lab m manual 2006

A member company of International Diagnostics Group plc

The

Microbiology

Manual

The

Microbiology

Manual

ContentsIntroduction i

ISO 9000:2001 Certification iv

Customised media service v

Manufacturing Process pages v- viiiThe process outline v

Quality criteria vi

The application of growth rate analysers pages vii-viii

Preparing Culture Media pages ix-xiiiDehydrated Media ix

storage ix

weighing out ix

pH of culture media ix

glassware x

addition of powder to water x

heat x

dispensing prior to sterilisation x

Autoclaving Culture Media x

total heat input x

measurement points xi

volume of medium xi

heat conductance of agar xi

load distribution / composition xi

agitation xi

equipment xi

Sterility Indicators xii

adhesive tape xii

Browne’s tubes xii

Spore indicators xii

Molten Media xii

re-melting agar xii

pouring plates xii

drying plates xii

Sterile Supplements xiii

Storage of Prepared Media xiii

Troubleshooting Guide xiii

Quality Control of Culture Media pages xiv – xxiThe ecometric technique xiv

Productivity ratio xv

Liquid media xv

Templates / suggested records pages xvi-xviii

Preservation of stock cultures xix

Laboratory accreditation xx

HACCP xxi

Microbiology Methods pages xxiii-xxviiSources of information xxiii

TVC xxiii

Coliforms / Enterobacteriaceae xxiii

E. coli

presence or absence xxiv

enumeration using membranes xxiv

enumeration without membranes xxiv

O157:H7 xxiv

Yeasts and moulds xxv

Staphylococcus aureus xxv

Pseudomonas spp. xxv

Bacillus cereus xxv

Clostridium perfringens xxvi

Listeria

FDA method xxvi

modified USDA method xxvi

Salmonella

semi-solid method xxvi

conventional method xxvii

Campylobacter xxvii

Culture Media Information pages xxix – 3.6Format and abbreviation guide xxix

Dehydrated media guide xxix

Media Range 1.1Alphabetical Listing of Products

New Media Products 1.1.1

Harlequin™ Chromogenic Media 2.1

Biomolecular Products 3.1

Captivate™ 4.1

Selective Supplements 5.1

Agars, Peptones Extracts and other Media Constituents 6.1

Sterile Additives and Ready Prepared Media 7.1

Indexes 8.1By product code 8.1

By product name 8.4

By organism 8.8

Request a copy of the manual Last page

iii

CONTENTS

v

Customised Media Service

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 for

global markets. Its continued programme of product

innovation and development has ensured that the Company

has been responsive to market trends and changes in custom

and practice. The increasing importance of regulatory

compliance has also been a significant factor in this process

of innovation.

From time to time, however, LAB M has been asked to

‘design’ media to meet specific customer applications. This

has evolved into a more proactive approach to resolving

customer needs:

The LAB M Customised Media Service.

What do we offer?

• Individually designed media, engineered to meet specific

customer requirements.

• Close client collaboration.

• Development programmes that are both cost and time

efficient.

• A process approach to development, commencing with

feasibility tests, through product trials and into routine

production.

• Customised packaging and labelling.

• Assurance of compliance with regulatory requirements.

• Total client confidentiality.

• Intellectual Property options.

• 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 to discuss your needs on a confidential basis.

RAW MATERIALSAgars, Peptones, Extracts, Dyes, Chemicals etc.

Each component is individually tested for suitability. Growth promoting components are selected with the help ofan automated growth rate analyser (Malthus Instruments)

PRODUCTIONWeighing, Milling, Blending

A production batch is made from raw materials of specifiedbatch number which have been pre-tested for compatibility.The components are individually milled to ensure uniform

particle size. Weighings are double checked before thecomponents are blended.

QUALITY CONTROLPhysical, Biological parameters. Comparison with previous

batch and competition

Quality control first checks that the batch is completelyblended, then a series of physical and biological tests are

performed to ensure the product meets the exactingstandards required by our customers. Comparisons withprevious and competitor’s batches are made. Results are

recorded and a reference sample stored.

BOTTLINGInto 500g sealed containers, or bulk containers at request

Automated equipment delivers pre-weighed amounts into containers which are hermetically sealed.

Each container is immediately labelled with product details,code and batch number.

CUSTOMERSLAB M products are dispatched all over the world tomicrobiologists in all types of laboratory. Strict batch

traceability in accordance with ISO9001 ensures we can recallall products if necessary, safeguarding your products/process.

LAB M Culture Media: The Process Outline

LAB M Culture Media – The quality criteria

Raw materialsPeptones and Extracts – Clarity, pH, moisture, growth promotingproperties with Gram positive and Gram negative organismsaerobically and anaerobically, freedom from toxicity. Compatibilitywith other components, haemolysis patterns, antibiotic antagonists.

Agar – Clarity, pH, gel strength, melting point, setting point, heavymetal content (particularly Ca++, Mg++) compatibility with othercomponents. Clarity on re-melt.

Bile Salts – Clarity, pH, thin layer chromatography, compatibilitywith other components.

Dyes & Chemicals – pH, chemical parameters, growth promotioninhibition, properties after incorporation into culture media.

Selection of ingredientsLAB M pioneered the use of impedance technology for the selectionof Culture Media ingredients.

We were the first company to utilise automated growth rate analysersto ensure only the most suitable ingredients available are chosen. Forexample, with the use of growth rate analysis, we can select thosepeptones that trigger the exponential growth of inoculated micro-organisms in the shortest possible time; this technology is also usefulin minimising batch to batch variation.

Further information on this technique is on pages 5 and 6.

ProductionAll components from specified batch numbers. All componentsweighed accurately and checked.

Components milled to uniform particle size. Components blended forspecified time, multiple samples taken to ensure thorough blending.

Quality controlPhysical – pH, clarity, gel strength, colour, heat stability, viscosity,redox.

Biological – Growth characteristics, productivity ratio, chemicalreactions and colour changes, comparison with previous batch andcompetition.

vi

Choosing peptones for enrichment media - peptone A is chosen because of a faster response time.The survival time of the test organisms is also taken into consideration.

The application of growth rateanalysers in the manufacture of

bacteriological culture mediaby W.A. Hyde and K. Denton

IntroductionOne of the fundamental performance parameters of a bacteriologicalculture medium is its ability to promote the early and rapid growth ofmicro-organisms. There have been many methods devised to assessparameters, most of which rely on the viable count.

The viable count gives information on the state of the culture at asingle moment in time but gives no indication of the growth rate,unless serial viable counts are performed. Methods based on theclassical method of Lister are used for estimating growth rate. Usingthis method he was able to plot the classic growth rate curve of abacterial culture.

Unfortunately this technique is both laborious and inaccurate.Halverston and Ziegler in 19331 showed the technique to be subjectto very large experimental errors. For example, with five tubescounted from each dilution, variation can be from -70 to +260 percent!

Other variations on the Lister technique for assessing substrateperformance are:

a) Agar dilution techniques;

b) Surface inoculation techniques such as the drop method of Milesand Misra2 and the ecometric technique of Mossel et al3;

c) The novel spiral plater technique4;

d) Photometric and nephelometric techniques.

Techniques a - c still suffer from giving a point source of informationin what is a dynamic analysis.

The nephelometric/photometric techniques are relatively insensitiveand give little information about the log phase, and they cannot beused with materials which produce turbidity.

The introduction of electrical methods into the field of culture mediaperformance analysis has provided microbiologists with the means toaccurately record development of a culture over a large section of thegrowth curve. The main electrical parameters measured, areconductance and impedance. There are advantages in measuringeither of these parameters, and these are outlined in the excellentbook by Eden, R. and G5.

Impedance/conductance techniquesIn 1898, Stewart showed that bacterial growth in fluid could bedetected by changes in the electrical properties of that fluid. Sincethis time, several workers have made use of conductance andimpedance measurements in the analysis of bacterial growth, finallycrystallising in the 1970’s with papers by Ur and Brown (1975)6 andCady (1975)7 describing the use of continuous impedance monitoringas a tool with wide potential in microbiology.

In 1977, workers at the Torry Research Station in Aberdeen producedequipment capable of accurately plotting the growth curve of bacteriain various fluid culture media using impedance techniques8.

The curves produced on the equipment could be directly related to thetotal viable count.

Multi-channel growth rate analysers are commercially available, egthe Malthus system9.

PeptonesThe peptones, infusions and extracts that are used by microbiologistsare manufactured from biological raw materials and these are subjectto variations and inconsistency (e.g. meat, milk, plants). These aremanufactured by acid or enzyme digestion processes which arecumbersome and difficult to control.

It is not surprising, therefore, that products described as ‘meatpeptone’, for example, are much more variable in performance thanreagent grade chemicals. (Figure 1) Because of this variation it isstandard practice for culture media manufacturers to test samples ofthese raw materials from many sources in order to maintain a supplyof products with suitable performance characteristics.

Normally pre-shipment samples will be evaluated, both alone andmade up into typical formulations, and both performance andcomparison with ‘stock’ material will be taken into account before adecision to purchase is made. Full quality control of the bulk materialis then also carried out.

LAB M introduced an eight channel Malthus Growth Rate Analyserinto its raw material selection and quality control procedures in 1982.This has enabled closer control of the performance characteristics ofthe individual peptones and culture media formulations availablefrom the company.

Method of use

The eight channel machine is used to compare the performance oftwo substrates inoculated with three to four organisms or whentesting finished product to compare the new batch of productagainst a stock batch, and against a competitor’s product.

The organisms are inoculated into 2ml or 10ml volumes of thesterile substrate in tubes which contain the electrodes. The tubes arethen placed in an accurately controlled water bath and connectionsare made between the electrodes and the analyser. The base lines foreach channel are set on the chart recorder and the growth curves foreach test are recorded.

The speed of the chart and the sensitivity of the instrument can beadjusted in order to accommodate the growth characteristics ofvarious organisms. Using this technique it is possible with someorganisms and substrates to obtain a result in less than half aworking day.

vii

Figure 1

Growth curve informationThere are several parameters which can be assessed from the growthcurve produced by the Malthus system. These include:

Lag time – The time that elapses before a change in electricalproperties can be detected. Rapid metabolism causing changes inelectrical properties may be detected before cell division begins.

Detection time or take off – When the curve shifts from the baselinelag phase into the log phase, this information can be used as ameasure of the number of organisms present in a sample with the aidof suitable calibration graphs (Figure 2).

Log phase - The log phase gradient is a measure of the rate ofmetabolism in the multiplying bacteria.

Maximum peak of the curve - This is related to the maximum‘number’ of bacteria achieved in the culture. The information can beused in a number of ways, for example:

a) The peptone with the shortest lag time can be chosen for thosemedia that are required to give rapid growth from a smallinoculum, e.g. sterility test media, blood culture media, andfermentation media.

b) For culture media that demand standardisation of performancefrom batch to batch, it is possible to choose peptones with aspecified lag time growth rate.

c) The maximum peak is of interest when choosing a raw materialfor production of large numbers of bacteria such as for vaccines.

As equipment based on impedance methods became established inthe microbiological quality control procedures of the food industry, anew aspect of culture media quality control is opened up, and needsto be tested for, these are the electrical properties of the product. Onlyby the use of impedance/conductance measuring equipment in qualitycontrol, can performance of the culture media on similar equipmentin customers’ laboratories be assured.

ConclusionElectrical methods of monitoring bacterial growth have been knownsince the latter part of the 19th century, however, only in the last fewyears with the development of micro-processors and sophisticatedelectronics are these methods beginning to reach their full potential inGrowth Rate Analysers.

In culture media manufacture the Growth Rate Analyser providesinformation that makes a significant contribution to improving boththe performance and standardisation of products manufactured fromvariable raw materials. (Figure 3)

References1. Halvorson, H.O., Ziegler, N.R. (1933a) J. Bact. 25, 101, (1933b) I.Bid., 26, 331, 559

2. Miles, A.A., Misra, S.S. (1938) J. hyg., Camb., 38, 732

3. Mossel, D.A.A., Van Rossem, F., Koopmans, M., Hendriks, M.,Verdouden M., Eelderink, I. (1980) J. Appl. Bact., 49, 439-454

4. Gilchrist, J.E., Donelly, C.B., Peeler, J.T., Delany, J.M. (1973)Appl. Microbiol. 25, 244-252

5. Eden, R. Firstenberg, Eden, G., Impedance Microbiology (1984)Research Studies Press, John Wiley and Sons, New York

6. Ur, A., Brown, D.F. J., (1975) New Approaches to theIdentification of Microorganisms (edited by C.G. Heden and T. Illeni)P.61-71 John Wiley and Sons, New York.

7. Cady, P. (1975) (edited by C.G. Heden and T.Illeni, New York).P.73-99, John Wiley and Sons, New York

8. Hobbs, G., Gibson, D. M. (1977) J. Appl. Biol. 43, 3

9. Malthus Instruments Ltd., Topley House, Wash Lane, Bury, Lancs.

First published in International Labmate 1987 Vol.12 Issue 2 pp 36-37

viii

Figure 2

Figure 3

Preparing Culture Media

Quality AssuredBefore each batch of LAB M Culture media is passed for sale itundergoes a rigorous quality control procedure to ensure it givesmaximum recovery and reproducibility. Reconstitution of media inthe user’s laboratory must be done with care to ensure the same highstandards of performance.

The following section outlines the correct procedures which willensure high quality reconstituted products, and suggests simplequality control techniques that can be used to check the performanceof prepared media.

Dehydrated Culture Media

StorageDehydrated media stored unopened under optimal conditions have ashelf life of 3-5 years but once the container is opened the contentsshould be used within six months. In-house quality control by theuser will help determine the condition of product in openedcontainers. The best conditions for storing dehydrated media are in acool, even temperature away from any sources of moisture such aswashing up areas or laboratory autoclaves and away from stronglight. Storage in a refrigerator is generally not recommended as thereis the risk of condensation on the container when it is brought out ofthe refrigerator.

TABLE 1 – Deterioration of SS Agar stored in various conditionsfor 6 months.

Storage conditions moisture gain %

Unopened bottle stored in cool,

dark, dry conditions 0

Loose cap, stored in light on bench 1.1

Loose cap, stored in light in autoclave room 4.4

The effect of the moisture gain on the performance of the agar can bequite 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. Thisdemonstrates the importance of ensuring the container lid is tightlyclosed 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 thePreparation of Agar Media. (1972). Am. J. Med. Tech. Vol. 38 No. 7.

When a container is opened for the first time the date should be notedon the container. Dehydrated media should not be used if it shows anysign of moisture gain i.e. become lumpy or discoloured. The lid onthe container should be replaced quickly after media has been takenout and closed tightly.

Weighing OutUsing a top-pan balance with an accuracy of ±0.1 gram the powdershould be spooned onto a weighing boat or clean beaker. Do not tipthe media out of the container as this will cause excess dust whichmay be irritant and will certainly need cleaning up. The componentsof some formulations can be irritant so the wearing of a face mask atthis stage is advisable.

WaterPurification by distillation or deionisation is advisable. It is importantthat the equipment is properly maintained; the output of ion resinsneed to be electronically monitored and microbial colonisation of theresin and tubing must be avoided. Storage vessels for purified watermust also be monitored for microbial colonisation. It is advisable touse only fresh purified water with a conductivity of less than 10microsiemens. Stored water tends to become acidic because itabsorbs atmospheric CO2. Tap water is not recommended because ofthe potential presence of heavy metal ions which can cause inhibitionand precipitation problems.

pH of culture mediaMeter Sensitive to one decimal place.

Electrodes Calomel, gel filled unbreakable, combinationelectrodes shielded with detachable shield forcleaning. Flat electrodes may be used, but someproblems have been found by users.

Short-term storage pH 4.0 bufferof electrode

Electrode faults Indicated by slow or erratic readings andinability to obtain two points on the meterwithout adjustment.

Cleaning of 0.5% pepsin in N HCl for 1-2 hours.electrode Wash in deionised water, soak in pH 7.0

buffer for 2 hours.

Daily checks Use pH 4.0 and pH 7.0 buffer and, if possible,pH 10.0.

Testing meter pH calibrating or checking attachment.(Available from BDH.).

Water Deionised or double-distilled water is onlyweakly dissociated. Measure conductivity < 10 micro Siemens. To check pH add 0.3mlsaturated KCl to 100ml water.

Culture media Acidity: Bile salts precipitated; H2S reactionsreactions affected depressed; sugar fermentation; antibiotics lessby pH active (aminoglycocides, cephalosporins,

macrolides).

Alkalinity: Potentiates aminoglycocides; sugar fermentation; antibiotics less active (fusidin, tetracycline, penicillins).

Media difficult to Ringers, Maximal Recovery Diluent pH because oflow ionic strength

Effect of Autoclaving or irradiation will lower the pHautoclaving or about 0.2 - 0.5 units, but not predictable.irradiation

Effect of volume Mixed volume loads should be avoided. Timeof media on pH allowance must be made for various volume

sizes of media to ensure that overheatingleading to acid formation and caramelisationdoes not occur. Further details are given underAutoclaving Culture Media.

Temperature These adjust for changes within the electrodecompensator only. They DO NOT compensate for changes in

ionisation of solution at high temperatures.

Optimum 20 - 25˚C. or according to mediatemperature to manufacturer’s instructions.pH culture media

Acceptable ± 0.2 is usual. This assumes 1 litre volumepH variations produced strictly according to media

manufacturer’s instructions.

Adjustment of pH Should not be necessary if all systems correct, of media e.g. water quality, balance, volume etc., and

sterilisation is carried out precisely tomanufacturer’s instructions.

ix

pH out of If pH needs to be adjusted becausetolerance manufacturer’s instructions cannot be followed,

titration of small additions of N/10 HCl. orN/10 NaOH into aliquots of media should bemade, media processed and pH measured whencooled and appropriate calculations carried out.

QUALITY CONTROL USING MICROBIOLOGICALPERFORMANCE CRITERIA ARE ESSENTIAL

GlasswareAll glassware must be undamaged and clean having been rinsed with purified water before storage. Borosilicate glass is preferable tosoda-glass because the latter may leach alkali into any solutioncontained in it.

Addition of Powder to WaterThere are a number of ways to do this, attempting to mix the waterand powder too quickly can cause the formation of lumps which aredifficult to disperse.

1. Pour approximately 1/3 of the volume of water required into a flask,add the powder slowly whilst constantly swirling, then addremaining water.

2. Pour the full amount of powder onto the full amount of water in aflask. Allow to stand for 10 minutes before swirling to mix.

3. Add the powder to the flask and slowly pour on the water withfrequent swirling.

NOTE: The water should always be measured before adding to theflask. Never heat water before adding to the medium.

HeatRemember that heat denatures the nutrients and the agar in culturemedia. Heat is necessary for sterilisation and for dissolving the agarif the medium is to be distributed into tubes or bottles prior tosterilisation. However these processes must be carefully controlled toensure the minimum possible heat input is used. Heat input is less inequipment that allows rapid heating and rapid cooling, e.g. mediapreparators. Media should never be left at high temperatures forprolonged periods for example holding in a 56˚C water bathovernight will noticeably reduce a culture medium’s performance andgel strength.

Dispensing prior to SterilisationMost broth media are readily soluble at room temperature or with theaid of gentle heat. Ensure a clear, well mixed, solution is obtainedbefore dispensing into final containers and sterilising. Agarcontaining media need to be brought to the boil to take the agar intosolution. This boiling should be done with frequent agitation toensure even heat distribution. As soon as the medium begins to boilit should be removed from the source of heat.

CAUTION: Agar media, particularly those with low agar content,may boil unexpectedly and overflow out of the flask. To prevent thisagitate frequently and gently as the medium approaches boiling andthe agar begins to dissolve. Allow the medium to cool to 47˚C beforedispensing with constant mixing into final containers for sterilisation.

Autoclaving culture media

To achieve optimum performance from reconstituted culture media itis important to ensure sufficient heat input to kill all spores whilstprotecting the medium from excessive heat input that would damagethe nutrient and gelling properties of the medium. In our experiencethe commonest cause of problems with culture media is the heatsterilisation, a dynamic process which has many variables that mustbe considered. These are:

Total Heat Input

The total heat input of a sterilisation cycle can be shown graphically.

The total time above 50˚C is important because both nutrients andagar will be undergoing a denaturation process. The higher thetemperature the faster the denaturation, i.e. 30 minutes at 121˚C willnoticeably affect the gel properties of most agars whilst the mediumcould be held at 50˚C for several hours before denaturation can bedetected. Total heat input should be controlled to protect themedium from denaturation. Prolonged heating and cooling canlead to excessive heat input.

The total time spent above 100˚C is important, because not only isthis the temperature at which spores are being killed, but it is thetemperature with the potential to cause most damage to the medium’sperformance. The aim of effective sterilisation is to ensure all sporesare destroyed with the minimum necessary heat input in order toensure no ill effects are caused to the medium. This can be done byusing the lethal rate equation or tables (Stumbo 1973). From a chartrecording of the sterilisation process (the recording must be from athermocouple placed inside the medium during processing), theportion of the graph between 100˚C and 121˚C for both the heatingand cooling stages is divided into 1 minute intervals, and using theequation or table, (shown below) can be converted into an equivalenttime at 121˚C. The time is added together and the total time removedfrom the holding time. This ensures that the heating process issufficient to kill the spores, but keeps damaging heat input to aminimum. Thus the instruction to ‘autoclave at 121˚C for 15 minutes’should be taken to mean ‘sterilise by an equivalent heat process to121˚C for 15 minutes’. Once calculated the new holding time can beused for future cycles, but only for the same volume of medium.Different types of media differ in heat penetration i.e. agars andbroths so the reduction in holding time must be calculated fordifferent volumes of both.

Lethal Rate Equation:L = Log-1 (T - 121.1)

Z

Where L = Equivalent time at 121˚C

T = Actual temperature for 1 minute

Z = Temperature coefficient (= 10˚C for spores)

Log-1 = antilog of the number. Using this equation, a lethal rate table can be produced: (see facing page)

x

Figure 4 Schematic diagram of heat input during sterilisation

TABLE 2 – Lethal Rate Table:

Measurement PointsSome autoclaves will measure chamber temperature, some willmeasure the temperature of the medium (by use of a thermocouple),some will measure both. It is important to remember that thetemperature of the medium will lag behind that of the chamber bothin heating up and cooling down.

Volume of MediumThe heating and cooling periods of large volumes of media are longerthan for small volumes of media. Therefore, the total heat treatmentin cycles, based on holding time alone, can be significantly greaterfor large volumes than for small volumes. Adjustments have to bemade to the fluid heat penetration time in the sterilisation cycleshown below. Brecker & Bridson gave the following guide times forheat penetration at 121˚C in glass bottles:

TABLE 3 – Heat Penetration Times

Volume Time to reach 121˚C

500ml 18 minutes

1 litre 22 minutes

2 litre 27 minutes

5 litre 37 minutes

The thickness of the glass and the shape of the container were notspecified; both these factors affect the rate of heat penetration. Thesetimes seem to be based on both autoclave and medium being heatedfrom cold to 121˚C without the effective ‘holding’ at 100˚C thatoccurs with modern pulsing autoclaves or with traditional autoclavesbefore the outlet valve is closed.

In our experience, with a 5 minute hold at 100˚C, a 1 litre Pyrex flaskof medium will attain 121˚C within 5 minutes of the chamberreaching that temperature. Be aware that a sterilisation cycle that is optimum for 1 litre of medium will be excessive for l00ml of medium.

Heat Conductance of AgarAgar is a very poor conductor of heat and heat penetration into agarcontaining media is significantly slower than into non-agarcontaining media. Brecker & Bridson showed that 500ml of agar in athin-walled bottle took 6 minutes longer to reach 121˚C than did500ml of water in an identical bottle. If a thermocouple is used in a500ml bottle of water to control the sterilisation of 500ml of agarmedium, the difference in heat penetration must be taken intoaccount. The poor heat conductivity of agar has significant effectswhen trying to sterilise large volumes of medium (e.g. 2 litres ormore). Pre-heating of the medium to get the agar into solution beforeautoclaving is recommended. Brecker & Bridson found that: “In 4litres of medium, with the agar settling undissolved to the bottom, thetemperature at the centre of the agar mass had not reached 121˚C 1 hour after the autoclave chamber had.”

Load Distribution / CompositionHeat penetration in an overloaded autoclave will be hampered as willevacuation of air. Ensure sufficient space is left between items in aload to allow free passage of steam. As covered previously, thevolume of medium in the bottles is directly related to the heat input.It is therefore impossible to properly control a cycle if mixed volumes(e.g. 1 litre flasks and 20ml bottles) are put together in the same load.Mixed volume loads should therefore be avoided if at all possible.

AgitationThe continuous agitation of a medium during sterilisation greatlyshortens the heating and cooling times. Modern media preparatorshave this ability which gives them a significant advantage overgeneral purpose autoclaves.

EquipmentThe equipment used for autoclaving varies from the very simple tovery complex, although they all operate by using steam underpressure to attain temperatures above 100˚C. The crudest autoclave isthe domestic pressure cooker. We consider the lack of proper controlsmake it unsuitable for routine use. Simple bench top autoclaves arelarger versions of pressure cookers but with the ability to measure thetemperature and pressure of the chamber. This equipment is suitablefor the sterilisation of small to medium sized volumes and the heatingand cooling periods are short. It is important to allow this type ofequipment to free steam before closing the outlet valve, otherwise,not all the air will be flushed out of the chamber, reducing theeffectiveness of the equipment.

Larger laboratory autoclaves now have separate steam generationfacilities allowing rapid heating and some models evacuate the air byvacuum and then inject the steam under pressure ensuring there areno air pockets causing cold spots. This type of equipment will havebuilt-in thermocouples and chart recorders capable of monitoringboth chamber and load temperatures throughout the cycle. This typeof equipment also incorporates a cooling jacket through which coldwater is run after the completion of the holding time. For safetyreasons the door will not open until the chamber has cooled to 80˚C- this can prolong cooling times.

Media preparators are dedicated to sterilising media. The mainadvantage of this equipment is that they agitate the medium speedingup heating and cooling and ensuring thorough mixing. They are wellequipped with both time and temperature controls. The cooling isbrought about by use of a water jacket. The only disadvantage ofmedia preparators is that they can only handle one medium at a timewith a cycle time of just under 1 hour. Corry et al made the followingcomment. “General purpose lab autoclaves are difficult to standardiseand more consistent results are obtained by purpose-built mediasterilisers.”

It is important that whatever equipment is used is properlymaintained to ensure accuracy of gauges.

xi

Figure 5 Schematic diagram showing the lag of mediumtemperature compared to chamber temperature.

Temp Time Temp Time

100 0.008 111 0.098

101 0.010 112 0.123

102 0.012 113 0.155

103 0.015 114 0.195

104 0.019 115 0.245

105 0.025 116 0.309

106 0.031 117 0.389

107 0.039 118 0.490

108 0.049 119 0.617

109 0.062 120 0.776

110 0.078 121 1.000

Temp = Temperature for 1 minute interval

Time = The equivalent time at 121˚C

References:Stumbo, C.R. (1973) Thermobacteriology in Food Processing. 2ndEdition. Academic Press, New York.

Bridson, E.Y. and Brecker, A. (1970) Design and Formulation ofMicrobial Culture Media. Methods in Microbiology Volume 39edited by Norris, S.R. and Ribbon, D.W. Academic Press, New York.

Gardner, J.F. and Peel, M.M. (1986) Introduction to Sterilisation andDisinfection. Churchill Livingstone.

Sterility indicators

Sterility indicators can be a useful tool to highlight deficiencies insterilising equipment, but only if they are used correctly and theirlimitations are fully understood. Use of an inappropriate indicator forthe product or equipment can lead to the mistaken belief that theprocess is being properly monitored. The major drawback of sterilityindicators is that even when used carefully for the correct application,they will show that the minimum conditions have been achieved, butgive no information about the total heat input. Consequently theygive no indication as to whether the medium has been subjected toexcessive heat resulting in loss of performance of the sterilisedproduct. Wherever possible an appropriate equivalent heat processshould be calculated for the load to be sterilised.

Three types of indicator are commonly used, physical, chemical andbiological, examples of which are given below:

Adhesive TapeBowie-Dick tape is classed as a physical indicator even though itrelies upon a chemical change to produce the black diagonal stripeswhich indicate the tape has been subjected to heat. More commonlyreferred to as autoclave tape, it was designed for use in thedemonstration of adequate steam penetration. A cross of the tape wasplaced on a sheet of steam permeable paper across a towel at the mid-point of a stack of towels, and this was examined aftersterilisation to make sure the diagonal stripes changed to blackuniformly along the tape cross. It is therefore only an indicator ofsteam penetration, as the stripes will change colour before the fullcycle is complete. In most of the applications to which Bowie-Dicktape is put in the microbiology laboratory, it will be a veryunsatisfactory method for demonstrating the achievement of sterileconditions.

Browne’s TubesThe Browne’s tube is a chemical indicator comprising a heat sensitivesolution within a glass tube, which changes colour from red to greenwhen the tube is subjected to a high temperature for the requiredlength of time. In this respect it is a more reliable indicator of sterilitythan Bowie-Dick tape, as it shows both temperature and time aresufficient for sterility, whereas Bowie-Dick tape merely indicates aheating process has occurred. However the tube only indicates thatthe correct conditions have been achieved at that particular site in thesteriliser. The use of multiple tubes throughout the load is moresatisfactory, but again no information is given about the total heatinput. Furthermore, the tubes can deteriorate on storage which maylead to premature colour change and false information as to theperformance of the sterilising equipment.

Spore IndicatorsThe spores of Bacillus stearothermophilus are extremely resistant toheat and can thus be used as a biological indicator of sterilisation.Spore strips prepared in the laboratory or commercially producedstrips, are placed in various parts of the load. After the cycle iscomplete they are removed aseptically and placed in a bottle ofthioglycollate medium or cooked meat medium, and incubated at55˚C for 7 days, during which they are examined for growth. Again

this type of indicator shows if the time and temperature requirementshave been achieved but not if excessive heat has been used. LikeBrown’s tubes, spore strips only show ‘spot’ conditions, and maydeteriorate on storage giving false results. However, the length oftime needed to obtain a result means they are inconvenient for routineuse.

All the above indicators are a compromise in the absence ofaccurate steriliser control, and none can demonstrateoverheating of the medium. The only way to ensure sterilisationwithout overheating, is to use thermocouples introduced into themedium being processed, linked to chart recorders, and inaccordance with a previously determined equivalent heat processfor the equipment and volume of media being sterilised.

Molten Media

Holding molten media in water baths at 47 ˚C or above for more thana few hours should be avoided. The extra heat input will damage boththe nutrient and gelling properties of the medium. Holding at 47˚Covernight will result in noticeable denaturation of agar gel. Ideally,media should be poured as soon as possible; holding at 47˚C for 4hours should be the maximum holding time any medium is subjectedto. The addition of extra agar to compensate for the reduction ingelling caused by overheating is not recommended as the overheatingwill have also damaged the nutrient properties.

Re-melting AgarMost media will stand re-melting once in a boiling water bath. Again,the total heat input into the medium has to be considered. If a mediumis likely to be allowed to set then re-melted, it is doubly importantthat holding at 47˚C in a water bath is minimised. Never re-melt anagar more than once.

Pouring PlatesMedia should be cooled, in a water bath, to 47˚C before pouring.Immediately before pouring the medium should be swirled gently toensure thorough mixing. Plates should contain at least 5mm. ofmedium if they are to be stored before use to minimise the effects ofthe drying that will occur during storage. Single vent Petri dishes losemoisture more slowly than triple vent dishes but they are also moreprone to condensation problems if not adequately dried. The amountof medium specified for poured plate techniques is generally lessbecause the medium is not stored in vented Petri dishes. Whenlayered plates are used the total depth should not be less than 5 mm.

NB: -It is important to note that the tubing through which the mediumis dispensed can retain inhibitory substances on the inner surfaces.This means it is necessary to use separate tubing for non-selectivemedia and for inhibitory/antibiotic supplemented media. Failure to dothis can result in traces of inhibitory substances being incorporatedinto non-selective agars, thereby reducing their ability to support thegrowth of fastidious organisms.

Drying PlatesIn order to achieve well isolated colonies on agar plates streaked forsingle colonies, it is necessary to make sure the agar is free fromsurface moisture by drying, but caution must be taken to ensure overdrying does not occur as this can be detrimental to the performanceof the agar. It is worth noting that numerous procedures in the stagesof preparation of agar plates can result in loss of moisture, and thesum of these can impair performance if not considered and kept to aminimum. Boiling of the medium to dissolve agar before autoclaving,autoclaving, and pouring at temperatures above 50˚C can all result inmoisture loss, and so media should only be boiled before autoclavingif absolutely necessary, the autoclaving carefully controlled in anequivalent heat process, and cooled to 47˚C before pouring.

xii

There are 2 commonly used methods of drying plates:

1. Carefully place the plate in the incubator with the mediumcontaining side up. Lift the base of the dish up and rest it on the lidas in the diagram. This allows excess moisture to evaporate whilstminimising the possibility of contaminating the agar.

2. Plates are allowed to stand (with lids on) overnight on the bench.The effectiveness of this technique will depend on the venting ofthe Petri dishes, the humidity of the surroundings and the amountof excess moisture present. The practice of incubating platesovernight could lead to excessive moisture loss and reducedmedium performance. To check for sterility, incubate arepresentative sample at the temperature and time parameters usedfor performing the test for which the medium is employed.

Sterile Supplements

The addition of sterile supplements is performed after the mediumhas been sterilised and cooled to approximately 47˚C. Most sterilesupplements such as blood, serum and antibiotics are denatured athigher temperatures. The supplement itself should be warmed up toroom temperature before adding to the medium and it should beadded whilst mixing to prevent the formation of ‘cold spots’ andpremature gelling of the agar. Care must be taken to observe strictaseptic precautions whilst adding the supplement. The supplementedmedium should be thoroughly mixed by swirling before any platesare poured. Any antibiotic supplement reconstituted but not usedshould be thrown away. Antibiotics vary widely in their stability oncereconstituted and can deteriorate rapidly - even deep frozen. Serumproducts are best stored frozen then thawed. It is wise to aliquotserum on arrival into suitable volumes to be frozen until required. Toproduce ‘chocolate’ or heated blood plates add the sterile blood to themedium at 80˚C or add at a lower temperature and gently re-heat withfrequent swirling until the medium ‘chocolates’. This chocolating ofthe blood destroys enzymes which would otherwise inactivate thenicotinamide adenosinedinucleotide (NAD or V factor) required forgrowth by Haemophilus spp.

Storage of prepared media

The shelf life of prepared media is dependent upon the compositionof the medium, the form in which it is stored and the conditions ofstorage. All media should be stored in the dark to prevent theformation of bacteriostatic and bactericidal substances (e.g.peroxides).

Each time a batch of medium is prepared some form of performancequality control should be carried out. It is unwise to use mediabeyond their minimum shelf life without repeating the quality controland comparing it with the initial result.

PlatesMost plates stored medium side up at 4˚C in the dark will have aminimum life of 7 days. This can be extended up to 3-4 weeks forsimple nutrient media by using some form of airtight packing. Platescontaining antibiotics have a shelf life governed by the stability of theantibiotics. Generally speaking it is unwise to extend the shelf life ofan antibiotic containing medium beyond 7 days. As a medium losesmoisture the ingredients of the medium will be concentrated makingselective media progressively more selective. A plate with an originalmedium depth of 5mm will have its ingredients concentrated 20% bythe time its gel has shrunk to a depth of 4 mm. Plates showing visiblesigns of shrinkage (drying) should not be used. Plates should bebrought up to room temperature before use to avoid any ‘thermal’shock to the bacteria. Any plates left on the bench for more than 8hours should be discarded as unsuitable for use.

Bottled MediaAny medium in an airtight capped container will have a longer shelflife than in a plate. Many simple nutrient media can be stored at 15-20˚C for 3 months in the dark. Indeed, many can be stored for longerbut we advise repeat of a simple Q.C. procedure after 3 months. If amedium is stored as a gel and then is re-heated before use, repeat ofa simple Q.C. procedure is recommended. Bottled media containingantibiotics will have shelf lives governed by the activity of theantibiotic.

Troubleshooting Guide

The laboratory that routinely quality controls the media it produceswill occasionally find it has produced a batch that is not up tostandard. The following is a list of potential problems and theirpossible causes:

FAULT POSSIBLE CAUSESoft Gel Excess heat, pH too low causing acid

hydrolysis, inaccurate weighing, inadequatemixing, agar not dissolved.

pH incorrect Contaminated glassware, impure water,overheating, chemical contamination, pH takenat wrong temperature, pH equipment faulty orpoorly standardised, deterioration of dehydratedmedium. (See specific section on pH of culturemedia)

Abnormal colour Impure water, dirty glassware, deterioration ofdehydrated medium, excess heat, pH wrong.

Darkening Excess heat, deterioration of dehydratedmedium.

Precipitation Excess heat, deterioration of dehydratedmedium, impure water or glassware.

Toxicity Excess heat (scorching or burning),deterioration of dehydrated medium.

Poor growth Contaminated water or glassware, deteriorationof dehydrated medium, incorrect weighing andmixing. Excess heat.

Poor selective or Contaminated water or glassware, incorrectdifferential weighing and mixing, deterioration ofproperties dehydrated medium. Excess heat.

xiii

Fig. 6 Rapid drying of plates

Lid

agar

Quality Control of Culture Media

The routine quality control of culture media is an essential ‘goodlaboratory practice’ necessary to maintain the standards andperformance of any bacteriological culture technique. More recentlyit is a requirement of many laboratory accreditation schemes such asUKAS, and CLAS etc.

We recommend the following: A full quality control when a newbatch of dehydrated medium is introduced into the laboratory. Thisfull Q.C. to be repeated every 3-4 months on opened containers. Ashort-form quality control when a new batch of medium isreconstituted from dehydrated media previously quality controlled. Ashort-form quality control whenever prepared media are used beyondtheir minimum shelf life or are re-heated more than once.

Keeping of records - both full and short-form quality controls - sothat trends, e.g. fall off in performance can be detected. Each mediumused in the laboratory should have its own quality control protocoland the necessary organisms should be maintained.

TechniquesThe parameters of growth on culture media are:

● Lag time

● Organisms grown from known inoculum

● Organisms inhibited from known inoculum

● Comparative growth with standard inoculum

● Comparative inhibition with standard inoculum

● Colony size

● Colonial appearance

In practice absolute measurements of growth are time-consuming orrequire sophisticated equipment whilst colonial appearance issubjective and difficult to record. Colony size is easily measured butis an insensitive indicator of performance. Comparative methods arethe most suitable ones for routine quality control of culture mediathey can be used for comparisons of growth and inhibition. Theecometric technique of Mossel is simple and gives numericalreadings 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.

The Ecometric TechniqueThis plating technique is simple enough to use in both full and short-form quality controls. It is based on streaking an inoculum toextinction. The results obtained can be compared with previousbatches of the same medium or with batches of the same mediumfrom different manufacturers. The results can also be compared withresults obtained using the same organisms on non-selective media.

Method1. Inoculate 5ml of Brain Heart Infusion Broth (LAB 49) with a

loopful of the chosen test organism (see individual products) andincubate for 4 hours.

2. Divide the plate to be tested into quarters designated A, B, C andD as shown below:

3. Charge a 1 microlitre loop with the incubated 4-hour culture andspread the test plate, going from Al - Bl - Cl - Dl - A2 - B2 etc.without flaming or recharging the loop and finish at D5.

4. Repeat this process with a control plate. For a batch of dehydratedmedia new to the laboratory, use a suitable non-inhibitory plate.For a new batch of plates from the laboratory stock of dehydratedmedium, use a plate of the same medium from the last batch tocheck consistency. Incubate both plates for 18 hours.

5. Note the last point at which growth occurs, on the test and controlplates, and record the segment and line, e.g. C4 or D5 etc. This isthe end point and can be used to calculate the absolute growthindex (AGI) and relative growth index (RGI) of a medium. TheAGI is obtained by noting the end point in Table 4. below and thisgives the AGI. e.g. if the end point is C4 then the AGI is 75.

TABLE 4 – Absolute Growth Index (AGI)

Al = 5 Bl = 10 Cl = 15 Dl = 20

A2 = 25 B2 = 30 C2 = 35 D2 = 40

A3 = 45 B3 = 50 C3 = 55 D3 = 60

A4 = 65 B4 = 70 C4 = 75 D4 = 80

A5 = 85 B5 = 90 C5 = 95 D5 = 100

The RGI is a comparison of the AGI of the test plate and that of thecontrol plate. For example:

Plate A—non-selective control—end point A5, AGI = 85

Plate B—selective agar test —end point C4, AGI = 75

Using the formula RGI = AGI Test x 100AGI Control

RGI = 75 x 10085

= 88.24 %

Thus, for this particular organism the test plate was 88.24% asefficient as the non-selective control plate. The performance of aselective agar can be thoroughly tested using an organism which ithas been designed to isolate and one which it is designed to inhibit.For the former the RGI should be as close to 100 as possible and thelatter as close to 0 as possible. The very nature of selective agars andthe bacteria which must be selected or repressed means thateffectiveness of different media will vary quite considerably. Forexample, Campylobacter Selective Agar (LAB 112) is a very goodselective medium as it will give high RGI’s for Campylobacter spp.and achieve suppression of bacteria without inhibiting the organismwhich is to be isolated. Examples of less effective selection occurswith Salmonella media, where there is a close relationship betweenpathogens (Salmonella and Shigella) and the other enteric organisms,making it difficult to inhibit one without reducing the isolation rate ofthe other. The onus is therefore on the end user to decide whichmedium is best for their use. Having found a medium which issuitable, use that as the yardstick to measure the performance of newbatches and to check that the efficiency of all media-makingprocesses are being maintained. It is important to note that theecometric method is a simple technique by which laboratories cancheck the media they are producing. However, it has to be performedwith care, as simple errors such as holding the inoculation loop at adifferent angle may introduce errors. This can be overcome by usinga spiral plater to inoculate the plates to ensure the plating method isidentical for test and control agars.

xiv

Figure 7 Schematic diagram of the ecometric method

Reference:Mossel D.A.A. et al (1983) Quality Assurance of Selective CultureMedia for Bacteria, Moulds and Yeasts: An Attempt at Standardisationat the International Level. J. Appl. Bacteriol. 54 313-327

Productivity Ratio (P.R.)Determining the productivity ratio of a medium is another way tocheck its performance related to a control medium, which should bea nutritious agar such as Tryptone Soy Agar (LAB 11). The inoculumused must be the same for both media and the P.R. is calculated bycounting the colonies on the test and control media:

P.R. = No. of colonies on test x dilution factor .No. of colonies on control x dilution factor

A simple method to obtain the P.R. of a medium is using the ModifiedMiles-Misra technique:

1. From an overnight culture of the test organism, prepared in BufferedPeptone Water (LAB 46), prepare a tenfold serial dilution inMaximum Recovery Diluent (LAB 103).

2. Divide the test plates into quadrants and mark each quadrant with thedilution to be used as shown below. Repeat with the control plates.

3. Starting with the highest dilution (10-8 in example below) placeone drop of the dilutions on the relevant quadrant. Repeat forcontrol plate.

4. Spread each drop over the quadrant and incubate the plates at 37˚Cfor 18 hours.

5. Count colonies at the lowest dilution they can be easily counted,

for both test and control. Calculate the P.R.

For example: Test = 20 colonies at 10 -3 dilution

Control = 25 colonies at 10 -3 dilution

P.R. = 20 x 103

25 x 103

P.R. = 2025

P.R. = 0.8

Thus in this example the test medium is 80% as efficient as thecontrol medium.

This technique is not as simple as the ecometric method and isprobably more usefully used to quality control broth media.However, it is less prone to error than the ecometric technique and itsaccuracy can be further increased by using duplicate plates.

Liquid MediaAnother simple technique can be used to quality control fluid mediasuch as Fastidious Anaerobe Broth (LAB 71) and FluidThioglycollate Medium (LAB 25).

1. From an overnight culture of the test organism, prepared in TryptoneSoy Broth (LAB 4), prepare a tenfold serial dilution (to 10-12) inMaximum Recovery Diluent (LAB 103).

2. Add 1ml of each dilution to 9ml of test and control broths.Incubate at 37˚C for 18 hours.

3. Examine the broths and note the highest dilution showing growth(turbidity of the broth).

This method can be used in conjunction with the Miles-Misratechnique to demonstrate recovery of known levels of CFU’s in brothmedia.

xv

Figure 8 Template for Miles-Misra plates

Mark the bottom of the plate as shown below, using a permanent marker pen.

Once the quadrants are marked, place the plate face-up on

the template below. Charge a lµl loop with a 4-hour broth

culture and spread the plate, going from

Al-Bl-Cl-Dl-A2-B2-etc. without flaming or recharging the

loop. Replace on template after incubation to read result.

xvi

Templates for the Ecometric Method

xvii

Suggested Quality ControlRecord Sheet

N.B. The same inoculum should be used for all plates. All plates must be spread at the same time, and incubated under identical conditions.

Quality Control Record (A)DATE: VOLUME OF WATER ADDED:

MEDIUM: CONDUCTIVITY OF WATER ADDED:

LAB CODE: METHOD OF STERILISATION:

BATCH NO: SUPPLEMENTS ADDED:

WEIGHT OF AGAR: EXPIRY DATE OF SUPPLEMENTS:

CONTROL MEDIUM USED:

Q.C. Organism

1

2

3

4

RG1AG1Test Control

End PointTest Control

Q.C. Organism

1

2

3

4

RG1AG1Test Control

End PointTest Control

MeanRG1

Quality Control Record (B)MEDIUM: DATE RECEIVED:

BATCH NO: DATE OPENED:

APPEARANCE OF POWDER WHEN OPENED:

xviii

Suggested Quality ControlRecord Sheet

N.B. Comparisons of relative growth index are only useful to show a trend in the condition of the medium. Variation between individual figures will occur due toinevitable inaccuracies of the technique.

Date RGI Appearance Comments

1234

1234

1234

1234

1234

1234

1234

1234

Preservation of Stock Cultures

The following method has been used in our laboratory for severalyears. It does not require ultra low temperature equipment, adomestic freezer at -20˚C will suffice.

Preservation of Bacteria using a Modified Glass Bead Technique

1. Grow the organism on an appropriate solid medium until a heavygrowth is obtained. Use several plates with organisms formingsmall colonies.

2. Remove growth from plate using a sterile cotton swab andemulsify in 10% w/v glycerol in Brain Heart Infusion Broth.

3. Carefully pipette the suspension on to sterile glass beads with ahole for threading, then place them in a plastic freezer tube.Replace the cap and tap the bottle to remove the bubbles from beadcentres.

4. Pipette off the excess fluid into phenolic disinfectant in a discardjar. Cap the tube, label and place in freezer.

5. To recover the organisms, remove the beads using forceps, roll onan appropriate medium and streak out to obtain single colonies.

6. There are potential problems with this technique which can beavoided:(i) The tubes should be duplicated and then stored in separate pots- one for routine access, the other as a back-up in a separate freezer. (ii) The frozen beads will soon defrost on the bench. A block ofaluminium or copper drilled out to take the plastic tubes willprevent thawing. The block is kept in the freezer along with thebeads, and removed wearing a glove.

A commercially available form of the above system called ProtectBeads is available from LAB M, please call for further details.Details of recommended QC strains of organisms are contained in theindividual entries for media. QC Assays, which combines certifiedmedia control cultures and Protect storage beads, is also available asa convenient kit.

Organism/Bacteria ATCC NCTC Other

Acinetobacter calcoaceticus 15309 5866

Aerococcus viridans 11563 8251

Aeromonas hydrophila 7966 8049 NCIMB 9240

Bacillus cereus 11778

Bacillus coagulans 7050 10334 NCIMB 9365

Bacillus licheniformis 14580 10341 NCIMB 9375

Bacillus megaterium 14581 10342 NCIMB 9376

Bacillus subtilis 5398

Bacteroides fragilis 25285 9343

Brocothrix thermosphacta 11509 10822 NCIMB 10018

Campylobacter coli 11366 CIP 7080

Campylobacter jejuni 11168

Campylobacter laridis 11352

Citrobacter freundii 6272

Clostridium bifermentans 506

Clostridium perfringens 8237

Clostridium perfringens 8238

Corynebacterium diptheriae 19409 3984

Enterobacter aerogenes 13048 10006

Enterobacter cloacae 13047 10005

Enterococcus faecalis 8043 775

Enterococcus faecium DSM 2918

Organism/Bacteria ATCC NCTC Other

Escherichia coli 11775 9001

Escherichia coli 10090

Escherichia coli NCIMB 9483

Escherichia coli O111 9111

Escherichia coli O157 12900 (non-toxigenic)

Haemophilus influenzae 10479

Hafnia alvei 13337 8109

Klebsiella oxytoca CMCC 2703

Lactobacillus acidophilus 19992

Lactobacillus brevis 14869

Lactobacillus bulgaricus 11842

Lactobacillus casei 7469

Lactobacillus plantarum 14917

Lactobacillus sake 15521

Lactobacillus viridescens 12706

Leuconostoc mesenteroides DSM 20241

Listeria monocytogenes 19111 7973 NCIMB 50007

Microbacterium flavum NCIMB 8707

Microbacterium lacticum NCIMB 8540

Micrococcus luteus 15307 2665

Moraxella sp. NCIMB 10762

Morganella morganii 8076h 235 NCIMB 235

Neisseria gonorrhoeae 19242 8375

Pediococcus damnosus 29258

Proteus mirabilis 25933

Pseudomonas aeruginosa 25668 10662

Pseudomonas fluorescens 10038

Pseudomonas fragi 10689

Pseudomonas putida 10936

Salmonella dublin 9676

Salmonella enteritidis 5188

Salmonella gallinarum 9240

Salmonella saint paul 6022

Salmonella senftenberg 10384

Salmonella typhi 8394

Salmonella typhimurium 13311 74

Salmonella virchow 5742

Serratia liquefaciens NCIMB 9321

Serratia marcescens 13880 10211

Shigella flexneri 29903

Shigella sonnei 29930

Staphylococcus aureus 25923 637

Staphylococcus aureus 6538

Staphylococcus aureus CMCC 2656

Staphylococcus epidermidis 19990 1466

Streptococcus bovis Kiel 27421

Streptococcus lactis 19435 6681 NCIMB 6681

Streptococcus pneumoniae 10319

Streptococcus pyogenes 8198

Streptococcus thermophilus 19258

Vibrio cholerae 11348

Vibrio fluvialis 11327

Vibrio parahaemolyticus 11344

Yersinia enterocolitica 9610 CCUG 11291

Yersinia enterocolitica CCUG 4588

Yersinia enterocolitica CCUG 4586

xix

TABLE – 5 Culture collection strains for the quality control ofculture media

Yeasts & Moulds ATTC NCYC CMI

Alternaria alternata 89343

Aspergillus amstelodami 17455

Aspergillus flavus 91856

Aspergillus niger NCIMB 50097

Aurobasidium cladosporoides 45534

Candida albicans 18804

Cladosporum cladosporoides 45534

Debaryomyces kloeckeri 10620

Fusarium moniliforme 61274

Hansenula anomala 432

Mucor racemosus 17364

Penicillium cyclopium 19795

Pichia burtonii 439

Rhizopus stolonifer 61269

Saccharomyces cerevisiae 79

Zygosaccharomyces rouxii 1522

Abbreviation key:

ATCC American Type Culture Collection, 12301 Parklawn DriveRockville, Maryland, USA.

CCUG Culture Collection, University of Goteborg, GuldhedsgatenlOA, 5-413 46, Goteborg, Sweden.

CMCC Colworth Microbiological Culture Collection, ColworthHouse, Sharnbrook, Bedford MK44 1 LQ, U. K. [strainsfrom this source will be deposited with NCIMB].

CMI Commonwealth Mycological Institute, Ferry Lane, Kew,Surrey, UK

CIP Collection de l’Institut Pasteur, Paris, France.

DSM Deutsche Sammlung von Mikroorganismen, (GermanCollection of Micro-organisms), Grisebachstrasse 8, D-3400, Gottingen, FRG.

KIEL Federal Dairy Research Institute, Hermann Weigmanstrasse1, Kiel, FRG.

NCIMB National Collections of Industrial and Marine Bacteria Ltd.,AURIS Business Centre, 23 St. Machar Drive, Aberdeen,AB2 1RY. Scotland.

NCTC National Collection of Type Cultures, Central Public HealthLaboratory, Colindale Avenue, London NW9 5HT, U. K.

NCYC National Collection of Yeast Cultures, Food ResearchInstitute,Colney Lane, Norwich NR4 7UA, U. K.

The above table is adapted from the original published by the IUMS - ICFMH Working party on Culture media. Int. J. FoodMicrobiol. 1987 5 297 -299.

Laboratory Accreditation

The accreditation of a microbial testing facility must be the goal of allinvolved in the production of goods or provision of services toconsumers. Accreditation is the means by which external assessmentensures that the facility, personnel, and methods of a testinglaboratory are appropriate, monitored and challenged within acontinuously improving quality system.

The impetus for laboratory accreditation has, in the past, differed byindustry sector; for example pharmaceutical laboratories have formany years been subjected to Good Laboratory Practice and regularinspection by bodies such as the FDA and MCA. However, morerecently, the benefits of accreditation are being applied in diagnosticand food testing laboratories, and probably the greatest area ofgrowth is the food industry.

The impetus for this growth is essentially the Food Safety Act of1990, which laid down the requirements for food manufacturers withregard to the provision of safe food, and introducing the ‘DueDiligence Defence’ whereby the defendant must show that allreasonable precautions which could have been taken to avoid anincident were indeed in place. It follows from this that ensuring thetesting laboratory is operating to agreed standards could beinterpreted as a reasonable precaution which should be taken.

There are a number of schemes which provide assessment andcertification of laboratory performance; UKAS, CLAS, MAFF, GLP,ISO9000, CPA. The choice of scheme is bewildering, but narroweddepending upon the nature of testing carried out, e.g. a food laboratorywould not use CPA (Clinical Pathology Accreditation scheme) andmay also consider GLP (Good Laboratory Practice) not targeted to itsspecific needs. In addition, other schemes are available and the choicemay continue to expand. The choice of scheme should not be takenlightly, and should take into consideration the demands of thelaboratory customers (internal and external), and industry practice.

Further consideration might be given to possible future legislation, aschanges between systems will almost certainly involve extra cost if itbecomes necessary. One driving force for any future regulations islikely to be The Official Control of Foodstuffs Directive (89/397)which is an EC Directive concerned with the establishment of theSingle Market in Foodstuffs, and sets out the requirements for officialfood testing laboratories. Part of this sets out the requirements foraccreditation, stating that this must be to the EN 45000 Series ofstandards. Currently only UKAS in the UK meet the criteria foraccreditation bodies laid down in European Standard EN 45003.Whilst these standards relate specifically to official food testinglaboratories, i.e. those laboratories who will be monitoring food withregard to enforcement of the law, it will reflect the best practice towhich all testing facilities should aspire.

xx

Hazard Analysis Critical Control Points (HACCP)

In the same way that European legislation is driving the issue oflaboratory accreditation, it also affects other areas where the expertiseof the food microbiologist is essential, and a prime example of this isin the use of HACCP in the production of food.

The European Directive on the Hygiene of Foodstuffs 93/43/EECincludes the requirement that all businesses shall develop a system inaccordance with the HACCP principles to identify and control foodhazards.

Developed in the USA to ensure the absolute safety of food used inthe NASA space programme (can you imagine suffering foodpoisoning in zero gravity?), it is a system which recognises thelimitations of end-product testing and identifies the points in theproduction process critical to producing a safe product. It does notreplace end product testing; it puts end product testing into the correctcontext as a validation that the control system is functioningcorrectly.

The hazards inherent in any food production process are specific tothat process in that company at that time, and so the HACCP systemused to control it also has to be designed specifically and reviewedregularly.

Like Quality Systems HACCP can only work with the backing of theentire organisation starting from the top and requires a team approachto implement. The team must be multi-disciplinary and typically willinclude personnel from production, engineering, microbiology, andhygiene.

The team will then follow the principles of HACCP to implement asystem which will ensure the safety of the product:

Hazard AnalysisThis is the identification of the hazards associated with theproduction of the product and the ranking of the risk involved witheach and involves the input of relevant members of the team with thenecessary technical background. Once hazards are agreed then theCCP’s can be identified.

Critical Control PointsThis is any process, location, practice, or raw material which, if notcontrolled, will allow the product to pose a threat to consumer of theproduct. For example, if a product must reach 70˚C on cooking,failure to do so would pose a threat to the consumer. This is a criticalcontrol point.

Establish Control CriteriaThe control criteria must be a parameter or practice which can bemonitored, adjusted and verified in a time scale relevant to theproduction of the food. For example, oven temperature can be setaccurately determined limits, monitored by probes and adjusted inreal time. With the advent of rapid technology the hygienic status ofsurfaces can be measured in real time and re-cleaning performed ifnecessary.

Establish Monitoring ProceduresThe parameters which ensure that the critical control point is actuallyin control of the safety of the product, which allow positive feedbackand rapid corrective action. These are generally physical (time,temperature) chemical (pH, biocide levels, aw, ATP) or visual(handling procedures, equipment). Documentation of all monitoringresults is essential.

Microbiology is seldom an effective monitoring tool due to the timeinvolved. However it may be useful for monitoring contaminationlevels of raw materials prior to use.

Establish Corrective Action PlansAgreed actions necessary to regain control if monitoring of a CCPindicates that it is not controlling the safety of the product.Documentation is again essential to show the ‘quality loop’ -monitoring > non-conformance > action > monitoring > control.

Verification ProceduresFurther actions taken to verify the monitoring of CCP’s, to ensure thatthe control is in place, and the monitoring parameters are correct. If aproduct fails verification when the monitoring process shows noproblem, it indicates a failure in the system and a review is necessaryto identify a missing CCP or other source of the problem.

End product testing falls into this category, as could measuring thelevel of microbial contamination of the manufacturing environment.

Auditing and ReviewThe system must be reviewed and audited at regular intervals toensure that nothing has changed which would affect the efficacy ofthe control process, or omission of important detail. This mustinclude audit of the documentation and monitoring records, as well asphysical examination of the whole procedure. As with verification,any areas of concern must be subject to the corrective action anddemonstrate the ‘quality loop’.

Like all systems, HACCP is only effective if implemented properlywith the co-operation of all employees involved. A poorly operatedsystem could make things appear under control, when the reality isthat potentially dangerous products may be produced.

Developing a HACCP system can be a time consuming and complexbusiness, and the above representation is vastly over-simplified. It istherefore advisable to seek professional help via research associationsor expert consultants with many years experience of developingHACCP systems.

xxi

xxii This page left intentionally blank

xxiii

Microbiology Methods

We have set out some of the more common methods in use, plus oneor two newer methods, indicating the LAB M products required toperform them. This is not intended to be a comprehensive list, norreplace other sources of information, but as a simple guide only.There are numerous sources of information regarding microbiologymethods, just some of which are listed below:

British Standards Institute, Customer Services, Linford Wood, MiltonKeynes, MK14 6LE. Numerous microbiological standards e.g.BS5763 Methods for Microbiological examination of food andanimal feeding stuffs. Part 11. Enumeration of Bacillus cereus.

Compendium of Methods for the Microbiological Examination ofFoods (1992) Third edition. Edited by Vanderzant, C. andSplittstoesser D.F. American Public Health Association. ISBN 0-87553-173-3

Food and Drug Administration. Bacteriological Analytical Manual8th edition (1995) AOAC International. ISBN 0-935584-59-5.

Foodborne Pathogens An Illustrated Text (1991) Varnam, A.H. andEvans, M.G. Wolfe Publishing (now Times Mirror International).ISBN 0-7234-1521-8).

Manual of Microbiological Methods for the Food and Drink Industry.Technical Manual No. 43 2nd edition. (1995) Campden &Chorleywood Food Research Association, Chipping Campden,Goucestershire, GL55 6LD.

Micro organisms in foods - Their significance and methods ofenumeration 2nd edition (1978) ICMSF edited by Thatcher, F.S. andClark, D.S. ISBN 0-8020-2293-6

Practical Food Microbiology. Methods for the Examination of Foodfor Micro organisms of Public Health Significance 2nd edition (1995)Edited by Roberts, D., Hooper, W. and Greenwood, M. Public HealthLaboratory Service ISBN 0-901144-36-3

Environment Agency: The Microbiology of Drinking Water (2002).Methods for the Examination of Water and Associated Materials.

Total Viable Count(Total Aerobic Count)

Prepare an appropriate 1:10 dilution of the samplee.g. 10g* sample + 90ml MRD (LAB 103)

||

Homogenise for 2 mins||

Prepare serial dilutions by adding 1ml of homogenate to 9ml of MRD (LAB 103)

||

Inoculate 1ml of appropriate dilutions into a sterile Petri dish andadd aseptically 15ml of molten, cooled:Plate Count Agar APHA (LAB 10) or

Plate Count Agar (LAB 149) orMilk Plate Count Agar (LAB 115) or

Milk Agar (LAB 19) orYeast Extract Agar (LAB 18)

||

Incubate at the appropriate temperature to count the requiredbacterial population:

30˚C for 48hrs (aerobic mesotroph count)21˚C for 5 days or 6.5˚C for 10 days (aerobic psycrotroph count)

55˚C for 48hrs (aerobic thermotroph count)||

Select plates with between 30 and 300 colonies for enumeration. If the highest dilution has more than this use 300 as the figure to

calculate the original count and express as ‘greater than…’||

Calculate the original count in the sample expressed as CFU’s per gram or ml (The number should be given in standard scientific

notation e.g. 2.4 x 103)This method can be adapted to perform an anaerobic count by

incubating in anaerobic conditions. However incubation times mayvary to those above and specific texts should be consulted.

Remember that facultative organisms will give a count for both theaerobic and anaerobic counts.

*For liquid samples use 10ml to 90ml No homogenisation is required prior to preparing serial dilutions.

Coliform/Enterobacteriaceae(Enumeration)


||



||

Inoculate 1ml of appropriate dilutions into a sterile Petri dish andadd aseptically 15ml of molten, cooled:

VRBA (LAB 31) (Coliform count)or VRBGA (LAB 88) (Enterobacteriaceae count)

When set, overlay with more of the same agar to cover surface||

Incubate at 37˚C for 24hr(some USA methods 35˚C, coliforms for dairy purposes 30˚C)

||

Count all colonies >0.5mm diameter present on the plate. Selectplates with between 15 and 150 colonies for enumeration. If the

highest dilution has more than this use 150 as the figure to calculatethe original count and express as ‘greater than…’

||

Calculate the original count in the sample expressed as CFU’s pergram or ml (The number should be given in standard scientific

notation e.g. 2.4 x 103)*For liquid samples use 10ml to 90ml

No homogenisation is required prior to preparing serial dilutions.

Escherichia coli (Presence or Absence)


||


To detect presence in 0.1g of product, inoculate 1ml of homogenateinto 10ml of single strength MacConkey Broth Purple (LAB 5) or

Brilliant Green Bile Broth (LAB 51) (with inverted Durham tubes).To detect presence in 1g of product, inoculate 10ml of double

strength medium with 10ml of homogenate||

Incubate at 37˚C for 24hr and examine for acid and gas(LAB 5) or turbidity and gas (LAB 51)

(30˚C is used in the dairy industry)||

If negative incubate for a further 24hrs, and examine again||

Subculture 0.1ml of positive broth into 10ml single strengthBrilliant Green Bile Broth (LAB 51), and

Tryptone Water (LAB 129)||

Incubate at 44 ± 0.5˚C for 24hrs and examine for turbidity and gas production

||

If negative incubate for a further 24hrs and examine again||

For all positive tubes add 2-4 drops of indole reagent to thematching Tryptone Water. Wait one minute and examine for a red

colour in the alcohol layer at the top of the broth, indicating apositive indole reaction

||

Record result as presumptive presence of E. coli in0.1g or 1g of product as appropriate


E. coli(Enumeration Using Membranes)


||


Prepare serial dilutions by adding 1ml of homogenate to 9ml ofMRD (LAB 103)

||

Inoculate membrane on the surface of MineralsModified Glutamate Agar (LAB 80A + LAB 80B)

+ 12g/litre of Agar No.2 (MC 6)||

Incubate at 37˚C for 4hrs then transfer membraneto a Tryptone Bile Agar plate (LAB 72)

||

Incubate with membrane uppermost at 44 ± 0.5˚C for 18-24hrs||

Put 2ml of Vracko-Sherris reagent into lid of dish, and placemembrane on top. Allow to soak in reagent for 5 min.

||

Dry membrane in natural daylight or expose to UV for 5-30 min.||

Indole positive colonies will turn pink. Count all pink colonies aspresumptive E. coli and calculate the original count in the sample,

expressed as CFU’s per gram or ml (The number should be given instandard scientific notation e.g. 2.4 x 103


E. coli (Enumeration Without Membranes)


||



||

Inoculate the surface of Harlequin™ Tryptone Bile GlucuronideAgar (HAL 3)

||

Incubate at 30˚C for 4hrs then transfer to 44 ± 0.5˚C for 18-24hrs||


E. coli O157:H7(Presence or Absence)

Add 25g sample to 225ml of Modified Tryptone Soy Broth(MTSB) (LAB 165)

||


Incubate at 42˚C for 6 and 24hrs||

Perform Captivate™

immunomagnetic separationat 6 and 24hrs

||

Inoculate 50µl of Captivate™ beads onto SMAC(LAB 161) and CT SMAC (LAB 161 + X161).Incubate plates at 37˚C for 24hrs. Alternatively

Harlequin™ SMAC-BCIG (HAL 6) can be used.||

Examine plates for sorbitol negative colonies||

Confirm as E. coli O157 by serology and biochemistry (commercial ID kits)

It should be noted that not all O157 isolates produce theverocytotoxin which is the mode of pathogenesis of the organism.

Furthermore, other serovars of E. coli can produce theverocytotoxin and therefore be pathogenic for humans.

xxiv

Yeasts and Moulds (Enumeration)


||



||

Inoculate 1ml of appropriate dilutions onto the surface of :Rose Bengal Chloramphenicol Agar (LAB 36), or

Malt Extract Agar (LAB 37), orOxytetracycline Glucose Yeast Extract Agar (LAB 89)

||

Incubate at 25˚C for 5 days||



Calculate the original count in the sample expressed as CFU’s per gram or ml (The number should be given in standard

scientific notation e.g. 2.49 x 103)It is always prudent to perform a simple wet preparation of suspectyeast colonies by emulsifying a colony in a drop of saline on a slideand placing a coverslip on top. Examine microscopically to confirmlarge, rounded, usually budding yeast cells as opposed to the much

smaller bacterial rods and cocci).*For liquid samples use 10ml to 90ml


Staphylococcus aureus (Enumeration)


||



||

Inoculate 0.1ml or 0.5ml of appropriate dilutions by spreading overthe entire surface of Baird-Parker medium (LAB 85 and X085), or

Baird-Parker medium with RPF supplement (X086)||

Incubate at 37˚C for 48hr||

Select plates with between 15 and 150 black colonies forenumeration. If the highest dilution has more than this use

150 as the figure to calculate the original count and express as ‘greater than…’

||

Confirm at least 5 colonies of each colony type present using slideor tube coagulase tests, or a proprietary kit.

||


notation e.g. 2.4 x 103), taking into account the ratio of coagulasepositive and negative colonies.

The advice to count all black colonies is a reflection of the variationof appearance of S. aureus (and other staphylococci) on Baird-Parker

Medium. The production of the ‘typical’ lecithinase and lipasereactions is not a stable characteristic, and in particular damagedorganisms may lose the ability to produce them. Moreover, other

staphylococci can mimic these reactions and so confirmation of allcolony types is necessary to reduce the risk of reporting false counts.


Pseudomonas species (Enumeration)


||



||

Inoculate 0.1ml or 0.5ml by spreading over the surface ofPseudomonas Agar (LAB 108) supplemented with CFC (X108)

||

Incubate at 25˚C for 48hrs||




notation e.g. 2.4 x 103)It is advisable to confirm growth on the plate as Pseudomonas sppby performing an oxidase test. Yeasts and moulds may grow on the

medium, and these may be differentiated by performing a wetpreparation and examining under the microscope for typical cells.


Bacillus cereus (Enumeration)


||



||

Inoculate 0.1ml or 0.5ml by spreading over the surface of BacillusCereus Medium (PREP, LAB 73 or PEMBA, LAB 193)

||


Examine the plate for colonies. If not clearly visible incubate for afurther 24hrs.

||

Count typical colonies (large, 2-4mm, rough, pink in colour, with or without precipitate in surrounding medium).

