Add Escherichia coli WDCM 00013
2 vials of X009 (X209) which have been dissolved in ethanol and Candida albicans WDCM 00054
autoclave at 121˚C for 10 minutes. Allow to cool to 45˚C before using Saccharomyces pastorianus NCYC 185
with poured plate technique. THIS MEDIUM MUST NOT BE RE- Lactobacillus fermetum ATCC 9338
AUTOCLAVED.
Appearance: Pale yellow, clear. Storage:
Dehydrated culture media: 10-25°C away from direct sunlight
pH: 6.6 ± 0.2
Poured plates: 7 days at 2-8°C in the dark

Yersinia Selective Agar
(Schiemann’s C.I.N. Agar)
LAB120
Description
This medium is based on the work of Schiemann. It is used for the
isolation and enumeration of Yersinia spp. from clinical samples and
from food. The selective components are sodium desoxycholate,
crystal violet, cefsulodin, irgasan and novobiocin. Yersiniae ferment
mannitol with an intense, localised, acid production in the centre of
the colony which produces a red ‘bull’s eye’ appearance. The ratio of
transparent border to red centre varies with serotype and environmental
strains may appear rough with an irregular edge. Most other enteric
bacteria, if they grow, produce a larger colony with a diffuse pinkish
centre and opaque outer zone.

Peptone Mixture 22.5
Mannitol 20.0
Sodium chloride 1.0
Sodium pyruvate 2.0
Neutral red 0.03
Agar No. 2 12.0

Allow to soak for 10 minutes, then bring to the boil for 1 minute only.
DO NOT AUTOCLAVE. Allow to cool to 47˚C add 2 ampoules C.I.N.
supplement X120. Mix well, pour plates.
Appearance: Red, clear.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: Y. enterocolitica WDCM 00038


dark.
colony size shape &
Y. enterocolitica 1.0-2.5 CV.E.G. Red centre Colony varies
with strain,
may be
rough &
irregular
Gram +ve
organisms no growth
References
Schiemann, D.A. (1979). Synthesis of a selective agar medium for
Yersinia enterocolitica. Can. J. Microbiol. 25: 1298-1304.
Schiemann, D.A. (1982). Development of a two step enrichment
procedure for recovery of Yersinia enterocolitica from food. Appl.
Eniviron. Microbiol. 43: 14-27.
Mossel, D.A.A. (1987). Cefsulodin Irgasan Novobiocin (C.I.N.) agar.
Int. J. Food. Microbiol. 5: 208, 209.

2. Harlequin™ Chromogenic Weigh 36.5 grams of powder, disperse in 1 litre of deionised water.
Allow the mixture to soak for 10 minutes, swirl to mix and then
Media sterilise the medium by bringing to the boil. Cool to 47°C, mix well
and dispense into Petri dishes.
The Harlequin™ chromogenic microbiological culture media range DO NOT REMELT OR AUTOCLAVE THIS MEDIUM.
has been developed to improve the isolation and identiication of a
range of microorganisms. Traditional culture media generally rely Appearance: Translucent straw gel.
on the fermentation of sugars or other biochemical reactions for pH: 7.2 ± 0.2
presumptive identiication of bacteria. Harlequin™ chromogenic
media provide a more speciic identiication by detection of speciic Minimum QC organisms:
enzymes produced by certain groups of bacteria. The advantage Salmonella typhimurium WDCM00031
of this type of media is that it can eliminate or reduce the need for Escherichia coli WDCM 00013
subculture and the performance of conirmatory biochemical tests to
determine the identity of some microorganisms. Storage of Prepared Medium: Plates - up to 7 days at 2 - 8°C in
Chromogenic substrates act as the substrate for speciic enzymes the dark.
and change colour due to the action of the enzyme. Lab M have Inoculation:
their own patented chromogenic compounds called the novel CHE Clinical: Streak for single colonies after selective enrichment in
(cyclohexenoesculetin) substrates which give the bacterial colony Selenite Broth.
a black non-diffusing colouration when hydrolysed by the enzyme Food: Streak for single colonies after selective enrichment.
involved in the presence of iron salts. Some of the CHE derivatives are
used in Harlequin™ media along with indolyl derivatives (5-bromo- Incubation: 37°C aerobically for 18 - 24 hours
4-choro-3-indolyl), which give a blue-green colour on cleavage.
Chromogenic media are most rapidly gaining acceptance as an
indicator for Escherichia coli. The β-D-glucuronide enzyme is colony size shape &
present in approximately 95% of E. coli and is uncommon in the organism (mm) surface colour other
other Enterobacteriaceae. This type of test is now used widely for Salmonella spp. 1.5 - 3.5 CV.E.G. Green (Black if
water and food microbiology. There is also an increase in interest β-galactosidase
for chromogenic Salmonella media as traditional tests have a very +ve)
poor speciicity resulting in many false positive results. The use of
chromogenic media simpliies Salmonella testing and saves much Shigella spp. 3.0 - 4.0 CV.E.G. Colourless (Black if
time in unnecessary conirmation tests. β-galactosidase
+ve)
E.coli PP - 2.5 CV.E.G. Black (No Growth)
Proteus spp. PP - 1.0 CV.E.G. Colourless (Fishy Odour)
Harlequin™ Salmonella ABC
References
HAL001 Perry, J.D., Ford, M., Taylor, J., Jones, A., Freeman, R., Gould,
F.K., (1999). ABC Medium, a New Chromogenic Agar for Selective
Description Isolation of Salmonella spp. J. Clin. Micro. 37: 766-768.
Salmonella spp. can be differentiated from other members of the
family Enterobacteriaceae by their ability to produce α-galactosidase
in the absence of β-galactosidase. This medium, developed for the
isolation of Salmonella spp. from food and clinical samples, utilises
Harlequin™ TBGA (TBX)
a dual chromogen system to visualise these enzyme activities. This (Tryptone Bile Glucuronide Agar)
medium will also detect Salmonella typhi and paratyphi.
The irst substrate, CHE-β-Gal, is enzymatically cleaved by HAL003
β-galactosidase producing organisms giving black colonies in the
presence of iron. Most Enterobacteriaceae are β-galactosidase Description
positive and these produce black colonies on Salmonella ABC. A medium developed for the simple enumeration of E. coli without
The second substrate, X-α-Gal, is hydrolysed by Salmonella spp. the need for membranes, or pre-incubation on Minerals Modiied
producing green colonies that are easily distinguished from the black Glutamate Medium. It is based upon the formulation of Tryptone Bile
or colourless colonies of other organisms. The medium is based on Agar, LAB072, the medium has been modiied by the addition of a
D.C.A Hynes and hence utilises sodium desoxycholate and sodium chromogenic substrate to detect the ß-glucuronidase enzyme, which
citrate as inhibitors. Isolation of Salmonella spp. by culture remains is highly speciic for E. coli*, and is detected by the MUG reagent in
the most reliable method of detection. However, most media are other formulations. The advantage of the chromogenic substrate is that
highly non-speciic and consequently place a heavy burden on the it requires no UV lamp to visualise the reaction, and it is concentrated
laboratory in terms of biochemical and serological conirmation within the colony, facilitating easier enumeration in the presence of
of suspect colonies. With improved speciicity, the ABC medium other organisms, or when large numbers are present on the plate.
dramatically reduces the need for ‘false positive’ screening, saving
labour and reducing consumable costs.
Beef Extract 5.0 Bile Salts No.3 1.5
Peptone 5.0 X-glucuronide 0.075
Sodium citrate 8.5 Agar 15.0
Agar 12.0
Weigh 36.5 grams of powder, disperse in 1 litre of deionised water and
X-α-Gal 0.08 allow the mixture to soak for 10 minutes. Swirl to mix and sterilise at
121°C for 15 minutes. Cool to 47°C and pour in to Petri dishes. Dry
CHE-β-Gal 0.3 the surface prior to inoculation.
Appearance: Straw, clear gel.
IPTG 0.03 pH: 7.2 ± 0.2
HarlequinTM Chromogenic Media 96

Minimum QC organisms: Escherichia coli This medium is a modiication of Sorbitol MacConkey Agar (SMAC).
WDCM 00013 (blue/green) The addition of the chromogenic substrate BCIG (5-bromo-4-chloro-
3-indoxyl-β-D-glucuronide) improves the speciicty of the medium.
E. coli O157:H7 is typically sorbitol negative and β-glucuronidase
Inoculation: Inoculate 0.5 ml of a 1:10 dilution of the sample and negative producing pale translucent colonies on this medium. The
spread over the entire surface of the plate. Further dilution may be majority of other E. coli strains are β-glucuronidase positive and
necessary if large numbers of E. coli are present, to ensure colonies sorbitol positive (pink/red colonies). A small percentage of E. coli
can be easily counted. are β-glucuronidase positive and sorbitol negative and thus appear
Incubation: 30°C for 4 hours, followed by 18 hours at 44°C. as blue/green colonies on this medium. Consequently this medium
Interpretation: Count all blue/green colonies as presumptive E. coli, can distinguish between non-O157 sorbitol negative E. coli and
calculate the cfu/g in the original material. A simple indole test can be the genuine toxigenic E. coli O157:H7. This reduces the number of
performed by placing one drop of Kovac’s reagent onto a colony and unnecessary conirmation tests that are performed. The medium can be
if positive, a red halo will appear in the medium around the colony. If made more selective by the addition of Ceixime Tellurite supplement
negative, then the halo will be white. X161 to prepare CT-SMAC. Most workers recommend the use of CT-
supplemented medium alongside unsupplemented medium to ensure
*96-97% of E. coli strains positive. A notable exception is E. coli maximum isolation of E. coli O157. This medium can also be useful
0157:H7. for the detection of other VTEC producing E. coli in conjunction with
speciically targetted IMS particles (Captivate™).
References
Dibb, W.L. and Bottolfsen, K.L. (1984). Evaluation of Rosco Typical Formula g/litre
Diagnostic ß-glucuronidase Tablets in the Identiication of Urinary
Isolates of Escherichia coli. Acta Path.Microbiol. Immunol. Scand. Peptone 20.0
Sect. B 92 261-264. Sorbitol 10.0
Hansen, W. and Yourassowsky, E. (1984). Detection of ß-glucuronidase
in Lactose Fermenting Members of the Family Enterobacteriaceae Bile Salts No. 3 1.5
and its Presence in Bacterial Urine Cultures. Sodium Chloride 5.0
J. Clin. Micro.20 (6) 1177-1179.
Robinson, B.J. (1984). Evaluation of a Fluorogenic Assay for BCIG 0.1
Detection of E. coli. App & Env. Microbiol.48 (2) 285-288. Neutral Red 0.03
Perez, J.L., Berrocal, C.I. and Berrocal, L. (1986). Evaluation of a
Commercial ß-glucuronidase Test for the Rapid and Economical Crystal Violet 0.001
Identiication of Escherichia coli. J.App.Bacteriol. 61 541-545. Agar 12.0
Raghubeer, E. and Matches, J.R. (1990). Temperature Range for
Growth of Escherichia coli Serotype 0157:H7 and Selected Coliforms Method for reconstitution
in E. coli Medium. J.Clin. Micro. 28 (4) 803-805. Weigh 48.6 grams of powder and add to 1 litre of de-ionised water.
Bolton, F.J. (1995) Personal Communication Allow to soak for 10 minutes, swirl to mix and sterilise by autoclaving
at 121°C for 15 minutes. Cool to 47°C, add 2 vials of X161 CT
supplement and pour plates. Dry the surface prior to inoculation.
Harlequin™ LB Agar Appearance: Pale red, light violet tinge.
pH: 7.1 ± 0.2
HAL004
Refer to the Biomolecular Section Minimum QC organisms:
Escherichia coli O157:H7 WDCM 00014 (non-toxigenic)
Escherichia coli WDCM00013
Harlequin™ SMAC-BCIG
Inoculation: From O157 Broth LAB 165, surface streak for single
(Sorbitol MacConkey Agar with BCIG) colonies.
Incubation: 37°C aerobically for 18-24 hr. Examine plates for
HAL006 sorbitol negative, β-glucuronide negative colonies. Conirm as
O157:H7 by serology, (commercial kits or antiserum available).
Description
This is a speciic substrate medium for the isolation of Escherichia
coli O157:H7, the primary serovar associated with haemorrhagic Growth Characteristics
colitis (HC) and haemolytic uraemic syndrome (HUS). Pathogenicity colony size shape &
of the organism is linked to the production of verocytotoxins (VT1 organism (mm) surface colour
and VT2), but it should be noted that not all strains of O157 produce
verocytotoxins, and that strains from other serovars can be toxin E. coli O157:H7 2.5 - 4.0 CV.E.G. Translucent
producers (e.g. O26, O103, O111, O113, O145). E. coli O157 has sorbitol -ve
been associated epidemiologically with food poisoning outbreaks β-glucuronide -ve
involving beef burgers and cold cooked meats. E. coli 2.5 - 4.0 CV.E.G. Pink/red or
sorbitol +ve purple centre
β-glucuronide +ve
E. coli 2.5 - 5.0 CV.E.G. Green or translucent
sorbitol -ve with green centre
β-glucuronide +ve
Note: Sorbitol positive toxigenic E. coli O157:H7 have been isolated and
appear as sorbitol positive and β-glucuronide positive on this medium.
References
1) Okrend, A.J.G., Rose, B.E., and Lattuada, C.P. (1990) Use of
5-Bromo-4-Chloro-3-Indoxyl-β-D-Glucuronide in MacConkey
Sorbitol Agar to Aid in the Isolation of Escherichia coli O157:H7
from Ground Beef. J.Food Protection 53 (11) 941-943
97 HarlequinTM Chromogenic Media

Harlequin™ E. coli/Coliform Medium References
1) Baylis, C.L., Patrick, M. (1999). Comparison of a range of
HAL008 Chromogenic media for enumeration of total Coliforms and
Escherichia coli in foods. Leatherhead International Technical Notes.
No.135: 99.
Description
This dual chromogenic substrate medium has been developed for the
simultaneous enumeration of Escherichia coli and coliforms in food
and environmental samples. The different colony types are simple to Harlequin™ mLGA
distinguish allowing rapid counting of both E. coli and coliforms on
a single medium. (Membrane Lactose Glucuronide Agar)
Based upon the formulation of Tryptone Bile Agar LAB072, the
medium has been modiied by the addition of two chromogenic HAL009
substrates, one to detect the ß-glucuronidase enzyme (X-glucuronide)
and another to detect the ß-galactosidase enzyme (magenta-ß-gal). Description
Typical E. coli strains possess both enzymes but only cleave the Traditionally, membrane Lauryl Sulphate Broth (mLSB) has been
X-glucuronide substrate, thereby producing blue-green colonies. used as the standard media for isolating coliforms (including E.
Typical coliforms, however, possess only the ß-galactosidase enzyme coli) from water potentially contaminated with sewage. Harlequin™
and produce rose-pink colonies. membrane Lactose Glucuronide Agar (mLGA) is a modiication of
The colony types are easily distinguishable, even in the presence mLSB aimed at reducing costs by reducing the number of ilters
of other organisms, or when large numbers are observed, making used per test sample and aiding in the recovery and identiication of
simultaneous enumeration of E. coli and coliforms a quick and simple coliforms and E. coli. The medium has been modiied from the mLSB
procedure. formulation by the incorporation of X-glucuronide, sodium pyruvate
N.B. This product is not available for sale in the USA. and agar. X-glucuronide is incorporated to allow for the presumptive
isolation of E. coli, sodium pyruvate aids the recovery of chlorine
Typical Formula g/litre stressed organisms and agar is incorporated to remove the need for
absorbent pads. This medium is recommended for the enumeration
Tryptone 20.0 of coliform bacteria and E. coli by a single membrane iltration
technique in The Microbiology of Drinking Water 2002 (previously
Bile Salts No.3 1.5 Report 71).
X-glucuronide 0.075
Magenta-β-galactoside 0.1
Peptone 39.0
Agar 15.0
Yeast Extract 6.0
Method for reconstitution Lactose 30.0
Allow to soak for 10 minutes, swirl to mix and sterilise by autoclaving Phenol Red 0.2
for 15 minutes at 121°C. Cool to 47°C and mix well before dispensing Sodium Lauryl Sulphate 1.0
Sodium Pyruvate 0.5
Appearance:
X-Glucuronide 0.2
Agar 10.0
Finished medium: clear, straw gel.
pH: 7.2 ± 0.2 Method for reconstitution
Minimum Q.C. organisms: soak for 10 minutes, swirl to mix, then sterilise by autoclaving at
115o C for 10 minutes. Cool to 47°C and mix well before dispensing
Enterobacter aerogenes WDCM 00175 into sterile Petri dishes. Dry the agar surface prior to use.
Staphylococcus aureus WDCM 00034 (Inhibition)
Appearance: Red, clear gel.
Storage of Prepared Medium: Plates - up to 7 days at 2 - 8°C in pH: 7.4 ± 0.2
the dark.
Growth Characteristics Escherichia coli WDCM 00013
colony size shape & Enterobacter aerogenes WDCM 00175
organism (mm) surface colour other Staphylococcus aureus WDCM 00034 (inhibition)
Escherichia coli 1.0 - 2.0 CV.E.G. Blue-Green Storage of Prepared Medium: Plates - up to 7 days at 2-8°C in the
dark.
Enterobacter 1.5 - 2.5 CV.E.G. Rose-Pink
aerogenes Inoculation: E. coli and coliform counts can be performed on
the same sample of water. The volume and dilution of test sample
Pseudomonas 0.5 - 1.0 F.CR.D/ Buff should be chosen so as the number of colonies on the membrane lies
aeruginosa CV.E.G. between 20 and 80. With waters expected to contain low numbers of
coliforms, a sample of 100ml should be iltered. For full methodology
Enterococcus faecalis No growth refer to The Microbiology of Drinking Water 2002 section 4 B - The
enumeration of coliform bacteria and E. coli by a single membrane
Staphylococcus aureus No growth iltration technique.
Incubation: 4 hours at 30 °C followed by 14 hours at 37 °C
Inoculation: Inoculate 0.5 ml of a 1:10 dilution of the sample and
spread over the entire surface of the plate. Further dilution may be Interpretation: Count all green-blue colonies as presumptive E. coli,
necessary if large numbers of E. coli and/or coliforms are present, to and all green-blue and yellow colonies as presumptive coliforms.
ensure colonies can be easily counted.
Incubation: 18 - 24 hours at 37° C
Interpretation: Count all blue-green colonies as presumptive E. coli,
and calculate the cfu/g. Count all rose-pink colonies as presumptive
coliforms, and calculate cfu/g.

Growth Characteristics Method for reconstitution
colony size shape & Weigh 70.5 grams of powder and disperse in 950mL of deionised
organism (mm) surface colour other water. Allow to soak for 10 minutes, swirl to mix and sterilise by
autoclaving for 15 minutes at 121°C. Cool to 48-50°C, and add 2 vials
Escherichia coli* 0.5 - 1.5 CV.E.G. Green Yellow if of reconstituted X072 supplement. Swirl to mix. Add 2 vials of X010
glucuronidase supplement (pre-heated to 48-50°C). Mix well with gentle end-over-
-ve
end mixing and dispense into Petri dishes. Dry the agar surface prior
Lactose 0.5 - 1.5 CV.E.G. Yellow
to use.
fermenters Appearance:
Non-lactose 0.5 - 1.5 CV.E.G. Red Powder: ine, free-lowing, homogeneous, buff
fermenters
Finished medium: opaque, cream-yellow gel
Staphylococcus aureus No growth
pH: 7.2 ± 0.2
(suppressed)
*96-97% of E. coli strains positive. A notable exception is E. coli O157:H7 Minimum Q.C. organisms:
References Escherichia coli WDCM 00013 (Inhibited)
Sartory, D.P. & Howard, L. (1992). A medium detecting
B-glucuronidase for the simultaneous membrane iltration enumeration Storage:
of Escherichia coli and coliforms from drinking water. Letters in Dehydrated culture media: 10-25°C
Applied Microbiology 15, 273-276.
Calabrese, J.P. & Bisssonette, G.K. (1990). Improved membrane
Inoculation: Surface inoculation - streak out to single colonies. This
iltration method incorporating catalase and sodium pyruvate
medium is highly selective and a heavy inoculum can be used.
for detection of chlorine stressed coliform bacteria. Applied and
Environmental Microbiology 56, 3558-3564. Incubation: 37°C aerobically for 48 hours.
Microbiology of Drinking Water 2002 section 4 B - Environment Interpretation
Agency. The enumeration of coliform bacteria and E. coli by a single
membrane iltration technique. colony size shape &
organism (mm) surface colour
Listeria monocytogenes 1-2 Round, Regular Blue to blue-
green, surrounded
Harlequin™ Listeria Chromogenic by opaque halo
Agar (ISO) Listeria spp. 1-2 Round, Regular Blue to blue-

green, without
HAL010 opaque halo
Description Isolates presumptively identiied as Listeria spp. and Listeria

monocytogenes must be subjected to further biochemical tests
Listeria Chromogenic Agar (according to the formulation of Ottaviani to conirm their identity. Some strains of Listeria ivanovii may
and Agosti) is a selective medium for the isolation and presumptive demonstrate lecithinase activity.
identiication of Listeria monocytogenes from foodstuffs and related
materials as described in ISO 11290-1:1997.
References
Lithium chloride in the base medium and supplementary ISO 11290-1:1997 Microbiology of food and animal feeding stuffs -
antimicrobial compounds Ceftazidime, Polymyxin, Nalidixic acid Horizontal method for the detection of Listeria monocytogenes - Part
and Cycloheximide provide the medium’s selectivity. Chromogenic 1: Detection method. Incorporating Amendment 1.
activity is as a result of a chromogenic substrate for the detection of
the β-glucosidase enzyme, common to all Listeria spp. and to a few
strains of Enterococci and Bacilli.
The speciic differential activity of this agar is obtained with a
proprietary lecithin substrate for the detection of the phospholipase
enzyme that will only be present in the L. monocytogenes colonies
growing on this media. This enzyme activity will result in a halo of
precipitation surrounding the target colonies.
With the combination of both the chromogenic and phospholipase
enzyme reactions, it is possible to differentiate Listeria monocytogenes
(blue colonies surrounded by an opaque halo) from other Listeria spp
(blue colonies without an opaque halo).

Meat Peptone 18.0
Tryptone 6.0
Yeast extract 10.0
Sodium chloride 5.0
Disodium hydrogen orthophosphate anhydrous 2.5
Sodium pyruvate 2.0
Glucose 2.0
Glycerophosphate 1.0
5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside 0.05
Agar 13.5

Harlequin™ CSIM (ISO) References
Harlequin™ Cronobacter sakazakii Isolation Medium (ISO) disease in infants”. Emerging Infect Dis 12 (8): 1185–9.
HAL012 Caubilla-Barron J & Forsythe S (2007). “Dry stress and survival time
of Enterobacter sakazakii and other Enterobacteriaceae in dehydrated
Description infant formula”. Journal Food Protection 13: 467-472.
Cronobacter sakazakii (formerly Enterobacter sakazakii) is a member “Enterobacter sakazakii infections associated with the use of powdered
of the Enterobacteriaceae family and has been associated with serious infant formula--Tennessee, 2001” (2002). MMWR Morb Mortal Wkly
outbreak infections in neonates (premature infants) which have been Rep 51 (14): 297–300.
fed on infant formula milk. Although rarely causing infections in Farmer JJ III, Asbury MA, Hickman FW, Brenner DJ, the
immunocompetent adults, C. sakazakii has been implicated in sepsis, Enterobacteriaceae Study Group (USA) (1980). “Enterobacter
meningitis and necrotising enterocolitis with a high death rate in sakazakii: a new species of “Enterobacteriaceae” isolated from clinical
neonates. This opportunistic pathogen is common in the environment specimens”. Int J Syst Bacteriol 30: 569–84.
ISO/TS 22964:2006(E) Milk and milk products – Detection of
post pasteurisation contamination and survival in spray dried milk
Enterobacter sakazakii.
products.
C. sakazakii appears to constitutively express high levels of Iversen C, Lehner A, Mullane N, et al (2007). “The taxonomy of
α-glucosidase. This enzyme hydrolyses the chromogenic substrate Enterobacter sakazakii: proposal of a new genus Cronobacter gen. nov.
5-bromo-4-chloro-3-indolyl-α-D-glucopyranoside present in the and descriptions of Cronobacter sakazakii comb. nov. Cronobacter
medium, producing green to blue-green coloured colonies. Other sakazakii subsp. sakazakii, comb. nov., Cronobacter sakazakii subsp.
Enterobacteriaceae such as E. coli do not express strong α-glucosidase malonaticus subsp. nov., Cronobacter turicensis sp. nov., Cronobacter
activity and appear colourless or purple due to the uptake of crystal muytjensii sp. nov., Cronobacter dublinensis sp. nov. and Cronobacter
violet. genomospecies 1”. BMC Evol Biol 7: 64.
The combination of sodium desoxycholate, crystal violet and elevated Iversen C, Mullane N, Barbara McCardell, et al (2008). “Cronobacter
incubation temperature produce a very selective and speciic medium. gen. nov., a new genus to accommodate the biogroups of Enterobacter
Non-Enterobacteriaceae may appear colourless or violet coloured sakazakii, and proposal of Cronobacter sakazakii gen. nov. comb.
(due to their inability to hydrolyse the chromogenic substrate) or are nov., C. malonaticus sp. nov., C. turicensis sp. nov., C. muytjensii sp.
inhibited by the selective components and incubation temperature. nov., C. dublinensis sp. nov., Cronobacter genomospecies 1, and of
This media formulation is currently recommended as part of the three subspecies, C. dublinensis sp. nov. subsp. dublinensis subsp.
isolation protocol under ISO/TS 22964:2006(E) for the isolation of nov., C. dublinensis sp. nov. subsp. lausannensis subsp. nov., and C.
Enterobacter sakazakii from milk and milk products. dublinensis sp. nov. subsp. lactaridi subsp. nov.”. IJSEM.
Lai KK (2001). “Enterobacter sakazakii infections among neonates,
Typical Formula g/litre infants, children, and adults. Case reports and a review of the
literature”. Medicine (Baltimore) 80 (2): 113–22.
Pancreatic peptone of casein 7.0
Yeast extract 3.0
Sodium chloride 5.0 Harlequin™ Cronobacter sakazakii
Sodium desoxycholate 0.6 Agar – DFI Formulation
(Druggan, Forsythe & Iversen)
5-bromo-4-chloro-3-indolyl-α-D-Glucoside 0.15
Crystal violet 0.002 HAL013
Agar 14.0
Description
Method for reconstitution Cronobacter sakazakii (formerly Enterobacter sakazakii) is a member
of the Enterobacteriaceae family and has been associated with serious
Weigh 29.75 grams of powder and disperse in 1 litre of deionised outbreak infections in neonates (premature infants) which have been
water. Allow to soak for 10 minutes, swirl to mix and sterilise by fed on infant formula milk. Although rarely causing infections in
autoclaving for 15 minutes at 121°C. Cool to 47°C and mix well immunocompetent adults, C. sakazakii has been implicated in sepsis,
before dispensing into Petri dishes. Dry the agar surface prior to use. meningitis and necrotising enterocolitis with a high death rate in
Appearance: neonates. This opportunistic pathogen is common in the environment
Powder: ine, free-lowing, homogeneous, buff post pasteurisation contamination and survival in spray dried milk
Finished medium: clear, purple gel products.
Based on the formulation described by Druggan, Forsythe and Iversen,
pH: 7.0 ± 0.2 Harlequin™ Cronobacter sakazakii Agar is a medium on which
Cronobacter sakazakii appears to constitutively express high levels
Minimum Q.C. organisms: of α-glucosidase. This enzyme hydrolyses the chromogenic substrate
Cronobacter sakazakii ATCC 12868 5-bromo-4-chloro-3-indolyl-α-D-glucopyranoside present in the
Enterobacter aerogenes WDCM 00175 medium, producing green coloured colonies. Other Enterobacteriaceae
Bacillus cereus WDCM 00001 such as E. coli do not express strong α-glucosidase activity and
Staphylococcus aureus WDCM 00034 appear colourless. Hydrogen sulphide producing organisms, such
as Salmonella, and Proteus spp. appear grey, brown or black on this
Hazard classiication: NR – Not regulated formulation due to the production of precipitated ferrous sulphate,
which results from the hydrogen sulphide produced by these organisms
Storage: interacting with ferric ions in the medium. This reaction prevents the
Dehydrated culture media: 10-25°C away from direct sunlight. weakly α-glucosidase positive Proteus vulgaris from appearing as
Prepared media: 7 days at 2-8°C in the dark. green on the medium.
Selectivity is achieved from the inclusion of sodium desoxycholate
Inoculation: Following selective enrichment in Modiied Lauryl
which serves to inhibit the growth of most Gram-positive organisms.
Sulphate Tryptose Broth Vancomycin Medium, streak onto HAL012
Harlequin™ Cronobacter sakazakii Isolation Medium (ISO).
Interpretation: After incubation the plate should be assessed for
typical colonies of C. sakazakii. Typical colonies are 1-3mm and are
green to blue-green.

