Handbook Pmb

e-Hand Book of Practicing Microbiologists

Dehydrated Culture Media n Bases n Supplements n Ready to use Media n Indicators & Stains n Test Kits

MicroxpressTM

First Edition

Published by: Tulip Diagnostics (P) Ltd. for the use of its esteemed customers globally.TM TMAccumix , is the brand name of Dehydrated Culture Media, Bases and Supplements manufactured by Microxpress .

TMMicroxpress is the brand name of Tulip Diagnostics (P) Ltd.’s microbiology products division.TM: Tradename owner Tulip Diagnostics (P) Ltd., Gitanjali, Tulip Block, Dr. A. A. Rego Bagh, Alto Santa Cruz, Bambolim Complex P.O., Goa - 403 202, INDIA.Tel.: (0832) 2458546 - 51, Fax: (0832) 2458544. E-mail: [email protected]. Website: www.tulipgroup.com

Quality assurance involves right application of right

analytical procedures at the right stages. High level of

quality assurance is a challenge for industries as well as

microbiological laboratories. Laboratories should

understand the requirements of industries properly and

industries should implement the procedures

recommended by laboratories meticulously.

These procedures should be sufficiently elaborate. At the

same time, they should be written in a language that

can be easily understood by technicians. The Handbook

of Practicing Microbiologists proves to be a paragon for

the same.

Ms. Minakshi Kulkarni,

B.Sc. (Chemistry),

Post Graduate Diploma in Food, Drug and Cosmetic

Analysis.

Lead accessor for ISO 9001:2000, ISO 14001:2004,

HACCP.

Ms. Minakshi Kulkarni,

Handbook for Practicing Microbiologists is a

comprehensive coverage of all the available methods

used for the qualitative and quantitative analysis of

various pharmaceutical, beverage and food products.

The methods documented in the manual will go a long

way in the annals of medical and environmental

research and revolutionize the modern concepts in

solving medical and environmental riddles.

Prof. M.S. Sarang,

M.Sc. (Microbiology),

Ex-Head, Department of Microbiology,

Wilson College, Mumbai.

Prof. M.S. Sarang,

Review of the e-Handbook of Practicing Microbiologists

After turning into an invitro major with undisputed

leadership in IVD both nationally and internationally,

Tulip Group has invested considerable time and

resources for the development of cutting edge

Microbiology Products and Disinfectants. These forays

have created tremendous interest with the users and

garnered instant market acceptance.

Tulip Group believes that in today's information based

society, knowledge and technical upgradation are key

elements for Tulip Group and its customers to achieve

their quality objectives consistently and effectively.

In the last decade or so, microbiological analysis in

various industries like Pharma, Food, Beverage,

Agriculture and Medicine have attained an important

status and have become the corner stones of quality

assurance.

This Hand Book of Practicing Microbiologists provides

a basic platform for Microbiologists from various

industries and covers the essential practical methods

employed in Industrial and Clinical Microbiology. The

Hand Book should find favour as a training tool as

well as a technical resource manual for Practicing

Microbiologists who endeavour to fulfill stringent

regulatory requirements across the industrial

spectrum, as well as for accurate clinical diagnosis.

F O R E W O R D

“A journey of a thousand miles begins with the first step.”

Quality policy...

The Quality Policy of Tulip Group of Companies is:

l To develop, manufacture and market state of the art, high

quality, user friendly products.

l To design and manufacture devices in such a way that when

used under the conditions and for the purpose intended,

they will not compromise, directly or indirectly, the clinical

conditions and safety of the products, the safety or health of

the users or where applicable, other persons, or the safety of

the property.

l To meet customer requirements and achieve customer

satisfaction.

l To meet regulatory requirements.

l To be market leader and trend setter in diagnostic and

laboratory testing.

Objectives:

l By periodically assessing customer and regulatory

requirements and up grading products, processes and

services.

l By adopting solutions for design and construction of device

conforming to safety principles taking into account the

generally acknowledged state of art.

l By emphasis on Research an Development of innovative

and new products.

l By implementing Good Manufacturing Practices.

l By adopting and implementing Quality Management

System adhering to international standards.

l By employing the best available personnel and training

them to update the skill and knowledge.

The Quality Policy of Tulip Group of Companies is:

Objectives:

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Microbiological Methods in Pharma Industry 01-21

Media Fill Run. 01

Microbiological analysis of water. 02

Sterility Testing. 04

Determining Biocontamination of surfaces. 08

Monitoring Microbial load from filling operators garment clothes / hand gloves on sterile room. 09

Microbiological Evaluation program for controlled Environment. 09

Microbiological Assay of Antibiotics. 11

Antimicrobial Effectiveness Testing (Preservative efficacy testing). 17

Validation of autoclaves and ovens using Biological Indicators. 17

Cup-plate Assay of Cyano-Cobalmin (Vitamin B ) using E.coli mutant 113 (D). 2012

Calcium Pantothenate Assay. 20

Microbiological Methods in Food Industry 22-81

Enumeration of Coliforms, Faecal Coliforms and of E.coli in Water in Sealed Containers and 22

Prepackaged Ice Using the Hydrophobic Grid-membrane Filter (HGMF) Method.

Microbiological Examination of Ice Cream and Ice Milk 24

Microbiological Examination of Cottage Cheese 27

Microbiological Examination of Egg Products and of Liquid Eggs 31

Microbiological Examination of Milk 34

Microbiological Examination of Mineral Water 35

Microbiological Examination of Milk Powder 39

Microbiological Examination of Froglegs 42

Microbiological Examination of Cocoa and Chocolate 45

Examination of Canned Tomatoes, Tomato Juice and Vegetable Juice, Tomato Puree, 48

Tomato Paste, Tomato Pulp and Tomato Catsup for Mould Filaments

Microbiological Examination of Cheese 55

Microbiological Examination of Water in Sealed Containers 62

(Excluding Mineral and Spring Water) and of Prepackaged Ice

Microbiological Examination of Foods for Aerobic Colony Counts (ACC) 66

Enumeration of Coliforms, Faecal Coliforms and of E. Coli in Water in Sealed Containers and 68

Prepackaged Ice Using the MPN Method

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Microbiological Methods in Pharma Industry 01-21

Microbiological Methods in Food Industry 22-81

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Proposed Official Method: Enumeration of Pseudomonas aeruginosa in Prepackaged Ice and 75

Water in Sealed Containers by the Hydrophobic Grid-Membrane Filter (HGMF) Technique

Detection of Food Poisoning by Clostridium botulinum and Its Toxins 77

Soil Microbiology 82-85

Isolation of Dermatophytes, other Fungi and Yeasts from Soil 82

Cultivation of those fungi and bacteria which are able to utilise sodium nitrate 83

as the sole source of nitrogen

Observation for chlamydospore production by Candida albicans and 84

for the maintenance of fungal stock cultures.

Isolation of Nitrogen Fixing Bacteria from Soil 84

Dilution and isolation of Phosphate Solubilizing Microorganisms from soil 85

Microbiological Methods in Beverage Industry 86-87

Detection and Enumeration of respiratory deficient yeast cells used in beverage. 86

Maintenance of 'Yeast' cultures which are used as 'Seed' for fermentation. 86

To check sterility of Beverage products (Beer, wine etc) by microfilteration technique. 86

Medical Microbiology 88-122

Introduction 88

Blood Culture 88

Upper respiratory tract infections, including throat, nose, ear and eye infections 89

Lower respiratory tract infections 91

Wound , skin and deep sepsis 105

Genital tract infections 108

Gastrointestinal infections 111

Urinary tract infections 116

Pyrexia of unknown origin (PUO) 121

Quantitative Analysis of culture media using Ecometric Method 123

Application 124-147

Microxpress Product List 148-176

Meningitis 118

Soil Microbiology 82-85

Microbiological Methods in Beverage Industry 86-87

Medical Microbiology 88-122

Quantitative Analysis of culture media using Ecometric Method 123

Application 124-147

Microxpress Product List 148-176

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits

A Window to Tulip Group...

INTRODUCTION

PRODUCT DEVELOPMENT

MANUFACTURING

Since its inception in 1988, Tulip Group of Companies comprising of eight

independent diagnostic companies, has emerged as a leading

manufacturer and marketer of in vitro diagnostic reagents and kits,

dehydrated culture media and high technology disinfectant products

nationally and internationally.

Well known for its innovative approach, the companies are owned,

managed and run by highly involved professionals.

The individual group companies specialize in research, development and

designing of specific systems and platforms in diverse technological areas

covering almost all areas of diagnostic relevance.

The Group believes in creating ‘better systems for diagnosis and

prevention’ and sets trends by innovating continuously.

While Tulip Diagnostics (P) Ltd. focuses on assay systems for

Immunohaematology, Haematology, Rheumatology, Infectious Diseases

and Haemostasis, its division Microxpress focuses on dehydrated culture

media, bases, supplements, reagents and tests kits for microbiology and

mycobacteriology.

Orchid Biomedical Systems, Qualpro Diagnostics, Zephyr Biomedicals,

focus on rapid membrane & ELISA based immunodiagnostic platforms for

Fertility, Infectious Diseases, Parasitology, Cancer and Cardiac Markers.

Coral Clinical Systems focuses on Clinical Biochemistry while its division

BioShields focuses on high technology disinfectants.

The products are manufactured in professionally set up modern facilities

complying to relevant FDA guidelines.

The innovativeness is fuelled by an inventive streak with an accent on

indigenous technology as a fundamental basis for product development

and designing of viable technological platforms for diagnosis.

Production systems have been devised around process flows to achieve

consistent product performance, batch to batch and stringent in coming, in

process QA ensure adherence to expected performance parameters

whereas finished QC benchmarked to standard reference materials

ensures accuracy of products.

INTRODUCTION

PRODUCT DEVELOPMENT

MANUFACTURING

QUALITY ASSURANCE

HUMAN RESOURCES

NATIONAL SALES

INTERNATIONAL PRESENCE

OPPORTUNITIES FOR COLLABORATION

The companies apply cGMP and GLP in force from time to time and all the

companies are ISO 9001:2000, ISO 13485 (2003), NF EN ISO 13485

(2004) compliant. Most products are already CE marked.

The company places great importance to talent garnering and skill

development. Inhouse training programmes are conducted at desired

frequency to develop functional proficiency, understanding processes and

imparting knowledge. Tulip Group team is constantly motivated to be

responsible and responsive to its customers and business.

The Company’s national business is built around twelve branch locations,

nationwide with product flow all over the country through a diverse and

efficient distributor network that guarantees product availability,

maintenance of cool chain and customer responsiveness.

The Company has a professional sales team of around 325 sales / service

professionals headquartered all over the country to carry forward its

customer contact and sales programme; with a customer base of over

15000 customers and 300 distributors.

Internationally the company channelizes its products and technology

through distributors, NGO’s and arrangements with other international

companies globally. The company also offers bulk, OEM and contract

manufacturing facilities to various international companies. Currently, the

company exports its products to over 50 countries worldwide, representing

over 45% of its turnover.

The company is constantly seeking distribution partners in un-represented

countries. It also seeks competent vendors for various biomaterials,

chemicals and instrumentation used in its manufacturing processes.

The company also seeks collaboration with like-minded companies who

are looking to commercialize their products and technologies in India

utilizing its deep resources and understanding of the Indian &

International business environment.

QUALITY ASSURANCE

HUMAN RESOURCES

NATIONAL SALES

INTERNATIONAL PRESENCE

OPPORTUNITIES FOR COLLABORATION

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Corporate Flowchart

Group

MICROXPRESS - A DIVISION OFTULIP DIAGNOSTICS (P) LTD.

· General biochemical identification tests

· Analytical reagents· Water testing solutions· Stains · One step presumptive

identification tests· Ready prepared media· Blood culture systems· Mycobacteriology

M y c o b a c t e r i a i d e n t i f i c a t i o n , isolation, staining and sensitivity testing.

· Instraprep range of r e a d y t o p o u r , steril ized pouched media

· Dehydra ted culture media

· Bases· S e l e c t i v e

supplements, agents and enrichments

· Immunohaematology· Haematology· Rheumatology· Infectious Diseases· Haemostasis· Instruments

· Parasitology· Fertility· Infectious Diseases

· Parasitology· Infectious Diseases· Cancer Markers· Cardiac Markers

· Infectious Diseases· ELISA based and rapid

membrane tests

· Clinical Biochemistry· Analytical Reagents· Stains

· Disinfection products

BIOSHIELDS - A DIVISION OFCORAL

CLINICAL SYSTEMS

TULIP DIAGNOSTICS (P) LTD.

ORCHID BIOMEDICALSYSTEMS

ZEPHYR BIOMEDICALS

QUALPRO DIAGNOSTICS

CORAL CLINICAL SYSTEMS

READY TO USE ACCUMIX



Certification...

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Microxpress

Certification... Microxpress

iv Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits

Media Fill RunIntroduction“Sterile” is a powerful word with harsh legal implications surrounding noncompliance. Global regulatory authorities would define sterile as 'free of viable organisms' and sterility assurance has become one of the most scrutinized area of Pharmaceutical and medical device manufacture. The favored method of Production of sterile Pharmaceutical Products includes a terminal sterilization process. Such as autoclaving or irradiation. Since it is not practical to examine every unit for conformation of sterility, terminal sterilization process use biological indicators (BIS) to provide levels of sterility assurance.

Aseptic processing used to produce sterile parenteral drug products and active Pharmaceutical ingredients involves the handling of pre-sterilized products in a highly controlled environment. Using the BI correlation approach is not applicable here, as aseptic processing involves ensuring a great deal of process control.

All efforts are made to minimize the risk of contamination.l Air in critical areas is supplied at point of use as (HEPA) filtered.l Positive air pressure is used to prevent ingress of air borne contamination.l Human intervention is kept to a minimum.l Cleaning is thorough and validated.l Disinfections practices are tight and validated.

Despite such measures, contamination is an ever present threat, since there will always be risk that materials and surfaces may carry organisms and influences in air filtration may pose risk. Routine sampling for sterility testing is not sensitive enough to detect such low level contamination. Samples are too small, and only gross contamination is likely to be detected. Pharmaceutical manufacturers therefore need other means of guaranteeing the quality of their product. This is why process stimulation (MEDIA FILLS) supported by environmental monitoring and other related processes are required. Media Fill is one of the best tools to demonstrate control of the process to the industry standard for allowable contamination levels.

Media Fills utilize culture media in place of product to evaluate contamination levels. During conducting experiments it is important that process stimulations are designed to accurately represent the aseptic process. The new FDA guidelines pay particular attention to this aspect of aseptic processing and it is becoming an area requiring more work and focus to satisfy the regulations. The Media Fill should be designed to mimic as closely as possible, the aseptic process used in practice. The Media Fill design is one element within the overall considerations to be made in the validation of an aseptic process. Areas of focus includel Facility and room designl Design of the filling machinel Process flow

Introduction

1Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits

Microbiological Methods in Pharma IndustryHand Book of

Practicing Microbiologists

l Heating, ventilation and air-conditioning design.l Trends in environmental monitoring data.l Contamination Control Programme.l Quality assurance and Quality Control systems.l Process stimulationsl Personnel training and qualification.

An appreciation of the many factors influencing the validation Programme allows a process stimulation to be effectively designed. Key elements in the stimulations to be taken into account include,

l Type of product being filtered.l Lot / Batch sizel Container and closure configuration.l Fill volumel Line speedl Operator shifts and fatiguel Filling line configurationl Sterile hold timesl Number of units filledl Acceptance criterial Run duration

Growth Media UsedThe selection of the correct growth medium to be used in the process stimulation is a very important step. The medium needs to support the growth of a wide variety of microorganisms, including aerobic bacteria, yeast and moulds. The broad range of organisms being looked for is consistent with organisms tracked through the firms Environmental Monitoring Programme. The FDA guidance notes the use of Soyabean Casein Digest Medium, also known as “Tryptone Soya Broth”.

As already noted the new FDA guidelines recommend that Media Fills mimic actual aseptic process as closely as possible. One of the main areas where this is implicated is where the culture medium is introduced into process. In the past manufacturers have made up and sterilized the medium outside of the controlled area and introduced it directly into the filling line. In order to more closely mimic the process, the culture medium should be filtered into the Process- Just as would occur to a liquid Pharmaceutical product. This created several concerns.

l Dehydrated Culture Media is usually supplied in a non-sterile form and carries a high bioburden. And thus contaminating the controlled area. Hence it would be preferential to source media that has been 'IRRADIATED'.

l Mycoplasma can be a concern with culture media even it is sterile filtered, therefore irradiation gives assurance that media is free from Mycoplasma.

Growth Media Used

Microbiological Analysis of Water

IntroductionWater is one of the most widely and abundantly used substance in Pharmaceutical manufacturing. It is required for a variety of purposes ranging from manufacturing processes to the preparation of the final dosage forms. The quality of water therefore assumes considerable importance.

Introduction

2 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits

RequirementsMateriall Sterile (Gamma Irradiated) SCDM [AM50925- 5k]l Soyabean Casein Digest Agar [AM1041/AM5091]l 0.22 micron membrane filterl vials/ampoules/bottles.l S.aureus ATCC 25923l E.coli ATCC 25922l S.pyogenes ATCC 19615l C.albicans ATCC 10231

Equipmentsl Autoclavel Incubatorl Ovenl Laminar Air flowl Membrane Holderl Air Sampler (Accubas AX1)

Procedurel Weigh required quantity of sterile SCDM (30.0 gms/lit) aseptically in sterile

distilled water.l Boil with frequent agitation to dissolve the powder completely l DO NOT AUTOCLAVE OR OVERHEAT.l Filter the medium using sterile 0.22 micron membrane filter.l Prepare filling line for media fill.l Fill the containers (vials / ampoules / bottles etc.) with filtered sterile

medium.l Quantity of containers to be filled should be as per maximum Lot size of the

product.l During Media Fill run carry out environmental monitoring of filling room

either by air sampler or exposing media plates.l Carry out 100% inspection of filled units before incubation. Any defects that

compromise the container closure or non-integral units are rejected.0 0

l Perform the incubation for 14 days at 20-35 C ( + 2.5 C ). Incubate the filled units in an inverted position for first half of the incubation period and then return to an upright position for the remainder.

l For growth promotion test, fill additional units at the end of the process filling. Inoculate with specific cultures like S.aureus, S.pyogenes, E.coli, C.albicans

RequirementsMaterial

Equipments

Procedure

respectively. These are then incubated under identical conditions as the process stimulation samples.

l Carry out checking of filled vials every day for microbial contamination during incubation period.

Interpretationl Test units should remain sterile (clear solution without turbidity) upto 14

days.l Growth Promotion Test should give characteristic growth with respective

organism.l Exposed plates of environmental monitoring should give microbial counts

within limit.

LimitsThe following table indicates the maximum permitted number of contaminated units per various Media-Fill “run sizes' to indicate a 0.1% contamination with a 95% Confidence Level.

Media Fill Units Contaminated Units Permitted

3000 0

4750 1

6300 2

7750 3

9150 4

10510 5

11840 6

13150 7

14430 8

15710 9

16960 10

References1. Innovations in Pharmaceutical Technology- Phil Smith2. Drugs and Health Products- Health Canada3. Aseptic Pharmaceutical Manufacturing- Michael Groves and Ram Murfy4. USP chapter <797>.

Interpretation

Limits

Media Fill Units Contaminated Units Permitted

References

Various types of water to be used in the manufacture of Pharmaceutical articles l Purified Waterl Water for injectionl Sterile water for injection



Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 3



Guidelines for microbial control in water for Pharmaceutical useThe criteria for controlling the microbial quality of purified water for injection vary according to method of production, distribution and storage. The purification system used for production of water of acceptable microbiological quality is also validated prior to initial production.

Guidelines for microbial control in water for Pharmaceutical useThe criteria for controlling the microbial quality of purified water for injection vary according to method of production, distribution and storage. The purification system used for production of water of acceptable microbiological quality is also validated prior to initial production.

Various types of water used for manufacturing is analyzed for microbiological parameters as per guidelines of IP/USP/BP.

Following tests area carried out during water testing1) Total aerobic microbial count2) E.coli3) Salmonella4) S.aureus5) Pseudomonas

RequirementsMateriall Soyabean Casein Digest Agar (AM1091/AM5091)l Saboraud’s Dextrose Agar (AM1087/ AM5087l MacConkey’s Broth (AM1062/ AM5062)l Brilliant Green Bile Broth / Selenite F Broth (AM1020/ AM5020)l Deoxycholate Citrate Agar / Bismuth Sulphite Agar A(AM1031/ AM5031)l TSI Agar / Urea Broth (AM1099/ AM5099) (AM51061)l Cetrimide Agar (AM1022/ AM5022)l Pseudomonas Agar for Pyocyanin(AM108414/ AM508414)l Pseudomonas Agar for Flourescein(AM108411/ AM508411)l Mannitol Salt Agar(AM1069/ AM5069)l Nutrient Broth (AM1077/ AM5077)l Rabbit Serum

Equipmentsl Laminar air Flowl Autoclavel Ovenl PH meterWater analysis is very important and critical test in the Pharma Industry and is conducted on daily basis.

ProcedureSAMPLING OF WATER

Guidelines for microbial control in water for Pharmaceutical use

Guidelines for microbial control in water for Pharmaceutical use


Equipments

Procedure

a) Wash the hands with soap and water. Rinse it with 70% alcohol.b) Use face mask while sampling.c) Clean the water collection point with 70% alcohol.d) Start the flow of water and allow it to run for 5 minutes.e) Collect a water sample in sterile container.f) Let the sample attain room temperature before testing.g) Carry out testing within 60 minutes after sampling without refrigeration.

1. For total aerobic microbial counta) Pipette out 1 ml sample in sterile petridish. To this add 20 ml sterile

0soyabean casein digest agar at 40-45 C. Mix it and allow to solidify. 0Incubate the plates at 30-35 C for 48 hours.

b) Pipette out 1 ml sample in sterile petridish. To this add 20 ml sterile 0Saboraud’s Dextrose agar at 40-45 C. Mix it and allow to solidify.

0Incubate the plates at 20-25 C for 48-72 hours.c) After incubation count the number of colonies from both plates and by

adding both count report the total aerobic microbial count.

2. For E. coli counta) Add 10 ml sample into 100 ml sterile nutrient broth.

0b) Incubate at 37 C for 18 -24 hours.c) Transfer 1 ml of above enrichment broth into 5 ml MacConkey’s broth.

0d) Incubate at 37 for 24-48 hours.e) Prepare positive control by inoculating a loopfull of 24 hours old culture

of E. coli to 5 ml sterile MacConkey’s broth.0f) Incubate at 37 C for 24-48 hours.

g) If acid and gas formation is observed in sample tube transfer 0.1 ml from this tube into 5 ml sterile MacConkey’s broth and 5 ml Peptone

0water each. Incubate at 44 C for 20- 24 hours.h) After incubation if acid and gas formation is noticed in MacConkey’s

broth presence of E. coli is confirmed.

3. For Salmonella0a) Add 10 ml of sample to 100 ml sterile Nutrient broth. Incubate at 37 C

for 24 hours.b) Transfer 1 ml of above enrichment broth to 10 ml sterile selenite broth or

010 ml of brilliant green broth. Incubate at 37 C for 24 hours.c) For positive control transfer a loopfull of 24 hours old culture of

0Salmonella abony into 10 ml of selenite broth. Incubate at 37 C for 48 hours.

d) From selective broth streak out a loopfull on either of deoxycholate 0citrate agar or bismuth sulphite agar. Incubate at 37 C for 24 hours.

e) If characteristic colonies are observed in sample, subculture onto TSI agar and inoculate into urea broth.

f) If characteristic growth is observed on TSI agar and absence of red colour in urea broth then it confirms the presence of Salmonella in the sample.

1. For total aerobic microbial count

2. For count

3. For Salmonella

E. coli




4. For Pseudomonasa) Transfer 10 ml of sample into 100 ml sterile fluid soyabean casein

0digest medium.Incubate at 37 C for 48 hours.b) Streak a loopfull of above enrichment broth onto cetrimide agar and

0incubate at 37 C for 48 hours. Streak a loopfull of 24 hours old culture of Pseudomonas aeruginosa on

0cetrimide agar plates and incubate at 37 C for 24 hours. If characteristic colonies are obtained carry out following tests

i) Streak out respective colonies on Pseudomonas agar medium for detection of flourescein and pyocyanin

ii) Perform gram-staining, if gram negative bacilli are observed then test is positive.

For S. aureusStreak a loopfull of enrichment culture from SCDM broth on Mannitol salt agar.

0Incubate at 37 C for 24 hours.Also streak a loopfull of 24 hours old culture of S. aureus on Mannitol salt agar.

0Incubate at 37 C for 24 hours.

If characteristic colonies are obtained from sample and also from positive control perform coagulase test.If coagulase test is positive then it confirms the presence of S. aureus in the sample.

Interpretationl E.coli - Gas and acid production, consider as positive test, Acid production is

indicated by change in colour of the medium red to yellow.No gas and no acid production consider as negative test.

4. For Pseudomonas

For

Interpretation

S. aureus

l Salmonella – Bismuth Sulphite Agar- Black colonies with metallic sheen consider as positive test.Deoxycholate Citrate Agar- Black coloured colonies consider as positive test.Absence of Standard colonies as negative test.TSI slant- Luxuriant growth with alkaline slant, Acidic butt, gas formation and H S production (blackening) is considered as positive test.2

Absence of standard growth pattern, consider as negative test.l Pseudomonas – colonies on Cetrimide agar is surrounded by a blue green

pigment, is considered as positive test.The presence of blue-green pigmentation or Fluorescence on Pseudomonas Agar Base is considered as growth positive test. Absence of this characteristic is considered as negative test.

l S.aureus- yellow coloured colonies on Mannitol Salt Agar, is considered as positive test.Absence of standard colonies is considered as negative test.

Limits (for purified water used in Pharma Industry)

1. TBC + TFC NMT 100 CFU/ml

2. E.coli Absent/10 ml

3. Salmonella Absent/10 ml

4. S.aureus Absent/10 ml

5. Pseudomonas Absent/10 ml

References1. IP 20072. USP-27

Limits (for purified water used in Pharma Industry)

References

Sterility Testing

IntroductionThe test for sterility are intended for detecting the presence of viable forms of micro-organisms in or on Pharmacopeial preparations. The test must be carried out under condition designed to avoid accidental contamination of the product during the test. Precautions taken for this purpose should not adversely affect any microorganisms which should be revealed in the test.

The working conditions in which the tests are performed should be monitored regularly by sampling the air and surfaces of the working area and by carrying out control tests. The tests are based upon the principle that if micro-organisms are placed in a medium which provides nutritive material and water, and kept at a favorable temperature, the organisms will grow and their presence can be indicated by a turbidity in the originally clear medium.

The test for sterility are designed to reveal the presence of micro-organisms in the

Introduction samples used in the test, interpretation of results is based on the assumption that the contents of every container in the batch had been tested, would also have complied with the tests. Since every container cannot be tested, a sufficient number of containers should be examined to give a suitable degree of confidence in the results of the test.

Test proceduresThe tests can be carried out using 2 methods.A] Membrane FiltrationB] Direct Inoculation.

A] Membrane Filtration Method:- is to be preferred where the substance being examined is (a) an oil, (b) an ointment (c) non bacteriostatic solid, not readily soluble in the culture medium, (d) a soluble powder or liquid that possesses instant bacteriostatic and fungistatic properties.

Test procedures




Sterility by Membrane FiltrationRequirements Materialsl Fluid peptic digest medium/peptone water (AM1079/AM5079)l Soyabean Casein Digest Medium. (AM1092/AM5092)l Fluid Thioglycollate Medium. (AM1045/AM5045)l Membrane filter 0.45 µm and 47 mm diameter.l Bacillus subtilis ATCC 6633l Candida albicans ATCC 10231

Equipmentsl Membrane filtration holder.l Laminar air flow.

o ol Incubator 20 C to 25 C.

o ol Incubator 30 C to 35 C.l Sterile pair of scissors and forceps.l Oil free vacuum pump.l Flask with attachment for vacuum pump tube.

Testing Procedurel Carry out sampling of product to be tested as per table 1.l Start laminar air flow.l Enter in sterility testing room following the proper gowning procedure.l Clean the exterior surface of vials with a suitable antimicrobial agent.l Arrange sterile filter assembly inside LAF.l Prepare each membrane by aseptically transferring a small quantity

(sufficient to moisten the membrane) of sterile fluid digest medium.l Then transfer aseptically combined quantities of the preparation being

examined. (Refer table No.2 and 3) filter the content by applying vacuum.l Wash the filter membrane by three successive quantities each of

approximately 100 ml of sterile fluid peptic digest medium.l After filtration aseptically remove the membrane from the holder, cut the

membrane in two halves. Carefully immerse one half into 100 ml of o oSoyabean Casein Digest Medium and incubate at 20 C to 25 C for 7 days.

Similarly also immerse the other half membrane into fluid thioglycollate o omedium and incubate at 30 C to 35 C for 7 days. Inspect tubes on every day

and record the observation.

Prepare the following controlsl Membrane Control-Filter the sterile Peptone water through sterile assembly

after filtration aseptically remove the membrane from the holder, cut the membrane in two halves. Carefully immerse one half into 100 ml of

o oSoyabean Casein Digest Medium and incubate at 20 C to 25 C for 7 days. Similarly also immerse the other half membrane into fluid thioglycollate

o omedium and incubate at 30 C to 35 C for 7 days. Inspect tubes on every day and record it.

Sterility by Membrane FiltrationRequirements Materials

Equipments

Testing Procedure

Prepare the following controls

l Laminar contol- remove the cotton swabs of both sterile media tubes and keep it open inside LAF for 10 minutes. Incubate the tubes at respective temperature for 7 days.

l Media control- Keep each of one sterile media tubes (without opening) at respective temperature for 7 days.

l Positive control- Inoculate sterile fluid thioglycollate medium with about 100 viable micro-organisms of Candida albicans and Bacillus subtilis individually.

o oIncubate at 30 C to 35 C, And soyabean casein digest medium with 100 viable micro-organisms of Bacillus subtilis, and Candida albicans

o oindividually. Incubate at 20 C to 25 C.

B] Direct Inoculation

Sterility Testing by Direct InoculationIn this method the quantity of the substance or preparation being examined which is to be used for inoculation in the culture media various according to quantity in each container. Product is directly inoculated into medium.

RequirementMateriall Soyabean Casein Digest Medium (AM1092/AM5092)l Fluid Thioglycollate Medium (AM1045/AM5045)l Bacillus Subtilis ATCC 6633l Candida albicans ATCC 10231

Euipmentsl Laminar air flow

0 0l Incubator 20 C to 25 C

0 0l Incubator 30 C to 35 Cl Sterile Pipettes/Sterile syringe

Procedurel Carry out Sampling of product to be tested as per Table I.l Start Laminar Air Flow.l Enter in sterile testing room with proper gowning procedure.l Clean the exterior surface of vials with suitable antimicrobial agent.l Aseptically transfer the specified volume of the material from each container

to SCDM and FTG individually. (refer Table 4 for quantity of product and volume of culture medium).

l Mix the material added with the medium but do not relate excessively.l Incubate the inoculate media for not less than 14 days, unless otherwise

0 0specified in monograph at 30 C - 35 C in case of Fluid Thioglycollte Medium 0 0and at 20 C - 25 C in the case of Soyabean Casein Digest medium.

l When the material being examined renders the medium turbid so that the presence or absence of microbial growth cannot be determined readily by visual examination transfer suitable portions of the medium to fresh tube of

B] Direct Inoculation

Sterility Testing by Direct Inoculation

RequirementMaterial

Euipments

Procedure




the same medium between the third or seventh day after the test is started. Continue incubation of the transfer vessels for not less than 7 additional days after the transfer and for a total of not less than 14 days.

l For oils and oily solutions. Use media to which have been added 0.1% w/v of (4-tert-octylphenoxy) Polyethoxyethannol, 1% w/v of Polysorbate 80 or other suitable emulsifying agent in an appropriate concentration, shown not to have any antimicrobial properties under the condition test.

Prepare the following Controlsl Laminar control: Remove the cotton plugs of both sterile media tubes and

keep it open inside the LAF for 10 minutes. Incubate the tubes at respective temperature for 14 days.

l Media Control- Keep each of one sterile media tubes (without opening) at respective temperature for 7 days.

l Positive Control- Inoculate sterile fluid thioglycollate medium with about 100 viable micro-organisms of Candida albicans and Bacillus subtilis individually.

o oIncubate at 30 C to 35 C,And soyabean casein digest medium with 100 viable micro-organisms of Bacillus subtilis, and Candida albicans

o oindividually. Incubate at 20 C to 25 C.

Prepare the following Controls

l Pipette control- Rinse the sterile pipette with both of the sterile media individually. Incubate the tubes at respective temperature.

Interpretationl At intervals during the incubation period and at its conclusion examine the

media for macroscopic evidence of microbial growth. If no evidence of growth is found, the preparation being examined passes the tests for sterility. If evidence of microbial growth is found, reserve the containers showing this and unless it is demonstrated by any other means that their presence is due to causes unrelated to the preparation being examined and hence that the test for sterility are invalid and may therefore be recommended, perform a retest using the same number of samples, volumes to be tested and the media as in the original test. If no evidence of microbial growth is then found the preparation being examined passes the test for sterility.

l Membrane control-should not show any change in colour or turbidity.l Laminar control should not show any change in colour or turbidity.l Media control should show not show any change in colour or turbidity.l Positive control should show presence of microbial growth. (Turbidity).

Interpretation

TABLE ITABLE I

Number of items in the batchNumber of items in the batch Minimum number of items recommended to be testedMinimum number of items recommended to be tested

1. Injectable preparations l Not more than 100 containers l 10% or 4 containers whichever is greater l More than 100 but not more than 500 containers l 10 containers.l More than 500 containers l 2% or 20 containers whichever is less.

2. Ophthalmic and other non-injectable preparationsl Not more than 200 containers l 5% or 2 containers whichever is greater.l More than 200 containers l 10 containers.

3. Surgical Dressingsl Not more than 100 packages l 10% or 4 packages whichever is greater.l More than 100 but not more than 500 packages l 10 packages.l More than 500 packages l 2% or 20 packages whichever is less.

4. Bulk solidsl Less than 4 containers l Each containerl 4 containers but not more than 50 containers l 20% or 4 containers whichever is greaterl More than 50 containers. l 2% or 10 containers whichever is greater.

1. Injectable preparations

2. Ophthalmic and other non-injectable preparations

3. Surgical Dressings

4. Bulk solids




TABLE IITABLE II

Quantity in each container of injectable preparationQuantity in each container of injectable preparation Minimum quantity to be used for each culture mediumMinimum quantity to be used for each culture medium

For Liquidsl Less than 1 ml l Total contents of a containerl 1 ml or more but less than 4 ml l Half the content of a containerl 4 ml or more but less than 20 ml l 2 mll 20 ml or more but less than 100 ml l 10% of the content of a container unless otherwise specified in the

monograph.l 100 ml or more l Not less than half the contents of a container unless otherwise

specified in the monograph

For Solidsl less than 50 mg l Total contents of a containerl 50 mg or more but less than 200 mg l Half the contents of a containerl 200 mg or more l 100 mg

For Liquids

For Solids

TABLE IIITABLE III

Type of preparationType of preparationQuantity to be mixed

(A)Quantity to be mixed

(A)

Quantity to be used for each culture medium

(B)

Quantity to be used for each culture medium

(B)

10 to 100 ml

1 to 10 g

5 to 10 ml

0.5 to 1 gNot less than 1 g*

l Ophthalmic solutions, other no-injectable liquid preparations.l Other preparations, preparation soluble in water or appropriate solvents,

insoluble preparations to be suspended or emulsified (ointments and creams)

l Absorbent cotton

* one portion




TABLE IVTABLE IV

Referencesth1. US Food and Drug adm, 1998, Bacteriological Analytical Manual, 8 Ed, Rev.

A, AOAC, International, Guithersburg, md.

References 2. IP, 2007, Ministry of Health and Family welfare, Govt of India, Vol.1.3. US Pharmacopeail Convention, Inc.2001. The United States Pharmacopoeail

25/NF 20-2002. The US Parmacopeial Convention, Inc-Rockville, md.

Quantity in each container of injectable preparation

Quantity in each container of injectable preparation

Minimum quantity to be used for each culture medium

Minimum quantity to be used for each culture medium

Minimum volume of culture medium (ml)Minimum volume of culture medium (ml)

For Liquidsl Less than 1 ml l Total contents of a container 15l 1 ml or more but less than 5 ml l Half the content of a container 20l 5 ml or more but less than 20 ml l 2 ml 20l 20 ml or more but less than 50 ml l 5 ml 40l 50 ml or more but less than 100 ml l 10 ml 80

For Solidsl less than 50 mg l Total contents of a container 40l 50 mg or more but less than 200 mg l Half the contents of a container 80l 200 mg or more l 100 mg 80

For Liquids

For Solids

Determining Biocontamination of Surfaces

IntroductionThis method provides guidelines on the determination of biocontamination of surfaces in situations, particularly risk zones, where biocontamination control is considered desirable or necessary. This measurement involves the collection of representative samples for the detection of viable particles that are present and that may need to be controlled or monitored. These methods might not give the total number of viable microorganisms present but, under controlled conditions, can give relevant and comparable results. These methods are applied routinely in the operational condition and, if appropriate, in as built and at rest conditions.

A count of organisms on a surface at a point in time is obtained by a contact device or a swab. A contact device can apply a solid nutrient medium of known area to the surface, which is then incubated. The resultant colonies give a mirror image “map” of the original viable units. A swab can be used to wipe a surface and the number of microorganisms removed by the swab can be counted.

Determination of Bioburden Count PlateRequirementsl Sterile contact plates (Nutrient Agar / Tryptone Soya Agar with lecithin and

Tween80).l Equipmentl Incubator

Introduction

Determination of Bioburden Count PlateRequirements

Procedurel Remove the wrapper of sterile contact plate.l Apply the plate on the surface (which is to be evaluated) in such away that

Nutrient Medium should touch the surface for a few seconds with a uniform and steady pressure to the whole area without allowing any circular or linear movement.

ol Incubate the one set of plates at 37 + 2 C for 24-48 hours and other at 20-

o25 C for 48-72 hours.l After incubation period count the number of colonies from both set.

Determination of Bioburden by SwabsRequirementsMaterialsl Sterile Swabsl Sterile Salinel Sterile plates of Tryptone Soya Agar with Lecithin and Tween 80.

(AM11031/AM51031)

l Autoclavel Laminar Air Flowl Incubator

Equipments

Procedure

Determination of Bioburden by SwabsRequirementsMaterials

Equipments




Standard Procedurel Carry out swab testing inside filling room.l Follow entry procedure to enter the filling room.l Take a sterile swab and moist it with sterile saline.l Swab approximately 25 square centimeters area from side benches, floor and

working platform of Laminar Air Flow.l Rotate the swab aseptically on Tryptone Soya Agar with Lecithin and Tween

80.o o

l Incubate the plates at 37 C + 2 C for 40-48 hours.l Count the number of CFU per plate.

Standard Procedure l Carry out testing once in a month.

Limitsl Class 100 (All surfaces including floor) not more than 3 CFU/Plate.l Class 10,000 (working surfaces) not more than 5 CFU/Plate.l Class 10,000 (floor) not more than 10 CFU/Plate.

References1. USP 27 (1116) Microbiological evaluation.2. IP 2007 Vol. I.

Limits

References

Monitoring Microbial load from filling operators garment clothes / hand gloves in sterile room

IntroductionAseptically processed products require manufactures to pay close attention to detail and maintain rigorous discipline and strict supervision of personnel in order to maintain the level of environmental quality appropriate for the sterility assurance of the product. Monitoring of personnel should be conducted before or after working in the processing area.

Materialsl Sterile Swabsl Sterile Salinel Nutrient Agar (AM1074/AM5074)l Sabouraud’s Dextrose Agar (AM1087/AM5087)

Equipmentsl LAFl Vortex Mixerl Autoclave

Procedurel Carry out swab testing inside filling room.l Follow proper procedure to enter in clean room.

Introduction

Materials

Equipments

Procedure

l Take a sterile swab and moist it with sterile saline.l Swab approximately 25 sq. cm area from filling operator clothes (Head gear,

shirt sleeves etc) and both hand gloves.l Transfer the swab aseptically into 2 ml sterile saline.l Disintegrate the swab on the vortex mixer.l Carry out bacterial count by plating above saline on Nutrient Agar.l Carry out yeast/mould count by plating above saline on Nutrient Agar and

Sabouraud’s Dextrose Agar.o o

l Incubate the N.A plates at 37 C + 2 C for 24-48 hours and Sabouraud’s o oDextrose Agar plates at 25 C + 2 C for 72 hours.

l Count the number of colonies from both plates and calculate the total CFU/ml of saline.

Limits

CFU/mlClass

Gloves Personnel Clothing

100 3 5

10,000 10 20

References1. USP-27 (1116) Microbiological Evaluation.

Limits

CFU/mlClass

Gloves Personnel Clothing

References

Microbiological Evaluation Program for controlled Environment

IntroductionMicrobial monitoring program for controlled environment should assess the effectiveness of cleaning and sanitization practices. Microbial monitoring regardless of how sophisticated the system may be, will not and need not identify and quantitative all microbial contaminants present in these controlled environment. However routine microbial monitoring should provide sufficient information to ascertain that the controlled environment is operating within an

Introduction adequate state of control.

Environmental microbial monitoring of clean rooms and same other controlled environments, when appropriate, should include quantitative of the microbial content of room air. The objective of the microbial monitoring program is to obtain representative estimation of bioburden of the environment.




In Pharmaceutical industry all manufacturing activities are carried out in clean areas as recommended by FDA guidelines.Different standards of clean room are used.Eg- Class 100, Class 10,000, Class 1,00,000

Class 100- all sterile fillings (Injectables/eye drops) are carried out.

Class 10,000- Manufacturing activities like blending mixing, filtration, stirring and punching of tablets.

Class 1,00,000- Air locks, Packing area, manufacturing of non-injectable product.

Environmental Monitoring is performed by two methods. 1) Environmental monitoring by settling plate.2) Environmental monitoring by using air sampler.

Environmental monitoring by settling plateRequirementsMateriall Soyabean Casein Digest Agar (AM1091/AM5091)l S.aureus ATCC 25923l C.albicans ATTC 10231

Equipmentsl Autoclavel Incubator

Procedurel Prepare sterile plates of Soyabean Casein Digest Agar.l Expose the plates in specified area as per respective protocol.

o ol After exposure as per specified time incubate one set at 37 C + 2 C and set

o oanother set at 25 -30 C for 5 days.l Count the number of colonies from both sets.

st1 set will give bacterial colonies.nd2 set will give Yeast/Mould colonies.

l Count the colonies from both sets and calculate final CFU value.o o o o

l Incubate one sterile plate of SCDA at 37 C + 2 C and another at 25 -30 C for 5 days. (Negative control).

l Incoculate one SCDA Plate with 24 hours old culture of S.aureus and incubate o oat 37 C + 2 C for 5 days. Inoculate another SCDA Plate with 24 hours old

oculture C.albicans and incubate at 25-30 C for 5 days. (Positive Control).

Interpretationl Negative Control Plates should remain sterile till end of the incubation period.l Positive Control Plates should give growth of standard colonies.

Environmental Monitoring is performed by two methods. 1) Environmental monitoring by settling plate.2) Environmental monitoring by using air sampler.

Environmental monitoring by settling plateRequirementsMaterial

Equipments

Procedure

Interpretation

Environmental Monitoring by using air samplerRequirementsMateriall Soyabean Casein Digest Agar (AM1091/AM5091).l S.aureus ATCC 25923l C.albicans ATCC 10231

Equipmentsl Air Sampler (Accubas AX1)l Autoclavel Incubator

Procedurel Prepare sterile plates of Soyabean Casein Digest Agar.l Mount the air sampler at specified area as per respective protocol.l Place the sterile SCDA Plate inside air sampler.l Set all parameters of air samplers (Sampling flow, Sampling quantity and

time).l Start the air sampler.

ol At the end of exposure remove the plate from air sampler and incubate at 37 C

o o o+ 2 C or 25 -30 C For 5 days.o o o o

l Incubate one sterile plate of SCDA at 37 C + 2 C and another at 25 -30 C for 5 days. (Negative control).

l Incoculate one SCDA Plate with 24 hours old culture of S.aureus and incubate o oat 37 C + 2 C for 5 days. Inoculate another SCDA Plate with 24 hours old

oculture C.albicans and incubate at 25-30 C for 5 days. (Positive Control).l At the end of incubation period count the number of colonies.

Interpretationl Negative Control Plates should remain sterile till end of the incubation period.l Positive Control Plates should give growth of standard colonies.

LimitsAir cleanliness guidelines in colony forming units in controlled environment. (Using a slit-to-Agar sampler or equivalent).

Class (U.S. Customary) CFU per cubic meter of air CFU per cubic feet of air

100 Less than 3 Less than 0.1

10,000 Less than 20 Less than 0.5

100,000 Less than 100 Less than 2.5

References1. USP 27 (1116) Microbiological Evaluation2. Fedral Standard 209E September 1992.

Environmental Monitoring by using air samplerRequirementsMaterial

Equipments

Procedure

Interpretation

Limits

Class (U.S. Customary) CFU per cubic meter of air CFU per cubic feet of air

References




Microbiological Assay of Antibiotics

IntroductionThe inhibition of microbial growth under standardized conditions may be utilized for demonstrating the therapeutic efficacy of antibiotics. Any subtle change in the antibiotic molecule which may not be detected by chemical methods will be revealed by a change in the antimicrobial activity and hence microbiological assays are very useful for resolving doubts regarding possible change in potency of antibiotics and their preparations.

The microbiological assay is based upon a comparison of the inhibition of growth of micro-organisms by measured concentrations of the antibiotics to be examined with that produced by known concentrations of a standard preparation of the antibiotic having a known activity. Two general methods are usually employed, the cylinder-plate (or cup-plate) method and the turbidimetric (or tube assay) method.

The cylinder-plate method (Method A) depends upon diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a Petri dish or plate to an extent such that growth of the added micro-organisms is prevented entirely in a zone around the cylinder containing a solution of the antibiotic. The turbidimetric method (Method B) depends upon the inhibition of growth of a microbial culture in a uniform solution of the antibiotic in a fluid medium that is favorable to its rapid growth in the absence of the antibiotic.

The assay is designed in such a way that the mathematical model on which the potency equation is based can be proved to be valid. If a parallel-line model is chosen, the two log dose-response lines of the preparation being examined and the standard preparation should be parallel; they should be rectilinear over the range of doses used in the calculation.

These conditions should be verified by validity test by a given probability. Other mathematical models, such as the slope ratio method, may be used provided that proof of validity is demonstrated.

Standard Preparation and Units of ActivityA Standard Preparation is an authentic sample of the appropriate antibiotic for which the potency has been precisely determined by reference to the appropriate international standard. The potency of the standard may be expressed in International Units in µg per mg of the pure antibiotic.

The Standard Preparations for India are maintained at the Central Drugs Laboratory, Calcutta. A Unit referred to in the official assays and tests, is the specific activity contained in such an amount of the respective Standard Preparations as is indicated by the Ministry of Health & Family Welfare, Government of India from time to time.

A Standard Preparation may be replaced by a working standard prepared by any

Introduction

Standard Preparation and Units of Activity

laboratory which should be compared at definite intervals under varying conditions with the standard.

Buffer solutions: Prepare as directed in Table 1. The buffers are sterilized after preparation and the pH specified in each case is that after sterilization.

Preparation of the standard solution: To prepare a stock solution, dissolve a quantity of the Standard Preparation of given antibiotic, accurately weighed, and previously dried where so indicated in Table 4, in the solvent specified in the table, and then dilute to the required concentration as indicated. Store in a refrigerator and use within the period indicated. On the day of assay, prepare from the stock solution five or more test dilutions, usually in the ratio 1:2.25 for Method A or smaller for Method B. Use the final diluent specified and a sequence such that the middle or median has the concentration specified in Table 4.

Preparation of the sample solution: From the information available for the substance being examined (the “unknown”), assign to it an assumed potency per unit weight or volume, and on this assumption prepare on the day of the assay a stock solution and test dilution as specified for each antibiotic in Table 3 but with the same final diluent as used for the Standard Preparation. The assay with 5 levels of the Standard requires only one level of the unknown at a concentration assumed equal to the median level of the Standard.

Test organisms: The test organisms for each antibiotic is listed in Table 3, together with its identification number in the American Type culture Collection (ATCC) and the National Collection of Type Cultures (NCTC) or the National Collection of Industrial Bacteria (NCIB). Maintain a culture on slants of the medium and under the incubation conditions specified in Table 5, and transfer weekly to fresh slants.

Preparation of inoculum: The method of preparation of the microbial suspensions for preparing the inoculum for the assay is given in Table 2. If the suspensions are prepared by these methods, growth characteristics are sufficiently uniform so that inoculum can be adequately determined.

Cylinder Plate Method ( Method A )RequirementsMateriall Antibiotic Assay Medium (No.1) AM1002/AM5002l Antibiotic Assay Medium (No.5) AM50031l Antibiotic Assay Medium (No.8) AM50032l Antibiotic Assay medium (No.11) AM1004/AM5004l Buffer Solutions (Refer table 1)l 24 hours old ATCC Cultures. (Refer Table 2)l Distilled waterl 0.1 M. HCl

Buffer solutions:

Preparation of the standard solution:

Preparation of the sample solution:

Test organisms:

Preparation of inoculum:

Cylinder Plate Method ( Method A )RequirementsMaterial




l Medium Al Medium 1

Equipmentsl Laminar air flowl Autoclavel Incubatorl Assay cylinders (Aluminum or stainless steel)

(O.D. 8mm + 0.1mm, I.D. 6mm + 0.1mm, length 10mm + 0.1mm)l Sterile Pipette

Testing Procedure:- Preparation of test organisml Maintain the test organisms on slants of Medium A and transfer to a fresh

slant once a week. Incubate the slants at the temperature indicated above for 24 hours. Using 3 ml of saline solution, wash the organism from the agar slant onto a large agar surface of Medium A such as a Roux bottle containing 250 ml of agar. Incubate for 24 hours at the appropriate temperature. Wash the growth from the nutrient surface using 50 ml of saline solution. Store the test organism under refrigeration. Determine the dilution factor which will give 25% light transmission at about 530 nm. Determine the amount of suspensions to be added to each 100 ml of agar of nutrient broth by use of test plates or test broth. Store the suspension under refrigeration.

l Proceed as described in Method 1 but incubate the Roux bottle for 5 days . Centrifuge and decant the supernatant liquid. Resuspend the sediment with

o50 to 70 ml of saline solution and heat the suspension for 30 minutes at 70 C. Wash the spore suspension three times with 50 to 70 ml of saline solution. Resuspend in 50 to 70 ml of saline solution and heat-shock again for 30 minutes. Use test plates to determine the amount of the suspension required for 100 ml of agar. Store the suspension under refrigeration.

l Maintain the test organism on 10-ml agar slants of Medium G. Incubate at o o32 to 35 for 24 hours. Inoculate 100 ml of nutrient broth. Incubate for 16 to

o18 hours at 37 and proceed as described in Method 1.l Proceed as described in Method 1 but wash the growth from the nutrient

surface using 50 ml of Medium 1 (prepared without agar) in place of saline solution.

l Prepare sterile medium appropriate to the assay. (Recommended by Pharmacopoeia)

l Add the requisite quantity of suspension of the micro-organism to the sterile o omedium at a temperature between 40 C - 50 C (Refer to Table 2 and 3)

l Immediately pour the inoculated medium (approximately 21 ml) into petri dish to give depth of 3 to 4 mm. (1 to 2 mm for nystatin) Ensure that the layers of medium are uniform in thickness, by placing the dishes or plate on a leveled surface.

l Using the appropriate buffer solutions (refer Table 1 and 4). Prepare stock solutions of know concentration of the standard preparation and solution of the corresponding assumed concentrations of the antibiotic to be examined.

Equipments

Testing Procedure:- Preparation of test organism

For one level assay with standard curve on the day of the assay. l Prepare from the stock solution, 5 dilutions (S1 to S5) representing five test

levels of the standard and increasing stepwise in the ratio of 4:5.l From the information available for the antibiotic preparation which is being

examined (the “unknown”) assign to it an assumed potency per unit weight or volume and on this assumption prepare on the day of the assay a stock solution with the same solvent as used for the standard. Prepare from this stock solution five dilutions to a concentration equal to the median level of the standard to give the sample solution.

l For preparing the standard curve use a total of 12 petridishes to accommodate 72 cylinders or cavities. Use a set of 3 plates (18 cylinders) for each dilution.

l On each of the three plates of a set fill alternate cylinders or cavities with solution S (Representing the median concentration of the standard solution) 3

and each of the remaining nine cylinders or cavities with one of the other four dilutions of the standard solution. Repeat the process for the other 3 dilutions of the standard solution.

l For each unknown preparation (to be examined) use a set of three plates (18 cylinders) and fill alternate cylinders or cavities with the sample solution and each of the remaining 9 cylinders or cavities with solution S .3

l Incubate the plates for about 18 hours at the specified temperature and measure the diameter or the zones of inhibition.

Estimation of PotencyAverage the readings of solution S and the readings of the concentration tested on 3

each set of three plates, and average also all 36 readings of solution S . The 3

average of the 36 readings of solution S is the correction point for the curve. Correct 3

the average value obtained for each concentration (S S S and S ) to the figure it 1, 2 4, 5

would be if the readings for solution S for that set of three plates were the same as 3

the correction point. Thus, in correcting the value obtained with any concentration, say S if the average of 36 readings of S is, for example, 18.0mm and the average 1, 3

of the S concentrations on one set of three plates is 17.8mm, the correction is + 3

0.2 mm. If the average reading of S is 16.0 mm, the corrected reading of S is 16.2 1 1

mm. Plot these corrected values including of the average of the 36 readings for solution S on two-cycle semilog paper, using the concentrations in Units or ?g per 3

ml ( as the ordinate logarithm scale) and the diameter of the zones of inhibition as the abscissa. Draw the straight response line either through these points by inspection or through the points plotted for highest and lowest zone diameters obtained by means of the following expressions:

L = 3a + 2b + c – e ; H = 3e + 2d + c – a

5 5

where L = the calculated zone diameter for the lowest concentration of the standard curve response line.

Estimation of Potency




H = the calculated zone diameter for the highest concentration of the standard curve response line.

c = average zone diameter of 36 readings of the reference point standard solution.

a, b, d, e = corrected average values for the other standard solutions, lowest to highest concentrations, respectively.

Average the zone diameters for the sample solution and for solutions S on the 3

plates used for the sample solution. If the sample gives a large average zone size than the average of the standard (solution S ) add the difference between them to 3 ,

the zone size of solution S of the standard response line. If the average sample 3

zone size is smaller than the standard values, subtract the difference between them from the zone size of solution S of the standard response line. From the response 3

line read the concentration corresponding to these corrected values of zone sizes. From the dilution factors the potency of the sample may be calculated.

Two Level Factorial AssayPrepare parallel dilutions containing 2 levels of the standard (S and S ) and the 1 2

unknown (U and U ). On each of four or more plates, fill each of its four cylinders or 1 2

cavities with a different test dilution, alternating standard and unknown. Keep the plates at room temperature and measure the diameters of the zones of inhibition.

Estimation of potency:- Sum the diameters of the zones of each dilution and calculate the % potency of the sample (in terms of the standard) from the following equation:

% potency = Antilog (2.0 + a log I)

wherein a may have a positive or negative value and should be used algebraically and where a = (U + U ) - (S + S )1 2 1 2

(U + U ) + (S - S )1 2 1 2

U and U are the sums of the zone diameters with solutions of the unknown of high 1 2

and low levels.

S and S are the sums of the zone diameters with solutions of the standard of high 1 2

and low levels.

I = ratio of dilutions.

If the potency of the sample is lower than 60 % or greater than 150 % of the standard, the assay is invalid and should be repeated using higher or lower dilutions of the same solution.

The potency of the sample may be calculated from the expression.

% potency x assumed potency of the sample100

Two Level Factorial Assay

Estimation of potency:-

Turbidometric or Tube Assay Method (Method B)RequirementMateriall Antibiotic Assay Medium (No.3) AM1003/AM5003l Buffer solutions (refer Table 1)l 24 hours old ATCC cultures (refer Table 2)l Distilled waterl 0.1 m. HCll Dilute formaldehyde solution

Equipmentsl Laminar Air Flowl Autoclavel Incubatorl Water bathl Spectro photo meter

ProcedureThe method has the advantage of a shorter incubation period for the growth of the test organism (usually 3 to 4 hours) but the presence of solvent residues or other inhibitory substances affects this assay more than the cylinder-plate assay and care should be taken to ensure freedom form such substances in the final test solutions. This method is not recommended for cloudy or turbid preparations.

Prepare five different concentrations of the standard solution for preparing the standard curve by diluting the stock solution of the Standard Preparation of the antibiotic (Table 3) and increasing stepwise in the ratio 4:5. Select the median concentration (Table 3) and dilute the solution of the substance being examined (unknown) to obtain approximately this concentration. Place 1 ml of each concentration of the standard solution and of the sample solution in each of the tubes in duplicate. To each tube add 9 ml of nutrient medium (as per pharmacopieal requirements) previously seeded with the appropriate test organism (Table 3).

At the same time prepare three control tubes, one containing the inoculated culture medium (culture control), another identical with it but treated immediately with 0.5 ml of dilute formaldehyde solution (blank) and a third containing uninoculated culture medium.

Place all the tubes, randomly distributed or in a randomized block arrangement, in an incubator or a water-bath and maintain them at the specified temperature (Table 4) for 3 to 4 hours. After incubation add 0.5 ml of dilute formaldehyde solution to each tube. Measure the growth of the test organism by determining the absorbance at about 530 nm of each of the solutions in the tubes against the blank.

Estimation of potency: Plot the average absorbance for each concentration of the

Turbidometric or Tube Assay Method (Method B)RequirementMaterial

Equipments

Procedure

Estimation of potency:




standard on semi-logarithm paper with the absorbance on the arithmetic scale and concentrations on the logarithm scale. Construct the best straight response line through the points either by inspection or by means of the following expressions:

L = 3a + 2b + c – e ; H = 3e + 2d + c – a 5 5

where L = the calculated absorbance for the lowest concentration of the standard response line.

H = the calculated absorbance for the highest concentration of the standard response line.

a, b, c, d, e = average absorbance values for each concentration of the standard response line lowest to highest respectively.

Plot the values obtained for L and H and connect the points. Average the absorbances for the sample and read the antibiotic concentration from the standard response line. Multiply the concentration by the appropriate dilution

factors to obtain the antibiotic content of the sample.

Table 1Buffer solutions are prepared by dissolving the following quantities of dipotassium hydrogen phosphate and potassium dihydrogen phosphate in sufficient water to produce 1000ml after adjusting the pH with 8M phosphoric acid or 10M potassium hydroxide.

Buffer Dipotassium Hydrogen Potassium Dihydrogen pH adjusted afterNumber Phosphate, K HPO (g) Phosphate, KH PO (g) sterilization to 2 4 2 4

1 2.0 8.0 6.0 ± 0.1

2 16.73 0.523 8.0 ± 0.1

3 --- 13.61 4.5 ± 0.1

4 20.0 80.00 6.0 ± 0.1

5 35.0 --- 10.5 ± 0.1*

6 13.6 4.0 7.0 ± 0.2

* After addition of 2 ml of 10 M KOH.

Table 1

Buffer Dipotassium Hydrogen Potassium Dihydrogen pH adjusted afterNumber Phosphate, K HPO (g) Phosphate, KH PO (g) sterilization to 2 4 2 4

Table 2 - Preparation of inoculumTable 2 - Preparation of inoculum

Test OragansimIncubation conditions Suggested

dilution factoroTemp. ( C) Time

Suggested inoculum composition

Amount (ml per 100 ml) Antibiotics assayed

Bacilllus cereus var. mycoides

Bacillus pumilus

Bacillus subtilis

Bodetella bronchiseptica

Klebsiella pneumoniae

Micrococcus luteus (9341)

Micrococcus luteus (10240)

Mycobacterium smegmatis

2Pseudomonas aeruginosa

Saccharomyces cerevisiae (9763)

Saccharomyces cerevisiae (2601)

Staphylococcus aureus

Staphylococcus epidermidis

32-35

32-35

32-35

32-35

36-37

32-35

32-35

36-37.5

36-37.5

29-31

29-31

32-35

32-35

5 days

5 days

5 days

24 hrs

24 hrs

24 hrs

24 hrs

48 hrs

24 hrs

48 hrs

48 hrs

24 hrs

24 hrs

---

---

---

1:20

1:25

1:40

1:35

As determined

1:25

As determined

As determined

1:20

1:40

As required

As required

As requiredAs requiredAs required

0.1

0.1

1.5

0.3

1.0

0.5

1.0

1.0

0.030.44.0

OxytetracyclineTetracycline

FramycetinKanamycin sulphate

FramycetinKanamycin BRifampicin

Polymycin B

Streptomycin

Erythromycin

Bacitracin

Bleomycin

Carbenicillin

Amphotericin B

Nystatin

AmikacinDoxycycline, Oxytetracycline

Kanamycin sulphate

GentamicinNeomycinNovobiocin

0.1

0.2




Table 3 - Test organisms for microbiological assay of antibioticsTable 3 - Test organisms for microbiological assay of antibiotics

AntibioticAntibiotic Test organismTest organism 1 ATCC No.1 ATCC No.2NCTC No.3(NCIB No.)

2NCTC No.3(NCIB No.)

Amikacin Staphylococcus aureus 29737 7447

Amphotericin B Saccharomyces cerevisiae 9763 10716

Bacitracin Micrococcus luteus 10240 7743

Bleomycin Mycobacterium smegmatis 607 -

Carbenicillin Pseudomonas aeruginosa 25619 -

Doxycycline Staphylococcus aureus 29737 7447

Erythromycin Micrococcus luteus 9341 (8553)

Framycetin Bacillus pumilus 14884 8241Bacillus subtilis 6633 8236,10400

Gentamicin Staphylococcus epidermidis 12228 (8853)

Kanamycin sulphate Bacillus pumilus 14884 8241Staphylococcus aureus 29737 7447

Kanamycin B Bacillus subtilis 6633 8236

Neomycin Staphylococcus epidermidis 12228 (8853)

Novobiocin Staphylococcus epidermidis 12228 (8853)

Nystatin Saccharomyces cerevisiae 2601 10716

Oxytetracycline Bacilllus cereus var. mycoides 11778 10320Staphylococcus aureus 29737 7447

Polymyxin B Bodetella bronchiseptica 4617 8344

Rifampicin Bacillus subtilis 6633 8236

Streptomycin Bacillus subtilis 6633 8236Klebsiella pneumoniae 10031 (9111)

Tetracycline Bacilllus cereus 11778 10320Staphylococcus aureus 29737 7447

16 Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits



Table 4 - Stock solutions and test dilutions of Standard PreparationsTable 4 - Stock solutions and test dilutions of Standard Preparations

Standard Stock SolutionStandard Stock Solution Test Dilution Test Dilution

Antibiotic Assay method Prior drying Initial solvent (further diluent if different)

(1) (2) (3) (4)

Final stock concentration

per ml

Use before (number of

days)(5) (6)

Final diluent Median dose µg or units

per ml

Incubation otemp ( C)

(7) (8) (9)

Amikacin

Amphotericin B

Bacitracin

Bleomycin

Carbenicillin

Doxycycline

Erythromycin

Framycetin

Gentamicin

Kanamycin sulphate

Kanamycin B

Neomycin

Novobiocin

NystatinOxytetracycline

Polymyxin B

Rifampicin

Streptomycin

Tetracycline

B

A

A

A

A

B

A

A

A

AB

A

A

A

AAB

A

A

AB

AB

No

Yes

Yes

Yes

No

No

Yes

Yes

Yes

NoNo

No

Yes

Yes

YesNoNo

Yes

No

YesYes

NoNo

Water7DMF

0.01M HCl8B6

B1

0.1M HCl

Methanol8(10 mg/ml) ,(B2)

B2

B2

B2Water

B2

B2

Ethanol9(10 mg/ml) ,(B2)

7DMF0.1M HCl0.1M HCl

Water, (B4)

Methanol

WaterWater

0.1M HCl0.1M HCl

1 mg

1 mg

100 Units

2 Units

1 mg

1 mg

1 mg

1 mg

1 mg

800 Units1000 Units

1000 Units

1 mg

1 mg

1000 Units1 mg1 mg

10,000 Units

1 mg

1 mg1 mg

1 mg1 mg

14

Same day

Same day

14

14

5

14

14

30

3030

30

14

5

Same day44

14

1

3030

14

Water

B5

B1

B6

B6

Water

B2

B2

B2

B2Water

B2

B2

B4

B4B3

Water

B4

B1

WaterWater

WaterWater

10 µg

1.0 µg

1.0 Unit

0.04 Unit

20 µg

0.1 µg

1.0 µg

1.0 µg

0.1 µg

0.8 Unit10 Units

1.0 Unit

1.0 µg

0.5 µg

20 Units2.5 µg

0.24 µg

10 Units

5.0 µg

1.0 µg30 µg

2.5 µg0.24 µg

32-35

29-31

32-35

32-35

36-37.5

35-37

35-37

30-35

36-37.5

37-3932-35

32-35

36-37.5

32-35

29-3132-3535-37

35-39

29-31

32-3535-37

32-3535-37

Medium A

Ingredients in grams per liter

Peptone 6.0

Pancreatic digest of casein 4.0

Yeast Extract 3.0

Beef Extract 1.5

Dextrose 1.0

Agar 15.0

Final pH 6.5-6.6

Medium A


Medium B


Peptone 6.0

Beef Extract 1.5

Agar 15.0

Glycerin 10.0

Sodium Chloride 3.0

Final pH 6.9-7.1

References: IP 2007.

Medium B


References: IP 2007.




Antimicrobial Effectiveness Testing (Preservative efficacy testing)

IntroductionAntimicrobial preservatives are substances added to non sterile dosage forms to protect them from microbiological growth or from micro-organisms that are introduced inadvertently during or subsequent to the manufacturing process. In the case of sterile articles packaged in a multiple dose containers, antimicrobial preservatives are added to inhibit the growth of micro-organism that may be introduced from repeatedly withdrawing individual doses.All useful antimicrobial agents are toxic substances. For maximum protection of patients, the concentration of the preservative shown to be effective in the final packaged product should be below a level may be toxic to human beings.The concentration of an added antimicrobial preservative can be kept at a minimum if the active ingredient of the formulations passes an intrinsic antimicrobial activity.

RequirementMateriall Soyabean Casein Digest Medium (AM1092 / AM5092)l Soyabean Casein Digest Agar. (AM1091 / AM5091)l Sabouraud Dextrose Agar. (AM1087 / AM5087)l Sabouraud Dextrose Broth. (AM1088 / AM5088)l Candida albicans ATCC 10231l Aspergillus niger ATCC16404l Escherichia coli ATCC 8739l Pseudomonas aeruginosa ATCC 9027l Staphylococcus aureus ATCC 6538l Saline Solutionl Saline Solution Containing 0.05% w/v of Polysorbate 80

Equipmentsl Laminar air Flowl Incubatorl Autoclave

Testing ProcedurePreparation of InoculumFrom a recently grown stock culture of each of the test organism, Subculture on the surface of Medium. For bacterial cultures use Soyabean Casein Digest Agar and

0 0incubate at 30 Cto 35 C for 18-24 hours and for fungal cultures use Sabouraud

Introduction

RequirementMaterial

Equipments

Testing ProcedurePreparation of Inoculum

0 0Dextrose Agar and incubate at 20 Cto 25 C for 48 to 96 hours.

Using sterile saline solution harvest the bacteria and C.albicans cultures and dilute 8suitably with sterile saline solution to bring the count to about 1x10 per ml.

Similarly harvest A.niger culture with sterile saline solution containing 0.05% w/v 8of polysorbate 80 and adjust the spore count to about 1x10 per ml with sterile

saline solution.

Alternately the stock culture organisms may be grown in a suitable liquid medium, for bacterial cultures and C.albicans use Soyabean Casein Digest Medium and for A.niger use Sabouraud Dextrose Broth. Harvest the cells by Centrifugation, wash it and resuspend in sterile saline to give the required microbial or spore count.

Determine the number of colony forming units (CFU) per ml in each suspension by spread plate technique or pour plate technique.

Inoculation of Productl Inoculate each original product container or product tube (when original

container is not suitable for inoculation with sterile syringe fitted with needle, transfer 20ml per capped bacteriological tube) with 0.1 ml of standardized microbial suspension per 20 ml of product. The final concentration should be

5 6 between 1x10 and 1x10 micro-organisms per ml of product.0 0

l Incubate the inoculate containers or tubes at 20 Cto 25 C. Determine the viable count (by the plate count method) at 7,14,21 and 28 days subsequent to inoculation.

l Record also any change observed in appearance.

Interpretationl The concentrations of viable bacteria are not more than 0.1% of the initial

thconcentrations by the 14 day.l The concentrations of viable yeasts and moulds remain at or below the initial

concentration during the first 14 days.l The concentration of each test microorganisms remain at or below these

thdesignated levels during the remainder of the 28 day test period.

References1. IP 2007.2. USP 27.

Inoculation of Product

Interpretation

References

Validation of autoclaves and Ovens using Biological Indicators

IntroductionBiological indicators are characterized and standardized preparations of specific micro-organisms having known stable high resistance to one or more sterilization procedures. A biological indicator is used to (a) assist in the qualification of the

Introduction physical operation of sterilizer, (b) develop and establish a validated sterilization process for a particular article and for the sterilization of equipment, materials and packaging components for aseptic processing, (c) monitor an established sterilization cycle and (d) revalidate established and documented sterilization




cycles.

A biological indicator is in one of two main forms, each of which incorporates a viable culture of a known species of micro-organisms. In one, the organisms (spores) are added to a carrier (disc or strip of filter paper, glass or plastic) and packed so as to maintain the integrity of the inoculated carrier but when used appropriately in the individual immediate package allows the sterilizing agent to exert its effect. In the other, the spores are added to representative units of the lot to be sterilized (inoculate product) or to similar units (inoculated similar product). An inoculated product should not adversely affect the performance characteristics of the viable spores. If the material to be sterilized is a liquid and if it is not practicable to add a biological indicator to selected units of the lot, viable spores may be added to a simulated product but in such a way that the resistance of the simulated product to the sterilization process does not differ from the resistance to sterilization of the product to be sterilized.

The following factors govern the choice of indicator organismsl The test strain should be stable and non-pathogenic.l The resistance of the test strain to the particular sterilization process should be

great compared with the resistance of all species of micro-organisms likely to contaminate the product including, where possible, the ambient flora in the production environment.

l The recovery of the test strain should be reproducible when cultivated under carefully standardized conditions.

A biological indicator used for monitoring of a sterilization process may not be suitable, and may even be satisfactory, for validation of sterilization cycles, which may differ in their needs for particular applications. The proportion of test organisms surviving the sterilization process should be quantified and related to the expected lethality of the process. The effective use of an indicator for the monitoring of a sterilization process requires a knowledge of the product being sterilsed and its components parts and a general idea of the probable types and numbers of micro-organism constituting the microbial burden in the product immediately prior to sterilization.

A biological indicator is characterized by the strain of test organisms constituting the microbial burden in the product immediately prior to sterilization.

A biological indicator is characterized by the strain of test organism, the total viable spore count per carrier (test piece of the indicator), the D-value (Decimal Reduction Value), the Z-value and the expiry date. Information on the recovery medium and the conditions of incubation should also be known. The D-value is a measure of the resistance of a micro-organisms to particular type of sterilization process. It is the value of the appropriate parameter of the process (duration or absorbed dose) requires to reduce the number of viable micro-organisms to 10% of the original number. In the case of steam sterilization, the D-value is expresses by the time in minutes at a defined temperature, e.g. D , D indicate the temperature of 121 170

sterilization. In the case of radiation sterilization, the D-value is expressed by the absorbed dose and subscripts are often used to show the log system used, e.g. D . 10

In the case of ethylene oxide sterilization, the D-value is expresses by the time in minutes and is only of significance under precisely defined sterilization conditions.

In case of steam and dry heat sterilizations, the Z-value relates the heat resistance of a micro-organism to changes in temperature. The Z-value is the change in temperature required to alter the D-value by a factor of 10.

Biological indicators with indeterminate labeled spore counts or without such labeled information at all, or with a vague description of the sterilization method for which the indicator is to be used, are unsatisfactory unless the user determines the required resistance characteristics and the total spore count per carrier with the necessary precision under the user’s sterilization conditions.

The selection of a biological indicator is critical and requires that due weight be given to a knowledge of the resistance of the indicator to the specific sterilization process so that when it is used within its performance characteristics it provides a challenge to the sterilization process that exceeds the challenge of the natural microbial burden in or on the product.

The indicator should be placed at the locations presumed or, wherever possible, found by previous physical measurements to be least accessible to the sterilizing agent. Even in placing the indicator in any selected location, attention should be paid to its positioning, e.g. vertical, sideways, to assure maximum penetration of the sterilizing substrate. The performance of a biological indicator is a function of both its initial viable spore count and the resistance of the viable spores to the sterilization process. It is therefore important that the indicator maintains its numbers of viable spores and resistance characteristics throughout its shelf-life.

Some characteristics of commercially available biological indicatorsSome characteristics of commercially available biological indicators

Sterilization Mode Example of a typical D-value

(minutes)

Minimum D values for selecting a suitable biological indicator

(minutes)

Minimum survival time

(minutes)

Kill time

(minutes)

0Dry Heat 160

0Moist Heat 121

1.9

1.9

Min 1.0Max 3.0Min 1.5Max 3.0

Min 4.0Max 14.0Min 4.5

Max 14.0

Min 10.0Max 12.0Min 13.5Max 32.0




Validation of AutoclavesMateriall Soyabean Casein Digest medium (AM1092 / AM5092)l Spore strip (Geobacillus sterothermophilus)l Glass vials

Eqiupments l Laminar Flow

0 0l Incubator (60 C + 2 C)l Autoclave

Procedurel Depending upon the loading capacity of the autocalve, the position of

exposure of the bacteriological spore strip are decided i.e, for small autoclave– two sites, for large autoclave – six sites.

l Place the bacteriological spore strips in a clean sterile test tube/vial as its two sites i.e. bottom and top of the fully loaded small autoclave and its six sites for larger autoclave.

0l Operate the respective Autoclaves as per the operating procedure (121 /15

PSI/151 min).l After autoclaving remove and transfer the strips aseptically in sterile 50 ml

SCDM. Carry out the operation inside LAF.l Label them accordingly.

0 0l Incubate at 60 C + 2 C for 7 days with a positive and negative control.

Note: 1) Positive control= Sterile 50 ml SCDM incubated with unexposed bacteriological spore strip to steam sterilization.

Note: 2) Negative control= Sterile 50 ml SCDM incubated without bacteriological spore strip.

l Observe for growth everyday till 7 days.l Validation using bacteriological spore strip is carried out once in 6 monthsl Sterilize all cultures before discarding.

Interpretationl Growth within 3 days in positive control vials, no growth in negative control

thvials and test vials till 7 day indicates proper steam sterilization.l Growth within 3 days in positive control vials, no growth in negative control

vials and growth in test vials in 7 days indicated failure in steam sterilization, hence faults have to be rectified.

l Autoclave should be validated again after correction.

Validation of OvensMaterial l Soyaben Casein Digest Medium (AM1092/AM5092)

Validation of AutoclavesMaterial

Eqiupments

Procedure

Interpretation

Validation of OvensMaterial

l Spore strip (Bacillus Atrophaeus)l Glass vials / petridishes

Equipmentsl Laminar Flow

0 0l Incubator (35 C + 2 C)l Oven

Procedurel Place the Bacillus Atrophaeus spore strips in a petri dishes / glass vials and

cover with aluminum foil.l Arrange petri dishes / glass vials in the fully loaded oven (for Big oven use 10

strips, for medium size oven use 6 strips and for small oven use 2 strips each).0 0

l Carry out standard cycle of sterilization (160 C for 2 hours / 180 C for 1 hours).

l Remove petri dishes containing spore strips from oven when temperature falls 0below 60 C and transfer it to Laminar airflow unit.

l Let them cool down to room temperature. Conduct further experiment under laminar air flow.

l Aseptically transfer each strip into separate tube containing 50 ml of sterile 0 0soyabean casein digest medium. Incubate the tubes at 35 C + 2 C for 7

days. Check the tubes for turbidity / pellicle formation every day till end of 7 days.

l Inoculate the unheated strip in another sterile Soyabean Casein Digest Medium tube under aseptic conditions that acts as positive control.

l Keep one tube containing sterile SCDM as negative control without adding spore strip.

l Incubate the test tubes along with positive control and negative control at 0 0(35 C + 2 C) for 7 days.

l Sterilize all cultures before discarding.

Interpretationl Positive control - Within 18-24 hours growth should be observedl Negative control – No growth should be present till the end of 7 days.l Test media tubes containing spore strips exposed to sterilization cycle should

not show any growth till the end of 7 days.

References1. IP 2007.2. USP 27 (55).

Equipments

Procedure

Interpretation

References




Cup-plate Assay of Cyano-Cobalmin (Vitamin B ) using E.coli mutant 113 (D)12

IntroductionTo determine the potency Vit B solution with comparison with standard Vit B12 12

solution- by Agar diffusion method. The term ‘Vitamin’ refers to an essential diotarcy factor which is required in small amounts and whose absence results in deficiency diseases. Various vitamins are produced by various micro-orgs. Yeast are the best producers. They are the normal flora of the intestine are known to produce B vitamins, Vit. B also known as Cyano – cobalmin or cobalmin Vit B is produced 12, 12

in considerable quantity by some species of Nocardia, Streptomyces, and many other bacteria belonging to the general Bacillus, Clostridia and many others. In general, Streptomyces olivacoous NRRL B 1125 is used. Vit B is a complex water 12

soluble vitamin which was originally obtained from liver as Cayano cobalminer or hydroxy combalmin, the deficiency of this vitamin produces permicious Anaemia, hence it is also called Anti-Pernicious agent.

Vit B is assayed by using E.coli-mutant 113.D. The potency of Vit B in any given 12 12

solution can be determined by observing the growth response of an auxtrophic organism, which is requiring Vit B for growth. This auxatroph should give a growth 12

response in proportion to the amount of Vit B added. The response is indicated by 12

a exhibition zone around the cup containing Vit B12.