Due to the large size of the colonies, count the dilution with lessthan 15 colonies on the plate if possible.

||


notation e.g. 2.4 x 103)This is only a presumptive count, as other Bacillus spp. will grow

on PREP agar (also known as MYP agar). Confirmation can bedone by biochemical methods, although this is not straight forward.

Use reference schemes such as Bergey’s DeterminativeBacteriology Volume 2 or Cowan and Steele. Alternatively a

proprietary kit for detection of specific enterotoxin may be used,and strains of other Bacillus spp. have been reported to produce

enterotoxin.*For liquid samples use 10ml to 90ml


xxv

Clostridium perfringens (Enumeration)


||



||

Inoculate 1ml of appropriate dilutions into a sterile Petri dish andadd aseptically 15ml of molten, cooled, Perfringens Agar (OPSP,

LAB 109) or Perfringens Agar (TSC, LAB 194).When set, overlaywith more of the same agar to cover surface

||

Incubate anaerobically at 37˚C for 24hrs

Examine plates for typical colonies||




notation e.g. 2.4 x 103)*For liquid samples use 10ml to 90ml


Isolation of Listeria species (the ‘FDA’ method)

Add 25g sample to 225ml of Listeria Enrichment Broth (LEB, LAB 138) or Buffered LEB (LAB 139)

||


Incubate at 30˚C for 24 and 48hrs||

Streak a loopful (10 µl) onto Oxford Agar (LAB 122) or PalcamAgar (LAB 148). Incubate plates at 30˚C for 24 and 48hrs.

||


Confirm colonies as Listeria spp. and speciate using biochemical tests

Colonies can be speciated using Listeriazym (T500), and a blood agar plate to determine haemolytic activity.

Some workers have reported improved isolation by using bothOxford and Palcam agars together, similar to the use of different

plating media for the isolation of Salmonella.

Isolation of Listeria species (the ‘modified USDA’ method)

Add 25g sample to 225ml of UVM I (LAB 155)||



Subculture 0.1ml of enriched UVM I into 10ml Fraser Broth (LAB 164) and incubate at 35˚C for 24 and 48hrs

||

Streak a loopful of Fraser Broth (10 µl) onto Oxford Agar (LAB 122) or Palcam Agar (LAB 148). Incubate plates at 30˚C for 24 and 48hrs.

||


Confirm colonies as Listeria spp. and speciate using biochemical tests

Colonies can be speciated using Listeriazym (T500), and a blood agar plate to determine haemolytic activity.

Some workers have reported improved isolation by using bothOxford and Palcam agars together, similar to the use of different

plating media for the isolation of Salmonella.

Isolation of Salmonella species (Semi-solid technology)

Add 25g sample to 225ml of Buffered Peptone Water (LAB 46)||

Homogenise for 2 minutes and incubate at 37˚C for 24hrs||

Subculture three drops of enriched BPW centrally onto the surfaceof MSRV (LAB 150) or Diassalm (LAB 537) plates in duplicate.

||

Incubate with the lid uppermost at 37˚C or 42˚C for 24hrs||

Examine for a spreading zone of growth. If no growth incubate fora further 24hrs

||

Subculture spreading growth to XLD (LAB 32) and Brilliant GreenAgar (LAB 34) to obtain pure growth for confirmation

by biochemical and serological methodsUse of a filter paper disc soaked in polyvalent H Salmonella

antiserum can increase the specificity of the media. Place the discmid way between the inoculation point and the edge of the dishbefore incubation. The motility of salmonellas will be inhibited

around the disc, whereas non-salmonellas will grow up to the disc.Use of the disc with Diassalm enhances the H2S reaction on this

medium. The area where the motility is inhibited by the disc has ahigh concentration of Salmonella and gives a good H2S reaction

indicated by blackening of the growth around the disc.

xxvi

Subculture 1ml of enriched BPW into 9ml of Selenite Cystine Broth(LAB 55A + LAB 44B). Incubate at 37˚C for 24 and 48hrs

||

Inoculate selective agars: Brilliant Green Agar (LAB 34) and XLD Agar (LAB 32)

||

Incubate at 37˚C for 24 hrs and examine for typical colonies||

Confirm suspect colonies by serological and biochemical methods

Subculture 0.1ml of enriched BPW into 10ml of RappaportVassiliadis (Soy) Broth (LAB 86). Incubate at 41.5 ± 0.5˚C* for 24hrs

||

Inoculate selective agars: Brilliant Green Agar (LAB 34) and XLD Agar (LAB 32)

||

Incubate at 37˚C for 24 hrs and examine for typical colonies||

Confirm suspect colonies by serological and biochemical methods

xxvii

Isolation of Salmonella species (conventional method)

Add 25g sample to 225ml of Buffered Peptone Water (LAB 46)||

Homogenise for 2 minutes and incubate at 37˚C for 24hrs

If Selenite cystine broth is not acceptable because of the use of sodium biselenite, then an alternative medium may be substituted, such as Mueller Kauffman Tetrathionate Broth (LAB 42) or Tetrathionate Broth (LAB 97). Both these formulations require the addition of chemicals not supplied by LAB M.

Typical colony descriptions for Brilliant Green Agar and XLD agar can be found under the individual listings in the main body of the catalogue,and are also available as colony cards which show typical colonies in full colour.

*It is critical that the incubation temperature does not exceed 43˚C as this can inhibit some salmonellas. To ensure this lower temperatures arerecommended: 41.5 ± 0.5˚C for incubators, 42 ± 0.1˚C for water baths. (Reference: Peterz M., Wiberg C., and Norberg P. 1989. The effect ofincubation temperature and magnesium chloride concentration on growth of Salmonella in home-made and in commercially available dehydratedRappaport-Vassiliadis broths. J. Appl. Bact. 66 523-528).

||

||

Isolation of Campylobacter species Add 25g sample to 75ml (or 1:4 w/v) of

Campylobacter Enrichment Broth (LAB 135)||


Incubate at 37˚C for 2-4 hrs followed by a further 14-44 hrs at 42˚C||

Streak a loopful (10µl) onto Campylobacter Blood Free Medium(LAB 112) and incubate at 37˚C for 40-48 hrs

||


Confirm colonies as Campylobacter spp. using biochemical tests

xxviii This page left intentionally blank

Format and abbreviation guide

Product Name(Alternative name or commonly used abbreviation)

PRODUCT CODE

Description:A brief outline which may include any of the following informationon the medium:

● HISTORY

● MECHANISMS

● APPLICATIONS

● RECOGNITION BY REGULATORY/ADVISORY BODIES

● ADVANTAGES

Formula: The product composition in grams per litre; minoradjustments to the published formula may be made to meetperformance criteria.

Method for reconstitutionDistilled water can be substituted for deionised water. “Allow to soak times” are not critical. If agar media are to be dispensed prior to sterilising, first bring to the boil to dissolve the agar.

Appearance: – of the finished cooled medium.

pH: at 20˚C. For agars, pour a small quantity into a universal bottle,allow to set and plunge the probe into the medium.

Minimum Q.C. organisms – for use every time a new batch ofprepared medium is reconstituted. This short form check should notbe confused with a full Q.C. of the medium. Where an organismshould show inhibition this could be complete or partial. Recordsshould be kept of these results to help recognise changes inperformance over a period of time.

Storage of Prepared Media – All prepared media should be storedin the dark. If a medium is to be used beyond the suggested shelf life,appropriate quality control should be performed to demonstrate thatthere has been no detectable fall off in performance.

Growth characteristicsAbbreviation key for colonial descriptions:

CV = convex CR = crenated

F = flat 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 formulae in this manual and on the product label areadhered to wherever possible. However it is occasionally necessaryto make minor adjustments to meet performance criteria.

Dehydrated culture mediaselection guide

Agars, Peptones and other culture media ingredientsMC 7 Acid Hydrolysed CaseinMC 2 Agar No. 1 Bacteriological-High ClarityMC 6 Agar No. 2 Bacteriological-General PurposeMC 29 Agar No. 4 Plant Tissue CultureMC 24 Bacteriological PeptoneMC 4 Balanced Peptone No. 1MC 19 Beef ExtractMC 25 Bile Salts No. 3MC 12 Columbia peptoneMC 33 FMV (Foot and Mouth Vaccine) peptoneMC 15 GelatineMC 13 Glucose (Dextrose)MC 402 IPTGMC 40 Lactalbumin HydrolysateMC 20 LactoseMC 23 Malt Extract PowderMC 22 Maltose monohydrateMC 14 MannitolMC 18 Meat PeptoneMC 406 MUGMC 9 Mycological PeptoneMC 10 Ox BileMC 11 Proteose Peptone AMC 27 Skim Milk PowderMC 17 Sodium ChlorideMC 26 Sodium DesoxycholateMC 16 Sodium ThioglycollateMC 3 Soy PeptoneMC 35 Special TryptoneMC 5 TryptoneMC 8 TryptoseMC 405 X-galMC 32 XyloseMC 1 Yeast Extract Powder

AnaerobesLAB 160 Brazier's CCEY AgarLAB 24 Cooked Meat GranulesLAB 90 Fastidious Anaerobe Agar (F.A.A.)LAB 71 Fastidious Anaerobe Broth (F.A.B.)LAB 25 Fluid Thioglycollate Medium (U.S.P.)LAB 109 Perfringens Agar (O.P.S.P.)LAB 194 Perfringens Agar (TSC)LAB 23 Reinforced Clostridial AgarLAB 22 Reinforced Clostridial Medium (Broth)LAB 64 Thioglycollate Medium (Brewer)

Biomolecular ProductssHAL 4 Harlequin™ LB AgarHAL 5 Harlequin™ LB Top AgarLAB 168 LB AgarLAB 174 LB Agar (Lennox)LAB 169 LB BrothLAB 173 LB Broth (Lennox)LAB 191 Luria Bertani (Hi-Salt) BrothLAB 182 NZCYM BrothLAB 181 NZY Broth (NZYM)LAB 177 Superbroth LAB 178 Superbroth with AgarLAB 183 Terrific BrothLAB 175 YPD BrothLAB 176 YPD with AgarLAB 180 2xYT AgarLAB 179 2xYT Broth

Blood Agar BasesLAB 28 Blood Agar BaseLAB 15 Blood Agar Base No. 2LAB 1 Columbia Agar BaseLAB 525 Eugon AgarLAB 90 Fastidious Anaerobe Agar (F.A.A.)LAB 11 Tryptone Soy Agar (U.S.P.)

xxix

Blood Culture Media

LAB 49 Brain Heart Infusion Broth LAB 71 Fastidious Anaerobe Broth (F.A.B.)LAB 4 Tryptone Soy Broth (USP)

Coliform/Enterobacteriaceae MediaLAB 51 Brilliant Green Bile 2% BrothLAB 91 E. E. Broth (Enterobacteriacae Enrichment Broth)LAB 60 Endo Agar LAB 61 Eosin Methylene Blue AgarHAL 8 Harlequin™ E. coli/Coliform MediumHAL 9 Harlequin™ mLGAHAL 3 Harlequin™ Tryptone Bile Glucuronide Agar (TBGA)LAB 126 Lactose BrothLAB 196 Lauryl Tryptose BrothLAB 45 MacConkey Agar No. 3LAB 5 MacConkey Broth (Purple)LAB 72 Tryptone Bile AgarLAB 573 V.R.B.A. with MUGLAB 31 Violet Red Bile Agar (VRBA)LAB 88 Violet Red Bile Glucose Agar (VRBGA)

Diagnostic Medical MicrobiologyLAB 121 Bromocresol Purple Lactose AgarLAB 6 C.L.E.D. (Bevis)-double indicatorLAB 41 C.L.E.D. (Mackey & Sandys)-single indicatorLAB 67 G.C. Agar Base HAL 7 Harlequin™ C.L.E.D.LAB 27 Hoyle's MediumLAB 123 Kirschner’s-T.B. enrichmentLAB 35 T.Y.C. Medium-investigation of dental cariesRefer to other sections for our full range of Medical Products.

Diluents/Isotonic solutionsLAB103 Maximum Recovery DiluentLAB 100 Ringer’s Solution (1/4 Strength)LAB 100ZRinger’s Solution (1/4 Strength) - TabletsLAB 101 Ringer’s Solution (Calgon)LAB 102 Ringer’s Solution (Thiosulphate)

Enteric PathogensLAB 167 Aeromonas AgarLAB 13 Bismuth Sulphite AgarLAB 34 Brilliant Green AgarLAB 46 Buffered Peptone Water - pre-enrichment brothLAB 112 Campylobacter Agar (Blood Free - Improved)LAB 135 Campylobacter Enrinchment BrothLAB 161 Sorbitol MacConkey Agar (SMAC)LAB 3 D.C.L.S.LAB 29 Desoxycholate Citrate Agar (DCA)LAB 65 Desoxycholate Citrate Agar (Hynes)LAB 537 DiassalmHAL 1 Harlequin™ Salmonella ABCHAL 6 Harlequin™ Sorbitol MacConkey Agar (SMAC-BCIG)LAB 110 Hektoen Enteric AgarLAB 116 M.L.C.B. AgarLAB 150 M.S.R.V.LAB 30 MacConkey Agar (with salt)LAB 2 MacConkey Agar (without salt)LAB 42 Mueller Kauffman Tetrathionate BrothLAB 165 O157 Broth (MTSB)LAB 86 Rappapport Vassiliadis MediumLAB 52 S.S. Agar (Salmonella Shigella Agar)LAB 44 Selenite BrothLAB 55 Selenite Cystine BrothLAB 96 T.C.B.S. Cholera MediumLAB 97 Tetrathionate Broth BaseLAB 32 XLD AgarLAB 120 Yersinia CIN Agar

Enterococci Test MediaLAB 106 Kanamycin Aesculin Azide AgarLAB 107 Kanamycin Aesculin Azide BrothLAB 166 Slanetz and Bartley (m Enterococcus Medium)

Food MicrobiologyLAB 167 Aeromonas AgarLAB 85 Baird-Parker MediumLAB 34 Brilliant Green Agar (Modified)LAB 46 Buffered Peptone WaterLAB 112 Campylobacter (Blood Free - Improved)LAB 135 Campylobacter Enrichment BrothLAB 105 China Blue Lactose AgarLAB 537 DiassalmLAB 136 Easter and Gibson Pre-enrichment BrothLAB 137 Easter and Gibson Salmonella MediumLAB 171 EC Medium (E. coli Medium)LAB 16 Flourescence Agar - Pseudomonas spp.LAB 164 Fraser BrothHAL 8 Harlequin™ E.coli/Coliform MediumHAL 2 Harlequin™ Listeria MediumHAL 6 Harlequin™ Sorbitol MacConkey Agar (SMAC-BCIG)HAL 3 Harlequin™ Tryptone Bile Glucuronide Agar (TBGA)LAB 196 Lauryl Tryptose BrothLAB 158 Liquid Baird-Parker MediumLAB 138 Listeria Enrichment BrothLAB 139 Listeria Enrichment Broth (Buffered)LAB 122 Listeria Isolation Medium (Oxford)LAB 172 Listeria Monocytogenes Blood Agar (LMBA)LAB 93 M.R.S. AgarLAB 94 M.R.S. BrothLAB 150 M.S.R.V.LAB 42 Mauller Kauffman Tetrathionate BrothLAB 116 MLCB AgarLAB 165 O157 Broth (MTSB)LAB 147 Orange Serum AgarLAB 73 P.R.E.P. Agar-cultivation of Bacillus cereusLAB 148 Palcam AgarLAB 144 Palcam BrothLAB 193 PEMBA Agar - Bacillus Cereus MediumLAB 194 Perfringens Agar (TSC)LAB 109 Perfringens Agar (O.P.S.P.)LAB 149 Plate Count AgarLAB 10 Plate Count Agar A.P.H.A.LAB 98 Potato Dextrose AgarLAB 108 Pseudomonas AgarLAB 86 Rappaport Vassiliadis Medium (Broth)LAB 23 Reinforced Clostridial Agar-enumeration of anaerobesLAB 55 Selenite Cystine BrothLAB 161 Sorbitol MacConkey AgarLAB 87 Sugar Free Agar-enumeration of organisms in butter and

similar productsLAB 97 Tetrathionate Broth Base A.P.H.A.LAB 155 U.V.M. BrothLAB 31 V.R.B.A.LAB 573 V.R.B.A. with MUGLAB 88 V.R.B.G.A.LAB 79 W.L. Agar-examination of worts, beers and yeast

culturesLAB 38 Wort Agar-yeasts in dairy and sugar productsLAB 99 Wort BrothLAB 32 XLD Agar

Identification MediaLAB 59 Kligler Iron AgarLAB 126 Lactose BrothLAB 54 Lysine Iron AgarLAB 104 Peptone WaterLAB 69 Simmons Citrate AgarLAB 53 Triple Sugar Iron AgarLAB 129 Tryptone WaterLAB 130 Urea AgarLAB 131 Urea Broth

Neutralising MediaLAB 188 D/E Neutralising AgarLAB 187 D/E Neutralising BrothLAB 186 D/E Neutralising Broth BaseLAB 185 Letheen Agar (AOAC)LAB 184 Letheen Broth (AOAC)LAB 189 Microbial Content Test Agar

xxx

Nutrient Media for general useLAB 48 Brain Heart Infusion AgarLAB 49 Brain Heart Infusion BrothLAB 525 Eugon AgarLAB 526 Eugon BrothLAB 8 Nutrient AgarLAB 68 Nutrient Broth 'E'LAB 8 Nutrient Broth No. 2LAB 62 Tryptose Phosphate BrothLAB 18 Yeast Extract Agar

Sensitivity Test MediaLAB 39 Mueller Hinton Agar (II)LAB 114 Mueller Hinton Broth (II)LAB 74 Nu-sens AgarLAB 12 Sensitivity Test AgarLAB 170 Susceptibility Test 'ISO' Agar

Staphylococci MediaLAB 85 Baird-Parker MediumLAB 95 DN'ase Test AgarLAB 158 Liquid Baird-Parker MediumLAB 7 Mannitol Salt AgarLAB 192 ORSIM - Oxacillin Resistant Staphylococci Isolation

MediumLAB 113Z Salt Meat Broth (Tablets)LAB 84 Single Step Staph Selective Agar (4S)-rapid method for

S. aureus

Streptococci Test MediaLAB 523 Azide Blood Agar BaseLAB 92 M17 AgarLAB 35 T.Y.C. MediumLAB 75 Todd Hewitt Broth

Sterility Test MediaLAB 25 Fluid Thioglycollate U.S.P.LAB 159 Malt Extract BrothLAB 14 Nutrient Broth No. 2 B.P.LAB 33 Sabouraud Liquid Medium U.S.P.LAB 11 Tryptone Soy Agar U.S.P.LAB 4 Tryptone Soy Broth U.S.P.

Transport MediumLAB 124 Amies with charcoalLAB 125 Amies without charcoalLAB 505 Carey-Blair Medium

Total Viable CountsLAB 19 Milk AgarLAB 115 Milk Plate Count AgarLAB 10 Plate Count Agar A.P.H.A.-standard methodsLAB 149 Plate Count Agar-for spiral platers or pour platesLAB 163 R2A MediumLAB 63 Tryptone Glucose Extract Agar A.P.H.A.LAB 11 Tryptone Soy AgarLAB 197 Water Plate Count Agar (ISO)LAB 18 Yeast Extract Agar

Water TestingHAL 9 Harlequin™ mLGALAB 126 Lactose BrothLAB 196 Lauryl Tryptose BrothLAB 5 MacConkey Broth (Purple)LAB 82 Membrame Lauryl Sulphate BrothLAB 80 Minerals Modified Glutamate BrothLAB 163 R2A MediumLAB 197 Water Plate Count Agar (ISO)Refer to other sections for our full range of Water Test Media.

Yeasts and MouldsLAB 117 Dermatophyte Test mediumLAB 37 Malt Extract AgarLAB 159 Malt Extract BrothLAB 89 O.G.Y.E. AgarLAB 98 Potato Dextrose AgarLAB 36 Rose Bengal Chloramphenicol AgarLAB 9 Sabouraud Dextrose AgarLAB 33 Sabouraud Liquid MediumLAB 111 Sabouraud Maltose AgarLAB 38 Wort AgarLAB 99 Wort BrothLAB 119 Yeast Extract Dextrose Chloramphenicol Agar

xxxi

1. Media Range

Aeromonas AgarBile Salt Irgasan Brilliant Green Agar

LAB 167DescriptionAeromonas Agar is a highly selective medium for the isolation ofAeromonas spp. from food, clinical and environmental samples.Based on the selective agents, brilliant green and irgasan, thismedium will not inhibit those strains of Aeromonas sensitive toampicillin used in other media.

Formulation g/litre

Beef Extract 5.0

Meat Peptone 5.0

Xylose 10.0

Bile Salts No.3 8.5

Sodium thiosulphate 5.44

Irgasan 0.005

Brilliant green 0.005

Neutral red 0.025

Agar 11.5

Appearance: Purple, Clear gel

pH: 7.0 ± 0.2

Method for reconstitutionWeigh 45.5 grams of powder and disperse in 1 litre of deionisedwater. Allow to soak for 10 minutes, swirl to mix and sterilise bybringing to the boil. Cool to 47˚C, mix well and dispense into Petridishes.

Inoculation: Faecal specimens: Inoculate surface of mediumdirectly, spreading for single colonies. Samples requiring enrichment:Inoculate alkaline peptone water and incubate at 37˚C for 18-24 hr.Subculture onto Aeromonas Agar, surface spreading for singlecolonies.

Incubation: Incubate plates aerobically at 37˚C for 18-24 hr.Examine for typical colonies and confirm as Aeromonas spp.

Storage: Poured plates: 7 days at 2-8˚C in the dark (may be extendedif moisture tight packaging used).

Minimum Q.C. organisms: Aeromonas hydrophilaNCIMB 9240E. coli NCIMB 50034 (inhibited)

ConfirmationTypical colonies (translucent pink colonies 0.5-3.0mm diameter)should be confirmed as presumptive Aeromonas spp. by performingan oxidase test and inoculating into Hugh & Leifsons O/F medium.Aeromonas spp. will give a positive oxidase reaction and demonstrateboth oxidative and fermentative metabolism. Pseudomonas spp. willalso be oxidase positive, but do not possess fermentative metabolism.An alternative method is to inoculate triple sugar iron tubes.Aeromonas will typically produce an acid butt (yellow) and analkaline or unchanged slant (red) whilst Pseudomonas spp. willremain unchanged in both the butt and slant. To fully identifycolonies as Aeromonas spp. the above tests should be supported usinga proprietary kit such as API 2ONE or Microbact 24E (other productsmay be available).

InterpretationOrganism Size Shape Colour

Aeromonas spp.* 0.5-3.0 CV.E.G Translucent pink

Pseudomonas spp. 0.5-1.0 CV.E.G Translucent pink

S.aureus No growth

E. coli No growth

*The selective nature of the medium may mean occasional strains do not grow,or grow poorly.

Amies Transport Medium without Charcoal

LAB 125

DescriptionThis medium is as LAB 124 without the charcoal. It is used whenmicroscopic examination of a film is an important part of theprocedure and the charcoal may interfere with interpretation.

Formula g/litre

Sodium chloride 3.0

Potassium chloride 0.2

Disodium hydrogen phosphate 1.15

Sodium thioglycollate 1.0

Calcium chloride 0.1

Magnesium chloride 0.1

Potassium phosphate 0.2

Agar No. 1 4.0

Method for reconstitutionWeigh 9.75 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to boil todissolve agar. Distribute into bijou bottles filling to shoulder.Constantly mix whilst distributing. Sterilise by autoclaving at 121˚C for 15 minutes. Screw caps down after autoclaving.

Appearance: Soft translucent gel.

pH: 7.1 ± 0.2

Minimum Q.C. organisms: N. gonorrhoeaeS. pyogenes

Storage of Prepared Medium: Capped containers up to 6 months at 15-20˚C in the dark.

Inoculation: As LAB 124.

Amies Transport Medium with Charcoal

LAB 124

DescriptionAmies introduced his modification of Stuarts transport medium toovercome a number of problems. Stuarts transport medium sufferedfrom overgrowth by coliforms that were capable of utilising sodiumglycerophosphate. Amies replaced the problem component with aninorganic phosphate buffer system. Calcium and magnesium salts areadded to control the permeability of the bacterial cell wall and thus prolong their survival. The addition of charcoal to the medium extended the survival time of Neisseria gonorrhoeae from 24 to 72 hours.

1.1

Formula g/litre

Charcoal-activated 10.0

Sodium chloride 3.0







Agar No. 1 4.0

Method for reconstitutionWeigh 19.75 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to boil todissolve agar. Distribute into bijou bottles filling to shoulder.Constantly mix whilst distributing. Sterilise by autoclaving at 121˚Cfor 15 minutes. Screw caps down after autoclaving.

Appearance: Soft gel with heavy concentration of evenly suspendedcharcoal.

Minimum Q.C. organisms: N. gonorrhoeaeS. pyogenes

Storage of Prepared Medium: Capped containers – up to 6 monthsat 15-20˚C in the dark.

pH: 7.2 ± 0.2

Inoculation: Push the swab containing the sample into the gel toapproximately one third of the gels depth. Cut off the unwanted swabstick then screw on the cap pushing the swab further down into thegel. Cap tightly and keep cool during transport to the laboratory.

ReferencesAmies, C.R. (1967). Can. J. Pub. Health. 58: 296-300. A modifiedformula for the preparation of Stuarts Transport Medium.

Anaerobe Identification Medium BaseLAB 66

DescriptionA medium introduced by Phillips in 1976 for testing the fermentationcapabilities of non-sporing anaerobes. The base is carbohydrate freeand nutritious.

Formula g/litre

Beef Extract 4.0

Peptone mixture 16.0

Sodium chloride 5.0

Agar No. 2 15.0

Method for reconstitutionWeigh 40 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and asepticallyadd 50-70ml of sterile defibrinated horse blood. Mix well and pour.Before use, flood the surface with 1ml of a sterile solution of thesubstrate under test.

Appearance: Blood agar plate.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: B. fragilis

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped Container – up to 3 months at 15-20˚C in the dark.

Inoculation: Heavily inoculate a small area of the plate with a

loopful of a fresh culture of the test organism. Up to 4 organisms perplate can be tested.

Incubation: 37˚C anaerobically for 24-48 hours. Recognition offermentation: Remove a small plug of agar from below the growth.Cover the plug with bromothymol blue indicator (0.04%). Colourchanges due to production of acidity will develop in a few secondsand should be viewed against a white background. Comparison withcontrols is useful, a plug taken from an area well away from anygrowth can be used as a negative control.

ReferencesPhillips K.D. (1976). A Simple and sensitive technique fordetermining the fermentation reactions of non-sporing anaerobes. J. Appl. Bact. 41: 325-328..

Aseptic Commissioning BrothLAB 157

DescriptionA general growth medium specifically designed for thecommissioning of aseptic filling machines in the pharmaceutical andallied industries. It is a simple formulation incorporating peptone,yeast extract and sucrose as energy sources, and phenol red toindicate growth of organisms producing acid. Gas production andturbidity also indicate growth and are indicated by bubbles inDurham’s tubes or distortion of plastic packaging. It is suitable forsterilisation by filtration, passing quickly through the filters, andreducing blockage.

Formula g/litre

Tryptone 5.0

Yeast Extract 2.5

Sucrose 5.0

Sodium chloride 5.0

Phenol red 0.005

Method for reconstitutionWeigh 17.5 grams of powder and disperse in 1 litre of deionisedwater. Allow to soak for 10 minutes, swirl to mix and heat gently todissolve. Sterilise at 121˚C for 15 minutes, or alternatively, byfiltration.

Appearance: Red, clear liquid.

pH: 7.2 + 0.2

Minimum Q.C. organisms: Escherichia coli (acid and gasproduction) NCIMB 50034Proteus spp. (turbidity, no acid or gas)

Inoculation: Dispense medium through filling line into the finalpackaging, or suitable container, as appropriate.

Incubation: As per routine sterility testing laid down inPharmacopoeias or in house methodology. Typically 20-25˚C for up to 14 days.

1.2

Bacillus Cereus MediumPhenol Red Egg Yolk Polymyxin Agar (P.R.E.P.)Mannitol Egg Yolk Polymyxin Agar

LAB 73

DescriptionIntroduced by Mossel and his co-workers in 1967 for the enumerationof Bacillus cereus in foods, this formula was shown to be the mosteffective for this purpose by Inal in 1972. Two reactions on thismedium differentiate B. cereus from other members of the Bacillusgroup, these are mannitol fermentation and lecithinase production.Mannitol fermentation on this medium produces a yellow colour, B. cereus is mannitol negative and produces red colonies. The lecithinase production of B. cereus is indicated by a whiteprecipitate around the colonies. Polymyxin is added to suppresscoliforms but some Proteus spp and Gram positive cocci may grow through.

Formula g/litre

Beef Extract 1.0

Balanced Peptone No. 1 10.0

D-Mannitol 10.0

Sodium chloride 10.0

Phenol red 0.025

Agar No. 1 15.0

Method for reconstitutionWeigh 46 grams of powder, disperse in 900ml of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and asepticallyadd 100ml of X073 egg yolk emulsion and 2 vials of X074Polymyxin.

Appearance: Pink, opaque gel.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: B. cereus NCIMB 50014E. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: – up to 7 days at 2-8˚C in the dark.

Inoculation: Surface, spreading or streaking for single colonies.

Incubation: 30˚C aerobically for 24-48 hours.

Growth Characteristicscolony size shape &

organism (mm) surface colour

B. cereus 3.0-4.0 F.CR.D. Pink, white halo

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

B. coagulans 2.0 F.CR.D. Yellow

B. licheniformis 2.0 F.Rz.D. Yellow

Proteus spp. 1.0 CV.E.G. Pink (swarms)

E. faecalis 0.5 CV.E.G. Yellow

E. coli no growth

S. aureus 1.0 CV.E.G. Yellow (white halo)

ReferencesInal, T.: Vergleictiende Untersuchungen über die Selektivmedien zumqualitativen und quantitativen Nachweis von Vacillus cereus inLebensmitteln.

Mitteilung I. : Fleischwritsch, 51: 1629-1632 (1971). IV. Mitteilung:Fleischwritsch, 52: 1160-1162 (1972).

Mossel, D.A.A., Koopman, M.J. and Jongerius, E. (1967).Enumeration of Bacillus cereus in foods. Appl. Microbiol. 15: 650-653. Thatcher, F.S., Clarke, D.S. (1978) Micro-organisms infoods. Volume 1 second edition. University of Toronto.

BS5763 Part 1L:1994. ISO7932:(1993) 3/100

Baird-Parker Medium BaseLAB 85

DescriptionBaird-Parker introduced this complex medium in 1962 to overcomethe problems of recovering damaged Staphylococcus aureus fromfoodstuffs. The medium is highly selective due to potassium telluriteand lithium chloride. The tellurite inhibits most coliforms and is also reduced by S. aureus to telluride giving typical black colonies.Glycine and sodium pyruvate are both utilised by staphylococci asgrowth factors, pyruvate also neutralises toxic peroxides that may beformed in the medium. Sulphamethazine may be added to inhibitProteus spp. The typical reactions of S. aureus are detected by the eggyolk emulsion: (1) lecithinase production - an opaque zone round the colony; (2) lipase production - a zone of clearing outside theopaque zone.

Colonies suspected of being S. aureus should be confirmed by thecoagulase test or by a latex agglutination kit.

Alternatively the base medium can be used with the RPF (RabbitPlasma Fibrinogen) supplement (X086). This supplement is a morespecific alternative to Egg Yolk Tellurite Emulsion (EYT) for thedirect detection of coagulase positive S. aureus. The use of the RPFsupplement overcomes several disadvantages of EYT: (1) S. aureusisolates on traditional Baird-Parker Medium have to be confirmedwith a separate coagulase reaction, (2) S. aureus does not always givetypical egg yolk reactions.

Formula g/litre

Tryptone 10.0

Beef Extract 7.5

Yeast Extract 1.0

Lithium chloride 5.0

Glycine 12.0

Sodium pyruvate 10.0

Agar No. 2 20.0

Method for reconstitutionWeigh 65.5 grams of powder, disperse in 1 litre of deionised water.Allow the medium to soak for 10 minutes, swirl to mix then steriliseby autoclaving at 121°C for 15 minutes. Cool to 47°C and add 50mlof X085 sterile egg yolk tellurite emulsion. Mix well and dispenseinto Petri dishes.

For Baird-Parker RPF medium

Weigh 5.9 grams of powder and disperse in to 90ml of deionisedwater. Allow the medium to soak for 10 minutes, swirl to mix thensterilise by autoclaving at 121°C for 15 minutes. Cool to 47°C andadd 1 vial of X086 RPF supplement. Mix well and pour into Petridishes.

Appearance: Cream/pale fawn, opaque with X085. Translucent, palestraw with X086.

pH: 6.8 ± 0.2

Minimum Q.C. organisms: S. aureus NCIMB 50080S. epidermidis NCIMB 50082E. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Plates – up to 3 days at 2-8˚C in the dark.

Inoculation: Surface spread.

Incubation: 37˚C aerobically for 48 hours.

1.3

Growth characteristics(with X085)colony size shape &

organism (mm) surface colour other

S. aureus 1.0-3.0 CV.E.G. Black Narrow opaquemargin surroundedby a 2-5 mm zone

of clearing

S. saprophyticus 0.5-2.0 CV.E.G. Black (poor growth)

Other Coagulase 0.5-1.0 CV.E.G. Black (no growth)negativestaphylococci

Proteus spp. 0.5-2.0 F.Rz.G Brown (no growth)Black

Bacillus spp. 0.5-1.0 F.Rz.D. Brown (no growth)

Entero- no growthbacteriaceae

Growth characteristics(with X086)colony size shape &


Coagulase 1.0-3.0 CV.E.G. White Narrow opaquepositive . Grey zone of fibrinS. aureus . Black precipitation

Coagulase 0.5-2.0 CV.E.G. White (poor growth)negative . Greystaphylococci BlackProteus spp. 0.5-2.0 F.Rz.G Brown (no growth)

Black

Bacillus spp. 0.5-1.0 F.Rz.D. Brown (no growth)

Entero- no growthbacteriaceae

ReferencesBaird-Parker, A.C. (1962). An improved diagnostic and selectivemedium for isolating coagulase positive staphylococci. J. Appl. Bact.25(1): 12-19.

Baird-Parker, A.C. and Davenport, E. (1965). The effect of Recoverymedium on the isolation of S. aureus after heat treatment and afterstorage of frozen or dried cells. J. Appl. Bact 28: 390-402.

Ten Broeke, R. (1976). The Staphylococcus medium of Baird-Parkerin practical use. The occurrence of coagulase-positive, egg yolk non-clearing staphylococci. Antonie van Leeuwenhoek 33: 220-236.

Smith, B.A. and Baird-Parker, A.C. (1964). The use ofsulphamethazine for inhibiting Proteus spp. on Baird-Parker’sisolation medium for Staphylococcus aureus. J. Appl. Bact 27(1): 78-82.

Beckers N J. et al (1984). Canad. J. Microbiol. 30: 470-474.

Sawhney D. (1986) J. Appl Bact. 61:149-155.

Bismuth Sulphite Agar(Wilson and Blair Medium)

LAB 13A + LAB 13B

DescriptionA modification of Wilson and Blair’s original medium for theisolation of Salmonella typhi and other Salmonella from clinicalsamples, sewage and other materials. The presence of bismuthsulphite and brilliant green make this medium highly selective. As the medium contains neither lactose nor sucrose it can be used todetect lactose and sucrose fermenting Salmonella.

Formula g/litre

Bismuth Sulphite Agar Base ‘A’ LAB 13a

Beef Extract 6.0


Ferric citrate BPC 0.4

Brilliant Green 0.01

Agar No. 2 20.0

Bismuth Chemical Mixture ‘B’ LAB 13b

Bismuth ammonium citrate 3.0

Sodium sulphite 5.0

Disodium phosphate 5.0

Glucose 5.0

Method for reconstitutionAgar Base ‘A’: Weigh 36.4 grams of powder and mix with 1 litre ofdeionised water. Sterilise for 15 minutes at 121˚C. Cool to 50˚Capprox. and add 100ml of Chemical Mixture ‘B’. Mix well and pourthin plates. Store at 4˚C for 3 days to mature, before use.

Chemical Mixture ‘B’: Suspend 18 grams of powder in 100ml of deionised water. Bring to boil over a tripod and gauze, and coolquickly in cold water. Add to 1 litre of Agar Base ‘A’ prepared as above.

Appearance: Pale green, opaque gel.

pH: 7.6 ± 0.2

Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Plates – store 3 days before use. Use within 7 days. Store at 2-8˚C in the dark.

Inoculation: Surface, streak out to single colonies.

Incubation: 37˚C for 24 hours aerobically.

Growth characteristicscolony size shape &


S. typhi 1.5-2.0 CV.E.G. Black Metallic sheenblack deposit in

medium. (H2S-vestrains green)

Other Salmonella spp. 1.0-2.5 CV.E.G. Black/ Metallic sheen

Green especially in heavy growth,

single colonies maygive rabbit eye

appearance

E. coli P.P.-1.0 CV.E.G. Green

Klebsiella spp. P.P-2.0 CV.E.G. Green

Citrobacter spp. 1.0-2.5 CV.E.G. Green (black centre)

Proteus spp. 1.0-2.5 CV.E.G. Green/ (black centre)

Brown

ReferencesWilson, W.J. and Blair, E.M. M’V (1926). A combination of bismuthand sodium sulphites affording an enrichment and selective mediumfor the typhoid-paratyphoid groups of bacteria. J. Pathol. Bacteriol.,29: 310-311.

International Journal of Food Microbiology (1987) 5:3:200-202.

I.C.M.S.F. (1978) Micro organisms in Foods I. Their significance andenumeration. 2nd edition Univ of Toronto Press. Speck M.L.

Compendium of methods for microbiological examination of foods.(1984) 2nd edition. American Public Health Association,Washington. 3/102

1.4

Blood Agar baseLAB 28

DescriptionAn inexpensive general purpose agar base which, with the addition of5% sterile blood, can be used to cultivate a wide range of microorganisms of clinical significance. Typical haemolysis patterns areobtained with this medium.

Formula g/litre

Beef Extract 10.0


Sodium chloride 5.0

Agar No. 2 12.0

Method for reconstitutionWeigh 37 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. Cool to 47˚C and add 5-7%sterile defibrinated blood. Mix by swirling the flask and pour intoPetri dishes.

Appearance: Dependent upon blood additive.

pH: 7.4 ± 0.2

Minimum Q.C. organisms: S. aureus. NCIMB 50080S. pyogenes ATCC 19615


Inoculation: Surface, streaking to single colonies.

Incubation: 37˚C aerobically, anaerobically or microaerobically for 24 hours.



S. aureus 0.5-1.5 CV.E.G. White- haemolyticGolden

S. pyogenes P.P.-1.0 CV.E.G. Grey beta haemolyticalpha haemolyticnon-haemolytic

S. pneumoniae P.P.-1.0 F.E.G. Grey alpha haemolyticdraughtsman

N. meningitidis P.P.-1.5 CV.E.G. Grey mucoid

E. coli 1.5-2.5 CV.E.G. Grey haemolytic

Ps. aeruginosa 0.5-3.0 F.CR.D. Grey many colonialforms

green pigment

C. perfringens 0.5-1.5 CV.CR.G. Grey Target haemolysisnon-haemolytic

B. fragilis 0.5-1.5 CV.E.G. Grey mucoid

P. anaerobius P.P.-0.5 CV.E.G. Grey-White

F. necrophorum P.P. CV.E.G. Trans- haemolyticparent

ReferencesCruikshank, R. (1972). Medical Microbiology. 11th edn. Livingstone,London

Blood Agar Base No. 2LAB 15

DescriptionA very rich agar base which, with the addition of blood, is capable ofgrowing delicate clinical pathogens. The medium gives colonialappearances, haemolysis patterns and pigment production ofdiagnostic value. When the blood is ‘chocolated’ the medium givesgood recovery of Haemophilus spp. The medium can be madeselective for various groups by the addition of appropriate antibioticmixtures eg:

Streptococci – Colistin/Oxolinic acid (XO13)Gardnerella spp. – Colistin/Oxolinic acid (XO11)C. perfringens – Neomycin (XO15) (XO16)Staphylococci/streptococci – Colistin/Naladixic acid (XO12)

Formula g/litre

Tryptose 15.0

Soy Peptone 2.5

Yeast Extract 5.0

Sodium chloride 5.0

Agar No. 2 12.0

Method for reconstitutionWeigh 39.5 grams of powder, disperse in 1 litre of deionised water.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, defibrinatedhorse or sheep blood. Mix well before pouring.

Appearance: Dependent upon blood additive.

pH: 7.4 ± 0.2

Minimum Q.C. organisms: S. aureus NCIMB 50080S. pyogenes ATCC 19615


Inoculation: Surface, streaking out to single colonies.

Incubation: 37˚C aerobically or microaerobically for 24 hours,anaerobically for 24 and 48 hours.



S. aureus 1.5-2.0 CV.E.G. White/ (haemolytic)Golden

S. pyogenes 1.0-1.5 CV.E.G. Grey beta haemolytic)(alpha or nonhaemolytic)

S. pneumoniae 0.5-1.0 F.E.G. Grey (draughtsman(alpha haemolytic)

(mucoid)(require CO2)

N. meningitidis 0.5-1.0 CV.E.G. Grey (May requireCO2)

E. coli 2.0-3.0 CV.E.G. Grey (haemolytic)

Ps. aeruginosa 1.0-3.0 F.CR.D. Grey (green pigment (haemolytic)

C. perfringens 1.0-2.5 CV.CR.-(E)G Grey “Target”-haemolysis

(non-haemolytic)

B. fragilis 1.0-1.5 CV.E.G. Grey non haemolytic

P. anaerobius 0.5-1.0 CV.E.G. White non haemolytic

1.5

Brain Heart Infusion AgarLAB 48

DescriptionA general purpose nutritious agar base. This medium was first usedfor the isolation of dental pathogens. The mixture of brain and heartinfusions is particularly useful in the isolation of Actinomyces israeliand Histoplasma capsulatum. With the addition of 7% defibrinatedblood the medium will support the growth of a wide range offastidious organisms, the phosphate buffer will help neutralise theacids produced from the utilisation of glucose and thus maintainviability. The medium is not recommended for the determination ofhaemolytic reactions because of the glucose content.

The use of porcine material in this product ensures there are noSpecified Risk Materials (SRM’S) with respect to TransmissibleSpongeform Encephalopathies (TSE’S).

Formula g/litre

Brain-Heart Infusion Solids (porcine) 17.5

Tryptose 10.0

Glucose 2.0

Sodium chloride 5.0


Agar No. 2 12.0

Method for reconstitutionWeigh 49 grams of powder, disperse in 1 litre of deionised water.Allow to stand for 10 minutes then swirl to mix. Sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C then pour intoPetri dishes.

Appearance: Pale Straw colour, clear gel.

pH: 7.4 ± 0.2

Minimum Q.C. organisms: S. aureus NCIMB 50080E. coli NCIMB 50034

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped container – up to 3 months at 15-20˚C in the dark.

Inoculation: Surface, streaking out to single colonies.

Incubation: Time and temperature to suit specimen/organisms.

Growth characteristics (with horse blood)colony size shape &


S. aureus 1.0-1.5 CV.E.G. White/Golden

other Staphylococci 0.5-1.5 CV.E.G. White/Yellow

S. pyogenes 0.5-1.0 CV.E.G. White

S. milleri P.P.-0.1 CV.E.G. Transp.(White)

E. faecalis 1.0-1.25 CV.E.G. Grey/Green

S. pneumoniae 0.5-1.0 F.E.G. Grey/Green

E. coli 2.0-3.0 CV.E.G. Grey

Pseudomonas 2.0-4.0 F.CR.D. Greyaeruginosa

ReferencesRoseburg. T., Epps, L.J. and Clarke, A.R. (1944). A study of theisolation, cultivation and pathogenicity of Actinomyces israelirecovered from the human mouth and from actinomycosis in man. J. inf. Dis., 74: 131-149.

Howell, E. (1948) Efficiency of methods of isolation of Histoplasmacapsulatum. Pbl. Hlth. Rep. 63: 173-178. 3/108

Brain Heart Infusion BrothLAB 49

DescriptionA rich isotonic infusion medium with tryptose (a mixture of meat andmilk peptones) providing a wide range of substrates. A lowconcentration of glucose is used to stimulate early growth. Themedium is lightly buffered to prevent the early death of some speciesdue to acid production. Organisms which produce significantamounts of acid may well overwhelm the buffering system and auto-sterilise. The medium is suitable for use as a blood culture medium oras an enrichment broth for fastidious organisms.

The use of porcine material in this product ensures there are noSpecified Risk Materials (SRM’S) with respect to TransmissibleSpongeform Encephalopathies (TSE’S).

Formula g/litre

Brain-Heart Infusion solids (porcine) 17.5

Tryptose 10.0

Glucose 2.0

Sodium chloride 5.0


Method for reconstitutionWeigh 37 grams of powder then disperse in 1 litre of deionised water.Allow to stand for 10 minutes then dissolve with gentle heat beforedispensing 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.

pH: 7.4 ± 0.2


Storage of Prepared Medium: Capped container – up to 3 monthsat 15-20˚C in the dark.

Inoculation: (as a blood culture medium). Using a minimum volumeof 50ml of medium add the blood to a dilution of from 1:10 to 1:20.Use in conjunction with an anaerobic culture medium e.g. FastidiousAnaerobe Broth LAB 71.

Incubation: 37˚C aerobically for 7 to 15 days.

Interpretation: Observe daily, subculture after 1, 2, 3, 7 and 15 daysor immediately on showing signs of growth.

ReferencesRosenow. E.C. (1919). Studies on selective localisation; focal infectionwith special reference to oral sepsis. J. Dent. Res. 1:205-267.

Brazier’s CCEY AgarLAB 160

DescriptionBrazier’s CCEY agar is the formulation currently used by theAnaerobe Reference Unit for the isolation of C.difficile, resultingfrom work initiated by Ken Phillips and Paul Levett, and completedby Jon Brazier.

Based upon the market leading anaerobe medium, FastidiousAnaerobe Agar, it incorporates additional ingredients to improve theisolation and differentiation of C.difficile from clinical specimens.

Cholic acid is present to promote spore germination followingalcohol shock treatment, and p-hydroxyphenylacetic acid to enhancethe production of p-cresol, a distinctive metabolite of C.difficile

Selectivity is achieved by addition of supplement X093 (cefoxitincycloserine) and egg yolk emulsion X073 is added to helpdifferentiate C.difficile from lecithinase positive clostridia. Finallythe addition of lysed horse blood optimises the recognition of colonyfluorescence when cultures are examined using UV light.

1.6

Formula g/litre

Peptone Mix 23.0

Sodium chloride 5.0

Soluble Starch 1.0

Agar 12.0

Sodium bicarbonate 0.4

Glucose 1.0

Sodium pyruvate 1.0

Cysteine HCl 0.5

Haemin 0.01

Vitamin K 0.001

L-arginine 1.0

Soluble pyrophosphate 0.25

Sodium succinate 0.5

Cholic acid 1.0

p-Hydroxyphenylacetic acid 1.0

Method for reconstitutionWeigh 48 grams of powder and add to 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix, and sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and asepticallyadd the following: 2 vials of X093, 40ml of Egg Yolk Emulsion X073and 10ml lysed horse blood. Mix well and pour into Petri dishes.

Appearance: Tan opaque gel.

pH: 7.0 + 0.2

Minimum Q.C. organisms: C. difficile E. coli (inhibition) NCIMB 50034

Storage of prepared medium: Plates – up to 7 days at 2-8˚C in the dark.

Inoculation: Surface streak untreated or alcohol shocked specimensfor single colonies.

Incubation: 37˚C for 24-48hrs under anaerobic conditions

Characteristics of C.difficile: Gray opaque flat colonies, raisedelevation, 2-3mm diameter, generally circular but tending to elongatein the direction of spreading, ground glass appearance and a rough,fimbriate edge. Lecithinase negative. Incubation longer than 48hrsmay result in a lighter gray or white centre to the colony. Phenolicodour due to the production of p-cresol. Colonies fluoresce yellow-green under UV light. Confirm by latex agglutination.

ReferencesBrazier J.S. (1993) Rôle of the Laboratory in Investigations ofClostridium difficile Diarrhoea. Clinical Infectious Diseases 16 (4)228-33.

Brilliant Green Agar (modified)(Phenol Red Brilliant Green Agar, BPLS)

LAB 34

DescriptionFirst introduced by Kristensen et al in 1925 as a selective medium forthe isolation of salmonellae (except S. typhi). The medium wasmodified by the Netherlands Institute for Public Health, Utrecht. Themodification was to increase the selectivity of the medium byincreasing the dye concentration. This formulation is quoted by theInternational Standards Organisation, standard European CommunityMethods, the American Public Health Association and theAssociation of Official Analytical Chemists. The medium is suitablefor subcultures from selective enrichment media. However becausethis medium is highly selective, small numbers of salmonellae maybe missed. This medium is definitely not recommended for S. typhi

and Shigella spp. Less inhibitory media such as X.L.D. and HektoenEnteric Agar will be useful in detecting salmonellae and shigellaeinhibited by Brilliant Green Agar.

Formula g/litre

Beef Extract 5.0


Yeast Extract 3.0


Sodium dihydrogen phosphate 0.6

Lactose 10.0

Sucrose 10.0

Phenol red 0.09


Agar No. 2 12.0

Method for reconstitutionWeigh 52 grams of powder and disperse in 1 litre of deionised water.Allow to soak for ten minutes and then bring to the boil with frequentswirling to dissolve the solids and cool to 47˚C in a water bath. Pourplates and dry the surface before inoculation. DO NOT remelt orautoclave: overheating causes precipitation of the medium. Storeplates away from light.

Appearance: Tan, clear gel.

pH: 6.9 ± 0.2



Inoculation: Surface streaking for single colonies, a heavy inoculumcan be used.

Incubation: 37˚C for 18-24 hours aerobically.



Salmonella spp. 1-1.5 CV.E.G. Pink (red zone incolonies medium)

S. typhi 1.0 CV.E.G. Pink/Red (may not grow)

E. coli no growth (0.5-1.0 yellowcolony)

Proteus spp no growth

Ps aeruginosa no growth (crenated small redcolonies)

Klebsiella spp. 1-1.5 CV.E.G. Green (yellow) (NG)colonies

Enterococcus spp. no growth

S. sonnei no growth (0.5 mm red)

ReferencesEdel, W. and Kamplemacher, E.H. (1968). Comparative studies onSalmonella isolation in eight European laboratories. Bull. Wld. Hlth.Org. 39: 487-491.

Edel, W. and Kamplemacher, E.H. (1969). Salmonella infections innine European laboratories using a standard technique. Bull Wld.Hlth. Org. 41: 297-306.

American Public Health Association (1966). Recommended Methodsfor the Microbiological Examination of Foods, 2nd end. (ed. J.M. Sharf) A.P.H.A. Washington.

Association of Official Analytical Chemists (AOAC) (1978)Bacteriological Analytical Manual, 5th edn., Washington D.C.

Pharmacopoeia of culture media for food microbiology. (1987). Int. J. Food Microbiol. 513: 245-247. 3/112

1.7

Brilliant Green Bile 2% BrothLAB 51

DescriptionA modification of MacConkey’s medium, formulated in 1926 byDunham and Schoenlein, for the recovery of coliform bacteria infoodstuffs and water. The brilliant green and bile inhibit most Grampositive organisms thus overcoming the problem of someClostridium spp. fermenting lactose and giving false positive results.

Formula g/litre


Lactose 10.0

Ox Bile 20.0


Method for reconstitutionWeigh 40 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then warm to dissolve.Dispense into tubes or bottles with inverted Durham tubes. Steriliseby autoclaving at 115˚C for 15 minutes.

Appearance: Green, clear.

pH: 7.4 ± 0.2

Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli NCIMB 50034

Storage of Prepared Medium: Capped containers – up to 1 monthat 2-8˚C in the dark.

Inoculation: Serial 1:10 dilutions of homogenised sample areinoculated into the broth in the proportion of 1ml sample to 9mlbroth. Ensure the Durham tube is free from gas bubbles beforecommencing inoculation. B.G.B. broth can be used at double strengthif required but cannot be sterilised by autoclaving, pasteurisationmust be used instead.

Incubation: E. coli and thermotrophs 44˚C for 18 hours aerobically.Mesopholic coliforms 32˚C for 24-48 hours aerobically.Psychrotrophic coliforms 4˚C for 10 days aerobically.

Interpretation: Turbidity, colour changes (to yellow or yellowishgreen) and production of gas are all presumptive evidence of thegrowth of organisms of the coli-aerogenes group. Confirmation byindole production in Tryptone Water LAB 129 (44˚C for E. coli).

ReferencesPharmacopoeia of Culture Media for Food Microbiology (1987). Int.J. Food Microbiol. 5:3:206-207.

American Public Health Association, American Water WorksAssociation and Water Pollution Control Federation, (1975),Standard Methods for the Examination of Water and Wastewater,14th ed., Washington D.C.

Association of Official Analytical Chemists (AOAC). BacteriologicalAnalytical Manual, 5th ed., Washington, D.C. Association of OfficialAnalytical Chemists. 1978.

Hausler, W. J. (ED) (1972). Standard Methods for the Examination ofDairy Products. 13th ed., Washington. D.C. American Public HealthAssociation.

Shane, M.S. (1947). Studies on false confirmed test using B.G.B. andcomparison studies on Lauryl Sulfate Tryptose Broth as presumptivemedium. J. Am. Water Works Assoc., 39: (4), 337.

Bromocresol Purple Lactose Agar(Drigalski agar)

LAB 121

DescriptionA non selective differential medium for the isolation and enumerationof Enterobacteriaceae from urine, water and food products. Lactosefermenting organisms produce yellow colonies, non lactosefermenters produce purple colonies.

Formula g/litre

Peptone mixture 7.4

Lactose 8.5

Bromocresol purple 0.025

Agar No. 1 12.0

Method for reconstitutionWeigh 28 grams of powder and disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Allow to cool to 47˚C then pourinto Petri dishes.

Appearance: Purple, clear agar.

pH: 6.8 ± 0.2

Minimum Q.C. organisms: E. coli. NCIMB 50034S. aureus NCIMB 50080


Inoculation: Surface, plating either over entire surface for colonycount or streak out to single colonies.




E. coli 1.5-2.0 CV.E.G. Yellow (N.L.F. – purple)

Klebsiella spp. 4.0-6.0 CV.E.G. Yellow (mucoid)

Citrobacter spp. 2.0-2.5 CV.E.G. Yellow

Proteus spp. 1.0-1.5 CV.E.G. Purple

Salmonella spp. 1.0-2.0 CV.E.G. Purple

S. aureus 0.5 CV.E.G. Cream (purple if N.L.F.)


ReferencesDrigalski, C. (1902). Uber ein Verfahren zum Nachweis derTyphusbacillen. Z. Hyg. Infekt. 39:283-300.

1.8

Buffered Listeria Enrichment BrothLAB 139

DescriptionA medium for the selective enrichment of food and environmentalsamples for Listeria spp, LAB 139 is a buffered version of the ‘FDA’broth LAB 138. The extra buffering capacity maintains the pH of theenrichment culture during incubation, ensuring optimum conditionsfor the recovery of Listeria spp.

Formula g/litre

Tryptone 17.0

Soy peptone 3.0

Sodium chloride 5.0

Dipotassium hydrogen phosphate 2.5

Glucose 2.5

Yeast Extract 6.0

Potassium dihydrogen phosphate 1.35


Method for reconstitutionWeigh 47 grams of powder and add to 1 litre of deionised water.Allow to soak for 10 minutes then swirl to mix and sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and add 2 vials ofX138 (X139/X539 can be used as an alternative) reconstituted in50% alcohol. Aseptically dispense into sterile tubes or bottles.

Appearance: Yellow, clear.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: L. monocytogenes NCIMB 50007E. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Capped containers – up to 14 days at2-8˚C in the dark.

Inoculation: Add 25 grams of sample to 225mls of Buffered ListeriaEnrichment Broth and homogenise.

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

Subculture: After 24 and 48 hours onto Listeria Isolation Medium –LAB 122.

Buffered Peptone WaterLAB 46

DescriptionA pre-enrichment medium design to help sublethally damagedsalmonellae recover before introducing them into a selectivemedium. This nutrient medium is free from inhibitors and is wellbuffered to maintain the pH at 7.2 for the incubation period. Sublethalinjury to salmonellae occurs in many food processes and this pre-enrichment step greatly increases recovery of these organisms.

Formula g/litre

Peptone 10.0

Sodium chloride 5.0



Method for reconstitutionWeigh 20 grams of powder and disperse in 1 litre of deionised water.Mix to dissolve then distribute into tubes or bottles. Sterilise byautoclaving at 121˚C for 15 minutes.

Appearance: Pale straw, clear liquid.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: E. coli NCIMB 50034


Inoculation: Add 25 grams of sample to 225ml of Buffered PeptoneWater and homogenise.

Incubation: Aerobically at 37˚C for 18-24 hours.

Subculture: 10ml aliquots in 100ml of Selenite Cystine Broth LAB 55 and 0.1ml into 10ml Rappaport Vassiliadis Medium LAB 86.

ReferencesEdel W. and Kampelmacher E.H. (1973). Bull. Wld Hlth Org. 48: 167-174.

Poemla P.K. and Silliker J.H. (1976) Salmonella in Compendium ofMethods for microbiological examination of foods. Am. Pub. HealthAss., Washington.

Campylobacter Blood Free Selective Medium(Modified CCDA-Improved)

LAB 112

DescriptionA blood free medium which will support the growth of most entericcampylobacters. The selective cocktail X112 (X212) makes themedium selective for C. jejuni. C. coli and C. laridis when incubatedat 37˚C. With this product incubation at 42˚C is no longer necessaryand higher recovery rates have been reported at 37˚C than at 42˚C.

The supplement X112 (X212) consists of cefoperazone andamphotericin and is superior to the selective cocktails of Skirrow,Butzler and Blazer-Wang all of which contain antibiotics shown to beinhibitors to C. coli. The colonial morphologies of Campylobacterspp. on this medium are distinctive.

Formula g/litre

Peptone blend 25.0

Bacteriological Charcoal 4.0

Sodium chloride 3.0

Sodium desoxycholate 1.0

Ferrous sulphate 0.25


Agar No. 2 12.0

Method for reconstitutionWeigh 45.5 grams of powder, disperse in 1 litre of deionised waterand allow to soak for 10 minutes. Swirl to mix, then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C then add 2 vials ofX112 supplement, mix well and pour into Petri dishes. Continuouslymix whilst pouring to prevent the charcoal settling.

Appearance: Black agar.

pH: 7.4 ± 0.2

Minimum Q.C. organisms: C. jejuniE. coli (inhibition) NCIMB 50034Candida albicans (inhibition)NCIMB 50010

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark.

Inoculation: C. jejuni, C. coli. C. laridis surface streaking to singlecolonies.

Incubation: 37˚C for 48 hours in an atmosphere of 5% oxygen, 10%carbon dioxide and 85% nitrogen. C. cinaedi and C. fennelliaerequire up to 7 days.

1.9



C. jejuni 2.0-3.0 F.E.G. Grey/White Efflorescent

(spreading moist)

C. coli 1.0-2.5 CV.E.G. Creamy MoistGrey

C. laridis 1.5-3.0 F.E.G. Grey (C.V. moist)

C. cinaedi 2.0-3.0 F.E.G. Pale Grey requires 7 days in

absence of X112

C. fenneliae 2.0-3.0 F.E.G. Pale Grey requires 7 days in

absence of X112

Other Enterobacteriacae – No growth if sensitive to cefoperazone.

ReferencesBolton F.J. Hutchinson D.N., Parker G. Reassessment of SelectiveAgars and Filtration Techniques for Isolation of CampylobacterSpecies from Feces. Eur.J. Clin. Microbiol. Infects. Dis. (1988) 7 p 155-160.

Bolton F. J. (1988) Personal Communication.

Bolton F.J. Hutchinson D.N., Parker G. Isolation of Campylobacter:What are we missing? J.Clin.Path. (1987) 40 p 702-703.

Goosens H., De. Boeck M., Coignau H., Vlaes L., Van Den Borre C.,Butzler J.P. Modified Selective Medium for Isolation ofCampylobacter spp from Feces: Comparison with Preston Medium, aBlood Free Medium, and a Filtration System. J.Clin. Micro. (1986)24 p 840-843.

Gun-Munro J., Rennie R.P., Thornley J.H. Richardson H.L., Hodge D., Lynch J. Laboratory and Clinical Evaluation of IsolationMedia for Campylobacter jejuni J. Clin Micro. (1987). 25 p 2274-2277.

Herbert G.A., Hollis D.G., Weaver R.E., Karmali M.A., Simor A.E.,Roscoe M., Fleming P.C., Smith, S.S. Lane J. Evaluation of a Blood-Free, Charcoal-Based, Selective Medium for the Isolation ofCampylobacter organisms from Faeces. J. Clin. Micro. (1986) 23 p456-459.

Campylobacter Enrichment Broth(Bolton Formulation)

LAB 135

DescriptionA selective enrichment broth for the isolation of Campylobacter spp.from food, environmental samples and faeces. The use of a selectiveenrichment broth enhances the recovery of sub-lethally damagedorganisms due to processing of foods, or if small numbers ofcampylobacters are present in heavily contaminated specimens. This broth has been shown to give appreciably better results thanPreston Broth.

Formula g/litre

Meat Peptone 10.0

Lactalbumin Hydrolsates 5.0

Yeast Extract 5.0

Sodium chloride 5.0

Haemin 10.0mg

Sodium pyruvate 0.5

α – ketoglutaric acid 1.0

Sodium metabisulphite 0.5

Sodium carbonate 0.6

Method for reconstitutionWeigh 27.6 grams of powder, disperse in 1 litre of deionised waterand allow to soak for 10 minutes. Swirl to mix and autoclave at 121˚Cfor 15 minutes. Cool to 47˚C, add 2 vials of selective supplementX131 ( X132 can be used as an alternative) reconstituted with 5ml of50% alcohol and 50ml of saponin lysed horse blood, mix well anddispense into sterile containers.

Appearance: Translucent, wine-red with a fine black suspension.

pH: 7.4 ± 0.2

Minimum Q.C. organisms: Campylobacter jejuniE. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Capped containers: 7 days at 2-8˚Cin 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. Forfaeces 1ml of a 10% suspension in Buffered Peptone Water LAB 46is added to 5ml of broth.

Incubation: Aerobically at 37˚C for 2-4 hours, followed by a further16-44 hours at 42˚C.

Subculture: Onto Campylobacter Blood Free Selective MediumLAB 112.

ReferencesBolton, F.J. Personal Communication.

Hunt J.M., Abeyta C., and Tran T. (1998) Chapter 7 Campylobacterin FDA Bacteriological Analytical Manual 8th Edition.

C.E.M.O. Agar Base(Contagious Equine Metritis Organism)

LAB 78

DescriptionThis medium is a selective isolation medium for Taylorellaequigenitalis the causative organism of contagious equine metritis.The medium is a sugar free base with a mixture of high grade caseinand soy peptones as nutrients and with L-cystine and sodium sulphiteas supplements and reducing agents. The medium is made selectivewith the addition of amphotericin (5 mg/L) and trimethoprim (10mg/L). Streptomycin (200 mg/L) can also be used but sensitivevariants of T. equigenitalis have been described.

Formula g/litre

Tryptone 15.0

Soy Peptone 5.0

Sodium chloride 5.0

Agar No. 2 12.0

L-Cystine 0.3

Sodium sulphite 0.2

Method for reconstitutionWeigh 37.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise by autoclavingat 121˚C for 15 minutes. Allow to cool to 80˚C, add 50ml of sterilehorse blood and allow to ‘chocolate’. Further cool to 47˚C beforeadding antibiotic selective agents. Mix well and pour into Petri dishes.

Appearance: Chocolated Blood Agar.

pH: 7.3 ± 0.2

Minimum Q.C. organisms: T. equigenitalisE. coli (inhibition) NCIMB 50034


Inoculation: Surface, streaking out for single colonies.

Incubation: 37˚C in 10% CO2 for 2-3 days.1.10



T. equigenitalis 0.1-1.0 CV.E.G. Cream colony sizevariation iscommon

ReferencesAtherton, J.G. (1978). Inhibition of the C.E.M. organism in mixedcultures. Vet. Rec. 432.

Mackintosh, M.E. (1981). Bacteriological techniques in the diagnosisof equine genital infections. Vet. Rec. 108, 52-55. Atherton, J.G.Personal Communication.

Fleming, M.P. Tribe. G. W. (1977). Vet. Rec. 101, 1470.

Cetrimide AgarU.S.P.

LAB 133

DescriptionA medium recommended by the United States Pharmacopoeia for theisolation of Pseudomonas aeruginosa from pharmacologicalpreparations. Subculture is carried out onto the medium afterenrichment in LAB 4 Tryptone Soy Broth. Cetrimide inhibits thegrowth of many micro organisms whilst allowing P. aeruginosa todevelop typical colonies which will fluoresce in ultraviolet light andproduce green pigment.

Formula g/litre

Pancreatic Digest of Gelatin 20.0


Potassium sulphate 10.0

Cetyl trimethylammonium bromide (cetrimide) 0.3

Agar 13.6

Method for reconstitutionWeigh 45.3 grams of powder, disperse in 1 litre of deionised water.Add 10ml of glycerol, allow to soak for 10 minutes then swirl to mix.Sterilise at 121˚C for 10 minutes.

Appearance: Opalescent, pale yellow agar.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: P. aeruginosa NCIMB 50067E. coli (inhibition) NCIMB 50034


Inoculation: Subculture from enrichment broth, streak out for singlecolonies.

Incubation: 30-35˚C aerobically for 24-48 hours.



P. aeruginosa 0.5-1.0 F.CR.D. green pigment (non pigment)green/yellowfluorescence

P. fluorescens 0.5 CV.R.E.G. green/yellow fluorescence

E. coli N.G.

S. aureus N.G.

Proteus spp. N.G.

ReferencesUnited States Pharmacopoeia XXI. 1985.

Brown V.I., Lowbury E.J.L. (1965). Use of an improved CetrimideAgar Medium and other culture methods for Pseudomonasaeruginosa. J. Clin. Pathol. 18, 752-756.

China Blue Lactose AgarLAB 105

DescriptionChina Blue Lactose Agar is a non-inhibitory medium for thedifferentiation and enumeration of bacteria in milk. The formulationis the same as that described by the Methodenkomission fürMilchivirtschaft. Lactose fermenters in milk form blue colonies. Themedium is non-inhibitory to the growth of cocci, making it a usefulmedium for the detection of streptococci and staphylococci as well ascoliform organisms.

Formula g/litre


Beef Extract 3.0

Lactose 10.0

Sodium chloride 5.0

Aniline blue 0.325

Agar No. 2 12.0

Method for reconstitutionWeigh 35 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and pour into Petridishes.

Appearance: Blue clear agar.

pH: 7.0 ± 0.2

Minimum Q.C. organisms: E. coli NCIMB 50034Staph. aureus NCIMB 50080


Inoculation: Surface, spread inoculum evenly over entire surface.


Interpretation: Count all colonies



E. coli 1.5-2.0 CV.E.G. Blue (colourless if NLF)

Klebsiella spp. 2.0-2.5 CV.E.G. Blue (mucoid)

Proteus spp. 0.5-2.5 F.Rz.G. colourless (spreading)

Salmonella spp. 2.0 CV.E.G. colourless

Shigella spp. 1.0 CV.E.G. colourless

Pseudomonas spp. 2.5-3.0 F.CR.D. colourless

S. aureus 0.5-1.0 CV.E.G. Pale Blue

S. epidermidis 0.5-1.0 CV.E.G. Blue

ReferencesMethodenbuch Band VI. Vergand Deutscher LandwirtschaftlicherUntersuchungs – und Forschugsanstallen.

1.11

C.L.E.D. (Bevis modification)(Cystine Lactose Electrolyte Deficient – Double Indicator)

LAB 6

DescriptionBevis modified Mackey and Sandys original medium by introducinga double indicator to improve the differentiation of lactose and nonlactose fermenting coliforms, staphylococci and streptococci. Theswarming of Proteus spp. is inhibited. LAB M C.L.E.D. will growmany of the more demanding streptococci of Lancefield groups A, B,C, G and F. This medium may not grow Pasteurella spp. or halophilicorganisms.

