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

TM

e-Hand Book of Practicing Microbiologists


First Edition

Review of the e-Handbook of Practicing Microbiologists


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. 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.

Published by: Tulip Diagnostics (P) Ltd. for the use of its esteemed customers globally. AccumixTM, is the brand name of Dehydrated Culture Media, Bases and Supplements manufactured by MicroxpressTM. MicroxpressTM 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: sales@tulipgroup.com. Website: www.tulipgroup.com

FOREWORD
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.

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

Quality policy...

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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.

Contents...

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Contents

A Window To Tulip Group Corporate Flowchart Our Certification


Microbiological Methods in Pharma Industry l Media Fill Run. l Microbiological analysis of water. l Sterility Testing. l Determining Biocontamination of surfaces. l Monitoring Microbial load from filling operators garment clothes / hand gloves on sterile room. l Microbiological Evaluation program for controlled Environment. l Microbiological Assay of Antibiotics. l Antimicrobial Effectiveness Testing (Preservative efficacy testing). l Validation of autoclaves and ovens using Biological Indicators. l Cup-plate Assay of Cyano-Cobalmin (Vitamin B12) using E.coli mutant 113 (D). l Calcium Pantothenate Assay. Microbiological Methods in Food Industry l Enumeration of Coliforms, Faecal Coliforms and of E.coli in Water in Sealed Containers and Prepackaged Ice Using the Hydrophobic Grid-membrane Filter (HGMF) Method. l Microbiological Examination of Ice Cream and Ice Milk l Microbiological Examination of Cottage Cheese l Microbiological Examination of Egg Products and of Liquid Eggs l Microbiological Examination of Milk l Microbiological Examination of Mineral Water l Microbiological Examination of Milk Powder l Microbiological Examination of Froglegs l Microbiological Examination of Cocoa and Chocolate l Examination of Canned Tomatoes, Tomato Juice and Vegetable Juice, Tomato Puree, Tomato Paste, Tomato Pulp and Tomato Catsup for Mould Filaments l Microbiological Examination of Cheese l Microbiological Examination of Water in Sealed Containers (Excluding Mineral and Spring Water) and of Prepackaged Ice l Microbiological Examination of Foods for Aerobic Colony Counts (ACC) l Enumeration of Coliforms, Faecal Coliforms and of E. Coli in Water in Sealed Containers and Prepackaged Ice Using the MPN Method

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01-21 01 02 04 08 09 09 11 17 17 20 20 22-81 22 24 27 31 34 35 39 42 45 48 55 62 66 68

Contents...

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Proposed Official Method: Enumeration of Pseudomonas aeruginosa in Prepackaged Ice and Water in Sealed Containers by the Hydrophobic Grid-Membrane Filter (HGMF) Technique l Detection of Food Poisoning by Clostridium botulinum and Its Toxins
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Contents

75 77 82-85 82 83 84 84 85 86-87 86 86 86 88-122 88 88 89 91 105 108 111 116 118 121 123 124-147 148-176

Soil Microbiology l Isolation of Dermatophytes, other Fungi and Yeasts from Soil l Cultivation of those fungi and bacteria which are able to utilise sodium nitrate as the sole source of nitrogen l Observation for chlamydospore production by Candida albicans and for the maintenance of fungal stock cultures. l Isolation of Nitrogen Fixing Bacteria from Soil l Dilution and isolation of Phosphate Solubilizing Microorganisms from soil Microbiological Methods in Beverage Industry l Detection and Enumeration of respiratory deficient yeast cells used in beverage. l Maintenance of 'Yeast' cultures which are used as 'Seed' for fermentation. l To check sterility of Beverage products (Beer, wine etc) by microfilteration technique. Medical Microbiology l Introduction l 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 Gastrointestinal infections l Urinary tract infections l Meningitis l Pyrexia of unknown origin (PUO) Quantitative Analysis of culture media using Ecometric Method Application Microxpress Product List

A Window to Tulip Group...

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QUALITY ASSURANCE 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. HUMAN RESOURCES 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. NATIONAL SALES The Companys 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. INTERNATIONAL PRESENCE Internationally the company channelizes its products and technology through distributors, NGOs 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. OPPORTUNITIES FOR COLLABORATION 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.

INTRODUCTION 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. PRODUCT DEVELOPMENT 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. MANUFACTURING 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.

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

About us...

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Corporate Flowchart

Group

TULIP DIAGNOSTICS (P) LTD. Immunohaematology Haematology Rheumatology Infectious Diseases Haemostasis Instruments

ORCHID BIOMEDICAL SYSTEMS Parasitology Fertility Infectious Diseases

ZEPHYR BIOMEDICALS Parasitology Infectious Diseases Cancer Markers Cardiac Markers

QUALPRO DIAGNOSTICS Infectious Diseases ELISA based and rapid membrane tests

CORAL CLINICAL SYSTEMS Clinical Biochemistry Analytical Reagents Stains

MICROXPRESS - A DIVISION OFTULIP DIAGNOSTICS (P) LTD.

BIOSHIELDS - A DIVISION OFCORAL CLINICAL SYSTEMS Disinfection products

READY TO USE General biochemical identification tests Analytical reagents Water testing solutions Stains One step presumptive identification tests Ready prepared media Blood culture systems Mycobacteriology Mycobacteria identification, isolation, staining and sensitivity testing. Instraprep range of ready to pour, sterilized pouched media

ACCUMIX
Dehydrated culture media Bases S e l e c t i v e supplements, agents and enrichments

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

Certification...

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iii

Certification...

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

Microbiological Methods in Pharma Industry

Hand Book of Practicing Microbiologists

Media Fill Run


Introduction 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 include l Facility and room design l Design of the filling machine l Process flow
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Heating, ventilation and air-conditioning design. Trends in environmental monitoring data. Contamination Control Programme. Quality assurance and Quality Control systems. Process stimulations 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,
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Type of product being filtered. Lot / Batch size Container and closure configuration. Fill volume Line speed Operator shifts and fatigue Filling line configuration Sterile hold times Number of units filled Acceptance criteria Run duration

Growth Media Used The 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.
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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'. Mycoplasma can be a concern with culture media even it is sterile filtered, therefore irradiation gives assurance that media is free from Mycoplasma.

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

Microbiological Methods in Pharma Industry

Hand Book of Practicing Microbiologists

Requirements Material l Sterile (Gamma Irradiated) SCDM [AM50925- 5k] l Soyabean Casein Digest Agar [AM1041/AM5091] l 0.22 micron membrane filter l vials/ampoules/bottles. l S.aureus ATCC 25923 l E.coli ATCC 25922 l S.pyogenes ATCC 19615 l C.albicans ATCC 10231 Equipments l Autoclave l Incubator l Oven l Laminar Air flow l Membrane Holder l Air Sampler (Accubas AX1) Procedure l 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

respectively. These are then incubated under identical conditions as the process stimulation samples. Carry out checking of filled vials every day for microbial contamination during incubation period.

Interpretation l 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. Limits The 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 3000 4750 6300 7750 9150 10510 11840 13150 14430 15710 16960 Contaminated Units Permitted 0 1 2 3 4 5 6 7 8 9 10

References 1. Innovations in Pharmaceutical Technology- Phil Smith 2. Drugs and Health Products- Health Canada 3. Aseptic Pharmaceutical Manufacturing- Michael Groves and Ram Murfy 4. USP chapter <797>.

Microbiological Analysis of Water


Introduction Water 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.
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Various types of water to be used in the manufacture of Pharmaceutical articles Purified Water Water for injection Sterile water for injection

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

Microbiological Methods in Pharma Industry

Hand Book of Practicing Microbiologists

Guidelines for microbial control in water for Pharmaceutical use The 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 use The 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 testing 1) Total aerobic microbial count 2) E.coli 3) Salmonella 4) S.aureus 5) Pseudomonas Requirements Material l Soyabean Casein Digest Agar (AM1091/AM5091) l Saborauds Dextrose Agar (AM1087/ AM5087 l MacConkeys 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 Equipments l Laminar air Flow l Autoclave l Oven l PH meter Water analysis is very important and critical test in the Pharma Industry and is conducted on daily basis. Procedure SAMPLING OF WATER

a) b) c) d) e) f) g)

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

1. For total aerobic microbial count a) Pipette out 1 ml sample in sterile petridish. To this add 20 ml sterile soyabean casein digest agar at 40-450 C. Mix it and allow to solidify. Incubate the plates at 30-350 C for 48 hours. b) Pipette out 1 ml sample in sterile petridish. To this add 20 ml sterile Saborauds Dextrose agar at 40-450 C. Mix it and allow to solidify. Incubate the plates at 20-250 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 count a) Add 10 ml sample into 100 ml sterile nutrient broth. b) Incubate at 370 C for 18 -24 hours. c) Transfer 1 ml of above enrichment broth into 5 ml MacConkeys broth. d) Incubate at 370 for 24-48 hours. e) Prepare positive control by inoculating a loopfull of 24 hours old culture of E. coli to 5 ml sterile MacConkeys broth. f) Incubate at 370 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 MacConkeys broth and 5 ml Peptone water each. Incubate at 440 C for 20- 24 hours. h) After incubation if acid and gas formation is noticed in MacConkeys broth presence of E. coli is confirmed. 3. For Salmonella a) Add 10 ml of sample to 100 ml sterile Nutrient broth. Incubate at 370 C for 24 hours. b) Transfer 1 ml of above enrichment broth to 10 ml sterile selenite broth or 10 ml of brilliant green broth. Incubate at 370 C for 24 hours. c) For positive control transfer a loopfull of 24 hours old culture of Salmonella abony into 10 ml of selenite broth. Incubate at 370 C for 48 hours. d) From selective broth streak out a loopfull on either of deoxycholate citrate agar or bismuth sulphite agar. Incubate at 370 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.

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

Microbiological Methods in Pharma Industry

Hand Book of Practicing Microbiologists

4. For Pseudomonas a) Transfer 10 ml of sample into 100 ml sterile fluid soyabean casein digest medium.Incubate at 370 C for 48 hours. b) Streak a loopfull of above enrichment broth onto cetrimide agar and incubate at 370 C for 48 hours. Streak a loopfull of 24 hours old culture of Pseudomonas aeruginosa on cetrimide agar plates and incubate at 370 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. aureus Streak a loopfull of enrichment culture from SCDM broth on Mannitol salt agar. Incubate at 370 C for 24 hours. Also streak a loopfull of 24 hours old culture of S. aureus on Mannitol salt agar. Incubate at 370 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. Interpretation l 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.

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 H2S production (blackening) is considered as positive test. Absence of standard growth pattern, consider as negative test. 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. 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. 2. 3. 4. 5. References 1. IP 2007 2. USP-27 TBC + TFC E.coli Salmonella S.aureus Pseudomonas NMT 100 CFU/ml Absent/10 ml Absent/10 ml Absent/10 ml Absent/10 ml

Sterility Testing
Introduction The test for sterility are intended for detecting the presence of viable forms of microorganisms 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 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 procedures The tests can be carried out using 2 methods. A] Membrane Filtration B] 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.

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Microbiological Methods in Pharma Industry

Hand Book of Practicing Microbiologists

Sterility by Membrane Filtration Requirements Materials l 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 m and 47 mm diameter. l Bacillus subtilis ATCC 6633 l Candida albicans ATCC 10231 Equipments l Membrane filtration holder. l Laminar air flow. o o l Incubator 20 C to 25 C. o o l 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 Procedure l 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 Soyabean Casein Digest Medium and incubate at 20oC to 25oC for 7 days. Similarly also immerse the other half membrane into fluid thioglycollate medium and incubate at 30oC to 35oC for 7 days. Inspect tubes on every day and record the observation. Prepare the following controls l 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 Soyabean Casein Digest Medium and incubate at 20oC to 25oC for 7 days. Similarly also immerse the other half membrane into fluid thioglycollate medium and incubate at 30oC to 35oC for 7 days. Inspect tubes on every day and record it.

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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. Media control- Keep each of one sterile media tubes (without opening) at respective temperature for 7 days. Positive control- Inoculate sterile fluid thioglycollate medium with about 100 viable micro-organisms of Candida albicans and Bacillus subtilis individually. Incubate at 30oC to 35oC, And soyabean casein digest medium with 100 viable micro-organisms of Bacillus subtilis, and Candida albicans individually. Incubate at 20oC to 25oC.

B] Direct Inoculation Sterility Testing by Direct Inoculation In 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. Requirement Material l Soyabean Casein Digest Medium (AM1092/AM5092) l Fluid Thioglycollate Medium (AM1045/AM5045) l Bacillus Subtilis ATCC 6633 l Candida albicans ATCC 10231 Euipments l Laminar air flow 0 0 l Incubator 20 C to 25 C 0 0 l Incubator 30 C to 35 C l Sterile Pipettes/Sterile syringe Procedure l 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 specified in monograph at 300C - 350C in case of Fluid Thioglycollte Medium and at 200C - 250C 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

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Microbiological Methods in Pharma Industry

Hand Book of Practicing Microbiologists

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. 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.