RequirementsMateriall B12 Maintenance Media (For E.coli mutant) AM 1008l B12 Assay Agar (using E.coli mutant culture) AM 1007l Sterile buffered distilled water pH-7. l Culture E.coli, 113-Davis, washed cells (10 hrs. old cultures and having O.D .

of 0.1).l Standard Vit B solution, Test Sample. 12

EquipmentsGlass ware: Sterile petridishes, sterile 10 ml. And 1 ml, pipette, sterile Pasteur pipette, cork borer (6 mm).

Procedurel Prepare Vit B standards 0.005, 0.01, 0.02, 0.03, 0.05, 0.00, 0.1 mg/ml in 12

Introduction


Equipments

Procedure

sterile buffered distilled water. l Similarly dilute the test solution, so that it falls in the range of the standard.l To 20 ml of sterile and cooled assay medium, add 0.5 ml. Of standardized

culture, mix and pour into sterile petridish. Punch out four Agar cups with a sterile Cork borer in each petridish.

l To the first petridish add 3 standards, 50 µl each into 3 separate cups and to ththe 4 cup add 50 µl dilution of the unknown corresponding approx. to 0.01

mg/ml.l To the second petridish add remaining 3 standards 50 µl each into 3 separate

thcups and to the 4 cup add 50 µl dilution of the unknown corresponding approximately to 0.04 mg/ml.

o ol Refrigerate at 4 C for ½ hr and then incubate at 37 C for 24 hours.l Measure the zone of exhibit of each standard and unknown dilution. Plot a

graph of zone size on the Y-axis against conc. of Vit B on X-axis. Calculate 12

the exact conc. of Vit B in the unknown from the graph.12

Standard inoculum preparationl E. coli 113-Davis mutant culture to be subcultured every fortnight on B12

Maintenance Media (For E.coli Mutant) AM 1008.l Use 10 hours old culture for inoculum preparation.l Transfer a loopfull of culture from Slant to 10 ml sterile saline.l Wash the cells 2-3 times with sterile saline.l After final washing, suspend the cells in sterile saline and adjust , the OD at

550 nm upto 0.1.

InterpretationZone of exhibition (excess growth as compared to mat growth of E.coli at the background) is observed around each cup containing known and unknown concentrations of Vitamin B12.

References1. IP 2007.2. US Pharmacopeail convention Inc. 2001, The United States Pharmacopeia

25/NF 20-2002.

Standard inoculum preparation

Interpretation

References

Calcium Panthothenate AssayIntroductionSerial dilutions of test and standard material is prepared and it is added into assay medium which does not contain Calcium Pantothenate. Medium tubes are inoculated with standard inoculum of Lactobacillus Plantarum culture and incubated at specified temperature. Inoculum culture grows in presence of Calcium Pantothenate and final turbidity is measured by reading transmittance using spectrophotometer.

Introduction RequirementMateriall Pantothenate assay media – Accumix - AM10784l Pantothenate culture Agar- Accumix- AM10786l Lactobacillus Plantarum – ATCC 8014l Standard Calcium Pantothenatel Phosphorus pentoxide

RequirementMaterial




l Acetic Acidl Sodium Acetate

Equipmentso o

l Incubator at 37 C + 2 C .l Pipettel Weighing Balancel Spectrophotometerl Autoclave

ProcedureInoculum Preparationl Lactobacillus plantarum to be subcultured every week on Pantothenate

culture agar.l Use less than one week old culture for inoculum preparation.l Transfer Lactobacillus Plantarum culture to a sterile tube containing 10 ml of

*culture medium.l Incubate this culture for 16 to 24 hours at any selected temperature between

o o o 30 - 37 but held constant to within + 0.5l The cell suspension so obtained is the inoculum.

*Cultural MediumTo each of a series of test tubes containing 5 ml of Pantothenate Assay Medium add 5.0 ml of **water containing 0.2 mg of Calcium Pantothenate per 5 ml. Plug with

ocotton/ cap the tubes, sterilize in an autoclave at 121 and cool.

**Water containing 0.2 mg of Calcium Pantothenate per 5 mlDilute 0.08 ml of ***Standard stock solution of Calcium Pantothenate to 100 ml in a clean dried volumetric flask.

***Standard Stock solution of Calcium PantothenateDissolve 50 mg of USP Calcium Pantothenate, previously dried and stored in the dark over phosphorus pentoxide (Silica bags) and accurately weighed in a dehumidified room, in about 500 ml of (D/W /DM/R.O) water in a 1000 ml volumetric flask. Add 10 ml 0.2 N acetic acid and 100 ml of sodium acetate solution (1 in 60), then dilute with water to volume.

Use immediately or store under toluene in a refrigerator.

Test Procedurel To series of test tubes containing 5 ml of Pantothenate Assay Medium add in

duplicates, 1.0 ml, 1.5 ml,2.0, 3.0 ml, 4.0 ml & 5.0 ml of **water containing 0.2 mg of Calcium Pantothenate per 5 ml. Add sufficient water to make 10 ml.

l To other 4 similar test tubes containing 5 ml of Pantothenate Assay Medium add sufficient water to make 10 ml.

l Cover the tubes of both series suitably to prevent contamination and heat in

Equipments

ProcedureInoculum Preparation

*Cultural Medium

**water containing 0.2 mg of Calcium Pantothenate per 5 ml

**Water containing 0.2 mg of Calcium Pantothenate per 5 ml***Standard stock solution

***Standard Stock solution of Calcium Pantothenate

Test Procedure

**water containing 0.2 mg of Calcium Pantothenate per 5 ml

oan autoclave at 121 C and for 5 minutes cool.l Add 10 µl of inoculum to each tube, except 2 of the 4 tubes containing no

Standard preparation to serve as the uninoculated blanks and mix. Incubate o o the tubes at a temperature between 30 C& 37 C.

ol Held constant to within +0.5 , until following 22 to 24 hours of incubation.l Determine the transmittance of the tubes in the following manner.l Mix the contents of each tube and transfer to a cuvette. Place the cuvette in a

spectrophometer that has been set at a specific wavelength between 540 nm & 660 nm.

l Read the transmittance when a steady state is reached.l This steady state is observed a few seconds after agitation, when the

galvanometer reading remains constant for 30 seconds or more.l Allow approximately the same time interval for the reading on each table.l With the transmittance set at 1.00 for the uninoculated blank, read the

transmittance of the inoculated blank.l With the transmittance set at 1.00 for the inoculated blank, read the

transmittance of the remaining tubes.l If there is evidence of contamination with a foreign microorganisms,

disregard the result of the assay.

CalculationPrepare a standard concentration – response curve as follows: For each level of the standard, calculate the response from the sum of the duplicate values of the transmittance as the difference, Plot this response on the ordinate of cross-section paper against the logarithm of the ml of Standard Preparation per tube on the abscissa using for the ordinate either an arithmetic or a logarithm scale, which ever gives the better approximation to a straight line. Draw the straight line or smooth curve that best fits the plotted points.

Calculate the response, y, adding together the two transmittances for each level of the Assay preparation. Read from the standard curve the logarithm of the volume of the Standard Preparation corresponding to each of those values of y that fall within the range of the lowest and highest points plotted for the standard. Substract from each logarithm so obtained the logarithm of the volume, in ml of the Assay preparation to obtain the difference, x, for each dosage level. Average the values of

1x for each of three or more dosage levels to obtain x = M , the log-relative potency of the Assay Preparation. Determine the quantity, in mg, of USP Calcium Pathothenate RS corresponding to the Calcium Pathothenate in the portion of material taken for Assay antilog:

1M = antilog (M + log3 R),

In which R is the number if mg of calcium pantothenate that was assumed to be present in each mg (or capsule or tablet) of the material taken for assay.

Reference1. USP 27 (111) Design and analysis of Biological assays.

Calculation

Reference


Microbiological Methods in Food IndustryHand Book of


Enumeration of Coliforms, Faecal Coliforms and E.coli in Water in Sealed Containers and Prepackaged Ice Using the Hydrophobic Grid-membrane Filter (HGMF) Method

IntroductionThe HGMF filter method is applicable to the enumeration of coliforms, faecal coliforms and aerogenic Escherichia coli in water sealed containers (including mineral and spring water) and prepackaged ice in accordance with the Regulations of the Food and Drugs Act.

The HGMF filter involves a technique where 100 ml of the sample is inoculated through a filter, incubated at a specific temperature and for a specific time on non-selective agar and subsequently on a selective agar. After counting typical colonies on the filter, the most probable number of growth units (MPNGU) present can be estimated from a standard statistical MPNGU table.

This method has been shown to produce satisfactory results with artificially contaminated water in sealed containers (including mineral and spring water).

The HGMF analysis takes 26-30 h and yields counts that are as high as, and more precise than, the Most Probable Number method. A single dilution gives an accurate count over a wide range of contamination levels. Counting precision may be better than on conventional plates or membrane filters because the HGMF reduces the effect of individual visual acuity on the count. If a low count is expected, the detection limit can be lowered by filtering more of the sample.

The HGMF method is capable of detecting coliforms that grow poorly or ferment lactose slowly in LST or BGLB media. Stressed organisms are resuscitated for 4 hrs on a non-selective medium before being exposed to selective growth conditions.

Materiall HGMF (1600 gird-cell, 0.45 µm pore size; available as ISO-GRID Membrane

Filters from Oxoid LTD, Nepean, Ont) or equivalent (i.e. Millipore Filters)l Membrane filter forcepsl Peptone water, 0.1% (PW) (AM1079, AM5079)l Nutrient agar (NA) plates, (AM1074, AM5074)l Selective agars (use one agar from each of the following groups)l For Coliformsl M-Endo Agar LES (AM106921, AM506921)l Violet Red Bile Agar (AM1107, AM5107)l For Faecal Coliforms to E.colil M-FC Agar Base (AM506923)l Chromogenic Coliform Agar (AM10251, AM50251)l EC Broth (AM1039, AM5039)l Lauryl Tryptose Broth (AM1053, AM5053)l Brilliant Green Bile Broth 2% (AM1020, AM5020)l E.coli ATCC 25922

Introduction

Material

l E.aerogenes ATCC 13048

Equipmentsl Spreadfilter with funnel (Filtaflex) or ISO-GRID filtration unit (Oxoid) or

equivalent.0 0

l Incubators capable of maintaining 35 C or 44.5 C.l Laminar Air Flow.l Autoclave.l Incubator.

ProcedureEach sample unit must be analyzed individually. Carry out the test in accordance with the following instructions:

1) Handling of Sample Unitsl Water in sealed containers-Do not store sample units for more than 25

0hrs before analysis. Store under refrigeration (0-5 C) conditions.l Prepackaged ice-

(a) If sample units are prepackaged in leak proof containers, thaw 0them in the containers under refrigeration (0-5 C) prior to analysis.

(b) If sample units are not in leak proof containers, transfer the ice aseptically to sterile plastic bags or other suitable sterile containers. Seal containers to prevent contamination, and thaw sample unites

0under refrigeration (0-5 C). DO NOT store thawed sample units for more than 6 hrs before analysis.

2) Perparation for Analysisl Prepare sterile peptone water (PW), nutrient agar (NA) plates and

selective agar plates.(Refer pack inserts provided by manufacturer).l Clean the surface of the working area with a suitable disinfectant.l Clearly label duplicate NA and selective agars with appropriate

identifying information.

3) Preparation of Dilutionsl As required, prepare a 1:10 dilution of the sample by aseptically adding

10 ml (the analytical unit) into 90 ml of the PW. Mix the dilutions by shaking the dilution bottle 25 times through a 30 cm arc in approximately 7 seconds.

l The HGMF will allow counts to be made from suspensions containing up to 5,000 organisms/ml. There normally should be no need to prepare further dilutions. If this is necessary, prepare succeeding decimal dilutions as required, using a separate sterile pipette for each transfer. Record the dilution (C) used for analysis.

Equipments

Procedure

1) Handling of Sample Units

2) Perparation for Analysis

3) Preparation of Dilutions




l Filter the total volume to be filtered in one operation; do not attempt to filter successive aliquots. Record the volume (V) filtered.

4) Filtrationl Agitate each sample or dilution bottle.l Handle HGMF with sterile forceps.l Following the manufacturer’s instruction for use of the filtration

apparatus, aseptically transfer 100 ml of the sample or pipette 1.0 ml of the required dilution and inoculate the HGMF. Open the filter valve until all liquid has passed through and aseptically remove the HGMF. Do in duplicate.

l Follow the manufacturer’s instructions for cleaning the filtration apparatus.

l Repeat with subsequent dilutions as required.

5) Plating and Incubationl Transfer the HGMF to the surface of a NA plate by rolling it onto the agar

0to avoid trapping air bubbles. Incubate plates at 35 C for 4 hrs. Plates should be placed in an inverted position in stacks of not more than two.

l Transfer the HGMF to the surface of a selective agar plate by rolling it 0onto the agar to avoid trapping air bubbles. Incubate plates at 35 C for

24+2 hrs. Plates should be placed in an inverted position in stacks of not more than two.

6) Counting and Scoring HGMFl Typical colonies:

(a) Coliforms: M-Endo Agar : pink to reddish growths (E.coli with green metallic sheen ) VRB Agar: purple-red growths.

(b) Faecal Coliforms to E.coli: l M-FC Agar: the blue growths are lactose-fermenting faecal coliforms

Chromgenic agar: purple, dark blue colonies.l Follow the manufacturer’s instructions for the use of automated and

manual counters.l HGMF will give accurate counts over a wider range than is possible with

plates. Count only those HGMF containing 20-1580 occupied grid cells.l Count 1 (one) for each grid-cell showing typical growth. (DO NOT count

the individual colonies if a grid-cell contains more than one typical colony). If a rough estimate indicated fewer than 200 occupied grid-cells, count the whole HGMF.

l For higher densities (up to 50 % occupied grid-cells), rotate the HGMF so that the center indicator lies either to the left or right. Count positive grid-cells (containing typical colonies) in the 4 rows immediately below the centre and in 4 rows immediately above the center (8 rows). Multiply this partial HGMF count by 5 estimate the score.

l Record as too numerous to count (TNTC) any HGMF for which all grid-cells are typical.

4) Filtration

5) Plating and Incubation

6) Counting and Scoring HGMF

l Record the scores of both the duplicate HGMF. If there are no typical grid cells, record the score as zero.

(7) Control CulturesPrepare positive and negative controls as follows:Positive Control:Use 24 hours old E.coli (ATCC 2592) cultures that is known to produce typical reactions on the selective Agar plates and is capable of fermenting Lactose to produce typical reactions on L-EMB Agar.

Negative Control:Use 24 hours old Enterobacter aerogenes (ATCC 13048) culture that does not produce “Positive” reactions on EMB Agar and is indole negative, Methyl red negative, Voges-Proskaver-positive and citrate positive.

(8) (a) Confirmation Steps for coliformsConfirm 5 typical colonies by inoculating growth from each colony into

0tubes of LT (one colony per tube) and incubate at 35 C for 24 to 48 hrs. Any gas positive LT tubes should be sub cultured to BGBB and incubated

0at 35 C for 24 to 48 hrs. Gas production in Brilliant Green Bile Broth within 48 hrs is a confirmed coliform test.

(b) Confirmation Steps for identification of E.coli (Faecal).Confirm 5 typical colonies by inoculating growth from each colony into

0tubes of LT (one colony per table) and incubate for 48 hrs at 35 C. Transfer one loopfull of growth from each tube to EC Broth (avoid

0transferring Pellicle). Incubate the tubes at 45 C for 24 hrs formation of gas in all the tubes at the end of 48 hrs constitutes a positive E.coli (Faecal) confirmation test.

(9) Reporting of resultsl Report average MPNGU as per total count from duplicate plates round-

3off to two significant figures (e.g, record 2850 as 2.9 x 10 ).l If the lowest dilution plated shows no typical grid-cells, the recovered

value will be the lower average obtainable with a given volume plated onto a given set of replicate HGMF, preceded by a “less than” (<) sign, e.g. for 1.0 ml and a set of duplicate HGMF (1 ml per HGMF) the value is <0.5. This figure should be multiplied by the dilution factor of the inoculum on the HGMF.

References1. Association of Official Analytical Chemists (AOAC). 1985. Officail final action

hydrophobic grid membrane filter method for detecting total coliforms, feacal coliforms and E.coli in foods. J.Assoc, Anal.Chem.68:481 .

2. Atlas, R.M.1997. Handbook of Microbiological Media. Second edition. L.C. Parks(editor). CRC Press Inc.

3. Brodsky, M.H., P.Entis, A.N.Sharpe and G.A. Jarvis. 1982. Enumeration of

(7) Control Cultures

Positive Control:

Negative Control:

(9) Reporting of results

References




indicator organisms in foods using the automated hydrophobic grid-membrane filter technique. J.Food Prot. 45:292-296.

4. Entis, P.1984. Enumeration of total coliforms, fecal coliforms and Echerichia coli in foods by hydrophobic grid membrane filter: collaborative study. J.Assoc. Offic. Anal. Chem.67:812-823.

5. Sharpe, A.N. and P.I.Peterkin.1988. Membrane filter food microbiology. Research Studies Press Ltd, Taunton, Somerset, U.K.

6. Health Canada. Appendices A,B,C and G. (Vol.1). Compendium of Analytical Methods.

Microbiological Examination of Ice Cream an Ice Milk

IntroductionThis method shall be used for the determination of total aerobic bacteria (Aerobic Colony Count) and coliform bacteria (Coliforms) in ice cream or ice milk.

Materiall Plate Count Agar (AM1081, AM5081).l Lauryl Tryptose Broth (AM1053, AM5053).l Brilliant Green Bile Broth 2 % (AM1020, AM5020).l Peptone Water 0.1% (AM1079, AM5079).

Equipmentsl Autoclave.l Laminar Air flow.l Incubator.

ProcedureEach sample unit shall be analyzed individually. The tests shall be carried out on the sample in accordance with the following instructions:

1) Handling of Sampling Unitsl Keep the sample units frozen in the laboratory before analyzing them.l Analyze the sample units as soon as possible after they have been

received at the laboratory.

2) Preparation of mediaThe following media, to be prepared and sterilized according to the manufacturer’s instructions, shall be used:l Plate Count (PC) agar.l Lauryl Tryptose (LT) broth.l Brilliant Green Bile Broth 2%.

3) Preparation of Dilutionsl Prepare sterile 0.1% peptone water diluent.l Combine portions from several locations within the frozen sample unit to

ensure a representative analytical unit of 11 (10)g. Weight or volume in brackets indicate alternative procedure for making dilutions.

l Prepare a 1:10 dilution of the ice milk by aseptically adding the analytical unit into 99(90) ml of the peptone water diluent.

l Mix the 1:10 dilution by shaking the dilution bottle 25 times in a 30 cm

Introduction

Material

Equipments

Procedure

1) Handling of Sampling Units

2) Preparation of media


arc in approximately 7 sec.l Check the pH of the dilution. If the pH is outside the range the range of

5.5 to 7.6, adjust to 7.0, with sterile NaOH or HCL.l Prepare succeeding dilutions as required to determine the ACC and the

number of Coliforms present in the ice cream by transferring 11(10) ml of the previous dilution into 99(90) ml of 0.1% peptone water diluent. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present.

4) Determination of the ACCThe medium used is PC agar prepared for making pour plates.l Agitate each dilution bottle to resuspend material.l Without delay, pipette 1 ml of each prepared dilution into each of two

appropriately marked Petri plates using a sterile pipette for each transfer.

0l Pour 12-15 ml of the tempered agar (40-45 C) into each plate and mix

contents by rotating and tilting.l Allow agar to solidify.l Plates shall be poured not late than 15 min, after preparation of

dilutions.0

l Incubate plates in an inverted position at 35 + 0.5 C for 48 + 2 hrs.l Avoid crowding or exercise stacking of plates in order to permit rapid

equilibration of plates with incubator temperature.l Count colonies promptly after the incubation temperature.

5) Determination of ColiformsPresumptive Testl The medium used is LT Broth, dispensed in 10 ml volumes into tubes

containing gas vials. (inverted Durham tubes).l Arrange LT Broth tubes in rows of fives, and mark them identifying the

sample, the sample unit and the dilution to be inoculated.l Inoculate each tube of a set of five tubes of single strength LT Broth with

1 ml of the 1:10 dilution (ice cream or milk suspension; see section 3,3, above). Repeat for each succeeding decimal solution as required.

l Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials.

0l Incubate the inoculated LT Broth tubes at 35 + 0.5 C for 24 + 2 hrs.

Examine for gas formation, record results, and on the same day, begin the confirmed test for all gas-positive tubes.

4) Determination of the ACC

5) Determination of ColiformsPresumptive Test




l Incubate gas-negative tubes for an additional 24 + 2 hrs, examine, record the number of gas-positive tubes, add to the result obtained in step e, above and begin the confirmed test for the additional gas-positive tubes.

l The absence of gas in all of the tubes at the end of 48 + 2 hrs of incubation constitutes a negative presumptive test.

6) Confirmed Testl The confirmatory medium used is BGBB Broth dispensed in 10 ml

volumes in tubes containing gas vials.l Submit all gas-positive LT Broth tubes to the confirmed test.l Shake or rotate the LT Broth tubes to mix the contents and transfer one

loopful form each positive LT Broth tube to a tube of the BGBB Broth (Avoid transferring pellicle).


0l Incubate the inoculated BGBB Broth tubes at 35 + 0.5 C for 24 + 2

hrs. Examine for gas formation, and record resultsl Incubate gas negative tubes for an additional 24 + 2 hrs, examine,

record the number of additional gas-positive tubes and add to the results obtained in previous step above.

l Formation of gas within 48 + 2 hrs of incubation constitutes a positive confirmed test.

7) Calculation of most probable numbers (MPN)Table A-1 shows the most probable numbers of coliform per 100 g or ml of test material corresponding to the number of gas-positive tubes in the coliform test. Table A-1 has been adapted from a conversion table prepared for the analysis of drinking water where 10,1.0 and 0.1 ml of the water under test are used as test portions. The table is equally appropriate if 10, 1.0 and 0.1 g or ml of a food constitutes the test portions in the tubes. When other sized portions of the test material are placed in the tubes, the MPN values obtained from Table A-1 has to be multiplied by an appropriate number, to correct for the actual amount of test material in the tubes, and also to obtain the MPN per g or ml as is usually done for foods, rather than per 100 ml (g), for which the values are given in the table. The volume of diluent added to the tubes (and which accompanies the sample) is ignored when calculating the MPN.

ExampleThe following inoculated tubes give a positive reading:1. 5 tubes with 10 ml of 1:10 dilution of test material-all 5 are positive.2. 5 tubes with 1ml of 1:10 dilution of test material-1 are positive.3. 5 tubes with 1 ml of 1:00 dilution of test material-none are positive.

The quantities (test portions) in each of the five tubes of the three dilution series represent 1,0.1 and 0.01 g or ml test material respectively.

6) Confirmed Test

7) Calculation of most probable numbers (MPN)

Example

However, since only 1/10 of these amounts were actually used in the analysis, the values of 33 obtained from Table A-1 must be multiplied by 10 giving 33 x 10 = 330 organisms per 100 g or ml of test material. Since the results have to be expressed per g or ml, the MPN value of 330 must be divided by 100. When higher dilutions are used, the same procedure is followed, but the multiplier (dilution factor) is enlarged to relate the amount of test material actually present to the values given for 10, 1.0 and 0.1 g or ml in Table A-1.

Dilution factor=Reciprocal of the dilution of the analytical unit.

For calculating the MPN, use the dilution factor of the middle set of the three dilutions selected.

To determine which consecutive dilutions to use, refer to the combinations shown below: (See also Table A-2 ).

1. If only 3 dilutions are made, use the results for those 3 dilutions to compute the MPN. Examples a and b.

2. If more than 3 dilutions are employed, use the results of only 3 consecutive dilutions. Select the highest dilution, for which all 5 tubes are positive and 2 subsequent higher dilutions. Examples c and d.

Determination n c m M

ACC 5 2 100,000 1,000,000

Coliforms 5 1 10 1,000

n = Number of sample units (subsamples) to be examined per lot.

c = Maximum number of sample units (subsamples) per lot which may have a bacterial concentration. Higher than the value for ‘m’ without violation of the Regualtion.

m = Maximum number of bacteria per g of ice cream or ice milk which is of no concern (acceptable level of contamination).

M = Maximum number of bacteria per g of ice cream or ice milk which if exceeded by any one sample. Unit (subsample), renders the lot under investigation in violation of the Regulation.

l If more than 3 dilutions are made, but none of the dilutions tested have all 5 tubes positive, use the first 3 dilutions. Example e.

l If a positive tube occurs in the dilution higher than the 3 chosen to rule (see no.3), the number of such positive tubes should be added to those of the next lower dilution. Example f.

l If the tubes of all sets of a dilution series are positive, choose the 3 highest dilutions of the series and indicate by a ‘greater than’ symbol (>) that the MPN is greater than the one calculated. Example g.




Refer to Table A-1 and look up the value which corresponds to the number of positive tubes obtained.

MPN/g or ml = No Microorganism x dilution factor of(Table A-1)/100 middle set of tubes

TABLE A-1Most Probable Number (MPN) of Bacteria Per 100 g (mL) of Test Material Using 5 Tubes 10,1 and 0.1 mL or g of Test Material

Pos* MPN Pos* MPN Pos* MPN Pos* MPN Pos* MPN Pos* MPN10;1;0.1 10;1;0,1 10;1;0,1 10;1;0,1 10;1;0,1 10;1;0,1

0 <1.8 100 2 200 4.5 300 7.8 400 13 500 23

001 1.8 101 4 201 6.8 301 11 401 17 501 31002 3.6 102 6 202 9.1 302 13 402 21 502 43003 5.4 103 8 203 12 303 16 403 25 503 58004 7.2 104 10 204 14 304 20 404 30 504 76005 9 105 12 205 16 305 23 405 36 505 95

010 1.8 110 4 210 6.8 310 11 410 17 510 33

011 3.6 111 6.1 211 9.2 311 14 411 21 511 46012 5.5 112 8.1 212 12 312 17 412 26 512 64013 7.3 113 10 213 14 313 20 413 31 513 84

014 9.1 114 12 214 17 314 23 414 36 514 110

015 11 115 14 215 19 315 27 415 42 515 130020 3.7 120 6.1 220 9.3 320 14 420 22 520 49021 5.5 121 8.2 221 12 321 17 421 26 521 70

022 7.4 122 10 222 14 322 20 422 32 522 95023 9.2 123 12 223 17 323 24 423 38 523 120

024 11 124 15 224 19 324 27 424 44 524 150025 13 125 17 225 22 325 31 425 50 525 180030 5.6 130 8.3 230 12 330 17 430 27 530 79

031 7.4 131 10 231 14 331 21 431 33 531 110

032 9.3 132 13 232 17 332 24 432 39 532 140033 11 133 15 233 20 333 28 433 45 533 180

034 13 134 17 234 22 334 31 434 52 534 210

035 15 135 19 235 25 335 35 435 59 535 250040 7.5 140 11 240 15 340 21 440 34 540 130

041 9.4 141 13 241 17 341 24 441 40 541 170042 11 142 15 242 20 342 28 442 47 542 220043 13 143 17 243 23 343 32 443 54 543 280044 15 144 19 244 25 344 36 444 62 544 350045 17 145 22 245 28 345 40 445 69 545 440050 9.4 150 13 250 17 350 25 450 41 550 240051 11 151 15 251 20 351 29 451 48 551 350052 13 151 17 252 17 352 32 452 56 552 540053 15 153 19 253 26 353 37 453 64 553 920054 17 154 22 254 29 354 41 454 72 554 1600

055 19 155 24 255 32 355 45 455 81 555 >1600

* Number of positive tubes with each of 3 volumes used.

TABLE A-1





TABLE A-2Dilutions to be used and calculations of MPN per g or mL of test material

10 1 0.1 0.01 0.001

a. 5/5** 5/5 2/5 5-5-2 540 1

b. 5/5 5/5 2/5 5-5-2 540 10

c. 5/5 5/5 2/5 2/5 5-2-2 95 100

d. 5/5 5/5 2/5 0/5 5-2-0 49 100

e. 2/5 2/5 1/5 0/5 2-2-1 12 10

f. 5/5 2/5 1/5 1/5*** 5-2-2 95 10

g. 5/5 5/5 5/5 5/5 5-5-5 >1600 100

* Dilutions to be used are shaded gray. ** No. of positive tubes/No. of tubes inoculated. ***

TABLE A-2

10 1 0.1 0.01 0.001

8) InterpretationThe tolerances as specified hereafter and representing the maximum count of total aerobic bacteria (Aerobic Colony Count), and the maximum probable incidence of coliform bacteria (Coliforms) in ice cream or in ice milk shall be applied in determining whether the tested lot of the product complies with Food and Drug Regulations.

9) LimitsThe maximum count of total aerobic bacteria permitted for each lot is that represented by an Aerobic.

8) Interpretation

9) Limits

Colony count not exceeding:l 1,00,000 per g in more than two of the five sample units, andl 1,00,000 per g in any of the five sample units, included in the sample

taken from a lot.

These tolerances are summarized in the above table.

Reference1. Official Method MFO-2 Health Protection Branch-Ottawa.Reference

Microbiological Examination of Cottage Cheese

IntroductionThis method shall be used for the determination of coliform bacteria (Coliforms) in cottage cheese in accordance with the Food and Drug Regulations.

Materiali) Lauryl Tryptose Broth (AM1053, AM5053)ii) Brilliant Green Bile Broth 2% (AM1020, AM5020)iii) 2% Sodium Citrate solution.

Equipmentsl Laminar Air Flowl Autoclave l Incubator

ProcedureEach sample unit shall be analyzed individually. The test shall be carried out in

Introduction

Material

Equipments

Procedure

accordance with the following instructions:

1) Collection of Samplesl A sample, consisting of five sample units drawn at random from each lot,

shall be taken.l Each sample unit shall consist of at least 100 g.l Collect original unopened containers wherever possible.l More than one sample unit may be collected from large institutional or

bulk containers when the total number of sample units required exceeds the number of containers in the lot. When the lot consists of containers smaller than 100 g, a sample unit will consist of more than one container (e.g. four 25 g containers in each sample unit).

l Employ aseptic techniques in collecting the sample units when sampling from bulk. Place each collected sample unit into a separate sterile container.

0l Keep sample units refrigerated (0-5 C) during transport.

1) Collection of Samples

Dilutions*Dilutions*

UndilutedUndiluted 1:101:10 1:1001:100 1:10001:1000

Amount of original test material (g or mL)Amount of original test material (g or mL)Combinationto be used

Combinationto be used

MPN fromTable A-1MPN fromTable A-1

Dilution factor on middledilution





2) Handling of Sample Units.0

l Keep sample units refrigerated (0-5 C) in the laboratory prior to analyzing them.

l Analyze the sample units as soon as possible after they have been received in the laboratory.

3) Preparation of MediaThe following media, prepared and sterilized according to the manufacturer’s instructions, shall be used:l Lauryl Tryptose (LT) Brothl Brilliant Green Bile Broth 2%

4) Preparation of Dilutions0

l Prepare sterile 2% sodium citrate solution and temper to 40-45 C.l Combine portions from several locations within the sample unit to

ensure a representative analytical unit of 11 (10)* g.l Weight and volume in brackets indicate alternate procedure for making

dilutions.l Prepare a 1:10 dilution of the cottage cheese by aseptically adding the

analytical unit to 99(90)* ml of the sodium citrate in a sterile blender jar. Blend for the minimum time required to produce a homogeneous suspension. To prevent overheating, blending time should not exceed 2.5 min.

l Check the pH of the suspension. If the pH is outside the range of 5.5 to 7.6, adjust to 7.0, with either sterile NaOH or HCL.

5) Determination of ColiformsI) Presumptive Testl The medium used is LT Broth, dispensed in 10 ml volumes into

tubes containing gas vials (inverted Durham tubes).l Arrange LT Broth tubes in rows of five, and mark them identifying

the sample, the sample unit and the dilution to be inoculated.l Inoculate each tube of a set of five tubes of single strength LT Broth

with 1 ml of the 1:10 dilution (cottage cheese homogenate). Repeat for each succeeding decimal dilutions as required


0 0l Incubate the inoculated LT Broth tubes at 35 C + 0.5 C for 24 +

2 hrs. Examine for gas formation, record results, and on the same day begin the confirmed test for all gas-positive tubes (see section II below)

l Incubate gas-negative tubes for an additional 24 + 2 hr, examine, record the number of gas-positive tubes, add to the results obtained in the previous step above, and begin the confirmed test for the additional gas-positive tubes.

l The absence of gas in all the tubes at the end of 48 + 2 hrs of incubation constitutes a negative presumptive test.

2) Handling of Sample Units.

3) Preparation of Media


5) Determination of ColiformsI) Presumptive Test

l Compute the “MPN” of presumptive coliforms per g of cottage cheese following the instructions in Part 5, to convert the number of gas-positive tubes to MPN values. Record results.

II) Confirmed Testl The conformity medium used is BGB Broth dispensed in 10 ml volumes

in tubes containing gas vials.l Submit all gas-positive LT Broth tubes to the confirmed test.l Shake or rotate the LT Broth tubes to mix the contents and transfer one

loopful from each positive LT Broth tube to a tube of the BGB Broth. (Avoid transferring pellicle).


0 0l Incubate the inoculated BGB Broth tubes at 35 C + 0.5 C for 24 + 2

hrs. Examine for gas formation, and record results.l Incubate gas-negative tubes for an additional 24 + 2 hrs, examine

record the number of gas-positive tubes, and add to the results obtained in the previous step above.

l Formation of gas within 48 + 2 hrs incubation constitutes a positive confirmed test.

l Compute the “MPN” of presumptive coliforms per g of cottage cheese following the instructions in Part 5, to convert the number of gas-positive tubes to MPN values. Record results.

6) Calculation of most probable numbers (MPN)Table A-1 shows the most probable numbers of coliform per 100 g ml of test material corresponding to the number of gas-positive tubes in the coliform test. Table A-1 has been adapted from a conversion table prepared for the analysis of drinking water where 10,1.0 and 0.1 ml of the water under test are used as test portions. The table is equally appropriate if 10, 1.0 and 0.1 g or ml of a food constitutes the test portions in the tubes. When other sized portions of the test material are placed in the tubes, the MPN values obtained from Table A-1 has to be multiplied by an appropriate number, to correct for the actual amount of test material in the tubes, and also to obtain the MPN per g or ml as is usually done for foods, rather than per 100 ml (g), for which the values are given in the table. The volume of diluent added to the tubes (and which accompanies the sample) is ignored when calculating the MPN.

ExampleThe following inoculated tubes give a positive reading:1. 5 tubes with 10 ml of 1:10 dilution of test material-all 5 are positive.2. 5 tubes with 1ml of 1:10 dilution of test material-1 are positive.3. 5 tubes with 1 ml of 1:00 dilution of test material-none are positive.

The quantities (test portions) in each of the five tubes of the three dilution series represent 1,0.1 and 0.01 g or ml respectively are used.

II) Confirmed Test


Example




However, since only 1/10 of these amounts were actually used in the analysis, the values of 33 obtained from Table A-1 must be multiplied by 10 giving 33 x 10 = 330 organisms per 100 g or ml of test material. Since the results have to be expressed per g or ml, the MPN value of 330 must be divided by 100. When higher dilutions are used, the same procedure is followed, but the multiplier (dilution factor) is enlarged to relate the amount of test material actually present to the values given for 10, 1.0 and 0.1 g or ml in Table A-1.

Dilution factor=Reciprocal of the dilution of the analytical unit.


To determine which consecutive dilutions to use, refer to the combinations shown below: (See also Table A-2 ).

1. If only 3 dilutions are made, use the results for those 3 dilutions to compute the MPN. Examples a and b.

2. If more than 3 dilutions are employed, use the results of only 3 consecutive dilutions. Select the highest dilution, for which all 5 tubes are positive and 2 subsequent higher dilutions. Examples c and d.


ACC 5 2 100,000 1,000,000



n = Number of sample units (subsamples) to be examined per lot.c = Maximum number of sample units (subsamples) per lot which may have a

bacterial concentration. Higher than the value for ‘m’ without violation of the Regulation.

m = Maximum number of bacteria per g of ice cream or ice milk which is of no concern (acceptable level of contamination).

M = Maximum number of bacteria per g of ice cream or ice milk which exceeded by any one sample. Unit (subsample), renders the lot under investigation in violation of the Regulation.


l If a positive tube occurs in the dilution higher than the 3 chosen to rule (see no.3), the number of such positive tubes should be added to those of the next lower dilution. Example f.

l If the tubes of all sets of a dilution series are positive, choose the 3 highest dilutions of the series and indicate by a ‘greater than’ symbol (>) that the MPN is greater than the one calculated. Example g.





0 <1.8 100 2 200 4.5 300 7.8 400 13 500 23

001 1.8 101 4 201 6.8 301 11 401 17 501 31002 3.6 102 6 202 9.1 302 13 402 21 502 43003 5.4 103 8 203 12 303 16 403 25 503 58004 7.2 104 10 204 14 304 20 404 30 504 76005 9 105 12 205 16 305 23 405 36 505 95

010 1.8 110 4 210 6.8 310 11 410 17 510 33

011 3.6 111 6.1 211 9.2 311 14 411 21 511 46012 5.5 112 8.1 212 12 312 17 412 26 512 64013 7.3 113 10 213 14 313 20 413 31 513 84

014 9.1 114 12 214 17 314 23 414 36 514 110

015 11 115 14 215 19 315 27 415 42 515 130020 3.7 120 6.1 220 9.3 320 14 420 22 520 49021 5.5 121 8.2 221 12 321 17 421 26 521 70

022 7.4 122 10 222 14 322 20 422 32 522 95

TABLE A-1






023 9.2 123 12 223 17 323 24 423 38 523 120

024 11 124 15 224 19 324 27 424 44 524 150025 13 125 17 225 22 325 31 425 50 525 180030 5.6 130 8.3 230 12 330 17 430 27 530 79

031 7.4 131 10 231 14 331 21 431 33 531 110

032 9.3 132 13 232 17 332 24 432 39 532 140033 11 133 15 233 20 333 28 433 45 533 180

034 13 134 17 234 22 334 31 434 52 534 210

035 15 135 19 235 25 335 35 435 59 535 250040 7.5 140 11 240 15 340 21 440 34 540 130

041 9.4 141 13 241 17 341 24 441 40 541 170042 11 142 15 242 20 342 28 442 47 542 220043 13 143 17 243 23 343 32 443 54 543 280044 15 144 19 244 25 344 36 444 62 544 350045 17 145 22 245 28 345 40 445 69 545 440050 9.4 150 13 250 17 350 25 450 41 550 240051 11 151 15 251 20 351 29 451 48 551 350052 13 151 17 252 17 352 32 452 56 552 540053 15 153 19 253 26 353 37 453 64 553 920054 17 154 22 254 29 354 41 454 72 554 1600

055 19 155 24 255 32 355 45 455 81 555 >1600




10 1 0.1 0.01 0.001

a. 5/5** 5/5 2/5 5-5-2 540 1

b. 5/5 5/5 2/5 5-5-2 540 10

c. 5/5 5/5 2/5 2/5 5-2-2 95 100

d. 5/5 5/5 2/5 0/5 5-2-0 49 100

e. 2/5 2/5 1/5 0/5 2-2-1 12 10

f. 5/5 2/5 1/5 1/5*** 5-2-2 95 10

g. 5/5 5/5 5/5 5/5 5-5-5 >1600 100

* Dilutions to be used are shaded gray. ** No. of positive tubes/No. of tubes inoculated.

TABLE A-2

10 1 0.1 0.01 0.001



Amount of original test material (g or mL)Amount of original test material (g or mL)Combinationto be used





7) InterpretationThe tolerances as specified hereafter and representing the maximum probable incidence of coliforms bacteria (Coliforms) in cottage cheese shall be applied

7) Interpretation in determining whether the tested lot of the product complies with the Food and Drugs Regulations.




8) LimitsThe maximum Most Probable Number (MPN) of coliform bacteria permitted for each lot is that represented by a coliform MPN not exceeding:

l 10 per g in more than one of the five sample units, andl 1,000 per g in any of the five sample units, included in the sample taken from

a lot.

These tolerances are summarized in the following table.



8) Limits


n = Number of sample units (subsamples) to be examined per lot. C = Maximum number of sample units (subsamples) per lot which may have a bacterial concentration higher than the value for ‘m’ without violation of the Regulation.

m = Maximum number of bacteria per g of cottage cheese which is of no concern (acceptable level of contamination).

M = Maximum number of bacteria per g of cottage cheese which if exceeded by any one sample unit (subsample) renders the lot under investigation in violation of the Regulation.

References1. Official Method MFO-4 Health Protection Branch - Ottawa. References

Microbiological Examination of Egg Products and of Liquid Eggs

IntroductionThis method shall be used for the determination of bacteria of the genus Salmonella in egg products and in liquid eggs, in accordance with the Food and drug Regulations.

Materiall Nutrient Broth (AM1077, AM5077)l Nutrient Agar (AM1074, AM5074)l Tetrathionate Brilliant Green Bile Broth (AM50953)l Bismuth Sulphite Agar (AM1013, AM5013)l MacConkey Agar (AM1059, AM5059)l Triple Sugar Iron Agar (AM1099, AM5099)l Lysine Iron Agar (AM10576, AM50576)l Urea Agar Base Christensen’s (AM1105, AM5105)l Brilliant Green Agar modified (AM1018, AM5018)l Salmonella Typhimurium ATCC 14028l Salmonella Identification Kit (20797001)

Equipmentl Laminar Air Flow.l Autoclave.l Incubator.

Procedure1) Collection of Samplesl A sample, consisting of ten sample units drawn at random from each lot,

shall be taken.l Each sample unit shall contain at least 100 g or ml.l Collect original unopened containers wherever possible.l More than one sample unit may be collected from large institutional or

bulk containers when the total number of sample units required exceeds

Introduction

Material

Equipment

Procedure1) Collection of Samples

the number of containers in the lot. When the lot consists of containers smaller than 100 g, a sample unit will consist of more than one container (e.g., four 25 g containers in each sample unit).


0l Keep unfrozen sample units refrigerated (0-5 C), and frozen sample

units frozen during transport.

2) Handling of Sample Units0

l Keep unfrozen sample units refrigerated (0-5 C), and frozen sample units frozen in the laboratory prior to analyzing them.

l Analyze the sample units as soon as possible after they have been received in the laboratory.

3) Preparation of MediaThe following media, which are to be prepared and sterilized according to the manufacturer’s instructions, shall be used:(1) Nutrient Broth (NB)(2) Selenite Cystine (SC) Broth(3) Tetrathionate Brilliant Green (TBG) Broth(4) Bismuth Sulfite (BS) Agar(5) Brilliant Green Agar Modified(6) MacConkey Agar (MA)(7) Triple Sugar Iron (TSI) Agar(8) Lysine Iron (LI) Agar(9) Urea Agar Base Christensen’s(10) Nutrient Agar (NA)

4) Non-Selective Enrichment (Pre-enrichment)l Thaw frozen sample units. Do not allow the temperature of any sample



4) Non-Selective Enrichment (Pre-enrichment)




0unit to exceed 45 C.l Withdraw a 25 g analytical unit form each 100 g sample unit. When a

sample unit consists of more than one container mix the contents of each container of the sample unit aseptically prior to obtaining the 25 g analytical unit. The analytical units may be composited. Since compositing presents difficulties in preparation and disposal of the materials, exercise care in handling bulk preparations.

l Suspend the individual analytical units or the composite unit(s) in nine times their weight of NB in pre-warmed, sterile blender jar.

l Blend the mixture at low speed for two min.l Check the pH of each blended analytical or composite unit. If the pH is

outside the range of 6.0 - 7.0, adjust to 7.0 with either sterile NaOH or HCl.

l Inoculate NB with a known culture of Salmonella and subsequently make transfers to all other media used in the analysis. This is the positive media control. Set up a negative control by incubating appropriate unioculated media during each step of the analysis.

l Incubate the inoculated pre-enrichment Broth(s) and the controls at 0 035 C + 0.5 C for 18-24 hrs. In no circumstances shall the incubation be

prolonged for more than 24 hrs.

5) Selective Enrichmentl Transfer 1 ml of the incubated pre-enrichment broth into each of 9 ml of

SC and TBG broths using a sterile pipette.0 0

l Incubate the SC and TBG broths for 24 + 2 hrs at 35 C + 0.5 C and at 0 043 C + 0.5 , respectively.

6) Selective Platingl After the incubation period, streak a loopful each of the selective

enrichment Broths onto BS Agar and BG Agar plates to obtain well isolated colonies. The Broth may also be streaked onto a third commercially available plating medium.

l It has been observed that BS Agar is inhibitory for Salmonella serotypes 0other than S.typhi unless it is refrigerated at 4 C for at least 24 hrs before

streaking. The possibility of an inhibitory effect of this medium should be taken into consideration.

0 0l Incubate plates at 35 C + 0.5 C for 24 + 2 hrs. It may be necessary to

incubate the BS Agar plates for 48 + 2 hrs.l Examine the plates after the incubation period for colonies indicative of

Salmonella. On BG Agar, such colonies are pink to fuchsia surrounded by red medium. On BS Agar, they are usually black, with or without a metallic sheen, with increasing time of incubation the surrounding medium is gradually blackened. It should be noted, however, that lactose-and/or sucrose-fermenting strains (eg. S. arizonae) may develop a coliform-like (greenish) appearance on BG Agar. A heavy growth of coliforms may mask the appearance of the Salmonella colonies. On BS Agar, some Salmonella types may form dark brown

5) Selective Enrichment

6) Selective Plating

rather than black colonies.l If there are no colonies indicative of Salmonella, on the plates bacteria

of the genus Salmonella are considered absent from the analytical or composite unit from the colonies originated.

7) Biochemical Screeningl Using an inoculating needle, pick colonies indicative of Salmonella from

the MA plates and inoculate the biochemical media listed in table 1. 0 0Incubate these media at 35 C + 0.5 C for 18-24 hrs. Cap the tubes

loosely to avoid the occurrence of erroneous results.l Other media may be used to observe the reactions listed in Table 1. If

additional biochemical information is desired, other reaction media or commercially available diagnostic kits may be used.

l If none of the isolates from a particular analytical or composite unit shows biochemical reactions suggestive of Salmonella, then bacteria of genus Salmonella are considered to be absent from the analytical or composite units from which the isolates originated. If Salmonella are suspected, proceed to serological testing.

l Use TSI or LI Agar cultures less than 72 hr old for the serological 0identification but store the cultures at 2-8 C if the serological

identification is not carried within 12 hrs after the incubation of the TSI or LI Agar cultures.

0 0l Incubate the inoculated NA slants at 35 C + 0.5 C for 24 + 2 hrs.l Use NA slant cultures less than 48 hrs old for the serological

0identification but store the cultures at 2-8 C if the serological identification is not carried out within 12 hrs after the incubation of the NA slant cultures.

8) Serological TestingI) Testing with somatic polyvalent antiserum (Group A-I, and Vi)l Mark the following sections on an agglutination plate: C+ (positive

control), C-(negative control), and T(test culture). If several cultures are tested at the time, identify several test areas. T , T , T , etc. 1 2 3

l Place one drop of physiological saline on each of the areas marked T and C+, and two drops on the area marked C-.

l Remove sufficient culture material from a biochemical test medium (from the slant area not the butt) or from an NA slant and prepare a heavy suspension in the test area(s) and in the negative control area.

l For the positive control, use a known Salmonella culture and make a suspension in the area marked C+.

l Prepare the somatic polyvalent antiserum as directed by the manufacturer and place one drop onto each of the test suspension areas and onto the positive control area.

l Mix each of the culture-saline-serum suspensions (test cultures and positive control), and the saline-culture mixture of the negative control with a sterile needle or loop. Tilt the slides back and forth for 1 min.

l Hold slides against a dark background and observe for agglutination.

7) Biochemical Screening

8) Serological TestingI) Testing with somatic polyvalent antiserum (Group A-I, and Vi)




Cultures usually agglutinate within1min.l False-positive reactions may occur, these can be resolves by further

testing with somatic grouping and with flagellar antisera.l The tests are invalidated if the negative control shows agglutination

(autoagglutination).

II) Testing with Somatic Grouping AntiseraIt is preferable to test the culture against somatic grouping antisera whenever possible; however the culture may be sent to a typing center for identification. The majority of the Salmonella isolated from foods belong to the groups B, C, D and E. It is important to recognize that unless a complete set of grouping sera is available some Salmonella may be missed. In such cases any culture possessing the biochemical reactions indicative of Salmonella should be sent to a typing center for identification.l Mark the following sections on an agglutination plate: C+ (positive

control), l Use a positive control culture for each individual group tested.l Place one drop of physiological saline on each of the area marked T and

C+, and two drops on the area marked C-.l Remove sufficient culture material from a biochemical test medium

(from the slant area, not the butt) or from an NA slant and prepare a heavy suspension in the test area(s) and in the negative control area.

l Prepare the grouping antisera as directed by the manufacturer, and

II) Testing with Somatic Grouping Antisera

place one drop onto each of the test suspension areas and onto the positive control area.

l Mix the culture-saline-serum suspension (test cultures and positive controls), and the saline-culture mixture of the negative control with a sterile needle or loop. Tilt the slides back and forth for 1 min.

l Hold slides against dark background and observe for agglutination. Cultures usually agglutinate within 1 min.

l If the culture-saline-serum mixture has not agglutinated, repeat procedure with another group antiserum.

l If the serological test is positive, the culture shall be sent to a Salmonella typing center for serotyping.

l The tests are invalidated if the negative control shows agglutination (autoagglutination).

l If all the serological tests performed on an isolate are negative but the original culture gave biochemical reactions indicative of Salmonella (see Table 1), that culture shall be sent to typing center for verification.

9) Interpretation / LimitsThe lot of egg products or of liquid eggs sampled shall be considered in compliance with the Food and Drug Regulations when bacteria of the genus Salmonella are not found in any of the 10 sample units analyzed (individually or as composites).

9) Interpretation / Limits

Table 1Minimal Biochemical Screening MediaTable 1

MediumMedium ReactionReaction ObservationsObservations Reaction shown by majority of SalmonellaReaction shown by majority of Salmonella

Triple Sugar Iron (TSI) Agar

Lactose and/or Sucrose utilization

Positive reaction: Yellow slantNegative reaction: Colour becomes more intensely red.

Negative (some strains may show a positive reaction)

Dextrose utilizationPositive reaction: yellow butt with or without gas formation

Negative reaction: Colour of butt remains unchangedPositive

H S production2

Positive reaction:Blackening of butt often extending into the slant Negative reaction: No Blackening

Positive Slow H S producers may be 2

encountered. If lactose positive Salmonella are present, the H S reaction 2

may be inhibited on TSI Agar

Gas FormationPositive reaction: Formation of gas pockets in the medium

Negative reaction: No gas pockets in the mediumPositive

Lysine Iron (LI) Agar H S production2 Same as in TSI Agar Positive

Lysine decarboxylasePositive reaction: Butt turns purple

Negative reaction: Yellow butt if dextrose is utilizedPositive

Lysine desaminasePositive reaction: Wine coloured slant

Negative reaction: No wine coloured slantNegative





Urea Agar Base Christensen’s *

Production of UreasePositive reaction: Slant pinkish red

Negative reaction: Colour of slant unchangedNegative

* although Lysine deaminase is used to distinguish Proteus from Salmonella, the urease test is a more reliable indicator for Proteus spp.

References1. Official Method MFO-6 Health Protection Branch Ottawa.

Microbiological Examination of Milk

IntroductionThis method shall be used for the determination of total aerobic bacteria (Aerobic Colony Count) in milk, partly (partially) skimmed milk, milk for manufacture into dairy products,) partly (partially) skimmed milk with added milk solids in accordance with Food and Drug Regulations, respectively.

Materiall Plate Count Agar. (AM1081, AM5081)l Peptone water 0.1% (AM1079, AM5079)

Equipmentl Laminar Air Flow Unit l Autoclavel Incubatorl Colony Counter

ProcedureEach sample unit shall be analyzed individually. The tests shall be carried out on the sample in accordance with the following instructions.

1) Collection of Samplesl A sample, consisting of five sample units drawn at random from each lot,

shall be taken.l Each sample unit shall consist of at least 100 ml.l Collect original unopened containers wherever possible.l More than one sample unit may be collected from large institutional or

bulk containers when the total number of sample units required exceeds the number of containers in the lot. When the lot consists of containers smaller than 100 ml, a sample unit will consist of more than one container (e.g. four 25 ml containers in each sample unit).


0l Keep sample units refrigerated (0-5 C ) during transport.

Introduction

Material

Equipment

Procedure


2) Handling of Sampling Units0

l Keep sample units refrigerated (0-5 C ) in the laboratory prior to analyzing them.

l Analyze the sample units as soon as possible after they have been received at the laboratory.

3) Preparation of mediaThe following medium, to be prepared and sterilized according to the manufacturer’s instructions, shall be used:l Plate Count (PC) Agar.

4) Preparation of Dilutionsl Prepare sterile 0.1% Peptone Water diluent.l Thoroughly mix each sample unit by shaking the container.l Prepare a 1:10 dilution of the “milk” by aseptically pipetting 11(10)

ml* (the analytical unit) of the milk into 99 (90) ml* of the diluent.

* Weight or volume in brackets indicate alternate procedure for making dilutions.

l Mix the 1:10 dilution by shaking the dilution bottle 25 times in a 30 cm arc in approximately 7 sec.

l Check the pH of the suspension. If the pH is outside the range of 5.5 to 7.6, adjust to 7.0, with either sterile NaOH or HCl.

l Prepare succeeding dilutions as required to determine the ATCC present in the “milk” by transferring 11(10) ml of the previous dilution into 99(90) ml of 0.1% Peptone Water diluent. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present.

5) Determination of the ACCThe medium used is PC Agar prepared for making pour plates.








Analysisl Agitate each dilution bottle to resuspend material.l Without delay, pipette 1 ml of each prepared dilution into each of two

appropriately marked Petri plates using a sterile pipette for each transfer.

0l Pour 12-15 ml of tempered Agar (40-45 C) into each plate and mix

contents by rotating and tilting.l Allow the Agar to solidify.l Plates shall be poured not later than 15 min after preparation of

dilutions.0 0

l Incubate plates in an inverted position at 35 C + 0.5 C for 48 + 2 hrs.l Avoid crowding or excessive stacking of plates in order to permit rapid

equilibration of plates with incubator temperature.l Count colonies promptly after the incubation period.l Select for counting those plated containing 30-300 colonies, including

pinpoint colonies. If counts do not fall within this range, select plates that have counts nearest to this range.

6) Recording Resultsl Calculate average count (arithmetic mean) of duplicate plates.l When reporting results, round-off the counts to two significant figures,

and record only the first two left hand digits (e.g: record 2,850 as 2,900).

l If the lowest dilution plated shows no colonies, report the count as the product of 0.5 x the dilution factor preceeded by a “less than” (<) sign.

l To compute the ACC, use the formula: N=A x D, where N is the number of colonies per g of product, A is the average count, and D is the respective dilution factor.

7) Interpretation / LimitsThe tolerance as specified hereafter and representing the maximum incidence of total aerobic bacteria (Aerobic Colony Count) in milk, partly (partially) skimmed milk or partly (partially) skimmed milk with added milk solids, shall be applied in determining whether the tested lot complies with the Food and Drug Regulations.

Analysis

6) Recording Results

7) Interpretation / Limits

The maximum count of total aerobic bacteria permitted for each lot is that represented by the Aerobic Colony not exceeding.

(1) 50,000 per ml in more than two of the five sample units, and(2) 1,000,000 per ml in any sample unit included in the sample taken from

a lot.

l The tolerance as specified hereafter and representing the maximum incidence of total aerobic bacteria in milk for manufacture into dairy products, shall be applied in determining whether the tested lot complies with the Food and Drug Regulations.

l The maximum count of total aerobic bacteria permitted for each lot is that represented by the Aerobic Colony Count not exceeding 2,000,000 per ml in any sample unit included in the sample taken from lot.

These tolerances are summarized in the following table.


ACC

1. Flavoured Milk 5 2 50,000 1,000,000

2. Milk for manufacture 5 0 2,000,000 2,000,000


bacterial concentration higher than the value for ‘m’ without violation of the Regulation.

m = Maximum number of bacteria per ml of flavoured milk which is of no concern (acceptable level of contamination).

M = Maximum number of bacteria per designated unit, which if exceeded by any one sample unit (subsample) renders the lot under investigation in violation of the Regulation.

References1. Official Method MFO-7 Health Protection Branch - Ottawa.


References

Microbiological Examination of Mineral Water

IntroductionThis method shall be used for the determination of coliform bacteria (Coliforms) in mineral water in accordance with the Food and Drug Regulations.

Materiall Lauryl Tryptose Broth (AM1053, AM5053).l Brilliant Green Bile Broth 2% (AM1020, AM5020).

Introduction

Material

Equipmentl Laminar Air Flow Unitl Incubatorl Autoclave

ProcedureEach of the 10 sample units shall be analyzed individually.

Equipment

Procedure




The tests shall be carried out in accordance with the following instructions:

1) Collection of Samplesl A sample, consisting of ten sample units drawn at random from each lot,

shall be taken.l Each sample unit shall consist of at least 100 ml.l Collect original unopened containers wherever possible.

0l Ship and store the sample units under refrigeration (<5 C) if more than

2 hrs elapse between collection and analysis. Do not freeze the sample units.

2) Handling of Sample Unitsl Do not store sample units for more than 24 hrs before analysis.

3) Preparation of MediaThe following media, to be prepared and sterilized according to the manufacturer’s instructions, shall be used.l Lauryl Tryptose Broth .l Brilliant Green Bile Broth 2% .

4) Preparation of Dilutionsl The undiluted sample units only are required.

5) Presumptive Testl The medium used is LT Broth, dispensed in 10 ml volumes into tubes

containing gas vials (inverted Durham tubes).l Arrange LT Broth tubes in rows of five, and mark them identifying the

sample, the sample unit and the dilution to be inoculated.l Inoculate each of five tubes of double strength LT Broth with 10 ml of the

undiluted sample unit, and inoculate each of five tubes of single strength LT Broth with 1 ml of the undiluted sample unit, and inoculate each of five tubes of LT Broth with 0.1 ml of each undiluted sample unit.


0 0l Incubate the inoculated LT Broth tubes at 35 C + 0.5 C for 24 + 2 hrs.

Examine for gas formation, record results, and on the same day begin the confirmed test for all gas-positive tubes.

l Incubate gas-negative tubes for an additional 24 + 2 hrs, examine record the number of gas-positive tubes, add to the result obtained in step above, and begin the confirmed test for the additional gas-positive tubes.

l The absence of gas in all of the tubes at the end of 48 + 2 hrs of incubation constitutes a negative presumptive test.

l Compute the “MPN” of presumptive coliforms per100 ml of mineral water following the instructions to convert the number of gas-positive tubes to MPN values. Record results.





5) Presumptive Test

6) Confirmed Testl The confirmatory medium used is BGLB Broth dispensed in 10 ml

volumes in tubes containing gas vials.l Submit all gas-positive LT Broth tubes to the confirmed test.l Shake or rotate the LT Broth tubes to mix the contents and transfer one

loopful from each positive LT Broth tube to a tube of the BGB Broth. (Avoid transferring pellicle).


0 0l Incubate the inoculated BGB Broth tubes at 35 C + 0.5 C for 24 + 2

hrs. Examine for gas formation, and record results.l Incubate gas-negative tubes for an additional 24 + 2 hrs, examine

record the number of additional gas-positive tubes and add to the results obtained.

l Formation of gas during 48 + 2 hrs incubation constitutes a positive confirmed test.

l Compute the “MPN” of confirmed Coliforms per 100 ml of mineral water, to convert the number of gas-positive tubes to MPN values. Record results.

7) Interpretation The tolerance as specified hereafter and representing the maximum probable incidence of coliform bacteria (Coliforms) in mineral water, shall be applied in determining whether the tested lot of the product complies with Food and Drug Regulations.

Coliform bacteria (Coliforms) shall be considered absent in a lot when not more than one of the 10 sample units taken from the lot is positive for Coliforms, and the MPN for that sample unit is not more than 10 Coliforms per 100 ml of the mineral water.

The tolerances are summarized in the following table:


Coliforms 10 1 0 10


bacterial concentration higher than the value for ‘m’ without violation of the Regulation.

m = Maximum number of bacteria per 100 ml of mineral water which is of no concern (acceptable level of contamination).

M = Maximum number of bacteria per 100 ml of mineral water which, if exceeded by any one sample unit (subsample), renders the lot under investigation in violation of the Regulation.

6) Confirmed Test

7) Interpretation






Table A-1 shows the most probable numbers of coliforms per 100 ml or g of test material corresponding to the number of gas-positive tubes in the coliform test.

Table A-1 has been adapted from a conversion table prepared for the analysis of drinking waters where 10, 1.0 and 0.1 ml of the water under test are used as test portions. table is equally appropriate if 10, 1.0, and 0.1 g of a solid food constitute the test portions in the tubes. When other sized portions are placed in the tubes, MPN values obtained from Table A-1 must be multiplied by an appropriate number, to correct for the ,and also to obtain the MPN per g (ml) as is usually done for foods, rather than per 100 ml (g), for which the values are given in the table. The volume of diluent added to the tubes (and which accompanies the test material) is ignored when calculating the MPN.

ExampleThe following inoculated tubes give a positive reading:(1) 5 tubes with 10 ml of 1:10 dilution of test material - all 5 are positive(2) 5 tubes with 1 ml of 1:10 dilution of test material - 1 is positive(3) 5 tubes with 1 ml of 1:100 dilution of test material - none are positive

The quantities in each of the five tubes of the three dilution series represent 1, 0.1 and 0.01 g (ml), respectively of the test material. According to Table A-1, a reading of 5-1-0 gives a value of 33 when 10, 1 and 0.1 g (ml) respectively are used. However, since only 1/10 of these amounts were actually used in the analysis, the value of 33 obtained from Table A-1 must be multiplied by 10 giving 33 x 10 = 330 organisms per 100 g (ml) of test material. Since the results have to expressed


Example

per g (ml), the MPN value is 330 this becomes a Health 1 concern if the product is distributed.


To determine which consecutive dilutions to use, refer to the combinations shown below: (See also Table A-2).l If only 3 dilutions are made, use the results for those 3 dilutions to compute

the MPN. Examples a and b.l If more than 3 dilutions are employed, use the results of only 3 consecutive

dilutions. Select the highest dilution (last dilution, i.e. dilution with the smallest quantity of product), in which all 5 tubes are positive and 2 subsequent higher dilutions. Examples c and d.


l If a positive tube occurs in the dilution higher than the 3 chosen to rule, the number of such positive tubes should be added to those of the next lower dilution. Example f.

l If the tubes of all sets of a dilution series are positive, choose the 3 highest dilutions of the series and indicate by a "greater than" symbol (>) that the MPN is greater than the one calculated. Example g.





000 <1.8 100 2 200 4.5 300 7.8 400 13 500 23

001 1.8 101 4 201 6.8 301 11 401 17 501 31002 3.6 102 6 202 9.1 302 13 402 21 502 43003 5.4 103 8 203 12 303 16 403 25 503 58004 7.2 104 10 204 14 304 20 404 30 504 76005 9 105 12 205 16 305 23 405 36 505 95

010 1.8 110 4 210 6.8 310 11 410 17 510 33

011 3.6 111 6.1 211 9.2 311 14 411 21 511 46012 5.5 112 8.1 212 12 312 17 412 26 512 64013 7.3 113 10 213 14 313 20 413 31 513 84

014 9.1 114 12 214 17 314 23 414 36 514 110

015 11 115 14 215 19 315 27 415 42 515 130

TABLE A-1






020 3.7 120 6.1 220 9.3 320 14 420 22 520 49021 5.5 121 8.2 221 12 321 17 421 26 521 70

022 7.4 122 10 222 14 322 20 422 32 522 95023 9.2 123 12 223 17 323 24 423 38 523 120

024 11 124 15 224 19 324 27 424 44 524 150025 13 125 17 225 22 325 31 425 50 525 180030 5.6 130 8.3 230 12 330 17 430 27 530 79

031 7.4 131 10 231 14 331 21 431 33 531 110

032 9.3 132 13 232 17 332 24 432 39 532 140033 11 133 15 233 20 333 28 433 45 533 180

034 13 134 17 234 22 334 31 434 52 534 210

035 15 135 19 235 25 335 35 435 59 535 250040 7.5 140 11 240 15 340 21 440 34 540 130

041 9.4 141 13 241 17 341 24 441 40 541 170042 11 142 15 242 20 342 28 442 47 542 220043 13 143 17 243 23 343 32 443 54 543 280044 15 144 19 244 25 344 36 444 62 544 350045 17 145 22 245 28 345 40 445 69 545 440050 9.4 150 13 250 17 350 25 450 41 550 240051 11 151 15 251 20 351 29 451 48 551 350052 13 151 17 252 17 352 32 452 56 552 540053 15 153 19 253 26 353 37 453 64 553 920054 17 154 22 254 29 354 41 454 72 554 1600

055 19 155 24 255 32 355 45 455 81 555 >1600








MPN permL or gMPN permL or g


10 1 0.1 0.01 0.001

a. 5/5** 5/5 2/5 5-5-2 540 5.4

b. 5/5 5/5 2/5 5-5-2 540 10 54

c. 5/5 5/5 2/5 2/5 5-2-2 95 100 95

d. 5/5 5/5 2/5 0/5 5-2-0 49 100 49

e. 2/5 2/5 1/5 0/5 2-2-1 12 10 1.2

f. 5/5 2/5 1/5 1/5*** 5-2-2 95 10 9.5

g. 5/5 5/5 5/5 5/5 5-5-5 >1600 100 >1600


Reference: 1. Official Method MFO-09 Health Protection Branch - Ottawa.

TABLE A-2

10 1 0.1 0.01 0.001

Reference:



Amount of original test material (g or mL)Amount of original test material (g or mL)




Microbiological Examination of Milk Powder

IntroductionThis method shall be used for the determination of the bacteria of the genus Salmonella in milk powder, with the Food and Drug Regulations.

Materiall Nutrient Broth (NB) (AM1077, AM5077). l Selenite Cystine (SC) broth (AM1044, AM5044)l Tetrathionate Brilliant Green (TBG) broth (AM50954)l Bismuth Sulfite (BS) agar (AM1013, AM 5013)l Brilliant Green Sulfa (BGS) agar (AM1018, AM5018)l MacConkey Agar (MA) (AM1058, AM5058)l Triple Sugar Iron (TSI) agar (AM1099, AM5099)l Lysine Iron (LI) agar (AM10576, AM50576)l Christensen's Urea (CU) agar (AM1105, AM5105)l Nutrient Agar (NA) (AM1074, AM5074)

Equipmentl Laminar Air Flow unit.l Autoclave.l Incubator.

ProcedureThe 20 sample units shall be analyzed individually or as two or more composites for determining the presence of bacteria of the genus Salmonella.

The test shall be carried out in accordance with the following instructions.

1) Collection of Samplesl A sample, consisting of 20 sample units drawn at random from each lot, shall

be taken.l Each sample unit shall consist of at least 100 g.l Collect original unopened containers wherever possible.l More than one sample unit may be collected from large institutional or bulk

containers when the total number of sample units required exceeds the number of containers in the lot. A sample unit will consist of more than one container when the lot consists of containers smaller than 100 g (e.g., four 25 g containers in each sample unit)

l Employ aseptic techniques in collecting the sample units when sampling from bulk.

l Place each collected sample unit into a separate sterile container.

2) Handling of Sample unitsl Analyze the sample units as soon as possible after they have been received in

the laboratory.

Introduction

Material

Equipment

Procedure


2) Handling of Sample units

3) Preparation of Media The following media, which are to be prepared and sterilized according to the manufacturer's instructions, shall be used: (1) Nutrient Broth (NB)(2) Selenite Cystine (SC) broth (3) Tetrathionate Brilliant Green (4) Bismuth Sulfite (BS) agar (5) Brilliant Green Sulfa (BGS) agar (6) MacConkey Agar (MA) (7) Triple Sugar Iron (TSI) agar (8) Lysine Iron (LI) agar (9) Christensen's Urea (CU) agar (10) Nutrient Agar (NA).

4) Non-selective Enrichment (Pre-enrichment) l Withdraw a 25 g analytical unit from each 100 g sample unit. When a

sample unit consists of more than 1 container mix the contents of each container of the sample unit aseptically prior to obtaining the 25 g analytical unit. The analytical units may be composited.

l Resuspend the individual analytical units or the composite unit(s) in nine times their weight of distilled water.

l Add brilliant green solution to obtain a final concentration of 1:50,000. l Shake the container to ensure uniform distribution of the

microorganisms present, in the suspension. l Check the pH of each suspended analytical or composite unit. If the pH is

outside the range of 6.0 to 7.0, adjust to 7.0. with either sterile NaOH or HCL.

l Inoculate NB with a known culture of Salmonella, and subsequently make transfers to all other media used in the analysis. This is the positive media control. Set up a negative control by incubating appropriate uninoculated media during each step of the analysis.

l Incubate the inoculated pre-enrichment broth(s) and the controls at o o35 C + 0.5 for 18-24 hrs. In no circumstances should the incubation

be prolonged for more than 24 hrs.

5) Selective Enrichment l Transfer 1 ml of the incubated pre-enrichment broth into each of 9 ml of

SC and TBG broths using a sterile pipette. o o

l Incubate the SC and TBG broths for 24 + 2 hrs at 35 C + 0.5 and at o o43 C + 0.5 , respectively.

6) Selective Plating l After the incubation period, streak a loopful of each of the selective

enrichment broths onto BS agar* and BGS agar plates to obtain well-isolated colonies. The broths may also be streaked onto a third


4) Non-selective Enrichment (Pre-enrichment)






commercially available plating medium. l It has been observed that BS agar is inhibitory for Salmonella serotypes

oother than S.typhi unless it is refrigerated at 4 C for at least 24 hrs before streaking. The possibility of an inhibitory effect of this medium should be taken into consideration.

o ol Incubate the plates at 35 C+ 0.5 C for 24 + 2 hrs. It may be necessary

to incubate the BS agar plates for 48 + 2 hrs. l Examine the plates after the incubation period for colonies indicative of

Salmonella. On BGS agar, such colonies are pink to fuchsia surrounded by red medium. On BS agar, they are usually black, with or without a metallic sheen; with increasing time of incubation the surrounding medium is gradually blackened. It should be noted however that lactose and/or sucrose-fermenting strains (e.g., S. arizonae) may develop a coliform-like (greenish) appearance on BGS agar. A heavy growth of coliforms may mask the appearance of the Salmonella colonies. On BS agar, some Salmonella types may form dark brown rather than black colonies.

l If there are no colonies indicative of Salmonella on the plates, bacteria of the genus Salmonella are considered absent from the analytical or composite unit from which the colonies originated.

7) Purification l Streak suspect colonies onto MA plates for purification.

o ol Incubate the agar plates at 35 C + 0.5 C for 24 + 2 hrs. l Observe MA plates after incubation period. Typical Salmonella colonies

are lactose-negative and will appear colourless. However, lactose-positive biotypes are known to occur and may appear pink.


the MA plates and inoculate the biochemical media listed in Table 1. o oIncubate these media at 35 C + 0.5 C for 18 - 24 hrs. Cap the tubes

loosely to avoid the occurrence of erroneous results. l Other media may be used to observe the reactions listed in Table 1. If

additional biochemical information is desired, other reaction-media or commercially available diagnostic kits may be used.

l If none of the isolates from a particular analytical or composite unit shows biochemical reactions indicative of Salmonella, then the bacteria of the genus Salmonella are considered to be absent from the analytical or composite unit from which the isolates originated. If Salmonella are suspected, proceed to serological testing.

l Use TSI or LI agar cultures less than 72 hrs old for the serological oidentification but store the cultures at 2-8 C if the serological

identification is not carried out within 12 hrs after the incubation of the TSI or LI agar cultures.

l If the serological identification is not performed within 72 hrs after the incubation period, streak suspect cultures onto NA slants.

7) Purification


o ol Incubate the inoculated NA slants at 35 C + 0.5 C for 24 + 2 hrs. l Use NA slant cultures less than 48 hr old for the serological identification

obut store the cultures at 2-8 C if the serological identification is not carried out within 12 hrs after the incubation of the NA slant cultures.

9) Serological Identification

I ) Testing with somatic polyvalent antiserum (Group A to I +Vi) (a) Mark the following sections on an agglutination plate: C+

(positive control), C-(negative control), and T (test culture). If several cultures are tested at the same time, mark several test areas T , T , T , etc. 1 2 3

(b) Place one drop of physiological saline on each of the areas marked T and C+, and two drops on the area marked C-.

(c) Remove sufficient culture material from a biochemical test medium used (the slant area not the butt) or from an NA slant, and prepare a heavy suspension in the test areas and in the negative control area.

(d) For the positive control, use a known Salmonella culture and make a suspension in the area marked C+.

(e) Prepare the somatic polyvalent antiserum as directed by the manufacturer, and place one drop onto each of the test suspension areas and onto the positive control area.

(f) Mix each of the culture-saline-serum suspensions (test cultures and positive control), and the saline-culture mixture of the negative control with a sterile needle or loop. Title the slides back and forth for 1 min.

(g) Hold the slides against a dark background and observe. Cultures usually agglutinate within 1 min.

(h) False positive reactions may occur; these can be resolved by further testing with somatic grouping and with flagellar antisera.

(i) The tests are invalidated if the negative control shows agglutination (autoagglutination).

II ) Testing with Somatic Grouping Antisera It is preferable to test the culture against somatic grouping anterisera whenever possible; however the culture may be sent to a typing centre for identification. The majority of Salmonella isolated from foods belong to groups B, C, D, and E. It is important to recognize that unless a complete set of grouping sera is available some Salmonella may be missed. In such cases any culture processing the biochemical reactions indicative of Salmonella should be sent to a typing centre for identification. (a) Mark the following sections on an agglutination plate: C+

(positive control) C- (negative control), and T (test culture). If several cultures are tested at the same time, identify several test areas T , T , T , etc. 1 2 3


I ) Testing with somatic polyvalent antiserum (Group A to I +Vi)

II ) Testing with Somatic Grouping Antisera

Table 1Minimal Biochemical Screening MediaTable 1








H S production2















(b) Use a positive control culture for each individual group tested, as in 3.8.1.d.

(c) Place one drop of physiological saline on each of the areas marked T and C+ and two drops on the area marked C-.

(d) Remove sufficient culture material from a bio-chemical test medium (from the slant area not the butt) or from an NA slant and prepare a heavy suspension in the test area(s) and in negative control area.

(e) Prepare the grouping antisera as directed by the manufacturer, and place one drop onto each of the test suspension areas and onto the positive control.

(f) Mix each of the culture-saline-serum suspensions (test cultures and positive controls), and the saline-culture mixture of the negative control with a sterile needle or loop. Tilt slides back and forth for 1 min.

(g) Hold the slides against a dark background and observe for

agglutination. Cultures usually agglutinate within 1 min. (h) If the culture-saline-serum mixture has not agglutinated, repeat

procedure with another group antiserum. (i) If the serological test is positive, the culture shall be sent to a

Salmonella typing centre for serotyping. (j) The tests are invalidated if the negative control shows

agglutination (autoagglutination). l If all of the serological tests performed on an isolate are negative but the

original culture gave biochemical reactions indicative of Salmonella (see Table 1), that culture shall be sent to a typing centre for verification.

10) Interpretation The lot of milk powder sampled shall be considered in compliance with the Food and Drug Regulations when bacteria of the genus Salmonella are not found in any of the 20 sample units analyzed (individually or as composites).

10) Interpretation

Christensen’sUrea Agar *




References1. Official Method MFO-12 Health Protection Branch Ottawa.




Microbiological Examination of Frog Legs

IntroductionThis method shall be used for the determination of the bacteria of the genus in froglegs in accordance the Food and Drug Regulations.

Materiall Selenite Cystine (SC) broth (AM1044, AM5044) l Nutrient Broth (NB) (AM1077, AM5077) l Tetrathionate Brilliant Green (TBG) broth (AM50954) l Bismuth Sulfite (BS) agar (AM1013, AM5013) l Brilliant Green Sulfa (BGS) agar (AM1018, AM5018)l MacConkey Agar (MA) (AM1059, AM5059) l Triple Sugar Iron (TSI) agar (AM1099, AM5099) l Lysine Iron (LI) agar (AM10576, AM50576)l Christensen's Urea (CU) agar (AM1105, AM5105)l Nutrient Agar (NA) (AM1074, AM5074) l Salmonella culturel NaOH or HCL

Equipmentl Laminar Air Flow Unit.l Incubator.l Autoclave.

ProcedureThe five sample units shall be analyzed individually or as one or more composite(s) for determining the presence of bacteria of the genus Salmonella .

The test shall be carried out in accordance with the following instructions:

1) Collection of Samplesl A sample, consisting of five sample units drawn at random from a lot,

shall be taken. (Table I).l Each sample unit shall consist of at least 25 g of whole froglegs.l Employ aseptic techniques in collecting the sample units.l Place each collected sample unit into a separate sterile container.l Keep sample units frozen during transport.

2) Handling of Sample Unitsl Keep sample units frozen in the laboratory prior to analyzing them.l Analyze sample units as soon as possible after receipt at the laboratory.

3) Preparation of MediaThe following media, to be prepared and sterilized according to the manufacturer's instructions, shall be used:(1) Nutrient Broth (NB)

Introduction

Material

Equipment

Procedure




(2) Selenite Cystine (SC) broth (3) Tetrathionate Brilliant Green (TBG) broth (4) Bismuth Sulfite (BS) agar (5) Brilliant Green Sulfa (BGS) agar (6) MacConkey Agar (MA) (7) Triple Sugar Iron (TSI) agar (8) Lysine Iron (LI) agar (9) Christensen's Urea (CU) agar (10) Nutrient Agar (NA)

4) (Pre-enrichment)l Thaw frozen sample units. Do not allow the temperature of any sample

ounit to exceed 45 C.l Place a minimum of 25g of whole froglegs (analytical unit) from each of

the sample units into a separate sterile container.l Alternatively, composite the analytical units and place each composite

into a separate sterile container.

Since compositing presents difficulties in preparation and disposal of the material, exercise care in handling bulk preparations.l Add sufficient NB to cover the froglegs.l Shake the container(s) to unite the contents.l Check the pH of each suspended analytical or composite unit. If the pH is

outside the range of 6.0 - 7.0, adjust to 7.0 with either sterile NaOH or HCl.

l Inoculate NB with a known culture of and subsequently make transfers to all other media used in the analysis. This is the positive media control. Set up a negative control by incubating appropriate uninoculated media during each step of the analysis.

l Incubate the inoculated pre-enrichment broth(s) and the controls at o o35 ± 0.5 C for 18-24 hrs. In no circumstances shall the incubation be


5) Selective Enrichment1) Transfer 1 ml of the incubated pre-enrichment broth into each of 9 ml of

SC and TBG broths using a sterile pipette.o o2) Incubate the SC broth and TBG broth for 24 ± 2 hrs at 35 C± 0.5 C

o oand at 43 C± 0.5 C, respectively.