Formula g/litre


Beef Extract 3.0

Tryptone 4.0

Lactose 10.0

L-Cystine 0.128

Bromothymol blue indicator 0.02

Andrade’s indicator 0.08

Agar No. 1 15.0

Method for reconstitutionWeigh 36 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. Cool to 47˚C and mix beforepouring.

Appearance: Green/blue, clear gel.

pH: 7.5 ± 0.2

Minimum Q.C. organisms: E. coliNCIMB 50034S. aureusNCIMB 50080


Inoculation method: Surface inoculation, either streaking for singlecolonies or spread evenly over entire surface for colony counts.




E. coli 2.0-3.0 CV.E.G. Yellow/ (Blue if Orange non-lactose

fermenter)

K. aerogenes 3.0-4.0 CV.E.G. Yellow/ mucoidOrange

Proteus spp. 2.0-3.0 CV.E.G. Blue

Ps. aeruginosa 1.0-4.0 F.CR.D. Blue (Green pigment & odour)

Shigella spp. 1.5-2.5 CV.E.G. Blue

Salmonella spp. 2.0-3.0 CV.E.G. Blue (Yellow-orange if lactose +ve)

S. aureus 1.0-1.5 CV.E.G. Yellow/ (Blue if Orange non-lactose

fermenting)

Other (Yellow ifstaphylococci 0.5-1.5 CV.E.G. Blue-White lactose

fermenting)

Enterococcus spp. 0.5 CV.E.G. Yellow-Orange

ReferencesBevis, T.D. (1968). A modified electrolyte-deficient culture medium.J. Med. Lab. Tech., 25: 38-41.

Mackey, J.P. and Sandys, G.H. (1966). Diagnosis of urinaryinfections, Brit.Med. J., 1: 1173.

Sandys, G.H. (1960). A new medium for preventing swarming ofProteus spp. with a description of a new medium suitable for use inroutine laboratory practice. J. Med.Lab. Tech., 17: 224-233.

C.L.E.D. Medium(Mackey and Sandys) (Cystine Lactose Electrolyte Deficient-Single Indicator)

LAB 41

DescriptionA medium for urine culture first described by Mackey and Sandys in1960. The absence of electrolytes inhibits the swarming of Proteusspp. Cystine is added for the benefit of those organisms which havea specific cystine requirement. Differentiation of lactose and nonlactose fermenters is achieved using bromothymol blue as pHindicator. This medium supports the growth of Streptococcuspyogenes and most other fastidious organisms that do not requireblood.

Formula g/litre


Beef Extract 3.0

Tryptone 4.0

Lactose 10.0

L-Cystine 0.128

Bromothymol blue indicator 0.02

Agar No. 1 15.0

Method for reconstitutionWeigh 36 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. Cool to 47˚C mix and distributeinto Petri dishes.

Appearance: Green/blue clear gel.

pH: 7.3 ± 0.2

Minimum Q.C. organisms: E. coli NCIMB 50034S. aureus NCIMB 50080


Inoculation: Surface inoculation either spreading for single coloniesor spread evenly over entire surface for colony counts.


1.12



E. coli 2.0-3.0 CV.E.G. Yellow (blue if nonlactose fermenters)

K. aerogenes 3.0-4.0 CV.E.G. Yellow (mucoid)

Proteus spp. 2.0-3.0 CV.E.G. Blue

Ps. aeruginosa 1.0-4.0 F.CR.D. Blue (green pigment& odour)

Shigella spp. 1.5-2.5 CV.E.G. Blue

Salmonella spp. 2.0-3.0 CV.E.G. Blue (yellow iflactose +ve)

Staph. aureus 1.0-1.5 CV.E.G. Yellow (blue if non-lactose

fermenting)

other Staphylococcus (yellow if spp. 0.5-1.5 CV.E.G. Blue-white lactose

fermenting)

Enterococcus spp. 0.5 CV.E.G. Yellow

ReferencesMackey, J.P. and Sandys, G.H. (1966). Diagnosis of urinaryinfections. Brit.Med.J. 1: 1173.

Guttman, D and Naylor, G.R.E. (1967). Dip-slide: an aid toquantitative urine culture in general practice. Brit.Med. J. 3: 343-345.

Columbia Agar BaseLAB 1

DescriptionA general purpose nutritious agar base formulated by Ellner et al.When further enriched by the addition of sterile blood, Columbiaagar can be used for the isolation of most clinically significantpathogens. The blood can be ‘chocolated’ if required. The mediumcan be made selective for various groups by the addition ofappropriate antibiotic mixtures eg:

Streptococci – Colistin/Oxolinic acid (X013)Gardnerella spp. – Colistin/Nalidixic acid (X011)C. perfringens – Neomycin (XO15) (X016)Campylobacters - (X214)Staphylococci/streptococci – Colistin/Naladixic acid (X012)

Formula g/litre

Columbia Peptone Mixture 23.0

Corn Starch 1.0

Sodium chloride 5.0

Agar No. 2 12.0

Method for reconstitutionWeigh 41 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. Cool to 48˚C and add 5-7%sterile, defibrinated horse or sheep blood. Mix well before pouring.

Appearance: Cherry red if blood is fresh and well oxygenated.

pH: 7.3 ± 0.2

Minimum Q.C. organisms: S. aureus NCIMB 50080S. pyogenes ATCC 19615


Inoculation: Surface plating, streaking out for single colonies.

Incubation: 37˚C aerobically or microaerobically for 24 hours.Anaerobically for 24 and 48 hours.



S. aureus 1.5-2.0 CV.E.G. White-Yellow Haemolytic

S. pyogenes 0.5-1.0 CV.E.G.(D) White α, β-haemolyticdependent on

strain

S. pneumoniae 0.5-1.5 F.E.G. Grey greenish discolouration

in medium, mucoidin H2/C02

Neisseria meningitidis 1.0-2.0 CV.E.G. trans/ (mucoid)

Grey

E. coli 2.0-3.0 CV.E.G. Opaque/ (haemolytic)Grey

Ps. aeruginosa 0.5-4.0 F.CR.D. Opaque many colonialGrey forms

(green pigment)(haemolytic)

(mucoid)

C. perfringens 1.5-2.0 CV.CR.G. Grey usually targethaemolysis

(non haemolytic)

B. fragilis 1.0-1.5 CV.E.G. Grey (mucoid)

P. anaerobius P.P.-0.5 CV.E.G. White/Grey

ReferencesEllner, P.D., Stoessel, C.J., Drakeford, E and Vasi, F. (1966). A newculture medium for medical bacteriology. Amer. J. Clin Pathol.,45:502-504.

Goldberg, R.L., and Washington, J.A., (1976). Comparison ofisolation of Haemophilus vaginalis (Corynebacterium vaginale) fromPeptone-Starch-Dextrose Agar and Columbia Colistin-Nalidixic AcidAgar. J. Clin. Microbiol., 4:245-247.

Thayer, D.D. and Martin, H. E. (1966). An improved medium for thecultivation of N. gonorrhoeae and N. meningitidis. Publ. Hlth.Report, 81:559-562.

Cooked Meat Granules(for Cooked Meat Medium)

LAB 24

DescriptionDried minced ox heart which has been trimmed of excess fat andprepared according to the Martin and Lekpers modification ofRobertsons original formulation. The medium gives good growth of mostorganisms and is especially useful in the recovery of fastidiousanaerobes. The medium is also suitable for prolonged storage of cultures.

Method for reconstitutionTo make Cooked Meat Medium the granules should be added toNutrient Broth (LAB 14) or Fastidious Anaerobe Broth (LAB 71) inthe ratio of 15 grams of granules to 200ml of broth. Cooked MeatMedium should be dispensed into containers allowing at least 20-25mm depth of meat particles and a broth supernatant of at least 10-15mm. To sterilise autoclave at 121˚C for 15 minutes in capped tubeswhich should be tightened after autoclaving to prevent re-oxygenation.

Medium prepared with Nutrient Broth LAB 14 should be re-steamed when used after a period of storage. Medium made withFastidious Anaerobe Broth LAB 71 will not require re-steaming after storage.

Storage of Prepared Medium: – up to 6 months at 15-20˚C in the dark.

ReferencesCruickshank, R. 1972. Medical Microbiology. 11th Edn. Livingstone,London.

1.13

Cooked Meat MediumLAB 127

DescriptionThis is a complete version of Robertson’s Cooked Meat Broth whichwas designed to grow all anaerobes found in battlefield woundsduring the First World War. The medium is based on LAB M CookedMeat Granules – LAB 24 and Nutrient Broth – LAB 14.

Formulation g/litre

Cooked meat granules 75.0

Beef Extract 10.0


Sodium chloride 5.0

Method for reconstitutionUse a calibrated scoop to distribute 0.9 gram amounts of granules intotubes or bottles. Add 10ml deionised water. Sterilise at 121˚C for 10minutes. Use proportionately more granules and water if greaterdepths of medium are required.

Appearance: Granules covered in slightly opalescent pale yellow liquid.

pH: 7.0 ± 0.2

Inoculation: Samples or swabs directly into the medium.

Incubation: 37˚C for mesophiles, appropriate temperature for thermophiles.

Interpretation: Reddening of meat – saccharolytic organism –Blackening and digestion – proteolytic organism.

ReferencesRobertson, M., (1916) J. Path. & Bact. 20 327-349.

Cooked Meat Medium TabletsLAB 24Z

DescriptionAs Cooked Meat Granules (LAB 24) with Nutrient Broth (LAB 14)incorporated and presented in tablet form.

Method for reconstitutionAdd 2 tablets to 10ml of deionised water in a narrow container. Allowto soak for 15 minutes then sterilise by autoclaving at 121˚C for 15minutes.

D.C.A.(Desoxycholate Citrate Agar)

LAB 29

DescriptionThis is Leifson’s original formulation of this selective medium for theisolation of Salmonella spp. and Shigella spp. from faeces andenvironmental samples. It has approximately half the quantity ofinhibitors used in the Hynes modification. The medium uses sodiumcitrate and sodium desoxycholate as inhibitors. Sodium thiosulphateis the substrate for the enzyme thiosulphate reductase being brokendown to form sulphite and hydrogen sulphide. The hydrogen sulphidereacts with the ferric ions to produce a black precipitate of ferroussulphide. This gives a typical black centre to the colonies of mostspecies of Salmonella.

Formula g/litre

Beef Extract 5.0


Lactose 10.0

Sodium citrate 5.0


Ferric citrate 1.0


Neutral Red 0.025

Agar No. 2 12.0

Method for reconstitutionWeigh 45.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix, then bring to the boil withfrequent stirring. When the medium boils up into the neck of theflask, quickly remove from the source of heat and allow the froth tosubside. Return to the heat and allow the foam to boil up into the neckof the flask once more. Remove at once and cool to 47˚C approx.before pouring plates. Dry the surface before inoculation. DO NOTREMELT OR AUTOCLAVE THIS MEDIUM.

Appearance: Pale pink, translucent, a fine precipitate ofdesoxycholate may be present which may clear if the pH is increasedby the growth of organisms.

pH: 7.0 ± 0.2



Inoculation: Surface, streaking for single colonies.




S. typhi 0.5. 1.5 CV.E.G. transp (black centre)Yellow

Other Salmonella spp. 1.5-2.0 CV.E.G. transp (black centre)

(Opaque) (clearing Yellow around colony)

S. sonnei 1.5-2.0 CV.E.G. transp (more opaque(pinkish) centre)

S. flexneri group 1.0-1.5 CV.E.G. transp-(pinkish)

S. dysenteriae 0.5-1.0 CV.E.G. transp

E. coli P.P.-1.5 CV.E.D. Red/Pink ppt in medium(uninhibited) (G)

Citrobacter spp. P.P.-2.0 CV.E.D. Red/Pink ppt in medium(G) (black centre)

Proteus spp. 1.0-2.0 CV.E.G. Yellow (black/grey centre)

fishy odour(clearing around

colony)

Pseudomonas spp. 0.5-1.5 CV.E.D. Yellow/ (green pigment)

(G) Pink

ReferencesHynes. M. (1942). The isolation of intestinal pathogens by selectivemedia. J. Path. Bact. 54. 193-207.

Liefson. E. (1935). New culture media based on Sodiumdesoxycholate for the isolation of intestinal pathogens and for theenumeration of colon bacilli in milk and water. J. Path. Bact. 40: 581-589.

1.14

D.C.A. Hynes(Desoxycholate Citrate Agar -Hyne’s modification)

LAB 65

DescriptionThis modification of Leifson’s D.C.A. medium was introduced in1942. The medium was designed to be more inhibitory to commensalflora whilst allowing for adequate growth of Salmonella spp andShigella spp. The citrate and desoxycholate levels are significantlyincreased. To keep the desoxycholate in solution the pH also had tobe increased. The medium still uses lactose fermentation andhydrogen sulphide production as differential indicators.

Formula g/litre

Beef Extract 5.0


Lactose 10.0


Sodium citrate 8.5

Ferric citrate 1.0


Neutral red 0.02

Agar No. 2 12.0

Method for reconstitutionWeigh 52 grams of powder, disperse in 1 litre of deionised water in atwo litre flask. Bring to the boil over a gauze, swirling frequently toprevent burning. Simmer for 30 seconds to dissolve. Cool to 47˚Cbefore pouring plates. Dry the surface before inoculation. DO NOTREMELT OR AUTOCLAVE THIS MEDIUM.

Appearance: Pink, clear, bile aggregates may appear on the surfaceon refrigeration.

pH: 7.4 ± 0.2







S. sonnei 1.0-2.0 CV.E.G.(D) Colourless- pale pink

S. flexneri 1.0-2.0 CV.E.G. Colourless

Salmonella spp. 1.0-4.0 CV.E.G. Colourless (black centre)

S. typhi 0.5-1.5 CV.E.G. Colourless (Black/greycentre)

E. coli P.P.-1.5 CV.CR.D. Red (No growth)

K. aerogenes 1.0-2.5 CV.E.G. Pink (mucoid)

Proteus spp 0.5-2.0 CV.E.G. Colourless (Yellow) fishy odour

P. aeruginosa 0.5-1.0 CV.CR.D. Colourless (green)

ReferencesHynes, M. (1942). The isolation of intestinal pathogens by selectivemedia. J. Path. Bact, 54: 193-207

D.C.L.S. Agar(Desoxycholate Citrate Lactose Sucrose Agar)

LAB 3

DescriptionA modification of Leifson’s D.C.A. medium which incorporatessucrose as an additional fermentable substrate to differentiate lactosenegative sucrose positive coliforms from Salmonella spp. Thismedium is unsuitable for the isolation of Yersinia spp. which aresucrose positive.

Formula g/litre


Beef Extract 3.0

Lactose 5.0

Sucrose 5.0

Sodium citrate 10.5



Agar No. 2 12.0

Neutral Red 0.03

Method for reconstitutionWeigh 50 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes then heat gently with frequent mixingand bring to the boil. Simmer for 1 minute to complete dissolution ofthe solids. Cool to 47˚C then distribute 20ml into 90mm Petri dishes.Dry the surface by partial exposure, before use. DO NOT REMELTOR AUTOCLAVE THIS MEDIUM.

Appearance: Pale Pink, clear.

pH: 7.2 ± 0.2



Inoculation: Surface plating, streaking out to single colonies.




S. typhi 0.5-1.0 CV.E.G. Trans.colourless

Other Salmonella spp. 1.5-2.0 CV.E.G. Slight

cloudycolourless

S. sonnei 1.5-2.0 CV.E.G. Trans. (More opaquePinkish centre)

S. flexneri 0.5-1.0 CV.E.G. Trans.Pinkish

S. dysenteriae 0.5 CV.E.G. Trans.colourless

E. coli P.P.-1.5 CV.E.G.(D) Red (ppt around(inhibited) colonies)

Citrobacter spp. P.P.-2.0 CV.E.D.(G) Red (ppt around(inhibited) colonies)

Proteus spp. 1.0-2.0 CV.E.G. Yellow (Fishy odour)

Pseudomonas spp. 0.5-1.0 CV.E.D. Yellow (Green pigment)

Pink

1.15

ReferencesHynes, M. (1942). The isolation of intestinal pathogens by selectivemedia. J. Path. Bact. 54: 193-207.

Leifson, E. (1935). New culture media based on sodiumdesoxycholate for the isolation of colon bacilli in milk and water. J.Path. Bact. 40: 581-589.

Dermatophyte Test Medium (D.T.M.)LAB 117

DescriptionA modification of the formulation of Taplin, Zaias, Rebell and Blankfor the detection of dermatophytic fungi. This medium helps in thedifferentiation between saprophytic and environmental fungi.

Formula g/litre


Glucose 40.0

Agar No. 2 12.0

Phenol Red 0.2

Method for reconstitutionWeigh 62 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes then bring to the boil with frequentstirring. Dissolve 2 vials of Chloramphenicol X009 (X209) in ethanoland add these to the agar, mix well and distribute into tubes oruniversal containers. Sterilise at 121˚C for 15 minutes, allow to coolin the sloped position.

Note: Do not exceed the times stated for sterilisation, overheatedacidified agar loses gel strength and the sugars are caramelised.

Appearance: Orange, clear gel.

pH: 5.5 ± 0.2

Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097Trichophyton sp.

Storage of Prepared Medium: Slopes – up to 1 month at 2-8˚C inthe dark.

Inoculation: Surface plating or stab inoculation.

Incubation: 22-25˚C aerobically for 10-14 days.

Interpretation: Dermatophytes appear as fluffy colonies, colourvaries with species, the medium is reddened. Fungi other thandermatophytes cause the medium to become yellow due to acidproduction. If incubation is prolonged the medium may becomereddened. Yeasts appear as white creamy colonies. Blastomyces,Histoplasma and Coccidiodes may also turn the medium red, thoughthese are rarely encountered in lesions associated with ring worm.

ReferencesTaplin, D., Zaias, N., Rebell, G., Blank, H. (1969). Isolation andrecognition of dermatophytes on a new medium. (DTM) Arch.Dermatol. 99: 203-209.

Dextrose Tryptone AgarLAB 20

DescriptionA medium for the enumeration of thermophilic spore bearers infoods. The medium was designed to detect the thermophilic bacteriacausing ‘flat sour’ spoilage of canned foods. The medium also detectsthe ‘flat sour’ organism Bacillus stearothermophilus in sugar andother sweetening agents used in the preparation of frozen dairy foods,cereals and other food products.

Formula g/litre

Tryptone 10.0

Glucose 5.0


Agar No. 2 12.0

Method for reconstitutionWeigh 27 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to the boil todissolve agar before dispensing in 20ml amounts for poured platetechnique. Sterilise by autoclaving at 121˚C for 15 minutes.

Appearance: Purple clear agar.

pH: 6.9 ± 0.2

Minimum Q.C. organisms: B. stearothermophilus


Inoculation: Pour plate technique, pre-heat sample by steaming for20 minutes if a spore count is required.

Incubation: For Thermophiles – Aerobically for 48 hours at 55˚C.For Mesophiles – Aerobically for 48-72 hours at 30-32˚C.

Interpretation: Count all colonies for total counts, count yellowcolonies for differential acid producer count. Non acid producingcolonies are grey to colourless.

Growth Characteristicsorganism colony shape & colour

size (mm) surface

B. stearothermophilus 2.0 Rz.D Yellow zone mauve centre

Bacillus spp. 1.5-3.0 Rz.D Mauve (Yellow halo)

S. aureus 0.5-1.5 CV.E.G. Yellow

E. coli 1.0-1.5 CV.E.G. Yellow

Klebsiella spp. 1.5-2.5 CV.E.G. Yellow (mucoid)

Enterococci 0.5 CV.E.G. Yellow

Proteus spp. 2.0-3.0 RzD Yellow (spreads)

ReferencesWilliams, O.B. (1963). Tryptone Medium for the Detection of FlatSour Spores. Food Research 1, (3): 217-221.

American Public Health Association. (1972). Standard Methods forthe Examination of Dairy Products. 13th Edn. Ed. W.J. HauslerA.P.H.A. Washington.

Tanner, F.W. (1946). The Microbiology of Food 2nd edn., GarrardPress, Champners.

Baumgartner, J.G. and Hersom, A.C. (1956). Canned Foods. 4th Edn.Churchill, London.

1.16

Diagnostic Semi-Solid SalmonellaAgar (Diassalm)According to Van Netten and Van der Zee et al

LAB 537

DescriptionDiassalm, as developed by Van Netten et al (1991), is a semi-soliddifferential medium for the isolation of Salmonella spp. from foodand water. It is an improved modification of MSRV (De Smedt andBolderdijk 1988) and SR (Perales and Audicana 1989) with regard tothe composition of the basal medium, selective system and theintroduction of a differential system.

The original basal medium was a commercially available sulphidemobility-indole medium (SIM BBL) (Blazevic 1968). LAB M havesubstituted their raw materials into Blazevic’s formula to create aricher base for Diassalm. Selectivity is achieved by the use ofmalachite green oxalate, magnesium chloride and novobiocin. Thediagnostic properties of Diassalm are based on the use of twoindicator systems; saccharose combined with bromocresol purple;and ferro-iron in combination with thiosulphate.

The efficiency of Diassalm is due to the ability of salmonellae tomove through the highly selective mobility medium in a Petri dish,whilst the double diagnostic system allows visualisation of motileand non-motile suspected salmonellae due to blacking zones againstthe turquoise background. Diassalm can be seeded after pre-enrichment or after 8hr enrichment in selective broth (De Smedt andBolderdijk 1987).

Formula g/litre

Tryptone 20.0

Meat Peptone 6.1

Ferrous ammonium sulphate 0.2


Sucrose 7.5

Lactose 0.5


Malachite green oxalate 0.037

Magnesium chloride anhyd. 11.0

Agar No.1 2.8

Method for reconstitutionWeigh 53.0 grams of powder, disperse in 1 litre of deionised water.Mix well, bring quickly to the boil. Allow to cool to 47˚C and add 1vial of Novobiocin supplement – X150 (10mg/vial). Mix well andpour plates. Nitrofurantoin may be used instead of Novobiocin toimprove the isolation of S. enteritidis.

Appearance: - Green transparent, soft gel

pH: 5.5 ± 0.2

Minimum QC organisms: Salmonella typhimuriumNCIMB 50076Proteus mirabilis (Inhibition)

Storage of Prepared Medium: Plates – up to 7 days: at 2-8˚C in the dark.

Inoculation: 3 drops (0.1ml) of 8 to 20hr. incubated pre-enrichment broth are inoculated in one spot in the centre of oneplate of Diassalm.

Incubation: At 42 ± 0.5 or 37˚C for 18-24 hours. Keep the liduppermost at all times.

InterpretationAfter incubation the plates are examined for a mobility zone with apurple/black colour change. When the mobility zone is absent, but thecentre is blackened, non-motile salmonellae may be present. A loopfull of the motile zone which is the farthest from the sample

inoculum (or the blackened centre if non-motile) is sub-cultured ontobrilliant green agar and XLD agar. Futher biochemical and serologicalidentification are performed according to recognised procedure.

Direct latex agglutination may also be carried out from the edge ofthe mobility zone.

ReferencesBlazevics, D.J. (1968) Appl. Microbiol. 16, 688

De Smedt J.M. et al 1987 J. Food Protection 50, 658

Perales, I and Audicana. Evaluation of semi-solid Rappaport mediumfor detection of Salmonellae in meat products. J. Food Protection 52,

Van Netten, P., Van de Moosdijk, A., Perales, I. and Mossel, D.A.A.Letters in Applied Microbiology

Van Netten, P., Van der Zee, H., and Van der Moosdijk, A., (1991).The use of diagnostic selective semi-solid medium for the isolation ofSalmonella enteritidis from poultry. Proceedings of the 10thSymposium on the quality of poultry meat, Spelderholt Beckbergen,pp. 59-67.

Van der Zee, H., and Van Netten, P., (1992). Diagnostic semi-solidmedia based on Rappaport-Vassiliadis Broth for the detection ofSalmonella spp. and S. enteritidis in foods. Proceedings of theInternational Symposium of Salmonella and Salmonellosis.

Van der Zee, H., (1992). Detection of Salmonella spp. with the use ofa standard method, diagnostic semi-solid agars and immunocapturekit. Proceedings Third World Congress Foodborne infections andintoxications, Berlin.

D.N.’ase AGARLAB 95

DescriptionDN’ase agar provides a convenient means of identifying potentiallypathogenic staphylococci, based on the ability of coagulase-positivespecies to split DNA. DN’ases produced by the organisms hydrolysethe DNA molecule to a mixture of smaller mono and polynucleotides. DiSalvo observed perfect correlation between coagulaseactivity and DN’ase production using S. aureus strains from clinicalspecimens. Other publications have also reported a close correlation.

Formula g/litre

Tryptone 20.0

Deoxyribonucleic acid (DNA) 2.0

Sodium chloride 5.0

Agar No. 2 12.0

Method for reconstitutionWeigh 39 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Allow to cool to 47˚C then pourinto Petri dishes.

Appearance: Pale cream, clear.

pH: 7.3 ± 0.2

Minimum Q.C. organisms: S. aureus NCIMB 50080S. epidermidis NCIMB 50082

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.

Inoculation: Use a heavy inoculum on a small area. Four or moreorganisms can be tested on one 90mm Petri dish.


Interpretation:Having obtained good growth flood the plate with 1N hydrochloricacid. This will precipitate the DNA in the medium. DN’ase producingorganisms will be surrounded by a clear area where the DNA hasbeen broken down into fractions which are not precipitated by theHydrochloric acid. Gram positive, catalase positive cocci that

1.17

produce DN’ase can be provisionally classified as S. aureus, andconfirmed by tube coagluase or thermostable DN’ase tests. DN.’aseis also produced by some Gram negative bacilli such as Serratiamarcescens, Pseudomonas aeruginosa. Some corynebacteria andstreptococci may also produce DN’ase.

ReferencesBaird-Parker, A. C. 1965. The classification of staphylococci andmicrococci from world-wide sources. J. Gen. Microbiol. 38, 363-387.Black, W. A., Hodgson, R. and McKechnie, A. 1971.

DiSalvo, J. W. 1958 Deoxyribunuclease and coagulase activity ofmicrococci. Med. Tech. Bull. U.S. Armed Forces Med. J. 9, 191.

Martin, W. J and Ewing, W. H. 1967. The deoxryibonuclease test asapplied to certain gram-negative bacteria. Can. J. Microbiol. 13, 616-618.

Messinova, O. V., Yusupova, D. V. and Shamsutdinov, N. S. 1963.Deoxyribonuclease activity of Corynebacterium and its relation tovirulence. Fed. Proc. 22, T1033.

Streitfeld, M. M., Hoffmann, E. M. and Janklow, H. M. 1962.Evaluation of extracellular deoxyribonuclease activity inPseudomonas. J. Bacteriol. 84, 77. Wannamaker, L. W. 1964.Streptococcal deoxryribonuclease, pp. 140-165. J. W. Uhr (ed.). TheStreptococcus, Rheumatic Fever, Glomerulophritis. Baltimore:Williams & Williams.

Weckman, B. G. and Catlin, B. W. 1957 Deoxryribonuclease activityof micrococci from clinical sources. J. Bacteriol. 73, 747-753.

Zierdt, C. H. and Golde, D. W. 1970. Deoxryribonuclease-positiveStaphylococcus epidermidis strains. Appl. Microbiol. 20(1), 54-57.

Easter and Gibson Pre-enrichment Broth

LAB 136

DescriptionA pre-enrichment broth for use in detection procedures utilisingconductance/impedance techniques. This broth is developed fromLAB 46 Buffered Peptone Water and will enhance the recovery ofsub-lethally damaged bacteria. Salmonella reduce T.M.A.O. toproduce a significant change in the medium that can be detected bythe conductance/impedance equipment.

Best results will be obtained by those customers using Pre-Enrichment Broth (LAB 136) and Salmonella Medium (LAB 137)bought from LAB M. These media are both manufactured with thesame peptones, so that organisms “switched on” to utilising thesubstrates in LAB 136 can carry on metabolising the same substratesin LAB 137, significantly shortening the lag time.

Formula g/litre

Tryptone 7.5

Meat Peptone 2.5

Sodium chloride 5.0


Potassium di-hydrogen phosphate 1.5

Mannitol 5.0

Method for reconstitutionWeigh 25 grams of powder, disperse in 1 litre of deionised water.Heat gently to dissolve supplement, dispense into appropriatecontainers, then sterilise by autoclaving at 121˚C for 15 minutes.

Appearance: Very pale yellow clear solution.

pH: 7.2 ± 0.2


Storage of Prepared Medium: Capped container – up to 1 month at15-20˚C in dark.

Inoculation: Homogenised food samples.


Sub-culture: Into LAB 137 Easter and Gibson Salmonella Medium.

ReferencesEaster M.C., Gibson D.M. (1985). Rapid and automated detection ofSalmonella by electrical measurements. J. Hyg. 94, 245-262.

Gibson D.M. (1987). Some modifications to the media for rapid,automated detection of salmonellas by conductance. J. Appl.Bacteriol. 63, 299-304.

Ogden I.D., Cann P.C. (1987). A modified conductance medium forthe detection of Salmonella spp. J. Appl. Bacteriol. 63, 459-464.

Easter and Gibson SalmonellaMedium

LAB 137

DescriptionA medium for the rapid detection of Salmonella spp byconductance/impedance techniques. This medium is based on LAB55 Selenite Cystine Broth and is supplemented with dulcitol andT.M.A.O. (trimethylamine-N-oxide). Salmonella reduce T.M.A.O. toT.M.A. (trimethylamine) and, in so doing, increase the conductivityof the medium which can be detected and measured by monitoringequipment such as Malthus.

Best results will be obtained by those customers using Pre-Enrichment Broth (LAB 136) and Salmonella Medium (LAB 137)bought from LAB M. These media are both manufactured with thesame peptones, so that organisms ‘switched on’ to utilising thesubstrates in LAB 136 can carry on metabolising the same substratesin LAB 137 significantly shortening the lag time.

Formula g/litre

Meat Peptone 2.5

Tryptone 2.5


Dulcitol 5.0


Method for reconstitutionWeigh 2.5 grams of powder, disperse in 100ml of deionised water.Add 1 vial of X137 T.M.A.O./Selenite supplement. Swirl to mix, heatto boiling to effect sterilisation. When cool add 1ml of stock solutionof L-cystine. Distribute into sterile conductance tubes or bottles. DO NOT AUTOCLAVE.

Stock solution of L-cystine0.1gm L-cystine dissolved in 15ml of normal NaOH – add to 100mlsterile distilled water. Keep refrigerated. Discard after 1 month.

Appearance: Clear reddish/orange solution. A slight precipitate may form.

pH: 7.2 ± 0.2


Storage of Prepared Medium: capped container – up to 1 month at15-20˚C in dark.

Inoculation: From LAB 136 Easter and Gibson Pre-EnrichmentBroth.

Incubation: 37˚C aerobically connected to monitoring equipment.

1.18

ReferencesEaster M. C. Gibson D. M. 1985. Rapid and automated detection ofSalmonella by electrical measurements. J. Hyg. 94, 245-262.

Gibson D.M. 1987. Some modifications to the media for rapidautomated detection of salmonellas by conductance. J.Appl.Bacteriol. 63, 299-304.

Ogden I.D., Cann P.C. 1987. A modified conductance medium for thedetection of Salmonella spp. J. Appl. Bacteriol. 63, 359-464.

E.E. Broth(Enterobacteriaceae Enrichment Broth)

LAB 91

DescriptionE.E. Broth is recommended as an enrichment medium whenexamining food and feedstuffs for Enterobacteriaceae. It is amodification of LAB 51 Brilliant Green Bile Broth, with an improvedbuffering capacity to encourage early growth and preventautosterilization. E.E. Broth uses glucose instead of lactose to makethe medium a test for all enterobacteria including non lactosefermenting organisms.

Formula g/litre


Dextrose 5.0



Bile Salts 20.0


Method for reconstitutionWeigh 43.5 grams of powder and add to 1 litre of deionised water.Swirl to dissolve, warm gently if necessary, then distribute intobottles or tubes and heat at 100˚C for 30 minutes only. Cool rapidly.OVERHEATING THIS MEDIUM WILL ADVERSELY AFFECTITS PERFORMANCE.


pH: 7.2 ± 0.2

Minimum Q.C. organisms: E. coli NCIMB 50034B. subtilis (inhibition)

Storage of Prepared Medium: capped containers – up to 3 monthsat 15-20˚C in the dark.

Inoculation: Add 1 part of sample suspension or dilution to 10 partsof medium.

Incubation: 44˚C for 18 hours for thermotrophs. 32˚C for 24-48hours for mesotrophs. 4˚C for 10 days for psychrotrophs.

Interpretation: Turbidity and a colour change to yellow-green ispresumptive evidence of Enterobacteriaceae. Subculture ontoconfirmatory media e.g. LAB 88 V.R.B.G.A. must be carried out.

ReferencesMossel, D. A. A., Visser, M. and Cornelissen, A. M. R. 1963. Theexamination of foods for Enterobacteriaceae using a test of the typegenerally adopted for the detection of salmonellae. J.Appl. Bacteriol.26, 444-452.

Mossel, D. A. E., Harrewijn, G. A. and Nesselrooy-van Zadelhoff, C.F. M. 1974. Standardisation of the selective inhibitory effect ofsurface active compounds used in media for the detection ofEnterobacteriaceae in food and water. Health Lab. Sci. 11, 260-267.Richard, N. 1982. Monitoring the quality of selective liquid media bythe official French dilution technique used for the bacteriologicalexamination of foods. In: Quality assurance and quality control ofmicrobiological culture media, edited by J. E. L. Corry, G.I.T.-VerlagDarmstadt, pp. 51-57.

Endo AgarLAB 60

DescriptionThis medium was developed in 1914 for the isolation of Salmonellatyphi; other media have since proved superior for this purpose, butEndo agar has a role as a coliform medium. It is recommended by theAmerican Public Health Association as a standard medium for theenumeration of coliforms in water and dairy products. In this mediumacetaldehyde is produced by coliforms and then fixed by the sulphiteto produce a metallic sheen with the basic fuchsin dye. Most entericGram negative organisms will grow well, whilst Gram positiveorganisms are mostly inhibited.

Formula g/litre


Lactose 10.0

Dipotassium phosphate 3.5

Sodium sulphite 2.5

Agar No. 1 15.0

Method for reconstitutionWeigh 41 grams of powder, disperse in 1 litre of deionised water. Add4ml of a 10% w/v alcoholic solution of basic fuchsins (95% ethylalcohol). Bring to the boil with frequent swirling to dissolve thesolids. Sterilise by autoclaving at 121˚C for 15 minutes. Cool to 47˚Cin a water bath before pouring. The precipitate typically associatedwith this medium should be dispersed by gentle swirling prior topouring the plates.

This medium is light sensitive and should therefore be stored in thedark, preferably under refrigeration. The medium will become darkred in colour if exposed to light.

Basic Fuchsin is a potential Carcinogen and care should be takenwhen handling it to avoid inhalation of the powdered dye andcontamination of the skin.

Appearance: Pale pink/orange

pH: 7.5 ± 0.2







E. coli 1.0-2.0 CV.E.G. Deep Red (Metallic sheen)

K. aerogenes 1.0-2.5 CV.E.G. Red (mucoid)

Proteus spp 2.0-3.0 CV.E.G. Pale Pinkcolourless

Ps. aeruginosa 0.5-1.0 F.CR.D. Pale Pink

Shigella spp 0.5-1.0 CV.E.G. Pale Pink

Salmonella spp 1.0-1.5

Gram positive no growth.organisms

ReferencesEndo, 1914, Centr. Bakt., Abt 1, Orig., 35: 109.

American Public Health Association, 1975. Standard Methods for theExamination of Water and Wastewater, 14th Edn. American PublicHealth Association, Inc. Washington D.C.

American Public Health Association, 1972. Standard Methods for theExamination of Dairy Products, 13th End., American Public HealthAssociation, Inc., Washington, D.C.

1.19

Eosin Methylene Blue Agar (Levine)LAB 61

DescriptionThis medium was introduced in 1916 by Holt-Harris and Teague todifferentiate Escherichia spp. and Aerobacter spp. It was modified byLevine in 1918 who removed sucrose from the formula and increasedthe lactose content. The distinctive metallic sheen produced by E. colion this medium is due to acid production resulting in an amidebonding between the eosin and methylene blue, other coliforms donot produce enough acid to cause this reaction. Eosin inhibits mostGram positive organisms. The prepared medium is sensitive to light.

Formula g/litre


Lactose 10.0


Monopotassium phosphate 1.3

Eosin Y 0.4

Methylene Blue 0.065

Agar No. 2 15.0

Method for reconstitutionWeigh 37.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 50˚C and agitate gentlyto ensure uniform distribution of the flocculant precipitate which is afeature of this medium before pouring into Petri dishes. STORE IN THE DARK.

Appearance: Blue/purple with a light precipitate.

pH: 6.8 ± 0.2





Growth Characteristicscolony shape &

organism size (mm) surface colour other

E. coli 2.0-3.0 CV.E.G. Blue Black (Metallic sheen)

Klebsiella spp. 3.0-4.0 CV.E.G. BrownBlue (mucoid)

Salmonella spp. 2.0-3.0 CV.E.G. Colourless

Shigella spp. 1.0-2.0 CV.E.G. Colourless

Candida spp. 0.5-1.5 CV.Rz.G.(D) White

S. aureus P.P. CV.E.G. Colourless

E. faecalis P.P. CV.E.G. Colourless

ReferencesAmerican Public Health Association, American Water WorksAssociation and Water Pollution Control Federation, (1975).Standard Methods for the Examination of Water and Wastewater,14th Edn., Washington, D.C. American Public Health Association.

Girolami, R.L. and Stamm, J.M. (1976). Inhibitory effect of light ongrowth supporting properties of Eosin Methylene Blue Agar. Appl.Environ. Microbiol., 31:1 141-142.

Haesler, W. J. (ed) (1972). Standard Methods for the Examination ofDairy Products, 13th edn., Washington, D.C., American PublicHealth Association.

Levine, M. (1918). Differentiation of E. coli and B. aerogenes on asimplified Eosin-Methylene Blue agar. J. Infect. Dis., 23: 43-47.

Fastidious Anaerobe Agar (F.A.A.)LAB 90

DescriptionA primary isolation medium capable of growing most clinicallysignificant anaerobes. Developed by LAB M, comparisons haveshown this medium to be superior to other formulations as a primaryisolation medium for fastidious organisms. The peptones includedhave been chosen for maximum growth stimulation. Starch andsodium bicarbonate act as de-toxification agents whilst haeminencourages pigment production in Porphyromonas melaninogenicus.Specific growth promoting agents are Cysteine for Fusobacteriumnecrophorum, Propionibacterium acne and Bacteriodes fragilis,arginine for Eubacterium spp. soluble pyrophosphate for Porph.gingivalis and Porph. asaccharolyticus. Pyruvate helps neutralisehydrogen peroxide and is also utilised by Veillionella spp. as anenergy source. Vitamin K and sodium succinate provide essentialgrowth factors for some anaerobes as does the 0.1% glucose. The lowlevel of glucose prevents the production of high levels of acids andalcohols which would inhibit colonial development.

Formula g/litre

Peptone mix 23.0

Sodium chloride 5.0

Soluble starch 1.0

Agar No. 2 12.0


Glucose 1.0

Sodium pyruvate 1.0

Cysteine HCl monohydrate 0.5

Haemin 0.01

Vitamin K 0.001

L-Arginine 1.0

Soluble pyrophosphate 0.25

Sodium succinate 0.5

Method for reconstitutionWeigh 46 grams of powder and add to 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C then asepticallyadd 5-10% of sterile defibrinated horse blood, mix well and pour intoPetri dishes. This medium can be made selective for various speciesof anaerobes by the addition of appropriate selective cocktails e.g.

Gram negative anaerobes X090, X290

Non-sporing anaerobes X091, X291

Actinomyces spp. X092

Clostridium difficile X093

Appearance: Red due to addition of blood. The blood will darken(reduce) because of the presence of reducing agents.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: B. fragilisP. anaerobious



Incubation: 37˚C anaerobically with 10% CO2 for 48 hours to 5 days.

1.20

Growth Characteristics (48 hours)colony shape &

organism size (mm) surface colour other

B. fragilis 1.0-2.0 CV.E.G. Grey

C. perfringens 1.0-2.0 CV.E.G. Grey ‘Target’haemolysis

(non haemolytic)

F. necrophorum 1.0-2.0 CV.E.G.(D) trans- (grey)parent (haemolytic)

Porphyromonas asaccharolyticus1.0-2.0 CV.E.G. Grey/

Brown (clearing)

B. ureolyticus 0.5 F.E.D. translucent pitting

Prop. acne 0.5 CV.E.G. White

Pept. anaerobius 0.5-2.0 CV.E.G. White/Grey

A. israeli 0.5-1.0 CV.E.G. White (‘molar tooth’)(smooth)

ReferencesBrazier, J.S. (1986). Yellow fluorescence of Fusobacteria Letters inApplied Microbiol. 2: 124-126.

Brazier, J.S. (1986). A note on ultra violet red fluorescence ofanaerobic bacteria in vitro. J. Appl. Bact. 60: 121-126.

Eley, A., Clarry, T., Bennett, K.W. (1989). Selective and differentialmedium for isolation of Bacteriodes ureolyticus from clinicalspecimens. European Journal of Clinical Microbiology, InfectiousDiseases. 8: 83-85.

Wade W. Griffiths, M. (1987). Comparison of Media for cultivationof subgingival bacteria. J. Dent. Res. 66: no. 4 abstract 334.

Heginbotham M., Fitzgerald T.C., and Wade W.G. (1990).Comparison of solid media for the culture of anaerobes. J. Clin. Path.43: 253-256.

Fastidious Anaerobe Broth (F.A.B.)LAB 71

DescriptionF.A.B. was developed by LAB M working in conjunction with themicrobiology department of a University of Manchester teachinghospital. The medium was designed to give optimum growth offastidious anaerobes and has found applications as a blood culturemedium and an enrichment broth for the isolation of anaerobes. Themedium is very rich in nutrients from the specially selected peptonemixture. Vitamin K. haemin and L-cysteine are all growth factorsrequired by some anaerobes. L-cysteine together with sodiumthioglycollate reduce the Eh of the medium and the agar contentinhibits absorption of oxygen and convection currents. Resazurin is aredox indicator. Several published evaluations show F.A.B. to be theliquid medium of choice for fastidious anaerobes.

Formula g/litre


Yeast Extract 10.0


Sodium chloride 2.5

Agar No. 1 0.75

L-Cysteine HCl 0.5

Resazurin 0.001


Haemin 0.005

Vitamin K 0.0005

Method for reconstitutionWeigh 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 agarthen dispense into screw cap containers. Sterilise by autoclaving at121˚C for 15 minutes. Tighten the caps as soon as possible afterautoclaving.

Appearance: Pale straw, clear, viscous. May have a narrow band ofred/purple at the surface due to action of oxygen on the resazurin. Ifthe medium is reddish this indicates too much oxygen has beenabsorbed, the medium should be reheated to deoxygenate. Do notreheat more than once.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: B. fragilis


Inoculation: If used as a blood culture medium a minimum dilutionof 1:10 should be used.

Incubation: 37˚C for 24-72 hours. Keep the container airtight.

Growth indicators: The broth may become turbid or individualcolonies may form suspended in the medium.

ReferencesGould, J.H., Duerden, B.I. (1983). Blood culture – current state andfuture prospects. J. Clin. Pathol. 36: 963-977.

Ganguli, G.A., O’Hare, W., Hyde, W.A. (1984). Rapid Detection ofBacteraemia by early subculture. J. Med. Microbiol. 17: 311-315.

Ganguli, L.A., Keaney, M.G.L., Hyde, W.A., Fraser, B.J. (1985).More Rapid identification of bacteraemia by manual rather thanradiometric methods. J. Clin. Pathol. 38: 1146-1149.

Junt, G.H., Price, E.H. (1982). Comparison of a home made bloodculture broth containing a papain digest of liver, with fourcommercially available media, for the isolation of anaerobes fromsimulated paediatoic blood cultures. J. Clin. Pathol. 35: 1142-1149.

Ganguli, L.A., Turton, L.J., Tillotson, G.S. (1982). Evaluation ofFastidious Anaerobe Broth as a blood culture medium. J. Clin. Pathol.35: 458-461.

Tillotson, G.S. (1981). Evaluation of ten commercial blood culturesystems to isolate a pryridoxal dependent streptococcus. J. Clin.Pathol. 34: 930-934.

Fluid Thioglycollate Medium (U.S.P.)LAB 25

DescriptionA medium for sterility tests, prepared according to the specificationof the United States Pharmacopeia. Aerobic and anaerobic organismsgrow well in this medium even from small inocula. In appropriatetubes or bottles the thioglycollate ensures adequate anaerobicconditions. The low level of agar reduces oxygen diffusion into themedium. The thioglycollate will also serve to inactivate anymercurial compounds used as preservatives.

Formula g/litre

Tryptone 15.0

L-Cystine 0.5

Glucose 5.5

Yeast Extract 5.0

Sodium chloride 2.5


Resazurin 0.001

Agar 0.75

1.21

Method for reconstitutionWeigh 29.75 grams, disperse in 1 litre of deionised water. Soak for 10minutes, swirl to mix, then bring to the boil to dissolve and dispense15ml into 6mm x 150mm tubes. Sterilise by autoclaving for 15minutes at 121˚C. Store at ambient temperature in the dark, but not inthe refrigerator. If more than 30% of the medium turns pink(oxidised) the Eh may be restored (once only) by heating in a boilingwater bath or by free-steaming.

Appearance: Pale straw colour, clear. Surface may be pink/blue dueto oxidation of Resazurin.

pH: 7.1 ± 0.2

Minimum Q.C. organisms: C. sporogenesS. aureus NCIMB 50080


Incubation: 30-35˚C aerobically for 14 days.

Growth indicators: Turbidity, colonies in medium.

ReferencesThe Pharmacopeia of the United States of America. 21st End. (1985).

Fluorescence AgarLAB 16

DescriptionThis medium is a modification of King, Ward and Raney’s mediumformulated for the demonstration of the fluorescein pigmentproduced by many strains of Pseudomonas. The pyocyanin pigmentproduced by most strains of Pseudomonas aeruginosa is alsoproduced. Can be made selective by the addition of selective agentssuch as X108 Cetrimide Fucidin Cephaloridine.

Formula g/litre



Magnesium sulphate 1.5

Agar No. 2 12.0

Method for reconstitutionWeigh 35 grams of powder, disperse in 1 litre of deionised watercontaining 10ml Glycerin B.P. Allow to soak for 10 minutes, swirl tomix then sterilise for 15 minutes at 121˚C. Mix well before pouring.Slant over a generous butt if required.

Appearance: Straw coloured, clear gel.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: Ps. aeruginosa NCIMB 50067


Inoculation: Surface spreading.

Incubation: 30-37˚C for 24 and 48 hours aerobically.



Ps. aeruginosa 0.5-2.5 F.CR.D. Grey (colony size variesGreen- with strain)

fluorescent-(non pigmented)pigment (mucoid)

Ps. fluorescens 1.0-2.5 F.CR.D. Greyfluorescent

pigment

ReferencesKing, E.O., Ward, M.K. and Raney, D.E. (1954). 2 simple media fordemonstration of pyocyanin and fluorescein, J. Lab. Clin Med., 44: 301-307.

Brown, V.I. and Lowbury, E.J.L. (1965). Use of an improvedcetrimide agar medium and other culture methods for Pseudomonasaeruginosa. J. Clin. Path. 18: 752-756.

Fraser BrothLAB 164

DescriptionDeveloped as a modification of UVM II medium, Fraser broth is asecondary enrichment broth for the isolation of Listeria spp., and issimilar to Palcam broth in that it contains aesculin to indicate thepresence of a potential Listeria isolate. It also contains lithiumchloride in an attempt to suppress the growth of enterococci in themedium (as does Palcam). Fraser broth may also be used as a primaryenrichment medium by incorporating 1/2 strength supplement intothe broth base (X164 or X564).

Formula g/litre


Yeast extract 5.0

Aesculin 1.0





Method for reconstitutionWeigh 55 grams of power and add to 1 litre of deionised water (addto 900ml if preparing 1/2 Fraser). Allow to soak for 10 minutes, swirlto mix and sterilise at 121˚C for 15 minutes. Cool to 47˚C and add 2vials of Fraser supplement X165 (or 2 vials of 1/2 Fraser supplementX164), mix well and aseptically dispense into sterile tubes or bottles.

Appearance: Straw opalescent broth with precipitate (clears onstorage).

pH 7.2 + 0.2

Minimum Q.C. organisms: Listeria sp NCIMB 50007e. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Bottles – up to 14 days at 2-8˚C.

Inoculation: 1/2 Fraser – Add 25g sample to 225ml of 1/2 Fraserbroth and homogenise Fraser – Subculture 0.1ml of primaryenrichment broth (UVM I or 1/2 Fraser) into 10ml of Fraser broth.

Incubation: 1/2 Fraser – 30˚C aerobically for 24hrs.

Fraser – 35˚C aerobically for 24hrs and 48hrs. Subculture ontoselective agars at 24 and 48hrs.

InterpretationBlackening of the broth indicates the presence of a potential Listeriaand should be subcultured onto Listeria isolation medium (Oxford)LAB122 or Palcam agar LAB148. All broths should be subculturedbefore discarding, irrespective of colour change.

ReferencesFraser J.A., and Sperber W.H., (1988) Rapid detection of Listeria sppin food and environmental samples by esculin hydrolysis. J.FoodProtection 51 (10) 762-765.

McClain D., and Lee W.H. (1989) FSIS method for isolation ofL.monocytogenes from processed meat and poultry products.Lab.Comm.No.57, Revised May 24, (1989). US Dept of Agric.FSIS,Microbiol. Div.

1.22

G.C. Agar BaseLAB 67

DescriptionA nutritious agar base described by Thayer and Martin for theisolation of Neisseria gonorrhoeae. The rich peptone mixture isenhanced by the use of corn starch to absorb toxic metabolites and abuffering system is used to maintain neutral pH. The medium is madeselective by the use of various antibiotic cocktails. Thayer and Martinoriginally recommended the use of vancomycin, colistin and nystatinV.C.N. but the addition of trimethoprim (X068, X268 V.C.N.T.) isuseful in preventing the swarming of proteus. More recently theemergence of vancomycin sensitive gonococci has made the NewYork City selective agents (lincomycin, colistin, amphotericin,trimethoprim X070, X270 LCAT) the combination of choice.Enrichment of the base is usually by the addition of lysed blood.Alternatively chocolated blood or haemoglobin powder and Thayerand Martin’s mixture of vitamins, amino acids and coenzymes can beused. The supplement X069, X269 can be added as this is withoutAmphotericin and this permits the growth of yeasts. The growthsupplement X271 can be added to this medium to aid in the isolationof Neisseria spp.

Formula g/litre

Special Peptone 15.0

Corn Starch 1.0

Sodium chloride 5.0



Agar No. 2 10.0

Method for reconstitutionWeigh 36 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 48˚C and add 50-70mlof lysed blood and 2 vials of X070 selective agent. Mix well and pourinto Petri dishes.

Appearance: Dependent on blood supplement used.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: N. gonorrhoeae ATCC CDC98E. coli (inhibition) NCIMB 50034



Incubation: 37˚C microaerobically for 24-48 hours.



N. gonorrhoeae 1.0-2.0 CV.E.G. Transparent variations incolony size

N. lactamica 1.0-2.0 CV.E.G. Grey

N. meningitidis 2.0-3.0 CV.E.G. Grey

B. catarrhalis 2.0-3.0 CV.E.G. Cream

Other organisms should not grow with the exception of antibioticresistant variants.

ReferencesYoung, H. 1978. Cultural diagnoses of gonorrhoea with modifiedNew York City (MNYC) medium. Brit. Journ. Ven. Dis. 54: 36-40:

Thayer, J. D. and Martin, J. E. 1966. Improved medium selective forthe cultivation of N. gonorrhoeae and N. Meningitidis: Public Healthrep. 81: 559-562.

Hektoen Enteric AgarLAB 110

DescriptionA medium developed at the Hektoen Institute in Chicago for theenhanced recovery of shigellae from clinical specimens. Thismedium has high levels of peptones and sugar which counteract someof the toxic effects of bile salts used to make the medium selective.This allows the shigellae to grow as well as the salmonellae. Salicinis fermented by many coliforms including those that do not fermentlactose and sucrose. The medium employs a double indicator systemsimilar to that used in LAB 6 C.L.E.D., (Bevis) and an H2S indicatorsystem similar to that used in LAB 32 XLD. Although intendedprimarily for clinical use this medium is quoted in B.S. 4285 assuitable for the examination of dairy products for salmonellae.

Formula g/litre

Meat Peptone 12.0

Yeast Extract 3.0

Lactose 12.0

Sucrose 12.0

Salicin 2.0

Bile Salts No. 3 7.0


Sodium chloride 5.0


Ammonium ferric citrate 1.5

Acid fuchsin 0.1

Bromothymol blue 0.065

Agar No. 1 14.0

Method for reconstitutionWeigh 76 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then heat gently and bringto the boil. Cool to 47˚C and pour plates. DO NOT AUTOCLAVEOR OVERHEAT THIS MEDIUM.


pH: 7.5 ± 0.2

Minimum Q.C. organisms: Salmonella sp. NCIMB 50076Shigella sp.E. coli (some inhibition)NCIMB 50034


Inoculation: Surface plating, streak out to single colonies.


1.23



H2S +ve 2-3 CV.E.G. Green+Salmonella Black

H2S -ve 2-3 CV.E.G. GreenSalmonella

S. sonnei 2-2.5 CV.E.G. Green (Rough)

S. flexneri 1.0-2.5 CV.E.G. Green

S. dysenteriae 1-2 CV.E.G. Green

E. coli 0.5-2 CV.E.G. Salmon ppt. (Rough)around (No growth)

colonies

Citrobacter spp. 1.0-2.0 CV.E.G. Salmon (Rough)

Klebsiella spp. 0.5-2.0 CV.E.G. Salmon (Mucoid)

Proteus spp. 1.0-2.0 CV.E.G. Green/ (No growthBlack brownish centre)centre

Pseudomonas spp. 0.5-1.5 F.Rz.D. Green (No growth)

ReferencesKing, S. and Metzger, W.I. (1967). A new medium for the isolation ofSalmonella and Shigella species. Bact. Proc. Am. Soc. Microbiol. 77.

King, S. and Metzger, W.I. (1968). A new plating medium for theisolation of enteric pathogens. Hektoen Enteric Agar, Appl.Microbiol., 16(4), 577.

King, S. and Metzger, W.I. (1968). A new plating medium for theisolation of enteric pathogens. II. Comparison of Hektoen Agar withSS and EMB agar. Appl. Microbiol., 16(4), 579.

Speck, M.L. (ed.). (1976). Compendium of Methods for theMicrobiological Examination of Food. Washington, D.C.: AmericanPublic Health Association.

Helicobacter Pylori MediumLAB 140

DescriptionA selective medium for the isolation of Helicobacter pylori, thecausative agent of chronic gastritis.

This campylobacter-like organism was described in 1983 colonisingthe gastric mucosa, a site previously thought to be sterile due to thelow PH. The high level of Urease production by this organismappears to be the major pathogenicity factor enabling it to withstandthe strongly acidic environment.

Helicobacter Pylori Medium is a modification of CCDA Medium forthe isolation of Campylobacter spp. Formulated by Bolton et al atPreston Public Health Laboratory, it incorporates a rich agar basesupplemented with horse serum to promote optimum growth, andVancomycin, Cefsulodin, and Amphotericin as selective agents.

Formula g/litre

Beef Extract 10.0

Meat Peptone 5.0

Tryptone 5.0

Sodium chloride 5.0

Charcoal 4.0

Acid Hydrolysed Casein 3.0

Ferrous sulphate 0.25



Agar no.2 12.0

Method for reconstitutionWeigh 45.0 grams of powder and disperse in 1 litre of deionisedwater. Soak for 10 minutes, swirl to mix and sterilise at 121˚C for 15minutes. Cool to 47˚C and add 100ml of Horse Serum, and 2 vials ofVCA supplement X040. Mix well and pour, continuing to mix whilstpouring to keep the charcoal in suspension.

Appearance: Black Agar

pH: 7.4 ± 0.2

Minimum Q.C. organisms: Helicobacter pylori S.aureus (inhibition) NCIMB 50080

Storage of prepared medium: Plates – up to 7 days at 2-8˚C the dark.

Inoculation: Surface streaking for single colonies.

Incubation: 37 ˚C for 72 hours.

Growth CharacteristicsOrganisms Colony Size Shape and Colour

(mm) Surface

H. pylori 1.0-1.5 CVEG Grey

B. catarrhalis 1.0-2.0 CVEG White/Cream

ReferencesBolton et al. Public Health Laboratory, Preston. Personalcommunication.

Hoyle’s MediumLAB 27

DescriptionA highly selective culture medium for the isolation anddifferentiation of Corynebacterium diphtheriae types gravis, mitisand intermedius. Hoyle’s medium gives rapid growth of all types ofC. diphtheriae, which results in most specimens giving adequategrowth with overnight incubation.

Formula g/litre

Beef Extract 10.0

Peptone 10.0

Sodium chloride 5.0

Agar 12.0

Method of ReconstitutionWeigh 37 grams of powder and disperse in 1 litre of deionised water.Allow to soak for 10 minutes, and sterilise by autoclaving at 121˚Cfor 15 minutes. Cool to 47˚C, add 50ml of lysed horse or sheep bloodand 10ml of X027 potassium tellurite solution. Mix well beforepouring.

Appearance: Dark Red, clear gel.

pH: 7.8 ± 0.2

InoculationSpread the entire surface with the swab or sample underinvestigation. Hoyle’s medium is very selective and spreading forsingle colonies using a wire loop is not necessary. Use of a non-selective blood agar alongside Hoyle’s is recommended.

Incubation: 37˚C for 18-48 hrs, aerobically

1.24

Interpretationcolony size shape &


C. diphtheriae 1.5-2.5 CV.CR.D Grey Colonies difficult var gravis (daisy head) to emulsify

C. diphtheriae 0.5-2.0 CV.E.G. Grey Easily var mitis (dark centre) emulsified

C. diphtheriae 0.5-1.0 CV.E.G. Greyvar intermedius (dark centre)

C. Ulcerans 1.0-1.5 CV.E.G. Grey Streptococcal (dark centre) appearance

in Gram stain

C. hoffmanii 0.5-1.0 CV.E.G. Black White/grey(confluent growth)colonies

C. xerosis 0.5-2.5 CV.E.G. Black

Streptococcus pp - 1.5 CV.E.G. Black Enterococci mayspp. be larger

H. influenzae pp - 1.5 CV.E.G. Grey/black Some strains no growth

Storage: Plates – up to 7 days at 2-8˚C

Minimum Q.C. Organisms C. diphtheriae var mitis(non-toxigenic) E. coli NCTC 10418 (inhibition)

Reference:Hoyle L. (1941) A Tellurite Blood Agar Medium for the RapidDiagnosis of Diphtheria. Lancet 1 175-176

176. Elek S.D. (1948) The Recognition of Toxigenic Bacterial Strainsin vitro. Brit. Med. J. 1 493-496.

Kanamycin Aesculin Azide Agar(K.A.A. Agar)

LAB 106

DescriptionA selective isolation and enumeration medium for enterococci(Lancefield group D streptococci) in food. Sodium azide andkanamycin provide the selective inhibition required whilst aesculinand iron salts form an indicator system for the presumptiveidentification of enterococci. Incubation at 42˚C will increase themedium’s selectivity.

Formula g/litre

Tryptone 20.0

Yeast Extract 5.0

Sodium chloride 5.0

Sodium citrate 1.0

Aesculin 1.0

Ferric ammonium citrate 0.5

Sodium azide 0.15

Kanamycin sulphate 0.02

Agar No. 1 10.0

Method for reconstitutionWeigh 43 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C, then dispense intoPetri dishes.

Appearance: Pale straw, clear.

pH: 7.0 ± 0.2

Minimum Q.C. organisms: E. faecalis NCIMB 50030E. coli (inhibition) NCIMB 50034


Inoculation: Surface, spread 0.1ml to 0.5ml over entire surface ofplate.

Incubation: 37˚C or 42˚C aerobically for 18-24 hours.

Interpretation: Count all white/grey colonies, approx 2mmdiameter, surrounded by a black halo to give presumptiveenterococcus/faecal streptococcus count.

ReferencesMossel, D.A.A., Bijken, P.H.G., Eelderink, I. and van Spreekens,K.A. (1978). Streptococci, edited by Skinner, F. A. and Quesnel, L.B. SAB Symposium Series No. 7 Academic Press,London.

Kanamycin Aesculin Azide Broth(K.A.A. Broth)

LAB 107

DescriptionAn enrichment and isolation medium for enterococci. The mediumcan be used with the M.P.N. technique to enumerate enterococci infood. This broth is identical to LAB 106 K.A.A. agar with theomission of the agar.

Formula g/litre

Tryptone 20.0

Yeast Extract 5.0

Sodium chloride 5.0

Sodium citrate 1.0

Aesculin 1.0


Sodium azide 0.15

Kanamycin sulphate 0.02

Method for reconstitutionWeigh 33 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, warm gently to dissolve completelythen disperse into tubes or bottles. Sterilise by autoclaving at 121˚Cfor 15 minutes.

Appearance: Light straw, clear.

pH: 7.0 ± 0.2

Minimum Q.C. organisms: E. faecalis NCIMB 50030E. coli (inhibition) NCIMB 50034


Inoculation: Inoculate tubes with decimal dilutions of foodsuspension.

Incubation: 37˚C or 42˚C aerobically for 18-24 hours.

Interpretation: Blackening of the medium suggests the presence ofenterococci/faecal streptococci.

ReferencesMossel, D.A.A., Bijken, P.H.G., Eelderink, I. and. van Spreekens.K.A. (1978). Streptococci, edited by Skinner, F.A. and Quesnel, L.B.SAB Symposium Series No. 7 Academic Press, London.

1.25

Kirchner’s T.B. MediumLAB 123

DescriptionThe cultivation of non-sputum specimens for mycobacteria requiresspecial attention. Specimens such as C.S.F., tissue or body fluidsobtained by surgical procedures are not easily repeated and maycontain small numbers of mycobacteria. It is important, therefore,that as large a portion of specimen as possible be inoculated intosuitable growth media.

Historically, such specimens were inoculated into guinea pigs;however improvements in culture methods have led to a situationwhere comparable results, can be obtained from the use of liquidculture media.

Selective Kirchner’s Medium is a liquid culture media, which is madeselective by the addition of an antibiotic cocktail, and has been shownto be sufficient in the recovery of mycobacteria from non-sputumspecimens.

Formula g/litre




Trisodium citrate 2.5

L-Asparagine 5.0

Phenol red 0.012

Method for reconstitutionWeigh 17.4 grams disperse in 1 litre deionised water, add 20mlGlycerol A.R. Allow the mixture to soak for 10 minutes, swirl to mixand sterilise by autoclaving at 121°C for 10 minutes. Allow themedium to cool and supplement with 1ml sterile heat in activatedhorse serum and 2 vials of X124, if required. Aseptically dispense in9ml volumes into sterile Macartney bottles.

Appearance: Pale red, clear fluid.pH: 7.0 ± 0.2

Minimum Q.C. organisms: M. tuberculosis(H37RV strain)E. coli (inhibition)NCIMB 50034Candida albicans.(inhibition) NCIMB 50010

Storage of Prepared Medium: up to 3 months at 2-8˚C in the dark.

Inoculation: Kirchner’s Medium is suitable for the cultivation ofmycobacteria from the following specimens:

Pre-treatment Required

Sputum, Urine & Faeces Concentration and acid oralkali decontamination.

C.S.F., Pus with no other bacteria None.

Tissue Disintegration followed byacid or alkali decontamination, if necessary.

The acidified or alkaline de-contaminated material may be addeddirectly to the Kirchner medium. The pH is then adjusted using theappropriate normal solution observing colour or indicator.

Although Kirchner’s Medium has been shown to give higherisolation rates than solid media, it is recommended that specimensshould also be inoculated onto Lowenstein Jensens slopes for thefollowing reasons: - Growth suitable for further characterisation maybe obtained more rapidly - Some Mycobacterium species includingM. intracellulare, M .kansasii and M. scrofulaceum may be inhibitedby the antibiotic cocktail.

Incubation: 37˚C aerobically for up to six weeks.

Growth Characteristicsorganism

M. tuberculosis white floccular ‘snowflake’ colonies

M. scrofulaceum- ‘ropey’ deposit

M. kansasii- fine granular deposit.

ReferencesKirchner, O., (1932). Erfahrungen bei diagnosticher Verwendung derTiefenkulter. Sbl fur Bact. Abt 1. Originale 124, 409-412.

Marks, J. (1972), Ending the routine guinea pig test. Tubercle (1972).53: 31-34.

Mitchison, D.A. Allen, B.W., Manickavasagar, D. (1983). SelectiveKirchner’s medium in the culture of specimens other than sputum formycobacteria. J.Clin. Path. 36: 1357-1361.

Kligler Iron AgarLAB 59

DescriptionA differential medium for the recognition of enteric pathogens bytheir ability to ferment glucose and/or lactose, and liberate sulphides.Fermentation liberates acid, with or without gas, turning phenol redindicator yellow. Fermentation of glucose only, is followed byreversion in pH on the slope, from initial acidity to final alkalinity(red colour), but not in the anaerobic conditions of the butt, whichremains acid (yellow). Fermentation of lactose as well as glucose,produces acidity in both slope and butt (yellow). Liberation ofsulphide results in the formation of iron sulphide (blackening ofeither slope or butt).

Formula g/litre

Peptone 20.0

Lactose 10.0

Glucose 1.0

Sodium chloride 5.0



Phenol red 0.025

Agar No. 2 12.0

Method of reconstitutionWeigh 49 grams of powder and mix with 1 litre of distilled water.Bring to the boil with frequent stirring to dissolve completely.Dispense into tubes and sterilise for 15 minutes at 121˚C. Cool in aslanted position such that slopes are formed over deep butts approx.3cm in depth.

Appearance: Reddish brown agar.

pH: 7.4±0.2

Minimum Q.C. Organisms Salmonella typhimuriumNCIMB 50076Pseudomonas aeruginosaNCIMB 50067

InoculationSubcultures for further identification are picked from the centre ofisolated colonies on selective media and streaked across the slant andstabbed deep into the butt of tubes of Kligler Iron Agar.


1.26

InterpretationOrganism Butt Slope Sulphide

Salmonella typhi Acid Alkaline +

S. paratyhi A + B Acid Alkaline -

Other Salmonella Acid/gas Alkaline +

E. coli Acid/gas Acid -

Proteus spp Acid/gas Alkaline +

Shigella sonnei Acid Alkaline -

S. flexneri Acid Alkaline -

Storage: Tightly capped containers - up to 3 months at 15-20˚C in thedark.

References:Kligler, I.J. (1917). A Simple Medium for the Differentiation ofMembers of the Typhoid - Paratyphoid Group. Am. J. Publ. Hlth,7:1042-1044.

Bailey, S.F. and Lacey, G.R. (1927). A modification of the KliglerLead Acetate Medium. J. Bact. 13:182-189.

Lactose BrothLAB 126

DescriptionA medium for the performance and confirmation of the PresumptiveTest for members of the coliform group in water and dairy products,recommended by the U.S.P.

Formula g/litre

Beef Extract 3.0

Gelatin Peptone 5.0

Lactose 5.0

Method for reconstitutionWeigh 13 grams of powder, disperse in 1 litre of deionised water, heatto dissolve then distribute into bottles with Durham tubes. Sterilise byautoclaving at 121˚C for 15 minutes.

Appearance: Straw coloured, clear.

pH: 6.9 ± 0.2



Inoculation: See methods for standard techniques.


Interpretation: Coliforms are presumptively identified by theirability to ferment lactose and produce gas within 48 hours at 35˚C.

ReferencesAmerican Public Health Association. (1975). Standard Methods forthe examination of water and waste water, 892. Washington. UnitedStates Pharmocopeia, XXI, 1985.

Liquid Baird-Parker MediumLAB 158

DescriptionDeveloped by Van Doorne et al in 1981, this medium is essentiallyBaird-Parker Agar LAB 085 without the agar and egg yolkcomponents. This medium was chosen for development to overcomethe problems of other selective enrichments and non-selectiveenrichments, in the isolation of Staphlococcus aureus.