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

Prepare the following Controls l 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. Incubate at 30oC to 35oC,And soyabean casein digest medium with 100 viable micro-organisms of Bacillus subtilis, and Candida albicans individually. Incubate at 20oC to 25oC. TABLE I Number of items in the batch 1. Injectable preparations l Not more than 100 containers l More than 100 but not more than 500 containers l More than 500 containers 2. Ophthalmic and other non-injectable preparations l Not more than 200 containers l More than 200 containers

Interpretation l 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).

Minimum number of items recommended to be tested

l l l

10% or 4 containers whichever is greater 10 containers. 2% or 20 containers whichever is less.

l l

5% or 2 containers whichever is greater. 10 containers.

3. Surgical Dressings l Not more than 100 packages l More than 100 but not more than 500 packages l More than 500 packages 4. Bulk solids l Less than 4 containers l 4 containers but not more than 50 containers l More than 50 containers.

l l l

10% or 4 packages whichever is greater. 10 packages. 2% or 20 packages whichever is less.

l l l

Each container 20% or 4 containers whichever is greater 2% or 10 containers whichever is greater.

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TABLE II Quantity in each container of injectable preparation For Liquids l Less than 1 ml l 1 ml or more but less than 4 ml l 4 ml or more but less than 20 ml l 20 ml or more but less than 100 ml
l

Minimum quantity to be used for each culture medium

l l l l l

100 ml or more

Total contents of a container Half the content of a container 2 ml 10% of the content of a container unless otherwise specified in the monograph. Not less than half the contents of a container unless otherwise specified in the monograph

For Solids l less than 50 mg l 50 mg or more but less than 200 mg l 200 mg or more

l l l

Total contents of a container Half the contents of a container 100 mg

TABLE III Type of preparation Quantity to be mixed (A) 10 to 100 ml Quantity to be used for each culture medium (B) 5 to 10 ml

l l

Ophthalmic solutions, other no-injectable liquid preparations. Other preparations, preparation soluble in water or appropriate solvents, insoluble preparations to be suspended or emulsified (ointments and creams) Absorbent cotton

1 to 10 g

0.5 to 1 g Not less than 1 g*

* one portion

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TABLE IV Quantity in each container of injectable preparation For Liquids l Less than 1 ml l 1 ml or more but less than 5 ml l 5 ml or more but less than 20 ml l 20 ml or more but less than 50 ml l 50 ml or more but less than 100 ml For Solids l less than 50 mg l 50 mg or more but less than 200 mg l 200 mg or more Minimum quantity to be used for each culture medium Minimum volume of culture medium (ml)

l l l l l

Total contents of a container Half the content of a container 2 ml 5 ml 10 ml

15 20 20 40 80

l l l

Total contents of a container Half the contents of a container 100 mg

40 80 80

References 1. US Food and Drug adm, 1998, Bacteriological Analytical Manual, 8th Ed, Rev. A, AOAC, International, Guithersburg, md.

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.

Determining Biocontamination of Surfaces


Introduction This 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 Plate Requirements l Sterile contact plates (Nutrient Agar / Tryptone Soya Agar with lecithin and Tween80). l Equipment l Incubator Procedure l 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. o l Incubate the one set of plates at 37 + 2 C for 24-48 hours and other at 20o 25 C for 48-72 hours. l After incubation period count the number of colonies from both set. Determination of Bioburden by Swabs Requirements Materials l Sterile Swabs l Sterile Saline l Sterile plates of Tryptone Soya Agar with Lecithin and Tween 80. (AM11031/AM51031) Equipments Autoclave Laminar Air Flow Incubator

l l l

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Standard Procedure l 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.

Carry out testing once in a month.

Limits l 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. References 1. USP 27 (1116) Microbiological evaluation. 2. IP 2007 Vol. I.

Monitoring Microbial load from filling operators garment clothes / hand gloves in sterile room
Introduction Aseptically 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. Materials l Sterile Swabs l Sterile Saline l Nutrient Agar (AM1074/AM5074) l Sabourauds Dextrose Agar (AM1087/AM5087) Equipments l LAF l Vortex Mixer l Autoclave Procedure l Carry out swab testing inside filling room. l Follow proper procedure to enter in clean room.
l l l l l l l l

Take a sterile swab and moist it with sterile saline. Swab approximately 25 sq. cm area from filling operator clothes (Head gear, shirt sleeves etc) and both hand gloves. Transfer the swab aseptically into 2 ml sterile saline. Disintegrate the swab on the vortex mixer. Carry out bacterial count by plating above saline on Nutrient Agar. Carry out yeast/mould count by plating above saline on Nutrient Agar and Sabourauds Dextrose Agar. Incubate the N.A plates at 37oC + 2oC for 24-48 hours and Sabourauds Dextrose Agar plates at 25oC + 2oC for 72 hours. Count the number of colonies from both plates and calculate the total CFU/ml of saline.

Limits Class 100 10,000 CFU/ml Gloves 3 10 Personnel Clothing 5 20

References 1. USP-27 (1116) Microbiological Evaluation.

Microbiological Evaluation Program for controlled Environment


Introduction Microbial 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 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.

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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 plate Requirements Material l Soyabean Casein Digest Agar (AM1091/AM5091) l S.aureus ATCC 25923 l C.albicans ATTC 10231 Equipments l Autoclave l Incubator Procedure l Prepare sterile plates of Soyabean Casein Digest Agar. l Expose the plates in specified area as per respective protocol. o o l After exposure as per specified time incubate one set at 37 C + 2 C and set o o another set at 25 -30 C for 5 days. l Count the number of colonies from both sets. 1st set will give bacterial colonies. 2nd 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 at 37oC + 2oC for 5 days. Inoculate another SCDA Plate with 24 hours old culture C.albicans and incubate at 25-30oC for 5 days. (Positive Control). Interpretation l Negative Control Plates should remain sterile till end of the incubation period. l Positive Control Plates should give growth of standard colonies.

Environmental Monitoring by using air sampler Requirements Material l Soyabean Casein Digest Agar (AM1091/AM5091). l S.aureus ATCC 25923 l C.albicans ATCC 10231 Equipments l Air Sampler (Accubas AX1) l Autoclave l Incubator Procedure l 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. o l 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 at 37oC + 2oC for 5 days. Inoculate another SCDA Plate with 24 hours old culture C.albicans and incubate at 25-30oC for 5 days. (Positive Control). l At the end of incubation period count the number of colonies. Interpretation l Negative Control Plates should remain sterile till end of the incubation period. l Positive Control Plates should give growth of standard colonies. Limits Air cleanliness guidelines in colony forming units in controlled environment. (Using a slit-to-Agar sampler or equivalent). Class (U.S. Customary) 100 10,000 100,000 CFU per cubic meter of air CFU per cubic feet of air Less than 3 Less than 0.1 Less than 20 Less than 0.5 Less than 100 Less than 2.5

References 1. USP 27 (1116) Microbiological Evaluation 2. Fedral Standard 209E September 1992.

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Microbiological Assay of Antibiotics


Introduction The 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 Activity A 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 g 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 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 ) Requirements Material l Antibiotic Assay Medium (No.1) AM1002/AM5002 l Antibiotic Assay Medium (No.5) AM50031 l Antibiotic Assay Medium (No.8) AM50032 l Antibiotic Assay medium (No.11) AM1004/AM5004 l Buffer Solutions (Refer table 1) l 24 hours old ATCC Cultures. (Refer Table 2) l Distilled water l 0.1 M. HCl

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l l

Medium A Medium 1

l l

Equipments l Laminar air flow l Autoclave l Incubator l 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 organism l 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 50 to 70 ml of saline solution and heat the suspension for 30 minutes at 70oC. 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 32o to 35o for 24 hours. Inoculate 100 ml of nutrient broth. Incubate for 16 to 18 hours at 37o 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 medium at a temperature between 40oC - 50oC (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.

For one level assay with standard curve on the day of the assay. 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. 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. 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. On each of the three plates of a set fill alternate cylinders or cavities with solution S3 (Representing the median concentration of the standard solution) 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. 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 S3. Incubate the plates for about 18 hours at the specified temperature and measure the diameter or the zones of inhibition.

Estimation of Potency Average the readings of solution S3 and the readings of the concentration tested on each set of three plates, and average also all 36 readings of solution S3 . The average of the 36 readings of solution S3 is the correction point for the curve. Correct the average value obtained for each concentration (S1, S2 S4, and S5) to the figure it would be if the readings for solution S3 for that set of three plates were the same as the correction point. Thus, in correcting the value obtained with any concentration, say S1, if the average of 36 readings of S3 is, for example, 18.0mm and the average of the S3 concentrations on one set of three plates is 17.8mm, the correction is + 0.2 mm. If the average reading of S1 is 16.0 mm, the corrected reading of S1 is 16.2 mm. Plot these corrected values including of the average of the 36 readings for solution S3 on two-cycle semilog paper, using the concentrations in Units or ?g per 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 ; 5 H = 3e + 2d + c a 5

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

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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 S3 on the plates used for the sample solution. If the sample gives a large average zone size than the average of the standard (solution S3), add the difference between them to the zone size of solution S3 of the standard response line. If the average sample zone size is smaller than the standard values, subtract the difference between them from the zone size of solution S3 of the standard response line. From the response 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 Assay Prepare parallel dilutions containing 2 levels of the standard (S1 and S2) and the unknown (U1and U2). On each of four or more plates, fill each of its four cylinders or 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 = (U1 + U2) - (S1 + S2) (U1 + U2) + (S1 - S2) U1 and U2 are the sums of the zone diameters with solutions of the unknown of high and low levels. S1 and S2 are the sums of the zone diameters with solutions of the standard of high 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 sample 100

Turbidometric or Tube Assay Method (Method B) Requirement Material l Antibiotic Assay Medium (No.3) AM1003/AM5003 l Buffer solutions (refer Table 1) l 24 hours old ATCC cultures (refer Table 2) l Distilled water l 0.1 m. HCl l Dilute formaldehyde solution Equipments l Laminar Air Flow l Autoclave l Incubator l Water bath l Spectro photo meter Procedure The 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

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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 ; 5 H = 3e + 2d + c a 5

factors to obtain the antibiotic content of the sample. Table 1 Buffer 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 after Number Phosphate, K2HPO4 (g) Phosphate, KH2PO4 (g) sterilization to 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. Suggested inoculum composition Amount (ml per 100 ml) As required As required As required As required As required 0.1 0.1 1.5 0.3 1.0 0.5 1.0 1.0 0.1 0.2 0.03 0.4 4.0 Antibiotics assayed Oxytetracycline Tetracycline Framycetin Kanamycin sulphate Framycetin Kanamycin B Rifampicin Polymycin B Streptomycin Erythromycin Bacitracin Bleomycin Carbenicillin Amphotericin B Nystatin Amikacin Doxycycline, Oxytetracycline Kanamycin sulphate Gentamicin Neomycin Novobiocin

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 Table 2 - Preparation of inoculum Test Oragansim Bacilllus cereus var. mycoides Bacillus pumilus Bacillus subtilis Bodetella bronchiseptica Klebsiella pneumoniae Micrococcus luteus (9341) Micrococcus luteus (10240) Mycobacterium smegmatis Pseudomonas aeruginosa2 Saccharomyces cerevisiae (9763) Saccharomyces cerevisiae (2601) Staphylococcus aureus Staphylococcus epidermidis Incubation conditions Temp. ( C) 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
o

where

Time 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

Suggested dilution factor ------1:20 1:25 1:40 1:35 As determined 1:25 As determined As determined 1:20 1:40

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Table 3 - Test organisms for microbiological assay of antibiotics Antibiotic Amikacin Amphotericin B Bacitracin Bleomycin Carbenicillin Doxycycline Erythromycin Framycetin Gentamicin Kanamycin sulphate Kanamycin B Neomycin Novobiocin Nystatin Oxytetracycline Polymyxin B Rifampicin Streptomycin Tetracycline Test organism Staphylococcus aureus Saccharomyces cerevisiae Micrococcus luteus Mycobacterium smegmatis Pseudomonas aeruginosa Staphylococcus aureus Micrococcus luteus Bacillus pumilus Bacillus subtilis Staphylococcus epidermidis Bacillus pumilus Staphylococcus aureus Bacillus subtilis Staphylococcus epidermidis Staphylococcus epidermidis Saccharomyces cerevisiae Bacilllus cereus var. mycoides Staphylococcus aureus Bodetella bronchiseptica Bacillus subtilis Bacillus subtilis Klebsiella pneumoniae Bacilllus cereus Staphylococcus aureus ATCC1 No. 29737 9763 10240 607 25619 29737 9341 14884 6633 12228 14884 29737 6633 12228 12228 2601 11778 29737 4617 6633 6633 10031 11778 29737 NCTC2 No. (NCIB3 No.) 7447 10716 7743 7447 (8553) 8241 8236,10400 (8853) 8241 7447 8236 (8853) (8853) 10716 10320 7447 8344 8236 8236 (9111) 10320 7447