6) Selective Platingl After the incubation period, streak a loopful of each of the selective

enrichment broths onto BS agar and BGS agar plates to obtain well-isolated colonies. The broths may also be streaked onto a third commercially available plating medium.

l It has been observed that BS agar is inhibitory for serotypes other than it

4) (Pre-enrichment)






ois refrigerated at 4 C for at least 24 hrs before streaking. The possibility of an inhibitory effect of this medium should be taken into consideration.

o ol Incubate the plates at 35 C± 0.5 C for 24 ± 2 hrs. It may be necessary

to incubate the BS agar plates for 48 ± 2 hrs.l Examine the plates after the incubation period for colonies indicative of

Salmonella. On BGS agar, such colonies are pink to fuchsia surrounded by red medium. On BS agar, they are usually black, with or without a metallic sheen; with increasing time of incubation the surrounding medium is gradually blackened. It should be noted, however, that lactose and/or sucrose-fermenting strains (eg. S. arizonae) may develop a coliform-like (greenish) appearance on BGS agar. A heavy growth of coliforms may mask the appearance of the Salmonella colonies. On BS agar, some Salmonella types may form dark brown rather than black colonies.

l If there are no colonies indicative of Salmonella on the plates, bacteria of the genus Salmonella are considered to be absent from the analytical or composite unit from which the colonies originated.

7) Purificationl Streak suspect colonies onto MA plates for purification.

o ol Incubate the agar plates at 35 C± 0.5 C for 24 ± 2 hrs.l Observe MA plates after the incubation period. Typical colonies are

lactose-negative and will appear colourless. However, lactose-positive biotypes are known to occur and may appear pink.


the MA plates and inoculate the biochemical media listed in Table 1. o oIncubate these media at 35 C± 0.5 C for 18 - 24 hrs. Cap the tubes

loosely to avoid the occurrence of erroneous results.l Other media may be used to observe the reactions listed in Table II. If

additional biochemical information is desired, other reaction-media or commercially available diagnostic kits may be used.

l If none of the isolates from a particular analytical or composite unit show biochemical reactions indicative of Salmonella, then bacteria of the genus Salmonella are considered to be absent from the analytical or composite unit from which the isolates originated. If Salmonella are suspected, proceed to serological testing.




o ol Incubate the inoculated NA slants at 35 C± 0.5 C for 24 ± 2 hrs.l Use NA slant cultures less than 48 hr old for the serological identification

obut store the cultures at 2-8 C if the serological identification is not

7) Purification


carried out within 12 hr after the incubation of the NA slant cultures.


I) Testing with somatic polyvalent antiserum (Groups A to I + Vi)(a) Mark the following sections on an agglutination plate:

C+(positive control), C-(negative control), and T(test culture). If several cultures are tested at the same time, mark several test areas T , T , T , etc.1 2 3

(b) Place one drop of physiological saline on each of the areas marked T and C+, and two drops on the area marked C-.

(c) Remove sufficient culture material from a biochemical test medium used (the slant area, not the butt) or from an NA slant, and prepare a heavy suspension in the test areas and in the negative control area.

(d) For the positive control, use a known culture and make a suspension in the area marked C+.

(e) Prepare the somatic polyvalent antiserum as directed by the manufacturer, and place one drop onto each of the test suspension areas and onto the positive control area.

(f) Mix each of the culture-saline-serum suspensions (test cultures and positive control), and the saline-culture mixture of the negative control with a sterile needle or loop. Tilt the slides back and forth for 1 min.

(g) Hold the slide against a dark background and observe for agglutination. Cultures usually agglutinate within 1 min.

(h) False positive reactions may occur; these can be resolved by further testing with somatic grouping and with flagellar antisera.

(i) The tests are invalidated if the negative control shows agglutination (autoagglutination).

II) Testing with Somatic Grouping AntiseraIt is preferable to test the culture against somatic grouping antisera whenever possible; however, the culture may be sent to a typing centre for identification. The majority of isolated from foods belong to groups B, C, D, and E.It is important to recognize that unless a complete set of grouping sera is available some may be missed. In such cases any culture possessing the biochemical reactions indicative of Salmonella should be sent to a typing centre for identification.(a) Mark the following sections on an agglutination plate:

C+(positive control), C-(negative control), and T(test culture). If several cultures are tested at the same time, mark several test areas T , T , T , etc.1 2 3

(b) Use a positive control culture for each individual group tested, as in d.


I) Testing with somatic polyvalent antiserum (Groups A to I + Vi)

II) Testing with Somatic Grouping Antisera




(c) Place one drop of physiological saline on each of the areas marked T and C+, and two drops on the area marked C-.

(d) Remove sufficient culture material from a bio-chemical test medium (from the slant area not the butt) or from an NA slant and prepare a heavy suspension in the test area(s) and in the negative control area.

(e) Prepare the grouping antisera as directed by the manufacturer and place one drop onto each of the test suspension area(s) and onto the positive control area.

(f) Mix each of the culture-saline-serum suspensions (test cultures and positive control), and the saline-culture mixture of the negative control) with a sterile needle or loop. Tilt the slides back and forth for 1 min.

(g) Hold the slide against a dark background and observe for agglutination. Cultures usually agglutinate within 1 min.

(h) If the culture-saline-serum mixture has not agglutinated, repeat procedure with another grouping antiserum.

(i) If the serological test is positive, the culture shall be sent to a centre for serotyping.

(j) The test is invalidated if the negative control shows agglutination (autoagglutination)

l If all of the serological tests performed on an isolate are negative but the original culture gave biochemical reactions indicative of (see Table 1), that culture shall be sent to a typing centre for

verification.

10) InterpretationThe lot of froglegs sampled shall be considered in compliance with Food and Drug Regulations when bacteria of the genus are not found in any of the five sample units analyzed (individually or as composites).

Minimal Biochemical Screening MediaTable I - Lot Structure1. When there are no identifiable lots, the amount of product present on the

premises shall be considered a lot.2. One exporter - one importer - if cartons or containers bear no markings,

numbers, etc., - sample as .3. One exporter - one importer - cartons or containers bear markings, numbers,

etc., which could be interpreted as indicating different production periods or different areas of production; where feasible -segregate by lots and sample each as a separate lot.

4. One exporter - one or more importers - if no markings, numbers, etc., sample quantity received by each importer as a "lot".

5. More than one exporter - one importer - sample quantity from each exporter as being a "lot" if segregation by exporter can be made, if not - sample as one lot.

6. One exporter (separate identifiable packers) - one importer - sample product from individual packers as individual lots if containers have "marks" or

10) Interpretation

Minimal Biochemical Screening MediaTable I - Lot Structure

Table IIMinimal Biochemical Screening MediaTable II








H S production2
















Christensen’sUrea (CU) agar *




Microbiological Examination of Cocoa and Chocolate

IntroductionThis method shall be used for the determination of the bacteria of the genus Salmonella in cocoa and chocolate, in accordance with the Food and Drug Regulations.

Materiall Nutrient Broth (AM1077, AM5077) l Selenite Cystine (SC) broth (AM1044, AM5044)l Tetrathionate Brilliant Green (TBG) broth (AM50954) l Bismuth Sulfite (BS) agar (AM1013, AM5013) l Brilliant Green Sulfa (BGS) agar (AM1018, AM5018) l MacConkey Agar (MA) (AM1059, AM5059)l Triple Sugar Iron (TSI) agar (AM1099, AM5099) l Lysine Iron (LI) agar (AM10576, AM50576) l Christensen's Urea (CU) agar (AM1105, AM5105) l Nutrient Agar (NA) (AM1074, AM5074) l Salmonella culturel Milkl NaOH or HCL

Equipmentl Laminar Air Flow.l Autoclave.l Incubator.

ProcedureThe ten sample units shall be analyzed individually or as one or more composite(s) for determining the presence of bacteria of the genus Salmonella.

The test shall be carried out in accordance with the following instructions:

1) Collection of Samples l A sample, consisting of ten sample units drawn at random from each lot,

shall be taken. l Each sample unit shall contain at least 100 g. l Collect original unopened containers wherever possible. l More than one sample unit may be collected from large institutional or

bulk containers when the total number of sample units required exceeds

Introduction

Material

Equipment

Procedure


the number of containers in the lot. When the lot consists of containers smaller than 100 g, a sample unit will consist of more than one container (e.g., four 25 g containers in each sample unit).


2) Handling of Sample Unitsl Analyze the sample units as soon as possible after they have been


3) Preparation of mediaThe following media, to be prepared and sterilized according to the manufacturer's instructions, shall be used: (1) Nutrient Broth (NB) (2) Selenite Cystine (SC) broth (3) Tetrathionate Brilliant Green (TBG) broth (4) Bismuth Sulfite (BS) agar (5) Brilliant Green Sulfa (BGS) agar (6) MacConkey Agar (MA) (7) Triple Sugar Iron (TSI) agar (8) Lysine Iron (LI) agar (9) Christensen's Urea (CU) agar (10) Nutrient Agar (NA)

4) Non-selective Enrichment (Pre-enrichment) l Prepare a 10% w/v of non-fat-dry-milk solution in distilled water

containing brilliant green at a final concentration of 1:50,000. Sterilize o oat 121 C and cool to 45 C.

a. Weigh 25 g (the analytical unit) from each sample unit into a separate blender jar and add 225 ml of non-fat-dry-milk solution, blend for the minimum time required to produce a homogeneous suspension and transfer the contents to a sterile container. To avoid overheating, blending time should not exceed 2.5 min.

b. The analytical units may be composited. Suspend the composite units in nine times their weight of non-fat-dry-milk solution and proceed as in the previous step.



4) Non-selective Enrichment (Pre-enrichment)




l Check the pH of each blended analytical or composite unit. If the pH is outside the range of 6.0 - 7.0, adjust to 7.0 with either sterile NaOH or HCl.

l Inoculate 10ml of the non-fat-dry-milk- solution prepared above, with a known culture of Salmonella, and subsequently make transfers to all other media used in the analysis. This is the positive media control. Set up a negative control by incubating appropriate uninoculated media during each step of the analysis.

l Incubate the inoculated pre-enrichment broth(s) and the controls at o o35 C + 0.5 C for 18-24 hrs. In no circumstances shall the incubation be


5) Selective Enrichmentl Transfer 1 ml of the incubated pre-enrichment broth into each of 9 ml of

SC and TBG broths using a sterile pipette. o o

l Incubate the SC broth and TBG broth for 24 + 2 hrs at 35 C+ 0.5 C and o oat 43 C + 0.5 C, respectively.

6) Selective Platingl After the incubation period, streak a loopful of each of the selective

enrichment broths onto BS agar and BGS agar plates to obtain well isolated colonies. The broths may also be streaked onto a third commercially available plating medium.

l It has been observed that BS agar inhibits Salmonella serotypes other othan S. typhi unless it is refrigerated at 4 C for at least 24 hr before

streaking. The possibility of an inhibitory effect of this medium should be taken into consideration.

o ol Incubate plates at 35 C+0.5 C for 24 + 2 hrs. It may be necessary to

incubate the BS agar plates for 48 + 2 hrs. l Examine the plates after the incubation period for colonies indicative of

Salmonella. On BGS agar, such colonies are pink to fuchsia surrounded by red medium. On BS agar, they are usually black, with or without a metallic sheen; with increasing time of incubation, the surrounding medium is gradually blackened. It should be noted however that lactose-and/or sucrose-fermenting strains (e.g., S. arizonae) may develop a coliform-like (greenish) appearance on BGS agar. A heavy growth of coliforms may mask the appearance of the Salmonella colonies. On BS agar, some Salmonella types may form dark brown rather than black colonies.

l If there are no colonies indicative of Salmonella on the plates, bacteria of the genus Salmonella are considered absent from the analytical or composite unit from which the colonies originated.

7) Purification l Streak suspect colonies onto MA plates for purification.

o ol Incubate the plates at 35 C + 0.5 C for 24 + 2 hrs.



7) Purification

l Observe the MA plates after the incubation period. Typical Salmonella colonies are lactose-negative and appear colourless. However, lactose-positive biotypes are known to occur and may appear pink.


the MA plates and inoculate the biochemical media listed in Table 1. o oIncubate these media at 35 C + 0.5 C for 18 - 24 hrs. Cap the tubes

loosely to avoid the occurrence of erroneous results. l Other media may be used to observe the reactions listed in Table I. If

additional biochemical information is desired, other reaction media or commercially available diagnostic kits may be used.

l If none of the isolates from a particular analytical or composite unit shows biochemical reactions suggestive of Salmonella, then bacteria of the genus Salmonella are considered to be absent from the analytical or composite units from which the isolates originated. If Salmonella are suspected, proceed to serological testing.




o ol Incubate the inoculated NA slants at 35 C + 0.5 C for 24 + 2 hrs. l Use NA slant cultures less than 48 hrs old for the serological

oidentification but store the cultures at 2-8 C if the serological identification is not carried out within 12 hrs after the incubation of the NA slant cultures.

9) Identification

I) Testing with somatic polyvalent antiserum (Group A to I + Vi). a. Mark the following sections on an agglutination plate:

C+(positive control), C-(negative control), and T(test culture). If several cultures are tested at the same time, mark several test areas T , T , T etc. 1 2 3

b. Place one drop of physiological saline on each of the areas marked T and C+, and two drops on the area marked C-.

c. Remove sufficient culture material from a biochemical test medium (from the slant area not the butt) or from an NA slant and prepare a heavy suspension in the test area(s) and in the negative control area.

d. For the positive control, use a known Salmonella culture and make a suspension in the area marked C+.

e. Prepare the somatic polyvalent antiserum as directed by the manufacturer and place one drop onto each of the test suspension


9) Identification

I) Testing with somatic polyvalent antiserum (Group A to I + Vi).




areas and onto the positive control area. f. Mix each of the culture-saline-serum suspensions (test cultures and

positive control), and the saline-test culture mixture of the negative control with a sterile needle or loop.

Tilt the slides back and forth for 1 min. g. Hold the slides against a dark background and observe for

agglutination. Cultures usually agglutinate within 1 min. h. False-positive reactions may occur; these can be resolved by further

testing with somatic grouping and with flagellar antisera. i. The tests are invalidated if the negative control shows

agglutination (autoagglutination).

II) Testing with Somatic Grouping Antisera It is preferable to test the culture against somatic grouping antisera whenever possible; however the culture may be sent to a typing centre for identification. The majority of the Salmonella isolated from foods belong to Groups B, C, D, and E. It is important to recognize that unless a complete set of grouping sera is available, some Salmonella may be missed. In such cases, any culture possessing the biochemical reactions indicative of Salmonella should be sent to a typing centre for identification. a. Mark the following sections on an agglutination plate:

C+(positive control), C-(negative control) and T(test culture). If several cultures are tested at the same time, mark several test areas T , T , T , etc. 1 2 3

b. Use a positive control culture for each individual group tested as in d.

c. Place one drop of physiological saline on each of the areas marked

T and C+, and two drops on the area marked C-. d. Remove sufficient culture material from a biochemical test medium

(from the slant area, not the butt) or from an NA slant and prepare a heavy suspension in the test area(s) and in the negative control area.

e. Prepare the grouping antisera as directed by the manufacturer, and place one drop onto each of the test suspension areas and onto the positive control area.

f. Mix each of the culture-saline-serum suspensions (test cultures and positive control), and the saline-test culture mixture of the negative control with a sterile needle or loop.

Tilt the slides back and forth for 1 min. g. Hold the slides against a dark background and observe for

agglutination. Cultures usually agglutinate within 1 min. h. If the culture-saline-serum mixture has not agglutinated, repeat

procedure with another grouping antiserum. i. If the serological test is positive, the culture shall be sent to a

Salmonella typing centre for serotyping. j. The tests are invalidated if the negative control shows

agglutination (auto-agglutination). l If all of the serological tests performed on an isolate are negative but the

original culture gave biochemical reactions indicative of Salmonella (see Table 1), that culture shall be sent to a typing centre for verification.

10) Interpretation The lot of cocoa or chocolate sampled shall be considered in compliance with the Food and Drug Regulations when bacteria of the genus Salmonella are not found in any of the ten sample units analyzed (individually or as composites).

10) Interpretation

Table IMinimal Biochemical Screening MediaTable I








H S production2
















Christensen’sUrea (CU) agar *




Reference1. Official Method MFO-11 Health Protection Branch - Ottawa.

Examination of Canned Tomatoes, Tomato Juice and Vegetable Juice, Tomato Puree, Tomato Paste, Tomato Pulp and Tomato Catsup for Mould Filaments

Introduction This method shall be used for the determination of mould filaments in canned tomatoes, tomato juice and vegetable juice, and in tomato puree, tomato paste, tomato pulp and tomato catsup, in accordance with the Food and Drug Regulations.

Materiala. Compound microscope, either binocular or monocular equipped withl mechanical stage. l condenser with iris diaphragm. l source of illumination. l two objectives - a 10 x (16 mm) for counting and a 20 x (8mm) for

confirmation. l 8 x - 12.5 x oculars. l The 10 x objective must be calibrated with the ocular to give a field

diameter of 1.382 mm.l The ocular must be equipped with a micrometer disk cross-ruled in sixths

of ocular diaphragm opening.b. Howard mould counting chamber or cell of the type with specifications as

outlined in Part 6, Diagram IIa or IIb and cover glass. c. Distilled water. d. Lint-free clean towel or cloth for drying Howard cell and cover glass. e. Bunsen burner. f. Spatula with a 5.0 mm flat blade. If the blade is not of this size it may be

ground down to the designated width and to a flat surface. With a glass

Introduction

Material

pencil, mark the blade l0.0 mm from the tip to give a working area of 50 sq. mm. The purpose of recommending this spatula is to standardize the quantity of product transferred from the sample to the Howard cell.

g. Dissecting needle. h. U.S. standard sieve no. 2 (for canned tomatoes). i. Wide mouth bottles with screw caps or other suitable containers (for canned

tomatoes, puree, pulp, paste and catsup). j. Spoon or other suitable utensil (for puree, pulp, paste and catsup). k. Refractometer (for puree, pulp and paste). l. Coarse filter paper or celluwipe (for puree, pulp and paste). m. Glass Slide.

ProcedureThe examination shall be carried out in accordance with the following instructions.1) Collection of Samples l A sample, consisting of six sample units drawn at random from each lot,

shall be taken. l Each sample unit shall contain at least 100 g or ml. l Employ aseptic techniques in collecting the sample units when sampling

from bulk. Place each collected sample unit into a separate, sterile container.

2) ApparatusI) Compound microscope, either binocular or monocular equipped with: l mechanical stage.

Procedure


2) Apparatus




l condenser with iris diaphragm. l source of illumination. l two objectives - a 10 x (16 mm) for counting and a 20 x (8mm) for

confirmation. l 8 x - 12.5 x oculars. l The 10 x objective must be calibrated with the ocular to give a field

diameter of 1.382 mm (Preparation of Microscope, section 3.2.1). l The ocular must be equipped with a micrometer disk cross-ruled in

sixths of ocular diaphragm opening (Preparation of Microscope, section 3.2.2).

II) Howard mould counting chamber or cell of the type with specifications as outlined in Part 6, Diagram IIa or IIb and cover glass. l Distilled water. l Lint-free clean towel or cloth for drying Howard cell and cover glass. l Bunsen burner. l Spatula with a 5.0 mm flat blade. If the blade is not of this size it

may be ground down to the designated width and to a flat surface. With a glass pencil, mark the blade l0.0 mm from the tip to give a working area of 50 sq. mm. The purpose of recommending this spatula is to standardize the quantity of product transferred from the sample to the Howard cell.

l Dissecting needle. l U.S. standard sieve no. 2 (for canned tomatoes). l Wide mouth bottles with screw caps or other suitable containers (for

canned tomatoes, puree, pulp, paste and catsup). l Spoon or other suitable utensil (for puree, pulp, paste and catsup). l Refractometer (for puree, pulp and paste). l Coarse filter paper or celluwipe (for puree, pulp and paste).

3) Preparation of Microscope I) Calibrate the 10 x objective with the ocular to give a field of view

diameter of 1.382 mm as follows: a. Using the 10 x objective and ocular(s) in the range of 8 - 12.5 x

measure the diameter of field of view with a stage micrometer or with the two parallel lines or circle measuring 1.382 mm scribed on a Howard cell.

b. If the field diameter is less than 1.382 mm use lower power ocular(s).

c. If the field diameter is greater, raise the height of the ocular(s) until the diameter coincides with 1.382 mm or make an accessory drop-in ocular diaphragm with aperture accurately cut to necessary size.

II) Equip microscope with a micrometer disk cross-ruled in sixths of ocular diaphragm opening as follows: a. Obtain or make a micrometer disk of suitable diameter to fit into

3) Preparation of Microscope

ocular (approximately 21 mm) and 1 mm thick. The disk should be marked with a centre grid made up of 36 small squares, six to each side of such a size that the length of six squares is equal to the diameter of the ocular diaphragm which has been adjusted to give a field diameter of 1.382 mm as in step 3.2.1, (Part 6, Diagram I).

b. To make the grid, calculate the width of grid (10 - 14 mm) that will coincide with 1.382 mm on stage. Mark width on micrometer disk, place disk in ocular and check that width coincides. If not, remove disk and change lines as necessary. Once the proper width has been determined, etch grid on micrometer disk with very fine lines making certain grid is centred on the disk.

III) Establish adequate light source for examination as follows: a. Locate and focus a mould filament with the microscope. b. Focus the light source into the condenser, adjust the height of the

condenser, the diameter of the iris diaphragm and the intensity of the light source to give clear uniform illumination such that there is sufficient light to see all particles but not so intense as to mask the characteristics of the mould.

c. Use a coloured filter if necessary to increase contrast of filaments.

4) Preparation of Sample Unitsl Each sample shall consist of six sample units of one container each as

outlined in section 2, Sampling. Each sample unit shall be analyzed separately.

l Examine each sample unit immediately after it is prepared. If there is any delay, the sample unit should be thoroughly shaken again prior to examination.

I) a. Tomato Juice and Vegetable Juice (i) Before opening, shake container (sample unit) 60 times in 30

sec through a 30 cm arc. (ii) Open container. If considerable foam is produced, pass the

flame of a Bunsen burner lightly over the surface to disperse the foam.

(iii) Proceed as in step 4 Preparation of Howard Mould Count Cell.(iv) Repeat procedure for remaining five sample units.

II) b. Canned Tomatoes (i) Before opening, shake container (sample unit) 60 times in 30

sec through a 30 cm arc. (ii) Open container. Drain liquid from canned tomatoes through a

no. 2 sieve into a suitable clean receptacle. (iii) Transfer liquid to a wide mouth bottle and screw lid on

securely. (iv) Continue as in the step above.

4) Preparation of Sample Units




III) c. Tomato Puree, Tomato Pulp and Tomato Paste (i) Open container (sample unit) and mix tomato product 60

times in 30 sec with a spoon or other suitable utensil. (ii) Transfer a small portion onto a coarse filter paper or celluwipe

and measure the refractive index of the filtrate. Removal of the pulp from tomato mixture does not affect the refractive index as it is based only on the soluble solids. If the pulp is not removed, a hazy image will be formed which is hard to centre and read.

(iii) Determine amount of distilled water to add to 100 ml of sample unit from Table I to give a final refractive index of

o o1.3448 -1.3454 at 20 C or 1.3442 -1.3448 at 25 C. (iv) Mix sample unit as in step (i), transfer 100 ml to a wide mouth

bottle, add required amount of distilled water, secure lid and repeat mixing.

(v) Measure refractive index as in step (ii) and correct if necessary.

(vi) Proceed for Preparation of Howard Mould Count Cell. (vii) Repeat procedure for remaining five sample units.

IV) d. Tomato Catsup (i) Open container (sample unit) and mix 60 times in 30 sec with

a spoon or other suitable instrument. (ii) Transfer a measured well mixed representative portion to a

wide mouth bottle. (iii) Dilute contents of bottle with an equal volume of distilled

water, secure lid and shake 60 times in 30 sec through a 30 cm arc.

(iv) Proceed for Preparation of Howard Mould Count Cell. (v) Repeat procedure for remaining five sample units.

5) Preparation of Howard Mould Count Cell (1) l Clean Howard cell and cover glass making certain central area of cell is

clean. l Rinse with distilled water, dry with a lint free cloth and pass lightly over a

Bunsen flame.l Determine adequate cleanliness of slide by placing cover glass in

position and pressing it firmly against the shoulders. If Newton's rings appear between each shoulder and the cover glass, and remain after pressure has been released, the slide is considered sufficiently clean. When the rings are formed they may be observed by holding the slide at such an angle that the light is reflected from the cover glass. These rings resemble a rainbow in colour and when properly formed are broken arcs of concentric circles. If Newton's rings are not formed re-wash slide and cover glass. Absence of Newton's rings indicates dirt preventing proper seating of cover glass on shoulders which results in chamber holding an incorrect volume of sample.

5) Preparation of Howard Mould Count Cell (1)

l Clean spatula and dissecting needle, rinse in distilled water, flame and cool.

l Prepare glass slide using technique (I) or (II) as follows

I) Inclined Cover Glass Technique (i) Remove cover from Howard cell. (ii) Dip spatula into well mixed sample up to 10 mm line and

transfer a sample portion to an area on the central disk (or rectangle) halfway between the centre and far edge, using a dissecting needle to facilitate the transfer. Do not allow the spatula or needle to touch the central disk, only the sample.

(iii) Rest one edge of the cover glass in a slanting position on the edges of the cell shoulders nearest the portion of test material.

(iv) Lower the cover glass slightly until it almost touches the test material on the disk; then, lower it rapidly but gently into place, so that the material spreads evenly over the entire surface of the disk.

(v) Do not lower the cover glass too rapidly, for in doing so, a portion of the sample may splash over onto one or both of the shoulders, thus ruining the mount. On the other hand, do not lower too gently, otherwise the test material will not spread evenly over the disk.

II) Parallel Cover Glass Technique (i) Remove cover from Howard cell. (ii) Dip spatula into well mixed sample up to 10 mm line and

transfer a sample portion onto the approximate centre of the disk, using a dissecting needle to facilitate the transfer. Do no allow the spatula or needle to touch the central disk, only the sample.

(iii) Hold the cover glass parallel to the surface of the central disk and lower it slowly until it just touches the sample portion.

(iv) While maintaining contact with the test sample, alternately raise and lower the cover glass very slightly 2 or 3 times; then, without stopping lower it rapidly but gently until it just touches the shoulders of the cell, so that the test portion spreads evenly over the entire surface of the disk.

III) Ensure the slide is characterized by: a. Sufficient material to fill area used for counting. b. Newton's rings visible. c. Even distribution of material on slide. Ensure sample portion

is taken from a thoroughly mixed sample. Otherwise, when cover glass is put in place, insoluble material, and consequently moulds, may be more abundant at the centre of the mount.

d. Absence of air bubbles.




IV) Discard any mount showing: a. Uneven distribution of material. b. Absence of Newton's rings. c. Liquid which has been drawn across the moat and between

the cover glass and shoulder. d. Numerous air bubbles.

V) Microscopical Examination l Place cell on microscope stage and examine at a

magnification of 90 -125 x with suitable illumination such that the diameter of each field of view is 1.382 mm (1.5 sq. mm) as outlined in Preparation of the Microscope (section 3.2). Use higher magnification (180 - 250 x) only for confirmation of mould.

l From each of 2 or more mounts examine at least 25 fields taken in such a manner as to be representative of all sections of the mount. The recognized procedure for examining a mount is to examine alternate fields in alternate rows throughout the entire area of the mount. To accomplish this, examine alternate fields horizontally across the slide preparation until 5 fields have been examined. Then move the mechanical stage vertically to the next alternate row and examine 5 more alternate fields in reverse horizontal direction. Repeat this process until 25 fields have been examined. If a field with an air bubble is encountered, move to another field unless mould is seen at first glance, because the field will contain insufficient sample. Otherwise never move the slide purposely to exclude or include mould filaments.

l Observe each field noting presence or absence of mould filaments as characterized in Part 6, Diagram III. If not

familiar with the diverse forms of mould, examine known moulds as follows: (i) Remove mouldy areas from fresh tomatoes infected with

various types of mould, boil in low count tomato juice to simulate actual conditions and examine microscopically.

(ii) Recognize the difference between various mould filaments and plant remnants such as tracheal tube thickenings, pieces of cell wall, lint or fabric segments.

(iii) Refer to one of several publications (2, 3, 4) for further clarification of mould and plant filaments.

(iv) It is not necessary to classify types of mould, only to positively identify mould filaments as characterized in Part 6, Diagram III.

l Count field as positive when the aggregate length of < 3 of the longest filaments present exceeds 1/6 diameter of field. These filaments may be separate or attached to each other. A clump or mass of mould has the same value as a single filament (Part 6, Diagram IV).

6) Calculation and Recording Results l Calculate proportion of positive fields from results of

examination of all observed fields for each sample unit. l Report results as a percentage of fields containing mould

filaments individually for each sample unit:

Number of positive fields/ x 100 = % positive fields per sample unitNumber of fields examined

and as an average for the whole sample:% average positive = % sample unit 1+%2+%3+%4+%5+%6

fields for whole sample 6

0Table I - DILUTION OF PUREE (PULP) FOR MOULD COUNT AT 20 C (2)0Table I - DILUTION OF PUREE (PULP) FOR MOULD COUNT AT 20 C (2)

Actual Refr. IndexActual Refr. Index Dilution FactorDilution Factor Amt. of Water to be Added to 100 ml of Sample UnitAmt. of Water to be Added to 100 ml of Sample Unit Total Volume of Diluted Sample UnitTotal Volume of Diluted Sample Unit

1.3462 1.145 14.5 114.5

1.3478 1.292 29.2 129.2

1.3494 1.440 44.0 144.0

1.3511 1.585 58.5 158.5

1.3527 1.730 73.0 173.0

1.3544 1.876 87.6 187.6

1.3560 2.024 102.4 202.4

1.3577 2.171 117.2 217.2

1.3593 2.322 132.2 232.2

1.3610 2.474 147.4 247.4

DIAGRAMSDIAGRAM I MICROMETER DISK

A: Length of grid that coincides with 1.382 mm on the microscope stageB: Proper area of field of viewC: Area of micrometer disk not visible through microscopeD: Diameter equal to 1.382 mm and cross ruled in sixths

DIAGRAMSDIAGRAM I MICROMETER DISK




7) Interpretation1) 4The tolerance as specified hereafter and representing the maximum

incidence of positive fields in canned tomatoes, tomato juice or vegetable juice, shall be applied in determining whether the tested lot of the product complies with Section B.11.016 of the Food and Drug Regulations. The maximum percentage of positive fields permitted for each lot is that represented by a percentage of positive fields not exceeding 25% in any sample unit included in the sample taken from a lot.

2) The tolerance as specified hereafter and representing the maximum incidence of positive fields in tomato puree, tomato paste, tomato pulp or tomato catsup, shall be applied in determining whether the tested lot of the product complies with Section B.11.017 of the Food and Drug Regulations. The maximum percentage of positive fields permitted for each lot is that represented by a percentage of positive fields not

7) Interpretation exceeding 50% in any sample unit included in the sample taken from a lot.

References 1. Horwitz, W. (ed.) 1980. Official Methods of Analysis of the Association of

Official Analytical Chemists. (44.096), Thirteenth edition. AOAC., Washington, D.C.

2. Continental Can Company. 1968. Mold Counting of Tomato Products. Continental Can Company Inc., Research and Development, Chicago, Illinois.

3. Gould, W.A. 1974. Tomato Production, Processing and Quality Evaluation. AVI Publishing Co., Inc., Westport, Connecticut.

4. American Can Company. 1957. The Howard Mold Count Method as Applied to Tomato Products. American Can Company, Research Division, Maywood, Illinois.

References

AA DD

CC

BB




A: Calibration circle, 1.382 mm diameterB: Area of liquid for mould countC: Cover glassD: Cover glassE: Two engraved parallel lines spaced 1.382 mm apart

F: Rectangle, 15 X 20 mmG: Moat

[1.382/2]2 X 3.1416 = 1.5 sq. mm., area of microscopic field1.5 X 0.1 = 0.15 cu. mm., volume of material in microscopic field

HOWARD MOULD COUNTING CHAMBER

DIAGRAM 11a

HOWARD MOULD COUNTING CHAMBER

DIAGRAM 11a

AA

CC

BB

BB

DD

the diameter of the field.F: This field is considered negative because only one filament is present which is

<1/6th the diameter of the field .G: This filed is considered positive because a clump of mould is present. It has the

same value as a single filament.H: This field is considered positive because a clump of mould is present even

though the longest three filaments are <1/6th the diameter of the field.

Reference1. Official Method MFO - 5 Health Protection Branch - Ottawa.Reference




Only filaments which have at least one of the following characteristics shall be classified as mould:A: Left side (and not right side); parallel walls of even intensity with both ends

definitely blunt.B: Parallel walls of even intensity with characteristic branching.C: Parallel walls of even intensity with characteristic granulation.

D: Parallel walls of even intensity with definite septation.E: Left side (and not right side); occasionally encountered, parallel walls of even

intensity with one end blunt and the other end rounded.F: Occasionally encountered, slowly tapering walls of even intensity with

characteristic granulation or septation.

A: This field is considered positive because the sum of the lengths of three separate filaments is >1/6th the diameter of the field.

B: This field is considered negative because the sum of the lengths of any three filaments is <1/6th the diameter of the field even though more than three separate filaments are present.

C: This field is considered positive because the sum of the lengths of three attached filaments is >1/6th the diameter of the field.

D: This field is considered negative because the sum of the lengths of three attached filaments is <1/6th the diameter of the field.

E: This field is considered positive because the length of one filament >1/6th

DIAGRAM III MOULD FILAMENTSDIAGRAM III MOULD FILAMENTS

DIAGRAM IV EXAMPLES OF FIELDS WITH MOULD FILAMENTSDIAGRAM IV EXAMPLES OF FIELDS WITH MOULD FILAMENTS

AA CCBB

EE

DD

FF

EE GGFF HH

CC

DD

AA

BB




Microbiological Examination of Cheese

IntroductionThis method shall be used for the determination of Escherichia coli and of Staphylococcus aureus in cheese, including cheese curd but excluding cottage cheese, made from either pasteurized or unpasteurized milk, in accordance with the Food and Drug Regulations.

Materiall Lauryl Tryptose (LT) broth LTB (AM1053, AM5053)l Escherichia Coli (EC) broth (AM1039,AM5039)l Levine's Eosin Methylene Blue (EMB) agar (AM1040, AM5040)l Nutrient agar (NA) (AM1074, AM5074) l IMViC media: a. Tryptone broth b. Buffered Glucose broth (AM1047/5047) c.

Simmons’s Citrate (SC) agar (AM1090, AM5090 l Baird-Parker (BP) agar (AM1011, AM5011) l Brain Heart Infusion (BHI) broth (AM1017, AM5017)l Trypticase Soy (TS) agar (AM11031, AM51031)l Blood agar (BA) (AM1014, AM5014)l Toluidine Blue-DNA agar (TDA) (AM10381)l Phenol Red Carbohydrate broth (AM1080, AM5080)l Tryptone Water (AM1104, AM5104)l 2% Sodium Citrate Solutionl Cheesel 0.1 % Peptone Water (AM1079/ AM5079)l E.coli ATCC 8739l E.coli identification Kit (20796001)l Kovac’s Indole Reagent(20700040)l Methyl Red Solution (20710040)l VP Reagent having 40% NaOH & 0.3% creatine (20680020)

Equipmentl Laminar Air Flow Unit.l Autoclave.l Incubator.l Water Bath.

ProcedureEach sample unit shall be analyzed individually. The test shall be carried out in accordance with the following instructions1) Collection of Samplesl 2.2.1 A sample, consisting of five sample units drawn at random from

each lot, shall be taken.l Each sample unit shall consist of at least 100 g. l Collect original unopened containers or packages wherever possible. l Employ aseptic techniques in collecting the sample units when sampling

Introduction

Material

Equipment

Procedure


bulk cheese. l Place each collected sample unit into a separate sterile container.

ol Keep sample units refrigerated (0-5 C) during transportation.

2) Handling of Sample Units o

l Keep sample units refrigerated (0-5 C) prior to anlaysis. l Analyze sample units as soon as possible after receipt at the laboratory.

3) Preparation of Media The following media, prepared and sterilized according to the manufacturers' instructions, shall be used: (1) Lauryl Tryptose (LT) broth (2) Escherichia Coli (EC) broth (3) Levine's Eosin Methylene Blue (EMB) agar (4) Nutrient agar (NA) (5) IMViC media: a. Tryptone broth b. Buffered Glucose broth c. Simmon's

Citrate (SC) agar (6) Baird-Parker (BP) agar (7) Brain Heart Infusion (BHI) broth (8) Trypticase Soy (TS) agar (9) Blood agar (BA) (10) Toluidine Blue-DNA agar (TDA) (11) Phenol Red Carbohydrate broth

4) Preparation of Dilutionso

l Temper sterile aqueous 2% sodium citrate to 40-45 C, and prewarm osterile blender jars to 40-45 C.

l Combine portions from several locations within the sample unit, to obtain a representative analytical unit of 11(10)* g.

l Prepare a 1: 10 dilution of cheese by adding the analytical unit to 99(90)* mL of the tempered sodium citrate solution in a prewarmed sterile blender jar. Blend for the minimum time required to produce a homogeneous suspension. To prevent overheating, blending time should not exceed 2.5 min.

l Check the pH of the suspension. If the pH is outside the range of 5.5 to 7.6, adjust to 7.0 with either sterile NAOH or HCl.

l Prepare succeeding dilutions as required to determine the total numbers of E. coli and S. aureus, by transferring 11(10)* mL of the previous dilution into 99(90)* mL of 0.1% sterile peptone water diluent. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present. * Weight and volume in brackets indicate alternative procedure for preparation of dilutions.







5) Determination of E. coliI) Presumptive Coliform Test

a. The medium used is LT broth, dispensed in 10 mL volumes into tubes containing gas vials (inverted Durham tubes).

b. Arrange LT broth tubes in rows of fives, and mark them identifying the sample, the sample unit and the dilution to be inoculated.

c. Inoculate LT broth with a culture of E. coli known to ferment lactose oand produce gas at 45 C to serve as a positive control, incubate,

and subsequently transfer into all media used at different stages of the procedure. Set up an uninoculated type of medium corresponding to each step in the procedure as a negative control.

d. Inoculate each tube of a set of five tubes of single strength LT broth with 1 mL of the 1:10 dilution (cheese homogenate). Repeat for each succeeding decimal dilution as required.

e. Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials.

o of. Incubate the inoculated LT broth tubes at 35 C± 0. 5 C for 24 ± 2 hrs. Examine for gas formation, and on the same day begin the presumptive E. coli (faecal coliform) test for all gas-positive tubes .

g. Incubate gas-negative tubes for an additional 24 ± 2 hrs, examine, record the number of additional gas-positive tubes and begin the presumptive E. coli (faecal coliform) test for the additional gas-positive tubes.

h. The absence of gas in all of the tubes at the end of 48 ± 2 hrs of incubation constitutes a negative test.

II) Determination of Presumptive E. coli (Faecal Coliforms) a. The medium used is EC broth, dispensed in 10 mL volumes in tubes

containing gas vials. b. Submit all gas-positive LT broth tubes to the presumptive E. coli

(faecal coliform) test. c . Shake or rotate the LT broth tubes to mix the contents and transfer

one loopful from each positive LT broth tube to a tube of EC medium. Avoid transferring pellicle.

d. Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials.

oe. Incubate the inoculated EC broth tubes in a water bath at 45 C± 00.2 C for 24 ± 2 hrs. Make certain that the water level in the bath

is above the level of the medium in the tubes. f. Examine for gas production and on the same day begin the E. coli

identification for all gas-positive tubes .g. Incubate gas-negative tubes for an additional 24 ± 2 hrs,

examine, record the number of gas-positive tubes above, and begin the E. coli identification for the additional gas-positive tubes.

5) Determination of E. coli IV) Identification of E. coli a. The media and reagents used, are:

(i) EMB agar dispensed in Petri plates. (ii) NA dispensed in Petri plates and as slants in tubes. (iii) EC broth dispensed in 10 mL volumes in tubes containing gas

vials. (iv) Tryptone broth dispensed in 5 mL volumes in tubes. (v) Buffered Glucose broth dispensed in 6 mL volumes in tubes. (vi) Simmon's Citrate (SC) agar dispensed as slants in tubes. (vii) Kovac's indole reagent consisting of pure amyl or isoamyl

alcohol, p-dimethylaminobenzaldehyde, and 12N HCl (analytical grade) at a ratio of 15:1: 5 or Ehrlich-Boehme's indole reagent consisting of p-dimethylaminobenzaldehyde, 96% ethanol, and 12N HCl at a ratio of 0.4:38:8.

(viii)Methyl red solution consisting of 0.1 g methyl red dissolved in 300 mL of 95% ethanol and diluted with distilled water to 500 mL.

(ix) VP reagent consisting of an aqueous solution of 40% NaOH and 0.3% creatin, or VP reagent consisting of 16% aqueous KOH and 6% a-naphthol in 95% ethanol.

b. From each gas-positive EC broth tube (see section 3.4.2, steps f., g., above), streak a loopful of culture onto a separate EMB agar plate.

o oc . Incubate the plates at 35 C± 0.5 C for 18 to 24 hrs and examine for colonies which are nucleated with or without a metallic sheen.

d. Select two such colonies from each plate and streak onto separate NA plates to obtain discrete colonies.

o oe. Incubate the NA plates at 35 C± 0.5 C for 24 ± 2 hrs, and from each of them, pick an isolated colony and streak onto a separate NA slant.

o of. Incubate the slants at 35 C± 0.5 C for 24 ± 2 hrs. g. From one of the two NA plates prepared (step d., above), transfer

inoculum into a separate tube of each of the EC broth and the IMViC media (for IMViC tests, see steps m., n., o., below).

h. If isolates picked from EMB agar and purified on NA plates are stored for more than 72 hrs before being subjected to the IMViC reactions, inoculate fresh NA slants from the slants prepared in step

o o e., above, incubate at 35 C± 0.5 C for p>24 ± 2 hrs, then inoculate the IMViC media from the freshly incubated slants.

oi. Incubate the inoculated EC broth and IMViC media at 35 C± 0.5 oC for p>24 ± 2 hr or, as indicated in step n., below, for 48 ± 2 hrs.

j. Examine the EC broth tubes after 24 ± 2 hrs for gas production and record results.

k. If the EC broth tubes are gas-negative, incubate for an additional 24 ± 2 hrs, examine, and record results. If no gas is produced within 48 ± 2 hrs, the isolate is not considered to be E. coli.




l. If gas is produced within 24 or 48 ± 2 hrs, make smear from the corresponding NA slant which was inoculated from the same colony (see steps e. and g., above), and stain by Gram's procedure. Examine microscopically and record results. If the organisms are not Gram-negative, nonsporeforming rods, they are not E. coli.

m. Indole (I)(i) Transfer inoculum from each isolate to be tested (see step g.,

above) into a separate tube of Tryptone broth. o o(ii) Incubate the inoculated tubes at 35 C± 0.5 C for 24 ± 2

hrs. (iii) Add 0.2-0.3 mL of either indole reagent (see step a., vii,

above) to each tube and shake the tube to mix the contents. (iv) Let the tube stand for 10 min and observe. A dark red colour in

the alcohol layer indicates a positive test. n. Methyl-Red Voges Proskauer Tests (MR & VP)

(i) Transfer inoculum from each isolate to be tested (see step g., above) into a separate tube of Buffered Glucose broth.

o o(ii) Incubate the inoculated tubes at 35 C± 0.5 C for 48 ± 2 hrs.

(iii) Pipette 1 mL from each incubated tube into a separate empty tube, and add 1 mL of VP reagent. Shake the tubes vigorously to aerate.

(iv) Let the tubes stand for 4 hrs and observe. The test is VP positive if an eosin pink colour develops within 4 hrs.

(v) Incubate the remainder of the Buffered Glucose broth for an o oadditional 48 ± 2 hrs at 35 C± 0.5 C. Add 5 drops of the

methyl red solution to each tube and shake the tubes to mix the contents. The test is positive if a red colour develops.

(vi) Alternatively, two separate tubes may be set up for the MR and VP tests; if this is done, an equal volume of VP reagent must be added to one of the tubes, five drops of the methyl red solution to the other.

o. Sodium Citrate Test (C)(i) Transfer inoculum from each isolate to be tested (see step g.,

above) onto a separate slant of SC agar. Use an inoculating needle and apply a light inoculum.

o o(ii) Incubate the inoculated slants at 35 C± 0.5 C for 48 ± 2 hrs and observe for growth. Visible growth is usually accompanied by a change of colour from green to deep blue.

p. The characteristic IMViC reaction pattern for E. coli is as follows: Indole (+ or -) Methyl red (+) Voges-Proskauer (-) Citrate (-)

q. If gas is produced in EC broth, and IMViC reactions characteristic of E. coli are obtained, the other isolate (see steps d., e., f., above)

need not be further tested. However, if no gas is produced in EC broth within 48 ± 2 hrs and/or the IMViC pattern is not characteristic of E. coli, the remaining isolate shall be tested for gas production in EC broth and for its IMViC reaction pattern. Repeat steps g. to o., above. If both isolates fail to produce gas in EC broth and/or produce IMViC reaction patterns not characteritic of E. coli, then E. coli is considered to be absent from the EC broth tube from which the isolates originated.

r. Compute the MPN of E. coli per g of cheese following the instructions in Part 5, on the basis of the number of gas-positive EC

obroth tubes, which were incubated at 35 C (see step i., above), and found to contain Gram-negative, non-sporeforming, rod-shaped bacteria, giving the IMViC reactions characteristic of E. coli (see step p., above).

6) Determination of S. aureusI) Presumptive Count

a. The selective agar used is BP agar. b. Pre-pour plates of BP agar and allow their surfaces to dry before

they are inoculated. c. Mark clearly each Petri plate to be used, identifying the sample, the

sample unit, and the dilution. d. Agitate each dilution bottle (see section 3.3.5 above) to resuspend

material. e. For pasteurized-milk cheese, distribute 2 mL of the 1:10 dilution

accurately over the surface of five BP plates by spreading 0.4 mL on each of the five plates.

f. For unpasteurized-milk cheese, spread 0.2 mL of the 1:10 dilution evenly over the surface of each of two BP plates.

g. From at least two subsequent dilutions, spread 0.2 mL evenly over the surface of each of two BP agar plates.

o oh. Invert the plates and incubate at 35 C± 0.5 C for 48 ± 2 hrs. i. The following two types of colonies are considered to be

presumptive S. aureus. Type 1: Convex, entire, shiny black surrounded by clear zones extending into the opaque medium. Type 2: Covex, entire, shiny black without clearly defined zones. Each colony type may shown grey-white margins around the colonies and/or opaque zones (double halos).

j. Count colonies immediately after the incubation period. k. Do not count black mucoid colonies larger than 2 mm in diameter,

or swarmers. l. Count the colonies of each type and record separately, but add

together to give the total presumptive count. m. Counting of the five plates of the 1:10 dilution

(i) If the number of all presumptive S. aureus colonies per plate is fewer than 20, add separately the number of each colony

6) Determination of S. aureus




type on all five plates to provide a count for each type per 2 mL (i.e. per 0.2 g cheese). Multiply each total by five to obtain the presumptive S. aureus count per g of cheese to obtain the total presumptive S. aureus count per g of cheese.

(ii) If the number of all presumptive S. aureus colonies per plate is greater than 20 but does not exceed 200, take two plates at random, and separately count the numbers of colonies of each type. For each type, compute the average presumptive S. aureus count per plate (per 0.4 mL). Mulitply each presumptive count by 25 to obtain the presumptive count per g of cheese. Add the two presumptive counts per g of cheese to obtain the total presumptive S. aureus count per g of cheese.

(iii) If the number of presumptive staphylococcal colonies on some of the five plates is < 20, but on others is = 20, proceed as in (i) above.

n. Counting of duplicate plates: (i) Select the duplicate plates of the dilution that yields a

combined presumptive S. aureus count between 20 and 200 colonies per plate. Count separately the colonies of each type and compute the average presumptive count for each type per plate (0.2 mL). Multiply each count by 5 and by the appropriate dilution factor, and record as the presumptive count of each of the two types per g of cheese. Add the two results, and record as the total presumptive S. aureus count per g of cheese.

o. Record negative presumptive counts as < 5 per g of cheese if five plates of the 1:10 dilution are used; or as < 2.5 x the dilution factor per g of cheese for duplicate plates.

II) Test for Coagulase Production a. The media and reagent to be used are:

(i) Non-selective media such as NA, TS agar or BA, dispensed as slants in tubes.

(ii) BHI broth dispensed in 1 mL volumes in tubes. (iii) Certified rabbit plasma containing EDTA dispensed in 0.5 mL

volumes in 12 x 100 mm tubes. b. From the replicate plates counted, select a number of each colony

type observed as follows to check for culture purity and for coagulase reaction.(i) When the total count per type for all the plates of a dilution is

less than five, pick all colonies of that type. (ii) When the total count per type for all plates of a dilution is

equal to or greater than five colonies, pick five colonies of that type at random.

c. Streak each colony picked onto a non-selective medium, so as to obtain discrete colonies.

o od. Incubate at 35 C± 0.5 C for 24 ± 2 hrs.

e. Make a smear from the growth of each isolate on the non-selective medium and stain with a simple stain (e.g., crystal violet). Observe microscopically for the presence of cocci.

f. If the isolates are composed of cocci only, transfer inoculum from each plate of non-selective medium into a separate tube of BHI broth. If an isolate is not pure, choose another colony from step c. above and repeat steps d. and e.

o og. Incubate at 35 C± 0.5 Cfor 18-24 hrs and observe for growth. h. Inoculate BHI broth with a culture of S. aureus known to be

coagulase-positive, to serve as a positive control. Incubate at o o35 C± 0.5 C for 18-24 hrs. Use uninoculated medium from the

same batch of BHI broth as a negative control. i. Transfer 0.2 mL of each BHI broth culture (see steps f. and h.,

above) into a separate tube of rabbit plasma and shake or rotate the tubes to mix thoroughly.

oj. Incubate the tubes at 35 C and examine after one hr. and after four hr. Do not shake the tubes during incubation. Negative tubes should be incubated overnight at room temperature and rechecked for a 3+ or 4+ reaction.

k. 3+ or 4+ reaction is confirmation that the isolate is S. aureus.

III) The Thermonuclease Test a. Perform the test for the presence of thermostable nuclease (TNase)

concurrently with the coagulase test in the following manner: b. Medium used is toluidine blue - DNA agar (TDA). c. Pipette 3.0 mL of molten TDA mixture to a microscope slide or a

plastic immunoplate; or pipette sufficient TDA to a flat bottom Petri plate to give a height of 1.5 mm.

d. With a cork borer or a suitably cut pasteur pipette, cut wells, 2 mm in diameter, approximately 12 mm apart, in the agar layer.

e. Remove agar plugs by aspiration. f. Heat broth cultures used for the determination of coagulase

production in a boiling water bath for 15 min., and cool rapidly under cold tap water.

g. Fill wells in the TDA with heated and subsequently cooled broth cultures

oh. Incubate the TDA slides or plates in a moist chamber at 35 C and examine after 4 hrs.

i. A bright pink halo extending = 1 mm beyond the perimeter of the well is indicative of nuclease activity. Plates giving doubtful reactions should be held at room temperature and re-examined the following morning.

j. Isolates are confirmed as S. aureus if: (i) they possess TNase activity and produce 3+ or 4+ degree of

coagulase reaction (EDTA rabbit plasma), or (ii) if they are TNase-negative but give a 4+ coagulase reaction.

k. With cultures that show a 2+ or lesser coagulase reaction but




which are positive for TNase activity, perform the following additional tests:

IV) Anaerobic utilization of Glucose a. Inoculate culture to be tested into a tube of carbohydrate

fermentation medium containing 0.5% glucose. ob . Overlay with sterile paraffin oil or Vaspar and incubate at 35 C for

18-24 hrs. c. Colour change indicating an acid reaction which is a positive test.

S. aureus gives a positive reaction.

V) Anaerobic utilization of mannitol a. Same as in 6 IV (a) except that the source of carbohydrate is

mannitol. b. S. aureus usually gives a positive reaction but some strains do not

ferment mannitol.

VI) Lysostaphin sensitivitya. The reagents used are:

(i) Phosphate Saline Buffer (0.02 M) pH 7.3-7.4. (ii) Lysostaphin solution containing 50 µg per mL of lysostaphin

in the phosphate buffer. b. Inoculate the culture to be tested into 0.2 mL of the phosphate

buffer and mix to obtain a homogeneous suspension. c. Transfer one half of the suspended cells to another tube (13 x 100

mm) and mix with 0.1 mL of the phosphate saline buffer. d. Add 0.1 mL of the lysostaphin solution to the original tube to give a

concentration of 25 µg lysostaphin per mL of cell suspension. oe. Incubate both tubes at 37 C for as long as 2 hrs.

f. If the turbidity clears in the tube containing cells plus lysostaphin, the test is positive. If clearing has not occurred in 2 hrs., the test is negative.

g. S. aureus gives a positive reaction. h. Run positive and negative controls simultaneously with the tests. i. If two of the three ancillary tests are positive, the isolate is

considered to be S. aureus.

VII) On the basis of the confirmatory tests for each of the two types of cultures, record the total number of S. aureus per g of cheese.

No. of type 1 colonies x presumptive countconfirmed as S. aureus type 1 colonies/g

----------------------------No. of type 1 colonies tested

Number of = PlusS. aureus/g No. of type 2 colonies x presumptive count

confirmed as S. aureus type 2 colonies/g---------------------------No. of type 2 colonies tested

7) Interpretation l The tolerance as specified hereafter and representing the maximum

probable incidence of Escherichia coli, and the maximum count of Staphylococcus aureus in cheese, including cheese curd but excluding cottage cheese, made from pasteurized milk, shall be applied in determining whether the tested lot of the product complies with Section B.08.048(1) of the Food and Drug Regulations.

(1) The maximum MPN of E. coli permitted for each lot is that represented by an E. coli MPN not exceeding: (a) 100 per g in more than two of five sample units, and (b) 2,000 per g in any one sample unit, included in the sample

taken from a lot. (2) The maximum count of S. aureus permitted for each lot is that

represented by a count of S. aureus not exceeding: (a) 100 per g in more than two of five sample units, and (b) 10,000 per g in any sample unit, included in the sample

taken from a lot.

l The tolerance as specified hereafter and representing the maximum probable incidence of Escherichia coli, and the maximum count of Staphylococcus aureus in cheese, made from unpasteurized milk, shall be applied in determining whether the tested lot of the product complies with the Food and Drug Regulations. (1) The maximum MPN of E. coli permitted for each lot is that

represented by an E. coli MPN not exceeding: (a) 500 per g in more than two of five sample units, and (b) 2,000 per g in any one sample unit, included in the sample

taken from a lot. (2) The maximum count of S. aureus permitted for each lot is that

represented by a count of S. aureus not exceeding: (a) 1,000 per g in more than two of five sample units, and (b) 10,000 per g in any sample unit, included in the sample

taken from a lot.

These tolerances are summarized in the following table:


1. Cheese made from Pasteurized Milk

E. coli 5 2 100 2,000

S. aureus 5 2 100 10,000

2. Cheese made from Unpasteurized Milk

E. coli 5 2 500 2,000

S. aureus 5 2 1,000 10,000

n = Number of sample units (subsamples) to be examined per lot.

7) Interpretation





c = Maximum number of sample units (subsamples) per lot which may have a bacterial concentration higher than the value for "m" without violation of the Regulation.

m = Maximum number of bacteria per g of cheese, which is of no concern (acceptable level of contamination).

M = Maximum number of bacteria per g of cheese, which if exceeded by any one sample unit (subsample), renders the lot under investigation in violation of the Regulation.

8) CALCULATION OF MOST PROBABLE NUMBERS (MPN) Table A-1 shows the most probable numbers of coliforms per 100 g or mL of test material corresponding to the number of gas-positive tubes in the coliform test. Table A-1 has been adapted from a conversion table prepared for the analysis of drinking waters where 10, 1.0 and 0.1 mL of the water under test are used as test portions. The table is equally appropriate if 10, 1.0 and 0.1 g of a solid food constitute the test positions in the tubes. When other sized portions of the test material are placed in the tubes, MPN values obtained from Table A-1 must be multiplied by an appropriate number, to correct for the actual amount of test material in the tubes, and also to obtain the MPN per g (mL) as is usually done for foods, rather than per 100 mL (g), for which the values are given in the table. The volume of diluent added to the tubes (and which accompanies the test material) is ignored when calculating the MPN.

Example: The following inoculated tubes give a positive reading: (1) 5 tubes with 10 mL of 1:10 dilution of test material - all 5 are positive (2) 5 tubes with 1 mL of 1:10 dilution of test material - 1 is positive (3) 5 tubes with 1 mL of 1:100 dilution of test material - none is positive

The quantities in each of the five tubes of the three dilution series represent 1, 0.1

8) CALCULATION OF MOST PROBABLE NUMBERS (MPN)

and 0.01 g (mL), respectively of the test material. According to Table A-1, a reading of 5-1-0 gives a value of 33 when 10, 1 and 0. 1 g (mL) respectively are used. However, since only 1/10 of these amounts were actually used in the analysis, the value of 33 obtained from Table A-1 must be multiplied by 10 giving 33 x 10 = 330 organisms per 100 g (mL) of test material. Since the results have to be expressed per g (mL), the MPN value is 330 ÷ 100 = 3.3. When higher dilutions are used, the same procedure is followed, but the multiplier (dilution factor) is enlarged to relate the amount of test material actually present to the values given for 10, 1.0 and 0.1 g (mL) in Table A-1. Dilution factor - Reciprocal of the dilution of the analytical unit. For calculating the MPN, use the dilution factor of the middle set of the three dilutions selected. To determine which consecutive dilutions to use, refer to the combinations shown below: (See also Table A- 2). 1. If only 3 dilutions are made, use the results of 3 dilutions to compute the

MPN. Examples a. and b. 2. If more than 3 dilutions are employed, use the results of only 3 consecutive

dilutions. Select the highest dilution in which all 5 tubes are positive and 2 subsequent higher dilutions. Examples c. and d.

3. If more than 3 dilutions are made, but none of the dilutions tested have all 5 tubes positive, use the first 3 dilutions. Example e.

4. If a positive tube occurs in the dilution higher than the 3 chosen to rule, the number of such positive tubes should be added to those of the next lower dilution. Example f.

5. If the tubes of all sets of a dilution series are positive, choose the 3 highest dilutions of the series and indicate by a "greater than" symbol (>) that the MPN is greater than the one calculated. Example g.

Refer to Table A-1 and look up the value which corresponds to the number of positive tubes obtained. MPN/g = No. of Microorganisms (Table A-1)/100 X dilution factor of middle set of tubes .

TABLE A-1Most Probable Number (MPN) of Bacteria Per 100 g (mL) of Test Material Using 5 Tubes With 10,1 and 0.1 mL or g of Test Material


000 <1.8 100 2 200 4.5 300 7.8 400 13 500 23

001 1.8 101 4 201 6.8 301 11 401 17 501 31002 3.6 102 6 202 9.1 302 13 402 21 502 43003 5.4 103 8 203 12 303 16 403 25 503 58004 7.2 104 10 204 14 304 20 404 30 504 76005 9 105 12 205 16 305 23 405 36 505 95

010 1.8 110 4 210 6.8 310 11 410 17 510 33

011 3.6 111 6.1 211 9.2 311 14 411 21 511 46012 5.5 112 8.1 212 12 312 17 412 26 512 64013 7.3 113 10 213 14 313 20 413 31 513 84

TABLE A-1









10 1 0.1 0.01 0.001

a. 5/5** 5/5 2/5 5-5-2 540 1 5.4

b. 5/5 5/5 2/5 5-5-2 540 10 54

c. 5/5 5/5 2/5 2/5 5-2-2 95 100 95

d. 5/5 5/5 2/5 0/5 5-2-0 49 100 49

e. 2/5 2/5 1/5 0/5 2-2-1 12 10 1.2

f. 5/5 2/5 1/5 1/5*** 5-2-2 95 10 9.5

g. 5/5 5/5 5/5 5/5 5-5-5 >1600 100 >1600


TABLE A-2

10 1 0.1 0.01 0.001








014 9.1 114 12 214 17 314 23 414 36 514 110

015 11 115 14 215 19 315 27 415 42 515 130020 3.7 120 6.1 220 9.3 320 14 420 22 520 49021 5.5 121 8.2 221 12 321 17 421 26 521 70

022 7.4 122 10 222 14 322 20 422 32 522 95023 9.2 123 12 223 17 323 24 423 38 523 120

024 11 124 15 224 19 324 27 424 44 524 150025 13 125 17 225 22 325 31 425 50 525 180030 5.6 130 8.3 230 12 330 17 430 27 530 79

031 7.4 131 10 231 14 331 21 431 33 531 110

032 9.3 132 13 232 17 332 24 432 39 532 140033 11 133 15 233 20 333 28 433 45 533 180

034 13 134 17 234 22 334 31 434 52 534 210

035 15 135 19 235 25 335 35 435 59 535 250040 7.5 140 11 240 15 340 21 440 34 540 130

041 9.4 141 13 241 17 341 24 441 40 541 170042 11 142 15 242 20 342 28 442 47 542 220043 13 143 17 243 23 343 32 443 54 543 280044 15 144 19 244 25 344 36 444 62 544 350045 17 145 22 245 28 345 40 445 69 545 440050 9.4 150 13 250 17 350 25 450 41 550 240051 11 151 15 251 20 351 29 451 48 551 350052 13 151 17 252 17 352 32 452 56 552 540053 15 153 19 253 26 353 37 453 64 553 920054 17 154 22 254 29 354 41 454 72 554 1600

055 19 155 24 255 32 355 45 455 81 555 >1600






IntroductionThis method shall be used for the determination of total aerobic bacteria (Aerobic Colony Count) and of coliform bacteria (Coliforms) in water in sealed containers, excluding mineral and spring water; and of coliforms in prepackaged ice, in accordance with the Food and Drug Regulations, respectively.

Materiall Plate Count (PC) agar (AM1081, AM5081) l Lauryl Tryptose (LT) broth (AM1053, AM5053) l Brilliant Green Lactose 2% Bile (BGLB) broth (AM1020, AM5020) l Peptone Water 0.1% (AM1079, AM5079)

Equipmentl Laminar Air Flow Unitl Incubatorl Autoclave

ProcedureFive sample units shall be analyzed individually for Aerobic Colony Count (ACC). Ten sample units shall be analyzed individually for coliforms.

The tests shall be carried out in accordance with the following instructions:

1) Collection of Samplesl A sample, consisting of ten sample units drawn at random from each lot,

shall be taken. l Each sample unit shall consist of at least 100 ml or g.l Collect original unopened container wherever possible.l Employ aseptic techniques in collecting the sample units when sampling

bulk ice. Place each collected sample unit into a separate sterile container.

l Ship and store the sample units of water in sealed containers under orefrigeration (< 0.5 C) if more than two hrs lapse between collection

and analysis. Do not freeze the sample units. l Do not allow sample units of prepackaged ice to thaw during shipment.

2) Handling of Sample Unitsa) Water in sealed containers l Do not store sample units for more than 24 hrs before analysis.

b. Prepackaged Ice l If sample units are prepackaged in leakproof containers, thaw

othem in the containers under refrigeration (0-5 C) prior to analysis. l If sample units are not in leakproof containers, transfer the ice

Introduction

Material

Equipment

Procedure



aseptically to sterile plastic bags or other suitable sterile containers. Seal containers to prevent contamination, and thaw

osample units under refrigeration (0-5 C). Do not store thawed sample units for more than six hr before analysis.

3) Preparation of Media The following media, prepared and sterilized according to the manufacturer's instructions, shall be used: l Plate Count (PC) agar l Lauryl Tryptose (LT) broth l Brilliant Green Lactose 2% Bile (BGLB) broth

4) Preparation of Dilutions (Water in sealed containers only)l Prepare sterile 0.1% peptone water diluent. l Thoroughly mix each sample unit by shaking the container.

*l Prepare a 1:10 dilution of the water by aseptically pipetting 11(10) of

*the "water" into 99(90) ml of the diluent. l Mix the 1:10 dilution by shaking the dilution bottle 25 times in a 30 cm

arc in approximately 7 sec.l Prepare subsequent dilutions as required to determine the ACC of the

water, by transferring 11(10)ml of the previous dilution into 99(90) ml of the diluent. *Weight and volume in brackets indicate alternate procedure for making dilutions.

l Shake all dilutions (as in step above) immediately prior to making transfers to ensure uniform distribution of the microorganisms present.

5) Determination of ACC Examine five sample units of the water. The medium used is PC agar, prepared for making pour plates.

a. Analysisl Agitate each dilution bottle to distribute uniformly the

microorganisms present. l Without delay, pipette 1 ml of the undiluted sample unit into each

of two appropriately marked Petri plates using a sterile pipette for each transfer. Repeat for each prepared dilution.

ol Pour 12-15 ml of tempered (40-45 C) agar into each plate and

mix contents by rotating and tilting.l Allow the agar to solidify.l Plates should be poured not later than 15 min after preparation of

dilutions. o o

l Incubate plates in an inverted position at 35 C± 0.5 C for 48 ±


4) Preparation of Dilutions (Water in sealed containers only)

5) Determination of ACC

a. Analysis

Microbial Examination of Water in Sealed Containers (Excluding Mineral and Spring Water) and of Prepackaged Ice




l The absence of gas in all of the tubes at the end of 48 ± 2 hrs of incubation constitutes a negative presumptive test.

l Compute the "MPN" of presumptive coliforms per 100 ml of water or of melted ice following the instructions in Part 5 to convert the number of gas-positive tubes to MPN values. Record results.

b. Confirmed Test l The confirmatory medium used is BGLB broth, dispensed in 10 ml

volumes into tubes containing gas vials. l Submit all gas-positive LT broth tubes to the confirmed test. l Shake or rotate the LT broth tubes to mix the contents, and with a

sterile loop, transfer one loopful from each positive LT broth tube to the BGLB broth. (Avoid transferring pellicle).


o o l Incubate the inoculated BGLB broth tubes at 35 C± 0.5 C for 24

± 2 hrs. Examine for gas formation and record results. l Incubate gas-negative tube for an additional 24 ± 2 hrs, re-

examine, record the number of additional gas-positive tubes and add to the result obtained in above.

l Formation of gas during 48 ± 2 hrs incubation constitutes a positive confirmed test.

l Compute the "MPN" of confirmed coliforms per 100 ml of the water in sealed containers or per 100 g of the ice following the instructions in Part 5 to convert the number of gas-positive tubes to MPN values. Record results.

7) Interpretation The tolerances as specified hereafter and representing the maximum total aerobic bacteria (Aerobic Colony Count) in water in sealed containers (excluding mineral and spring water), and the maximum probable incidence of coliform bacteria (Coliforms) in water in sealed containers and in prepackaged ice, shall be applied in determining whether the tested lot of the product complies with the Food and Drug Regulations.

a. The maximum count of total aerobic bacteria permitted for each lot of water in sealed containers is that represented by an Aerobic Colony Count not exceeding: l 100 per ml in more than two of the five sample units, and l 10,000 per ml in any sample unit, included in the sample taken

from the lot.

b. Coliform bacteria (Coliforms) shall be considered absent in a lot when not more than one of the 10 sample units taken from the lot is positive for coliforms, and the MPN for that sample unit is not more than 10 coliform per 100 ml of the water in sealed containers or per 100 g of the pre-packaged ice.

b. Confirmed Test

2 hrs. l Avoid crowding or excessive stacking of plates in order to permit

rapid equilibration of plates with incubator temperature.l Count colonies promptly after the incubation period. l Select for counting those plates containing 30-300 colonies,

including pinpoint colonies. If counts do not fall within this range, select plates that have counts nearest to this range.

b. Recording Results l Calculate the average count (arithmetic mean) of duplicate plates,

following the examples in Table 5-1 of "Standard Methods for the Examination of Dairy Products", A.P.H.A., 14th Edition (E.H. Marth, Jr., Editor. 1978).

l When reporting results, round-off the counts to two significant figures and record only the first two left hand digits. (e.g., record 2,850 as 2,900).

l If the lowest dilution plated shows no colonies, report the count as the product of 0.5 x the dilution factor preceeded by a "less than" (<) sign.

l To compute the ACC, use the formula: N=AxD, where N is the number of colonies per g or ml of product, A is the average count, and D is the respective dilution factor.

6) Determination of coliforms Examine 10 sample units of water or ice.

a. Presumptive Test l The medium used is LT broth dispensed in 10 ml volumes into tubes

containing gas vials (inverted Durham tubes). l Arrange LT broth tubes in rows of five, and mark them, identifying

the sample, the sample unit and the dilution to be inoculated. l Inoculate each of five tubes of double strength LT broth with 10 ml

of the undiluted sample unit (see section above), and inoculate each of five tubes of single-strength LT broth with 1 ml of the undiluted sample unit, and inoculate each of five tubes of LT broth with 0.1 ml of the undiluted sample unit.

l Mix inoculum and medium by gently shaking or rotating the tubes but avoid entrapping air in the gas vials.

o ol Incubate the inoculated LT broth tubes at 35 C± 0.5 C for 24 ±

2 hrs. Examine for gas production, record results, and on the same day begin the confirmatory test for all gas positive tubes. (See section 3.5.2 below)

l Incubate gas-negative tubes for an additional 24 ± 2 hrs, record the number of gas-positive tubes, add to the results obtained in step (e) above, and begin the confirmed test for the additional gas-positive tubes.

b. Recording Results

6) Determination of coliforms

a. Presumptive Test




c. The tolerances are summarized in the following table:


1 For water in sealed containers

Aerobic Colony Count 5 2 100 10,000

Coliforms 10 1 0 10

2 For prepackaged ice

Coliforms 10 1 0 10

n = Number of sample units (subsamples) to be examined per lot. c = Maximum number of sample units (subsamples) per lot which may have a

bacterial concentration higher than the value for 'm' without violation of the Regulation.

m = Maximum number of bacteria per designated unit*, which is of no concern (acceptable level of contamination).

M = Maximum number of bacteria per designated unit*, which if exceeded by any one sample unit (subsample), renders the lot under investigation in violation of the Regulation.

* per ml for the Aerobic Colony Count per 100 ml or g for Coliforms.

8) Calculation Of Most Probable Numbers (MPN)Table A-1 shows the most probable numbers of coliforms per 100 ml or g corresponding to the number of gas-positive tubes in the coliform test. Table A-1 has been adapted from a conversion table prepared for the analysis of drinking water where 10, 1.0 and 0.1 ml of the water under test are used as test portions. The table is equally appropriate if 10, 1.0, and 0.1 g or ml of a food constitute the test portions in the tubes. When other sized portions of the test material are placed in the tubes, the MPN values obtained from Table A-1 has to be multiplied by an appropriate number, to correct for the actual amount of test material in the tubes, and also to obtain the MPN per g or ml as is usually done for foods, rather than per 100 ml (g), for which the values are given in the table. The volume of diluent added to the tubes (and which accompanies the sample) is ignored when calculating the MPN.

ExampleThe following inoculated tubes give a positive reading:(1) 5 tubes with 10 ml of 1:10 dilution of test material - all 5 are positive (2) 5 tubes with 1 ml of 1:10 dilution of test material - 1 is positive (3) 5 tubes with 1 ml of 1:100 dilution of test material - none are positive


1 For water in sealed containers

2 For prepackaged ice

8) Calculation Of Most Probable Numbers (MPN)

Example

The quantities (test portions) in each of the five tubes of the three dilution series represent 1, 0.1 and 0.01 g or ml test material respectively.

According to Table A-1, a reading of 5-1-0 gives a value of 33 if 10, 1 and 0.1 g or ml respectively are used. However, since only 1/10 of these amounts were actually used in the analysis, the value of 33 obtained from Table A-1 must be multiplied by 10 giving 33 x 10 = 330 organisms per 100 g or ml of test material. Since the results have to be expressed per g or ml, the MPN value of 330 must be divided by 100. When higher dilutions are used, the same procedure is followed, but the multiplier (dilution factor) is enlarged to relate the amount of test material actually present to the values given for 10, 1.0 and 0.1 g or ml in Table A-1.

Dilution factor = Reciprocal of the dilution of the analytical unit.


To determine which consecutive dilutions to use, refer to the combinations shown below: (See also Table A-2). 1. If only 3 dilutions are made, use the results for those 3 dilutions to compute

the MPN. Examples a and b. 2. If more than 3 dilutions are employed, use the results of only 3 consecutive

dilutions. Select the highest dilution (last dilution, i.e. dilution with the smallest quantity of product) in which all 5 tubes are positive and 2 succeeding higher dilutions. Examples c and d.

3. If more than 3 dilutions are made, but none of the dilutions tested have all 5 tubes positive, use the first 3 dilutions. Example e.

4. If a positive tube occurs in the dilution higher than the 3 chosen to rule, the number of such positive tubes should be added to those of the next lower dilution. Example f.

5. If the tubes of all sets of a dilution series are positive, choose the 3 highest dilutions of the series and indicate by a "greater than" symbol (>) that the MPN is greater than the one calculated. Example g.


MPN/100 ml (g) = No. of Microorganisms x dilution factor of middle (Table A-1) set of tubes.






000 <1.8 100 2 200 4.5 300 7.8 400 13 500 23

001 1.8 101 4 201 6.8 301 11 401 17 501 31002 3.6 102 6 202 9.1 302 13 402 21 502 43003 5.4 103 8 203 12 303 16 403 25 503 58004 7.2 104 10 204 14 304 20 404 30 504 76005 9 105 12 205 16 305 23 405 36 505 95

010 1.8 110 4 210 6.8 310 11 410 17 510 33

011 3.6 111 6.1 211 9.2 311 14 411 21 511 46012 5.5 112 8.1 212 12 312 17 412 26 512 64013 7.3 113 10 213 14 313 20 413 31 513 84

014 9.1 114 12 214 17 314 23 414 36 514 110

015 11 115 14 215 19 315 27 415 42 515 130020 3.7 120 6.1 220 9.3 320 14 420 22 520 49021 5.5 121 8.2 221 12 321 17 421 26 521 70

022 7.4 122 10 222 14 322 20 422 32 522 95023 9.2 123 12 223 17 323 24 423 38 523 120

024 11 124 15 224 19 324 27 424 44 524 150025 13 125 17 225 22 325 31 425 50 525 180030 5.6 130 8.3 230 12 330 17 430 27 530 79

031 7.4 131 10 231 14 331 21 431 33 531 110

032 9.3 132 13 232 17 332 24 432 39 532 140033 11 133 15 233 20 333 28 433 45 533 180

034 13 134 17 234 22 334 31 434 52 534 210

035 15 135 19 235 25 335 35 435 59 535 250040 7.5 140 11 240 15 340 21 440 34 540 130

041 9.4 141 13 241 17 341 24 441 40 541 170042 11 142 15 242 20 342 28 442 47 542 220043 13 143 17 243 23 343 32 443 54 543 280044 15 144 19 244 25 344 36 444 62 544 350045 17 145 22 245 28 345 40 445 69 545 440050 9.4 150 13 250 17 350 25 450 41 550 240051 11 151 15 251 20 351 29 451 48 551 350052 13 151 17 252 17 352 32 452 56 552 540053 15 153 19 253 26 353 37 453 64 553 920054 17 154 22 254 29 354 41 454 72 554 1600

055 19 155 24 255 32 355 45 455 81 555 >1600


TABLE A-1












10 1 0.1 0.01 0.001

a. 5/5** 5/5 2/5 5-5-2 540 1 5.4

b. 5/5 5/5 2/5 5-5-2 540 10 54

c. 5/5 5/5 2/5 2/5 5-2-2 95 100 95

d. 5/5 5/5 2/5 0/5 5-2-0 49 100 49

e. 2/5 2/5 1/5 0/5 2-2-1 12 10 1.2

f. 5/5 2/5 1/5 1/5*** 5-2-2 95 10 9.5

g. 5/5 5/5 5/5 5/5 5-5-5 >1600 100 >1600

* Dilutions to be used are shaded gray. *** No. of positive tubes/No. of tubes inoculated.

Reference1. Official Method MFO - 15 Health Protection Branch - Ottawa.

TABLE A-2

10 1 0.1 0.01 0.001

Reference




Microbiological Examination of Foods for Aerobic Colony Counts (ACC)IntroductionThis method shall be used for the determination of total aerobic bacteria (Aerobic Colony Count or ACC) in pasteurized milk and cream and other non-fermented dairy products, frozen dairy products (ice cream and ice milk), butter, milk powders and other dairy product powders, and milk for manufacture into dairy products to determine compliance with the requirements of the Food and Drug Regulations.

Materiall Peptone Water diluent (0.1%) (AM1079, AM5079) l Plate Count agar (PC) (AM1081, AM5081)l Control cultures: use ATCC cultures or equivalent l Sterile 1N NaOH and 1N HCl

Equipmentl Thermometer, calibrated and certifiedl Incubator, 35°Cl Autoclavel Laminar Air Flowl Stomacher, blender or equivalentl pH meter or paper capable of distinguishing 0.3 to 0.5 pH units within a

range of 5.0 to 8.0

Introduction

Material

Equipment

ProcedureEach sample unit shall be analyzed individually. The tests shall be carried out on the sample in accordance with the following instructions:

1) Collection of Samplesl A sample, consisting of 20 sample units drawn at random from each lot,

shall be taken.l Each sample unit shall consist of at least 100 g.l Collect original unopened containers wherever possible.l More than one sample unit may be collected from large institutional or

bulk containers when the total number of sample units required exceeds the number of containers in the lot. A sample unit will consist of more than one container when the lot consists of containers smaller than 100 g (e.g., four 25 g containers in each sample unit)

l Employ aseptic techniques in collecting the sample units when sampling from bulk.