Tryptone 15.0
Soya peptone 5.0
Sodium chloride 5.0
X-α-D-glucopyranoside 0.1
Agar 15.0

Allow to soak for 10 minutes, swirl to mix and sterilise by autoclaving
for 15 minutes at 121°C. Cool to 47°C and mix well before dispensing
Appearance:
Finished medium: clear, straw-coloured gel
pH: 7.3 ± 0.2
Storage:
Incubation: Incubate plates at 37+1°C for 24 hours.
Interpretation: Cronobacter sakazakii appear as green or pale
green with a green ‘bullseye’ centre and 1-2.5mm in size. Other
organisms generally appear black (if hydrogen sulphide producers)
or colourless.
References
disease in infants”. Emerging Infect Dis 12 (8): 1185–9.
Caubilla-Barron J & Forsythe S (2007). “Dry stress and survival time
of Enterobacter sakazakii and other Enterobacteriaceae in dehydrated
infant formula”. Journal Food Protection 13: 467-472.
“Enterobacter sakazakii infections associated with the use of powdered
infant formula--Tennessee, 2001” (2002). MMWR Morb Mortal Wkly
Rep 51 (14): 297–300.
Farmer JJ III, Asbury MA, Hickman FW, Brenner DJ, the
Enterobacteriaceae Study Group (USA) (1980). “Enterobacter
sakazakii: a new species of “Enterobacteriaceae” isolated from
clinical specimens”. Int J Syst Bacteriol 30: 569–84.
Iversen C, Druggan P & Forsythe S (2004). “A selective differential
medium for Enterobacter sakazakii, a preliminary study”. International
Journal of Food Microbiology 96 (2): 133-139.
Iversen C, Lehner A, Mullane N, et al (2007). “The taxonomy of
Enterobacter sakazakii: proposal of a new genus Cronobacter gen. nov.
and descriptions of Cronobacter sakazakii comb. nov. Cronobacter
sakazakii subsp. sakazakii, comb. nov., Cronobacter sakazakii subsp.
malonaticus subsp. nov., Cronobacter turicensis sp. nov., Cronobacter
muytjensii sp. nov., Cronobacter dublinensis sp. nov. and Cronobacter
genomospecies 1”. BMC Evol Biol 7: 64.
Iversen C, Mullane N, Barbara McCardell, et al (2008). “Cronobacter
gen. nov., a new genus to accommodate the biogroups of Enterobacter
sakazakii, and proposal of Cronobacter sakazakii gen. nov. comb.
nov., C. malonaticus sp. nov., C. turicensis sp. nov., C. muytjensii sp.
nov., C. dublinensis sp. nov., Cronobacter genomospecies 1, and of
three subspecies, C. dublinensis sp. nov. subsp. dublinensis subsp.
nov., C. dublinensis sp. nov. subsp. lausannensis subsp. nov., and C.
dublinensis sp. nov. subsp. lactaridi subsp. nov.”. IJSEM.
Lai KK (2001). “Enterobacter sakazakii infections among neonates,
infants, children, and adults. Case reports and a review of the
literature”. Medicine (Baltimore) 80 (2): 113–22.

3. Harmonised Pancreatic digest of casein 15.0
Pharmacopoeia (USP/EP/JP) Papaic digest of soya bean 5.0
Sodium chloride 5.0
Buffered Sodium Chloride-Peptone Agar 15.0
Solution pH 7.0 (USP/EP/JP) Method for reconstitution
HP017 Disperse 40 grams of powder in 1 litre of deionised water. Allow
Description 121oC for 15 minutes. Cool to 47°C and mix well before dispensing
A diluent recommended by the Harmonised European Pharmacopoeia into sterile Petri dishes. Dry the agar surface prior to use.
for the microbiological examination of non-sterile pharmaceutical
products. The medium is also used to prepare and dilute test suspensions Appearance:
of the microbiological strains described in the Harmonised European Powder: ine, free-lowing, homogeneous, buff
Pharmacopoeia. Conforms to USP/EP/JP performance speciication.
The low level of peptone lessens the physiological shock experienced Finished medium: straw gel, clear to slight haze
by micro-organisms when suspended in a diluent. The dual phosphate pH: 7.3 ± 0.2
components create a buffered environment and sodium chloride
maintains the osmotic balance. Minimum Q.C. organisms:
Typical Formula g/litre Bacillus subtilis ATCC 6633
Potasium dihydrogen phosphate 3.6 Pseudomonas aeruginosa ATCC 9027
Disodium hydrogen phosphate dihydrate 7.2 Aspergillus brasiliensis ATCC 16404
Sodium chloride 4.3
Peptone (meat or casein) 1.0
Storage:
Method for reconstitution Dehydrated culture media: 10-25°C away from direct sunlight.
Disperse 16.1 grams of powder in 1 litre of deionised water. Allow to Prepared media: 7 days at 2-8°C in the dark.
soak for 10 minutes then swirl to mix. Distribute into suitable vessels Use: The media is used for the preparation and maintenance of test
and sterilise at 121°C for 15 minutes. strains used in the growth promotion test, suitability of the counting
methods and negative controls as described in the Harmonised
Appearance: European Pharmacopoeia. It is also a supporting plating medium for
Powder: ine, free-lowing, homogeneous, white/buff various protocols described in the microbial enumeration test section
of the Harmonised European Pharmacopoeia.
Finished medium: colourless, clear to slight haze
pH: 7.0 ± 0.2 Interpretation:
Cultural response is speciic to the test micro-organism. Refer
Minimum Q.C. organisms: to speciic guidelines as deined in the Harmonised European
Staphylococcus aureus ATCC 6538 Pharmacopoeia.
Bacillus subtilis ATCC 6633 References
Clostridium sporogenes ATCC 19404 European Pharmacopoeia 8th Edition
Casein Soya Bean Digest Broth
(USP/EP/JP)
Storage:
HP002
Prepared media: 7 days at 2-8°C in the dark. Description
Use: Test suspensions are made by inoculating the diluent with A medium recommended by the Harmonised European Pharmacopoeia
cultivated micro-organisms. These suspensions must be used within for the cultivation of a wide range of micro-organisms. Conforms to
2 hours if held at room temperature or 24 hours if stored at 2-8°C. USP/EP/JP performance speciication. The medium is also commonly
No marked increase or decrease in original colony forming unit count referred to as tryptone (or tryptic) soy broth and abbreviated to
should be observed. Refer to the appropriate test protocol for further TSB. Enzymatic digests of casein and soya bean act as a source of
guidance. nitrogen and glucose is a carbon source in the form of a fermentable
carbohydrate. Sodium chloride maintains the osmotic balance and
Interpretation: dipotassium hydrogen phosphate acts as a buffering agent.
Refer to speciic guidelines as deined in the Harmonised European
Pharmacopoeia. Typical Formula g/litre
References Pancreatic digest of casein 17.0
European Pharmacopoeia 8th Edition Pancreatic digest of soya bean 3.0
Sodium chloride 5.0
Casein Soya Bean Digest Agar Dipotasium hydrogen phosphate 2.5
(USP/EP/JP) Glucose monohydrate 2.5
HP016 Method for reconstitution
Description Disperse 30 grams of powder in 1 litre of deionised water. Allow to
A medium recommended by the Harmonised European Pharmacopoeia soak for 10 minutes then swirl to mix. Distribute into suitable vessels
for the cultivation of a wide range of micro-organisms. Conforms to and sterilise at 121°C for 15 minutes.
USP/EP/JP performance speciication. The medium is also commonly
referred to as tryptone (or tryptic) soy agar and abbreviated to TSA. Appearance:
Enzymatic digests of casein and soya bean act as a source of nitrogen Powder: ine, free-lowing, homogeneous, buff
and amino acid and sodium chloride maintains the osmotic balance.
Finished medium: Straw, clear to slight haze
pH: 7.3 ± 0.2
Harmonised Pharmacopoeia 102

Minimum Q.C. organisms: Minimum Q.C. organisms:
Staphylococcus aureus ATCC 6538 Pseudomonas aeruginosa ATCC 9027
Bacillus subtilis ATCC 6633 Escherichia coli ATCC 8739
Storage:
Hazard classiication: NR – Not regulated Dehydrated culture media: 10-25°C away from direct sunlight.
Storage: Prepared media: 7 days at 2-8°C in the dark.
Dehydrated culture media: 10-25°C away from direct sunlight. Inoculation: According to the European Pharmacopoeia 8.0
Prepared media: 7 days at 2-8°C in the dark. subculture is performed from enrichment in casein soya bean digest
broth onto the agar surface.
Use:
Incubation:
According to the sterility protocol deined in the Harmonised
European Pharmacopoeia the samples are incubated in portions of the Incubate at 30-35°C for 18-72 hours.
medium at 20-25°C for 14 days. No growth of micro-organisms is
required for a sterility pass. Growth Characteristics
According to the growth promotion test deined in the Harmonised Shape &
European Pharmacopoeia Bacillus subtilis, Candida albicans and Organism surface Colour Other
Aspergillus brasiliensis are inoculated (with not more than 100 CFU) P. aeruginosa CV.E.G. Yellow/green Fluorescent under
and incubated for not more than 3 days in the case of bacteria, and not UV light
more than 5 days in the case of fungi. The media is suitable if a clearly
visible growth of the micro-organisms occurs.
The media is also used for the preparation of the test micro-organisms References
Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus European Pharmacopoeia 8th Edition
subtilis. Refer to the speciic strain and preparation for incubation time
and temperature.
The media is also used for sample preparation and pre-incubation for
the test for speciied micro-organisms. Refer to individual guidelines
Columbia Agar
for each testing protocol.
(USP/EP/JP)
Interpretation:
HP012
Growth is indicated by turbidity. Refer to speciic guidelines as
Description
deined in the Harmonised European Pharmacopoeia.
A medium recommended by the Harmonised European
References Pharmacopoeia for isolation and identiication of Clostridia from non-
European Pharmacopoeia 8th Edition sterile pharmaceutical products. Conforms to USP/EP/JP performance
speciication. Originally described as a general purpose nutritious
agar base by Ellner et al. at Columbia University that can be enriched
by the addition of sterile blood. The peptone mixture and yeast extract
Cetrimide Agar provides a source of nitrogen, essential vitamins and amino acids.
The starch provides a carbon source and sodium chloride maintains
(USP/EP/JP) osmotic balance. The Harmonised European Pharmacopoeia states
that where necessary, gentamicin sulfate at a concentration of 20mg/L
HP010 can be added after sterilisation to reduce the growth of non-target
Description organisms. According to the Harmonised European Pharmacopoeia,
Reinforced Medium for Clostridia is used as a selective enrichment
A medium recommended by the Harmonised European Pharmacopoeia broth, with subculture performed onto Columbia Agar.
for the isolation and identiication of Pseudomonas aeruginosa, in non-
sterile pharmaceutical samples. Conforms to USP/EP/JP performance
speciication. Gelatin is a source of nitrogen whilst glycerol acts as a Typical Formula g/litre
carbon source. Cetrimide is a quarternary ammonium compound that
inhibits the growth of a wide range of Gram-positive and some Gram- Pancreatic digest of casein 10.0
negative micro-organisms. Magnesium chloride and dipotassium Meat peptic digest 5.0
sulphate improve the production of pyoverdin and pyocyanin pigments
that combine to give Pseudomonas aeruginosa characteristic green Heart pancreatic digest 3.0
colonies. According to the Harmonised European Pharmacopoeia,
subculture is carried out onto the medium after enrichment in Casein Yeast extract 5.0
Soya Bean Digest Broth. Maize starch 1.0
Typical Formula g/litre Sodium chloride 5.0

Pancreatic digest of gelatin 20.0 Agar 15.0
Magnesium chloride 1.4 Method for reconstitution
Dipotassium sulphate 10.0 Disperse 44 grams of powder in 1 litre of deionised water. Allow to
soak for 10 minutes, swirl to mix then sterilise by autoclaving at 121oC
Cetrimide 0.3 for 15 minutes. Cool to 45-50°C and mix well before dispensing into
sterile Petri dishes. Dry the agar surface prior use.
Agar 13.6
Appearance:
Method for reconstitution Powder: ine, free-lowing, homogeneous, buff
Disperse 45.3 grams of powder in 1 litre of deionised water. Allow to
soak for 10 minutes, add 10mL of glycerol, swirl to mix and boil to Finished medium: straw clear gel
dissolve. Sterilise by autoclaving atg121oC for 15 minutes. Cool to pH: 7.3 ± 0.2
47°C and mix well before dispensing into sterile Petri dishes. Dry the
agar surface prior to use. Minimum Q.C. organisms:
Appearance:
Finished medium: translucent, pale straw gel
pH: 7.2 ± 0.2
103 Harmonised Pharmacopoeia

Hazard classiication: NR – Not regulated Hazard classiication: Xi - Irritant
Storage: Storage:
Dehydrated culture media: 10-25°C away from direct sunlight. Dehydrated culture media: 10-25°C away from direct sunlight.
Prepared media: 7 days at 2-8°C in the dark. Prepared media: 7 days at 2-8°C in the dark.
Inoculation: According to the European Pharmacopoeia 8.0 Inoculation: According to the Harmonised European Pharmacopoeia,
subculture is performed from enrichment in Reinforced Medium for a volume corresponding to 1g of the sample prepared in casein soya
Clostridia onto the agar surface. bean digest broth pre enrichment is transferred to Enterobacteria
Enrichment Broth-Mossel.
Incubation:
Incubation:
Growth Characteristics Growth Characteristics

Shape & Expected result in Expected result on
Organism surface Colour Other Organism EE Broth VRBGA subculture
C. sporogenes CV.E.G. Buff/cream The occurrences of E. coli Turbid growth Growth, typical colonies
anaerobic rods (with
or without endospores)
P. aeruginosa Turbid growth Growth, typical colonies
giving a negative
catalase reaction
indicates the presence S. aureus No visible growth No growth
of Clostridia
References
References
Ellner, P.D., Stoessel, C.J., Drakeford, E. and Vasi, F. (1966). A new
culture medium for medical bacteriology. Amer J. Clin Pathol. 45.
502-504. Fluid Thioglycollate Medium
European Pharmacopoeia 8th Edition (USP/EP/JP)
HP001
Description
Enterobacteria Enrichment Broth - A medium recommended by the Harmonised European
Mossel (USP/EP/JP) Pharmacopoeia for sterility testing. Conforms to USP/EP/JP
performance speciication. Casein and yeast extract provide a source
HP003 of nitrogen, essential vitamins and amino acids. The glucose provides
a carbon source and sodium chloride maintains osmotic balance.
Description L-Cystine and sodium thioglycollate act as reducing agents to create
A medium recommended by the Harmonised European Pharmacopoeia an anaerobic environment and maintain a low Eh. This is aided by
for the selective enrichment of bile-tolerant Gram-negative bacteria the low level of agar which reduces the oxygen permeability through
from non-sterile pharmaceutical samples. Conforms to USP/EP/JP the medium. Resazurin is an oxidation indicator which turns from
performance speciication. Gelatin peptone provides nitrogen, while colourless to red/pink when oxidised. Sodium thioglycollate also
glucose is a source of fermentable carbohydrate, facilitating eficient serves to inactivate mercurial compounds. If after sterilisation more
growth of most Enterobacteria, including non-lactose fermenting than the upper one third of the medium has become red/pink it may be
organisms, such as Salmonella species. Potassium dihydrogen restored once by heating in a water bath or in free-lowing steam until
phosphate and disodium hydrogen phosphate act as a buffer system to the colour disappears. Ensure the media is cooled quickly and prevent
encourage early growth and prevent auto sterilisation. Brilliant green the introduction of non-sterile air into the containers.
and ox bile are selective agents, inhibiting non-target competitive
organisms. According to the Harmonised European Pharmacopoeia,
Enterobacteria Enrichment (EE) Broth - Mossel is used as a selective Typical Formula g/litre
enrichment broth, with subculture performed onto Violet Red Bile L-Cystine 0.5
Glucose Agar (VRBGA).
Agar 0.75
Typical Formula g/litre Sodium chloride 2.5
Pancreatic digest of casein 10.0 Glucose anhydrous 5.0
Glucose monohydrate 5.0 Yeast extract 2.5
Dehydrate ox bile 20.0 Pancreatic digest of casein 15.0
Potassium dihydrogen phosphate 2.0 Sodium thioglycollate 0.3
Disodium hydrogen phosphate dehydrate 8.0 Resazurin 0.001
Brilliant green 5.0 Method for reconstitution

Method for reconstitution soak for 10 minutes, swirl to mix and bring to the boil. Distribute into
Disperse 45 grams of powder in 1 litre of deionised water. Allow to suitable vessels and sterilise at 121°C for 15 minutes.
soak for 10 minutes, swirl to mix and distribute into bottles or tubes.
Appearance:
Heat at 100oC for 30 minutes in a boiling water bath or lowing steam
and cool immediately. DO NOT AUTOCLAVE. Powder: ine, free-lowing, homogeneous, buff
Appearance: Finished medium: straw, clear to slight haze with red/pink layer at the
Powder: ine, free-lowing, homogeneous, buff to light green top of the medium
Finished medium: green, clear to slight haze pH: 7.1 ± 0.2
pH: 7.2 ± 0.2
Minimum Q.C. organisms: Staphylococcus aureus ATCC 6538
Escherichia coli ATCC 8739 Bacillus subtilis ATCC 6633
Pseudomonas aeruginosa ATCC 9027 Pseudomonas aeruginosa ATCC 9027
Staphylococcus aureus ATCC 6538 Candida albicans ATCC 10231

Hazard classiication: NR – Not regulated Hazard classiication: NR – Not regulated
Storage: Storage:
Dehydrated culture media: 10-25°C away from direct sunlight. Dehydrated culture media: 10-25°C away from direct sunlight.
Prepared media: 7 days at 2-8°C in the dark. Prepared media: 7 days at 2-8°C in the dark.
Use: According to the sterility protocol deined in the Harmonised subculture is performed from enrichment in MacConkey Broth onto
European Pharmacopoeia the samples are incubated in portions of the the agar surface.
medium at 30-35°C for 14 days. No growth of micro-organisms is Incubation:
required for a sterility pass.
According to the growth promotion test deined in the Harmonised
European Pharmacopeia, Clostridium sporogenes, Pseudomonas
aeruginosa and Staphylococcus aureus are inoculated (with not more
than 100 CFU) and incubated for not more than 3 days. The media Growth Characteristics
is suitable if a clearly visible growth of the micro-organisms occurs.
Organism Colour Other
Interpretation: Lactose fermenting bile Pink Red precipitation
tolerant bacteria
Growth is indicated by turbidity, refer to speciic guidelines as deined
in the Harmonised European Pharmacopoeia. Non-lactose fermenting Colourless No precipitate
bile tolerant bacteria
References
References
MacConkey Agar
MacConkey Broth
(USP/EP/JP)
HP006 (USP/EP/JP)
Description HP005
A medium recommended by the Harmonised European Description
Pharmacopoeia for isolation and identiication of Escherichia coli A medium recommended by the Harmonised European Pharmacopoeia
from non-sterile pharmaceutical products. Conforms to USP/EP/JP for the selective enrichment of Escherichia coli from non-sterile
performance speciication. Gelatin serves as source of carbon and pharmaceutical samples. Conforms to USP/EP/JP performance
nitrogen. Lactose is a fermentable carbohydrate and sodium chloride speciication. Gelatin peptone provides a source of nitrogen, while
maintains the osmotic balance. Bile salts and crystal violet act as lactose is a fermentable carbohydrate. Ox bile acts a selective agent
selective agents inhibiting many Gram-positive bacteria. Escherichia inhibiting most Gram-positive organisms and bromocresol purple
coli can ferment lactose to produce acid which results in a pH drop. acts as a pH indicator. A colour change from purple to yellow
This is indicated by neutral red resulting in pink colonies. Enough indicates growth of a bile-tolerant, lactose-fermenting organism
acid production will cause the precipitation of bile salts resulting in such as Escherichia coli. According to the Harmonised European
a bile precipitate or halo around lactose fermenting bacteria. Non- Pharmacopoeia, MacConkey Broth is used as a selective enrichment
lactose fermenting bacteria such as Salmonella spp. grow but remain broth, with subculture performed onto MacConkey Agar.
colourless with no bile precipitate. According to the Harmonised
European Pharmacopoeia, MacConkey Broth is used as a selective Typical Formula g/litre
enrichment broth, with subculture performed onto MacConkey Agar.
Pancreatic digest of gelatin 20.0
Typical Formula g/litre Lactose monohydrate 10.0
Pancreatic digest of gelatin 17.0 Dehydrate ox bile 5.0
Peptones (meat and casein) 3.0 Bromocresol purple 0.01
Lactose monohydrate 10.0 Method for reconstitution
Sodium chloride 5.0 Weigh 35 grams of powder, and add to 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix. Distribute into bottles or
Bile salts 1.5 tubes, and sterilise at 121°C for 30 minutes
Agar 13.5 Appearance:
Neutral red 0.03 Powder: ine, free-lowing, homogeneous, buff to slight yellow
Crystal violet 0.001 Finished medium: purple, clear to slight haze

pH: 7.3 ± 0.2
Disperse 50 grams of powder in 1 litre of deionised water. Allow to Minimum Q.C. organisms:
soak for 10 minutes then swirl to mix. Heat to boiling. Sterilise by Escherichia coli ATCC 8739
autoclaving at 121oC for 15 minutes. Cool to 47°C and mix well Staphylococcus aureus ATCC 6538
before dispensing into sterile Petri dishes. Dry the agar surface prior
to use. Hazard classiication: Xi - Irritant
Appearance:
Storage:
Powder: ine, free-lowing, homogeneous, buff to slight red/purple
Finished medium: red/purple, clear gel Prepared media: 7 days at 2-8°C in the dark.
pH: 7.1 ± 0.2
1mL of casein soya bean digest broth pre enrichment is transferred to
Minimum Q.C. organisms: 100mL of MacConkey broth.
Incubation:
Incubate at 42-44°C for 24-48 hours

Growth Characteristics Growth Characteristics
Organism Acid Gas Shape &
Bile-tolerant lactose fermenters Positive Positive Organism surface Colour Other
(eg Escherichia coli) Staphylococcus aureus CV.E.G Yellow Yellow/white
colonies surrounded
Bile-tolerant non-lactose fermenters Negative Negative by a yellow zone
(eg Salmonella typhimurium)
References
References European Pharmacopoeia 8th Edition
Mannitol Salt Agar Potato Dextrose Agar

(USP/EP/JP) (USP/EP/JP)
HP009 HP015
Description
Description
A medium recommended by the Harmonised European Pharmacopoeia
A medium recommended by the Harmonised European Pharmacopoeia for the cultivation of fungi and speciically for the preparation of
for isolation and identiication of Staphylococcus
HP009 aureus from the Aspergillus brasiliensis test strain. Conforms to USP/EP/JP
non-sterile pharmaceutical products. Conforms to USP/EP/JP performance speciication. The medium is commonly abbreviated to
performance speciication. The peptone and beef extract provide a PDA. The extract from potato and dextrose provide a nutritionally
source of nitrogen, essential vitamins and amino acids. The mannitol rich base that encourages mould sporulation and pigment production.
is a fermentable carbohydrate and the high level of sodium chloride
provides a selective environment favourable to Staphylococcus
aureus. Phenol red acts as a pH indicator changing from red to yellow Typical Formula g/litre
when acid is produced by the fermentation of mannitol. The majority Potato extract 4.0*
of S. aureus ferment mannitol producing yellow colonies, occasional
strains of coagulase-negative Staphylococci may also ferment *(equivalent to 200g of Infusion from potatoes)
mannitol. It is necessary to conirm the identity of presumptive S.
aureus colonies by other means e.g. coagulase, protein A, DN’ase, Dextrose 20.0
thermonuclease or latex agglutination. According to the Harmonised Agar 15.0
European Pharmacopoeia, subculture is carried out onto the medium
after enrichment in Casein Soya Bean Digest Broth. Method for reconstitution
Disperse 39 grams of powder in 1 litre of deionised water. Allow
Typical Formula g/litre to soak for 10 minutes, swirl to mix then sterilise by autoclaving at
121oC for 15 minutes. Cool to 47°C and mix well before dispensing
Pancreatic digest of casein 5.0 into sterile Petri dishes. Dry the agar surface prior to use.
Peptic digest of animal tissue 5.0 Appearance:
Beef extract 1.0 Powder: ine, free-lowing, homogeneous, buff
D-Mannitol 10.0 Finished medium: straw gel, clear to slight haze
Sodium chloride 75.0 pH: 5.6 ± 0.2
Agar 15.0 Minimum Q.C. organisms:

Phenol red 0.025 Aspergillus brasiliensis ATCC 16404
Hazard classiication: NR - Not Regulated
soak for 10 minutes, swirl to mix and bring to the boil, then sterilise Storage:
by autoclaving at 121oC for 15 minutes. Cool to 47°C and mix well Dehydrated culture media: 10-25°C away from direct sunlight.
before dispensing into sterile Petri dishes. Dry the agar surface prior
to use. Prepared media: 7 days at 2-8°C in the dark.
Appearance: Use: The media is used for the preparation and maintenance of
fungal test strains used in the growth promotion test, suitability of
Powder: ine, free-lowing, homogeneous, buff to slight red
the counting methods and negative controls as described in the
Finished medium: red, clear gel Harmonised European Pharmacopoeia.
pH: 7.4 ± 0.2 Interpretation:
Cultural response is speciic to the test micro-organism, refer to speciic
Minimum Q.C. organisms: guidelines as deined in the Harmonised European pharmacopoeia.
Staphylococcus aureus ATCC 6538 References
Hazard classiication: NR - Not Regulated
Storage:
subculture is performed from enrichment in casein soya bean digest
broth onto the agar surface.
Incubation:

Rappaport Vassiliadis Salmonella Reinforced Medium for Clostridia
Enrichment Broth (USP/EP/JP) (USP/EP/JP)
HP007 HP011
A medium recommended by the Harmonised European A medium recommended by the Harmonised European
Pharmacopoeia for the selective enrichment of Salmonella from Pharmacopoeia for the selective enrichment of Clostridia from non-
non-sterile pharmaceutical samples. Conforms to USP/EP/JP sterile pharmaceutical samples. Conforms to USP/EP/JP performance
performance speciication. Malachite green and magnesium chloride speciication. The medium is also commonly referred to as Reinforced
act as selective agents, combined with high osmotic pressure and Clostridial Medium and abbreviated to RCM. Peptone, beef and yeast
low pH to effectively inhibit non-target competitive organisms. Soya extract provide a source of nitrogen, essential vitamins and amino acids.
peptone provides a source of nitrogen and potassium phosphate Starch aids the detoxiication of harmful metabolites and glucose is a
acts as a buffer. Some Salmonella (S. typhi and S. choleraesuis) fermentable carbohydrate. Sodium chloride provides osmotic balance
are known to be sensitive to Malachite green and as such may fail and sodium acetate acts as a buffer. L-Cysteine act as reducing agents
to grow. If these organisms are suspected an alternative selective to create an anaerobic environment and maintain a low Eh. This is
enrichment broth, (e.g. LAB202 Mueller-Kauffmann Tetrathionate aided by the low level of agar which reduces the oxygen permeability
novobiocin Broth - MKTTn) should be used in parallel. This through the medium. According to the Harmonised European
formulation is hygroscopic and will produce a slight exothermic Pharmacopoeia, Reinforced Medium for Clostridia is used as a selective
reaction when mixed with water. According to the Harmonised enrichment broth, with subculture performed onto Columbia Agar.
European Pharmacopoeia, Rappaport Vassiliadis Salmonella
Enrichment Broth is used as a selective enrichment broth, with Typical Formula g/litre
subculture performed onto Xylose Lysine Deoxycholate (XLD) agar.
Beef extract 10.0
Typical Formula g/litre Peptone 10.0
Soya peptone 4.5 Yeast extract 3.0
Magnesium chloride anhydrous * 13.58 Soluble starch 1.0
*equivalent to 29.0g of Magnesium Chloride Hexahydrate Glucose monohydrate 5.0
Sodium chloride 8.0 Cysteine hydrochloride 0.5
Dipotassium phosphate 0.4 Sodium chloride 5.0
Potassium dihydrogen phosphate 0.6 Sodium acetate 3.0
Malachite green 0.036 Agar 0.5