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Table 4 - Stock solutions and test dilutions of Standard Preparations Standard Stock Solution
Antibiotic Assay method Prior drying Initial solvent (further diluent if different) Final stock concentration per ml

Test Dilution
Use before (number of days) Final diluent Median dose g or units per ml Incubation temp (oC)

(1) Amikacin Amphotericin B Bacitracin Bleomycin Carbenicillin Doxycycline Erythromycin Framycetin Gentamicin Kanamycin sulphate Kanamycin B Neomycin Novobiocin Nystatin Oxytetra cycline Polymyxin B Rifampicin Streptomycin Tetracycline

(2) B A A A A B A A A A B A A A A A B A A A B A B

(3) No Yes Yes Yes No No Yes Yes Yes No No No Yes Yes Yes No No Yes No Yes Yes No No

(4) Water DMF7 0.01M HCl B68 B1 0.1M HCl Methanol (10 mg/ml)8,(B2) B2 B2 B2 Water B2 B2 Ethanol (10 mg/ml)9,(B2) DMF7 0.1M HCl 0.1M HCl Water, (B4) Methanol Water Water 0.1M HCl 0.1M HCl

(5) 1 mg 1 mg 100 Units 2 Units 1 mg 1 mg 1 mg

(6) 14 Same day Same day 14 14 5 14 14 30 30 30 30 14 5 Same day 4 4 14 1 30 30 1 4

(7) Water B5 B1 B6 B6 Water B2 B2 B2 B2 Water B2 B2 B4 B4 B3 Water B4 B1 Water Water Water Water

(8) 10 g 1.0 g 1.0 Unit 0.04 Unit 20 g 0.1 g 1.0 g 1.0 g 0.1 g 0.8 Unit 10 Units 1.0 Unit 1.0 g 0.5 g 20 Units 2.5 g 0.24 g 10 Units 5.0 g 1.0 g 30 g 2.5 g 0.24 g

(9) 32-35 29-31 32-35 32-35 36-37.5 35-37 35-37 30-35 36-37.5 37-39 32-35 32-35 36-37.5 32-35 29-31 32-35 35-37 35-39 29-31 32-35 35-37 32-35 35-37

1 mg 1 mg 800 Units 1000 Units 1000 Units 1 mg 1 mg 1000 Units 1 mg 1 mg 10,000 Units 1 mg 1 mg 1 mg 1 mg 1 mg Medium B

Medium A Ingredients Peptone Pancreatic digest of casein Yeast Extract Beef Extract Dextrose Agar Final pH in grams per liter 6.0 4.0 3.0 1.5 1.0 15.0 6.5-6.6

Ingredients Peptone Beef Extract Agar Glycerin Sodium Chloride Final pH References: IP 2007.

in grams per liter 6.0 1.5 15.0 10.0 3.0 6.9-7.1

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Antimicrobial Effectiveness Testing (Preservative efficacy testing)


Introduction Antimicrobial 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. Requirement Material l 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 10231 l Aspergillus niger ATCC16404 l Escherichia coli ATCC 8739 l Pseudomonas aeruginosa ATCC 9027 l Staphylococcus aureus ATCC 6538 l Saline Solution l Saline Solution Containing 0.05% w/v of Polysorbate 80 Equipments l Laminar air Flow l Incubator l Autoclave Testing Procedure Preparation of Inoculum From 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 incubate at 300Cto 350C for 18-24 hours and for fungal cultures use Sabouraud Dextrose Agar and incubate at 200Cto 250C for 48 to 96 hours. Using sterile saline solution harvest the bacteria and C.albicans cultures and dilute suitably with sterile saline solution to bring the count to about 1x108 per ml. Similarly harvest A.niger culture with sterile saline solution containing 0.05% w/v of polysorbate 80 and adjust the spore count to about 1x108 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 Product l 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 between 1x105 and 1x106 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. Interpretation l The concentrations of viable bacteria are not more than 0.1% of the initial concentrations by the 14th 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 designated levels during the remainder of the 28th day test period. References 1. IP 2007. 2. USP 27.

Validation of autoclaves and Ovens using Biological Indicators


Introduction Biological 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 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

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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 organisms l 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. D121, D170 indicate the temperature of 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. D10 . 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 users 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 indicators Sterilization Mode Example of a typical D-value (minutes) Dry Heat 1600 Moist Heat 1210 1.9 1.9 Minimum D values for selecting a suitable biological indicator (minutes) Min 1.0 Max 3.0 Min 1.5 Max 3.0 Minimum survival time (minutes) Min 4.0 Max 14.0 Min 4.5 Max 14.0 Kill time (minutes) Min 10.0 Max 12.0 Min 13.5 Max 32.0

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Validation of Autoclaves Material l Soyabean Casein Digest medium (AM1092 / AM5092) l Spore strip (Geobacillus sterothermophilus) l Glass vials Eqiupments l Laminar Flow 0 0 l Incubator (60 C + 2 C) l Autoclave Procedure l 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. 0 l 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 0 l 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 months l Sterilize all cultures before discarding. Interpretation l Growth within 3 days in positive control vials, no growth in negative control vials and test vials till 7th 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 Ovens Material l Soyaben Casein Digest Medium (AM1092/AM5092)

l l

Spore strip (Bacillus Atrophaeus) Glass vials / petridishes

Equipments l Laminar Flow 0 0 l Incubator (35 C + 2 C) l Oven Procedure l 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 below 600 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 soyabean casein digest medium. Incubate the tubes at 350 C + 20 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 (350 C + 20 C) for 7 days. l Sterilize all cultures before discarding. Interpretation l Positive control - Within 18-24 hours growth should be observed l 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. References 1. IP 2007. 2. USP 27 (55).

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Cup-plate Assay of Cyano-Cobalmin (Vitamin B12) using E.coli mutant 113 (D)
Introduction To determine the potency Vit B12 solution with comparison with standard Vit B12 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. B12, also known as Cyano cobalmin or cobalmin Vit B12 is produced 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 B12 is a complex water 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 B12 isassayed by using E.coli-mutant 113.D. The potency of Vit B12 in any given solution can be determined by observing the growth response of an auxtrophic organism, which is requiring Vit B12 for growth. This auxatroph should give a growth response in proportion to the amount of Vit B12 added. The response is indicated by a exhibition zone around the cup containing Vit B12. Requirements Material l B12 Maintenance Media (For E.coli mutant) AM 1008 l B12 Assay Agar (using E.coli mutant culture) AM 1007 l 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 B12 solution, Test Sample. Equipments Glass ware: Sterile petridishes, sterile 10 ml. And 1 ml, pipette, sterile Pasteur pipette, cork borer (6 mm). Procedure Prepare Vit B12 standards 0.005, 0.01, 0.02, 0.03, 0.05, 0.00, 0.1 mg/ml in
l l

l l

sterile buffered distilled water. Similarly dilute the test solution, so that it falls in the range of the standard. 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. To the first petridish add 3 standards, 50 l each into 3 separate cups and to the 4th cup add 50 l dilution of the unknown corresponding approx. to 0.01 mg/ml. To the second petridish add remaining 3 standards 50 l each into 3 separate cups and to the 4th cup add 50 l dilution of the unknown corresponding approximately to 0.04 mg/ml. Refrigerate at 4oC for hr and then incubate at 37oC for 24 hours. 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 B12 on X-axis. Calculate the exact conc. of Vit B12 in the unknown from the graph.

Standard inoculum preparation l 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. Interpretation Zone 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. References 1. IP 2007. 2. US Pharmacopeail convention Inc. 2001, The United States Pharmacopeia 25/NF 20-2002.

Calcium Panthothenate Assay


Introduction Serial 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. Requirement Material l Pantothenate assay media Accumix - AM10784 l Pantothenate culture Agar- Accumix- AM10786 l Lactobacillus Plantarum ATCC 8014 l Standard Calcium Pantothenate l Phosphorus pentoxide

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l l

Acetic Acid Sodium Acetate

Equipments o o l Incubator at 37 C + 2 C . l Pipette l Weighing Balance l Spectrophotometer l Autoclave Procedure Inoculum Preparation l 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 30o - 37o but held constant to within + 0.5o l The cell suspension so obtained is the inoculum. *Cultural Medium To 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 ml. cotton/ cap the tubes, sterilize in an autoclave at 121o and cool. **Water containing 0.2 mg of Calcium Pantothenate per 5 ml Dilute 0.08 ml of ***Standard stock solution of Calcium Pantothenate to 100 ml in a clean dried volumetric flask. ***Standard Stock solution of Calcium Pantothenate Dissolve 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 Procedure l 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 ml. 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

l l l

l l l l l l

an autoclave at 121oC and for 5 minutes cool. Add 10 l of inoculum to each tube, except 2 of the 4 tubes containing no Standard preparation to serve as the uninoculated blanks and mix. Incubate the tubes at a temperature between 30oC& 37oC. Held constant to within +0.5o, until following 22 to 24 hours of incubation. Determine the transmittance of the tubes in the following manner. 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. Read the transmittance when a steady state is reached. This steady state is observed a few seconds after agitation, when the galvanometer reading remains constant for 30 seconds or more. Allow approximately the same time interval for the reading on each table. With the transmittance set at 1.00 for the uninoculated blank, read the transmittance of the inoculated blank. With the transmittance set at 1.00 for the inoculated blank, read the transmittance of the remaining tubes. If there is evidence of contamination with a foreign microorganisms, disregard the result of the assay.

Calculation Prepare 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 x for each of three or more dosage levels to obtain x = M1, 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: M = antilog (M1 + 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. Reference 1. USP 27 (111) Design and analysis of Biological assays.

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Enumeration of Coliforms, Faecal Coliforms and E.coli in Water in Sealed Containers and Prepackaged Ice Using the Hydrophobic Grid-membrane Filter (HGMF) Method
Introduction The 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 nonselective 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. Material l HGMF (1600 gird-cell, 0.45 m pore size; available as ISO-GRID Membrane Filters from Oxoid LTD, Nepean, Ont) or equivalent (i.e. Millipore Filters) l Membrane filter forceps l 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 Coliforms l M-Endo Agar LES (AM106921, AM506921) l Violet Red Bile Agar (AM1107, AM5107) l For Faecal Coliforms to E.coli l 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
l

E.aerogenes ATCC 13048

Equipments l 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. Procedure Each sample unit must be analyzed individually. Carry out the test in accordance with the following instructions: 1) Handling of Sample Units l Water in sealed containers-Do not store sample units for more than 25 hrs before analysis. Store under refrigeration (0-50C) conditions. l Prepackaged ice(a) If sample units are prepackaged in leak proof containers, thaw them in the containers under refrigeration (0-50C) 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 under refrigeration (0-50C). DO NOT store thawed sample units for more than 6 hrs before analysis. 2) Perparation for Analysis l 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 Dilutions l 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.

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

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

4) Filtration l Agitate each sample or dilution bottle. l Handle HGMF with sterile forceps. l Following the manufacturers 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 manufacturers instructions for cleaning the filtration apparatus. l Repeat with subsequent dilutions as required. 5) Plating and Incubation l Transfer the HGMF to the surface of a NA plate by rolling it onto the agar to avoid trapping air bubbles. Incubate plates at 350C 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 onto the agar to avoid trapping air bubbles. Incubate plates at 350C for 24+2 hrs. Plates should be placed in an inverted position in stacks of not more than two. 6) Counting and Scoring HGMF l 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 manufacturers 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 gridcells, 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 gridcells (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 gridcells are typical.

(7) Control Cultures Prepare 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 coliforms Confirm 5 typical colonies by inoculating growth from each colony into tubes of LT (one colony per tube) and incubate at 350C for 24 to 48 hrs. Any gas positive LT tubes should be sub cultured to BGBB and incubated at 350C 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 tubes of LT (one colony per table) and incubate for 48 hrs at 350C. Transfer one loopfull of growth from each tube to EC Broth (avoid transferring Pellicle). Incubate the tubes at 450C 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 results l Report average MPNGU as per total count from duplicate plates roundoff to two significant figures (e.g, record 2850 as 2.9 x 103). 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. References 1. 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

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indicator organisms in foods using the automated hydrophobic gridmembrane 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


Introduction This method shall be used for the determination of total aerobic bacteria (Aerobic Colony Count) and coliform bacteria (Coliforms) in ice cream or ice milk. Material l 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). Equipments l Autoclave. l Laminar Air flow. l Incubator. Procedure Each 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 Units l 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 media The following media, to be prepared and sterilized according to the manufacturers instructions, shall be used: l Plate Count (PC) agar. l Lauryl Tryptose (LT) broth. l Brilliant Green Bile Broth 2%. 3) Preparation of Dilutions l 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
l l

arc in approximately 7 sec. 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. 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 ACC The 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. 0 l 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 Coliforms 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 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. 0 l 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.