Other selective broth media suffer from the potential of inhibiting sublethally damaged S. aureus cells where the salt content is greater than40g/l. Even Giolitti Cantoni Medium may be inhibitory to somestrains of S. aureus.

Non selective enrichments have been suggested, but are not ideal dueto potential inhibition of S. aureus by microbial antagonism in amixed bacterial population.

Liquid Baird-Parker Medium is the ideal solution for detecting lownumbers (<20/g) of S. aureus as it has been shown to give acceptableselectivity whilst being non-inhibitory to injured cells.

Formulation g/litre

Meat Peptone 8.0

Tryptone 2.0

Beef Extract 5.0

Yeast Extract 1.0


Glycine 12.0


Method for ReconstitutionDouble Strength Medium: Weigh 86 grams of powder and dispersein 1 litre of deionised water. Soak for 10 minutes, mix to dissolve anddispense 10ml amounts in tubes. Sterilise at 121˚C for 15 minutes.Before use, heat to 100˚C in a water bath or steamer for 15 minutesto remove oxygen, cool and add 0.2ml of 1% potassium telluritesolution to each tube.

Single Strength Medium: Weigh 43 grams of powder and proceedas above. When cool, add 0.1ml of 1% potassium tellurite solution toeach tube.

Appearance: Clear straw broth.

pH: 6.8 ± 0.2.

Minimum Q.C. organisms: S. aureus NCIMB 50080E. coli (inhibition) NCIMB 50034

Storage of prepared medium: Up to 1 month at 2-8˚C (withouttellurite added).

Inoculation: Double strength; 10ml of sample homogenate to eachtube. Single strength; 1ml of sample homogenate to each tube.

Incubation: 37˚C anaerobically for 48 hours. Anaerobic conditionscan be achieved using an anerobic jar (container caps must be loosefitting) or by overlaying the surface of the broth with 1cm of moltenagar or liquid parafin.

Interpretation: Culture tubes showing growth (blackening of themedium and turbidity) onto Baird-Parker Agar LAB 085.

ReferencesVan Doorne, H., Baird, R.M., Hendriksz, D.T., Van der Kreek, D.M.,Pauwels, H.P. (1981) Liquid Modification of Baird-Parker’s Mediumfor the Selective Enrichment of Staphylococcus aureus. Antonie VanLeeuwenhoek 47 267-278.

1.27

Listeria Enrichment BrothLAB 138

DescriptionA medium for the selective enrichment of food and environmentalsamples for Listeria spp. first described in 1987 by J. Lovett. Themedium offers more rapid enrichment than the low temperatureenrichment techniques. This medium is now recommended by theCommission of European Communities and the International DairyFederation for the examination of soft cheeses for Listeriamonocytogenes. The medium is incubated at 30˚C and utilisesacriflavine, nalidixic acid and cyclohexamide as selective agents.

Formula g/litre

Tryptone 17.0

Soy Peptone 3.0

Sodium chloride 5.0


Glucose 2.5

Yeast Extract 6.0

Method for reconstitutionWeigh 36 grams of powder and add to 1 litre of deionised water.Allow to soak for 10 minutes then swirl to mix and sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and add 2 vials ofX138 (X139/X539 can be used as an alternative) reconstituted in50% alcohol. Aseptically dispense into sterile tubes or bottles.


pH: 7.3 ± 0.2


Storage of Prepared Medium: Capped containers – up to 14 days at2-8˚C in the dark.

Inoculation: Add 25 grams of sample to 225mls of ListeriaEnrichment Broth and homogenise.

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

Subculture: After 24 and 48 hours onto Listeria Isolation Medium –LAB 122.

ReferencesLovett, J. Frances, D.W. Hunt, J.M. J. Food Protect. 50: 188-192.Bolton, F. J. Personal Communication Public Health Laboratory,Preston U.K.

Listeria Isolation Medium(Oxford Formulation)

LAB 122

DescriptionA selective identification medium for the isolation of Listeriamonocytogenes from food and clinical material. Columbia agar is thenutrient base to which selective inhibitors have been added. Lithiumchloride is used to inhibit enterococci and acriflavine to inhibit someGram negative and Gram positive species. Further selective agentsmay be added after autoclaving to increase the selectivity; these arecolistin, fosfomycin, cefotetan and cyclohexamide. Aesculin isincluded in the formula as a differential indicator. L. monocytogeneswill hydrolyse aesculin to aesculutin which reacts with the iron salt togive a black precipitate around the colonies.

LAB M’s formulation has been used to successfully isolate Listeriafrom such diverse products as chicken giblets and dairy cheeses. Theadvisability of using this medium at two levels of selectivity has beenrecognised.

Formula g/litre

Columbia Agar Base 41.0

Aesculin 1.0



Method for reconstitutionWeigh 57.5 grams of powder. Add to 1 litre of deionised water. Allowto soak for 10 minutes, swirl to mix then sterilise by autoclaving at121˚C for 15 minutes. Allow to cool to 47˚C, add 2 vials of selectivesupplement X122 (X123 can be used as an alternative), mix well andpour plates.

Appearance: Pale yellow, slightly opaque gel.

pH: 7.2 ± 0.2



Inoculation: Surface, streak out to single colonies. This medium ishighly selective, a heavy inoculum can be used.




L. monocytogenes 0.5-1.0 CV.E.G. Grey/ Black/brownGreen around colonies

diffusion

Gram-ve Bacilli No growth

Enterococci p.p. 0.5 CV.E.G. Black Usually no growth

ReferencesGarayzabal, J.F.F., Rodriguez, L.D., Boland, J.A.V., Cancelo, J.L.B.,Fernandez, G.S. (1986). Listeria monocytogenes dans le laitpasteurise. Can. J. Microbiol. 32: 149-150.

Donnelly. C.W., Gregory J. Baigent (1986). Method for flowcytometric detection of Listeria monocytogenes in milk. Appl. &Environ. Microbiol.Oct. 689-695.

Bolton, C.F.J. Preston P.M.L. Personal communication. Lovett, J.Francis, D.W. Hunt. J.M. (1987). Listeria monocytogenes in rawmilk: Detection, Incidence and Pathogenicity. Journ. Food Protect.Vol. 50. No. 3: 188-192.

Van Netten, P., Van de Van, A., Perales, I., Mossel, P.A.A. (1988). Aselective and diagnostic medium for use in enumeration of Listeriaspp. in foods. International Journal of Food Microbiology 6:187-198.

1.28

M17 AgarLAB 92

DescriptionA medium for the enumeration of lactic streptococci (Streptococcuslactis, Streptococcus cremoris, Streptococcus diacetylactis) in dairyproducts. The medium can also be used to investigate thebacteriophage susceptibilities of these organisms. Anotherapplication is for the enumeration of Streptococcus thermophilus inyoghurts.

Formula g/litre

Balanced Peptone 5.0

Soy Peptone 5.0

Yeast Extract 2.5

Beef Extract 5.0

Lactose 5.0

Sodium glycerophosphate 19.0


Ascorbic acid 0.5

Agar No. 2 15.0

Method for reconstitutionWeigh 57.2 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to boil todissolve agar before dispensing in 15ml aliquots. Sterilise byautoclaving at 115˚C for 20 minutes.

Appearance: Pale straw, translucent agar.

pH: 7.1 ± 0.2

Minimum Q.C. organisms: S. lactis


Inoculation: Pour plate technique.

Incubation: 30˚C for 48-72 hours for mesophilic streptococci, 37˚Cfor 48 hours for Streptococcus thermophilus.

Interpretation: Count all colonies. Streptococci form colonies of 1-2mm in diameter.

ReferencesTerzaghi, B.E. Sandine, W.E.. (1975). Improved medium for lacticStreptococci and their Bacteriophages. Appl. Microbiol. 29 No. 6 pp807-813.

MacConkey Agar(With Salt)

LAB 30

DescriptionA selective medium for the isolation of bile tolerant organisms fromfaeces, urine, sewage and foodstuffs. Bile-tolerant Gram positiveorganisms as well as Gram negative organisms will grow on thismedium. This formula is recommended by W.H.O. and other bodiesfor the examination of water and milk. Some strains of Proteus spp.will spread on this medium making interpretation difficult, for thisreason LAB 2 MacConkey Agar (without salt) may be preferred as itis less prone to this phenomenon.

Formula g/litre

Peptone 20.0

Lactose 10.0

Bile Salts 5.0

Sodium chloride 5.0

Neutral red 0.05

Agar No. 2 12.0

Method for reconstitutionWeigh 52 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and mix wellbefore pouring into Petri dishes.

Appearance: Pink/red, clear

pH: 7.4 ± 0.2





Growth Characteristicsapprox. size shape &


E. coli 2.0-3.0 CV.E.G. Red (ppt around colony)

K. aerogenes 2.0-4.0 CV.E.G. Pink/Red (mucoid)

Citrobacter spp. 2.0-4.0 CV.E.G. Pink/Red (ppt aroundcolony)

Proteus spp. 1.5-2.5 CV.E.G. Yellow(spreading)

Salmonella spp. 1.5-2.5 CV.E.G. Colourless

Shigella spp. 1.0-2.0 CV.E.G. Colourless(pink)

Transp.

S. aureus 0.5-2.0 CV.E.G. White/ (dependent onPink lactose

fermentationOrange and pigmentOpaque production)

Enterococcus spp. P.P.-0.5 CV.E.G. Pink/Deep

RedOpaque

P. aeruginosa 1.0-3.0 F.CR.D. Transp. (colonial-Pinkish variation)

ReferencesEnvironment Agency: The Microbiology of Drinking Water (2002).Methods for the Examination of Water and Associated Materials.

1.29

World Health Organisation (1971). International Standards forDrinking Water. 3rd Edn. W.H.O., Geneva.

Taylor, E.W. (1958). The Examination of Water and Water Supplies.7th Edn. Churchill, London.

Cruikshank, R. (1973). A Guide to the Laboratory Diagnosis andControl of Infection. Medical Microbiology. 12th Edn. Churchill.

MacConkey Agar(without salt)

LAB 2

DescriptionA medium first introduced by MacConkey in 1905 for the isolationand differentiation of lactose and non lactose fermenting entericbacteria. The medium has since been modified to improve therecovery of staphylococci and enterococci, it is used for culturing awide range of clinical material and has applications in food, waterand dairy bacteriology.

Formula g/litre

Mixed Peptones 20.0

Lactose 10.0

Bile 5.0

Neutral red 0.05

Agar No. 2 13.5

Method for reconstitutionWeigh 48.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. Cool to 47˚C and mix wellbefore pouring plates. Prior to inoculation, dry the surface of the agarby partial exposure at 37˚C.

Appearance: Pink/red, clear.

pH: 7.4 ± 0.2



Inoculation: Surface inoculation, streaking for single colonies.




E. coli 2.0-3.0 CV.E.G. Red (non lactosefermenting

yellow)

K. aerogenes 3.0-4.0 CV.E.G. Pink-Red (mucoid)

Proteus spp. 2.0-3.0 CV.E.G. Yellow fishy odour

Ps. aeruginosa 2.0-4.0 F.CR.D. Pink- characteristicYellow- odour if green(Green)

Shigella spp. 1.0-3.0 CV.E.G. Yellow

Salmonella spp. 2.0-3.0 CV.E.G. Yellow

S. aureus 0.5-1.0 CV.E.G. Pink- (lactose-negative)Orange

Enterococcus spp. 0.5 CV.E.G. Pink-Deep Red

ReferencesMacConkey, A.T. (1905) Lactose-fermenting bacteria in faeces.J.Hyg. (Camb), 5: 333-379.

MacConkey, A. T. (1908) Bile salt media and their advantages insome bacteriological examinations, J.Hyg. (Camb.), 8: 322-341.


World Health Organisation (1971), International Standards forDrinking Water, 3rd Edn. W.H.O., Geneva. Taylor, E.W. (1958). The Examination of Water Supplies, 7th Edn. Churchill, London.

MacConkey Agar No. 3LAB 45

DescriptionA modification recommended by the W.H.O. and the AmericanPublic Health Association for the isolation of Enterobacteriaceaefrom waters and sewage. The medium has been made more selectivethan MacConkey’s original formula by the use of crystal violet aswell as bile salts. Gram positive organisms will not grow on thismedium.

Formula g/litre

Peptone 20.0

Lactose 10.0


Sodium chloride 5.0

Neutral red 0.03

Crystal violet 0.001

Agar No. 2 15.0

Method for reconstitutionWeigh 51.5 grams of powder and add to 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise for 15minutes at 121˚C. Cool to 47˚C and pour into Petri dishes. Dry thesurface before inoculation.

Appearance: Pale red slight violet tinge.

pH: 7.1 ± 0.2

Minimum Q.C. organisms: E. coli NCIMB 50034Ent. faecalis (inhibition)NCIMB 50030

Storage of prepared medium: Plates – up to 7 days at 2-8˚C in the dark.





E. coli 3.0-4.0 CV.E.G. Red (red ppt around(D) colony)

Kleb. aerogenes 4.0-5.0 CV.E.G. Pink-red (mucoid)

Proteus spp. 3.0-4.0 CV.E.G. Pale- (fishy odour)yellow

Ps. aeruginosa 1.0-1.5 F.CR.D. Yellow-green

Shigella spp. 2.0-3.0 CV.E.G. Pale- (pink)(D) yellow

Staph. aureus No growth

Other Staphylococcus spp. No growth

Enterococcus spp. No growth

1.30

ReferencesAmerican Public Health Association (1950). Diagnostic Proceduresand Reagents. 3rd edn. A.P.H.A., New York.

American Public Health Association (1946). Standard Methods forthe examination of Water and Sewage. 9th edn. A.P.H.A., New York.

MacConkey Broth (Purple)

LAB 5

DescriptionThis medium is used in the detection and enumeration of faecalcoliforms (37˚C) and E. coli (44˚C). The replacement of neutral redused in the original formulation by bromocresol purple makes thecolour change caused by acid producing organisms easier to read.

Formula g/litre

Peptone 20.0

Lactose 10.0

Bile Salts 5.0


Method for reconstitutionWeigh 35 grams of powder, disperse in 1 litre of deionised water. Mixwell and dispense into tubes or bottles with inverted Durham tubes.Sterilise by autoclaving for 15 minutes at 121˚C. Prepare doublestrength broth (70g/l) if 50ml or 10ml amounts of inoculum are to beadded to equal volumes of broth. Prepare single strength broth (35g/l)if 1ml or 0.1ml amounts of inoculum are to be added to 10ml of broth.

Appearance: Purple, clear.

pH: 7.3 ± 0.2

Minimum Q.C. organisms: E. coli NCIMB 50034B. subtilis (inhibition)


Incubation: 37˚C aerobically for coliforms, 44˚C aerobically for E. coli. Use Durham tubes to detect gas production for E. coli.

Growth Indicators: Turbidity, gas production. Lactose-fermentingorganisms cause a colour change from purple to yellow.

ReferencesMinistry of Health (1937). Bacteriological Tests for Graded Milk,Memo 139/Foods. H.M.S.O., London.

Minister of Health, Public Health Laboratory Service WaterCommittee (1969). The Bacteriological Examination of WaterSupplies, 4th Edn. report No. 71. H.M.S.O., London.

World Health Organisation (1971). International Standards forDrinking Water, 3rd Edn, W.H.O., Geneva.

Malt Extract AgarLAB 37

DescriptionAn acidic medium which will support the growth of most yeasts andmoulds whilst inhibiting most bacteria. It was first described byThom and Church in 1926 in a study of Aspergillus spp. claiming thehigh carbohydrate content ensured rapid growth. Selectivity can beincreased by further lowering the pH with the addition, aftersterilisation, of XO37 Lactic Acid. It should be noted that excessheating of this medium together with its low pH can easily result inhydrolysis of the agar gel producing soft plates.

Formula g/litre

Malt Extract 30.0

Mycological Peptone 5.0

Agar No. 2 15.0

Method for reconstitutionWeigh 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 for10 minutes. If the addition of XO37 Lactic Acid is required thisshould be done after sterilisation. One 5ml vial of XO37 will lowerthe pH of 250ml of medium to 3.5-4.0. Cool to 47˚C before makingadditions and pouring plates.

Appearance: Pale brown/straw, clear.

pH: 5.4 ± 0.2 (if XO37 is added pH 3.5-4.0)

Minimum Q.C. organisms: Candida spp. NCIMB 50010

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.

Inoculation: Pour plate technique or surface streaking for singlecolonies.

Incubation: 25˚C aerobically for 5 days.



Candida albicans 4 CV.E.D. White

Candida krusei 10 F.CR.D. White

Penicillium notatum 25 Green (white/yellow -

velvet strain dependent)

Aspergillus niger 25 White (yellow/blackborder, centre)blackcentre

ReferencesGalloway, L.D. and Burgess, R. (1952). Applied Mycology andBacteriology, Leonard Hill, London. Thom and Church, 1926. The Aspergilli.

1.31

Malt Extract BrothLAB 159

DescriptionA liquid medium of low pH for the growth of yeasts and moulds,typically employed as part of sterility testing protocols for variousproducts. The high carbohydrate content of the medium ensures rapidgrowth of yeasts and moulds.

Formula g/litre

Malt Extract 17.0


Method for reconstitutionWeigh 20g of powder and disperse in 1 litre of deionised water. Allowto soak for 10 minutes, swirl to dissolve and dispense into finalcontainers. Sterilise by autoclaving at 115˚C for 10 minutes.

Appearance: Pale brown/straw, clear broth.

pH 5.4 + 0.2

Inoculation: Inoculate samples direct into tubes of brothaccording to the particular method being employed.

Incubation: 25˚C (or 37˚C) for up to 7 days aerobically,depending upon protocol used. Subculture turbid tubes onto solidmedia for identification of growth.

Minimum QC organism: Candida albicans NCIMB 50010Aspergillus niger NCIMB 50097

ReferencesGalloway, L.D. and Burgess, R. (1952) Applied Mycology andBacteriology, 3rd ed, Leonard Hill, London pp 54 & 57.

Mannitol Salt AgarLAB 7

DescriptionA medium for staphylococci which is selective because the highsodium chloride level inhibits most other species with the exceptionof halophilic vibrios. The majority of Staphylococcus aureus fermentmannitol producing yellow colonies, occasional strains of coagulasenegative staphylococci may also ferment mannitol. It is necessary toconfirm the identity of presumptive S. aureus colonies by othermeans e.g. coagulase, protein A, DN’ase, thermonuclease or latexagglutination. This medium can be made selective for methicillinresistant S. aureus by the addition of X207 (methicillin supplement).

Formula g/litre

Beef Extract 1.0



D-Mannitol 10.0

Agar No. 2 12.0

Phenol Red 0.025

Method for reconstitutionWeigh 108 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, mix by swirling. Sterilise byautoclaving for 15 minutes at 121˚C. Cool to 47˚C before pouringinto Petri dishes.

Appearance: Red, clear gel.

pH: 7.4 ± 0.2

Minimum Q.C. organisms: S. aureusE. coli (inhibition)


Inoculation Method: Surface plating, streak out for single colonies.




S. aureus 1.5-2.0 CV.E.G. BrightYellow

Other Staphylococci 1.0-1.5 CV.E.G. White or (some ferment

Yellow mannitol)

Enterobacteriaceae no growth

Vibrio spp. & other 1.5-4.0 various usually (yellow ifhalophiles Pink mannitol +ve)

ReferencesAmerican Public Health Association (1966). Recommended Methodsfor Microbiological Examination of Foods, 2nd Edn. (ed. J.M. Sharf)A.P.H.A. Washington.

Davis, J.G., (1959). Milk Testing 2nd edn, Dairy Industries, London.

Maximum Recovery Diluent(Peptone/Saline diluent)

LAB 103

DescriptionAn osmotically controlled solution which is an alternative to, and areplacement for, 1/4 strength Ringer’s LAB 100. The presence of alow level of peptone lessens the physiological shock normallyexperienced by bacterial cells when they are introduced to a diluentsuch as Ringer’s Solution. The level of peptone is such thatmultiplication of the organisms is not possible in the time in whichthe sample will be present in the diluent (1-2 hours). This formula isrecommended by ISO 6887: BS5763.

Formula g/litre

Peptone 1.0

Sodium chloride 8.5

Method for reconstitutionDissolve 9.5 grams of powder in 1 litre of deionised water, heatgently to dissolve then distribute into final containers. Sterilise byautoclaving at 121˚C for 15 minutes.

Appearance: Clear fluid.


ReferencesStraka, R.P. and Stokes, J.L. (1957). Rapid destruction of bacteria incommonly used diluents and its elimination. Appl. Microbiol. 5: 21-25.

ISO 6887. (1983). Microbiology-General guidance for thepreparation of dilutions for microbiological examination. BS5763Part 6. Preparation of dilutions.

1.32

Membrane Lauryl Sulphate BrothLAB 82

DescriptionThis medium superseded Membrane Enriched Teepol broth whenShell Chemicals withdrew Teepol 610 from sale. Sodium laurylsulphate was found to be an adequate reproducible substitute and thismedium is recommended for the enumeration of coliform andorganisms in water and sewage.

Formula g/litre

Peptone 39.0

Yeast Extract 6.0

Lactose 30.0

Phenol red 0.2

Sodium lauryl sulphate 1.0

Method for reconstitutionWeigh 76.2 grams of powder, disperse in 1 litre of deionised water.Distribute into screw cap containers and sterilise by autoclaving at115˚C for 10 minutes.

Appearance: Red, clear solution.

pH: 7.4 ± 0.2



Inoculation: E. coli and coliform counts should be made on separatesamples of water. The volumes should be chosen so as the number ofcolonies on the membrane lies between 10 and 100. With watersexpected to contain less than 1 coliform per ml, a sample of 100mlshould be filtered. The membrane filter should be placed faceupwards on a pad soaked in Membrane Lauryl Sulphate Broth, afterfiltration. These membranes should be incubated in a container whichdoes not allow evaporation to occur. Water tight metal containersplaced in an accurate water bath are required for incubation ofmembranes at 44˚C.

Incubation: E. coli 4 hours at 30˚C 14 hours at 44˚C; Coliforms 4hours at 30˚C 14 hours at 35˚C.

Interpretation: No colonies:- assume a nil count. Small colonies ofan intermediate colour:- return to incubation for a full period.

E. coli: Yellow-coloured colonies from membranes incubated at 44˚Cshould be subcultured to Lactose Broth LAB 126 and TryptoneWater, LAB 129 to confirm gas and indole production respectively,after 24 hours incubation at 44˚C.

Coliform organisms: Yellow colonies from membranes incubated at35˚C or 37˚C should be subcultured into Lactose Broth LAB 126.After 48 hours incubation at 37˚C a result should be obtainedregarding the production of gas.

Full details of the methodology can be found in The BacteriologicalExamination of Water Supplies 71, 1969.

ReferencesBurnham, N.P. (1967). Proc. Soc. Wat. Treat, Exam. 16:40.


Windle Taylor, E. (1961) Glutamic acid medium, 40th Ann Rep. Div.Water Exam. Met. Water Board London pp 18-22.

Milk AgarLAB 19

DescriptionAn approved formulation for the enumeration of micro-organisms inmilk, rinse waters and dairy products. With the addition of a further5 g/l Agar No. 1 the medium is suitable for the preparation of Roll-Tubes using established mechanical equipment. Also see Milk PlateCount Agar LAB 115. Milk Agar can also be used with the P-INCsupplement (X019, X219) for accelerated shelf-life determination ofdairy products, (see page 5.8).

Formula g/litre

Yeast Extract 3.0

Peptone 5.0

Antibiotic Free Skim Milk Powder 1.0

Agar No. 1 15.0

Method for reconstitutionWeigh 24 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 15 minutes, swirl to mix then sterilise for 15minutes at 121˚C. Cool to 45˚C before mixing with sample dilutions.

Appearance: White, opalescent gel.

pH: 7.2 ± 0.2




Incubation: Aerobically at 30˚C for 72 hours.

ReferencesMinistry of Health (1937). Bacteriological Tests for Graded Milk. Memo 139/Foods H.M.S.O., London. British Standard 4285:Methods of Microbiological Examination for Diary Purposes.

1.33

Milk Plate Count AgarLAB 115

DescriptionA medium recommended by the British Standards Institute and theInternational Organisation for Standardisation for the enumeration ofviable bacteria in milk and other dairy products.

Formula g/litre

Tryptone 5.0

Yeast Extract 2.5

Dextrose 1.0

Antibiotic Free Skim Milk Powder 1.0

Agar No. 1 10.0

Method for reconstitutionWeigh 19.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to the boil todissolve the agar. Allow to cool to 47˚C and dispense into suitablecontainers. Sterilise by autoclaving at 121˚C for 15 minutes.

Appearance: Pale cream, opalescent gel.

pH: 6.9 ± 0.2





Interpretation: Count all colonies. Calculate back to determineviable organisms per ml

ReferencesBritish Standards Institute. (1984). BS 4285 Section 1.2.International Organisation for Standardisation Draft InternationalStandard. (1982) ISO/DIS 6610. D.I.N. 10192.

Minerals Modified Glutamate MediumLAB 80A & LAB 80B

DescriptionThis medium was developed for use with the Most Probable NumbersTechnique (M.P.N.) for the enumeration of coliforms in watersupplies. The medium is an improved version of the chemicallydefined glutamic acid medium described by Gray in 1964. Theproduct is supplied in two parts because it has been shown thatseparating the sodium glutamate from the base improves its stability.

Formula g/litre

LAB 80a (double strength)

Lactose 20.0

Sodium formate 0.5

L-Cystine 0.04

L(-) Aspartic acid 0.048

L(+) Arginine 0.04

Thiamine 0.002

Nicotinic acid 0.002

Pantothenic acid 0.002

Magnesium sulphate (MgSO4.7H20) 0.2


Calcium chloride (CaCl2.2H2O) 0.02



LAB 80b

Glutamic acid (sodium salt) 12.7

Method for reconstitutionDouble strength: Dissolve 22.7 grams of base medium (LAB 80a)together with 12.7 grams of sodium glutamate (LAB 80b) in 1 litre ofdeionised water containing 5 grams of ammonium chloride eg BDHcat no. 27149. Dispense 10ml and 50ml volumes into tubes withinverted Durham tube.

Single strength: Dissolve in 11.35 grams of base medium (LAB 80a)together with 6.35 grams of sodium glutamate (LAB 80b) in 1 litre ofdistilled water containing 2.5 grams ammonium chloride. Dispense5ml volumes into tubes with inverted Durham tubes.

Sterilise by autoclaving for 10 minutes at 115˚C, alternatively heat to100˚C for 30 minutes on three successive days.

Appearance: Purple, clear solution.

pH: 6.7 ± 0.2



Inoculation: Use the Most Probable Number technique. With 10mland 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.


Interpretation Tubes showing the production of acid (medium turnsyellow) and gas in the Durham’s tube are considered presumptivepositive. Each presumptive positive tube should be subcultured toBrilliant Green Bile Broth LAB 51 with Durham tube and incubatedat 44˚C for 24 hours and examined for gas production. A tube of Tryptone Water LAB 129 should also be inoculated and incubatedat 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 evidenceof E. coli.

ReferencesGray, R.D. (1964). An improved formate lactose glutamate mediumfor the detection of Escherichia coli and other coliform organisms inwater. J. Hyg. Camb. 62: 495-508.

PHLS Water Sub-Committee. (1958). A comparison betweenMacConkey broth and Glutamic acid media for the detection ofcoliform organisms in water. J. Hyg. Camb. 56: 377-388.

PHLS Standing Committee on Bacteriological Examination of WaterSupplies. (1968). Comparison of MacConkey Broth, Teepol Brothand Glutamic Acid Media for the enumeration of Coliform organismsin water. J. Hyg. Camb. 66: 67-87.


1.34

M.L.C.B. Agar(Mannitol Lysine Crystal Violet Brilliant Green Agar)

LAB 116

DescriptionA medium for the selective isolation of Salmonella spp. (with theexception of S. typhi and S. paratyphi A) from food and faeces.Salmonella colonies are recognised by distinctive colonialappearance and H2S production and like the Bismuth Sulphite Agarof Wilson & Blair, this medium will detect lactose and sucrosefermenting strains. Some problems may occur with H2S negativestrains, eg S. pullorum, S. senftenberg, S. sendai and S. berta. Thismedium should not be used to detect S. typhi and S. paratyphi A, asthese strains are more susceptible to the brilliant green dye.

Formula g/litre

Yeast Extract 5.0

Tryptone 5.0

Meat Peptones 7.0

Sodium chloride 4.0

Mannitol 3.0

L-Lysine HCL 5.0




Crystal violet 0.01

Agar No. 2 15.0

Method for reconstitutionWeigh 49 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to the boil withfrequent agitation to completely dissolve the powder. Cool to 47˚Cand pour plates. DO NOT AUTOCLAVE OR OVERHEAT.

Appearance: Pale purple, translucent gel.

pH: 6.8 ± 0.2

Minimum Q.C. organisms: Salmonella spp. NCIMB 50076E. coli (inhibition) NCIMB 50034


Inoculation: Surface plating, streaking for single colonies.Inoculation can be carried out directly, or from enrichment broths.Because of the low selectivity of this medium the inoculum shouldnot be heavy, and it is recommended that this medium should be usedin conjunction with other more selective media.




Salmonella spp. 2.0-3.0 CV.E.G. Black

Salmonella spp. 2.0-3.0 CV.E.G. Pale(H2S negative)

Proteus spp. 1.0-2.0 CV.E.G. Grey brown

Shigella spp. mainlyinhibited

Citrobacter spp. 0.5-2.5 CV.E.G. Mainlyinhibitedpale may

have blackcentre

E. coli mainly inhibited

Klebsiella spp mainly inhibited

Gram positive organisms mainly inhibited

ReferencesInove et al. 66th meeting of the Japanese Vet. Medicine Society.

M.R.S. Agar(de Man, Rogosa and Sharpe Agar)

LAB 93

DescriptionThis medium was developed for the cultivation and enumeration ofLactobacillus spp. from various sources and is intended as asubstitute for Tomato Juice Agar. The medium is suitable for mostlactic acid bacteria. When acidified to pH 5.4 M.R.S. Agar can beused to enumerate Lactobacillus bulgaricus in yoghurts. Tween® 80,sodium acetate, magnesium and manganese sulphates act as growthstimulants.

Formula g/litre

Mixed Peptones 10.0

Yeast Extract 5.0

Beef Extract 10.0

Glucose 20.0


Sodium acetate 5.0

Ammonium citrate 2.0


Manganese sulphate 0.05

Tween® 80 1.08

Agar No. 1 15.0

Method for reconstitutionWeigh 70 grams of powder and add 1 litre of deionised water. Allowto soak for 10 minutes, swirl to mix. Heat to dissolve and sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and adjust pH ifthe acidified medium is required.

Appearance: Light amber, clear.

pH: 6.4 ± 0.2

Minimum Q.C. organisms: Lactobacillus acidophilus

1.35

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped containers – up to 1 month at 15-20˚C in the dark.

Inoculation: Surface, spread to cover surface, or use pour platetechnique.

Incubation: 25˚C microaerobically for 2-5 days.

Interpretation: Count all colonies exhibiting typical morphology.



Lactobacillus 0.5-2.5 F.E.G. White Roughacidophilus

Lactobacillus sake 0.5-1.0 F.E.G. White

Streptococcus lactis 0.5-1.5 CV.E.G. White

Lactobacillus bulgaricus 1.0-1.3 CV.E. G. White

Referencesde Man, J.C.,. Rogosa, M and Sharpe, M.E. (1960). A medium for thecultivation of lactobacilli. J. Appl. Bacteriol. 23: 130-135.

M.R.S. Broth(de Man, Rogosa and Sharpe Broth)

LAB 94

DescriptionA medium for the cultivation and enumeration of Lactobacillus spp.this product has the same formulation as LAB 93 M.R.S. agar withthe omission of agar. The medium can be used for confirmatory testson organisms isolated on M.R.S. agar. The medium can also be usedfor enumeration by the Miles and Misra technique.

Formula g/litre

Mixed Peptones 10.0

Yeast Extract 5.0

Beef Extract 10.0

Glucose 20.0


Sodium acetate 5.0



Tween® 80 1.08

Ammonium citrate 2.0

Method for reconstitutionWeigh 55 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then warm to completelydissolve solids. Dispense into suitable tubes or bottles then steriliseby autoclaving at 121˚C for 15 minutes.

Appearance: Light amber, clear.

pH: 6.4 ± 0.2

Minimum Q.C. organisms: Lactobacillus acidophilus


Inoculation: Either with suspect colonies from M.R.S. agar or withserial dilutions of test material.

Incubation: 25˚C microaerobically for 2-5 days.

Interpretation: For enumeration purposes count tubes showing signsof growth as positive.

Referencesde Man, J.C., Rogosa, M. and Sharpe, M.E. (1960). A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-135.

MSRV(Semi-solid Rappaport Medium)

LAB 150

DescriptionMSRV was developed in 1986 by De Smedt, Bolderdijk and Rappoldas a rapid means of Salmonella detection. The medium, based uponRappaport Vassiliadis broth, is inoculated directly from the pre-enrichment medium, in the centre of the plate. Motile organismsspread from the centre in the semi-solid agar, but non-salmonellas areinhibited by the selective agents.

After overnight incubation the use of polyvalent salmonella antiseraor a latex kit can confirm the presence of a Salmonella. Alternatively,a paper disc wetted with polyvalent H antiserum can be placed 1/3 ofthe way from the edge of the dish, and will signal the presence of aSalmonella by inhibiting the mobility of the organism around thedisc.

Using this medium De Smedt and Bolderdijk have reported thepossibility of detecting Salmonella in 24hrs (1987)

Formula g/litre

Tryptone 2.3

Meat Peptone 2.3


Sodium chloride 7.3



Malachite green 0.037

Agar No. 1 2.5

Method for reconstitutionWeigh 31.5 grams of powder and disperse in 1 litre of deionisedwater. Soak for 10 minutes, swirl to mix and bring to the boil. Coolto 47˚C. and add 2 vials of X150 novobiocin supplement (10mg/vial).Mix well before dispensing.

Appearance: Turquoise/blue, clear, soft gel.

pH: 5.2 0.2

Minimum Q.C organisms: Salmonella typhimuriumNCIMB 50076E. coli (inhibition) NCIMB 50034

Storage of prepared medium: Plates – up to 7 days at 4˚C.

Inoculation: From pre-enrichment broth (6-24hrs) adding 0.1ml tothe centre of the plate.

Incubation: 37˚C. or 42 ±0.5˚C. for 18-24 hours. Keep lid uppermostat all times.

Interpretation: A spreading growth indicates a Salmonella may bepresent, substantiated if a disc with polyvalent H antiserum has beenadded and is inhibiting the zone. This should be confirmed bysubculturing from the edge of the mobility zone onto XLD andbrilliant green agar and performing biochemical and serological tests.Direct latex agglutination may be carried out from the edge of themobility zone.

ReferencesDe Smedt, J.M. and Bolderdijk, R.F. (1987): ‘One Day Detection ofSalmonella from Foods and Environmental Samples by MobilityEnrichment’. Fifth International Symposium on Rapid Methods andAutomation in Microbiology and Immunology, Florence (1987).Brixia Academic Press.

1.36

De Smedt, J.M. and Bolderdijk, R.F., Rappold H. andLautenschlaeger, D. Rapid Salmonella Detection in Foods inMobility Enrichment on a Modified Semi-Solid Rappaport-Vassiliadis Medium. Journal of Food Protection 49 510-514. (1986).

De Smedt, J.M. and Bolderdijk, R.F. Dynamics of SalmonellaIsolation with Modified Semi-Solid Rappaport-Vassiliadis Medium.Journal of Food Protection 50 658-661. (1987).

De Smedt, J.M. and Bolderdijk, R.F. Collaborative Study of theInternational Office of Cocoa. Chocolate and Sugar Confectionery onthe Use of Mobility Enrichment for Salmonella Detection in Cocoaand Chocolate. Journal of Food Protection 53 659-664. (1990).

Goossens, H., Wauters, G., De Boeck, M., Janssens, M., and Butzler,J.P. Semi-solid selective mobility enrichment medium for isolation ofSalmonella from faecal specimens J. Clin. Microbiol 19 940-941.(1984).

Mueller Hinton Agar IILAB 39

DescriptionA medium for antimicrobial sensitivity testing by the disc diffusionmethod. This medium, used in the technique of Bauer and Kirby, hasbeen adopted by the National Committee for Clinical LaboratoryStandards (NCCLS) in the USA as the definitive method forsusceptibility testing. The medium has a very low thymine andthymidine content, making it suitable for trimethoprim andsulphonamide testing, controlled to ensure correct zone sizes withaminoglycoside and tetracyline antibiotics. The medium wasoriginally formulated as a heat labile protein free medium for theisolation of pathogenic Neisseriaceae.

Formula g/litre

Beef Extract 2.0


Starch 1.5

Agar No. 1 17.0

Calcium ions 50-100mg/litre

Magnesium ions 20-35mg/litre

Method for reconstitutionWeigh 38 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise at 121˚C for15 minutes. Cool to 47˚C, mix well and pour plates.


pH: 7.3 ± 0.1

Minimum Q.C. organisms: E. coli ATCC 25922S. aureus (antibiotic sensitivityzones) ATCC 25923


Inoculation: Surface, inoculum as described by N.C.C.L.S.

Incubation: As recommended by methodology for particularorganisms and antibiotics by NCCLS.

ReferencesMueller, J.H. and Hinton, J. (1941). Protein-free medium for primaryisolation of gonococcus and meningococcus. Proc. Soc. Exp. Biol.and Med., 48: 330-333.

Goodale, W.I., Gould, G. and Schwab, L. (1943). LaboratoryIdentification of sulphonamide resistant gonococcic infection. J.Am.Med. Ass., 123: 547-549.

American Public Health Association. (1950). Diagnostic Proceduresand Reagents. 3rd edn., A.P.H.A., New York.

NCCLS. (1986). Performance standards for antimicrobialsusceptibility testing – second informational supplement.

Mueller Hinton Broth IILAB 114

DescriptionThis medium is the broth version of Mueller Hinton Agar. It is anantagonist free medium for use in the tube dilution technique for thedetermination of antibiotic M.I.C. values. The medium is carefullystandardised to meet N.C.C.L.S. standards for antimicrobialsusceptibility tests on bacteria which grow aerobically.

Formula g/litre

Beef Extract 2.0


Starch 1.5

Calcium ions 50 mg/litre

Magnesium ions 20 mg/litre

Method for reconstitutionWeigh 21 grams powder, disperse in 1 litre distilled water. Allow tosoak for 10 minutes, swirl to mix then heat gently to dissolve.Distribute into tubes or bottles, and sterilise at 121˚C for 10 minutes.


pH: 7.4 ± 0.2

Minimum Q.C. organisms: S. aureus ATCC 25923E. coli ATCC 25922(M.I.C. values)


Inoculation: Standard inocula are required. As described by NCCLS.

Incubation: As recommended by methodology for particularorganisms and antibiotics by NCCLS.

ReferencesMacFaddin, J. (1985). Media for isolation cultivation, identificationmaintenance of medical bacteria. Williams & Williams, Baltimore.

N.C.C.L.S.-M7-A. (1985). Methods for dilution antimicrobiolsusceptibility tests for bacteria that grow aerobically. ApprovedStandard.

1.37

Mueller Kauffman Tetrathionate BrothLAB 42

DescriptionA selective enrichment broth for salmonellae first described byMueller in 1923 then modified by Kauffman in 1935 with theaddition of Brilliant Green and Ox Bile increasing its selectivity.Organisms which can utilise tetrathionate, such as most Salmonella,flourish. However some salmonellas will be missed in this mediumeither because they are sensitive to Brilliant Green or cannot utilisetetrathionate (included in this group is S. typhi.) This medium is usedin the standard European Community Salmonella Isolation Procedureand in International Standards Organisation (ISO) Methods.

Formula g/litre

Tryptone 7.0

Soy Peptone 2.3

Sodium chloride 2.3

Calcium carbonate 25.0

Sodium thiosulphate anhydrous 40.7

Ox Bile 4.75

Method for reconstitutionWeigh 82 grams of powder, disperse in 1 litre of deionised water.Bring to the boil and cool to below 45˚C. Prior to use, add 30ml 1Niodine solution (or 19ml of a solution containing iodine 20g,potassium iodine 25g, distilled water 100ml) and 9.5ml of brilliantgreen 0.1% w/v solution. Mix well and dispense into sterilecontainers, keeping the chalk in suspension.

Appearance: Green, turbid solution which precipitates on standing.

pH: 7.8 ± 0.2


Storage of Prepared Medium: Capped container – up to 7 days at2-8˚C in the dark.

Inoculation: 1 part sample to 9 parts medium or 1 part pre-enrichment medium to 10 parts medium.


Subculture: Subculture onto selective agars. e.g. B.G.A. LAB 34,XLD LAB 32, M.L.C.B. LAB 116.

ReferencesMueller. (1923). Un nouveau milieu d’enrichissment pour larecherche du bacille typhique et des paratyphiques C.R. Soc. Biol(Paris) 89: 434. (1975).

International Organisation for Standardisation. Meat and meatproducts. Detection of salmonellae (Reference method).

ISO 3565 (E). International Organisation for StandardisationMicrobiology-1981 General guidance on methods for the detection ofSalmonella. ISO 6579(E).

International Organisation for Standardisation. Milk and milkproducts- 1985. Detection of Salmonella. ISO 6785(E).

Edel, W. and Kampelmacher, E.H. (1968). Comparative studies onSalmonella isolations in eight European laboratories, Bull. Wld. Hlth.Org. 41: 297-306.

Kauffman, F., (1935). Weitere Erfahrungen mit dem kombiniertenAnreicherungsverfahren fur Salmonellabacillen. Z. Hyg. 117: 26-32.

van Leusden, F.M., van Schothorst, M. and Beckers, H.J. (1982). Thestandard Salmonellae isolation method. In: Isolation andidentification methods for food poisoning organisms, edited byCorry, J.E.L., Roberts, D. and Skinner, F.A. SAB Technical Series,No. 17 35-49. Academic Press, London.

Nu Sens AgarLAB 74

DescriptionThis is a sensitivity test medium developed by LAB M in response torequests for a nutritionally rich sensitivity medium. This medium willgrow all organisms not having a specific need for blood, givingreproducible zone sizes. It is composed of specially selected peptoneswith a small amount of glucose, solidified with a very pure agar andis free from antagonists.

Formula g/litre


Peptone 7.5

Beef Extract 5.0

Sodium chloride 5.0

Glucose 2.0

Agar No. 1 15.0

Method for reconstitutionWeigh 36.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and pour plates.

Appearance: Transparent, almost colourless.

pH: 7.4 ± 0.2

Minimum Q.C. organisms: S. aureus NCTC 6571E. coli NCTC 10418(antibiotic sensitivity zones)


Inoculation method: Joan Stokes method, surface inoculum for semiconfluent growth, or breakpoint technique.


ReferencesEricsson, H.M., Sherris, J.C. (1971). Antibiotic sensitivity testing.Report of an international collaborative study. Acta. Pathol.Microbiol. Scand. Suppl. 217: 1-90.

Garrod, L.P. and Waterworth, P.M. (1969). Effect of mediumcomposition on the apparent sensitivity of Pseudomonas aeruginosato gentamicin. J. Clin. Pathol. 22: 534-538.

1.38

Nutrient AgarLAB 8

DescriptionA general purpose medium for the cultivation of organisms that arenot demanding in their nutritional requirements e.g. organisms thatcan be isolated from air, water, dust etc. Nutrient Agar is suitable forteaching and demonstration purposes, it is isotonic and can beenriched with biological fluids such as sterile blood and egg yolk.

Formula g/litre

Peptone 5.0

Beef Extract 3.0

Sodium chloride 8.0

Agar No. 2 12.0

Method for reconstitutionWeigh 28 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. Cool to 47˚C, mix well thenpour plates.

Appearance: Buff, opalescent gel.

pH: 7.3 ± 0.2


Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped containers – up to 3 months at 15-20˚C in the dark.

Inoculation: Surface streaking for single colonies.

Incubation: Temperature and time to suit organisms. Usuallyaerobic.



S. aureus 1.0-2.0 CV.E.G. White-Yellow

other Staphylococcus 0.5-2.0 CV.E.G. White-spp. Yellow

Strep. pyogenes P.P.-0.5 CV.E.G. Transp.

E. coli 1.5-2.5 CV.E.G. Grey

Proteus spp. spreading - Grey fishy odour

Klebsiella spp. 2.0-4.0 CV.E.G. Grey mucoid

Bacillus spp. 2.0-6.0 various Grey may spread

Ps. aeruginosa 2.0-4.0 F.CR.D. Grey-Green odour ifpigmented

Nutrient Broth No. 2 B.P.LAB 14

DescriptionA general purpose broth which can be used for sterility testing foraerobic organisms as recommended by the British Pharmacopoeia.This broth can also be used as the suspending medium for cookedmeat granules for the cultivation of anaerobic organisms.

Formula g/litre

Beef Extract 10.0


Sodium chloride 5.0

Method for reconstitutionWeigh 25 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then dispense into tubesor bottles, and sterilise for 15 minutes at 121˚C.


pH: 7.3 ± 0.2




ReferencesBritish Pharmacopoeia. (1973). H.M.S.O., London. Cruikshank, R.(1972). Medical Microbiology. 11th edn. Livingstone, London.

Nutrient Broth “E”LAB 68

DescriptionAn inexpensive broth for the growth of nutritionally non-demandingorganisms. Ideal for teaching purposes.

Formula g/litre

Beef Extract 1.0

Yeast Extract 2.0

Peptone 5.0

Sodium chloride 5.0

Method for reconstitutionWeigh 13 grams of powder, add to1 litre of deionised water. Heat todissolve then dispense into bottles or tubes. Sterilise by autoclavingat 121˚C for 15 minutes.


pH: 7.4 ± 0.2



Inoculation and incubation: To suit chosen organism.

Growth indicator: Turbidity.

1.39

O157 Broth MTSB(Modified Tryptone Soy Broth)

LAB 165

DescriptionModified tryptone soy broth has emerged as the medium of choice forthe enrichment of E. coli O157:H7 in red meats1,2. As concernregarding this organism has grown due to the severity of the diseasesyndromes caused, and the increase in foodborne infection3, so too hasthe need to optimise methods for its efficient isolation. Symptomsstart with severe stomach cramps and watery, bloody diarrhoea, and apercentage of individuals infected will develop Haemolytic UraemicSyndrome (HUS) leading to acute renal failure4. In a comparison of 4different selective broth media, MTSB was the most productive andselective for the isolation of E. coli O157:H7. MTSB is made selectivefor O157:H7 by including bile salts in the dehydrated medium, and theaddition of novobiocin supplement (X150).

Formulation g/litre

Tryptone 17.0

Sodium chloride 5.0

K2HPO4 4.0

Dextrose 2.5

Soy Peptone 3.0

Bile Salts No.3 1.5

Method for reconstitutionWeigh 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 for15 minutes. Cool to 47˚C and add 2 vials of Novobiocin supplementX150. Mix well and distribute aseptically into sterile containers.

Appearance: Clear straw broth

pH: 7.2 ± 0.2

Minimum QC organisms: E. coli O157:H7(non-toxigenic) NCTC 12900E. coli NCIMB 50034(inhibition)

Inoculation: Add 25g sample to 225ml of supplemented MTSB andhomogenise for 2 minutes.

Incubation: 42˚C aerobically for 24hrs. Subculture onto CT-SMAC(LAB161 plus X161) or SMAC-BCIG (HAL 6) and examine for non-sorbitol fermenting colonies and/or glucuronidase negativeorganisms. Some workers1 recommend the use of animmunomagnetic separation step after 6hrs incubation.

Interpretation: Turbidity in the broth indicates growth. All brothsshould be subcultured to selective media whether turbid or not.

References1) Bolton, E.J., Crozier, L., Williamson, J.K. (1995) Optimisation ofmethods for the isolation of Escherichia coli O157 from beefburgers.PHLS Microbiology Digest 12 (2) 67-70.

2) Willshaw, G.A., Smith, H.R., Roberts, D., Thirlwell, J., Cheasty, T., Rowe, B. (1993) Examination of raw beef products forthe presence of verocytotoxin producing Escherichia coli,particularly those of serogroup O157. J.Appl.Bacteriol. 75 420-426.

3) Sharp, J.C.M., Coia, J.E., Curnow, J., Reilly, W.J. (1994)Escherichia coli O157 infections in Scotland. J.Med.Microbiol 40 3-9.

4) Doyle, M.P. (1991) Escherichia coli O157:H7 and itssignificancein foods. Int.J.Food Microbiol. 12 289-302.

Orange Serum AgarLAB 147

DescriptionA medium developed for the investigation of organisms involved inthe spoilage of citrus products including fruit juices and citrusconcentrates. The low pH of these products restricts the growth oforganisms to those capable of tolerating an acid environment such asyeasts and moulds and bacteria belonging to the genera Bacillus,Lactobacillus, Leuconostoc, Streptococcus and Clostridium. Byhaving a low pH and incorporating orange extract, Orange SerumAgar is the ideal isolation medium.

Formula g/litre

Tryptone 10.0

Yeast Extract 3.0

Orange Extract 5.0

Glucose 4.0

Di-potassium phosphate 3.0

Agar No.2 7.0

Method for reconstitutionWeigh 42 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes. Bring to the boil swirling frequently,cool to 47˚C. and dispense into containers. Sterilise by autoclaving at115˚C. for 15 minutes.

Appearance: Amber, slightly opalescent gel.

pH: 5.5 ± 0.2

Minimum Q.C. organisms L. acidophilusPen. roquefortii

Storage of Prepared Medium: Plates – up to 7 days at 4˚C. in thedark. Capped containers – up to 3 months at 15-20˚C. in the dark.


Incubation: 3 days at 30˚C. for bacteria, 5 days at 30˚C for yeastsand moulds.

Interpretation: Count bacterial colonies and yeast/mouldsseparately. Calculate the colony forming units (C.F.U.’s) per ml of thesample allowing for dilution factors.

ReferencesHays, G.L. (1951) The isolation, cultivation and identification oforganisms which have caused spoilage in frozen concentrated orangejuice. Proc. Florida State Hort. Soc.

Hays, G.L. and Reister, D.W. (1952) The control of ‘off-odour’spoilage in frozen concentrated orange juice. Food Tech 6 p386.

Murdock, D.I., Folinazzo, J.F., and Troy, V.S. (1952) Evaluation ofplating media for citrus concentrates. Food Tech. 6 p181.

1.40

Oxytetracycline-Glucose-Yeast ExtractAgar Base(O.G.Y.E.)

LAB 89

DescriptionA selective medium for the enumeration of yeasts and moulds infood, introduced by Mossel in 1970. Unlike many selective media foryeasts O.G.Y.E. has a neutral pH and it has been shown to give betterrecovery rates than those media with a low pH. Oxytetracycline isused to inhibit bacteria, certain high protein foods may reduce theeffectiveness of this antibiotic as a selective agent. Rose BengalChloramphenicol LAB 36 is recommended in these instances.

Formula g/litre

Yeast Extract 5.0

Dextrose 20.0

Biotin 0.001

Agar No. 2 12.0

Method for reconstitutionWeigh 37 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 115˚C for 10 minutes. Cool to 47˚C and asepticallyadd two vials of X089 Oxytetracycline selective supplement. Mixthoroughly and pour into Petri dishes.

Appearance: Pale yellow, clear.

pH: 7.0 ± 0.2

Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097Saccharomyces cerevisiae

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark. Capped containers – up to 1 month at 4˚C in the dark.

Inoculation: Surface spreading or pour plate.




Candida spp. 3.0-4.0 CV.E.D. Cream

Candida krusei 7.0-8.0 F.CR.D. White

S. cerevisiae 3.0-4.0 CV.E.D. White

Pen. notatum 1.5 Green/blue centre

Pen. flavescens 1.5-2.0 Yellow centre

Aspergillus niger 3.0-5.0 Yellow with black centre

Rhizopus spp. confluent Aerial hyphae withblack conidia

Tetracycline resistant bacteria may grow, colonial morphologydependent upon species.

ReferencesMossel, D.A.A. et al. (1970). O.G.Y.E. for Selective Enumeration ofMoulds and Yeast in Foods and Clinical Material. J. Appl. Bact. 35:454-457.

Banks, J.G., Board, R.G. (1987). Some factors influencing therecovery of yeasts and moulds from chilled foods. Int. J. FoodMicrobiol. 4: 197-206.

Palcam Agar Base(Polymyxin, Acriflavine, Lithium chloride, Ceftazidine,Aesculin, Mannitol)

LAB 148DescriptionPalcam Agar was developed by Van Netten et al in 1989 as animproved selective differential medium for the isolation of Listeriamonocytogenes from food, clinical and environmental specimens.

Improved selectivity is achieved by the combination of antibioticsupplements and microaerobic incubation, whilst the double indicatorsystem of aesculin hydrolysis and mannitol fermentation aidsdifferentiation of Listeria spp from enterococci and staphylococciwhich can be confused with Listeria spp on other types of culturemedia.

Formula g/litre

Columbia Peptone Mix 23.0

Sodium chloride 5.0

Corn Starch 1.0

Yeast Extract 3.0

Glucose 0.5

Mannitol 10.0

Aesculin 0.8



Phenol red 0.08

Agar No. 2 12.0

Method for reconstitutionWeigh 71 grams of powder and disperse in 1 litre of deionised water.Soak for 10 minutes, swirl to mix then sterilise by autoclaving at121˚C for 15 minutes. Cool to 47˚C and add 2 vials of P.A.C.Supplement – X144. Mix thoroughly and pour into Petri dishes.

Appearance: Red, translucent

pH: 7.2 ± 0.2

Minimum Q.C. organisms L. monocytogenesNCIMB 50007E. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Plates – up to 7 days at 4˚C in the dark.

Inoculation: 0.1ml of sample selectively enriched in Palcam Broth(or other enrichment medium) spread over surface of plate.

Incubation: 30˚C aerobically or microaerobically for 24-48 hours.



L. monocytogenes 1.5-2.0mm F.E.D. Grey/green Black halo,(draughtsman

colonies).

Other Listeria spp. 0.5-2.0mm F.E.D. Grey/green Black halo,

(draughtsmancolonies).

Enterococci Inhibited - - (Small yellowcolonies withyellow/green

halo).

Staphylococci Inhibited - - (Small white/yellow colonies

with yellow/green halo)

Bacillus spp. Inhibited - - -

1.41

ReferencesVan 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(Liquid, Polymyxin, Acriflavine, Lithium chloride,Ceftazidime, Aesculin, Mannitol)

LAB 144

DescriptionDeveloped by Van Netten et al (1989) L-Palcam is a selectivedifferential medium for the enrichment of Listeria spp. in food,environmental and clinical samples. It is unique amongst Listeriaenrichment media in that it contains an indicator system (aesculin)which will signal the presence of a possible Listeria by abrowning/blackening of the broth; the result being the indication of apotential problem up to 48 hours before growth on plating media canbe observed.

Formula g/litre

Columbia Peptone Mix 23.0

Yeast Extract 5.0

Peptonised Milk 5.0

Sodium chloride 5.0

Mannitol 5.0

Aesculin 0.8


Phenol red 0.08


Method for ReconstitutionWeigh 54.4 grams of powder and disperse in 1 litre of deionisedwater. Soak for 10 minutes, swirl to mix and autoclave at 121˚C for15 minutes. Cool to 47˚C and add two vials of X144. Mix well anddispense into sterile tubes or bottles.

Appearance: Clear red broth

pH: 7.2 ± 0.2


Storage of prepared medium: Capped containers – up to 7 days at 4˚C.

Inoculation: Sample or pre-enriched sample added to the broth in theratio 1 : 10.

Incubation: 30˚C for 24 hours and 48 hours.

Subculture: Onto Palcam Agar – LAB 148. If low numbers ofListeria are present the medium may not produce the brown blackcolour. All tubes should be subcultured onto selective agar before asample is scored as negative.

ReferencesVan 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.

Peptone WaterLAB 104

DescriptionA general purpose growth medium that can be used as a base forcarbohydrate fermentation studies. The medium has a high level oftryptone making it suitable for use in the indole test. Adjustment ofthe pH to 8.4 makes the medium an enrichment broth for Vibrio spp.

Formula g/litre

Peptone 5.0

Tryptone 5.0

Sodium chloride 5.0

Method for reconstitutionWeigh 15 grams of powder, and disperse in 1 litre of deionised water.Allow to dissolve then distribute into final containers. Sterilise byautoclaving at 121˚C for 15 minutes. If sterile additions are to bemade to this medium e.g.: carbohydrates, the volume of water forreconstitution must be reduced accordingly. A pH indicator may beadded to detect acid production from carbohydrate utilisation.

Appearance: Clear, colourless.

pH: 7.2 ± 0.2



Inoculation: A light inoculum from a pure culture.

Incubation: According to organism.

ReferencesBergey’s Manual of Systematic Bacteriology, Vol. 1, (1984).Williams and Wilkins, Baltimore/London.

MacFadden, J.F. (1983). Biochemical Tests for the Identification ofMedical Bacteria, 2nd edn. Williams and Wilkins, Baltimore/London.

Perfringens Agar (O.P.S.P.)

LAB 109

DescriptionThis medium was developed by Handford in 1974 to overcome someof the problems associated with enumerating Clostridium perfringensin foods. The medium is buffered and utilises sodium metabisulphiteand liver extract as sources of H2S with ferric ammonium citrate asthe indicator. The medium is made selective with the addition ofX109 Sulphadiazine and X110 Oleandomycin/Polymyxinsupplements.

Formula g/litre

Tryptone 15.0

Yeast Extract 5.0

Soy Peptone 5.0

Liver Extract 7.0



Tris buffer 1.5

Agar No. 2 10.0

1.42

Method for reconstitutionWeigh 45.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes then sterilise by autoclaving at 121˚Cfor 15 minutes. Allow to cool to 47˚C before adding 2 vials each ofselective supplements X109 and X110.


pH: 7.3 ± 0.2

Minimum Q.C. organisms: C. perfringens NCIMB 50027E. coli NCIMB 50034(inhibition)

Storage of prepared medium: Use on day of preparation.

Inoculation: Pour plates, 1ml sample plus 9ml medium. When setoverlay with sterile medium.

Incubation: 37˚C anaerobically for 24 hours.

Interpretation: Count large black colonies, presumptively identifiedas C. perfringens.

ReferencesHandford, P.M. (1974). A new medium for the detection andenumeration of C. perfringens in foods. J. Appl. Bact. 37: 559-570.

Shahidi, S.A. and Ferguson, A.R. (1971). A new quantitative andconfirmatory medium for C. perfringens in food. Appl. Microbiol.21: 500-506.

Marshall, R.S., Steenberger, J.F. and McClung, L.S. (1965). Appl.Microbiol. 13: 559. A rapid technique for the enumeration ofC. perfringens.

Pharmacopoeia of culture media for food microbiology. (1987). Int.J. Food Microbiol. 5: 3: 240-241.

Plate Count AgarLAB 149

DescriptionA medium designed for use with the spiral plating system and othersurface inoculation techniques. The formula is equivalent to A.P.H.A.Plate Count Agar and is suitable for the determination of total viablecounts in food products by surface count and pour plate methods.

Formula g/litre

Tryptone 5.0

Yeast Extract 2.5

Glucose 1.0

Agar No. 2 12.0

Method for reconstitutionWeigh 20.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C then pour intoPetri dishes.

Appearance: Pale straw colour, clear.

pH: 7.0 ± 0.2

Minimum Q.C. organisms: S. epidermidis NCIMB 50082E. coli NCIMB 50034


Inoculation: Surface, or pour plate.

Incubation: 30˚C aerobically for 48 hours for aerobic mesotrophcount. 6˚C aerobically for 10 days for aerobic psychrotroph count.55˚C aerobically for 48 hours for aerobic thermotroph count.

Interpretation: Count all colonies or use spiral plating colony countequipment.

ReferencesReasoner, D.J., Geldreich, E.E. (1985) A New Medium for theEnumeration and Subculture of Bacteria from potable water. App &Env. Microbiol. Jan. 1985 p.1-7.

American Public Health Association (1985) Standard Methods for theEnumeration of Water and Wastewater. 16th Edition. AmericanPublic Health Association Inc. Washington D.C.


Plate Count Agar (A.P.H.A.)(Standard Methods Agar, Tryptone Glucose Yeast Agar)

LAB 10

DescriptionFormulated to A.P.H.A. specifications, this medium is used forestablishing total viable counts for aerobes in food, dairy and waterbacteriology. The product uses agar of very high gel strength in orderthat it can be used in pour-plate as well as surface inoculationtechniques. The product can be remelted prior to use although itshould not be held for a prolonged period in the molten stage.

Formula g/litre

Tryptone 5.0

Yeast Extract 2.5

Glucose 1.0

Agar No. 1 15.0

Method for reconstitutionWeigh 23.5 grams of powder, disperse in 1 litre of deionised water.Bring to the boil with frequent stirring to dissolve. Dispense intotubes and sterilise by autoclaving at 121˚C for 15 minutes. Cool to44-46˚C for not more than 3 hours prior to use.

ROLL-TUBES. Add an additional 10g/litre Agar No. 1 prior toreconstitution of the medium.

Appearance: Pale straw coloured, clear gel.

pH: 7.0 ± 0.2



Inoculation method: Pour plate technique or surface inoculation.

Incubation: 30˚C aerobically for 48 hours for aerobic mesotrophcount. 6˚C aerobically for 10 days for aerobic psychrotroph count.55˚C aerobically for 48 hours for aerobic thermotroph count.

Interpretation: Count all colonies and calculate the number oforganisms (or ‘colony forming units’ c.f.u.) per ml of sampleallowing for dilution factors.

ReferencesAmerican Public Health Association (1972). Standard Methods forthe Examination of Dairy Products. 13th edn. (ed. Hausler, W.J.)A.P.H.A., Washington.

American Public Health Association (1966). Recommended Methodsfor the Microbiological Examination of Foods, 2nd edn. (ed. Sharf,J.M.) A.P.H.A., Washington.

American Public Health Association (1976). Standard Methods forthe Examination of Water and Waste Water, 14th edn. (ed.. Franson,M.A) A.P.H.A., Washington.

1.43

Potato Dextrose AgarLAB 98

DescriptionPotato Dextrose Agar is recommended by the American PublicHealth Association for the enumeration of yeasts and moulds inexamination of dairy products, soft drinks, dried and frozen foods andother types of product. Depending on whether the medium is to beused as a selective or non-selective agar it can be used with orwithout acidification.

Formula g/litre

Potato Extract 4.0

Dextrose 20.0

Agar No. 1 15.0

Method for reconstitutionWeigh 39 grams of powder, disperse in 1 litre of deionised water, thensterilise at 121˚C for 15 minutes. Mix well before pouring into sterilePetri dishes. In certain cases it may be desirable to lower the pH ofthe medium to 3.5 in order to suppress bacterial growth. This can bedone by adding 10ml of sterile 10% Lactic Acid X037, to one litre ofPotato Dextrose Agar LAB 98. This addition must be afterautoclaving and cooling to 47˚C. Once the pH has been lowered themedium may not be heated again without resultant loss of gelstrength caused by agar hydrolysis.

Appearance: Translucent white agar.

pH: 5.6 ± 0.2 (3.5-4.0 if X037 is added)

Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097Saccharomyces cerevisiae


Inoculum: Pour plate technique.




Candida spp. 2.0 C.V.E.D. White

Candida krusei 2.0 F.Rz.D. Grey/White

Tryc. mentagrophytes 4.0 Fluffy YellowWhite obverse

Tryc. verrucusum 5.0 Fluffy YellowWhite obverse

Toro. glabrata 3.0 C.V.E.G. White

Asp. niger 4.0 Black spores Yellowcentre obverse

White surround

Pen. notatum 4.0 Green spores Greencentre

White surround obverse

ReferencesAssociation of Official Analytical Chemists (AOAC). BacteriologicalAnalytical Manual, 5th ed. (1978). Washington D.C. Hausler, W.J. (ed.).

Standard Methods for the Examination of Dairy Prod. 14th edn.,Washington D.C.: American Public Health Association, (1976).

Pseudomonas Agar Base (C.F.C./C.N. Agar)

LAB 108

DescriptionThe base medium is a modification of King’s medium A which usesmagnesium and potassium salts to enhance production of thepigments pyocyanin (green) and fluorescein (detected by U.V./bluelight). The medium is made selective for Pseudomonas aeruginosaby the addition of X107 C.N. supplement. Alternatively the mediumcan be made selective for Pseudomonas species generally by theaddition of X108 C.F.C. supplement. This medium can be madeselective for the isolation of Burkholderia cepacia by the addition ofX140.

Formula g/litre


Gelatin Peptone 16.0

Potassium sulphate 10.0


Agar No. 2 11.0

Method for reconstitutionWeigh 48.4 grams of powder and disperse in 1 litre of deionisedwater. Add 10ml of glycerol. Sterilise by autoclaving at 121˚C for 15minutes. Allow the medium to cool to 47˚C then add the contents of2 vials of either X107 C.N. supplement or X108 C.F.C. supplement.Mix well and pour into Petri dishes.

Appearance: Pale straw, opaque.

pH: 7.1 ± 0.2

Minimum Q.C. organisms: P. aeruginosa NCIMB 50067E. coli (inhibition) NCIMB 50034


Inoculation: Surface, spread 0.1 to 0.5ml of sample over entiresurface.

Incubation: 25-30˚C aerobically for 48 hours.

Interpretation: Count all colonies as Pseudomonas species.Colonies that exhibit the pyocyanin and fluorescein pigments countas P. aeruginosa.


organism (mm) surface colour fluorescence

Ps. aeruginosa 2.0-3.0 CV.Cr.D. Green/Blue yes

P. fluorescens 2.0-3.0 CV.Cr.D. Yellow yes

P. fragi 1.0-3.0 CV.Cr.D. Grey no

P. maltophila 2.0-3.0 CV.Cr.G. Grey no

P. putida 2.0-3.0 F.Cr.D. Grey no

ReferencesBurton, M.O., Campbell, J.J.R. and Eagles, B.A. (1948). The mineralrequirement 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 mediafor the demonstration of pyocyanin and fluorescein. 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.

1.44

R2A MediumLAB 163

DescriptionR2A medium was developed to determine the bacterial count inpotable waters during treatment and distribution, and has been shownto give significantly higher counts than plate count agar (PCA) orsimilar high-nutrient media. The standard plate count (SPC) methodusing PCA provides an enumeration of bacteria which grow best at,or near, body temperature and this estimation at best may correlate tothe coliforms present in the sample. However, there will be apopulation of heterotrophic bacteria which cannot grow at all underthe conditions of the SPC method or may grow so slowly that thecolonies fail to reach a size detectable to the eye in the 48-hincubation period. In order to enumerate this section of the bacterialpopulation in water, a medium of low nutritional content (R2A) andextended incubation times are required. R2A medium isrecommended by the Environment Agency, Methods for theExamination of Waters and Associated Materials, and StandardMethods for the Enumeration of Water and Wastewater.

Formulation g/litre

Yeast Extract 0.5

Meat Peptone 0.5

Casamino acids 0.5

Glucose 0.5

Starch 0.5

Dipotassium hydrogen Phosphate 0.3


Sodium pyruvate 0.3

Agar No.2 15.0

pH: 7.2 + 0.2

Appearance: Clear opalescent gel

Method for reconstitutionWeigh 18 grams of powder and disperse in 1 litre of deionised water.Swirl to mix and sterilise at 121˚C for 15 minutes. (If required, bringto the boil to dissolve the agar, and pour into smaller volumes beforesterilising). Cool to 44-46˚C for not more than 3 hours before use.

Inoculation: Pour 15ml into a Petri dish containing 1ml of sample,mix well and allow to set. Pour a further 10ml as an overlay and againallow to set. Alternatively it may be used as a spread plate,inoculating 0.1ml onto the plate and spreading over the entire surfaceof the medium. It can also be used with membrane filters if required.

Incubation: When plates have set, incubate at 22˚C for 5-7 days or30˚C for 3 days. Other incubation temperatures between 20˚C and28˚C may be used.

Interpretation: Count all colonies and report the number of bacteriain the original sample as the heterotrophic plate count.

Minimum QC organisms: Pseudomonas fluorescens,Aeromonas hydrophila

ReferencesReasoner, D.J., Geldreich, E.E. (1985) A New Medium for theEnumeration and Subculture of Bacteria from potable water. App &Env. Microbiol. Jan. 1985 p. 1-7.

American Public Health Association (1985) Standard Methods for theEnumeration of Water and Wastewater. 16th Edition. AmericanPublic Health Association Inc. Washington DC.