Weigh 27.14 grams of powder, and add to 1 litre of deionised water. Disperse 38 grams of powder in 1 litre of deionised water. Allow to
Swirl to dissolve. Distribute into bottles or tubes, and sterilise at soak for 10 minutes, swirl to mix and bring to the boil. Distribute into
115°C for 15 minutes. suitable vessels and sterilise at 121°C for 15 minutes.
Appearance: Appearance:
Powder: ine, free-lowing, homogeneous, buff to light green Powder: ine, free-lowing, homogeneous, buff
Finished medium: blue, clear to slight haze Finished medium: straw, clear to slight haze
pH: 5.2 ± 0.2 pH: 6.8 ± 0.2
Minimum Q.C. organisms: Minimum Q.C. organisms:

Salmonella enterica subsp. enterica serovar Clostridium sporogenes ATCC 19404
Typhimurium ATCC 14028
Salmonella enterica subsp. enterica serovar Hazard classiication: NR - Not regulated
Abony ATCC 6017 Storage:
Hazard classiication: Xi - Irritant Prepared media: 7 days at 2-8°C in the dark.
Storage: Inoculation: According to the Harmonised European Pharmacopoeia,
a sample is prepared with and without heat treatment and transferred
Dehydrated culture media: 10-25°C away from direct sunlight. to Reinforced Medium for Clostridia.
Incubation:
0.1mL of Casein Soya Bean Digest Broth pre-enrichment is transferred Incubate at 30-35°C for 48-72 hours
to 10mL of Rappaport Vassiliadis Salmonella Enrichment Broth.
Incubation: Growth Characteristics
Expected result Expected result on
Organism in RCM Columbia Agar subculture
Clostridia spp. Growth Growth, typical colonies
Expected result in Expected result on
References
Organism RV Broth XLD subculture European Pharmacopoeia 8th Edition
Salmonella spp. Turbid growth Growth, typical colonies
S. aureus No visible growth No growth
References

Sabouraud Dextrose Agar Typical Formula g/litre
Dextrose 20.0
(USP/EP/JP)
Peptic digest of animal tissue 5.0
HP014
Description Pancreatic digest of casein 5.0
A medium recommended by the Harmonised European Pharmacopoeia Method for reconstitution
for the isolation and identiication of Candida albicans from non- Disperse 30 grams of powder in 1 litre of deionised water. Allow to
sterile pharmaceutical samples. Conforms to USP/EP/JP performance soak for 10 minutes then swirl to mix. Distribute into suitable vessels
speciication. The medium is commonly abbreviated to SDA. The and sterilise at 121°C for 15 minutes.
medium is also used for the preparation and maintenance of fungal
test strains as described by the Harmonised European Pharmacopoeia. Appearance:
The peptone digests and dextrose provide a nutritious base for Powder: ine, free-lowing, homogeneous, buff
luxuriant fungal growth and the acidic pH affords selectivity
against bacteria. Due to the high carbohydrate content and low pH Finished medium: straw, clear to slight haze
this medium is highly sensitive to overheating, which will cause a pH: 5.6 ± 0.2
drop in the gel strength. According to the Harmonised European
Pharmacopoeia, Sabouraud Dextrose Broth is used as an enrichment
Typical Formula g/litre Aspergillus brasiliensis ATCC 16404
Dextrose 40.0 Hazard classiication: NR - Not regulated
Peptic digest of animal tissue 5.0 Storage:
Pancreatic digest of casein 5.0 Dehydrated culture media: 10-25°C away from direct sunlight.
Agar 15.0 Prepared media: 7 days at 2-8°C in the dark.
Method for reconstitution a quantity corresponding to 1g or 1mL of the sample is use to inoculate
Disperse 65 grams of powder in 1 litre of deionised water. Allow to 100mL of Sabouraud Dextrose Broth.
soak for 10 minutes, swirl to mix then sterilise by autoclaving at 121oC
Incubation:
for 15 minutes. Cool to 45-50°C and mix well before dispensing into
sterile Petri dishes. Dry the agar surface prior to use. Incubate at 30-35°C for 3-5 days
Appearance:
Expected result Expected result on
Finished medium: straw gel, clear to slight haze Organism in SDB SDA subculture
pH: 5.6 ± 0.2 Candida albicans Growth Growth, white colonies
References
Candida albicans ATCC 10231 European Pharmacopoeia 8th Edition
Hazard classiication: NR - Not regulated Violet Red Bile Glucose Agar

Storage:
(USP/EP/JP)
Prepared media: 7 days at 2-8°C in the dark. HP004
Description
subculture is performed from enrichment in Sabouraud Dextrose A medium recommended by the Harmonised European Pharmacopoeia
Broth onto the agar surface. for isolation and identiication of bile-tolerant Gram-negative bacteria
from non-sterile pharmaceutical products. Conforms to USP/EP/JP
Incubation: performance speciication. Yeast extract provides vitamins and gelatin
Incubate at 30-35°C for 24-48 hours serves as source of carbon and nitrogen. Glucose is a fermentable
carbohydrate and sodium chloride maintains the osmotic balance.
Bile salts and crystal violet act as selective agents inhibiting many
Growth Characteristics Gram-positive bacteria. The formulation is a modiication of Violet
Red Bile Agar by Mossel which substitutes lactose for glucose.
Shape &
Enterobacteriaceae, such as Escherichia coli and Salmonella spp.,
Organism surface Colour Other are able to ferment glucose. This produces acid which results in a pH
Candida albicans CV.E.D. White Characteristic odour drop indicated by neutral red resulting in pink colonies. Enough acid
production will cause the precipitation of bile salts resulting in a bile
References precipitate or halo around glucose fermenting bacteria. Non-glucose
European Pharmacopoeia 8th Edition fermenting bile tolerant bacteria such as Psuedomonas aeruginosa
grow but remain colourless with no bile precipitate. According to the
Harmonised European Pharmacopoeia, Enterobacteria Enrichment
Broth-Mossel is used as a selective enrichment broth, with
Sabouraud Dextrose Broth subculture performed onto Violet Red Bile Glucose Agar (VRBGA).
(USP/EP/JP) Typical Formula g/litre

HP013 Yeast extract 3.0
Description
Pancreatic digest of gelatin 7.0
A medium recommended by the Harmonised European
Pharmacopoeia for the enrichment of Candida albicans from non- Bile salts 1.5
sterile pharmaceutical samples. Conforms to USP/EP/JP performance
Sodium chloride 5.0
speciication. The medium is also used for the cultivation of fungal test
strains as described by the Harmonised European Pharmacopoeia. The Glucose monohydrate 10.0
peptone digests and dextrose provide a nutritious base for luxuriant
fungal growth and the acidic pH affords selectivity against bacteria. Agar 15.0
Due to the high carbohydrate content and low pH this medium is highly
sensitive to overheating. According to the Harmonised European Neutral red 0.03
Pharmacopoeia, Sabouraud Dextrose Broth is used as an enrichment Crystal violet 0.002

soak for 10 minutes then swirl to mix. Heat to boiling. Cool to 47°C Xylose 3.5
and mix well before dispensing into sterile Petri dishes. Dry the agar
L-Lysine 5.0
Appearance: Lactose monohydrate 7.5
Powder: ine, free-lowing, homogeneous, buff to slight red/purple Sucrose 7.5

Finished medium: red/purple, clear gel Sodium chloride 5.0
pH: 7.4 ± 0.2 Yeast extract 3.0
Phenol red 0.08
Pseudomonas aeruginosa ATCC 9027 Agar 13.5
Escherichia coli ATCC 8739 Sodium deoxycholate 2.5
Sodium thiosulfate 6.8
Storage:
Dehydrated culture media: 10-25°C away from direct sunlight. Method for reconstitution
Prepared media: 7 days at 2-8°C in the dark. Disperse 55 grams of powder in 1 litre of deionised water. Allow to
Inoculation: According to the European Pharmacopoeia 8.0 subculture soak for 10 minutes then swirl to mix. Heat to boiling. Cool to 50°C
is performed from enrichment in Enterobacteria Enrichment Broth- and mix well before dispensing into sterile Petri dishes. Dry the agar
Mossel onto the agar surface. surface prior to use.
Appearance:
Incubation:
Powder: ine, free-lowing, homogeneous, buff to slight red
Finished medium: red, clear gel
Growth Characteristics pH: 7.4 ± 0.2

Organism Colour Other
Glucose-fermenting bile-tolerant Pink Red precipitate
Gram-negative bacteria
Salmonella typhimurium ATCC 14028
Non glucose-fermenting bile- Colourless No precipitate Salmonella abony ATCC 6017
tolerant Gram-negative bacteria
References Storage:
Mossel, D.A.A. Media for Enterobacteriaceae (1985) International Dehydrated culture media: 10-25°C away from direct sunlight.
Journal of Food Microbiology, 2 (1-2), pp. 27-32. Prepared media: 7 days at 2-8°C in the dark.
European Pharmacopoeia 8th Edition Inoculation: According to the European Pharmacopoeia 8.0
subculture is performed from enrichment in Rappaport Vassiliadis
Salmonella Enrichment Broth onto the agar surface
Incubation:
Xylose Lysine Deoxycholate Agar Incubate at 30-35°C for 18-48hours
(USP/EP/JP)
HP008
Shape &
Description
Organism surface Colour Other
A medium recommended by the Harmonised European Pharmacopoeia
for isolation and identiication of Salmonella from non-sterile Salmonella spp. CV.E.G. Red Black or black centered
colonies
pharmaceutical products. Conforms to USP/EP/JP performance
speciication. Originally formulated by Taylor to differentiate enteric
pathogens, the agar is widely used as the preferred differential medium References
for Salmonella spp. The medium is void of peptones but instead uses Taylor, W. I. 1965. Isolation of shigellae. I. Xylose lysine agars:
yeast extract as a carbon, nitrogen and vitamin source and xylose, new media for isolation of enteric pathogens. Am. J. Clin. Pathol. 44
lactose and sucrose are fermentable carbohydrates. Salmonella are (4):471-475.
able to ferment xylose to produce acid but not lactose or sucrose. European Pharmacopoeia 8th Edition
When the xylose is exhausted Salmonella will decarboxylate lysine
shifting the pH back to neutral. At near neutral pH, Salmonella
can reduce sodium thiosulfate producing hydrogen sulide which
creates a complex with ferric ammonium citrate to produce black
or black centred colonies. Other organisms are able decarboxylate
lysine but acid production from the fermentation of lactose and
sucrose keeps the pH too acidic for H2S production. Selectivity is
achieved through the incorporation of sodium deoxycholate and
phenol red acts as a pH indicator. According to the Harmonised
European Pharmacopoeia, Rappaport Vassiliadis Salmonella
Enrichment Broth is used as a selective enrichment broth, with
subculture performed onto Xylose Lysine Deoxycholate (XLD) agar.

Minimum QC organisms - ß-gal reaction
4. Biomolecular Products Escherichia coli DH5a
(ATCC 53868) Lac Z+ve
®
(black)
LB Agar YPD Agar
Escherichia coli DH5a, Lac Z
LB Broth YPD Broth -ve (remains cream)
Harlequin™ LB agar* 2 x YT Agar Storage of Prepared Medium: Plates – up to 7 days at 2-8°C in the
Harlequin™ LB Top* 2 x YT Broth dark.
Inoculation: Typically surface spread over plate to detect cream
LB Agar (Lennox) NZY Broth colonies indicating disruption of β-complementation. Alternatively,
LB Broth (Lennox) NZCYM Broth spread for single colonies if required.
Incubation: 37°C aerobically, for 16-18 hours. The colour of the
Terriic Broth Luria Bertani Agar (Hi-Salt) colonies will substantially increase with prolonged incubation (up to
24 hours).
*Available from Lab M in the UK only. Outside the UK Harlequin LB Interpretation: Examine for the presence of cream colonies, which
agar is available from Sigma-Aldrich as S-Gal LB agar (C4478). indicates a successful insertion of the target DNA.
Lab M’s Biomolecular products form the basis of gene reporter

References
assays that employ enzyme substrates, such as X-gal and our patented Miller, J.H. (1972). Experiments in Molecular Genetics. Cold Spring
CHE-gal, to indicate inactivation of α-complementation. These Harbour Laboratory. Cold Spring Harbour New ,York.
products are formulated to promote the growth of the recipient and Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning:
donor cells used in DNA insertion technology. More importantly, they A Laboratory Manual, 2nd ed., Cold Spring Harbour Laboratory. Cold
are formulated to provide optimum conditions for plasmid retention Spring Harbour New, York.
or bacteriophage reproduction and survival.
As different applications have varied requirements of the culture
medium used, Lab M offer a range of media types. Some are of standard
formulation whilst others are modiied to enhance the performance of LB Agar
speciic applications. This variety allows the researcher to choose the
appropriate medium for the application being used. LAB168
Lab M have formulated unique versions of LB Agar and LB Top
Agar which incorporate the patented water soluble chromogen CHE- Description
β-gal into the complete medium. This improves colour deinition of A nutritious medium designed for rapid bacterial growth, typically
α-complemented colonies compared to the standard X-β-gal plate used in molecular biology procedures e.g. in the detection of phage
and removes the need for hazardous chemicals in the preparation of or plasmid transformed bacteria and the maintenance of recombinant
the medium. Therefore we have produced a safe, fast and easy way to strains. This agar contains the required concentration of sodium
differentiate between lac+and lac- colonies. Simply add water to the chloride to promote replication of plasmids.
powder and autoclave. All products are available directly from Lab
M in the UK. Typical Formula g/litre
Tryptone 10.0
Harlequin™ LB Agar Yeast Extract 5.0

HAL004
Agar 15.0
Description
A nutritious molecular biology medium containing the novel Method for reconstitution
chromogen CHE-galactoside to enable rapid, safe and unambiguous Weigh 40.0 grams of powder and disperse in 1 litre of deionised water.
detection of plasmid transformed bacteria. The CHE-galactoside Swirl to mix and sterilise by autoclaving at 121°C for 15 minutes.
replaces the traditional X-gal substrate, simplifying the technique Cool to 47°C and add ilter sterilised antibiotic if required. Pour into
as there is no preparation of stock solutions in dimethyl formamide sterile Petri dishes and allow the medium to set. Dry the surface prior
or dimethyl sulphoxide and surface application of the chromogen to to inoculation.
the medium. The intense black colour of the colonies gives a sharper
contrast between lac and lac colonies, giving improved colony
- +
Addition of Substrate
detection compared to blue X-gal stained colonies.
Prepare the X-Gal solution by dissolving in DMF, to give a
concentration of 20mg/ml Once dissolved, spread 40µl as a surface
Typical Formula g/litre layer over the top of the agar and allow to dry. Also spread 4µl of a
Tryptone 10.0 solution of IPTG (200mg/ml). Alternatively, use Harlequin™ LB agar
complete (HAL004), which already contains the enzyme substrate
Yeast Extract 5.0 and inducer. This eliminates the potentially hazardous use of DMF
and prevents variation in the colour of ß-complemented colonies due
Sodium chloride 10.0 to differences in substrate concentration.
CHE-galactoside 0.3
IPTG 0.03 pH: 7.0 ± 0.2
Minimum QC organisms - ß-gal reaction:
Agar 12.0 Escherichia coli DH5a
(ATCC 53868)
Lac Z+ve (black if CHE-gal
is present in the medium)
Weigh 37.8 grams of powder and disperse in 1 litre of deionised Escherichia coli DH5a, Lac Z
water. Swirl to mix and sterilise by autoclaving at 121°C for 15 -ve (remains cream even in the
minutes. Cool to 47°C and add appropriate ilter sterilised antibiotic
presence of CHE-gal)
if required. Pour into sterile Petri dishes, allow the medium to set and
dry the surface prior to inoculation.
pH: 7.0 ± 0.2
Biomolecular Products 110

Storage of Prepared Medium: Plates – up to 7 days at 2-8°C in the J.A., Smith, J.G. and Struhl. (1994). Current protocols in molecular
dark. biology. Vol. 1. Current protocols, New York. N.Y.
Inoculation: Typically surface spread over plate to detect cream Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning:
colonies indicating disruption of ß-complementation. Alternatively, A Laboratory Manual, 2nd ed., Cold Spring Harbour Laboratory. Cold
spread for single colonies if required. Spring Harbour New, York.
Incubation: 37°C aerobically, for 16-18 hours. If a chromogenic
substrate is used, the colour of the colonies will substantially increase
with prolonged incubation (up to 24 hours).
Interpretation: Using the base medium alone, all colonies will
LB Broth
appear cream. Alternatively, if a chromogen is included, examine for
the presence of cream colonies, which indicates a successful insertion
LAB169
of the target DNA.
Description
References A nutrient broth primarily used for the growth and maintenance of
Miller, J.H. (1972). Experiments in Molecular Genetics. Cold Spring Escherichia coli. Used as the primary propagation step for donor
Harbour Laboratory. Cold Spring Harbour New ,York. or recipient cells, when further work is to be performed on LB
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Agar. This broth contains a high level of sodium chloride to aid the
Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbour maintenance of plasmids. If working with temperate bacteriophages,
Laboratory. Cold Spring Harbour New, York. such as lambda, the addition of magnesium sulphate (MgSO4.7H2O)
at 2 grams per litre is recommended to promote phage absorption.

LB Agar (Lennox) Tryptone 10.0
LAB174 Yeast Extract 5.0
Description
This is a nutritionally rich medium containing half the sodium chloride
level of LB agar (LAB168). This allows the researcher to select the
optimum salt concentration for his experiment. This medium can also Weigh 25.0 grams of powder and disperse in 1 litre of deionised water.
be used for plasmid replication experiments. Nutritionally rich media Allow the mixture to soak for 10 minutes, swirl to mix and sterilise by
are required for molecular biology applications as the strains used autoclaving at 121°C for 15 minutes.
are often derived from Escherichia coli K12, which is deicient in B
vitamin production. Appearance: Straw, clear liquid.
pH: 7.0 ± 0.2
Minimum QC organisms: Escherichia coli DH5
Tryptone 10.0 (ATCC 53868)
Yeast Extract 5.0
Sodium chloride 5.0 at 15-20°C in the dark.
Agar 15.0 Inoculation: As per normal techniques, using a pure culture of
donor/recipient cells.
Method for reconstitution Incubation: 37°C aerobically for 16-18 hours.
Weigh 35.0 grams of powder and disperse in 1 litre of deionised water. Interpretation: Examine all tubes for turbidity, indicating growth.
Allow the mixture to soak for 10 minutes, swirl to mix and sterilise
by autoclaving at 121°C for 15 minutes. Cool to 47°C and add ilter References
sterilised antibiotic as required. Pour into sterile Petri dishes and allow Miller, J.H. (1972). Experiments in Molecular Genetics. Cold Spring
the medium to set. Dry the surface prior to inoculation. Harbour Laboratory. Cold Spring Harbour New ,York.
Sambrook, J., Fritsch, E.F. and Maniatis. T. (1989). Molecular
Addition of Substrate Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbour
Prepare the X-Gal solution by dissolving in DMF, to give a Laboratory. Cold Spring Harbour New, York.
concentration of 20mg/ml. Once dissolved, spread 40µl as a surface
layer over the top of the agar and allow to dry. Also spread 4µl of a
solution of IPTG (200mg/ml).
LB Broth (Lennox)
pH: 7.0 ± 0.2 LAB173
Minimum QC organisms - ß-gal reaction: Description
Escherichia coli DH5a This is a nutrient broth containing half the sodium chloride level of
(ATCC 53868) LB Broth (LAB169), this allows for the addition of calcium chloride,
Lac Z+ve (black if CHE-gal required in some applications for eficient phage adsorption to the cell
is present in the medium) e.g. phage P1. This medium can also be used for plasmid replication
Escherichia coli DH5a, Lac Z experiments. Chloramphenicol can be added to achieve high plasmid
-ve (remains cream even in the copy number by inhibiting chromosomal replication.
present of CHE-gal)
Storage of Prepared Medium: Plates – up to 7 days at 2-8°C in the Tryptone 10.0
dark.
Inoculation: Surface; either spread over entire surface for colony Yeast Extract 5.0
count or streaking for single colonies. Sodium chloride 5.0
Interpretation: Using the base medium alone, all colonies will Method for reconstitution
appear cream. Alternatively, if a chromogen is included, examine for Weigh 20.0 grams of powder and disperse in 1 litre of deionised water.
the presence of cream colonies, which indicates a successful insertion Allow the mixture to soak for 10 minutes, swirl to mix and sterilise by
of the target DNA. autoclaving at 121°C for 15 minutes.
References Appearance: Straw, clear liquid.
Lennox, E.S. (1955). Transduction of linked genetic characters of the
host by bacteriophage P1. Virology 1, 190. pH: 7.5 ± 0.2
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman.
111 Biomolecular Products

Minimum QC organisms: Escherichia coli DH5 NZCYM Broth
(ATCC 53868)
LAB182
Storage of Prepared Medium: Capped containers – up to 3 months Description
This is an improved medium for increased yields of the phage Lambda.
Inoculation: As per normal techniques, using a pure culture of donor/ This formulation includes a higher concentration of essential elements
recipient cells. for increased bacterial growth. To encourage multi phage insertion
Incubation: 37°C aerobically for 16-18 hours. into the host cell, it is recommended to add 0.2% maltose (prepare a
Interpretation: Examine all tubes for turbidity, indicating growth. 20% solution and add 1ml per 100ml of medium), which promotes
expression of LamB (lambda receptor). If maltose is added, do not use
References this medium to create phage stocks, as binding of phage particles to
membrane fragments will occur because of increased LamB density.
Lennox, E.S. (1955). Transduction of linked genetic characters of the
host by bacteriophage P1. Virology 1, 190.
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman.
J.A., Smith, J.G. and Struhl. (1994). Current protocols in molecular Enzymatic casein digest 10.0
biology. Vol. 1. Current protocols, New York. N.Y.
Acid hydrolysed casein 1.0
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning:
A Laboratory Manual, 2nd ed., Cold Spring Harbour Laboratory. Cold Yeast extract 5.0
Spring Harbour New, York.
Magnesium Sulphate 2.0
Sodium chloride 5.0
Luria Bertani (Hi-Salt) Broth
LAB191 Weigh 23.0 grams of powder and disperse in 1 litre of deionised
Description dispense into inal containers. Sterilise by autoclaving at 121°C for
A nutritious medium designed for rapid bacterial growth, typically 15 minutes.
used in the detection of phage or plasmid transformed bacteria. This
broth is formulated to LB Broth (LAB169), but has a higher pH for Appearance: Straw, clear liquid.
different applications. pH: 7.0 ± 0.2
Tryptone 10.0 (ATCC 53868)
Yeast Extract 5.0 Storage of Prepared Medium: Capped containers – up to 3 months
Sodium chloride 10.0 at 15-20°C in the dark.
Inoculation: Mix a fresh overnight culture of host cells with
Method for reconstitution bacteriophage and use to inoculate NZCYM Broth.
Incubation: 37°C aerobically.
dispense into inal containers. Sterilise by autoclaving at 121°C for References
15 minutes.
Blattner, F., et al., (1977) Science 196, 161.
pH: 7.5 ± 0.2
NZY Broth (NZYM)
(ATCC 53868)
LAB181
Storage of Prepared Medium: Capped containers – up to 3 months Description
Designed for increased replication of phage Lambda. To encourage
Inoculation: Dependent upon application. multi phage insertion into the host cell, it is recommended that 0.2%
Incubation: 37°C aerobically. maltose be added (prepare a 20% solution and add 1ml per 100ml
of medium), which promotes expression of LamB (lambda receptor).
If maltose is added, the medium should not be used to create phage
stocks, as binding of phage particles to membrane fragments will
occur because of increased LamB density.

Enzymatic casein digest 10.0
Yeast extract 5.0
Magnesium Sulphate 2.0
Sodium chloride 5.0

dispense into inal containers. Sterilise by autoclaving at 121°C for
15 minutes.
Appearance: Straw, clear liquid.
pH: 7.0 ± 0.2

Minimum QC organisms: Escherichia coli DH5 YPD Agar
(ATCC 53868)
LAB176
at 15-20°C in the dark. Description
Inoculation: Mix a fresh overnight culture of host cells with A nutritious medium used as an alternative to YPD Broth, where a
bacteriophage and use to inoculate NZY Broth. solid base is required. Glucose is included to promote rapid growth.
Incubation: 37°C aerobically for 16-18 hours. Typical Formula g/litre

References Tryptone 20.0
Blattner, F., et al., (1977) Science 196, 161. Yeast extract 10.0
Glucose 20.0
Terriic Broth Agar 17.0
LAB183 Method for reconstitution

Description water. Allow the mixture to soak for 10 minutes, swirl to mix and
A nutritious medium that will support high bacterial cell densities, sterilise by autoclaving at 121°C for 15 minutes. Cool to 47°C and
usually resulting in increased yields of DNA and recombinant add ilter sterilised antibiotic as required. Pour into sterile Petri dishes
proteins. The formulation requires the addition of glycerol to complete and allow to set. Dry the surface prior to inoculation.
the formulation.
Appearance: Dark straw, clear liquid.
Typical Formula g/litre pH: 6.5 ± 0.2
Tryptone 12.0
Minimum QC organisms: Saccharomyces cerevisiae
Yeast extract 24.0
Di Potassium phosphate 9.4 Inoculation: Surface; either spread over entire surface for colony
Potassium di phosphate 2.2 count or streaking for single colonies.
Glycerol 4.0/8.0 ml
(added after autoclaving). Interpretation: Yeasts will grow as cream colonies, size dependent
upon inoculum density.
water. Add 4.0 or 8.0 ml of glycerol, swirl to mix and dispense into YPD Broth
inal containers. Allow the mixture to soak for 10 minutes, swirl to
dissolve and sterilise by autoclaving at 121°C for 15 minutes. LAB175
Description
pH: 7.0 ± 0.2 A nutritious broth base recommended for the maintenance and
propagation of yeasts widely used in gene insertion techniques.
Minimum QC organisms: Escherichia coli DH5 Glucose is included to promote rapid growth.
(ATCC 53868)
at 15-20°C in the dark. Tryptone 20.0
Inoculation: Inoculate with a pure culture of the host strain containing Yeast extract 10.0
the required recombinant plasmid.
Glucose 20.0
Incubation: 37°C aerobically.
References
Tartoff, C.D. and Hobbs, C.A. (1987). Improved media for growing water. Allow the mixture to soak for 10 minutes, swirl to mix and
plasmid and cosmid clones. Bethesda Res. Lab. Focus, 9:205. dispense into inal containers. Sterilise by autoclaving at 121°C for
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: 15 minutes.
A Laboratory Manual, 2nd ed., Cold Spring Harbour Laboratory. Cold
Spring Harbour New, York. Appearance: Dark straw, clear liquid.
pH: 6.5 ± 0.2
Minimum QC organisms: Saccharomyces cerevisiae

Inoculation: As per normal techniques, using a pure culture of strain
to be cultivated.
Interpretation: Examine all tubes for turbidity, indicating growth.
113 Biomolecular Products

2xYT Agar
LAB180
Description
An agar version of 2xYT broth, for the growth of host cells of
ilamentous single stranded bacteriophages e.g. the M13 phage.

Tryptone 16.0
Yeast extract 10.0
Sodium chloride 5.0
Agar 15.0

sterilise by autoclaving at 121°C for 15 minutes. Cool to 47°C, pour
into sterile Petri dishes and allow the medium to set. Dry the surface
prior to inoculation.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: Escherichia coli DH5

(ATCC 53868).
Storage of Prepared Medium: Plates – up to 7 days at 2-8°C in

the dark.
Inoculation: dependent upon application.
Incubation: 37˚C aerobically.
2xYT Broth
LAB179
Description
A nutritious liquid medium formulated to promote the growth of
host cells, thereby encouraging increased replication and yield from
ilamentous single stranded bacteriophages (such as the M13 phage).

Tryptone 16.0
Yeast extract 10.0
Sodium chloride 5.0

sterilise by autoclaving at 121°C for 15 minutes.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: Escherichia coli DH5

(ATCC 53868).

Inoculation: Mix fresh overnight culture of host cells with
bacteriophage and use to inoculate 2xYTBroth.
Incubation: 37°C aerobically, typically for 4-5 hours to reduce risk
of selecting deletion mutants.
Interpretation: Concentrate bacterial cells by centrifugation and
transfer supernatant containing bacteriophage to a fresh tube, being
careful not to disturb the pellet formed. The resulting bacteriophage
stock can be stored at +4°C or -20°C.