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

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 ACC Coliforms n 5 5 c 2 1 m 100,000 10 M 1,000,000 1,000

6) Confirmed Test l 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). l Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials. 0 l Incubate the inoculated BGBB Broth tubes at 35 + 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 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. 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 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.

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 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. 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. 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.

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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 (Table A-1)/100

dilution factor of middle set of tubes

TABLE A-1 Most 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* 10;1;0.1 0 001 002 003 004 005 010 011 012 013 014 015 020 021 022 023 024 025 030 031 032 033 034 035 040 041 042 043 044 045 050 051 052 053 054 055 MPN <1.8 1.8 3.6 5.4 7.2 9 1.8 3.6 5.5 7.3 9.1 11 3.7 5.5 7.4 9.2 11 13 5.6 7.4 9.3 11 13 15 7.5 9.4 11 13 15 17 9.4 11 13 15 17 19 Pos* 10;1;0,1 100 101 102 103 104 105 110 111 112 113 114 115 120 121 122 123 124 125 130 131 132 133 134 135 140 141 142 143 144 145 150 151 151 153 154 155 MPN 2 4 6 8 10 12 4 6.1 8.1 10 12 14 6.1 8.2 10 12 15 17 8.3 10 13 15 17 19 11 13 15 17 19 22 13 15 17 19 22 24 Pos* 10;1;0,1 200 201 202 203 204 205 210 211 212 213 214 215 220 221 222 223 224 225 230 231 232 233 234 235 240 241 242 243 244 245 250 251 252 253 254 255 MPN 4.5 6.8 9.1 12 14 16 6.8 9.2 12 14 17 19 9.3 12 14 17 19 22 12 14 17 20 22 25 15 17 20 23 25 28 17 20 17 26 29 32 Pos* 10;1;0,1 300 301 302 303 304 305 310 311 312 313 314 315 320 321 322 323 324 325 330 331 332 333 334 335 340 341 342 343 344 345 350 351 352 353 354 355 MPN 7.8 11 13 16 20 23 11 14 17 20 23 27 14 17 20 24 27 31 17 21 24 28 31 35 21 24 28 32 36 40 25 29 32 37 41 45 Pos* 10;1;0,1 400 401 402 403 404 405 410 411 412 413 414 415 420 421 422 423 424 425 430 431 432 433 434 435 440 441 442 443 444 445 450 451 452 453 454 455 MPN 13 17 21 25 30 36 17 21 26 31 36 42 22 26 32 38 44 50 27 33 39 45 52 59 34 40 47 54 62 69 41 48 56 64 72 81 Pos* 10;1;0,1 500 501 502 503 504 505 510 511 512 513 514 515 520 521 522 523 524 525 530 531 532 533 534 535 540 541 542 543 544 545 550 551 552 553 554 555 MPN 23 31 43 58 76 95 33 46 64 84 110 130 49 70 95 120 150 180 79 110 140 180 210 250 130 170 220 280 350 440 240 350 540 920 1600 >1600

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

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TABLE A-2 Dilutions to be used and calculations of MPN per g or mL of test material Dilutions* 1:10 1:100 Undiluted Amount of original test material (g or mL) 10 1 0.1 0.01 5/5** 5/5 2/5 5/5 5/5 2/5 5/5 5/5 2/5 5/5 5/5 2/5 2/5 2/5 1/5 5/5 2/5 1/5 5/5 5/5 5/5 ** No. of positive tubes/No. of tubes inoculated. 1:1000 0.001 5-5-2 5-5-2 5-2-2 5-2-0 2-2-1 5-2-2 5-5-5 540 540 95 49 12 95 >1600 Combination to be used MPN from Table A-1 Dilution factor on middle dilution 1 10 100 100 10 10 100

a. b. c. d. e. f. g.

2/5 0/5 0/5 1/5*** 5/5 ***

* Dilutions to be used are shaded gray.

8) Interpretation The 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) Limits The maximum count of total aerobic bacteria permitted for each lot is that represented by an Aerobic.

Colony count not exceeding: l 1,00,000 per g in more than two of the five sample units, and l 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. Reference 1. Official Method MFO-2 Health Protection Branch-Ottawa.

Microbiological Examination of Cottage Cheese


Introduction This method shall be used for the determination of coliform bacteria (Coliforms) in cottage cheese in accordance with the Food and Drug Regulations. Material i) Lauryl Tryptose Broth (AM1053, AM5053) ii) Brilliant Green Bile Broth 2% (AM1020, AM5020) iii) 2% Sodium Citrate solution. Equipments l Laminar Air Flow l Autoclave l Incubator Procedure Each sample unit shall be analyzed individually. The test shall be carried out in accordance with the following instructions: 1) Collection of Samples l 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. 0 l Keep sample units refrigerated (0-5 C) during transport.

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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 Media The following media, prepared and sterilized according to the manufacturers instructions, shall be used: l Lauryl Tryptose (LT) Broth l Brilliant Green Bile Broth 2% 4) Preparation of Dilutions 0 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 Coliforms I) 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 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 l Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials. 0 0 l 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.

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 Test l 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). l Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials. 0 0 l 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 gaspositive 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. 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 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.

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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 ).

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 l

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 ACC Coliforms n 5 5 c 2 1 m 100,000 10 M 1,000,000 1,000

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. 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. 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 (Table A-1)/100 x dilution factor of middle set of tubes

TABLE A-1 Most 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* 10;1;0.1 0 001 002 003 004 005 010 011 012 013 014 015 020 021 022 MPN <1.8 1.8 3.6 5.4 7.2 9 1.8 3.6 5.5 7.3 9.1 11 3.7 5.5 7.4 Pos* 10;1;0,1 100 101 102 103 104 105 110 111 112 113 114 115 120 121 122 MPN 2 4 6 8 10 12 4 6.1 8.1 10 12 14 6.1 8.2 10 Pos* 10;1;0,1 200 201 202 203 204 205 210 211 212 213 214 215 220 221 222 MPN 4.5 6.8 9.1 12 14 16 6.8 9.2 12 14 17 19 9.3 12 14 Pos* 10;1;0,1 300 301 302 303 304 305 310 311 312 313 314 315 320 321 322 MPN 7.8 11 13 16 20 23 11 14 17 20 23 27 14 17 20 Pos* 10;1;0,1 400 401 402 403 404 405 410 411 412 413 414 415 420 421 422 MPN 13 17 21 25 30 36 17 21 26 31 36 42 22 26 32 Pos* 10;1;0,1 500 501 502 503 504 505 510 511 512 513 514 515 520 521 522 MPN 23 31 43 58 76 95 33 46 64 84 110 130 49 70 95

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Pos* 10;1;0.1 023 024 025 030 031 032 033 034 035 040 041 042 043 044 045 050 051 052 053 054 055

MPN 9.2 11 13 5.6 7.4 9.3 11 13 15 7.5 9.4 11 13 15 17 9.4 11 13 15 17 19

Pos* 10;1;0,1 123 124 125 130 131 132 133 134 135 140 141 142 143 144 145 150 151 151 153 154 155

MPN 12 15 17 8.3 10 13 15 17 19 11 13 15 17 19 22 13 15 17 19 22 24

Pos* 10;1;0,1 223 224 225 230 231 232 233 234 235 240 241 242 243 244 245 250 251 252 253 254 255

MPN 17 19 22 12 14 17 20 22 25 15 17 20 23 25 28 17 20 17 26 29 32

Pos* 10;1;0,1 323 324 325 330 331 332 333 334 335 340 341 342 343 344 345 350 351 352 353 354 355

MPN 24 27 31 17 21 24 28 31 35 21 24 28 32 36 40 25 29 32 37 41 45

Pos* 10;1;0,1 423 424 425 430 431 432 433 434 435 440 441 442 443 444 445 450 451 452 453 454 455

MPN 38 44 50 27 33 39 45 52 59 34 40 47 54 62 69 41 48 56 64 72 81

Pos* 10;1;0,1 523 524 525 530 531 532 533 534 535 540 541 542 543 544 545 550 551 552 553 554 555

MPN 120 150 180 79 110 140 180 210 250 130 170 220 280 350 440 240 350 540 920 1600 >1600

* Number of positive tubes with each of 3 volumes used. TABLE A-2 Dilutions to be used and calculations of MPN per g or mL of test material Dilutions* 1:10 1:100 Undiluted Amount of original test material (g or mL) 10 1 0.1 0.01 5/5** 5/5 2/5 5/5 5/5 2/5 5/5 5/5 2/5 5/5 5/5 2/5 2/5 2/5 1/5 5/5 2/5 1/5 5/5 5/5 5/5 ** No. of positive tubes/No. of tubes inoculated. in determining whether the tested lot of the product complies with the Food and Drugs Regulations. 1:1000 0.001 5-5-2 5-5-2 5-2-2 5-2-0 2-2-1 5-2-2 5-5-5 540 540 95 49 12 95 >1600 Combination to be used MPN from Table A-1 Dilution factor on middle dilution 1 10 100 100 10 10 100

a. b. c. d. e. f. g.

2/5 0/5 0/5 1/5*** 5/5

* Dilutions to be used are shaded gray.

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

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

10 per g in more than one of the five sample units, and 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. Determination Coliforms n 5 c 1 m 10 M 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 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. References 1. Official Method MFO-4 Health Protection Branch - Ottawa.

Microbiological Examination of Egg Products and of Liquid Eggs


Introduction This 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. Material l 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 Christensens (AM1105, AM5105) l Brilliant Green Agar modified (AM1018, AM5018) l Salmonella Typhimurium ATCC 14028 l Salmonella Identification Kit (20797001) Equipment l Laminar Air Flow. l Autoclave. l Incubator. Procedure 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 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 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). Employ aseptic techniques in collecting the sample units when sampling from bulk. Place each collected sample unit into a separate sterile container. Keep unfrozen sample units refrigerated (0-50C), and frozen sample units frozen during transport.

2) Handling of Sample Units 0 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 Media The following media, which are to be prepared and sterilized according to the manufacturers 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 Christensens (10) Nutrient Agar (NA) 4) Non-Selective Enrichment (Pre-enrichment) l Thaw frozen sample units. Do not allow the temperature of any sample

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l l l

unit to exceed 450C. 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. Suspend the individual analytical units or the composite unit(s) in nine times their weight of NB in pre-warmed, sterile blender jar. Blend the mixture at low speed for two min. 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. 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. Incubate the inoculated pre-enrichment Broth(s) and the controls at 350C + 0.50C for 18-24 hrs. In no circumstances shall the incubation be prolonged for more than 24 hrs.

rather than black colonies. 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.

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. 0 0 l Incubate the SC and TBG broths for 24 + 2 hrs at 35 C + 0.5 C and at 0 0 43 C + 0.5 , respectively. 6) Selective Plating l 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 other than S.typhi unless it is refrigerated at 40C for at least 24 hrs before streaking. The possibility of an inhibitory effect of this medium should be taken into consideration. 0 0 l 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
32

7) Biochemical Screening l Using an inoculating needle, pick colonies indicative of Salmonella from the MA plates and inoculate the biochemical media listed in table 1. Incubate these media at 350C + 0.50C 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 identification but store the cultures at 2-80C if the serological identification is not carried within 12 hrs after the incubation of the TSI or LI Agar cultures. 0 0 l 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 identification but store the cultures at 2-80C if the serological identification is not carried out within 12 hrs after the incubation of the NA slant cultures. 8) Serological Testing I) 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. T1, T2, T3, etc. 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.

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l l

Cultures usually agglutinate within1min. False-positive reactions may occur, these can be resolves by further testing with somatic grouping and with flagellar antisera. 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 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 Table 1 Minimal Biochemical Screening Media Medium Triple Sugar Iron (TSI) Agar Reaction Lactose and/or Sucrose utilization Dextrose utilization

l l l l

place one drop onto each of the test suspension areas and onto the positive control area. 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. Hold slides against dark background and observe for agglutination. Cultures usually agglutinate within 1 min. If the culture-saline-serum mixture has not agglutinated, repeat procedure with another group antiserum. If the serological test is positive, the culture shall be sent to a Salmonella typing center for serotyping. The tests are invalidated if the negative control shows agglutination (autoagglutination). 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 / Limits The 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).

Observations Positive reaction: Yellow slant Negative reaction: Colour becomes more intensely red. Positive reaction: yellow butt with or without gas formation Negative reaction: Colour of butt remains unchanged Positive reaction:Blackening of butt often extending into the slant Negative reaction: No Blackening Positive reaction: Formation of gas pockets in the medium Negative reaction: No gas pockets in the medium Same as in TSI Agar Positive reaction: Butt turns purple Negative reaction: Yellow butt if dextrose is utilized Positive reaction: Wine coloured slant Negative reaction: No wine coloured slant

Reaction shown by majority of Salmonella Negative (some strains may show a positive reaction) Positive Positive Slow H2S producers may be encountered. If lactose positive Salmonella are present, the H2S reaction may be inhibited on TSI Agar Positive Positive Positive Negative

H2S production

Gas Formation Lysine Iron (LI) Agar H2S production Lysine decarboxylase Lysine desaminase

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Medium Urea Agar Base Christensens *

Reaction Production of Urease

Observations Positive reaction: Slant pinkish red Negative reaction: Colour of slant unchanged

Reaction shown by majority of Salmonella Negative

* although Lysine deaminase is used to distinguish Proteus from Salmonella, the urease test is a more reliable indicator for Proteus spp. References 1. Official Method MFO-6 Health Protection Branch Ottawa.