2) Handling of Sampling Unitsl Keep frozen sample units frozen in the laboratory before analyzing

them. Refrigerate shelf-stable products (0-5°C).l Analyze the sample units as soon as possible after they have been


Procedure






3) Preparation of Dilutionsl Prepare sterile 0.1% peptone water diluent.l Combine portions from several locations within the frozen sample unit to

ensure a representative analytical unit of 11 (10) g.NOTE: Weight or volume in brackets indicates alternate procedure for making dilutions.

l Prepare a 1:10 dilution of the sample by aseptically adding the analytical unit into 99(90) mL of the peptone water diluent.

l Mix the 1:10 dilution by shaking the dilution bottle 25 times in a 30 cm arc in approximately 7 sec.

l Check the pH of the dilution. If the pH is outside the range of 5.5 to 7.6, adjust to 7.0, with either sterile 1N NaOH or 1N HCl.

l Prepare succeeding dilutions as required to determine the ACC in the sample by transferring 11(10) mL of the previous dilution into 99(90) mL of 0.1% peptone water diluent. Shake all dilutions (as in step 6.2.4, above) immediately prior to making transfers to ensure uniform distribution of the microorganisms present. Use a separate sterile pipette for making each transfer.

l Make similar dilutions using the control culture.

4) Determination of the ACCThe medium used is PC agar prepared for making pour plates.

I) Analysis : Do the following for dilutions prepared from the food and the control culture.l Agitate each dilution bottle to resuspend material. l Without delay, pipette 1 mL of each prepared dilution into each of

two appropriately marked Petri plates using a sterile pipette for each transfer.

l Pour 12-15 mL of tempered agar (40-45°C) into each plate and mix contents by rotating and tilting.

l Allow the agar to solidify.



I) Analysis :

l Plates shall be poured not later than 15 min. after preparation of dilutions.

l Incubate plates in an inverted position at 35°C for 48 ± 2hr. Incubate an uninoculated plate, as a negative control.

l Avoid crowding or excessive stacking of plates in order to permit rapid equilibration of plates with incubator temperature.

l Count colonies promptly after the incubation period. l Select for counting those plates containing 20-200 colonies,

including pinpoint colonies. If counts do not fall within this range, select plates that have counts nearest to this range.

II) Recording Resultsa. Calculate average count (arithmetic mean) of duplicate plates,

following the examples in any microbiological reference, such as Chapter 6 of "Standard Methods for the Examination of Dairy Products" (7.1).

b. When reporting results round-off the counts to two significant figures, and record only the first two left hand digits (e.g., record 2,850 as 2,900).

c. If the lowest dilution plated shows no colonies, report the count as the product of 0.5 the dilution factor preceded by a "less than" (<) sign.

d. To compute the ACC, use the formula: N = A, where N is the number of colonies per g of product, A is the average count, and D is the respective dilution factor.

InterpretationThe tolerances as specified hereafter and representing the maximum count of total aerobic bacteria (Aerobic Colony Count) in the foods listed in Table I shall be applied in determining whether the tested lot of the product complies with the listed Sections of the Food and Drug Regulations.

II) Recording Results

Interpretation

Table I - Criteria and sampling plans for ACC in specific foodsTable I -

DeterminationDetermination FoodFood

ACC

Criteria: No. ofsample units

(n)

Criteria: No. ofsample units

(n)

Criteria: AcceptanceNumber

(c)

Criteria: AcceptanceNumber

(c)

Criteria: Concentrationof Microorganisms

(m)

Criteria: Concentrationof Microorganisms

(m)

Criteria: Maximum Concentration

of Microorganisms(M)

Criteria: Maximum Concentration

of Microorganisms(M)

5 2 10,000 25,000Pasteurized milk and cream and other non-fermented dairy products

Frozen dairy products (ice cream and ice milk), butter, milk powders and other dairy product powders

Milk for manufacture into dairy products

5 2 10,000 50,000

5 0 50,000




Lot: A batch or production unit which may be identified by the same code. When there is no code identification, a lot may be considered as (a) that quantity of product produced under essentially the same conditions, at the same establishment and representing no more than one day's production; or (b) the quantity of the same variety of product from one and the same manufacturer available for sampling at a fixed location.

n: The number of sample units usually but not always selected at random from a lot and examined in order to satisfy the requirements of a particular acceptance plan used. This is the sample.

m: The numerical value of "m" represents acceptable concentrations of microorganisms usually per g or mL. In a 2-class plan, "m" separates sample units of acceptable and defective quality; in a 3-class plan, "m" separates sample units of acceptable quality from those of marginally acceptable quality. The "m" values listed in the table are based on levels achievable under GMP.

M: (Only in a 3-class plan), the numerical value of "M" represents unacceptable concentrations of microorganisms, usually per g or mL, that indicate a (potential) health or injury hazard, imminent spoilage or gross insanitation; "M" separates sample units of marginally acceptable quality from those of defective quality. A value determined for any one sample unit of a sample that is greater than that of "M" renders the pertaining lot unacceptable.

c: The maximum allowable number of marginally acceptable sample units. "c" is the acceptance number of a plan. When this number is exceeded, the lot becomes unacceptable. The method described above, being comprised of 6 pages and identified as MFO-24 and dated July 2002, is hereby designated the "Official Method" referred to in Sections B.08.011 of the Regulations of the Food and Drug Act for the microbiological examination of pasteurized milk and cream and other non-fermented dairy products, frozen dairy products (ice cream and ice milk), butter, milk powders and other dairy product powders, and milk for manufacture into dairy products.

Reference1. Official Method MFO-24.Reference

Enumeration of Coliforms, Faecal Coliforms and E. Coli in Water in Sealed Containers and Prepackaged Ice Using the MPN Method

IntroductionThe Most Probable Number (MPN) method is applicable to the enumeration of coliforms, faecal coliforms and aerogenic Escherichia coli in water in sealed containers (including mineral and spring water) and prepackaged ice in accordance with the Regulations of the Food and Drugs Act.

The MPN procedure involves a multiple tube fermentation technique where three or more decimal dilutions of the sample are inoculated into tubes of broth medium and incubated at a specific temperature and for a specific time. The method is progressive; i.e., first determining the presence of coliforms in the tubes, then determining if these tubes also contain faecal coliforms, and then confirming whether E. coli is present. Based on the number of tubes indicating the presence / absence of the three groups of organisms, the most probable number present can be estimated from a standard statistical MPN table.

This method has been shown to produce satisfactory results with naturally-contaminated and artifically-contaminated water in sealed containers (including mineral and spring water) and prepackaged ice.

The presence of coliforms, faecal coliforms and aerogenic E. coli in water may be determined by means of the MPN procedure. Briefly, this method involves serially diluting out the target organisms in the sample, in 5-replicate aliquots, to

Introduction extinction. The probable level of the target organisms is then statistically estimated from an MPN table.

Gas production is used as an indication of ability to ferment lactose from LST broth (presumptive coliform test); gas production from BGLB broth is considered

o confirmation of coliform presence; gas production at 45 C from EC broth is used as confirmation of faecal coliform presence; and appearance of typical nucleated, dark-centred colonies with or without metallic sheen when positive EC broths are streaked onto L-EMB agar are indicative of E. coli. The typical colonies on L-EMB agar must be confirmed by further biochemical tests to pr ove the presence of E. coli.

Materiall Peptone Water (0.1%) (AM1079, AM5079)l Lauryl Tryptose broth (AM1053, AM5053)l Brilliant Green Bile broth 2% (AM1020, AM5020)l Escherichia coli (EC) broth (AM1039, AM5039)l Levine's Eosin Methylene Blue (L-EMB) agar (AM1040, AM5040)l Endo Agar (AM1041, AM5041)l MacConkey agar with crystal violet, Nacl and 0.15% Bile Salt. (AM1059,

AM5059)l Nutrient Agar (NA) (AM1074, AM5074)

Material




l EMB Agar (AM10391/AM50391)l Simmon’s Citrate Agar (AM1090, AM5090)l E.coli idenification kit (20796001)l Kovac’s Indole Reagent (20700040)l Methyl Red Solution (20710040)l VP Reagent (20680020)l Control cultures (use ATCC cultures or equivalent):

Positive control(s): E. coli that is known to produce gas at 45°C and is capable of fermenting lactose to produce typical reactions on L-EMB agar; if using EC-MUG, a strain that is known to produce -glucuronidase

EMB / IMViC negative control: Enterobacter aerogenes or an equivalent gram negative rod that does not produce "positive" reactions on EMB and is indole-negative, methyl red -negative, Voges-Proskauer-positive, and citrate positive.

MPN broths negative control: Salmonella berta or an equivalent gram negative rod that is gas-negative in MPN broths and in the secondary EC broth.

NOTE: Some strains of E. aerogenes will give false-positive reactions in the MPN broths (LT, BGB and EC broths) by producing a small gas bubble. Therefore, use S. berta or an equivalent culture for these broths and E. aerogenes or an equivalent culture for EMB agar and IMViC tests.

Equipmentl Covered water baths, with circulating system to maintain temperature of

45°C. Water level should be above the medium in immersed tubes.l Thermometer, calibrated and traceablel Incubator, 35°Cl Autoclavel Laminar Air Flow

Procedure Each sample unit must be analyzed individually. Carry out the test in accordance with the following instructions:

1) Collection of Samplesl Each sample unit shall consist of at least 500 ml or g.l Do not allow sample units of prepackaged ice to thaw during shipment.

2) Handling of Sample Unitsi) Water in sealed containers

Equipment

Procedure



l Do not store sample units for more than 24 hrs before analysis. oStore under refrigeration (0-5 C) conditions.

ii) Prepackaged Icel If sample units are prepackaged in leakproof containers, thaw

othem in the containers under refrigeration (0-5 C) prior to analysis. l If sample units are not in leakproof containers, transfer the ice

aseptically to sterile plastic bags or other suitable sterile containers. Seal containers to prevent contamination, and thaw sample units under refrigeration (0-5°C). Do not store thawed sample units for more than 6 h before analysis.

3) Preparation for Analysisl Keep ready sterile peptone water.l Clean the surface of the working area with a suitable disinfectant.l Arrange LT broth tubes in rows of five and mark them identifying the

sample unit and the dilution to be inoculated.

4) Preparation of Sample and Initial Set-up l Inoculate each of separate sets of five tubes of LT broth with each dilution

to be tested, according to the scheme, as follows.Inoculate each of the five tubes of 10 mL double strength LT broth (first row) with 10 mL of the undiluted water sample. Inoculate each of the five tubes of 10 mL single strength LT broth (second row) with 1 mL undiluted water. Inoculate each of the five tubes of 10 mL single strength LT broth (third row) with 0.1 mL of undiluted water.

l Follow incubation of LT and confirmation steps for coliforms, faecal coliforms and E. coli as required, and record results as MPN per 100 mL of water, by following the instructions in Section 5 (Calculation of MPN).

5) Incubation of LT Brothl In order to verify growth conditions in the elevated temperature water

baths, inoculate one LT broth tube with the MPN broths positive control and one LT broth tube with the MPN negative control, for each bath used. Transfer into all media used at different stages of the procedure. Set up an uninoculated tube of medium corresponding to each step in the procedure as a media control.


l Incubate the inoculated LT broth tubes at 35 °C for 24 ± 2 hrs. Examine for gas formation (gas formation may be either a gas bubble or effervescence), record results, and begin the confirmed coliform, faecal coliform, and E. coli tests for all gas-positive tubes, as required.

l Incubate gas-negative tubes for an additional 24 ± 2 hrs, examine, record the number of additional gas-positive tubes, add to the result obtained in earlier step and begin the confirmed coliform, faecal coliform and E. coli tests for the additional gas-positive tubes, as required.

3) Preparation for Analysis

4) Preparation of Sample and Initial Set-up

5) Incubation of LT Broth





6) Confirmation Steps for Determination of Coliformsl Use BGB broth 2% dispensed in 10 mL volumes in tubes containing gas

vials.l Shake or rotate the positive LT broth tubes to mix the contents and

transfer one loopful from each tube to a tube of BGB broth (avoid transferring pellicle). Sterile wood applicator sticks or other appropriate transfer devices may be used for making the transfers.


0l Incubate the inoculated BGB broth tubes at 35 C for 24 ± 2 hrs.

Examine for gas formation (gas bubble or effervescence) and record results.

l Incubate gas-negative tubes for an additional 24 ± 2 hrs, re-examine, record the numbers of additional gas-positive tubes and add to the result obtained above.

l Formation of gas during 48 ± 4 hrs incubation constitutes a positive confirmed test.

l Compute the MPN of Confirmed Coliforms per 100 mL of water by referring Table III.

7) Confirmation Steps for Determination of Faecal Coliformsl Use EC broth, dispensed in 10 mL volumes in tubes containing gas vials.l Shake or rotate the positive LT broth tubes to mix the contents and

transfer one loopful from each tube to a tube of EC broth (avoid transferring pellicles). Sterile wood applicator sticks or other appropriate transfer devices may be used for making the transfers.


l Incubate the inoculated EC broth tubes in a water bath at 45 °C for 24 ± 2 hrs. Maintain the water level in the bath at least 1 cm above the level of the medium in the tubes.

l Examine for gas production (gas bubble or effervescence), record results, and begin on the same day E. coli identification for all gas-positive tubes.

l Incubate gas-negative tubes for an additional 24 ± 2 hrs, examine, record the number of additional gas-positive tubes and begin the E. coli identification for the additional gas-positive tubes.


l Formation of gas during 48 ± 4 hrs incubation constitutes a positive faecal coliform test.

l Compute faecal coliform MPN per 100 mL of water following the instructions.

6) Confirmation Steps for Determination of Coliforms

7) Confirmation Steps for Determination of Faecal Coliforms

8) Confirmation Steps for Identification of E. colil Gently shake each gas-positive EC broth tube and streak a loopful of the

culture onto a L-EMB or Endo agar plate.0

l Incubate the plates at 35 C for 18 to 24 hrs, and examine for typical non-mucoid, nucleated, dark-centred colonies with or without a metallic sheen which are indicative of E. coli.

l If the colonies are well isolated on L-EMB or Endo agar plates, pick one typical colony and streak onto a non-selective agar such as NA (EMB or MacConkey can also be used). Circle one other typical colony on EMB before storing the plates at 4°C, to be taken to non-selective media if the initial colony does not confirm as E. coli. Incubate at 35°C for 18-24 hrs. Use these cultures for further confirmation.

l If the colonies are not well isolated on L-EMB or Endo agar plates, pick two typical colonies and re-streak onto EMB to obtain discrete colonies. Select one well isolated typical colony from one of the EMB plates and streak onto a non-selective agar such as NA (EMB or MacConkey can also be used). Refrigerate the second EMB plate in case it is needed at a later point. Incubate as above and use these cultures for further confirmation.

l GIMViCFrom the streaked plates (NA, EMB or MacConkey), transfer inoculum into a separate tube of each of EC broth and the IMViC media. Collectively they are referred to as the GIMViC media, where the "G"-medium is the secondary EC broth, "I" -medium is Tryptone broth, "M"- and "V"-medium is Buffered Glucose broth, and "C"-medium is Simmon's Citrate agar. If GIMViC tests are not carried out within 96 h of inoculating the non-selective agar, prepare fresh plates or slants prior to inoculating the GIMViC media.

Inoculate one tube of each of the GIMViC media for each of the isolates to be identified. Inoculate IMViC positive and negative controls into each of the IMViC media and MPN positive and negative controls into secondary EC broth.

Alternatively, IMViC tests may be done using any commercially available testing system.

Gas Production at 45.0 °C (G)Incubate inoculated tubes of G medium (EC broth) in a water bath at 45.0 °C for 24 ± 2 hrs. Examine for gas production. If no gas has been produced, incubate for an additional 24 ± 2 hrs and re-examine. Record results.

Indole (I)Incubate inoculated tubes of Tryptone or tryptophane broth at 35 °C for 24 ± 2 hrs. Add indole reagent (commercially available) to each tube following manufacturer's instructions. A dark red colour in the alcohol

8) Confirmation Steps for Identification of E. coli

GIMViC

Gas Production at 45.0 °C (G)

Indole (I)




layer indicates a positive test. An orange colour probably indicates the presence of skatole and may be reported as a ± reaction. A yellow colour would be considered negative.

Methyl-Red (MR) and Voges-Proskauer (VP) Tests (MVi)Inoculate 2 tubes of Buffered Glucose broth and incubate at 35 °C for 48 ± 2 hrs. Use MR and VP reagents (commercially available) following manufacturer's instructions. The test is VP-positive if an eosin pink colour develops after 5-10 minutes. The MR test is positive if a red colour

Methyl-Red (MR) and Voges-Proskauer (VP) Tests (MVi)

develops, and negative if a yellow colour develops.

Simmon's Citrate Test (C)In inoculating the slants of SC agar, use a straight needle and apply a light inoculum. Use care to avoid transferring nutrients together with inoculum as these nutrients (carbon) could lead to the development of a

0blue colour and an incorrect interpretation. Incubate the slants at 35 C for 48 ± 2 hrs and observe for growth. Visible growth (positive reaction) is usually accompanied by a change of colour from green to deep blue.

Simmon's Citrate Test (C)

Table I GIMViC Pattern for E. coli Biotypes

0Gas at 45 C Indole Methyl Red Voges Proskauer Citrate

G I M V C

Type I + + + - -

Type II (Anaerogenic) - - + - -

Table I

Gas at 45 C Indole Methyl Red Voges Proskauer Citrate0

Table II** Differentiation of Commonly Occuring Coliforms

0Gas in EC broth at 45 C Indole test Methyl Red test Voges Proskauer test Growth on Citrate

Escherichia coli

Type I (typical) + + + - -

Type II (Anaerogenic) - - + - -

Intermediates

Type I - - + -* +

Type II - + + -* +

Enterobacter aerogenes

Type I - - - + +

Type II - + - + +

Enterobacter cloacae

Irregular - - + +

Type II + - + - -

Type VI + - - + +

Irregular other types Reactions variable

* Weak positive reactions are occasionally found.

Table II**

Gas in EC broth at 45 C Indole test Methyl Red test Voges Proskauer test Growth on Citrate0

InterpretationThe characteristic GIMViC reaction pattern for E. coli is given in Table I. If necessary, commonly occurring coliforms may be differentiated by using the data in Table II. If characteristic reactions for E. coli are obtained with GIMViC tests, the other isolate need not be further tested. However, if the

Interpretation first isolate gives a non-characteristic IMViC pattern, test the second isolate for its GIMViC reaction pattern. Repeat confirmation steps. If both isolates fail to produce IMViC reaction patterns characteristic of E. coli, then E. coli is considered to be absent from the tube of primary EC broth from which the isolates originated.




5) Calculation of MPNsTable A-III shows the most probable numbers of coliforms per 100 ml or g corresponding to the number of gas-positive tubes in the coliform test.

Table A-III has been adapted from a conversion table prepared for the analysis of drinking water where 10, 1.0 and 0.1 ml of the water under test are used as test portions. The table is equally appropriate if 10, 1.0, and 0.1 g or ml of a food constitute the test portions in the tubes. When other sized portions of the test material are placed in the tubes, the MPN values obtained from Table A-III has to be multiplied by an appropriate number, to correct for the actual amount of test material in the tubes, and also to obtain the MPN per g or ml as is usually done for foods, rather than per 100 ml (g), for which the values are given in the table. The volume of diluent added to the tubes (and which accompanies the sample) is ignored when calculating the MPN.

ExampleThe following inoculated tubes give a positive reading:(1) 5 tubes with 10 ml of 1:10 dilution of test material - all 5 are positive(2) 5 tubes with 1 ml of 1:10 dilution of test material - 1 is positive (3) 5 tubes with 1 ml of 1:100 dilution of test material - none are positive

The quantities (test portions) in each of the five tubes of the three dilution series represent 1, 0.1 and 0.01 g or ml test material respectively.

According to Table A-III, a reading of 5-1-0 gives a value of 33 if 10, 1 and 0.1 g or ml respectively are used. However, since only 1/10 of these amounts were actually used in the analysis, the value of 33 obtained from Table A-III must be multiplied by 10 giving 33 x 10 = 330 organisms per 100 g or ml of test material. Since the results have to be expressed per g or ml, the MPN value of 330 must be divided by 100. When higher dilutions are used, the same procedure is followed, but the

5) Calculation of MPNs

Example

multiplier (dilution factor) is enlarged to relate the amount of test material actually present to the values given for 10, 1.0 and 0.1 g or ml in Table A-III.

Dilution factor = Reciprocal of the dilution of the analytical unit.


To determine which consecutive dilutions to use, refer to the combinations shown below: (See also Table A-IV). l If only 3 dilutions are made, use the results for those 3 dilutions to compute

the MPN. Examples a and b. l If more than 3 dilutions are employed, use the results of only 3 consecutive

dilutions. Select the highest dilution (last dilution, i.e. dilution with the smallest quantity of product) in which all 5 tubes are positive and 2 succeeding higher dilutions. Examples c and d.


l If a positive tube occurs in the dilution higher than the 3 chosen to rule, the number of such positive tubes should be added to those of the next lower dilution. Example f.

l If the tubes of all sets of a dilution series are positive, choose the 3 highest dilutions of the series and indicate by a "greater than" symbol (>) that the MPN is greater than the one calculated. Example g.

Refer to Table A-III and look up the value which corresponds to the number of positive tubes obtained. MPN/100 ml (g) = No. of Microorganisms x dilution factor of

(Table A-III) middle set of tubes.

TABLE A-IIIMost Probable Number (MPN) of Bacteria Per 100 g (mL) of Test Material Using 5 Tubes 10,1 and 0.1 mL or g of Test Material


000 <1.8 100 2 200 4.5 300 7.8 400 13 500 23

001 1.8 101 4 201 6.8 301 11 401 17 501 31002 3.6 102 6 202 9.1 302 13 402 21 502 43003 5.4 103 8 203 12 303 16 403 25 503 58004 7.2 104 10 204 14 304 20 404 30 504 76005 9 105 12 205 16 305 23 405 36 505 95

010 1.8 110 4 210 6.8 310 11 410 17 510 33

011 3.6 111 6.1 211 9.2 311 14 411 21 511 46012 5.5 112 8.1 212 12 312 17 412 26 512 64013 7.3 113 10 213 14 313 20 413 31 513 84

TABLE A-III











014 9.1 114 12 214 17 314 23 414 36 514 110

015 11 115 14 215 19 315 27 415 42 515 130020 3.7 120 6.1 220 9.3 320 14 420 22 520 49021 5.5 121 8.2 221 12 321 17 421 26 521 70

022 7.4 122 10 222 14 322 20 422 32 522 95023 9.2 123 12 223 17 323 24 423 38 523 120

024 11 124 15 224 19 324 27 424 44 524 150025 13 125 17 225 22 325 31 425 50 525 180030 5.6 130 8.3 230 12 330 17 430 27 530 79

031 7.4 131 10 231 14 331 21 431 33 531 110

032 9.3 132 13 232 17 332 24 432 39 532 140033 11 133 15 233 20 333 28 433 45 533 180

034 13 134 17 234 22 334 31 434 52 534 210

035 15 135 19 235 25 335 35 435 59 535 250040 7.5 140 11 240 15 340 21 440 34 540 130

041 9.4 141 13 241 17 341 24 441 40 541 170042 11 142 15 242 20 342 28 442 47 542 220043 13 143 17 243 23 343 32 443 54 543 280044 15 144 19 244 25 344 36 444 62 544 350045 17 145 22 245 28 345 40 445 69 545 440050 9.4 150 13 250 17 350 25 450 41 550 240051 11 151 15 251 20 351 29 451 48 551 350052 13 151 17 252 17 352 32 452 56 552 540053 15 153 19 253 26 353 37 453 64 553 920054 17 154 22 254 29 354 41 454 72 554 1600

055 19 155 24 255 32 355 45 455 81 555 >1600




Dilution factor of middledilution

Dilution factor of middledilution


TABLE A-IVDilutions to be used and calculations of MPN per g or mL of test material

10 1 0.1 0.01 0.001

a. 5/5** 5/5 2/5 5-5-2 540 1 5.4

b. 5/5 5/5 2/5 5-5-2 540 10 54

c. 5/5 5/5 2/5 2/5 5-2-2 95 100 95

d. 5/5 5/5 2/5 0/5 5-2-0 49 100 49

e. 2/5 2/5 1/5 0/5 2-2-1 12 10 1.2

f. 5/5 2/5 1/5 1/5*** 5-2-2 95 10 9.5

g. 5/5 5/5 5/5 5/5 5-5-5 >1600 100 >1600

* Dilutions to be used are shaded gray. *** No. of positive tubes/No. of tubes inoculated.

TABLE A-IV

10 1 0.1 0.01 0.001




Interpretation/Limits The tolerance as specified hereafter and representing the maximum probable incidence of coliform bacteria (Coliforms) and E. coli in water in sealed containers and prepackaged ice, shall be applied in determining whether the tested lot of the product complies with the Regulations of the Food and Drugs Act.

Coliform bacteria (Coliforms) shall be considered absent in a lot when not more than one of the 5 sample units taken from the lot is positive for Coliforms, and the

Interpretation/Limits MPN for that sample unit is not more than 10 Coliforms per 100 mL of water in sealed containers and prepackaged ice.

E. coli shall be considered absent in a lot when none of the five sample units taken from the lot is positive.

The tolerances are summarized in the following table:

Table V: Criteria and sampling plans for Coliforms and E.coliTable V:


Coliforms

No. ofSample Units

(n)

No. ofSample Units

(n)

AcceptanceNumber

(c)

AcceptanceNumber

(c)

Concentrationof Microorganisms

(m)


(m)

Maximum Concentrationof Microorganisms

(M)


(M)

5 1 0* 10Water in sealed containers and Prepackaged ice

CriteriaCriteria

E. coli 5 0 0* -Water in sealed containers and Prepackaged ice

* means less than the Lower Limit of Detection (LLD) of the method, and, in reality, means <1.8 for MPN methods and <1 for membrane filter methods.

Lot: A batch or production unit which may be identified by the same code. When there is no code identification, a lot may be considered as (a) that quantity of product produced under essentially the same conditions, at the same establishment and representing no more than one day's production; or (b) the quantity of the same variety of product from one and the same manufacturer available for sampling at a fixed location.


m: The numerical value of "m" represents acceptable concentrations of microorganisms, usually per g or mL. In a 2-class plan (as for Salmonella), "m" separates sample units of acceptable and defective quality; in a 3-class plan, "m" separates sample units of acceptable quality from those of marginally acceptable quality. The "m" values listed in the table are based on levels achievable under GMP.


c: The maximum allowable number of marginally acceptable sample units. "c" is the acceptance number of a plan. When this number is exceeded, the lot becomes unacceptable.

References 1. American Public Health Association. 2001. Compendium of Methods for the

Microbiological Examination of Foods; Fourth Edition. Frances P. Downes and Keith Ito (eds.). American Public Health Association, Washington, D.C.

2. American Public Health Association. 1992. Standard Methods for the Examination of Dairy Products; 16th Edition. R.T. Marshall (ed.). American Public Health Association Inc., Washington, D.C.

3. International Commission on Microbiological Specifications for Foods. 1978. Microorganisms in Foods; Their Significance and Method of Enumeration; Second Edition; University of Toronto Press.

4. McGuire, O.E. 1964. Wood Applicators for the Confirmatory Test in Bacteriological Analysis of Water. Public Health Reports. 79: 812-814.

5. Powers, E.M. and T.G. Latt. 1977. Simplified 48-Hour IMViC Test: an Agar Plate Method. Appl. Environ. Microbiol. 34: 274-279.

6. American Public Health Associsation. 1998. Standard Methods for the Examination of Water and Waste Water; Twentieth Edition. Lenore.S. Clesceri, A.E. Greenberg and A.D. Eaton, (eds.). American Public Health Association, Inc., Washington, D.C.

7. Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition. L.C. Parks (editor). CRC Press Inc.

8. Official Method MFO-18 Health Protection Branch - Ottawa.

References




Proposed Official Method: Enumeration of Pseudomonas aeruginosa in Prepackaged Ice and Water in Sealed Containers by the Hydrophobic Grid-Membrane Filter (HGMF) Technique

IntroductionThe method shall be used for the enumeration of in water in sealed containers (including mineral and spring water) to determine compliance with the requirements of the Regulations of the Food and Drugs Act.

This method has been shown to produce satisfactory results with naturally-contaminated and artifically-contaminated water in sealed containers (including mineral and spring water) in HPFB studies.

The hydrophobic grid-membrane filter (HGMF) method uses commercially available media. A single dilution accommodates a wide range of contamination levels. Counting precision may be better than on conventional membrane filters. Further identification can be done using commercially available identification kits and biochemical reactions.

Materials l Pseudomonas Isolation agar (AM108417, AM508417)l Cetrimide agar (AM1022, AM5022) l Pseudomonas agar for Pyocyanin (AM108414, AM508414)l Pseudomonas agar for Fluorescein (AM108411, AM508411)l King's Medium A Base (AM50491)l King's Medium B Base (AM50492)l Soyabean Casein Digest Agar (AM1091, AM5091)l Skim Milk agar (optional) (AM10901, AM50901)l Oxidase Reagent (20690040)l Control cultures (use ATCC cultures or equivalent): positive control: P.

aeruginosa (ATCC 27853) negative control: e.g., Escherichia coli (ATCC 25922)

Equipmentl HGMF (1600 grid-cell, 0.45 µm pore size; available as ISO-GRID Membrane

Filters from Oxoid Ltd.) or equivalent.l Membrane filter forceps .l Spreadfilter with funnel or ISO-GRID filtration unit l Incubator capable of maintaining 35°C. A second incubator may be required

depending on the selective agar chosen; e. g., 42°C. l Manual or automated colony counting devices (optional).

Procedure Analyze each sample unit individually. Carry out the test in accordance with the following instructions: Each Sample Unit shall contain at least 500 ml.

Introduction

Materials

Equipment

Procedure

1) Handling of Sample Units l In the laboratory prior to analysis, keep sample units refrigerated (0-

o5 C). l Analyze sample units as soon as possible after receipt in the laboratory.

2) Preparation for Analysis l Have ready sterile Soyabean Casein Digest Agar plates (SCDA) and the 2

selective agars of choice. l Clean the surface of the working area with a suitable disinfectant. l Clearly label duplicate SCDA and selective agars with appropriate

identifying information. l The HGMF will allow counts to be made from suspensions containing up

to 5,000 organisms/mL. There normally should be no need to prepare dilutions.

3) Filtration l Agitate each sample to resuspend material that may have settled out. l Handle HGMF with sterile membrane filter forceps. l Following the manufacturer's instructions for use of the filtration

apparatus, pour 100 mL of the analytical unit into it. Open the filter valve until all liquid has passed through and aseptically remove the HGMF. Do in duplicate.

l It is recommended that a suspension of a known P. aeruginosa culture be used as a positive control and organisms of another genus, (e.g., Escherichia coli) be used as the negative control. Make suitable dilutions of the positive and negative controls and filter as above.

l Follow the manufacturer's instructions for cleaning the filtration apparatus.

4) Plating and Incubation 7.4.1 Transfer each HGMF to the surface of a SCDA plate, rolling it onto the agar to avoid trapping air bubbles. Incubate plates in stacks of not more than three, at 35°C for 4 hrs. After 4 hrs, transfer the two HGMF to the two selective agars of choice. Incubate the plates at 35°C for 22-24 hrs.

a) Typical colonies mPAC - Pseudomonas Isolation Agar – Pseudomonas aeruginosa are green or blue green.Cetrimide - P. aeruginosa are blue, blue-green or yellow-green pigmented colonies. Pseudomonas Agar for Pyocyanin - Pseudomonas spp. are blue-green or brown pigmented colonies and may fluorescence. Pseudomonas Agar for Fluorecein - Pseudomonas spp. are blue-green


2) Preparation for Analysis

3) Filtration

4) Plating and Incubation

mPAC

Cetrimide

Pseudomonas Agar for Pyocyanin

Pseudomonas Agar for Fluorecein




or brown pigmented colonies and fluorescence.

King's A Medium - Pseudomonas spp. are yellow-green pigmented colonies and may fluorescence.

King's B Medium - Pseudomonas spp. are yellow-green pigmented colonies and may fluorescence.

5) Counting HGMF l Follow manufacturer's instructions for the use of automated or manual

counters. Typical colonies in the HGMF grid-cells are presumptive P.aeruginosa. Confirm as below.

6) Confirmation l If HGMFs do not contain growth, record test results as <1/100 mL. l If growth is present, inoculate growth from five of the presumptive

P.aeruginosa grid-cells (or typical colonies) from each of the selective media onto SCDA and incubate for 18-24 h at 35°C.

l If the plates are overgrown, pick and re-streak the colonies onto selective

King's A Medium

King's B Medium

5) Counting HGMF

6) Confirmation

agar, incubate at 35°C for 22-24 hours and if presumptive colonies are isolated follow instructions below.

l Perform the oxidase test on suspected yellow to green pigmented colonies.

l Perform a Gram stain on oxidase positive strains. Confirm only Gram-negative, oxidase positive colonies.

l Confirm up to five Gram-negative, oxidase positive colonies from each selective agar using biochemical tests or rapid identification kits.

7) Interpretation l The tolerances as specified hereafter and representing the maximum

total P.aeruginosa in water in sealed containers (including mineral and spring water), shall be applied in determining whether the tested lot of the product complies with the Regulations of the Food and Drugs Act.

l Pseudomonas aeruginosa shall be considered absent in a lot when not more than one of the 5 sample units taken from the lot is positive for Pseudomonas aeruginosa, and the count for that sample unit is not more than 2 Pseudomonas aeruginosa per 100 mL of water in sealed containers.

7) Interpretation

Table I: Criteria and sampling plans for Pseudomonas aeruginosaTable I:


Pseudomonas aeruginosa

No. ofSample Units

(n)

No. ofSample Units

(n)

AcceptanceNumber

(c)

AcceptanceNumber

(c)


(m)


(m)


(M)


(M)

5 1 0* 2Water in sealed containers

CriteriaCriteria

The tolerance is summarized in the following table: * means less than the Lower Limit of Detection (LLD) of the method, and, in reality, means <1 for membrane filter methods. Lot: A batch or production unit which may be identified by the same code. When

there is no code identification, a lot may be considered as (a) that quantity of product produced under essentially the same conditions, at the same establishment and representing no more than one day's production; or (b) the quantity of the same variety of product from one and the same manufacturer available for sampling at a fixed location.


m: The numerical value of "m" represents acceptable concentrations of microorganisms, usually per g or mL. In a 2-class plan (as for Salmonella), "m" separates sample units of acceptable and defective quality; in a 3-class plan, "m" separates sample units of acceptable quality from those of marginally acceptable quality. The "m" values listed in the table are based on levels achievable under GMP.


c: The maximum allowable number of marginally acceptable sample units. "c" is the acceptance number of a plan. When this number is exceeded, the lot becomes unacceptable.

References 1. Atlas, R.M. 1997 . Second edition. L.C. Parks (editor). CRC Press Inc. 2. Sharpe, A.N. and P.I. Peterkin. 1988. . Research Studies Press. Taunton,

Somerset, U.K. 3. Warburton, D.W., B. Bowen and A. Konkle. 1994. The survival and recovery

of and its effect upon salmonellae in water: methodology to test bottled water in Canada. Can. J. Microbiol. 40:987-992.

4. Proposed Official Method MFO-195. Health Products & Food Branch - Ottawa.

References




Detection of Food Poisoning by Clostridium botulinum and its Toxins

IntroductionClostridium botulinum is an anaerobic, gram-positive, spore-forming, rod-shaped bacterium that produces the most potent poison known, a protein of characteristic neurotoxicity. Severe food poisoning, botulism, results from the consumption of botulinum toxin produced in food in which this organism has grown.

Antigenic types of C. botulinum are defined by the toxins they produce and each antigenic toxin type is neutralized completely by the homologous antitoxin only and cross-neutralization by heterologous antitoxin types is absent or minimal. The seven recognized C. botulinum types are designated A, B, C, D, E, F and G. Five of these apparently produce only one type of toxin but all are given type designations corresponding to the sole or major type of toxin produced. Type C produces predominately C1 toxin with lesser amounts of C2 or only C2, and type D produces predominately type D toxin along with smaller amounts of C2 toxin. The production of more than one type of toxin may be a more common phenomenon than previously realized. There is a slight reciprocal cross-neutralization of types E and F, and recently strains of C. botulinum have been identified which produce a mixture of toxins consisting mostly of the dominant type of toxin plus small amounts of different types of toxins, e.g. Ab, Af and Bf.

Botulism as a type of food poisoning in humans is rare. But the case fatality remains relatively high. In the United States from 1899 through 1995, 1,026 outbreaks of botulism were recorded. These involved 2,444 cases and caused 1,040 deaths.

Of outbreaks in which the toxin type was determined, 446 were due to type A, 117 to type B, 149 to type E and 6 to type F . The implicated foods of two outbreaks contained both A and B toxins. The limited number of reports of C or D toxin to be the causative agent of human botulism have not received general acceptance. All except types F and G, about which little is known are important causes of animal botulism. Human botulism also may result from wounds infected with C. botulinum in which the organism grows and elaborates its toxin, but this is a rare occurrence. Gastrointestinal symptoms are usually absent in such cases.

Infant botulism, first recognized as a distinct clinical entity in 1976, is now the most common form of human botulism reported in the United States. It affects infants 12 months of age or less, with 95% of cases occurring between 2 and 26 weeks of life. This form of botulism results from growth and neurotoxin production by C. botulinum within the intestinal tract of infants rather then from the ingestion of performed toxin. It is usually caused by C. botulinum types A or B, but a few cases have been reported as being caused by other toxin types. Infant botulism has been diagnosed in most states of the United States and in every populated continent except Africa. As of January 1994, 1,270 hospitalized cases of infant botulism had been reported world wide. Of these 1,206 (95.0%) occurred in the United States.

Introduction In infant botulism, constipation almost always precedes the characteristic signs of neuromuscular paralysis by a few days or weeks. Illness varies greatly in severity. Some infants show only mild weakness, lethargy are reduced feeding and do not require hospitalization. Severe symptoms, such as generalized muscle weakness, weakened suck and swallowing, faint cry and diminished gag reflex with a pooling of oral secretions are more commonly reported. Generalized muscle weakness and loss of head control reaches such a degree that some infants appear “floopy’. Approximately half of all (hospitalized) patients require endotracheal intubation and mechanical breathing support at some point during their hospital stay. High quality intensive care is responsible for a case-fatality ratio that is <1%. The administration of the recently developed human Botulism Immune Globulin (BIG) shortens the mean hospital stay by over 50%.

Definitive diagnosis of infant botulism depends on the demonstration of toxin and / or organisms in the feces. C. botulinum has been recovered from patients’ feces for as long as 5 months after onset of illness and toxin for as long as 4 months. Although testing of serum is very useful for establishing the diagnosis of botulism in adults, it is of limited value in infants. In a recently reported study, toxin was found in the serum of only 9 of 67 (13%) culture-positive infant botulism patients. Honey is a common source of C. botulinum spores implicated in infant botulism. In studies of honey, up to 13% of the test samples contained low numbers of C. botulinum spores. For this reason the U.S FDA, the U.S CDC, the American Academy of Pediatrics, as well as several honey industry groups have all recommended that honey not be fed to infants under the age of 1 year.

The organisms C. botulinum is distributed widely in soils and in the sediments of oceans and lakes, so that there is a diversity of sources for food contamination. The finding of type E organisms in aquatic environments by many investigators correlates with the tracing of most cases of type E botulism to contaminated fish or other sea foods. Types A and B are most commonly encountered terrestrially, and the primary vehicles of botulism caused by these two types, are foods commonly contaminated with soils. In the US these foods have been primarily home-canned vegetables, but in Europe home canned meat products also have been important vehicles for intoxication.

C. botulinum Isolates are further subdivided into four distinct groups by properties other than toxin antigenic types, with each group composed of strains of different types but having similar cultural and physiological characteristics. Group I includes all strains of type A plus the proteolytic strains of types B and F; Group II includes all strains of type E plus the non-proteolytic strains of types B and F; Group III includes all strains of types C and D; Group IV contains the proteolytic but non-saccharolytic type G. A tentative fifth group containing strains of C.butyricum and C.baratii that produce botulinum toxins type E and F, respectively is under consideration. All type A strains and some B and F strains are proteolytic, whereas all type E strains and




the remaining B and F strains are non-proteolytic. Type G shows slow proteolytic activity. Optimum temperature for growth and toxin production of the proteolytic

0strains is close to 35 C, while that for nonproteolytic strains is approximately 26 0to 28 C. Non- proteolytic types B, E, and F can produce toxin at refrigeration

0temperature (3 to 4 C). Toxins of the nonproteolytic strains do not manifest maximum potemtial toxicity until activated with trypsin; toxins of the proteolytic strains generally occur in fully, or close to fully, activated form. These and other differences are important in epidemiological and laboratory investigations of botulism outbreaks.

Measures to prevent botulism from foods include reduction of the microbial contamination level, acidification, reduction of moisture level, and destruction of all C. botulinum spores in the food. Heat is the most common method of destruction; properly processed canned foods do not contain viable C. botulinum. The greater incidence of botulism from home-canned foods than from commercially canned foods undoubtedly reflects the commercial canner’s greater awareness and better control of the destructive heating required.

A certain food may contain viable C. botulinum spores and still not cause botulism. As long as the organisms do not grow, toxin is not synthesized. Many foods satisfy the nutritional requirements of C. botulinum, but not all provide the necessary anaerobic conditions. Many canned foods and many meat and fish products meet both nutritional and anaerobic requirements for growth of C. botulinum. However, growth in otherwise suitable foods is prevented if the product, naturally or by design, is acidic (pH = less than 4.6), has low water activity (A < 0.9), a high w

sodium chloride concentration (5% for non-proteolytics; 10% for proteolytics), an inhibitory sodium nitrite concentration (100-200 ppm used in conjunction with other inhibitors), or two or more of these factors in combination. Unless

0temperature is strictly controlled and kept below 3 C, refrigeration will not prevent growth and toxin formation by non-proteolytic strains. Moreover, the usual vehicles of botulism are foods processed to prevent spoilage and are not normally refrigerated. Botulinum toxin is heat-liable, therefore, botulism can be prevented by thoroughly heating all processed foods prior to consumption, e.g. boiling for 10 minutes before serving.

Material l Cooked Meat Medium (AM1030, AM5030)l Gel Phosphate Bufferl Tryptone Glucose Extract Broth (AM1102, AM5102)l Gram Stain Kit (20750020)l Anaerobic Agar (AM1000, AM5000)l Egg Yolk Emulsion (AS010)

Equipmentl Incubatorl Autoclave

Material

Equipment

l Laminar Air Flowl Refrigeratorl Sterile Motor and Pestle

ProcedureSamplingI) Foods

Suspect foods should be refrigerated until tested, except for unopened canned foods, which, unless badly swollen and in danger or bursting, need not be refrigerated.

Before testing, record such identifying data as product, manufacturer or home canner, source, type of container and size, labeling, manufacturer’s batch, lot, or production code, and condition container.

Clean and mark the container with laboratory identification number or symbol, disinfect with an alcohol disinfectant, and open aseptically for sampling. Carefully avoid aerosols.

Check for normal ingredients which, by their presence or concentration in the product, could be lethal for mice by the intraperitoneal route of administration, e.g., a high salt concentration (anchovies) or high sugar concentration (heavy syrups).

II) Clinical SpecimensAll clinical specimens should be collected as soon as botulism is suspected and before botulinum antitoxin is administered.

1. Serum (Adult patient). Collect enough blood (approx. 50 ml) to provide at least 20 ml of serum for toxin neutralization tests. Allow blood to clot in the refrigerator; centrifuge, and remove serum to a sterile vial or test

0tube with a leak proof cap. Examine immediately or refrigerate at 4 C. Examination of post-treatment (8-12 hrs) serum is also helpful to evaluate antitoxin therapy.

2. Feces. Collect 25 g of the patient’s feces (as much as possible from infants) in a sterile, unbreakable, leak-proof container. Preferably, use a screw-cap wide-mouth plastic bottle. Seal caps with waterproof tape.

0Cardboard containers are not acceptable. Refrigerate specimens at 4 C until examined. A “soap-suds’ enema should not be given before the feces are collected, since the soap may inactivate the toxin. If a passed stool is not available, the physician should be requested to obtain a specimen using a sterile water enema.

3. Miscellaneous clinical specimens. Specimens, such as vomits, gastric aspirates, cerebrospinal fluid or tissues obtained at autopsy should be

0colleted in sterile leak-proof containers and refrigerated at 4 C .

ProcedureSamplingI) Foods

II) Clinical Specimens




Procedure for identifying viable C. botulinumPreparation of various samples1) Opening Canned Foods

Sanitize the uncoded end of the can with an effective alcohol disinfectant. Allow a contact time of a few minutes, then remove the disinfectant and wipe the sanitized area with a sterile, dry towel. If the can is swollen, position the can so that the side seam is away from the analyst. A container with buckled ends should be chilled before opening and flamed with extreme caution to avoid bursting the can. Flame- sterilize the sanitized can end with a Bunsen burner by directing the flame down onto the can until the visible moisture film evaporates. Avoid excessive flaming which may cause scorching and blackening of the inside enamel coating. Remove a disc of metal from the center area of the flamed end with a sterile, sanitary can opener. Remove a disc about 5 cm in diameter, except from cans which are 202 diameter where a 3-cm disc is satisfactory.

2) Solid FoodsTransfer solid foods with little or no liquid aseptically to a sterile mortar. Add an equal amount of gel phosphate buffer solution and grind with a sterile pestle in preparation of media inoculation. Alternatively, small pieces of the product may be inoculated directly into the enrichment broth using sterile forceps, or placed in a stomacher bag and pummeled with an equal volume of gel phosphate buffer.

3) Liquid FoodsInoculate liquid foods directly into the culture media, using sterile pipettes.

4) Reserve Test SampleAfter culturing, aseptically remove a reserve portion of the test sample to a sterile jar for later tests.

5) Examining a Product for Appearance and OdorVisually, note any evidence of decomposition, but do not taste the product under any circumstance. Record observations.

6) Preparation of Enrichment Culturesl Before inoculation, heat broth media in flowing steam or boiling water

for 15 mins. After heating, cool rapidly to room temperature in cold water without agitation.

l Inoculation of enrichment media. Inoculate 1 to 2 g of solid or macerated food or 1 to 2 ml of liquid food per 15 ml of enrichment broth. Inoculate duplicate tubes of CMM and duplicate tubes of Tryptone Glucose Extract Broth.

0 0 0l Incubate the CMM at 35 C and the TGE broth at 26 C to 28 C.l After 5 days of incubation, examine each culture for turbidity, the

production of gas, and the digestion of the meat particles. Note the odor. Examine the cultures microscopically by a wet mount preparation under

Procedure for identifying viable C. botulinumPreparation of various samples

high-power, phase-contrast microscope, or a stained smear (Gram stain, crystal violet, methylene blue) with bright field illumination. Observe the morphology of the bacteria and note the presence of clostridial cells, the occurrence and relative extent of sporulation, and the location of spores within the cells.

l Test each enrichment culture for toxin.l For pure culture isolation, gently mix and transfer 2 ml of the culture at

peak sporulation to a sterile screw-cap tube and refrigerate.

7) Isolation of Pure Culturesl The possibility of isolating C. botulinum in pure culture from a mixed

flora in the enrichment culture is greatly improved if spores are present. To 1 or 2 ml of enrichment culture showing some sporulated cells (or the retained test sample) add an equal volume of absolute ethanol in a sterile screw-cap tube. Mix the alcohol with the culture and incubate the mixture at room temperature for 1 hour, after which, this mixture is plated as described below.

l An alternative procedure to the alcohol method is to heat 1 to 2 ml of the enrichment culture sufficiently to destroy the vegetative cells but not the spores of C. botulinum present. A simple distinction, with some exception, is based on the origin of the product investigated, if the product is of aquatic origin the organism would be of the nonproteolytic types, for products of terrestrial origin the organism would be of the proteolytic types. For a nonproteolytic type, do not use heat, for a

0proteolytic type, heat at 80 C for 10 to 15 min.l Streak the alcohol-or heat-treated culture on petri dishes containing

anaerobic agar in order to obtain well separated colonies. Dilution of the culture may be necessary before plating in order to select well-isolated colonies. To prevent spreading of the colonies, the plates must be well dried. Alternatively, untreated enrichment cultures or stools can be streaked directly to isolate C. botulinum on one of the selective differential plating media recently developed.

0l Incubate the inoculated plates anaerobically at 35 C for about 48 hrs. A

Case anaerobic jar, Gaspak or other anaerobic systems are adequate to obtain anaerobiosis.

l After anaerobic incubation, select about 10 well-separated and typical colonies from each plate. Colonies of C. botulinum may be raised or flat, smooth or rough; they commonly show spreading and have an irregular edge. On Anaerobic medium containing egg yolk the colonies usually exhibit a surface iridescence when examined by oblique light. This luster zone is due to lipase activity and is often referred to as a pearly layer; it usually extends beyond but follows the irregular contour of the colony. Besides the pearly layer, colonies of C. botulinum types C, D and E are ordinarily surrounded by a zone (2 to 4 mm) of a yellow precipitate caused by lecithinase activity. Colonies of types A and B generally show a smaller zone of precipitation. However, considerable difficulty in selecting toxin producing colonies may be experienced since certain




other members of the genus Clostridium, which do not elaborate toxin, produce colonies with characteristics similar to those of C. botulinum.

l Inoculate each colony into a tube of sterile broth with a sterile transfer loop. For nonproteolytic C. botulinum, inoculate broth; for the proteolytic types, inoculate CMM. For orientation concerning the type of organism apply the same reasoning suggested above. Incubate the inoculated tubes for 5 days as previously described; then test for toxin. Restreak the toxin producing culture in duplicate on egg yolk agar medium. Incubate

0one plate anaerobically and the other aerobically at 35 C for 48 hours. If colonies typical of C. botulinum are found only on the plate incubated anaerobically, and no growth is found on the plate incubated aerobically, the culture may be considered pure. Failure to isolate C. botulinum from at least one of the colonies selected means that its presence in the mixed flora of the enrichment culture is at very low level. Sometimes the numbers can be increased enough to permit isolation by repeated serial transfers through additional enrichment steps.

l Store the pure culture in the sporulated state under refrigeration.

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22. Silas, J.C.,J.A, Carpenter, M.K. Hamdy, and M.A.Harrison.1958. Selective and differential medium for detecting Clostridium botulinum. Appl.Environ. Microbiol.50:1110-1111.

23. Smith, L.D. S., and H.Sugiyama. 1988. Botulism: The organism, its toxin, the nddisease, 2 ed. Charles C.Thomas, Sprongfield, Ill.

nd24. Stumbo, C.R.1973. Thermobacteriology in food processing, 2 ed. Academic Press, New York.

25. Szabo, E.A.,J.M.Pemberton, and P.M.Desmarchelier.1993. Detection of the genes encoding botulinum neurotoxin types A to E by the polymerase chain




reaction. Appl. Environ. Microbiol. 59:3011-3020.26. Takeshi, K.,Y.Fujinaga, K.Inoue, H.Nakajima, K.Oguma, T.Ueno,

H.Sunagawa, and T.Ohyama.1996. Simple method for detection of Clostridium botulinum type A to F neurotoxin genes by polymerase chain reaction. Microbiol. Immunol. 40(1):5-11.

27. U.S.HEW/PHS/CDC.1979. Botulism in the United States 1899-1977. Publ.

No.(CDC) 74-8279, U.S. Dep. Of Health, Ed., and Welfare, Public Health Serv., Washington, D.C.plus the incidence reports presented at the annual meetings of the Interagency Botulism Research Coordianting Committee (BRCC).

th 28. U.S.HHS/PHS/FDA.1995. Bacteriological analytical manual, 8 ed. Assoc. of Off. Anal. Chem., Gaithersburg, Md.


Soil MicrobiologyHand Book of


Isolation of Dermatophytes, other Fungi and Yeasts from Soil

Procedurel Prepare serial dilutions of soil sample using sterile D.W.l Inoculate each specimen in duplicate.l Incubate one set of media aerobically at 22-25°C and the other set at 35°C

for 5-30 days. Loosen the caps of tubes and ensure adequate moisture for the plates to compensate for loss of water vapour.

l Examine every 2-4 days. l Describe each specific type of colony morphology and sub-culture to

appropriate media for further identification tests.l Prepare positive control by inoculating Candida albicans ATCC 10231 and

Aspergillus niger ATCC 16404 and incubate at 22-25°C for 5 days.l Prepare negative control by uninoculating medium.

Interpretation

Positive control Expected results

Candida albicans ATCC 10231 Good growth; cream coloniesAspergillus niger ATCC 16404 White mycelium; black spores

Negative control

Uninoculated medium No change

Some of the pathogenic fungi may produce infective spores which are are easily dispersed into the laboratory. Such organisms should be examined only within a protective cabinet.

References1. Carlier Gwendoline I. M. (1948) Brit. J. Derm. Syph. 60. 61-63. 2. Hodges R. S. (1928) Arch. Derm. Syph., New York, 18. 852.3. Sabouraud R. (1910) 'Les Teignes', Masson, Paris.4. Georg Lucille K., Ajello L. and Papageorge Calomira (1954) . J. Lab. Clin.

Med. 44. 422-428.5. Ajello Libero (1957) . J. Chron. Dis. 5. 545-551.6. Williams Smith H. and Jones J. E. T. (1963) J. Path. Bact. 86. 387-412.7. Hantschke D. (1968) Mykosen. 11. 113-115.8. Dolan C. T. (1971) Appl. Microbiol. 21. 195-197.9. Pagano J., Levin J. G. and Trejo W. (1957-58) Antibiotics Annual 1957-58,

137-143.10. Kutscher A. H., Seguin L., Zegarelli E. V., Rankow R. M., Mercadante J. and

Piro J. D. (1959a) J. Invest. Derm. 33. 41-47.11. Kutscher A. H., Seguin L., Zegarelli E. V., Rankow R. M., Campbell J. B. and

Mercadante J. (1959b) Antibiotics and Chemotherapy 9. 649-659.12. Sinski J. T. (1960) J. Invest Dermat. 35. 131-133.13. Ridley M. F. (1960) Australian J. Dermat. 5. 209-213.14. McDonough E. S., Georg L. K., Ajello L. and Brinkman S. (1960) Mycopath.

Mycol. Appl. 13. 113-116.

DO NOT SEAL THE PLATES.

Procedure

DO NOT SEAL THE PLATES.

Interpretation

Positive control Expected results

References

IntroductionCarlier showed that the medium gives reliable results with Microsporum audouini, Microsporum canis, Trichophyton mentagrophytes, Trichophyton flavum, Trichophyton rubrum and Candida albicans. Sabouraud Dextrose Agar may be

2used in place of the Standard American medium of Hodges . The fungi maintain their typical cultural appearance and thus may be readily identified according to

3the standard macroscopic characters described by Sabouraud . The medium is often used with antibiotics for the isolation of pathogenic fungi from

4material containing large numbers of other fungi or bacteria. Georg et al. aseptically added 0.5g cycloheximide, 20,000 units penicillin and 40,000 units streptomycin to each litre of autoclaved, cooled medium. Cryptococcus neoformans, Aspergillus fumigatus and Allescheria boydii are sensitive to cycloheximide; Actinomyces bovis and Nocardia asteroides are sensitive to penicillin and streptomycin. Alternatively, one may add 0.4g chloramphenicol and 0.05g cycloheximide to each litre of reconstituted medium before autoclaving

5(Ajello ). The same micro-organisms are sensitive to this new combination - see Dermasel Selective Supplement SR0075.

6Williams Smith & Jones employed Sabouraud Dextrose Agar, containing 20,000 units penicillin and 0.04g neomycin per litre, for the count of yeasts in the

7alimentary tract of the pig. Hantschke used colistin, novobiocin and cycloheximide 8to isolate Candida albicans. Dolan used gentamicin, chloramphenicol and

cycloheximide for the selective isolation of pathogenic fungi.Sabouraud Dextrose Agar may also be used as the basis of a Pagano-Levin

9medium for the isolation of Candida albicans. 0.1g of triphenyltetrazolium chloride (as a filter sterilised solution) is added to each litre of autoclaved molten medium cooled to 55°C. The medium is usually made inhibitory to most non-pathogenic fungi and bacteria by the addition of antibiotics as above. After incubation for 3 days at 25°C, Candida albicans colonies are unpigmented or pale pink whilst other Candida species and other fungi form deeper pink or red colonies. The test is adequate for screening purposes but other diagnostic criteria should also

10,11,12,13be utilised for the identification of Candida albicans .

Materiall Sabouraud Dextrose Agar (AM1087/AM5087)l Distilled waterl Candida albicans ATCC 10231l Aspergillus niger ATCC 16404

Equipmentl Autoclavel Incubatorl Laminar Air Flow

Introduction

Material

Equipment




IntroductionCzapek Dox Agar is a medium containing sodium nitrate as the sole source of nitrogen, it is one of the most useful solid media for the general cultivation of fungi.

1Dawson employed Czapek Dox Agar (Modified) in her technique for the identification of Candida albicans by chlamydospore formation in primary cultures.Identification was usually possible within 24 hours.

2Smith cited the following recommendations for the use of Czapek Dox Agar for 3 4taxonomic studies: by Thom and Church for Aspergillus; by Thom and by Raper

5 6and Thom for Penicillium; and by Wakesman for actinomycetes.

Materiall Czapek Dox Agar (AM10301/AM50301)l Distilled waterl Aspergillus niger ATCC 9642l Candida albicans ATCC 10231


ProcedureGeneral Cultivationl To avoid excessive condensation cool the molten medium to 50°C before

pouring approximately 12ml into each 9cm diameter Petri dish. Store the poured plates in an inverted position and inoculate using a needle or wire, with the plate still inverted in order to avoid scattering stray fungal spores over the surface of the medium. Time and temperature of incubation vary considerably according to the species being cultivated. As a general guide, incubate for 1-2 weeks at 25°C. Most Penicillium species have an optimum growth temperature between 20° and 25°C, whilst many Aspergillus species grow best at about 30°C. However, different fungi grow over a wide range of

2temperatures; Aspergillus fumigatus grows well at 50°C (Smith ) and 7,8Cladosporium herbarum will grow on meat at -6°C .

l Prepare positive control by inoculating Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404 and incubate at 22-25°C for 5 days.

l Prepare negative control by uninoculating medium.

Introduction

Material

Equipment

ProcedureGeneral Cultivation

Cultivation of those fungi and bacteria which are able to utilise sodium nitrate as the sole source of nitrogen

Identificationof Candida albicans1. Using an inoculating needle (previously flamed, cooled and rubbed against

the swab) cut across and through the medium in a Czapek Dox Agar plate to the base of the petri dish. With the same needle, raise the medium along the whole of one side of the cut - so that the inoculum is spread between the agar and the base of the dish.

2. Incubate the inoculated plates for 24 hours at 28°C.3. Using a low-power objective, microscopically examine the unopened plates

for chlamydospores through the base of each dish. Alternatively, remove the tops of the dishes, and examine through the top of the medium.

4. If no chlamydospores are seen, incubate for a further 24 hours and re-examine.

Interpretation

Positive control Expected Results

Aspergillus niger ATCC 9642 White / yellow mycelium, black sporesCandida albicans ATCC 10231 Good growth, cream coloured colonies

Negative Control

Uninoculated medium No change

References1. Dawson Christine O. (1962) Saboutaudia 1. 214-219.2. Smith G. (1960) An Introduction to Industrial Mycology 5th ed., Edward

Arnold Ltd. London.3. Thom C. and Church M. B. (1926) ‘The Aspergilli’ Williams and Wilkins Co.

Baltimore.4. Thom C. (1930) ‘The Aspergilli’ Williams and Wilkins Co. Baltimore.5. Raper K. B. and Thom C. (1949) ‘Manual of the Penicillia’ Williams and

Wilkins Co. Baltimore.6. Wakesman S. A. (1931) ‘Principles of Soil Microbiology’ Bailliere Tindall and

Cox, London.7. Brooks F. T. and Kidd M. N. (1921) Specia Report No. 6, Food Invest Board,

DSIR, London. 8. Brooks F. T. and Handsford C. G. (1922) Trans. Brit. Mycol. Soc. 8. 113-142.

Identificationof Candida albicans

Interpretation

Positive control Expected Results

Negative Control

References




IntroductionCorn Meal Agar is a well established mycological medium which is a suitable substrate for chlamydospore production by and the maintenance of fungal stock cultures. When grown on this medium, microscopic examination of shows the characteristic chlamydospore production which is an accepted criterion for the

1identification of this species. Prospero and Reyes investigated the use of corn meal agar, soil extract agar, and purified polysaccharide medium for the morphological identification of Candida albicans. Out of 290 yeast colonies isolated on Sabouraud agar, corn meal agar stimulated the production of chlamydospores in 149 colonies (51%), soil extract agar in 103 (36%) and purified polysaccharide medium in 94 (32%). The addition of `Tween 80’ (e.g. 1%) to Corn Meal Agar

2,3,4,5,6greatly enhances the development of chlamydospores on the medium . 7Mackenzie found that all 163 isolates of obtained from laboratories in the United

8Kingdom produced chlamydospores on Corn Meal Agar but Dawson using only 27 isolates of Candida albicans, found that Czapek Dox Agar and rice infusion agar were slightly superior for chlamydospore production. Corn meal agar is a nutritionally impoverished medium and so may be employed for the maintenance of stock cultures of fungi, especially the black-pigmented varieties. The addition of glucose (0.2g% w/v) to Corn Meal Agar will enhance the chromogenesis of some

9species of Trichophyton e.g. Trichophyton rubrum .

Material1) Corn Meal Agar (AM10301 / AM50301).2) 0.001% Trypan Blue Solution.3) Candida albicans ATCC 10231.4) Candida krusei ATCC 6258.5) Sabouraud Dextrose Agar (AM1087 / AM5087).

Equipmentl Autoclavel Incubatorl Laminar Air flow

ProcedureA single Petri dish containing Corn Meal Agar may be used to identify four or five different colonies of Candida grown on Sabouraud Dextrose Agar. Using a straight wire, pick a colony off the surface of the latter medium and make a deep cut in the

Introduction

Material

Equipment

Procedure

Observation for chlamydospore production by Candida albicans and for the maintenance of fungal stock cultures

Corn Meal Agar (i.e. a horizontal furrow). Repeat for each colony. Place a flamed sterile coverslip over the line of inoculum. After incubation for 24 to 48 hours at 22°C, the streaks are examined microscopically, through the cover slip, using a low power objective. Along such streaks, Candida albicans produces mycelium-bearing ball-like clusters of budding cells and the characteristic thick-walled round

9chlamydospores . The addition of 0.001g % w/v Trypan blue to Corn Meal Agar provides a contrasting background for the observation of characteristic

10morphological features of yeast cultures .

Prepare positive control by inoculating Candida albicans ATCC 10231 and prepare negative control by inoculationg Candida krusei ATCC 6258 and incubationg for 24 to 48 hours at 22°C.

Interpretation

Positive Control Expected Results

Chlamydospore Production

Candida albicans ATCC 10231 Good growth; white colonies and chlamydospores.

Negative Control

Candida krusei ATCC 6258 Good growth; white / cream colonies, no chlamydospores.

References1. Prospero Magdalene T. and Reyes A. C. (1955) Acta Mel. Phillipina 12(2).

69-74.2. Rosenthal S. A. and Furnari D. (1958) J. Invest. Derm. 31. 251-253.3. Kelly J. P. and Funigiello (1959) J. Lab. Clin. Med. 53. 807-809.4. Walker L. and Huppert M. (1959) Am. J. Clin. Path. 31. 551-558.5. Walker L., Huppert M. and Woods A. (1960). Am. J. Clin. Path. 33. 190-194.6. Gordon M.A. and Little G. N. (1962-63) . Sabouraudia 2. 171-175.7. Mackenzie D. W. R. (1962) J. Clin. Path. 15(6). 563-565.8. Dawson Christine O. (1962) Sabouraudia 1 (4). 214-219.9. Conant N. F., Smith D. T., Baker R. D., Callaway J. L. and Martin D. S. (1971)

Manual of Clinical Mycology, 3rd Edn. W. B. Saunders, Philadelphia, USA.10. Washington J. A. (1981) Laboratory Procedures in Clinical Microbiology,

Springer-Verlag, New York, USA.

Interpretation

Positive Control Expected Results

Chlamydospore Production

Negative Control

References

Isolation of Nitrogen Fixing Bacteria from SoilIntroductionNitrogen fixing bacteria are capable of taking gaseous nitrogen and combining it with hydrogen to make ammonia. The plant can use fixed nitrogen for growth.

Introduction Thus nitrogen fixing bacteria increases the soil productivity. To isolate these bacteria a medium free from nitrogen is required. Jensen's Medium and Jensen's Broth are based on same Principle for isolation of Nitrogen Fixing Bacteria.




Materiall Jensen's Broth (AM50485)l Jensen's Medium (AM10486/AM50486)l Rhizobium leguminosarum ATCC 10004l Rhixobium meliloti ATCC 9930


Procedurel Prepare 20 ml sterile Jensen’s Broth tubes.l Prepare sterile Jensen’s Medium Plates.l Add aseptically 1-2 gms of soil sample into Jensen’s broth.

Material

Equipment

Procedure

0l Incubate tubes for 7 days at 30 C.l Check for growth and then transfer a loopful of enriched medium on Jensen’s

Medium (Agar).0

l Incubate plates for 7 days at 30 C.l Observe characteristic colonies.l Prepare positive control by inoculating Rhizobium leguminosarum ATCC

01004 and Rhixobium meliloti ATCC 9930 cultures and incubating at 30 C for 7 days.

InterpretationPositive control will give luxuriant growth.

Reference1. Vincent J. M. 1970, A Manual for Practical Study of Root Nodule Bacteria P-

194 IBP Hand Book, Backwell Scientific Publication Oxford.

Interpretation

Reference

Detection and Isolation of Phosphate Solubilizing Microoraganisms from soil

IntroductionBoth inorganic and organic phosphates exits in soil. The organic phosphorus containing compounds are derived from plants and microorganisms. Phosphate dissolving soil microorganisms play part in correcting phosphorous balance of crop plants. Many fungi and bacteria are potential solubilizers of bound phosphates. So they are used in phosphate dissolving culture preparations (Biofertilizers).

Materiall Pikovaskaya's Agar (AM508092/AM108092)l Distilled waterl Aspergillus niger ATCC (16404)l Pseudomonas aeruginosa ATCC (27853)l Bacillus Subtilis ATCC (6633)

Equipmentl Incubatorl Autoclavel Laminar Air Flow

Procedurel Make serial dilutions of soil sample in distilled water.l Select appropriate dilution and plate out 1ml on sterile Pikovaskaya's Agar

plate by spread plate technique.

Introduction

Material

Equipment

Procedure

0l Incubate plates at 35 C for 48 hours.l Observe characteristic colonies and compare with growth response of positive

control.For Positive control streak following cultures on Pikovaskaya's Agar and incubate at

035 C for 48 hours.Aspergillus niger ATCC (16404)Pseudomonas aeruginosa ATCC (27853)Bacillus Subtilis ATCC (6633)

InterpretationPositive control culture response

Organism (ATCC) Growth Phosphate Solubilization

Aspergillus niger (16404) Luxuriant +

Pseudomonas aeruginosa (27853) Luxuriant +

Bacillus Subtilis (6633) Good (+)

Key + = Clear zone surrounding the colony.(+) = Moderate clear zone surrounding the colony.

References1. Sundara Rao W.V.B. And Sinha M. K. 1963, Ind. J. Agri Science 33.272.2. N. S. Subba Rao, 1977 soil microorganisms and plant growth Oxford and IBH

Publishing Co., New Delhi.

Interpretation

Organism (ATCC) Growth Phosphate Solubilization

References


Microbiological Methods in Beverage IndustryHand Book of


Detection and Enumeration of respiratory deficient yeast cells used in beverage

IntroductionRespiratory deficient yeast cells are those cells that lack certain respiratory enzymes. They often appear as small colonies on solid medium. During fermentation it shows poor growth and also produce unfavorable flavour. This causes less yield and also change in flavour. Hence these type of yeast cells cannot be used for production.

The test for determining respiratory deficient cells base on the fact that if respiratory enzymes are present in yeast cells, it will reduce colourless tetrazolium salt to insoluble form triphenyl formant and produce a red pigmented colonies, while respiratory deficient cells due to lack of respiratory enzyme cannot reduce tetrazolium salt and form colourless colonies.

Materiall Wort Agar (AM1111, AM5111)l TTC Solution 1% (AS0271)l Agar Powder (AB001)

Equipmentl Autoclave

Introduction

Material

Equipment

l Incubatorl Oven

Procedurel Grow yeast cells on Wort Agar by spread plate technique or pour plate

technique.l Prepare (100 ml) 1.5% sterile agar.

0l Cool it to 45-50 C and add 1 vial (10 ml) of 1% TTC solution. Mix it uniformly.l Pour 15-20 ml of TTC Agar on yeast growth obtained on Wort Agar. Yeast

colonies to be completely over laid.l Let the plate to be solidified.

0l Incubate at 30 C for 1 hour.l Count white colonies and red colonies.

InterpretationNormal respiratory sufficient colonies turn pink to red while respiratory deficient colonies remain white.

Procedure

Interpretation

Maintenance of 'Yeast' cultures which are used as 'Seed' for fermentation

IntroductionAll beverage products are manufactured by fermentation process using specific yeast or mould strains. These microbial strains contribute specific flavour and taste to the product. Therefore it is very critical to maintain purity of the culture during preservation.

Materiall Wort Agar (AM1111, AM5111)

Equipment l Laminar Air Flow Unit

Introduction

Material

Equipment

l Autoclavel Incubatorl Refrigerator

Procedurel Prepare 10 ml slants of Wort Agar Mediuml Subculture from ‘Mother culture’ or previously preserved slant on ‘Wort Agar’

slants.0

l Incubate at 30 C for 40-48 hours.0

l Confirm the specific morphological character of culture and store at 2-8 C.

Procedure

To check sterility of Beverage products (Beer, wine etc.) by microfilteration technique

IntroductionAll beverage products are manufactured by fermentation process using specific yeast or mould strains. These microbial strains contributed specific flavour and taste to the product. Therefore it is very important that finished product should be free from unwanted microbes otherwise these microbes can alter the original flavour and taste of the product.

Introduction To ensure the absence of unwanted microflora during manufacturing process at various stages product is monitored by performing sterility test by microfiltration technique.

Materiall WL Nutrient Agar (AM51092)l Raka- Ray Agar Base (AM10844)

Material


Microbiological Methods in Beverage IndustryHand Book of


l WL Differential Agar (AM1109/ AM5109)l Lysine Medium Base (AM10577/ AM50577)l Lactobacillus MRS Agar (AM1051/ AM5051)l 0.22 micron Filter paper.

Equipmentl Membrane holderl Autoclavel Laminar Air Flowl Incubatorl Oil free vacuum pump.

Procedurel Collect the samples in sterile bottles.l Conduct the experiment inside Laminar Air Flow unit.

Equipment

Procedure

l Arrange sterile membrane holder stand inside LAF unit.l Moist the filter membrane by filtering sterile distilled water.l Filter 100 ml sample through membrane with the aid of oil free vacuum

pump.l After filtration remove the membrane aseptically using sterile forcep and

mount it on sterile agar plate.l Use respective type of agar medium as per requirement.l Incubate the plates at respective temperature and period as mentioned in

table I.l At the end of incubation period check for specific colonies.

Interpretation / LimitAgar plates should not exhibit typical standard colonies, Refer Table I for colonical characteristics.

Interpretation / Limit

Name of Media WL Nutrient AgarName of Media WL Nutrient AgarIncubation

0Temp. 30 CIncubation Temp. 30 C0

Incubation Period 40-48 hours

Incubation Period 40-48 hours

Cultural CharacteristcsCultural Characteristcs

Table 1Table 1

1) S.cerevisiae-> Good - Luxuraint2) E.coli -> Fair - Good3) L.Fermentum-> Fair - Good4) P.mirabilis-> Fair - Good

WL Differential Agar 035 C 48 hours

Lysine Medium Base 025 C 7 Days

1) E.coli-> Luxuriant2) L.fermentum-> Luxuriant3) P.mirabilis-> Luxuriant4) S.cerevisiae-> Inhibited

Lactobacillus MRS Agar

Raka-Ray Agar Base

035 C

025-30 C

18-24 hours

4-7 days

1) Pichia fermentans- > Luxuriant2) L.plantarum-> Luxuriant

1) L.fermentum-> Good2) E.coli-> Inhibited


Medical MicrobiologyHand Book of


Introduction

The clinician uses the laboratory to assist in diagnosis and management of the patient due to following reasons.

l To confirm a clinical impression or establish a diagnosis.l To rule out a diagnosis.l To monitor therapy.l To establish prognosis.l To Screen for or detect disease.

Clinical microbiologists are part of the health care team and serve an important role in the diagnosis, management and prevention of infections in patients.

The microbiologists can also assist the patient care facility in saving money by helping l To identify and control nosocomial (Hospital acquired) pathogens and to

quickly track organisms resistant to antimicrobial agents.l To coordinate the antimicrobial agents tested in the laboratory with those

selected by the institutions pharmacy and therapeutics committee.

This manual presents an overview of the products and techniques (Simplified testing procedure) involved in diagnosis of infectious diseases.

Types of samples received in the clinical laboratoryl BLOOD CULTURE.l UPPER RESPIRATORY TRACT INFECTIONS, INCLUDING THROAT, NOSE, EAR

AND EYE INFECTIONS.l LOWER RESPIRATORY TRACT INFECTIONS.l WOUND, SKIN, AND DEEP SEPSIS.l GENITAL TRACT INFECTIONS.l URINARY TRACT INFECTIONS.l MENINGITIS.l GASTROINTESTINAL INFECTIONS.l PYREXIA OF UNKNOWN ORIGIN.

Blood Culture

INTRODUCTIONCulture of patients blood is one of the most important investigations in clinical microbiology, for the demonstration of septicaemia or bacteriemia indicates that there is an immediate threat to the patients life and an urgent need for appropriate antibiotic therapy.

Blood culture is requested mainly in two clinical situations1. Where the possibility of septiciaemia or bacteriaemia is suggested by the

presence of fever, shock or other symptoms occurring in association with a known or suspected local infection such as sepsis in a surgical wound, puerperal sepsis, pneumonia, meningitis, osteomyelitis or endocarditis; and

2. Where it is one of the procedures required in the investigation of a fever difficult to diagnose, because of the absence of signs of a specific infection or local infective lesion that is a pyrexia of unknown origin (PUO).

REQUIRED REAGENTS/MEDIASl BHI supplemented with 0.05% SPS (20660700 / 20660200)l Glucose broth supplemented with 0.05% SPS (20661700 / 20662700) l Soyabean casein digest broth supplemented with 0.05% SPS (20670700 /

20670200)l Cooked meat medium (AM1030/AM5030) (BIS formula-

AM103011/AM503011)l Thioglycollate brothl Diphasic broth (Castaneda system)

INTRODUCTION

REQUIRED REAGENTS/MEDIAS

l Blood agar base (AM1014/AM5014), OR

l Blood agar base (Ready prepared media-250ml) (20500006)l Modified Gram’s Stain Kit (20750020/20750021)

REQUIRED MATERIALS1. Sterile hand gloves.2. Face mask. 3. Sterile plating loops (10µl). 4. Activated 2% glutaraldehyde solution. 5. Cedar wood oil. 6. Glass slide.7. 70% isopropyl alcohol in water with 1% iodine or 1-2% chlorhexidine.

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens.

0 03. Incubator with air plus 5-10% CO at 37 C±2 C. 20 04. Incubator with Nitrogen or Hydrogen plus 5-10% CO at 37 C±2 C. 2

5. Autoclave.

PROCEDUREa. Collection of sample:

1. Wear sterile gloves.

REQUIRED MATERIALS

REQUIRED EQUIPMENTS

PROCEDURE




2. Disinfect the venepuncture site on the patient’s skin by applying 70% isopropyl alcohol in water with 1% iodine or 1-2% chlorhexidine for at least 1min and allow to dry.

3. With Precautions to avoid touching and recontaminating the venepuncture site, take the sample of blood.

b. Quantity of innoculum:1. To maximize the chances of detecting scanty bacteria, a larger volume

example 10-20ml, of blood should be collected half of it should be inoculated into each of a set of two culture bottles.

2. Dilution of blood in the culture medium should be 1in 5 to 1in 10.3. For infants and small children 1to 5ml of blood should be taken for

bacterial culture. Quantities less than 1ml may not be adequate to detect pathogens.

c. Innoculation:Inoculate the blood into more than one of the following culture bottle 1. BHI supplemented with 0.05% SPS 2. Glucose broth supplemented with 0.05% SPS 3. Soyabean casein digest broth supplemented with 0.05% SPS4. Cooked meat medium5. Thioglycollate broth6. Diphasic broth (Castaneda system)

d. Incubation:l To encourage the growth of strict aerobes such as yeast and

Pseudomonas aeruginosa, vent the bottle with a sterile , cotton plugged needle.

l The atmosphere in commercially prepared blood culture bottles is usually at low oxidation-reduction potential, allowing most facultative and obligate anaerobes to grow.

e. Recovery:1. First recovery to be carried out within 4-8 hours of incubation on two

plates of Blood agar.0

l Incubate one plate in air plus 5-10% CO , at 37 C for 18-24 2

hours.l Incubate second plate anaerobically in nitrogen or hydrogen plus

05-10% CO , at 37 C for 18-24 hours.2

Simultaneously examining the gram film using commercially available modified grams stain kit.

2. Second recovery to be carried out at 18-24 hours of incubation .l Third recovery to be carried out at 4-7 days of incubation.

(Note: If the presence of meningococcus or haemophilus seems likely e.g in suspected septicaemic meningitis, a heated blood agar (chocolate agar) plate may be substituted for the aerobic plain blood agar plates).

References1. Basic Laboratory in Clinical Bacteriology, J.Vandepitte, K.Engbaek, P.Piot,

C.C.Heuck, W.H.O. Geneva, 1991.th2. Diagnostic Microbiology, Bailey & Scott, 9 Ed., Mosby 1994, Ellen Jo Baron,

Lance R. Peterson.3. Practical Medical Microbiology, Mackie & McCartney, Vol 1, Microbial

thInfections, 13 Ed., Churchill Livingston 1978, Edited by J.P. Duguid, B.P. Marmion, R.H.A. Swain.

th4. Practical Medical Microbiology, Mackie & McCartney, Vol. 2, 13 Ed., Churchill Livingston 1989, Edited by J.G. Collee, J.P. Duguid, A.G. Fraser, B.P.Marmion.

nd5. Handbook of Microbiological Media, Ronald M. Atlas, Lawrence C.Parks, 2 Ed., 1997.