Listeria spp. are able to hydrolyse the aesculin to form aesculetin,
5. µPREP™ which reacts with the FAC resulting in a black precipitate and a
visible positive reaction. However, Enterococci can also perform
Ready-to-Reconstitute Media this reaction, so further plating is required onto an isolation medium
such as Harlequin™ Listeria Chromogenic Agar ISO (HAL010) or
µPREP™ is a line of bagged, sterile dehydrated microbiological culture Pinnacle™ Listeria Chromogenic Agar ISO (PIN001). Selectivity is
media that are made ready to use simply by adding water; no autoclaving provided by lithium chloride, acrilavine and nalidixic acid.
required.
Using Lab M’s proven dehydrated culture media formulations, µPREP™ Typical Formula g/litre
is designed primarily for high throughput laboratories where speed,
convenience, reliability and cost-effectiveness are high priorities, and Peptone mixture 15.0
where storage space is often at a premium. µPREP™ Half Fraser Broth
ISO (+FAC) has the added advantage that all supplements, including Yeast extract 5.0
ferric ammonium citrate normally added manually after autoclaving, are Aesculin 1.0
added, meaning Technicians do not need to add any supplements at all.
µPREP™ BPW (ISO) and µPREP™ Half Fraser Broth ISO (+FAC) are
supplied sterile in boxes of 10 highly robust bags, each of which makes Potassium dihydrogen phosphate 1.35
20 litres of prepared medium.
Socium chloride 20.0
µPREP™ bags are connected to a reverse osmosis (RO)/deionised
water supply and the water is sterilised as it is pumped via a membrane Lithium chloride 3.0
ilter into the bag.
Acrilavin 0.0125
Nalidixic acid 0.01
µPREP™ BPW (ISO)
MPB001
Description Method for reconstitution
Formulated to ISO 6579, Buffered Peptone Water (ISO) is a Each µPREP™ Half Fraser Broth ISO (+FAC) bag contains suficient
pre-enrichment medium designed to help sublethally damaged media to prepare 20 litres complete Half Fraser Broth. µPREP™ bags
salmonellae recover before introducing them into a selective medium. should be hydrated by addition of 20 litres RO/deionised water via a
This nutrient medium is free from inhibitors and is well buffered membrane ilter.
to maintain pH 7.0 for the incubation period. Sublethal injury to
salmonellae occurs in many food processes and this pre-enrichment
step greatly increases recovery of these organisms. Minimum Q.C. organisms:
Typical Formula g/litre Enterococcus faecalis WDCM 00087
Enzymatic digest of casein 10.0
Sodium chloride 5.0 Storage
Disodium hydrogen phosphate (anhydrous) 3.6* Bag (as supplied): store in the dark at 10-25oC
Bag (reconstituted): store in the dark at 10-25oC for up to 72 hours
Potassium dihydrogen phosphate 1.5 (providing asepsis is maintained)
*Equivalent to 9.0g of disodium hydrogen phosphate dodecahydrate
References
Method for reconstitution Fraser, J.A. and Sperber, W.H. (1988). Rapid detection of Listeria
spp in food and environmental samples by esculin hydrolysis. J.
Each µPREP™ BPW (ISO) bag contains suficient media to prepare Food Protect. 51, No.10, 762-765.
20 litres BPW (ISO). µPREP™ bags should be hydrated by addition
of 20 litres RO/deionised water via a membrane ilter. McClain, D. and Lee, W.H. (1989). FSIS method for isolation of
L. monocytogenes from processed meat and poultry products. Lab.
Minimum Q.C. organisms: Comm.No.57, Revised May 24, (1989). US Dept of Agric.FSIS,
Escherichia coli WDCM 00013 Microbiol. Div.
Salmonella Typhimurium WDCM 00031
ISO 11290-1:1996+A1:2004, Microbiology of food and animal
Staphylococcus aureus WDCM 00034 feeding stuffs. Horizontal method for the detection and enumeration
Listeria monocytogenes WDCM 00021 of Listeria monocytogenes. Detection method
Storage
Bag (as supplied): store in the dark at 10-25oC
Bag (reconstituted): store in the dark at 10-25oC for up to 72 hours
(providing asepsis is maintained)
µPREP™ Accessories
References
BS EN ISO 6579:2002 Microbiology of food and animal feeding µPREP™ Filter Unit
stuffs – Horizontal method for the
detection of Salmonella spp. (Incorporating Corrigendum No. 1) MPA001
Used with Lab M µPREP™ bags, the µPREP™ Filter Unit is a
device for the ilter-sterilisation of reverse osmosis (RO)/deionised
water. The µPREP™ Filter Unit should be sterilised prior to use.
µPREP™ Half Fraser Broth ISO µPREP™ Filter Units may be sterilised and reused for upto 100
litres or 5 illed (20 litre) bags.
(+FAC)
MPB004
Description
µPREP™ Quick Connectors
µPrep™ Half Fraser Broth ISO (+FAC) is a sterile, ready to
reconstitute primary enrichment medium for the isolation of Listeria MPA002
spp. from foodstuffs formulated according to ISO 11290. Used with Lab M µPREP™ bags, the µPREP™ Quick Connectors
may be attached to tubing sets for the dispensing of reconstitued
media. The µPREP™ Quick Connectors should be attached to
tubing and sterilised prior to use. µPREP™ Quick Connectors may
be rinsed, resterilised and reused.µPREP™ Filter Units may be
sterilised and reused for upto 100 litres or 5 illed (20 litre) bags.
115 µPREP™ Ready-To-Reconstitute Media

6. Remove magnet from rack or tubes from the rack and add 1ml
6. Captivate™ of wash. Cap and resuspend particles by inverting several times.
7. Repeat separation and wash steps 4-6 twice more. Finally
Immunomagnetic Separation resuspend particles in 100µl of wash.
8. Remove 50µl of the complexed, resuspended particles to the
Captivate ™ is a range of antibody coated paramagnetic particles for plating media, streaking for single colonies. Incubate plates at
the speciic immunomagnetic separation (IMS) of microorganisms. 37°C for 18-24 hours and examine for typical colonies.
This patented technology consists of microscopic paramagnetic
particles. The beads have a magnetite core and a “ceramic” zirconium Phosphate Buffered Saline plus Polysorbate .
oxide coating. The beads are manufactured by a high speed blending
process and typically cover a size diameter range of 1-4 µm, with a Typical Formula g/litre
2.5µm average size. Sodium chloride 8.0
Puriied antibodies to surface components of the target microorganism
are covalently coupled to the bead. With careful antibody selection, a
highly speciic separation system for microorganisms is produced. Disodium hydrogen phosphate 1.15
The pre-coated beads are designed for the IMS of target bacteria from Potassium dihydrogen phosphate 0.2
enrichment cultures. A sample is taken from a ilter stomacher bag
and incubated with the Captivate ™ beads for 30 minutes. The bead/ Polysorbate 20. 0.5
microorganism complexes are then removed from the solution by
placing the sample tube in a Captivate separator rack (CAP100-12P). pH: 7.0 ± 0.2
This separates them from the background organisms and interfering
materials. The complexes are then washed using a PBS/ Polysorbate Dissolve the components in deionised water and check the pH.
20 wash buffer to remove non-speciically bound material. The beads Sterilise the solution by autoclaving at 121°C for 15 min. Allow the
can then be plated out onto the appropriate selective agar media and solution to cool and check the pH. Store in the dark and use within
incubated as described. one month.
The IMS technique will increase the sensitivity and speciicity of the
methodology and, in most circumstances, results can be achieved 24
hours earlier than standard protocols.
These products can also serve as a capture system for rapid detection
systems. Captivate™ O157
Special Notes on IMS Techniques.
There are important factors that affect the performance of IMS
CAP001
techniques. Thorough mixing of the particles and sample allied with Description
eficient recovery of the beads from the sample matrix is paramount
to the success of this technique. Care must be taken not to aspirate Captivate ™ O157 are magnetisable particles coated with speciic
the sample vigorously as this can result in the loss of captured target antibody intended for the isolation of E. coli O157:H7 from food,
organisms. Certain sample types (e.g. very fatty, particulate and animal feeds, beverages, pharmaceutical or environmental samples.
viscous samples) can interfere with bead recovery. To counter-act The particles help to concentrate O157:H7 cells in mixed culture
this interference, samples can be diluted in PBS-Polysorbate e.g. reducing the probability of missing low numbers or overgrowth of
1:2-1:4, reducing the effect of the matrix and allowing more eficient O157:H7 colonies by competing lora. In fact, immunomagnetic
bead recovery. Alternatively with problem samples, after the initial separation is now regarded as the gold standard method for isolation
magnetic separation the incomplete removal of the sample (i.e. of E. coli O157:H7 from food and environmental samples.
remove 800µl) and continuation of the wash protocol as described E. coli O157:H7 is the primary serovar associated with food borne
can minimise bead losses. gastrointestinal infection, resulting in self-limiting diarrhoea,
that can lead to serious disease conditions such as haemorrhagic
colitis, haemolytic uraemic syndrome (HUS) and thrombotic
Captivate Product Speciication
™ thrombocytopaenic purpura (TTP).
The organism itself is associated with raw meats and unpasteurised
milk1, probably due to the implication of farm animals and particularly
Working concentration: Typically 5mg/ml cattle as carriers of E. coli O157:H7. Large outbreaks have been
recorded in the United States from consumption of unpasteurised
Fe3O4content: 29-33% w/w
apple juice (apple cider) possibly as a result of using apples which
Antibody: Particles coated with high have fallen to the ground where the potential for contamination with
avidity, afinity puriied the organism exists3,4.
and absorbed polyclonal
antibodies to cell surface Enrichment Protocol for E. coli O157:H7.
antigens.
The recommended protocol for the isolation of E. coli O157:H7
Speciicity: Reacts with target organism. employs a 6 hour enrichment step at 42°C in modiied Tryptone Soy
Average size: 2.5 µm (typical range 1-8µm) Broth (mTSB, LAB165) plus novobiocin (X150) followed by IMS
(see below) and plating onto Sorbitol MacConkey Agar (LAB161 or
Formulation: Particles are suspended in HAL006) supplemented with or without the addition of ceixime and
PBS plus1% BSA pH potassium tellurite (X161)5-8. It is also recommended that a further
7.3-7.5 and 0.09% azide as IMS and inoculation of SMAC plates is performed after incubation
preservative. of the sample for 24 hours. Alternative enrichment protocols using
Storage: 8°C (may be shipped at different media have been described e.g. Buffered Peptone Water
ambient) (LAB046) plus VCC (X546)5-8.
Shelf life: 2 years.
Follow the Captivate ™ protocol as outlined earlier in points 1 to 8.
Interpretation: Examine the SMAC and CTSMAC plates for

Generic Captivate ™ IMS Procedure typical E. coli O157 non-sorbitol fermenting colonies that are
1. Add 20µl of well mixed Captivate™ particles to a suitable smooth and circular, 1-3 mm in diameter that are colourless to pale
micro tube (1.5 - 2.5ml volume). orange. Conirm the colony identity with commercially available
latex agglutination kits or antisera.
2. To this add 1ml of the enrichment culture taking care to avoid
transfer of sample debris. Product Presentation
3. Cap tube tightly and rotamix the suspension for 30 minutes at Captivate ™ O157 is available in packs of 50 test, product code
room temperature. CAP001-050 and 250 test, product code CAP001-250. Materials
required, but not provided, include phosphate buffered saline-
4. Insert tube into magnetic separator rack for 3 minutes to Polysorbate 20, pipettes and tips, stomacher machine and bags,
concentrate the beads to a pellet. Gently invert the rack several magnetic separator rack and culture media. Magnetic separating racks
times to aid pelleting of the beads. (CAP-100-12P) and rotating mixers (CAP101-58) are also available
5. Carefully aspirate the supernatant from the tube and cap without from LAB M.
removing particles, taking care to avoid splashing.
CaptivateTM IMS 116

Reference
1) Padhye, N.V., and Doyle, M.P. (1992). Escherichia coli O157:H7:
Captivate™ O145
Epidemiology, Pathogenesis and Methods for Detection in Food.
J.Food.Prot. 55, 555-565. CAP 006
2) Martin, M.L. et al (1986) Isolation of Escherichia coli from cattle Magnetised particles, coated with antibodies speciic to Escherichia
associated with two cases of hemolytic syndrome. Lancet ii 1043. coli O145.
3) Besser, R.E. et al (1993) An outbreak of diarrhoea and hemolytic
uremic syndrome from Escherichia coli O157:H7 in fresh pressed
apple cider. JAMA 259 2217-2220
4) McCarthy, M. (1996) E. coli O157:H7 outbreak in USA traced to Captivate™ O104
apple juice. Lancet 348 1299.
5)Wright, D.J., Chapman, P.A. and Siddons, C.A. (1994). CAP 007
Immunomagnetic separation as a sensitive method for the isolation
of Escherichia coli O157 from food samples. Epidemiology and Magnetised particles, coated with antibodies speciic to Escherichia
Infection 113, 31-39. coli O104.
6) Bolton, F.J.; Crozier, L.; Williamson, J.K. (1995) New technical
approaches to Escherichia coli O157. PHLS Microbiol. Dig. 12 67-
71. Captivate™ O121
7) Vernozy-Rozand, C. (1997). Detection of Escherichia coli O157
and other VTEC in food. Journal of Applied Microbiology. 82, 537- CAP 008
551.
Magnetised particles, coated with antibodies speciic to Escherichia
8) Ogden, I.D.; Hepburn, N.F.; & MacRae, M. (2001). The optimisation
coli O121.
of media used in the immunomagnetic separation methods for the
detection of Escherichia coli O157 in foods. J. Appl.Mic. 91, 373-
379.
Captivate™ O45
CAP 009
Non-O157 Shiga Toxin-Producing
Escherichia coli (STEC) coli O45.
Since E. coli O157:H7 was irst identiied, shiga-toxin producing
Escherichia coli (STEC, also referred to as VTEC - verocytotoxin-
producing E. coli) of various serotypes have become an ncreasing
concern for public health. Numerous outbreaks have been attributed
Captivate™ O91
to STEC serotypes and symptoms may includes bloody or acute
diarrhoea, and/or the development in some patients of Haemolytic CAP 010
Ureamic Syndrome (HUS) and Thrombotic Thrombocytopenic
Purpura (TTP). Magnetised particles, coated with antibodies speciic to Escherichia
coli O91.
Non-O157 VTEC are associated with the following: fresh meat;
ready-to-eat fermented meats (e.g. salami, pepperoni); fresh produce
(ready-to eat pre-cut vegetables and sprouted seeds); raw and low
heat-treated milk and derived dairy products; and in the hides and
leeces of cattle, sheep and goats.
Reference methods, including ISO/TS 13136:2012 and MLG

5B.05, include immunomagnetic capture and separation for the
Captivate Accessories
key serogroups. This is because the different serogroups cannot
be differentiated on agar without this technique. Subsequently
immunomagnetic separation results in improved rates of isolation. Captivate™ Separator Rack
Lab M offer an extensive range of STEC IMS solutions based on the CAP 100-12P
key O-groups identiied, but also emerging O-groups implicated in
outbreaks and sporadic human diseases. Magnetic rack for use in the Captivate™ isolation protocol. 12
tube capacity.
Captivate™ O26
CAP 003
coli O26. Custom Coating Service
A coating service is available for coating our IMS reagent with
alternative antibodies. Prices will be calculated on an individual
Captivate™ O111 basis.
CAP 004 For more information please contact our Technical Department
Magnetised particles, coated with antibodies speciic to Escherichia Tel: +44 (0) 161 820 3833
coli O111.
Fax: +44 (0) 161 820 5383
Email: [email protected]
Captivate™ O103
CAP 005
coli O103.
117 CaptivateTM IMS

Interpretation
7. Pinnacle™ colony size shape &
Pre-Poured Plates organism

Listeria monocytogenes
(mm)
1-2
surface
Round, Regular
colour
Blue to blue-
green, surrounded
by opaque halo
The Pinnacle™ brand is a new line of ready-to-use plated culture Listeria spp. 1-2 Round, Regular Blue to blue-
medium, providing Lab M’s high quality DCM prepared as ready-to-use green, without
plates. opaque halo
Plates are poured under a stringent quality management system in Isolates presumptively identiied as Listeria spp. and Listeria
a GMP environment and prepared in a clean room environment via a monocytogenes must be subjected to further biochemical tests
combination of different sized media preparators. Upon cooling plates to conirm their identity. Some strains of Listeria ivanovii may
are packed using a horizontal low wrapping machine. demonstrate lecithinase activity.
All plates are tested against a stringent quality system following ISO References
11133:2014, which has been extended by Lab M to include batch-to-
batch testing along with an enhanced panel of culture strains and more ISO 11290-1:1997 Microbiology of food and animal feeding stuffs -
rigorous acceptance criteria. Horizontal method for the detection of Listeria monocytogenes - Part
1: Detection method. Incorporating Amendment 1.
All plates are stored prior to despatch in a dedicated cold room
environment before shipping to customers upon QC release.
Pinnacle™ LCA Listeria Chromogenic

Agar (ISO) Pinnacle™ TBGA (TBX)
PIN001
PIN002
Based on Lab M’s established Harlequin™ Listeria Chromogenic A prepared medium based on Lab M’s existing Harlequin™ Tryptone
Agar ISO (HAL010), according to the formulation of Ottaviani and Bile Glucuronide Agar (TBGA), product reference HAL003. A
Agosti), Pinnacle™ LCA is a ready-prepared selective medium for the medium developed for the simple enumeration of E. coli without
isolation and presumptive identiication of Listeria monocytogenes the need for membranes, or pre-incubation on Minerals Modiied
from foodstuffs and related materials as described in ISO 11290- Glutamate Medium. Based upon the formulation of Tryptone Bile
1:1997. Agar, LAB072, the medium has been modiied by the addition of a
chromogenic substrate to detect the β-glucuronidase enzyme, which
Lithium chloride in the base medium and supplementary antimicrobial is highly speciic for E. coli*, and is detected by the MUG reagent
compounds Ceftazidime, Polymyxin, Nalidixic acid and Amphotericin in other formulations. The advantage of the chromogenic substrate
B provide the medium’s selectivity. Chromogenic activity is as a result is that it requires no UV lamp to visualise the reaction, and it is
of a chromogenic substrate for the detection of the β-glucosidase concentrated within the colony, facilitating easier enumeration in the
enzyme, common to all Listeria spp. and to a few strains of Enterococci presence of other organisms, or when large numbers are present on
and Bacilli. the plate.
The speciic differential activity of this agar is obtained with a Typical Formula g/litre
proprietary lecithin substrate for the detection of the phospholipase
enzyme that will only be present in the L. monocytogenes colonies Tryptone 20.0
growing on this media. This enzyme activity will result in a halo of
precipitation surrounding the target colonies. Bile Salts No.3 1.5
X-glucuronide 0.075
With the combination of both the chromogenic and phospholipase
enzyme reactions, it is possible to differentiate Listeria monocytogenes Agar 15.0
(blue colonies surrounded by an opaque halo) from other Listeria spp
(blue colonies without an opaque halo). Deionised water 1000ml
Typical Formula g/litre Minimum QC organisms: Escherichia coli

WDCM 00013 (blue/green)
Meat Peptone 18.0
Tryptone 6.0 Inoculation: Inoculate 0.5 ml of a 1:10 dilution of the sample and
spread over the entire surface of the plate. Further dilution may be
Yeast extract 10.0 necessary if large numbers of E. coli are present, to ensure colonies
can be easily counted.
Incubation: 30°C for 4 hours, followed by 18 hours at 44°C.
Sodium chloride 5.0 Interpretation: Count all blue/green colonies as presumptive E. coli,
Disodium hydrogen orthophosphate anhydrous 2.5 calculate the cfu/g in the original material. A simple indole test can be
performed by placing one drop of Kovac’s reagent onto a colony and
Sodium pyruvate 2.0 if positive, a red halo will appear in the medium around the colony. If
negative, then the halo will be white.
Glucose 2.0
*96-97% of E. coli strains positive. A notable exception is E. coli
Glycerophosphate 1.0 0157:H7.
References
5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside 0.05 Dibb, W.L. and Bottolfsen, K.L. (1984). Evaluation of Rosco
Agar 13.5 Diagnostic ß-glucuronidase Tablets in the Identiication of Urinary
Isolates of Escherichia coli. Acta Path.Microbiol. Immunol. Scand.
Deionised water 1000ml Sect. B 92 261-264.
Hansen, W. and Yourassowsky, E. (1984). Detection of ß-glucuronidase
Minimum Q.C. organisms: in Lactose Fermenting Members of the Family Enterobacteriaceae
Listeria monocytogenes WDCM 00021 and its Presence in Bacterial Urine Cultures.
Escherichia coli WDCM 00013 (Inhibited) J. Clin. Micro.20 (6) 1177-1179.
Robinson, B.J. (1984). Evaluation of a Fluorogenic Assay for
Detection of E. coli. App & Env. Microbiol.48 (2) 285-288.
Pinnacle™ Pre-Poured Plates 118

Perez, J.L., Berrocal, C.I. and Berrocal, L. (1986). Evaluation of a
Commercial ß-glucuronidase Test for the Rapid and Economical
Pinnacle™ mLGA
Identiication of Escherichia coli. J.App.Bacteriol. 61 541-545. (Membrane Lactose Glucuronide Agar)
Raghubeer, E. and Matches, J.R. (1990). Temperature Range for
Growth of Escherichia coli Serotype 0157:H7 and Selected Coliforms PIN004
in E. coli Medium. J.Clin. Micro. 28 (4) 803-805. Description
Bolton, F.J. (1995) Personal Communication
Pinnacle™ membrane Lactose Glucuronide Agar (mLGA) is a
medium for the detection and identiication of E. coli and coliforms
from water samples.
Traditionally, membrane Lauryl Sulphate Broth (mLSB) has been

Pinnacle™ CSIM (ISO) used as the standard media for isolating coliforms (including E.
Cronobacter sakazakii Isolation Medium (ISO) coli) from water potentially contaminated with sewage. Harlequin™
membrane Lactose Glucuronide Agar (mLGA) is a modiication of
PIN003 mLSB aimed at reducing costs by reducing the number of ilters
used per test sample and aiding in the recovery and identiication of
Description coliforms and E. coli. The medium has been modiied from the mLSB
formulation by the incorporation of X-glucuronide (BCIG), sodium
Cronobacter sakazakii (formerly Enterobacter sakazakii) is a member pyruvate and agar.
of the Enterobacteriaceae family and has been associated with serious
outbreak infections in neonates (premature infants) which have been X-glucuronide is a chromogenic substrate which detects the
fed on infant formula milk. Although rarely causing infections in β-glucuronidase enzyme - highly speciic for E. coli* - and allows
immunocompetent adults, C. sakazakii has been implicated in sepsis, for the presumptive isolation of E. coli. Sodium pyruvate aids the
meningitis and necrotising enterocolitis with a high death rate in recovery of chlorine stressed organisms and agar is incorporated to
neonates. This opportunistic pathogen is common in the environment remove the need for absorbent pads.
post pasteurisation contamination and survival in spray dried milk Using Lab M ‘s established Harlequin™ mLGA, this medium is
products. recommended for the enumeration of coliform bacteria and E. coli
by a single membrane iltration technique in The Microbiology of
C. sakazakii appears to constitutively express high levels of Drinking Water 2009 (previously Report 71).
α-glucosidase. This enzyme hydrolyses the chromogenic substrate
5-bromo-4-chloro-3-indolyl-α-D-glucopyranoside present in the *96-97% of E. coli strains positive. A notable exception is E. coli
medium, producing green to blue-green coloured colonies. Other O157:H7.
Enterobacteriaceae such as E. coli do not express strong α-glucosidase
activity and appear colourless or purple due to the uptake of crystal Typical Formula g/litre
violet.
Peptone 39.0
The combination of sodium desoxycholate, crystal violet and elevated
incubation temperature produce a very selective and speciic medium. Yeast Extract 6.0
Non-Enterobacteriaceae may appear colourless or violet coloured Lactose 30.0
(due to their inability to hydrolyse the chromogenic substrate) or are
inhibited by the selective components and incubation temperature. Phenol Red 0.2
Using Lab M ‘s established Harlequin™ CSIM (ISO), this media
formulation is currently recommended as part of the isolation protocol Sodium Lauryl Sulphate 1.0
under ISO/TS 22964:2006(E) for the isolation of Enterobacter Sodium Pyruvate 0.5
sakazakii from milk and milk products.
X-Glucuronide 0.2
Typical Formula g/litre Agar 10.0
Pancreatic peptone of casein 7.0 Deionised Water 1000mL
Yeast extract 3.0
Sodium chloride 5.0 Escherichia coli WDCM 00013
Sodium desoxycholate 0.6 Enterobacter aerogenes WDCM 00175
Staphylococcus aureus WDCM 00034 (inhibition)
5-bromo-4-chloro-3-indolyl-α-D-Glucoside 0.15
Crystal violet 0.002 Inoculation: E. coli and coliform counts can be performed on
the same sample of water. The volume and dilution of test sample
Agar 14.0 should be chosen so as the number of colonies on the membrane lies
Deionised Water 1000mL between 20 and 80. With waters expected to contain low numbers of
coliforms, a sample of 100ml should be iltered. For full methodology
refer to The Microbiology of Drinking Water 2002 section 4 B - The
Minimum Q.C. organisms: enumeration of coliform bacteria and E. coli by a single membrane
Cronobacter sakazakii ATCC 12868 iltration technique.
Enterobacter aerogenes WDCM 00175 Incubation: 4 hours at 30 °C followed by 14 hours at 37 °C
Bacillus cereus WDCM 00001
Staphylococcus aureus WDCM 00034 Interpretation: Count all green-blue colonies as presumptive E. coli,
and all green-blue and yellow colonies as presumptive coliforms.
Inoculation: Following selective enrichment in Modiied Lauryl
Sulphate Tryptose Broth Vancomycin Medium, streak onto HAL012
Harlequin™ Cronobacter sakazakii Isolation Medium (ISO).
Interpretation: After incubation the plate should be assessed for
typical colonies of C. sakazakii. Typical colonies are 1-3mm and are
green to blue-green.
119 Pinnacle™ Pre-Poured Plates

Pinnacle™ Salmonella ABC Pinnacle™ Columbia Agar Base (25ml
ill)
PIN005
Description PIN006
Pinnacle™ Salmonella ABC is a selective medium for the isolation of Description
Salmonellae from food samples and is based on Lab M‘s established A nutritious agar base capable of growing a wide variety of
Harlequin™ Salmonella ABC. microorganisms. Lab M’s Columbia Agar Base is available in a non-
standard ill volume of 25ml and is suitable for use in a number of
Salmonella spp. can be differentiated from other members of the applications.
family Enterobacteriaceae by their ability to produce α-galactosidase
in the absence of β-galactosidase. This medium utilises a dual
chromogen system to visualise these enzyme activities. Salmonella Typical Formula g/litre
ABC will also detect Salmonella typhi and paratyphi.
Columbia Peptone Mixture 25.1
The irst substrate, CHE-β-Gal, is enzymatically cleaved by ß- Soluble Starch 1.0
galactosidase producing organisms giving black colonies in the
presence of iron. Most Enterobacteriaceae are β-galactosidase Sodium Chloride 5.0
positive and these produce black colonies on Salmonella ABC.
The second substrate, X-α-Gal, is hydrolysed by Salmonella spp. Agar 12.0
producing green colonies that are easily distinguished from the black Deionised Water 1000mL
or colourless colonies of other organisms. The medium is based on
D.C.A Hynes and hence utilises sodium desoxycholate and sodium
citrate as inhibitors. Isolation of Salmonella spp. by culture remains Minimum Q.C. organisms:
the most reliable method of detection. However, most media are Pseudomonas aeruoginosa ATCC 9027
highly non-speciic and consequently place a heavy burden on the Staphylococcus aureus ATCC 6538
laboratory in terms of biochemical and serological conirmation Escherichia coli ATCC 8739
of suspect colonies. With improved speciicity, the ABC medium
dramatically reduces the need for ‘false positive’ screening, saving
labour and reducing consumable costs.
Pinnacle™ Blood Agar No. 2 (25ml
ill)
Beef Extract 5.0
PIN007
Peptone 5.0
Description
Sodium citrate 8.5 A very rich agar base which, with the addition of blood, is capable
Sodium desoxycholate 5.0 of growing delicate clinical pathogens. The medium gives colonial
appearances, haemolysis patterns and pigment production of
Agar 12.0 diagnostic value. Lab M’s Blood Agar No.2 is available in a non-
standard ill volume of 25ml and is suitable for use in a number of
X-α-Gal 0.08 applications.
CHE-β-Gal 0.3
Tryptose 15.0
IPTG 0.03
Deionised Water 1000mL Soy Peptone 2.5
Yeast Extract 5.0
Minimum QC organisms: Sodium Chloride 5.0
Escherichia coli WDCM 00013 Agar No. 2 12.0
Deionised Water 1000mL
colony size shape & Minimum Q.C. organisms:
organism (mm) surface colour other Streptococcus pyogenes NCTC 8198
Salmonella spp. 1.5 - 3.5 CV.E.G. Green (Black if Escherichia coli ATCC 25922
β-galactosidase Staphylococcus aureus ATCC 25923
+ve) Enterococcus faecalis ATCC 29212
Pseudomonas aeruiginosa ATCC 27853
Shigella spp. 3.0 - 4.0 CV.E.G. Colourless (Black if
β-galactosidase
+ve)
E.coli PP - 2.5 CV.E.G. Black (No Growth)
Proteus spp. PP - 1.0 CV.E.G. Colourless (Fishy Odour)
References
Perry, J.D., Ford, M., Taylor, J., Jones, A., Freeman, R., Gould,
F.K., (1999). ABC Medium, a New Chromogenic Agar for Selective
Isolation of Salmonella spp. J. Clin. Micro. 37: 766-768.