Microbiological Examination of Milk


Introduction This 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. Material l Plate Count Agar. (AM1081, AM5081) l Peptone water 0.1% (AM1079, AM5079) Equipment l Laminar Air Flow Unit l Autoclave l Incubator l Colony Counter Procedure Each sample unit shall be analyzed individually. The tests shall be carried out on the sample in accordance with the following instructions. 1) Collection of Samples l 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). l Employ aseptic techniques in collecting the sample units when sampling from bulk. Place each collected sample unit into a separate sterile container. 0 l Keep sample units refrigerated (0-5 C ) during transport. 2) Handling of Sampling 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 at the laboratory. 3) Preparation of media The following medium, to be prepared and sterilized according to the manufacturers instructions, shall be used: l Plate Count (PC) Agar. 4) Preparation of Dilutions 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 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 l l

Mix the 1:10 dilution by shaking the dilution bottle 25 times in a 30 cm arc in approximately 7 sec. 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. 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 ACC The medium used is PC Agar prepared for making pour plates.

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Analysis 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. 0 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. 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 Results l 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 / Limits The 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.

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. 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. Determination ACC 1. Flavoured Milk 2. Milk for manufacture n 5 5 c 2 0 m 50,000 2,000,000 M 1,000,000 2,000,000

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 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. References 1. Official Method MFO-7 Health Protection Branch - Ottawa.

Microbiological Examination of Mineral Water


Introduction This method shall be used for the determination of coliform bacteria (Coliforms) in mineral water in accordance with the Food and Drug Regulations. Material l Lauryl Tryptose Broth (AM1053, AM5053). l Brilliant Green Bile Broth 2% (AM1020, AM5020). Equipment l Laminar Air Flow Unit l Incubator l Autoclave Procedure Each of the 10 sample units shall be analyzed individually.

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The tests 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 consist of at least 100 ml. l Collect original unopened containers wherever possible. 0 l 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 Units l Do not store sample units for more than 24 hrs before analysis. 3) Preparation of Media The following media, to be prepared and sterilized according to the manufacturers instructions, shall be used. l Lauryl Tryptose Broth . l Brilliant Green Bile Broth 2% . 4) Preparation of Dilutions l The undiluted sample units only are required. 5) 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, 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. l Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials. 0 0 l 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.

6) Confirmed Test l 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). l Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials. 0 0 l 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: Determination Coliforms n 10 c 1 m 0 M 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 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.

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8) Calculation of most probable numbers (MPN) 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. 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 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

per g (ml), the MPN value is 330 this becomes a Health 1 concern if the product is distributed. 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). 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 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, 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 (Table A-1)/100 x dilution factor of middle set of tubes

TABLE A-1 Most 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* 10;1;0.1 000 001 002 003 004 005 010 011 012 013 014 015 MPN <1.8 1.8 3.6 5.4 7.2 9 1.8 3.6 5.5 7.3 9.1 11 Pos* 10;1;0,1 100 101 102 103 104 105 110 111 112 113 114 115 MPN 2 4 6 8 10 12 4 6.1 8.1 10 12 14 Pos* 10;1;0,1 200 201 202 203 204 205 210 211 212 213 214 215 MPN 4.5 6.8 9.1 12 14 16 6.8 9.2 12 14 17 19 Pos* 10;1;0,1 300 301 302 303 304 305 310 311 312 313 314 315 MPN 7.8 11 13 16 20 23 11 14 17 20 23 27 Pos* 10;1;0,1 400 401 402 403 404 405 410 411 412 413 414 415 MPN 13 17 21 25 30 36 17 21 26 31 36 42 Pos* 10;1;0,1 500 501 502 503 504 505 510 511 512 513 514 515 MPN 23 31 43 58 76 95 33 46 64 84 110 130

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Pos* 10;1;0.1 020 021 022 023 024 025 030 031 032 033 034 035 040 041 042 043 044 045 050 051 052 053 054 055

MPN 3.7 5.5 7.4 9.2 11 13 5.6 7.4 9.3 11 13 15 7.5 9.4 11 13 15 17 9.4 11 13 15 17 19

Pos* 10;1;0,1 120 121 122 123 124 125 130 131 132 133 134 135 140 141 142 143 144 145 150 151 151 153 154 155

MPN 6.1 8.2 10 12 15 17 8.3 10 13 15 17 19 11 13 15 17 19 22 13 15 17 19 22 24

Pos* 10;1;0,1 220 221 222 223 224 225 230 231 232 233 234 235 240 241 242 243 244 245 250 251 252 253 254 255

MPN 9.3 12 14 17 19 22 12 14 17 20 22 25 15 17 20 23 25 28 17 20 17 26 29 32

Pos* 10;1;0,1 320 321 322 323 324 325 330 331 332 333 334 335 340 341 342 343 344 345 350 351 352 353 354 355

MPN 14 17 20 24 27 31 17 21 24 28 31 35 21 24 28 32 36 40 25 29 32 37 41 45

Pos* 10;1;0,1 420 421 422 423 424 425 430 431 432 433 434 435 440 441 442 443 444 445 450 451 452 453 454 455

MPN 22 26 32 38 44 50 27 33 39 45 52 59 34 40 47 54 62 69 41 48 56 64 72 81

Pos* 10;1;0,1 520 521 522 523 524 525 530 531 532 533 534 535 540 541 542 543 544 545 550 551 552 553 554 555

MPN 49 70 95 120 150 180 79 110 140 180 210 250 130 170 220 280 350 440 240 350 540 920 1600 >1600

* Number of positive tubes with each of 3 volumes used. TABLE A-2 Dilutions to be used and calculations of MPN per g or mL of test material Undiluted 10 5/5** Dilutions* 1:10 1:100 Amount of original test material (g or mL) 1 0.1 0.01 5/5 2/5 5/5 5/5 2/5 5/5 5/5 2/5 5/5 5/5 2/5 2/5 2/5 1/5 5/5 2/5 1/5 5/5 5/5 5/5 ** No. of positive tubes/No. of tubes inoculated. 1:1000 0.001 5-5-2 5-5-2 5-2-2 5-2-0 2-2-1 5-2-2 5-5-5 540 540 95 49 12 95 >1600 Combination to be used MPN from Table A-1 Dilution factor on middle dilution 10 100 100 10 10 100 MPN per mL or g 5.4 54 95 49 1.2 9.5 >1600

a. b. c. d. e. f. g.

2/5 0/5 0/5 1/5*** 5/5

* Dilutions to be used are shaded gray.

Reference: 1. Official Method MFO-09 Health Protection Branch - Ottawa.

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Microbiological Examination of Milk Powder


Introduction This method shall be used for the determination of the bacteria of the genus Salmonella in milk powder, with the Food and Drug Regulations. Material l 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) Equipment l Laminar Air Flow unit. l Autoclave. l Incubator. Procedure The 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 Samples l 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 units l Analyze the sample units as soon as possible after they have been received in the laboratory. 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 35oC + 0.5o 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 o 43 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 wellisolated colonies. The broths may also be streaked onto a third

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l l

commercially available plating medium. It has been observed that BS agar is inhibitory for Salmonella serotypes other than S.typhi unless it is refrigerated at 4oC for at least 24 hrs before streaking. The possibility of an inhibitory effect of this medium should be taken into consideration. Incubate the plates at 35oC+ 0.5oC for 24 + 2 hrs. It may be necessary to incubate the BS agar plates for 48 + 2 hrs. 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. 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.

l l

Incubate the inoculated NA slants at 35oC + 0.5oC for 24 + 2 hrs. Use NA slant cultures less than 48 hr old for the serological identification but store the cultures at 2-8oC 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 T1, T2, T3, etc. (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 T1, T2, T3, etc.

7) Purification l Streak suspect colonies onto MA plates for purification. o o l 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, lactosepositive biotypes are known to occur and may appear pink. 8) Biochemical Screening l Using an inoculating needle, pick colonies indicative of Salmonella from the MA plates and inoculate the biochemical media listed in Table 1. Incubate these media at 35oC + 0.5oC 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 identification but store the cultures at 2-8oC 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.
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(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 Table 1 Minimal Biochemical Screening Media Medium Triple Sugar Iron (TSI) Agar Reaction Lactose and/or Sucrose utilization Dextrose utilization

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). 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).

Observations Positive reaction: Yellow slant Negative reaction: Colour becomes more intensely red. Positive reaction: yellow butt with or without gas formation Negative reaction: Colour of butt remains unchanged Positive reaction:Blackening of butt often extending into the slant Negative reaction: No Blackening Positive reaction: Formation of gas pockets in the medium Negative reaction: No gas pockets in the medium Same as in TSI Agar Positive reaction: Butt turns purple Negative reaction: Yellow butt if dextrose is utilized Positive reaction: Wine coloured slant Negative reaction: No wine coloured slant Positive reaction: Slant pinkish red Negative reaction: Colour of slant unchanged

Reaction shown by majority of Salmonella Negative (some strains may show a positive reaction) Positive Positive Slow H2S producers may be encountered. If lactose positive Salmonella are present, the H2S reaction may be inhibited on TSI Agar Positive Positive Positive Negative Negative

H2S production

Gas Formation Lysine Iron (LI) Agar H2S production Lysine decarboxylase Lysine desaminase Christensens Urea Agar * Production of Urease

* although Lysine deaminase is used to distinguish Proteus from Salmonella, the urease test is a more reliable indicator for Proteus spp. References 1. Official Method MFO-12 Health Protection Branch Ottawa.

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Microbiological Examination of Frog Legs


Introduction This method shall be used for the determination of the bacteria of the genus in froglegs in accordance the Food and Drug Regulations. Material l 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 culture l NaOH or HCL Equipment l Laminar Air Flow Unit. l Incubator. l Autoclave. Procedure The 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 Samples l 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 Units l 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 Media The 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) (Pre-enrichment) l Thaw frozen sample units. Do not allow the temperature of any sample unit to exceed 45oC. 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 35o 0.5oC for 18-24 hrs. In no circumstances shall the incubation be prolonged for more than 24 hrs. 5) Selective Enrichment 1) Transfer 1 ml of the incubated pre-enrichment broth into each of 9 ml of SC and TBG broths using a sterile pipette. 2) Incubate the SC broth and TBG broth for 24 2 hrs at 35oC 0.5oC and at 43oC 0.5oC, 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 wellisolated 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

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l l

is refrigerated at 4oC for at least 24 hrs before streaking. The possibility of an inhibitory effect of this medium should be taken into consideration. Incubate the plates at 35oC 0.5oC for 24 2 hrs. It may be necessary to incubate the BS agar plates for 48 2 hrs. 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. 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.

carried out within 12 hr after the incubation of the NA slant cultures. 9) Serological Identification 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 T1, T2, T3, etc. (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 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 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 T1, T2, T3, etc. (b) Use a positive control culture for each individual group tested, as in d.

7) Purification l Streak suspect colonies onto MA plates for purification. o o l 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. 8) Biochemical Screening l Using an inoculating needle, pick colonies indicative of Salmonella from the MA plates and inoculate the biochemical media listed in Table 1. Incubate these media at 35oC 0.5oC 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. l Use TSI or LI agar cultures less than 72 hrs old for the serological identification but store the cultures at 2-8oC 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. o o l 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 but store the cultures at 2-8oC if the serological identification is not

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(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 Table II Minimal Biochemical Screening Media Medium Triple Sugar Iron (TSI) Agar Reaction Lactose and/or Sucrose utilization Dextrose utilization

verification. 10) Interpretation The 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 Media Table I - Lot Structure 1. 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

Observations Positive reaction: Yellow slant Negative reaction: Colour becomes more intensely red. Positive reaction: yellow butt with or without gas formation Negative reaction: Colour of butt remains unchanged Positive reaction:Blackening of butt often extending into the slant Negative reaction: No Blackening Positive reaction: Formation of gas pockets in the medium Negative reaction: No gas pockets in the medium Same as in TSI Agar Positive reaction: Butt turns purple Negative reaction: Yellow butt if dextrose is utilized Positive reaction: Wine coloured slant Negative reaction: No wine coloured slant

Reaction shown by majority of Salmonella Negative (some strains may show a positive reaction) Positive Positive Slow H2S producers may be encountered. If lactose positive Salmonella are present, the H2S reaction may be inhibited on TSI Agar Positive Positive Positive Negative

H2S production

Gas Formation Lysine Iron (LI) Agar H2S production Lysine decarboxylase Lysine desaminase

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Medium Christensens Urea (CU) agar *

Reaction Production of Urease

Observations Positive reaction: Slant pinkish red Negative reaction: Colour of slant unchanged

Reaction shown by majority of Salmonella Negative

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

Microbiological Examination of Cocoa and Chocolate


Introduction This 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. Material l 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 culture l Milk l NaOH or HCL Equipment l Laminar Air Flow. l Autoclave. l Incubator. Procedure The 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 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). Employ aseptic techniques in collecting the sample units when sampling from bulk. Place each collected sample unit into a separate sterile container.