6. Data on file: Tulip Diagnostics (P) Ltd.

References

Upper respiratory tract infections, including throat, nose, ear and eye infections

INTRODUCTIONThe commonest respiratory infections are localized in the oropharynx, nasopharynx and nasal cavity, causing sore throat, nasal discharge and often fever, but the throat pathogens may also spread to infect the larynx, causing hoarseness, the middle ear, causing otitis media with earache, a paranasal sinus, causing sinusitis with pain in the face or head, and the eye causing conjunctivitis or keratitis. The upper respiratory tract may also be involved in wider respiratory or generalized infections such as whooping cough, influenza, measles and infectious mononucleosis. In most cases the primary infection is viral, though the causal virus is generally not demonstrated, and there is often concomitant carriage or secondary infection with

INTRODUCTION one of the potential bacterial pathogens commonly present in the nasopharynx, e.g. pneumococcus, Haemophilus influenzae, Staphylococcus aureus and Streptococcus pyogenes. Drug resistant coliform bacilli or yeasts may come to dominate the throat flora in patients receiving antibiotics, but their presence is generally of little pathological significance.

1. REQUIRED REAGENTS/MEDIASStuart transport medium (AM1094/AM5094)l Blood agar base (AM1014/AM5014 )

ORl Blood agar base (Ready prepared media-250ml) (20500006). l Stuart transport medium (AM1094/AM5094)

1. REQUIRED REAGENTS/MEDIAS




l 6mm disk containing 1 unit of benzyl penicillinl 6mm disk containing 0.1 unit of bacitracinl 6mm disk containing 50 unit nystatinl 6mm disk containing 20µg amphotericin l 6mm disk containing 2 µg of amoxycillin or ampicillinl Candida identification kit (20794001)

2. REQUIRED MATERIALS1. Sterile hand gloves .2. Face mask. 3. Sterile plating loops(10µl). 4. Activated 2% glutaraldehyde solution.5. Cedar wood oil, glass slide. 6. 70% isopropyl alcohol in water with 1% iodine or 1-2% chlorhexidine. 7. Plain, albumen-coated or charcoal coated sterile cotton wool swab.

3. REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens.

0 03. Incubator with air plus 5-10% CO at 37 C±2 C. 20 04. Incubator with Nitrogen or Hydrogen plus 5-10% CO at 37 C±2 C.2

5. Autoclave.

4. PROCEDUREa. Collection of sample throat swab, nasal swab. Ear swab and eye swab:

1. Wear sterile gloves.2. With a plain, albumen-coated or charcoal coated sterile cotton

wool swab collect the exudates as much as possible from the area that is inflamed or bears exudates.

3. Replace the swab in its tube with care not to soil the rim.4. It should be taken for testing within a hour or should be placed in a

0refrigerator at 4 C until testing or if it has to be transported it should be submitted in a tube of Stuart transport medium.

b. Innoculation:Direct sensitivity methodl Rub the swab rotating over a large “well” areas, about one third of

the surface on each of blood agar plates. and heated blood agar plates

l Streak the wells with a loop over the remainder of the plate.l Place a 6mm disk containing 1 unit of benzyl penicillin on the well

area of one blood agar plate and a disk containing 0.1 unit of bacitracin on that of the second blood agar plate and a disk containing 50 unit nystatin on third blood agar plate and a disk

2. REQUIRED MATERIALS

3. REQUIRED EQUIPMENTS

4. PROCEDURE

Direct sensitivity method

containing 20µg amphotericin on fourth blood agar plate.l Place a 6mm disk containing 2µg of amoxycillin or ampicillin on

the well area of heated blood agar plate.

c. Incubation:0i) Incubate one plate in air plus 5-10% CO , at 37 C for 18-24 2

hours.ii) Incubate second plate anaerobically in nitrogen or hydrogen plus

05-10% CO , at 37 C for 18-24 hours.2

d. Interpretation:l Zones of b-haemolysis, stronger on aerobic blood agar plate than

in anaerobic agar plate with resistant to penicillin and sensitive to 2 µg of amoxycillin or ampicillin on heated blood agar plate. ---- Haemolytic haemophili.

l Zones of b-haemolysis, larger and clearer in anaerobic blood agar plate than in aerobic agar plate with sensitivity to both penicillin (zone diameter >16mm) and bacitracin (zone >12mm) ---- S.pyogenes.

l Growth on aerobic blood agar plate after 48 hours of incubation with small opaque white colonies, typically with short pointed ‘rootlets’ projecting from their margins ---- Candida albicans.

(Note: Sensitivity of Candida albicans to be checked for 50 unit nystatin & 20µg amphotericin and later subjected to confirmatory test using commercially available identification kit).








References




Lower Respiratory Tract Infections

INTRODUCTIONUnlike most regions of the upper respiratory tract, the trachea, bronchi and lungs are normally free from colonization with commensal and potentially pathogenic bacteria, but when there defences are upset they are liable to be invaded by organisms from the throat. They are also susceptible to primary infection with various inhaled pathogens, such as the tubercle and whooping cough bacilli.

The commonest infections are acute tracheobronchitis, acute exacerbations of chronic bronchitis, and the pneumonias.

In many or most cases the primary infection is caused by a virus, e.g. rhinovirus,myxovirus, adenovirus or respirator syncytial virus, but there is often a secondary infection with a bacterial pathogen from the nasopharynx, most commonly pneumococcus or haemophilus influenzae. Other secondary invaders of the lower tract include Staphylococcus aureus , which may cause fatal pneumonia after influenza, coliform bacilli and Pseudomonas aeruginosa, Branhamella catarrhalis, Candida albicans and Aspergillus fumigatus. The staphylococcus, coliforms and candida are found particularly in hospitalized patients treated with antibiotics to which these organism are resistant.

Other organisms that may cause primary infection in the bronchial tract or lungs are Mycoplasma pneumoniae, which is the commonest , Legionella pneumophila, Chlamydia psittaci B and Coxiella burneti. The protozoon Pneumocystis carini is liable to cause diffuse infection of the lungs in persons who are immunosuppressed, e.g. patients infected with human immunodeficiency virus.

SPECIMENS1. Type of specimen

1. Sputum2. Bronchial swabs and aspirates3. Blood

2. Collection of specimenFor the best chance of success, specimens of sputum and blood for culture, Pleural fluid from bronchial aspirate for adenosine deaminase should be collected before the start of any antibiotic therapy. In suspected atypical pneumonia an initial blood sample for serology should be taken and for suspected pulmonary tuberculosis initial pleural fluid sample for adenosine deaminase test should be taken at the same early stage

1. SputumThe material from lower respiratory tract infections most commonly submitted for bacteriological examination is sputum, a mixture of bronchial secretion and inflammatory exudate coughed up into the mouth and expectorated. There are however difficulties both in collecting a suitable sample and

INTRODUCTION

SPECIMENS

1. Sputum

interpreting the results of its culture. In some infections , e.g. those due to mycoplasma or legionella, there is often a lack of secretion and sputum cannot be obtained.

**Collection of sputum sample should include the following advice:l Make the collection in a disposable, wide-mouthed, screw- capped

plastic container of about 100ml capacity.l If possible, collect the sputum before any antibiotic therapy is begun,

and when the patient first coughs on waking in the morning.

l Instruct the patient to wait until he feels material coughed into his throat and then to work it forward into his mouth and spit it directly into the opened container, trying to avoid spilling over the rim. At once tightly screw on the cap of the container. Wipe off any spilled material on its outside with a tissue moistened with disinfectant (2% glutaraldehyde), but take care not to let any disinfectant enter the container.

l If the patient has difficulty in coughing sputum into his mouth, ask a physiotherapist to pummel his chest. This exercise often causes exudates to move in the bronchi and stimulate productive coughing.

l Deliver the specimen to the laboratory as quickly as possible, preferably within 2 hrs, for delicate pathogens such as pneumococcus and haemophilus may die out during any longer delay.

2. Bronchial swabs and aspiratesA specimen of bronchial secretion is collected by some means that prevents its contact with the throat mouth.

Such collection may be done by transtracheal puncture and aspiration or by the use of a protected swab passed through a bronchoscope into the bronchi .

Direct aspiration of secretion through a bronchoscope, e.g. by bronchial lavage, is unsatisfactory as the inside of the bronchoscope is liable to become soiled with throat secretion.

However transtracheal aspiration and bronchial swabbing require anaesthesia of the patient and the attention of skilled medical staff, and for these reasons are generally not performed. Nevertheless they may be attempted for the diagnosis of unusual or obscure infections. In all cases of suspected pulmonary tuberculosis a sample of pleural fluid should be taken for adenosine deaminase test (ADA-MTB Kit -20306015 ) before antibiotics are given. Lung infection with Mycobacterium tuberculosis show high ADA value of the pleural fluid.

3. Blood cultureIn all cases of suspected pneumonia a sample of blood should be taken for

2. Bronchial swabs and aspirates

3. Blood culture




culture before antibiotics are given. Lung infections are commonly associated with bacteriaemia and it may be possible to culture from the blood a delicate pathogen whose growth is suppressed in cultures of sputum contaminated with salivary organisms. Moreover, the finding of a bacterium in the blood is strong evidence that it has been infecting the lungs and is not merely a throat organism contaminating sputum. (NOTE: Refer BLOOD CULTURE for details)

4. EXAMINATION FOR TUBERCULOSISIn communities where tuberculosis is moderately or very common, every specimen of sputum received in the laboratory should be screened for tubercle bacilli, regardless of whether the physician requests the examination. Examination for tuberculosis requires the following steps.l STAINING OF SPUTUM.l DECONTAMINATION AND CONCENTRATION OF SPUTUM.l CULTURING .l SENSITIVITY TESTING TOWARDS PRIMARY AND SECONDARY LINE

DRUGS.l IDENTIFICATION/DIFFERENTIAL TESTING.l ADA TESTING OF PLEURAL FLUID.

1. * STAINING OF SPUTUMSUMMARYAcid Fast Bacilli staining is the first line microscopic procedure performed in detection of Mycobacterium tuberculosis.

PRINCIPLECarbol fuchsin forms acid insoluble complex with the mycolic acid present on the Acid Fast Bacilli and renders red / pinkish red color to them as they resist decolorization by strong acid .

REQUIRED REAGENTS1. Mycostain (20307100)2. Acid fast decolouriser (20308500)3. Novachrom (20302125)

REQUIRED MATERIALS1. Sterile hand gloves.2. Face mask. 3. Sterile plating loops (5ml measuring cylinder).4. Activated 2% glutaraldehyde solution. 5. Cedar wood oil. 6. Glass slide.

REQUIRED EQUIPMENTSBiosafety hood with Bunsen burner, Microscope with oil immersion lens, Autoclave.

4. EXAMINATION FOR TUBERCULOSIS

1. * STAINING OF SPUTUMSUMMARY

PRINCIPLE

REQUIRED REAGENTS

REQUIRED MATERIALS

REQUIRED EQUIPMENTS

SPECIMEN COLLECTION REFER**PROCEDUREa. Preparation of smear:l Place the specimen under test on a clean, scratch-free, preferably

new glass slide using a sterile plating loop.l Homogenize and evenly spread the sample with the loop by tracing

concentric circles well separated to cover approximately 1/3 of the whole area of the glass slide to form a fairly thick uniform smear.

l When the smear is completed, plunge the inoculating loop into liquid disinfectant (2% Glutaraldehyde) and shake to remove any sputum, then flame sterilize the loop.

l Air dry the smear .l Flame the edges of the slide and place it on a drying rack.l The slide is then air dried and heat fixed by passing three times

through a flame. (NOTE: While passing the smear slide through the flame, ensure that the side opposite the smear is facing the flame)

b. Staining procedure :Method I - (With Mycostain-20307100 and Acid fast decolouriser-20308500)l Place the slide over a staining rack at a suitable height for applying

heat.l Mix well and add Mycostain (A) over the smear to cover it

completely.l Allow the stain to stand for 5 minutes with the application of heat.

(NOTE: The slide may be heated with a torch prepared by twisting a small piece of cotton wool on to the tip of an inoculating wire and soaking it in methylated spirit before lighting. Heat the slide with the flame from the torch. When steam rises from the slide, remove and extinguish the torch. After about 1 minute recharge the torch with methylated spirit, relight it , and again heat the slide until steam rises from the slide. Continue in this way for 5 minutes).

l Leave the slide to cool for another 2 minutes.l Wash the slide with water and wipe the downward lower surface of

the slide where carbon has settled with a clean tissue paper.l With the aid of a dropper, cover the slide with » 1-2 ml of ACID

FAST DECOLORIZER (25% sulphuric acid)-20308500 and leave it for 1 minute, until the red color of the smear changes to yellowish brown. Wash the slide under running tap water. Repeat the procedure until smear appears colourless. This takes » 5-10 minutes depending on the thickness of the smear.

l Finally counter stain the smear. Mix well and add MYCOSTAIN (B) with the stain dropper, to cover the smear.

l Allow MYCOSTAIN (B) to stand for 15-20 seconds and wash the smear again under running tap water.

l Air dry and observe under oil immersion (magnification 100X).

PROCEDUREa. Preparation of smear:

b. Staining procedure :Method I - (With Mycostain-20307100 and Acid fast decolouriser-20308500)




Interpretation:l Presence of pink to red colored slender Bacilli - Smear Acid Fast

Bacilli positive.l Absence of pink to red colored slender Bacilli - Smear Acid Fast

Bacilli negative.l Pus cells and other bacteria stain purple to blue color.

Grading of results:After 5 minutes of examination covering about 100 fields, report the results as follows:

Number of Acid Fast Bacilli Observed Report

No Acid Fast Bacilli Negative

1-10 Acid Fast Bacilli Actual number

>10 Acid Fast Bacilli +

Masses of Acid Fast Bacilli in several fields ++

Method II- (With Novachrom -20302125)l Place the slide over a staining rack .l Mix well and add Novachrom AFB sain (A) over the smear to cover it

completely.l Keep for 6 minutes and then rinse with plenty of water slowly to

remove excess of Novachrom AFB stain (A)l Tilt slide to drain , mix well and add Novachrom AFB stain (B) over

the smear to cover it completely.l Keep for 6 minutes and then rinse the smear once more with plenty

of water slowly to remove excess of Novachrom AFB stain (B).l Air dry and observe under oil immersion (magnification 100X).

Interpretation:l Presence of pink to red colored slender Bacilli - Smear Acid Fast

Bacilli positive.l Absence of pink to red colored slender Bacilli - Smear Acid Fast

Bacilli negative.l Pus cells and other bacteria stain purple to blue color.

Grading of results:After 5 minutes of examination covering about 100 fields, report the results as follows:


No Acid Fast Bacilli Negative

1-10 Acid Fast Bacilli Actual number

>10 Acid Fast Bacilli +

Masses of Acid Fast Bacilli in several fields ++

Interpretation:


Method II- (With Novachrom -20302125)

Interpretation:


2. DECONTAMINATION AND CONCENTRATION OF SPUTUMSUMMARYBefore inoculation on to culture media, specimens such as sputum which are contaminated with bacteria other than Mycobacteria must be treated by a method that kills the other bacteria but not the Mycobacteria. Various methods of decontamination have been advocated, the most used of which have the additional advantages of homogenizing the specimen and concentrating the mycobacteria in a centrifuged deposit that serves as the inoculum for cultures.

REQUIRED REAGENTSLyfectol (20301012)

REQUIRED MATERIALS1. Sterile hand gloves.2. Face mask. 3. Sterile plating loops(10µl). 4. Activated 2% glutaraldehyde solution. 5. Cedar wood oil. 6. Glass slide. 7. 5ml measuring cylinder.8. 15-25ml universal container.

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens. 3. Centrifuge at 3000-4000g. 4. 5ml measuring cylinder. 5. Vortex mixer. 6. Autoclave.

PRINCIPLEProper decontamination and concentration of specimen containing normal microbial flora are crucial to detection of Mycobacterium tuberculosis.

Acid fast bacilli trapped in mucoid portion of sputum are released by mucolytic action of N-acetyl L-cysteine. NaOH decontaminates other microorganisms and final wash with phosphate buffer ensures that specimen is at optimum pH for staining and culturing.

SPECIMEN COLLECTION REFER**PROCEDUREThe procedure mentioned below is for 2.5 ml of the sputum sample. In case of variation in quantity of specimen used , process using proportionate amounts of reagent, mucolytic and disinfection reagent.

2. DECONTAMINATION AND CONCENTRATION OF SPUTUMSUMMARY

REQUIRED REAGENTS

REQUIRED MATERIALS

REQUIRED EQUIPMENTS

PRINCIPLE

PROCEDURE




PREPARATION OF MUCOLYTIC REAGENTl Bring the reagents to room temperature.l Add »12.5mg of N-acetyl L-cysteine to 2.5ml of 2% NaOH.l Mix to dissolve.l The mucolytic reagent can be used within 24 hours of preparation, if

stored at 2-8 ºC.

****PROCESSING OF SPECIMENl Take approximately 2.5ml of the specimen in a clean sterile 15-25ml

universal container.l Add 2.5ml of the mucolytic reagent and close the container tightly with a

screw cap fitted with an intact liner.l Mix well by gently vortexing at every 5 minutes interval for 20 minutes.l After 20 minutes, unscrew the cap of the container carefully and add 5ml

of phosphate buffer.l Close again the container tightly as in step 2.l Mix well and centrifuge for 25 minutes at 3000-4000g.l After centrifugation unscrew the cap of the container with the content

carefully and discard the supernatant gently in an activated 2% Glutaraldehyde solution, taking care as not to disturb the pellet at the bottom.

l To the pellet at the bottom add 0.1ml of sterile saline and resuspend the contents

l Use this suspended material for microscopy (Acid fast bacilli staining refer*), Acid fast bacilli culture.

3. CULTURINGSUMMARYThe optimal medium for tubercle bacilli are Lowenstein jensen’s solidified egg based medium with glycerol , Kirchner selective medium, Middle brook agar and broth with enriched supplement (for M.tuberculosis) or Lowenstein jensen’s solidified egg based medium with pyruvate for (M.bovis). As tubercle bacilli are obligate aerobes, there cultures are grown on the surface of slope medium in a bottle also containing sufficient air to provide oxygen for their respiration. The bottle is kept sealed with a tightly applied screw cap to prevent the medium drying up during the long period of incubation.

REQUIRED REAGENTS1. Combicult (20303001).2. Mycocult (20304006).3. Middle brook 7H9 Agar Base (AM506927).4. Middle brook OADC Growth supplement (AS0181).5. Middle brook 7H10 Broth.

REQUIRED MATERIALS1. Sterile hand gloves.2. Face mask.

PREPARATION OF MUCOLYTIC REAGENT

3. CULTURINGSUMMARY

REQUIRED REAGENTS

REQUIRED MATERIALS

3. Sterile plating loops(10µl).4. Activated 2% glutaraldehyde solution. 5. Cedar wood oil. 6. Glass slide. 7. Micropippete (100µl - 500µl). 8. Sterile micropipette tips.

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens. 3. Vortex mixer, Incubator at 37±0.5ºC. 4. Autoclave.

PRINCIPLEThe Combicult, Mycocult, Middle brook7H9 agar, Middle brook 7H10 broth medium supports the growth of Mycobacterium tuberculosis.

The malachite green in Mycocult, Middle brook7H9 agar, not only has an inhibitory effect on growth of organisms other than Mycobacterium tuberculosis, but also provides the desired colour contrast for easy identification of Mycobacterium tuberculosis colonies.

The gold standard for primary isolation of Mycobacterium tuberculosis is the use of liquid media in conjunction with solid media (Combicult). Liquid media also support higher detection rates especially with specimen material containing small number of bacilli. Simultaneous inoculation of solid media and liquid media yields significantly higher recovery rates for Mycobacterium tuberculosis growth as compared to when each media is used independently.

SPECIMEN PREPARATION REFER **** (As in decontamination and concentration of sputum)PROCEDUREl Bring the required media for inoculation to room temperature.l Label the medium slant appropriately.l Draw 10µl of the decontaminated and concentrated specimen from the

reconstituted pellet with a sterile calibrated loop and place it on the respective medium aseptically.

l Close the medium cap tightly and incubate at 37±0.5ºC.l Observe for growth weekly till 8 weeks.

INTERPRETATION OF RESULTSl Growth on Mycocult, Middle brook7H9 agar : Colonies may be detected

from third week onwards upto eight weeks. The colonies are characterized by rough granular buff colored growth, which has an initial size of 1-3mm and full-grown size of 5-8mm.

l Growth in Kirchner liquid media, Middle brook broth : Growth in this medium is characterized by fluffy growth to small granules. The granules

REQUIRED EQUIPMENTS

PRINCIPLE

PROCEDURE

INTERPRETATION OF RESULTS




sediment to the bottom.

4. SENSITIVITY TESTINGSUMMARYInadequate chemotherapy, irregularity of treatment and use of improper antitubercular regimen lead to high failure rates of antitubercular treatment. As result, the prevalence of chronic patients discharging drug resistant organisms increases. Alarming figures of drug resistance in newly detected patients are being reported, mainly from developing countries. This calls for sensitivity testing of antibiotic sensitivity invitro prior to starting therapy.

REQUIRED REAGENTS1. Sensicult primary (20305101)2. Sensicult secondary (6 drugs) (20305201)3. Sensicult secondary (10 drugs) (20305202)

REQUIRED MATERIALS1. Sterile hand gloves.2. Face mask.3. Sterile plating loops (10µl).4. Activated 2% glutaraldehyde solution.5. Cedar wood oil. 6. Glass slide.7. Micropippete (100µl - 500µl).8. Sterile micropipette tips.

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens.3. Vortex mixer.4. Incubator at 37±0.5ºC. 5. Autoclave.

PRINCIPLEDue to increase in drug resistant strains of Mycobacterium tuberculosis and increasing failure rates of antitubercular drug regimens, it is desirable to start antitubercular therapy only after sensitivity assay of the most suitable drug against particular isolate infecting the patient.

PROCEDUREINNOCULUM PREPARATION:1. Take a loopful asceptically from the Mycobacterium tuberculosis colony

grown.2. Transfer it aseptically to the screw capped bottle containing 0.1ml of

sterile saline and glass beads, for inoculum preparation.

4. SENSITIVITY TESTINGSUMMARY

REQUIRED REAGENTS

REQUIRED MATERIALS

REQUIRED EQUIPMENTS

PRINCIPLE

PROCEDUREINNOCULUM PREPARATION:

3. Close cap tightly and subject the contents of the bottle to mechanical shaking (vortex) for 10 minutes.

4. Keep standing for 10 minutes before opening the bottle.5. Dilute this in saline to match Mc Farland 0.5 Standard. This is seed

culture -1.6. Further dilute to 1: 100 with saline.This is seed culture-2.7. Mix well and use both this as inoculum as mentioned below.8. Discard the container with glass beads in 2% activated Glutaraldehyde

solution.

INNOCULATIONl Bring the primary /secondary drug containing Lowenstein-jensen media

panel for MTB sensitivity tests slants to room temperature .l Apply 100µl from the seed culture-1 to each drug slant and 100µl from

the seed culture -2 to Lowenstein Jensen slant.l Close the cap tightly and incubate at 37±0.5ºC.l Observe for the growth till 8 weeks every alternate days.

INTERPRETATION OF RESULTSAs and when there is sufficient growth on control (>20-30 colonies) observe the growth in drug containing media.l Sensitive if no growth.l Resistant if growth observed.

5. IDENTIFICATION/DIFFERENTIAL TESTSSUMMARYMany a times, Mycobacterium other than tuberculosis (MOTT) may be the cause of disease in human and other animals. Various biochemical and biological criteria have been used to identify and differentiate M. tuberculosis from MOTT. The identification of Mycobacteria to the species level is important because of clinical significance; some species are pathogenic while others are not. Knowledge of species is also critical in order to provide adequate patient management because specific antimycobacterial drugs are required against different pathogenic Mycobacteria species. Differentiation of M.tuberculosis is possible on the basis of 1. Rate and Temperature of growth.2. Pigment production. 3. Colonial characteristics and morphology. 4. Catalase reactions.5. Nitrate activity.6. Niacin. 7. Thiophen-2-carboxylic acid hydrazide(TCH) sensitivity test. 8. PNB sensitivity test.9. Tween 80 hydrolysis.

INNOCULATION


5. IDENTIFICATION/DIFFERENTIAL TESTSSUMMARY




PROCEDUREI. CATALASE

PRINCIPLEMost species of Mycobacteria, with the exception of isoniazide-resistant strains of M.tuberculosis and M.gastri, produce the intracellular enzyme catalase. Catalase splits hydrogen peroxide into water and oxygen.

Catalase can be detected and measured in two ways:l Room temperature method.l Heat stable (pH 7/68ºC ) method.

Room Temperature MethodUntreated catalase enzyme produced by Mycobacteria reduces hydrogen peroxide to water and oxygen. This is as bubbling of oxygen, which occurs following the addition of 50-100µl of the Tween-Peroxide reagent to the 3-4 week old culture growth obtained on the solid plate or slant.

Heat Stable (pH 7/68ºC ) method.Certain Mycobateria loose catalase activity when suspended in pH7.0 bufer and heated to 68ºC. These include M.tuberculosis and most members of M.tuberculosis complex, M.bovis, M.gastri and some strains of M.merinum and M.avium complex.

REQUIRED REAGENTS1. Catalase Detection Kit (20403020).

REQUIRED MATERIALS1. Sterile hand gloves.2. Face mask.3. Activated 2% glutaraldehyde solution.4. Micropippete (100µl - 500µl).5. Sterile micropipette tips.6. Test tube stand.7. Screw cap test tubes (16x125mm).

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Water bath or constant temperature block heater.3. Autoclave.

SPECIMEN COLLECTION3-4 weeks old cultures obtained from solid media only should be used for testing.

TEST PROCEDUREi. PREPARATION OF 1ML TWEEN - PEROXIDE REAGENT

Note: Tween - Peroxide reagent is to be prepared fresh, immediately

PROCEDUREI. CATALASE

PRINCIPLE

Room Temperature Method

Heat Stable (pH 7/68ºC ) method.

REQUIRED REAGENTS

REQUIRED MATERIALS

REQUIRED EQUIPMENTS

SPECIMEN COLLECTION

TEST PROCEDUREi. PREPARATION OF 1ML TWEEN - PEROXIDE REAGENT

prior to performing the test. Should any reagent remain after performing the test, discard the same.l Just prior to testing, mix 0.5ml of Tween-80 solution to 0.5ml of

hydrogen peroxide in a sterile clean test tube.l Gently swirl to mix.

ii. INNOCULATION AND OBSERVATIONRoom Temperature Method:l Add 50-100µl of the Tween-Peroxide reagent to the 3-4 week old

culture growth obtained on the solid plate or slant.l Observe for the formation of bubbles. Formation of bubbles may

take 5minutes in some cases.

Heat Stable (pH 7/68ºC ) method:l Label the required number of screw cap tubes to correspond with

the cultures to be tested.l Open the screw cap tubes and with a sterile pipette, add 0.5ml of

phosphate buffer pH 7.0 to each tube.l With a sterile loop/spade, emulsify several colonies from the

culture into the phosphate buffer pH 7.0.l A fresh disposal loop should be used for each inoculation.l Cap the tubes and place the tubes containing emulsified colonies in

a water bath or constant temperature block heater at 68ºC for 20 minutes. (Adherence to temperature and time are critical for obtaining accurate results.)

l After 20 minutes, remove the tubes and allow to cool to R.T. on a test tube stand.

l Add to each tube 0.5ml of Tween-Peroxide reagent using a pipette.l Observe for the formation of bubbles appearing on the surface of

the liquid. Hold tubes for 20 minutes before discarding as negative.

Note: Room Temperature method and Heat Stable (pH 7/68ºC) method can be performed on the same slant or plate of the culture. Scrape and remove the colonies for Heat stable method prior to performing the Room Temperature test.

INTERPRETATION OF RESULTSRoom Temperature Method:l Immediate profuse bubbles formed - Positive (Rapid).l Few slow formation of bubbles - Positive (slow).l No bubbling obtained until 5 minutes - Negative.

Heat Stable (pH 7/68ºC ) method:l Formation of bubbles - Positive.l No bubbling until 20 minutes - Negative.

ii. INNOCULATION AND OBSERVATIONRoom Temperature Method:

Heat Stable (pH 7/68ºC ) method:

INTERPRETATION OF RESULTSRoom Temperature Method:

Heat Stable (pH 7/68ºC ) method:




II. TWEEN-80 HYDROLYSISPRINCIPLEThe commonly non-pathogenic, slow growing scotochromogens and non-chromogens produce a lipase that is able to hydrolyse Tween-80 into oleic acid and sorbitol, while the saprophytic species of these groups are unable to hydrolyse Tween-80.

Intact Tween-80 binds neutral indicator to give an amber coloured complex.

When Tween-80 is hydrolyzed, it can no longer bind with neutral red indicator. The neutral red indicator assumes its normal configuration at pH 7, which is pink-red in colour.

REQUIRED REAGENTSTween 80 Hydrolysis Kit (20402100).

REQUIRED MATERIALS1. Sterile hand gloves.2. Face mask.3. Activated 2% glutaraldehyde solution.4. Micropippete (100µl - 500µl).5. Sterile micropipette tips.6. Test tube stand.7. Screw cap test tubes (16x125mm).

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner.2. Water bath /incubator at 37±2ºC. 3. Autoclave.

SPECIMEN COLLECTIONSlow growing cultures obtained from solid media can be inoculated on the Tween-80 substrate as soon as sufficient growth is obtained for transfer.

TEST PROCEDUREi. PREPARATION OF TWEEN - HYDROLYSIS REAGENTl Bring the concentrated mixed TWEEN-HYDROLYSIS reagent to

room temperature prior to use.l Use the required number of sterile screw capped tubes/bottles of

size 16x125mm containing 2ml of sterile distilled water.l Aseptically pipette out in each test tube 100µl of the concentrated

mixed TWEEN-80 HYDROLYSIS reagent. l Slightly warm (50ºC) this solution to enable complete dissolution

of Tween-80 by gently swirling.l Close the screw cap tightly. l The amber coloured solution thus obtained is Tween-Hydrolysis

Substrate reagent.

II. TWEEN-80 HYDROLYSISPRINCIPLE

REQUIRED REAGENTS

REQUIRED MATERIALS

REQUIRED EQUIPMENTS

SPECIMEN COLLECTION

TEST PROCEDUREi. PREPARATION OF TWEEN - HYDROLYSIS REAGENT

l Allow the reagent to cool prior to testing.

ii. INNOCULATIONl Label the required number of sterile screw caps tube(s) containing

Tween-Hydrolysis substrate reagent, to correspond with the culture identity, to be tested and a blank without any culture.

l Using a 3mm sterile loop, draw a loopful of the culture to be tested and inoculate the corresponding labeled Tween-Hydrolysis substrate reagent, following aseptic conditions. A fresh disposable loop should be used for each inoculation.

l Close the screw cap of the inoculated Tween-Hydrolysis Substrate reagent and incubate at 35-37ºC.

l Observe the change in colour of the Tween-Hydrolysis Substrate reagent from amber to pink-red at 24 hours, 5 days and 10-12 days. Always compare with the blank to see change in colour.

l Do not shake the Tween-Hydrolysis Substrate reagent while reading.

INTERPRETATION OF RESULTSl Change in colour, of the Tween-Hydrolysis Substrate reagent in the screw

cap tube and not the cells, from amber to pink-red is a positive test result.

l Results are to be read at 24 hours, recorded and positive tubes to be discarded.

III. NIACIN DROP TESTPRINCIPLEThe utilization of protein in the culture medium during growth, by Mycobacterium tuberculosis and not by Mycobacterium bovis results in the production of niacin or nicotinic acid. Niacin therefore accumulates in the medium in which Mycobacterium tuberculosis grows.

KSCN + Chloramine T Cyanogen chloride

Niacin + Cyanogen chloride g-carboxy-glutaconic aldehyde

g-carboxy-glutaconic aldehyde formation of Schiff base(Canary yellow colour)

REQUIRED REAGENTSNiacin drop test Kit (20401050).

REQUIRED MATERIALS1. Sterile hand gloves.2. Face mask.3. Activated 2% glutaraldehyde solution.4. Micropippete (100µl - 500µl).

ii. INNOCULATION


III. NIACIN DROP TESTPRINCIPLE

REQUIRED REAGENTS

REQUIRED MATERIALS




5. Sterile micropipette tips.6. Test tube stand.7. Screw cap test tubes(16x125mm).8. White background card or board,sterile.9. Sterile isotonic solution.

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner.2. Centrifuge. 3. Autoclave.

SPECIMEN COLLECTIONThree to four week old viable cultures on drug free L.J media or Middle brook 7H10 containing more than 50 colonies should be used for testing.

TEST PROCEDUREl Allow the reagents from the kit to attain R.T.(20-25ºC) prior to testing.l To the culture slant or quadrant on drug free medium add 1ml of sterile

distilled water or sterile isotonic saline.l If the growth on the medium is confluent (dense), lightly puncture the

surface with the pipette so that the sterile distilled water or sterile isotonic saline is in contact with the medium.

l Allow the sterile distilled water or sterile isotonic saline to remain in contact with the culture medium for 30 minutes, at R.T.(20-25ºC). When the culture is in a tube, place the tube in a slanted position so that the sterile distilled water or sterile isotonic saline overlays over the colonies.

l Remove the liquid extract from the culture into a sterile, clean-labeled screw cap test tube and centrifuge at 3000 r.p.m. for 15 minutes to obtain a clear supernatant.

l Transfer 0.6 ml of the supernatant into another clean,sterile,labeled screw cap test tube.

l Twist the caps of NIACIN Reagents; R1, R2, R3 and R4 in the clockwise direction to pierce open the dropper nozzle of the respective bottles.

l To the labeled screw cap test tube with the supernatant, add the following reagents in the same sequence as mentioned below.1. One drop of Reagent (R1) and mix by gentle swirling. 2. One drop of Reagent (R2) and mix by gentle swirling.3. One drop of Reagent (R3) and mix by gentle swirling.4. One drop of Reagent (R4) and mix by gentle swirling.5. Close the screw cap test tube and allow to stand on a test tube rack

at R.T.6. Observe for colour development from colourless to yellow using a

white background.it is recommended to compare the colour in the tubes with 0.5 ml distilled water and 0.5 ml niacin cut-off concentration solution, prepared as mentioned below.

7. Record and report the readings immediately and definitely at 15

REQUIRED EQUIPMENTS

SPECIMEN COLLECTION

TEST PROCEDURE

minutes.8. Replace the caps and restore the NIACIN reagents at 2-8ºC

immediately after use.

INTERPRETATION OF RESULTSl Yellow colour developed within 15 minutes is indicative of positive

reaction.l No colour development till 15 minutes is indicative of negative reaction.

IV. PNB SENSITIVITY TESTPRINCIPLEPNB SENSITIVITY TEST is used to identify if an isolate belongs to Mycobacterium tuberculosis complex or nontuberculous mycobacteria.

M. tuberculosis and M. bovis and their variants are sensitive to para - nitrobenzoic acid (PNB) and fail to grow on L.J mdium with PNB.

Nontuberculous bacteria are generally resistant to PNB and show abundant growth on an L.J. medium with PNB.

REQUIRED REAGENTS1. PNB Sensitivity Test Kit (20408006).2. Mycocult Kit (20304006).3. Mycocult PY (20314006).

REQUIRED MATERIALS1. Sterile hand gloves. 2. Face mask. 3. Activated 2% glutaraldehyde solution. 4. Micropippete (100µl - 500µl).5. Sterile micropipette tips.6. Test tube stand.7. Screw cap test tubes(16x125mm).8. Sterile 1ml glass bottles with glass beads.9. Mcfarland standard No. 1.10. Sterile isotonic solution.

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Water bath /incubator at 37±2ºC. 3. Vortex mixer.4. Autoclave.

SPECIMEN COLLECTIONThree to four week old viable cultures on drug free solid media should be used for testing.


IV. PNB SENSITIVITY TESTPRINCIPLE

REQUIRED REAGENTS

REQUIRED MATERIALS

REQUIRED EQUIPMENTS

SPECIMEN COLLECTION




TEST PROCEDURE IF THE PRIMARY ISOLATE IS SUSPECTED TO BE M.tuberculosis:l Draw a loopful of bacterial colonies from 3-4 weeks old cultures

obtained on a drug free solid medium.l Transfer these colonies into a sterile 1ml glass bottle containing 0.1ml

sterile distilled water or sterile saline with glass beads.l Cap the bottle tightly and subject the contents of the bottle to mechanical

shaking (vortexing) for 10 minutes to homogenize the uspension.l Keep the bottle in standing position for 10 minutes before opening.l Dilute the turbidity so obtained to match McFarland Standard No.1. This

is the isolate inoculum.0

l Bring the PNB SENSITIVITY TEST kit to room temperature (20-25 C) prior to inoculation. Retrieve one slant/vial from the PNB SENSIVITY TEST kit per isolate to be tested.

l Similarly, also retrieve 2 drug free L.J. control slants containing glycerol per isolate to be tested .

l Appropriately label the slant of PNB SENSITIVITY TEST and the two drug free L.J. control slants containing glycerol per isolate with the patient number /i.d.

l Inoculate 0.1ml of the isolate inoculum with the pipette on to the following:1. One PNB SENSITIVITY TEST vial.2. Two drug free L.J. control slants containing glycerol.

l Close all the caps tightly and incubate the three slants as follows:

Slants Temp. Incubation specifications Examine at0PNB slant 37 C Internally illuminated 3, 7, 14 and

incubator 21 days0L.J. control slant 1 37 C Internally illuminated 3, 7, 14 and

incubator 21 days0L.J. control slant 2 25 C Dark incubator 3, 7, 14 and

21 days

l When growth is evident on the drug free L.J. control slant incubated at 037 C, examine all the three slants for pigmentation.

TEST PROCEDURE IF THE PRIMARY ISOLATE IS SUSPECTED TO BE M.bovis:l Draw a loopful of bacterial colonies from 3-4 weeks old cultures



shaking (vortexing) for 10 minutes to homogenize the unspension.l Keep the bottle in standing position for 10 minutes before opening.l Dilute the turbidity so obtained to match McFarland Standard No.0.5.

This is the isolate inoculum.0

l Bring the PNB SENSITIVITY TEST kit to room temperature (20-25 C)

TEST PROCEDURE IF THE PRIMARY ISOLATE IS SUSPECTED TO BE :

Slants Temp. Incubation specifications Examine at

TEST PROCEDURE IF THE PRIMARY ISOLATE IS SUSPECTED TO BE :

M.tuberculosis

M.bovis

prior to inoculation. Retrieve one slant/vial from the PNB SENSIVITY TEST kit per isolate to be tested.

l Similarly, also retrieve 2 drug free L.J. control slants containing sodium pyruvate per isolate to be tested .

l Appropriately label the slant of PNB SENSITIVITY TEST and the two drug free L.J. control slants containing sodium pyruvate per isolate with the patient number /i.d.

l Inoculate 0.1ml of the isolate inoculum with the pipette on to the following:1. One PNB SENSITIVITY TEST vial.2. Two drug free L.J. control slants containing glycerol.

l Close all the caps tightly and incubate the three slants as follows:

Slants Temp. Incubation specifications Examine at0PNB slant 37 C Internally illuminated 3, 7, 14 and

incubator 21 days0L.J. control slant 1 37 C Internally illuminated 3, 7, 14 and

incubator 21 days0L.J. control slant 2 25 C Dark incubator 3, 7, 14 and

21 days

l When growth is evident on the drug free L.J. control slant incubated at 037 C, examine all the three slants for pigmentation.

INTERPRETATION OF RESULTSMembers of the Mycobacterium tuberculosis complex can be identified on the following basis:

0l Those that do not grow within three days at 37 C.

0l Those that do not grow at 25 C.l Those that do not grow on the PNB slant.l Those that do not produce yellow or orange pigmentations in the dark

and even after exposure to light.

V. TCH SENSITIVITY TESTPRINCIPLETCH SENSITIVITY TEST is used to distinguish M.bovis from other non-chromogenic slow growing mycobacteria including M.tuberculosis.

M.bovis is sensitive to low concentrations of Thiophen-2-carboxylic Acid Hydrazide (TCH), whereas M.tuberculosis and other species of mycobacteria are resistant.

REQUIRED REAGENTSTCH Sensitivity Test Kit (20404003).

REQUIRED MATERIALS1. Sterile hand gloves.

Slants Temp. Incubation specifications Examine at


V. TCH SENSITIVITY TESTPRINCIPLE

REQUIRED REAGENTS

REQUIRED MATERIALS




2. Face mask. 3. Activated 2% glutaraldehyde solution. 4. Micropippete (100µl - 500µl). 5. Sterile micropipette tips.6. Test tube stand. 7. Screw cap test tubes (16x125mm). 8. Sterile 1ml glass bottles with glass beads. 9. Mcfarland standard No. 1. 10. Sterile isotonic solution.

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Water bath /incubator at 37±2ºC. 3. Vortex mixer. 4. Autoclave.

SPECIMEN COLLECTIONThree to four week old viable cultures on drug free solid media should be used for testing.

TEST PROCEDURE l Draw a loopful of bacterial colonies from 3-4 weeks old cultures



shaking (vortexing) for 10 minutes to homogenize the suspension.l Keep the bottle in standing position for 10 minutes before opening.l Dilute the turbidity so obtained to match McFarland Standard No.0.5.

This is the isolate inoculum.0

l Bring the TCH SENSITIVITY TEST kit to room temperature (20-25 C) prior to inoculation.

l Appropriately label each set of TCH SENSITIVITY TEST slants with the patient number /i.d.

l Inoculate 0.1ml of the isolate inoculum with the pipette on to each of the following:1. R1- L.J. slants with TCH 10mg/litre (Sodium pyruvate and glycerol

free).2. R2- L.J. slants with Sodium pyruvate (TCH and glycerol free ).3. R3- L.J. slants with glycerol (TCH and Sodium pyruvate free ).

0 0l Close all the caps tightly and incubate the slants at 37 C±2 C for three

weeks.l Discard the glass bottle with glass beads in activated 2%

Glutaraldehyde solution for two hours prior to incineration.l After three weeks of incubation, read and record the results as follows:

REQUIRED EQUIPMENTS

SPECIMEN COLLECTION

TEST PROCEDURE


Slant R1 R2 R3 Results

No growth Growth No growth M. bovisObservations

Growth No growth Growth M. tuberculosis

VI. NITRATE REDUCTION KITPRINCIPLESome species of bacteria including Mycobacteria can be differentiated on the basis of their ability to reduce nitrate present in the medium to nitrite or nitrogen gases.

The presence of nitrite in the medium is indicated by the change in colour of the strip to pale pink or deep red or violet color. No change in colour of the strip only indicates that nitrite is not present in the medium.

There may be two explanations for this observation:l The nitrate may not have been reduced and hence the strain is nitrate

negative.l The nitrate may have been reduced to nitrite, and further the nitrite has

then been completely reduced to nitric oxide, nitrous oxide, or nitrogen which will not react with the Nitrite detection strips: the strain is nitrate -positive.

Any test medium that gives a negative result for the nitrite detection strip must be further tested to determine which of the two interpretations is correct. This is done by adding a small amount of zinc dust to all negative tests. The zinc dust will catalyze the reduction of nitrate to nitrite chemically. Thus, if the nitrate has not been reduced by the organisms. i.e., the strain are nitrate negative, it will be reduced by the zinc dust and the nitrite detection strip will show colour change from colourless to pink/red/violet. If no colour change in the Nitrite detection strip is observed after the addition of zinc dust, the organism have not only reduced nitrate to nitrite, but have further reduced nitrite to nitrogenous gases; these organism are to be reported as nitrate positive.

REQUIRED REAGENTSNitrate Reduction Kit (20405025).

REQUIRED MATERIALS1. Sterile hand gloves.2. Face mask. 3. Activated 2% glutaraldehyde solution. 4. Micropippete (100µl - 500µl). 5. Sterile micropipette tips. 6. Test tube stand. 7. Screw cap test tubes (16x125mm).


VI. NITRATE REDUCTION KITPRINCIPLE

REQUIRED REAGENTS

REQUIRED MATERIALS




8. Mcfarland Standard No. 10.9. Sterile 1ml screw capped glass vial with glass beads. 10. Sterile isotonic solution.

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Water bath /incubator at 37±2ºC. 3. Vortexing mixer. 4. Autoclave.

SPECIMEN COLLECTION AND PREPARATION1. Retrieve the require 3-4 weeks L. J. media/middlebrook slants of

MTBH37RV strain containing approximately more than 100 colonies.2. Transfer aseptically 9-10 loopful of the culture into a sterile 1ml screw

capped glass vial containing 0.1ml of sterile saline with glass beads. (Note: Care should be taken to transfer only the colonies and not the egg based media/agar media as these may give false turbidity resulting in false negative results.)

3. Cap the vial tightly and subject the contents of the bottle to mechanical shaking (vortexing) for 4-5 minutes to homogenize the suspension.

4. Keep the bottle in standing position for 10minutes before opening.5. Dilute the turbidity so obtained to match Mcfarland Standard No. 10.

TESTING PROCEDUREMethod Il In a clean sterile test tube aseptically pipette out 0.5ml of nitrate

substrate reagent (R1 reagent).l Aseptically emulsify 300µl to 400µl of MTB culture suspension

matching Mcfarland standard 10 in 0.5ml of nitrate substrate reagent (R1 reagent).

l Shake by hand to mix and incubate at 37±2ºC for 3 hours.l Retrieve the required number of nitrite detection strips.l Dip the Nitrite detection strip into the incubated test substrate for the

solution to be just absorbed on the reaction pad.ORPut one drop (10µl -25µl) of the incubated test substrate for the solution to be just absorbed on the reaction pad.

l Observe for colour change from colourless to pink/red/violet at 30-60 seconds.

l If no colour has developed, add a pinch of zinc dust, for confirmation in the incubated test substrate solution and carry out step 6 and 7.

Method IIl In a clean sterile test tube aseptically pipette out 0.5ml of nitrate

substrate reagent (R1 reagent).l Aseptically emulsify 100µl of MTB culture suspension matching

Mcfarland standard 10 in 0.5ml of nitrate substrate reagent (R1

REQUIRED EQUIPMENTS

SPECIMEN COLLECTION AND PREPARATION

TESTING PROCEDUREMethod I

Method II

reagent)l Shake by hand to mix and incubate at 37±2ºC for 16-24 hours.l Retrieve the required number of nitrite detection strips.l Dip the Nitrite detection strip into the incubated test substrate for the

solution to be just absorbed on the reaction pad.ORPut one drop (10µl -25µl) of the incubated test substrate for the solution to be just absorbed on the reaction pad.

l Observe for colour change from colourless to pink/red/violet at 30-60 seconds.

l If no colour has developed, add a pinch of zinc dust, for confirmation in the incubated test substrate solution and carry out step 6 and 7.


Colour change observedResultDirectly with nitrite With Nitrite detection strip

detection strip on addition of zinc dust

Pale pink or deep --- Nitrate positive strainred or violet

No colour change No colour change Nitrate positive strain

No colour change Pale pink or deep red Nitrate negative strainor violet

VII. BIOCHEMICAL TESTSUMMARYTuberculosis occurs worldwide and is rampant in many countries. Though curable, its infection is on the rise. The most specific test is the positive bacterial culture of a patient's sputum sample. This is cumbersome and time consuming, X-rays, smears for AFB and Tuberculin tests though comparatively rapid are not conclusive. Adenosine Deaminase (ADA) is an enzyme widely distributed in mammalian tissues, particularly in T Lymphocytes. Increases levels of ADA are found in various forms of tuberculosis making it a marker for the same. Though ADA is also increased in various infectious diseases like infectious Mononucleosis, Typhoid, Viral Hepatitis, initial stages of HIV, and in cases of malignant tumors, the same can be rules out clinically.

ADA-MTBPRINCIPLEAdenosine Deaminase hydrolyses adenosine to ammonia and inosine. The ammonia further reacts with a phenol and hypochlorite in an alkaline medium to form a blue indophenol complex with sodium nitroprusside acting as a catalyst. Intensity of the blue coloured indophenol complex formed is directly proportional to the amount of ADA present in the sample.

ADA2Adenosine+H O --------> Ammonia + Inosine


Colour change observedResultDirectly with nitrite With Nitrite detection strip

detection strip on addition of zinc dust

VII. BIOCHEMICAL TESTSUMMARY

ADA-MTBPRINCIPLE




AlkalineAmmonia+Phenol+Hypochlorite --------> Blue Indophenol Complex

Medium

REQUIRED REAGENTSADA-MTB Test Kit (20306015).

REQUIRED MATERIALS1. Sterile hand gloves. 2. Face mask. 3. Activated 2% glutaraldehyde solution. 4. Micropippete (500µl - 1000µl) and (10µl - 500µl). 5. Sterile micropipette tips. 6. Test tube stand.7. Test tubes.

REQUIRED EQUIPMENTS1. Water bath /incubator at 37±2ºC.2. Spectrophotometer/colorimeter with a filter of 570-630 nm wave

length. 3. Autoclave.

SPECIMEN COLLECTION AND PREPARATION.Collect specimen prior to use of antimicrobial agent. Wherever possible, indicate clearly the patient is on antitubercular drugs.

CSF: Collect as much as possible in a syringe, clean skin with alcohol before aspirating specimen. Body fluids: Disinfect the site and collect specimen with aseptic precautions. Serum, Plasma: No special preparation of the patient is required prior to sample collection by approved techniques. It is recommended to use fresh sample specimen for testing. Do not use haemolyzed, contaminated or turbid sample specimens. Fresh EDTA, citrate heparinised or oxalate anticoagulated plasma specimens are suitable for performing the test.

TEST PROCEDUREl Reagent Preparation1. Reagent L1(Buffer reagent), L2 (Adenosine Reagent) and Standard are

ready to use. 02. Adenosine Reagent (L2) may from crystals at 2-8 C. Dissolve the same

0 0by gently warming (37 C - 50 C) the reagent for some time before use. 3. Dilute both the Phenol Reagent (L3) & Hypochlorite Reagent (L4) to a

ratio of 1:5 with distilled water before use (1 part reagent + 4 parts of distilled water) these are working reagents.

4. The working Phenol Reagent and working Hypochlorite Reagent are 0stable for at least 6 months when stored at 2-8 C in tightly closed

bottles.

REQUIRED REAGENTS

REQUIRED MATERIALS

REQUIRED EQUIPMENTS

SPECIMEN COLLECTION AND PREPARATION.

TEST PROCEDUREReagent Preparation

l TESTING1) Bring all reagents and samples to room temperature before use.2) Set the spectrophotometer filter at 570-630 nm (Hg 578 or 623 nm) at

037 C.3) Pipette into clean dry test tubes labeled Blank (B), Standard (S), Sample

Blank (SB) and Test (T) as follows

Addition Sequence B S SB T

(ml) (ml) (ml) (ml)

Buffer Reagent 0.20 0.20 - -

Adenosine Reagent - - 0.20 0.20

Deionised water 0.20 - - -

Standard - 0.20 - -

Sample - - - 0.20

04) Mix well and incubate at 37 C for exactly 60 minutes, and then add the following:

Working Phenol Reagent 1.00 1.00 1.00 1.00

Sample - - - -

Working Hypochlorite 1.00 1.00 1.00 1.00Reagent

05) Mix well and incubate at 37 C for 15 minutes or at R.T. For 30 minutes.6) Measure the absorbance of the Blank (Abs. B), Standard (Abs.S),

Sample Blank (Abs.SB) and Test (Abs.T) against distilled water.

7) Calculations Abs.T-Abs.SB

Total ADA activity in U/L = ------------------------ x 50Abs.S-Abs.B

8) LinearityThe procedure is linear upto 150 U/L. If values exceed this limit dilute the sample with deionised water and repeat the assay.Calculate the value using the appropriate dilution factor.

INTERPRETATION OF RESULT:REFERENCE VALUES

Serum, Plasma, Pleural, Normal <30 U/LPericardial Suspect 30U/L to 40 U/L& Ascitic Fluids Strong Suspect >40U/L to 60 U/L

Positive >60U/L

CSF Normal <10U/LPositive >10U/L

It is recommended that each laboratory establish its own normal range representing its patient population.

TESTING

Addition Sequence B S SB T

(ml) (ml) (ml) (ml)

INTERPRETATION OF RESULT:REFERENCE VALUES




Examination for common infectionsSummaryAll sputa should be examined for pneumococcus, haemophilus and the other aerobic pathogens that commonly infect the bronchi and lungs. The recommended procedures are as follows.

Examination for common infection involves following steps:-1) Visual Observation:

Note whether the specimen contains opaque green-yellow pus. Do not examine specimens consisting of clear, watery saliva.

2) Homogenization: SummaryThere are advantages in homogenizing the specimen before making films and cultures. Most sputa are inhomogeneous. The purulent material, which contains most of the relevant pathogens, is usually embedded in clear mucoid secretion, and if the specimen is not first homogenized it is difficult to separate out a purulent portion for filming and culture. If the specimen is homogenized, every drop and loopful of it will contain some of the pathogens present. Moreover, the homogenized material is suitable for quantitative examinations.

REQUIRED REAGENTS/MEDIASSolution of dithiothreitol (Sputolysin/Calbiochem).

REQUIRED MATERIALS1. Sterile hand gloves. 2. Face mask. 3. Activated 2% glutaraldehyde solution.

REQUIRED EQUIPMENTS1. Vortex mixer.

0 02. Incubator at 37 C±2 C. 3. Machine that tilts to and fro.

PROCEDUREl Mix and incubate equal volumes of the sputum and a solution of

dithiothreitol (e.g. Sputolysin, Calbiochem).l With dithiothreitol, either mix rapidly on a vortex mixer for 15 seconds

and stand for 15 minutes at ambient temperature orl Preferably, mix gently and continuously on a machine that tilts to and fro

0placed in an incubator for 30 min at 37 C.l Take the homogenized sputum for microscopy, and culture.

3) MicroscopyREQUIRED REAGENTS/MEDIASModified grams stain kit (20750020 / 20750021).

Examination for common infectionsSummary

1) Visual Observation:

2) Homogenization: Summary


REQUIRED MATERIALS

REQUIRED EQUIPMENTS

PROCEDURE

3) MicroscopyREQUIRED REAGENTS/MEDIAS

REQUIRED MATERIALS1. Sterile hand gloves.2. Face mask. 3. Sterile plating loops(10µl). 4. Activated 2% glutaraldehyde solution. 5. Cedar wood oil. 6. Glass slide.

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens. 3. Autoclave.

PROCEDURE1. Gram Stainl Make a smear of the homogenized sputum or a purulent portion of

the sputum if it is not homogenised. l Stain by commercially available Modified grams stain kit. Follow

manufacturers instruction.l And examine with oil-immersion.

Observationl First note the presence and relative numbers of polymorphs and

squamous epithelial cells.l Next note whether there is a wide diversity of bacterial forms,

suggesting salivary contaimination, or the predominance of one potentially panthogenic form, e.g. Gram-positive diplococci (probably pneumococci), small slender Gram-negative bacilli (probably haemophili), or clustered Gram-positive cocci (probably Staphylococcus aureus.

Interpretationl If there are less than 10 polymorphs per square, the specimen is

probably mainly saliva.l If more, it is probably derived from an infected site in the lower

respiratory tract.l Numerous staphylococci is particularly significant, as it indicated

that treatment with a ß-lactamase resistant penicillin, such as flucloxacillin, is urgently required.

2. Wet filml Place approximately 100µl of homogenized sputum on a clean

scratch free glass slide.l Slowly place the cover slip on top.l Mount under microscope and examine with 10x and later 40x lens.l Carefully search for the presence of conidiophores.

REQUIRED MATERIALS

REQUIRED EQUIPMENTS

PROCEDUREGram Stain

Observation

Interpretation

2. Wet film




Observationl Note the presence of sporing heads.

Interpretationl The presence of such sporing heads indicates that the fungus is

growing in the bronchial tract.l While the observation of a few colonies growing in a culture of the

sputum may reflect only the recent inhalation of spores from the environment.

4) CultureSUMMARYA semi-quantitative method of culture is recommended, so that the presence

6of a potential pathogen in only small numbers, e.g. Less than 10 /ml sputum, may either be ignored or be reported to the physician as probably representing contamination of the specimen from the throat. If, however, antibiotic treatment had been started before the specimen was taken, or if special considerations apply, as in cystic fibrosis, the presence of a potential pathogen in small numbers should not be ignored.

REQUIRED REAGENTS/MEDIASl Blood agar base (AM1014/AM5014).

ORl Blood agar base (Ready prepared media - 250ml) (20500006).l Fildes digest agar.l Sabouraud dextrose agar (AM1014/AM5014).

ORl Blood agar base (Ready prepared media-250ml) (20600006).l Malt extract agar (AM1087/AM5087).l Nutrient broth (AM1077/AM5077).

ORl Nutrient broth (Ready prepared media-5ml) (20500006).l Peptone Water (AM1079/AM5079).

ORl Peptone Water (Ready prepared media-5ml) (20572006).l 50 mg optochin disk.l 1 unit benzyl pencillin disk.l 2 µg amoxycillin disk.l Gram negative bacteria identification test kit (20791001).l Neisseria identification kit (20795001).l Staph identification kit (20792001).l Candida identification kit Microxpress (20794001).

REQUIRED MATERIALS1. Sterile hand gloves. 2. Face mask. 3. Sterile plating loops(10µl).

Observation

Interpretation

4) CultureSUMMARY


REQUIRED MATERIALS

4. Activated 2% glutaraldehyde solution.5. Glass spreader. 6. 70% Isopropyl alcohol. 7. Sterile forceps.

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner.2. Autoclave.

PROCEDURE1. Dilute the 1-in-2 homogenized sputum a further 1 in 100 in Sterile

Nutrient Broth/Peptone water2. Inoculate a 0.005 ml loopful of the dilution on to each culture plate.

Blood AgarHeated blood (chocolate) agarFildes digest agarSabouraud dextrose agar ORMalt extract agar

3. The inoculum should be spread confluently over half of the plate and streaked out over the other half

4. Place 50 µg optochin disk, and 1 unit benzyl pencillin disk onto confluently seeded area of blood plate

5. Place 50 unit nystatin disk, onto confluently seeded area of Sabouraud dextrose agar or Malt extract agar.

6. Place 2 µg amoxycillin disk onto confluently seeded area of Fildes digest agar.

INCUBATION1. Incubate one set of Blood Agar, Heated blood (chocolate) agar, Fildes

0digest agar at 37 C for 18-24 hrs in humid air plus 5-10% CO , and re-2

incubate if the colonies are then still small and indistinct. nd 02. 2 set of each agar should be incubated at 37 C for 2-4 days

anaerobically. 3. Incubate one set of Sabouraud dextrose agar OR Malt extract agar at 35-

037 C for 2 days.nd 04. And 2 set of Sabouraud dextrose agar OR Malt extract agar at 28 C for

10 days.

OBSERVATIONObserve for growth and zone of inhibition.

INTERPRETATION1. The growth on the whole area of the plate of 25 or more colonies of the

6 same potential pathogen will then indicate that 10 or more of that pathogen were present in each millilitre of the original sputum.

2. Candida may be recognized as small opaque cream-coloured colonies with spiky projections on blood agar.

REQUIRED EQUIPMENTS

PROCEDURE

INCUBATION

OBSERVATION

INTERPRETATION




3. Sensitive to the respective antibiotics if zone of inhibition observed.4. Resistant to the respective antibiotics if no zone of inhibition observed.

Note: 1. Heated-blood ('chocolate') agar, may give better growth of pneumococcus and haemophilus,

2. Fildes digest agar, gives a very distinctive growth of H.influenzae and tends to suppress the growth of many salivary commensal bacteria.

3. If required further identification may be carried out to confirm Staphylococcal, Pseudomonas, Neisseria , Branhamella and other coliforms with commercially available identification kit.




References




st6. Tuberculosis; A Clinical HandBook, 1 Edition 1995, Edited by L.I. Lutwick.7. Clinical Diagnosis & Management by Laboratory Methods, Todd, Sanford &

thDavidsohn, 17 Edition 1998, Edited by john Bernard Henry.8. Tuberculosis Case Finding and Chemotherapy, K, Toman, World Health

Organisation, Geneva, 1979.9. Bombay Hospital Journal ; Drug Resistance in Tuberculosis; by Lina Deodhar

et al. April 1999.10. Laboratory methods for clinical and public health-Mycobacteriology, U.S.

Dept of health, education and welfare.11. Data on file: Tulip Diagnostics (P) Ltd.

Wound, Skin & Deep Sepsis

SUMMARYWound infections may be endogenous exogenous. Endogenous infections, or auto infections, are caused by organisms that have been leading a commensal existence elsewhere in the patient's body; for example, an abdomin surgical wound may become infected with organisms from the large bowel after an operation involving incision of the colon. In exogenous infections the source of the infecting organism outwith the body of the patient who becomes infected; cross-infection is a particular example of exogenous infection in which the caused organism is spread from person to person. Infection may occur after accidental or intentional trauma of the skin or other tissue; the latter type is often called 'surgical' or 'postoperative sepsis.

Infection of a wound is difficult to define and no clear rules can be given to distinguish it from contamination and colonization. Wounds and other open lesions are liable to contamination with a multiplicity of organisms from the surfaces and environment; the contaminating organisms are at first generally present in relatively small numbers, as originally introduced and need not subsequently multiply. Infection occurs when one or more of the contaminants evades the clearing effect of the host's defences, replicates in large numbers, and attacks and harms the host's tissues. In the case of a commensal or low-grade pathogen, the multiplication may cause little or no harm to the host and may best be described as colonization. Whether harmful infection or harmless colonization is dependent on the virulence of the organisms and the local condition is therefore important in assessing the significance of bacteriological findings.

SUMMARY REQUIRED REAGENTS/MEDIASBlood agar base (AM1014/AM5014). ORBlood agar base (Ready prepared media-250ml) (20500006).Modified grams stain kit (20750020 / 20750021).Stuart transport medium (AM1094/AM5094).Mycostain kit and Acidfast decolorizer (20307100 / 20308500).Immunofluorescent stain.Nutrient broth (AM1077/AM5077).ORNutrient broth (Ready prepared media-5ml) (20500006).Peptone Water (AM1079/AM5079).ORPeptone Water (Ready prepared media-5ml) (20572006).MacConkey Agar (AM1061/AM5061).ORMacConkey Agar (Ready prepared media-250ml) (20550006/10550006).CLED Agar (AM1026/AM5026).ORCLED Agar (Ready prepared media-250ml) (20520006/10520006).PNPG Blood Agar (0.43 gm p-nitrophenyl glycerol or blood agar containing 2-3 times extra agar).Gram negative bacteria identification kit (20791001).Staph identification kit (20792001). Strept identification kit (20793001).

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l Homogenize in a tissue grinder with a little sterile Nutrient Broth/Peptone water broth.

Cooked meat medium (AM1030/AM5030) (BIS formula - AM103011/AM503011)

REQUIRED MATERIALS1. Sterile hand gloves. 2. Face mask. 3. Sterile plating loops (10µl). 4. Activated 2% glutaraldehyde solution. 5. Cedar wood oil. 6. Glass slide. 7. 70% isopropyl alcohol in water with 1% iodine or 1-2% chlorhexidine. 8. Plain, albumen-coated or charcoal coated sterile cotton wool swab. 9. Glass spreader. 10. 70% Isopropyl alcohol.11. Sterile forceps.

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Microscope. 3. Autoclave.


Wear sterile gloves.With a plain, albumen-coated or charcoal coated sterile cotton wool swab collect the exudates as much as possible from the area that is inflamed or bears exudates. Replace the swab in its tube with care not to soil the rim.It should be taken for testing within a hour or should be placed in a

0refrigerator at 4 C until testing or if it has to be transported it should be submitted in a tube of Stuart transport medium Collect pus or exudate in a small screw capped bottle, a firmly stopperred tube or syringe or a sealed capillary tube.Extra swab should be placed and broken into a cooked meat broth immediately it has been taken from the lesion in the ward or at operation.Note: Pathogens grow in directly seed broth and in subcultures from it.Collect the blood sample for culturing (refer blood culture for details) if the patient is flexible or in shock, or it seems possible that his local infection is accompanied by a bacteriaemia.Collect fragments of excised tissue removed at wound toilet, or curettings from infected sinuses and other tissues, should be sent in a sterile container without fixative.

REQUIRED MATERIALS

REQUIRED EQUIPMENTS


Fluid AspiratesAspirates specimen from joints, pleural, pericardial and peritonial l Collect aseptically, and centrifuge to deposit the cells and bacteria.l Discard the supernatant into disinfectant (2% activated glutaraldehyde).l Examine for tuberculosis as described in (Examination for tuberculosis).

Peritoneal dialysisMany patients with renal failure are now treated by the procedure of pertioneal dialysis, which exposes them to the risk of bacteria being introduced into the peritoneum and causing a serious peritonitis.l Centrifuge the effluent as described for fluid aspirates.

b. Laboratory ExaminationThe basic procedures usually include:-l A naked eye examination of the specimen.l The microscopical examination of a Gram film and a wet film.l Culture on aerobic and anaerobic blood agar plates on MacConkey agar

and in cooked-meat broth.l Gas chromatography may be performed directly on liquid specimens to

indicate the presence of anaerobes.

1. Naked-eye ExaminationNote the appearance of a specimen of pus or exudate on initial examination.

Interpretationl The pus of a staphylococcal lesion is typically creamy and thick in

consistency, with pus cells evident on microscopy.l That of a Streptococcus pyogenes infection is generally straw-

coloured and watery, with lysis of pus cells seen on microscopy.l That of proteus infection has a fishy smell.l That of pseudomonas infection a sweet,musty odour and often a

blue pigmentation .l Pus containing anaerobic organisms often has an offensive putrid

smell.l That of actinomycosis often contains small microcolonies that

appear as 'sulphur granules'.l In some fungal infections such as mycentoma, black or brown

granules may be present.l The pus of an amoebic abscess is said to resemble anchovy sauce.

2. Microscopy1. Gram Stainl Make a smear of the swab exudates /centrifuged deposit on a

clean scratch free glass slide.l Stain by commercially available modified grams stain. Follow

manufacturers instruction.

Fluid Aspirates

Peritoneal dialysis

b. Laboratory Examination

1. Naked-eye Examination

Interpretation

2. Microscopy1. Gram Stain




l And examine with oil-immersion.

Observationl Note the presence and relative numbers of polymorphs and

bacteria.l Pay particular attention to the numbers and variety of

different morphological forms of Gram-positive and Gram-negative bacteria.

Interpretationl Gram positive cocci in typical clusters or chains may suggest a

staphylococcal or streptococcal infection.l The appearance of Gram-positive diplococci may be given by

either pneumococci or enterococci. Faintly staining Gram-negative rods are sometimes missed if much background debris is heavily counterstained.

l Gram-variable filaments of actinomyces may appear like chains of cocci and their fragments as diphtheroid bacilli.

l Many Gram-positive clostridia appear as Gram-positive forms in pus.

2. Wet filml Place approximately 100µl of swab exudates/centrifuged

deposit on a clean scratch free glass slide (note: if dry dilute with sterile saline).

l Slowly place the cover slip on top.l Mount under microscope and examine with 10x and later 40x

lens.

Observationl Carefully search for the presence of fungi, motile bacteria and

uric acid crystals.

Interpretationl Presence of uric acid crystals, may be responsible for the

condition of septic arthritis in the absence of any infection.l Presence of fungal - Fungal infection.l Presence of motile bacteria - Bacterial infection.

(Note: Darkground microscopy of a wet film is useful in the diagnosis of primary syphilis.)

Observation

Interpretation

2. Wet film

Observation

Interpretation

3. Ziehl-Neelsen methodl Make a smear of the swab exudates/centrifuged deposit on a

clean scratch free glass slide.l Stain by commercially available Mycostain kit and Acidfast

decolorizer. Follow manufacturers instruction.l And examine with oil-immersion.

(Note: 1. A smear stained by the Ziehl-Neelsen method should be examined when the clinical circumstances suggest that the tubercle bacillus, another mycobacterium or a nocardia may be present, e.g. In chronic and neck abscesses. 2. Immunofluorescent staining allows the prompt identification of pathogenes for which specific antisera are available, e.g. Some pathogenic clostridia.)

3. Culture1) Inoculate the specimen on the following media l Blood Agarl MacConkey Agar or CLED Agarl Cooked Meat Brothl PNPG Blood Agar (0.43 gm p-nitrophenyl glycerol or blood

agar containing 2-3 times extra agar).2) Place following antibiotics disks on to blood agar plate prior to

incubation.l One unit benzyl penicillin and 10 µg gentamicin disk on one

plate.l 5 µg metronidazole and 50 µg neomycin on another blood

agar plate.3) Incubatel One plate with One unit benzyl penicillin and 10 µg

0 0gentamicin aerobically air plus 5-10% CO at 37 C + 2 C, 2

l Second plate with 5 µg metronidazole and 50 µg neomycin 0anaerobically in nitrogen / hydrogen plus 5-10% CO at 37 C 2

0+ 2 C,0 0

l Remaining media incubate aerobically at 37 C + 2 C, l All should be incubated for a period of 24-48 hours.

Note:- In case of indication of slow growing pathogen like some species of Actinomyces or Bacteroides it should be further incubated for a period of 7 days.

3. Ziehl-Neelsen method

3. Culture




MediaMedia Incubation Incubation GrowthGrowth SensitivitySensitivity

ObservationObservation

Blood Agar

MaConkey Agar with sodium taurocholate

CLED Agar

Cooked Meat Broth

PNPG Blood agar with 2-3 times the usual concentration of Agar

Showing zones of inhibition or no zones of inhibition towards respective antibiotics disks

Aerobically


Anaerobically


Aerobically

1) Growth/No growth2) Growth with or without lysis of RBC's

1) Growth/No growth2) Growth-Pink colonies or colourless

colonies

1) Growth/No growth2) Growth with or without lysis of RBC's

1) Growth/No growth2) Growth-Pink colonies or Blue

colourless coloniesAerobically

Aerobically Growth/No growth

Aerobically Growth/No growth

IdentificationThe obtained cultures should be purified and subjected to following identification test.1) Coagulase2) Lance field grouping3) Biochemical identification for Staph, Strept and Gram negative

coliforms with commercially available biochemical identification test kit.

Interpretation and ReportingA pure growth of a recognized pathogen obtained (as per the identification) from a wound or closed abscess is easily interpreted as significant and will be reported.

In case of mixed cultures grown from superficial lesions contaminated with commensal and saprophytic organisms it is disregarded as insignificant. The result may than be reported on following lines. “ Many

Identification

Interpretation and Reporting

mixed faecal and skin bacteria present', without giving identities or antibiotic sensitivities.








References

Genital Tract InfectionsIntroductionThe laboratory approach to the diagnosis of genital tract infections is best considered in relation to the sex of the patient. Although some of the specific

Introduction infections, e.g gonorrhoea, syphilis and chlamydial infection, are common to both-sexes, there are usually differences in the presenting symptoms and the sites and the sites and methods of collection of specimens in these infections. Moreover,




some other infections, e.g. Vaginitis and uterine sepsis, are confined to women.

Genital infections in WomenSummaryThese include urethritis, vaginitis, vaginosis, genital ulceration, cervicitis, uterine sepsis, salpingitis, ophoritis and the condition recognized as pelvic in inflammatory disease.

REQUIRED REAGENTS/MEDIASl Blood agar base (AM1014/AM5014).

ORl Blood agar base (Ready prepared media-250ml) (20500006).l Modified grams stain kit (20750020 / 20750021).l Stuart transport medium (AM1094/AM5094).l Trichomonas transport medium.l Sabouraud dextrose agar (AM1087/AM5087).

ORl Sabouraud dextrose agar (20600006/10600006).l Malt extract agar (AM1067/AM5067).l Peptone starch dextrose agar.l Modified New York City selective medium.l Blood agar containing metronidazole 2.5 g/litre.l Candida identification test kit (20794001).l Neisseria identification kit (20795001).l Amies transport medium.l 5 µg metronidazole disk.l 50 µg neomycin disk.l PPLO broth.l Horse serum.l Urea.l Phenol red indicator.

REQUIRED MATERIALS1. Sterile hand gloves. 2. Face mask. 3. Sterile plating loops (10µl). 4. Activated 2% glutaraldehyde solution. 5. Cedar wood oil, glass slide. 6. 70% isopropyl alcohol in water with 1% iodine or 1-2% chlorhexidine. 7. Plain, albumen-coated or charcoal coated sterile cotton wool swab. 8. Glass spreader. 9. 70% Isopropyl alcohol. 10. Sterile forceps.

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Microscope.

Genital infections in WomenSummary


REQUIRED MATERIALS

REQUIRED EQUIPMENTS

3. Autoclave. 0 04. Incubator with air plus 5% CO at 37 C + 2 C.2

0 05. Anaerobic Incubator CO at 37 C + 2 C. 2

PROCEDURECollection of specimen Collect two swabs of the following one for making films and other for seeding cultures.l For diagnosis of vaginitis , vaginosis or uterine sepsis collect a high vaginal

swab.l Insert the swab into the upper part of the vagina and rotate there before

withdrawing it, so that exudate is collected from the upper as well as the lower vaginal wall.

l For examination of gonococci collect an endocervical swab.1. Use vaginal speculum to provide a clear sight of the cervix and rub the

swab in and around the introitus of the cervix and withdraw without contamination from the vaginal wall.

2. Take swabs from any exudate discharged from the meatus of the urethra, or a Bartholin's gland and also rectal and pharyngeal.

3. Place the swabs for culture in tubes of Amies transport medium/ Stuart transport media for delivery to the laboratory.

l For examination for Trichomonas, collect swab from the vagina and cervix.1. Place swab in clear Trichomonas transport medium for microscopy and

possibly culture.

Microscopy1. Gram Stainl Make a smear of the swab exudates on a clean scratch free glass slidel Stain by commercially available modified grams stain kit. Follow

manufacturers instruction.l And examine with oil-immersion.

Observation and Interpretationl Gram positive yeast cells- Candidosisl Gram positive hyphae- Pseudomyceliuml Small Gram negative or Gram variable bacilli of diptheroid morphology-

anaerobic vaginosis.l Gram negative diplococci from endocervical swab- gonococci infection.(Note:- Positive smears must be confirmed by culture).

l Place approximately 100µl of swab exudates on a clean scratch free glass slide(note: if dry dilute with sterile saline)

l If enough fluid cannot be expressed add a little sterile saline.l Slowly place the cover slip.l Mount under microscope and examine with 10x and later 40x lens.

2. Wet film.

PROCEDURECollection of specimen

Microscopy1. Gram Stain

Observation and Interpretation

2. Wet film.




Observation and InterpretationObserve under 10x and 40x dry objectives for:l Presence of rounded or pear shaped Trichomonas showing jerky motility.l Presence of polymorphs or the yeast and Hyphal form of Candida.

3. Culture1) Inoculate the specimen on the following medial Blood Agar

2) Place following antibiotics disks on to blood agar plate prior to incubation.l 5 µg metronidazole and 50 µg neomycin.

3) Incubate0 0

l One plate in a humid aerobically air plus 5% CO at 37 C + 2 C .20 0

l Second plate anaerobically with CO at 37 C + 2 C.2

Observationl Examine after 18-24 hours and again after reincubation for another 24

hours.(Note: Gardnerella vaginalis, anaerobic cocci and bacilli, and candida may show very little growth after 18-24hours.)

Interpretationl Candidosis - Appearance of white colonies with spiky projections and

growth in the inhibition zones around antibacterial disks.l Gardnerella vaginalis - Growth in 48 hours of numerous very small

colonies on both the aerobic and the anaerobic blood agar plate.l Neisseria gonorrhoea - Good growth on blood agar plate incubated in a

0 0humid aerobically air plus 5% CO at 37 C + 2 C .2

(Note: 1. When the clinical features or the appearances in the Gram smear suggest that there may be a. Gonococcal infection - the specimen should be inoculated additionally

on to a plate of moist heated-blood ('cholate') agar and a plate of the 0modified New York City selective medium for incubation at 35- 37 C in

air plus 5-10% CO2.

b. Anaerobic vaginosis - it is helpful to inoculate the specimen also on to peptone-starch-dextrose agar

c. Candidosis - it is helpful to inoculate the specimen also on to Sabouraud dextrose agar or malt extract agar and placing a 50 unit nystatin disk and a 20µg amphotericin disk.)

d. Uterine sepsis - the specimen should be inoculated on to Macconkey and into cooked meat broth as well as on both blood agar plate and examined for pyogenic bacteria as described for wound swabs.

e. Actinomycotic infection- anaerobic culture should be continued for 7 days and the specimen should be seeded in to an additional plate of

Observation and Interpretation

3. Culture

Observation

Interpretation

selective medium, e.g. Blood agar containing metronidazole 2.5 g/litre.)

Identification:The obtained cultures should be purified and subjected to following identification test.1) Starch hydrolyses on peptone starch dextrose agar - (Gardnerella

vaginalis -positive)2) Biochemical identification for Candida and Neisseria gonorrhea with

commercially available identification kit.

Non-specific genital infection (NSGI).1. Chlamydia trachomatisl Scrappings of epithelial cells from the cervix and urethra should be

collected on microscope slides for the identification of elementary chlamydial bodies and larger inclusions in the cells' cytoplasm by immunofluorescence with specific, preferably monoclonal antoserum.

l Scrappings may otherwise be examined in an enzyme-linked immunosorbent assay (ELISA) test.

l Scrappings collected in antibiotic-free transport medium for culture in irradiated McCoy cells.

2. Mycoplasma infection:l Ureaplasma can be cultured from cervical or vaginal swabs and the

centrifuged deposit from the first 30-40 ml of voided urine.

Note:- Their isolation and identification is aided by their ability to hydrolyse urea.

Test Procedurel Make 10 fold dilutions of the specimen in small (0.9 ml) volumes of

liquid medium comprising PPLO broth, horse serum, urea and phenol red indicator.

1l Express the exudate from the swab into 0.9 ml medium to obtain a 10 ,

dilution.6

l Prepare further dilutions up to 10 .

Incubation0

l Incubate at 37 C and examine for a colour change each day up to 7 days.

Observationl With colour changes resulting from ureaplasma growth the medium

remains clear.l Any turbidity is a sign of bacterial contamination which invalidates the

result.

Identification:

Non-specific genital infection (NSGI).1. Chlamydia trachomatis

2. Mycoplasma infection:

Note:-

Test Procedure

Incubation

Observation




3. Syphilis and herpes:-Examine the exudate by darkground microscopy for the presence of Treponemes.

In suspected genital herpes, fluid from any vesicle and exudate collected from the base of ulcers should be placed in virus transport medium and submitted for culture for herpes simplex virus.

Genital infections in menSummaryThe infections in men are mostly caused by the same organisms as in women, but are seldom asymptomatic.

Collection of Specimens:-1. Direct collectionl Collect the urethral discharge milked from the urethra directly on to

slides for examination in Gram-stained films for gonococci.l And inoculate immediately on to warmed plated of heated-blood agar

and selective medium for the culture of gonococci.

For transportation:-l Collect exudate as much as possible on a swab, plunged the swab into a

tube of Amies transport.