Pinnacle™ Legionella GVPC Medium Interpretation
(ISO) Organism
shape &
surface Colour Other
(Glycine Vancomycin Polymixin Cycloheximide)
Legionella spp. CV.E.G Grey/white Ground glass apperance
PIN008 Several species (including

L. anisa & L. bozemanii)
exhibit blue-white
Description luorescence under long-
wave UV light
Buffered Charcoal Yeast Extract (BCYE) medium is a basal agar for
the isolation of Legionella species from water samples. It is based
on the charcoal and yeast extract formulation of Feeley et al., with Note - Legionella colonies are often grey/white in colour but can
the addition of ACES (N-2-acetamido-2-aminoethanesulphonic acid) exhibit a wide range of colours.
buffer and α-ketoglutarate to enhance performance, as described by
References
Edelstein. Selective agents can be added to produce GVPC medium.
This medium is recommended under ISO 11731:1998 for the isolation Feeley, J.C., Gibson, R.J. et al. (1979). Journal of Clinical
of Legionella species from water and conforms to the performance Microbiology 10: 437-441
requirements of BS EN ISO 11133:2014.
Pesculle, A.H., Feeley, J.C. et al. (1980). Journal of Infectious Disease
Yeast extract provides a source of carbon and nitrogen, while activated 141: 727-732
charcoal acts as a detoxifying agent resulting in increased recovery
from water samples. Iron (III) pyrophosphate provides iron necessary Edelstein, P.H. (1981). Journal of Clinical Microbiology 14: 298-303
for the growth of Legionella species. ACES buffer and potassium
hydroxide are used to maintain pH and improve recovery. L-cysteine, BS EN ISO 11133:2014 Microbiology of food, animal feed and water
for which Legionella spp. are auxotrophic, and α-ketoglutarate, found – Preparation, production, storage and performance testing of culture
to improve recovery of Legionella species, are also incorporated into media
the agar.
ISO 11731:1998 Water quality –Detection and enumeration of
A number of other organisms commonly present in water samples Legionella
are also able to grow well on basal BCYE medium. Therefore, a
combination of selective agents is incorporated to produce glycine ISO 11731-2:2004 Water quality – Detection and enumeration of
Vancomycin Polymyxin Cycloheximide medium, which is typically Legionella – Part 2: Direct membrane iltration method for waters
used for primary isolation of Legionella species. The mixture of with low bacterial counts
glycine, vancomycin, and polymyxin B inhibits or suppresses most
non-target bacterial species, both Gram-positive and Gram–negative,
including common contaminants such as Enterococci, coliforms, and
Pseudomonas species, while cycloheximide suppress the growth of
yeasts and moulds. Pinnacle™ Legionella BCYE Medium
Typical Formula g/litre (ISO)
Yeast extract 10.0 (Buffered Charcoal Yeast Extract)
Activated charcoal 2.0
PIN009
ACES buffer 10.0
Description
Potassium hydroxide 2.8
Buffered Charcoal Yeast Extract (BCYE) medium is a basal agar for
Iron (III) pyrophosphate 0.25 the isolation of Legionella species from water samples. It is based
on the charcoal and yeast extract formulation of Feeley et al., with
Agar 14.0 the addition of ACES (N-2-acetamido-2-aminoethanesulphonic acid)
α-Ketoglutarate 1.0 buffer and α-ketoglutarate to enhance performance, as described by
Edelstein. This medium is recommended under ISO 11731:1998 for
L-Cysteine hydrochloride 0.4 the isolation of Legionella species from water and conforms to the
Glycine 3.0 performance requirements of BS EN ISO 11133:2014.
Vancomycin hydrochloride 0.001 Yeast extract provides a source of carbon and nitrogen, while activated
charcoal acts as a detoxifying agent resulting in increased recovery
Polymixin B sulphate 80,000iu
from water samples. Iron (III) pyrophosphate provides iron necessary
Cycloheximide 0.08 for the growth of Legionella species. ACES buffer and potassium
hydroxide are used to maintain pH and improve recovery. L-cysteine,
Deionised water 1000mL
for which Legionella spp. are auxotrophic, and α-ketoglutarate, found
to improve recovery of Legionella species, are also incorporated into
the agar.
Legionella pneumophila WDCM 00107 or WDCM
00180 Typical Formula g/litre
Legionella anisa WDCM 00106 Yeast extract 10.0
Pseudomonas aeruginosa WDCM 00025 or WDCM
00026 Activated charcoal 2.0
Escherichia coli WDCM 00012 or WDCM 00013 ACES buffer 10.0
Enterococcus faecalis WDCM 00009 or WDCM
00087 Potassium hydroxide 2.8
Inoculation: According to ISO 11731:1998, each concentrated water
Iron (III) pyrophosphate 0.25
sample is tested after undergoing three protocols; Untreated, heat
treatment and acid treatment. In all cases 0.1mL to 0.5mL of each Agar 14.0
sample portion is spread onto the agar surface with a sterile spreader.
α-Ketoglutarate 1.0
Incubation: After insuring inocula has been absorbed invert the
plates and incubate at 36±1°C for up to 10 days. L-Cysteine hydrochloride 0.4
Deionised water 1000mL
121 Pinnacle™ Pre-Poured Plates

Legionella pneumophila WDCM 00107 or WDCM
00180
Legionella anisa WDCM 00106
Inoculation: According to ISO 11731:1998, presumptive Legionella

colonies from GVPC agar plates are subcultured onto BCYE for
further conirmation.
Incubation: Incubate at 36±1°C for at least 2 days
Interpretation
shape &
Organism surface Colour Other
Legionella spp. CV.E.G Grey/white Ground glass apperance
Several species (including
L. anisa & L. bozemanii)
exhibit blue-white
luorescence under long-
wave UV light
Note - Legionella colonies are often grey/white in colour but can

exhibit a wide range of colours.
References
Feeley, J.C., Gibson, R.J. et al. (1979). Journal of Clinical
Microbiology 10: 437-441
Pesculle, A.H., Feeley, J.C. et al. (1980). Journal of Infectious Disease

141: 727-732
Edelstein, P.H. (1981). Journal of Clinical Microbiology 14: 298-303
BS EN ISO 11133:2014 Microbiology of food, animal feed and water

– Preparation, production, storage and performance testing of culture
media
ISO 11731:1998 Water quality –Detection and enumeration of

Legionella
ISO 11731-2:2004 Water quality – Detection and enumeration of

Legionella – Part 2: Direct membrane iltration method for waters
with low bacterial counts

Colistin & Nalidixic Acid Selective
8. Lyophilised Media Supplements Supplement
Presentation and Shelf Life X011
Lab M lyophilised supplements are presented in packs of 10 vials, COLISTIN, NALIDIXIC ACID for the isolation of G. vaginalis
and for the majority of the supplements each vial is suficient for from clinical material.
500ml of medium. Larger and smaller volumes are indicated for
relevant products. Suitable for addition to LAB001 Columbia Agar or LAB015 Blood
Agar Base No. 2 to produce a selective isolation medium.
The shelf life of freeze-dried supplements is 2-3 years provided
they are stored in a refrigerator at 2-8˚C. The shelf life of each Final Concentration mg/litre
product is detailed on the individual product literature and labelling.
Once rehydrated the stability of antibiotics varies greatly and will Colistin 10
determine the shelf life of the prepared agars and broths. For this
reason any unused, rehydrated, supplement should be discarded, Nalidixic acid 15
as even deep-freezing may not prevent the rapid degradation of the Add 1 vial X011 to 500ml medium
antibiotics. To ensure the correct level of selective supplements the
entire vial contents must be added to the stated volume of cooled,
Rehydrate contents of vial with 5ml sterile deionised water. Add
molten medium.
aseptically to sterilised medium cooled to 47˚C, together with any
Rehydration other additives, mix gently and pour.
Vials should be rehydrated aseptically using a pipette charged with Reference:
the appropriate volume of the speciied diluent for the particular
Goldberg, R.L., Washington, J.A. II (1976). “Comparison of Isolation
supplement being added. The supplement should be rehydrated,
of Haemophilus vaginalis (Corynebacterium vaginalae) from
withdrawn and added to the medium in a single process, followed by
Peptone-Starch-Dextrose Agar and Columbia, Colistin, Nalidixic
immediate disposal of the pipette into an approved container.
Acid Agar. J. Clin. Microbiol. 4(3): 245.
Addition
Most antibiotics are heat labile, and so to prevent a reduction of Colistin & Nalidixic Acid
potency the medium should be cooled to 47-50˚C (as speciied
for each individual product), by holding in a water bath set at this X012
temperature.
Once the supplement has been added the medium must be gently COLISTIN, NALIDIXIC ACID for the preparation of Columbia
but thoroughly mixed to ensure that the selective agents are evenly C.N.A. medium.
distributed. Failure to do this will result in a range of concentrations A medium selective for Gram positive cocci is obtained when this
in the plates/bottles and consequent inconsistency in results. Media antibiotic mixture is added to LAB001 Columbia Agar.
should not be held for an additional period at 47-50˚C, but poured
immediately. The shelf life of supplemented media is governed by Final Concentration mg/litre
the stability of the added components, and is generally shorter than
unsupplemented agars and broths. For information on the shelf life of Colistin 10
prepared media consult the individual product listings in the previous
section of the manual. Nalidixic acid 10
Add 1 vial X012 to 500ml medium

Chloramphenicol Supplement aseptically to sterilised medium cooled to 47˚C, together with any
other additives, mix gently and pour.
X009 Reference:
Ellner, P.D., Stossel, C.I., Drakeford, E., Vasi, F. (1966). “A new
CHLORAMPHENICOL for the selective isolation of yeasts and culture medium for medical bacteriology.” Amer. J. Clin. Path. 45:
moulds from food, environmental and clinical specimens. 502.
For larger volumes X209 is available (1 vial per 1L)
Chloramphenicol’s broad antibiotic spectrum suppresses most
contaminating bacteria allowing the yeasts and moulds to grow. It
can be added to such media as LAB009 Sabouraud Dextrose Agar, Colistin & Oxolinic Acid
LAB036 Rose Bengal Chloramphenicol Agar, LAB037 Malt Extract
Agar and LAB117 Dermatophyte Test Medium to increase their X013
selectivity whilst not lowering the pH. Reduction of pH will increase
the selectivity of a yeast and mould medium but will also inhibit some COLISTIN, OXOLINIC ACID for the selective isolation of
yeasts as well as having a deleterious effect on the agar gel. streptococci from clinical material.
When added to LAB001 Columbia agar or LAB015 Blood Agar Base
Final Concentration mg/litre No. 2, X013 renders the medium selective for streptococci. Alteration
in haemolysis patterns may occur when azide or crystal violet are
Chloramphenicol 100 employed as selective agents but this does not occur with X013.
Final Concentration mg/litre
Rehydrate contents of vial with 5ml of Ethyl or Methyl alcohol. Add Colistin 10
aseptically to sterilised medium cooled to 47˚C, mix gently and pour.
Oxolinic acid 5
References:
Jervis, B. (1973). Rose Bengal Chlortetracycline agar with other
media for the selective isolation and enumeration of moulds and Rehydrate contents of vial with 5ml of sterile deionised water. Add
yeasts in foods. J. Appl. Bact. 36 Pages 723-727. aseptically to sterilised medium cooled to 47˚C together with other
additives, mix gently and pour.
Reference:
Petts, D. (1984). Colistin - Oxolinic Acid - Blood Agar: a new selective
medium for streptococci. J. Clin. Microbiol. 19: 4-7.
123 Lyophilised Media Supplements

Neomycin 75mg P-INC Supplement (PNVC)
X015 X019
NEOMYCIN 75 for the isolation of Clostridium spp. and other PENICILLIN, NISIN, CRYSTAL VIOLET, for accelerated shelf
anaerobes. life determination of dairy products.
When added to blood agar the resulting medium will allow the growth
of clostridia, most Bacteriodes fragilis strains and some anaerobic For larger volumes X219 is available (1 vial per 1L)
cocci. The Pre-incubation test uses a selective mixture to inhibit Gram
positive organisms whilst allowing the growth of Gram negative
Final Concentration mg/litre bacteria, the main cause of post-pasteurisation contamination and a
Neomycin 75 major factor in determining the shelf life of the product. The technique
is also useful for monitoring plant hygiene.
Reconstitute each vial by the addition of 5ml of sterile deionised
water. Add aseptically to sterilised medium cooled to 47˚C, mix Penicillin 20,000iu/litre
gently and pour.
Nisin 40,000iu/litre m
Crystal violet 2.0
Add 1 vial of X019 to 200ml of Milk Agar LAB019
Neomycin 100mg
Rehydrate contents of 1 vial with 5ml sterile deionised water. Add
X016 aseptically to sterilised medium cooled to 47˚C, mix thoroughly and
pour plates.
NEOMYCIN 100 for the selective isolation of Clostridium spp. Method A
When added to egg yolk medium this supplement will allow the Pre-incubate test material at 21˚C for 24hr. Prepare suitable dilution
growth of clostridia whilst inhibiting other lecithinase producing series, and inoculate Milk Agar plates containing P-INC supplement.
organisms. Incubate at 21˚C for 24hr, and count all colonies (some may be small,
use of a hand lens is recommended). Calculate the CFU/ml and using
Final Concentration mg/litre the tables of Grifith’s et al the shelf life can be determined.
Neomycin 100 Method B

Rehydrate X219 with 1ml of deionised water only, add 0.1ml to the
Add 1 vial X016 to 500ml medium test material and incubate at 20˚C for 24hr. Prepare suitable dilution
Reconstitute each vial by the addition of 5ml of sterile deionised series, and inoculate Milk Agar plates. Proceed as for Method A
water. Add aseptically to sterilised medium cooled to 47˚C, mix above.
gently and pour.
VCNT Selective Supplement

Kanamycin 75mg X068
X018 V.C.N.T. VANCOMYCIN, COLISTIN, NYSTATIN,
TRIMETHOPRIM for Thayer Martin Medium.
KANAMYCIN 75 for the selective isolation of Clostridium spp.
and other anaerobes. The addition of trimethoprim in V.C.N.T. inhibits the swarming of
Proteus spp. which occasionally make interpretation dificult.
An alternative to X015. Kanamycin is more inhibitory to anaerobic
cocci.
Final Concentration mg/litre Vancomycin 3
Kanamycin 75 Colistin 7.5

Nystatin 12.5
Add 1 vial X018 to 500ml medium Trimethoprim 5
Reconstitution as X015, X016.
References: Add 1 vial X068 to 500ml medium
Lowbury, C.J.L., Lilly, H.A. (1955). A selective plate medium for Cl.
welchi. J. Path. & Bact. 70: 105. Rehydrate contents of vial with 5ml sterile deionised water. Add
Collee, J.G., Watt, B. (1971). Changing approaches to the sporing aseptically to sterilised medium cooled to 47°C together with other
anaerobes in medical microbiology. Spore Research ed. A. N. additives, mix gently and pour.
Barkeer.
Reference:
Sutter, V.L., Citron, D.M., Edelstein, M.A.C., Finegold, S.M. (1985). Thayer, J.D. and Martin, J.E. (1966). Improved medium selective for
Wadsworth Anaerobic Bacteriology Manual 4 ed. Star publishers,
Belmont, California. the cultivation of N. gonorrhoeae and N. meningitidis. Public Health
rep. 81: 559-562.
Wren, M.W.D. (1980). Multiple selective media for the isolation of
anaerobic bacteria from clinical specimens. J. Clin. Path. 33: 61-65
Lyophilised Media Supplements 124

LCAT Selective Supplement Polymyxin B
X070 X074
L.C.A.T. LINCOMYCIN, COLISTIN, AMPHOTERICIN, POLYMYXIN for the isolation of B. cereus from foods.
TRIMETHOPRIM for the isolation of Neisseria spp. from clinical For larger volumes X274 is available (1 vial per 2L)
material.
L.C.A.T. is often preferred to X068 V.C.N.T. for the isolation of N. Suitable for the preparation of LAB073 Bacillus cereus Medium
gonorrhoeae because of the emergence of vancomycin sensitive (P.R.E.P.). The addition of X073 sterile egg yolk emulsion is also
strains. The antifungal agent amphotericin is more readily soluble and required.
therefore a more active antifungal than nystatin. L.C.A.T. is quoted
as the selective agent for New York City G.C. agar but can readily be Final Concentration
substituted for V.C.N. or V.C.N.T. in Thayer Martin G.C. agar.
Polymyxin B 8mg/litre = 64,000i.u/litre
Lincomycin 1
Colistin 6 Rehydrate contents of vial with 5ml of sterile deionised water. Add
aseptically to sterilised medium cooled to 47˚C together with egg
Amphotericin 1 yolk emulsion, mix gently and pour.
Trimethoprim 6.5 Reference:
Add 1 vial X070 to 500ml medium Micro-organisms in Food. Ed. Thatcher, F.S., Clarke, D.F. published
by Univ. of Toronto Press.
Rehydrate contents of vial with 5ml sterile 25% alcohol in water. Add
aseptically to sterilised medium cooled to 47˚C together with other
additives, mix gently and pour. RPF Supplement
Reference:
Young, H. (1978). Cultural Diagnosis of Gonorrhoea with modiied X086
N.Y.C. Medium. Brit. Journ. Ven. Dis. 54: 36-40.
RPF Supplement
Rabbit Plasma Fibrinogen Supplement
Description
Polymyxin & Ceftazidime Selective Bovine ibrinogen, rabbit plasma, trypsin inhibitor and potassium
Supplement tellurite for the isolation of Staphylococcus aureus.
For addition to LAB285 Baird-Parker Medium Base (ISO) and
X072 LAB085 Baird-Parker Medium Base.
POLYMYXIN B, CEFTAZIDIME supplement for the isolation of Final Concentration amount / vial amount / litre
Listeria monocytogenes.
For addition to LAB172, LMBA Bovine Fibrinogen 0.375g 3.75g
Rabbit Plasma 2.5ml 25ml
Trypsin Inhibitor 2.5mg 25mg
Polymyxin B 10
Potassium Tellurite 2.5mg 25mg
Ceftazidime 20
Add 1 vial X072 and 1 vial of X072N to 500ml medium. Add 1 vial of X086 to 90mL of medium.
Rehydrate contents of vial by the addition of 10mL sterile deionised
Rehydrate contents of vial by the addition of 5ml of sterile deionised water. Add aseptically to sterilised media (cooled to 47oC), mix gently
water. Add aseptically to sterilised medium cooled to 47°C, mix to evenly distribute and pour.
gently and pour.
Oxytetracycline Supplement
Nalidixic Acid Selective Supplement X089
X072N OXYTETRACYCLINE for O.G.Y.E. medium.
NALIDIXIC ACID supplement for the isolation of Listeria For use with LAB089 Oxytetracycline Glucose Yeast Extract Agar
monocytogenes. for the enumeration of yeasts and moulds from foodstuffs. Highly
For addition to LAB172, LMBA proteinaceous foods and incubation above 30˚C will inactivate
oxytetracycline.
Nalidixic acid 40
Oxytetracycline 100
Add 1 vial X072N and 1 vial of X072 to 500ml medium.
Rehydrate contents of vial by the addition of 5 ml of sterile deionised Rehydrate contents of vial with 5ml sterile deionised water. Add
water. Add aseptically to sterilised medium cooled to 47°C, mix aseptically to sterilised medium cooled to 47˚C, mix gently and pour.
gently and pour.
References:
Mossel, D.A.A., et al. (1970). O.G.Y.E. for the selective enumeration
of moulds and yeasts in food and clinical material. J. Appl. Bact. 35:
454-457.

Nalidixic Acid & Vancomycin CFC Supplement
X108
X090
MODIFIED C.F.C. – CEPHALOTHIN, FUCIDIN, CETRIMIDE
NALIDIXIC ACID, VANCOMYCIN for the isolation of Gram for the selective isolation of Pseudomonas spp.
negative anaerobes from clinical material.
When added to LAB108 Pseudomonas Agar, to prepare C.F.C.
Suitable for use with LAB090 Fastidious Anaerobe Agar. When used
medium this supplement can be used to select pseudomonads from
with other blood agar bases, e.g. LAB001 Columbia Agar, further
food and environmental samples.
enrichment of the medium with haemin and menadione is beneicial.
For larger volumes X223 is available (1 vial per 2L)
Nalidixic acid 10 Final Concentration mg/litre
Vancomycin 2.5 Cephalothin 50

Fucidin 10
Cetrimide 10
Rehydrate contents of vial with 5ml sterile deionised water. Add Add 1 vial X108 to 500ml medium
additives, mix gently and pour. Rehydrate contents of vial with 5ml of sterile 50% alcohol. Add
aseptically to sterilised medium cooled to 47˚C, mix gently and pour.
Reference:
Reference:
Wren, M.W.D., 1980. J. Clin. Path. 33: 61-65. Multiple Selective
Media for the isolation of anaerobic bacteria. Mead, G.C. and Adams, B.W. (1977). Br. Poult. Sci. 18: 661-667
Cycloserine & Cefoxitin

Sulphadiazine Supplement
X093
X109
CYCLOSERINE, CEFOXITIN for the isolation of Clostridium
dificile from clinical materials. SULPHADIAZINE. SEE ALSO X110
Suitable for use with LAB090 Fastidious Anaerobe Agar. For use with LAB109 Perfringens agar to prepare O.P.S.P. for the
selective isolation of Clostridium perfringens from foodstuffs.
D-Cycloserine 250 Final Concentration mg/litre
Sulphadiazine 100
Cefoxitin 8
Add 1 vial X109 and 1 vial X110 to 500ml medium
Rehydrate contents of vials with 5ml of sterile deionised water. Add
Rehydrate contents of vial with 5ml of water. Add aseptically to aseptically to sterilised medium cooled to 47˚C, mix gently and pour.
sterilised medium cooled to 47˚C together with other additives, mix
gently and pour. Reference:
Reference: Handford, P.M. (1974). J. Appl. Bact. 37, 559-570.
George, W.L., Sutter, V.L., Citron, D., Finegold, S.M. (1976). Selective
and differential medium for isolation of Clostridium dificile.
Oleandomycin & Polymyxin
Supplement
CN Supplement X110
X107
OLEANDOMYCIN PHOSPHATE, POLYMYXIN. SEE ALSO
C.N. CETRIMIDE, NALIDIXIC ACID for the isolation of X109
Pseudomonas aeruginosa. For use with LAB109 Perfringens agar to prepare O.P.S.P. for the
selective isolation of Clostridium perfringens from foodstuffs.
Suitable for use with LAB108 Pseudomonas Agar to make the
medium selective for Ps. aeruginosa.
Oleandomycin 0.5
Cetrimide 200
Polymyxin 10,000 i.u./litre
Nalidixic acid 15 Add 1 vial X109 and 1 vial X110 to 500ml medium
Rehydrate contents of vial with 5ml of sterile deionised water. Add Rehydrate contents of vials with 5ml of sterile deionised water. Add
aseptically to sterilised medium cooled to 47˚C, mix gently and pour. aseptically to sterilised medium cooled to 47˚C, mix gently and pour.
Reference: Reference:
Goto, S., Enomoto, S. 1970. Jap. J. Microbiol. 14: 65-72. Handford, P.M. (1974). J. Appl. Bact. 37, 559-570.

Cefoperazone & Amphotericin CIN Selective Supplement
Selective Supplement
X120
X112 C.I.N. - CEFSULODIN, IRGASAN, NOVOBIOCIN for the
CEFOPERAZONE, AMPHOTERICIN for the isolation of isolation of Yersinia spp. from clinical and environmental
Campylobacter spp. from clinical, environmental and food material.
samples. For addition to LAB120 Yersinia C.I.N. Agar Base used in the
For larger volumes X212 is available (1 vial per 1L) selective isolation of Y. enterocolitica.
Suitable for use with LAB112 Campylobacter Selective Medium Final Concentration mg/litre
(blood free) or with blood agar media. Incubation at 37˚C gives better Cefsulodin 15
results than at 42˚C and is generally more convenient.
Irgasan 4
Final Concentration mg/litre Novobiocin 2.5
Cefoperazone 32 Add 1 vial X120 to 500ml medium
Amphotericin 10
Rehydrate contents of vial with 5ml of 30% sterile alcohol. Add
Add 1 vial X112 to 500ml medium aseptically to sterilised medium cooled to 47˚C, mix gently and pour.
Rehydrate contents of vial with 5ml of sterile deionised water. Add

References:
aseptically to sterilised medium cooled to 47˚C, mix gently and pour. Schiemann, D.A. (1979). Synthesis of a selective medium of Yersinia
enterocolitica. Can. J. Microbiol. 25 (2): 1298.
Reference: Schiemann, D.A. (1980). Isolation of toxigenic Yersinia enterocolitica
Bolton, F.J., Hutchinson, D.N., Parker, G. (1988). Reassessment from retail pork products. J. Food Prot. 43: 360.
of Selective Agars and Filtration Techniques for Isolation of Schiemann, D.A. (1982). Development of a two-step enrichment
Campylobacter Species from Faeces. Eur. J. Clin. Microbiol. Infect. procedure for recovery of Yersinia enterocolitica from food. Appl.
Dis. 7: 155-160. Microbiol. 43 (1): 14.
Modiied Preston Campylobacter CNCAF Selective Supplement

Supplement
X123
X114
Cefotetan, Natamycin, Colistin, Acrilavine, Fosfomycin (CNCAF)
Modiied Preston Campylobacter Supplement for the isolation of Selective Supplement
Campylobacter spp. Description
For use with LAB015 Blood Agar Base supplemented with 5% lysed For the isolation of Listeria monocytogenes from environmental,
horse blood or LAB014 Nutrient Broth No.2 supplemented with 5% clinical and food samples. This supplement may be added to LAB122
lysed horse blood and X115 Campylobacter Growth Supplement. Listeria Isolation Medium Oxford, LAB206 Listeria Isolation Media
(Oxford).
Final Concentration Per vial Conc. in
medium
Formulation Per vial Concentration in medium
Rifampicin 5mg 10mg/L LAB122 / LAB206
Polymyxin B 2500 IU 5000 IU Cefotetan 1 mg 2 mg/L
Trimethoprim 5mg 10mg/L Natamycin 12.5 mg 25 mg/L
Amphotericin B 5mg 10mg/L Colistin 10 mg 20 mg/L
Rehydration Acrilavine 2.5 mg 5 mg/L

Wearing latex gloves aseptically reconstitute the contents of the vial
with 5ml of sterile 50% ethanol using a sterile pipette. Ensure contents Fosfomycin 5 mg 10 mg/L
of vial are well mixed before addition to culture media.
Vials per litre of medium 2
Appearance
Campylobacter Growth Supplement White to off-white lyophilised tablet.
Reconstitution
X115 Contents of the vial should be reconstituted by the addition of sterile
50% ethanol in water. Add aseptically to sterilised medium cooled to
Campylobacter growth supplement for the isolation of 47oC. Mix gently then pour.
Camylobacter spp.
Suitable for use with LAB014 Modiied Preston Campylobacter
Medium (Nutrient Broth No. 2)
Formulation Per vial Conc. in

medium
(LAB014)
Sodium pyruvate 0.125 g 0.25 g
Sodium metabisulphite 0.125 g 0.25 g
Ferrous sulphate 0.125 g 0.25 g
Wearing latex gloves aseptically reconstitute the contents of the vial

with 5 ml of sterile deionised water using a sterile pipette.