Rappaport Vassiliadis (R.V.) SingleComponent Enrichment Broth

LAB 86

IntroductionRappaport Vassiliadis Broth (R10 modification) was born out of along series of experiments carried out to determine the correct levelsof malachite green and magnesium chloride that would allowSalmonella to multiply freely yet still inhibit the other entericorganisms.

This formulation has been shown to be superior to MuellerKauffmann and Selenite Broth for the isolation of Salmonella frommeat products.

The development work carried out on the formulation shows that it isextremely efficient in detecting small numbers of Salmonella inheavily contaminated products. This formulation is very hygroscopicand will produce a slight exothermic reaction when mixed with water.

Formula g/litre

Soy Peptone 4.5

Sodium chloride 7.2



Magnesium chloride anhydrous 13.58

Malachite green 0.033

Method for reconstitutionWeigh 26.8 grams powder, disperse in 1 litre of deionised water, swirlto mix, when dissolved dispense in 10ml volumes in screw cappedbottles and sterilise by autoclaving at 115˚C for 15 minutes.

Appearance: Clear, green fluid.

pH: 5.2 ± 0.2

Minimum Q.C. organisms: E. coli (inhibited) NCIMB 50034S. typhimurium NCIMB 50076

Storage of Prepared Medium: Capped container – 6 months at 2-8˚C

Inoculation: From pre-enrichment broth in the proportions of 1-partinoculum to 99 parts R.V. Broth. Sub-culture onto either XLD Agar,M.L.C.B. Agar or other salmonella selective agars.

Incubation: 41.5 ± 0.5˚C for 24 hours (incubator) or 42 ± 0.1˚C for24hrs (water bath).

ReferencesVassiliadis, P., (1983) The Rappaport Vassiliadis (R.V.) EnrichmentMedium for the Isolation of salmonellas: An overview J. Appl.Bacteriol. 56 69-76.

Vassiliadis, P., Mavromatti, CH. Efstratiou, M. and Chronas, G.(1985). A note on the stability of Rappaport-Vassiliadis EnrichmentMedium J. Appl. Bacteriol. 59 143-145.

Bolton, F.G., Preston, P.H.L. Personal communication.

Int. J. Food Micro. Pharmacopoeia of culture media for FoodMicrobiology.

Peterz, M., Wiberg, C., and Norberg, P. 1989. The effect of incubationtemperature and magnesium chloride concentration on growth ofSalmonella in home-made and in commercially available dehydratedRappaport-Vassiliadis broths.

1.45

Reinforced Clostridial AgarLAB 23

DescriptionThis is a solidified version of R.C.M. (LAB 22) and can be used forthe enumeration of anaerobes by pour plate, shake tube or membranefiltration methods. When solidified in tubes or bottles with minimalhead space it can be used for anaerobic culture without the need foranaerobic atmosphere.

Formula g/litre

Yeast Extract 3.0

Beef Extract 10.0

Peptone 10.0

Glucose 5.0

Soluble Starch 1.0

Sodium chloride 5.0

Sodium acetate 3.0

L-Cysteine hydrochloride 0.5

Agar No. 2 12.0

Method for reconstitutionWeigh 49.5 grams of powder, disperse in 1 litre of deionised water,allow to soak for 10 minutes, swirl to mix then sterilise for 15minutes at 121˚C. Cool to 47˚C. and distribute into sterile dishes ortubes containing decimal dilutions of the sample under test.

Appearance: Pale straw, translucent gel.

pH: 6.8 ± 0.2

Minimum Q.C. organisms: C. perfringens NCIMB 50027


Inoculation: Pour plate technique or tube culture.

Incubation: 30˚C for up to 72 hours. Anaerobic conditions for pourplate. Count as early as possible as prolonged incubation may resultin the medium being disrupted due to gas production.

Interpretation: Count all colonies as presumptive clostridia.

ReferencesMiller, N.J., Garrett, O.W. and Prickett, P.S. (1939). Anaerobictechnique – a modified deep agar shake. Food Res. 4: 447-451.

Ingram, M. and Barnes, E.M. (1956). A simple modification of thedeep shake tube for counting anaerobic bacteria. Lab. Pract. 5: 145.

Reinforced Clostridial Medium (R.C.M.)

LAB 22

DescriptionThis medium was formulated by Hirsch and Grinstead to recoversmall numbers of Clostridium spp. from a variety of sources. Variousworkers have reported its efficiency with many products andspecimens, R.C.M. is rich, non-selective and uses cysteinehydrochloride and glucose as reducing agents. The small amount ofagar reduces diffusion of oxygen through the fluid.

Formula g/litre

Yeast Extract 3.0

Beef Extract 10.0

Peptone 10.0

Soluble Starch 1.0

Glucose 5.0

L-Cysteine hydrochloride 0.5

Sodium chloride 5.0

Sodium acetate 3.0

Agar No. 1 0.75

Method for reconstitutionWeigh 38 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to the boil todissolve. Distribute 25ml into 1oz Universal containers. Sterilise for15 minutes at 121˚C.


pH: 6.8 ± 0.2



Inoculation: Homogenised food sample to a ratio of 1:10 with R.C.M.

Incubation: 30˚C for up to 72 hours.

Growth IndicatorsTurbidity, colonies in medium, gas production.

ReferencesHirsch, A. and Grinstead, E. (1954). Methods for the growth andenumeration of anaerobic spore-formers from cheese. J. Dairy Res.21: 101-110.

Barnes, E.M. and Goldberg, H.S. (1962). The isolation of anaerobicGram-negative bacteria from poultry. J. Appl. Bact. 25: 94-106.

1.46

Ringer’s Solution (1/4 strength)LAB 100 LAB 100Z (TABLETS)

DescriptionAn osmotically controlled solution for the preparation of suspensionsof food samples and for use as a diluent in dilution techniques forbacterial enumeration. The solution can also be used in the samplingof food production apparatus by the rinse and swab method.

Formula g/litre





Method for reconstitutionLAB 100 – Dissolve 2.5 grams of powder in 1 litre of deionisedwater, when completely dissolved dispense into containers asrequired and sterilise by autoclaving at 121˚C for 15 minutes.

LAB 100Z – Dissolve 1 tablet in 500ml of deionised water. Proceed as for LAB 100.

Ringer’s Solution (Calgon)LAB 101

DescriptionFor use with calcium alginate swabs. The sodium hexametaphosphatein the formula will enable the alginate swabs to completely dissolve.This ensures total release of all the organisms taken up by the swabto increase the accuracy of a quantitative bacterial count.

Formula g/litre





‘Calgon’ (Sodium hexametaphosphate) 10.0

Method for reconstitutionDissolve 12.5 grams of powder in 1 litre of deionised water. Dispense10ml amounts into screw cap containers and sterilise by autoclavingat 121˚C for 15 minutes.

Ringer’s Solution (Thiosulphate)LAB 102

DescriptionAn isotonic rinse for use in hygiene studies on plant and equipmentthat have been treated with chlorine disinfectants. 100ml of LAB MThiosulphate Ringer Solution will neutralise 7mg of chlorine.

Formula g/litre





Method for reconstitutionDissolve 2.8 grams of powder in 1 litre of deionised water. Dispenseinto containers as required then sterilise by autoclaving at 121˚C for15 minutes.

ReferencesMin. of Health, (1937). Bacteriological Tests for Graded Milk. Memo139/Foods. H.M.S.O. London. Davis, J.G. (1956). LaboratoryControl of Dairy Plant. Dairy Industries, London. Higgins, M.(1950). Comparison of recovery rate of organisms from cotton wooland calcium alginate wool swabs. Mon. Bull. Min. Health, P.H.L.S.,9: 50-51.

Rose Bengal Chloramphenicol Agar Base

LAB 36

DescriptionA selective medium for the enumeration of moulds and yeasts infoods. The original formulation of Jarvis (1973) usedchlortetracycline, this has been substituted by chloramphenicolbecause of superior selectivity. The Rose Bengal dye is taken up bythe growing colonies making them easier to see and inhibiting theirspreading. Rose Bengal becomes increasingly toxic on exposure tolight so it is important to store plates in the dark.

Formula g/litre


Dextrose 10.0



Rose bengal 0.05

Agar No. 2 12.0

Method for reconstitutionWeigh 28.5 grams of powder, disperse in 1 litre of deionised water,allow to soak for 10 minutes, swirl to mix then sterilise for 15minutes at 121˚C. Allow to cool to 47˚C then add 2 vials of X009(X209). Chloramphenicol (or 2 vials X089 oxytetracycline). Mixwell, then pour into Petri dishes. This medium should be protectedfrom light. Chloramphenicol may be added before autoclaving.

Appearance: Deep purple/red, clear.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097Sacchromyces cerevisiae E. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped Container – up to 1 month at 2-8˚C in the dark.

Inoculation: Surface spreading.

Incubation: 25˚C aerobically for 24 hours to 5 days.



Rhizopus spp. 13.5 Fluffy White

Mucor spp. 12.5 Fluffy White

Fusarium spp. 10 Fluffy White

Aspergillus flavus 8 Flat & Yellow/ hyphaehyphae Green

Candida spp. 1.5 CV.E.G. White

Sacchromyces spp. 1.0 CV.E.G. White

E. coli no growth

Ent. faecalis no growth

B. subtilis no growth

Staphylococcus spp. no growth

1.47

ReferencesJarvis, B. (1973). Comparison of an improvised Rose-BengalChlortetracycline Agar with other media for the selective isolationand enumeration of moulds and yeasts in food. J. Appl. Bact. 36: 723-727.

Overcast, W.W. and Weakley, D.L. (1969). An aureomycin rose-bengal agar for the enumeration of yeast and mould in cottage cheese.J.Milk and Fd.Tech. 32: 442-445.

Banks, J.G. Board, R.G. (1987). Some factors influencing therecovery of yeasts and moulds from chilled foods. Int. J. FoodMicrobiol. 4: 197-206.

Sabouraud Dextrose AgarLAB 9

DescriptionIntroduced by Sabouraud in 1910 as a selective medium for fungi andyeasts. The acidic pH (5.6) of this medium inhibits many species ofbacteria. The medium can be made more selective by the addition ofchloramphenicol supplement (X009) (X209). Diagnostic features,such as sporing structures and pigmentation are well developed onthis medium. Because of its low pH this medium is very sensitive tooverheating which will soften the agar and caramelise thecarbohydrate.

Formula g/litre


Dextrose 40.0

Agar No. 2 12.0

Method for reconstitutionWeigh 62 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. NOTE: The gel strength of themedium may diminish if recommended sterilising time ortemperature is exceeded.

Cool to 47˚C, mix well then pour plates.

Appearance: Buff opalescent gel.

pH: 5.6 ± 0.2

Minimum Q.C. organisms: Candida sp. NCIMB 50010E. coli (inhibition) NCIMB 50034


Inoculation method: Surface streaking for single colonies or stabmethod.

Incubation: Aerobically, yeasts 37˚C for 48 hours; fungi 25-30˚C forup to 3 weeks.



C. albicans 0.5-2.0 CV.E.D. White Yeasty smell

C. krusei 1.0-3.0 F.CR.D. Grey- Yeasty smellWhite

T. rubrum 25 White- Reverse-fluffy shades of

red

T. mentagraphytes 25 White- Reverse-fluffy yellow-

orange

M. canis 25 White- reverse-centre- yellow-yellowradial

E. floccosum 25 White- Reverse-fluffy tan

ReferencesSabouraud, R. (1910). Les Teignes Paris. Pagano. J., Levin, J.D. andTrejo, W. (1957-8). Diagnostic medium for the differentiation ofspecies of Candida. Antibiotics Annual, 137-143.

Sabouraud Liquid Medium U.S.P.(Fluid Sabouraud Medium)

LAB 33

DescriptionA liquid sterility test medium for the detection of yeasts, moulds andacidophilic bacteria in pharmaceutical products. This mediumconforms with the United States Pharmacopeia. It can also be used asa growth medium for the determination of fungistatic activity inpharmaceutical products.

Formula g/litre

Pancreatic digest of casein 5.0

Peptic digest of fresh meat 5.0

Glucose 20.0

Method for reconstitutionWeigh 30 grams of powder, disperse in 1 litre of deionised water,allow to soak for 10 minutes, swirl to mix. Heat to dissolve, thendispense into final containers before sterilising at 121˚C for 15minutes.

Appearance: Pale straw clear.

pH: 5.7 ± 0.2



Inoculation: As recommended in the U.S.P.

Incubation: 22-25˚C aerobically for 10 days.

ReferencesThe Pharmacopeia of the United States of America. XVII (1965).

Sabouraud Maltose AgarLAB 111

DescriptionThis is a modification of Sabouraud Dextrose Agar, substitutingmaltose for dextrose, recommended by the American Public HealthAssociation.

Formula g/litre

Balanced Peptone 10.0

Maltose 40.0

Agar No. 2 12.0

Method for reconstitutionWeigh 62 grams of powder, disperse in 1 litre of deionised water,allow to soak for 10 minutes, swirl to mix then autoclave at 121˚C for15 minutes. Do not overheat or the agar gel will be softened and thecarbohydrate will be caramelised. This medium may be madeselective by the addition of 2 ampoules X009 Chloramphenicolselective supplement which may be added either before or afterautoclaving. Cool to 47˚C and mix well before pouring plates.

Appearance: Cream/yellow, translucent.

pH: 5.6 ± 0.2

1.48



Inoculation: Surface streaking or stab inoculum.

Incubation: Aerobic; yeasts 37˚C for 48 hours; other fungi 25˚C forup to 3 weeks.



C. albicans 0.5-2.0 CV.E.D. White Yeasty smell

C. krusii 1.0-3.0 F.CR.D. Grey- Yeasty smellWhite

T. rubrum 25mm White- Reverse-fluffy shades of

Red

T.mentagraphytes 25mm White- Reverse-fluffy yellow-

orange

M. canis 25mm White- Reverse-centre yellow-yellow orangeradial

E. floccosum 25mm White- Reverse-fluffy tan

ReferencesSabouraud, R. (1910). Les Teignes. Paris. Pagano, J., Levin, J. D. andTrejo, W. (1957-8) Diagnostic medium for the differentiation ofspecies of Candida. Antibiotics Annual. 137-143.

Salt Meat Broth TabletsLAB 113Z

DescriptionAn enrichment medium for the isolation of staphylococci fromheavily contaminated samples. It can also be used for the isolation ofhalophilic micrococci which contaminate hides and raw salt.

Formula g/litre

Beef Extract 10.0

Meat Peptone 5.0

Tryptone 5.0

Cooked Meat Granules 30.0

Sodium Chloride 100.0

Method for reconstitutionAdd 2 tablets to 10ml of deionised water in a narrow container. Allowto soak for 15 minutes then sterilise by autoclaving at 121˚C for 15minutes.

Appearance: Straw broth over meat particles

pH: 7.6 ± 0.2

Minimum Q.C. organisms: S. aureus NCIMB 50080E. coli (inhibition) NCIMB 50034


Inoculation: Up to 1g of sample to 10ml of medium.

Incubation: 37˚C aerobically. Subculture at 24 and 48 hours.

Subculture: On to Baird-Parker Medium LAB 85, Mannitol SaltAgar LAB 7 or 4S agar LAB 84.

Selenite BrothLAB 44A & LAB 44B

DescriptionA medium for the selective enrichment of salmonellae from faeces,food and sewage. First described by Leifson in 1936 the medium is apeptone lactose broth, moderately buffered, which utilises sodiumbiselenite as a selective agent. This medium can be incubated atvarious temperatures from 35-43˚C to vary the selectivity.Subcultures should be performed after no more than 24 hoursincubation as there is an increasing loss of selectivity if incubation isprolonged.

Formula g/litre

Selenite Broth Base LAB 44A:

Peptone 5.0

Lactose 4.0

Sodium phosphate 10.0

Sodium biselenite LAB 44B:

Sodium hydrogen selenite 4.0

Method for reconstitutionDissolve 4 grams of sodium biselenite in 1 litre of cold deionisedwaer. Add 19 grams of Selenite Broth Base and warm to dissolve.Distribute into tubes or bottles and sterilise for 5-10 minutes in aboiling water bath, or by free steaming. DO NOT AUTOCLAVE.

Appearance: Pale orange/red with slight precipitate (overheatingwill cause excessive precipitate and loss of selectivity).

pH: 7.1 ± 0.2 (complete medium)



Inoculation: Approximately 0.5-1 gram of sample per 10ml tube.

Incubation: Up to 24 hours aerobically at 35-43˚C.

Subculture: Onto two or more selective agars.

ReferencesLeifson, E. (1936). New selenite enrichment media for isolation oftyphoid and paratyphoid (Salmonella) bacilli. Amer. J.Hyg. 24: 423-432.

MacFaddin, J.F. (1985). Media for the isolation, cultivation,identification of Medical Bacteria Vol 1. Williams and Wilkins,Baltimore.

1.49

WARNING: SODIUM BISELENITE

Toxic by inhalation and ingestion.Danger of cumulative effects. Causessevere burns. After contact with skinwash with water immediately. If youfeel unwell seek medical advice.

Selenite Cystine BrothLAB 55A & 44B

DescriptionThis formulation is a result of the investigation of North and Bartramin 1953. They examined the effect of varying concentrations ofcystine and phosphate on the recovery of salmonellae in egg productsusing selenite broth. It was found that the addition of 10micrograms/ml of cystine to Leifson’s selenite broth enhancedrecovery of salmonellae.

Formula g/litre

Selenite Cystine Broth Base LAB 55A


Lactose 4.0

Sodium phosphate 10.0

L-Cystine 0.01

Sodium biselenite LAB 44B

Sodium hydrogen selenite 4.0

Method for reconstitutionDissolve 4 grams of Sodium biselenite (LAB 44B) in 1 litre ofdeionised water. Add 19 grams of Selenite Cystine Broth Base andheat to dissolve. Distribute into tubes or bottles, and sterilise for 10minutes in a boiling water bath, or steamer. DO NOT AUTOCLAVETHIS MEDIUM.

Appearance: Pale straw colour, clear with slight precipitate. (A brickred precipitate indicates overheating).

pH: 7.0 ± 0.2



Inoculation: Add sample to broth in the ratio of 1:10. Use a pre-enrichment broth if damaged organisms are to be recovered.

Incubation: 37˚C for 24-48 hours aerobically. Subculture ontoSalmonella selective media.

ReferencesInternational Organisation for Standardization. Microbiology (1981).General guidance on methods for the detection of Salmonella. ISO, 6579-1981.

International Organization for Standardization. Milk and milkproducts (1985). Detection of Salmonella. ISO 6785-2985 (E).

ICMSF, (1978). Micro-organisms in foods. 1. Their significance andmethods of enumeration, 2nd edn., University of Toronto Press,Toronto, Ont.

Leifson, E. (1936). New selenite enrichment media for the isolationof typhoid and paratyphoid (Salmonella) bacilli. Am, J. Hyg. 24, 423-432.

North, W.R. and. Bartram, M.T. (1953). The efficiency of selenitebroth of different compositions in the isolation of Salmonella. Appl.Microbiol. 1, 130, 134.

Speck, M.L. (1984). Compendium of methods for themicrobiological examination of foods, 2nd edn., American PublicHealth Association.

Sensitivity Test Agar (S.T.A.)

LAB 12

DescriptionA medium formulated for antibiotic susceptibility testing by the JoanStokes technique. S.T.A. is inhibitor-free, very rich and includesvarious nucleotides to enable fastidious organisms to be tested. It isnecessary to add lysed or ‘chocolated’ blood for some organisms.

Formula g/litre

Peptone-Infusion Solids 21.5

Starch 0.6

Sodium chloride 5.0

Disodium citrate 1.0

Adenine sulphate 0.01

Guanine hydrochloride 0.01

Uracil 0.01

Xanthine 0.01

Aneurine hydrochloride 0.01

Uridine 0.1

Agar No. 2 12.0

Method for reconstitutionWeigh 40 grams of powder, disperse in 1 litre of deionised water,allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. To prepare blood agar cool to45˚C and add 7% lysed blood or 6% defibrinated blood according topreference. Mix well then pour plates.

Appearance: Dependent upon the blood additive.

pH: 7.4 ± 0.2

Minimum Q.C. organisms: S. aureus NCTC 6571E. coli NCTC 10418(antibiotic sensitivity zones)


Inoculation method: Surface, according to technique.

Incubation: 37˚C, atmosphere to suit organisms metabollicrequirements.

Interpretation: There are no defined zone sizes as in MuellerHinton, but all antibiotics should give adequate zone sizes whencompared to controls using standard organisms, e.g. S. aureus NCTC6571, E. coli NCTC 10418, Ps. aeruginosa NCTC 10662.

ReferencesStokes, E.J. (1968). Clinical Bacteriology 3rd edn. Arnold, London.Committee of the A.C.P. (1965). Report on the Antibiotic Sensitivitytest trial organised by the bacteriology committee of the Associationof Clinical Pathologists. J.Clin. Pathol., 18: 1-5.

Hanus, F.J. Sands, J.G. and Bennett, E.O. (1967). Antibiotic activityin the presence of agar. Appl. Microbiol., 15: 31-34.

Bechtle, R.M. and Scherr, G.H. (1958). A new agar for in vitroantimicrobial sensitivity testing. Antibiot. Chemother., 8: 599-606.

1.50

WARNING: SODIUM BISELENITE

Toxic by inhalation and ingestion.Danger of cumulative effects. Causessevere burns. After contact with skinwash with water immediately. If youfeel unwell seek medical advice.

Simmons Citrate AgarLAB 69

DescriptionA medium devised by Simmons in 1926 to help in the differentiationof enteric bacteria and in the isolation of fungi. CertainEnterobacteriacae have the ability to utilize citrate as the sole sourceof carbon and utilize inorganic ammonium salts as the sole source ofnitrogen resulting in an increase in alkalinity. Bromothymol Blue isused as a pH indicator.

Formula g/litre


Ammonium dihydrogen phosphate 1.0


Sodium citrate 2.0

Sodium chloride 5.0


Agar No. 2 15.0

Method for reconstitutionWeigh 24 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then heat to dissolve theagar and solids. Dispense into tubes or bottles then sterilise byautoclaving at 121˚C for 15 minutes. Allow to set as slopes.

Appearance: Green, opalescent.

pH: 6.9 ± 0.2

Minimum Q.C. organisms: E. coli NCIMB 50034C. freundii NCTC 9750


Inoculation: Streak on surface and stab into the butt.

Incubation: 37˚C aerobically for 24-48 hours, with loose caps toallow gaseous exchange.

Interpretation: Utilisation of citrate and ammonium salts results ingrowth and a change in colour of the medium from green to blue.

Growth Characteristicsorganism growth colour of medium

(most) Salmonella spp yes blue

S. typhi no green

S. cholerasuis no green

S. gallinarum no green

S. paratyphi A no green

Serratia spp. yes blue

Shigella spp. no green

Yersinia spp. no green

Klebsiella spp. yes blue

Proteus mirabilis yes blue

P. vulgaris yes blue

Providencia rettgeri no green

P. providenciae no green

E. coli no green

C. freundii yes blue

ReferencesSimmons, J.S. (1926). A Culture medium for differentiatingorganisms of typhoid - colon aerogenes groups and for isolation ofcertain fungi. J.Inf. Dis. 39: 209-215.

Koser, S.A. (1923). Utilisation of the salts of organic Acids by theColon-aerogenes group. J. Bact. 8: 493-520.

MacFaddin, J.F. (1983). Biochemical Tests for Identification ofMedical Bacteria. Williams and Wilkins.

Single Step Staphyloccocus SelectiveAgar (4-S medium)

LAB 84

DescriptionThis staphylococcal isolation and identification medium is based onthe ability of S. aureus to grow and produce lecithinase in thepresence of a high sodium chloride concentration and potassiumtellurite at 42˚C. The high temperature inhibits the production oflipase (which causes clearing of egg protein) allowing the effect oflecithinase (which causes opacity of egg protein) to be more easilydetected. For the recovery of heat stressed staphylococci a 3 hourpreincubation in Brain Heart Infusion Broth LAB 49 at 37˚C isrecommended.

Formula g/litre

Tryptone 4.0

Yeast Extract 3.0

Dextrose 10.0


Agar No. 2 13.0

Method for reconstitutionWeigh 80 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 48˚C and addaseptically 30ml X073 egg yolk emulsion and 1ml X027 3.5%potassium tellurite solution. Mix well then pour plates.

Appearance: Pale cream, opaque.

pH: 6.7 ± 0.2

Minimum Q.C. organisms: S. aureus NCIMB 50080S. epidermidis NCIMB 50082

Storage of Prepared Medium: Plates- up to 7 days at 2-8°C in the dark.

Inoculation: Surface spreading or streak out to single colonies.




S. aureus 0.5-1.0 CV.E.G. Grey (Opaque zone ofppt around colony)

S. epidermidis 0.5 CV.E.G. White/ (No ppt zone)grey

S. faecalis P.P. CV.E.G. Grey (Usually nogrowth)

Most other organisms are inhibited by this medium at 42˚C.

ReferencesMintzer-Morgenstern, L. and Katzenelson, E. (1982). A SimpleMedium for Isolation of Coagulase-Positive Staphylococci in aSingle Step. J. Food Protect. 45:3:218-22.

1.51

Slanetz and Bartley Medium(Membrane Enterococcus Agar)

LAB 166

DescriptionThis medium was originally described by Slanetz and Bartley for theenumeration of enterococci from water samples using a membranefiltration technique, but it may also be used as a spread plate for theexamination of other sample types. Enterococci reduce tetrazoliumchloride to the insoluble red dye formazan, producing colonies whichare dark red or maroon on the surface of the membrane or agar. Thisreaction is not exclusive to enterococci, and the count at this stageshould be considered presumptive. Colonies may be confirmed asenterococci by demonstrating aesculin hydrolysis using KanamycinAesculin Azide Agar LAB 106

Formula g/litre

Tryptose 20.0

Yeast Extract 5.0

Glucose 2.0


Sodium azide 0.4

2,3,4 Tetrazolium chloride 0.1

Agar 12.0

Method for reconstitutionWeigh 43.5 grams of powder and mix with 1 litre of deionised water.Bring to the boil with frequent stirring to dissolve completely. Coolto 47˚C and pour into sterile Petri dishes. DO NOT AUTOCLAVE,OVERHEAT, OR LEAVE FOR GREATER THAN 4hr AT 47˚C.

Appearance: Rose coloured gel.

pH: 7.2 ± 0.2

Mininmum QC organisms: Enterococcus faecalisNCIMB 50030 Escherichia coli NCIMB 50034 (inhibition).

Storage: Plates – upto 7 days at 2-8˚C. Storage in bottles is notrecommended as re-melting the medium will cause damage.

InoculationWater: Filter 100ml of the water through a suitable membrane, andplace this on the surface of a properly dried Slanetz and Bartley plate.

Other samples: Dilute as necessary and spread 0.5ml over the surfaceof the plate using a spreader, and allow to soak into the agar.

IncubationWater: at 37˚C for 48hr if testing potable waters or processed foods.At 37˚C for 4hr then 44˚C for 44hr if testing untreated waters or rawmaterials.

InterpretationCount all red and maroon colonies as presumptive enterococci.Confirmation of isolates can be achieved by demonstration of apositive aesculin reaction on KAAA LAB106.

Reference:Slanetz, L.W., and Bartley, C.H. (1957) J.Bact. 74 591-595.


Sorbitol MacConkey Agar(SMAC, CT-SMAC)

LAB 161DescriptionThis is a selective differential medium for the isolation ofEscherichia coli 0157:H7, the primary serovar associated withhaemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS).Pathogenicity of the organism is linked to the production ofverocytotoxins (VT1 and VT2), but it should be noted that not allstrains of O157:H7 produce verocytotoxins, and that strains fromother serovars can be toxin producers (e.g. O26, O111, O113, O145).

O157:H7 has been associated epidemiologically with food poisoningoutbreaks involving beefburgers and cold cooked meats. The mediumis a modification of MacConkey Agar No. 3 with the substitution ofthe fermentable carbohydrate from lactose to sorbitol. O157:H7 issorbitol negative and produces translucent colonies whereas mostother E. coli strains are sorbitol positive and so produce pink/redcolonies. Selectivity of the medium can be increased by addingCefixime-Tellurite (C.T.) supplement X161.

Formula g/litre

Peptone 20.0

Sorbitol 10.0

Bile salts no.3 1.5

Sodium chloride 5.0

Neutral red 0.03


Agar 12.0

Method for reconstitutionWeigh 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 byautoclaving at 121˚C for 15 minutes. Cool to 47˚C, add 2 vials of CTsupplement X161, and pour plates. Dry the surface prior toinoculation.

Appearance: Pale red, slight violet tinge.

pH: 7.1 + 0.2

Minimum QC organisms: E. coli O157:H7 (non-toxigenic)NCTC 12900 (translucent) E. coli NCIMB 50034 (Pink/red) Ent. faecalisNCIMB 50030 (inhibition)


Inoculation: Surface streak for single colonies.

Incubation: 37˚C aerobically for 18-24 hr.

Growth Characteristics:Organism Size (mm) Shape Colour

E. coli O157:H7 2.5 – 4.0 CV.E.G Translucent

Other E. coli 2.5 – 4.0 CV.E.G Pink/red

Sorbitol +ve organisms 2.5 – 5.0 Any Pink/red

References

Law, D., Ganguli, L.A., Donohue-Rolfe, A., Acheson, D.W.K. (1992) J. Med. Micro. 36 198-202.

Hitchins, A.D., Hartman, P.A., and Todd, E.C.D. (1992) in“Compendium of methods for the microbiological examination offoods” Ch.24. Published by American Public Health Association.

Varnam, A.H., Evans, M.G., (1991) Foodborne Pathogens anIllustrated Text. Published by Wolfe Publishing Ltd.

Riley, L.W. (1991) Ann. Rev. Micro. 41 383-408.

Riley, L.W. et al (1983) New Eng. J. Med 308 681-685.

Salmon, R.L., Farrel, I.D., Hutchinson, J.G.P. (1989) Epid. Inf. 103 249-254.1.52

S.S. Agar(Salmonella Shigella Agar)

LAB 52

DescriptionThis medium is a modification of Leifson’s DCA Medium firstdescribed in 1941 by Mayfield and Goeber shortly before Hynesdescribed his modification of DCA. The selectivity of the mediumwas increased by the addition of extra bile salts, sodium citrate andthe addition of brilliant green dye. There is also the extra thiosulphategiving good H2S production which reduces the ferrous ammoniumsulphide giving black centred colonies with H2S positive organisms.

The selectivity of this medium can be such that it was suggested byTaylor et al in 1965 to be unsuitable for the isolation of Shigellaspecies. Greater understanding of the selection mechanisms involvedenable us to adjust the reaction and allow the more delicate Shigellato grow without unduly impairing the medium’s selective properties.

Formula g/litre

Beef Extract 5.0


Lactose 10.0


Sodium citrate 8.5


Ferric citrate 1.0

Brilliant Green 0.00033

Neutral Red 0.025

Agar No. 2 13.5

Method for reconstitutionWeigh 60 grams of powder, disperse in 1 litre of deionised water, andallow to soak for 10 minutes. Swirl to mix, then bring to the boil, andallow to cool to 47˚C. Mix well then pour plates. Dry the surfacebefore incubation. DO NOT AUTOCLAVE THIS MEDIUM.

Appearance: Pale Pink, clear.

pH: 7.0 ± 0.2



Inoculation method: Surface plating, streaking for single colonies.




E. coli 0.1-2.0 CV.E.D. Red Red ppt aroundcolonies

(No growth)

K. aerogenes 0.1-3.0 CV.E.G. Pink-Red (No growth)

Proteus spp. 1.0-2.0 CV.E.G. Yellow (grey centre)(fishy odour)

Ps. aeruginosa 0.1-1.0 CV.CR.D. Pink (Green pigment)Yellow

Salmonella spp. 2.0-3.0 CV.E.G. Yellow (black centre)

Shigella spp. 0.5-2.0 CV.E.G. Pink-Yellow

Gram positive organisms – no growth.

ReferencesIsenberg, H.D., Kominos, S., and Siegel, M. (1969). Isolation ofsalmonellae and shigellae from an artificial mixture of fecal bacteria.Appl. Microbiol., 18: 4, 656-659.

Leifson, E. (1935). New culture media based on sodiumdesoxycholate for the isolation of intestinal pathogens and for theenumeration of colon bacilli in milk and water. J. Pathol. Bacteriol.,40: 581-589.

Taylor, W.I., Harris, B. (1965) Isolation of shigellae. II. Comparisonof plating media and enrichment broths. Am. J. Clin. Pathol. 44: 4,476-479.

Sugar Free AgarLAB 87

DescriptionA formula described by the International Dairy Federation for theenumeration of psychrotrophic and mesophilic Gram negative rods inbutter and other dairy products. The Gram negative rods are able todeaminate proteins as a carbon source, whilst some enterococci areinhibited by this formula. The medium conforms to the formulationof the International Dairy Federation (I.D.F.).

Formula g/litre

Gelatin Peptone 7.5

Tryptone 7.5

Sodium chloride 5.0

Agar No. 1 14.0

Method for reconstitutionWeigh 34 grams of powder and disperse in 1 litre of deionised water.Allow to soak for 10 minutes, boil to dissolve and disperse into tubesor flasks. Sterilise by autoclaving at 121˚C for 15 minutes.

Appearance: Light straw, clear.

pH: 7.6 ± 0.2



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 –aerobically.

Interpretation: Count colonies.

ReferencesInternational Dairy Federation (1964). International standard count of contaminating organisms in butter. International Standard FIL-IDF30.

Ritter, P. and Eschmann, K.H. (1966). Alimenta 5(2), 43-45. Thomas, S. B. (1969). J. Appl. Bact. 32, 269-296.

Mossel, D.A.A., Krol, B. and Moerman, P.C. (1972). Alimenta 11(2),51-60.

1.53

T.C.B.S. Cholera Medium(Thiosulphate Citrate Bile Salts Sucrose Agar)

LAB 96

DescriptionT.C.B.S. is designed for the selective isolation of Vibrio species,particularly V. cholerae. The formulation was developed byKobayashi, Enomoto, Sakazaki and Kuwahara and inhibits most ofthe Enterobacteriaceae for at least 24 hours. Therefore heavyinoculation of the medium is possible.

Formula g/litre

Yeast Extract 5.5

Peptone Mix 10.0


Sodium citrate 10.0

Bile salts 9.0

Sucrose 17.0


Ferric citrate 1.0


Thymol blue 0.04

Agar No. 1 15.0

Method of reconstitutionWeigh 88 grams of powder and add to 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to the boil. Coolto 47˚C and pour into Petri dishes. DO NOT AUTOCLAVE OROVERHEAT THIS MEDIUM.

Appearance: Dark green clear agar.

pH: 8.6 ± 0.2

Minimum Q.C. organisms: V. cholerae (type F)E. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Plates – up to 7 days at 4˚C in thedark. Capped containers – up to 1 month at 15-20˚C in the dark.

Inoculation: Surface plating with a heavy inoculum, streak out tosingle colonies.




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 orGreen

V. alginolyticus 3.0-5.0 CV.E.G. Yellow

V. metschnikovii 2.0-4.0 CV.E.G. Yellow

V. fluvialis 2.0-3.0 CV.E.G. Yellow

V. vulnificus 2.0-3.0 CV.E.G. Yellow

V. mimicus 2.0-3.0 CV.E.G. Green

Enterococci 1.0 CV.E.G. Yellow

Proteus spp. 1.0 F.CR.G. Green/Yellow

Ples. shigelloides P.P. Green

ReferencesKobayashi, T., Enomoto, S., Sakazaki, R. and Kuwahara, S. (1963).Jap. Bacteriol 18: 10-11, 387-391.

Furniss, A.L., Lee J.V. and Donovan, T.J. (1978). The Vibrios, PHLSMonograph No. 11.

Tetrathionate Broth Base A.P.H.A.LAB 97

DescriptionA selective enrichment broth for the growth of Salmonella typhi andother Salmonella spp. from faeces, foods etc. It conforms to theformulation recommended by the American Public HealthAssociation for use in the examination of dairy products and foodsfor salmonellae. Organisms which reduce tetrathionate, such assalmonellae, proliferate in the medium, whilst most enteric organismsare inhibited. Certain members of the Proteus group will also reducetetrathionate thereby impairing the performance of the medium insome cases. To overcome this, Novobiocin may be added to themedium at a level of 40 microgram/ml before addition of the iodine.Gram-positive organisms are inhibited by the inclusion of bile salts.

Formula g/litre


Bile Salts 1.0

Calcium carbonate 10.0


Method for reconstitutionWeigh 46 grams of powder and add to 1 litre of deionised water.Bring to the boil with frequent swirling to fully dissolve the medium.Cool to 45˚C and add 20ml of iodine solution prepared as indicatedbelow. Mix well before dispensing into bottles and continue swirlingwhilst dispensing to avoid the calcium carbonate sedimenting. Forthe best results the medium should be used the same day as prepared.

Iodine solution: Dissolve 5g of potassium iodide and 6g of iodinecrystals in 20ml of distilled water.

Appearance: Turbid white.

pH: 8.4 ± 0.2


Storage of Prepared Medium: Capped containers – up to 7 days at4˚C in the dark (without iodine solution).

Inoculation: Add 1 part of sample suspension or inoculated pre-enrichment medium to 9 parts of Tetrathionate Broth.

Incubation: 12-24 hours at 37˚C.

Subculture: Onto LAB 34 Brilliant Green Agar and either LAB 32XLD or LAB 110 Hektoen Enteric or other Salmonella selectivemedia.

ReferencesStandard methods for the Examination of Dairy products, 10thEdition. APHA, (1953).

1.54

Thioglycollate Medium (Brewer)LAB 64

DescriptionThis is the original formula introduced by Brewer in 1940 as a clearmedium for the cultivation of anaerobes. It has found applications asa sterility test medium and as a blood culture medium although it hasbeen superseded by Fluid Thioglycollate LAB 25 and FastidiousAnaerobe Broth LAB 71 for these purposes.

The agar makes the medium viscous slowing down the permeation ofoxygen and any convection currents. Sodium thioglycollate acts as areducing agent and also neutralises the bacteriostatic properties ofmercurial compounds. Methylene blue is a redox indicator which iscolourless at low Eh but turns green on exposure to oxygen.

Formula g/litre

Beef Extract 1.0

Yeast Extract 2.0


Dextrose 5.0

Sodium chloride 5.0


Methylene blue 0.002

Agar No. 1 1.0

Method for reconstitutionWeigh 20 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes then bring to the boil with gentleagitation to dissolve the solids. Distribute into screw top containersleaving minimal headspace. Sterilise by autoclaving at 121˚C for 15minutes. Tighten caps as soon as possible after autoclaving.

Appearance: Straw coloured, translucent, viscous liquid which mayhave a green surface due to contact with oxygen. If the medium hasa diffuse green tinge it should not be used until the oxygen has beendriven off by holding in a boiling water bath for 5 minutes. Do notreheat more than once.

pH: 7.2 ± 0.2



Inoculation: Ensure adequate dispersal of the inoculum in the broth.

Incubation: 37˚C for 24-72 hours.

Growth Indicators: A diffuse turbidity or individual colonies.

ReferencesBrewer, J.H. (1940). Clear liquid mediums for the culture ofanaerobes. J. Amer. Med. Ass. 115: 598-600.

Todd Hewitt BrothLAB 75

DescriptionA nutritious broth medium formulated by Todd and Hewitt for theproduction of antigenic streptococcal haemolysin. Todd Hewitt Brothis also used to cultivate streptococci prior to serological grouping.The use of a fermentable sugar in the formulation leads to theproduction of acid which would normally inactivate the haemolysin.This is prevented by the inclusion of buffers to maintain the pH of themedium thus preserving the haemolysin, as well as promoting thegrowth of pneumococci.

Formula g/litre

Infusion from fat-free minced meat 10.0

Tryptone 20.0

Dextrose 2.0


Sodium chloride 2.0

Disodium phosphate anhydrous 0.4

Method for ReconstitutionWeight 36.4grams of powder and disperse in 1 litre of deionisedwater. Allow to soak for 10 minutes, swirl to mix and warm todissolve. Dispense into 10ml volumes in screw capped containers andsterilise by autoclaving at 121˚C for 15 minutes.

Appearance: Pale straw, clear broth.

pH: 7.8 ± 0.2

Inoculation: Pick a well isolated colony for subculture into ToddHewitt Broth.

Incubation: 37˚C for 18-48hrs, aerobically.

Storage: Capped containers - up to 3 months at 15-20oC in the dark.

Minimum Q.C. Organisms Streptococcus pyogenesATCC 19615.

Reference:Todd, E.W., and Hewitt, L.F., (1932) A New Culture Medium for theProduction of Antigenic Streptococcal Haemolysin. J. Path. Bact. 35 (1) 973-974.

Updyke, E.,L., and Nickle, M.I. (1954) A Dehydrated Medium for thePreparation of Type Specific Extracts of Group A Streptococci. Appl.Microbiol. 2 117-118.

1.55

Triple Sugar Iron AgarLAB 53

DescriptionThis is a modification of the Krumwiede and Kohn medium of 1917which differentiates some of the Enterobacteriaceae on the basis offour reactions; fermentation of lactose, glucose and sucrose and H2Sproduction. This medium should be used in conjunction with a ureasetest to eliminate Proteus spp. when screening for Salmonella spp.

Formula g/litre

Beef Extract 3.0

Yeast Extract 3.0


Sodium chloride 5.0

Lactose 10.0

Sucrose 10.0

Glucose 1.0

Ferric citrate 0.3


Phenol red 0.025

Agar No. 2 12.0

Method for reconstitutionWeigh 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 withfrequent swirling to dissolve the solids. Distribute into tubes andsterilise at 121˚C for 15 minutes. Allow to set as a slope ensuring thatthe slant is over a butt approximately 3cm deep.

Appearance: Reddish-orange gel.

pH: 7.4 ± 0.2

Minimum Q.C. organisms: E. coli NCIMB 50034Ps. aeruginosa NCIMB 50067


Inoculation: A heavy inoculum is streaked over the surface of theslope and stabbed into the butt.


InterpretationSlant/butt Colour Utilisation

Alkaline/acid Red/yellow Glucose only fermented Peptones utilised

Acid/acid Yellow/yellow Glucose fermented Lactose + or sucrose fermented

Alkaline/alkaline Red/Red Neither glucose, lactose, nor sucrose fermented

Peptones utilised

Organism Butt Slant Sulphideproduction

Shigella dysenteriae NC

S. sonnei Acid or -

S. flexneri Alk.

Salmonella typhi Acid NC +

S. paratyphi Acid NC -S. cholerasuis Gas

S. typhimurium AcidS. enteritidis Gas NC +S. pullorum

S. gallinarum Acid NC +

E. coliEnterobacter Acid Acid -aerogenes GasE. cloacae

Proteus mirabilis Acid Acid +Gas

Providencia rettgeri Acid NC -

NC = No Change

ReferencesAmerican Public Health Association (1963). Diagnostic Proceduresand Reagents, 4th edn., A.P.H.A., New York.

American Public Health Association (1966). Recommended Methodsfor the Microbiological Examination of Foods. 2nd edn., A.P.H.A.,New York.

Edwards, P.R. and Ewing, W.H. (1962). Identification ofEnterobacteriaceae. Burgess Publishing Co., Minneapolis.

1.56

Tryptone Bile AgarLAB 72

DescriptionFirst introduced by Delaney, McCarthy and Grasso in 1962 as amethod for detecting faecal coliforms in water supplies based on theproduction of indole on a bile medium at 44˚C. The idea was appliedto foodstuffs by Anderson and Baird-Parker in 1975. The inoculum isplaced onto the membrane on a resuscitation agar and incubated at37˚C for 4 hours. The membrane is then transferred to a TryptoneBile Agar plate and incubated at 44˚C: after incubation the membraneis flooded with indole reagent. Indole positive colonies produce a redcolour on the membrane and are easily counted.

Formula g/litre

Tryptone 20.0


Agar No. 2 15.0

Method for reconstitutionWeigh 36.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and pour into Petridishes.


pH: 7.2 ± 0.2


Storage of Prepared Medium: Plates – up to 4 days at 2-8˚C in thedark. Capped container – up to 1 month at 2-8˚C in the dark.

Inoculation: 1ml of a 1:10 dilution of homogenised sample onto amembrane. The recommended membranes are 85mm in diameterwith a 0.45 micron pore size manufactured from cellulose esters.

Incubation: 4 hours at 37˚C on Nutrient Agar LAB 8 or MineralsModified Glutamate Medium LAB 80A and 80B plus agar – then 18-24 hours on Tryptone Bile Agar at 44˚C.

Indole reagent: 5% p-dimethylaminobenzaldehyde in N-HC1(Vracko & Sherris 1963).

Indole reaction: Pipette 1-2ml of reagent into Petri dish lid, removemembrane with forceps and place on reagent. Allow to stand for 5minutes for reaction to develop, then dry in sunlight to ‘fix’ thecolour. Count all pink colonies as E. coli.

Growth characteristicscolony size shape & indole reaction on

organism (mm) surface membrane

E. coli 1.0-3.0 CV.E.G. positive – pink colour

otherEnterobacteriaceae 0.5-2.0 CV.E.G. negative – no colour

Klebsiella spp. no growth

Pseudomonas spp. no growth

Staphylococcus spp. no growth

Bacillus spp. no growth

ReferencesAnderson, J.M., Baird-Parker, A.C. (1975). A rapid and direct methodfor enumerating Eschericia coli biotype I in food. J. Appl. Bact. 39:111-117.

Delaney, J.E., McCarthy, J.A. & Grasso, R.J. (1962). Measurement ofE. coli type I by the membrane filter technique Wat. Sewage Wks.109, 289.

Baird, R.M., Corry, J.E.L., Curtis, G.D.U. (1988). Pharmacopoeia ofculture media for food microbiology. Int. J. Food Microbiol. 276-277.

Tryptone Bile Glucuronide AgarLAB 162

Refer to Harlequin™ HAL 3

Tryptone Glucose Extract AgarLAB 63

DescriptionA plate count agar suggested by the American Public HealthAssociation (A.P.H.A.) for estimation of total viable counts in foodand dairy products. This medium is also recommended by theAssociation of Official Analytical Chemists (A.O.A.C.).

Formula g/litre

Beef Extract 3.0

Tryptone 5.0

Glucose 1.0

Agar No. 1 15.0

Method for reconstitutionWeigh 24 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then boil to dissolvebefore distributing into tubes or bottles. Sterilise at 121˚C for 15minutes.

Appearance: Pale straw colour, clear.

pH: 7.0 ± 0.2


Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark. Capped containers – up to 3 months at 15-20˚C in the dark.


Incubation: 30˚C aerobically for 48 hours for aerobic mesophilecount. 6˚C aerobically for 10 days for aerobic psychrotroph count.55˚C aerobically for 48 hours for aerobic thermophile count.

ReferencesAssociation of Official Analytical Chemists (AOAC). (1995).Bacteriological Analytical Manual, 8th ed.: Association of OfficialAnalytical Chemists.

Hausler, W.J. (Ed.) (1976). Standard Methods for the Examination ofDairy Products, 14th edn.: American Public Health Association.

Speck, M.J. (Ed.) (1976). Compendium of Methods for theMicrobiological Examination of Foods.: American Public HealthAssociation.

1.57

Tryptone Soy Agar (U.S.P.)(Soybean Casein Digest Medium U.S.P.)

LAB 11

DescriptionA general purpose agar which will support the growth of a wide rangeof micro organisms. The formula conforms with that laid down by theUnited States Pharmacopeia for sterility testing. The medium can beused for phage typing, colicine typing and for testing the X and Vfactor requirements of Haemophilus spp.

Formula g/litre

Tryptone (Casein Digest USP) 15.0

Soy Peptone 5.0

Sodium chloride 5.0

Agar No. 2 12.0

Method for reconstitutionWeigh 37 grams of powder, disperse in 1 litre of deionised water,allow to soak for 10 minutes, swirl to mix then sterilise for 15minutes at 121˚C. Cool to 47˚C, mix well and then pour plates.


pH: 7.3 ± 0.2

Minimum Q.C. organisms: E. coli NCIMB 50034S. epidermidis NCIMB 50082

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark. Capped containers – up to 3 months at 15-20˚C in the dark.

Inoculation: Surface plating.

Incubation: Time and temperature to suit organisms, usuallyaerobic.



S. aureus 1.0-1.5 CV.E.G. White-Yellow

Other staphylococci 1.0-1.5 CV.E.G. White- strain-dependent

Yellow

Strep. pyogenes PP-0.5 CV.E.G. Transp.

S. pneumoniae PP CV.E.G. Transp. (CO2)

Ent. faecalis 0.5-1.0 CV.E.G. Grey-White

Klebsiella spp. 2.0-3.0 CV.E.G. White mucoid

Ps. aeruginosa 0.5-3.0 F.CR.D. Grey- (marked strainGreen variation)

ReferencesUnited States Pharmacopeia 21st edition. (1985).

Blair, J.E. and Carr, M. (1953). The bacteriophage typing ofstaphylococci. J. Infect. Dis. 93: 1-13.

Examination of Dairy Products. A.P.H.A., New York.

Tryptone Soy Broth (U.S.P.)(Soybean Casein Digest Medium U.S.P.)

LAB 4

DescriptionA general purpose nutritious broth capable of growing a wide rangeof bacteria and fungi. The medium is recommended by the UnitedStates Pharmacopeia for the sterility testing of a wide range ofpharmaceutical products. The medium is also widely used for bloodcultures although the high carbohydrate level may cause rapid growthand subsequent death of acid-producing organisms.

Formula g/litre

Tryptone (Casein Digest USP) 17.0

Soy Peptone 3.0

Sodium chloride 5.0


Dextrose 2.5

Method for reconstitutionWeigh 30 grams of powder, disperse in 1 litre of deionised water.Swirl to mix and warm if necessary to dissolve. Dispense into tubesor flasks and sterilise at 121˚C for 15 minutes. Do not exceedtemperature.


pH: 7.3 ± 0.2



Incubation: 20-25˚C aerobically for 14 days, for sterility tests. 37˚Caerobically for 14 days for blood cultures.

Growth indicators: Turbidity or precipitate.

ReferencesUnited States Pharmacopeia 21st edition (1985).

1.58

Tryptone WaterLAB 129

DescriptionA substrate for the testing of an organism’s ability to produce indolefrom tryptophan. The indole test is frequently used in the classificationof coliform organisms. This product is preferable to peptone waterLAB 104 because it has a higher content of tryptophan.

Formula g/litre

Tryptone 10.0

Sodium chloride 5.0

Method for reconstitutionWeigh 15 grams of powder, disperse in 1 litre of deionised water.Heat to dissolve then distribute into screw cap bottles. Sterilise byautoclaving at 121˚C for 15 minutes.

Appearance: Colourless, clear.

pH: 7.5 ± 0.2



Inoculation: From pure culture.

Incubation: 37˚C for 24-48 hours.

Interpretation: Indole positive organisms will give a distinct colourchange when either Kovac’s or Ehrlich’s indole reagent is added.

ReferencesAmerican Public Health Association. (1955). American Water WorksAssociation 10th edn. 391-392.

MacFaddin, J. (1983). Biochemical tests for the identification ofmedical bacteria. 2nd edn. Williams & Wilkins, Baltimore.

Tryptose Phosphate BrothLAB 62

DescriptionThis is a versatile nutritionally rich buffered glucose broth. Themedium is a general purpose broth that has been used as a bloodculture medium, a supplement for animal cell culture, and with theaddition of sodium azide 0.025% as a selective medium forstreptococci in dairy products.

Formula g/litre

Tryptose 20.0

Dextrose 2.0

Sodium chloride 5.0


Method for reconstitutionWeigh 29.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, heat to dissolve solids then distributeinto final containers. Sterilise by autoclaving at 121˚C for 15 minutes.

Appearance: Very pale straw, clear.

pH: 7.3 ± 0.2



Inoculation: For blood culture work dilute sample at least 1:10 in broth.

Incubation: Dependent on application.

ReferencesAmerican Public Health Association (1948). Standard Method for theExamination of Dairy Products, 10th edn. A.P.H.A., New York.

American Public Health Association, (1950). Diagnostic Proceduresand Reagents, 3rd edn., A.P.H.A., New York.

T.Y.C. Medium(Tryptone Yeast Cystine)

LAB 35

DescriptionA medium designed by J. D. de Stoppelaar in 1967 to differentiateStreptococcus sanguis (frequently found in dental plaque) fromStreptococcus mutans (implicated in dental caries). The medium usesa high sucrose content to promote the formation of specific glucansby S. mutans thus forming distinctive colonies. It can be madeselective by the addition of 0.2 units per ml of Bacitracin.

Formula g/litre

Tryptone 15.0

Yeast Extract 5.0

L-Cystine 0.2

Sodium sulphite 0.1

Sodium chloride 1.0

Disodium phosphate anhydrous 0.8


Sodium acetate anhydrous 12.0

Sucrose 50.0

Agar No. 2 12.0

Method for reconstitutionWeigh 98 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix, then sterilise for 15minutes at 121˚C. Cool to 47˚C, mix and pour plates.

Appearance: White, translucent gel.

pH: 7.3 ± 0.2

Minimum Q.C. organisms: S. mutans ATCC 25175



Incubation: 37˚C for 4-5 days in an atmosphere of 90% H2, 10%CO2.

1.59



Strep. mutans 1.0-3.0 ‘heaped’ Yellow Crumbles whenType A colony touched with wire

granularsurface

irregularedge

Type B 1.0-3.0 As Type A Grey white soft consistency,

white precip in agar(glistening drop)

S. sanguis 1.0-3.0 Convex White very rubberyglossy (glistening drop)

crenated

Referencesde Stoppelaar, J.D. van de Houte, J. and de Moor, C.E. (1967). Thepresence of dextran forming bacteria resembling Streptococcus bovisand Streptococcus sanguis in dental plaque. Arch. Oral Biol. 12:1199-1201.

de Stoppelaar, J.D. (1971). Streptococcus mutans, Streptococcussanguis and dental caries. Thesis, Rijksuniversiteit, Utrecht.

Emilson, C.G., Bratthall, D. (1976). Growth of Streptococcus mutanson various selective media. Journal of Clinical Microbiology 4: 95-98.

Gold, O.G., Jordon, H.V., Van Houte, J. (1973). A selective mediumfor Streptococcus mutans. Archives of Oral Biology 19: 1357-1364.

Ikeda, T., Sandham, H.J. (1972). A high-sucrose medium for theidentification of Streptococcus mutans. Archives of Oral Biol. 4: 781-783.

Wade, W.G., Alldred, M. J., Walker, D.M. (1986). J. Med. Microbiol.22: 319-323. An improved medium for isolation of Streptococcusmutans.

Urea Agar Base(Christensen)

LAB 130

DescriptionThis is a modification of Christensen’s urea base for the detection ofrapid urease production by Proteus spp. Other enterobacteria willsplit the urea, but this will be delayed. This delay is achieved by theincorporation of glucose and the introduction of a buffering systeminto the medium. The indicator for ammonia production is phenolred.

Formula g/litre

Peptone 1.0

Glucose 1.0

Sodium chloride 5.0



Phenol red 0.012

Agar No. 1 12.0

Method for reconstitutionWeigh 2.1 grams of powder, disperse in 95ml of deionised water.Allow to soak for 10 minutes, swirl to mix, then sterilise at 121˚C for15 minutes. Allow to cool to 47˚C, add aseptically 5ml sterile ureasolution X130/X135. Distribute into sterile bottles and slopes, allowto set in the sloped position.

Appearance: Yellow/pale pink, translucent.

pH: 6.8 ± 0.2

Minimum Q.C. organisms: Proteus spp.E. coli NCIMB 50034

Storage of Prepared Medium: Capped container – up to 1 month at2-8˚C in the dark.

Inoculation: Pure culture using straight wire for stab/streaktechnique.

Incubation: 37˚C for 4-6 hours or overnight, aerobically.

Interpretation: Production of red colour in under 6 hours is positivefor rapid urease production.

Organism Growth CharacteristicsProteus spp. Red colour 4-6 hours

Citrobacter spp. Red colour 18-24 hours

Klebsiella spp. Red colour 18-24 hours

Staphylococcus spp. Red colour 24-48 Hours

Helicobacter pylori Red colour 30 minutes

ReferencesChristensen, W.B. (1946). Urea decomposition as a means ofdifferentiating Proteus and paracolon cultures from each other andfrom Salmonella and Shigella types. J. Bacteriol. 52: 461-466.

Urea Broth Base(Christensen)

LAB 131

DescriptionThis is a liquid version of Christensen’s medium (LAB 130)introduced by Maslen in 1952. This modification allows inoculationby Pasteur pipette, and it is easier to detect contamination in a fluidrather than in a slope. Maslen also claimed that it is easier to detectpositive results.

Formula g/litre

Peptone 1.0

Glucose 1.0



Sodium chloride 5.0

Phenol red 0.004

Method for reconstitutionWeigh 0.9 grams of powder, add to 95ml of deionised water. Swirl tomix then sterilise by autoclaving at 121˚C for 15 minutes. Allow tocool to 47˚C then add aseptically 5ml of X130/X135 sterile ureasolution. Distribute into sterile screw cap bijou bottles.


pH: 6.8 ± 0.2

Minimum Q.C. organisms: Proteus sp.E. coli NCIMB 50034


Inoculation: Fluid culture by pasteur pipette or straight wire frompure growth.

Incubation: 37˚C for 4-6 hours – preferably in a water bath for mostrapid growth, aerobically.

Interpretation: The production of a red colour in under six hours isa positive result for rapid urease.

1.60

Organism Growth CharacteristicsProteus spp. Red colour 4-6 hours

Corynebacterium hoffmani Red colour 18-24 hours

C. ulcerans Red colour 18-24 hours

Some strains Citrobacter Red colour 18-24 hours

Klebsiella ,, ,,

Escherichia ,, ,,

Yersinia ,, ,,

Staphylococcus ,, ,,

Pasteurella multocida Red colour 18-24 hours

ReferencesMaslen L.G.C. (1952). Routine use of liquid urea medium for identifying Salmonella and Shigella organisms. J. Brit. Med. 2: 545-546.

UVM BaseLAB 155

DescriptionUVM (University of Vermont Medium) Base is a two stage selectiveenrichment broth for the isolation of Listeria from meat products andenvironmental swabs, and forms the basis of the USDA method. Theoriginal method has been modified to replace the second stage broth(UVM II) with Fraser broth LAB164 (McClain & Lee 1989).

Formula g/litre

Tryptone 5.0

Meat Peptone 5.0

Beef Extract 5.0

Yeast Extract 5.0




Aesculin 1.0

pH: 7.4 ± 0.2

Appearance: Straw opalescent broth

Method for reconstitutionWeigh 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 for15 minutes. Cool to 47˚C and add 2 vials of UVM I supplement(X155/X555) or UVM II supplement (X156) as required. Mix welland distribute into sterile tubes or bottles.

Inoculation: UVM I – Add 25g sample to 225ml of UVM I andhomogenise. UVM II – Subculture 0.1ml of enriched UVM I into10ml UVM II.

Incubation: UVM I – 30˚C aerobically for 24 hrs. Subculture ontoselective agars and into UVM II. UVM II – 30˚C for 24 and 48 hrs.Subculture onto selective agars at 24 and 48 hrs. Incubate selectiveagars for 24 and 48 hrs.

Minimum QC organism: Listeria sp. NCIMB 50007E. coli (inhibition) NCIMB50034

ReferencesMcClain D., and Lee W.H. (1989) FSIS method for isolation ofL.monocytogenes from processed meat and poultry products.Lab.Comm.No.57, Revised May 24, 1989. US Dept of Agric.FSIS,Microbiol. Div.

Warburton D.W. et al (1991) A Canadian comparative study ofmodified versions of the FDA and USDA methods for the detectionof L.monocytogenes. J.Food Protection 54 (9) 669-676.

Violet Red Bile Agar (V.R.B.A.)

LAB 31

DescriptionA medium for the enumeration of coliform organisms in food anddairy products. The selectivity of the medium is due to the presenceof bile salts and crystal violet. Lactose fermenters produce red/purplecolonies often surrounded by a halo of the same colour. Non lactosefermenters produce pale colonies. Selectivity can be increased byincubation at 42-44˚C.

Formula g/litre

Yeast Extract 3.0


Sodium chloride 5.0


Lactose 10.0

Neutral red 0.03


Agar No. 2 12.0

Method for reconstitutionWeigh 38.5 grams of powder, disperse in 1 litre of deionised water.Dissolve by bringing to the boil with frequent swirling of the flask toprevent overheating. Autoclaving is not necessary. Cool to 45˚C anddistribute into bottles or tubes. If held molten in a water bath, usewithin 3 hours.

Appearance: Light purple-violet, clear gel.

pH: 7.4 ± 0.2

Minimum Q.C. organisms: E. coli NCIMB 50034S. epidermidis (inhibition)NCIMB 50082


Inoculation: Pour plate (with or without overlay) or surface spread.

Incubation: 37˚C for 18-24 hours for ‘coliforms’; 4˚C for 10 daysfor psychrotrophs; 32˚C for 24-48 hours for mesotrophs; 42˚C for 18hours for thermotrophs.

Interpretation: Count all red/purple colonies > 0.5mm in diameter.Calculate the number of coliforms in original sample.

ReferencesAmerican Public Health Association (1972), Standard Methods forthe Examination of Dairy Products. 13th edn. (ed. W.H. Hausler),A.P.H.A., Washington.

American Public Health Association (1966). Recommended Methods for the Microbiological Examination of Foods. 2nd edn. (ed. J.M. Sharf) A.P.H.A., Washington.

Davis, J.G. (1951). Milk Testing Dairy Industries, London.

Mossel, D.A.A., Eelderink, I. and Sutherland, J.P. (1977).Development and use of single, ‘polytropic’ diagnostic tubes for theapproximate taxonomic grouping of bacteria, isolated from foods,water and medicinal preparations. Zbl. Bakt. Hyg. I., Orig., A 278,66-79.

Mossel, D.A.A., Eelderink, I., Koopmans, M. and van Rossem, F.(1979). Influence of carbon source, bile salts and incubationtemperature on the recovery of Enterobacteriaceae from foods usingMacConkey type agars. J. Food Protec. 42, 470-475.

Mossel, D.A.A., van der Zee, H., Hardon, A.P. and van Netten, P.(1986). The enumeration of thermotropic types amongst theEnterobacteriaceae colonizing perishable foods. J. Appl. Bacteriol.60, 289-295.

1.61

Violet Red Bile Glucose Agar(V.R.B.G.A.)

LAB 88

DescriptionA modification of Violet Red Bile Agar LAB 31 introduced byMossel in 1978. V.R.B.A. LAB 31 contains lactose which isfermented by members of the coli/aerogenes group, this mediumgives a ‘coliform’ count. V.R.B.G.A. LAB 88 has substituted lactosewith glucose. Glucose is fermented by all members of theEnterobacteriaceae thus V.R.B.G.A. gives a presumptiveEnterobacteriaceae count. Bile salts and crystal violet are used toinhibit Gram positive and non-enteric organisms. The overlayprocedure ensures anaerobic conditions and suppresses the growth ofnon-fermentative Gram negative bacteria.

Formula g/litre

Yeast Extract 3.0


Sodium chloride 5.0


Glucose 10.0

Neutral red 0.03


Agar No. 2 12.0

Method for reconstitutionWeigh 38.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes then swirl to mix. Bring to the boil withfrequent swirling to prevent overheating. Further sterilisation is notrequired. 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

Minimum Q.C. organisms: E. coli NCIMB 50034S. epidermidis (inhibition)NCIMB 50082

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark. 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.

ReferencesPharmacopoeia 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 ofa modified MacConkey agar medium for the selective growth andenumeration of Enterobacteriacaea. J. Bacteriol. 84: 381.

W. L. Nutrient Agar(Wallerstein Laboratory)

LAB 79

DescriptionThis medium was developed by Green and Gray in 1950 for theisolation and enumeration of yeasts, moulds and bacteria in thebrewing process. The medium has a pH of 5.5 which is optimum forBrewers yeast and will allow the growth of a wide range of organismsincluding Enterobacteriaceae, Flavobacterium, Lactobacillus andPediococcus spp. as well as yeasts and moulds. If a process involvingbakers or distillers yeast is under examination the pH should beadjusted to 6.5. The medium may be adapted to detect bacteria onlyby the addition of 0.004 g/litre of Actidione to suppress the yeasts.

Formula g/litre

Yeast Extract 4.0

Tryptone 5.0

Dextrose 50.0





Ferric chloride 0.0025


Bromocresol green 0.022

Agar No. 2 15.0

Method for reconstitutionWeigh 75 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. If adjustment of pH to 6.5 isrequired used 1% sodium bicarbonate.


pH: 5.5 ± 0.2

Minimum Q.C. organisms: S. cerevisiae.

Storage of Prepared Medium: Plates – up to 7 days at 4˚C in thedark. Capped container – up to 1 month at 4˚C in the dark.

Inoculation: Surface plating or pour plate.

Incubation: 30˚C aerobically for 48 hours (bacteria); 20˚Caerobically for 48 hours (yeasts).

Interpretation: Count all colonies. Calculate organisms per ml inoriginal sample.

ReferencesGreen, S.R. and Gray, P.P. (1950). Differential Procedure Applicableto 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.

1.62

Wort AgarLAB 38

DescriptionA medium for the enumeration of yeasts and moulds in butter,developed by Parfitt in 1933. The medium can be modified to enableit to isolate osmophilic yeasts from soft drinks and sugar products bythe addition of high concentrations of sucrose and glucose.

Formula g/litre

Malt Extract 15.0

Peptone 0.78

Maltose 12.75

Dextrin 2.75


Ammonium chloride 1.0

Agar No. 2 15.0

Method for reconstitutionWeigh 48.3 grams of powder and disperse in 1 litre of deionisedwater. Add 2.35ml of glycerol. Allow to soak for 10 minutes, swirl tomix then sterilise for 15 minutes at 121°C. Use 60 grams per litre ifrequired for inoculation by plate streaking with a wire loop. Do not exceed time or temperature of sterilisation. If osmophilicmodification is required add 48.3 grams of powder to 1 litre of asolution containing 35% w/v sucrose and 10% w/v glucose thensterilise at 108˚C (5 p.s.i.) for 20 minutes.

Appearance: Light Brown, translucent.

pH: 5.0 ± 0.2


Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark. Capped container – up to 1 month at 15-20˚C in the dark.

Inoculation: Pour plate or surface spread.




Candida spp. 4.0 CV.E.G White

Fungi Varies withspecies

S. cerevisiae 2.0-3.0 Varies with Creamstrain

ReferencesParfitt, E.H. (1933). The influence of media upon the yeast andmould count of butter. J. Dairy Sci. 16: 141-147.

Scarr, M.P. (1959). Selective media used in the microbiologicalexamination of sugar products. J. Sci. Fd. Agric. 10: 678-681.

Wort BrothLAB 99

DescriptionA broth version of the medium LAB 38 Wort Agar developed byParfitt for the enumeration of yeasts and moulds, in butter. Themedium can be modified for the isolation of osmophilic yeasts fromsoft drinks and sugar products by the addition of high concentrationsof sucrose and glucose.

Formula g/litre

Malt Extract 15.0

Peptone 0.78

Maltose 12.75

Dextrin 2.75


Ammonium chloride 1.0

Method for reconstitutionWeigh 33.3 grams of powder and disperse in 1 litre of deionisedwater, add 2.35ml of glycerol. Allow to soak for 10 minutes, swirl tomix then sterilise by autoclaving at 121˚C for 15 minutes. Ifosmophilic version is required disperse 33.3 grams of powder in 1litre of a solution of 35% w/v sucrose and 10% w/v glucose thensterilise at 108˚C (5 p.s.i.) for 20 minutes.

Appearance: Light Brown, translucent.

pH: 4.8 ± 0.2




1.63

X.L.D. Agar(Xylose Lysine Decarboxylase Agar)

LAB 32

DescriptionThis medium was introduced by Taylor in 1965 to improve therecovery and recognition of Shigella spp, and has proved to be anexcellent medium for Salmonella spp. The medium is low in nutrientsand relies on a small amount of sodium desoxycholate for selectivity.The indicator system is novel and complex. Most enteric organismsexcept Shigella, will ferment xylose to produce acid. However thesalmonellae will also decarboxylate the lysine to keep the pH neutral.At near neutral pH Salmononella can produce H2S from the reductionof thiosulphate producing black or black centred colonies.Citrobacter spp. can also decarboxylate lysine, however, the acidproduced by fermentation of both lactose and sucrose will keep thepH too acid for H2S to be produced.