2) Handling of Sample Units l Analyze the sample units as soon as possible after they have been received at the laboratory. 3) Preparation of media The 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 at 121oC and cool to 45oC. 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.

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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. 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. Incubate the inoculated pre-enrichment broth(s) and the controls at 35oC + 0.5oC for 18-24 hrs. In no circumstances shall the incubation be prolonged for more than 24 hrs.

Observe the MA plates after the incubation period. Typical Salmonella colonies are lactose-negative and appear colourless. However, lactosepositive biotypes are known to occur and may appear pink.

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 broth and TBG broth for 24 + 2 hrs at 35 C+ 0.5 C and o o at 43 C + 0.5 C, 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 commercially available plating medium. l It has been observed that BS agar inhibits Salmonella serotypes other than S. typhi unless it is refrigerated at 4oC for at least 24 hr before streaking. The possibility of an inhibitory effect of this medium should be taken into consideration. o o l 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 o l Incubate the plates at 35 C + 0.5 C for 24 + 2 hrs.

8) Biochemical Screening l Using an inoculating needle, pick colonies indicative of Salmonella from the MA plates and inoculate the biochemical media listed in Table 1. Incubate these media at 35oC + 0.5oC 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. l Use TSI or LI agar cultures less than 72 hrs old for the serological identification but store the cultures at 2-8oC 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. o o l 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 identification but store the cultures at 2-8oC 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 T1, T2, T3etc. 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

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areas and onto the positive control area. 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). f. 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 T1, T2, T3, etc. 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 Table I Minimal Biochemical Screening Media Medium Triple Sugar Iron (TSI) Agar Reaction Lactose and/or Sucrose utilization Dextrose utilization Observations

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). 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).

Reaction shown by majority of Salmonella Negative (some strains may show a positive reaction) Positive Positive Slow H2S producers may be encountered. If lactose positive Salmonella are present, the H2S reaction may be inhibited on TSI Agar Positive

Positive reaction: Yellow slant Negative reaction: Colour becomes more intensely red. Positive reaction: yellow butt with or without gas formation Negative reaction: Colour of butt remains unchanged Positive reaction:Blackening of butt often extending into the slant Negative reaction: No Blackening Positive reaction: Formation of gas pockets in the medium Negative reaction: No gas pockets in the medium

H2S production

Gas Formation

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Medium Lysine Iron (LI) Agar

Reaction H2S production Lysine decarboxylase Lysine desaminase

Observations Same as in TSI Agar Positive reaction: Butt turns purple Negative reaction: Yellow butt if dextrose is utilized Positive reaction: Wine coloured slant Negative reaction: No wine coloured slant Positive reaction: Slant pinkish red Negative reaction: Colour of slant unchanged

Reaction shown by majority of Salmonella Positive Positive Negative Negative

Christensens Urea (CU) agar *

Production of Urease

* although Lysine deaminase is used to distinguish Proteus from Salmonella, the urease test is a more reliable indicator for Proteus spp. Reference 1. 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. Material a. Compound microscope, either binocular or monocular equipped with l 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 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. Dissecting needle. U.S. standard sieve no. 2 (for canned tomatoes). Wide mouth bottles with screw caps or other suitable containers (for canned tomatoes, puree, pulp, paste and catsup). Spoon or other suitable utensil (for puree, pulp, paste and catsup). Refractometer (for puree, pulp and paste). Coarse filter paper or celluwipe (for puree, pulp and paste). Glass Slide.

g. h. i. j. k. l. m.

Procedure The 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) Apparatus I) Compound microscope, either binocular or monocular equipped with: l mechanical stage.

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l l l l l l

condenser with iris diaphragm. source of illumination. two objectives - a 10 x (16 mm) for counting and a 20 x (8mm) for confirmation. 8 x - 12.5 x oculars. 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). The ocular must be equipped with a micrometer disk cross-ruled in sixths of ocular diaphragm opening (Preparation of Microscope, section 3.2.2).

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 Units l 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.

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 dropin 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

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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 1.3448 -1.3454 at 20oC or 1.3442 -1.3448 at 25oC. (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.

l l

Clean spatula and dissecting needle, rinse in distilled water, flame and cool. 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.

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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. 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 unit Number 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

Table I - DILUTION OF PUREE (PULP) FOR MOULD COUNT AT 200C (2) Actual Refr. Index 1.3462 1.3478 1.3494 1.3511 1.3527 1.3544 1.3560 1.3577 1.3593 1.3610 Dilution Factor 1.145 1.292 1.440 1.585 1.730 1.876 2.024 2.171 2.322 2.474 Amt. of Water to be Added to 100 ml of Sample Unit 14.5 29.2 44.0 58.5 73.0 87.6 102.4 117.2 132.2 147.4 Total Volume of Diluted Sample Unit 114.5 129.2 144.0 158.5 173.0 187.6 202.4 217.2 232.2 247.4

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7) Interpretation 1) 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

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.

DIAGRAMS DIAGRAM I MICROMETER DISK

B C

A: B: C: D:

Length of grid that coincides with 1.382 mm on the microscope stage Proper area of field of view Area of micrometer disk not visible through microscope Diameter equal to 1.382 mm and cross ruled in sixths

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HOWARD MOULD COUNTING CHAMBER DIAGRAM 11a

A B C

A: B: C: D: E:

Calibration circle, 1.382 mm diameter Area of liquid for mould count Cover glass Cover glass Two engraved parallel lines spaced 1.382 mm apart

F: Rectangle, 15 X 20 mm G: Moat [1.382/2]2 X 3.1416 = 1.5 sq. mm., area of microscopic field 1.5 X 0.1 = 0.15 cu. mm., volume of material in microscopic field

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DIAGRAM III MOULD FILAMENTS

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.

DIAGRAM IV EXAMPLES OF FIELDS WITH MOULD FILAMENTS

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

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. Reference 1. Official Method MFO - 5 Health Protection Branch - Ottawa.

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Microbiological Examination of Cheese


Introduction This 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. Material l 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. Simmonss 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 Solution l Cheese l 0.1 % Peptone Water (AM1079/ AM5079) l E.coli ATCC 8739 l E.coli identification Kit (20796001) l Kovacs Indole Reagent(20700040) l Methyl Red Solution (20710040) l VP Reagent having 40% NaOH & 0.3% creatine (20680020) Equipment l Laminar Air Flow Unit. l Autoclave. l Incubator. l Water Bath. Procedure Each sample unit shall be analyzed individually. The test shall be carried out in accordance with the following instructions 1) Collection of Samples l 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
l l

bulk cheese. Place each collected sample unit into a separate sterile container. Keep sample units refrigerated (0-5 oC) 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 Dilutions o l Temper sterile aqueous 2% sodium citrate to 40-45 C, and prewarm o sterile 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.

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5) Determination of E. coli I) 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 and produce gas at 45 oC 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. f. Incubate the inoculated LT broth tubes at 35 oC 0. 5 oC 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. e. Incubate the inoculated EC broth tubes in a water bath at 45 oC 0.20C 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.

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. c . Incubate the plates at 35 oC 0.5 oC 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. e. Incubate the NA plates at 35 oC 0.5 oC for 24 2 hrs, and from each of them, pick an isolated colony and streak onto a separate NA slant. f. Incubate the slants at 35 oC 0.5 oC 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 e., above, incubate at 35 oC 0.5 oC for p>24 2 hrs, then inoculate the IMViC media from the freshly incubated slants. i. Incubate the inoculated EC broth and IMViC media at 35 oC 0.5 o C 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.

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l.

m.

n.

o.

p.

q.

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. Indole (I) (i) Transfer inoculum from each isolate to be tested (see step g., above) into a separate tube of Tryptone broth. (ii) Incubate the inoculated tubes at 35 oC 0.5 oC 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. 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. (ii) Incubate the inoculated tubes at 35 oC 0.5 oC 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 additional 48 2 hrs at 35 oC 0.5 oC. 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. 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. (ii) Incubate the inoculated slants at 35 oC 0.5 oC for 48 2 hrs and observe for growth. Visible growth is usually accompanied by a change of colour from green to deep blue. The characteristic IMViC reaction pattern for E. coli is as follows: Indole (+ or -) Methyl red (+) Voges-Proskauer (-) Citrate (-) 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)

r.

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. 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 broth tubes, which were incubated at 35 oC (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. aureus I) 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. h. Invert the plates and incubate at 35 oC 0.5 oC 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
57

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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. d. Incubate at 35 oC 0.5 oC for 24 2 hrs.
58

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. g. Incubate at 35 oC 0.5 oCfor 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 35oC 0.5 oC 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. j. Incubate the tubes at 35 oC 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 h. Incubate the TDA slides or plates in a moist chamber at 35 oC 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

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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. b . Overlay with sterile paraffin oil or Vaspar and incubate at 35 oC 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 sensitivity a. The reagents used are: (i) Phosphate Saline Buffer (0.02 M) pH 7.3-7.4. (ii) Lysostaphin solution containing 50 g 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 g lysostaphin per mL of cell suspension. e. Incubate both tubes at 37 oC 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 count confirmed as S. aureus type 1 colonies/g ---------------------------No. of type 1 colonies tested Number of = Plus S. 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: Determination n 1. Cheese made from Pasteurized Milk E. coli 5 S. aureus 5 2. Cheese made from Unpasteurized Milk E. coli 5 S. aureus 5 c 2 2 2 2 m 100 100 500 1,000 M 2,000 10,000 2,000 10,000

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

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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

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-1 Most 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 Pos* 10;1;0.1 000 001 002 003 004 005 010 011 012 013 MPN <1.8 1.8 3.6 5.4 7.2 9 1.8 3.6 5.5 7.3 Pos* 10;1;0,1 100 101 102 103 104 105 110 111 112 113 MPN 2 4 6 8 10 12 4 6.1 8.1 10 Pos* 10;1;0,1 200 201 202 203 204 205 210 211 212 213 MPN 4.5 6.8 9.1 12 14 16 6.8 9.2 12 14 Pos* 10;1;0,1 300 301 302 303 304 305 310 311 312 313 MPN 7.8 11 13 16 20 23 11 14 17 20 Pos* 10;1;0,1 400 401 402 403 404 405 410 411 412 413 MPN 13 17 21 25 30 36 17 21 26 31 Pos* 10;1;0,1 500 501 502 503 504 505 510 511 512 513 MPN 23 31 43 58 76 95 33 46 64 84

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Pos* MPN Pos* MPN 10;1;0.1 10;1;0,1 014 9.1 114 12 015 11 115 14 020 3.7 120 6.1 021 5.5 121 8.2 022 7.4 122 10 023 9.2 123 12 024 11 124 15 025 13 125 17 030 5.6 130 8.3 031 7.4 131 10 032 9.3 132 13 033 11 133 15 034 13 134 17 035 15 135 19 040 7.5 140 11 041 9.4 141 13 042 11 142 15 043 13 143 17 044 15 144 19 045 17 145 22 050 9.4 150 13 051 11 151 15 052 13 151 17 053 15 153 19 054 17 154 22 055 19 155 24 * Number of positive tubes with each of 3 volumes used.

Pos* 10;1;0,1 214 215 220 221 222 223 224 225 230 231 232 233 234 235 240 241 242 243 244 245 250 251 252 253 254 255

MPN 17 19 9.3 12 14 17 19 22 12 14 17 20 22 25 15 17 20 23 25 28 17 20 17 26 29 32

Pos* 10;1;0,1 314 315 320 321 322 323 324 325 330 331 332 333 334 335 340 341 342 343 344 345 350 351 352 353 354 355

MPN 23 27 14 17 20 24 27 31 17 21 24 28 31 35 21 24 28 32 36 40 25 29 32 37 41 45

Pos* 10;1;0,1 414 415 420 421 422 423 424 425 430 431 432 433 434 435 440 441 442 443 444 445 450 451 452 453 454 455

MPN 36 42 22 26 32 38 44 50 27 33 39 45 52 59 34 40 47 54 62 69 41 48 56 64 72 81

Pos* 10;1;0,1 514 515 520 521 522 523 524 525 530 531 532 533 534 535 540 541 542 543 544 545 550 551 552 553 554 555

MPN 110 130 49 70 95 120 150 180 79 110 140 180 210 250 130 170 220 280 350 440 240 350 540 920 1600 >1600

TABLE A-2 Dilutions to be used and calculations of MPN per g or mL of test material Undiluted 10 5/5** Dilutions* 1:10 1:100 Amount of original test material (g or mL) 1 0.1 0.01 5/5 2/5 5/5 5/5 2/5 5/5 5/5 2/5 5/5 5/5 2/5 2/5 2/5 1/5 5/5 2/5 1/5 5/5 5/5 5/5 ** No. of positive tubes/No. of tubes inoculated. 1:1000 0.001 5-5-2 5-5-2 5-2-2 5-2-0 2-2-1 5-2-2 5-5-5 540 540 95 49 12 95 >1600 Combination to be used MPN from Table A-1 Dilution factor on middle dilution 1 10 100 100 10 10 100 MPN per mL or g 5.4 54 95 49 1.2 9.5 >1600

a. b. c. d. e. f. g.