Prostatitis suspected:-l NO spontaneous discharge form the urethra.l Massage the prostate per rectum which may express some exudate for

3. Syphilis and herpes:-

Genital infections in menSummary

Collection of Specimens:-1. Direct collection

For transportation:-

Prostatitis suspected:-

examination.l The examination of a chancre requires the careful collection of exudate

and its preparation for darkground microscopy.l A specimen of clotted venous blood should be collected for serological

examination.

Laboratory examinations:-Procedure is similar as for genital infection in women.

Note: The interpretation of results is usually easier in male than female infections, for specimens of urethral discharge and exudate from ulcers are less likely to be contaminated with organisms from the perineum.








Laboratory examinations:-

Note:

References

Gastrointestinal InfectionsSUMMARYThe commonest specimens examined for gastrointestinal infections are those of faeces from patients with diarrhoea, with or without abdominal plain or vomiting. Formed stools may be submitted from patients suspected of having enteric fever, helminthiasis or the subclinical carriage of an intestinal pathogen, and clotted blood may be submitted for serological examination for suspected enteric fever or an intestinal virus infection.

In infants under 3 years old, many cases of gastroenteritis are caused by rotaviruses. In patients treated with antibiotics etc, for prophylaxis during intestinal surgery. Serve enterocolitis may be caused by a drug-resistant strain of Staphylococcus aureus and a life-threathening pseudomembranous colitis by Clostridium difficile. Milder, simple diarrhoea often follows prolonged treatment with any of a variety of antibiotics which deranges the bowel flora and prediposes to superinfection with various drug-resistant bacteria. Candida albicans or Cyrptosporidium.

SUMMARY Particularly in childhood, diarrhoea may be caused by infections elsewhere than in the gastrointestinal tract, e.g. By respiratory, urinary and septicaemia infections, and by vertain ono-infective conditions such as the food allergies.

REQUIRED REAGENTSl Preston campylobacter enrichment broth.l Preston Agar Base (AM10831/AM50831).l Preston Selective Supplement (AS 50231).l Campylobacter selective medium.l Deoxycholate citrate agar (DCA) (AM 1031/AM 5031).l Xylose Lysine Dextrose Agar (XLD) (AM 1112/AM 5112).l Selinite F Broth (AM 1089/AM 5089).l Buffered glycerol saline transport medium.l Phosphate buffered saline.

orl 0.1% Peptone Water.

REQUIRED REAGENTS




l Nutrient agar (20570006/10570006 /AM1074/AM5074).or

l Urea agar.l MacConkey agar (AM1061/AM5061).l Blood agar (20500006 /AM1014/AM5014).l 6% NaCl nutrient agar (AM1074, AM5074).l Mannitol salt agar (AM1069, AM5069). l Poly-myxin B.l Selective medium of Holbrook and Anderson.l Modified Gram’s Stain Kit (20750020/20750021).l TCBS agar (AM1095/AM5095).l Alkaline peptone water (AM1910/AM5910) .l Oxidase reagent (20690040).l Peptone water with phenol red and glucose (AM1080/AM5080).l Peptone water with phenol red and sucrose (AM1080/AM5080). l Alkaline peptone water (AM1910/AM5910). l CLED-(AM1026/AM5026 and 20520006,10520006).l Tellurite taurocholate gelatin agar.l 1% sodium dimethyl-p-phenylenediamine monohydrochloride.

REQUIRED MATERIALS1. Sterile hand gloves. 2. Screw- capped glass or plastic container with a 'spoon' projecting from the

underside of the cap of about 25ml capacity. 3. Face mask. 4. Sterile plating loops (10µl). 5. Activated 2% glutaraldehyde solution.6. Cedar wood oil.7. Glass slide.

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens. 3. Autoclave. 4. Incubator at 37°C±2°C.

Collection of specimenl Obtain a specimen of faeces . l Collect a specimen of venous blood for blood culture if enteric fever is

suspected, l Collect a sample of the suspect food if food poisoning is suspected because a

cluster of cases are related to the eating of a common foodstuff. l Collect paired acute and convalescent samples of clotted blood for serological

examinations, at an interval of about 10 days in suspected enteric fever and 2-4 weeks in suspected viral infection.

REQUIRED MATERIALS

REQUIRED EQUIPMENTS

Collection of specimen

Faecesl Make the collection in a disposable, wide-mouthed, screw- capped glass or

plastic container with a 'spoon' projecting from the underside of the cap of about 25ml capacity.

l Collect faeces passed into a clean bedpan, unmixed with urine or disinfectant or from the surface of heavily soiled toilet paper.

l Collect 1-2 ml of faeces on the spoon and insert it, carried on the spoon, into the bottle.

l Take care not to soil the rim or outside of the bottle.l Apply the cap tightly. l Do not collect several spoonfuls or attempt to fill the container.l Transmit the specimen quickly to the laboratory.l If delay is unavoidable, and particularly when the weather is warm, collect the

faeces in a container holding about 6 ml buffered glycerol saline transport medium.

Examination of faeces1) Naked eye observation

The faecal sample should first be inspected with the naked eye for l Its consistency whether formed or fluid.l The presence of mucus, pus and blood indicative of severe dysentery.l The presence of helminths.

2) CulturingPreparation of faecal suspensionDilute the portion of a solid specimen (0.3 gms) in 3 ml of Phosphate buffered saline or 0.1% Peptone Water.

FOR CULTURING OF CAMPYLOBACTERl Inoculate one or two loopfuls of the faecal suspension on a plate of

campylobacter selective medium, e.g. The Skirrow or Preston medium. (Note: If the faeces is more than 24 hours old, inoculate one or two loopfuls into a tube of Preston campylobacter enrichment broth,

0incubate for 24 hours at 42-43 C, and then subculture on to a plate of Campylobacter selective medium.)

Incubation0

l Incubate for 48 hours at 42-43 C under microaerophilic conditions 5-6% O , 7-10% CO in H or N2 2 2 2 .

FOR CULTURING OF SALMONELLA AND SHIGELLAl Seed a plate of selective medium, e.g Deoxycholate citrate agar (DCA)

and XLD and a tube of one enrichment broth. e.g Selinite F Brothl Inoculate one or two loopfuls of the faecal suspension in to the DCA

plate, stroking out with care to yield many separate colonies, and Inoculate one or two drops of the suspension into the selenite F broth,

Faeces

Examination of faeces1) Naked eye observation

2) Culturing

FOR CULTURING OF CAMPYLOBACTER

Incubation

FOR CULTURING OF SALMONELLA AND SHIGELLA




Incubation0

l Incubate aerobically for 18-24 h at 37 C.

Observationl Inspect the plate for pale (non-lactose-fermenting) enterobacterial

colonies on DCA.l Pick and prepare a pure subculture (e.g. On a nutrient agar or urea agar

slope) from each of any different morphological types of pale colonies.l Observe the Selenite F broth tubes for Turbidity/Growth.l If Turbidity/Growth observed streak out a loopful of the selenite culture

on DCA.l Incubate this plate overnight and examine for pale colonies as before.l Pick one colony as cleanly as possible and plate it out on MacConkey

agar to obtain pure well separated pale colonies the following day. Use this pure culture for identification.

(Note: 1. Xylose lysine deoxycholate (XLD) agar has considerable advantages as a primary plating medium for faeces and its use instead of DCA to S. dysenteriae and S.flexneri, and it distinguishes most salmonellas (red colonies with black centres) from shigellas (red colonies without black centres) and most non-pathogenic coliforms (yellow colonies). 2. The chances of isolating a scanty salmonella or shigella will be increased if additional selective and enrichment media are seeded.)

OTHER FOOD-POISONING BACTERIAINTRODUCTIONIf the epidemiological circumstances suggest that the patient has been involved in an outbreak of food-poisoning and if the sample of his faeces has been collected within 3 days of the start for his illness, examine the sample for Clostridium perfringens, Staphylococcus aureus, Bacillus cereus and Vibrio parahaemolyticus as well as for campylobacter, salmonella and shigella. If possible also obtain a sample of the suspected food-stuff for culture .

FOR CLOSTRIDIUM PERFRINGENSNote: Clostridium perfringens is the causative of outbreaks due to well cooked foods stuff as their spores are more likely to survive cooking. Culture should therefore be done by a semi-quantitative method, for faeces collected from patients at the height of the illness commonly contain 1000000 or more C.perfringens per gram and it is only the finding of such high counts that should be reported as probably significant.

FOR STAPHYLOCOCCUS AUREUSPlate out a few loopfuls of a 1 in 10 saline suspension of the faeces on plates of 1. Blood agar (AM1014/AM5014), 2. MacConkey agar (AM1061/AM5061) 3. Selective medium. e.g. 6% NaCl nutrient agar (AM1074, AM5074), 4. Mannitol salt agar, plus 1250 units poly-myxin B/litre.

Incubation

Observation

OTHER FOOD-POISONING BACTERIAINTRODUCTION

FOR

FOR

CLOSTRIDIUM PERFRINGENS

STAPHYLOCOCCUS AUREUS

Incubation0Incubate aerobically for 18-24 hrs at 37 C and examine for colonies of S.aureus.

Note:- In an outbreak, send subcultures from each patient to a refernce laboratory for phagetyping and tests for enterotoxin production.

FOR BACILLUS CEREUSNote:- Bacillus cereus causative is particularly when the outbreak is attributed to the eating of a rice dish.

Inoculate loopfuls of faecal suspension on to plates of 1. Blood agar 2. MacConkey agar 3. Selective medium of Holbrook and Anderson

Incubation0Incubate aerobically for 18-24 hrs at 37 C.

ObservationLook for large rough pale colonies on MacConkey agar and blue colonies surrounded by a precipitate on Holbrook and Anderson's medium .

IdentificationIdentify them by their appearance in a Gram film with commercially available modified grams stain kit.

FOR VIBRO PARAHAEMOLYTICUSNote:- Particularly in outbreaks attribute to raw sea fish and shellfish, plate a few loopfuls of a 1 in 10 suspension of faeces on1) Thiosulphate citrate bile sucrose (TCBS) agar (AM1095/AM5095) and

0incubate aerobically for 18-24 hrs at 37 C.2) Also inoculate a portion of the faecal suspension into an equal volume of

double strength alkaline (pH 8.8) peptone water, incubate for 18-24 hrs at 0 025 C, then subculture on to a TCBS plate and incubate for 18-24 hrs at 37 C.

ObservationInspect the plates for large (2-5 mm) blue or green, (non-sucrose-fermenting) colonies.

IdentificationDemonstrate by 1. Gram filming with commercially available modified grams stain kit.2. That they are oxidase positive with commercially available oxidase reagent. 3. Other biochemical identification test as follows:l Suspend a colony in 3% NaCl solution and inoculate drops of the

suspension into following media for identifying V.parahaemolyticus. l Peptone water with phenol red and glucose.

Incubation

Note:-

FOR Note:-

Incubation

Observation

Identification

FOR Note:-

Observation

Identification

BACILLUS CEREUS

VIBRO PARAHAEMOLYTICUS




l Peptone water with phenol red and sucrose l Alkaline peptone water with 8% NaCl l Alkaline peptone water without 8% NaCll CLED

Note: V.parahaemolyticus ferments glucose but not sucrose with production of acid but not gas. V.parahaemolyticus grows in Alkaline peptone water with 8% NaCl but not in Alkaline peptone water without 8% NaCl and on CLED.

INFANTILE GASTROENTERITISMost cases appear to be viruses particularly rotaviruses, which only rarely cause gastrogenteritis in children more than a few years old or in adults.

ENTEROPATHOGENIC ESCHERICHIA COLISummaryThere is still no test for enteropathogenic properties suitable for routine application to faecal isolates. It is therefore probably wisest not to examine faeces for E.coli in sporadic cases of infantile diarrhoea, but only to do so when there is an outbreak among infants and other common intestinal pathogens have not been found.

Procedure1) Plate a loopful of faecal suspension on a blood agar plate and MacConkey

0plate and incubate 18-24 h at 37 C.2) Test from 3-10 E.coli-like colonies from the blood agar-(2050006/

AM1014/AM5014) plate for slide agglutination within 1 min with pools of polyvalent sera for enteropathogenic serotypes of E.coli.

3) If any colony gives a strong reaction with one of the pools,l Inoculate the remainder of it on to a nutrient agar slope and incubate the

slope overnight. l Prepare a dense suspension from the slope culture in saline and test it by

slide agglutination with monovalent sera to identify the K antigen.0

l Then heat the suspension for 1 hr at 100 C, cool it and repeat the slide tests with the monovalent sera to identify the O antigen.

l Confirm the O serotype by demonstrating tube agglutination to the titre of the serum.

ENTEROTOXIGENIC Escherichia coli SummaryStrains of E.coli producing heat-stable or heat-labile enterotoxin may cause diarrhoea in adults and children who have not previously encountered them and should be sought particularly in cases of 'traveller's diarrhoea'.

Procedure1. Plate a loopful of faecal suspension on a blood agar plate and MacConkey

0plate and incubate 18-24 h at 37 C.2. Test from 3-10 E.coli-like colonies from the blood agar-(2050006/

AM1014/AM5014) plate for slide. agglutination within 1 min with pools of

INFANTILE GASTROENTERITIS

ENTEROPATHOGENIC Summary

Procedure

ENTEROTOXIGENIC Summary

Procedure

ESCHERICHIA COLI

Escherichia coli

polyvalent sera for enterotoxigenic serotypes of E.coli.

MICROSCOPY FOR PROTOZOA, CYSTS AND OVAIntroductionUsually a faeces sample is not examined microscopically unless the clinical particulars or failure to demonstrate an alternative pathogen suggests that the patient's illness may be due to amoebiasis, giardiasis, balantidiosis, crytosporidiosis or helminthiasis.

Procedurel Prepare a wet film of a concentrate of the faeces .l Examine for protozoa, protozoal cysts and helminth ova at 10X, 40X .l Stain film for the oocysts of cryptospordium. l Apply an adhesive, Cello tape-tipped swab to the perianal skin and examine

microscopically for threadworm (enterobius) ova.

MICROSCOPY FOR Staphylococcal enterocolitisSummaryWhen this life-threatening condition is suspected, e.g. In a patient with severe diarrhoea following intestinal surgery and antibiotic treatment, a bacteriological diagonsis is urgently required.

ProcedureMake a Gram-stained film of the faeces with commercially available modified grams stain kit and examine it for very numerous Gram-positive cocci largely replacing the normal mixed bacterial flora.

Note: Immediately report the finding to physician.

OTHER INTESTINAL PATHOGENSIntroductionCulture of faeces for other intestinal pathogens should be attempted when the circumstances suggest they may be present.

For Clostridium difficileAlways examine for this organism in cases in which pseudomembranous colotis is suspected. The faeces may be tested directly for cytotoxin.

For Vibro choleraeIntroductionThis important pathogen is not indigenous in most countries, but its presence should be sought in patients suffering from diarrhoea just after returning by air from a country in which cholera is known to be present.

Procedure1) Culture the faces in alkaline peptone water on TCBS agar and on tellurite

taurocholate gelatin agar.

MICROSCOPY FOR PROTOZOA, CYSTS AND OVAIntroduction

Procedure

MICROSCOPY FOR Summary

Procedure

OTHER INTESTINAL PATHOGENSIntroduction

For

For Introduction

Procedure

Staphylococcal enterocolitis

Clostridium difficile

Vibro cholerae




2) Identify yellow (sucrose-fermenting) colonies on TCBS agar as V.cholerae by demonstrating l The organisms are motile l Gram-negative vibrios with commercially available modified Gram Stain

Kit (20750020/20750021).l Oxidase positive.l And capable of growth on CLED and other salt-free media.

3) Identify a classical cholera strain of V.cholerae by demonstrating that it reacts with V.cholerae O1 antiserum in a slide agglutination test.

4) Recognize non-O1 (non-cholera) strains of V.cholerae by their failure to react with O1 antiserum while giving slide agglutination with V.cholerae H antiserum.

5) Send a subculture to a reference laboratory for confirmation of an O1 strain and O sero-typing of a non-O1 strain.

For Aeromonas hydrophilaProcedurel Innoculate on to selective agar such as sheep blood agar with 15 mg

ampicillin/litre.0

l Incubate medium for 24-48 hours at 37 C, l Flood the plate with 1% sodium dimethyl-p-phenylenediamine

monohydrochloride .

Observationl Recognize the aeromonas colonies by their purple-black colour and

surrounding narrow zone of haemolysis. l Pick quickly to subculture for identifying tests.

For Plesiomonas shigelloidesInoculate on to MacConkey and DCA media.

ObservationGrowth on MacConkey and DCA media, usually as pale, but sometimes pink colonies.

Note: Distinguished from those of Shigella and Escherichia coli by their oxidase-positive reaction.

For Yersinia enterocoliticaProcedure1) Inoculate several drops of 1 in 10 suspension of the faeces in buffered saline

into a tube of selenite F broth or phosphate-buffered saline, pH 7.6 and hold 0the culture for up to 6 weeks at 4 C.

2) Inoculate a loopful of the faecal suspension, and at weekly intervals

For Aeromonas hydrophilaProcedure

Observation

For Plesiomonas shigelloides

Observation

Note:

For Yersinia enterocoliticaProcedure

thereafter loopfuls of the selenite broth culture, on to plates of DCA or Yersinina selective agar base with Yersinina selective supplement.

Incubation0Incubate for 24 hrs at 32 C.

ObservationInspect the DCA plates for pale (non-lactose-fermenting) colonies and the Yersinia selective agar plates for colonies with dark red centers. Identify the organisms as Y.enterocolitica by demonstrating

0l It is motile at 22 C.

0l Hydrolyses urea at 35 C.

ReportingAntibiotic sensitivities should not be reported to the physician. A note of the sensitivity results should be kept in the laboratory and if the physician inquires about them, the circumstances in which antibiotics therapy may be beneficial rather than harmful may be discussed with him.

Negative findings should be reported in terms only of the organisms that were sought and not found. e.g 'No campylobacter, salmonella or shigella isolated', and not in general terms, e.g. 'No pathogens found', for the latter type of report may suggest that examinations were made for all possible kinds of pathogens, including viruses, protozoa, fungi and helminths.

Any isolation of an infectious enteric pathogen should be notified at once to the local public health authority to prompt the investigation of outbreaks and the institution of preventive measures.








Incubation

Observation

Reporting

References




Urinary Tract Infections

SummarySamples of urine from patients with suspected infections of the urinary tract are the most numerous, e.g. 30-40%, of the different kinds of specimens received in most clinical laboratories. The schedule for their routine examination should therefore be carefully determined with a view to obtaining the necessary diagnostic information with the greatest possible economy of labour resources.

REQUIRED REAGENTSl CLED Agar (2052006/10520006) OR (AM1026/AM5026)l Chrom UTI Agar (AM10254/AM50254)l Easybact (20201012)l Mueller Hinton Agar (AM1071/AM5071 and 20560006,10560006).l Amoxycillin/ampicillin (25mg disk)l Cephalexin (30 mg) diskl Nalidixic acid (30 mg) disk l Ciprofloxacin (5 mg) diskl Nitrofurantin (50 mg) diskl Trimethoprim (2.5 mg) disk l Amoxcycillin (20 mg) + clavulante (10 mg) Amoxyclav (30 mg) diskl Cefaroxime (30 mg) diskl Gentamicin (10 mg) diskl Mcfarland 0.5 standard (20701040)l Modified Gram Stain Kit.

REQUIRED MATERIALS1. Sterile hand gloves. 2. Face mask. 3. Sterile plating loops(10µl). 4. Activated 2% glutaraldehyde solution. 5. Cedar wood oil. 6. Glass slide. 7. Glass spreader. 8. Micropipette and tips (100µl - 1000µl).

Summary

REQUIRED REAGENTS

REQUIRED MATERIALS

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens.

0 03. Incubator at 37 C+ 2 C. 4. Autoclave.

PROCEDURE COLLECTION OF SAMPLEProcedure for collection of clean catch midstream urine samples.The objective is to collect a specimen, which reflect as much as possible only the urine present in the uniary bladder.Thus a clean midstream void is recommended. Instruct the patient as follows:1) Wash and clean the private parts with a dilute soap. Remove all traces of soap

by washing with large quantity of water. Wipe dry.2) Void out, into the toilet, the first stream of urine. This will flush out dead

epithelial cells of the urinary bladder, microparticulates and normal microbial flora, which may have collected in the urine. Then hold the remaining urine in the bladder.

3) Next, void the second stream of urine aspectically into the EASYBACT vial right upto the brim. Again hold the remaining urine in the bladder.

4) Lastly, void out the remaining third stream, into the toilet.5) This procedure ensures that the urine is voided as three discreet segments.

(First stream to flush out contaminants, second stream as the clean midstream for test).

Follow manufacturers instructions as mentioned in the Test Procedure for performance of the test.

(Note: Semi-quantitative culture of the urine is carried out to determine whether it contains a potentially pathogenic bacterium in numbers sufficient to identify it as the causal infecting organism ('significant bacteriuria').

IDENTIFICATION OF BACTERIAThe pathogenic organisms can be identified as per the colour chart provided with the EASYBACT kit and the table mentioned below:

REQUIRED EQUIPMENTS

PROCEDURE COLLECTION OF SAMPLE

IDENTIFICATION OF BACTERIA

E. coli Proteus Klebsiella Candida Pseudomonas Streptococci Staphylococci

Pink Green Dull White Colourless Pink PinkColoniesRegular Regular Large Mucous Regular Convex Regular Regular

Medium Pink/Red Blue/Grey Green/Grey Pink Blue/Green Pink/Red Pink/Red

(Note: For confirmation and identification of the isolated organism it is recommended to perform gram stains and biochemical identification using commercially available Biochemical identification kits.




(Note: Microscopical examination of a wet film of uncentrifuged urine can be carried out to determine whether polymorphs ('pus cells') are present in numbers indicative of infection in the urinary tract)

ANTIBIOTIC SENSITIVITY TEST2 Methods:1) Direct Sensitivity2) Indirect Sensitivity

1) Direct SensitivityThis is carried out if prior microscopy has indicated that infection may be present.l Flood inoculate the urine on to CLED/Chrom UTI Agar plates. (Note:-

CLED & Chrom UTI Agar plates helps in primary differentaition of urine culture).

l Dry the surface.l Place the following antibiotics- 4 antibiotics per plate.

1) Amoxycillin/ampicillin (25mg disk)2) Cephalexin (30 mg)3) Nalidixic acid (30 mg)4) Ciprofloxacin (5 mg)5) Nitrofurantin (50 mg)6) Trimethoprim (2.5 mg)

l Place the antibiotics disk containing amoxcycillin (20 mg) + clavulante (10 mg) Amoxyclav (30 mg) on a separate plate with a B-lactose producing strain of E.coli as control organism.

l For patients in hospital place following antibiotic disk.1) Cefaroxime (30 mg)2) Gentamicin (10 mg)

0 0l Incubate the plates at 37 C+ 2 C for 18-24 hours.

ObservationObserve for zone of inhibition.

InterpretationInterpret as resistant/sensitive.

2) Indirect SensitivityThis is carried out after isolating a pure culture from the specimen.l Dilute the inoculum of a pure culture to match Mcfarland 0.5 standard.l Add 200 µl on each plate Muller Hinton Agar and spread it evenly with a

sterile glass spreaderl Dry the surface.

ANTIBIOTIC SENSITIVITY TEST

1) Direct Sensitivity

Observation

Interpretation

2) Indirect Sensitivity

l Place the following antibiotics- 4 antibiotics per plate.1) Amoxycillin/ampicillin (25mg disk)2) Cephalexin (30 mg)3) Nalidixic acid (30 mg)4) Ciprofloxacin (5 mg)5) Nitrofurantin (50 mg)6) Trimethoprim (2.5 mg)

l Place the antibiotics disk containing amoxcycillin (20 mg) + clavulante (10 mg) Amoxyclav (30 mg) on a separate plate with a B-lactose producing strain of E.coli as control organism.

l For patients in hospital place following antibiotic disk.1) Cefaroxime (30 mg)2) Gentamicin (10 mg)

0 0l Incubate the plates at 37 C+ 2 C for 18-24 hours.

ObservationsObserve for zone of inhibition.

InterpretationInterpret as resistant/sensitive.

Note:- It is generally of little value to identify the species in the mixed cultures of faecal-type bacteria that are commonly obtained from patients with indwelling catheters. Antibiotics are of little use in the treatment of such infections. The findings may be reported as 'many mixed bacteria'.







6. Detection, Prevention and Management of urinary Tract Infections, C.M. thKunin, 4 Edition, 1987.


Observations

Interpretation

References




Meningitis

There is urgent need for the laboratory diagnosis of suspected meningitis, for bacterial meningitis is life-threatening and requires appropriate antibiotic therapy at the earliest possible moment.

REQUIRED REAGENTS

Blood agar-7. Cooked meat broth.

2. Fresh sterile screw-capped containers.

The clinical signs of meningeal irritation always suggest infection of the meninges, but they sometimes occur in association with certain other acute infections not involving the meninges (meningismus) and with certain non-infective conditions such as subarachnoid haemorrhage. Infants, moreover, may have meningitis without the usual localizing signs. Laboratory examinations therefore have an important role in establishing whether or not there is meningitis as well as in determining the causal organisms in cases of infective meningitis. Every patient suspected of having meningitis should have a specimen of cerebrospinal fluid (CSF) examined in the laboratory.

1. Glacial acetic acid.2. Propan 1, 2 diol.3. Crystal violet 1% in water.4. Distilled water.5. Modified grams stain kit (20750020 / 20750021).6. 20500006 (AM1014/AM5014).

8. Glucose detection kit.9. Protein detection kit. 10. Mycostain (20307100).11. Acid Fast Decolouriser (20308500).12. Mycocult (20304006).13. ADA-MTB (20306015). 14. Korthoff's medium.15. Stuarts medium.16. Ellinghausen & McCullough's medium.

REQUIRED MATERIALS1. Sterile hand gloves.

3. Face mask. 4. Sterile plating loops(10µl). 5. Activated 2% glutaraldehyde solution. 6. Cedar wood oil. 7. Glass slide. 8. Modified Fuchs-Rosenthal slide chamber. 9. Cover slip.

REQUIRED REAGENTS

REQUIRED MATERIALS

REQUIRED EQUIPMENTS1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens. 3. Autoclave. 4. Incubator with microaerophilic condition at 42°C-43°C. 5. Incubator at 37°C±2°C.

CSFThe procedure for collection of CSF should be attempted only by physicians well trained in its performance and rigorous aseptic precautions must be observed to prevent the introduction of infection. l Make the collection in a

l

l

l

l

l

l

l

l

l

PROCEDURECOLLECTION OF SPECIMENS

fresh sterile screw-capped containers.(Note:These should not be containers that have been cleaned and sterilized after previous use for other purposes. Such re-used containers may contain bacteria from a previous specimen, e.g. Urine or a culture, which, although killed by the sterilization procedure, may be seen in a Gram-stained film of the CSF and lead to the issuing of an erroneous preliminary report based on the findings in the film.)The patient should lie on his side with his back overhanging the edge of a firm couch or bed. His head, flexed forwards, should be on the same level as his sacrum, and his knees should be drawn up. Disinfect the skin at the interspace between the third and fourth lumbar vertebrae, which is at the level of a line joining the highest points of the iliac crests. with Alcoholic iodine solution. Anesthetize by the intra dermal and subcutaneous injection of a little local anesthetic. Use a sterile hollow lumbar puncture needle containing an occlusive style, wearing sterile gloves, push the needle deeply between the third and fourth lumbar spines, either in the mid line or slightly to one side of it, so that the tip of the needle, with its bevel downwards, passes slightly headwards into the spinal canal,which it should reach at a depth of 4-6 cm. Withdraw the stylet, check for first drop of CSF appear within a few seconds. If no fluid appears replace and push the needle a little further on. If the needle strikes bone withdraw a short distance and pressed in again in a different direction. Allow fluid to fall drop by drop into the container, until 3-5 ml has been collected, withdraw the needle and supply occlusive dressing to the puncture site. (Note: Removal of larger volume may lead to headache, when there is increased intracranial pressure. the fluid may tend to spurt out, in which case the withdrawal should be quickly checked, for a large sudden removal of fluid

REQUIRED EQUIPMENTS

PROCEDURECOLLECTION OF SPECIMENSCSF




may draw down the cerebellum into the foramenmagnum and compress the medulla.)Allow the patient to lie down for several hours afterwards.Dispatch the specimen to the laboratory as quickly as possible, delay may result in the death of delicate pathogens, such as meningococci, and the disintegration of leucocytes. Do not be keep in a refrigerator, which tends to kill H.influenzae. If delay for a few hours is unavoidable, the specimen is best kept in an incubator at

Normal CSF -If clear and colourless like water. Previous cerebral haemorrhage -If yellow color .Infected CSF - If turbid and blood contaminated .

If turbid and blood contaminated, the specimen should be further examined for the following:

Cell count.Gram film.Culture. Glucose and protein contents.Presence of haemophilus, meningococcal or pneumococcal antigens.

Normal CSF - 0-5 leucocytes/mm , mainly lymphocytes.(Note: In neonates up to 30/mm

mmmm

mm

l

l

l

l037 C.

Blood cultureBlood for cultures should be collected at the same time as the CSF, if possible before antibiotics are given.

LABORATORY EXAMINATION OF CSFVisual ExaminationExamined with the naked eye for the presence of turbidity and any sign of contamination with blood from the puncture wound.

Interpretationl

l

l

l

l

l

l

l

Cell countMicroscopyl Mix well uncentrifuged fluid and place in a slide counting chamber (Procedure

of counting chamber refer below)l Count the leucocytes .l Note the relative numbers of polymorphs and lymphocytes and the number of

erythrocytes in specimens contaminated with blood.

Interpretation:-3

l3 , mainly polymorphs. )

3 l Purulent meningitis -100-3000 leucocytes/ mostly polymorphs.

3l Aseptic meningitis-10-500 leucocytes/ , mostly lymphocytes, though

polymorphs may predominate in the earliest acute stage of the illness. 3

l Tuberculous meningitis - 100-500 leucocytes/ , mostly lymphocytes.

Blood culture

LABORATORY EXAMINATION OF CSFVisual Examination

Interpretation

Cell countMicroscopy

Interpretation:-

Note:- If the CSF is heavily blood stained, it is not worthwhile to attempt to make a cell count, for numerous leucocytes derived from the blood will be present. But if there is only slight contamination with blood, the leucocytes and erythrocytes should be encountered separately. The findings of leucocytes in numbers greatly in excess of 1 per 1000 erythrocytes, the approximate ratio in blood, will suggest the presence of meningitis.

When examining the wet film of CSF for the cell count, care should be taken not to mistake the rare presence of yeasts or amoebae for leucocytes.

COUNTING CHAMBERIntroduction The cell count is usually performed in a modified Fuchs-Rosenthal slide chamber, which has a film depth of 0.2 mm between the counting surface of the slide and the overlying coverslip.

The counting surface is marked with triple lines into nine large squares, each 1 3 in area and subdivided into 16 small squares. The volume of fluid in the film

overlying five large squares is thus 1 and the count of the cells on five squares is 3thus the count per .

DilutionCSF that is clear or only slightly turbid should be examined undiluted, but when the specimen is highly turbid and its cell count very high, it may be necessary to dilute it 1 in 10 or 1 in 100 before examination.

When separate counts are to be made of the leucocytes and erythrocytes, 0.85% NaCl solution should be used as diluent.

If, however, the presence of large numbers of erythrocytes makes the recognition and counting of the leucocytes difficult. The dilution should be done with a counting fluid which lyses the erythrocytes and stains the nuclei of the leucocytes. A suitable fluid contains acetic acid and crystal violet, e.g.Glacial acetic acid 1 mlPropan 1, 2 diol 2.5 mlCrystal violet 1% in water 1.5 mlDistilled water 100 ml

Procedure1) Make sure the surfaces of the counting chamber and its coverslip are clean

and dry. Press the coverslip on to the support areas at the sides of the counting surface until broad bands of rainbow colours (Newton's rings) appear and indicate that close and even contrast has been made.

2) Gently but thoroughly mix the diluted or undiluted uncentrifuged CSF. Take up about 0.2 ml in the capillary end of a Pasteur pipette. Carefully apply the tip of the pipette to the counting surface at the edge of the coverslip and allow the fluid to run into the chamber so that it fills the whole chamber without the

mm

mm

COUNTING CHAMBERIntroduction

Dilution

Procedure




presence of bubbles of air and yet does not spill over to the support areas on either side. Newton's rings should still be apparent.

3) First inspect the area of the counting grid with the low power of the microscope. Defocus the condenser to make the unstained cells clearly visible. Then count the cells with a x 40 dry objective (magnification x 300 or x 400). Count the cells on five of the large squares. Include in the count any cells that overlap the innermost line of the triple-lined border on the left-hand and distal sides of the square and exclude from the count the cells that overlap the border on the right and proximal sides. Take care not to count erythrocytes as leucocytes.

4) Add together the counts for the five large squares and, in the case of diluted 3specimens, multiply by the dilution factor to get the count per mm .

Note: If there is any difficulty in differentiating polymorphs and lymphocytes in the counting chamber, make a film of the cellular deposit after the specimen has been centrifuged, fix with heat, stain with methylene blue, Leishman or carbol thionine and examine by oil immersion to asses the relative numbers of the two types of leucocytes.

Gram film of CSFCentrifuge the deposit and make a thick film within an area of about 10mm diameter of the deposit on a clean glass slide and stain by Gram's method with commercially available Modified grams stain kit.

NoteWhen the CSF is highly turbid and proteinaceous, part of the film should be thin, for sometimes a wholly thick film, although dried and fixed by heat, becomes washed off the slide in the course of staining.

ObservationA very careful search for bacteria should be made particularly in areas of the film where there are plenty of leucocytes, and the search should be continued for at least 10 min before accepting the result as negative.

NoteThe findings of bacterial forms resembling meningococci, pneumococci, haemophili, coliform bacilli, streptococci or listeria should at once be reported to the physician, for different antibiotics are preferred for treatment of the different infections: e.g. Benzylepenicillin (or chloramphenicol) for meningococcus and pneumococcus; chloramphenicol for haemophilus; chloramphenicol, ampicillin oro cotrimoxazole for coliforms; benzylpenicillin or chloramphenicol for group B streptococci; and ampicillin or chloramphenicol for listeria.

CULTURE OF CSFProcedureImmediately after centrifugation of the CSF and the removal of some of the deposit

Gram film of CSF

Note

Observation

Note

CULTURE OF CSFProcedure

for the Gram film, Seed the remainder of the deposit, on to following culture media, e.g l

l

l

Note

Incubation0 037 C+ 2 C.

Observationl Growth /No growth.l If no growth is apparent after 18-24 hours incubation, incubate the plates for

another day, inspect for growth. l If the plate cultured remains free from growth and turbidity develops in the

cooked meat broth then carry out (a) Gram staining with commercially available Modified Grams stain kit and (b) Sub culture on to 1) Blood Agar 2) heated blood agar plates, incubated aerobically and anaerobically at

0 037 C+ 2 C.

Biochemical TestsTest the supernatant from the Centrifuged CSF for its content of glucose and protein with commercially available glucose and protein detection kits.

Interpretationl Normal CSF contains 2.2-4 mmol glucose/litre (about 60 % of the plasma

glucose value) and 0.15-0.4 g protein/litre (in neonates up to 1.5 g protein/litre).

l In purulent bacterial meningitis the glucose concentration is reduced (0.2 mmol/lit) and the protein concentration increased (0.5-3.0 g/litre).

l In aseptic (viral) meningitis the glucose concentration is normal and the protein concentration raised a little (0.5-1 g/litre).

Bacterial AntigensA rapid indication of the type of infection is obtained by the performance of a coagglutination test or counter immunoelectrophoresis test on the CSF (or blood serum) to demonstrate the presence of the antigens of meningococci of serotype A,B or C or the capsular antigens of the commoner types of pneumococci or Piteman's type b of Haemophilus influenzae with commercially available coagglutination test kit.

A plate of blood agar.A plate of heated-blood ('chocolate') agar for incubation in humid air plus 5-10 % CO .2

A tube of cooked meat broth.

When there may be a brain abscess, possibly due to bacteroides or anaerobic cocci, a further blood agar plate should be seeded for incubation 2-5 days in an anaerobic atmosphere with 5-10% CO . 2

Incubate 18-24 hrs at

Note

Incubation

Observation

Biochemical Tests

Interpretation

Bacterial Antigens




Viral meningitis

l

l

l

0l 70 C until inoculation into cell cultures, l Submit throat swab in viral transport medium for culturing of mumps virus. l Faeces may be cultured for echo, coxsackie and polio virus.

Tuberculous meningitisIn suspect of tuberculous infectionl Prepare a smear of centrifuged deposit of the CSF and examine in a Ziehl-

Neelsen stained film for acid-fast bacilli (commercially available ZN hot stain-Mycostain) and culture on one or two slopes of Lowenstein-Jensen media available commercially.

l Carry out ADA testing of CSF with commercially available ADA Kit.

Leptospiral meninggitisIn suspect of leptospiral infection l Collect paired sera and demonstrate a rising titre of leptospiral antibodies in

the serovar-specific microscopical agglutination test or the genus specific complement-fixation and sensitized erythrocyte tests.

In the presence of aseptic meningitis isolate the virus from the CSF, a throat swab or a specimen of faeces, and examine paired sera for viral antibodies.

Note:Keep CSF at -

Viral meningitis

Note:

Tuberculous meningitis

Leptospiral meninggitis

MicroscopyObserve the CSF under dark-ground illumination for motile leptospira.

CultureInnoculate the CSF or Blood into Korthoff's or stuarts or Ellinghausen & McCullough's medium.

Incubation and observationIncubate at 28°C-30°C and examine periodically up to 6 weeks before discarding it as negative.








Microscopy

Culture

Incubation and observation

References

Pyrexia Of Unknown Origin (PUO)

The term PUO is generally applied to any febrile illness lasting more than a few days, the cause of which is obscure due to the absence of obvious specific or localizing signs and symptoms. In these conditions there is thus a special need for a laboratory diagnosis to guide the choice of appropriate therapy.

Laboratory diagnosis of PUO infections:-The following procedures should be considered, Tests should be done for the more likely infections and then, if these are negative, tests for the less likely should be done.1) Blood culture should always be attempted. A first specimen should be

collected before antibiotics are given, and several specimens collected on separate occasions should be examined before a negative result is accepted. (Refer blood culture for detail procedure)

2) Specimens of urine, throat secretion, sputum (if present) and faeces should be examined for the common pathogenic bacteria, and faces should be examined for protozoa, cysts and helminthic ova.(Refer UTI Infection, Lower Respiratory tract infection and Gastrointestinal

Laboratory diagnosis of PUO infections:-

infection for detail procedure)

4) Haematological investigations should be done to detect leucocytosis, suggestive of a cryptic absces; eosinophilia, suggestive of helminthasis; and atypical lymphocytes, suggestive of infectious mononuleosis.

5) A tuberculin test and a chest X-ray should be done to detect tuberculosis.6) Thick and thin blood films should be examined for malaria, leishmaniasis,

trypanosomiasis and filariasis in travelers returned from countries in which these infections are present.

3) Paired sera should be collected for serological tests for antibody responses to a range of possible pathogens, e.g. Cytomegalovirus, hepatitis B virus, influenza virus, infectious mononucleosis virus, chlamydia, coxiella, leptospria, mycoplasma, brucella, legionella, leptospira, syphilis spirochaete, toxoplasma, aspergillus and other fungi and entamoeba. The antistreptolysin-O (ASO) test should be done for cryptic S. pyogenes infection. The first specimen should be taken as early in the illness as possible and the second 2-4 weeks later.







thInfections, 13 Ed., Churchill Livingston 1978, Edited by J.P. Duguid, B.P.

References Marmion, R.H.A. Swain.th4. Practical Medical Microbiology, Mackie & McCartney, Vol. 2, 13 Ed.,

Churchill Livingston 1989, Edited by J.G. Collee, J.P. Duguid, A.G. Fraser, B.P.Marmion.




Hand Book of Practicing Microbiologists

Quantitative Analysis of culture media using Ecometric Method

Introduction Culture Media play a pivotal role in any Microbiology Laboratory. They are widely employed for isolation, identification and sensitivity testing of different Pathogenic microorganisms. Most of the laboratories usually prepare their own media for routine diagnosis as well as research purposes. However to ensure that the media is of good quality and capable of giving satisfactory results, proper quality management system is essential. For that purpose certain parameters of media prepared should be monitored.

Growth supporting characters is the most important parameter while conducting quality control of media. In practice the absolute measurements of growth of microorganisms are either time consuming or require sophisticated instrument. Colony size may be used to see the performance but it is again an intensive indicator. Colony characteristics are subjective and very difficult to record. So method like 'ECOMETRIC' gives us comparative data, and is therefore suitable for routine quality control of microbiological performance of culture as well as inhibition characteristics of media.

Requirements1) 1 l nichrome loop2) 5 ml vials of non selective medium3) Soyabean Casein Digest Medium (AM1092/AM5092)4) Tryptone Glucose Extract Broth (AM1102/AM5102)5) Brain Heart infusion Broth (AM1017/AM5017)

Procedurel Inoculate 5ml of sterile non-selective medium with 10 l culture suspension or

one loopfull of chosen test organism (less than 100 colonies) and incubate for o o o o 4 hours at 37 C + 2 C for bacteria and 25 C - 30 C for mould and yeast

culture.l Divide the plate to be tested into 4 quarters designated A,B,C and D as per

figure below:

Introduction

Requirements

Procedure

Figure Il Charge 1ìl loop with the incubated 4-hour culture and streak the test plate,

going from A1-B1-C1-D1- A2-B2 etc without flaming or recharging the loop and finish at D5.

l Repeat this process with a control plate. The control used for comparing should be either Competitors products or previously approved batches of the same media.

l Incubate the plates at required temperature and period. After incubation note the last point at which growth occurs on the test and control plates and record the segment or line. e.g.- C4 or D5.

l This is the end point and is used to calculate absolute growth index (AGI) and relative growth index (RGI).

Calculationl Refer table1 and note down values of absolute growth index end points for

test and controll Calculate RGI using formula

Relative growth Index = AGI test x 100AGI control

Table1 Absolute Growth Index (AGI)

A1=5 B1=10 C1=15 D1=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

Limitsl For efficiency activity RGI should be Minimum 70%l For inhibition activity RGI should be 0%

References1. Indian Journal of Medical Microbiology (2005) 23(3): 159 -163.2. Krisher K, Callihan DR, Jones RN, Luper DC, Miller JM, Sharp SE, et al. Quality

Control for commercially prepared Microbiological Culture Media; Approved rdStandards-3 edn. NCCLS M22-A3, 2004:24.

3. Anonymous. Quality Control of Culture Media. In: Culture Media Mannual LAB M. (IDG Ltd. , Unites Kingdom) 2002.P. 16-8.

4. Aarora DR. Quality Assurance Microbiology. Indian J Med Microbiol 2004; 22:81-6.

5. Weenk GH, vd Brink J, Meeuwissen J, van Oudenallen A, van Schievan RR, A standard protocol for the quality control of microbiology media. Int J Food Microbiol 1992; 17:183-98.

Figure I

Calculation

Limits

ReferencesAA BB

DD CC

1122

3344

55 55

5555

4433

2211

44 4433 33

22 2211 11

AP

PLI

CA

TIO

NS

Food

and

Bev

erag

e A

naly

sis

PR

OD

UC

TS

Bacillus cereus

cil

Aduric and Thermophiic atour rFl SSpoeformers

Cpyleramobact

u Clostridim

Coliforms

Listeria

omers

Mesophilic Sporefr r (Aeobic)

Oiorganis

smophilc sm

PsycotrophiOsmshrc rgani

Salmonelal

hi Sgella

toc Saphyloccus

tococcus/nSrept

Eterococcus Tot e CountalPlat

Viio br

Yeasts d Mouldsan

Yersinia erocoltcaentii

esophic ormers

MliSporef naerobi(A

c)

ron ultsporefISphie ormers

Proteolyticmicroorganisms

Bac

illus

Cer

eus

Aga

r B

ase

(AM

1009

/AM

5009

)

Bai

rd P

arke

r Aga

r B

ase

(AM

1011

/AM

5011

)

BP

Su

lph

a S

up

ple

men

t* (

AS

005)

Eg

g Y

olk

Em

uls

ion

* (A

010)

Eg

g Y

olk

Tel

luri

te E

mu

lsio

n*

(AS

011)

Po

tass

ium

Tel

luri

te 3

.5%

* (A

S02

3)

Bile

Esc

ulin

Aga

r (A

M10

12/A

M50

12)

Bis

mut

h S

ulph

ite A

gar

(AM

1013

/AM

5013

)

Blo

od A

gar

Bas

e (A

M10

14/A

M50

14)

Bra

in H

eart

Infu

sion

Aga

r (A

M10

16/A

M50

16)

Bra

in H

eart

Infu

sion

Bro

th (

AM

1017

/AM

5017

)

Bril

liant

Gre

en A

gar,

Mod

ified

(A

M10

18/A

M50

18)

Su

lph

a S

up

ple

men

t* (

AS

027)

Bril

liant

Gre

en B

ile B

roth

2%

(A

M10

20/A

M50

20)

Ch

lora

mp

hen

ico

l Yea

st G

luco

se A

gar

* (A

M10

24/A

M50

24)

Col

ombi

a B

lood

Aga

r B

ase

(AM

1029

/AM

5029

)

Sta

ph

-Str

epto

Su

pp

lem

ent*

(A

S02

5)

Str

epto

Su

pp

lem

ent*

(A

S02

6)

Coo

ked

Mea

t Med

ium

(A

M10

30/A

M50

30)

DN

ase

Test

Aga

r B

ase

(AM

1038

1)

Deo

xych

olat

e C

itrat

e A

gar

(AM

1031

/AM

5031

)

Dex

tros

e Tr

ypto

ne A

gar

(AM

1036

/AM

5036

)

Dex

tros

e Tr

ypto

ne B

roth

(A

M10

37/A

M50

37)

Diff

eren

tial R

einf

orce

d C

lost

ridia

l Bro

th (

AM

1038

/AM

5038

)

EC

Bro

th (

AM

1039

/AM

5039

)

l

l

l

l

l

l

ll

ll

l

l

l

l

l

ll

l

l

ll

l

l

l

ApplicationHand Book of


Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits124

AP

PLI

CA

TIO

NS

Food

and

Bev

erag

e A

naly

sis

PR

OD

UC

TS

acius Bllcereus

Aciduriand Tli

c hermophic FatSour Sporef

l ormers

CampylobacterCrum lostidi

Cf oliorms

Leri istaesophic ormers

MliSporef (Aerobic)

Omophiic organismssl

PsycotrophiOranismshrc g

Salmonella

Shigelal

Stoc aphyloccusStrtococcus/Ent

eperococcus Tot le Count

alPat

i Vibro

oulYeasts and Mds

ni Yersia enterocolitica

Mesophilc poreformersiS

(Anaerobic)

Iron Sultsporefphie ormers

rotytcsm

Peoli microorganis

EM

B A

gar,L

evin

e (A

M10

40/A

M50

40)

Flu

id L

acto

se M

ediu

m (

AM

1042

/AM

5042

)

Flu

id S

abou

raud

Med

ium

(A

M10

43/A

M50

43)

Flu

id S

elen

ite C

ystin

e M

ediu

m (

AM

1044

/AM

5044

)

Flu

id T

hiog

lyco

llate

Med

ium

(A

M10

45/A

M50

45)

Hea

rt In

fusi

on A

gar

(AM

1048

1/A

M50

481)

Hea

rt In

fusi

on B

roth

(A

M10

482/

AM

5048

2)

Iron

Sul

phite

Aga

r (A

M10

483/

AM

5048

3)

Kar

mal

i Cam

pylo

bact

er A

gar

Bas

e (A

M10

49/A

M50

49)

Cam

pyl

ob

acte

r S

elec

tive

Su

pp

lem

ent

wit

h H

emin

(K

arm

ali)

, Mo

dif

ied

* (A

S00

7)

Klig

ler

Iron

Aga

r (A

M10

50/A

M50

50)

Lact

obac

illi M

RS

Aga

r (A

M10

51/A

M50

51)

Lact

ose

Bro

th (

AM

1052

/AM

5052

)

Laur

yl T

rypt

ose

Bro

th (

AM

1053

/AM

5053

)

List

eria

Iden

tific

atio

n A

gar

Bas

e, P

ALC

AM

(A

M10

55/A

M50

55)

Lis

teri

a S

elec

tive

Su

pp

lem

ent

*(A

S01

8)

Mac

Con

key

Aga

r w

ith C

ryst

al V

iole

t, N

aCl a

nd 0

.15%

Bile

Sal

ts

(AM

1058

/AM

5058

)

Mac

Con

key

Bro

th w

ith N

eutr

al R

ed (

AM

1064

/AM

5064

)

Mac

Con

key

Bro

th P

urpl

e w

ith B

rom

ocre

sol P

urpl

e (A

M10

63/A

M50

63)

Mal

onat

e B

roth

Ew

ing

Mod

ified

(A

M10

65/A

M50

65)

Mal

t Aga

r (A

M10

66/A

M50

66)

Mal

t Ext

ract

Aga

r (A

M10

67/A

M50

67)

Mal

t Ext

ract

Bro

th (

AM

1068

/AM

5068

)

Man

nito

l Mot

ility

Tes

t Med

ium

(A

M10

681/

AM

5068

1)

l ll

l

ll

l

ll

ll

l

l

ll

l

l

l l

l

ll

ll

l l ll

l

l

ll l

l




AP

PLI

CA

TIO

NS

Food

and

Bev

erag

e A

naly

sis

PR

OD

UC

TS

acius Bllcereus

Aciduriand Tli

c hermophic FatSour Sporef

l ormers

CampylobacterClrium ostdi

Cf oliorms

Lieri staMesophilc poreformers

iS

(Aerobic)

Osmophilic organisms

ycots

Pshrrophic OrganismSalmonella

Shigelal

StaphylococcusStreptococcus/Entocus

eroccTot Plte Count

ala

io Vibr

oulYeastsand Mds

ni Yersia enterocolitica

Mesophilc poreformersiS

(Anaerobic)

Iron Sulphite sporeforers m

rotytcism

Peoli mcroorganis

Man

nito

l Sal

t Aga

r (A

M10

69/A

M50

69)

MR

-VP

Med

ium

(A

M10

71/A

M50

71)

Nut

rient

Aga

r (A

M10

74/A

M50

74)

Nut

rient

Aga

r pH

6.8

(A

M10

75/A

M50

75)

Nut

rient

Bro

th (

AM

1077

/AM

5077

)

Pla

te C

ount

Aga

r (A

M10

81/A

M50

81)

Pot

ato

Dex

tros

e A

gar

(AM

1082

/AM

5082

)

Pot

ato

Dex

tros

e B

roth

(A

M10

83/A

M50

83)

Rei

nfor

ced

Clo

strid

ial A

gar

(AM

1085

/AM

5085

)

Sab

oura

ud D

extr

ose

Aga

r (A

M10

87/A

M50

87)

Sab

oura

ud D

extr

ose

Bro

th (

AM

1088

/AM

5088

)

Sel

enite

F B

roth

(A

M10

89/A

M50

89)

Sim

mon

s C

itrat

e A

gar

(AM

1090

/AM

5090

)

Ski

m M

ilk A

gar

(AM

1090

1/A

M50

901)

Soy

abea

n C

asei

n D

iges

t Aga

r (A

M10

91/A

M50

91)

Soy

abea

n C

asei

n D

iges

t Med

ium

(A

M10

92/A

M50

92)

SS

Aga

r (A

M10

93/A

M50

93)

TC

BS

Aga

r (A

M10

95/A

M50

95)

Tetr

athi

onat

e B

roth

Bas

e, H

ajna

(A

M10

96/A

M50

96)

Trip

le S

ugar

Iron

(T

SI)

Aga

r (A

M10

99/A

M50

99)

Tryp

tone

Glu

cose

Ext

ract

Aga

r (A

M11

01/A

M51

01)

Tryp

tone

Glu

cose

Ext

ract

Bro

th (

AM

1102

/AM

5102

Tryp

tone

Pho

spha

te B

roth

(A

M11

03/A

M51

03)

Tryp

tone

Wat

er (

AM

1104

/AM

5104

)

l

ll

ll

l

ll

l

l

ll

ll

ll

ll

l l

l

l l

l ll

l

l

ll

ll

ll

ll

ll

ll

ll

l

l

ll

ll

ll

ll

l

l ll




0*

- S

tore

bet

wee

n 2-

8C

.

AP

PLI

CA

TIO

NS

Food

and

Bev

erag

e A

naly

sis

PR

OD

UC

TS

acis Bllucereus

cia Tl

Aduric ndhermophiic FatSour poref

l Sormers

Campyler obact

umClostridi

f Coliorms

e Listriaeo

ormers

Msphilic Sporef erobi (Ac)

Oophiiorganismssmlc

PsycotrophiOsmshrc rgani

Salmonella

hia Sgell

Stoc aphyloccusStococcus/nt

reptEerococcus Tot le Count

alPat

i Vibro

Yeasts and MouldsYersinia erocolitica

ent

Mesophic poreformersliS

(Anaerobic)

Iron ultsporefSphie ormers

rytcsm

Poteoli microorganis

Ure

a B

roth

Bas

e (A

M11

06/A

M51

06)

Ure

a 40

%*

(AS

028)

Vio

let R

ed B

ile A

gar

(AM

1107

/AM

5107

)

Vog

el J

ohns

on A

gar

Bas

e (A

M11

08/A

M51

08)

Po

tass

ium

Tel

luri

te 1

%*

(AS

022)

XLD

Aga

r (A

M11

12/A

M51

12)

Yeas

t Ext

ract

Aga

r (A

M11

13/A

M51

13)

Yeas

t Mal

t Aga

r (A

M11

14/A

M51

14)

Yers

inia

Sel

ectiv

e A

gar

Bas

e (A

M11

16/T

M11

6005

)

Yer

sin

ia S

elec

tive

Su

pp

lem

ent

*(A

S02

9)

ll

ll

l

l

ll

l

l

l




Dai

ry A

naly

sis

ciupeBalls Scies he (and otrspore beer ars)

AP

PLI

CA

TIO

NS

PR

OD

UC

TS

Coliforms

aerobiAn

c cis

Miroorgansm (iCostdiIncludng lria)

Lactobacilli

st Lieriao

monocytgenes

loahSamnell/Sigella

PatStandard le Count

ococcusStaphyl

Sreptococcus/t

Enterococcus

& dYeastMoul

nictca

Yersia enteroolii

Campyloer bact

eaiProtolytic ctivty

Azi

de B

lood

Aga

r B

ase

(AM

1006

/AM

5006

)

Bac

illus

Cer

eus

Aga

r B

ase

(AM

1009

/500

9)

Po

lym

yxin

B S

elec

tive

Su

pp

lem

ent*

(A

S02

1)

Eg

g Y

olk

Su

pp

lem

ent*

(A

S01

0)

Bai

rd P

arke

r Aga

r B

ase

(AM

1011

/AM

5011

)

B P

Su

lph

a S

up

ple

men

t* (

AS

005)

Eg

g Y

olk

Em

uls

ion

* (A

010)

Eg

g Y

olk

Tel

luri

te E

mu

lsio

n*

(AS

011)

Po

tass

ium

Tel

luri

te 3

.5%

* (A

S02

3)

Bile

Esc

ulin

Aga

r (

AM

1012

/AM

5012

)

Bis

mut

h S

ulph

ite A

gar

(AM

1013

/AM

5013

)

Bra

in H

eart

Infu

sion

Aga

r (A

M10

16/A

M50

16)

Bra

in H

eart

Infu

sion

Bro

th (

AM

1017

/AM

5017

)

Bril

liant

Gre

en A

gar

Mod

ified

(A

M10

18/A

M50

18)

Su

lph

a S

up

ple

men

t* (

AS

027)

Bril

liant

Gre

en B

ile B

roth

2%

(A

M10

20/A

M50

20)

Chl

oram

phen

icol

Yea

st G

luco

se A

gar

(AM

1024

/AM

5024

)

Coo

ked

Mea

t Med

ium

(A

M10

30/A

M50

30)

Diff

eren

tial R

einf

orce

d C

lost

ridia

l Bro

th (

AM

1038

/AM

5038

)

EC

Bro

th (

AM

1039

/AM

5039

)

EM

B A

gar,

Levi

ne (

AM

1040

/AM

5040

)

End

o A

gar

(AM

1040

/AM

5040

)

Flu

id L

acto

se M

ediu

m (

AM

1042

/AM

5042

)

Flu

id S

elen

ite C

ystin

e M

ediu

m (

AM

1044

/AM

5044

)

Iron

Sul

phite

Aga

r (A

M10

483/

AM

5048

3)

Lact

obac

illus

MR

S A

gar

(AM

1051

/AM

5051

)

l

l

l

l

l

l

ll

l

l

l

l l

l l l ll l

l

l




Dai

ry A

naly

sis

cipBallus Secies h (and oter spore be earrs)

AP

PLI

CA

TIO

NS

PR

OD

UC

TS

Colifoms r

aerobiAn

c cis

Miroorgansm (iCodiIncludng lstria)

Lactobacilli

st Lieriamonocytogenes

l Samonella/Shigella

atStandard Ple Count

oocsStaphylccu

Sreptococcus/tnterococcus

E

& dYeast Moul

ni erocoltca

Yersiaentii

ampyleC

obactr

Proteolytic activity

Lact

ose

Bro

th (

AM

1052

/AM

5052

)

Laur

yl T

rypt

ose

Bro

th (

AM

1053

/AM

5053

)

List

eria

Iden

tific

atio

n A

gar

Bas

e, P

ALC

AM

, (A

M10

55/A

M50

55)

Lis

teri

a S

elec

tive

Su

pp

lem

ent*

(A

S01

8)

Mac

Con

key

Aga

r w

ith C

ryst

al V

iole

t, N

aCl a

nd 0

.15%

Bile

S

alts

(A

M10

59/A

M50

59)

Mac

Con

key

Bro

th w

ith B

rom

ocre

sol P

urpl

e (A

M10

63/A

M50

63)

Mal

onat

e B

roth

, Ew

ing

Mod

ified

(A

M10

65/A

M50

65)

Mal

t Aga

r (A

M10

66/A

M50

66)

Mal

t Ext

ract

Aga

r (A

M10

67/A

M50

67)

Man

nito

l Sal

t Aga

r (A

M10

69/A

M50

69)

MR

-VP

Med

ium

(A

M10

71/A

M50

71)

Nut

rient

Aga

r (A

M10

74/A

M50

74)

Nut

rient

Bro

th (

AM

1077

/AM

5077

)

Pla

te C

ount

Aga

r (A

M10

81/A

M50

81)

Pot

ato

Dex

tros

e A

gar

(AM

1082

/AM

5082

)

Pot

ato

Dex

tros

e B

roth

(A

M10

83/A

M50

83)

Pre

sto

n S

elec

tive

Su

pp

lem

ent

(Cam

pyl

ob

acte

r S

elec

tive

S

up

ple

men

t IV

, Mo

dif

ied

) (A

S23

1)

Rei

nfor

ced

Clo

strid

ial A

gar

(AM

1085

/AM

5085

)

Sim

mon

s C

itrat

e A

gar

(AM

1090

/509

0)

Ski

m M

ilk A

gar

(AM

1090

1/A

M50

901)

Soy

abea

n C

asei

n D

iges

t Aga

r (A

M10

91/A

M50

91)

Soy

abea

n C

asei

n D

iges

t Med

ium

(A

M10

92/A

M50

92)

SS

Aga

r (A

M10

93/A

M50

93)

Tetr

athi

onat

e B

roth

Bas

e, H

ajna

(A

M10

96/A

M50

96)

Tom

ato

Juic

e A

gar

(AM

1097

/AM

5097

)

l l

l

ll

l

l ll

l l

l

ll

l

ll

l

l

l l

l

l

ll

l

ll

l

l

l l

l




Dai

ry A

naly

sis

cilus pecieBalSs other p (and sore bearers)

AP

PLI

CA

TIO

NS

PR

OD

UC

TS

olf Ciorms

Aaerobic nicogisms

Mroran (ncling Costidia)

Iudlr

obalLactcili

Lsteri iaonogens

mocyte

lmonela/hilSa

lSgela

SandPlatetdar o Cunt

Saphlococcusty

eococcus/Strptnt Eerococcus

Ye & Mouldast

nicca

Yersia enterooliti

Campylobacter

Poeoliactvityrtytc i

Tom

ato

Juic

e A

gar,

Spe

cial

(A

M10

98/A

M50

98)

Trip

le S

ugar

Iron

(T

SI)

Aga

r (A

M10

99/A

M50

99)

Tryp

tone

Glu

cose

Ext

ract

Aga

r (A

M11

01/A

M51

01)

Tryp

tone

Pho

spha

te B

roth

(A

M11

03/A

M51

03)

Tryp

tone

Wat

er (

AM

1104

/AM

5104

)

Ure

a A

gar

Bas

e, C

hris

tens

en (

AM

1105

/AM

5105

)

Ure

a B

roth

Bas

e (A

M11

06/A

M51

06)

Ure

a 40

%*

(AS

028)

Vio

let R

ed B

ile A

gar

Bas

e (A

M11

07/A

M51

07)

Vog

el J

ohns

on A

gar

Bas

e (A

M11

08/A

M51

08)

Po

tass

ium

Tel

luri

te 1

%*

(AS

022)

XLD

Aga

r (A

M11

12/A

M51

12)

Yers

inia

Sel

ectiv

e A

gar

Bas

e (A

M11

16/T

M11

6005

)

Yer

sin

ia S

elec

tive

Su

pp

lem

ent*

(A

S02

9)

l

ll

l

l

l ll

ll

l

ll

l

l

l

l

Ure

a 40

%*

(AS

028)




Pha

rmac

euti

cal A

naly

sis

AP

PLI

CA

TIO

NS

PR

OD

UC

TS

Aiotc Asay ntbiis a Medi

col E.i

oPeudomnas

s

l Samonellae

Staphylococcus

TtlAeroicoa b Mroial Couticb

n

oYeast and Muld Count

iiy SterltTesting

Vitamn Assy ia

obaclCount

Lactilus

Mic

rob

ial L

imit

Tes

t

Alte

rnat

ive

Thi

ogly

colla

te M

ediu

m (

AM

1900

/AM

5900

)

Ant

ibio

tic A

ssay

Med

ium

A (

No.

1, S

eed

Aga

r) (

AM

1002

/AM

5002

)

Ant

ibio

tic A

ssay

Med

ium

C (

No.

3, A

ssay

Bro

th)

(AM

1003

/AM

5003

)

Ant

ibio

tic A

ssay

Med

ium

No

11 (

Neo

myc

in, E

ryth

rom

ycin

Ass

ay A

gar)

(A

M10

04/A

M50

04)

Ant

ifung

al A

ssay

Aga

r (A

M10

041/

AM

5004

1)

B12

Ass

ay A

gar

usin

g E

.col

i Mut

ant C

ultu

re (

AM

1007

/AM

5007

)

B12

Ass

ay M

ediu

m (

AM

1007

1)

B12

Mai

nten

ance

Med

ium

for

E.c

oli M

utan

t (A

M10

08/A

M50

08)

Bai

rd P

arke

r Aga

r B

ase

(AM

1011

/AM

5011

)

B P

Su

lph

a S

up

ple

men

t* (

AS

005)

Eg

g Y

olk

Em

uls

ion

* (A

S01

0)

Eg

g Y

olk

Tel

luri

te E

mu

lsio

n*

(AS

011)

Po

tass

ium

Tel

luri

te 3

.5%

* (A

S02

3)

Bis

mut

h S

ulph

ite A

gar

(AM

1013

/AM

5013

)

Bril

liant

Gre

en A

gar

Mod

ified

(A

M10

18/A

M50

18)

Su

lph

a S

up

ple

men

t* (

AS

027)

Cet

rimid

e A

gar

Bas

e (A

M10

22/A

M50

22)

Nal

idix

ic S

elec

tive

Su

pp

lem

ent*

(A

S02

0)

Deo

xych

olat

e C

itrat

e A

gar

(AM

1031

/AM

5031

)

EM

B A

gar,

Levi

ne (

AM

1040

/AM

5040

)

Flu

id L

acto

se M

ediu

m (

AM

1042

/AM

5042

)

Flu

id S

elen

ite C

ystin

e M

ediu

m (

AM

1044

/AM

5044

)

Flu

id T

hiog

lyco

llate

Med

ium

(A

M10

45/A

M50

45)

Glu

cose

Yea

st E

xtra

ct A

gar

(AM

1048

/AM

5048

)

Lact

ose

Bro

th (

AM

1052

/AM

5052

)

Mac

Con

key

Aga

r w

ith C

ryst

al V

iole

t,NaC

l and

0.1

5% B

ile S

alts

(A

M10

59/A

M50

59)

l

l l l

l

l l l

l

l l

l

l

l ll l

l

l

l l




Pha

rmac

euti

cal A

naly

sis

AP

PLI

CA

TIO

NS

PR

OD

UC

TS

Antibiotic Assay Mda ei

c E oli .

udomonasPse

lmonelaeSa

l

aococcusStphyl

oTotal Aerbic roil uMicbaCont

eaodYast nd Mul Count

Serility etigtTsn

iamssyVtin Aa

oLactobacillus Cunt

Mic

rob

ial L

imit

Tes

t

Mac

Con

key

Bro

th w

ith B

rom

ocre

sol P

urpl

e (A

M10

63/A

M50

63)

Man

nito

l Sal

t Aga

r (A

M10

69/A

M50

69)

Nut

rient

Bro

th (

AM

1077

/AM

5077

)

Pep

tone

Wat

er (

AM

1079

/AM

5079

)

Pot

ato

Dex

tros

e A

gar

(AM

1082

/AM

5082

)

Sab

oura

ud C

hlor

amph

enic

ol A

gar

(AM

1086

/AM

5086

)

Sab

oura

ud D

extr

ose

Aga

r (A

M10

87/A

M50

87)

Sel

enite

F B

roth

(A

M10

89/A

M50

89)

Soy

abea

n C

asei

n D

iges

t Aga

r (A

M10

91/A

M50

91)

Soy

abea

n C

asei

n D

iges

t Med

ium

(A

M10

92/A

M50

92)

Tetr

athi

onat

e B

roth

Bas

e, H

ajna

(A

M10

96/A

M50

96)

Trip

le S

ugar

Iron

(T

SI)

Aga

r (A

M10

99/A

M50

99)

Ure

a B

roth

Bas

e (A

M11

05)

Ure

a 40

%*

(AS

028)

Vog

el J

ohns

on A

gar

Bas

e (A

M11

08/A

M51

08)

Po

tass

ium

Tel

luri

te 1

%*(

AS

022)

XLD

Aga

r (A

M11

12/A

M51

12)

l

l

ll

l

l

l

l

l

l

ll

ll

l l l

l

l

0*

- S

tore

bet

wee

n 2-

8C

.




AP

PLI

CA

TIO

NS

Wat

er a

nd W

aste

wat

er A

naly

sis

PR

OD

UC

TS

CTotal Plate ount

toc Saphyloccus

P MNConfirmed

P resumptveMNP

i

Identii& ifcaton ii Test

Bochemcals

Selective soli Iaton

Pre-Enrichment/ Selective Enrichment

Pseudomonas

Legionella Species

Klebsiella

reptFecal Stococci

phetiColiage Decton

DfiiofF iferentaton ecal& lCof

Nonfeca liorm

F iormsecalColf

MP ple TestNCometd

MP Cied TestNonfrm

MP Presumptive TesN

t

Cltridia os

Viio br

oulYeasts and MdsYersinia nterocoliica

Et

Tota

l col

iform

Sal

mon

ella

/Shi

gella

Qua

litat

ive

Test

sQ

uant

itativ

e Te

sts

Asp

argi

ne P

rolin

e B

roth

(A

M10

05/A

M50

05)

Azi

de B

lood

Aga

r B

ase

(AM

1006

/AM

5006

)

Bai

rd P

arke

r Aga

r B

ase

(AM

1011

/AM

5011

)

BP

Su

lph

a S

up

ple

men

t* (

AS

005)

Eg

g Y

olk

Em

uls

ion

* (A

010)

EY

Tel

luri

te E

nri

chm

ent*

(A

S01

1)

Po

tass

ium

Tel

luri

te 3

.5%

* (A

S02

3)

Bee

f Ext

ract

Aga

r (A

M10

111/

AM

5011

1)

Bile

Esc

ulin

Aga

r B

ase

(AM

1012

/AM

5012

)

Bis

mut

h S

ulph

ite A

gar

(A

M10

13/A

M50

13)

Bra

in H

eart

Infu

sion

Aga

r (A

M10

16/A

M50

16)

Bra

in H

eart

Infu

sion

Bro

th (

AM

1017

/AM

5017

)

Bril

liant

Gre

en A

gar

Mod

ified

(A

M10

18/A

M50

18)

Su

lph

a S

up

ple

men

t* (

AS

027)

Bril

liant

Gre

en B

ile B

roth

2%

(A

M10

20/A

M50

20)

Cet

rimid

e A

gar

Bas

e (

AM

1022

/AM

5022

)

Nal

idix

ic S

elec

tive

Su

pp

lem

ent*

(A

S02

0)

Cet

rimid

e B

roth

(A

M10

23/A

M50

23)

Cor

n M

eal A

gar

(AM

1030

1/A

M50

301)

Deo

xych

olat

e C

itrat

e A

gar

(AM

1031

/AM

5031

)

Diff

eren

tial R

einf

orce

d C

lost

ridia

l Bro

th (

AM

1038

/AM

5038

)

EC

Bro

th (

AM

1039

/AM

5039

)

EM

B A

gar,

Levi

ne (

AM

1040

/AM

5040

)

End

o A

gar

(AM

1041

/AM

5041

)

l

ll l l

l

l

l l

ll

ll

l l

l

l

l

ll

ll

l

Campyler obact




AP

PLI

CA

TIO

NS

Wat

er a

nd W

aste

wat

er A

naly

sis

Tot Plte Countala

Staphylococcus

MPNConfirmed

ptveMPNPresumi

dent&

Iification Test

Biochemicals

Seltive soliecIaton

Pre-Echment/nri

Selective Echmentnri

Pseudomonas

Leginella Speieso

c

Klebsiella

Fecal Streptococci

etiColiphage Decton

fiiofF

Diferentaton ecal& fNonfecal Coliorm

iormsFecalColf

P MNCompleted Test

MP istNConfrmed Te

MP Presumptve TesN

it

Clria ostdi

Viio br

Yeasts and MouldsYerinia nterocolii

sEtca

Tota

l col

iform

Sal

mon

ella

/Shi

gella

Qua

litat

ive

Test

sQ

uant

itativ

e Te

sts

Flu

id L

acto

se M

ediu

m (

AM

1042

/AM

5042

)

Flu

id S

elen

ite C

ystin

e M

ediu

m (

AM

1044

/AM

5044

)

Klig

ler

Iron

Aga

r (A

M10

50/A

M50

50)

Lact

ose

Bro

th (

AM

1052

/AM

5052

)

Laur

yl T

rypt

ose

Bro

th (

AM

1053

/AM

5053

)

Legi

onel

la A

gar

Bas

e (A

M10

54/A

M50

54)

Leg

ion

ella

Gro

wth

Su

pp

lem

ent*

(A

S01

6)

Leg

ion

ella

Sel

ecti

ve S

up

ple

men

t* (

AS

017)

Mac

Con

key

Aga

r w

ith C

ryst

al V

iole

t, N

aCl a

nd w

ith 0

.15%

B

ile S

alts

(A

M10

59/A

M50

59)

Mac

Con

key

Aga

r w

ithou

t Cry

stal

Vio

let a

nd w

ith 0

.15%

B

ile S

alts

(AM

1060

/AM

5060

)

Mac

Con

key

Aga

r w

ithou

t Cry

stal

Vio

let,

NaC

l and

with

0.

5% S

odiu

m T

auro

chol

ate

(AM

1061

/AM

5061

)

Mac

Con

key

Bro

th w

ith N

eutr

al R

ed (

AM

1064

/AM

5064

)

Mac

Con

key

Bro

th P

urpl

e W

/Bro

moc

reso

l

Mal

onat

e B

roth

Ew

ing,

Mod

ified

(A

M10

65/A

M50

65)

Mal

t Aga

r (A

M10

66/5

066)

Mal

t Ext

ract

Aga

r (A

M10

67/5

067)

Man

nito

l Mot

ility

Tes

t Med

ium

(A

M10

681/

AM

5068

1)

Man

nito

l Sal

t Aga

r (A

M10

69/A

M50

69)

MR

-VP

Med

ium

(A

M10

71/A

M50

71)

Nut

rient

Aga

r pH

6.8

(A

M10

75/A

M50

75)

Nut

rient

Bro

th (

AM

1077

/AM

5077

)

Pla

te C

ount

Aga

r (A

M10

81/A

M50

81)

Pre

ston

Aga

r B

ase

(AM

1083

1/A

M50

831)

l ll

l

l

ll

l

ll

l l l

l

ll

l

l l

ll

l

l

ll

l

l

l

l

l

Pur

ple

(AM

1063

/AM

5063

)PR

OD

UC

TS

Campylobacter




0*

- S

tore

bet

wee

n 2-

8C

.

AP

PLI

CA

TIO

NS

Wat

er a

nd W

aste

wat

er A

naly

sis

PR

OD

UC

TS

Tot e CountalPlat

Stoc aphyloccus

MP CiedNonfrm

MP PresumptiveN

Identification & Biochemical Tests

Selective Isolation

re-EchmentPnri/ iEchmentSelectve nri

seudom Ponas

lSesLegionela peci

Klebil sela

F treptciecalSococ

Colphage etectioniD

Diferentiation of Fecal &f

Nonecal Coliformf

Fecal Coliforms

MPN Completed Test

MPNConfirmed Test

ptve TesMPNPresumit

a Clostridi

i Vibro

Ys Mouldseastand

nierocolYersia Entitica

Tota

l col

iform

Sal

mon

ella

/Shi

gella

Qua

litat

ive

Test

sQ

uant

itativ

e Te

sts

Campylobacter

Pre

sto

n S

elec

tive

Su

pp

lem

ent

(Cam

pyl

ob

acte

r S

elec

tive

S

up

ple

men

t IV

, Mo

dif

ied

) (A

S02

31)

Pse

udom

onas

Aga

r B

ase

(AM

1084

/AM

5084

)

Cet

rin

ix S

up

ple

men

t* (

AS

008)

CF

C S

up

ple

men

t* (

AS

009)

Rei

nfor

ced

Clo

strid

ial A

gar

(AM

1085

/AM

5085

)

Sel

enite

F B

roth

(A

M10

89/A

M50

89)

Sim

mio

ns C

itrat

e A

gar

(AM

1090

/AM

5090

)

Soy

abea

n C

asei

n D

iges

t Aga

r (A

M10

91/A

M50

91)

Soy

abea

n C

asei

n D

iges

t Med

ium

(A

M10

92/A

M50

92)

SS

Aga

r (A

M10

93/A

M50

93)

TC

BS

Aga

r (A

M10

95/A

M50

95)

Tetr

athi

onat

e B

roth

Bas

e, H

ajna

(A

M10

96/A

M50

96)

Trip

le S

ugar

Iron

Aga

r (

AM

1099

/AM

5099

)

Tryp

tone

Glu

cose

Ext

ract

Aga

r (A

M11

01/A

M51

01)

Tryp

tone

Wat

er (

AM

1104

/AM

5104

)

Ure

a A

gar

Bas

e, C

hris

tens

en (

AM

1105

/AM

5105

)

Ure

a 40

%*

(AS

028)

Ure

a B

roth

Bas

e (A

M11

06/A

M51

06)

Ure

a 40

%*

(AS

028)

Vio

let R

ed B

ile A

gar

(AM

1107

/AM

5107

)

XLD

Aga

r (A

M11

12/A

M51

12)

Yeas

t Mal

t Aga

r (A

M11

14/A

M51

14)

Yeas

t Mal

t Bro

th (

AM

1115

/AM

5115

)

Yers

inia

Sel

ectiv

e A

gar

Bas

e (A

M11

16/T

M51

16)

Yer

sin

ia S

elec

tive

Su

pp

lem

ent*

(A

S02

9)

l

l

ll

ll

l

l ll

l

l

ll

ll

ll

l

ll

ll

l

l

ll

l l

l




AP

PLI

CA

TIO

NS

Vet

erin

ary

Test

ing

MdsYeast& oul

Viio br

Stococcirept

toc Saphylocci

Salmonella/Shigella

Pseudomonas

actumMycoberi

Listeria

G urpose CultureeneralP

a Medi

Coliforms

Brucella

naerobes A

(Including Clostridia)

PR

OD

UC

TSA

zide

Blo

od A

gar

Bas

e (A

M10

06/A

M50

06)

Bai

rd P

arke

r Aga

r B

ase

(AM

1011

/AM

5011

)

BP

Su

lph

a S

up

ple

men

t* (

AS

005)

Eg

g Y

olk

Em

uls

ion

*(A

010)

Eg

g Y

olk

Tel

luri

te E

mu

lsio

n*

(AS

011)

Po

tass

ium

Tel

luri

te 3

.5%

* (A

S02

3)

Bis

mut

h S

ulph

ite A

gar

(AM

1013

/AM

5013

)

Bra

in H

eart

Infu

sion

Aga

r (A

M10

16/A

M50

16)

Bra

in H

eart

Infu

sion

Bro

th (

AM

1017

/AM

5017

)

Bril

liant

Gre

en A

gar

Mod

ified

(A

M10

18/A

M50

18)

Su

lph

a S

up

ple

men

t* (

AS

027)

Cet

rimid

e A

gar

Bas

e (A

M10

22/A

M50

22)

Nal

idix

ic S

elec

tive

Su

pp

lem

ent*

(A

S02

2)

Col

umbi

a B

lood

Aga

r B

ase

(AM

1029

/AM

5029

)

Bru

cella

Sel

ecti

ve S

up

ple

men

t* (

AS

006)

Sta

ph

-Str

epto

Su

pp

lem

ent*

(A

S02

5)

Str

epto

Su

pp

lem

ent*

(A

S02

6)

Coo

ked

Mea

t Med

ium

(A

M10

30/A

M50

30)

Cor

n M

eal A

gar

(AM

1030

1/A

M50

301)

DN

ase

Test

Aga

r B

ase

(AM

1038

1)

EM

B A

gar,

Levi

ne (

AM

1040

/504

0)

End

o A

gar

(AM

1041

/AM

5041

)

ll

l

l

l l

l

l

ll

l

l ll

l

l l




AP

PLI

CA

TIO

NS

Vet

erin

ary

Test

ing

MdsYeast& oul

Viio br

Stococcirept

toc Saphylocci

Salmonella/Shigella

Pseudomonas

actumMycoberi

Listeria

G urpose CultureeneralP

a Medi

Coliforms

Brucella

naerobes A

(Including Clostridia)

PR

OD

UC

TSLi

ster

ia Id

entif

icat

ion

Aga

r B

ase

PALC

AM

(A

M10

55/A

M50

55)

Flu

id S

elen

ite C

ystin

e M

ediu

m (

AM

1044

/AM

5044

)

Flu

id T

hiog

lyco

llate

Med

ium

(A

M10

45/A

M50

45)

Hea

rt In

fusi

on A

gar

(AM

1048

1/A

M50

481)

Lis

teri

a S

elec

tive

Su

pp

lem

ent*

(A

S01

8)

Live

r In

fusi

on A

gar

(AM

1055

1/A

M50

551)

Live

r In

fusi

on B

roth

(A

M10

552/

AM

5055

2)

Low

enst

ein

Jens

en M

ediu

m B

ase

(AM

1057

/AM

5057

)

Mac

Con

key

Aga

r B

ase

(AM

1058

/AM

5058

)

Mal

t Aga

r (A

M10

66/A

M50

66)

Mal

t Ext

ract

Aga

r (A

M10

67/A

M50

67)

Man

nito

l Sal

t Aga

r (A

M10

69/A

M50

69)

MR

-VP

Med

ium

(A

M10

71/A

M50

71)

Pot

ato

Dex

tros

e A

gar

(AM

1082

/AM

5082

)

Pse

udom

onas

Aga

r B

ase

(AM

1084

/AM

5084

)

Cet

rin

ix S

up

ple

men

t* (

AS

008)

CF

C S

up

ple

men

t* (

AS

009)

Rei

nfor

ced

Clo

strid

ial A

gar

(AM

1085

/AM

5085

)

Sab

oura

ud D

extr

ose

Aga

r (A

M10

87/A

M50

87)

Sab

oura

ud D

extr

ose

Bro

th (

AM

1088

/AM

5088

)

Sel

enite

F B

roth

(A

M10

89/A

M50

89)

Sim

mon

s C

itrat

e A

gar

(AM

1090

/AM

5090

)

l

l

ll l

l l

l

l

l l

l

ll

l

l

l

l

l l

l

ll




AP

PLI

CA

TIO

NS

Vet

erin

ary

Test

ing

Y Mdseast&oul

i Vibro

Streptococci

Stoc aphylocci

ala/lSmonellShigela

Pseudomonas

Mycobactum eri

Listeria

General Purpose Culture Media

Cf oliorms

rucel Bla

Anaerobes ncli (Iudng ostdi Clria)

PR

OD

UC

TSS

oyab

ean

Cas

ein

Dig

est A

gar

(AM

1091

/AM

5091

)

Soy

abea

n C

asei

n D

iges

t Med

ium

(A

M10

92/A

M50

92)

SS

Aga

r (A

M10

93/A

M50

93)

TC

BS

Aga

r (A

M10

95/A

M50

95)

Tetr

athi

onat

e B

roth

Bas

e, H

ajna

(A

M10

96/A

M50

96)

Trip

le S

ugar

Iron

(T

SI)

Aga

r (A

M10

99/A

M50

99)

Tryp

tose

Aga

r (A

M11

041/

AM

5104

1)

Ure

a A

gar

Bas

e, C

hris

tens

en (

AM

1105

/AM

5105

)

Ure

a 40

%*

(AS

028)

Vio

let R

ed B

ile A

gar

(AM

1107

/AM

5107

)

Vog

el J

ohns

on A

gar

Bas

e (

AM

1108

/AM

5108

)

Po

tass

ium

Tel

luri

te 1

%*

(AS

022)

XLD

Aga

r (A

M11

12/A

M51

12)

Yeas

t Mal

t Aga

r (A

M11

14/A

M51

14)

Yeas

t Mal

t Bro

th (

AM

1116

/AM

5116

)

l l

l

l

l

ll

l

ll

l

l

l

l l

0*

- S

tore

bet

wee

n 2-

8C

.