CVTN Supplement Cepacia Selective Supplement
X132 X140
Cefoperazone, Vancomycin, Trimethoprim, Natamycin
(CVTN) Selective Supplement TICARCILLIN, POLYMYXIN, for the isolation of Burkholderia
(Pseudomonas) cepacia
Description Suitable for use with LAB108 pseudomonas selective agar, or speciic
For the isolation of Campylobacter spp. from food and environmental selective bases such as that described by Gilligan et al.
samples by the enrichment broth technique. Developed for use with
LAB135 Campylobacter Enrichment Broth. Gives higher isolation Final Concentration mg/litre
rates than Preston Broth and does not require modiied atmosphere Ticarcillin 100
incubation.
Polymyxin 300,000 iu/litre
medium Add 1 vial to 500ml of medium
(LAB135)
Cefoperazone 10mg 20mg/L Rehydrate contents of vial with 5ml sterile deionised water. Add
aseptically to sterilised medium cooled to 47˚C, mix well and pour.
Vancomycin 10mg 20mg/L Reference:
Gilligan, P.H., Gage, P.A., Bradshaw, L.M., Schidlow, D.V., DeCicco,
Trimethoprim 10mg 20mg/L
B.T. (1985) Isolation medium for the recovery of Pseudomonas
cepacia from respiratory secretions of patients with cystic ibrosis.
Natamycin 12.5mg 25mg/L J.Clin.Microbiol. 22 (1) 5-8.
Vials per litre of medium 2
Appearance
White to off-white lyophilised tablet.
PAC Selective Supplement
Reconstitution
Rehydrate contents of vial with 5ml sterile deionised water. Add X144
aseptically to sterilised medium cooled to 47oC. Mix gently and
dispense into sterile containers. P.A.C. supplement for the enrichment and isolation of Listeria spp
from food and environmental samples.
For the addition to LAB144 Palcam Broth and Lab 148 Palcam Agar
Final concentration mg/litre

NAN Selective Supplement Polymyxin 10
Acrilavine 5
X139
Ceftazidime 20
N.A.N. NALIDIXIC ACID, ACRIFLAVINE, NATAMYCIN.
Add 1 vial of X144 to 500ml of Palcam Broth or Agar
An alternative natamycin based supplement for the selective
enrichment broth culture of Listeria spp. Rehydrate contents of vial with 5ml sterile deionised water. Add
For addition to LAB 138, Listeria Enrichment Broth and LAB139, aseptically to sterilised medium cooled to 47˚C, along with other
Buffered Listeria Enrichment Broth. additives, mix well and pour.
Nalidixic acid 40 Novobiocin

Acrilavine 15
X150
Natamycin 25
NOVOBIOCIN for the enrichment of E. coli O157:H7 from food,
Add 1 vial of X139 to 500ml medium. environmental and clinical samples.
For the addition to LAB165 O157 Broth MTSB
Rehydrate contents of vial by the addition of 5ml of sterile deionised
water. Add aseptically to sterilised medium cooled to 47°C, mix Final concentration mg/litre
gently and dispense.
Novobiocin 20
Add 1 vial of X150 to 500ml of O157 Broth MTSB.

UVM I Supplement ORSIM Selective Supplement
X155 X192
UVMI. Supplement for the primary enrichment of Listeria spp OXACILLIN, POLYMYXIN B supplement for the isolation of
from food and environmental samples. Methicillin Resistant Staphylococcus aureus (MRSA).
For addition to LAB 155 UVM Broth Base For addition to LAB192, ORSIM (Oxcacillin Resistant Staphylococcus
Isolation Medium).
Nalidixic acid 20 Final Concentration mg/litre
Acrilavine 12 Oxacillin 2
Add 1 vial of X155 to 500ml of UVM Broth Base Polymyxin B 25,000 I.U
Rehydrate contents of vial with 5ml sterile deionised water. Add Add 1 vial of X192 to 500ml medium.
Rehydrate contents of vial by the addition of 5ml of sterile deionised
water. Add aseptically to sterilised medium cooled to 47°C, mix
gently and dispense.
Ceixime Tellurite Supplement
X161
CEFIXIME TELLURITE supplement for the isolation of E. coli Polymyxin B
O157:H7 from food, environmental and clinical samples.
X193
For the addition to LAB161 Sorbitol MacConkey Agar (SMAC) or
HAL006 (BCIG-SMAC). POLYMYXIN B for the isolation of Bacillus cereus from foods.
The addition of X073, Egg Yolk Emulsion, is also required. For
Final concentration mg/litre addition to LAB193, PEMBA Bacillus cereus Medium.
Ceixime 0.05
Potassium tellurite 2.5
Add 1 vial of X161 to 500 ml of Sorbitol MacConkey Polymyxin B 100,000 IU
Agar (SMAC) or HAL006 (BCIG-SMAC) Add 1 vial X193 to 500ml medium
Rehydrate contents of vial with 5ml sterile deionised water. Add Rehydrate contents of vial by the addition of 5ml of sterile deionised
aseptically to sterilised medium cooled to 47˚C, mix well and pour. water. Add aseptically to sterilised medium cooled to 47°C, mix
gently and pour.
Half Fraser Supplement

D-Cycloserine Supplement
X164
X194
HALF FRASER supplement for the primary enrichment of
Listeria spp from food and environmental samples. D-CYCLOSERINE supplement for the isolation of Clostridium
For larger volumes X564 is available (1 vial per 2.25 L) perfringens from foods.
For addition to LAB164 Fraser Broth Base For use with LAB194, Perfringens Agar Base (TSC).
Final Concentration mg/litre Final Concentration mg/litre

Ferric ammonium citrate 500 D-Cycloserine 400
Acrilavine 12.5 Add 1 vial of X194 to 500mls medium.
Nalidixic acid 10
Rehydrate contents of vial by the addition of 5mls of sterile deionised
Add 1 vial of X164 to 450ml of Fraser Broth Base water. Add aseptically to sterilised medium cooled to 47°C, mix
Add 1 vial of X564 to 2.25 litres of Fraser Broth Base gently and pour.
Rehydrate contents of vial with 2ml 50% methanol (5ml for X564).
Add aseptically to sterilised medium cooled to 47˚C, mix well and
pour. GVPC Selective Supplement
X195
Fraser Supplement GVPC Selective Supplement - Glycine, Vancomycin,
X165 Polymyxin B, Cycloheximide
FRASER supplement for the secondary enrichment of Listeria Description
spp from food and environmental samples. A selective supplement developed for use with LAB195 BCYE
For addition to LAB164 Fraser Broth Base Legionella Isolation Medium for the isolation of Legionella spp.
X195 GVPC Supplement should be used in conjunction with X196
Final Concentration mg/litre BCYE Growth Supplement.
Ferric ammonium citrate 500
Acrilavine 25
Nalidixic acid 20
Add 1 vial of X165 to 500ml of Fraser Broth Base
Rehydrate contents of vial with 2ml 50% methanol. Add aseptically to

sterilised medium cooled to 47˚C, mix well and pour.

medium
Chloramphenicol Supplement
(LAB195)
X209
Glycine 1500mg 3000mg/L
CHLORAMPHENICOL for the selective isolation of yeasts and
Vancomycin hydrochloride 0.5mg 1mg/L moulds from food, environmental and clinical specimens.
For smaller volumes X009 is available (1 vial per 500ml)
Polymyxin B sulphate 39600 IU 79200 IU/L
Chloramphenicol’s broad antibiotic spectrum suppresses most
Cycloheximide 40mg 80mg/L contaminating bacteria allowing the yeasts and moulds to grow. It
can be added to such media as LAB009 Sabouraud Dextrose Agar,
Vials per litre of medium 2 LAB036 Rose Bengal Chloramphenicol Agar, LAB037 Malt Extract
Agar and LAB117 Dermatophyte Test Medium to increase their
Appearance selectivity whilst not lowering the pH. Reduction of pH will increase
the selectivity of a yeast and mould medium but will also inhibit some
Off-white lyophilised tablet. yeasts as well as having a deleterious effect on the agar gel.
T - Toxic Final Concentration mg/litre
Reconstitution Chloramphenicol 100
aseptically to sterilised medium cooled to 47 oC. Mix gently and Add 1 vial X209 to 1 litre medium
dispense into sterile containers.
References Rehydrate contents of vial with 5ml of Ethyl or Methyl alcohol. Add
International Standard. ISO 11731:1998(E). Water quality- Detection aseptically to sterilised medium cooled to 47˚C, mix gently and pour.
and enumeration of Legionella
References:
Jervis, B. (1973). Rose Bengal Chlortetracycline agar with other
media for the selective isolation and enumeration of moulds and
yeasts in foods. J. Appl. Bact. 36 Pages 723-727.
BCYE Growth Supplement
X196
BCYE Growth Supplement Cefoperazone & Amphotericin
Description
Selective Supplement
L-Cysteine Hydrochloride and α-Ketoglutarate BCYE growth X212
supplement for the isolation of Legionella spp. For addition to
LAB195 BCYE Legionella Isolation Medium CEFOPERAZONE, AMPHOTERICIN for the isolation of
Campylobacter spp. from clinical, environmental and food
samples.
Formulation mg/litre
L-Cysteine Hydrochloride 400
α-Ketoglutarate 1000 Suitable for use with LAB112 Campylobacter Selective Medium
(blood free) or with blood agar media. Incubation at 37˚C gives better
Add 1 vial per 500mL of sterilised medium as appropriate. results than at 42˚C and is generally more convenient.
References Final Concentration mg/litre

ISO 11731:1998(E) Water quality- Detection and enumeration of Cefoperazone 32
Legionella
Amphotericin 10
Add 1 vial X212 to 1 litre medium
Rehydrate contents of vial with 5ml of sterile deionised water. Add

BCYE Growth Supplement (no aseptically to sterilised medium cooled to 47˚C, mix gently and
pour.
L-Cysteine)
Reference:
X197 Bolton, F.J., Hutchinson, D.N., Parker, G. (1988). Reassessment
of Selective Agars and Filtration Techniques for Isolation of
BCYE Growth Supplement (no L-cysteine) Campylobacter Species from Faeces. Eur. J. Clin. Microbiol. Infect.
Dis. 7: 155-160.
Description
α-Ketoglutarate growth supplement for the presumptive identiication
of Legionella spp. For addition to LAB195 BCYE Legionella Isolation
Medium.
α-Ketoglutarate 1000
Add 1 vial per 500mL of sterilised medium as appropriate.
References
ISO 11731:1998(E) Water quality- Detection and enumeration of
Legionella

VPT (Skirrow’s) Selective Supplement products. Hannah Research Inst.
Grifiths, M.W., and Phillips, J.D., and Muir, D.D. (1986) Aust. J.
X214 Dairy Technol. 41, 77-79.
VANCOMYCIN, POLYMYXIN, TRIMETHOPRIM, to make

Skirrow’s medium for the isolation of Campylobacter spp.
Suitable for use with LAB001 Columbia Agar or other blood agar CFC Supplement
bases, supplemented with lysed horse blood.
X223
MODIFIED C.F.C. – CEPHALOTHIN, FUCIDIN, CETRIMIDE
Vancomycin 10 for the selective isolation of Pseudomonas spp.
Polymyxin 2500 iu/litre When added to LAB108 Pseudomonas Agar, to prepare C.F.C.
medium this supplement can be used to select pseudomonads from
Trimethoprim 5 food and environmental samples.
Add 1 vial of X214 to 1 litre of medium
Rehydrate contents of vial with 5ml sterile deionised water. Add Final Concentration mg/litre
aseptically to sterilised medium cooled to 47˚C, along with other
additives, mix well and pour. Cephalothin 50
Fucidin 10
Reference:
Skirrow, M.B. (1977) British Medical Journal 2 11-9. Cetrimide 10
Add 1 vial X223 to 2,000ml medium
Rehydrate contents of vial with 5ml of sterile 50% alcohol. Add
P-INC Supplement - PNCV aseptically to sterilised medium cooled to 47˚C, mix gently and pour.
X219 Reference:
Mead, G.C. and Adams, B.W. (1977). Br. Poult. Sci. 18: 661-667
PENICILLIN, NISIN, CRYSTAL VIOLET, for accelerated shelf
life determination of dairy products.
The Pre-incubation test uses a selective mixture to inhibit Gram GC Growth Supplement
positive organisms whilst allowing the growth of Gram negative
bacteria, the main cause of post-pasteurisation contamination and a X271
major factor in determining the shelf life of the product. The technique
is also useful for monitoring plant hygiene. GROWTH SUPPLEMENT, to improve the isolation of Neisseria
spp. from selective media.
Final Concentration mg/litre For addition to GC agar base LAB067.
Penicillin 20,000iu/litre Final Concentration mg/litre

Nisin 40,000iu/litre m L-cystine 11
Crystal violet 2.0 L-cysteine 259
Add 1 vial of X219 to 1 litre of Milk Agar LAB019 Thiamine HCl 0.03
Ferric nitrate 0.2
Co-Carboxylase 1
Rehydrate contents of 1 vial with 5ml sterile deionised water. Add NAD 1.0
aseptically to sterilised medium cooled to 47˚C, mix thoroughly and
pour plates. Guanine HCl 0.3
Method A Adenine 10
Pre-incubate test material at 21˚C for 24hr. Prepare suitable dilution L-glutamine 100
series, and inoculate Milk Agar plates containing P-INC supplement. PABA 0.13
Incubate at 21˚C for 24hr, and count all colonies (some may be small,
use of a hand lens is recommended). Calculate the CFU/ml and using Vitamin B12 0.1
the tables of Grifith’s et al the shelf life can be determined.
Add 1 vial to 1 litre of medium
Method B
Rehydrate X219 with 1ml of deionised water only, add 0.1ml to the Rehydrate contents of vial with 5ml sterile deionised water. Add
test material and incubate at 20˚C for 24hr. Prepare suitable dilution aseptically to sterilised medium cooled to 47˚C, along with other
series, and inoculate Milk Agar plates. Proceed as for Method A additives, mix well and pour.
above.
References:
Grifiths, M.W., and Phillips, J.D. (1985) J.Appl.Bact. 57, 107.
Grifiths, M.W., and Phillips, J.D., and Muir, D.D. (1980) J. Soc.
Dairy Technol. 33, 8.
Grifiths, M.W., and Phillips, J.D., and Muir, D.D. (1984) Rapid
detection of post-pasteurised contamination. Hannah Research Inst.
Bulletin No.10.
Grifiths, M.W., and Phillips ,J.D., and Muir, D.D. (1984) Dairy Ind.
Int. 50 (3) 25
Grifiths, M.W., and Phillips, J.D., and Muir, D.D. (1984) Post-
pasteurisation contamination - the major cause of failure of fresh dairy

Polymyxin B Half Fraser Supplement
X274 X564
POLYMYXIN for the isolation of B. cereus from foods. HALF FRASER supplement for the primary enrichment of
Listeria spp from food and environmental samples.
Suitable for the preparation of LAB073 Bacillus cereus Medium
(P.R.E.P.). The addition of X073 sterile egg yolk emulsion is also For addition to LAB164 Fraser Broth Base
required. For smaller volumes X164 is available (1 vial per 450ml)
For smaller volumes X074 is available (1 vial per 500ml) Final Concentration mg/litre
Final Concentration Ferric ammonium citrate 500
Polymyxin B 8mg/litre = 64,000i.u/litre Acrilavine 12.5
Add 1 vial X074 to 500ml medium Nalidixic acid 10
Add 1 vial X274 to 2L medium Add 1 vial of X564 to 2.25 litres of Fraser Broth Base
Rehydrate contents of vial with 5ml of sterile deionised water. Add Rehydrate contents of vial with 2ml 50% methanol (5ml for X564).
aseptically to sterilised medium cooled to 47˚C together with egg Add aseptically to sterilised medium cooled to 47˚C, mix well and
yolk emulsion, mix gently and pour. pour.
Reference:
Micro-organisms in Food. Ed. Thatcher, F.S., Clarke, D.F. published
by Univ. of Toronto Press.
Nalidixic Acid
X291
NALIDIXIC ACID for the isolation of non-sporing anaerobes
from clinical material.
Suitable for use with LAB090 Fastidious Anaerobe Agar. When
used with other blood agar bases, e.g. LAB001 Columbia Agar,
further enrichment of the medium with haemin, menadione and
sodium pyruvate is beneicial. The addition of Polysorbate 80, which,
enhances the growth of anaerobic cocci, to the medium is required for
N.A.T. medium. The Polysorbate 80 may be added before sterilisation
at a concentration of 0.1%.

Nalidixic acid 10
Add 1 vial X291 to 1 litre medium
Rehydrate contents of each vial with 5ml sterile deionised water. Add
additives, mix gently and pour.
Reference:
Wren, M.W.D., 1980. J. Clin. Path. 33: 61-65. Multiple Selective
Media for the isolation of anaerobic bacteria.
VCC Supplement
X546
V.C.C. Supplement for the selective enrichment of E. coli O157:H7
from food and other samples.
For use with Buffered Peptone Water LAB046

Vancomycin 8.0
Ceixime 0.05
Cefsulodin 10.0
Add 1 vial of X546 to 2.25 litres of LAB046
Rehydrate the contents of one vial with 20ml of sterile deionised

water. Add aseptically to sterilised medium cooled to 47˚C. Mix well
and dispense into 225ml aliquots.

Typical Analysis
9. OrganoTone™ Media
Constituents Gel strength (Nikan) 650-1000g/m2
Colourimetry (1.5% soln at 65˚C) > 0.28 at 340nm
Sourcing > 0.02 at 525nm

The Lab M range of media constituents are selected on the basis of Melting point > 85˚C
quality and performance from the world’s leading suppliers. It is a
deliberate policy not to invest in our own peptone manufacturing Setting point 32-35˚C
facility in order to allow our microbiologist freedom to choose the pH 6.5-7.4
best ingredients available on the international market.
Moisture < 10%
Agars
Total ash < 3%
A range of agars are offered to suit all microbiological applications.
Koch originally used gelatin to solidify culture media, but the superior Calcium < 0.02%
properties of agar resulted in its universal adoption as the gelling Magnesium < 0.02%
agent of choice. Careful selection of agars is vital as they can interact
with nutrient components in a beneicial or deleterious manner. Sodium chloride < 1.0%
Iron < 0.01%
Peptones and Extracts
Like agars, peptones and extracts are biologically variable products Insoluble ash < 0.1%
requiring careful selection. They provide the amino acids and peptides Sulphate 1.5%
required by micro-organisms for growth as well as other vital growth
factors such as minerals, vitamins and nucleic acid fractions. Salmonella Absent
To ensure we use only the best available peptones and extracts these TVC < 10 3/g
materials are exhaustively tested. Growth parameters are obtained
by classical microbiological techniques and by automated growth Spores < 2/g
rate analysis. Chemical and physical properties are also closely
monitored. Lab M can select speciic peptones for special purposes
such as vaccine production and fermentation processes. If more
information is required on special services please contact Lab M or
your local agent.
Agar No. 2 Bacteriological
MC006
Acid Hydrolysed Casein
A bacteriological agar which gives a irm gel at working concentrations
MC007 of 1.0 to 1.5% which is reasonably clear. This agar is recommended
for all culture media except sensitivity testing media and those where
A soluble protein hyrolysate obtained by digesting casein with hot absolute clarity is advantageous.
acid. It is almost free from growth factors, vitamins and antagonists,
and these qualities make it suitable for use as a protein source in Typical Analysis
media for antibiotic and vitamin assays.
Gel strength (Nikan) 650-1000g/m2
Typical Analysis Colourimetry (1.5% soln at 65˚C) > 0.3 at 340nm
Appearance cream/white powder >0.04 at 525nm
Solubility in water at 5% total Appearance cream/white powder
Clarity clear and colourless Melting point >85˚C
pH of 2% solution 6.0 ± 0.5 Setting point 32-35˚C
Total Nitrogen 8.3% ± 0.5 pH 6.5-7.4

Total Amino Nitrogen 6.1% ± 0.5 Moisture <12%
Total ash <3.5%
Calcium < 0.5%
Agar No. 1 Bacteriological Magnesium < 0.1%
Sodium chloride <1.0%
MC002
Iron < 0.01%
A high clarity agar with good gelling properties and a low concentration
Insoluble ash < 0.1%
of metal ions. This agar is suitable for all bacteriological purposes
including sensitivity testing and pour plate techniques. A irm gel is Sulphate < 3.0%
obtained at working concentrations of 1.0 to 1.5%. No signiicant
precipitation is observed on reheating or prolonged holding at 65˚C. Salmonella Absent
TVC < 103/g
Spores < 2/g
133 Media Constituents

Agar No.4 Beef Extract
Plant Tissue Culture Grade
MC019
MC029 This complements the nutritive properties of peptones in culture
media and is often used as an added enrichment. Beef extract can be
Lab M Agar No.4 has been selected speciically for use as a gelling
used as a direct replacement for meat peptones and, as it contains no
agent in plant tissue culture techniques. The product is selected
carbohydrates, can be used as a component of media for fermentation
primarily on gel strength, a parameter of particular importance for this
studies.
application, and then tested to ensure it meets the parameters set by a
major plant producer. The agar contains no nutrients for plant growth
and is designed to be incorporated into classical formulations such as Typical Analysis
Murashige and Skoog, as well as customer’s own formulations.
Appearance light brown powder
Typical analysis Solubility in water at 5% total
Ash 2.30% Clarity clear, light brown colour
Acid Insoluble Ash 0.16% pH of 2% solution 7.0 ± 0.2
Calcium 0.31% Total Nitrogen 12.0% ± 0.5
Magnesium 0.12% Amino Nitrogen 1.6% ± 0.5
Iron 0.018%
Total Nitrogen 0.15%
Recommended Concentration 0.75 - 1.5% Bile Salts No. 3
Melting Point 88-91˚C MC025
Setting Point 32-33˚C
A reined bile salt, comprising mainly sodium cholate and sodium
Mesh 80 desoxycholate. It is used as a selective agent in culture media such as
pH (1.5% at 20˚C) 7.0 ± 0.2 Violet Red Bile Agar for the enumeration of coliforms, MacConkey
Agar No. 3 for the isolation and differentiation of enteric organisms,
Gel Strength (1.5% W/V) >700g/cm 2 and SS Agar for the isolation of enteric pathogens. This fraction of
bile is highly active, allowing maximum selection of organisms of
enteric origin at relatively low concentrations (0.15%).
Typical Analysis
Bacteriological Peptone
Appearance white powder
MC024
Solubility in water at 2% total
An economical source of nutrients provided by a balanced mixture
of meat peptones and tryptone. The growth requirements of most non Clarity clear
fastidious organisms will be fulilled by the range of amino acids, pH of a 2% solution 8.0 ± 0.5
peptides and proteoses in this mixture.
Typical Analysis
Appearance cream powder FMV (Foot and Mouth Vaccine)

Solubility in water at 5% total Peptones
Clarity clear, pale straw colour
MC033
pH of 2% solution 7.2 ± 0.2
Description
Total Nitrogen 12% ± 0.5 A special blend of peptones developed for use in the production of
Amino Nitrogen 5% ± 0.5 Foot and Mouth vaccine.
Typical analysis:
Balanced Peptone No. 1 Appearance cream powder

MC004
Clarity clear, straw colour
A rich mixture of tryptone and meat peptones which fulills the
nutritional demands of a wide variety of micro-organisms. This pH of 2% solution 7.0 ± 0.2 Total
peptone is used in many Lab M culture media formulations.
Nitrogen 10.0 - 14.5%
Typical Analysis Amino Nitrogen 2.5 - 5.5%
Appearance beige powder

Total Nitrogen 12.8% ± 0.5
Amino Nitrogen 5.1% ± 0.5
Media Constituents 134

Gelatin Powder Lecithin
(Soya Bean Lecithin)
MC015
MC041
Description
A collagenous protein used for the solidiication of culture media and Description
for the detection and differentiation of certain proteolytic bacteria. A deoiled, powdered soybean lecithin for use in microbiological
culture media.
Typical Analysis:
Composition
Appearance buff crystalline powder A mixture of polar (phospho- and glyco-) lipids and a small amount
pH 5.7 of carbohydrates.
Acetone insolubles min. 97%
Oil max. 1.5%
Phosphatidylcholine 20-24%
Glucose (Dextrose) Phosphatidylethanolamine 18-22%
Phosphatidylinositol 12-15%
MC013 Phosphatidic acid 4-7%
Fatty acids (total content) approx. 50%
Description including saturated fatty acids, monounsaturated fatty acids &
Glucose for use in microbiological culture media. polyunsaturated fatty acids
Typical analysis:
IPTG (Isopropyl-β-D-
pH (1% aqueous solution) 6-7
Thiogalactopyranoside)
Total plate count <1000/g
MC402 Solubility in:
Description water dispersible
Isopropyl-β-D-Thiogalactopyranoside (IPTG) is used as an inducer fats / oils soluble
of the lac Z operon. Incorporation of this compound into media
containing X-β-Galactoside (MC405), or equivalent chromogenic
compound, enhances the colour development of organisms capable
Appearance
of fermenting lactose. This combination of compounds is especially Fine, beige powder
useful in transformation experiments involving Escherichia coli where Hazard classiication
disruption of the lac Z operon is used as a marker for DNA insertion. NR – Not regulated
Storage
Store at 10-25˚C. Do not store above 25˚C.
Lactalbumin Hydrolysate This component is suitable for use in the preparation of microbiological
culture media. Other uses and applications are subject to validation by
MC040 the end user.
Description
Lactalbumin is a protein removed from the whey, left after removal
of casein from milk. Lactalbumin hydrolysate is a pancreatic digest of
Liver Digest
these proteins, containing high levels of essential amino acids. It can
be used for tissue culture media and for production of vaccines of viral MC034
origin. Other uses include growth of lactobacilli, clostridial spores and
certain fermentation procedures. Liver digest is prepared by the controlled hydrolysis of liver. It is rich
in vitamins and essential amino acids and has excellent nutritional
properties and especially favours the growth of strict anaerobes and
Typical analysis:
other fastidious microorganisms. Due to its capacity to stimulate
sugar metabolism in saccharolytic organisms it is perfectly suited
Appearance cream powder for the growth of a broad range of organisms such as Clostridium,
Solubility in water at 5% total Leuconostoc, Bacillus, homo and hetero-fermentative Lactic acid
bacteria, as well as yeasts and ilamentous moulds.
Typical Analysis
Total Nitrogen 12.0% ± 0.5 Appearance light brown powder
Amino Nitrogen 5.5 ± 0.5 Solubility in water at 5% total
Clarity clear, brown colour
pH of 2% solution 6.0 - 7.0
Lactose Total Nitrogen 9.5 - 11.5%
MC020 Amino Nitrogen 4.0 - 6.0%
Description Storage
Lactose for use in microbiological culture media. Dehydrated culture media: 10-25o C away from direct sunlight.

Malt Extract MUG (4-Methylumbelliferyl-β-
MC023 D-Glucuronide)
A water soluble extract of malted barley suitable for use in the MC406
cultivation of yeasts and moulds. Malt extract has a very high
carbohydrate content and consequently is very sensitive to over Description
heating which will cause a darkening of the medium. MUG is a luorogenic compound used for the speciic detection of E.
coli in bacteriological culture media. MUG reagent is cleaved by the
Typical Analysis enzyme glucuronidase to release luorescent 4-methylumbelliferone
that can be detected under long wave UV light (366nm) as a blue/
Appearance yellow/brown powder green luorescence. MUG can be incorporated into a range of culture
media to enhance detection of E. coli.
Clarity clear, light brown colour
pH of 2% solution 5.2 ± 0.2 Mycological Peptone
Maltose 55% MC009
Other Carbohydrates 40%
A mixture of peptones with a high carbohydrate content suitable for
Protein 5% the rapid growth and colonial development of yeasts and moulds.
Bacterial growth is inhibited by the low pH of this peptone.
Typical Analysis
Maltose Monohydrate Appearance beige powder
MC022 Solubility in water at 5% total
Description Clarity clear, pale straw colour
Maltose for use in microbiological culture media.
Total Nitrogen 13% ± 0.5
Mannitol Amino Nitrogen 1.4% ± 0.5
MC014
Description Ox Bile
D-Mannitol for use in microbiological culture media.
MC010
Description
Meat Peptone
Ox bile is a dehydrated, puriied fresh bile used as a selective agent
MC018 in bile media, such as Brilliant Green Bile 2% Broth (LAB051).
Description
A highly nutritious enzymatic digest of meat for use in microbiological Proteose Peptone A
culture media
MC011
Typical Analysis:
An enzymatic digest of meat adapted to encourage the production of
Appearance cream powder toxins by Corynebacterium diphtheriae, staphylococci, salmonellae,
and clostridia. This peptone is highly nutritious and suitable for use in
Solubility in water at 5% total culture media for fastidious organisms such as Neisseria, Haemophilus
Clarity clear, pale straw colour and Pasteurella species.
pH of 2% solution 6.7 - 7.3 Typical Analysis

Total Nitrogen 11.0 - 13.0%
Appearance cream powder
Amino Nitrogen 2.5 - 5.0%
Clarity clear, light straw colour

Skim Milk Powder Soy Peptone
MC027 MC003
A bacteriological grade of thermophile free spray dried skim milk. Prepared using the enzyme papain to digest soyabean meal, this
Used in Milk Plate Count Agar LAB115 and in media for diagnostic peptone is a rich source of nutrients with a high carbohydrate content.
tests involving the digestion or coagulation of casein and the Most organisms will grow rapidly in this peptone but some bacteria
fermentation of lactose. Recommended working concentration 10%. will produce high levels of acid leading to auto-sterilisation unless an
adequate buffering system is incorporated.
Typical Analysis
Typical Analysis
Clarity opaque white suspension Appearance cream powder
Total Nitrogen 5.3% ± 0.5 Solubility in water at 5% total
Lactose 48.0% ± 0.5 Clarity clear, straw colour

Sodium Chloride (Bacteriological) Amino Nitrogen 1.6% ± 0.5
MC017
Description
Sodium chloride for use in microbiological culture media.
Tryptone
MC005
Sodium Desoxycholate An enzymatic hydrolysate of casein, rich in peptones and amino acids
(including tryptophane). This peptone can be utilised by most bacteria
as a growth substrate.
MC026
Sodium desoxycholate is a speciic bile acid, derived from Typical Analysis
deconjugated bile salts. Leifson showed that desoxycholic acid had
the most inhibitory effect on bacterial growth, and that this could Appearance cream powder
be enhanced by the removal of magnesium ions by chelating with Solubility in water at 5% total
sodium citrate. These components comprise the selective agents in
DCA, DCA (Hynes) and DCLS. Clarity clear, pale straw colour
Typical Analysis pH of 2% solution 7.2 ± 0.5

Moisture <5% Tryptose
Heavy metals <20 ppm
MC008
Sodium cholate <2%
A blend of peptones suitable for the cultivation of most fastidious
organisms including Neisseria gonorrhoeae, Streptococcus milleri
and Brucella spp. especially where rapid or profuse growth is
Sodium Thioglycollate required such as in blood culture media and blood agars.
MC016 Typical Analysis

Appearance beige powder
Description
Sodium thioglycollate for use in bacteriological culture media. It is Solubility in water at 5% total
used to lower the oxidation-reduction potential of the medium and for
Clarity clear, light straw colour
the neutralisation of mercurial compound preservatives.