2/5 0/5 0/5 1/5*** 5/5

* Dilutions to be used are shaded gray.

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Microbial Examination of Water in Sealed Containers (Excluding Mineral and Spring Water) and of Prepackaged Ice
Introduction This 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. Material l 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) Equipment l Laminar Air Flow Unit l Incubator l Autoclave Procedure Five 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 Samples l 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 refrigeration (< 0.5 oC) 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 Units a) 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 them in the containers under refrigeration (0-5 oC) 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 oC). 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. Analysis l 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. o l 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

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l l l

2 hrs. Avoid crowding or excessive stacking of plates in order to permit rapid equilibration of plates with incubator temperature. Count colonies promptly after the incubation period. 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.

l l

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

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

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c. The tolerances are summarized in the following table: Determination n 1 For water in sealed containers Aerobic Colony Count 5 Coliforms 10 2 For prepackaged ice Coliforms 10 c 2 1 1 m 100 0 0 M 10,000 10 10

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. 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 (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. Refer to Table A-1 and look up the value which corresponds to the number of positive tubes obtained. MPN/100 ml (g) = No. of Microorganisms (Table A-1) x dilution factor of middle set of tubes.

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. 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 are positive

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TABLE A-1 Most 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* 10;1;0.1 000 001 002 003 004 005 010 011 012 013 014 015 020 021 022 023 024 025 030 031 032 033 034 035 040 041 042 043 044 045 050 051 052 053 054 055 MPN <1.8 1.8 3.6 5.4 7.2 9 1.8 3.6 5.5 7.3 9.1 11 3.7 5.5 7.4 9.2 11 13 5.6 7.4 9.3 11 13 15 7.5 9.4 11 13 15 17 9.4 11 13 15 17 19 Pos* 10;1;0,1 100 101 102 103 104 105 110 111 112 113 114 115 120 121 122 123 124 125 130 131 132 133 134 135 140 141 142 143 144 145 150 151 151 153 154 155 MPN 2 4 6 8 10 12 4 6.1 8.1 10 12 14 6.1 8.2 10 12 15 17 8.3 10 13 15 17 19 11 13 15 17 19 22 13 15 17 19 22 24 Pos* 10;1;0,1 200 201 202 203 204 205 210 211 212 213 214 215 220 221 222 223 224 225 230 231 232 233 234 235 240 241 242 243 244 245 250 251 252 253 254 255 MPN 4.5 6.8 9.1 12 14 16 6.8 9.2 12 14 17 19 9.3 12 14 17 19 22 12 14 17 20 22 25 15 17 20 23 25 28 17 20 17 26 29 32 Pos* 10;1;0,1 300 301 302 303 304 305 310 311 312 313 314 315 320 321 322 323 324 325 330 331 332 333 334 335 340 341 342 343 344 345 350 351 352 353 354 355 MPN 7.8 11 13 16 20 23 11 14 17 20 23 27 14 17 20 24 27 31 17 21 24 28 31 35 21 24 28 32 36 40 25 29 32 37 41 45 Pos* 10;1;0,1 400 401 402 403 404 405 410 411 412 413 414 415 420 421 422 423 424 425 430 431 432 433 434 435 440 441 442 443 444 445 450 451 452 453 454 455 MPN 13 17 21 25 30 36 17 21 26 31 36 42 22 26 32 38 44 50 27 33 39 45 52 59 34 40 47 54 62 69 41 48 56 64 72 81 Pos* 10;1;0,1 500 501 502 503 504 505 510 511 512 513 514 515 520 521 522 523 524 525 530 531 532 533 534 535 540 541 542 543 544 545 550 551 552 553 554 555 MPN 23 31 43 58 76 95 33 46 64 84 110 130 49 70 95 120 150 180 79 110 140 180 210 250 130 170 220 280 350 440 240 350 540 920 1600 >1600

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

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TABLE A-2 Dilutions to be used and calculations of MPN per g or mL of test material Dilutions* 1:10 1:100 Undiluted Amount of original test material (g or mL) 10 1 0.1 0.01 5/5** 5/5 2/5 5/5 5/5 2/5 5/5 5/5 2/5 5/5 5/5 2/5 2/5 2/5 1/5 5/5 2/5 1/5 5/5 5/5 5/5 1:1000 0.001 5-5-2 5-5-2 5-2-2 5-2-0 2-2-1 5-2-2 5-5-5 540 540 95 49 12 95 >1600 Combination to be used MPN from Table A-1 Dilution factor on middle dilution 1 10 100 100 10 10 100 MPN per mL or g 5.4 54 95 49 1.2 9.5 >1600

a. b. c. d. e. f. g.

2/5 0/5 0/5 1/5*** 5/5

* Dilutions to be used are shaded gray.

*** No. of positive tubes/No. of tubes inoculated.

Reference 1. Official Method MFO - 15 Health Protection Branch - Ottawa.

Microbiological Examination of Foods for Aerobic Colony Counts (ACC)


Introduction This 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. Material l 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 Equipment l Thermometer, calibrated and certified l Incubator, 35C l Autoclave l Laminar Air Flow l Stomacher, blender or equivalent l pH meter or paper capable of distinguishing 0.3 to 0.5 pH units within a range of 5.0 to 8.0 Procedure Each sample unit shall be analyzed individually. The tests shall be carried out on the sample in accordance with the following instructions: 1) Collection of Samples l 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 Units l Keep frozen sample units frozen in the laboratory before analyzing them. Refrigerate shelf-stable products (0-5C). l Analyze the sample units as soon as possible after they have been received at the laboratory.

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3) Preparation of Dilutions l 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 ACC The 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 (40-45C) into each plate and mix contents by rotating and tilting. l Allow the agar to solidify. Table I - Criteria and sampling plans for ACC in specific foods Determination Food Criteria: No. of sample units (n) 5 5

l l l l l

Plates shall be poured not later than 15 min. after preparation of dilutions. Incubate plates in an inverted position at 35C for 48 2hr. Incubate an uninoculated plate, as a negative control. Avoid crowding or excessive stacking of plates in order to permit rapid equilibration of plates with incubator temperature. Count colonies promptly after the incubation period. 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 Results a. 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. Interpretation The 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.

Criteria: Acceptance Number (c) 2 2

Criteria: Concentration of Microorganisms (m) 10,000 10,000

Criteria: Maximum Concentration of Microorganisms (M) 25,000 50,000

ACC

Pasteurized milk and cream and other nonfermented dairy products Frozen dairy products (ice cream and ice milk), butter, milk powders and other dairy product powders Milk for manufacture into dairy products

50,000

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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. Reference 1. Official Method MFO-24.

Enumeration of Coliforms, Faecal Coliforms and E. Coli in Water in Sealed Containers and Prepackaged Ice Using the MPN Method
Introduction The 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 naturallycontaminated 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 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 confirmation of coliform presence; gas production at 45oC 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. Material l 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)

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l l l l l l l

EMB Agar (AM10391/AM50391) Simmons Citrate Agar (AM1090, AM5090) E.coli idenification kit (20796001) Kovacs Indole Reagent (20700040) Methyl Red Solution (20710040) VP Reagent (20680020) Control cultures (use ATCC cultures or equivalent): Positive control(s): E. coli that is known to produce gas at 45C 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 indolenegative, methyl red -negative, VogesProskauer-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.

Do not store sample units for more than 24 hrs before analysis. Store under refrigeration (0-5oC) conditions. ii) Prepackaged Ice l If sample units are prepackaged in leakproof containers, thaw them in the containers under refrigeration (0-5oC) 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 (0-5C). Do not store thawed sample units for more than 6 h before analysis.
l

3) Preparation for Analysis l 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 Broth l 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 Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials. 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.

Equipment l Covered water baths, with circulating system to maintain temperature of 45C. Water level should be above the medium in immersed tubes. l Thermometer, calibrated and traceable l Incubator, 35C l Autoclave l Laminar Air Flow Procedure Each sample unit must be analyzed individually. Carry out the test in accordance with the following instructions: 1) Collection of Samples l 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 Units i) Water in sealed containers

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The absence of gas in all of the tubes at the end of 48 4 hrs of incubation constitutes a negative presumptive test.

6) Confirmation Steps for Determination of Coliforms l 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. l Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials. 0 l 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 Coliforms l 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 Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials. 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 The absence of gas in all of the tubes at the end of 48 4 hrs of incubation constitutes a negative presumptive test. 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.

8) Confirmation Steps for Identification of E. coli l 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 4C, to be taken to non-selective media if the initial colony does not confirm as E. coli. Incubate at 35C 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 GIMViC From 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

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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 Table I GIMViC Pattern for E. coli Biotypes Gas at 450C G + Indole I + -

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 blue colour and an incorrect interpretation. Incubate the slants at 35 0C 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.

Type I Type II (Anaerogenic)

Methyl Red M + +

Voges Proskauer V -

Citrate C -

Table II** Differentiation of Commonly Occuring Coliforms Gas in EC broth at 450C Type I (typical) Type II (Anaerogenic) Type I Type II Type I Type II + Indole test Methyl Red test Escherichia coli + + + Intermediates + + + Enterobacter aerogenes + Enterobacter cloacae + + Reactions variable Voges Proskauer test -* -* + + + + Growth on Citrate + + + +

Irregular Type II + Type VI + Irregular other types * Weak positive reactions are occasionally found.

Interpretation The 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

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.

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5) Calculation of MPNs Table 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. 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 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

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. 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-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 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, 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-III Most 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* 10;1;0.1 000 001 002 003 004 005 010 011 012 013 MPN <1.8 1.8 3.6 5.4 7.2 9 1.8 3.6 5.5 7.3 Pos* 10;1;0,1 100 101 102 103 104 105 110 111 112 113 MPN 2 4 6 8 10 12 4 6.1 8.1 10 Pos* 10;1;0,1 200 201 202 203 204 205 210 211 212 213 MPN 4.5 6.8 9.1 12 14 16 6.8 9.2 12 14 Pos* 10;1;0,1 300 301 302 303 304 305 310 311 312 313 MPN 7.8 11 13 16 20 23 11 14 17 20 Pos* 10;1;0,1 400 401 402 403 404 405 410 411 412 413 MPN 13 17 21 25 30 36 17 21 26 31 Pos* 10;1;0,1 500 501 502 503 504 505 510 511 512 513 MPN 23 31 43 58 76 95 33 46 64 84

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Pos* MPN Pos* MPN 10;1;0.1 10;1;0,1 014 9.1 114 12 015 11 115 14 020 3.7 120 6.1 021 5.5 121 8.2 022 7.4 122 10 023 9.2 123 12 024 11 124 15 025 13 125 17 030 5.6 130 8.3 031 7.4 131 10 032 9.3 132 13 033 11 133 15 034 13 134 17 035 15 135 19 040 7.5 140 11 041 9.4 141 13 042 11 142 15 043 13 143 17 044 15 144 19 045 17 145 22 050 9.4 150 13 051 11 151 15 052 13 151 17 053 15 153 19 054 17 154 22 055 19 155 24 * Number of positive tubes with each of 3 volumes used.

Pos* 10;1;0,1 214 215 220 221 222 223 224 225 230 231 232 233 234 235 240 241 242 243 244 245 250 251 252 253 254 255

MPN 17 19 9.3 12 14 17 19 22 12 14 17 20 22 25 15 17 20 23 25 28 17 20 17 26 29 32

Pos* 10;1;0,1 314 315 320 321 322 323 324 325 330 331 332 333 334 335 340 341 342 343 344 345 350 351 352 353 354 355

MPN 23 27 14 17 20 24 27 31 17 21 24 28 31 35 21 24 28 32 36 40 25 29 32 37 41 45

Pos* 10;1;0,1 414 415 420 421 422 423 424 425 430 431 432 433 434 435 440 441 442 443 444 445 450 451 452 453 454 455

MPN 36 42 22 26 32 38 44 50 27 33 39 45 52 59 34 40 47 54 62 69 41 48 56 64 72 81

Pos* 10;1;0,1 514 515 520 521 522 523 524 525 530 531 532 533 534 535 540 541 542 543 544 545 550 551 552 553 554 555

MPN 110 130 49 70 95 120 150 180 79 110 140 180 210 250 130 170 220 280 350 440 240 350 540 920 1600 >1600

TABLE A-IV Dilutions to be used and calculations of MPN per g or mL of test material Undiluted 10 5/5** Dilutions* 1:10 1:100 Amount of original test material (g or mL) 1 0.1 0.01 5/5 2/5 5/5 5/5 2/5 5/5 5/5 2/5 5/5 5/5 2/5 2/5 2/5 1/5 5/5 2/5 1/5 5/5 5/5 5/5 1:1000 0.001 5-5-2 5-5-2 5-2-2 5-2-0 2-2-1 5-2-2 5-5-5 540 540 95 49 12 95 >1600 Combination to be used MPN from Table A-1 Dilution factor of middle dilution 1 10 100 100 10 10 100 MPN per mL or g 5.4 54 95 49 1.2 9.5 >1600

a. b. c. d. e. f. g.