Pro

duct

s fo

r B

rew

ery

and

Ferm

enta

tion

Ana

lysi

s

AP

PLI

CA

TIO

NS

gar MaAedi

Liquid Media

Coliforms

lliLactobaci

Fer

men

tati

on

Pro

cess

Flu

id L

acto

se M

ediu

m (

AM

1042

/AM

5042

)

Lact

obac

illus

MR

S A

gar

(AM

1051

/AM

5051

)

Mac

Con

key

Aga

r w

ith C

ryst

al V

iole

t, N

aCl a

nd 0

.15%

Bile

Sal

ts (

AM

1059

/AM

5059

)

Mac

Con

key

Bro

th P

urpl

e w

ith B

rom

ocre

sol P

urpl

e (A

M10

63/A

M50

63)

Mal

t Aga

r (A

M10

66/A

M50

66)

WL

Diff

eren

tial A

gar

(AM

1109

/AM

5109

)

WL

Nut

rient

Bro

th (

AM

1110

/AM

5110

)

Wor

t Aga

r (A

M11

11/A

M51

11)

Yeas

t Mal

t Aga

r (A

M11

14/A

M51

14)

l

l

l l

l l

ll

ll l

Bre

wer

y A

nal

ysis

PR

OD

UC

TS

0*

- S

tore

bet

wee

n 2-

8C

.




Pro

duct

s fo

r M

edic

al a

nd

Res

earc

h In

stit

utes

AP

PLI

CA

TIO

NS

PR

OD

UC

TS

Yrsa eocoticeininterlia

Yeast & Moulds

Vibrio

Streptococcus

Saphyocustlocc

Shigella

Slmonelaa

l

Proteus

Peu sdomonas

Neisseria

Mcopmaylas

Mycobacteria

Lisia ter

Legionella

Kebel lsila

Haemophilis

E co .li

Corynebacterium

Cltri osdia

ca Bruell

Bordella te

tdesBaceroi

0C

- C

ultiv

atio

n, E

- E

nric

hmen

t, I -

Iden

tific

atio

n, N

- N

on-S

elec

tive

and

S -

Sel

ectiv

e Is

olat

ion.

* -

Sto

re b

etw

een

2-8

C.

Ana

erob

ic A

gar

(AM

1000

/AM

5000

)

Azi

de B

lood

Aga

r B

ase

(AM

1006

/AM

5006

)

Bac

tero

ides

Bile

Esc

ulin

Aga

r (A

M10

10/A

M50

10)

Bac

tero

ides

Sel

ecti

ve S

up

ple

men

t* (

AS

001)

Bai

rd P

arke

r Aga

r B

ase

(AM

1011

/AM

5011

)

B P

Su

lph

a S

up

ple

men

t* (

AS

005)

Eg

g Y

olk

Em

uls

ion

* (A

S01

0)

Eg

g Y

olk

Tel

luri

te E

mu

lsio

n*

(AS

011)

Po

tass

ium

Tel

luri

te 3

.5%

* (A

S02

3)

Bis

mut

h S

ulph

ite A

gar

(AM

1013

/AM

5013

)

Blo

od A

gar

Bas

e (A

M10

14/A

M50

14)

Bor

det G

engo

u A

gar

Bas

e (A

M10

15/A

M50

15)

Bo

rdet

ella

Sel

ecti

ve S

up

ple

men

t* (

AS

004)

Bra

in H

eart

Infu

sion

Bro

th (

AM

1017

/AM

5017

)

Bril

iiant

Gre

en A

gar

Mod

ified

(A

M10

18/A

M50

18)

Su

lph

a S

up

ple

men

t* (

AS

027)

Bril

liant

Gre

en B

ile A

gar

(AM

1019

/AM

5019

)

Bril

liant

Gre

en B

ile B

roth

2%

(A

M10

20/A

M50

20)

Cet

rimid

e A

gar

Bas

e (A

M10

22/A

M50

22)

Nal

idix

ic S

elec

tive

Su

pp

lem

ent*

(A

S02

0)

Cet

rimid

e B

roth

(A

M10

23/A

M50

23)

Chl

oram

phen

icol

Yea

st G

luco

se A

gar

(AM

1024

/AM

5024

)

CLE

D A

gar

with

And

rade

indi

cato

r (A

M10

26/A

M50

26)

CLE

D A

gar

with

Bro

mot

hym

ol B

lue

(AM

1027

/AM

5027

)

Coa

gula

se M

anni

tol A

gar

Bas

e (A

M10

28/A

M50

258)

Col

umbi

a B

lood

Aga

r B

ase

(AM

1029

/AM

5029

)

C

CC

C/I/

S

S

S

C/N

C/N

C/S

E

S

N E

S C

S

NN

NN

NN

N

NN

NN

NN

N

S

SS

S

Campylobacter Selective Islation o




Pro

duct

s fo

r M

edic

al a

nd

Res

earc

h In

stit

utes

AP

PLI

CA

TIO

NS

PR

OD

UC

TS

Yersnia eterocoliticain

oYeast & Mulds

Vibior

epccStrtocous

Staphyloccus oc

g Shiella

Salmonella

te Prous

Pseuomasdon

eie Nssria

Mycopasma l

obiMycactera

Listeria

gilLeonela

Klebiela sl

aeopsHmhili

E. coil

oracuCynebterim

Cltriia osd

Brucla el

e Bordtella

Bactides ero

0C

- C

ultiv

atio

n, E

- E

nric

hmen

t, I -

Iden

tific

atio

n, N

- N

on-S

elec

tive

and

S -

Sel

ectiv

e Is

olat

ion.

* -

Sto

re b

etw

een

2-8

C.

amy Cplobacter Seion

lectve Isolati

Bru

cella

Sel

ecti

ve S

up

ple

men

t* (

AS

006)

Cam

pyl

ob

acte

r S

elec

tive

Su

pp

lem

ent

wit

h H

emin

(K

arm

ali)

M

od

ifie

d*

(AS

007)

Sta

ph

-Str

epto

Su

pp

lem

ent*

(A

S02

5)

Str

epto

Su

pp

lem

ent*

(A

S02

6)

Coo

ked

Mea

t Med

ium

(A

M10

30/A

M50

30)

Deo

xych

olat

e C

itrat

e A

gar

(AM

1031

/AM

5031

)

Diff

eren

tial R

einf

orce

d C

lost

ridia

l Bro

th (

AM

1038

/AM

5038

)

EC

Bro

th (

AM

1039

/AM

5039

)

EM

B A

gar

(AM

1039

1/A

M50

391)

EM

B A

gar,

Levi

ne (

AM

1040

/AM

5040

)

EM

Bro

th (

AM

1040

1/A

M50

401)

End

o A

gar

(AM

1041

/AM

5041

)

Flu

id L

acto

se M

ediu

m (

AM

1042

/AM

5042

)

Flu

id S

abou

raud

Med

ium

(A

M10

43/A

M50

43)

Flu

id S

elen

ite C

ystin

e M

ediu

m (

AM

1044

/AM

5044

)

Flu

id T

hiog

lyco

llate

Med

ium

(A

M10

45/A

M50

45)

G.C

Aga

r B

ase

(AM

1046

/AM

5046

)

G.C

Su

pp

lem

ent*

(A

S01

2)

Hae

mo

glo

bin

So

luti

on

* (A

S01

4)

Klig

ler

Iron

Aga

r (A

M10

50/A

M50

50)

Laur

yl T

rypt

ose

Bro

th (

AM

1053

/AM

5053

)

Legi

onel

la A

gar

Bas

e (A

M10

54/A

M50

54)

Leg

ion

ella

Gro

wth

Su

pp

lem

ent*

(A

S01

6)

Leg

ion

ella

Sel

ecti

ve S

up

ple

men

t* (

AS

017)

List

eria

Iden

tific

atio

n A

gar

Bas

e,PA

LCA

M (

AM

1055

/AM

5055

)

E/C

SS

C

S

C/S

C/S

NN

N

C/S

C/S

NN

N

EE

E

E

EE

C

C/S

C

II

II

I

E

S

S




Pro

duct

s fo

r M

edic

al a

nd

Res

earc

h In

stit

utes

AP

PLI

CA

TIO

NS

PR

OD

UC

TS

Yersnia eterocoliticain

soYeat & Mulds

Vibior

epccStrtocous

Staphyloccus oc

g Shiella

Salmonella

te Prous

Pseuomasdon

eie Nssria

Mycopasma l

obriMycactea

Listeria

gilLeonela

Klebsielal

aeopisHmhil

E. coil

oreacuCynbterim

Clstriia od

Bruclla e

e Bordtella

Bactides ero

0C

- C

ultiv

atio

n, E

- E

nric

hmen

t, I -

Iden

tific

atio

n, N

- N

on-S

elec

tive

and

S -

Sel

ectiv

e Is

olat

ion.

* -

Sto

re b

etw

een

2-8

C.

amy Cplobacter Setion

lecve Isolati

Lis

teri

a S

elec

tive

Su

pp

lem

ent*

(A

S00

8)

Loef

fler

Med

ium

Bas

e (A

M10

56/A

M50

56)

Ho

rse

Ser

um

* (A

S01

5)

Low

enst

ein

Jens

en M

ediu

m B

ase

(AM

1057

/AM

5057

)

Gru

ft M

yco

bac

teri

al S

up

ple

men

t* (

AS

013)

Mac

Con

key

Aga

r w

ith C

ryst

al V

iole

t, N

aCl a

nd 0

.15%

Bile

Sal

ts(A

M10

59/A

M50

59)

Mac

Con

key

Aga

r w

ithou

t Cry

stal

Vio

let,

NaC

l and

with

0.5

%

Sod

ium

Tau

rach

olat

e (A

M10

61/A

M50

61)

Mac

Con

key

Bro

th P

urpl

e w

ith B

rom

ocre

sol P

urpl

e (A

M10

63/A

M50

63)

Mal

onat

e B

roth

Ew

ing

Mod

ified

(A

M10

65/A

M50

65)

Mal

t Aga

r (A

M10

66/A

M50

66)

Mal

t Ext

ract

Aga

r (A

M10

67/A

M50

67)

Mal

t Ext

ract

Bro

th (

AM

1068

/AM

5068

)

Man

nito

l Sal

t Aga

r (A

M10

69/A

M50

69)

MR

-VP

Med

ium

(A

M10

70/A

M50

70)

Myc

opla

sma

Aga

r B

ase

(PP

LO A

gar)

(A

M10

73/A

M50

73)

Myc

op

lasm

a E

nri

chm

ent

Su

pp

lem

ent*

(A

S01

9)

Ho

rse

Ser

um

* (A

S01

5)

Pot

ato

Dex

tros

e A

gar

(AM

1082

/AM

5082

)

Pot

ato

Dex

tros

e B

roth

(A

M10

83/A

M50

83)

Pre

ston

Aga

r B

ase

(AM

1083

1/A

M50

831)

Pse

udom

onas

Aga

r B

ase

(AM

1084

/AM

5084

)

Cet

rin

ix S

up

ple

men

t* (

AS

008)

CF

C S

up

ple

men

t* (

AS

009)

Rei

nfor

ced

Clo

strid

ial A

gar

(AM

1085

/AM

5085

)

C/N

C/N

NN

NN

NN

NN

NN

N

N II

C C C

S

II

I

C/S

C/N

C/N

S

S

C




Pro

duct

s fo

r M

edic

al a

nd

Res

earc

h In

stit

utes

AP

PLI

CA

TIO

NS

PR

OD

UC

TS

eoc

Yersinianteroclitia

Yeasodst & Mul

Vibrio

Strepoccstocu

hs

Stapylococcu

Shigla el

mlSalonela

Proteus

u Psedomonas

Neisea sri

o Mycplasma

Mcobteriyaca

Listeria

Legiell ona

b Klesiella

Haeoplis mhi

o E. cli

Cornebacriuytem

Clostridia

Brucella

rdelaBotel

Bacteroides

0C

- C

ultiv

atio

n, E

- E

nric

hmen

t, I -

Iden

tific

atio

n, N

- N

on-S

elec

tive

and

S -

Sel

ectiv

e Is

olat

ion.

* -

Sto

re b

etw

een

2-8

C.

Campybac loter Setie Isaionlecvolt

Sab

oura

ud C

hlor

amph

enic

ol A

gar

(AM

1086

/AM

5086

)

Sab

oura

ud D

extr

ose

Aga

r (A

M10

87/A

M50

87)

Sab

oura

ud D

extr

ose

Bro

th (

AM

1088

/AM

5088

)

Sel

enite

F B

roth

(A

M10

89/A

M50

89)

Sim

mon

s C

itrat

e A

gar

(AM

1090

/AM

5090

)

SS

Aga

r (A

M10

93/A

M50

93)

TC

BS

Aga

r (A

M10

95/A

M50

95)

Tetr

athi

onat

e B

roth

Bas

e, H

ajna

(A

M10

96/A

M50

96)

Trip

le S

ugar

Iron

(T

SI)

Aga

r (A

M10

99/A

M50

99)

Tryp

tic D

iges

t Bro

th (

AM

1100

/AM

5100

)

Tryp

tone

Pho

spha

te B

roth

(A

M11

03/A

M51

03)

Tryp

tone

Wat

er (

AM

1104

/AM

5104

)

Ure

a A

gar

Bas

e, C

hris

tens

en (

AM

1105

/AM

5105

)

Ure

a 40

%*

(AS

028)

Ure

a B

roth

Bas

e (A

M11

06/A

M51

06)

Ure

a 40

%*

(AS

028)

Vio

let R

ed B

ile A

gar

(AM

1107

/AM

5107

)

Vog

el J

ohns

on A

gar

Bas

e (A

M11

08/A

M51

08)

Po

tass

ium

Tel

luri

te 1

%*

(AS

022)

XLD

Aga

r (A

M11

12/A

M51

12)

Yeas

t Mal

t Aga

r (A

M11

14/A

M51

14)

Yeas

t Mal

t Bro

th (

AM

1115

/AM

5115

)

Yers

inia

Sel

ectiv

e A

gar

Bas

e (A

M11

16/A

M51

16)

Yer

sin

ia S

elec

tive

Su

pp

lem

ent*

(A

S02

9)

S C/N

C/N

E

II

II

I

SS

S

E

II

II

C

E I II

II

II

II

N

S

SS

C/N

C/N

C/S




Pro

duct

s fo

r B

ioch

emic

al T

ests

e e UraseTst

Methyl Red Tet s

liMaonate Utlization

Lctina Testeihse

dIndoleProuction

Hdelpdyrogn Suhie duio Proctn

lssHaemoyi Test

Escli yrolsisunHdy

CraeUtiiztionitt la

ntnCarbo Utilizaio

i Media Wth More Than One Carbohydrate

Meda WihOneit Cabohydrt rae

Md ihteiaWtou abyd Crohrates

AP

PLI

CA

TIO

NS

PR

OD

UC

TS

Car

bohy

drat

e F

erm

enta

tion

0*

- S

tore

bet

wee

n 2-

8C

.

Deoxyribonuclease Test

And

rade

Pep

tone

Wat

er(A

M10

01/A

M50

01)

Bai

rd P

arke

r Aga

r B

ase(

AM

1011

/AM

5011

)

BP

Su

lph

a S

up

ple

men

t* (

AS

005)

Eg

g Y

olk

Em

uls

ion

* (A

S01

0)

Eg

g Y

olk

Tel

luri

te E

mu

lsio

n*

(AS

011)

Po

tass

ium

Tel

luri

te 3

.5%

* (A

S02

3)

Bile

Esc

ulin

Aga

r (A

M10

12/A

M50

12)

Blo

od A

gar

Bas

e (A

M10

14/A

M50

14)

Bra

in H

eart

Infu

sion

Aga

r (A

M10

16/A

M50

16)

Bro

mot

hym

ol B

lue

Lact

ose

Aga

r (A

M10

21/A

M50

21)

Chr

iste

nsen

Citr

ate

Aga

r(A

M10

25/A

M50

25)

Col

umbi

a B

lood

Aga

r B

ase

(AM

1029

/AM

5029

)

DN

ase

Test

Aga

r B

ase

(AM

1038

1)

Hea

rt In

fusi

on A

gar

(AM

1048

1/A

M50

481)

Sta

ph

-Str

epto

Su

pp

lem

ent*

(A

S02

5)

Str

epto

Su

pp

lem

ent*

(A

S02

6)

Klig

ler

Iron

Aga

r (A

M10

50/A

M50

50)

Mal

onat

e B

roth

, Ew

ing

Mod

ified

(A

M10

65/A

M50

65)

MR

-VP

Med

ium

(A

M10

71/A

M50

71)

Pep

tone

Wat

er (

AM

1079

/AM

5079

)

Pep

tone

Wat

er w

ith P

heno

l Red

(A

M10

80/A

M50

80)

Sim

mon

s C

itrat

e A

gar

(AM

1090

/AM

5090

)

l

l

l

l l

l

ll

l

l

l

ll

ll

l

l l

ll




Pro

duct

s fo

r B

ioch

emic

al T

ests

Uease es rTt

Mtyl e TetehRds

MalnaeUtiizationot l

astLecithinse Te

Indle Productono

i

de Hyrogn Sulphide o Productin

Hemolsis esayTt

ilssEsculn Hydroyi

CraeUitnitt tlizaio

Carbon Utilization

Meda WihMoe Tanit rh On arbhydate

eCor

Med ihOeiaWt n abhyd Crorate

i Media Wthout Carbohydrates

AP

PLI

CA

TIO

NS

PR

OD

UC

TS

Car

bohy

drat

e F

erm

enta

tion

0*

- S

tore

bet

wee

n 2-

8C

.

Doxyribouclease esen

Tt

Soy

abea

n C

asei

n D

iges

t Aga

r (A

M10

91/A

M50

91)

Trip

le S

ugar

Iron

(T

SI)

Aga

r (A

M10

99/A

M50

99)

Tryp

tone

Wat

er (

AM

1104

/AM

5104

)

Ure

a A

gar

Bas

e, C

hris

tens

en (

AM

1105

/AM

5105

)

Ure

a 40

%*

(AS

028)

Ure

a B

roth

Bas

e (A

M11

06/A

M51

06)

Ure

a 40

%*

(AS

028)

l

ll

l

l l




AP

PLI

CA

TIO

NS

Cos

met

ic A

naly

sis

PR

OD

UC

TS

Yeast & Mould Cnts ou

Y Mdseast&oul

tl Seriity Test

Staphyloci occ

Pseudomonas

nt Eerobacteriaceae

Bacillus

Bactal Counteri

s

Anaerobes ncludi (ing ostdium)Clri

An

ae

rob

ic A

ga

r (A

M1

00

0/A

M5

00

0)

Ba

ird

Pa

rke

r A

ga

r B

ase

(A

M1

011

/AM

50

11)

BP

Su

lph

a S

up

ple

me

nt*

(A

S0

05

)

Eg

g Y

olk

Em

uls

ion

* (A

S0

10

)

Eg

g Y

olk

Te

llu

rite

Em

uls

ion

* (A

S0

11

)

Po

tas

siu

m T

ellu

rite

3.5

%*

(AS

02

3)

Brilli

an

t G

ree

n B

ile B

roth

2%

(AM

10

20

/AM

50

20

)

Bro

mo

thym

ol B

lue

La

cto

se

Ag

ar

(AM

10

21

/50

21

)

Ce

trim

ide

Ag

ar

Ba

se

(A

M1

02

2/A

M5

02

2)

Na

lid

ixic

Se

lec

tiv

e S

up

ple

me

nt*

(A

S0

20

)

Ce

trim

ide

Bro

th (

AM

10

23

/50

23

)

CL

ED

Ag

ar

with

An

dra

de

In

dic

ato

r (A

M1

02

6/A

M5

02

6)

CL

ED

Ag

ar

with

Bro

mo

thym

ol B

lue

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M1

02

7/A

M5

02

7)

Co

oke

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ea

t M

ed

ium

(A

M1

03

0/A

M5

03

0)

Diff

ere

ntia

l Re

info

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lostr

idia

l Bro

th (

AM

10

38

/AM

50

38

)

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ase

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st A

ga

r B

ase

(A

M1

03

81

)

EM

B A

ga

r, L

evin

e (

AM

10

40

/AM

50

40

)

En

do

Ag

ar

(AM

10

41

/AM

50

41

)

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id L

act

ose

Me

diu

m (

AM

10

42

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50

42

)

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id T

hio

gly

co

llate

Me

diu

m (

AM

10

45

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50

45

)

Ma

cC

on

ke

y A

ga

r w

ith C

rysta

l Vio

let, N

aC

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d 0

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% B

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alts

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M1

05

9/A

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9)

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cC

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ga

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10

60

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50

60

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lon

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win

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od

ifie

d (

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10

65

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50

65

)

Ma

lt A

ga

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M1

06

6/A

M5

06

6)

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M1

06

7/A

M5

06

7)

l

l

l

l

l l

l l

l l

l

l l l

ll

l l l

l l




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PLI

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met

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naly

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OD

UC

TS

M Yeast & ould Counts

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acis Bllu

Bacterial Counts

Anaerobes (including Costrdium)li

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lt E

xtr

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roth

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M1

06

8/A

M5

06

8)

Ma

nn

itol S

alt

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ar

(AM

10

69

/AM

50

69

)

MR

-VP

Me

diu

m (

AM

10

70

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50

70

)

Nu

trie

nt A

ga

r (A

M1

07

4/A

M5

07

4)

Nu

trie

nt B

roth

(A

M1

07

7/A

M5

07

7)

Pla

te C

ou

nt A

ga

r (A

M1

08

1/A

M5

08

1)

Po

tato

De

xtro

se A

ga

r (A

M1

08

2/A

M5

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2)

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om

on

as A

ga

r B

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M1

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4/A

M5

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4)

Ce

trin

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up

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me

nt*

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S0

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)

CF

C S

up

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me

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09

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ar

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10

85

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50

85

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bo

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Ag

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10

87

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50

87

)

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bo

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AM

10

88

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50

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ya

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sein

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ar

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91

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50

91

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ya

be

an

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sein

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diu

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AM

10

92

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50

92

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le S

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TS

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ga

r (A

M1

09

9/A

M5

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ge

l Jo

hn

so

n A

ga

r B

ase

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51

08

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tas

siu

m T

ellu

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* (A

S0

22

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Ye

ast

Ma

lt A

ga

r (A

M11

14

/AM

511

4)

l

l

l

l l

l

l

l

l

ll

l

ll l

l

l

l

l

0*

- S

tore

bet

wee

n 2-

8C

.




Microxpress Product ListHand Book of


DEHYDRATED CULTURE MEDIAAM5908 Acetate Differential Agar

A medium for the differentiation of Shigella species from Escherichia coli.

AM5909 AK Agar No. 2 (Sporulating Agar) ( Arret and Kirshbaum Medium)A medium for preparation of spore inoculum of Bacillus subtilis ATCC 6633 used for the detection of antibiotic residues in milk and dairy products.

A medium for enrichment of Vibrio species.

A medium for enrichment of Vibrio species in compliance with ISO specification ISO / DIS 8914 : 1990.

AM1912 Alkaline Peptone Water BISAM5912 A medium for enrichment of Vibrio species in compliance with BIS

specification IS 5887 (Part 5) 1976, reaffirmed 1986.

AM1900 Alternative Thioglycollate MediumAM5900 A medium recommended for sterility testing of certain biological

products, which may be turbid or viscous.

AM5000 A medium recommended for cultivation of anaerobic microorganisms.

AM1001 Andrade Peptone WaterAM5001 A basal medium to which various carbohydrates may be added to

study fermentation reactions, particularly of members of the Enterobacteriaceae.

AM10011 Andrade Peptone water BISA basal medium to which various carbohydrates may be added to study fermentation reactions in compliance with BIS specification I S:5887 ( Part 1, Part4 and Part5)

AM1002 Antibiotic Assay Medium A (No 1) (Seed Agar)AM5002 A medium for determining antibiotic potency by microbiological

assay techniques as per USP/IP.

AM1910 Alkaline Peptone WaterAM5910

AM5911 Alkaline Peptone Water ISO

AM1000 Anaerobic Agar

DEHYDRATED CULTURE MEDIAAM5908 Acetate Differential Agar

AM5909 AK Agar No. 2 (Sporulating Agar) ( Arret and Kirshbaum Medium)

AM1910 Alkaline Peptone WaterAM5910

AM5911 Alkaline Peptone Water ISO

AM1912 Alkaline Peptone Water BISAM5912

AM1900 Alternative Thioglycollate MediumAM5900

AM1000 Anaerobic AgarAM5000

AM1001 Andrade Peptone WaterAM5001

AM10011 Andrade Peptone water BIS

AM1002AM5002

Antibiotic Assay Medium A (No 1) (Seed Agar)

AM1003 Antibiotic Assay Medium C (No 3) (Assay Broth)AM5003 A medium for determining antibiotic potency by microbiological

assay techniques as per USP/IP.

AM50031 Antibiotic Assay Medium E (No.5) (Streptomycin Assay Agar with yeast Extract)A medium for determining antibiotic potency by microbiological assay techniques as per USP/IP.

AM50032 Antibiotic Assay Medium F (No.8) (Base Agar with low pH)A medium for determining antibiotic potency by microbiological assay techniques as per USP/IP.

AM1004 Antibiotic Assay Medium No 11(Neomycin, Erythromycin Assay Agar)

AM5004 A medium for determining antibiotic potency by microbiological assay techniques as per USP.

AM500411 Antibiotic Assay Medium G (No.19)A medium for determining antibiotic potency by microbiological assay techniques as per USP/IP.

AM500412 Antibiotic Assay Medium No.32A medium for determining antibiotic potency by microbiological assay techniques as per USP.

AM10041 Antifungal Assay AgarAM50041 A medium recommended for assaying the antifungal activity.

AM50042 Artificial Sea WaterA medium for cultivation of marine organisms.

AM50043 Ashby’s Glucose AgarA medium for cultivation of Azotobacter species by using glucose as carbon source.

AM50044 Ashby’s Mannitol AgarA medium for cultivation of Azotobacter species by using mannitol as carbon source.

AM1005 Asparagine Proline BrothAM5005 A medium for cultivation of Pseudomonas aeruginosa by

membrane filtrationtechnique.

AM1003AM5003

AM50031 Antibiotic Assay Medium E (No.5) (Streptomycin Assay Agar with yeast Extract)

AM1004

AM5004

AM10041AM50041

AM50042 Artificial Sea Water

AM1005AM5005

Antibiotic Assay Medium C (No 3) (Assay Broth)

AM50032 Antibiotic Assay Medium F (No.8) (Base Agar with low pH)

Antibiotic Assay Medium No 11(Neomycin, Erythromycin Assay Agar)

AM500411 Antibiotic Assay Medium G (No.19)

AM500412 Antibiotic Assay Medium No.32

Antifungal Assay Agar

AM50043 Ashby’s Glucose Agar

AM50044 Ashby’s Mannitol Agar

Asparagine Proline Broth

CAT. NO. DESCRIPTION CAT. NO. DESCRIPTION CAT. NO. DESCRIPTION CAT. NO. DESCRIPTION


0 0 0Store Dehydrated Media below 30 C, Store all Supplements and Media between 2-8 C, Store below -20 C.* * **





AM1006 Azide Blood Agar BaseAM5006 A medium for selective isolation and cultivation of Staphylococcus

and Streptococcus species and when supplemented with blood for hemolytic reactions.

AM50061 Azide Dextrose BrothA selective medium for detection and cultivation of Streptococci in water, sewage, milk and other materials.

AM1007 B Assay Agar (using E. coli mutant culture)12

A medium for microbiological assay of vitamin B by the cup plate 12

or disc plate method.

AM10071 B Assay Medium (using E. coli mutant and Lactobacillus 12

leichmannii culture)A medium for determining vitamin B concentration by the 12

microbiological assay technique.

AM1008 B Maintenance Media (for E.coli Mutant)12

A medium for the propagation, cultivation and maintenance of E. coli mutant 113-3 D, ATCC 11105, which is the test organism in vitamin B assay.12

AM1009 Bacillus Cereus Agar BaseAM5009 A selective medium for isolation and enumeration of Bacillus

cereus.Polymixin B Selective Supplement AS021Egg Yolk Emulsion AS010

AM1010 Bacteroides Bile Esculin AgarAM5010 A medium for selective isolation, cultivation and presumptive

identification of Bacteroides fragilis group.Bacteroides Selective Supplement AS001

AM1011 Baird Parker Agar BaseAM5011 A medium with added supplements for selective isolation and

enumeration of coagulase positive Staphylococci from clinical and non-clinical specimens. Egg Yolk Tellurite Emulsion AS011Egg Yolk Emulsion AS010Potassium Tellurite 3.5% AS023B P Sulpha Supplement AS005

*

*

*

**

*

**

**

AM1006AM5006

AM1007

AM10071

AM1008

AM1009AM5009

Polymixin B Selective Supplement AS021Egg Yolk Emulsion AS010

AM1010AM5010

Bacteroides Selective Supplement AS001

AM1011AM5011

Egg Yolk Tellurite Emulsion AS011Egg Yolk Emulsion AS010Potassium Tellurite 3.5% AS023B P Sulpha Supplement AS005

Azide Blood Agar Base

AM50061 Azide Dextrose Broth

B Assay Agar (using mutant culture)

B Assay Medium (using mutant and

culture)

B Maintenance Media (for Mutant)

Bacillus Cereus Agar Base

Bacteroides Bile Esculin Agar

Baird Parker Agar Base

E. coli12

E. coli Lactobacillus 12

leichmannii

E.coli12

*

*

*

**

*

**

**

AM101111 Baird Parker Agar Base IPAM501111 A medium with added supplements for selective isolation and

enumeration of coagulase positive Staphylococci from clinical and non-clinical specimens in compliance with IP.Egg Yolk Emulsion AS010Potassium Tellurite 1% AS022

AM101112 Baird Parker Agar Base USPAM501112 A medium with added supplements for selective isolation and

enumeration of coagulase positive Staphylococci from food and other specimens in compliance with USP.Egg Yolk Emulsion AS010Potassium Tellurite 1% AS022

AM101113 Baird Parker Agar Base (Agar medium O) EPAM501113 A medium with added supplements for selective isolation and

enumeration of coagulase positive Staphylococci from food and other specimens in compliance with EP.Egg Yolk Emulsion AS010Potassium Tellurite 1% AS022

AM101114 Baird Parker Agar Base (Agar medium O) BPAM501114 A medium with added supplements for selective isolation and

enumeration of coagulase positive Staphylococci from food and other specimens in compliance with BP.Egg Yolk Emulsion AS010Potassium Tellurite 1% AS022

AM101115 Baird Parker Agar Base BISAM501115 A medium with added supplements for selective isolation and

enumeration of coagulase positive Staphylococci from food and other materials in compliance with BIS specifications IS: 5887 ( part II ) 1976.Egg Yolk Tellurite Emulsion AS011Egg Yolk Emulsion AS010Potassium Tellurite 3.5% AS023

AM10111 Beef Extract AgarAM50111 A general purpose nutrient medium, which supports the growth of

not particularly fastidious bacteria.

AM1012 Bile Esculin AgarAM5012 A differential medium for isolation and presumptive identification

of Group D Streptococci / Enterococci from foods.

**

**

**

**

**

*

AM101111 Baird Parker Agar Base IPAM501111

AM101112 Baird Parker Agar Base USPAM501112

AM101113 Baird Parker Agar Base (Agar medium O) EPAM501113

AM101114 Baird Parker Agar Base (Agar medium O) BPAM501114

AM101115 Baird Parker Agar Base BISAM501115

Agar

Bile Esculin Agar

Egg Yolk Emulsion AS010Potassium Tellurite 1% AS022




Egg Yolk Tellurite Emulsion AS011Egg Yolk Emulsion AS010Potassium Tellurite 3.5% AS023

AM10111 Beef ExtractAM50111

AM1012AM5012

**

**

**

**

**

*






microorganisms including bacteria, yeasts and moulds.

AM1017 Brain Heart Infusion BrothAM5017 A highly nutritious general-purpose liquid medium for cultivation

of a variety of fastidious and non-fastidious microorganisms, including aerobic and anaerobic bacteria from a variety of clinical and non-clinical specimens.

AM1018 Brilliant Green Agar, ModifiedAM5018 A medium for selective isolation of Salmonella other than S. typhi

from clinical and non-clinical samples.Sulpha Supplement AS027

AM10181 Brilliant Green Agar, Modified IPAM50181 A medium for selective isolation of Salmonella other than S. typhi

from foods, dairy products and other samples in compliance with IP.

AM10182 Brilliant Green Agar, Modified USPAM50182 A medium for selective isolation of Salmonella other than S. typhi

from foods, dairy products and other samples in compliance with USP.

AM10183 Brilliant Green Agar, Modified ( Agar Medium L) EPAM50183 A medium for selective isolation of Salmonella other than S. typhi

from foods, dairy products and other samples in compliance with EP.

AM10184 Brilliant Green Agar, Modified ( Agar Medium L) BPAM50184 A medium for selective isolation of Salmonella other than S. typhi

from foods, dairy products and other samples in compliance with BP.

AM1019 Brilliant Green Bile AgarAM5019 A medium for isolating, differentiating and enumerating coliform

bacteria.

AM1020 Brilliant Green Bile Broth 2%AM5020 A medium for detection of coliforms in water and waste water, food,

milk and dairy products as well as in other materials of sanitary importance.

AM1021 Bromothymol Blue Lactose AgarAM5021 A medium for detection and isolation of pathogenic Staphylococci.

*

AM1017AM5017

AM1018AM5018

Sulpha Supplement AS027

AM1019AM5019

AM1020AM5020

AM1021AM5021

Brain Heart Infusion Broth

Brilliant Green Agar, Modified

AM10181 Brilliant Green Agar, Modified IPAM50181

AM10182 Brilliant Green Agar, Modified USPAM50182

AM10183 Brilliant Green Agar, Modified ( Agar Medium L) EPAM50183

AM10184 Brilliant Green Agar, Modified ( Agar Medium L) BPAM50184

Brilliant Green Bile Agar

Brilliant Green Bile Broth 2%

Bromothymol Blue Lactose Agar

*

AM501211 Bile Esculin Agar ISOA differential medium for isolation and presumptive identification of Yersinia enterocolitica in compliance with ISO specification ISO 10273, 1994.

AM501212 Bile Esculin Azide AgarA medium for selective isolation and presumptive identification of faecal Streptococci.

AM10121 Bile Salt AgarAM50121 A medium used for the isolation and enumeration of enteric bacilli.

AM10122 Bile Salt Agar BISAM50122 A medium used for the isolation and enumeration of bile tolerant

enteric bacilli in compliance with BIS specification IS:5887 (Part5) 1976 reaffirmed 1986.

AM1013 Bismuth Sulphite AgarAM5013 A highly selective medium for isolation of Salmonella species,

particularly S.typhi from clinical and non-clinical specimens.

AM10131 Bismuth Sulphite Agar IP (Twin Pack)AM50131 A highly selective medium for isolation of Salmonella species,

particularly S.typhi from sewage and other specimens in compliance with IP.

AM10132 Bismuth Sulphite Agar Medium USPAM50132 A highly selective medium for isolation of Salmonella species,

particularly S.typhi from sewage and other specimens in compliance with USP.

AM1014 Blood Agar BaseAM5014 A non-selective general-purpose medium to which blood may be

added for use in isolation and cultivation of Streptococci and other fastidious pathogenic organisms like Neisseria, etc. and also for detection of haemolytic activity.

AM1015 Bordet Gengou Agar BaseAM5015 A medium for the detection and isolation of Bordetella pertussis

and Bordetella parapertussis from clinical specimens.Bordetella Selective Supplement AS004

AM1016 Brain Heart Infusion AgarAM5016 A general-purpose medium for cultivation of a wide variety of

*

AM501211 Bile Esculin Agar ISO

AM501212 Bile Esculin Azide Agar

AM10122 Bile Salt Agar BISAM50122

Bismuth Sulphite Agar

AM10131 Bismuth Sulphite Agar IP (Twin Pack)AM50131

AM10132 Bismuth Sulphite Agar Medium USPAM50132

Blood Agar Base

Bordet Gengou Agar Base

Brain Heart Infusion Agar

AM10121 Bile Salt AgarAM50121

AM1013AM5013

AM1014AM5014

AM1015AM5015

Bordetella Selective Supplement AS004

AM1016AM5016

*






AM10211 Buffered Peptone WaterAM50211 A medium used for pre-enrichment of injured Salmonella species

from clinical and non- clinical specimens.

AM10212 Buffered Peptone Water BISAM50212 A medium used for pre-enrichment of injured Salmonella species

from clinical and non- clinical specimens in compliance with BIS specification IS :5887 (Part3) 1999.

AM10213 Buffered Peptone Water ISOAM50213 A medium used for pre-enrichment of injured Salmonella species

from clinical and non- clinical specimens in compliance with ISO specification 657 9: 2002.

AM50214 Buffered Peptone Water with NaClA medium used for pre-enrichment of injured Salmonella species from clinical and non- clinical specimens .

AM50215 Buffered Peptone Water with NaCl IPA medium used for pre-enrichment of injured Salmonella species from clinical and non- clinical specimens in compliance with IP.

AM50216 Buffered Peptone Water with NaCl EPA medium used for pre-enrichment of injured Salmonella species from clinical and non- clinical specimens in compliance with EP.

AM50217 Buffered Peptone Water with NaCl BPA medium used for pre-enrichment of injured Salmonella species from clinical and non- clinical specimens in compliance with BP.

AM50218 Campylobacter Agar BaseA medium for selective isolation of Campylobacter species from clinical and non-clinical samples.Campylobacter Supplement-I (Blaser-Wang) AS0061Campylobacter Supplement-III (Skirrow) AS0071

AM50219 Candida MediumA medium for selective isolation and cultivation of Candida species.

AM50220 Carbohydrate Consumption Broth BaseA medium for cultivation and differentiation of Listeria species.

AM1022 Cetrimide Agar Base AM5022 A selective medium for isolation of Pseudomonas aeruginosa from

**

AM10211 Buffered Peptone WaterAM50211

AM10212 Buffered Peptone Water BISAM50212

AM10213 Buffered Peptone Water ISOAM50213

AM50214 Buffered Peptone Water with NaCl

AM50215 Buffered Peptone Water with NaCl IP

AM50216 Buffered Peptone Water with NaCl EP

AM50217 Buffered Peptone Water with NaCl BP

AM50218 Campylobacter Agar Base

Campylobacter Supplement-I (Blaser-Wang)Campylobacter Supplement-III (Skirrow)

AM50219 Candida Medium

AM50220 Carbohydrate Consumption Broth Base

Cetrimide Agar Base

**

AS0061 AS0071

AM1022AM5022

clinical and non-clinical specimens.Nalidixic Selective Supplement AS020

AM10221 Cetrimide Agar Base IPAM50221 A selective medium for isolation of Pseudomonas aeruginosa from

clinical and non-clinical specimens in compliance with IP.

AM10222 Cetrimide Agar Base Medium USPAM50222 A selective medium for isolation of Pseudomonas aeruginosa from

clinical and non-clinical specimens in compliance with USP.

AM10223 Cetrimide Agar Base ( Agar Medium N) EPAM50223 A selective medium for isolation of Pseudomonas aeruginosa from

clinical and non-clinical specimens in compliance with EP.

AM10224 Cetrimide Agar Base ( Agar Medium N) BPAM50224 A selective medium for isolation of Pseudomonas aeruginosa from

clinical and non-clinical specimens in compliance with BP.

AM1023 Cetrimide BrothAM5023 A medium for selective cultivation of Pseudomonas aeruginosa.

AM50231 Chapman Stone AgarA medium for selective isolation of Staphylococci from clinical and non-clinincal specimens.

AM1024 Chloramphenicol Yeast Glucose AgarAM5024 A selective medium for isolation and enumeration of yeasts and

moulds in milk and milk products.

AM1025 Christensen Citrate AgarAM5025 A medium for differentiation of enteric pathogens and coliforms on

the basis of citrate utilization.

AM10251 Chromogenic Coliform AgarAM50251 A chromogenic medium with sodium lauryl sulphate recommended

for simultaneous detection of Escherichia coli and total coliforms in water and food samples.

AM10252 Chromogenic E. coli AgarAM50252 A chromogenic medium for detection and enumeration of

Escherichia coli in foods without further confirmation on membrane filter or by indole reagent.

*

*

*

*

Nalidixic Selective Supplement AS020

AM1023AM5023

AM1024AM5024

AM1025AM5025

AM10251 Chromogenic Coliform AgarAM50251

AM10252 Chromogenic AgarAM50252

*

AM10221 Cetrimide Agar Base IPAM50221

AM10222 Cetrimide Agar Base Medium USPAM50222

AM10223 Cetrimide Agar Base ( Agar Medium N) EPAM50223

AM10224 Cetrimide Agar Base ( Agar Medium N) BPAM50224

Cetrimide Broth

AM50231 Chapman Stone Agar

Chloramphenicol Yeast Glucose Agar

Christensen Citrate Agar

*

*

*E. coli


AM10253 Chromogenic Enterococci BrothAM50253 A chromogenic medium for identification and differentiation of

Enterococci from water samples.

AM10254 Chromogenic UTI AgarAM50254 A chromogenic differential medium for presumptive identification

of microorganisms mainly causing urinary tract infections.

AM1026 C.L.E.D Agar with Andrade IndicatorAM5026 A medium for isolation, enumeration and presumptive

identification of microorganisms from urine, giving good colonial differentiation.

AM1027 C.L.E.D Agar with Bromothymol BlueAM5027 A medium for isolation, enumeration and presumptive

identification of microoganisms from urine.

AM1028 Coagulase Mannitol Agar BaseAM5028 A medium with added plasma for isolation and differentiation of

Staphylococci from clinical specimens or for classifying pure cultures.

AM1029 Columbia Blood Agar BaseAM5029 A basal medium for preparation of blood and chocolate agar and

for various selective and identification media in isolating and cultivating fastidious microorganisms.Brucella Selective Supplement, Modified AS006Staph-Strepto Supplement AS025Strepto Supplement AS026

AM50291 Columbia Agar Base (Medium Q) EPA medium for detection of Clostridium perfringens from pharmaceutical products in compliance with EP.

AM50292 Columbia Agar Base (Medium Q) BP A medium for detection of Clostridium perfringens from pharmaceutical products in compliance with BP.

AM1030 Cooked Meat Medium AM5030 A medium for cultivation and maintenance of aerobes and

anaerobes, especially Clostridium species.

AM103011 Cooked Meat Medium BIS AM503011 A medium for cultivation and maintenance of aerobes and

*

*

**

*

AM10253 Chromogenic Enterococci BrothAM50253

AM10254 Chromogenic UTI AgarAM50254

AM1026AM5026

AM1027AM5027

AM1028AM5028

AM1029AM5029

Brucella Selective Supplement, Modified AS006Staph-Strepto Supplement AS025Strepto Supplement AS026

AM1030AM5030

*

*

C.L.E.D Agar with Andrade Indicator

C.L.E.D Agar with Bromothymol Blue

Coagulase Mannitol Agar Base

Columbia Blood Agar Base

AM50291 Columbia Agar Base (Medium Q) EP

AM50292 Columbia Agar Base (Medium Q) BP

Cooked Meat Medium

AM103011 Cooked Meat Medium BIS AM503011

**

*





anaerobes, especially Clostridium species in compliance with BIS specification IS: 5887 ( Part2) 1976.

AM10301 Corn Meal AgarAM50301 A general purpose medium for cultivation of fungi.

AM10302 Czapek Dox AgarAM50302 A semisynthetic medium for general cultivation of fungi, yeasts and

soil bacteria.

AM10303 Deoxycholate AgarAM50303 A medium for direct differential count of coliforms in dairy products

and for isolastion of enteric pathogens from rectal swabs, faeces and other pathological specimens.

AM1031 Deoxycholate Citrate AgarAM5031 A selective medium for isolation of enteric pathogens particularly

Salmonella and Shigella species.

AM10311 Deoxycholate Citrate Agar IPAM50311 A selective medium for isolation of enteric pathogens particularly

Salmonella and Shigella species in compliance with IP.

AM10312 Deoxycholate Citrate Agar ( Agar Medium J) EPAM50312 A selective medium for isolation of enteric pathogens in compliance

with EP.

AM50313 Deoxycholate Citrate Agar ( Agar Medium J) BPA selective medium for isolation of enteric pathogens in compliance with BP.

AM1032 Dextrose AgarAM5032 A medium used with or without blood for the cultivation of wide

variety of microorganisms.

AM1033 Dextrose BrothAM5033 A medium for the cultivation of fastidious microorganisms and for

detecting gas production from enteric bacilli.

AM1034 Dextrose Peptone AgarAM5034 A general-purpose medium for the cultivation of a wide variety of

microorganisms.

AM10301 Corn Meal AgarAM50301

AM1031AM5031

AM1032AM5032

AM1033AM5033

AM1034AM5034

AM10302 Czapek Dox AgarAM50302

AM10303 Deoxycholate AgarAM50303

Deoxycholate Citrate Agar

AM10311 Deoxycholate Citrate Agar IPAM50311

AM10312 Deoxycholate Citrate Agar ( Agar Medium J) EPAM50312

AM50313 Deoxycholate Citrate Agar ( Agar Medium J) BP

Dextrose Agar

Dextrose Broth

Dextrose Peptone Agar






AM1035 Dextrose Peptone BrothAM5035 A general-purpose medium for the cultivation of a wide variety of

microorganisms.

AM1036 Dextrose Tryptone AgarAM5036 A medium for cultivation and enumeration of mesophilic and

thermophilic aerobic microorganisms associated with food spoilage.

AM1037 Dextrose Tryptone BrothAM5037 A medium for enrichment of mesophilic and thermophilic

organisms associated with food spoilage.

AM50371 Dey - Engley Neutralizing Agar (D/E Agar Disinfectant Testing)A medium used in disinfectant testing where the neutralization of antiseptics and disinfectant is important for determining its bactericidal activity.

AM50372 Dey - Engley Neutralizing Broth (D/E Broth Disinfectant Testing)A medium used in disinfectant testing where the neutralization of antiseptics and disinfectant is important for determining its bactericidal activity.

AM1038 Differential Reinforced Clostridial BrothAM5038 A medium for cultivation of Clostridia from water.

AM503811 Differential Reinforced Clostridial Broth Base ISOA medium for cultivation of Clostridia from water, in compliance with ISO specification ISO 6461- 1: 1986.

AM10381 DNase Test Agar BaseA differential medium for detection of deoxyribonuclease activity to aid in the identification of bacteria and fungi isolated from clinical specimens especially Staphylococci.

AM50382 Double Sugar Agar, RussellA medium for differentiation of gram- negative enteric bacilli on the basis of their ability to ferment dextrose and lactose with or without gas formation.

AM1039 E. C. Broth0AM5039 A medium for detection of coliform bacteria at 35 C and E. coli at

0an elevated temperature of 44.5 or 45.5 C.

AM1035AM5035

AM1036AM5036

AM1037AM5037

AM1038AM5038

AM10381 DNase Test Agar Base

AM1039AM5039

Dextrose Peptone Broth

Dextrose Tryptone Agar

Dextrose Tryptone Broth

AM50371 Dey - Engley Neutralizing Agar (D/E Agar Disinfectant Testing)

AM50372 Dey - Engley Neutralizing Broth (D/E Broth Disinfectant Testing)

Differential Reinforced Clostridial Broth

AM503811 Differential Reinforced Clostridial Broth Base ISO

AM50382 Double Sugar Agar, Russell

E. C. Broth

AM503911 E. C. Broth ISOA medium recommended for selective enumeration of presumptive Escherichia coli by MPN technique in compliance with ISO specification ISO 7251 : 1993.

AM503912 EC D AgarA medium for selective isolation of coliforms, specially Escherichia coli in water and food by membrane filter technique.

AM103913 E.E.Broth , MosselAM503913 A medium for selective enrichment of Enterobacteriaceae in

bacteriological examination of foods.

AM503914 E.E.Broth , Mossel (Enrichment Broth Medium E ) EPA medium for selective enrichment of Enterobacteriaceae in bacteriological examination of foods in accordance with EP.

AM503915 E.E.Broth , Mossel (Enrichment Broth Medium E ) BPA medium for selective enrichment of Enterobacteriaceae in bacteriological examination of foods in accordance with BP.

AM503916 E.E.Broth , Mossel with OxgallA medium for selective enrichment of Enterobacteriaceae in bacteriological examination of foods.

AM503917 Eijkman Lactose BrothA medium for detection and differentiation of Escherichia coli from other coliforms on the basis of their ability to librate gas from lactose.

AM10391 EMB AgarAM50391 A slightly selective and differential medium recommended for

isolation, cultivation and differentiation of gram-negative enteric bacilli from clinical and non-clinical specimens.

AM1040 E M B Agar, LevineAM5040 A slightly selective and differential medium for isolation,

enumera t ion and d i f f e ren t ia t ion o f members o f Enterobacteriaceae.

AM104011 E M B Agar, Levine IP (Levine Eosin-Methylene Blue Agar Medium)AM504011 A slightly selective and differential medium for isolation,

enumera t ion and d i f f e ren t ia t ion o f members o f Enterobacteriaceae in compliance with IP.

AM503911 E. C. Broth ISO

AM503912 EC D Agar

AM103913 E.E.Broth , MosselAM503913

AM503914 E.E.Broth , Mossel (Enrichment Broth Medium E ) EP

AM503915 E.E.Broth , Mossel (Enrichment Broth Medium E ) BP

AM503916 E.E.Broth , Mossel with Oxgall

AM503917 Eijkman Lactose Broth

E M B Agar, Levine

AM104011 E M B Agar, Levine IP (Levine Eosin-Methylene Blue Agar Medium)AM504011

AM10391 EMB AgarAM50391

AM1040AM5040






AM104012 E M B Agar, Levine USPAM504012 A slightly selective and differential medium for isolation,

enumera t ion and d i f f e ren t ia t ion o f members o f Enterobacteriaceae in compliance with USP.

AM104013 E M B Agar, Levine BISAM504013 A slightly selective and differential medium for isolation,

enumera t ion and d i f f e ren t ia t ion o f members o f Enterobacteriaceae in compliance with BIS specification IS:5401(1969) and IS 5887 ( Part 1) 1976.

AM10401 EMB BrothAM50401 A slightly selective and differential medium recommended for

isolation, cultivation and differentiation of gram-negative enteric bacilli from clinical and non-clinical specimens.

AM1041 Endo AgarAM5041 A differential and slightly selective medium for detection of

coliforms and other enteric microorganisms.

AM10411 Fluid Casein Digest Soya Lecithin Medium ( Twin Pack) IPAM50411 A medium for detection of microbes on sanitized surfaces in

compliance with IP.

AM10412 Fluid Casein Digest Soya Lecithin Medium ( Twin Pack) USP.AM50412 A medium for detection of microbes on sanitized surfaces in

compliance with USP.

AM1042 Fluid Lactose MediumAM5042 A medium for detection of coliforms and the study of lactose

fermentation by common bacteria.

AM10421 Fluid Lactose Medium IPAM50421 A medium for detection of coliforms and the study of lactose

fermentation by common bacteria in compliance with IP.

AM1043 Fluid Sabouraud MediumAM5043 A liquid medium for sterility testing of moulds and lower bacteria in

pharmaceutical preparations.

AM1044 Fluid Selenite Cystine Medium (Twin pack)AM5044 A selective enrichment medium for isolation of Salmonellae from

faeces, foods, pharmaceutical articles, water and other materials of sanitary importance.

AM104012 E M B Agar, Levine USPAM504012

AM104013 E M B Agar, Levine BISAM504013

Endo Agar

AM10411 Fluid Casein Digest Soya Lecithin Medium ( Twin Pack) IPAM50411

AM10412 Fluid Casein Digest Soya Lecithin Medium ( Twin Pack) USP.AM50412

Fluid Lactose Medium

Fluid Lactose Medium IP

Fluid Sabouraud Medium

Fluid Selenite Cystine Medium (Twin pack)

AM10401 EMB BrothAM50401

AM1041AM5041

AM1042AM5042

AM10421AM50421

AM1043AM5043

AM1044AM5044

AM10441 Fluid Selenite Cystine Medium IP (Twin pack)AM50441 A selective enrichment medium for isolation of Salmonellae from

faeces, foods, pharmaceutical articles, water and other materials of sanitary importance in compliance with IP.

AM10442 Fluid Selenite Cystine Medium USP (Twin pack)AM50442 A selective enrichment medium for isolation of Salmonellae from

faeces, foods, pharmaceutical articles, water and other materials of sanitary importance incompliance with USP.

AM50443 Fluid Selenite Cystine Medium ISO (Twin pack)A selective enrichment medium for isolation of Salmonellae from faeces, foods, pharmaceutical articles, water and other materials of sanitary importance in compliance with ISO specification ISO 6579 : 1993.

AM1045 Fluid Thioglycollate MediumAM5045 A medium for sterility testing of biologicals and cultivation of

aerobes, anaerobes and microaerophiles.

AM10451 Fluid Thioglycollate Medium IPAM50451 A medium for sterility testing of biologicals and cultivation of

aerobes, anaerobes and microaerophiles in compliance with IP.

AM10452 Fluid Thioglycollate Medium USPAM50452 A medium for sterility testing of biologicals and cultivation of

aerobes, anaerobes and microaerophiles in compliance with USP.

AM10453 Fluid Thioglycollate Medium EPAM50453 A medium for sterility testing of biologicals and cultivation of

aerobes, anaerobes and microaerophiles in compliance with EP.

AM10454 Fluid Thioglycollate Medium BPAM50454 A medium for sterility testing of biologicals and cultivation of

aerobes, anaerobes and microaerophiles in compliance withBP.

AM50455 Fraser Broth BaseA medium recommended as a primary as well as secondary enrichment medium for the isolation and enumeration of Listeria monocytogenes from foods, environmental specimens and animal feeds.Fraser Selective Supplement AS0112Fraser Supplement AS0114

**

AM10441 Fluid Selenite Cystine Medium IP (Twin pack)AM50441

AM10442 Fluid Selenite Cystine Medium USP (Twin pack)AM50442

AM50443 Fluid Selenite Cystine Medium ISO (Twin pack)

Fluid Thioglycollate Medium

AM10451 Fluid Thioglycollate Medium IPAM50451

AM10452 Fluid Thioglycollate Medium USPAM50452

AM10453 Fluid Thioglycollate Medium EPAM50453

AM10454 Fluid Thioglycollate Medium BPAM50454

AM50455 Fraser Broth Base

Fraser Selective SupplementFraser Supplement

AM1045AM5045

AS0112 AS0114

**






AM50456 Fraser Broth Base ISOA medium recommended as a primary as well as secondary enrichment medium for the isolation and enumeration of Listeria monocytogenes in compliance with ISO specifications ISO 11290: 1996.Fraser Selective Supplement ISO AS0113Fraser Supplement AS0114

AM50457 Fraser Secondary Enrichment Broth BaseA medium for the isolation, cultivation and enrichment of Listeria monocytogenes from foods, environmental specimens and animal feeds.Fraser Enrichment Supplement AS0111Fraser Selective Supplement AS0112

AM1046 G.C.Agar BaseAM5046 A medium with various additives to isolate and cultivate Gonococci

and other fastidious organisms.G.C.Supplement AS012Haemoglobin Powder Soluble AS014

AM50461 Glucose AgarA medium for determining the fermentation reaction of presumptive Enterobacteriaceae.

AM1047 Glucose BrothAM5047 A medium in glucose fermentation studies where pH indicator is

not desired.

AM10471 Glucose Salt Teepol Broth (Twin Pack)AM50471 A medium for enrichment of Vibrio parahaemoliticus and marine

isolates in compliance with BIS specification IS: 5887 (Part 5) 1976 reaffirmed 1986.

AM1048 Glucose Yeast Extract AgarAM5048 A medium for enumeration of Lactobacilli in pharmaceutical

preparations.

AM10481 Heart Infusion AgarAM50481 A general purpose medium used in the cultivation of a wide range

of microorganisms from a variety of clinical and non-clinical specimens.

**

**

**

AM50456 Fraser Broth Base ISO

Fraser Selective Supplement ISOFraser Supplement

AM50457 Fraser Secondary Enrichment Broth Base

Fraser Enrichment SupplementFraser Selective Supplement

G.C.Agar Base

AM50461 Glucose Agar

Glucose Broth

AM10471 Glucose Salt Teepol Broth (Twin Pack)AM50471

Glucose Yeast Extract Agar

**

**

**

AS0113 AS0114

AS0111 AS0112

AM1046AM5046

G.C.Supplement AS012Haemoglobin Powder Soluble AS014

AM1047AM5047

AM1048AM5048

AM10481 Heart Infusion AgarAM50481

AM10482 Heart Infusion BrothAM50482 A medium used for cultivating fastidious microorganisms.

AM104821 Hektoen Enteric AgarAM504821 A selective medium for detection and isolation of pathogenic

intestinal bacteria including Salmonella and Shigella from clinical and non-clinical specimens.

AM104822 Hottinger BrothA medium for cultivation of less fastidious microorganisms and determination of indole production.

AM104823 Hoyle Medium BaseAM504823 A selective medium for isolation and differentiation of

Corynebacterium diptheriae.Potassium Tellurite 3.5% AS023

AM104824 Inactivator Broth (Twin Pack)AM504824 A medium for detection and isolation of bacterial contamination

from clean surfaces and accidently contaminated raw material samples of pharmaceutical formulations.

AM10483 Iron Sulphite AgarAM50483 A medium for the detection of thermophilic anaerobic organisms

causing sulphide spoilage in foods.

AM10484 IUT Medium BaseAM50484 A medium for cultivation of Mycobacterium tuberculosis.

AM50485 Jensen ‘s BrothA medium for detection and cultivation of nitrogen fixing bacteria.

AM10486 Jensen’s MediumAM50486 A medium for detection and cultivation of nitrogen fixing bacteria.

AM1049 Karmali Campylobacter Agar BaseAM5049 A blood free medium for selective isolation of thermotolerant

Campylobacter species from clinical and non-clinical specimens.Campylobacter Selective Supplement with Hemin (Karmali) Modified AS007

AM50491 King’s Medium A BaseA medium for isolation, cultivation and pigment production of Pseudomonas species.

*

*

AM10482 Heart Infusion BrothAM50482

AM10483 Iron Sulphite AgarAM50483

AM1049AM5049

Campylobacter Selective Supplement with Hemin (Karmali) Modified AS007

AM104821 Hektoen Enteric AgarAM504821

AM104822 Hottinger Broth

AM104823 Hoyle Medium BaseAM504823

Potassium Tellurite 3.5% AS023

AM104824 Inactivator Broth (Twin Pack)AM504824

AM10484 IUT Medium BaseAM50484

AM50485 Jensen ‘s Broth

AM10486 Jensen’s MediumAM50486

Karmali Campylobacter Agar Base

AM50491 King’s Medium A Base

*

*






AM50492 King’s Medium B BaseA medium for isolation, cultivation and pigment production of Pseudomonas species.

AM50493 Kirchner Medium Base, Modified A medium for cultivation of Mycobacterium tuberculosis.Horse Serum AS 015

AM1050 Kligler Iron AgarAM5050 A differential medium for differentiation of members of

Enterobacteriaceae on the basis of their ability to ferment dextrose and lactose and to produce hydrogen sulphide.

AM50501 Kligler Iron Agar ISOA differential medium recommended for identification of Pseudomonas species and members of Enterobacteriaceae on the basis of their ability to ferment dextrose and lactose and to produce hydrogen sulphide in compliance with ISO specification ISO/ DIS/ 13720: 1995.

AM1051 Lactobacillus MRS AgarAM5051 A medium for isolation and cultivation of all Lactobacillus species.

AM50511 Lactobacillus MRS Agar ISOA medium recommended by ISO committee for isolation and enumeration of lactic acid bacteria from meat and meat products.

AM1052 Lactose BrothAM5052 A medium for the detection of coliforms, as a pre-enrichment broth

for Salmonellae and in the study of lactose fermentation in general.

AM10521 Lactose Broth IPAM50521 A medium for the detection of coliforms in water, food and dairy

products in compliance with IP.

AM10522 Lactose Broth (Broth Medium D) EPAM50522 A medium for the detection of coliforms in water, food and dairy

products in compliance with EP.

AM10523 Lactose Broth (Broth Medium D) BPAM50523 A medium for the detection of coliforms in water, food and dairy

products in compliance with BP.

**

*

*

AM50492 King’s Medium B Base

AM50493 Kirchner Medium Base, Modified

Kligler Iron Agar

AM50501 Kligler Iron Agar ISO

Lactobacillus MRS Agar

AM50511 Lactobacillus MRS Agar ISO

Lactose Broth

AM10521 Lactose Broth IPAM50521

AM10522 Lactose Broth (Broth Medium D) EPAM50522

AM10523 Lactose Broth (Broth Medium D) BPAM50523

Horse Serum AS 015

AM1050AM5050

AM1051AM5051

AM1052AM5052

**

*

*

AM10524 Lactose Broth BISAM50524 A medium for the detection of coliforms in water, food and dairy

products in compliance with BIS specification IS: 5041 -1969.

AM50525 Lactose Sulphite Broth Base A medium recommended for detection and enumeration of Clostridium perfringens in pharmaceutical products.

AM50526 Lactose Sulphite Broth Base (Medium R) EP A medium recommended for detection and enumeration of Clostridium perfringens in pharmaceutical products in compliance with EP.

AM50527 Lactose Sulphite Broth Base ( Broth Medium R) BPA medium recommended for detection and enumeration of Clostridium perfringens in pharmaceutical products in compliance with BP.

AM1053 Lauryl Tryptose BrothAM5053 A medium for detection of coliform organisms in materials of

sanitary importance.

AM10531 Lecithin AgarAM50531 A medium for detection of bacterial contamination of surfaces in

unprotected and protected areas.

AM50532 Lee’s AgarA medium for differentiation and enumeration of yoghurt starter bacteria.

AM50533 Lee’s Multi-differential AgarA medium for the cultivation, enumeration and identification of brewery bacteria.

AM1054 Legionella Agar BaseAM5054 A medium with added supplements for cultivation of Legionella

species.Legionella Growth Supplement AS016Legionella Selective Supplement AS017

AM1055 Listeria Identification Agar Base (PALCAM)AM5055 A medium with added supplement for selective isolation and

identification of Listeria species.Listeria Selective Supplement (PALCAM) AS018

*

**

*

AM10524 Lactose Broth BISAM50524

AM50525 Lactose Sulphite Broth Base

AM50526 Lactose Sulphite Broth Base (Medium R) EP

AM50527 Lactose Sulphite Broth Base ( Broth Medium R) BP

Lauryl Tryptose Broth

AM50531

AM50532 Lee’s Agar

AM50533 Lee’s Multi-differential Agar

Legionella Agar Base

Listeria Identification Agar Base (PALCAM)

AM1053AM5053

AM10531 Lecithin Agar

AM1054AM5054

Legionella Growth Supplement AS016Legionella Selective Supplement AS017

AM1055AM5055

Listeria Selective Supplement (PALCAM) AS018

*

**

*






AM105511 Listeria Identification Broth Base (PALCAM)AM505511 A medium with added supplement for selective isolation and

identification of Listeria species.Listeria Selective Supplement (PALCAM) AS018

AM105512 Listeria Oxford Medium BaseAM505512 A medium for isolation of Listeria species from pathological

specimens.Oxford Listeria Supplement AS0201Listeria Moxalactam Supplement AS0171

AM10551 Liver Infusion AgarAM50551 A medium for the cultivation of Brucella and other pathogenic

bacteria.

AM10552 Liver Infusion BrothAM50552 A medium for the cultivation of highly fastidious microorganisms,

particularly Brucella species and anaerobes.

AM1056 Loeffler Medium BaseAM5056 A medium with added horse serum for cultivation of

Corynebacterium diphtheriae from clinical specimens and in pure cultures.Horse Serum AS015

AM1057 Lowenstein Jensen Medium BaseAM5057 A medium supplemented with eggs for cultivation and isolation of

Mycobacterium species, especially M. tuberculosis.Gruft Mycobacterium Supplement AS013Egg Yolk Emulsion AS010

AM10571 Luria AgarAM50571 A medium recommended for the cultivation and maintenance of

recombinant strains of Escherichia coli and for routine cultivation of not particularly fastidious microorganisms.

AM10572 Luria BrothAM50572 A medium recommended for the cultivation and maintenance of

recombinant strains of Escherichia coli and for routine cultivation of not particularly fastidious microorganisms.

AM50573 Luria Bertani Agar, MillerA medium recommended for the cultivation and maintenance of Escherichia coli in molecular biology procedures and for routine

*

**

**

**

AM105511AM505511

Listeria Selective Supplement (PALCAM) AS018

Oxford Listeria Supplement AS0201Listeria Moxalactam Supplement AS0171

AM10551AM50551

AM10552AM50552

AM1056AM5056

Horse Serum AS015

AM1057AM5057

Gruft Mycobacterium Supplement AS013Egg Yolk Emulsion AS010

AM10571 Luria AgarAM50571

AM10572AM50572

Listeria Identification Broth Base (PALCAM)

AM105512 Listeria Oxford Medium BaseAM505512

Liver Infusion Agar

Liver Infusion Broth

Loeffler Medium Base

Lowenstein Jensen Medium Base

Luria Broth

AM50573 Luria Bertani Agar, Miller

*

**

**

**

cultivation of not particularly fastidious microorganisms.

AM50574 Luria Bertani Broth, MillerA medium recommended for the cultivation and maintenance of Escherichia coli in molecular biology procedures and for routine cultivation of not particularly fastidious microorganisms.

AM10575 LPM Agar BaseA medium for isolation and cultivation of Listeria monocytogenes from food and dairy products.Moxalactam Supplement AS0182

AM10576 Lysine Iron AgarAM50576 A medium for differentiation of enteric organisms specially

Salmonella species, based on their ability to decarboxylate or deaminate lysine and production of hydrogen sulphide.

AM10577 Lysine Medium BaseAM50577 A medium for isolation and enumeration of wild yeasts in pitching

yeasts.Potassium Lactate 50% ( 10ml/Vial) AS0211

AM1058 MacConkey Agar BaseAM5058 A medium for studying carbohydrate fermentation reactions of

coliforms by adding the desired carbohydrate.

AM10581 MacConkey Agar Base Medium USPAM50581 A medium for studying carbohydrate fermentation reactions of

coliforms by adding the desired carbohydrate in compliance with USP.

AM1059 MacConkey Agar with crystal violet, NaCl, and 0.15% Bile SaltsAM5059 A slightly selective and differential medium for the detection of

coliforms and other enteric pathogens.

AM50591 MacConkey Agar with crystal violet, NaCl, and 0.15% Bile Salts IPA slightly selective and differential medium for the detection of coliforms and other enteric pathogens in compliance with IP.

AM50592 MacConkey Agar with crystal violet, NaCl, and 0.15% Bile Salts USPA slightly selective and differential medium for the detection of coliforms and other enteric pathogens in compliance with USP.

*

*

AM50574 Luria Bertani Broth, Miller

AM10575 LPM Agar Base

Moxalactam Supplement

AM10576 Lysine Iron AgarAM50576

AM10577 Lysine Medium BaseAM50577

50% ( 10ml/Vial)

MacConkey Agar Base

MacConkey Agar Base Medium USP

MacConkey Agar with crystal violet, NaCl, and 0.15% Bile Salts

AM50591 MacConkey Agar with crystal violet, NaCl, and 0.15% Bile Salts IP

AM50592 MacConkey Agar with crystal violet, NaCl, and 0.15% Bile Salts USP

*

*

AS0182

Potassium Lactate AS0211

AM1058AM5058

AM10581AM50581

AM1059AM5059






AM50593 MacConkey Agar with crystal violet, NaCl, and 0.15% Bile Salts (Agar Medium H) EPA slightly selective and differential medium for the detection of coliforms and other enteric pathogens in compliance with EP.

AM50594 MacConkey Agar with crystal violet, NaCl, and 0.15% Bile Salts (Agar Medium H) BPA slightly selective and differential medium for the detection of coliforms and other enteric pathogens in compliance with BP.

AM1060 MacConkey Agar without crystal violet and with 0.15% Bile SaltsAM5060 A medium for selective isolation and differentiation of lactose

fermenting and non-lactose fermenting enteric bacteria.

AM50601 MacConkey Agar without crystal violet and with 0.5% Bile SaltsA medium for isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacteria.

AM1061 MacConkey Agar without crystal violet, NaCl, and with 0.5% Sodium Taurocholate

AM5061 A medium for cultivation and differentiation of enteric bacteria and potentially pathogenic gram-positive organisms while restricting swarming of Proteus species.

AM1062 MacConkey Broth Double Strength with Neutral RedAM5062 A medium for primary isolation of coliforms from large samples

such as water and waste water.

AM10621 MacConkey Broth Double Strength with Neutral Red BISAM50621 A medium for primary isolation of coliforms from large samples

such as water and waste water in compliance with BIS specification IS : 5887, ( Part-1 and Part-2) 1976 and IS 5401- 1969.

AM1063 MacConkey Broth Purple with BCPAM5063 A medium for the presumptive identification of coliforms and for

cultivating gram-negative, lactose fermenting bacilli from a variety of samples like water, milk & food.

AM10631 MacConkey Broth Purple with BCP IPAM50631 A medium for the presumptive identification of coliforms and for

cultivating gram-negative, lactose fermenting bacilli from a variety of samples like water, milk and food in compliance with IP.

AM50593 MacConkey Agar with crystal violet, NaCl, and 0.15% Bile Salts (Agar Medium H) EP

AM50594 MacConkey Agar with crystal violet, NaCl, and 0.15% Bile Salts (Agar Medium H) BP

MacConkey Agar without crystal violet and with 0.15% Bile Salts

AM50601 MacConkey Agar without crystal violet and with 0.5% Bile Salts

MacConkey Agar without crystal violet, NaCl, and with 0.5% Sodium Taurocholate

MacConkey Broth Double Strength with Neutral Red

AM10621 MacConkey Broth Double Strength with Neutral Red BISAM50621

MacConkey Broth Purple with BCP

AM10631 MacConkey Broth Purple with BCP IPAM50631

AM1060AM5060

AM1061

AM5061

AM1062AM5062

AM1063AM5063

AM10632 MacConkey Broth Purple with BCP(Broth Medium G) EPAM50632 A medium for the presumptive identification of coliforms and for

cultivating gram-negative, lactose fermenting bacilli from a variety of samples like water, milk and food in compliance with EP.

AM10633 MacConkey Broth Purple with BCP (Broth Medium G) BPAM50633 A medium for the presumptive identification of coliforms and for

cultivating gram-negative, lactose fermenting bacilli from a variety of samples like water, milk and food in compliance with BP.

AM10634 MacConkey Broth Purple with BCP BISAM50634 A medium for the presumptive identification of coliforms and for

cultivating gram-negative, lactose fermenting bacilli from a variety of samples like water, milk and food in compliance with BIS specification IS : 5401 - 1969 and IS : 5887 ( Part 1 and Part 2) 1976.

AM10635 MacConkey Broth Purple with BCP ISOAM50635 A medium for the presumptive identification of coliforms and for

cultivating gram-negative, lactose fermenting bacilli from a variety of samples like water, milk and food in compliance with ISO specification ISO / DIS 9308 - 2, 1990.

AM1064 MacConkey Broth with Neutral RedAM5064 A standard medium for primary isolation as well as presumptive

identification of coli-aerogenes group in food and water.

AM10641 MacConkey Broth with Neutral Red BISAM50641 A standard medium for enrichment and enumeration of coliforms in

compliance with BIS specification IS: 5887 (Part 1 and Part 2) 1976.

AM1065 Malonate Broth Ewing ModifiedAM5065 A medium for differentiation of Enterobacteriaceae on the basis of

malonate utilization.

AM1066 Malt AgarAM5066 A medium for isolating and cultivating yeasts and moulds from

food and dairy products and carrying stock cultures of the same.

AM1067 Malt Extract AgarAM5067 A medium for enumeration, cultivation and isolation of yeasts and

moulds.

AM10632 MacConkey Broth Purple with BCP(Broth Medium G) EPAM50632

AM10633 MacConkey Broth Purple with BCP (Broth Medium G) BPAM50633

AM10634 MacConkey Broth Purple with BCP BISAM50634

AM10635 MacConkey Broth Purple with BCP ISOAM50635

MacConkey Broth with Neutral Red

AM10641 MacConkey Broth with Neutral Red BISAM50641

Malonate Broth Ewing Modified

Malt Agar

Malt Extract Agar

AM1064AM5064

AM1065AM5065

AM1066AM5066

AM1067AM5067






AM1068 Malt Extract BrothAM5068 A medium for detection of yeasts, moulds and aciduric

microorganisms.

AM10681 Mannitol Motility Test MediumAM50681 A semisolid medium suitable for determining motility and

mannitol fermentation.

AM1069 Mannitol Salt AgarAM5069 A selective medium for isolation and identification of

Staphylococcus aureus from clinical and non-clinical specimens.

AM10693 Mannitol Salt Agar IPAM50693 A selective medium for isolation and identification of

Staphylococcus aureus from clinical and non-clinical specimens in compliance with IP.

AM10694 Mannitol Salt Agar Medium USPAM50694 A selective medium for isolation and identification of

Staphylococcus aureus from clinical and non-clinical specimens in compliance with USP.

AM10695 Mannitol Salt BrothAM50695 A selective medium for isolation and identification of

Staphylococcus aureus from clinical and non-clinical specimens.

AM 10691 Marine Agar 2216 ( Zobell Marine Agar)AM50691 A medium for isolation and enumeration of heterotrophic marine

bacteria.

AM10692 Marine Broth2216 ( Zobell Marine Broth)AM50692 A medium for cultivation of heterotrophic marine bacteria.

AM106921 M-Endo Agar LESAM506921 A medium for enumeration of coliforms in water using a two step

membrane filter technique.

AM106922 M-Endo BrothAM506922 A medium for estimation of coliforms in water using a membrane

filter technique.

AM506923 M-FC Agar BaseA medium for detection and enumeration of faecal coliforms using

0membrane filter technique at higher temperature (44.5 C)

AM1068AM5068

AM10681AM50681

AM1069AM5069

Malt Extract Broth

Mannitol Motility Test Medium

Mannitol Salt Agar

AM10693 Mannitol Salt Agar IPAM50693

AM10694 Mannitol Salt Agar Medium USPAM50694

AM10695 Mannitol Salt BrothAM50695

AM 10691 Marine Agar 2216 ( Zobell Marine Agar)AM50691

AM10692 Marine Broth2216 ( Zobell Marine Broth)AM50692

AM106921 M-Endo Agar LESAM506921

AM106922 M-Endo BrothAM506922

AM506923 M-FC Agar Base

Rosolic Acid AS0232

AM506924 M-FC Broth BaseA medium for detection and enumeration of faecal coliforms using

0membrane filter technique at higher temperature (44.5 C)Rosolic Acid AS0232

AM506925 M-(HPC) Heterotrophic Plate Count Agar BaseA medium for enumeration of heterotrophic microorganisms from water samples using membrane filter technique.