X-β-Galactoside
(5-Bromo-4-Chloro-3-Indolyl-β-D-
Galactopyranoside)
MC405
Description
X-β-galactoside (5-Bromo-4-chloro-3-indolyl-β-D-galactoside) is
used as a chromogenic substrate for the detection of organisms capable
of fermenting lactose (lac Z-positive organisms). In combination
with IPTG (MC402), X-β-Galactoside can be used in transformation
experiments involving Escherichia coli where disruption of the lac Z
operon is used as a marker for DNA insertion.
Yeast Extract Powder

MC001
Prepared by the autolysis of Saccharomyces cerevisae under
thermostatically controlled conditions to protect the B vitamins and
other heat labile constituents. This extract provides a mixture of
amino acids, peptides, vitamins and carbohydrates making it suitable
for many applications.
Typical Analysis
Appearance yellow powder
Clarity clear, pale yellow

139
TYPICAL ANALYSIS
MC007 MC024 MC004 MC009 MC011 MC003 MC005 MC008 MC001

Acid Hydrolysed Bacteriological Balanced Mycological Proteose Soy Peptone Tryptone Tryptose Yeast
Casein Peptone Peptone No. 1 Peptone Peptone A Extract
Total Nitrogen 8.3% 12.0% 12.5% 12.6% 12.0% 9.0-9.4% 12.8% 12.7% 10.5%
Total AminoNitrogen 6.1% 5.0% 5.1% 1.4% 5.8% 1.6-1.8% 4.8% 4.9% 5.3%
Lab M Peptones
Amino N/Total N. 73.4% 41.7% 40.8% 11.1% 48.0% 17.7% 37.5% 38.7% 50.4%
Total Amino Acid Assay (mg/g)
Lysine 53.5 62.0 75.3 38.9 48.2 43.1 79.7 77.5 49.0
Histidine 18.7 21.0 26.8 12.7 15.6 16.2 31.2 29.0 14.0
Arginine 22.1 32.0 41.0 55.0 50.6 39.2 37.5 39.2 27.0
Media Constituents
Aspartic Acid 44.1 69.0 58.4 69.9 63.9 74.4 62.8 60.6 52.0
Threonine 23.5 40.0 31.1 17.7 27.5 21.7 36.9 34.0 33.0
Serine 30.0 40.0 40.3 26.2 36.2 27.0 50.3 45.3 34.0
Glutamic Acid 130.0 160.0 138.9 103.5 100.2 110.0 184.0 161.5 73.0
Proline 52.2 46.0 61.0 74.5 55.6 28.0 82.1 71.6 26.0
Glycine 11.1 29.0 44.5 105.9 83.38 22.6 15.6 30.0 25.0
Alanine 19.0 39.0 38.1 49.9 52.3 23.1 26.9 32.5 51.0
Cystine 1.1 1.0 1.1 2.7 8.4 5.3 2.2 1.6 6.0
Valine 35.2 45.0 45.3 22.9 35.2 23.7 59.2 52.3 37.0
Methionine 10.4 8.0 19.1 7.0 12.3 6.2 25.0 22.1 9.0
Isoleucine 27.9 33.0 43.8 18.7 23.3 26.8 58.5 51.1 73.0
Leucine 30.9 65.0 65.3 32.0 55.5 38.6 83.5 74.4 73.0
Tyrosine 12.7 12.0 9.8 12.8 13.3 16.8 14.7 12.2 12.0
Phenylalanine 17.0 34.0 31.8 20.2 27.2 22.7 42.4 37.1 25.0
Tryptophane - 3.0 4.9 1.9 4.6 3.7 6.6 5.7 9.0

Potassium Lactate
10. Sterile Additives
X035
Sterile additive products are offered in ready-to-use format. Each Description
product has been prepared with an appropriate sterilisation procedure,
e.g. aseptic preparation, iltration or irradiation, which will not affect A sterile solution of 10% potassium lactate for use with LAB201
the performance of the product. All sterile reagents should be stored Lysine Medium, for the isolation and enumeration of wild yeasts in
at 2-8ºC away from light. pitching yeasts.
The ready prepared media offer a cost effective alternative to
preparation of large quantities of small volume dispensed media. Directions
Add 1 vial X035 (5ml) Potassium Lactate to 500ml (10ml/litre)
hydrated LAB201 Lysine Medium prior to sterilisation.
HAL010 Listeria Selective Diagnostic
Supplement
X010
Lactic Acid 10%
Description
X036
A selective diagnostic supplement containing cycloheximide, nalidixic Description
acid and phosphatidylinostiol for the isolation and presumptive A sterile solution of 10% lactic acid added to culture media to reduce
identiication of Listeria monocytogenes. For use with HAL010 the pH, in order to suppress bacterial growth.
Harelquin™ Listeria Chromogenic Agar.
For larger volumes X037 is available (5ml)
For larger volumes X210 is available (2.5 litres per vial)
Directions
Add 5 vials (5ml) to 1 litre of Malt Extract Agar LAB037.
Final Concentration
Add 10 vials (10ml) to 1 litre of Potato Dextrose Agar LAB098.
Concentration Per vial Conc. in medium
X010 (mg/litre) Add 1 vial (1ml) to 1 litre of Lysine Agar LAB201.
Addition of X037 should be carried out after sterilisation and cooling
Cycloheximide 25 mg 50 the medium to 47ºC.
Nalidixic acid 10 mg 20
Phosphatidylinositol ~300 mg ~600 Lactic Acid 10%
Instructions for use X037
Pre-heat X010 to 48-50°C and aseptically add to sterilised medium
cooled to 48-50°C. Description
Add 1 vial X010 and 1 vial of X072 to 475mL medium A sterile solution of 10% lactic acid added to culture media to reduce
References the pH, in order to suppress bacterial growth.
ISO 11290-1:1997 Microbiology of food and animal feeding stuffs - For smaller volumes X036 is available (1ml)
Horizontal method for the detection of Listeria monocytogenes - Part Directions
Add 1 vial (5ml) to 1 litre of Malt Extract Agar LAB037.
Add 2 vials (10ml) to 1 litre of Potato Dextrose Agar LAB098.
Addition of X037 should be carried out after sterilisation and cooling
Potassium Tellurite Solution 3.5% the medium to 47ºC.
X027
Description Potassium Tellurite Solution 1%
A sterile solution of 3.5% potassium tellurite. A selective agent for
addition to Hoyles’s Medium (Modiied) (LAB027), for the selective X043
isolation and differentiation of Corynebacterium diphtheriae.
Directions Description
Add 1 vial (2ml) to 200ml of Hoyle’s Medium (LAB027). Potassium Tellurite Solution for use with Modiied Giolitti and
Cantoni Broth (ISO) LAB219 for the detection and enumeration of
coagulase-positive staphylococci.
Potassium Lactate Formulation Per vial Conc. in medium
X034 Potassium tellurite 10 g/L 0.1 g/L
Description
A sterile solution of 10% potassium lactate for use with LAB201 Addition
Lysine Medium, for the isolation and enumeration of wild yeasts in Aseptically add to media tempered to 44-47ºC suficient volume of
pitching yeasts. X043 1% potassium tellurite to give a inal concentration of 0.1g/L.
For larger volumes X035 is available (1 vial per 500ml) For example, add 0.1ml X043 to 9ml of single strength base or add
0.2ml X043 to 10ml of double strength base.
Directions
Appearance
Add 1 vial X034 (1ml) Potassium Lactate to 100ml (10ml/litre)
hydrated LAB201 Lysine Medium prior to sterilisation. Clear, colourless liquid
References
ISO 6888-3:2003 Microbiology of food and animal feeding stuffs
- Horizontal method for the enumeration of coagulase-positive
staphylococci (Staphylococcus aureus and other species) - Part 3:
Detection & MPN technique for low numbers.
Sterile Additives 140

Egg Yolk Emulsion HAL010 Listeria Selective Diagnostic
X073
Supplement
For larger volumes X573 is available (500ml) X210
Description
Description
A sterile emulsion of egg yolks for use in bacteriological culture
media. It may be added directly to nutrient media for the identiication A selective diagnostic supplement containing cycloheximide, nalidixic
of Clostridium, Bacillus and Staphylococcus species by their lipase acid and phosphatidylinostiol for the isolation and presumptive
and/or lecithinase activity. identiication of Listeria monocytogenes. For use with HAL010
Harelquin™ Listeria Chromogenic Agar.
Presented in 100ml bottles, add 100ml to 900ml of Bacillus cereus
medium (PREP and PEMBA, LAB073 and LAB193), 40 ml to For smaller volumes X010 is available (500 mL per vial)
Brazier’s CCEY Agar LAB160, or 50ml to Blood Agar Base LAB028
containing Fildes extract and serum. Final Concentration
Technique Concentration Per vial Conc. in medium
X210 (mg/litre)
For detection of lecithinase activity (especially in the investigation of
`bitty cream’ conditions) add 0.5 or 1.0ml of the emulsion to 10ml of Cycloheximide 125 mg 50
sterile Blood Agar Base (LAB028) or Nutrient Broth No.2 (LAB014).
In order to clear the medium, raise the inal salt concentration by the Nalidixic acid 50 mg 20
addition of 1% of sodium chloride. After incubation for up to 5 days at
35ºC, lecithinase-producers render the broth opalescent, whilst, on the Phosphatidylinositol ~1500 mg ~600
solid medium, the colonies are surrounded by opaque zones.
Instructions for use
Pre-heat X210 to 48-50°C and aseptically add to sterilised medium
cooled to 48-50°C.
Egg Yolk Tellurite Emulsion Add 1 vial of X210 and 5 vials of X072 to 2375mL medium.
X085 References
ISO 11290-1:1997 Microbiology of food and animal feeding stuffs -
Description Horizontal method for the detection of Listeria monocytogenes - Part
A sterile egg yolk emulsion containing potassium tellurite for use
in Baird-Parker Medium Base (ISO) LAB285 or Baird-Parker
Medium Base LAB085.
Potassium tellurite 0.20% w/v
Egg Yolk Emulsion
Egg yolk emulsion 20% X573
Final concentration of tellurite in medium is 0.01% w/v. For smaller volumes X073 is available (100ml)
Description
Appearance A sterile emulsion of egg yolks for use in bacteriological culture
Yellow, opaque solution with resuspendable deposit. media. It may be added directly to nutrient media for the identiication
Storage of Clostridium, Bacillus and Staphylococcus species by their lipase
and/or lecithinase activity.
Once opened store at 2-8°C. A deposit may form during storage. This
is normal and will not affect the product. Mix well before use. Presented in 100ml bottles, add 100ml to 900ml of Bacillus cereus
medium (PREP and PEMBA, LAB073 and LAB193), 40 ml to
Method for reconstitution Brazier’s CCEY Agar LAB160, or 50ml to Blood Agar Base LAB028
Add 50mL (5%) X085 to 1 litre of sterilised media, tempered to containing Fildes extract and serum.
48oC.
Technique
For detection of lecithinase activity (especially in the investigation of
`bitty cream’ conditions) add 0.5 or 1.0ml of the emulsion to 10ml of
sterile Blood Agar Base (LAB028) or Nutrient Broth No.2 (LAB014).
Urea Solution 40% In order to clear the medium, raise the inal salt concentration by the
addition of 1% of sodium chloride. After incubation for up to 5 days at
X130 35ºC, lecithinase-producers render the broth opalescent, whilst, on the
solid medium, the colonies are surrounded by opaque zones.
Description
A sterile solution of 40% urea, for addition to Urea Broth Base
(LAB131) and Urea Agar Base (LAB130) for the detection of urease
production by Proteus spp.
For larger volumes X135 is available (100ml)
Directions
Add 1 vial X130 (5ml) to 95ml of Urea Broth Base (LAB131) and
Urea Agar Base (LAB130).
Urea Solution 40%

X135
Description
A sterile solution of 40% urea, for addition to Urea Broth Base
(LAB131) and Urea Agar Base (LAB130) for the detection of urease
production by Proteus spp.
For smaller volumes X130 is available (5ml)
Directions
Add 5ml to 95ml of Urea Broth Base (LAB131) and Urea Agar Base
(LAB130).
141 Sterile Additives

NOTES
142
8. INDEXES
By product code
Dehydrated Culture Media LAB067 GC Agar Base Page 42

LAB068 Nutrient Broth ‘E’ Page 65
LAB001 Columbia Agar Base Page 26 LAB069 Simmons Citrate Agar Page 79
LAB002 MacConkey Agar (without salt) Page 53 LAB071 Fastidious Anaerobe Broth (FAB) Page 39
LAB003 D.C.L.S. Agar Page 29 LAB072 Tryptone Bile Agar Page 84
(Desoxycholate Citrate Lactose Sucrose)
LAB073 PREP (Bacillus Cereus Medium) Page 14
LAB004 Tryptone Soy Broth (USP/EP JP) Page 85
LAB075 Todd Hewitt Broth Page 83
(Soybean Casein Digest Medium)
LAB077 ColourScreen™ MLSTB-MT (ISO) Page 59
LAB005 MacConkey Broth Purple Page 54
LAB078 C.E.M.O. Agar Page 24
LAB006 C.L.E.D. Medium (Bevis modiication) Page 26
(Contagious Equine Metritis Organism)
LAB007 Mannitol Salt Agar Page 55
LAB079 W.L. Nutrient Agar Page 91
LAB008 Nutrient Agar Page 64 (Wallerstein Laboratory)
LAB009 Sabouraud Dextrose Agar Page 76 LAB080A Minerals Modiied Glutamate Medium Page 58
LAB010 Plate Count Agar APHA Page 70 LAB080B Sodium Glutamate Page 58
LAB011 Tryptone Soy Agar Page 85 LAB081 CSEB - Cronobacter sakazakii Page 27
(Soybean Casein Digest Medium) Enrichment Broth
LAB012 Sensitivity Test Agar (STA) Page 78 LAB082 Membrane Lauryl Sulphate Broth Page 56
LAB013A Bismuth Sulphite Agar Base ‘A’ Page 17 LAB085 Baird-Parker Medium Base Page 14
LAB013B Bismuth Sulphite Chemical Mixture ‘B’ Page 17 LAB086 Rappaport Vassiliadis Medium (RVS) Page 74
LAB014 Nutrient Broth No.2 Page 65 LAB087 Sugar Free Agar Page 81
LAB015 Blood Agar Base No. 2 Page 18 LAB088 Violet Red Bile Glucose Agar (VRBGA) Page 90
LAB018 Yeast Extract Agar (Yeastrel Milk Agar) Page 94 LAB089 Oxytetracycline Glucose Yeast Extract Page 67
LAB019 Milk Agar Page 57 Agar (O.G.Y.E)
LAB020 Dextrose Tryptone Agar Page 32 LAB090 Fastidious Anaerobe Agar (FAA) Page 39
LAB023 Reinforced Clostridial Agar Page 74 LAB091 E.E. Broth Page 36
(Enterobacteriaceae Enrichment)
LAB027 Hoyles Medium (Modiied) Page 44
LAB092 M17 Agar Page 52
LAB028 Blood Agar Base Page 18
LAB093 MRS Agar Page 60
LAB029 Desoxycholate Citrate Agar Page 28
LAB094 MRS Broth Page 62
LAB030 MacConkey Agar (with salt) Page 52
LAB095 DN’ase Agar Page 34
LAB031 Violet Red Bile Agar (VRBA) Page 89
LAB096 Cholera T.C.B.S. Medium Page 82
LAB032 XLD Agar Page 92
LAB097 Tetrathionate Broth (APHA) Page 82
LAB034 Brilliant Green Agar (Modiied) Page 20
LAB098 Potato Dextrose Agar Page 71
LAB035 TYC Medium (Tryptone Yeast Cystine) Page 87
LAB099 Wort Broth (Modiied) Page 92
LAB036 Rose Bengal Chloramphenicol Agar Base Page 76
LAB100Z Ringers Solution 1/4 Strength Tablets Page 76
LAB037 Malt Extract Agar Page 55
LAB103 Maximum Recovery Diluent Page 56
LAB038 Wort Agar Page 91
LAB104 Peptone Water Page 69
LAB039 Mueller Hinton Agar Page 63
LAB106 Kanamycin Aesculin Azide Agar Page 45
LAB041 C.L.E.D. Medium (single indicator) Page 25
(complete)
LAB044A Selenite Broth Base Page 77
LAB107 Kanamycin Aesculin Azide Broth Page 46
LAB044B Sodium Biselenite Page 78 (complete)
LAB045 MacConkey Agar No.3 Page 54 LAB108 Pseudomonas Agar Base Page 71
LAB046 Buffered Peptone Water Page 22 LAB109 Perfringens Agar (O.P.S.P.) Page 69
LAB048 Brain Heart Infusion Agar Page 19 LAB110 Hektoen Enteric Medium Page 44
LAB049 Brain heart Infusion Broth Page 19 LAB111 Sabouraud Maltose Agar Page 77
LAB051 Brilliant Green Bile 2% Broth Page 21 LAB112 Campylobacter Blood Free Medium Page 23
LAB052 S.S. Agar (Salmonella Shigella Agar) Page 80 (mCCDA)
LAB053 Triple Sugar Iron Agar Page 83 LAB114 Mueller Hinton Broth Page 63
LAB054 Lysine Iron Agar Page 51 LAB115 Milk Plate Count Agar Page 57
LAB055A Selenite Cystine Broth Base Page 78 LAB116 MLCB Agar Page 58
LAB059 Kligler Iron Agar Page 46 LAB117 Dermatophyte Test Medium (D.T.M.) Page 31
LAB060 Endo Agar Page 37
LAB061 Eosin Methylene Blue Agar (Levine) Page 37
LAB062 Tryptose Phosphate Broth Page 86
LAB063 Tryptone Glucose Extract Agar Page 85
LAB064 Thioglycollate Medium (Brewer) Page 83
LAB065 Desoxycholate Citrate Agar (Hynes) Page 29
143
LAB119 Yeast Extract Dextrose Chloramphenicol Page 94 LAB198 Raka-Ray No.3 Agar Page 72
Agar LAB199 Raka-Ray No.3 Agar Page 73
LAB120 Yersinia Selective Agar Page 95 (Increased Gel Strength)
(Schiemann’s C.I.N. Agar) LAB200 Yeast & Mould Agar Page 94
LAB121 Bromocresol Purple Lactose Agar Page 22 LAB201 Lysine Agar Page 51
LAB122 Listeria Isolation Medium (Oxford) Page 49 LAB202 Mueller Kaufmann Tetrathionate Page 64
LAB126 Lactose Broth Page 47 Novobiocin Broth (MKTTn)
LAB129 Tryptone Water Page 86 LAB203 R2A Broth Page 72
LAB130 Urea Agar Base Page 87 LAB204 Buffered Peptone Water (ISO) Page 22
(Christensen’s Urea Agar Base) LAB205 Tryptone Soy Broth (without dextrose) Page 86
LAB131 Urea Broth Base Page 88 LAB206 Listeria Isolation Media Page 50
LAB135 Campylobacter Enrichment Broth Page 23 LAB207 Bile Aesculin Agar Page 17
LAB209 Rhamnose MacConkey (VTEC O26) Agar Page 75
LAB138 Listeria Enrichment Broth Page 48
LAB211 Half Fraser BrothPLUS Page 43
LAB139 Buffered Listeria Enrichment Broth Page 22
LAB212 Fraser BrothPLUS Page 42
LAB144 PALCAM Broth Page 68
LAB215 Columbia II Agar Base Page 27
LAB147 Orange Serum Agar Page 66
LAB216 MacConkey Agar No.2 Page 53
LAB148 PALCAM Agar Base Page 67
LAB217 DRBC Agar Page 34
LAB149 Plate Count Agar Page 70
LAB218 DG18 Agar Page 32
LAB150 MSRV medium Page 62
(Semi Solid Rappaport Medium) LAB219 Modiied Giolitti and Cantoni Broth (ISO) Page 59
LAB155 UVM Base Medium Page 88 LAB220 DRCM (ISO) Differential Reinforced Page 35
(University of Vermont) Clostridial Medium
LAB159 Malt Extract Broth Page 55 LAB221 XLT4 Agar Page 93
LAB160 Brazier’s CCEY Agar Page 19 LAB222 Iron Sulphite Agar Page 45
LAB161 Sorbitol MacConkey Agar Page 80 LAB223 MRS Agar (ISO) Page 61
LAB163 R2A Medium Page 72 LAB224 Alkaline Saline Peptone Water (ISO) Page 13
LAB164 Fraser Broth Page 41 LAB285 Baird-Parker Medium Base (ISO) Page 15
LAB165 O157 Broth (MTSB) Page 65 LAB425 Fluid Thioglycollate Medium (Clear) Page 40
LAB166 Slanetz & Bartley Medium Page 79 LAB505 Cary Blair Medium Page 24
(Membrane Enterococcus Agar) LAB525 Eugon Agar Page 38
LAB167 Aeromonas Agar Page 13 LAB526 Eugon Broth Page 38
LAB168 L.B. Agar Page 102 LAB537 Diagnostic Semi Solid Salmonella Agar Page 33
LAB169 LB Broth Page 103 (Diassalm)
LAB170 Susceptibility Test (Iso) Agar Page 81 LAB573 Violet Red Bile Agar with MUG Page 89
(VRBA with MUG)
LAB171 E.C. Medium Page 36
LAB589 LEE Broth - Listeria Express Enrichment Page 49
LAB172 Listeria Monocytogenes Blood Agar Page 50
Broth
(LMBA)
LAB173 LB Broth (Lennox) Page 103
LAB174 L.B. Agar (Lennox) Page 103 Harlequin™ Dehydrated Culture Media
LAB175 YPD Broth Page 113
LAB176 YPD Agar Page 113 HAL001 Harlequin™ Salmonella ABC Medium Page 96
LAB179 2xYT Medium Page 106 HAL003 Harlequin™ TBGA Page 96
LAB180 2xYT with Agar Page 106 HAL004 Harlequin™ LB Agar Page 97, 110
LAB181 NZY Broth Page 112 HAL006 Harlequin™ SMAC-BCIG Page 97
LAB182 NZCYM Broth Page 112 HAL008 Harlequin™ E.coli / Coliform Medium Page 98
LAB183 Terriic Broth Page 113 HAL009 Harlequin™ mLGA Page 98
LAB184 Letheen Broth Page 48 HAL010 Harlequin™ Listeria Chromogenic Agar Page 99
(ISO 11290)
LAB185 Letheen Agar Page 47
HAL012 Harlequin™ CSIM (ISO) - Cronobacter Page 100
LAB186 D.E. Neutralising Broth Base Page 31
sakazakii Isolation Medium
LAB187 D.E. Neutralising Broth Page 30
HAL013 Harlequin™ CSA-DFI - Cronobacter Page 100
LAB188 D.E. Neutralising Agar Page 29 sakazakii Agar - DFI formulation
LAB189 Microbial Content Test Agar (M.C.T.A.) Page 57
LAB191 LB Broth (Hi-Salt) Page 104
Harmonised Pharmacopoeia
LAB192 O.R.S.I.M. (Oxacllin Resistant Page 66 HP001 Fluid Thioglycollate Medium (USP/EP/JP) Page 104
Staphylococci Isolation Medium)
HP002 Casein Soya Bean Digest Broth (USP/EP/JP) Page 102
LAB193 PEMBA (Bacillus Cereus Medium) Page 68
HP003 Enterobacteria Enrichment Broth Page104
LAB194 Perfringens Agar (T.S.C.) Page 69
– Mossel (USP/EP/JP)
LAB195 BCYE Legionella Isolation Medium Page 16
HP004 Violet Red Bile Glucose Agar (USP/EP/JP) Page 108
LAB196 Lauryl Tryptose Broth Page 47
HP005 MacConkey Broth (USP/EP/JP) Page 105
LAB197 Water Plate Count Agar Page 90
HP006 MacConkey Agar (USP/EP/JP) Page 105
144
HP007 Rappaport Vassiliadis Salmonella Page 107 µPREP™ Ready-To-Reconstitute Media
Enrichment Broth (USP/EP/JP)
HP008 Xylose Lysine Deoxycholate Agar Page 109 MPB001 µPrep™ BPW (ISO) Page 115
(USP/EP/JP) MPB004 µPrep™ Half Fraser Broth ISO (+FAC) Page 115
HP009 Mannitol Salt Agar (USP/EP/JP) Page 106 MPA001 µPrep™ Filter Unit Page 115
HP010 Cetrimide Agar (USP/EP/JP) Page 103 MPA002 µPrep™ Quick Connectors Page 115
HP011 Reinforced Medium for Clostridia Page 107
(USP/EP/JP)
CaptivateTM
HP012 Columbia Agar (USP/EP/JP) Page 103
HP013 Sabouraud Dextrose Broth (USP/EP/JP) Page 108 CAP001 Captivate™ O157 Page 116
HP014 Sabouraud Dextrose Agar (USP/EP/JP) Page 108 CAP003 Captivate™ O26 Page 117
HP015 Potato Dextrose Agar (USP/EP/JP) Page 106 CAP004 Captivate™ O111 Page 117
HP016 Casein Soya Bean Digest Agar (USP/EP/JP) Page 102 CAP005 Captivate™ O103 Page 117
HP017 Buffered Sodium Chloride-Peptone Solution Page 102 CAP006 Captivate™ O145 Page 117
pH 7.0 (USP/EP/JP) CAP007 Captivate™ O104 Page 117
CAP008 Captivate™ O121 Page 117
Media Constituents CAP009 Captivate™ O45 Page 117
CAP010 Captivate™ O91 Page 117
MC001 Yeast Extract Powder Page 138 CAP 100-12P Captivate™ Separator Rack Page 117
MC002 Agar No.1 Bacteriological Page 133
MC003 Soy Peptone Page 137
MC004 Balanced Peptone No.1 Page 134 Pinnacle™ Pre-Poured Plates
MC005 Tryptone Page 137
PIN001 Pinnacle™ LCA Listeria Chromogenic Page 118
MC006 Agar No.2 Bacteriological Page 133 Agar (ISO)
MC007 Acid Hydrolysed Casein Page 133 PIN002 Pinnacle™ TBGA (TBX) Page 118
MC008 Tryptose Page 137 PIN003 Pinnacle™ CSIM (ISO) Page 119
MC009 Mycological Peptone Page 136 PIN004 Pinnacle™ mLGA Page 119
MC010 Ox Bile Page 136 PIN005 Pinnacle™ Salmonella ABC Page 120
MC011 Proteose Peptone A Page 136 PIN006 Pinnacle™ Columbia Agar Base Page 120
MC013 Glucose (Dextrose) Page 135 (25ml ill)
MC014 Mannitol (D-Mannitol) Page 136 PIN007 Pinnacle™ Blood Agar No. 2 Page 120
MC015 Gelatin Powder Page 135 (25ml ill)
MC016 Sodium Thioglycollate Page 137 PIN008 Pinnacle™ Legionella GVPC Medium (ISO) Page 121
MC017 Sodium Chloride (Bacteriological) Page 137 PIN009 Pinnacle™ Legionella BCYE Medium (ISO) Page 121
MC018 Meat Peptone Page 136
MC019 Beef Extract Page 134
Supplements & Additives
MC020 Lactose Page 135
MC022 Maltose Monohydrate Page 136 X009 Chloramphenicol supplement Page 123
MC023 Malt Extract Page 136 X010 HAL010 Listeria selective diagnostic Page 140
MC024 Bacteriological Peptone Page 134 supplement
MC025 Bile Salts No.3 Page 134 X011 Colistin / Nalidixic acid selective Page 123
supplement
MC026 Sodium Desoxycholate Page 137
X012 Colistin & Nalidixic acid selective Page 123
MC027 Skim Milk Powder Page 137
supplement
MC029 Agar No.4 - Plant Tissue Culture Grade Page 134
X013 Colistin & Oxolinic acid selective Page 123
MC033 Tryptose No. 2 Page 134 supplement
MC034 Liver Digest Page 135 X015 Neomycin 75mg Page 124
MC040 Lactalbumin Hydrolysate Page 135 X016 Neomycin 100mg Page 124
MC041 Lecithin Page 135 X018 Kanamycin 75mg Page 124
MC402 IPTG (Isopropyl thiogalacatopyranoside) Page 135 X019 P-INC supplement (PNCV) Page 124
dioxane free
X027 Potassium Tellurite 3.5% Page 140
MC405 X-gal (X-B-Galactoside) Page 138
X034 Potassium Lactate Page 140
MC406 MUG Page 136
X037 Lactic acid 10% 5ml per 500ml Page 140
(4-methylumbelliferyl-B-D-glucuronide)
X043 Potassium Tellurite solution 1% Page 140
X068 V.C.N.T. selective supplement Page 124
X070 L.C.A.T. selective supplement Page 125
X072 Polymyxin, Ceftazidime selective supplement Page 125
X072N Nalidixic acid selective supplement Page 125
X073 Egg Yolk Emulsion Page 141
X074 Polymixin B Page 125
X085 Egg Yolk Tellurite Page 141
145
X086 RPF Supplement Page 125
X089 Oxytetracycline supplement Page 125
X090 Nalidixic Acid & Vancomycin Page 126
X093 Cycloserine/Cefoxitin selective supplement Page 126
X107 CN supplement Page 126
X108 CFC supplement Page 126
X109 Sulphadiazine supplement Page 126
X110 Oleandomycin / Polymixin supplement Page 126
X112 Cefoperazone /Amphotericin selective Page 127
supplement
X114 Modiied Preston Campylobacter supplement Page 127
X115 Campylobacter growth supplement Page 127
X120 CIN selective supplement Page 127
X123 C.N.C.A.F. selective supplement Page 127
X130 Urea 40% 5ml per 95ml Page 141
X132 CVTN supplement Page 128
X135 Urea 40% 100 ml Page 141
X139 N.A.N. selective supplement Page 128
X140 Cepacia selective supplement Page 128
X144 P.A.C. selective supplement Page 128
X150 Novobiocin selective supplement Page 128
X155 UVM I supplement Page 129
X161 Ceixime Tellurite supplement Page 129
X164 Half Fraser Supplement Page 129
X165 Fraser Supplement Page 129
X192 O.R.S.I.M. selective supplement Page 129
X194 D-Cycloserine supplement Page 129
X195 GVPC selective supplement Page 129
X196 BCYE growth supplement Page 130
X197 BCYE growth supplement (no L-cysteine) Page 130
X209 Chloramphenicol supplement Page 130
X210 HAL010 Listeria selective diagnostic Page 141
supplement
X212 Cefoperazone/Amphotericin selective Page 130
supplement
X214 VPT selective supplement Page 131
X219 P-INC supplement (PNCV) Page 131
X223 CFC Supplement Page 131
X271 GC growth supplement Page 131
X291 Nalidixic Acid (1 vial per litre) Page 131
X546 VCC supplement Page 131
146
By product name
Dehydrated Culture Media LAB060 Endo Agar Page 37