Formula g/litre

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




Method for reconstitutionWeigh 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 boilwith frequent stirring, and transfer immediately to a 47˚C water bath.Pour into plates as soon as the medium has cooled. Protracted boilingor prolonged holding at elevated temperature induces precipitation.

Appearance: Light rose, clear gel.

pH: 7.4 ± 0.2







Salmonella spp. 1.0-2.5 CV.E.G. Trans. (clearing of acidblack ppt of coliforms)centre

Shigella sonnei 1.5-2.5 CV.E.G. Pink

S. flexneri 1.0-2.0 CV.E.G. Pink

S. dysenteriae 0.5-1.5 CV.E.G. Pink

E. coli 0.5-1.5 CV.E.G.(D) Yellow inhibited (pptaround colony)

Citrobacter spp. 1.0-1.5 CV.E.G.(D) Yellow (black centre)

Proteus spp 1.0-2.5 CV.E.G. Trans. Pink fishy odour(blackcentre)

ReferencesTaylor, W.I. (1965). Isolation of shigellae. I. Xylose Lysine Agars:New media for the isolation of enteric pathogens. Am. J. Clin.Pathol., 44: 471-475.

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.Comparison of new and traditional media with stool specimens. Am.J. Clin. Pathol., 48: 350-355.

Taylor, W.I., and Schelhart, D. (1967). Isolation of shigellae. IV.Comparison of plating media with stools. Am. J. Clin. Pathol., 48:356-362.

Yeast Extract Agar(Yeastrel Milk Agar)

LAB 18

DescriptionA nutrient agar corresponding to the Standard Formulation for theplate count of micro-organisms in water and dairy products. Thismedium is also useful for teaching and demonstration purposes usingnon-fastidious organisms.

Formula g/litre

Yeast Extract 3.0


Agar No. 1 15.0

Method for reconstitutionWeigh 23 grams of powder, disperse in 1 litre of deionised water.Free steam or boil to dissolve. Mix well, and dispense into containers.Sterilise for 15 minutes at 121˚C.

To prepare Yeastrel Milk Agar add 10ml of fresh milk beforeautoclaving.

Appearance: Pale straw, clear gel.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: E. coli NCIMB 50034S. epidermidis NCIMB 50082


Inoculation: Pour plate technique or surface spreading.

Incubation: 30˚C aerobically for 48 hours for aerobic mesophilecount. 6˚C aerobically for 10 days for aerobic psychrotroph count.55˚C aerobically for 48 hours for aerobic thermophile count.

ReferencesEnvironment Agency: The Microbiology of Drinking Water (2002).Methods for the Examination of Water and Associated Materials.

British Standard 4285: Methods of Microbiological Examination forDairy Purposes.

1.64

Yeast Extract DextroseChloramphenicol Agar

LAB 119

DescriptionA selective medium for the enumeration of yeasts and moulds in milkand other dairy products. The formulation meets the requirements ofthe International Milk Union (1980), the International Organisationfor Standardisation (I.S.O.) and the British Standards Institute(B.S.I.). The medium is said to have superior storage properties toO.G.Y.E. and also has the advantage of incorporating an autoclavablesupplement.

Formula g/litre

Yeast Extract 5.0

Dextrose 20.0

Agar No. 1 15.0

Method for reconstitutionWeigh 40 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to boil. Add 2 vials of X009 (X209) which have been dissolved in ethanoland autoclave at 121˚C for 10 minutes. Allow to cool to 45˚C beforeusing with poured plate technique. THIS MEDIUM MUST NOT BE RE-AUTOCLAVED.

Appearance: Pale yellow, clear.

pH: 6.6 ± 0.2

Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097S. cerevisiaeE. coli (inhibition) NCIMB 50034


Incubation: 25˚C for 5 days, aerobically.


Interpretation: Count all colonies.

ReferencesEngel, G. (1982). Verglich verschieden Nährböden zum quantitativenNachweis von Hefen und Schimmelpilzen in Milch undMilchprodukten. Milchwiss. 37: 727-730.

International Organisation for Standardization (ISO): Milk and milk products – enumeration of yeasts and moulds – colony counttechnique at 25˚C – standard method ISO/DIS 6611.

International Milchwirtschaftsverband: Milch und Milchprodukten –Zählung von Hefen und Schimmelpilzen (Kolonieählung bel 25 C). –International IMV Standard 94: (1980) in Milchwiss. 36: 220-222.

Normenausschufl Lebensmittel und landwirtshaft. Produkte in DIN Deutsches Institut für Normung e.V. MikrobiologischeMilchuntersuchung. Bestimmung der Anzahl von Hefen undSchimmelpilzen. Reference method DIN 10186.

British Standards Institute. B.S. 4285. Section 3.6: (1986).

Yersinia Selective Agar(Schiemann’s C.I.N. Agar)

LAB 120

DescriptionThis medium is based on the work of Schiemann. It is used for theisolation and enumeration of Yersinia spp. from clinical samples andfrom food. The selective components are sodium desoxycholate,crystal violet, cefsulodin, irgasan and novobiocin. Yersiniae fermentmannitol with an intense, localised, acid production in the centre ofthe colony which produces a red ‘bull’s eye’ appearance. The ratio oftransparent border to red centre varies with serotype andenvironmental strains may appear rough with an irregular edge. Mostother enteric bacteria, if they grow, produce a larger colony with adiffuse pinkish centre and opaque outer zone.

Formula g/litre

Peptone Mixture 22.5

Mannitol 20.0

Sodium chloride 1.0


Sodium pyruvate 2.0


Neutral red 0.03


Agar No. 2 12.0

Method for reconstitutionWeigh 58 grams of powder, disperse in 1 litre of deionised water.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 ampoulesC.I.N. supplement X120. Mix well, pour plates.

Appearance: Red, clear.

pH: 7.4 ± 0.2

Minimum Q.C. organisms: Y. enterocoliticaE. coli (inhibition) NCIMB 50034






Y. enterocolitica 1.0-2.5 CV.E.G. Red centre Colony varieswith strain,

may berough &irregular

Citrobacterspp. 2.5-3.0 CV.E.G. Pale pink (may

not grow)colony

Gram +veorganisms no growth

ReferencesSchiemann, D.A. (1979). Synthesis of a selective agar medium forYersinia enterocolitica. Can. J. Microbiol. 25: 1298-1304.

Schiemann, D.A. (1982). Development of a two step enrichmentprocedure 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.

1.65

1.1 New Media Products

Azide Blood Agar BaseLAB 523

DescriptionThis is a selective medium for the isolation of Gram-positiveorganisms from clinical and non-clinical specimens. Sodium azidehas a bacteriostatic effect on the majority of Gram-negative bacteriabut permits the growth of Gram-positive bacteria such as streptococci and some strains of staphylococci. The medium can besupplemented with blood making it suitable for isolating, cultivatingand determining haemolytic reactions of fastidious pathogens.

Azide blood agar is recommended by the American Public HealthAssociation for the isolation of streptococci from cheese.

Formula g/litre

Tryptose 10.0

Beef Extract 3.0

Sodium chloride 5.0

Sodium Azide 0.2

Agar 12.0

Method for reconstitutionWeigh 30.2 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix andsterilise by autoclaving at 121°C for 15 minutes. To prepare azideblood agar, cool to 47°C and add sterile defibrinated blood to aconcentration of 5%. Pour in to sterile Petri dishes, allow the mediumto set and dry the surface prior to inoculation.

Appearance: Light to medium amber, slightly opalescent gel. With5% blood, medium is cherry red, opaque.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: Enterococcus faecalis NCIMB 50030Staphylococcus aureus NCIMB 50080Escherichia coli NCIMB 50034 (inhibition)

Storage of Prepared Medium: Plates – up to 7 days at 2-8°C in the dark.

Inoculation: Streak for isolation of single colonies.

Incubation: 35°C ± 2°C aerobically, anearobically or underconditions of increased CO2 (5-10%) for 18-24 and 40-48 hours ofincubation.

Interpretation: Examine plates for growth and haemolytic reactions.

Precautions: Proteus and Escherichia species may not always beinhibited on this formulation. Some strains of Gram-positive bacteriamay be encountered that fail to grow or grow poorly on this medium.

Anaerobic incubation will enhance haemolysis reactions.

ReferencesEdwards, S.J. (1933). The diagnosis of Streptococcus mastitis bycultural methods. J. Comp. Pathol. Ther. 46(4) 211-217.

Syndar, M.L. and Lichstein H.C. (1940). Sodium azide as aninhibition of Gram-negative bacteria. J. Infect. Dis 67(2) 113-115.

Lichstein, H.C. and Synder M.L. (1941). The inhibition of thespreading growth of Proteus and other bacteria to permit the isolationof associated streptococci. J. Bact. 42(5) 653-664.

American Public Health Association (1978) Standard Methods for theExamination of Dairy Products. 14th Edn. APHA Inc. New York.

Packer, R.A. (1943). The use of sodium azide (NaN3) as an inhibitionsubstance of Gram-negative bacteria. J.Infect. Dis. 67. 113.

Mossel, D.A.A., Diepen, H.M.J.van and De Bruin A.S. (1957). J. Appl. Bact. 20(2) 265-272.

Wood, R.L. (1965) Amer. J. Vet. Res 26. 1303-1308.

Dale, C.N. (1940) J. Bact. 40. 228-231.

Bohm, K.H. (1971) Z.bl. Bakt. I. Orig. 218. 330-334.

Cary-Blair MediumLAB 505

DescriptionCary-Blair medium is a transport medium for the collection andshipment of clinical specimens based on the formulation of Cary andBlair. The low nutrient content of the medium and the inclusion ofphosphate buffer prevents bacterial overgrowth by E. coli,Citrobacter freundii and Klebsiella aerogenes, which can occur inother transport medium containing sodium glycerophosphate. Thelow oxidation-reduction potential of the medium ensures bacterialsurvival over long periods.

Cary and Blair reported recovery of cholera vibrios up to 22 days,Salmonella and Shigella after 49 days and Yersinia pestis up to 75days storage at 28°C. Survival of Vibrio parahaemolyticus has beenreported after a 35-day period at 70-80°F.

The medium may be prepared as a pre-reduced anaerobic sterilisedmedium (PRAS) by the Holdeman and Moore method.

Formula g/litre



Sodium chloride 5.0


Agar 5.6

Method for reconstitutionWeigh 13.3 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix thenbring to the boil, with mixing, to dissolve the agar. Distribute intobijou bottles and sterilise by immersing in free-steam for 15 minutes.Allow the medium to cool and tighten the screw caps to prevent waterloss.

For transport of fastidious anaerobic bacteria prepare the medium asdirected and fill into long narrow screw capped tubes, or to the neckof the Bijou bottle.

Appearance: Colourless soft gel.

pH: 8.4 ± 0.2

Minimum Q.C. organisms: Shigella sonnei ATCC® 25931Vibrio furnissi NCTC 11218

Storage of Prepared Medium: Store away from light at 2-8°C or atroom temperature (22-25°C) for up to 19 months.

Inoculation: Use sterile, cotton-tipped swab on wooden sticks tocollect the specimen. Push the swab down one third of the depth ofthe medium and cut the stick. Screw the cap firmly on the bottle.Label the bottle and send to the testing laboratory without delay.

ReferencesCary, 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 forshipment 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.

1.1.1

NEW

NEW

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 LaboratoryManual, Virginia Polytechnic Institute Anaerobe Laboratory, 3rd Ed.

D/E Neutralising Agar(Dey & Engley)

LAB 188

DescriptionD/E Neutralising Agar is used to neutralise and determine thebactericidal activity of antiseptics and disinfectants. Developed byDey and Engley, this agar neutralises a broad spectrum ofantimicrobial chemicals, producing better results than those obtainedusing alternatives such as Letheen Agar. Complete neutralisation isrequired to prevent false results arising from disinfectant carryover.D/E Neutralising Agar is used as the plating medium when testingdisinfectants using D/E Neutralising Broth and D/E NeutralisingBroth Base. It can also be used to test disinfectants by a disc diffusionmethod. D/E Neutralising Agar contains thioglycollate to neutralisemercurial compounds, sodium thiosulphate to neutralise iodine andchlorine and sodium bisulphite to neutralise formaldehyde andgluteraldehyde. Lecithin is included to neutralise quaternaryammonium compounds and Tween® 80 neutralises phenols,hexachlorophene, formalin, and combined with lecithin, ethanol.Bromocresol purple allows detection of growth via a colour changefrom purple to yellow when organisms ferment the glucose containedin the medium.

Formula g/litre

Glucose 10.0

Lecithin 7.0


Tween® 80 5.0

Tryptone 5.0

Sodium bisulphite 2.5

Yeast extract 2.5



Agar 15.0

Method for reconstitutionWeigh 54.0 grams of powder and disperse in 1 litre of deionisedwater. Allow to soak for 10 minutes. Sterilise by autoclaving at 121°Cfor 15 minutes. Cool to 47°C and pour into sterile Petri dishes andallow to set.

Appearance: Purple opaque gel.

pH: 7.6 ± 0.2

Minimum Q.C. organisms: Bacillus subtilis NCIMB 8054Escherichia coli NCIMB 12210Pseudomonas aeruginosaNCIMB 12469Salmonella typhimuriumNCIMB 13284 Staphylococcus aureus NCIMB 12702

Storage of Powder: Store at 2-8°C in the dark. Formulation is veryhygroscopic, keep container tightly closed after use.

Storage of Prepared Medium: Plates – up to 7 days at 2-8°C in thedark.

Inoculation: Consult appropriate references as this product is used inseveral procedures.

Incubation: 37°C aerobically for 24-48 hours.

Interpretation: Count all colonies for total counts, count yellowcolonies for differential acid producer count. Non-acid producingcolonies are grey to colourless.

ReferencesRoberts, D., Hooper, W. and Greenwood, M., (1995). Methods for the examination of food for micro-organisms of public healthsignificance, 2nd edition, section 5.10, Practical Food Microbiology.Butler & Tanner. ISBN 0 901144 36 3.

Engley Jr., F.B. and Dey, B.P. (1970). A universal neutralisingmedium for antimicrobial chemicals. Presented at the ChemicalSpecialities Manufacturing Association (CSMA) Proceedings, 56thMid Year Meeting.

Dey, B.P. and Engley Jr., F.B. (1995). Comparison of Dey and Engley(D/E) Neutralising medium to Lethhen medium and StandardsMethods Medium for recovery of Staphylococcus aureus fromsanitised surfaces. J. Ind. Microbiol. 14:21-25.

Curry, A.S., Graf, J.G. and McEwen Jr., G.N. (ed.) (1993) CFTAMicrobiology Guidelines. The Cosmetic, Toiletry and FragranceAssociation, Washington, D.C.

D/E Neutralising Broth(Dey & Engley)

LAB 187

DescriptionD/E Neutralising Broth is used to neutralise and determine thebacteriocidal activity of antiseptics and disinfectants. Developed byDey and Engley, D/E Neutralising Broth neutralises a broad spectrumof antimicrobial chemicals, producing better results than thoseobtained using alternatives such as Letheen Broth, ThioglycollateMedium and Neutralising Buffer. Complete neutralisation is requiredto prevent false results arising from disinfectant carryover. Whenused with D/E Neutralising Broth Base the action of the antimicrobialagent can be assessed, i.e. whether it is bacteriostatic or hasbactericidal properties. The procedure is based upon D/ENeutralising Broth Base being deficient of all neutralising agents,therefore the potency of the disinfectant is not diminished afteraddition to the medium. Whereas, when disinfectant is added to theD/E Neutralising Broth, its activity is neutralised allowing for thedetection of any bacteria presence. D/E Neutralising Broth containsthioglycollate to neutralise mercurial compounds, sodiumthiosulphate to neutralise iodine and chlorine and sodium bisulphiteto neutralise formaldehyde and gluteraldehyde. Lecithin is includedto neutralise quaternary ammonium compounds and Tween® 80neutralises phenols, hexachlorophene, formalin, and combined withlecithin, ethanol. Bromocresol purple allows detection of growth viaa colour change from purple to yellow when organisms ferment theglucose contained in the medium.

Formula g/litre

Glucose 10.0

Lecithin 7.0


Tween® 80 5.0

Tryptone 5.0

Sodium bisulphite 2.5

Yeast extract 2.5



Method for reconstitutionWeigh 39.0 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix anddispense into final containers. Sterilise by autoclaving at 121°C for15 minutes.

1.1.2

NEW

NEW

Appearance: Purple opaque liquid.

pH: 7.6 ± 0.2


Storage of Powder: Store at 2-8°C in the dark. The formulation isvery hygroscopic therefore keep the container tightly closed after use.

Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20°C in the dark.



Interpretation: Examine all tubes for increased turbidity, formationof a pellicle or a colour change from purple to yellow, indicatingbacterial growth.

References

Roberts D., Hooper, W. and Greenwood, M., (1995). Methods for the examination of food for micro-organisms of public healthsignificance, 2nd edition, section 5.10, Practical Food Microbiology.Butler & Tanner. ISBN 0 901144 36 3.



Curry, A.S., Graf, J.G. and McEwen Jr., G.N. (ed.) (1993) CFTAMicrobiology Guidelines. The Cosmetic, Toiletry and FragranceAssociation, Washington, D.C.

D/E Neutralising Broth Base(Dey & Engley)

LAB 186

DescriptionD/E Neutralising Broth Base is a nutritious medium deficient of allneutralising agents. Therefore when a test disinfectant is added to thebroth, the potency is undiminished. Developed for use with Dey andEngley’s Neutralising Broth (LAB 187), incorporating D/ENeutralising Broth Base into the test procedure allows the user todifferentiate between bacteriostatic and bactericidal activity, and todetect viable organisms that remain after treatment. Its use isrecommended in disinfectant evaluation, environmental samplingand water-miscible cosmetics in accordance with Cosmetic, Toiletryand Fragrance Association (CTFA) guidelines.

Formula g/litre

Glucose 10.0

Tryptone 5.0

Yeast extract 2.5


Method for reconstitutionWeigh 17.5 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to dissolve,and dispense into final containers. Sterilise the medium by

autoclaving at 121°C for 15 minutes.

Appearance: Purple clear liquid.

pH: 7.6 ± 0.2






Interpretation: Examine all tubes for turbidity, indicating growth.

ReferencesRoberts, D., Hooper, W. and Greenwood, M., (1995). Methods for the examination of food for micro-organisms of public healthsignificance, 2nd edition, section 5.10, Practical Food Microbiology.Butler & Tanner. ISBN 0 901144 36 3.



Curry, A.S., Graf, J.G. and McEwen Jr., G.N. (ed.) (1993) CFTAMicrobiology Guidelines. The Cosmetic, Toiletry and FragranceAssociation, Washington, D.C..

EC Medium(Escherichia coli Medium)

LAB 171

DescriptionEC Medium (Escherichia coli Medium) is a selective enrichmentbroth designed for the isolation of coliforms, including E. coli, fromwater and food samples. It is the recommended medium of theAmerican Public Health Association (APHA) and the AOAC.

EC Medium is made selective for coliforms by the inclusion of BileSalts No.3 in the dehydrated medium. The selective nature of thismedium ensures that the growth of non-coliform bacteria isminimised. The medium is buffered by the addition of potassiumphosphates and osmotically balanced by sodium chloride. Themedium is used at 37°C for coliform organisms and 45.5°C isrecommended for the isolation E. coli.

Formula g/litre

Tryptone 20.0

Lactose 5.0

K2HPO4 4.0

KH2PO4 1.5

Sodium chloride 5.0


1.1.3

NEW

NEW

Method for reconstitutionWeigh 37.0 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix.Dispense into tubes of appropriate volume and, where applicable, addDurham tubes. Sterilise by autoclaving at 121°C for 15 minutes.

Appearance: Clear straw broth.

pH: 6.9 ± 0.2

Minimum Q.C. organisms: Escherichia coli NCIMB 50034 Enterococcus faecalis NCIMB 50030 (inhibition)Bacillus subtilis NCIMB 13061 (inhibition)


Inoculation: Coliforms: Follow the methods and procedures asstated in Standard Methods for the Examination of Water andWastewater and Compendium of Methods for the MicrobiologicalExamination of Foods.

Incubation: 45.5°C for 18-24 hours aerobically for E. coli and 37°Cfor 18-24 hours, aerobically for coliforms.

Interpretation: Turbidity of broth and gas collection in the Durhamtube indicates the presumptive growth of organisms from the coli-aerogenes group. All broths should be sub-cultured onto selectivemedia whether turbid or not.

ReferencesAmerican Public health Association, (1980). Standards Methods forthe Examination of Water and Wastewater, 15th Edition, AmericanPublic Health Association, Inc., Washington, D.C.

American Public health Association, (1976). Compendium ofMethods for the Microbiological Examination of Foods, AmericanPublic Health Association, Inc., Washington, D.C.

Association of Official Analytical chemists. (1995). Bacteriologicalanalytical 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.

Eugon Agar(Eugonic Agar)

LAB 525Description

Eugon Agar is used for the cultivation of a wide variety ofmicroorganisms, particularly in mass cultivation procedures. Themedium is prepared according to the formulation of Vera and wasdeveloped to obtain eugonic (luxuriant) growth of fastidiousmicroorganisms. The medium can be used with additions to enhanceits performance with certain microorganisms, e.g. Eugon Agarsupplemented with 5% sterile defibrinated blood will enable thegrowth of pathogenic fungi such as Nocardia, Histoplasma andBlastomyces.

Niven reported Eugon Agar for the detection of lactic acid bacteria incured meats and recommended it for investigating spoilage in meats.Harrison and Hansen employed the medium for plate counts of theintestinal flora of turkeys and Frank showed its use for thegermination of anaerobic spores pasteurised at 104°C. Eugon Agar isalso specified in the APHA Compendium of Methods for theMicrobiological Examination of Food.

The high sugar content of this medium dictates that it not suitable asa base for haemolytic reactions.

Formula g/litre

Tryptose 15.0

Soy Peptone 5.0

Dextrose 5.5

L-Cystine 0.7

Sodium chloride 4.0

Sodium sulphite 0.2

Agar 15.0

Method for reconstitutionWeigh 45.4 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix andthen sterilise by autoclaving at 121°C for 15 minutes. Cool to 47°C before the addition of supplements or pouring into sterile Petri dishes.

Appearance: Light amber clear gel, may contain a slight precipitate.

pH: 7.0 ± 0.2

Minimum QC organisms: Aspergillus niger NCIMB 50097Candida albicans NCIMB 50010Lactobacillus fermentum ATCC® 9388Streptococcus pyogenes NCIMB 13285

Storage of Prepared Medium: Plates can be stored up to 7 days at2-8°C in the dark.

Inoculation: For the examination of clinical specimens for bacteriaand fungi refer to the appropriate published references. For theexamination of food for the examination of bacteria and fungi refer tostandard methods.

Incubation: 35°C ± 2°C for up 72 ± 4 hours for bacteria. 30°C ± 2°Cfor up 72 ± 4 hours for fungi.

Interpretation: Refer to appropriate references and procedures.

ReferencesVera, H.D. (1947). The ability of peptones to support surface growthof lactobacilli. J. Bacteriol. 54:14.

MacFaddin, J.D. (1985). Media for the isolation-cultivation-identification-maintenance of medical bacteria. 301-303. vol. 1.Williams & Wilkens, MD.

Niven (1949). J. Bacteriol. 58:633.

Harrison, A.P.Jr. and Hansen, P.A. (1950). The bacterial flora of thececal feces of healthy turkeys. J. Bacteriol. 59. 197.

Frank, H.A. (1955). The influence of various media on spore countdeterminations of a putrefactive anaerobe. J. Bacteriol. 70:269.

Vanderzant, C. and Splittstoesser, D.F. (ed.). (1992). Compendium ofmethods for the microbiological examination of food, 3rd ed.American Public Health Association, Washington, D.C.

Isenberg, H.D. (ed.) (1992). Clinical microbiological procedureshandbook, American Society for Microbiology, Washington, D.C.

Murray, P.R. et al (ed) (1995). Manual of Clinical Microbiology, 6thed. American Society for Microbiology, Washington, D.C.

Association of Official Analytical Chemists. (1995). Bacteriologicalanalytical manual, 8th ed. AOAC International, Gaithersburg, MD.

1.1.4

NEW

Eugon Broth(Eugonic Broth)

LAB 526Description

This is the broth version of Eugon Agar (LAB525) for the cultivationof a wide variety of microorganisms, particularly in mass cultivationprocedures. The medium is prepared according to the formulation ofVera and was developed to obtain eugonic (luxuriant) growth offastidious microorganisms. The medium can be used with additionsto enhance its performance with certain microorganisms e.g. EugonBroth supplemented with 5% sterile defibrinated blood the mediumwill support the growth of pathogenic fungi such as Nocardia,Histoplasma and Blastomyces.

Formula g/litre

Tryptose 15.0

Soy Peptone 5.0

Dextrose 5.5

L-Cystine 0.7

Sodium chloride 4.0

Sodium sulphite 0.2

Method for reconstitutionWeigh 30.4 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix andsterilise by autoclaving at 121°C for 15 minutes. Cool before theaddition of enrichments and aseptically dispense into appropriatecontainers.

Appearance: Light amber solution, may contain a slight precipitate.

pH: 7.0 ± 0.2

Minimum QC organisms: Aspergillus niger NCIMB 50097Candida albicans NCIMB 50010Lactobacillus fermentum ATCC® 9388Streptococcus pyogenes NCIMB 13285

Storage of Prepared Medium: Store the prepared medium at 2-8°C.

Inoculation: For the examination of clinical specimens for bacteriaand fungi refer to the appropriate published references.

Incubation: 35°C ± 2°C for up 72 ± 4 hours for bacteria. 30°C ± 2°Cfor up 72 ± 4 hours for fungi.

Interpretation: Refer to appropriate references and procedures.

ReferencesVera, H.D. (1947). The ability of peptones to support surface growthof lactobacilli. J. Bacteriol. 54:14.

MacFaddin, J.D. (1985). Media for the isolation-cultivation-identification-maintenance of medical bacteria. 301-303. vol. 1.Williams & Wilkens, MD.

Isenberg, H.D. (ed.) (1992). Clinical microbiological procedureshandbook, American Society for Microbiology, Washington, D.C.

Murray, P.R. et al (ed) (1995). Manual of Clinical Microbiology, 6thed. American Society for Microbiology, Washington, D.C.

Lauryl Tryptose Broth(Lauryl Sulphate Broth, LTB, LSB)

LAB 196

DescriptionLauryl Tryptose Broth is a selective medium for the detection ofcoliforms in water, dairy products and other foods. The AmericanPublic Health Authority (APHA) recommend Lauryl Tryptose Brothfor the Most Probable Number Presumptive Test of coliforms inwaters, effluent or sewage and as a confirmation test of lactosefermentation with gas production from milk samples and for thedetection of coliforms in foods.

Lauryl Tryptose Broth is prepared according to the formulation ofMallmann and Darby. Mallmann and Darby showed that tryptose at a concentration of 2% increased the early logarithmic growth phase when compared to meat peptone. These researchers addedphosphate buffers and sodium chloride, which improved gasproduction by "slow lactose fermenting" organisms. Sodium laurylsulfate was incorporated as a selective agent for the inhibition of non-coliform organisms.

This medium can also be used with the addition of MUG (4-methylumbelliferyl-β-D-glucuronide) according to the ISO Standard11866-2 to give enhanced detection of Escherichia coli.

Formula g/litre

Tryptose 20.0

Lactose 5.0

Sodium chloride 5.0



Sodium lauryl sulphate 0.1

Method for reconstitutionWeigh 35.6 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix anddispense into tubes or bottles containing inverted Durham tubes.Sterilise by autoclaving at 121°C for 15 minutes.

Appearance: straw, clear liquid.

pH: 6.8 ± 0.2

Minimum Q.C. organisms: Escherichia coli NCIMB 50034Enterobacter aerogenesNCIMB 50029Staphylococcus aureusNCIMB 50080 (inhibition)

Storage of Prepared Medium: Store the prepared medium at roomtemperature (18-22°C), in the dark.

Inoculation: Inoculate the medium in accordance with standardmethods or laboratory policy.

Incubation: 35°C ± 2°C for 24 and 48 hours.

Interpretation: After incubation at 35°C for 24 hours examine forturbidity and gas production. If no gas has formed incubate for afurther 24 hours and re-examine.

Turbidity in the medium accompanied by the formation of gas within48 hours is a presumptive result for the presence of coliforms. Theresults should be confirmed by standard testing methods.

1.1.5

NEW NEW

ReferencesAmerican Public Health Association (1980) Standard Methods for theExamination of Water and Wastewater. 15th Edn. APHA Inc.Washington DC.

American Public Health Association (1978) Standard Methods for the Examination of Dairy Products. 14th Edn. APHA Inc.Washington DC.

American Public Health Association (1976) Standard Methods for theExamination of Foods. 15th Edn. APHA Inc. Washington DC.

Mallmann, W.L. and Darby, C.W. (1941) Am. J. Pub. Hlth. 31. 127-134.

ISO Standard 11866-2 Milk and Milk Products –Enumeration ofpresumptive Escherichia coli – part 2: Most probable numbertechnique using 4-methyl umbelliferyl-β-D-glucuronide.

Letheen Agar (AOAC)(Tryptone Glucose Extract Agar with Lecithin and Polysorbate 80)

LAB 185

DescriptionLetheen Agar is used for evaluating the bactericidal activity ofquaternary ammonium compounds, and is used with Letheen Broth to determine the suitability of preservatives for use in cosmeticformulations, as specified by the American Society for Testing andMaterials (ASTM), Standard Test Method for Preservatives in Water-Containing Cosmetics. Letheen Agar is a modification of TryptoneGlucose Extract (TGE) Agar, and is formulated to neutralisequaternary ammonium compounds used in testing of germicidalactivity, the importance of which was first described by Weber andBlack in 1948. The addition of Tween® 80 means Letheen Agar alsoneutralises phenols, hexachlorophene, formalin and ethanol (in thepresence of lecithin). Letheen Agar also allows calculation of colonyforming units to be assessed, when used with a hygiene swabbingprotocol and will ensure against disinfectant carry-over from theswabbing diluent/medium.

Formula g/litre

Dextrose 1.0

Tryptone 5.0

Beef extract 3.0

Lecithin 1.0

Tween® 80 7.0

Agar 15.0

Method for reconstitutionWeigh 32.0 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix, andthen sterilise by autoclaving at 121°C for 15 minutes. Cool to 47°Cand pour into sterile Petri dishes and allow the medium to set.

Appearance: Straw, opalescent gel.

pH: 7.0 ± 0.2

Minimum Q.C. organisms: Escherichia coli NCIMB 9517Staphylococcus aureusNCIMB 9518

Storage of Dehydrated Medium: Store at 2-8°C in the dark.Formulation is very hygroscopic, keep container tightly closed afteruse.

Storage of Prepared Medium: Plates – up to 7 days at 2-8°C in thedark.

Inoculation: Preservative testing - From the dilutions of product inLetheen Broth, subculture to Letheen Agar using a pour platetechnique, or surface inoculation.

Hygiene swabbing – Subculture from the swab diluent using a pour

plate or surface inoculation to allow calculation of the colonyforming units (cfu) for the area swabbed.


Interpretation: Count all colonies and calculate the number of cfuper ml of sample allowing for dilution factors, or the cfu of the areaswabbed (typically 25cm2).

ReferencesWeber, G.R. and Black, L.A. (1948). Relative efficiencies ofquaternary inhibitors. Soap and Sanit. Chem. 24: 134-139.

American Society for Testing Materials. (1998). Standard TestMethod for Preservatives in Water-Containing Cosmetics. E640-78.Annual Book of ASTM Standards, Philadelphia, PA.

Association of Analytical Chemists. (1995). Official methods ofanalysis, 16th edition, section 6.Association of Official AnalyticalChemists, Washington, D.C.

Roberts, D., Hooper, W., and Greenwood, M. (1995). Methods for theexamination of food for micro-organisms of public healthsignificance, 2nd edition, section 5.10, Practical Food Microbiology.Butler & Tanner. ISBN 0 901144 36 3..

Letheen Broth (AOAC)LAB 184

DescriptionLetheen Broth is primarily used for the assessing the bactericidalactivity of quaternary ammonium compounds, and for determiningthe phenol co-efficient of cationic surfactants as recommended by theOfficial Methods of Analysis of the Association of Official AnalyticalChemists (AOAC). It is also used in hygiene swabbing protocolswhere it is necessary to neutralise quaternary ammonium compounds.A modification of FDA Broth, Letheen Broth contains lecithin toneutralise quaternary ammonium compounds and Tween® 80 toneutralise phenols, hexachlorophene, formalin and (with lecithin)ethanol. Letheen Broth is easily prepared and has a clear appearanceaiding in visual inspection for growth. The American Society forTesting Materials (ASTM) specifies the use of Letheen Broth in theStandard Test Method for Preservatives in Water ContainingCosmetics.

Formula g/litre

Peptone 10.0

Beef extract 5.0

Sodium chloride 5.0

Lecithin 0.7

Tween® 80 5.0

Method for ReconstitutionWeigh 25.7 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to dissolve anddispense into final containers. Sterilise by autoclaving at 121°C for15 minutes.

Appearance: Straw, clear liquid.

pH: 7.0 ± 0.2

Minimum Q.C. organisms: Escherichia coliNCIMB 9517Staphylococcus aureusNCIMB 9518



Inoculation: There are a variety of methods which use Letheen Brothand the appropriate references should be consulted. For example:

Phenol co-efficient testing – Subculture from disinfectant dilutionsinto 10ml volumes of Letheen Broth.

1.1.6

NEW

NEW

Hygiene swabbing – Swab measured area or specific equipment andplace in 10ml volume of Letheen Broth. Area to be swabbed andvolume of medium may vary depending upon swabbing protocolused.


Interpretation: Examine all tubes for turbidity or as stipulated in themethod.

ReferencesAmerican Society for Testing Materials, (1998). Standard TestMethod for Preservatives in Water-Containing Cosmetics. E640-78.Annual Book of ASTM Standards, Philadelphia, PA.

Association of Analytical Chemists, (1995). Official methods ofanalysis, 16th edition, section 6. Association of Official AnalyticalChemists, Washington, D.C.

Roberts, D., Hooper, W. and Greenwood, M. (1995). Methods for theexamination of food for micro-organisms of public healthsignificance, 2nd edition, section 5.10, Practical Food Microbiology.Butler & Tanner. ISBN 0 901144 36 3.

Listeria Monocytogenes Blood Agar (LMBA)

LAB 172

DescriptionListeria monocytogenes Blood Agar (LMBA) has been developed forthe specific detection and enumeration of L. monocytogenes in foodsamples. This medium has been shown to improve the isolation rateof L. monocytogenes in ready to eat foods by up to 22%.

L. monocytogenes is the only important human pathogen among thespecies of Listeria currently recognised. It is distinguished from L. innocua on LMBA using colonial appearance and haemolysis.Studies have shown that the most commonly isolated species oflisteria, other that L. monocytogenes from food and processingenvironments is L. innocua.

L. monocytogenes can be found in all main categories of products e.g.dairy, meat and poultry. The symptoms of infection with thisorganism include fever, generalised aches and pains, sore throat,diarrhoea and abdominal pains. In severe cases pneumonia,septicaemia and meningitis may develop. Pregnant women areparticularly susceptible to listeriosis, due to immune suppression. L. monocytogenes can cross the placenta causing abortion, still birth,or meningitis of the new born.

LMBA contains lithium chloride in concentrations that inhibit the growth of enterococci yet allow good haemolysis by L. monocytogenes. LBMA is supplemented by polymyxin plusceftazidine (X072) and nalidixic acid (X072N) to suppress competingflora such as members of the bacillus group and staphylococci.

The addition of donated sheep blood (defibrinated with sodiumcitrate) to LMBA allows differentiation between haemolytic and non-haemolytic stains of Listeria. The use of sheep blood is standardmethodology for Listeria testing. However, ingredients in selectiveagars can result in partial lyses or darkening of the blood supplement.The use of citrated sheep blood prevents this and allowsdifferentiation of L. monocytogenes from other haemolytic Listeriaspecies e.g. L. seeligeri and L. ivanovii, due to its distinctivehaemolytic pattern. L. seeligeri is rarely isolated from foods andproduces very weak haemolysis, whilst L. ivanovii produces widezones of haemolysis compared to the narrow zone of L.monocytogenes and is an animal rather than human pathogen.

LBMA is a cost effective method by which to specifically isolate andenumerate L. monocytogenes

Formula g/litre

Tryptone 15.0

Soy peptone 5.0

Sodium chloride 5.0


Magnesium sulphate (3/4H2O) 3.8

Agar 15.0

Method for ReconstitutionWeigh 53.8 grams of powder and disperse into 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix thensterilise at 121°C for 15 minutes. Cool to 47°C, and aseptically addcitrated sheep blood to 5%, 2 vials of XO72 supplement and 2 vialsof XO72N supplement. Mix well, and pour into sterile Petri dishesand allow to set.

Appearance: Opaque and blood red.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: Listeria monocytogenesNCIMB 50007.Listeria innocua NCTC 11288.Escherichia coli NCIMB 50034.Enterococcus faecalis NCIMB 50030.


Inoculation: Surface plating, streaking out to single colonies, fromthe enrichment broth. For enumeration, 0.1ml of neat or 10-1 dilutionof the food sample is spread over the entire surface of the plate. Usemultiple plates if the volume required is greater than 0.1 ml.

Incubation: Aerobically at 37°C for 24-48 hours.

Interpretation:

Organism Shape and Surface Colour & Haemolysis

L. monocytogenes C.V.E.G. Cream, narrow zone of β haemolysis

L. ivanovii C.V.E.G. Cream, wide zone of β haemolysis

L. innocua C.V.E.G. Cream, no haemolysis

L. seeligeri C.V.E.G. Cream, very weak β haemolysis

Enterococcus faecalis No growth

Escherichia coli No growth

ReferencesJohansson, T (1998). Enhanced detection and enumeration of Listeriamonocytogenes from foodstuffs and food processing environments.International Journal of Food Microbiology, 40; 77-85.

Jay, J.M. (1996). Prevalence of Listeria spp. in meat and poultryproducts. Food Control, 7; 209-214.

Kozak, J., Balmer, T., Byrne, R. and Fisher, K. (1996). Prevalence ofListeria monocytogenes in foods: incidence in dairy products. FoodControl, 7; 215-222.

1.1.7

NEW

1.1.8

Lysine Iron AgarLAB 54

DescriptionThis is a differential medium for the detection of salmonellae andother enteric pathogens, by means of lactose fermentation, lysinedecarboxylase activity and hydrogen sulphide production.Salmonella strains (including Salmonella arizona) which fermentlactose and produce black colonies on Bismuth Sulphite Agar(LAB13) can be recognised by the alkaline reaction (purple colour)produced throughout the medium, together with blackening due tosulphide production. Enteric organisms that do not decarboxylatelysine yield an alkaline slant over an acid butt (yellow). Thus nodistinction between Shigella and E. coli is possible and Triple SugarIron Agar (LAB53) is recommended in parallel. Proteus andProvidencia cultures characteristically produce a distinctive red slantover an acid butt since these organisms deaminate lysine but withoutsulphide production. Salmonella arizona strains which produce pinkto red colonies on bile salt media are often overlooked in outbreaksof food poisoning, however the use of Bismuth Sulphite Agar withsubculture into Lysine Iron Agar allows determination of theirpresence.

Formula g/litre


Yeast Extract 3.0

Glucose 1.0

L-Lysine 10.0

Ferric Ammonium Citrate 0.5


Bromocresol Purple 0.02

Agar No. 2 12.0

Method for reconstitutionWeigh 31.5 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix andbring to the boil, with frequent stirring to dissolve completely.Dispense into tubes and sterilise by autoclaving at 121°C for 15minutes. Cool in a slanted position such that slopes are formed overdeep butts approx. 3cm in depth.

Appearance: Clear purple gel.

pH: 6.7 ± 0.2

Storage of Prepared Medium: Tightly capped containers – up to 3months at 15-20°C in the dark.

Inoculation: Subcultures for further identification are picked fromthe centre of isolated colonies on selective media and streaked acrossthe slant and stabbed into the butt of tubes of Lysine Iron Agar.


Growth CharacteristicsOrganism Butt Slant Sulphide Production

Salmonella arizona Alkaline Alkaline +

Salmonella Alkaline Alkaline +

Salmonella paratyphi Acid Acid -

Enterobacter aerogenes)

Klebsiella ) Alkaline Alkaline -

Hafnia )

Serratia )

Citrobacter Acid Alkaline +

Escherichia coli Acid (NC) Alkaline -

Shigella Acid Alkaline -

Proteus Acid ‘red’ -

Providencia Acid ‘red’ -

ReferencesEdwards, P.R. and Fife, M.A. (1961). Lysine iron agar in thedetection of Arizona cultures. Appl. Microbiol. 9:478-480.

Edwards, P.R. and Ewing, W.H. (1964). Identification ofEnterobacteriaceae. Burgess Publishing Co. Minn.

Microbial Content Test Agar (MCA)(Tryptone Soy Agar with Lecithin and Tween® 80 (TSALT))

(Casein Soy Peptone Agar with Lecithin and Polysorbate 80)

LAB 189

DescriptionThe use of Microbial Content Test Agar (MCA) is recommended forthe detection of microorganisms on surfaces sanitised withquaternary ammonium compounds, phenolic compounds andformalin. The medium is a modification of Tryptone Soy Agar withadded neutralising compounds lecithin and Tween® 80. It isrecommended for determining the hygiene status of containers,equipment and work areas treated with disinfectants or othersanitisers. The addition of Lecithin and Tween® 80 in the formulainactivates some preservatives that may inhibit bacterial growth,reducing "preservative carryover". The formulation is recommendedfor Aerobic Plate Count (Microbial Limit Test) for water misciblecosmetic products containing preservatives. Lecithin is included toneutralise quaternary ammonium compounds and Tween® 80 isincorporated to neutralise phenols, hexachlorophene, formalin andwith lecithin, ethanol.

Formula g/litre

Tryptone 15.0

Soy Peptone 5.0

Sodium Chloride 5.0

Tween® 80 5.0

Lecithin 0.7

Agar No.2 15.0

Method for ReconstitutionWeigh 45.7 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix andsterilise by autoclaving at 121°C for 15 minutes. Cool to 47°C andpour into sterile Petri dishes and allow the medium to set.

Appearance: Straw opalescent gel.

pH: 7.3 ± 0.2

Minimum Q.C. organisms: Escherichia coli ATCC 11229.Staphylococcus aureus ATCC 6538P.

Storage of Powdered Medium: Store at 2-8°C in the dark.Formulation is very hygroscopic, keep container tightly closed after use.


Inoculation: Consult the appropriate references as this product isused in several procedures.


Interpretation: Count all colonies and calculate the number ofcolony forming units, (cfu) per ml of sample allowing for dilutionfactors.

NEW

NEW

1.1.9

ReferencesOrth, D.S. (1993) Handbook of Cosmetic Microbiology. MarcelDekker, Inc., New York, NY.

Brummer, B. (1976). Influence of possible disinfectant transfer onStaphylococcus aureus plate counts after contact sampling. App.Environ. Microbiol. 32:80-84.

ORSIM(Oxacillin Resistant Staphylococci Isolation Medium)

LAB 192

DescriptionThe over-prescription of therapeutic antibiotics in recent years isthought to be a contributing factor in the rising numbers of multi-resistant bacteria being encountered. One such bacterium, MRSA(multi resistant Staphylococcus aureus), is particularly prevalentwithin the hospital environment, and is well recognised as a pathogenamongst immuno-compromised patients. In this situation, earlydetection is vital to ensure the individual’s survival. This media is animproved version of the highly regarded Mannitol Salt Agar(LAB007) and incorporates an enhanced indicator system usinganiline blue and mannitol fermentation. This combination producesintense blue colonies as presumptive MRSA, which are unmistakableamongst mixed cultures and easily visualised against the mediabackground. To complement this, ORSIM possesses a refinedselectivity, derived from a reduction in the salt level to 55g/L, and theintroduction of Lithium chloride at 5g/L. This chemical mixture stillprovides the required inhibition towards competing organisms, whilstensuring optimal recovery of MRSA, even at low numbers. Tocomplete the medium, the selective supplement X192 is included.This contains two antibiotics, oxacillin to inhibit multi sensitiveStaphylococcus aureus (the cause of false positives), and polymyxinB to suppress other halophillic bacteria such as Proteus spp.

Formula g/litre

Peptone 11.8

Yeast Extract 9.0

Mannitol 10.0



Aniline blue 0.2

Agar 12.5

Method for ReconstitutionWeigh 103.5 grams of powder and disperse into 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix thensterilise at 121°C for 15 minutes. Cool to 47°C, and aseptically add 2 vials of X192 supplement. Mix well, and pour into sterile Petri dishes.

Appearance: Straw/grey gel.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: Staphylococcus aureus (MRSA Strain) NCTC 11940Staphylococcus aureus (MSSA Strain) NCIMB 50080(inhibition).


Inoculation: Take a swab sample from a suspected infection andapply the swab end directly to the surface of a supplemented plate ofORSIM 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 forintense blue colonies and confirm using either coagulase/latexagglutination and Penicillin binding protein 2’ test (PBP2’). Onceconfirmed, all positive plates should be discarded safely.

*Typical strains of MRSA will be detected within 24 hours on thismedium. However, some strains may require longer incubation, so allnegative plates should be re-incubated for a further 24 hours*.

ReferencesOrth, D.S. (1993) Handbook of Cosmetic Microbiology. MarcelDekker, Inc., New York, NY.

Brummer, B. (1976). Influence of possible disinfectant transfer onStaphylococcus aureus plate counts after contact sampling. App.Environ. Microbiol. 32:80-84.

PEMBA(Bacillus Cereus Medium)

LAB 193

DescriptionThis medium is based on the highly specific and sensitive PEMBAmedium. It is used for the isolation and enumeration of Bacilluscereus. This formulation specifically enhances egg yolk precipitationand sporulation of Bacillus cereus. The bromothymol blue pHindicator gives clear visualisation of alkaline mannitol non-fermenting colonies and egg yolk precipitation indicative of B.cereus. The selectivity is provided by the polymyxin B supplement(X193) and provides excellent results for the majority of sampletypes.

Microscopic examination of presumptive B. cereus colony canconfirm identity by presence of lipid globules in vegetative cells.

Formula g/litre

Peptone 1.0

Mannitol 10.0

Sodium chloride 2.0





Agar 15.0

Method for ReconstitutionWeigh 41g of powder and disperse in 950ml of deionised water, allowthe mixture to soak for 10 minutes, swirl to mix and sterilize byautoclaving for 15 minutes at 121ºC for 15 minutes. Cool to 47ºC andadd two vials of X193 and 50ml of Egg Yolk Emulsion (X073) mixwell and pour the plates. Dry the agar surface before inoculation.

Appearance: Yellow and opaque.

pH: 7.2 ± 0.2

Minimum Q.C. organisms: Bacillus cereus NCIMB 50014.Escherichia coli NCIMB 50034 (inhibition).

Storage of Prepared Medium: up to 7 days at 2-8ºC in the dark.

Inoculation: Surface spreading or streaking for single colonies.

Incubation: 30ºC aerobically for 24-48 hours.

NEW

NEW

1.1.10

ReferencesHolbrook, 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.

Perfringens Agar Base (TSC)(Tryptose Sulphite Cycloserine (TSC) Agar)

LAB 194

DescriptionPerfringens Agar Base is a nutrient medium to which egg yolkemulsion (X073) and cycloserine (X194) are added for thepreparation of Tryptose Sulphite Cycloserine (TSC) Agar. Sodiummetabisulphite and ferric ammonium citrate are used as an indicatorof sulphite reduction by Clostridium perfringens. The reduction ofsulphite by Cl. perfringens produces black colonies and the egg yolkemulsion incorporated into the media detects the lecithinase activityof this bacteria. However not all strains produce lecithinase andtherefore black lecithinase positive and black lecithinase negativecolonies should be considered as presumptive Cl. perfringens.

Formula g/litre

Tryptose 15.0

Soy Peptone 5.0

Beef extract 5.0

Yeast extract 5.0



Agar 14.0

Method for reconstitutionWeigh 46.0 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix andsterilise by autoclaving at 121°C for 10 minutes. Allow the mediumto cool to 47°C and supplement with 2 vials of X194 (cycloserine)and 50ml of egg yolk emulsion (X073), mix well and pour into sterilePetri dishes. The egg yolk emulsion is omitted for preparation of EggYolk Free TSC Agar and Egg Yolk Free TSC Agar should be used foran overlay medium.

Appearance: Straw, clear gel or pale yellow opaque gel.

pH: 7.6 ± 0.2

Minimum Q.C. organisms: Clostridium perfringensNCIMB 50027Escherichia coli NCIMB 50034 (inhibition)


Inoculation: For a spread plate inoculate the agar plate with 0.1mlaliquots of an appropriate serial dilution of the homogenised testsample and overlay if required. For a pour plate mix 1ml aliquots ofan appropriate serial dilution of the homogenised test sample withapproximately 20 ml of TSC plus egg yolk emulsion. For full detailsrefer to appropriate references and standard method protocols.

Incubation: 35°C ± 2°C anaerobically for 18-24 hours.

Interpretation: Count all black colonies with or without a halo aspresumptive C. perfringens. Further confirmation should be carriedout according to standard method protocols e.g. nitrate reduction,lactose fermentation, gelatin liquefaction and absence of motility.

ReferencesShahidi, S.A. and Furguson, A.R. (1971). Appl. Microbiol. 21. 500-506.

Harmon, S.M., Kauttar, D.A. and Peeler, J.T. (1971). Appl.Microbiol. 22. 688-692.

Hauschild, A. H. W. and Hilsheimar R. (1973). Appl. Microbiol. 27.78-82.

Hauschild A.H.W. and Hilsheimar, R. (1973). Appl. Microbiol. 27.521-526.

Hauschild, A.H.W. et al (1977). Can. J. Microbiol. 23. 884-892.

Labbe, R. G. and Harmon, S.M. (1992). Compendium of methods forthe microbiological examination of foods, 3rd ed 623-635. American Public Health Association, Washington, D.C.

Rhodehamel, E.J. and Harmon, S.M. (1995). BacteriologicalAnalytical Manual 8th ed. 16.01-16.06 AOAC International,Gaithersberg, MD.

Andrews, W. (1995) Official methods of analysis AOACInternational 16th ed. 1-119. AOAC International, Arlington, VA.

Susceptibility Test ‘ISO’ AgarLAB 170

DescriptionSusceptibility Testing ‘Iso’ Agar is a semi-defined medium forantimicrobial susceptibility (sensitivity) testing (AST), in which theundefined elements are maintained at minimum levels. Theantimicrobial susceptibility test is utilised in epidemiological studiesand in determining the appropriate usage of antimicrobials in theclinical environment. The response of clinical isolates toantimicrobials, and the detection of microbial resistance, allows forprecise and rapid treatment. The AST is performed to detailedstandards, the results of which must be reproducible; a major factoris the medium on which it is performed.

The presence of antagonists in the medium e.g. thymidine and metalions, have a detrimental effect on results obtained. The addition ofthymidine for the growth of dependant strains antagonises theantimicrobial action of Trimethoprim and sulphonamides and resultsin false resistance results. Metal ions can exert known antagonisticeffects on a number of antibiotics. Therefore the anion and cationcontent of the medium must be regulated to prevent adverse effectson performance.

Susceptibility Testing ‘Iso’ Agar is produced having a stable mineralcontent, the presence of minimum antagonistic elements, and aconstant isotonic pH (preventing the blocking or enhancement ofantimicrobials), thereby ensuring production of optimum zones ofmicrobial inhibition.

This medium will support the growth of the majority of pathogensrequiring susceptibility testing, without the addition of supplements. However, certain organisms such as some streptococci,staphylococci, Enterobacteriaceae and Neisseria may require theaddition of intrinsic growth factors e.g. lysed horse blood, thymidine,thiamine and menadione. However these supplements can introduceerrors as they can affect the activity of certain antibiotics andconsequently their affects must assessed before use.

GROWTH CHARACTERISTICS

Organism colony shape & coloursize (mm) surface

B. cereus 3.0-4.0 F.CR.D Blue white halo

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

B. coagulans 2.0 F.CR.D Yellow

B. licheniformis 2.0 F.CR.D Yellow

Proteus spp. 1.0 F.Rz.D. Blue (swarms)


E. coli no growth

S. aueus 1.0 CV.E.G. Yellow white halo

NEW

NEW

1.1.11

Formula g/litre

Peptone Mixture 16.0

Glucose 2.0

Starch 1.0

Sodium chloride 2.8

Na2HPO4 0.4

Sodium glycerophosphate 0.22

Sodium gluconate 0.1

Sodium acetate 1.0

Uridine 0.3

Defined Chemical Mixture 0.078

Agar 12.0

Method for ReconstitutionWeigh 35.9 grams of powder and disperse in 1 litre of deionisedwater. Swirl to mix and sterilise by autoclaving at 121°C for 15minutes. Cool to 47°C and if required add 5-7% sterile lysed horseblood. Pour into sterile petri dishes and allow to set. Cool to 47°C,mix well and dispense into petri dishes.

Appearance: Straw clear gel.

pH: 7.3 ± 0.2

Minimum Q.C. organisms: Escherichia coli NCTC 12241Staphylococcus aureus NCTC 12981Pseudomonas aeruginosaNCTC 12934Enterococcus faecalis NCTC 12697Haemophilus influenzaeNCTC 12699Streptococcus pneumoniaeATCC 49619Neisseria gonorrhoeae NCTC 12700.

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.

Inoculation: Surface, inoculum as described by standard methods.

Incubation: As stipulated in the BSAC methodology.

ReferencesEricsson, H.M., Sherris, J.C. (1971). Antibiotic Sensitivity Testing.Report of an International Collaborative Study. Acta. Pathol.Microbiol. Scand. Sect B Suppl.; 217: 1-90.

Amato, R.F., Thornberry, C., (1979). Calcium and Magnesium inMueller Hinton Agar and their influence on disc diffusionsusceptibility results. Current Microbiol. 2: 135-138.

Hawkey, P.M., Birkenhead, D., Kerr, K.G., Newton, K.E., Hyde,W.A. (1993). Effect of divalent cations in bacteriological media onthe susceptibility of Xanthomonas maltophila to imipenem withspecial reference to zinc ions. J. Antimicrobial Chermother; 31: 181-183.

Garrod, L.P., Waterworth, P.M. (1969). Effect of mediumcomposition on the apparent sensitivity of pseudomonas aeruginosato gentimicin. J. Clin. Pathol; 22: 534-538.

Duncan, I.B.R. (1974). Susceptibility of 1500 isolates ofPseudomonas aeruginosa to gentimicin, carbenecillin, colistin, andpolymyxin B. Antimicrobial Agents and Chermother; (Jan) 9-15.

Violet Red Bile Agar with MUG(V.R.B.A. with MUG)

LAB 573

DescriptionViolet Red Bile Agar with MUG (Methylumbelliferyl-β-D-glucuronide) is a medium for the simultaneous enumeration ofcoliform organisms and Escherichia coli in food and dairy products.The selectivity of the medium is due to the presence of bile salts andcrystal violet. Lactose fermenters produce red/purple colonies oftensurrounded by a halo of bile precipitate. Escherichia coli producered/purple fluorescent colonies due to the fermentation of lactose andproduction of the enzyme glucuronidase, which hydrolyses MUG toyield the fluorescent compound methylumbelliferone, detectable bylong-wave UV light. Non-lactose fermenters produce pale colonies.

Standard Methods procedures specify VRBA with MUG fordetecting E. coli in food and dairy products by fluorescence.

Formula g/litre

Yeast extract 3.0

Balanced peptone No.1 7.0

Sodium chloride 5.0


Lactose 10.0

Neutral red 0.03


Agar No. 2 12.0

MUG, 4 methylumbelliferyl-ß-D-glucuronide 0.1

Method for reconstitutionWeigh 38.6 grams of powder, disperse in 1 litre of deionised water.Allow the mixture to soak for 10 minutes, swirl to mix and thensterilise the medium, with frequent mixing, by bringing to the boil.Cool to 47°C and distribute into bottles or tubes. If held molten in awater bath, use within 3 hours.

Appearance: Light purple-violet clear gel.

pH: 7.4 ± 0.2

Minimum Q.C. organisms: Escherichia coli NCIMB 50034Enterobacter aerogenesNCIMB 50029Staphylococcus aureus NCIMB 50080

Storage of Prepared Medium: Plates - up to 7 days at 2 - 8°C in thedark. Capped containers up to 1 month at 15-20°C in the dark.

Inoculation: Pour plate method (with or without overlay) or surfacespread.

Incubation: 37°C for 18-24 hours for ‘coliforms’; 4°C for 10 daysfor psychrotrophs; 32°C for 24-48 hours for mesotrophs; 42°C for 18hours for thermotrophs.

Interpretation: Examine plates for growth and fluorescence. Countall red/purple colonies > 0.5mm in diameter as coliforms and allfluorescent colonies as presumptive E. coli. Calculate the number ofcoliforms and E. coli in the original sample.

ReferencesChristen, G.L., Davidson, P.M., McAllistair, J.S. and Roth, L.A.(1993). Coliform and other indicator bacteria. 247-269. R.T. Marshall(ed.) Standard methods for the microbiological examination of dairyproducts, 16th ed. American Public Health Association, Washington,D.C.

Hitchens, A.D., Hartman, P.A. and Todd, E.C.D. (1992) Coliforms-Escherichia coli and its toxins. C. Vanderzant and D. F. Splittstoesser(ed.). Compendium of methods for the microbiological examinationof foods, 3rd ed. American Public Health Association, Washington,D.C.

NEW

(as recommended by theBritish Society forAntimicrobialChemotherapy (BSAC))

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. AOACInternational, Gaithersburg, MD.

Feng, P.C.S. and Hartman, P.A. (1982). Fluorogenic assays for immediate confirmation of Escherichia coli. Appl. Environ.Microbiol. 43:1320-1329.

Chang, G.W., Brill, J. and Lum, R. (1989). Proportion of β-D-glucuronidase negative Escherichia coli in human fecal samples.Appl. Environ. Microbiol. 55:335-339.

Hansen, W. and Yourassowsky, E. (1984). Detection of β-D-glucuronidase in lactose fermenting members of the familyEnterobacteriaceae and its presence in bacterial urine cultures. J. Clinical Microbiol. 20:1177-1179.

Kilian, M. and Bulow, P. (1976). Rapid diagnosis ofEnterobacteriaceae. Acta. Pathol. Microbiol. Scand. Sect. B.84:245-251.

Mates, A. and Shaffer, M. (1989). Membrane filtration differentiationof E. coli from coliforms in the examination of water. J. Appl.Microbiol. 67:343-346.

Damare, J.M., Campbell, D.F. and Johnston, R.W. (1985). Simplifieddirect plating method for enhanced recovery of Escherichia coli infood. Journal of Food Science. 50:1736-1746.

Water Plate Count Agar (ISO)LAB 197

DescriptionA nutritious non-selective medium which conforms to ISO 6222:1999(E) Water quality - Enumeration of culturable micro-organisms - Colony count by inoculation in a nutrient agar culturemedium. The estimation of overall numbers of microorganisms canbe used for the assessment and surveillance of water quality. Colonycounts are useful for assessment of ground water integrity and theefficiency of water treatment processes. They also give an indicationof the cleanliness and integrity of the distribution system. They canbe used to assess the suitability of a water supply for the preparationof food and drink, thus avoiding contamination of the product withspoilage organisms. The main value of colony counts lies in thedetection of changes in water supply quality from those expected,based on frequent long term monitoring. A sudden increase in thenumbers can be a warning of pollution and can call for immediateremedial action

Formula g/litre

Tryptone 6.0

Yeast Extract 3.0

Agar 15.0

Method for reconstitutionWeigh 24.0 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix thensterilise at 121°C for 15 minutes. Cool to 47°C before use.


pH: 7.2 ± 0.2

Minimum Q.C. organisms: Staphylococcus epidermidisNCIMB 50082Escherichia coli NCIMB 50034


Inoculation: Pour plate technique or surface inoculation.

Incubation: Aerobically at 36°C ± 2°C for 44 ± 4 hours and 22°C ±2°C for 68 ± 4 hours.

Interpretation: Count all colonies and calculate the number oforganisms (or 'colony forming units' c.f.u.) per ml of sample allowingfor dilution factors.

ReferencesISO 6222 (1999) Water quality - Enumeration of culturablemicroorganisms - Colony count by inoculation in a nutrient agarmedium.

1.1.12

NEW

2. Harlequin™ ChromogenicMedia

The Harlequin™ chromogenic microbiological culture media rangehas been developed to improve the isolation and identification of arange of microorganisms. Traditional culture media generally rely onthe fermentation of sugars or other biochemical reactions forpresumptive identification of bacteria. Harlequin™ chromogenicmedia provide a more specific identification by detection of specificenzymes produced by certain groups of bacteria. The advantage ofthis type of media is that it can eliminate or reduce the need forsubculture and the performance of confirmatory biochemical tests todetermine the identity of some microorganisms.

Chromogenic substrates act as the substrate for specific enzymes andchange colour due to the action of the enzyme. LAB M have theirown patented chromogenic compounds called the novel CHE(cyclohexenoesculetin) substrates which give the bacterial colony ablack non-diffusing colouration when hydrolysed by the enzymeinvolved in the presence of iron salts. Some of the CHE derivativesare used in Harlequin™ media along with indolyl derivatives (5-bromo-4-choro-3-indolyl), which give a blue-green colour oncleavage.

Chromogenic media are most rapidly gaining acceptance as anindicator for Escherichia coli. The β-D-glucuronide enzyme ispresent in approximately 95% of E. coli and is uncommon in theother Enterobacteriaceae. This type of test is now used widely forwater and food microbiology. There is also an increase in interest forchromogenic Salmonella media as traditional tests have a very poorspecificity resulting in many false positive results. The use ofchromogenic media simplifies Salmonella testing and saves muchtime in unnecessary confirmation tests.

Harlequin™ Salmonella ABC (Freeman Formulation)

HAL 1Description

Salmonella spp. can be differentiated from other members of thefamily Enterobacteriaceae by their ability to produce α-galactosidasein the absence of β-galactosidase. This medium, developed for theisolation of Salmonella spp. from food and clinical samples, utilisesa dual chromogen system to visualise these enzyme activities. Thismedium will also detect Salmonella typhi and paratyphi.

The first substrate, CHE-β-Gal, is enzymatically cleaved by β-galactosidase producing organisms giving black colonies in thepresence of iron. Most Enterobacteriaceae are β-galactosidasepositive and these produce black colonies on Salmonella ABC. Thesecond substrate, X-α-Gal, is hydrolysed by Salmonella spp.producing green colonies that are easily distinguished from the blackor colourless colonies of other organisms. The medium is based onD.C.A Hynes and hence utilises sodium desoxycholate and sodiumcitrate as inhibitors. Isolation of Salmonella spp. by culture remainsthe most reliable method of detection. However, most media arehighly non-specific and consequently place a heavy burden on thelaboratory in terms of biochemical and serological confirmation ofsuspect colonies. With improved specificity, the ABC mediumdramatically reduces the need for ‘false positive’ screening, savinglabour and reducing consumable costs.

Formula g/litre

Beef Extract 5.0

Peptone 5.0

Sodium citrate 8.5


Agar 12.0

X-α-Gal 0.08

CHE-β-Gal 0.3


IPTG 0.03

Method for reconstitutionWeigh 36.5 grams of powder, disperse in 1 litre of deionised water.Allow the mixture to soak for 10 minutes, swirl to mix and thensterilise the medium by bringing to the boil. Cool to 47°C, mix welland dispense into Petri dishes.

DO NOT REMELT OR AUTOCLAVE THIS MEDIUM.

Appearance: Translucent straw gel.

pH: 7.2 ± 0.2

Minimum QC organisms: Salmonella typhimurium NCIMB 50076Escherichia coli NCTC 9111

Storage of Prepared Medium: Plates - up to 7 days at 2 - 8°C in the dark.

Inoculation:Clinical: Streak for single colonies after selective enrichment in Selenite Broth.Food: Streak for single colonies after selective enrichment.

Incubation: 37°C aerobically for 18 - 24 hours

References

Perry, J.D., Ford, M., Taylor, J., Jones, A., Freeman, R., Gould, F.K.,(1999). ABC Medium, a New Chromogenic Agar for SelectiveIsolation of Salmonella spp. J. Clin. Micro. 37: 766-768.

2.1

NEW


Organism Colony Shape & Colour OtherSize (mm) Surface

Salmonella spp. 1.0 - 2.0 C.V.E.G Green (Black if β-galactosidase+ve)

Shigella spp. 1.0 - 2.0 C.V.E.G Colourless (Black if β-galactosidase +ve)

E. coli pp - 1.5 C.V.E.G Black (No Growth)

K. aerogenes 1.0 - 2.5 C.V.E.G Black (Mucoid)

Proteus spp. 0.5 - 2.0 C.V.E.G Colourless (Fishy Odour)

P. aeruginosa. 0.5 - 1.0 CV.CR.D Colourless (Green)

Harlequin™ Listeria MediumHAL 2

Description

This is a selective identification medium for the isolation of Listeriaspp. from food and clinical materials based on the Oxfordformulation. The aesculin hydrolysis reaction is replaced by acombination of the novel chromogen, CHE-glucoside, and ferrousgluconate. This reaction results in the formation of an intense blackpigment that is retained within the Listeria colonies. The retention ofthe pigment against the clear agar makes screening for suspectcolonies very easy. Lithium chloride is incorporated as an inhibitor ofenterococci and further selectivity is achieved by adding theantibiotic supplement X122 or X123 after sterilisation of themedium. In a comparative study, using naturally contaminated foodsamples, the Listeria Novel Substrate Medium gave a 34% increasein positive isolations over the traditional Listeria Isolation Medium(Oxford Formulation).

Formula g/litre

Peptone mix 30.0

Meat extracts 5.7

Starch 1.0

Sodium chloride 5.0


Ferrous gluconate 1.0

Sodium pyruvate 0.5

CHE-glucoside 0.25

Agar 15.0

Method for reconstitutionWeigh 73.5 grams of powder, disperse in 1 litre of deionised water.Allow the mixture to soak for 10 minutes, swirl to mix and thensterilise by autoclaving at 121°C for 15 minutes. Allow the mediumto cool to 47°C, add 1 vial of selective supplement X122 or X123,mix well and pour into Petri dishes.

Appearance: Yellow, slightly opaque gel.

pH: 7.0 ± 0.2

Minimum QC organisms: L. monocytogenes NCIMB 50007E. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Plates - up to 7 days at 2-8 °C in the dark.


Incubation: 30°C aerobically for a minimum of 22 hours (samplesshould not be considered negative until plates have been incubatedfor a minimum of 44 hours).

References

Smith, P.A., Mellors, D., Holroyd, A., Gray, C. (2001). A newchromogenic medium for the isolation of Listeria spp. Letters inApplied Microbiology. 32. 78-82.

Poster presentation, The 9th Meeting of the Microbiological MethodsInnovations Forum. Campden and Chorleywood Food ResearchAssociation.

Harlequin™ TBGA (TBX)(Tryptone Bile Glucuronide Agar)

HAL 3Description

A medium developed for the simple enumeration of E. coli withoutthe need for membranes, or pre-incubation on Minerals ModifiedGlutamate Medium. It is based upon the formulation of Tryptone BileAgar, LAB 072, the medium has been modified by the addition of achromogenic substrate to detect the ß-glucuronidase enzyme, whichis highly specific for E. coli*, and is detected by the MUG reagent inother formulations. The advantage of the chromogenic substrate isthat it requires no UV lamp to visualise the reaction, and it isconcentrated within the colony, facilitating easier enumeration in thepresence of other organisms, or when large numbers are present onthe plate.

Formula g/litre

Tryptone 20.0

Bile Salts No.3 1.5

X-glucuronide 0.075

Agar 15.0

Method for reconstitutionWeigh 36.5 grams of powder, disperse in 1 litre of deionised waterand allow the mixture to soak for 10 minutes. Swirl to mix andsterilise at 121°C for 15 minutes. Cool to 47°C and pour in to Petridishes. Dry the surface prior to inoculation.

Appearance: Straw, clear gel.

pH: 7.2 ± 0.2

Minimum QC organisms: Escherichia coli NCIMB 50034 (blue/green)Enterobacter aerogenes NCIMB 50029 (cream)

Inoculation: Inoculate 0.5 ml of a 1:10 dilution of the sample andspread over the entire surface of the plate. Further dilution may benecessary if large numbers of E. coli are present, to ensure coloniescan be easily counted.