AM506926 M-(HPC) Heterotrophic Plate Count Broth BaseA medium for enumeration of heterotrophic microorganisms from water samples using membrane filter technique.

AM506927 Middlebrook 7H9 Agar BaseA medium for isolation, cultivation and sensitivity testing of Mycobacterium tuberculosis.Middlebrook OADC Growth Supplement AS0181

AM506928 Modified Rappaport Vassiliadis MediumA medium for selective enrichment of Salmonella from environmental and food specimens.

AM506929 Modified Rappaport Vassiliadis Medium for Water Testing BISA medium for selective enrichment of Salmonella from water, environmental and food specimens in compliance with BIS specification IS 15187:2002.

AM506930 Modified Rappaport Vassiliadis Medium for Water Testing ISOA medium for selective enrichment of Salmonella from water, environmental and food specimens in compliance with ISO specification ISO/DIS 6579:1993 and IS 5887 (Part 3): 1999.

AM506931 Modified Teepol Broth( Twin Pack) ISOA medium for selective isolation and identification of enteric lactose fermenting bacteria in compliance with ISO specification ISO 9308-1: 1990.

AM506932 Modified Tergitol 7 Agar Base ISOA medium for enumeration, differentiation and selective isolation of coliform bacteria in water by membrane filter method in compliance with ISO specification ISO 9308 - 1 : 1990.TTC Solution 1% AS0271

*

*

*

*

Rosolic Acid

AM506924 M-FC Broth Base

Rosolic Acid

AM506925 M-(HPC) Heterotrophic Plate Count Agar Base

AM506926 M-(HPC) Heterotrophic Plate Count Broth Base

AM506927 Middlebrook 7H9 Agar Base

Middlebrook OADC Growth Supplement

AM506928 Modified Rappaport Vassiliadis Medium

AM506929 Modified Rappaport Vassiliadis Medium for Water Testing BIS

AM506930 Modified Rappaport Vassiliadis Medium for Water Testing ISO

AM506931 Modified Teepol Broth( Twin Pack) ISO

AM506932 Modified Tergitol 7 Agar Base ISO

TTC Solution 1%

*

*

*

*

AS0232

AS0232

AS0181

AS0271






AM506933 Motility Test MediumA medium for detection of bacterial motility.

AM506934 Motility Test Medium ( Edwards and Ewing) BISA medium for testing of motility of enteric bacteria in compliance with BIS specifications IS: 5887 (Part 1 and Part5) 1976 reaffirmed, 1986.

AM1070 M R -V P MediumAM5070 A medium for the differentiation of coli-aerogenes group by means

of the Methyl Red and Voges Proskauer reactions.

AM10701 M R -V P Medium BISAM50701 A medium for the differentiation of coli-aerogenes group by means

of the Methyl Red and Voges Proskauer reactions in compliance with BIS specification IS 5887 ( Part 1,4,5) 1999.

AM10702 M R -V P Medium ISOAM50702 A medium for the differentiation of coli-aerogenes group by means

of the Methyl Red and Voges Proskauer reactions in compliance with ISO specification ISO6579:2002.

AM1071 Mueller Hinton AgarAM5071 A medium for antimicrobial disk diffusion susceptibility testing of

common, rapidly growing bacteria by the Bauer-Kirby method.

AM1072 Mueller Hinton BrothAM5072 A medium for determining the antimicrobial susceptibility of

bacteria by the tube dilution method.

AM1073 Mycoplasma Agar Base (PPLO Agar)AM5073 A medium when supplemented with nutritive enrichments for

isolation and cultivation of Mycoplasma species.Mycoplasma Enrichment Supplement AS019Horse Serum AS 015

AM10731 Nitrate Broth BISAM50731 A medium for detection of nitrate reduction by bacteria. Also

recommended for the enumeration of Bacillus cereus in compliance with BIS specification IS:5887 (Part4), 1976.

AM10732 Nitrate Broth ISOAM50732 A medium for detection of nitrate reduction by bacteria. Also

recommended for the enumeration of Bacillus cereus in compliance

***

AM506933 Motility Test Medium

AM506934 Motility Test Medium ( Edwards and Ewing) BIS

M R -V P Medium

M R -V P Medium BISAM50701

AM10702 M R -V P Medium ISOAM50702

Mueller Hinton Agar

Mueller Hinton Broth

Mycoplasma Agar Base (PPLO Agar)

AM10731 Nitrate Broth BISAM50731

AM10732 Nitrate Broth ISOAM50732

AM1070AM5070

AM10701

AM1071AM5071

AM1072AM5072

AM1073AM5073

Mycoplasma Enrichment Supplement AS019Horse Serum AS 015

***

with ISO specification ISO/DIS 7932,1993.

AM50733 Nitrofurantoin Broth BaseA medium for enrichment and isolation of Pseudomonas species.

AM1074 Nutrient AgarAM5074 A general purpose culture medium for the cultivation of bacteria,

which may also be enriched by incorporating 10% v/v sterile blood or other biological fluids.

AM10741 Nutrient Agar IPAM50741 A general purpose culture medium for the cultivation of

microorganisms in compliance with IP.

AM50742 Nutrient Agar 1.5% ISOA general purpose culture medium for the cultivation of fastidious bacteria after enrichment by incorporating 10% v/v sterile blood or other biological fluids, in compliance with ISO specification ISO / DIS 13720: 1995.

AM1075 Nutrient Agar pH 6.8AM5075 A medium for the cultivation of a wide variety of bacteria and for

the enumeration of microorganisms in water, sewage, faeces and other materials.

AM1076 Nutrient Agar with 1% PeptoneAM5076 A general purpose medium for the examination of water and dairy

products.

AM1077 Nutrient BrothAM5077 A general purpose culture medium for the cultivation of bacteria,

which may also be enriched by incorporating 10% v/v sterile blood or other biological fluids.

AM1078 Nutrient Broth with 1% Peptone AM5078 A general purpose medium for the examination of water and dairy

products.

AM10781 Nutrient Broth Medium IPAM50781 A general purpose medium for aerobes in compliance with IP.

AM50782 Orange Serum AgarA medium for isolation, cultivation and enumeration of acid tolerant microorganisms present in citrus products.

AM50733 Nitrofurantoin Broth Base

Nutrient Agar

AM10741 Nutrient Agar IPAM50741

AM50742 Nutrient Agar 1.5% ISO

Nutrient Agar pH 6.8

Nutrient Agar with 1% Peptone

Nutrient Broth

Nutrient Broth with 1% Peptone

AM10781 Nutrient Broth Medium IPAM50781

AM50782 Orange Serum Agar

AM1074AM5074

AM1075AM5075

AM1076AM5076

AM1077AM5077

AM1078AM5078






AM50804 Phenol Red Lactose Broth ISOA medium for lactose fermentation studies of coliforms in compliance with ISO specification ISO 9308 -1: 1990.

AM50805 Phenol Red Maltose Broth A medium for maltose fermentation studies of microorganisms.

AM50806 Phenol Red Mannitol BrothA medium for mannitol fermentation studies of microorganisms.

AM50807 Phenol Red Sorbitol BrothA medium for sorbitol fermentation studies of microorganisms.

AM50808 Phenol Red Sucrose BrothA medium for sucrose fermentation studies of microorganisms.

AM50809 Phenol Red Xylose BrothA medium for xylose fermentation studies of microorganisms.

AM108091 Phenylalanine AgarAM508091 A medium for differentiation of Proteus and Providencia from other

Enterobacteriaceae on the basis of deamination of phenylalanine.

AM108092 Pikovskaya’s AgarAM508092 A medium for detection of phosphate solubilizing soil

microorganisms.

AM1081 Plate Count Agar (Standard Methods Agar)AM5081 A medium for obtaining microbial plate counts from milk and dairy

products, foods, water and other materials of sanitary importance.

AM10811 Plate Count Agar BISAM50811 A medium for obtaining microbial plate counts from milk and dairy

products, foods, water and other materials of sanitary importance , in compliance with BIS Specification IS: 5402- 1969.

AM10812 PNY MediumAM50812 A medium for cultivation and isolation of Lactobacillus species.

AM1082 Potato Dextrose AgarAM5082 A medium for the cultivation and enumeration of yeasts and

moulds from dairy and food products.

AM50804 Phenol Red Lactose Broth ISO

AM50805 Phenol Red Maltose Broth

AM50806 Phenol Red Mannitol Broth

AM50807 Phenol Red Sorbitol Broth

AM50808 Phenol Red Sucrose Broth

AM50809 Phenol Red Xylose Broth

AM108091 Phenylalanine AgarAM508091

AM108092 Pikovskaya’s AgarAM508092

AM1081 Plate Count Agar (Standard Methods Agar)

AM10811 Plate Count Agar BISAM50811

AM10812 PNY MediumAM50812

Potato Dextrose Agar

AM5081

AM1082AM5082

AM50783 Orange Serum BrothA medium for cultivation of acid tolerant microorganisms present in citrus products.

AM10784 Pantothenate Assay MediumA medium for microbiological assay of Pantothenic acid or its salts using Lacttobacillus plantarum test.

AM10785 Pantothenate Assay Medium, AOACA medium recommended by AOAC for microbiological assay of Pantothenic acid or its salts using Lacttobacillus plantarum ATCC 8014.

AM10786 Pantothenate Culture AgarA medium for culturing Lacttobacillus plantarum ATCC 8014.

AM10787 Pantothenate Inoculum BrothA medium used in preparation of inoculum for pantothenate assay.

AM1079 Peptone WaterAM5079 A non-selective medium for cultivating non-fastidious organisms

and a base for carbohydrate fermentation media.

AM10791 Peptone Water BISAM50791 A non-selective medium for cultivating non-fastidious organisms

and a base for carbohydrate fermentation media in compliance with BIS specification IS:5887 ( Part 1) : 1976.

AM1080 Peptone Water with Phenol RedAM5080 A non-selective medium for cultivating non-fastidious organisms

and a base for carbohydrate fermentation media.

AM50801 Peptone Water with Phenol Red ISOA medium for studying fermentation ability of Yersinia enterocolitica, in compliance with ISO specification ISO /DIS 10273 : 1994.

AM50802 Phenol Red Dextrose Broth A medium for dextrose fermentation studies of microorganisms.

AM50803 Phenol Red Lactose Broth A medium for lactose fermentation studies of microorganisms.

AM50783 Orange Serum Broth

AM10784 Pantothenate Assay Medium

AM10785 Pantothenate Assay Medium, AOAC

AM10786 Pantothenate Culture Agar

AM10787 Pantothenate Inoculum Broth

Peptone Water

AM10791 Peptone Water BISAM50791

Peptone Water with Phenol Red

AM50801 Peptone Water with Phenol Red ISO

AM50802 Phenol Red Dextrose Broth

AM50803 Phenol Red Lactose Broth

AM1079AM5079

AM1080AM5080






AM10821 Potato Dextrose Agar USPAM50821 A medium for the cultivation and enumeration of yeasts and

moulds from dairy and food products in compliance with USP.

AM1083 Potato Dextrose BrothAM5083 A medium for the cultivation and enumeration of yeasts and

moulds from dairy and food products.

AM10831 Preston Agar BaseAM50831 A medium with added supplements recommended for selective

isolation of thermotolerant Campylobacter species especially Campylobacter jejuni and Campylobacter coli.Preston Selective Supplement (Campylobacter Selective Supplement IV, Modified ) AS0231

AM1084 Pseudomonas Agar BaseAM5084 A medium with added supplements for the selective isolation of

Pseudomonas species.CFC Supplement AS009Cetrinix Supplement AS008

AM108411 Pseudomonas Agar ( For Fluorescein)AM508411 A medium for enhancement of fluorescein production by

Pseudomonas species.

AM108412 Pseudomonas Agar ( For Fluorescein ) IPAM508412 A medium for enhancement of fluorescein production by

Pseudomonas species in compliance with IP.

AM108413 Pseudomonas Agar ( For Fluorescein ) USPAM508413 A medium for enhancement of fluorescein production by

Pseudomonas species in compliance with USP.

AM108414 Pseudomonas Agar ( For Pyocyanin ) AM508414 A medium for enhancement of pyocyanin production by

Pseudomonas species.

AM108415 Pseudomonas Agar ( For Pyocyanin ) IPAM508415 A medium for enhancement of pyocyanin production by

Pseudomonas species in compliance with IP.

AM108416 Pseudomonas Agar ( For Pyocyanin ) USPAM508416 A medium for enhancement of pyocyanin production by

Pseudomonas species in compliance with USP.

*

**

AM10821 Potato Dextrose Agar USPAM50821

Potato Dextrose Broth

Pseudomonas Agar Base

AM108411 Pseudomonas Agar ( For Fluorescein)AM508411

AM108412 Pseudomonas Agar ( For Fluorescein ) IPAM508412

AM108413 Pseudomonas Agar ( For Fluorescein ) USPAM508413

AM108414 Pseudomonas Agar ( For Pyocyanin ) AM508414

AM108415 Pseudomonas Agar ( For Pyocyanin ) IPAM508415

AM108416 Pseudomonas Agar ( For Pyocyanin ) USPAM508416

AM1083AM5083

AM10831 Preston Agar BaseAM50831

Preston Selective Supplement Selective Supplement IV, Modified ) AS0231

AM1084AM5084

CFC Supplement AS009Cetrinix Supplement AS008

*

**

(Campylobacter

AM108417 Pseudomonas Isolation Agar BaseAM508417 A medium for selective isolation and identification of

Pseudomonas aeruginosa from clinical and non-clinical specimens

AM10841 R-2A Agar AM50841 A medium for enumeration of heterotrophic bacteria in treated

potable water.

AM50842 R-2A Agar (Agar Medium S) EPA medium for enumeration of heterotrophic bacteria in treated potable water in compliance with EP.

AM50843 R-2A Agar (Agar Medium S) BPA medium for enumeration of heterotrophic bacteria in treated potable water in compliance with BP.

AM10844 Raka - Ray Agar Base ( Lactic Acid Bacteria Selective Agar Base)A selective medium for the isolation of lactic acid bacteria in beer and brewing processes.Lactic Supplement AS0151

AM10845 Raka - Ray No. 3 Broth Base ( Lactic Acid Bacteria Selective Broth Base)A selective medium for the isolation of lactic acid bacteria in beer and brewing processes.Lactic Supplement AS0151

AM50846 Reddys Differential Agar, Modified (Lactic Streak Agar)A medium for the qualitative and quantitative differentiation of lactic Streptococci.

AM1085 Reinforced Clostridial AgarAM5085 A medium for the cultivation and enumeration of Clostridia and

other anaerobes.

AM10851 Reinforced Clostridial Broth USPAM50851 A medium for cultivation and enumeration of Clostridia and other

anaerobes in compliance with USP.

AM50852 Reinforced Clostridial Broth ( Medium P) EPA medium for cultivation and enumeration of Clostridia and other anaerobes in compliance with EP.

*

*

AM108417 Pseudomonas Isolation Agar BaseAM508417

AM50842 R-2A Agar (Agar Medium S) EP

AM50843 R-2A Agar (Agar Medium S) BP

AM10844 Raka - Ray Agar Base ( Lactic Acid Bacteria Selective Agar Base)

Lactic Supplement AS0151

AM10845 Raka - Ray No. 3 Broth Base ( Lactic Acid Bacteria Selective Broth Base)

Lactic Supplement AS0151

AM50846 Reddys Differential Agar, Modified (Lactic Streak Agar)

Reinforced Clostridial Agar

AM10851 Reinforced Clostridial Broth USPAM50851

AM50852 Reinforced Clostridial Broth ( Medium P) EP

AM10841 R-2A Agar AM50841

AM1085AM5085

*

*






AM50853 Reinforced Clostridial Broth ( Medium P ) BPA medium for cultivation and enumeration of Clostridia and other anaerobes in compliance with BP.

AM50854 Rogosa SL AgarA medium for cultivation of oral, vaginal and faecal Lactobacilli.

AM50855 Rogosa SL BrothA medium for cultivation of oral, vaginal and faecal Lactobacilli.

AM50856 Rose Bengal Agar BaseA medium for selective isolation and enumeration of yeasts and moulds from environmental materials and food stuffs.Chloramphenicol Selective Supplement AS00911

AM10857 Rose Bengal Chloramphenicol AgarAM50857 A medium selective isolation and enumeration of yeasts and

moulds from environmental materials and food stuffs .

AM1086 Sabouraud Chloramphenicol AgarAM5086 A medium for selective cultivation of yeasts and moulds.

AM50861 Sabouraud Chloramphenicol Agar IPA medium for selective cultivation of yeasts and moulds in compliance with IP.

AM50862 Sabouraud Chloramphenicol Agar EPA medium for selective cultivation of yeasts and moulds in compliance with EP.

AM50863 Sabouraud Chloramphenicol Agar BPA medium for selective cultivation of yeasts and moulds in compliance with BP.

AM1087 Sabouraud Dextrose AgarAM5087 A general-purpose medium for the cultivation of yeasts, moulds

and aciduric bacteria.

AM10871 Sabouraud Dextrose Agar IPAM50871 A general-purpose medium for the cultivation of yeasts, moulds

and aciduric bacteria in compliance with IP.

AM10872 Sabouraud Dextrose Agar USPAM50872 A general-purpose medium for the cultivation of yeasts, moulds

*

*

*

*

*

*

AM50853 Reinforced Clostridial Broth ( Medium P ) BP

AM50854 Rogosa SL Agar

AM50855 Rogosa SL Broth

AM50856 Rose Bengal Agar Base

AM10857 Rose Bengal Chloramphenicol AgarAM50857

Sabouraud Chloramphenicol Agar

AM50861 Sabouraud Chloramphenicol Agar IP

AM50862 Sabouraud Chloramphenicol Agar EP

AM50863 Sabouraud Chloramphenicol Agar BP

Sabouraud Dextrose Agar

AM10871 Sabouraud Dextrose Agar IPAM50871

AM10872 Sabouraud Dextrose Agar USPAM50872

Chloramphenicol Selective Supplement AS00911

AM1086AM5086

AM1087AM5087

*

*

*

*

*

*

and aciduric bacteria in compliance with USP.

AM1088 Sabouraud Dextrose BrothAM5088 A general-purpose medium for the cultivation of yeasts, moulds

and aciduric bacteria.

AM50881 Sabouraud Dextrose Broth USPA general-purpose medium for the cultivation of yeasts, moulds and aciduric bacteria in compliance with USP.

AM50882 Sabouraud Dextrose Broth EPA general-purpose medium for the cultivation of yeasts, moulds and aciduric bacteria in compliance with EP.

AM50883 Sabouraud Glucose Agar with Antibiotics IPA medium for selective cultivation of yeasts and moulds in compliance with IP.

AM50884 Sabouraud Glucose Agar with Antibiotics (Agar Medium C) EPA medium for selective cultivation of yeasts and moulds in compliance with EP.

AM50885 Sabouraud Glucose Agar with Antibiotics (Agar Medium C) BPA medium for selective cultivation of yeasts and moulds in compliance with BP.

AM1089 Selenite F Broth (Twin pack)AM5089 An enrichment medium for the isolation of Salmonella species from

faeces, urine, water, foods and other materials of sanitary importance.

AM10891 Selenite F Broth IP (Twin pack)AM50891 An enrichment medium for the isolation of Salmonella species from

faeces, urine, water, foods and other materials of sanitary importance in compliance with IP.

AM50892 SIM MediumA medium for determination of H S production, indole formation 2

and motility of enteric bacteria.

AM1090 Simmons Citrate AgarAM5090 A medium for the differentiation of gram-negative bacteria on the

basis of citrate utilization.

AM1088AM5088

AM50881

AM50882

AM1089AM5089

AM10891

AM50892

AM1090AM5090

Sabouraud Dextrose Broth

Sabouraud Dextrose Broth USP

Sabouraud Dextrose Broth EP

AM50883 Sabouraud Glucose Agar with Antibiotics IP

AM50884 Sabouraud Glucose Agar with Antibiotics (Agar Medium C) EP

AM50885 Sabouraud Glucose Agar with Antibiotics (Agar Medium C) BP

Selenite F Broth (Twin pack)

Selenite F Broth IP (Twin pack)AM50891

SIM Medium

Simmons Citrate Agar






AM109011 Simmons Citrate Agar BISAM509011 A medium for the differentiation of gram-negative bacteria on the

basis of citrate utilization in compliance with BIS specification IS: 5887 ( Part 1) - 1976 reaffirmed 1986.

AM10901 Skim Milk AgarAM50901 A medium for cultivation and enumeration of microorganisms in

milk and dairy products.

AM1091 Soyabean Casein Digest Agar (Antibiotic Assay Medium No 36) (Tryptone Soya Agar)

AM5091 A general-purpose medium for the isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms.

AM10911 Soyabean Casein Digest Agar (Casein Soyabean Digest Agar) IPAM50911 A general-purpose medium for the isolation and cultivation of a

wide variety of fastidious and non-fastidious microorganisms in compliance with IP.

AM10912 Soyabean Casein Digest Agar Medium USPAM50912 A general-purpose medium for the isolation and cultivation of a

wide variety of fastidious and non-fastidious microorganisms in compliance with USP.

AM10913 Soyabean Casein Digest Agar (Agar Medium B) (Casein Soyabean Digest Agar) EP

AM50913 A general-purpose medium for the isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms in compliance with EP.

AM10914 Soyabean Casein Digest Agar (Agar Medium B) (Casein Soyabean AM50914 Digest Agar) BP

A general-purpose medium for the isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms in compliance with BP.

AM1092 Soyabean Casein Digest Medium (Antibiotic Assay Medium No 37)AM5092 (Tryptone Soya Broth)

A general-purpose medium for the isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms.

AM10921 Soyabean Casein Digest Medium IPAM50921 A general-purpose medium for the isolation and cultivation of a

wide variety of fastidious and non-fastidious microorganisms in compliance with IP.

AM109011 Simmons Citrate Agar BISAM509011

Soyabean Casein Digest Agar (Antibiotic Assay Medium No 36) (Tryptone Soya Agar)

AM10911 Soyabean Casein Digest Agar (Casein Soyabean Digest Agar) IPAM50911

AM10912 Soyabean Casein Digest Agar Medium USPAM50912

AM10913 Soyabean Casein Digest Agar (Agar Medium B) (Casein Soyabean Digest Agar) EP

AM50913

AM10914 Soyabean Casein Digest Agar (Agar Medium B) (Casein Soyabean AM50914 Digest Agar) BP

Soyabean Casein Digest Medium (Antibiotic Assay Medium No 37)(Tryptone Soya Broth)

AM10921 Soyabean Casein Digest Medium IPAM50921

AM10901 Skim Milk AgarAM50901

AM1091

AM5091

AM1092AM5092

AM10922 Soyabean Casein Digest Medium USPAM50922 A general-purpose medium for the isolation and cultivation of a

wide variety of fastidious and non-fastidious microorganisms in compliance with USP.

AM50923 Soyabean Casein Digest Medium (Broth Medium A) (Casein Soyabean Digest Broth)EPA general-purpose medium for the isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms in compliance with EP.

AM50924 Soyabean Casein Digest Medium (Broth Medium A) (Casein Soya Bean Digest Agar) BPA general-purpose medium for the isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms in compliance with BP.

AM50925 Soyabean Casein Digest Medium, Sterile PowderAM50925-5K A medium for evaluation of sterility in manufacturing process.

AM1093 SS AgarAM5093 A differential and selective medium for isolation of Salmonella and

some Shigella species from clinical and non-clinical specimens.

AM50931 SS Agar, ModifiedA differential and selective medium for isolation of Salmonella and many Shigella species from clinical and non-clinical specimens.

AM10932 Staphylococcus Agar No. 110 (Gelatin Mannitol Salt Agar)AM50932 A medium for selective isolation and differentiation of pathogenic

Staphylococci.

AM50933 Streptococcus Selection AgarA medium for selective isolation and enumeration of Streptococci species .

AM50934 Streptococcus Selection BrothA medium for selective isolation and cultivation of Streptococci species .

AM1094 Stuart Transport MediumAM5094 A medium for collecting, transporting and preserving

microbiological specimens, particularly Neisseria species and other fastidious organisms.

AM10922 Soyabean Casein Digest Medium USPAM50922

AM50923

AM50924

AM50925 Soyabean Casein Digest Medium, Sterile Powder

SS Agar

AM50931 SS Agar, Modified

AM10932 Staphylococcus Agar No. 110 (Gelatin Mannitol Salt Agar)AM50932

AM50933 Streptococcus Selection Agar

AM50934 Streptococcus Selection Broth

Stuart Transport Medium

Soyabean Casein Digest Medium (Broth Medium A) (Casein Soyabean Digest Broth)EP

Soyabean Casein Digest Medium (Broth Medium A) (Casein Soya Bean Digest Agar) BP

AM50925-5K

AM1093AM5093

AM1094AM5094






food and dairy products prior to isolation.

AM10961 Tetrathionae Broth Base IPAM50961 A medium for selective enrichment of Salmonella in compliance

with IP.

AM10962 Tetrathionate Broth Base Medium USPAM50962 A medium for selective enrichment of Salmonella in compliance

with USP.

AM50963 Tinsdale Agar BaseA medium for primary isolation and identification of Corynebacterium diptheriae. Diptheria Virulence Supplement (Part A & B)

AM1097 Tomato Juice AgarAM5097 A medium for cultivation and enumeration of Lactobacillus species.

AM1098 Tomato Juice Agar, SpecialAM5098 A medium for cultivation and enumeration of acidophilic

microorganisms from clinical and non-clinical specimens.

AM1099 Triple Sugar Iron AgarAM5099 A medium for identification of gram negative enteric bacilli on the

basis of dextrose, lactose and sucrose fermentation and hydrogen sulphide production.

AM10991 Triple Sugar Iron Agar IPAM50991 A medium for identification of gram negative enteric bacilli on the

basis of dextrose, lactose and sucrose fermentation and hydrogen sulphide production in compliance with IP.

AM10992 Triple Sugar Iron Agar USPAM50992 A medium for identification of gram negative enteric bacilli on the

basis of dextrose, lactose and sucrose fermentation and hydrogen sulphide production in compliance with USP.

AM10993 Triple Sugar Iron Agar (Agar Medium M) EPAM50993 A medium for identification of gram negative enteric bacilli on the

basis of dextrose, lactose and sucrose fermentation and hydrogen sulphide production in compliance with EP.

*

*

*

AS0091

AM10961 Tetrathionae Broth Base IPAM50961

AM10962 Tetrathionate Broth Base Medium USPAM50962

AM50963 Tinsdale Agar Base

Tomato Juice Agar

Tomato Juice Agar, Special

Triple Sugar Iron Agar

AM10991 Triple Sugar Iron Agar IPAM50991

AM10992 Triple Sugar Iron Agar USPAM50992

AM10993 Triple Sugar Iron Agar (Agar Medium M) EPAM50993

Diptheria Virulence Supplement (Part A & B)

AM1097AM5097

AM1098AM5098

AM1099AM5099

*

*

*

AS0091

AM50941 TB Broth BaseA medium for cultivation of Mycobacterium tuberculosis.

AM1095 TCBS AgarAM5095 A selective medium for isolating and cultivating vibrios causing

cholera and food poisoning from clinical and non-clinical specimens.

AM509511 Teepol Broth (Twin Pack )A medium for selective isolation and identification of lactose fermenting enteric bacteria.

AM10951 Tergitol-7 Agar BaseAM50951 A selective medium for the detection of coliform bacteria and for

early detection of E.coli in water analysis.TTC Solution 1% AS0271

AM10952 Tergitol-7 Agar Base BISAM50952 A selective medium for the detection of coliform bacteria and for

early detection of E.coli in water analysis in compliance with BIS specification IS 5887 (Part1) 1976 reaffirmed 1986. TTC Solution 1% AS 0271

AM50953 Tergitol-7 BrothA selective medium for the detection of coliform bacteria and for early detection of E.coli in water analysis.

AM50954 Tetrathionate Brilliant Green Bile BrothA medium for isolation and identification of Salmonella.

AM50955 Tetrathionate Bile Brilliant green Broth IPA medium for isolation and identification of Salmonella in compliance with IP .

AM50956 Tetrathionate Bile Brilliant green Broth (Broth Medium I) EP

AM50957 Tetrathionate Bile Brilliant green Broth (Broth Medium I) BPA medium for isolation and identification of Salmonella in compliance with BP.

AM1096 Tetrathionate Broth Base, HajnaAM5096 A medium for selective enrichment of Salmonella, particularly in

*

*

A medium for isolation and identification of Salmonella in compliance with EP.

AM50941 TB Broth Base

TCBS Agar

AM509511 Teepol Broth (Twin Pack )

AM10951 Tergitol-7 Agar BaseAM50951

AM10952 Tergitol-7 Agar Base BISAM50952

TTC Solution 1%

AM50953 Tergitol-7 Broth

AM50954 Tetrathionate Brilliant Green Bile Broth

AM50955 Tetrathionate Bile Brilliant green Broth IP

AM50956 Tetrathionate Bile Brilliant green Broth (Broth Medium I) EP

AM50957 Tetrathionate Bile Brilliant green Broth (Broth Medium I) BP

Tetrathionate Broth Base, Hajna

AM1095AM5095

TTC Solution 1% AS0271

AS 0271

AM1096AM5096

*

*






AM10994 Triple Sugar Iron Agar BISAM50994 A medium for identification of gram negative enteric bacilli on the

basis of dextrose, lactose and sucrose fermentation and hydrogen sulphide production in compliance with BIS specifications IS 5887 (Part 3) 1999.

AM50995 Triple Sugar Iron Agar ISOA medium for identification of gram negative enteric bacilli on the basis of dextrose, lactose and sucrose fermentation and hydrogen sulphide production in compliance with ISO specifications ISO/DIS 6579:1993.

AM1100 Tryptic Digest Broth (Field’s Tryptic Digest Broth)AM5100 A medium for cultivation of fastidious microorganisms.

AM11001 Tryptone AgarAM51001 A general purpose medium for growth of non fastidious

microorganisms.

AM11002 Tryptone Glucose Beef Extract Agar (TGB Agar)AM51002 A medium for enumeration of bacteria in water, air, milk and dairy

products.

AM1101 Tryptone Glucose Extract AgarAM5101 A medium for enumeration of bacteria in water, air, milk and dairy

products.

AM1102 Tryptone Glucose Extract BrothAM5102 A general purpose enrichment medium for a wide variety of

microorganisms.

AM1103 Tryptone Phosphate BrothAM5103 A medium for enrichment of enteropathogenic E. coli.

AM11031 Tryptone Soya Agar with Lecithin and Tween 80AM51031 ( Soyabean Casein Digest Agar with Lecithin and Tween 80)

A medium recommended for validation of cleanliness on surfaces of containers, equipment surfaces and water miscible cosmetics.

AM51032 Tryptone Soya Yeast Extract Agar ISOA medium for confirmation of Listeria in Henry’s light, in compliance with ISO specifications ISO 10560: 1993.

*

AM10994 Triple Sugar Iron Agar BISAM50994

AM50995 Triple Sugar Iron Agar ISO

Tryptic Digest Broth (Field’s Tryptic Digest Broth)

Tryptone Agar

AM11002 Tryptone Glucose Beef Extract Agar (TGB Agar)AM51002

Tryptone Glucose Extract Agar

Tryptone Glucose Extract Broth

Tryptone Phosphate Broth

AM51031

AM51032 Tryptone Soya Yeast Extract Agar ISO

AM1100AM5100

AM11001AM51001

AM1101AM5101

AM1102AM5102

AM1103AM5103

AM11031 Tryptone Soya Agar with Lecithin and Tween 80*

AM51033 Tryptone Soya Yeast Extract Broth ISOA medium for confirmation of Listeria in Henry’s light, in compliance with ISO specifications ISO 10560: 1993.

AM1104 Tryptone WaterAM5104 A medium for detection of indole production especially by

coliforms.

AM110411 Tryptone Water without Sodium ChlorideAM510411 A medium for detection of Vibrio cholerae and Vibrrio

parahaemolyticus in compliance with BIS specifications IS: 5887 (Part 5) 1976 reaffirmed 1986.

AM11041 Tryptose AgarAM51041 A medium for cultivation of Brucella species and other fastidious

microorganisms.

AM11042 Universal Beer Agar (UB Agar)A medium for cultivation of microrganisms related to brewing industry.

AM1105 Urea Agar Base, ChristensenAM5105 A medium with added urea for detection of urease production,

particularly by the genus Proteus.Urea 40% AS028

AM11051 Urea Agar Base, Christensen BISAM51051 A medium with added urea for detection of urease production,

particularly by the genus Proteus in compliance with BIS specification IS:5887 (Part 1), 1976 and IS: 5887 (Part 3): 1999.Urea 40% AS028

AM51052 Urea Agar Base, Christensen ISOA medium with added urea for detection of urease production, particularly by the genus Proteus in compliance with ISO specification ISO 6579:1993.Urea 40% AS028

AM1106 Urea Broth BaseAM5106 A medium with added urea for the detection of urease production,

to differentiate Proteus species from Salmonella and Shigella species.Urea 40% AS028

*

*

*

*

AM51033 Tryptone Soya Yeast Extract Broth ISO

Tryptone Water

AM110411 Tryptone Water without Sodium ChlorideAM510411

Tryptose Agar

AM11042 Universal Beer Agar (UB Agar)

Urea Agar Base, Christensen

AM11051 Urea Agar Base, Christensen BISAM51051

AM51052 Urea Agar Base, Christensen ISO

Urea Broth Base

AM1104AM5104

AM11041AM51041

AM1105AM5105

Urea 40% AS028

Urea 40% AS028

Urea 40% AS028

AM1106AM5106

Urea 40% AS028

*

*

*

*






AM51061 Urea Broth IPA medium with added urea for the detection of urease production, to differentiate Proteus species from Salmonella and Shigella species in compliance with IP.

AM1107 Violet Red Bile AgarAM5107 A selective medium for the detection and enumeration of coliforms .

AM11071 Violet Red Bile Agar BISAM51071 A selective medium for the detection and enumeration of coliforms

in compliance with BIS specification IS : 5401- 1969.

AM51072 Violet Red Bile BrothA medium for detection and enumeration of coliforms from water and food.

AM51073 Violet Red Bile Glucose Agar.A medium for enumeration of Enterobacteriaceae.

AM51074 Violet Red Bile Glucose Agar without Lactose ISOA medium for enumeration of Enterobacteriaceae in compliance with ISO specification ISO 7402 : 1993

AM51075 Violet Red Bile Glucose Agar with Lactose (Agar Medium F) EPA medium for enumeration of Enterobacteriaceae in compliance with EP.

AM51076 Violet Red Bile Glucose Agar with Lactose (Agar Medium F) BPA medium for enumeration of Enterobacteriaceae in compliance with BP.

AM1108 Vogel Johnson Agar Base w/o TelluriteAM5108 A medium with the addition of potassium tellurite for isolation of

Staphylococcus aureus from clinical and non-clinical specimens.Potassium Tellurite 1% AS022

AM11081 Vogel Johnson Agar Base w/o Tellurite IPAM51081 A medium with the addition of potassium tellurite for isolation of

Staphylococcus aureus from clinical and non-clinical specimens in compliance with IP.Potassium Tellurite 1% AS022

AM11082 Vogel Johnson Agar Base w/o Tellurite USPAM51082 A medium with the addition of potassium tellurite for isolation of

*

*

*

AM51061 Urea Broth IP

Violet Red Bile Agar

AM11071 Violet Red Bile Agar BISAM51071

AM51072 Violet Red Bile Broth

AM51073 Violet Red Bile Glucose Agar.

AM51074 Violet Red Bile Glucose Agar without Lactose ISO

AM51075 Violet Red Bile Glucose Agar with Lactose (Agar Medium F) EP

AM51076 Violet Red Bile Glucose Agar with Lactose (Agar Medium F) BP

Vogel Johnson Agar Base w/o Tellurite

AM11081 Vogel Johnson Agar Base w/o Tellurite IPAM51081

AM11082 Vogel Johnson Agar Base w/o Tellurite USPAM51082

*

*

*

AM1107AM5107

AM1108AM5108

Potassium Tellurite 1% AS022

Potassium Tellurite 1% AS022

Staphylococcus aureus from clinical and non-clinical specimens in compliance with USP.Potassium Tellurite 1% AS022

AM51083 Wilson Blair Agar BaseA medium base for isolation and differentiation of Salmonella serotype Typhi.

AM1109 W.L. Differential AgarAM5109 A medium for selective isolation and enumeration of bacteria

encountered in breweries and industrial fermentation.

AM51091 W.L. Differential BrothA medium for selective isolation and enumeration of bacteria encountered in breweries and industrial fermentation.

AM51092 WL Nutrient AgarA medium for enumeration and cultivation of yeasts, moulds and bacteria encountered in brewing and fermentation processes.

AM1110 W.L. Nutrient BrothAM5110 A medium for cultivating yeasts, moulds and bacteria encountered

in brewing and fermentation processes.

AM1111 Wort AgarAM5111 A medium for cultivation and enumeration of yeasts.

AM1112 X.L.D. AgarAM5112 A moderately selective medium for isolation and differentiation of

Salmonella and Shigella.

AM51121 X.L.D. Agar IPA moderately selective medium for isolation and differentiation of Salmonella andShigella.in compliance with IP.

AM51122 X.L.D. Agar Medium USPA moderately selective medium for isolation and differentiation of Salmonella and Shigella.in compliance with USP.

AM51123 X.L.D. Agar ( Agar Medium K) EPA moderately selective medium for isolation and differentiation of Salmonella and Shigella in compliance with EP.

*Potassium Tellurite 1% AS022

Wilson Blair Agar Base

AM1109AM5109

AM1110AM5110

AM1111AM5111

AM1112AM5112

*

AM51083

W.L. Differential Agar

AM51091 W.L. Differential Broth

AM51092 WL Nutrient Agar

W.L. Nutrient Broth

Wort Agar

X.L.D. Agar

AM51121 X.L.D. Agar IP

AM51122 X.L.D. Agar Medium USP

AM51123 X.L.D. Agar ( Agar Medium K) EP






AM51124 X.L.D. Agar (Agar Medium K) BPA moderately selective medium for isolation and differentiation of Salmonella and Shigella in compliance with BP.

AM51125 X.L.D. Agar, Modified ISOA moderately selective medium for isolation and differentiation of Salmonella and Shigella.in compliance with ISO specification ISO 6579:2002.

AM51126 Yeast and Mould BrothA medium for isolation and cultivation of yeasts and moulds.

AM1113 Yeast Extract AgarAM5113 A highly nutritive medium for cultivation of a wide variety of

bacteria.

AM1114 Yeast Malt AgarAM5114 A medium for isolation and cultivation of yeasts, moulds and

aciduric bacteria.

AM1115 Yeast Malt BrothAM5115 A medium for isolation and cultivation of yeasts, moulds and

aciduric bacteria.

AM51151 Yeast Mannitol Agar with 1.5% AgarA medium for cultivation, Isolation and enumeration of soil microorganisms like Rhizobium species.

AM51152 Yeast Mannitol Agar with Congo RedA medium for cultivation of soil microorganisms like Rhizobium species.

AM51153 Yeast Mannitol BrothA medium for cultivation of Rhizobium species.

AM1116 Yersinia Selective Agar BaseAM5116 A medium with added supplements for isolation and enumeration

of Yersinia enterocolitica from clinical and non-clinical specimens.Yersinia Selective Supplement AS029*

AM51124 X.L.D. Agar (Agar Medium K) BP

AM51125 X.L.D. Agar, Modified ISO

AM51126 Yeast and Mould Broth

Yeast Extract Agar

Yeast Malt Agar

Yeast Malt Broth

AM51151 Yeast Mannitol Agar with 1.5% Agar

AM51152 Yeast Mannitol Agar with Congo Red

AM51153 Yeast Mannitol Broth

Yersinia Selective Agar Base

AM1113AM5113

AM1114AM5114

AM1115AM5115

AM1116AM5116

Yersinia Selective Supplement AS029*

MEDIA BASESAB001 Agar Powder

For use as a solidifying agent in microbiological culture media.

AB002 Beef Extract PowderA nutritious extract used in the preparation of a variety of culture media for the cultivation of a wide variety of microorganisms.

AB003 Brain Heart Infusion PowderA nutritious ingredient used in the preparation of a variety of culture media for the cultivation of a wide variety of fastidious microorganisms.

AB004 Casein Enzymic Hydrolysate (Tryptone)An ingredient used in the preparation of variety of culture media such as sterility testing media, diagnostic media and media for biochemical characterization.

AB005 Fish PeptoneA nutritious ingredient used in culture media for the cultivation of a variety of bacteria and fungi.

AB0051 Gelatin PeptoneFor use in culture media, especially for non-fastidious microorganisms. Also used in fermentation studies.

AB006 Heart Infusion PowderA nutritious ingredient used in preparation of culture media for cultivation of fastidious microorganisms.

AB007 Liver Extract PowderA nutritious extract used in the preparation of culture media for cultivation of fastidious bacteria.

AB008 Malt Extract PowderAn ideal ingredient used in the preparation of culture media for the cultivation of yeasts and moulds.

AB009 Meat Extract PowderA nutritious extract used as a ingredient in the preparation of culture media for cultivation of a wide variety of fastidious microorganisms.

MEDIA BASESAB001 Agar Powder

AB002 Beef Extract Powder

AB003 Brain Heart Infusion Powder

AB004 Casein Enzymic Hydrolysate (Tryptone)

AB005 Fish Peptone

AB0051 Gelatin Peptone

AB006 Heart Infusion Powder

AB007 Liver Extract Powder

AB008 Malt Extract Powder

AB009 Meat Extract Powder

0 0 0Store Dehydrated Media below 30 C, Store all Supplements and Media between 2-8 C, Store below -20 C.* * ** 0Store Media Bases below 30 C.





AB010 Mycological Peptone (Peptone M)A nutritious ingredient used in the preparation of culture media for cultivation of yeasts and moulds.

AB011 Peptone, BacteriologicalA nutritious ingredient used in the preparation of culture media for the cultivation of a wide variety of bacteria and fungi.

AB012 Peptone SpecialA nutritious enzymatic preparation used in the preparation of culture media for cultivation of a wide variety of fastidious microorganisms.

AB0121 Peptonized MilkA refined enzymatic digest of milk solids used in the preparation of culture media suitable for the cultivation of lactobacilli, yeasts and moulds.

AB013 Proteose PeptoneA nutritious ingredient used in the preparation of culture media employed for cultivation of a wide variety of microorganisms and in producing bacterial toxins.

AB014 Soya PeptonePapaic digest of soyabean meal, used in the preparation of culture media for the cultivation of many fastidious microorganisms, including fungi.

AB015 TryptoseAn enzymatic hydrolysate of protein that can replace meat infusion used in the preparation of culture media for the cultivation of many fastidious microorganisms.

AB0151 Veg PeptoneVeg Peptone is an enzymic hydrolysate of vegetable proteins containing a mixture of peptides and amino acids that gives comparable growth promoting properties as animal origin peptone, used in the preparation of a variety of culture media for the cultivation of microorganisms.

AB016 Yeast Extract PowderA nutritious extract used in the preparation of culture media for the cultivation of a wide variety of microorganisms.

AB010 Mycological Peptone (Peptone M)

AB011 Peptone, Bacteriological

AB012 Peptone Special

AB0121 Peptonized Milk

AB013 Proteose Peptone

AB014 Soya Peptone

AB015 Tryptose

AB0151 Veg Peptone

AB016 Yeast Extract Powder

MEDIA SELECTIVE SUPPLEMENTS, AGENTS AND ENRICHMENTS*

*

*

*

*

*

*

*

*

AS001 Bacteroides Selective Supplement An antibiotic supplement recommended for selective isolation of Bacteroides species. Bacteroides Bile Esculin Agar AM1010/5010

AS002 Bile salts (Ox Bile)A selective agent used in microbiological culture media to inhibit gram-positive Microorganisms.

AS003 Bile Salts No.3A selective agent used in microbiological culture media to inhibit gram-positive microorganisms.

AS004 Bordetella Selective SupplementAn antibiotic supplement recommended for selective isolation of Bordetella pertussis.Bordet Gengou Agar Base AM1015/5015

AS005 BP Sulpha SupplementAn antibiotic supplement recommended for use in Baird Parker Agar Base to suppress the growth of Proteus species.Baird Parker Agar Base AM1011/5011

AS006 Brucella Selective Supplement, ModifiedAn antibiotic supplement recommended for selective isolation of Brucella species in milk.Columbia Blood Agar Base AM1029/5029

AS0061 Campylobacter selective supplement (Blaser - Wang)An antibiotic supplement recommended for selective isolation of Campylobacter species. Campylobacter Agar Base AM 50218

AS007 Campylobacter Selective Supplement with Hemin (Karmali), ModifiedAn antibiotic supplement recommended for the isolation of thermotolerant Campylobacter species. Karmali Campylobacter Agar Base AM1049/5049

AS0071 Campylobacter supplement (Skirrow)An antibiotic supplement recommended for selective isolation of Campylobacter species. Campylobacter Agar Base AM 50218

MEDIA SELECTIVE SUPPLEMENTS, AGENTS AND ENRICHMENTS*

*

*

*

*

*

*

*

*

AS001 Bacteroides Selective Supplement

Bacteroides Bile Esculin Agar AM1010/5010

AS002 Bile salts (Ox Bile)

AS003 Bile Salts No.3

AS004 Bordetella Selective Supplement

Bordet Gengou Agar Base AM1015/5015

AS005 BP Sulpha Supplement

Baird Parker Agar Base AM1011/5011

AS006 Brucella Selective Supplement, Modified

Columbia Blood Agar Base AM1029/5029

AS0061 Campylobacter selective supplement (Blaser - Wang)

Campylobacter Agar Base AM 50218

AS007 Campylobacter Selective Supplement with Hemin (Karmali), Modified

Karmali Campylobacter Agar Base AM1049/5049

AS0071 Campylobacter supplement (Skirrow)

Campylobacter Agar Base AM 50218

0Store Media Bases below 30 C. * **0 0 Store all Supplements, Agents and Enrichments between 2-8 C, Store below -20 C.





AS008 Cetrinix SupplementAn antibiotic supplement recommended for the selective isolation of Pseudomonas species.Pseudomonas Agar Base AM1084/5084

AS009 CFC SupplementAn antibiotic supplement recommended for the selective isolation of Pseudomonas species.Pseudomonas Agar Base AM1084/5084

AS00911 Chloramphenicol Selective SupplementAn antibiotic supplement recommended for yeasts and moulds. Rose Bengal Agar Base AM50856

AS0091 Diptheria Virulence Supplement (Part A & Part B)A selective supplement recommended for the isolation and presumptive identification of Corynebacterium diptheriae.Tinsdale Agar Base AM50693

AS010 Egg Yolk Emulsion (100 ml/vial)A sterile, stabilized emulsion of egg yolk recommended for use in culture media.Bacillus Cereus Agar Base AM1009/5009Baird Parker Agar Base AM1011/5011Baird Parker Agar Base IP AM101111/501111Baird Parker Agar Base USP AM101112/501112Baird Parker Agar Base EP AM101113/501114Baird Parker Agar Base BP AM101114/501114Baird Parker Agar Base BIS AM101115/501115Mannitol Salt Agar Base AM1069/5069

AS011 Egg Yolk Tellurite Emulsion ( 100 ml /vl)A sterile, stabilized tellurite emulsion of egg yolk recommended for identification of Staphylococcus species.Baird Parker Agar Base AM1011/5011Baird Parker Agar Base BIS AM101115/501115

AS0111 Fraser Enrichment SupplementAn antibiotic supplement recommended for the selective isolation, cultivation and identification of Listeria monocytogenes from foods and environmental specimens.Fraser Secondary Enrichment Broth Base AM 50457

*

*

*

*

*

*

*

AS008 Cetrinix Supplement

Pseudomonas Agar Base AM1084/5084

AS009 CFC Supplement

Pseudomonas Agar Base AM1084/5084

AS00911 Chloramphenicol Selective Supplement

AS0091 Diptheria Virulence Supplement (Part A & Part B)

Tinsdale Agar Base AM50693

AS010 Egg Yolk Emulsion (100 ml/vial)

Bacillus Cereus Agar Base AM1009/5009Baird Parker Agar Base AM1011/5011Baird Parker Agar Base IP AM101111/501111Baird Parker Agar Base USP AM101112/501112Baird Parker Agar Base EP AM101113/501114Baird Parker Agar Base BP AM101114/501114

Mannitol Salt Agar Base AM1069/5069

AS011 Egg Yolk Tellurite Emulsion ( 100 ml /vl)

Baird Parker Agar Base AM1011/5011Baird Parker Agar Base BIS AM101115/501115

AS0111 Fraser Enrichment Supplement

Fraser Secondary Enrichment Broth Base AM 50457

*

*

*

*

*

*

*

Rose Bengal Agar Base AM50856

Baird Parker Agar Base BIS AM101115/501115

AS0112 Fraser Selective SupplementAn antibiotic supplement recommended for the selective isolation, cultivation and identification of Listeria monocytogenes from foods and environmental specimens.Fraser Secondary Enrichment Broth Base AM 50457Fraser Broth Base AM 50455

AS0113 Fraser Selective Supplement ISOAn antibiotic supplement recommended by ISO committee for the selective isolation, cultivation and identification of Listeria monocytogenes from foods, animal feeds and environmental specimens.Fraser Broth Base ISO AM50456

AS0114 Fraser Supplement An antibiotic supplement recommended for the selective isolation, cultivation and identification of Listeria monocytogenes from foods, animal feeds and environmental specimens. Also recommended by ISO committee.Fraser Broth Base AM 50455Fraser Broth Base ISO AM 50456

AS012 G.C. SupplementAn enrichment and antibiotic supplement recommended for the selective isolation of pathogenic Neisseria.G.C.Agar Base AM1046/5046

AS013 Gruft Mycobacterium SupplementAn enrichment and antibiotic supplement recommended for the selective cultivation of mycobacteria.Lowenstein Jensen Medium Base AM1057/5057

AS014 Haemoglobin Powder Soluble (100 gms/Vial)An enrichment supplement whose 2% w/v solution is autoclavable.GC Agar Base AM1046/5046

AS015 Horse SerumAn enrichment supplement recommended for isolation and cultivation of Mycoplasma, Trichomonas, Streptococcus species and C.diphtheriae.Mycoplasma Agar Base AM1073/5073Loeffler Medium Base AM1056/5056Kirchner Medium Base, Modified AM50493

*

*

*

*

*

*

**

AS0112

AS012 G.C. Supplement

G.C.Agar Base AM1046/5046

AS013 Gruft Mycobacterium Supplement

Lowenstein Jensen Medium Base AM1057/5057

AS014 Haemoglobin Powder Soluble (100 gms/Vial)

GC Agar Base AM1046/5046

AS015 Horse Serum

Mycoplasma Agar Base AM1073/5073Loeffler Medium Base AM1056/5056Kirchner Medium Base, Modified AM50493

Fraser Selective Supplement

Fraser Secondary Enrichment Broth Base AM 50457Fraser Broth Base AM 50455

AS0113 Fraser Selective Supplement ISO

Fraser Broth Base ISO AM50456

AS0114 Fraser Supplement

Fraser Broth Base AM 50455Fraser Broth Base ISO AM 50456

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* **0 0 Store all Supplements, Agents and Enrichments between 2-8 C, Store below -20 C.





AS0151 Lactic SupplementA supplement for selective isolation of lactic acid bacteria in beer and brewing procedures.Raka Ray Agar Base (Lactic Acid Bacteria Selective Agar Base) AM10844Raka Ray No. 3 Broth Base (Lactic Acid Bacteria Selective Broth Base) AM10845

AS016 Legionella Growth SupplementAn enrichment supplement used for enhancing growth of Legionella species.Legionella Agar Base AM1054/5054

AS017 Legionella Selective SupplementAn antibiotic supplement recommended for the selective isolation of Legionella species.Legionella Agar Base AM1054/5054

AS0171 Listeria Moxalactam SupplementAn antibiotic supplement recommended for the isolation of Listeria monocytogenes from mixed flora.Listeria Oxford Medium Base AM105512/505512

AS018 Listeria Selective SupplementAn antibiotic supplement recommended for the selective isolation and identification of Listeria monocytogenes.Listeria Identification Agar Base (PALCAM) AM1055/5055Listeria Identification Broth Base (PALCAM) AM105511/505511

AS0181 Middlebrook OADC Growth SupplementAn enrichment supplement recommended for cultivation of Mycobacteria .Middlebrook 7H9 Agar Base A. AM506927

AS0182 Moxalactam SupplementAn antibiotic supplement recommeded for selective isolation and cultivation of LPM Agar Base AM 10575

AS019 Mycoplasma Enrichment SupplementAn enrichment and antibiotic supplement recommended for the selective isolation of Mycoplasma species.Mycoplasma Agar Base AM1073/5073

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AS0151 Lactic Supplement

Raka Ray Agar Base (Lactic Acid Bacteria Selective Agar Base) AM10844

AS016 Legionella Growth Supplement

Legionella Agar Base AM1054/5054

AS017 Legionella Selective Supplement

Legionella Agar Base AM1054/5054

AS0171 Listeria Moxalactam Supplement

AS018 Listeria Selective Supplement

Listeria Identification Agar Base (PALCAM) AM1055/5055

AS0181 Middlebrook OADC Growth Supplement

Middlebrook 7H9 Agar Base A. AM506927

AS019 Mycoplasma Enrichment Supplement

Mycoplasma Agar Base AM1073/5073

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*

*

*

Raka Ray No. 3 Broth Base (Lactic Acid Bacteria Selective Broth Base) AM10845

Listeria Oxford Medium Base AM105512/505512

Listeria Identification Broth Base (PALCAM) AM105511/505511

AS0182 Moxalactam Supplement

LPM Agar Base AM 10575

AS020 Nalidixic Selective SupplementAn antibiotic supplement recommended for the selective isolation of Pseudomonas aeruginosa.Cetrimide Agar Base AM1022/5022

AS0201 Oxford Listeria SupplementAn antimicrobial supplement recommended for selective isolation of Listeria species. Listeria Oxford Medium Base AM105512/505512

AS021 Polymixin B Selective SupplementAn antibiotic supplement recommended for the selective isolation of B. cereus.Bacillus Cereus Agar Base AM1009/5009

AS0211 Potassium Lactate 50% (10ml / Vial)A filter sterilized supplement recommended for isolation and enumeration of wild Yeasts in pitching yeasts.Lysine Medium Base AM10577/50577

AS022 Potassium Tellurite 1% (10ml /vl)A filter sterilized supplement recommended for the selective isolation of Staphylococci.Baird Parker Agar Base IP AM101111/501111Baird Parker Agar Base USP AM101112/501112Baird Parker Agar Base EP AM101113/501114Baird Parker Agar Base BP AM101114/501114Vogel Johnson Agar Base W/O Tellurite AM1108/5108Vogel Johnson Agar Base W/O Tellurite IP AM11081/51081Vogel Johnson Agar Base W/O Tellurite USP AM11082/51082

AS023 Potassium Tellurite 3.5% (10ml/vl) A filter sterilized supplement recommended for the selective isolation of Staphylococci. Baird Parker Agar Base AM1011/5011Baird Parker Agar Base BIS AM 101115/501115Hoyle Medium Base AM 104823/504823

AS0231 Preston Selective Supplement (Campylobacter Selective Supplement IV, Modified)A supplement recommended for the selective isolation of Campylobacter species.Preston Agar Base AM 10831/50831

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AS020 Nalidixic Selective Supplement

Cetrimide Agar Base AM1022/5022

AS0201

AS021 Polymixin B Selective Supplement

Bacillus Cereus Agar Base AM1009/5009

AS0211

AS022 Potassium Tellurite 1% (10ml /vl)

Baird Parker Agar Base IP AM101111/501111Baird Parker Agar Base USP AM101112/501112Baird Parker Agar Base EP AM101113/501114Baird Parker Agar Base BP AM101114/501114Vogel Johnson Agar Base W/O Tellurite AM1108/5108Vogel Johnson Agar Base W/O Tellurite IP AM11081/51081Vogel Johnson Agar Base W/O Tellurite USP AM11082/51082

AS023 Potassium Tellurite 3.5% (10ml/vl)

Baird Parker Agar Base AM1011/5011Baird Parker Agar Base BIS AM 101115/501115Hoyle Medium Base AM 104823/504823

AS0231 Preston Selective Supplement (Campylobacter Selective Supplement IV, Modified)

Preston Agar Base AM 10831/50831

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Oxford Listeria Supplement

Listeria Oxford Medium Base AM105512/505512

Potassium Lactate 50% (10ml / Vial)

Lysine Medium Base AM10577/50577

* **0 0 Store all Supplements, Agents and Enrichments between 2-8 C, Store below -20 C.





AS0232 Rosolic Acid (0.1 gms per vial)A supplement recommended for selective isolation of coliform bacteria.M-FC Agar Base AM506923MFC Broth Base AM506924

AS024 Sodium DeoxycholateA sodium salt of deoxycholic acid, used in culture media to inhibit gram-positive microorganisms.

AS025 Staph-Strepto SupplementAn antibiotic supplement recommended for the selective isolation of Streptococcus species.Columbia Blood Agar Base AM1029/5029

AS026 Strepto SupplementAn antibiotic supplement recommended for the selective isolation of Streptococcus species.Columbia Blood Agar Base AM1029/5029

AS027 Sulpha SupplementAn antibiotic supplement recommended for the selective isolation of Salmonella species.Brilliant Green Agar Modified AM1018/5018

AS0271 TTC Solution 1 % ( 10 ml per vial)A filter sterilized solution recommended for the detection of microbial growth using TTC (2,3,5-Triphenyl Tetrazolium Chloride) reduction.Modified Tergitol 7 Agar Base ISO AM 506932Tergitol 7 Agar Base AM10951/50951Tergitol 7 Agar BaseBIS AM10952/50952

AS028 Urea 40% (5ml /vl)A filter sterilized supplement for detection of urease activity.Urea Agar Base, Christensen AM1105/5105Urea Agar Base, Christensen BIS AM11051/51051Urea Agar Base, Christensen ISO AM51052Urea Broth Base AM1106/5106

AS029 Yersinia Selective SupplementAn antibiotic supplement recommended for the selective isolation of Yersinia Enterocolitica.Yersinia Selective Agar Base AM1116/5116

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AS0232 Rosolic Acid (0.1 gms per vial)

M-FC Agar Base AM506923MFC Broth Base AM506924

AS024 Sodium Deoxycholate

AS025 Staph-Strepto Supplement


AS026 Strepto Supplement


AS027 Sulpha Supplement

Brilliant Green Agar Modified AM1018/5018

AS0271 TTC Solution 1 % ( 10 ml per vial)

Modified Tergitol 7 Agar Base ISO AMTergitol 7 Agar Base AM10951/50951

AS028 Urea 40% (5ml /vl)

Urea Agar Base, Christensen AM1105/5105

Urea Broth Base AM1106/5106

AS029 Yersinia Selective Supplement

Yersinia Selective Agar Base AM1116/5116

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506932

Tergitol 7 Agar BaseBIS AM10952/50952

Urea Agar Base, Christensen BIS AM11051/51051Urea Agar Base, Christensen ISO AM51052

READY TO USEWATER QUALITY TESTING KITS

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PRESUMPTIVE IDENTIFICATION TESTS**

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READY PREPARED MEDIA

20409001 PA coliform Test KitFor the detection of presence or absence of coliform bacteria from water samples.

20410001 Rapid Coliform Test KitFor rapid detection of E.coli and coliforms from water samples on the basis of enzyme substrate reaction.

20420001 Rapid Enterococci Test KitFor rapid identification and differentiation of enterococci from water samples.

20430010 Rapid H S Test Kit2

For simultaneous detection of Salmonella, Vibrio species, Citrobacter and E.coli from water samples.

20440010 PYR Test KitPYR Test to differentiate between group A Streptococci and Enterococci.

20450010 Presumptive Identification Test Kit for E.coliTest for fluorescence detection of E.coli on the basis of enzyme substrate reaction.

20460010 Test Kit for E.coliTest for presumptive identification of E.coli on the basis of enzyme substrate reaction and Indole test.

20470010 Test Kit for Esculin HydrolysisTest for detection of Esculin Hydrolysis.

20480010 Test Kit for MR-VPTest for detection of acid and acetoin production and for differentiation of Enterobacteriaceae.

20490010 Test Kit for Phenylalanine DeaminationTest for differentiation of Proteus, Providencia from enteric bacilli.

20501006 Alkaline Peptone WaterA medium for enrichment of microorganisms.

READY TO USEWATER QUALITY TESTING KITS

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PRESUMPTIVE IDENTIFICATION TESTS**

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READY PREPARED MEDIA

20409001 PA coliform Test Kit

20410001 Rapid Coliform Test Kit

20420001 Rapid Enterococci Test Kit

20430010 Rapid H S Test Kit

20440010 PYR Test Kit

20450010 Presumptive Identification Test Kit for

20460010 Test Kit for

20470010 Test Kit for Esculin Hydrolysis

20480010 Test Kit for MR-VP

20490010 Test Kit for Phenylalanine Deamination

20501006 Alkaline Peptone Water

2

E.coli

E.coli

* **0 0 Store all Supplements, Agents and Enrichments between 2-8 C, Store below -20 C. * ** 0 Store at R.T., Store betweeb 2-8 C.





20502006 BHI BrothA highly nutritious general purpose liquid medium for cultivation of variety of fastidious and non-fastidious microorganisms, including aerobic and anaerobic bacteria.

20503006 Bile BrothA medium for cultivation of members of the Enyerobacteriaceae.

20500006 Blood Agar Base10500006 A non-selective general-purpose medium to which blood may be

added for use in isolation and cultivation fastidious organisms and detecting hemolytic activity.

20510006 Brain Heart Infusion Agar10510006 A general-purpose medium for cultivation of a wide variety of

microorganisms including bacteria, yeasts and moulds.

20520006 C.L.E.D Agar with Andrade Indicator10520006 A medium for isolation, enumeration and presumptive

identification of microorganisms from urine, giving good colonial differentiation.

20521006 Columbia Blood Agar Base10521006 A basal medium for preparation of blood and chocolate agar and

for various selective and identification media in isolating and cultivating fastidious microorganisms.

20530006 E M B Agar, Levine10530006 A slightly selective and differential medium for isolation,

enumerat ion and d i f fe ren t ia t ion o f members o f Enterobacteriaceae.

20531006 Glucose BrothA medium used for cultivation and fermentation studies of microorganisms.

20532006 Glucose Phosphate BrothA medium for differentiation of coli- aerogenes group by means of MethylRed and Voges Proskauer reactions.

20533006 Hartley BrothA medium for cultivation of wide variety of bacteria from blood specially fastidious Streptococci and Corynebacterium diptheriae.

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20502006 BHI Broth

20503006 Bile Broth

20500006 Blood Agar Base10500006

20510006 Brain Heart Infusion Agar10510006

20520006 C.L.E.D Agar with Andrade Indicator10520006

20521006

20530006 E M B Agar, Levine10530006

20531006 Glucose Broth

20532006 Glucose Phosphate Broth

20533006 Hartley Broth

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Columbia Blood Agar Base10521006

20540006 Mac Conkey Agar with Crystal violet, NaCL, and 0.15% Bile Salts 10540006 A slightly selective and differential medium for the detection of

coliforms and other enteric pathogens.

20550006 Mac Conkey Agar without crystal violet, NaCL, and with 0.5% Sodium Taurocholate

10550006 A medium for cultivation and differentiation of enteric bacteria and potentially pathogenic gram-positive organisms while restricting swarming of Proteus species.

20560006 Mueller Hinton Agar 10560006 A medium for antimicrobial susceptibility testing of common,

rapidly growing microorganisms by the Bauer-Kirby method.

20570006 Nutrient Agar10570006 A medium for cultivation of a wide variety of less fastidious

microorganisms which can be enriched by the addition of blood or other biological fluids

20571006 Nutrient BrothA general purpose medium for cultivation of bacteria.

20572006 Peptone WaterA non selective medium for cultivating non- fastidious organisms.

20580006 Plate Count Agar(Standard Methods Agar)10580006 A medium for obtaining microbial plate counts from milk and dairy

products, foods, water and other materials of sanitary importance.

20590006 Potato Dextrose Agar10590006 A medium for cultivation and enumeration of yeasts and moulds

from dairy and other food products.

20600006 Sabouraud Dextrose Agar10600006 A general-purpose medium for cultivation of yeasts, moulds and

aciduric bacteria.

20601006 Selenite BrothAn enrichment medium for the isolation of Salmonella species from faeces, urine, water, foods and other material of sanitary importance.

20610006 Simmons Citrate Agar10610006 A medium for differentiation of gram-positive bacteria on the basis

of citrate utilization.

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20540006 Mac Conkey Agar with Crystal violet, NaCL, and 0.15% Bile Salts 10540006

20550006 Mac Conkey Agar without crystal violet, NaCL, and with 0.5% Sodium Taurocholate

10550006

20560006 Mueller Hinton Agar10560006

20570006 Nutrient Agar10570006

20571006 Nutrient Broth

20572006 Peptone Water

20580006 Plate Count Agar(Standard Methods Agar)10580006

20590006 Potato Dextrose Agar10590006

20600006 Sabouraud Dextrose Agar10600006

20601006 Selenite Broth

20610006 Simmons Citrate Agar10610006

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* ** 0 Store at R.T., Store betweeb 2-8 C.





20620006 Soyabean Casein Digest Agar (Antibiotic Assay Medium No 36) (Tryptone Soya Agar)

10620006 A general purpose medium for isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms.

20621006 Soyabean Casein Digest Medium(Antibiotic Assay Medium No 37) (Tryptone Soya Broth)A general purpose medium for isolation and cultivation of a wide variety of fastidious and non-fastidious Microorganisms.

20622006 SS Agar10622006 A differential and selective medium for isolation of Salmonella and

some Shigella species from clinical and non-clinical specimens.

20630006 Triple Sugar Iron Agar10630006 A medium for identification of gram negative enteric bacilli on the

basis of dextrose, lactose and sucrose fermentation and hydrogen sulphide production.

20640006 Violet Red Bile Agar10640006 A selective medium for the detection and enumeration of coliforms.

20650006 X.L.D Agar10650006 A moderately selective medium for isolation and differentiation of

Salmonella and Shigella.

20660700 BHI-Supplemented with 0.05% SPS20660200 For qualitative detection of microorganisms in blood.

20661700 Glucose Broth - Supplemented with 0.05% SPS20662700 For qualitative detection of microorganisms in blood.

20670700 Soyabean Casein Digest Broth- Supplemented with 0.05% SPS20670200 For qualitative detection of microorganisms in blood.

20104600 A medium for isolation and differentiation of urinary pathogens on the basis of lactose fermentation.

20102300 MacConkey Agar20102600 A medium for selective isolation and differentiation of coliforms

and other enteric pathogens.

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BLOOD CULTURING SYSTEMS**

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INSTAPREP*

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20104300 CLED Agar

20620006 Soyabean Casein Digest Agar (Antibiotic Assay Medium No 36) (Tryptone Soya Agar)

10620006

20621006 Soyabean Casein Digest Medium(Antibiotic Assay Medium No 37) (Tryptone Soya Broth)

20630006 Triple Sugar Iron Agar10630006

20640006 Violet Red Bile Agar10640006

20650006 X.L.D Agar10650006

20660700 BHI-Supplemented with 0.05% SPS20660200

20661700 Glucose Broth - Supplemented with 0.05% SPS

20670700 Soyabean Casein Digest Broth- Supplemented with 0.05% SPS20670200

20104300 CLED Agar20104600

20102300 MacConkey Agar20102600

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BLOOD CULTURING SYSTEMS**

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INSTAPREP*

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20622006 SS Agar10622006

20662700

20105300 Mueller Hinton Agar20105600 A medium for antimicrobial susceptibility testing of common and

rapidly growing microorganisms by the disk diffusion technique.

20101300 Nutrient Agar20101600 A medium for cultivation of a wide variety of less fastidious

microorganisms which can be enriched by the addition of blood or other biological fluids.

20103300 Sabouraud Dextrose AgarA medium for cultivation of yeasts, moulds and aciduric bacteria.

20106300 SS AgarA medium for differential and selective isolation of Salmonella and some Shigella species from pathological specimens and suspected foodstuffs.

20308500 Acid Fast DecolorizerPre-diluted, ready to use 25% sulphuric acid solution for decolourization of acid fast smears for the screening of M. tuberculosis and M. leprae.

20306015 ADA-MTBFor the determination of adenosine deaminase activity in serum, plasma & biological fluids.

20403020 Catalase Detection KitFor differentiation of isoniazide resistant strains of M. tuberculosis and M. gastri from genus mycobacterium based on catalase activity.

20303001 CombicultCombipack of solid and liquid medium for mycobacterium tuberculosis isolation.

20301012 LyfectolMucolytic, disinfectant, specimen pretreatment and buffering system for AFB staining and culture.

20304006 MycocultReady to use L J. solid medium for Mycobacterium tuberculosis isolation.

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MYCOBACTERIOLOGY*

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20105300 Mueller Hinton Agar20105600

20101300 Nutrient Agar20101600

20103300 Sabouraud Dextrose Agar

20106300 SS Agar

20308500 Acid Fast Decolorizer

20306015 ADA-MTB

20403020 Catalase Detection Kit

20303001 Combicult

20301012 Lyfectol

20304006 Mycocult

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MYCOBACTERIOLOGY*

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20314006 Mycocult PY Ready to use L.J solid medium with sodium pyruvate for isolation of Mycobacterium bovis.

20307100 MycostainAcid fast stain set for screening of M.tuberculosis and M.leprae.

20406003 Mycovue NRALJ slants with nitrate substrate for detection of nitrate reduction by M. tuberculosis.

20401050 Niacin Drop TestFor differentiation of M. tuberculosis from M.bovis based on niacin production.

20405025 Nitrate Reduction KitFor determination of nitrate reduction in cultures.

20302125 NovachromRapid two step cold AFB stain.

20408006 PNB Sensitivity Test Ready to use L J. solid media with para nitro benzoic (PNB) acid for differentiating primary isolates into M.tuberculosis complex and non tuberculous mycobacteria.

20305101 Sensicult PrimaryPrimary drug containing L-J media panel for MTB sensitivity tests.

20305201 Sensicult Secondary (6 drugs)Secondary drug containing L-J media panel for MTB sensitivity tests.

20305202 Sensicult Secondary (10 drugs)Secondary drug containing L-J media panel for MTB sensitivity tests.

20407001 Sensivue Primary Drug susceptibility (primary anti-tubercular drugs) test for M.tuberculosis with a nitrate reductase assay using proportion method.

20407101 Sensivue SecondaryDrug susceptibility (secondary anti-tubercular drugs) test for

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20314006 Mycocult PY

20307100 Mycostain

20406003 Mycovue NRA

20401050 Niacin Drop Test

20405025 Nitrate Reduction Kit

20302125 Novachrom

20408006 PNB Sensitivity Test

20305101 Sensicult Primary

20305201 Sensicult Secondary (6 drugs)

Sensicult Secondary (10 drugs)

20407001 Sensivue Primary

20407101 Sensivue Secondary

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20305202

M.tuberculosis with a nitrate reductase assay using proportion method.

20404003 TCH Sensitivity TestFor differentiation of M. tuberculosis from M. bovis.

20402100 Tween 80 HydrolysisBiochemical test for differentiation of saprophytic species of mycobacteria.

20680020 Barritt Reagent A, Barritt Reagent B, CreatineFor Voges Proskauer (VP) Test.

20690040 Gordon McLeod Reagent (Oxidase Reagent)An oxidase reagent to detect the presence of an enzyme cytochrome oxidase found in some bacteria.

20700040 Kovacs' Reagent (Indole)For Indole Test, particularly useful in the identification of E.coli.

20701040 McFarland Standard No. 0.5Turbidity standard, used in antimicrobial susceptibility testing.

20710040 Methyl Red IndicatorTo detect the ability of an organism to produce and maintain stable acid end products formed from glucose fermentation.

20720025 Nitrite Detection Kit For differentiating and identifying various types of bacteria by their ability to reduce nitrate.

20730040 PYR Reagent For detection of pyroglutamate aminopeptidase activity in group A streptococci and enterococci.

20740040 TDA Reagent For phenylalanine deamination reaction in differentiating Proteus from other members of Enterobacteriaceae.

20750020 Modified Gram's Stain Kit (containing A, B, and C)20750021 To differentiate between gram-negative and gram-positive

bacteria.

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ANALYTICAL REAGENTS AND INDICATORS**

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READYMADE STAINING SOLUTIONS*

20404003 TCH Sensitivity Test

20402100 Tween 80 Hydrolysis

20680020 Barritt Reagent A, Barritt Reagent B, Creatine

20690040 Gordon McLeod Reagent (Oxidase Reagent)

20700040 Kovacs' Reagent (Indole)

20701040 McFarland Standard No. 0.5

20710040 Methyl Red Indicator

20720025 Nitrite Detection Kit

20730040 PYR Reagent

20740040 TDA Reagent

20750020 Modified Gram's Stain Kit (containing A, B, and C)20750021

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ANALYTICAL REAGENTS AND INDICATORS**

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READYMADE STAINING SOLUTIONS*






Stains for Fungi20760040 Lactophenol Cotton Blue

For wet mount and staining of fungi20770040 Lactophenol Picric Acid

For wet mount and staining of fungi20780040 Picric Acid

For staining of fungi

20790001 Enterobacteriaceae Identification Test KitA panel of 12 tests for differentiation of Enterobacteriaceae species (Kit contains sterile broth for Indole, Methyl red, Voges Proskauer, Citrate utilization, and 8 different carbohydrates-Glucose, Adonitol, Arabinose, Lactose, Sorbitol, Mannitol, Rhamnose, Sucrose). Reagents supplied with the Kit: Kovac's reagent for Indole test, Methyl Red Indicator, Baritt Reagent A, Baritt Reagent B and Creatine for VP test.

20791001 Gram- Negative Bacteria Identification Test KitA panel of 12 tests for identification of Gram-negative rods (Kit contains sterile broth for Lysine utilization, Ornithine decarboxylation, Urease detection, Phenylalanine deamination (TDA), Nitrate reduction, H S production, Citrate utilization and 5 2

different carbohydrates-Glucose, Adonitol, Arabinose, Lactose, Sorbitol). Reagents supplied with the kit: TDA Reagent, Nitrite Detection Strip, Zinc Dust.

20792001 Staph Identification KitA panel of 12 tests for identification of Staphylococcus species (Kit contains sterile broth for Voges Proskauer test, Phosphatase detection, ONPG utilization, Arginine utilization, Urease detection, and 7 different carbohydrates- Arabinose, Lactose, Mannitol, Sucrose, Raffinose, Trehalose, Maltose. Reagents supplied with the kit: Baritt Reagent A, Baritt Reagent B and Creatine for VP test.

20793001 Strep Identification KitA panel of 12 tests for identification of Streptococcus species (Kit contains sterile broth for Esculin hydrolysis, Voges Proskauer test, Arginine utilization, PYR hydrolysis, ONPG utilization and 7 different carbohydrates-Glucose, Arabinose, Sorbitol, Mannitol, Sucrose, Raffinose, Ribose). Reagents supplied with the kit: Baritt Reagent A, Baritt Reagent B and Creatine for VP test, PYR Reagent.

20794001 Candida Identification KitA panel of 12 tests for identification of Candida species (Kit

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BIOCHEMICAL IDENTIFICATION KITS**

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Stains for Fungi20760040 Lactophenol Cotton Blue

20770040 Lactophenol Picric Acid

20780040 Picric Acid

20790001 Enterobacteriaceae Identification Test Kit

Reagents supplied with the Kit:

20791001 Gram- Negative Bacteria Identification Test Kit

Reagents supplied with the kit:

20792001 Staph Identification Kit


20793001 Strep Identification Kit


20794001 Candida Identification Kit

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BIOCHEMICAL IDENTIFICATION KITS**

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contains sterile broth for Urease detection and 11 different carbohydrates-Melibiose, Lactose, Maltose, Sucrose, Raffinose, Galactose, Trehalose, Cellobiose, Inositol, Xylose, Dulcitol).

20795001 Neisseria Identification KitA panel of 12 tests for identification of Neisseria species (Kit contains sterile broth for Urease detection, Voges Proskauer test, Oxidase detection, Catalase detection, Nitrate reduction, ONPG utilization, and 6 different carbohydrates-Glucose, Maltose, Lactose, Sucrose, Fructose, Mannose). Reagents supplied with the kit: Baritt Reagent A, Baritt Reagent B and Creatine for VP test, Gordon McLeod Reagent (Oxidase Reagent), Nitrite Detection Strip, Zinc Dust.

20796001 E.coli Identification KitA panel of 12 tests for identification of E.coli (Kit contains sterile broth for MR test, Voges Proskauer test, Citrate utilization, Indole test, Glucuronidase utilization, Nitrate reduction, ONPG utilization, Lysine decarboxylation and 4 different carbohydrates-Glucose, Lactose, Sucrose, Sorbitol). Reagents supplied with the kit: Baritt Reagent A, Baritt Reagent B and Creatine for VP test, Methyl Red Indicator, Nitrite Detection Strip, Zinc Dust, Kovac's Reagent for Indole test.

20797001 Salmonella Identification KitA panel of 12 tests for the identification of Salmonella species (Kit contains sterile broth for MR test, Voges Proskauer test, Citrate utilization, Urease detection, H S production, ONPG utilization, 2

Lysine decarboxylation and 5 different carbohydrates-Arabinose, Lactose, Maltose, Sorbitol, Dulcitol). Reagents supplied with the kit: Barritt Reagent A, Baritt Reagent B and Creatine for VP test, Methyl Red Indicator.

20798001 Listeria Identification KitA panel of 12 tests for identification of Listeria species (Kit contains sterile broth for Esculin Hydrolysis, Voges Proskauer test, Nitrate reduction, Methyl Red test, Catalase detection, and 7 different carbohydrates-Glucose, Xylose, Lactose, Mannitol, Rhamnose, Alpha-Methyl-D-Mannoside, Ribose). Reagents supplied with the kit: Baritt Reagent A, Baritt Reagent B and Creatine for VP test, Methyl Red Indicator, Nitrite Detection Strip, Zinc Dust.

20201012 EasybactChromogenic, differential, semi-quantitative bacteriuria collection and screening system.

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URINE CULTURE**

20795001 Neisseria Identification Kit


20796001 E.coli Identification Kit


20797001 Salmonella Identification Kit


20798001 Listeria Identification Kit


20201012 Easybact

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URINE CULTURE**


Countries Around the World to which Tulip Group Products are Exported

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