LAB061 Eosin Methylene Blue Agar (Levine) Page 37
LAB179 2xYT Medium Page 114 LAB525 Eugon Agar Page 38
LAB180 2xYT with Agar Page 114 LAB526 Eugon Broth Page 38
LAB167 Aeromonas Agar Page 13 LAB090 Fastidious Anaerobe Agar (FAA) Page 39
LAB224 Alkaline Saline Peptone Water (ISO) Page 13 LAB071 Fastidious Anaerobe Broth (FAB) Page 39
LAB073 Bacillus Cereus Medium (P.R.E.P.) Page 14 HP001 Fluid Thioglycollate Medium (USP/EP/JP) Page 41, 104
LAB085 Baird-Parker Medium Base Page 14 LAB425 Fluid Thioglycollate Medium (Clear) Page 40
LAB285 Baird-Parker Medium Base (ISO) Page 15 LAB164 Fraser Broth Page 41
LAB195 BCYE Legionella Isolation Medium Page 16 LAB212 Fraser Broth PLUS
(ISO) Page 42
LAB207 Bile Aesculin Agar Page 17 LAB067 GC Agar Base Page 42
LAB013A Bismuth Sulphite Agar Base ‘A’ Page 17 LAB219 Giolitti and Cantoni Broth (ISO) Modiied Page 59
LAB013B Bismuth Sulphite Chemical Mixture ‘B’ Page 17 LAB195 GVPC Legionella Isolation Medium Page 43
LAB028 Blood Agar Base Page 18 LAB211 Half Fraser BrothPLUS Page 43
LAB015 Blood Agar Base No. 2 Page 18 LAB110 Hektoen Enteric Page 44
LAB048 Brain Heart Infusion Agar Page 19 LAB027 Hoyles Medium (Modiied) Page 44
LAB049 Brain Heart Infusion Broth Page 19 LAB222 Iron Sulphite Agar Page 45
LAB034 Brilliant Green Agar (Modiied) Page 20 LAB106 Kanamycin Aesculin Azide Agar Page 45
LAB051 Brilliant Green Bile 2% Broth Page 21 (complete)
LAB121 Bromocresol Purple Lactose Agar Page 22 LAB107 Kanamycin Aesculin Azide Broth Page 46
(complete)
LAB139 Buffered Listeria Enrichment Broth Page 22
LAB059 Kligler Iron Agar Page 46
LAB046 Buffered Peptone Water Page 22
LAB168 L.B. Agar Page 110
LAB204 Buffered Peptone Water (ISO) Page 22
LAB174 L.B. Agar (Lennox) Page 111
LAB078 C.E.M.O. Agar Page 24
(Contagious Equine Metritis Organism) LAB126 Lactose Broth Page 47
LAB006 C.L.E.D. Medium (Bevis modiication) Page 26 LAB196 Lauryl Tryptose Broth Page 47
LAB041 C.L.E.D. Medium (single indicator) Page 25 LAB169 LB Broth Page 111
LAB112 Campylobacter Blood Free Medium Page 23 LAB191 LB Broth (Hi-Salt) Page 112
(mCCDA) LAB173 LB Broth (Lennox) Page 111
LAB135 Campylobacter Enrichment Broth Page 23 LAB185 Letheen Agar Page 47
LAB184 Letheen Broth Page 48
LAB505 Cary Blair Medium Page 24
LAB589 LEE Broth - Listeria Express Enrichment Page 49
HP010 Cetrimide Agar Page 25, 103
Broth
LAB130 Christensen’s Urea Agar Base Page 87
LAB138 Listeria Enrichment Broth Page 48
LAB160 Clostridium dificile (Brazier’s CCEY) Page 19
LAB122 Listeria Isolation Medium (Oxford) Page 49
Agar Base
LAB206 Listeria Isolation Media Page 50
LAB001 Columbia Agar Base Page 26
LAB172 Listeria Monocytogenes Blood Agar Page 50
LAB215 Columbia II Agar Base Page 27
(LMBA)
LAB081 CSEB - Cronobacter sakazakii Page 27
LAB201 Lysine Agar Page 51
Enrichment Broth
LAB054 Lysine Iron Agar Page 51
LAB003 D.C.L.S. Agar Page 29
(Desoxycholate Citrate Lactose Sucrose) LAB092 M17 Agar Page 52
LAB188 D.E. Neutralising Agar Page 29 LAB030 MacConkey Agar (with salt) Page 52
LAB187 D.E. Neutralising Broth Page 30 LAB002 MacConkey Agar (without salt) Page 53
LAB186 D.E. Neutralising Broth Base Page 31 LAB216 MacConkey Agar No.2 Page 53
LAB117 Dermatophyte Test Medium (D.T.M.) Page 31 LAB045 MacConkey Agar No.3 Page 54
LAB029 Desoxycholate Citrate Agar Page 28 LAB005 MacConkey Broth Purple Page 54
LAB065 Desoxycholate Citrate Agar (Hynes) Page 29 LAB037 Malt Extract Agar Page 55
LAB020 Dextrose Tryptone Agar Page 32 LAB159 Malt Extract Broth Page 55
LAB218 DG18 Agar Page 32 LAB007 Mannitol Salt Agar Page 55
LAB537 Diagnostic Semi Solid Salmonella Agar Page 33 LAB103 Maximum Recovery Diluent Page 56
(Diassalm) LAB082 Membrane Lauryl Sulphate Broth Page 56
LAB095 DN’ase Agar Page 34 LAB189 Microbial Content Test Agar (M.C.T.A.) Page 57
LAB217 DRBC Agar Page 35 LAB019 Milk Agar Page 57
LAB220 DRCM (ISO) Differential Reinforced Page 34 LAB115 Milk Plate Count Agar Page 57
Clostridial Medium LAB080A Minerals Modiied Glutamate Medium Page 58
LAB171 E.C. Medium Page 36 LAB116 MLCB Agar Page 58
LAB091 E.E. Broth Page 36 LAB219 Modiied Giolitti and Cantoni Broth (ISO) Page 59
(Enterobacteriaceae Enrichment)
147
LAB077 ColourscreenTM MLSTB - MT (ISO) Page 59 LAB064 Thioglycollate Medium (Brewer) Page 83
LAB093 MRS Agar Page 60 LAB075 Todd Hewitt Broth Page 83
LAB223 MRS Agar (ISO) Page 61 LAB053 Triple Sugar Iron Agar Page 83
LAB094 MRS Broth Page 62 LAB072 Tryptone Bile Agar Page 84
LAB150 MSRV medium Page 62 LAB063 Tryptone Glucose Extract Agar Page 85
(Semi Solid Rappaport Medium) LAB011 Tryptone Soy Agar Page 85
LAB039 Mueller Hinton Agar Page 63 (Soybean Casein Digest Medium)
LAB114 Mueller Hinton Broth Page 63 LAB004 Tryptone Soy Broth (USP/EP/JP) Page 85
LAB202 Mueller Kaufmann Tetrathionate Page 64 (Soybean Casein Digest Medium)
Novobiocin Broth (MKTTn) LAB205 Tryptone Soy Broth (without dextrose) Page 86
LAB008 Nutrient Agar Page 64 LAB129 Tryptone Water Page 86
LAB068 Nutrient Broth ‘E’ Page 65 LAB062 Tryptose Phosphate Broth Page 86
LAB014 Nutrient Broth No.2 Page 65 LAB035 TYC Medium (Tryptone Yeast Cystine) Page 87
LAB182 NZCYM Broth Page 112 LAB130 Urea Agar Base (Christensen’s Urea Base) Page 87
LAB181 NZY Broth Page 112 LAB131 Urea Broth Base Page 88
LAB165 O157 Broth (MTSB) Page 65 LAB155 UVM Base Medium Page 88
LAB192 O.R.S.I.M. (Oxacllin Resistant Page 66 (University of Vermount)
Staphylococci Isolation Medium) LAB031 Violet Red Bile Agar (VRBA) Page 89
LAB147 Orange Serum Agar Page 66 LAB573 Violet Red Bile Agar with MUG Page 89
LAB089 Oxytetracycline Glucose Yeast Extract Page 67 (VRBA with MUG)
Agar (O.G.Y.E) LAB088 Violet Red Bile Glucose Agar (VRBGA) Page 90
LAB148 PALCAM Agar Base Page 67 LAB197 Water Plate Count Agar Page 90
LAB144 PALCAM Broth Page 68 LAB079 W.L. Nutrient Agar Page 91
LAB193 PEMBA (Bacillus Cereus Medium) Page 68 (Wallerstein Laboratory)
LAB104 Peptone Water Page 69 LAB038 Wort Agar Page 91
LAB109 Perfringens Agar (O.P.S.P.) Page 69 LAB099 Wort Broth (Modiied) Page 92
LAB194 Perfringens Agar (T.S.C.) Page 69 LAB032 XLD Agar Page 92
LAB149 Plate Count Agar Page 70 LAB221 XLT4 Agar Page 93
LAB010 Plate Count Agar APHA Page 70 LAB200 Yeast & Mould Agar Page 94
LAB098 Potato Dextrose Agar Page 71 LAB018 Yeast Extract Agar (Yeastrel Milk Agar) Page 94
LAB073 PREP (Bacillus Cereus Medium) Page 14 LAB119 Yeast Extract Dextrose Chloramphenicol Page 94
Agar
LAB108 Pseudomonas Agar Base Page 71
LAB120 Yersinia Selective Agar Page 95
LAB203 R2A Broth Page 72 (Schiemann’s C.I.N. Agar)
LAB163 R2A Medium Page 72 LAB176 YPD Agar Page 113
LAB198 Raka-Ray No.3 Agar Page 72 LAB175 YPD Broth Page 113
LAB199 Raka-Ray No.3 Agar Page 73
(Increased Gel Strength)
Harlequin™ Dehydrated Culture Media
LAB086 Rappaport Vassiliadis Medium (RVS) Page 74
LAB023 Reinforced Clostridial Agar Page 74 HAL013 Harlequin™ CSA-DFI Page 100
HP011 Reinforced Medium for Clostridia Page 75, 107 HAL012 Harlequin™ CSIM (ISO) Page 100
(USP/EP/JP) HAL008 Harlequin™ E.coli / Coliform Medium Page 98
LAB209 Rhamnose MacConkey (VTEC O26) Agar Page 75 HAL004 Harlequin™ LB Agar Page 97, 110
LAB100Z Ringers Solution 1/4 Strength Tablets Page 76 HAL010 TM
Harlequin Listeria Chromogenic Agar Page 99
LAB036 Rose Bengal Chloramphenicol Agar Base Page 76 (ISO 11290)
LAB052 S.S. Agar (Salmonella Shigella Agar) Page 80 HAL009 Harlequin™ mLGA Page 98
LAB009 Sabouraud Dextrose Agar Page 76 HAL001 HarlequinTM Salmonella ABC Medium Page 96
HP013 Sabouraud Dextrose Broth (USP/EP/JP) Page 77, 108 HAL006 HarlequinTM SMAC-BCIG Page 97
LAB111 Sabouraud Maltose Agar Page 77 HAL003 Harlequin™ TBGA Page 96
LAB044A Selenite Broth Base Page 77
LAB055A Selenite Cystine Broth Base Page 78
Harmonised Pharmacopoeia
LAB012 Sensitivity Test Agar (STA) Page 78
HP017 Buffered Sodium Chloride-Peptone Solution Page 10
LAB069 Simmons Citrate Agar Page 79
pH 7.0 (USP/EP/JP)
LAB166 Slanetz & Bartley Medium Page 79
HP016 Casein Soya Bean Digest Agar (USP/EP/JP) Page 102
(Membrane Enterococcus Agar)
HP002 Casein Soya Bean Digest Broth (USP/EP/JP) Page 102
LAB080B Sodium Glutamate Page 58
HP010 Cetrimide Agar (USP/EP/JP) Page 103
LAB044B Sodium Biselenite Page 78
HP012 Columbia Agar (USP/EP/JP) Page 103
LAB161 Sorbitol MacConkey Agar Page 80
HP003 Enterobacteria Enrichment Broth Page104
LAB087 Sugar Free Agar Page 81
– Mossel (USP/EP/JP)
LAB170 Susceptibility Test (Iso) Agar Page 81
HP001 Fluid Thioglycollate Medium (USP/EP/JP) Page 104
LAB096 T.C.B.S. Cholera Medium Page 82
HP006 MacConkey Agar (USP/EP/JP) Page 105
LAB183 Terriic Broth Page 113
HP005 MacConkey Broth (USP/EP/JP) Page 105
LAB097 Tetrathionate Broth (APHA) Page 82
148
HP009 Mannitol Salt Agar (USP/EP/JP) Page 106 CaptivateTM
HP015 Potato Dextrose Agar (USP/EP/JP) Page 106
HP007 Rappaport Vassiliadis Salmonella Page 107 CAP009 Captivate™ O45 Page 117
Enrichment Broth (USP/EP/JP) CAP010 Captivate™ O91 Page 117
HP011 Reinforced Medium for Clostridia Page 107 CAP005 Captivate™ O103 Page 117
(USP/EP/JP) CAP007 Captivate™ O104 Page 117
HP014 Sabouraud Dextrose Agar (USP/EP/JP) Page 108 CAP004 Captivate™ O111 Page 117
HP013 Sabouraud Dextrose Broth (USP/EP/JP) Page 108 CAP008 Captivate™ O121 Page 117
HP004 Violet Red Bile Glucose Agar (USP/EP/JP) Page 108 CAP006 Captivate™ O145 Page 117
HP008 Xylose Lysine Deoxycholate Agar Page 109 CAP001 Captivate™ O157 Page 116
(USP/EP/JP) CAP003 Captivate™ O26 Page 117
CAP 100-12P Captivate™ Separator Rack Page 117
Media Constituents
Pinnacle™ Pre-Poured Plates
MC007 Acid Hydrolysed Casein Page 133
MC002 Agar No.1 Bacteriological Page 133 PIN007 Pinnacle™ Blood Agar No. 2 Page 120
MC006 Agar No.2 Bacteriological Page 133 (25ml ill)
MC029 Agar No.4 - Plant Tissue Culture Grade Page 134 PIN006 Pinnacle™ Columbia Agar Base Page 120
(25ml ill)
MC024 Bacteriological Peptone Page 134
PIN003 Pinnacle™ CSIM (ISO) Page 119
MC004 Balanced Peptone No.1 Page 134
PIN001 Pinnacle™ LCA Listeria Chromogenic Page 118
MC019 Beef Extract Page 134 Agar (ISO)
MC025 Bile Salts No.3 Page 134 PIN009 Pinnacle™ Legionella BCYE Medium (ISO) Page 121
MC033 Tryptose No. 2 Page 134 PIN008 Pinnacle™ Legionella GVPC Medium (ISO) Page 121
MC015 Gelatin Powder Page 135 PIN004 Pinnacle™ mLGA Page 119
MC013 Glucose (Dextrose) Page 135 PIN005 Pinnacle™ Salmonella ABC Page 120
MC402 IPTG Page 135 PIN002 Pinnacle™ TBGA (TBX) Page 118
(Isopropyl-β-D-Thiogalactopyranoside)
MC040 Lactalbumin Hydrolysate Page 135
Supplements & Additives
MC020 Lactose Page 135
X010 HAL010 Listeria selective Page 140
MC041 Lecithin Page 135 diagnostic supplement
MC034 Liver Digest Page 135 X210 HAL010 Listeria selective Page 140
MC023 Malt Extract Page 136 diagnostic supplement
MC022 Maltose Monohydrate Page 136 X196 BCYE growth supplement Page 130
MC014 Mannitol (D-Mannitol) Page 136 X197 BCYE growth supplement Page 130
(no L-Cysteine)
MC018 Meat Peptone Page 136
X123 C.N.C.A.F. selective supplement Page 127
MC406 MUG Page 136
(4-Methylumbelliferyl-β-D-Glucuronide) X115 Campylobacter growth supplement Page 127
MC009 Mycological Peptone Page 136 X161 Ceixime Tellurite supplement Page 129
MC010 Ox Bile Page 136 X112 Cefoperazone & Amphotericin Page 127
selective supplement
MC011 Proteose Peptone A Page 136
X212 Cefoperazone & Amphotericin Page 130
MC027 Skim Milk Powder Page 137
selective supplement
MC017 Sodium Chloride (Bacteriological) Page 137
X140 Cepacia selective supplement Page 128
MC026 Sodium Desoxycholate Page 137
X108 CFC supplement Page 126
MC016 Sodium Thioglycollate Page 137
X223 CFC Supplement Page 131
MC003 Soy Peptone Page 137
MC005 Tryptone Page 137
MC008 Tryptose Page 137
X120 CIN selective supplement Page 127
MC405 X-gal (X-β-Galactoside) Page 138
X107 CN supplement Page 126
MC001 Yeast Extract Powder Page 138
X012 Colistin & Nalidixic acid Page 123
X011 Colistin / Nalidixic acid Page 123
µPREP™ Ready-To-Reconstitute Media selective supplement
X013 Colistin & Oxolinic acid Page 123
MPB001 µPrep™ BPW (ISO) Page 115
X132 CVTN supplement Page 128
MPB004 µPrep™ Half Fraser Broth ISO (+FAC) Page 115
X093 Cycloserine & Cefoxitin Page 126
MPA001 µPrep™ Filter Unit Page 115
X194 D-Cycloserine supplement Page 129
MPA002 µPrep™ Quick Connectors Page 115
X073 Egg Yolk Emulsion Page 141
X085 Egg Yolk Tellurite Page 141
X165 Fraser Supplement Page 129
X271 GC growth supplement Page 131
X195 GVPC selective supplement Page 129
149
X018 Kanamycin 75mg Page 124
X070 L.C.A.T. selective supplement Page 125
X037 Lactic acid 10% 5ml per 500ml Page 140
X114 Modiied Preston Campylobacter Page 127
supplement
X139 N.A.N. selective supplement Page 128
X291 Nalidixic Acid (1 vial per litre) Page 131
X090 Nalidixic Acid & Vancomycin Page 126
X072N Nalidixic acid selective supplement Page 125
X016 Neomycin 100mg Page 124
X015 Neomycin 75mg Page 124
X150 Novobiocin Page 128
X192 O.R.S.I.M. selective supplement Page 129
X110 Oleandomycin & Polymixin supplement Page 126
X089 Oxytetracycline supplement Page 125
X144 P.A.C. selective supplement Page 128
X072 Polymyxin, Ceftazidime selective Page 125
supplement
X034 Potassium Lactate Page 140
X043 Potassium Tellurite 1% Page 140
X027 Potassium Tellurite 3.5% Page 140
X086 RPF Supplement Page 125
X109 Sulphadiazine supplement Page 126
X130 Urea 40% 5ml per 95ml Page 141
X135 Urea 40% 100 ml Page 141
X155 UVM I supplement Page 129
X068 V.C.N.T. selective supplement Page 124
X546 VCC supplement Page 131
X214 VPT (Skirrow’s) selective supplement Page 131
150
NOTES
151
NEWS
LAB M LAUNCHES PINNACLE™ GVPC LAB M JOINS NEOGEN
AND BCYE FOR LEGIONELLA TESTING
August 2015 saw the acquisition of Lab M by worldwide
We are proud to expand our Pinnacle™ range with the food and animal safety specialists, Neogen Corporation
addition of pre-poured plates for Legionella GVPC (NASDAQ: NEOG). Lab M’s existing range of microbial
and BCYE. Both are ISO formulation compliant and testing and diagnostic products complement Neogen’s
performance compliant according to BS EN ISO food safety capability perfectly. The future looks bright for
11133:2014 to support laboratories to meet their QC both companies as they drive forward their R&D and new
requirements, including the revised panel of non-target
product development efforts to provide new and innovative
organisms and the complete inhibition of Enterococcus
solutions for food safety, water, industrial and clinical
faecalis. This supports laboratories for their irst UKAS
markets.
accreditation following the implementation of the new
BS EN ISO 11133:2014.
Lab M’s current facility will be maintained, and its
FOLLOWING THE REVISED ISO 11133:2014 operations will be managed by Neogen’s Scotland-
based Neogen Europe subsidiary. Neogen Europe
Microbiology laboratories testing food, animal feed or services the Europe, Middle East and Africa (EMEA)
water samples using ISO formulations are now required territories and has over 160 employees.
to follow the latest BS EN ISO 11133:2014 standard
which applies to QC testing criteria. This impacts any labs
preparing their media in-house, but also to media
manufacturers such as ourselves. Not only are Lab M
compliant to the new standard, but our SOP’s are written
in a way to ensure we go above and beyond these criteria
to ensure our customers have peace-of-mind when
purchasing our products.
LAB M GAINS ISO 17025 ACCREDITATION FROM

UKAS
We’re pleased to announce that the quality control

laboratory of our facilities in Heywood, has been granted
BS EN ISO 17025:2005 accreditation by UKAS. Lab M’s
schedule of accreditation covers both the physical and
microbiological performance testing of our ready-to-use
Pinnacle™ media range, with our accredited methods
including pH, sterility, ill volume, qualitative performance
testing and quantitative performance testing.
All methods are based on the new requirements of
BS EN ISO 11133:2014.
LAB M’S HARMONISED PHARMACOPOEIA

RANGE IS NOW AVAILABLE
We have enhanced our pharmacopoeia range to ensure

that all media listed in the European pharmacopoeia 8.0
volume 1 (2014) are available in our portfolio. This range
of dehydrated culture media is formulated to and
performance tested by the requirements speciied within the ™
European pharmacopoeia, enabling laboratories to comply
to the standard which is harmonised with the equivalent
A Neogen® Company
chapters of the United States (USP) and Japanese 8829
pharmacopoeias (JP).
FOR MORE INFORMATION E-mail: [email protected] Web: www.labm.com
152
Local distributor stamp:
INTERNATIONAL MEDICAL PRODUCTS

Waversesteenweg 1110, box 7
1160 Brussels - BELGIUM
T: +32 (0)2 660 50 75 - F: +32 (0)2 660 20 98

[email protected] - www.intermed.be
™ 1 Quest Park, Moss Hall Road, Heywood, Lancashire, BL9 7JJ, UK

Tel: +44 (0) 161 820 3833 Fax: +44 (0) 161 820 5383
A Neogen® Company E-mail: [email protected] Web: www.labm.com NE8499

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MICROBIOLOGY

THE GATEWAY TO MICROBIOLOGY ™

Lab M specialises in microbiological culture media.

Lab M has an international reputation for the quality of its

Lab M has since evolved and relocated to a new purpose built

The Microbiology Manual Version 10

From time to time, however, Lab M has been asked to ‘design’

• Close client collaboration.

• Development programmes that are both cost and time

• A process approach to development, commencing with

• Commitment to on-going production.

• Quality products and service.

This is a unique service that differentiates us from our

Preparing Culture Media Weighing Out

Preservation of Bacteria using Protect ™

LAB072 Tryptone Bile Agar

HAL004 Harlequin™ LB Agar LAB098 Potato Dextrose Agar

LAB011 Tryptone Soy Agar Diluents/ Isotonic solutions

LAB044 Selenite Broth PIN005 Pinnacle™ Salmonella ABC Medium Prepared

LAB055 Selenite Cystine Broth LAB149 Plate Count Agar

LAB114 Mueller Hinton Broth PIN004 Pinnacle™ mLGA Prepared

LAB012 Sensitivity Test Agar LAB108 Pseudomonas Agar Base

Sodium thiosulphate 5.44

Storage: Dehydrated culture media: 10-25°C.

13 Dehydrated Culture Media

Sub-culture B. subtilis 2.0-3.0 F.CR.D. Yellow

Dehydrated Culture Media 14

15 Dehydrated Culture Media

Add one vial per 500mL of sterilised medium as appropriate.

Dehydrated Culture Media 16

Typical Formula g/litre Method for reconstitution

Minimum Q.C. organisms:

17 Dehydrated Culture Media

Dehydrated Culture Media 18

Typical Formula g/litre

LAB048 Tryptose 10.0

19 Dehydrated Culture Media

Storage of prepared medium: Plates – up to 7 days at 2-8˚C in Growth characteristics

Dehydrated Culture Media 20

Method for reconstitution Method for reconstitution

21 Dehydrated Culture Media

LAB139 pH: 7.2 ± 0.2

Tryptone 17.0 References

Incubation: 30˚C aerobically for up to 48 hours.

Buffered Peptone Water Appearance:

Dehydrated Culture Media 22

Minimum Q.C. organisms: Campylobacter jejuni

Storage of Prepared Medium: Capped containers: 7 days at 2-8˚C

23 Dehydrated Culture Media

Typical Formula g/litre Method for reconstitution

Dehydrated Culture Media 24

Typical Formula g/litre Beef Extract 3.0

Pancreatic digest of gelatin 20.0 Tryptone 4.0

Magnesium chloride 1.4 Lactose 10.0

Dipotassium sulphate 10.0 L-Cystine 0.128

Cetrimide 0.3 Bromothymol blue indicator 0.02

Agar 13.6 Agar No. 1 15.0

Method for reconstitution Method for reconstitution

25 Dehydrated Culture Media