Incubation: 30°C for 4 hours, followed by 18 hours at 44°C.

Interpretation: Count all blue/green colonies as presumptive E. coli,calculate the cfu/g in the original material. A simple indole test can beperformed by placing one drop of Kovac’s reagent onto a colony andif positive, a red halo will appear in the medium around the colony. Ifnegative, then the halo will be white.

*96-97% of E. coli strains positive. A notable exception is E. coli0157:H7.

References

Dibb, W.L. and Bottolfsen, K.L. (1984). Evaluation of RoscoDiagnostic ß-glucuronidase Tablets in the Identification of UrinaryIsolates of Escherichia coli. Acta Path.Microbiol. Immunol. Scand.Sect. B 92 261-264.

Hansen, W. and Yourassowsky, E. (1984). Detection of ß-glucuronidase in Lactose Fermenting Members of the FamilyEnterobacteriaceae and its Presence in Bacterial Urine Cultures. J. Clin. Micro.20 (6) 1177-1179.

Robinson, B.J. (1984). Evaluation of a Fluorogenic Assay forDetection of E. coli. App & Env. Microbiol.48 (2) 285-288.

2.2



L. monocytogenes 0.5 - 1.0 CV.E.G Black Draughtsman or CV.CR.D colonies

Bacillus spp. 1.0 - 1.5 CV.CR.D Grey centre with buff periphery

Gram +ve cocci p.p - 1.0 CV.E.G Buff Pale Greyenterococci Usually no

growth

NEW

NEW

2.3

Perez, J.L., Berrocal, C.I. and Berrocal, L. (1986). Evaluation of aCommercial ß-glucuronidase Test for the Rapid and EconomicalIdentification of Escherichia coli. J.App.Bacteriol. 61 541-545.

Raghubeer, E. and Matches, J.R. (1990). Temperature Range forGrowth of Escherichia coli Serotype 0157:H7 and SelectedColiforms in E. coli Medium. J.Clin. Micro. 28 (4) 803-805.

Bolton, F.J. (1995) Personal Communication.

LB AgarHAL 4

Refer to the Biomolecular Section

LB Top AgarHAL 5

Refer to the Biomolecular Section

Harlequin™ SMAC-BCIG (Sorbitol MacConkey Agar with BCIG)

HAL 6Description

This is a specific substrate medium for the isolation of Escherichiacoli O157:H7, the primary serovar associated with haemorrhagiccolitis (HC) and haemolytic uraemic syndrome (HUS). Pathogenicityof the organism is linked to the production of verocytotoxins (VT1and VT2), but it should be noted that not all strains of O157 produceverocytotoxins, and that strains from other serovars can be toxinproducers (e.g. O26, O103, O111, O113, O145). E. coli O157 hasbeen associated epidemiologically with food poisoning outbreaksinvolving beef burgers and cold cooked meats.

This medium is a modification of Sorbitol MacConkey Agar(SMAC). The addition of the chromogenic substrate BCIG (5-bromo-4-chloro-3-indoxyl-β-D-glucuronide) improves the specificty of themedium. E. coli O157:H7 is typically sorbitol negative and β-glucuronidase negative producing pale translucent colonies on thismedium. The majority of other E. coli strains are β-glucuronidasepositive and sorbitol positive (pink/red colonies). A small percentageof E. coli are β-glucuronidase positive and sorbitol negative and thusappear as blue/green colonies on this medium. Consequently thismedium can distinguish between non-O157 sorbitol negative E. coliand the genuine toxigenic E. coli O157:H7. This reduces the numberof unnecessary confirmation tests that are performed. The mediumcan be made more selective by the addition of Cefixime Telluritesupplement X161 to prepare CT-SMAC. Most workers recommendthe use of CT-supplemented medium alongside unsupplementedmedium to ensure maximum isolation of E. coli O157. This mediumcan also be useful for the detection of other VTEC producing E. coli in conjuncion with specifically targetted IMS particles(Captivate™).

Formula g/litre

Peptone 20.0

Sorbitol 10.0


Sodium Chloride 5.0

BCIG 0.1

Neutral Red 0.03

Crystal Violet 0.001

Agar 12.0

Method for reconstitutionWeigh 48.6 grams of powder and add to 1 litre of de-ionised water.Allow to soak for 10 minutes, swirl to mix and sterilise byautoclaving at 121°C for 15 minutes. Cool to 47°C, add 2 vials ofX161 CT supplement and pour plates. Dry the surface prior toinoculation.

Appearance: Pale red, light violet tinge.

pH: 7.1 ± 0.2

Minimum QC organisms: Escherichia coli O157:H7 NCTC 12900 (non-toxigenic)Escherichia coli NCIMB 50034Enterococcus faecalis NCIMB50030 (inhibition)

Inoculation: From O157 Broth LAB 165, surface streak for singlecolonies.

Incubation: 37°C aerobically for 18-24 hr. Examine plates forsorbitol negative, β-glucuronide negative colonies. Confirm asO157:H7 by serology, (commercial kits or antiserum available).

Note: Sorbitol positive toxigenic E. coli O157:H7 have been isolated and appearas sorbitol positive and β-glucuronide positive on this medium. To ourknowledge these isolates are limited to a small geographical area in Germany.

References1) 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:H7from Ground Beef. J.Food Protection 53 (11) 941-943

Harlequin™ CLEDHAL 7

Description

Harlequin™ C.L.E.D. is a chromogenic medium for the improvedisolation and differentiation of urinary tract pathogens. The superiorgrowth qualities of Harlequin™ C.L.E.D. mean that it can also beused to improve differentiation of mixed growth in specimens wheremore fastidious organisms may be expected. Escherichia coli isresponsible for the majority of urinary tract infections (U.T.I.'s), withother Gram-negative organisms such as Proteus mirabilis,Pseudomonas aeruginosa, Klebsiella pneumoniae, Morganella spp.,Enterobacter spp., and Citrobacter spp. often encountered. Gram-positive organisms involved in U.T.I.'s include Staphylococcusaureus, Staphylococcus saprophyticus, enterococci, and LancefieldGroup B streptococci. Traditional C.L.E.D. (Cystine LactoseElectrolyte Deficient) formulations rely upon Lactose fermentation todifferentiate organisms. However this does not allow discriminationbetween E. coli and the other coliforms that cause U.T.I.'s. InHarlequin™ C.L.E.D., lactose has been replaced by a doublechromogen system, which allows clear differentiation of many of thekey pathogens involved.

X-glucuronide is incorporated to produce characteristic green E. colicolonies (96-97% of strains are positive for ß-glucuronidase, theenzyme required to split this chromogenic compound). Othercoliforms will produce colourless colonies or, if they possess the ß-


Organism colony shape & coloursize (mm) surface

E. coli O157:H7 2.5-4.0 CV.E.G. Translucentsorbitol -veβ-glucuronide -ve

E. coli 2.5-4.0 CV.E.G. Pink/red orsorbitol +ve purple centreβ-glucuronide +ve

E. coli 2.5-5.0 CV.E.G. Green or translucentsorbitol -ve with green centreβ-glucuronide +ve

NEW

NEW

NEW

NEW

glucosidase enzyme, black colonies will be produced. This colour isdue to the action of this enzyme on the second chromogenic substrateCHE-glucoside. Glucosidase positive organisms include enterococciand Klebsiella spp. Further discrimination is achieved by the additionof phenylalanine which is deaminated by Proteus spp. and relatedorganisms, producing brown colonies (may also have a brown halosurrounding). In parallel trials Harlequin™ C.L.E.D. isolated moreclinically significant isolates than traditional C.L.E.D. formulationand an alternative chromogenic formulation(1).

Formula g/litre

Balanced Peptone No.1 6.5

Beef Extract 4.0

Meat Peptone 4.0

Yeast Extract 7.0

L-Cystine 0.11

CHE-glucoside 0.2

X-glucuronide 0.075

Ferrous Gluconate 0.95

Phenylalanine 0.2

Agar 20.0

Method for reconstitutionWeigh 43.0 grams of powder, disperse in 1 litre of deionised waterand allow the mixture to soak for 10 minutes. Swirl to mix andsterilise at 121°C for 15 minutes. Cool to 47°C and pour into sterilePetri dishes. Allow the medium to set. Dry the surface prior toinoculation.

Appearance: Tan, clear gel.

pH: 7.3 ± 0.2

Minimum Q.C. organisms: Escherichia coli NCIMB 50034Staphylococcus aureus NCIMB 50080

Storage of Powdered Medium: Plates - up to 7 days at 2 - 8°C in the dark.

Inoculation: Surface inoculation, either spreading for singlecolonies, or using a 1µl loop for semi-quantitative enumeration.

Incubation: 18 - 24 hours at 37° C

References1) Perry, J.D. - Personal Communication (data on file)

Harlequin™ E. coli/Coliform MediumHAL 8

Description

This dual chromogenic substrate medium has been developed for thesimultaneous enumeration of Escherichia coli and coliforms in foodand environmental samples. The different colony types are simple todistinguish allowing rapid counting of both E. coli and coliforms ona single medium. Based upon the formulation of Tryptone Bile Agar,LAB 072, the medium has been modified by the addition of twochromogenic substrates, one to detect the β-glucuronidase enzyme(X-glucuronide) and another to detect the β-galactosidase enzyme(magenta-β-gal). Typical E. coli strains possess both enzymes andproduce blue-purple colonies (a combination of the blue and magentapigments produced by the cleavage of the chromogenic compounds).Typical coliforms, however, possess only the β-galactosidase enzymeand produce red-magenta colonies. The colony types are easilydistinguishable, even in the presence of other organisms, or whenlarge numbers are observed, making simultaneous enumeration of E.coli and coliforms a quick and simple procedure.

Formula g/litre

Tryptone 20.0

Bile Salts No.3 1.5

X-glucuronide 0.075

Magenta-β-galactoside 0.1

Agar 15.0

Method for reconstitutionWeigh 36.6 grams of powder, disperse in 1 litre of deionised waterand allow the medium to soak for 10 minutes. Swirl to mix andsterilise at 121°C for 15 minutes. Cool to 47°C and pour in to sterilePetri dishes. Allow the medium to set. Dry the surface prior toinoculation.


pH: 7.2 ± 0.2

Minimum Q.C. organisms: Escherichia coli NCIMB 50034Enterobacter aerogenesNCIMB 50029Staphylococcus aureus NCIMB 50080 (Inhibition)

Storage of Powdered Medium: Plates - up to 7 days at 2 - 8°C in the dark.

2.4



Escherichia coli 1.0 - 2.0 CV.E.G Blue-Purple

Enterobacter aerogenes 1.5 - 2.5 CV.E.G Rose-Pink

Pseudomonas aeruginosa 0.5 - 1.0 F.CR.D/

CV.E.G Buff

Enterococcus faecalis No growth

Staphylococcus aureus No growth



Escherichia coli 1.0-2.5 CV.E.G. Green Colourless (if ß-glucuronidase-ve)

Klebsiella spp. 1.5-3.0 CV.E.G. Black Mucoid

Proteus spp. 1.0-2.5 CV.E.G. Brown Colourless if (halo) weak

phenylalanine reaction

Salmonella spp 1.0-2.5 CV.E.G. Colourless

Staphylococci 0.5-1.5 CV.E.G. Cream/white

Pseudomonas Aeruginosa 0.5-1.5 F.CR.D Colourless

Enterococci p.p.-2.0 CV.E.G. Black

Group B streptococci p.p.-2.0 CV.E.G. Cream Green (if ß-

glucuronidase +ve)

Candida spp. p.p.-1.0 CV.E.D. White

Enterobacter spp. 1.5-3.0 CV.E.G. Black

Citrobacter spp 1.0-2.5 CV.E.G. Black Black centre, colourless surround

NEW

Inoculation: Inoculate 0.5 ml of a 1:10 dilution of the sample andspread over the entire surface of the plate. Further dilution may benecessary if large numbers of E. coli and/or coliforms are present, toensure colonies can be easily counted.

Incubation: 18 - 24 hours at 37° C

Interpretation: Count all blue-purple colonies as presumptive E. coli, and calculate the cfu/g. Count all blue-purple and magentacolonies as presumptive coliforms, and calculate cfu/g.

References1) Baylis, C.L., Patrick, M. (1999). Comparison of a range ofChromogenic media for enumeration of total Coliforms andEscherichia coli in foods. Leatherhead International Technical Notes.No.135: 99.

Harlequin™ mLGA(Membrane Lactose Glucuronide Agar)

HAL 9Description

Traditionally, membrane Lauryl Sulphate Broth (mLSB) has beenused as the standard media for isolating coliforms (including E. coli)from water potentially contaminated with sewage. Harlequin™membrane Lactose Glucuronide Agar (mLGA) is a modification ofmLSB aimed at reducing costs by reducing the number of filters usedper test sample and aiding in the recovery and identification ofcoliforms and E. coli. The medium has been modified from themLSB formulation by the incorporation of X-glucuronide, sodiumpyruvate and agar. X-glucuronide is incorporated to allow for thepresumptive isolation of E. coli, sodium pyruvate aids the recovery ofchlorine stressed organisms and agar is incorporated to remove theneed for absorbent pads. This medium is recommended for theenumeration of coliform bacteria and E. coli by a single membranefiltration technique in The Microbiology of Drinking Water 2002(previously Report 71).

Formula g/litre

Peptone 39.0

Yeast Extract 6.0

Lactose 30.0

Phenol Red 0.2

Sodium Lauryl Sulphate 1.0

Sodium Pyruvate 0.5

X-Glucuronide 0.2

Agar 10.0

Method for reconstitutionWeigh 87.0 grams of powder, disperse in 1 litre of deionised waterand allow the mixture to soak for 10 minutes. Swirl to mix andsterilise at 121°C for 15 minutes. Cool to 47°C and pour into sterilePetri dishes.

Appearance: Red, clear gel.

pH: 7.4 ± 0.2

Minimum Q.C. organisms: Escherichia coli NCIMB 50034Enterobacter aerogenesNCIMB 50029Staphylococcus aureus NCIMB 50080 (inhibition)

Storage of Powdered Medium: Plates - up to 7 days at 2-8°C in the dark.

Inoculation: E. coli and coliform counts can be performed on thesame sample of water. The volume and dilution of test sample shouldbe chosen so as the number of colonies on the membrane lies between20 and 80. With waters expected to contain low numbers ofcoliforms, a sample of 100ml should be filtered. For fullmethodology refer to The Microbiology of Drinking Water 2002section 4 B - The enumeration of coliform bacteria and E. coli by asingle membrane filtration technique.

Incubation: 4 hours at 30 °C followed by 14 hours at 37 °C

Interpretation: Count all green-blue colonies as presumptive E. coli,and all green-blue and yellow colonies as presumptive coliforms.

*96-97% of E. coli strains positive. A notable exception is E. coli 0157:H7

ReferencesSartory, D.P. & Howard, L. (1992). A medium detecting B-glucuronidase for the simultaneous membrane filtrationenumeration of Escherichia coli and coliforms from drinking water.Letters in Applied Microbiology 15, 273-276.

Calabrese, J.P. & Bisssonette, G.K. (1990). Improved membranefiltration method incorporating catalase and sodium pyruvate fordetection of chlorine stressed coliform bacteria. Applied andEnvironmental Microbiology 56, 3558-3564.

Microbiology of Drinking Water 2002 section 4 B - EnvironmentAgency. The enumeration of coliform bacteria and E. coli by a singlemembrane filtration technique.

2.5


Organism colony shape & colour othersize (mm) surface

Escherichia coli* 0.5 - 1.5 CV.E.G Green Yellow if glucuronidase-ve

Lactose fermenters 0.5 - 1.5 CV.E.G Yellow

Non-lactose fermenters 0.5 - 1.5 CV.E.G Red

Staphylococcus No growth aureus (suppressed)

NEW

2.6 This page left intentionally blank

)

3. Biomolecular Products

LB Agar YPD Agar

LB Broth YPD Broth

Harlequin™ LB agar* Superbroth with Agar

Harlequin™ LB Top* 2 x YT Agar

LB Agar (Lennox) 2 x YT Broth

LB Broth (Lennox) NZY Broth

Superbroth NZCYM Broth

Terrific Broth Luria Bertani Agar (Hi-Salt)

*Available from LAB M in the UK only. Outside the UK HarlequinLB agar is available from Sigma-Aldrich as S-Gal LB agar (C4478).

LAB M’s Biomolecular products form the basis of gene reporterassays that employ enzyme substrates, such as X-gal and our patentedCHE-gal, to indicate inactivation of α-complementation. Theseproducts are formulated to promote the growth of the recipient anddonor cells used in DNA insertion technology. More importantly,they are formulated to provide optimum conditions for plasmidretention or bacteriophage reproduction and survival.

As different applications have varied requirements of the culturemedium used, LAB M offer a range of media types. Some are ofstandard formulation whilst others are modified to enhance theperformance of specific applications. This variety allows theresearcher to choose the appropriate medium for the applicationbeing used.

LAB M have formulated unique versions of LB Agar and LB TopAgar which incorporate the patented water soluble chromogen CHE-β-gal into the complete medium. This improves colourdefinition of α-complemented colonies compared to the standard X-β-gal plate and removes the need for hazardous chemicals in thepreparation of the medium. Therefore we have produced a safe, fastand easy way to differentiate between lac+ and lac- colonies. Simplyadd water to the powder and autoclave. All products are availabledirectly from LAB M in the UK.

Harlequin™ LB Agar HAL 4

DescriptionA nutritious molecular biology medium containing the novelchromogen CHE-galactoside to enable rapid, safe and unambiguousdetection of plasmid transformed bacteria. The CHE-galactosidereplaces the traditional X-gal substrate, simplifying the technique asthere is no preparation of stock solutions in dimethyl formamide ordimethyl sulphoxide and surface application of the chromogen to themedium. The intense black colour of the colonies gives a sharpercontrast between lac- and lac+ colonies, giving improved colonydetection compared to blue X-gal stained colonies.

Formula g/litre

Tryptone 10.0

Yeast Extract 5.0


CHE-galactoside 0.3

IPTG 0.03


Agar 12.0

Method for reconstitutionWeigh 37.8 grams of powder and disperse in 1 litre of deionised

water. Swirl to mix and sterilise by autoclaving at 121°C for 15minutes. Cool to 47°C and add appropriate filter sterilised antibioticif required. Pour into sterile Petri dishes, allow the medium to set anddry the surface prior to inoculation.


pH: 7.0 ± 0.2

Minimum QC organisms - ß-gal reactionEscherichia coli DH5a (ATCC® 53868) Lac Z+ve (black) Escherichia coli DH5a, Lac Z -ve (remains cream)


Inoculation: Typically surface spread over plate to detect creamcolonies indicating disruption of β-complementation. Alternatively,spread for single colonies if required.

Incubation: 37°C aerobically, for 16-18 hours. The colour of thecolonies will substantially increase with prolonged incubation (up to24 hours).

Interpretation: Examine for the presence of cream colonies, whichindicates a successful insertion of the target DNA.

ReferencesMiller, J.H. (1972). Experiments in Molecular Genetics. Cold SpringHarbour Laboratory. Cold Spring Harbour New ,York.

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring HarbourLaboratory. Cold Spring Harbour New, York.

Harlequin™ LB Top Agar HAL 5

DescriptionA nutritious molecular biology medium containing the novelchromogen CHE-galactoside to enable rapid, safe and unambiguousdetection of phage transformed bacteria.

Formula g/litre

Tryptone 10.0

Yeast Extract 5.0


CHE-galactoside 1.0

IPTG 0.03


Agar 4.0

Method for reconstitutionWeigh 32 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 before seeding the medium with required strains.


pH: 7.0 ± 0.2

Storage of Prepared Medium: Storage of capped medium in bottlesfor up to 1 month at room temperature in the dark.

ReferencesMiller, J.H. (1972). Experiments in Molecular Genetics. Cold SpringHarbour Laboratory. Cold Spring Harbour New,York.


3.1

NEW

NEW

LB Agar LAB 168

DescriptionA nutritious medium designed for rapid bacterial growth, typicallyused in molecular biology procedures e.g. in the detection of phageor plasmid transformed bacteria and the maintenance of recombinantstrains. This agar contains the required concentration of sodiumchloride to promote replication of plasmids.

Formula g/litre

Tryptone 10.0

Yeast Extract 5.0


Agar 15.0

Method for reconstitutionWeigh 40.0 grams of powder and disperse in 1 litre of deionisedwater. Swirl to mix and sterilise by autoclaving at 121°C for 15minutes. Cool to 47°C and add filter sterilised antibiotic if required.Pour into sterile Petri dishes and allow the medium to set. Dry thesurface prior to inoculation.

Addition of SubstratePrepare the X-Gal solution by dissolving in DMF, to give aconcentration of 20mg/ml Once dissolved, spread 40µl as a surfacelayer over the top of the agar and allow to dry. Also spread 4µl of asolution of IPTG (200mg/ml). Alternatively, use Harlequin™ LBagar complete (HAL004), which already contains the enzymesubstrate and inducer. This eliminates the potentially hazardous useof DMF and prevents variation in the colour of ß-complementedcolonies due to differences in substrate concentration.


pH: 7.0 ± 0.2

Minimum QC organisms - ß-gal reaction:Escherichia coli DH5a (ATCC® 53868)Lac Z+ve (black if CHE-gal ispresent in the medium)Escherichia coli DH5a, Lac Z -ve (remains cream even in thepresence of CHE-gal)


Inoculation: Typically surface spread over plate to detect creamcolonies indicating disruption of ß-complementation. Alternatively,spread for single colonies if required.

Incubation: 37°C aerobically, for 16-18 hours. If a chromogenicsubstrate is used, the colour of the colonies will substantially increasewith prolonged incubation (up to 24 hours).

Interpretation: Using the base medium alone, all colonies willappear cream. Alternatively, if a chromogen is included, examine forthe presence of cream colonies, which indicates a successful insertionof the target DNA.



LB Agar (Lennox)LAB 174

DescriptionThis is a nutritionally rich medium containing half the sodiumchloride level of LB agar (LAB168). This allows the researcher toselect the optimum salt concentration for his experiment. Thismedium can also be used for plasmid replication experiments.Nutritionally rich media are required for molecular biologyapplications as the strains used are often derived from Escherichiacoli K12, which is deficient in B vitamin production.

Formula g/litre

Tryptone 10.0

Yeast Extract 5.0

Sodium chloride 5.0

Agar 15.0

Method for reconstitutionWeigh 35.0 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix andsterilise by autoclaving at 121°C for 15 minutes. Cool to 47°C andadd filter sterilised antibiotic as required. Pour into sterile Petri dishesand allow the medium to set. Dry the surface prior to inoculation.

Addition of SubstratePrepare the X-Gal solution by dissolving in DMF, to give aconcentration of 20mg/ml. Once dissolved, spread 40µl as a surfacelayer over the top of the agar and allow to dry. Also spread 4µl of asolution of IPTG (200mg/ml).


pH: 7.0 ± 0.2

Minimum QC organisms - ß-gal reaction:Escherichia coli DH5a (ATCC® 53868)Lac Z+ve (black if CHE-gal ispresent in the medium)Escherichia coli DH5a, Lac Z -ve (remains cream even in thepresent of CHE-gal)


Inoculation: Surface; either spread over entire surface for colonycount or streaking for single colonies.


Interpretation: Using the base medium alone, all colonies willappear cream. Alternatively, if a chromogen is included, examine forthe presence of cream colonies, which indicates a successful insertionof the target DNA.

ReferencesLennox, E.S. (1955). Transduction of linked genetic characters of thehost 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 inmolecular biology. Vol. 1. Current protocols, New York. N.Y.


3.2

NEW NEW

LB Broth LAB 169

DescriptionA nutrient broth primarily used for the growth and maintenance ofEscherichia coli. Used as the primary propagation step for donor orrecipient cells, when further work is to be performed on LB Agar.This broth contains a high level of sodium chloride to aid themaintenance of plasmids. If working with temperate bacteriophages,such as lambda, the addition of magnesium sulphate (MgSO4.7H2O)at 2 grams per litre is recommended to promote phage absorption.

Formula g/litre

Tryptone 10.0

Yeast Extract 5.0


Method for reconstitutionWeigh 25.0 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix andsterilise by autoclaving at 121°C for 15 minutes.


pH: 7.0 ± 0.2

Minimum QC organisms: Escherichia coli DH5 (ATCC® 53868)


Inoculation: As per normal techniques, using a pure culture ofdonor/recipient cells.




Sambrook, J., Fritsch, E.F. and Maniatis. T. (1989). MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring HarbourLaboratory. Cold Spring Harbour New, York.

LB Broth (Lennox)LAB 173

DescriptionThis is a nutrient broth containing half the sodium chloride level ofLB Broth (LAB169), this allows for the addition of calcium chloride,required in some applications for efficient phage adsorption to thecell e.g. phage P1. This medium can also be used for plasmidreplication experiments. Chloramphenicol can be added to achievehigh plasmid copy number by inhibiting chromosomal replication.

Formula g/litre

Tryptone 10.0

Yeast Extract 5.0

Sodium chloride 5.0



pH: 7.5 ± 0.2



Inoculation: As per normal techniques, using a pure culture ofdonor/recipient cells.



ReferencesLennox, E.S. (1955). Transduction of linked genetic characters of thehost 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 inmolecular biology. Vol. 1. Current protocols, New York. N.Y.


Luria Bertani (Hi-Salt) Broth LAB 191

DescriptionA nutritious medium designed for rapid bacterial growth, typicallyused in the detection of phage or plasmid transformed bacteria. Thisbroth is formulated to LB Broth (LAB169), but has a higher pH fordifferent applications.

Formula g/litre

Tryptone 10.0

Yeast Extract 5.0




pH: 7.5 ± 0.2


Storage of Prepared Medium: Plates up to 7 days at 2-8°C in the dark.

Inoculation: Dependent upon application.

Incubation: 37°C aerobically.

3.3

NEW

NEW

NEW

NZCYM BrothLAB 182

DescriptionThis is an improved medium for increased yields of the phageLambda. This formulation includes a higher concentration ofessential elements for increased bacterial growth. To encourage multiphage insertion into the host cell, it is recommended to add 0.2%maltose (prepare a 20% solution and add 1ml per 100ml of medium),which promotes expression of LamB (lambda receptor). If maltose isadded, do not use this medium to create phage stocks, as binding ofphage particles to membrane fragments will occur because ofincreased LamB density.

Formula g/litre

Enzymatic casein digest 10.0

Acid hydrolysed casein 1.0

Yeast extract 5.0

Magnesium Sulphate 2.0

Sodium chloride 5.0



pH: 7.0 ± 0.2



Inoculation: Mix a fresh overnight culture of host cells withbacteriophage and use to inoculate NZCYM Broth.


References

Blattner, F., et al., (1977) Science 196, 161.

NZY Broth (NZYM)LAB 181

DescriptionDesigned for increased replication of phage Lambda. To encouragemulti phage insertion into the host cell, it is recommended that 0.2%maltose be added (prepare a 20% solution and add 1ml per 100ml ofmedium), which promotes expression of LamB (lambda receptor). Ifmaltose is added, the medium should not be used to create phagestocks, as binding of phage particles to membrane fragments willoccur because of increased LamB density.

Formula g/litre

Enzymatic casein digest 10.0

Yeast extract 5.0

Magnesium Sulphate 2.0

Sodium chloride 5.0



pH: 7.0 ± 0.2



Inoculation: Mix a fresh overnight culture of host cells withbacteriophage and use to inoculate NZY Broth.


References

Blattner, F., et al., (1977) Science 196, 161.

SuperbrothLAB 177

DescriptionA broth medium incorporating high levels of nutritious peptone andyeast extract, which is designed for increased bacteriophage lambda replication and yield. Superbroth is predominantly used forproducing liberated phage stocks.

Formula g/litre

Tryptone 35.0

Yeast extract 20.0

Sodium chloride 5.0



pH: 7.5 ± 0.2



Inoculation: Mix fresh overnight culture of host cells withbacteriophage and use to inoculate the Superbroth.

Incubation: 37°C aerobically until lysis occurs.

Interpretation: Examine tubes for lysis (clearing of broth) andconcentrate liberated phage by normal procedures.

References

Bolstein, D., et al. (1975) J. Mol. Biol. 91. 439.

Superbroth with AgarLAB 178

DescriptionAn agar version of the extremely rich Superbroth medium, used forthe producing high yields of bacteriophage Lambda (λ).

Formula g/litre

Tryptone 35.0

Yeast extract 20.0

Sodium chloride 5.0

Agar 15.0

3.4

NEW

NEW

NEW

NEW

Method for reconstitutionWeigh 75.0 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix andsterilise by autoclaving at 121°C for 15 minutes. Cool to 47°C andadd filter sterilised antibiotic as required. Pour into sterile Petri dishesand allow to set. Dry the surface prior to inoculation.


pH: 7.5 ± 0.2



Inoculation: Dependent upon application.


References

Bolstein, D., et al. (1975) J. Mol. Biol. 91. 439.

Terrific BrothLAB 183

DescriptionA nutritious medium that will support high bacterial cell densities,usually resulting in increased yields of DNA and recombinantproteins. The formulation requires the addition of glycerol tocomplete the formulation.

Formula g/litre

Tryptone 12.0

Yeast extract 24.0

Di Potassium phosphate 9.4

Potassium di phosphate 2.2

Glycerol 4.0/8.0 ml(added after autoclaving).

Method for reconstitutionWeigh 47.6 grams of powder and disperse in 1 litre of deionisedwater. Add 4.0 or 8.0 ml of glycerol, swirl to mix and dispense intofinal containers. Allow the mixture to soak for 10 minutes, swirl todissolve and sterilise by autoclaving at 121°C for 15 minutes.


pH: 7.0 ± 0.2



Inoculation: Inoculate with a pure culture of the host straincontaining the required recombinant plasmid.


References

Tartoff, C.D. and Hobbs, C.A. (1987). Improved media for growingplasmid and cosmid clones. Bethesda Res. Lab. Focus, 9:205.


YPD AgarLAB 176

DescriptionA nutritious medium used as an alternative to YPD Broth, where asolid base is required. Glucose is included to promote rapid growth.

Formula g/litre

Tryptone 20.0

Yeast extract 10.0

Glucose 20.0

Agar 17.0

Method for reconstitutionWeigh 67.0 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix andsterilise by autoclaving at 121°C for 15 minutes. Cool to 47°C andadd filter sterilised antibiotic as required. Pour into sterile Petri dishesand allow to set. Dry the surface prior to inoculation.

Appearance: Dark straw, clear liquid.

pH: 6.5 ± 0.2

Minimum QC organisms: Saccharomyces cerevisiae

Inoculation: Surface; either spread over entire surface for colonycount or streaking for single colonies.


Interpretation: Yeasts will grow as cream colonies, size dependentupon inoculum density.

YPD BrothLAB 175

DescriptionA nutritious broth base recommended for the maintenance andpropagation of yeasts widely used in gene insertion techniques.Glucose is included to promote rapid growth.

Formula g/litre

Tryptone 20.0

Yeast extract 10.0

Glucose 20.0


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 strainto be cultivated.



3.5

NEW

NEW

NEW

2xYT AgarLAB 180

DescriptionAn agar version of 2xYT broth, for the growth of host cells offilamentous single stranded bacteriophages e.g. the M13 phage.

Formula g/litre

Tryptone 16.0

Yeast extract 10.0

Sodium chloride 5.0

Agar 15.0

Method for reconstitutionWeigh 46.0 grams of powder and disperse in 1 litre of deionisedwater. Allow the mixture to soak for 10 minutes, swirl to mix andsterilise by autoclaving at 121°C for 15 minutes. Cool to 47°C, pourinto sterile Petri dishes and allow the medium to set. Dry the surfaceprior to inoculation.


pH: 7.0 ± 0.2

Minimum Q.C. organisms: Escherichia coli DH5 (ATCC® 53868).


Inoculation: dependent upon application.

Incubation: 37˚C aerobically.

2xYT BrothLAB 179

DescriptionA nutritious liquid medium formulated to promote the growth of hostcells, thereby encouraging increased replication and yield fromfilamentous single stranded bacteriophages (such as the M13 phage).

Formula g/litre

Tryptone 16.0

Yeast extract 10.0

Sodium chloride 5.0



pH: 7.0 ± 0.2

Minimum Q.C. organisms: Escherichia coli DH5 (ATCC® 53868).


Inoculation: Mix fresh overnight culture of host cells withbacteriophage and use to inoculate 2xYTBroth.

Incubation: 37°C aerobically, typically for 4-5 hours to reduce riskof selecting deletion mutants.

Interpretation: Concentrate bacterial cells by centrifugation andtransfer supernatant containing bacteriophage to a fresh tube, beingcareful not to disturb the pellet formed. The resulting bacteriophagestock can be stored at +4°C or -20°C.

3.6

NEW

NEW

4. Captivate™

Captivate™ is a range of antibody coated paramagneticparticles for the specific ImmunoMagneticSeparation (IMS) ofmicroorganisms.

This patented technology consists of microscopic paramagneticparticles. The beads have a magnetite core and a "ceramic" zirconiumoxide coating. The beads are manufactured by a high speed blendingprocess and typically cover a size diameter range of 1-4 µm, with a2.5µm average size.

Purified antibodies to surface components of the targetmicroorganism are covalently coupled to the bead. With carefulantibody selection, a highly specific separation system formicroorganisms is produced.

The pre-coated beads are designed for the IMS of target bacteria fromenrichment cultures. A sample is taken from a filter stomacher bagand incubated with the Captivate™ beads for 30 minutes. Thebead/microorganism complexes are then removed from the sample byplacing the sample in a magnetic concentrator device. This separatesthem from the background organisms and interfering materials. Thecomplexes are then washed using a PBS/Tween® 20 wash buffer toremove non-specifically bound material. The beads can then beplated out onto the appropriate selective agar media and incubated asdescribed.

The IMS technique will increase the sensitivity of the methodologyand, in most circumstances, results can be achieved 24 hours earlierthan standard protocols.

These products can also serve as a capture system for rapid detectionsystems.

Special Notes on IMS Techniques.

There are important factors that affect the performance of IMStechniques. Thorough mixing of the particles and sample allied withefficient recovery of the beads from the sample matrix is paramountto the success of this technique. Care must be taken not to aspirate thesample vigorously as this can result in the loss of captured targetorganisms. Certain sample types (e.g. very fatty, particulate andviscous samples) can interfere with bead recovery. To counter-act thisinterference, samples can also be diluted in PBS-Tween® e.g. 1:2-1:4,reducing the effect of the matrix and allowing more efficient beadrecovery. Alternatively with problem samples, after the initialmagnetic separation the incomplete removal of the sample (i.e.remove 800µl) and continuation of the wash protocol as describedcan minimise bead losses.

Captivate™ Product Specification

Working concentration: Typically 5mg/ml

Fe3O4 content: 29-33% w/w

Antibody: Particles coated with high avidity, affinity purified and absorbed polyclonal antibodies to cell surface antigens.

Specificity: Reacts with target organism.

Average size: 2.5 µm (typical range 1-8µm)

Formulation: Particles are suspended in PBS plus1% BSA pH 7.3-7.5and 0.09% azide as preservative.

Storage: 8°C (may be shipped at ambient)

Shelf life: 2 years.

Captivate™ O157CAP 1

Description

Captivate™ O157 are magnetisable particles coated with specificantibody intended for the isolation of E. coli O157:H7 from food,animal feeds, beverages, pharmaceutical or environmental samples.The particles help to concentrate O157:H7 cells in mixed culturereducing the probability of missing low numbers or overgrowth ofO157:H7 colonies by competing flora. In fact, immunomagneticseparation is now regarded as the gold standard method for isolationof E. coli O157:H7 from food and environmental samples.

E. coli O157:H7 is the primary serovar associated with food bornegastrointestinal infection, resulting in self-limiting diarrhoea, that can lead to serious disease conditions such as haemorrhagiccolitis, haemolytic uraemic syndrome (HUS) and thromboticthrombocytopaenic purpura (TTP).

The organism itself is associated with raw meats and unpasteurisedmilk1, probably due to the implication of farm animals andparticularly cattle as carriers of E. coli O157:H7. Large outbreakshave been recorded in the United States from consumption ofunpasteurised apple juice (apple cider) possibly as a result of usingapples which have fallen to the ground where the potential forcontamination with the organism exists3,4.

Enrichment Protocol for E. coli O157:H7.

The recommended protocol for the isolation of E. coli O157:H7employs a 6 hour enrichment step at 42°C in modified Tryptone SoyBroth (mTSB, LAB 165) plus novobiocin (X150) followed by IMS(see below) and plating onto Sorbitol MacConkey Agar (LAB 161 orHAL006) supplemented with or without the addition of cefixime andpotassium tellurite (X161)5-8. It is also recommended that a furtherIMS and inoculation of SMAC plates is performed after incubationof the sample for 24 hours. Alternative enrichment protocols usingdifferent media have been described e.g. Buffered Peptone Water(LAB 46) plus VCC (X546)5-8.

Generic Captivate™ IMS Procedure1) Add 20µl of well mixed Captivate™ particles to a suitable

micro-tube (1.5 - 2.5ml volume).

2) To this add 1ml of the enrichment culture taking care to avoid transfer of sample debris.

3) Cap tube tightly and rotamix the suspension for 30 minutes at room temperature.

4) Insert tube into magnetic separator rack for 3 minutes to concentrate the beads to a pellet. Gently invert the rack several times to aid pelleting of the beads.

5) Carefully aspirate the supernatant from the tube and cap withoutremoving particles, taking care to avoid splashing.

6) Remove magnet from rack or tubes from the rack and add 1ml ofwash. Cap and resuspend particles by inverting several times.

7) Repeat separation and wash steps 4-6 twice more. Finally resuspend particles in 100µl of wash.

8) Remove 50µl of the complexed, resuspended particles to the plating media, streaking for single colonies. Incubate plates at 37°C for 18-24 hours and examine for typical colonies.

Phosphate Buffered Saline plus Tween®.

Formula g/litre

Sodium chloride 8.0




Tween® 20. 0.5

pH: 7.0 ± 0.2Dissolve the components in deionised water and check the pH.Sterilise the solution by autoclaving at 121°C for 15 min. Allow thesolution to cool and check the pH. Store in the dark and use withinone month.

4.1

NEW

Interpretation: Examine the SMAC and CTSMAC plates for typical E. coli O157 non-sorbitol fermenting colonies that are smooth and circular, 1-3 mm in diameter that are colourless to pale orange. Confirm the colony identity with commercially availablelatex agglutination kits or antisera.

Product Presentation

Captivate™ O157 is available in packs of 50 test, product codeCAP001-050 and 250 test, product code CAP001-250. Materialsrequired, but not provided, include phosphate buffered saline-Tween® 20, pipettes and tips, stomacher machine and bags,magnetic separator rack and culture media. Magnetic separatingracks (CAP-100-12P) and rotating mixers (CAP101-58) are alsoavailable from LAB M..

Reference1) Padhye, N.V., and Doyle, M.P. (1992). Escherichia coli O157:H7:Epidemiology, Pathogenesis and Methods for Detection in Food.J.Food.Prot. 55, 555-565.

2) Martin, M.L. et al (1986) Isolation of Escherichia coli from cattleassociated with two cases of hemolytic syndrome. Lancet ii 1043.

3) Besser, R.E. et al (1993) An outbreak of diarrhoea and hemolyticuremic syndrome from Escherichia coli O157:H7 in fresh pressedapple cider. JAMA 259 2217-2220

4) McCarthy, M. (1996) E. coli O157:H7 outbreak in USA traced toapple juice. Lancet 348 1299.

5)Wright, D.J., Chapman, P.A. and Siddons, C.A. (1994).Immunomagnetic separation as a sensitive method for the isolation of Escherichia coli O157 from food samples. Epidemiologyand Infection 113, 31-39.

6) Bolton, F.J.; Crozier, L.; Williamson, J.K. (1995) New technicalapproaches to Escherichia coli O157. PHLS Microbiol. Dig. 12 67-71.

7) Vernozy-Rozand, C. (1997). Detection of Escherichia coli O157and other VTEC in food. Journal of Applied Microbiology. 82, 537-551.

8) Ogden, I.D.; Hepburn, N.F.; & MacRae, M. (2001). Theoptimisation of media used in the immunomagnetic separationmethods for the detection of Escherichia coli O157 in foods. J.Appl.Mic. 91, 373-379.

4.2





Description

There is now growing concern that VTEC’s could be playing a moresignificant role in human disease than currently estimated. Currentmicrobiological methods do not permit the sensitive isolation of non-O157-VTEC. Unlike E. coli O157:H7, which is sorbitol negative, thisgroup does not appear to have any common distinct biochemicalproperties. Therefore using a screening isolation medium such asSorbitol MacConkey Agar does not help in the isolation of theseorganisms.

To address this basic problem LAB M has developed a quartet ofindividual immunomagnetic separation reagents; O26, O103, O111and O145 to aid the isolation of the other common serotypes ofverotoxigenic E. coli (VTEC). Therefore, in combination with theCaptivate™ O157 reagent, the "top five" VTEC as identified bythe World Health Organisation can be targeted with these reagents.The IMS step should greatly increase the chance of isolating theseorganisms.

Enrichment methods are currently being developed by researchersand consequently there are no standard protocols to recommend.Bearing this in mind, we suggest users try the same enrichmentprotocol that we recommend for E. coli O157 at 37°C and 42°C with a generic E. coli plating medium such as Tryptone BileGlucuronide Agar (HAL 3) or TBA (LAB 72) plus MUG (MC406).SMAC-BCIG (HAL 6) has also been used for this application as itcontains the glucuronide chromogen, which the majority of E. colireact with. Some workers have recommended the use of"Enterohaemolysin agar" sheep blood agar for detection of VTEC.Verocytotoxin production has been shown to be closely linked withthe enterohaemolytic phenotype.

ReferencesBielaszewska, M. & Karch, H. (2000). Non-O157:H7 Shiga toxin(verocytotoxin)-producing Escherichia coli strains: epidemiologicalsignificance and microbiological diagnosis. World Journal ofMicrobiology and Biotechnology. Vol 16, 8-9, 711-718.

Beutin, L., Montenegro, M.A., Orskov, I., Prada, J., Zimmerman, S.and Stephan, R. (1989). Close association of verocytotoxin (shiga-like toxin) production and enterohaemolysin production in strains ofEsherichia coli. Journal of Clinical Microbiology 27, 2559-2564.

Captivate™ SalmonellaCAP 2

Description

This product is designed to capture and concentrate the commonserotypes of Salmonella involved in human and animal disease fromenriched samples. The particles are coated with affinity purifiedpolyclonal antibody directed towards common somatic and flagellarantigens. This gives an excellent high avidity broad spectrum IMSreagent for the capture of salmonellas. Due to the large variation inSalmonella serotypes and antigen expression there is naturally straindependent variation in the capture efficiency.

Custom Coating Service

A coating service is available for coating our IMS reagent withalternative antibodies. Prices will be calculated on anindividual basis.

For more information please contact: Dr. D.S. Illingworth.

Tel: +44 (0)161 797 5729

Fax: +44 (0)161 762 9322

Email: [email protected]

4.3

NEW

NEW

NEW

NEW NEW

5. Selective Supplements

Presentation and Shelf LifeLAB M lyophilised supplements are presented in packs of 10 vials,and for the majority of the supplements each vial is sufficient for500ml of medium. Larger and smaller volumes are indicated forrelevant products.

The shelf life of freeze-dried supplements is 2-3 years provided theyare stored in a refrigerator at 2-8˚C. Once rehydrated the stability ofantibiotics varies greatly and will determine the shelf life of theprepared agars and broths. For this reason any unused, rehydrated,supplement should be discarded, as even deep-freezing may notprevent the rapid degradation of the antibiotics. To ensure the correctlevel of selective supplements the entire vial contents must be addedto the stated volume of cooled, molten medium.

RehydrationVials should be rehydrated aseptically using a sterile needle andsyringe charged with 5ml of the specified diluent for the particularsupplement being added. The supplement should be rehydrated,withdrawn and added to the medium in a single process, followed byimmediate disposal of the syringe into an approved container. Underno circumstances attempt to re-sheath an exposed needle.

If sterile needles and syringes are not readily available, the rubberstopper may be completely removed and, using careful aseptictechnique, rehydrate the supplement using a sterile pipette.

AdditionMost antibiotics are heat labile, and so to prevent a reduction ofpotency the medium should be cooled to 47˚C by holding in a waterbath set at this temperature.

Once the supplement has been added the medium must be gently butthoroughly mixed to ensure that the selective agents are evenlydistributed. Failure to do this will result in a range of concentrationsin the plates/bottles and consequent inconsistency in results. Theshelf life of supplemented media is governed by the stability of theadded components, and is generally shorter than unsupplementedagars and broths. For information on the shelf life of prepared mediaconsult the individual product listings in the previous section of themanual.

AnaerobesX090, X290

NALIDIXIC ACID, VANCOMYCIN for the isolation of Gramnegative anaerobes from clinical material.

Suitable for use with LAB 90 Fastidious Anaerobe Agar. When usedwith other blood agar bases, e.g. LAB 1 Columbia Agar, furtherenrichment of the medium with haemin and menadione is beneficial.

Final Concentration mg/litre

Nalidixic acid 10

Vancomycin 2.5

Add 1 vial X090 to 500ml medium

Add 1 vial X290 to 1 litre medium

Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C together with otheradditives, mix gently and pour.

Reference:Wren, M.W.D., 1980. J. Clin. Path. 33: 61-65. Multiple SelectiveMedia for the isolation of anaerobic bacteria.

X091, X291NALIDIXIC ACID for the isolation of non-sporing anaerobesfrom clinical material.

Suitable for use with LAB 90 Fastidious Anaerobe Agar. When usedwith other blood agar bases, e.g. LAB 1 Columbia Agar, furtherenrichment of the medium with haemin, menadione and sodiumpyruvate is beneficial. The addition of Tween® 80, which, enhancesthe growth of anaerobic cocci, to the medium is required for N.A.T.medium. The Tween® 80 may be added before sterilisation at aconcentration of 0.1%.


Nalidixic acid 10



Rehydrate contents of each vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C together with otheradditives, mix gently and pour.

Reference:Wren, M.W.D., 1980. J. Clin. Path. 33: 61-65. Multiple SelectiveMedia for the isolation of anaerobic bacteria.

X092METRONIDAZOLE, NALIDIXIC ACID for the isolation ofActinomyces spp. from clinical material.

Suitable for use with LAB 90 Fastidious Anaerobe Agar. Themetronidazole will suppress the growth of most other anaerobes.


Metronidazole 10

Nalidixic acid 30

Add 1 vial X092 to 500ml medium.

Rehydrate contents of each vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C together with otheradditives, mix gently and pour.

Reference:Stannard, A.E., National Hospital for Nervous Diseases, London.Personal Communication.

X015, X215NEOMYCIN 75 for the isolation of Clostridium spp. and otheranaerobes.

When added to blood agar the resulting medium will allow thegrowth of clostridia, most Bacteriodes fragilis strains and someanaerobic cocci.


Neomycin 75



Reconstitute each vial by the addition of 5ml of sterile deionisedwater. Add aseptically to sterilised medium cooled to 47˚C, mixgently and pour.

5.1

X016NEOMYCIN 100 for the selective isolation of Clostridium spp.

When added to egg yolk medium this supplement will allow thegrowth of clostridia whilst inhibiting other lecithinase producingorganisms.


Neomycin 100


Reconstitution as X015.

X018KANAMYCIN 75 for the selective isolation of Clostridium spp.and other anaerobes.

An alternative to X015. Kanamycin is more inhibitory to anaerobiccocci.


Kanamycin 75


Reconstitution as X015, X016.

References:Lowbury, C.J.L., Lilly, H.A. (1955). A selective plate medium for Cl.welchi. J. Path. & Bact. 70: 105.

Collee, J.G., Watt, B. (1971). Changing approaches to the sporinganaerobes in medical microbiology. Spore Research ed. A. N.Barkeer.

Sutter, V.L., Citron, D.M., Edelstein, M.A.C., Finegold, S.M. (1985).Wadsworth Anaerobic Bacteriology Manual 4 ed. Star publishers,Belmont, California.

Wren, M.W.D. (1980). Multiple selective media for the isolation ofanaerobic bacteria from clinical specimens. J. Clin. Path. 33: 61-65.

Bacillus cereusX074

POLYMYXIN for the isolation of B. cereus from foods.

Suitable for the preparation of LAB 73 Bacillus cereus Medium(P.R.E.P.). The addition of X073 sterile egg yolk emulsion is alsorequired.

Final Concentration

Polymyxin B 8mg/litre = 64,000i.u/litre


Rehydrate contents of vial with 5ml of sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C together with eggyolk emulsion, mix gently and pour.

Reference:Micro-organisms in Food. Ed. Thatcher, F.S., Clarke, D.F. publishedby Univ. of Toronto Press.

X193POLYMYXIN B for the isolation of Bacillus cereus from foods.

The addition of X073, Egg Yolk Emulsion, is also required. Foraddition to LAB 193, PEMBA Bacillus cereus Medium.


Polymyxin B 100,000 IU


Rehydrate contents of vial by the addition of 5ml of sterile deionisedwater. Add aseptically to sterilised medium cooled to 47°C, mixgently and pour.

Campylobacter speciesX112, X212

CEFOPERAZONE, AMPHOTERICIN for the isolation ofCampylobacter spp. from clinical, environmental and foodsamples.

Suitable for use with LAB 112 Campylobacter Selective Medium(blood free) or with blood agar media. Incubation at 37˚C gives betterresults than at 42˚C and is generally more convenient.


Cefoperazone 32

Amphotericin 10



Rehydrate contents of vial with 5ml of sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix gently and pour.

Reference:Bolton, F.J., Hutchinson, D.N., Parker, G. (1988). Reassessment ofSelective Agars and Filtration Techniques for Isolation ofCampylobacter Species from Faeces. Eur. J. Clin. Microbiol. Infect.Dis. 7: 155-160.

X214VANCOMYCIN, POLYMYXIN, TRIMETHOPRIM, to makeSkirrow’s medium for the isolation of Campylobacter spp.

Suitable for use with LAB001 Columbia Agar or other blood agarbases, supplemented with lysed horse blood.


Vancomycin 10

Polymyxin 2500 iu/litre

Trimethoprim 5

Add 1 vial of X214 to 1 litre of medium

Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, along with otheradditives, mix well and pour.

Reference:Skirrow, M.B. (1977) British Medical Journal 2 11-9.

5.2

NEW

X131C.V.T.C. CEFOPERAZONE, VANCOMYCIN,TRIMETHOPRIM, CYCLOHEXIMIDE. For the isolation ofCampylobacter spp. from food and environmental samples bythe enrichment broth technique.

Developed for use with LAB 135 Campylobacter Enrichment Broth.Gives higher isolation rates than Preston broth and does not requiremodified atmosphere incubation.


Cefoperazone 20

Vancomycin 20

Trimethoprim 20

Cycloheximide 50


Rehydrate contents of vial with 5ml of sterile 50% alcohol. Addaseptically to sterilised medium cooled to 47˚C, mix gently anddispense into sterile containers.

Reference:Bolton, F.J., Preston., P.H.L.S. Personal communication, (1989).

X132C.V.T.N. CEFOPERAZONE, VANCOMYCIN,TRIMETHOPRIM, NATAMYCIN.

An alternative natamycin based supplement for the selectiveenrichment broth culture of Campylobacter spp.

For addition to LAB 135, Campylobacter Enrichment Broth.


Cefoperazone 20

Vancomycin 20

Trimethoprim 20

Natamycin 25


Rehydrate contents of vial by the addition of 5ml of sterile deionisedwater. Add aseptically to sterilised medium cooled to 47°C, mixgently and dispense.

Clostridium difficileX093

CYCLOSERINE, CEFOXITIN for the isolation of Clostridiumdifficile from clinical materials.

Suitable for use with LAB 90 Fastidious Anaerobe Agar.


D-Cycloserine 250

Cefoxitin 8


Rehydrate contents of vial with 5ml of water. Add aseptically tosterilised medium cooled to 47˚C together with other additives, mixgently and pour.

Reference:George, W.L., Sutter, V.L., Citron, D., Finegold, S.M. (1976).Selective and differential medium for isolation of Clostridiumdifficile.

Clostridium perfringensX109, X110

SULPHADIAZINE (X109). OLEANDOMYCIN PHOSPHATE,POLYMYXIN (X110).

For use with LAB 109 Perfringens agar to prepare O.P.S.P. for theselective isolation of Clostridium perfringens from foodstuffs.


Sulphadiazine 100

Oleandomycin 0.5

Polymyxin 10,000 i.u./litre

Add 1 vial X109 and 1 vial X110 to 500ml medium

Rehydrate contents of vials with 5ml of sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix gently and pour.

Reference:Handford, P.M. (1974). J. Appl. Bact. 37, 559-570.

X194D-CYCLOSERINE supplement for the isolation of Clostridiumperfringens from foods.

For use with LAB 194, Perfringens Agar Base (TSC).


D-Cycloserine 400

Add 1 vial of X194 to 500mls medium.

Rehydrate contents of vial by the addition of 5mls of sterile deionisedwater. Add aseptically to sterilised medium cooled to 47°C, mixgently and pour.

Escherichia coliX150

NOVOBIOCIN for the enrichment of E. coli O157:H7 from food,environmental and clinical samples.

For the addition to LAB165 O157 Broth MTSB

Final concentration mg/litre

Novobiocin 20

Add 1 vial of X150 to 500ml of O157 Broth MTSB.

Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix well and pour.

5.3

NEW

NEW

X161CEFIXIME TELLURITE supplement for the isolation of E. coliO157:H7 from food, environmental and clinical samples.

For the addition to LAB 161 Sorbitol MacConkey Agar (SMAC) orHAL 6 (BCIG-SMAC).


Cefixime 0.05

Potassium tellurite 2.5

Add 1 vial of X161 to 500 ml of Sorbitol MacConkeyAgar (SMAC) or HAL 6 (BCIG-SMAC)


X546V.C.C. Supplement for the selective enrichment of E. coli0157:H7 from food and other samples.

For use with Buffered Peptone Water LAB 46


Vancomycin 8.0

Cefixime 0.05

Cefsulodin 10.0

Add 1 vial of X546 to 2.25 litres of LAB 46

Rehydrate the contents of one vial with 20ml of sterile deionisedwater. Add aseptically to sterilised medium cooled to 47˚C. Mix welland dispense into 225ml aliquots.

Gardnerella vaginalisX011

COLISTIN, NALIDIXIC ACID for the isolation of G. vaginalisfrom clinical material.

Suitable for addition to LAB 1 Columbia Agar or LAB 15 BloodAgar Base No. 2 to produce a selective isolation medium.


Colistin 10

Nalidixic acid 15


Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, together with anyother additives, mix gently and pour.

Reference:Goldberg, R.L., Washington, J.A. II (1976). “Comparison of Isolationof Haemophilus vaginalis (Corynebacterium vaginalae) fromPeptone-Starch-Dextrose Agar and Columbia, Colistin, NalidixicAcid Agar. J. Clin. Microbiol. 4(3): 245.

Gram Positive CocciX012

COLISTIN, NALIDIXIC ACID for the preparation of ColumbiaC.N.A. medium.

A medium selective for Gram positive cocci is obtained when thisantibiotic mixture is added to LAB 1 Columbia Agar.


Colistin 10

Nalidixic acid 10


Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, together with anyother additives, mix gently and pour.

Reference:Ellner, P.D., Stossel, C.I., Drakeford, E., Vasi, F. (1966). “A newculture medium for medical bacteriology.” Amer. J. Clin. Path. 45: 502.

Haemophilus influenzaeX260

BACITRACIN for the isolation of Haemophilus influenzae.

Suitable for use with Columbia blood agar base and other blood agarssupplemented with heated (“chocolated”) blood.


Bacitracin 75

Add 1 vial of X260 to 1 litre of medium.

Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium with heated blood cooled to 47˚C,mix well and pour.

Helicobacter pyloriX040

VANCOMYCIN, CEFSULODIN, AMPHOTERICIN, for theisolation of Helicobacter pylori.

For addition to Helicobacter pylori medium LAB 140


Cefsulodin 10

Vancomycin 10

Amphotericin 20

Add 1 vial to 500ml medium


5.4

Impedance MicrobiologyX137

T.M.A.O. Selenite for inclusion in Easter and Gibson SalmonellaDetection Medium LAB 137.

The growth of Salmonella in the medium reduces T.M.A.O. toT.M.A. and in so doing, significantly increases the conductivity of themedium. The incorporation of sodium biselenite makes the mediumselective for salmonellae.

Final Concentration g/litre

T.M.A.O. (Trimethylamine-N-oxide) 5.0

Sodium biselenite 4.0


Reconstitute contents with 5ml of sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C. Swirl to mix thendispense into sterile containers.

References:Easter, M.C., Gibson, D.M. (1985). Rapid and automated detection ofSalmonella by electrical measurements, J. Hyg. 94: 245-262.

Gibson, D.M. (1987). Some modifications to the media for rapidautomated detection of salmonellas by conductance. H. Appl.Bacteriol. 63: 299-304.

Odgen, I.D., Cann, D.C. (1987). A modified conductance medium forthe detection of Salmonella spp. J. Appl. Bacteriol. 63: 359-464.

ListeriaX122

C.C.C.A.F. CEFOTETAN, CYCLOHEXIMIDE, COLISTIN,ACRIFLAVINE, FOSFOMYCIN, for the isolation of Listeriamonocytogenes from environmental, clinical and food samples.

For addition to LAB 122 Listeria Isolation Medium or HAL 2Harlequin™ Listeria Medium.


Cefotetan 2

Cycloheximide 400

Colistin 20

Fosfomycin 10

Acriflavine 5

Add 1 vial of X122 to 500ml of LAB 122.

Add 1 vial of X122 to 1 litre of HAL 2.

Reconstitute contents of vial by the addition of sterile 50% ethanol inwater. Add aseptically to sterilised medium cooled to 47˚C, mixgently then pour.

Reference:Curtis, et al. (1989). A selective differential medium for the isolationof Listeria monocytogenes. Lett. in Appl. Microbiol. 8: 95-98.

X138N.A.C. NALIDIXIC ACID, ACRIFLAVINE, CYCLOHEXIMIDEfor the selective enrichment broth culture of Listeriamonocytogenes.

For addition to LAB 138 Listeria Enrichment Broth recommended bythe F.D.A. for Listeria isolation from food and environmentalsamples and LAB 139 Buffered Listeria Enrichment Broth.


Nalidixic acid 40

Cycloheximide 50

Acriflavine 15

Add 1 vial of X138 to 500ml medium

Reconstitute contents of vial by the addition of sterile 50% ethanol inwater. Add aseptically to sterilised medium cooled to 47˚C, mixgently then pour.

Reference:Lovett et al. (1987). Listeria monocytogenes in raw milk: detectionincidence and pathogenicity. J. Food Protect. 50: 188-192.

X144P.A.C. supplement for the enrichment and isolation of Listeriaspp from food and environmental samples.

For the addition to LAB 144 Palcam Broth and Lab 148 Palcam Agar


Polymyxin 10

Acriflavine 5

Ceftazidime 20

Add 1 vial of X144 to 500ml of Palcam Broth or Agar


X164, X5641/2 FRASER supplement for the primary enrichment of Listeriaspp from food and environmental samples.

For addition to LAB 164 Fraser Broth Base


Ferric ammonium citrate 500

Acriflavine 12.5

Nalidixic acid 10

Add 1 vial of X164 to 450ml of Fraser Broth Base

Add 1 vial of X564 to 2.25 litres of Fraser Broth Base

Rehydrate contents of vial with 2ml 50% methanol (5ml for X564).Add aseptically to sterilised medium cooled to 47˚C, mix well and pour.

5.5

X072POLYMYXIN B, CEFTAZIDIME supplement for the isolationof Listeria monocytogenes.

For addition to LAB 172, LMBA


Polymyxin B 10

Ceftazidime 20

Add 1 vial X072 and 1 vial of X072N to 500ml medium.


X072NNALIDIXIC ACID supplement for the isolation of Listeriamonocytogenes.

For addition to LAB 172, LMBA


Nalidixic acid 40

Add 1 vial X072N and 1 vial of X072 to 500ml medium.

Rehydrate contents of vial by the addition of 5 ml of sterile deionisedwater. Add aseptically to sterilised medium cooled to 47°C, mixgently and pour.

X165FRASER supplement for the secondary enrichment of Listeriaspp from food and environmental samples.

For addition to LAB 164 Fraser Broth Base


Ferric ammonium citrate 500

Acriflavine 25

Nalidixic acid 20

Add 1 vial of X165 to 500ml of Fraser Broth Base

Rehydrate contents of vial with 2ml 50% methanol. Add asepticallyto sterilised medium cooled to 47˚C, mix well and pour.

X155, X555 UVMI. Supplement for the primary enrichment of Listeria sppfrom food and environmental samples.

For addition to LAB 155 UVM Broth Base


Nalidixic acid 20

Acriflavine 12

Add 1 vial of X155 to 500ml of UVM Broth Base

Add 1 vial of X555 to 2.25 litres of UVM Broth Base

Rehydrate contents of vial with 5ml sterile deionised water (10ml forX555). Add aseptically to sterilised medium cooled to 47˚C, mix welland pour.

X156UVMII. Supplement for the secondary enrichment of Listeria sppfrom food and environmental samples.

For the addition to LAB 155 UVM Broth Base


Nalidixic acid 20

Acriflavine 25

Add 1 vial of X156 to 500ml of UVM Broth Base

Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix well and pour

X123C.N.C.A.F. CEFOTETAN, NATAMYCIN, COLISTIN,ACRIFLAVINE, FOSFOMYCIN.

An alternative natamycin supplement for the isolation of Listeria spp.from environmental, clinical and food samples.

For addition to LAB 122 Listeria Isolation Medium.


Cefotetan 2

Natamycin 25

Colistin 20

Fosfomycin 10

Acriflavine 5


Rehydrate the contents of vial by the addition of 5ml of steriledeionised water. Add aseptically to sterilised medium cooled to 47°C,mix gently and pour.

X139,X539N.A.N. NALIDIXIC ACID, ACRIFLAVINE, NATAMYCIN.

An alternative natamycin based supplement for the selectiveenrichment broth culture of Listeria spp.

For addition to LAB 138, Listeria Enrichment Broth and LAB139,Buffered Listeria Enrichment Broth.


Nalidixic acid 40

Acriflavine 15

Natamycin 25

Add 1 vial of X139 to 500ml medium.

Add 1 vial of X539 to 2.25 L medium.

Rehydrate contents of vial by the addition of 5ml of sterile deionisedwater (10ml for X539). Add aseptically to sterilised medium cooledto 47°C, mix gently and dispense.

5.6

NEW

NEW

NEW NEW

Mycobacterium tuberculosisX124

P.T.T.A. POLYMYXIN B, TICARCILLIN, TRIMETHOPRIM,AMPHOTERICIN supplement for the isolation ofMycobacterium tuberculosis from clinical samples.

For addition to LAB 123 Kirchner’s T.B. Medium.


Polymyxin B 200,000 I.U.

Ticarcillin 100

Trimethoprim 10

Amphotericin 10


Rehydrate contents of vial by the addition of 5 ml of sterile deionisedwater. Add aseptically to sterilised medium cooled to 47°C, mixgently and dispense.

Neisseria gonorrhoeaeX070, X270

L.C.A.T. LINCOMYCIN, COLISTIN, AMPHOTERICIN,TRIMETHOPRIM for the isolation of Neisseria spp. from clinicalmaterial.

L.C.A.T. is often preferred to X068 V.C.N.T. for the isolation of N. gonorrhoeae because of the emergence of vancomycin sensitivestrains. The antifungal agent amphotericin is more readily soluble andtherefore a more active antifungal than nystatin. L.C.A.T. is quoted asthe selective agent for New York City G.C. agar but can readily besubstituted for V.C.N. or V.C.N.T. in Thayer Martin G.C. agar.


Lincomycin 1

Colistin 6

Amphotericin 1

Trimethoprim 6.5



Rehydrate contents of vial with 5ml sterile 25% alcohol in water. Addaseptically to sterilised medium cooled to 47˚C together with otheradditives, mix gently and pour.

Reference:Young, H. (1978). Cultural Diagnosis of Gonorrhoea with modifiedN.Y.C. Medium. Brit. Journ. Ven. Dis. 54: 36-40.

X069, X269L.C.T. LINCOMYCIN, COLISTIN, TRIMETHOPRIM. Avariant of L.C.A.T. with the amphotericin omitted to permit thegrowth of yeasts.

Concentrations and rehydration as L.C.A.T.



X068, X268V.C.N.T. VANCOMYCIN, COLISTIN, NYSTATIN,TRIMETHOPRIM for Thayer Martin Medium.

The addition of trimethoprim in V.C.N.T. inhibits the swarming ofProteus spp. which occasionally make interpretation difficult.


Vancomycin 3

Colistin 7.5

Nystatin 12.5

Trimethoprim 5



Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47°C together with otheradditives, mix gently and pour.

Reference:Thayer, J.D. and Martin, J.E. (1966). Improved medium selective forthe cultivation of N. gonorrhoeae and N. meningitidis. Public Healthrep. 81: 559-562.

X271GROWTH SUPPLEMENT, to improve the isolation of Neisseriaspp. from selective media.

For addition to GC agar base LAB 67.


L-cystine 11

L-cysteine 259

Thiamine HCl 0.03

Ferric nitrate 0.2

Co-Carboxylase 1

NAD 1.0

Guanine HCl 0.3

Adenine 10

L-glutamine 100

PABA 0.13

Vitamin B12 0.1

Add 1 vial to 1 litre of medium


5.7

NEW

Pseudomonas speciesX108

MODIFIED C.F.C. – CEPHALOTHIN, FUCIDIN, CETRIMIDEfor the selective isolation of Pseudomonas spp.

When added to LAB 108 Pseudomonas Agar, to prepare C.F.C.medium this supplement can be used to select pseudomonads fromfood and environmental samples.


Cephalothin 50

Fucidin 10

Cetrimide 10


Rehydrate contents of vial with 5ml of sterile 50% alcohol. Addaseptically to sterilised medium cooled to 47˚C, mix gently and pour.

Reference:Mead, G.C. and Adams, B.W. (1977). Br. Poult. Sci. 18: 661-667

X107C.N. CETRIMIDE, NALIDIXIC ACID for the isolation ofPseudomonas aeruginosa.

Suitable for use with LAB 108 Pseudomonas Agar to make themedium selective for Ps. aeruginosa.


Cetrimide 200

Nalidixic acid 15


Rehydrate contents of vial with 5ml of sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix gently and pour.

Reference:Goto, S., Enomoto, S. 1970. Jap. J. Microbiol. 14: 65-72.

X140TICARCILLIN, POLYMYXIN, for the isolation of Burkholderia(Pseudomonas) cepacia

Suitable for use with LAB 108 pseudomonas selective agar, orspecific selective bases such as that described by Gilligan et al.


Ticarcillin 100

Polymyxin 300,000 iu/litre

Add 1 vial to 500ml of medium


Reference:Gilligan, P.H., Gage, P.A., Bradshaw, L.M., Schidlow, D.V., DeCicco,B.T. (1985) Isolation medium for the recovery of Pseudomonascepacia from respiratory secretions of patients with cystic fibrosis.J.Clin.Microbiol. 22 (1) 5-8.

Pre-Incubation Test (P-INC)X019, X219

PENICILLIN, NISIN, CRYSTAL VIOLET, for accelerated shelflife determination of dairy products.

The Pre-incubation test uses a selective mixture to inhibit Grampositive organisms whilst allowing the growth of Gram negativebacteria, the main cause of post-pasteurisation contamination and amajor factor in determining the shelf life of the product.The technique is also useful for monitoring plant hygiene.


Penicillin 20,000iu/litre

Nisin 40,000iu/litre

Crystal violet 2.0

Add 1 vial of X019 to 200ml of Milk Agar LAB019

Add 1 vial of X219 to 1 litre of Milk Agar LAB019

Rehydrate contents of 1 vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix thoroughly andpour plates.

Method APre-incubate test material at 21˚C for 24hr. Prepare suitable dilutionseries, and inoculate Milk Agar plates containing P-INC supplement.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 usingthe tables of Griffith’s et al the shelf life can be determined.

Method BRehydrate X219 with 1ml of deionised water only, add 0.1ml to thetest material and incubate at 20˚C for 24hr. Prepare suitable dilutionseries, and inoculate Milk Agar plates. Proceed as for Method Aabove.

References:Griffiths, M.W., and Phillips, J.D. (1985) J.Appl.Bact. 57, 107.

Griffiths, M.W., and Phillips, J.D., and Muir, D.D. (1980) J. Soc.Dairy Technol. 33, 8.



Griffiths, M.W., and Phillips, J.D., and Muir, D.D. (1984) Rapiddetection of post-pasteurised contamination. Hannah Research Inst.Bulletin No.10.

Griffiths, M.W., and Phillips ,J.D., and Muir, D.D. (1984) Dairy Ind.Int. 50 (3) 25

Griffiths, M.W., and Phillips, J.D., and Muir, D.D. (1984) Post-pasteurisation contamination - the major cause of failure of freshdairy products. Hannah Research Inst.

Griffiths, M.W., and Phillips, J.D., and Muir, D.D. (1986) Aust. J.Dairy Technol. 41, 77-79.

5.8

SalmonellaX150

NOVOBIOCIN, for the isolation of Salmonella using semi-solidtechnology.

For addition to LAB 150 MSRV and LAB 537 Diassalm


Novobiocin 20 (MSRV)

Novobiocin 10 (Diassalm)

Add 1 vial to 500ml (MSRV)

Add 1 vial to 1 litre (Diassalm)


References:De Smedt, J.M., and Bolderdijk, R.F., (1986) Dynamics ofsalmonella isolation with modified semi-solid Rappaport Vassiliadismedium. J.Food Protection 50 658-661

Van Netten, P., Van Der Zee H., and Van Der Moosdijk A., (1991) Theuse of diagnostic selective semi-solid medium for the isolation ofSalsmonella enteritidis from poultry. Proceedings of the 10thsymposium on the quality of poultry meat. Spelderholt Beckbergen56-67.

StaphylococciX085

EGG YOLK TELLURITE

A sterile emulsion of egg yolk and potassium tellurite for use as aselective and differential agent in Baird-Parker Medium Base LAB85. The complete medium is selective for S.aureus, and the additionof egg yolk tellurite aids differentiation of this organism from otherscapable of growing on the agar.

Presented in 100ml bottles with a tellurite concentration of 0.2% togive a final concentration in the complete medium of 0.01% (w/v).Add 50ml to 1 litre of Baird-Parker Medium Base.

X086RPF: BOVINE FIBRINOGEN, RABBIT PLASMA, TRYPSININHIBITOR, POTASSIUM TELLURITE supplement for theisolation of Staphylococcus aureus.

For addition to LAB 85 Baird-Parker Medium.


Bovine Fibrinogen 0.375

Rabbit Plasma 2.5ml

Trypsin Inhibitor 2.5

Potassium Tellurite 2.5



X207METHICILLIN, for the isolation of Methicillin ResistantS.aureus (MRSA)

Suitable for use with LAB 7 Mannitol salt agar.


Methicillin 4

Add 1 vial of X207 to 1 litre of medium


X192OXACILLIN, POLYMYXIN B supplement for the isolation ofMethicillin Resistant Staphylococcus aureus (MRSA).

For addition to LAB 192, ORSIM (Oxcacillin ResistantStaphylococcus Isolation Medium).


Oxacillin 2

Polymyxin B 25,000 I.U


Rehydrate contents of vial by the addition of 5ml of sterile deionisedwater. Add aseptically to sterilised medium cooled to 47°C, mixgently and dispense.

StreptococciX013

COLISTIN, OXOLINIC ACID for the selective isolation ofstreptococci from clinical material.

When added to LAB 1 Columbia agar or LAB 15 Blood Agar BaseNo. 2, X013 renders the medium selective for streptococci. Alterationin haemolysis patterns may occur when azide or crystal violet areemployed as selective agents but this does not occur with X013.


Colistin 10

Oxolinic acid 5


Rehydrate contents of vial with 5ml of sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C together with otheradditives, mix gently and pour.

Reference:Petts, D. (1984). Colistin - Oxolinic Acid - Blood Agar: a newselective medium for streptococci. J. Clin. Microbiol. 19: 4-7.

5.9

NEW

NEW

Yeasts and MouldsX009, X209

CHLORAMPHENICOL for the selective isolation of yeasts andmoulds from food, environmental and clinical specimens.

Chloramphenicol’s broad antibiotic spectrum suppresses mostcontaminating bacteria allowing the yeasts and moulds to grow. It canbe added to such media as LAB 9 Sabouraud Dextrose Agar, LAB 36 Rose Bengal Chloramphenicol Agar, LAB 37 Malt ExtractAgar and LAB 117 Dermatophyte Test Medium to increase theirselectivity whilst not lowering the pH. Reduction of pH will increasethe selectivity of a yeast and mould medium but will also inhibitsome yeasts as well as having a deleterious effect on the agar gel.


Chloramphenicol 100



Rehydrate contents of vial with 5ml of Ethyl or Methyl alcohol. Addaseptically to sterilised medium cooled to 47˚C, mix gently and pour.

References:Jervis, B. (1973). Rose Bengal Chlortetracycline agar with othermedia for the selective isolation and enumeration of moulds andyeasts in foods. J. Appl. Bact. 36 Pages 723-727.

X089OXYTETRACYCLINE for O.G.Y.E. medium.

For use with LAB 89 Oxytetracycline Glucose Yeast Extract Agar forthe enumeration of yeasts and moulds from foodstuffs. Highlyproteinaceous foods and incubation above 30˚C will inactivateoxytetracycline.


Oxytetracycline 100


Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix gently and pour.

References:Mossel, D.A.A., et al. (1970). O.G.Y.E. for the selective enumerationof moulds and yeasts in food and clinical material. J. Appl. Bact. 35:454-457.

YersiniaX120

C.I.N. - CEFSULODIN, IRGASAN, NOVOBIOCIN for theisolation of Yersinia spp. from clinical and environmentalmaterial.

For addition to LAB 120 Yersinia C.I.N. Agar Base used in theselective isolation of Y. enterocolitica.


Cefsulodin 15

Irgasan 4

Novobiocin 2.5


Rehydrate contents of vial with 5ml of 30% sterile alcohol. Addaseptically to sterilised medium cooled to 47˚C, mix gently and pour.

References:Schiemann, D.A. (1979). Synthesis of a selective medium of Yersiniaenterocolitica. Can. J. Microbiol. 25 (2): 1298.

Schiemann, D.A. (1980). Isolation of toxigenic Yersiniaenterocolitica from retail pork products. J. Food Prot. 43: 360.

Schiemann, D.A. (1982). Development of a two-step enrichmentprocedure for recovery of Yersinia enterocolitica from food. Appl.Microbiol. 43 (1): 14.

5.10

6. Agars, Peptones, Extracts &Other Media Constituents

SourcingThe Lab M range of media constituents are selected on the basis ofquality and performance from the world’s leading suppliers. It is adeliberate policy not to invest in our own peptone manufacturingfacility in order to allow our microbiologist freedom to choose thebest ingredients available on the international market.

AgarsA range of agars are offered to suit all microbiological applications.Koch originally used gelatin to solidify culture media, but thesuperior properties of agar resulted in its universal adoption as thegelling agent of choice. Careful selection of agars is vital as they caninteract with nutrient components in a beneficial or deleteriousmanner.

Peptones and ExtractsLike agars, peptones and extracts are biologically variable productsrequiring careful selection. They provide the amino acids andpeptides required by micro-organisms for growth as well as othervital growth factors such as minerals, vitamins and nucleic acidfractions.

To ensure we use only the best available peptones and extracts thesematerials are exhaustively tested. Growth parameters are obtained byclassical microbiological techniques and by automated growth rateanalysis. Chemical and physical properties are also closelymonitored. LAB M can select specific peptones for special purposessuch as vaccine production and fermentation processes. If moreinformation is required on special services please contact LAB M oryour local agent.

Acid Hydrolysed CaseinMC 7

A soluble protein hyrolysate obtained by digesting casein with hotacid. It is almost free from growth factors, vitamins and antagonists,and these qualities make it suitable for use as a protein source inmedia for antibiotic and vitamin assays.

Typical Analysis

Appearance cream/white powder

Solubility in water at 5% total

Clarity clear and colourless

pH of 2% solution 6.0 ± 0.5

Total Nitrogen 8.3% ± 0.5

Total Amino Nitrogen 6.1% ± 0.5

Agar No. 1MC 2

A high clarity agar with good gelling properties and a lowconcentration of metal ions. This agar is suitable for allbacteriological purposes including sensitivity testing and pour platetechniques. A firm gel is obtained at working concentrations of 1.0 to1.5%. No significant precipitation is observed on reheating orprolonged holding at 65˚C.

Typical Analysis

Gel strength (Nikan) 650-1000g/m2

Colourimetry (1.5% soln at 65˚C) > 0.28 at 340nm

> 0.02 at 525nm

Melting point > 85˚C

Setting point 32-35˚C

pH 6.5-7.4

Moisture < 10%

Total ash < 3%

Calcium < 0.02%

Magnesium < 0.02%

Sodium chloride < 1.0%

Iron < 0.01%

Insoluble ash < 0.1%

Sulphate 1.5%

Salmonella Absent

TVC < 103/g

Spores < 2/g

Agar No. 2MC 6

A bacteriological agar which gives a firm gel at workingconcentrations of 1.0 to 1.5% which is reasonably clear. This agar isrecommended for all culture media except sensitivity testing mediaand those where absolute clarity is advantageous.

Typical Analysis

Gel strength (Nikan) 650-1000g/m2

Colourimetry (1.5% soln at 65˚C) > 0.3 at 340nm

>0.04 at 525nm

Appearance cream/white powder

Melting point >85˚C

Setting point 32-35˚C

pH 6.5-7.4

Moisture <12%

Total ash <3.5%

Calcium < 0.5%

Magnesium < 0.1%

Sodium chloride <1.0%

Iron < 0.01%

Insoluble ash < 0.1%

Sulphate < 3.0%

Salmonella Absent

TVC < 103/g

Spores < 2/g

6.1

Agar No.4Plant Tissue Culture Grade

MC 29LAB M Agar No.4 has been selected specifically for use as a gellingagent in plant tissue culture techniques. The product is selectedprimarily on gel strength, a parameter of particular importance forthis application, and then tested to ensure it meets the parameters setby a major plant producer. The agar contains no nutrients for plantgrowth and is designed to be incorporated into classical formulationssuch as Murashige and Skoog, as well as customer’s ownformulations.

Typical analysis

Ash 2.30%

Acid Insoluble Ash 0.16%

Calcium 0.31%

Magnesium 0.12%

Iron 0.018%

Total Nitrogen 0.15%

Recommended Concentration 0.75 - 1.5%

Melting Point 88-91˚C

Setting Point 32-33˚C

Mesh 80

pH (1.5% at 20˚C) 7.0 ± 0.2

Gel Strength (1.5% W/V) >700g/cm2

Bacteriological PeptoneMC 24

An economical source of nutrients provided by a balanced mixture ofmeat peptones and tryptone. The growth requirements of most nonfastidious organisms will be fulfilled by the range of amino acids,peptides and proteoses in this mixture.

Typical Analysis

Appearance cream powder


Clarity clear, pale straw colour


Total Nitrogen 12% ± 0.5

Amino Nitrogen 5% ± 0.5

Balanced Peptone No. 1MC 4

A rich mixture of tryptone and meat peptones which fulfills thenutritional demands of a wide variety of micro-organisms. Thispeptone is used in many LAB M culture media formulations.

Typical Analysis

Appearance beige powder





Amino Nitrogen 5.1% ± 0.5

Beef ExtractMC 19

This complements the nutritive properties of peptones in culturemedia and is often used as an added enrichment. Beef extract can beused as a direct replacement for meat peptones and, as it contains nocarbohydrates, can be used as a component of media for fermentationstudies.

Typical Analysis

Appearance light brown powder


Clarity clear, light brown colour




Bile Salts No. 3MC 25

A refined bile salt, comprising mainly sodium cholate and sodiumdesoxycholate. It is used as a selective agent in culture media such asViolet Red Bile Agar for the enumeration of coliforms, MacConkeyAgar No. 3 for the isolation and differentiation of enteric organisms,and SS Agar for the isolation of enteric pathogens. This fraction ofbile is highly active, allowing maximum selection of organisms ofenteric origin at relatively low concentrations (0.15%).

Typical Analysis

Appearance white powder


Clarity clear

pH of a 2% solution 8.0 ± 0.5

6.2

Columbia PeptoneMC12

DescriptionA blend of plant, meat and milk protein hydrolysates developed toprovide the nutritional requirements for a wide range of organisms.

Typical Analysis:




pH of 2% solution 6.7 - 7.3

Total Nitrogen 11.2 - 13.2%

Amino Nitrogen 4.2 – 6.2%

FMV (Foot and Mouth Vaccine)Peptones

MC33

DescriptionA special blend of peptones developed for use in the production ofFoot and Mouth vaccine.

Typical analysis:



Clarity clear, straw colour



Amino Nitrogen 2.5 - 5.5%

GelatineMC15

DescriptionA collagenous protein used for the solidification of culture media and for the detection and differentiation of certain proteolyticbacteria.

Typical Analysis:

Appearance buff crystalline powder

pH 5.7

Glucose (Dextrose)MC13

DescriptionGlucose for use in microbiological culture media.

IPTG (Isopropyl thiogalactopyranoside)dioxane free

MC402

DescriptionIPTG is used as an inducer of the lac Z operon. Incorporation of this compound into media containing X-β-galactoside (MC405), orequivalent chromogenic compound, enhances the colourdevelopment of organisms capable of fermenting lactose. Thiscombination of compounds is especially useful in transformationexperiments involving Escherichia coli where disruption of the lac Zoperon is used as a marker for DNA insertion.

Lactalbumin HydrolysateMC40

DescriptionLactalbumin is a protein removed from the whey, left after removalof casein from milk. Lactalbumin hydrolysate is a pancreatic digestof these proteins, containing high levels of essential amino acids. Itcan be used for tissue culture media and for production of vaccines ofviral origin. Other uses include growth of lactobacilli, clostridialspores and certain fermentation procedures.

Typical analysis:






Amino Nitrogen 5.5 ± 0.5

LactoseMC20

DescriptionLactose for use in microbiological culture media.

Liver DigestMC 34

Liver digest is prepared by the controlled hydrolysis of liver. It is richin vitamins and essential amino acids and has excellent nutritionalproperties and especially favours the growth of strict anaerobes andother fastidious microorganisms. Due to its capacity to stimulatesugar metabolism in saccharolytic organisms it is perfectly suited forthe growth of a broad range of organisms such as Clostridium,Leuconostoc, Bacillus, homo and hetero-fermentative Lactic acidbacteria, as well as yeasts and filamentous moulds.

Typical Analysis

Appearance light brown powder


Clarity clear, brown colour




6.3

NEW

NEW

NEW

NEW

NEW

NEW

NEW

Malt ExtractMC 23

A water soluble extract of malted barley suitable for use in thecultivation of yeasts and moulds. Malt extract has a very highcarbohydrate content and consequently is very sensitive to overheating which will cause a darkening of the medium.

Typical Analysis

Appearance yellow/brown powder


Clarity clear, light brown colour


Maltose 55%

Other Carbohydrates 40%

Protein 5%

Maltose MonohydrateMC22

DescriptionMaltose for use in microbiological culture media.

MannitolMC14

DescriptionD-Mannitol for use in microbiological culture media.

Meat PeptoneMC18

DescriptionA highly nutritious enzymatic digest of meat for use inmicrobiological culture media

Typical Analysis:







MUG (4-methylumbelliferyl-ββ-D-glucuronide)

MC406

DescriptionMUG is a fluorogenic compound used for the specific detection of E. coli in bacteriological culture media. MUG reagent is cleaved by the enzyme glucuronidase to release fluorescent 4-methylumbelliferone that can be detected under long wave UVlight (366nm) as a blue/green fluorescence. MUG can beincorporated into a range of culture media to enhance detection of E. coli. This product is available in 1g amounts.

Mycological PeptoneMC 9

A mixture of peptones with a high carbohydrate content suitable forthe rapid growth and colonial development of yeasts and moulds.Bacterial growth is inhibited by the low pH of this peptone.

Typical Analysis





Total Nitrogen 13% ± 0.5


Ox BileMC10

DescriptionOx bile is a dehydrated, purified fresh bile used as a selective agentin bile media, such as Brilliant Green Bile 2% Broth (LAB 51).

Proteose Peptone AMC 11

An enzymatic digest of meat adapted to encourage the production oftoxins by Corynebacterium diphtheriae, staphylococci, salmonellae,and clostridia. This peptone is highly nutritious and suitable for usein culture media for fastidious organisms such as Neisseria,Haemophilus and Pasteurella species.

Typical Analysis



Clarity clear, light straw colour




6.4

NEW

NEW

NEW

NEW

NEW

Skim Milk PowderMC 27

A bacteriological grade of thermophile free spray dried skim milk.Used in Milk Plate Count Agar LAB 115 and in media for diagnostictests involving the digestion or coagulation of casein and thefermentation of lactose. Recommended working concentration 10%.

Typical Analysis


Clarity opaque white suspension


Lactose 48.0% ± 0.5

Sodium ChlorideMC17

DescriptionSodium chloride for use in microbiological culture media.

Sodium ThioglycollateMC16

DescriptionSodium thioglycollate for use in bacteriological culture media. It isused to lower the oxidation-reduction potential of the medium and forthe neutralisation of mercurial compound preservatives.

IRRITANT

Keep away from oxidising agents.

Irritating to eyes, respiratory system and skin.

Wear suitable protective clothing and eye/face protection.

In case of contact, drench with water.

For eyes give prolonged irrigation with water.

Obtain immediate medical attention.

Sodium DesoxycholateMC26

Sodium desoxycholate is a specific bile acid, derived fromdeconjugated bile salts. Leifson showed that desoxycholic acid hadthe most inhibitory effect on bacterial growth, and that this could beenhanced by the removal of magnesium ions by chelating withsodium citrate. These components comprise the selective agents inDCA, DCA (Hynes) and DCLS.

Typical Analysis




Moisture <5%

Heavy metals <20 ppm

Sodium cholate <2%

Soy PeptoneMC 3

Prepared using the enzyme papain to digest soyabean meal, thispeptone is a rich source of nutrients with a high carbohydrate content.Most organisms will grow rapidly in this peptone but some bacteriawill produce high levels of acid leading to auto-sterilisation unless anadequate buffering system is incorporated.

Typical Analysis



Clarity clear, straw colour




Special TryptoneMC35

DescriptionSpecial tryptone is a high quality source of peptides produced byenzymatic digest of casein. It is a uniform, high quality peptoneproviding superior growth characteristics. Special tryptone is arefined hydrolysate, with very high solubility and clarity in solution.It is recommended for laboratory media and fermentation, especiallywhere high clarity of solution is required.

Typical Analysis:







TryptoneMC 5

An enzymatic hydrolysate of casein, rich in peptones and amino acids(including tryptophane). This peptone can be utilised by mostbacteria as a growth substrate. This peptone conforms to the U.S.P.requirements for a pancreatic digest of casein.

Typical Analysis







6.5

NEW

NEW

NEW

TryptoseMC 8

A blend of peptones suitable for the cultivation of most fastidiousorganisms including Neisseria gonorrhoeae, Streptococcus milleriand Brucella spp. especially where rapid or profuse growth isrequired such as in blood culture media and blood agars.

Typical Analysis



Clarity clear, light straw colour




Yeast Extract PowderMC 1

Prepared by the autolysis of Saccharomyces cerevisae underthermostatically controlled conditions to protect the B vitamins andother heat labile constituents. This extract provides a mixture ofamino acids, peptides, vitamins and carbohydrates making it suitablefor many applications.

Typical Analysis

Appearance yellow powder


Clarity clear, pale yellow




X-ββ-Galactoside (X-gal)MC405

DescriptionX-β-galactoside (X-gal or 5-Bromo-4-chloro-3-indolyl-β-D-galactoside) is used as a chromogenic substrate for the detection oforganisms capable of fermenting lactose (lac Z positive organisms).In combination with IPTG (MC402), X-gal can be used intransformation experiments involving Escherichia coli wheredisruption of the lac Z operon is used as a marker for DNA insertion.

XyloseMC32

DescriptionXylose for use in microbiological culture media.

6.6

NEW

NEW

6.7

TYPICAL ANALYSIS

MC 7 MC24 MC 4 MC 9 MC 11 MC 3 MC 5 MC 8 MC 1Acid 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%

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

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

LAB M Peptones

7. Sterile Additives and ReadyPrepared Media

AvailabilitySterile additive products are offered in ready-to-use format. Eachproduct has been prepared with an appropriate sterilisationprocedure, e.g. aseptic preparation, filtration or irradiation, whichwill not affect the performance of the product. All sterile reagentsshould be stored at 2-8ºC away from light.

The ready prepared media offer a cost effective alternative topreparation of large quantities of small volume dispensed media.

Cooked Meat with FABP069

Description:(100x10ml)

A culture medium useful for the recovery of fastidious anaerobicbacteria. This medium is also useful for prolonged storage of culturesand for the growth of a wide range of organisms.

Cooked Meat with Nutrient BrothP002


A medium for the growth of a wide range of organisms especiallyanaerobic bacteria.

Nutrient Agar SlopesP007


A medium for the short term maintenance and transport of a widerange of organisms.

Nutrient BrothP008


A medium for the growth of a wide range of organisms.

Peptone WaterP009


A general purpose growth medium.

Resuscitation BrothP081


A general medium for the resuscitation of microorganisms.

Selenite BrothP011E


A medium for the selective enrichment of salmonellae from faeces,food and sewage.

Selenite Cystine Broth P012E


A modification of the original selenite medium for the isolation ofsalmonellae.

Transport Medium (Amies Charcoal)P013


A modified version of Stuarts’s transport medium.

Trichomonas MediumP018


A medium for the cultivation of Trichomonas vaginalis and Candidaspp.

Urea Agar SlopeP015


A medium for the detection of urease activity by Proteus spp.

Serum BrothP017


Can be used as a basal medium for sugar fermentation tests forNeisseria.

7.1

Egg Yolk EmulsionX073

Description

A sterile emulsion of egg yolks for use in bacteriological culturemedia. It may be added directly to nutrient media for theidentification of Clostridium, Bacillus and Staphylococcus species bytheir lipase and/or lecithinase activity.

Presented in 100ml bottles, add 100ml to 900ml of Bacillus cereusmedium, or 50ml to Blood Agar Base LAB 28 containing Fildesextract and serum.

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 ofsterile Blood Agar Base (LAB 28) or Nutrient Broth No.2 (LAB 14).In order to clear the medium, raise the final salt concentration by theaddition of 1% of sodium chloride. After incubation for up to 5 daysat 35ºC, lecithinase-producers render the broth opalescent, whilst, onthe solid medium, the colonies are surrounded by opaque zones.

Egg Yolk Tellurite EmulsionX085

Description

A sterile egg yolk emulsion containing potassium tellurite for use inBaird-Parker Medium (LAB 85). Baird-Parker Medium is widelyused in the food industry for the detection of pathogenicstaphylococci. Baird-Parker plates containing Egg Yolk TelluriteEmulsion should be protected from dehydration by storing in vapourproof packaging.

Directions

Add 50ml to 1 litre of Baird-Parker Medium LAB 85.

(50ml Egg Yolk Tellurite Emulsion contains the equivalent of 3ml of3.5% potassium tellurite. This is the amount recommended for 1 litreof Baird-Parker Medium, i.e. concentration in X085 is 0.21% w/v.Final concentration in Baird-Parker Medium is 0.01% w/v.)

Lactic Acid 10%X037

Description

A sterile solution of 10% lactic acid added to culture media to reducethe pH, in order to suppress bacterial growth.

Directions

Add 1 vial (5ml) to 1 litre of Malt Extract Agar LAB 37.

Add 2 vials (10ml) to 1 litre of Potato Dextrose Agar LAB 98.

Addition of X037 should be carried out after sterilisation and coolingthe medium to 47ºC.

Potassium Tellurite Solution 3.5%X027

Description

A sterile solution of 3.5% potassium tellurite. A selective agent foraddition to Hoyles’s Medium (LAB 27), for the selective isolationand differentiation of Corynebacterium diphtheriae.

Directions

Add 1 vial (2ml) to 200ml of Hoyle’s Medium (LAB 27).

Urea Solution 40%X130/X135

Description

A sterile solution of 40% urea, for addition to Urea Broth Base (LAB 131) and Urea Agar Base (LAB 130) for the detection of urease production by Proteus spp.

Directions

Add 1 vial X130 (5ml) to 95ml of Urea Broth Base (LAB 131) andUrea Agar Base (LAB 130). X135 contains 100ml of solution forproduction of larger volumes of Urea Broth or Agar.

7.2

CAP 1 Captivate™ O157 4.1

CAP 2 Captivate™ Salmonella 4.3





HAL 1 Harlequin™ Salmonella ABC 2.1

HAL 2 Harlequin™ Listeria Medium 2.2, 5.5

HAL 3 Harlequin™ TBGA (TBX) formerly LAB 162 xxiv, 2.2, 4.3

HAL 4 Harlequin™ LB Agar 2.3, 3.1, 3.2

HAL 5 Harlequin™ LB Top Agar 2.3, 3.1

HAL 6 Harlequin™ SMAC-BCIG xxiv, 1.40, 2.3, 4.1, 4.3, 5.4

HAL 7 Harlequin™ CLED 2.3

HAL 8 Harlequin™ E. coli/Coliform Medium 2.4

HAL 9 Harlequin™ mLGA 2.5

LAB 1 Columbia Agar Base 1.13, 5.1, 5.4, 5.9

LAB 2 MacConkey Agar (without salt) 1.29, 1.30

LAB 3 DCLS Agar 1.15

LAB 4 Tryptone Soy Broth USP xv, 1.58

LAB 5 MacConkey Broth Purple xxiv, 1.31

LAB 6 CLED Medium (Bevis-double indicator) 1.12, 1.23

LAB 7 Mannitol Salt Agar 1.32, 1.49, 5.9

LAB 8 Nutrient Agar 1.39, 1.57

LAB 9 Sabouraud Dextrose Agar 1.48

LAB 10 Plate Count Agar APHA xxiii, 1.43

LAB 11 Tryptone Soy Agar USP xv, 1.58

LAB 12 Sensitivity Test Agar (STA) 1.50

LAB 13A Bismuth Sulphite Agar Base A 1.4, 1.1.8

LAB 13B Bismuth Sulphite Agar Base B 1.4, 1.1.8

LAB 14 Nutrient Broth No. 2 BP 1.13, 1.14, 1.39, 7.2

LAB 15 Blood Agar Base No. 2 1.5, 5.9

LAB 16 Fluorescence Agar 1.22

LAB 18 Yeast Extract Agar xxiii, 1.64

LAB 19 Milk Agar xxiii, 1.33, 5.8

LAB 20 Dextrose Tryptone Agar 1.16

LAB 22 Reinforced Clostridial Medium (Broth) 1.46

LAB 23 Reinforced Clostridial Agar 1.46

LAB 24 Cooked Meat Granules 1.13, 1.14

LAB 24Z Cooked Meat Medium Tablets 1.14

LAB 25 Fluid Thioglycollate Medium USP xv, 1.21, 1.55

LAB 27 Hoyle’s Medium 1.24, 7.2

LAB 28 Blood Agar Base 1.5, 7.2

LAB 29 Desoxycholate Citrate Agar (DCA) 1.14

LAB 30 MacConkey Agar (with salt) 1.29

LAB 31 Violet Red Bile Agar (VRBA) xxiii, 1.61, 1.62

LAB 32 XLD Agar xxvi, xxvii, 1.64

LAB 33 Sabouraud Liquid Medium USP 1.48

LAB 34 Brilliant Green Agar (Modified) xxvi, xxvii, 1.7

LAB 35 TYC Medium 1.59

LAB 36 Rose Bengal Chloramphenicol Agar xxv, 1.41,

1.47, 5.10

LAB 37 Malt Extract Agar xxv, 1.31, 5.10, 7.2

LAB 38 Wort Agar 1.63

LAB 39 Mueller Hinton Agar II 1.37

LAB 41 CLED Medium (single indicator) 1.12

LAB 42 Mueller Kauffman Tetrathionate Broth xxvii, 1.38

LAB 44A Selenite Broth Base 1.49

LAB 44B Sodium Biselenite xxvii, 1.49, 1.50

LAB 45 MacConkey Agar No. 3 1.30, 1.52

LAB 46 Buffered Peptone Water xxvi, xxvii, 1.9,

1.10, 4.1, 5.4

LAB 48 Brain Heart Infusion Agar 1.6

LAB 49 Brain Heart Infusion Broth xiv, xix, 1.6, 1.51

LAB 51 Brilliant Green Bile 2% Broth xxiv, 1.8, 1.19,

1.34, 6.4

LAB 52 SS Agar (Modified) ix, 1.53

LAB 53 Triple Sugar Iron Agar 1.56, 1.1.8

LAB 54 Lysine Iron Agar 1.1.8

LAB 55A Selenite Cystine Broth Base xxvii, 1.50

LAB 59 Kligler Iron Agar 1.26

LAB 60 Endo Agar 1.19

LAB 61 Eosin Methylene Blue Agar (Levine) 1.20

LAB 62 Tryptose Phosphate Broth 1.59

LAB 63 Tryptone Glucose Extract Agar 1.57

LAB 64 Thioglycollate Medium (Brewer) 1.55

LAB 65 Desoxycholate Citrate Agar (Hynes) 1.15

LAB 66 Anaerobe Identification Medium Base 1.2

LAB 67 GC Agar Base 1.23, 5.7

LAB 68 Nutrient Broth ‘E’ 1.39

LAB 69 Simmons Citrate Agar 1.51

LAB 71 Fastidious Anaerobe Broth (FAB) xv, 1.13, 1.21, 1.55

LAB 72 Tryptone Bile Agar xxiv, 1.57, 2.2, 4.3

LAB 73 Bacillus Cereus Medium (PREP) xxv, 1.3, 5.2, 7.2

LAB 74 Nusens Agar 1.38

LAB 75 Todd Hewitt Broth 1.55

LAB 78 CEMO Agar 1.10

LAB 79 WL Nutrient Agar 1.62

LAB 80A Minerals Modified Glutamate Medium xxiv, 1.34, 1.57

LAB 80B Sodium Glutamate xxiv, 1.34, 1.57

LAB 82 Membrane Lauryl Sulphate Broth 1.33

LAB 84 Single Step Staph Selective Agar (4S) 1.49, 1.51

LAB 85 Baird-Parker Medium xxv, 1.3, 5.9, 7.2

LAB 86 Rappaport Vassiliadis Medium (RVS) xxvii, 1.45

LAB 87 Sugar Free Agar 1.53

LAB 88 Violet Red Bile Glucose

Agar (VRBGA) xxiii, 1.19, 1.62

LAB 89 Oxytetracycline Glucose Yeast

Extract Agar xxv, 1.41, 5.10

LAB 90 Fastidious Anaerobe Agar (FAA) 1.20, 5.1, 5.3

LAB 91 E.E. Broth 1.19

LAB 92 M17 Agar 1.29

LAB 93 MRS Agar 1.35, 1.36

LAB 94 MRS Broth 1.36 8.1

By product code (V1.0)

8. INDEXES

LAB 95 DN’ase Agar 1.17

LAB 96 TCBS Cholera Medium 1.54

LAB 97 Tetrathionate Broth (APHA) xxvii, 1.54

LAB 98 Potato Dextrose Agar 1.44, 7.2

LAB 99 Wort Broth 1.63

LAB 100 Ringer’s Solution 1/4 Strength ix, 1.32, 1.47

LAB 100ZRinger’s Solution 1/4 Strength Tablets ix, 1.47

LAB 101 Ringer’s Solution (Calgon) ix, 1.47

LAB 102 Ringer’s Solution (Thiosulphate) ix, 1.47

LAB 103 Maximum Recovery Diluent ix, xv, xxiii-xxvi, 1.32

LAB 104 Peptone Water 1.42, 1.59

LAB 105 China Blue Lactose Agar 1.11

LAB 106 Kanamycin Aesculin Azide Agar (complete) 1.25, 1.52

LAB 107 Kanamycin Aesculin Azide Broth (complete) 1.25

LAB 108 Pseudomonas Agar Base xxv, 1.44, 5.8

LAB 109 Perfringens Agar OPSP xxvi, 1.42, 5.3

LAB 110 Hektoen Enteric Medium 1.23

LAB 111 Sabouraud Maltose Agar 1.48

LAB 112 Campylobacter Blood Free

(modified CCDA) xiv, xxvii, 1.9, 1.10, 5.2

LAB 113Z Salt Meat Broth Tablets 1.49

LAB 114 Mueller Hinton Broth II 1.37

LAB 115 Milk Plate Count Agar xxiii, 1.33, 1.34, 6.5

LAB 116 MLCB Agar 1.35

LAB 117 DTM Dermatophyte Test Medium 1.16, 5.10

LAB 119 Yeast Extract Dextrose

Chloramphenicol Agar 1.65

LAB 120 Yersinia CIN Agar Base 1.65, 5.10

LAB 121 Bromocresol Purple Lactose Agar 1.8

LAB 122 Listeria Isolation Medium (Oxford) xxvi, 1.22,

1.28, 5.5, 5.6

LAB 123 Kirchner’s T.B. Enrichment Medium 1.26, 5.7

LAB 124 Amies Transport Medium With Charcoal 1.1

LAB 125 Amies Transport Medium Without Charcoal 1.1

LAB 126 Lactose Broth 1.27, 1.33

LAB 127 Cooked Meat Medium 1.14

LAB 129 Tryptone Water xxiv, 1.8, 1.33, 1.34, 1.59

LAB 130 Urea Agar Base 1.60, 7.2

LAB 131 Urea Broth Base 1.60, 7.2

LAB 133 Cetrimide Agar 1.11

LAB 135 Campylobacter Enrichment

Broth (Bolton) xxvii, 1.10, 5.3

LAB 136 Easter - Gibson Pre-enrichment Medium 1.18

LAB 137 Easter - Gibson Salmonella Medium 1.18, 5.5

LAB 138 Listeria Enrichment Broth (FDA) xxvi, 1.28,

5.5, 5.6

LAB 139 Buffered Listeria Enrichment Broth xxvi, 1.9, 5.5, 5.6

LAB 140 Helicobacter Pylori Agar Base 1.24, 5.4

LAB 144 Palcam Broth 1.42, 5.5

LAB 147 Orange Serum Agar 1.40

LAB 148 Palcam Agar Base xxvi, 1.22, 1.41, 1.42, 5.5

LAB 149 Plate Count Agar xxiii, 1.43

LAB 150 MSRV Medium xxvi, 1.36, 5.9

LAB 155 UVM Base xxvi, 1.61, 5.6

LAB 157 Aseptic Commissioning Broth 1.2

LAB 158 Liquid Baird-Parker Medium 1.27

LAB 159 Malt Extract Broth 1.32

LAB 160 Brazier’s CCEY Agar 1.6

LAB 161 Sorbitol MacConkey Agar xxiv, 1.40, 1.52,

2.3, 4.1, 4.3, 5.4

LAB 162 Now HAL 3 (TBGA) 2.2

LAB 163 R2A Medium 1.45

LAB 164 Fraser Broth xxvi, 1.22, 1.61, 5.5, 5.6

LAB 165 O157 Broth MTSB xxiv, 1.40, 2.3, 4.1, 5.3

LAB 166 Slanetz & Bartley Medium 1.52

LAB 167 Aeromonas Agar 1.1

LAB 168 LB Agar 3.2

LAB 169 LB Broth 3.3

LAB 170 Susceptibility Test ‘ISO’ Agar 1.1.10

LAB 171 EC Medium 1.1.3

LAB 172 Listeria Monocytogenes Blood

Agar (LMBA) 1.1.7, 5.6

LAB 173 LB Broth (Lennox) 3.3

LAB 174 LB Agar (Lennox) 3.2

LAB 175 YPD Broth 3.5

LAB 176 YPD Agar 3.5

LAB 177 Superbroth 3.4

LAB 178 Superbroth with Agar 3.4

LAB 179 2 x YT Broth 3.6

LAB 180 2 x YT Agar 3.6

LAB 181 NZY Broth (NZYM) 3.4

LAB 182 NZCYM Broth 3.4

LAB 183 Terrific Broth 3.5

LAB 184 Letheen Broth (AOAC) 1.1.6

LAB 185 Letheen Agar (AOAC) 1.1.6

LAB 186 D/E Neutralising Broth Base 1.1.3

LAB 187 D/E Neutralising Broth 1.1.2, 1.1.3

LAB 188 D/E Neutralising Agar 1.1.2

LAB 189 Microbial Content Test Agar (MCA) 1.1.8

LAB 191 Luria Bertani (Hi Salt) Broth 3.3

LAB 192 ORSIM (Oxacillin Resistant Staphylococci

Isolation Medium) 1.1.9, 5.9

LAB 193 PEMBA (Bacillus Cereus Medium) xxv, 1.1.9, 5.2, 7.2

LAB 194 Perfringens Agar Base (TSC) xxvi, 1.1.10, 5.3

LAB 196 Lauryl Tryptose Broth 1.1.5

LAB 197 Water Plate Count Agar (ISO) 1.1.12

LAB 505 Cary Blair Medium 1.1.1

LAB 523 Azide Blood Agar Base 1.1.1

LAB 525 Eugon Agar 1.1.4

LAB 526 Eugon Broth 1.1.5

LAB 537 Diassalm xxvi, 1.17, 5.9

LAB 573 Violet Red Bile Agar with MUG 1.1.11

MC 1 Yeast Extract 6.6, 6.7

MC 2 Agar No. 1 Bacteriological - High Clarity 6.1

MC 3 Soy Peptone 6.5, 6.7

MC 4 Balanced Peptone No.1 6.2, 6.7

MC 5 Tryptone 6.5, 6.7

MC 6 Agar No. 2 Bacteriological - Gen. Purpose xxiv, 6.1

MC 7 Acid Hydrolysed Casein 6.1, 6.7

MC 8 Tryptose 6.6, 6.7

MC 9 Mycological Peptone 6.4, 6.7

MC 10 Ox Bile 6.4

8.2

MC 11 Proteose Peptone A 6.4, 6.7

MC 12 Columbia Peptone 6.3

MC 13 Glucose (Dextrose) 6.3

MC 14 Mannitol 6.4

MC 15 Gelatine 6.3

MC 16 Sodium Thioglycollate 6.5

MC 17 Sodium Chloride 6.5

MC 18 Meat Peptone 6.4

MC 19 Beef Extract 6.2

MC 20 Lactose 6.3

MC 22 Maltose Monohydrate 6.4

MC 23 Malt Extract 6.4

MC 24 Bacteriological Peptone 6.2, 6.7

MC 25 Bile Salts No. 3 6.2

MC 26 Sodium Desoxycholate 6.5

MC 27 Skim Milk Powder 6.5

MC 29 Agar No. 4 - Plant Tissue Culture Grade 6.2

MC 32 Xylose 6.6

MC 33 FMV Peptone 6.3

MC 34 Liver Digest 6.3

MC 35 Special Tryptone 6.5

MC 40 Lactalbumin Hydrolysate 6.3

MC 402 IPTG 6.3, 6.6

MC 405 X-gal 6.3, 6.6

MC 406 MUG 1.1.5, 4.3, 6.4

P002 Cooked meat with nutient broth 7.1

P007 Nutrient Agar Slopes 7.1

P008 Nutrient Broth 7.1

P009 Peptone Water 7.1

P011E Selenite Broth 7.1

P012E Selenite Cystine Broth 7.1

P013 Transport Medium (Amies Charcoal) 7.1

P015 Urea Agar Slope 7.1

P017 Serum Broth 7.1

P018 Trichomonas Medium 7.1

P069 Cooked meat with FAB 7.1

P081 Resuscitation broth 7.1

X009 Chloramphenicol 1.16, 1.47, 1.48, 1.65, 5.10

X011 Colistin, Nalidixic Acid 1.5, 1.13, 5.4

X012 Colistin, Nalidixic Acid 1.5, 1.13, 5.4

X013 Colistin, Oxolinic Acid 1.5, 1.13, 5.9

X015 Neomycin 75 1.5, 1.13, 5.1, 5.2

X016 Neomycin 100 1.5, 1.13, 5.2

X018 Kanamycin 75 5.2

X019 P-INC Supplement 1.33, 5.8

X027 Potassium Tellurite Solution 3.5% 1.24, 1.51, 7.2

X037 Lactic Acid 10% 1.31, 1.44, 7.2

X040 VCA 1.24, 5.4

X068 VCNT (for Thayer Martin) 1.23, 5.7

X069 LCT (for New York medium) 1.23, 5.7

X070 LCAT (for New York medium) 1.23, 5.7

X072 Polymyxin B, Ceftazimide 1.1.7, 1.1.9, 5.6

X072N Nalidixic Acid 1.1.7, 5.6

X073 Egg Yolk Emulsion 1.3, 1.6, 1.7, 1.51, 1.1.9, 1.1.10, 5.2, 7.2

X074 Polymyxin B 1.3, 5.2

X085 Egg Yolk Tellurite xxv, 1.3, 1.4, 5.9, 7.2

X086 RPF xxv, 1.3,1.4, 5.9

X089 Oxytetracycline (for OGYE) 1.41, 1.49, 5.10

X090 Nalidixic Acid, Vancomycin 1.20, 5.1

X091 Nalidixic Acid 1.20, 5.1

X092 Metronidazole, Nalidixic Acid 1.20, 5.1

X093 Cycloserine, Cefoxitin 1.6, 1.7, 1.20, 5.3

X107 CN (for Pseudomonas. aeruginosa) 1.44, 5.8

X108 CFC (for Pseudomonas spp) xxv, 1.22, 1.44, 5.8

X109 Sulphadiazine 1.42, 5.3

X110 Oleandomycin, Polymyxin 1.42, 5.3

X112 Cefoperazone, Amphotericin 1.9, 5.2

X120 CIN Selective Supplements 1.65, 5.10

X122 CCCAF (for Oxford medium) 1.28, 2.2, 5.5

X123 CNCAF (for Oxford medium) 1.28, 2.2, 5.6

X124 PTTA (T.B. supplement) 1.26, 5.7

X130 Urea solution 40% 5ml 1.60, 7.2

X131 CVTC 1.10, 5.3

X132 CVTN 1.10, 5.3

X135 Urea solution 40% 100ml 1.60, 7.2

X137 TMAO Selenite 1.18, 5.5

X138 NAC (for FDA broth) 1.9, 1.28, 5.5

X139 NAN 1.9, 1.28, 5.6

X140 Burkholderia (Pseudomonas) cepacia

Selective Supplement 1.44, 5.8

X144 PAC (for Palcam Media) 1.41, 1.42, 5.5

X150 Novobiocin (for MSRV/

Diassalm/O157 Broth) 1.17, 1.36, 1.40, 4.1, 5.3, 5.9

X155 UVM I Supplement 1.61, 5.6

X156 UVM II Supplement 1.61, 5.6

X161 Cefixime Tellurite (for SMAC/SMAC-BCIG) xxiv, 1.40,1.52, 2.3, 4.1, 5.4

X164 Fraser Supplement (1/2 strength) 1.22, 5.5

X165 Fraser Supplement (full strength) 1.22, 5.6

X192 Oxacillin, Polymyxin B 1.1.9, 5.9

X193 Polymyxin B 1.1.9, 5.2

X194 Cycloserine 1.1.10, 5.3

X207 Methicillin (for MRSA) 1.32, 5.9

X209 Chloramphenicol 1.16, 1.47, 1.48, 1.65, 5.10

X212 Cefoperazone, Amphotericin 1.9, 5.2

X214 Skirrows 1.13, 5.2

X215 Neomycin 75 1.5, 1.13, 5.1

X219 P-INC Supplement 1.33, 5.8

X260 Bacitracin 5.4

X268 VCNT (for Thayer Martin) 1.23, 5.7

X269 LCT (for New York medium) 1.23, 5.7

X270 LCAT (for New York medium) 1.23, 5.7

X271 GC Growth Supplement 1.23, 5.7

X290 Nalidixic Acid, Vancomycin 1.20, 5.1

X291 Nalidixic Acid 1.20, 5.1

X539 NAN 1.9, 1.28, 5.6

X546 VCC Supplement 4.1, 5.4

X555 UVM I Supplement 1.61, 5.6

X564 Fraser Supplement (1/2 strength) 1.22, 5.5

8.3

Acid Hydrolysed Casein MC 7 6.1, 6.7

Aeromonas Agar LAB 167 1.1

Agar No. 1 Bacteriological - High Clarity MC 2 6.1

Agar No. 2 Bacteriological - Gen. Purpose MC 6 xxiv, 6.1

Agar No. 4 - Plant Tissue Culture Grade MC 29 6.2

Amies Transport Medium With Charcoal LAB 124 1.1

Amies Transport Medium Without Charcoal LAB 125 1.1

Anaerobe Identification Medium Base LAB 66 1.2

Aseptic Commissioning Broth LAB 157 1.2

Azide Blood Agar Base LAB 523 1.1.1

Bacillus Cereus Medium (PREP) LAB 73 xxv, 1.3,5.2, 7.2

Bacillus Cereus Medium (PEMBA) LAB 193 xxv, 1.1.9,5.2, 7.2

Bacitracin X260 5.4

Bacteriological Peptone MC24 6.2, 6.7

Baird-Parker Medium LAB 85 xxv, 1.3, 5.9,7.2

Balanced Peptone No. 1 MC4 6.2, 6.7

Beef Extract MC19 6.2

Bile Salts No. 3 MC 25 6.2

Bismuth Sulphite Agar Base A LAB 13A 1.4, 1.1.8

Bismuth Sulphite Agar Base B LAB 13B 1.4, 1.1.8

Blood Agar Base LAB 28 1.5, 7.2

Blood Agar Base No. 2 LAB 15 1.5, 5.9

BPLS LAB 34 xxvi, xxvii, 1.7

Brain Heart Infusion Broth LAB 49 xiv, xix,

1.6, 1.51

Brazier’s CCEY Agar LAB 160 1.6

Brilliant Green Agar (Modified) LAB 34 xxvi,

xxvii, 1.7

Brilliant Green Bile 2% Broth LAB 51 xxiv, 1.8,

1.19. 1.34, 6.4

Bromocresol Purple Lactose Agar LAB 121 1.8

Buffered Listeria Enrichment Broth LAB 139 xxvi,

1.9, 5.5, 5.6

Buffered Peptone Water LAB 46 xxvi, xxvii,

1.9, 1.10, 4.1, 5.4

Burkholderia (Pseudomonas) cepaciaSelective Supplement X140 5.8

Campylobacter Blood Free (modified CCDA) LAB 112 xiv, xxvii,

1.9, 1.10, 5.2

Campylobacter Enrichment Broth LAB 135 xxvii,

1.10, 5.3

Captivate™ O157 CAP 1 4.1




Captivate™ Salmonella CAP 2 4.3


Cary Blair Medium LAB 505 1.1.1

CCCAF (for Oxford medium) X122 1.28, 2.2, 5.5

Cefixime Tellurite

(for SMAC/SMAC-BCIG) X161 xxiv, 1.40, 1.52,

2.3, 4.1, 5.4

Cefoperazone, Amphotericin X112 1.9, 5.2

Cefoperazone, Amphotericin X212 1.9, 5.2

CEMO Agar LAB 78 1.10

Cetrimide Agar LAB 133 1.11

CFC (for Pseudomonas spp) X108 xxv, 1.22,

1.44, 5.8

China Blue Lactose Agar LAB 105 1.11

Chloramphenicol X009 1.16, 1.47,

1.48, 1.65, 5.10

Chloramphenicol X209 1.16, 1.47, 1.48,1.65, 5.10

CIN Selective Supplements X120 1.65, 5.10

CLED Medium (Bevis-double indicator) LAB 6 1.12, 1.23

CLED Medium (single indicator) LAB 41 1.12

CN (for Pseudomonas aeruginosa) X107 1.44, 5.8

CNCAF (for Oxford Medium) X123 1.28, 2.2, 5.6

Colistin, Nalidixic Acid X011 1.5, 1.13, 5.4

Colistin, Nalidixic Acid X012 1.5, 1.13, 5.4

Colistin, Oxolinic Acid X013 1.5, 1.13, 5.9

Columbia Agar Base LAB 1 1.13, 5.1,

5.4, 5.9

Columbia Peptone MC 12 6.3

Cooked Meat Granules LAB 24 1.13, 1.14

Cooked Meat Medium LAB 127 1.14

Cooked Meat Medium Tablets LAB 24Z 1.14

Cooked meat with FAB P069 7.1

Cooked meat with nutrient broth P002 7.1

CVTC X131 1.10, 5.3

CVTN X132 1.10, 5.3

Cycloserine X194 1.1.10, 5.3

Cycloserine, Cefoxitin X093 1.6, 1.7, 1.20, 5.3

D/E Neutralising Agar LAB 188 1.1.2

D/E Neutralising Broth LAB 187 1.1.2, 1.1.3

D/E Neutralising Broth Base LAB 186 1.1.3

DCLS Agar LAB 3 1.15

Desoxycholate Citrate Agar (DCA) LAB 29 1.14

Desoxycholate Citrate Agar (Hynes) LAB 65 1.15

Dextrose Tryptone Agar LAB 20 1.16

Diassalm LAB 537 xxvi,

1.17, 5.9

DN’ase Agar LAB 95 1.17

DTM Dermatophyte Test Medium LAB 117 1.16, 5.10

E.E. Broth LAB 91 1.19

Easter - Gibson Pre-enrichment Medium LAB 136 1.18

Easter - Gibson Salmonella Medium LAB 137 1.18, 5.5

EC Medium LAB 171 1.1.3

Egg Yolk Emulsion X073 1.3, 1.6, 1.7,1.51, 1.1.9, 1.1.10,

5.2, 7.2

Egg Yolk Tellurite X085 xxv, 1.3, 1.4,5.9, 7.2

Endo Agar LAB 60 1.19

Eosin Methylene Blue Agar (Levine) LAB 61 1.20

Eugon Agar LAB 525 1.1.4

Eugon Broth LAB 526 1.1.5

Fastidious Anaerobe Agar (FAA) LAB 90 1.20, 5.1, 5.3

Fastidious Anaerobe Broth (FAB) LAB 71 xv, 1.13,

1.21, 1.558.4

By product name

Fluid Thioglycollate Medium USP LAB 25 xv, 1.21, 1.55

Fluorescence Agar LAB 16 1.22

FMV Peptone MC 33 6.3

Fraser Broth LAB 164 xxvi, 1.22,

1.61, 5.5, 5.6

Fraser Supplement (1/2 strength) X164 1.22, 5.5

Fraser Supplement (1/2 strength) X564 1.22, 5.5

Fraser Supplement (full strength) X165 1.22, 5.6

GC Agar Base LAB 67 1.23, 5.7

GC Growth Supplement X271 1.23, 5.7

Gelatine MC 15 6.3

Glucose (Dextrose) MC 13 6.3

Harlequin™ CLED HAL 7 2.3

Harlequin™ E. coli/Coliform Medium HAL 8 2.4

Harlequin™ LB Agar HAL 4 2.3, 3.1, 3.2

Harlequin™ LB Top Agar HAL 5 2.3, 3.1

Harlequin™ Listeria Medium HAL 2 2.2, 5.5

Harlequin™ mLGA HAL 9 2.5

Harlequin™ Salmonella ABC HAL 1 2.1

Harlequin™ SMAC-BCIG HAL 6 xxiv, 1.40, 2.3,

4.1, 4.3, 5.4

Harlequin™ TBGA (TBX) HAL 3 xxiv, 2.2, 4.3

Hektoen Enteric Medium LAB 110 1.23

Helicobacter Pylori Agar Base LAB 140 1.24, 5.4

Hoyle’s Medium LAB 27 1.24, 7.2

IPTG MC 402 6.3, 6.6

Kanamycin 75 X018 5.2

Kanamycin Aesculin Azide

Agar (complete) LAB 106 1.25, 1.52

Kanamycin Aesculin Azide

Broth (complete) LAB 107 1.25

Kirchner’s T.B. Enrichment Medium LAB 123 1.26, 5.7

Kligler Iron Agar LAB 59 1.26

Lactalbumin Hydrolysate MC 40 6.3

Lactic Acid 10% X037 1.31, 1.44, 7.2

Lactose MC 20 6.3

Lactose Broth LAB 126 1.27, 1.33

Lauryl Tryptose Broth LAB 196 1.1.5

LB Agar LAB 168 3.2

LB Agar (Lennox) LAB 174 3.2

LB Broth LAB 169 3.3

LB Broth (Lennox) LAB 173 3.3

LCAT (for New York medium) X070 1.23, 5.7

LCAT (for New York medium) X270 1.23, 5.7

LCT (for New York medium) X069 1.23, 5.7

LCT (for New York medium) X269 1.23, 5.7

Letheen Agar (AOAC) LAB 185 1.1.6

Letheen Broth (AOAC) LAB 184 1.1.6

Liquid Baird-Parker Medium LAB 158 1.27

Listeria Enrichment Broth (FDA) LAB 138 xxvi, 1.28,

5.5, 5.6

Listeria Isolation Medium (Oxford) LAB 122 xxvi, 1.22,

1.28, 5.5, 5.6

Listeria Monocytogenes Blood

Agar (LMBA)LAB 172 1.1.7, 5.6

Liver Digest MC 34 6.3

Luria Bertani (Hi Salt) Broth LAB 191 3.3

Lysine Iron Agar LAB 54 1.1.8

M17 Agar LAB 92 1.29

MacConkey Agar (without salt) LAB 2 1.29, 1.30

MacConkey Agar (with salt) LAB 30 1.29

MacConkey Agar No. 3 LAB 45 1.30, 1.52

MacConkey Broth Purple LAB 5 xxiv, 1.31

Malt Extract MC 23 6.4

Malt Extract Agar LAB 37 xxv, 1.31,

5.10, 7.2

Malt Extract Broth LAB 159 1.32

Maltose monohydrate MC 22 6.4

Mannitol MC 14 6.4

Mannitol Salt Agar LAB 7 1.32,

1.49, 5.9

Maximum Recovery Diluent LAB 103 ix, xv, xxiii-

xxvi, 1.32

Meat Peptone MC 18 6.4

Membrane Lauryl Sulphate Broth LAB 82 1.33

Methicillin (for MRSA) X207 1.32, 5.9

Metronidazole, Nalidixic Acid X092 1.20, 5.1

Microbial Content Test Agar (MCA) LAB 189 1.1.8

Milk Agar LAB 19 xxiii, 1.33, 5.8

Milk Plate Count Agar LAB 115 xxiii, 1.33,

1.34, 6.5

Minerals Modified Glutamate Medium LAB 80A xxiv, 1.34,

1.57

MLCB Agar LAB 116 1.35

Modified Tryptone Soy Broth LAB 165 xxiv, 1.40,2.3, 4.1, 5.3

MRS Agar LAB 93 1.35, 1.36

MRS Broth LAB 94 1.36

MSRV Medium LAB 150xxvi, 1.36, 5.9

Mueller Hinton Agar II LAB 39 1.37

Mueller Hinton Broth II LAB 114 1.37

Mueller Kauffman Tetrathionate Broth LAB 42 xxvii, 1.38

MUG MC 406 1.1.5, 4.3, 6.4

Mycological Peptone MC 9 6.4, 6.7

NAC (for FDA broth) X138 1.9, 1.28, 5.5

Nalidixic Acid X072N 1.1.7, 5.6

Nalidixic Acid X091 1.20, 5.1

Nalidixic Acid X291 1.20, 5.1

Nalidixic Acid, Vancomycin X090 1.20, 5.1

Nalidixic Acid, Vancomycin X290 1.20, 5.1

NAN X139 1.9, 1.28, 5.6

NAN X539 1.9, 1.28, 5.6

Neomycin 100 X016 1.5, 1.13, 5.2

Neomycin 75 X015 1.5, 1.13,

5.1, 5.2

Neomycin 75 X215 1.5, 1.13, 5.1

Novobiocin (for MSRV/Diassalm

/O157 Broth) X150 1.17, 1.36,

1.40, 4.1, 5.3, 5.9

Nusens Agar LAB 74 1.38

Nutrient Agar LAB 8 1.39, 1.57

Nutrient Agar Slopes P007 7.1

Nutrient Broth P008 7.1

Nutrient Broth ‘E’ LAB 68 1.39

Nutrient Broth No. 2 BP LAB 14 1.13, 1.14,

1.39, 7.2

8.5

NZCYM Broth LAB 182 3.4

NZY Broth (NZYM) LAB 181 3.4

O157 Broth MTSB LAB 165 xxiv, 1.40,

2.3, 4.1, 5.3

Oleandomycin, Polymyxin X110 1.42, 5.3

Orange Serum Agar LAB 147 1.40

ORSIM LAB 192 1.1.9, 5.9

Oxacillin, Polymyxin B X192 1.1.9, 5.9

Ox Bile MC 10 6.4

Oxytetracycline (for OGYE) X089 1.41, 1.49, 5.10

Oxytetracycline Glucose Yeast

Extract Agar LAB 89 xxv, 1.41, 5.10

PAC (for Palcam media) X144 1.41, 1.42, 5.5

Palcam Agar Base LAB 148 xxvi, 1.22,

1.41, 1.42, 5.5

Palcam Broth LAB 144 1.42, 5.5

PEMBA LAB 193 xxv, 1.1.9,5.2, 7.2

Peptone Water LAB 104 1.42, 1.59

Peptone Water P009 7.1

Perfringens Agar Base (TSC) LAB 194 xxvi,

1.1.10, 5.3

Perfringens Agar OPSP LAB 109xxvi, 1.42, 5.3

P-INC Supplement X019 1.33, 5.8

P-INC Supplement X219 1.33, 5.8

Plate Count Agar LAB 149 xxiii, 1.43

Plate Count Agar APHA LAB 10 xxiii, 1.43

Polymixin C, Ceftazimide X072 1.1.7, 5.6

Polymyxin B X074 1.3, 5.2

Polymyxin B X193 1.1.9, 5.2

Potassium Tellurite Solution 3.5% X027 1.24, 1.51, 7.2

Potato Dextrose Agar LAB 98 1.44, 7.2

PREP LAB 73 xxv, 1.3, 5.2,7.2

Proteose Peptone A MC 11 6.4, 6.7

Pseudomonas Agar Base LAB 108 xxv,

1.44, 5.8

PTTA (T.B. Supplement) X124 1.26, 5.7

R2A Medium LAB 163 1.45

Rappaport Vassiliadis Medium (RVS) LAB 86 xxvii, 1.45

Reinforced Clostridial Agar LAB 23 1.46

Reinforced Clostridial Medium (Broth) LAB 22 1.46

Resuscitation broth P081 7.1

Ringer’s Solution (Calgon) LAB 101 ix, 1.47

Ringer’s Solution (Thiosulphate) LAB 102 ix, 1.47

Ringer’s Solution 1/4 Strength LAB 100 ix, 1.32, 1.47

Ringer’s Solution 1/4 Strength Tablets LAB 100Z ix, 1.47

Rose Bengal Chloramphenicol Agar LAB 36 xxv, 1.41,

1.47, 5.10

RPF X086 xxv, 1.3, 1.4, 5.9

Sabouraud Dextrose Agar LAB 9 1.48

Sabouraud Liquid Medium USP LAB 33 1.48

Sabouraud Maltose Agar LAB 111 1.48

Salt Meat Broth Tablets LAB 113Z 1.49

Selenite Broth P011E 7.1

Selenite Broth Base LAB 44A 1.49

Selenite Cystine Broth P012E 7.1

Selenite Cystine Broth Base LAB 55A xxvii, 1.50

Sensitivity Test Agar (STA) LAB 12 1.50

Serum Broth P017 7.1

Simmons Citrate Agar LAB 69 1.51

Single Step Staph Selective Agar (4S) LAB 84 1.49, 1.51

Skim Milk Powder MC 27 6.5

Skirrows X214 1.13, 5.2

Slanetz & Bartley Medium LAB 166 1.52

Sodium Biselenite LAB 44B xxvii,

1.49, 1.50

Sodium Chloride MC 17 6.5

Sodium Desoxycholate MC 26 6.5

Sodium Glutamate LAB 80B xxiv, 1.34,

1.57

Sodium Thioglycollate MC 16 6.5

Sorbitol MacConkey Agar LAB 161 xxiv, 1.40,

1.52, 2.3, 4.1, 4.3, 5.4

Soy Peptone MC 3 6.5, 6.7

Special Tryptone MC 35 6.5

SS Agar (Modified) LAB 52 ix, 1.53

Sugar Free Agar LAB 87 1.53

Sulphadiazine X109 1.42, 5.3

Superbroth LAB 177 3.4

Superbroth with Agar LAB 178 3.4

Susceptibility Test ‘ISO’ Agar LAB 170 1.1.10

TCBS Cholera Medium LAB 96 1.54

Terrific Broth LAB 183 3.5

Tetrathionate Broth (APHA) LAB 97 xxvii, 1.54

Thioglycollate Medium (Brewer) LAB 64 1.55

TMAO Selenite X137 1.18, 5.5

Todd Hewitt Broth LAB 75 1.55

Transport Medium (Amies Charcoal) P013 7.1

Trichomonas Medium P018 7.1

Triple Sugar Iron Agar LAB 53 1.56, 1.1.8

Tryptone MC 5 6.5, 6.7

Tryptone Bile Agar LAB 72 xxiv, 1.57,

2.2, 4.3

Tryptone Glucose Extract Agar LAB 63 1.57

Tryptone Soy Agar USP LAB 11 xv, 1.58

Tryptone Soy Broth USP LAB 4 xv, 1.58

Tryptone Water LAB 129 xxiv, 1.8,

1.33, 1.34, 1.59

Tryptose MC 8 6.6, 6.7

Tryptose Phosphate Broth LAB 62 1.59

TYC Medium LAB 35 1.59

Urea Agar Base LAB 130 1.60, 7.2

Urea Agar Slope P015 7.1

Urea Broth Base LAB 131 1.60, 7.2

Urea solution 40% 5ml X130 1.60, 7.2

Urea solution 40% 100ml X135 1.60, 7.2

UVM Base LAB 155 xxvi,

1.61, 5.6

UVM I Supplement X155 1.61, 5.6

UVM I Supplement X555 1.61, 5.6

UVM II Supplement X156 1.61, 5.6

VCA X040 1.24, 5.4

VCC Supplement X546 4.1, 5.4

VCNT (for Thayer Martin) X068 1.23, 5.7

VCNT (for Thayer Martin) X268 1.23, 5.7

Violet Red Bile Agar (VRBA) LAB 31 xxiii,

8.6

1.61, 1.62

Violet Red Bile Agar with MUG LAB 573 1.1.11

Violet Red Bile Glucose Agar (VRBGA) LAB 88 xxiii, 1.19,

1.62

Water Plate Count Agar (ISO) LAB 197 1.1.12

WL Nutrient Agar LAB 79 1.62

Wort Agar LAB 38 1.63

Wort Broth LAB 99 1.63

X-gal MC 405 6.3, 6.6

XLD Agar LAB 32 xxvi,

xxvii, 1.64

Xylose MC 32 6.6

Yeast Extract MC 1 6.6, 6.7

Yeast Extract Agar LAB 18 xxiii, 1.64

Yeast Extract Dextrose

Chloramphenicol Agar LAB 119 1.65

Yersinia CIN Agar Base LAB 120 1.65, 5.10

YPD Broth LAB 175 3.5

YPD Agar LAB 176 3.5

2 x YT Broth LAB 179 3.6

2 x YT Agar LAB 180 3.6

8.7

8.8

Actinomyces 1.6, 1.20, 5.1

Aeromonas xvii, 1.1

Aspergillus niger xviii, 1.31, 1.32

Bacillus cereus xvii, xxiii, 1.3, 5.2,1.1.9, 7.2

Bacillus stearothermophilis x, 1.16

Bacteroides fragilis xvii, 1.1, 1.2, 1.20, 5.1

Blastomyces 1.16, 1.1.4, 1.1.5

Campylobacter xii, xvii, xxv, 1.9, 1.10, 5.2, 5.3

Candida xviii

Citrobacter xvii

Clostridium perfringens xvii, xxiv, 1.5, 1.13, 1.20, 1.42, 1.46,

1.1.10, 5.1, 5.2, 5.3, 7.2

Clostridium difficile 1.6, 1.20, 5.1, 5.2, 5.3

Coccidiodes 1.16

Coliforms xxi, 1.8, 1.11, 1.12, 1.19, 1.20, 1.27, 1.30, 1.31, 1.33,

1.34, 1.57, 1.59, 1.61, 1.1.5, 1.1.11, 2.2, 2.3

Corynebacterium xvii, 1.24, 7.2

Dermatophytes 1.16

Enterobacteriacieae xxi, 1.8, 1.19, 1.30, 1.51, 1.56, 1.62

Enterococcus xvii, 1.25, 1.52

Escherichia coli xvii, xxii, 1.20, 1.31, 1.57, 1.59, 1.1.3, 1.1.5,

1.1.11, 2.2, 2.3, 2.5, 3.1, 3.2, 3.3, 3.4, 3.5, 4.3

Escherichia coli O157:H7 xvii, xxii, 1.40, 1.52,

1.1.3, 2.4, 4.1, 5.3, 5.4

Eubacterium 1.20

Fusobacterium 1.20

Gardnerella 1.5, 1.13, 5.4

Haemophilus xi, xvii, 1.58, 5.4

Helicobacter 1.24, 5.4

Histoplasma 1.6, 1.16, 1.1.4, 1.1.5

Klebsiella xvii

Lactobacillus xvii, 1.35, 1.36, 1.40, 1.62

Listeria xvii, xxiv, 1.9,1.22, 1.28, 1.41, 1.42,

1.61, 1.1.6, 2.2, 5.5, 5.6

Micrococcus xvii, xxvii, 1.49

Mycobacterium 1.26, 5.6

Neisseria xvii, 1.1, 1.13, 1.23, 1.37, 5.7, 7.1

Peptostreptococcus 1.20

Porphyromonas 1.20

Propionibacterium 1.20

Proteus xvii, 1.12, 1.60, 7.1

Pseudomonas xvii, xxiii, 1.11, 1.22, 1.44, 5.8

Saccharomyces xvii, 3.6

Salmonella vii, xii, xvii, xxiv, xxv, 1.4, 1.7, 1.9, 1.14,

1.15, 1.17, 1.18, 1.23, 1.35, 1.36, 1.38,

1.45, 1.49, 1.50, 1.53, 1.54, 1.56, 1.64,

1.1.8, 2.4, 4.3, 5.5, 5.9, 7.1

Salmonella typhi xvii, 1.4, 1.19, 1.54

Serratia xvii

Shigella xii, xvii, 1.14, 1.15, 1.23, 1.53, 1.64

Staphylococcus aureus xvii, xxiii, 1.3, 1.5, 1.13, 1.17, 1.27, 1.32,

1.49, 1.51, 1.1.9, 5.4, 5.9, 7.2

Staphylococcus epidermidis xvii, 1.5, 1.13, 5.4

Streptococcus xvii, 1.5, 1.11, 1.12, 1.13, 1.29, 1.40

1.55, 1.59, 1.1.1, 5.4, 5.9

Taylorella 1.10

Trichomonas 7.1

TVC xxi, 1.33, 1.34, 1.43, 1.45, 1.57, 1.64, 1.1.12

Vibrio xvii, 1.42, 1.54

Yeasts and Moulds xviii, 1.31, 1.32, 1.41, 1.44, 1.47, 1.48, 1.63,

1.65, 5.10, 7.2

Yersinia xvii, 1.65, 5.10

By organism

To request a copy of ‘The Microbiology Manual’, please complete and return the form below to LAB M.Forms can be faxed back to +44 (0)161 762 9322 or posted to the address below:

Customer Services Department

IDG (UK) Limited.

Topley House

52 Wash Lane

BURY

Lancashire BL9 6AU

To receive updates to the manual as they are issued, please complete the form below, ticking the update box. Please make anote of the version of the manual which you are currently using by entering on the form below the version number whichcan be found at the top of the Product code index (e.g. V1.0) at the back of the manual.

Request a copy of the ‘Microbiology Manual’

New manual ❑ Register for manual updates ❑ Current manual version:

Name:

Job Title:

Company Name:

Company Address:

Postcode:

Tel. Number:

Fax Number:

Email address:

No. of tests per week?

Is Pathogen testing carried out on-site? Yes ❑ No ❑

Who is your current preferred supplier for culture media?

lab m manual 2006

Documents