2/5 0/5 0/5 1/5*** 5/5

* Dilutions to be used are shaded gray.

*** No. of positive tubes/No. of tubes inoculated.

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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 Table V: Criteria and sampling plans for Coliforms and E.coli Determination Food

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:

No. of Sample Units (n) Coliforms E. coli Water in sealed containers and Prepackaged ice Water in sealed containers and Prepackaged ice 5 5

Acceptance Number (c) 1 0

Criteria Concentration of Microorganisms (m) 0* 0*

Maximum Concentration of Microorganisms (M) 10 -

* 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. 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 (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. 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. 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.

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Proposed Official Method: Enumeration of Pseudomonas aeruginosa in Prepackaged Ice and Water in Sealed Containers by the Hydrophobic Grid-Membrane Filter (HGMF) Technique
Introduction The 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 naturallycontaminated 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) Equipment l HGMF (1600 grid-cell, 0.45 m 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 35C. A second incubator may be required depending on the selective agar chosen; e. g., 42C. 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. 1) Handling of Sample Units l In the laboratory prior to analysis, keep sample units refrigerated (05oC). 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 35C for 4 hrs. After 4 hrs, transfer the two HGMF to the two selective agars of choice. Incubate the plates at 35C 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

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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 35C. l If the plates are overgrown, pick and re-streak the colonies onto selective Table I: Criteria and sampling plans for Pseudomonas aeruginosa Determination Food No. of Sample Units (n) Pseudomonas aeruginosa Water in sealed containers 5
l l l

agar, incubate at 35C for 22-24 hours and if presumptive colonies are isolated follow instructions below. Perform the oxidase test on suspected yellow to green pigmented colonies. Perform a Gram stain on oxidase positive strains. Confirm only Gramnegative, oxidase positive colonies. 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.

Acceptance Number (c) 1

Criteria Concentration of Microorganisms (m) 0*

Maximum Concentration of Microorganisms (M) 2

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. 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 (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.

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. 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-19 5. Health Products & Food Branch - Ottawa.

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Detection of Food Poisoning by Clostridium botulinum and its Toxins


Introduction Clostridium 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. 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

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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 strains is close to 350C, while that for non- proteolytic strains is approximately 26 to 280C. Non- proteolytic types B, E, and F can produce toxin at refrigeration temperature (3 to 40C). Toxins of the non- proteolytic 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 canners 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 w < 0.9), a high 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 temperature is strictly controlled and kept below 30C, 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 Buffer l Tryptone Glucose Extract Broth (AM1102, AM5102) l Gram Stain Kit (20750020) l Anaerobic Agar (AM1000, AM5000) l Egg Yolk Emulsion (AS010) Equipment l Incubator l Autoclave

l l l

Laminar Air Flow Refrigerator Sterile Motor and Pestle

Procedure Sampling I) 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, manufacturers 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 Specimens All 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 tube with a leak proof cap. Examine immediately or refrigerate at 40C. Examination of post-treatment (8-12 hrs) serum is also helpful to evaluate antitoxin therapy. 2. Feces. Collect 25 g of the patients 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. Cardboard containers are not acceptable. Refrigerate specimens at 40C 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 colleted in sterile leak-proof containers and refrigerated at 40C .

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Procedure for identifying viable C. botulinum Preparation of various samples 1) 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 Foods Transfer 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 Foods Inoculate liquid foods directly into the culture media, using sterile pipettes. 4) Reserve Test Sample After culturing, aseptically remove a reserve portion of the test sample to a sterile jar for later tests. 5) Examining a Product for Appearance and Odor Visually, note any evidence of decomposition, but do not taste the product under any circumstance. Record observations. 6) Preparation of Enrichment Cultures l 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 0 l 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

l l

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. Test each enrichment culture for toxin. 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 Cultures l 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 proteolytic type, heat at 800C 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. 0 l 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

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other members of the genus Clostridium, which do not elaborate toxin, produce colonies with characteristics similar to those of C. botulinum. 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 one plate anaerobically and the other aerobically at 350C 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. Store the pure culture in the sporulated state under refrigeration.

References 1. Aranda, E., M.M. Rodriguez, M.A. Asensio, and J.J. Cordoba,1997. Detection of clostridium botulinum types A, B, E and F in foods by PCR and DNA probe. Lett. Appl. Microbiol.25:186-190. 2. Arnon, S.S.1998. Infant botulism, p.1570-1577. In R.D. Feigen and J.D. Cherry (eds), Textbook of pediatric infectious diseases, 4th ed. W.B.Saunders, Philadelphia,Pa. 3. Arnon,S.S.1995. Botulism as an intestinal toxemia, p.257-271. In M.J.Blaser, P.Smith, J.I.Ravdin, H.B.Greenberg, and R.L.Guerrant (eds.), Infections of the gastrointestinal tract. Raven Press, new York. 4. Arnon, S.S.1993. Clinical trial of human botulism immune globulin, p.477482. In B.R. DasGupta (ed.), Botulism and tetanus neutrotoxins: Neurontransmissions and biochemical aspects. Plenum Press, New York. 5. Doellgast, G.J.,M.X.Triscott, G.A, Beard, J.D.Bottoms, T.Cheng, B.H.Roh, M.G.Roman, P.A. Hall, and J.E.Brown, 1993. Sensitive enzyme-linked immunosorbent assay for detection of Clostridium botulinum neurotoxins A,B, and E using signal amplification via enzyme-linked coagulation assay. J.Clin. Microbiol. 31:2402-2409. 6. Dowell, V.R., and T.M. Hawkins. 1974. Laboratory methods in anaerobic bacteriology, CDC Laboratory Manual. PHS Publ. No1803,U.S. Dept. of Health, Ed., and Welfare. U.S. Public Health Serv., Washington, D.C. 7. Fach, P.,D.Hauser. J.P. Guillou and M.R.Popoff.1998. Polymerase chain reaction for the rapid identification of Clostridium botulinum type A strains and detection in Food samples. J.Appl. Bacteriol 75:234-239. 8. Fach, P., M.Gilbert, R.Griffais, J.P.Guillou, and M.R.Popoff.1995, PCR and gene probe identification of botulinum neurotoxin A, B, E, F, and G-producing Clostridium spp, and evaluation in food samples Appl. Environ. Microbial, 61:389-392.

9. Ferreira, J.L., M.K.Hamdy, S.G.McCay, M.Hemhill, N.Kirma, B.R.Baumstark, 1994. Detection of Clostridium botulinum type Fusing the polymerase chain reaction. Mol.Cell Probes 8:365-373. 10. Ferreira, J.L.1997.ORA/NCTR/CDC, Initiative for Development of an ELISA Method for the Detection of type A, B, C, E, and F Clostridium botulinum Toxin. Food and Drug Administration. LIB#4093. 11. Franciosa, G.,J.L.Ferreira, and C.L. Hatheway, 1994 Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR: Evidence of unexpressed type B toxin genes in type A toxigenic organisms. J.Clin. Microbiol. 63:1911-1917. 12. Hatheway, C.L,.1979.Laboratory procedures for cases of suspected infant botulism. Rev. Infect.Dis:1:647-651. 13. Hatheway,C.L., and L.McCroskey.1987. Examination of feces and serum for diagnosis of infant botulism in 336 patients. J.Clin. Microbiol. 25:23342338. 14. Hauschild, A.H.W.,R.Hilsheimer, K.F.Weiss, and R.B.Burke.1988. Clostridium botulinum in honey, syrups, and dry infant cereals. J.Food Prot.51:892-894. 15. Herzberg, M.(ed.).1970. Toxic microorganisms: Mycotoxins, Botulism. Proceedings of the 1st U.S-Japan Comp. Prog. In Natural resources. U.S.Dep. of the Interior, Washington, D.C. 16. Ingram, M., and T.A.Roberts (eds).1967. Botulism 1966. Chapman and Hall. London, England. 17. International Commission on Microbiological Specifications for Foods. 1978. Microorganisms in Foods 1,2nd University of Toronto Press, Toronto,Canada. 18. Lewis, K.H., and K.Cassel, Jr(eds.). 1964, Botulism, Proceedings of a Symposium. U.S.Dept. of Health, Ed., and Welfare. Public Health Serv., Washington, D.C. 19. Mills, D.C., T.F.Midura, and S.S.Arnon. 1985. Improved selective medium for the isolation of lipase-positive Clostridium botulinum from feces of human infants. J.Clin. Microbiol. 21:947-950. 20. Paisley,J.W.,B.A.Lauer, and S.S Arnon.1995. A second case of infant botulism caused by Clostridium baratii. Pediatr. Infect. Dis.J.14:912-914. 21. Roman, M.G.,J.Y.Humber, P.A.Hall, N.R.Reddy, H.M.Solomon, M.X. Triscott, G.A. Beard, J.D. Bottoms, T.Cheng, and G.J.Doellgast. 1994. Amplified immunoassay ELISA-ELCA for measuring Clostridium botulinum type E neurotoxin in fish fillets. J.Food Prot. 57:985-990. 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 disease, 2nd ed. Charles C.Thomas, Sprongfield, Ill. 24. Stumbo, C.R.1973. Thermobacteriology in food processing, 2nd 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

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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). 28. U.S.HHS/PHS/FDA.1995. Bacteriological analytical manual, 8th ed. Assoc. of Off. Anal. Chem., Gaithersburg, Md.

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Soil Microbiology

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Isolation of Dermatophytes, other Fungi and Yeasts from Soil


Introduction Carlier 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 used in place of the Standard American medium of Hodges2. The fungi maintain their typical cultural appearance and thus may be readily identified according to the standard macroscopic characters described by Sabouraud3. The medium is often used with antibiotics for the isolation of pathogenic fungi from material containing large numbers of other fungi or bacteria. Georg et al.4 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 (Ajello5). The same micro-organisms are sensitive to this new combination - see Dermasel Selective Supplement SR0075. Williams Smith & Jones6 employed Sabouraud Dextrose Agar, containing 20,000 units penicillin and 0.04g neomycin per litre, for the count of yeasts in the alimentary tract of the pig. Hantschke7 used colistin, novobiocin and cycloheximide to isolate Candida albicans. Dolan8 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 medium9 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 55C. The medium is usually made inhibitory to most nonpathogenic fungi and bacteria by the addition of antibiotics as above. After incubation for 3 days at 25C, 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 be utilised for the identification of Candida albicans10,11,12,13. Material l Sabouraud Dextrose Agar (AM1087/AM5087) l Distilled water l Candida albicans ATCC 10231 l Aspergillus niger ATCC 16404 Equipment l Autoclave l Incubator l Laminar Air Flow Procedure l Prepare serial dilutions of soil sample using sterile D.W. l Inoculate each specimen in duplicate. l Incubate one set of media aerobically at 22-25C and the other set at 35C for 5-30 days. Loosen the caps of tubes and ensure adequate moisture for the plates to compensate for loss of water vapour. DO NOT SEAL THE PLATES. 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 22-25C for 5 days. l Prepare negative control by uninoculating medium. Interpretation Positive control Candida albicans ATCC 10231 Aspergillus niger ATCC 16404 Negative control Uninoculated medium

Expected results Good growth; cream colonies White mycelium; black spores 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. References 1. 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.

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Cultivation of those fungi and bacteria which are able to utilise sodium nitrate as the sole source of nitrogen
Introduction Czapek 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. Dawson1 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. Smith2 cited the following recommendations for the use of Czapek Dox Agar for taxonomic studies: by Thom and Church3 for Aspergillus; by Thom4 and by Raper and Thom5 for Penicillium; and by Wakesman6 for actinomycetes. Material l Czapek Dox Agar (AM10301/AM50301) l Distilled water l Aspergillus niger ATCC 9642 l Candida albicans ATCC 10231 Equipment l Autoclave l Incubator l Laminar Air Flow Procedure General Cultivation l To avoid excessive condensation cool the molten medium to 50C 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 25C. Most Penicillium species have an optimum growth temperature between 20 and 25C, whilst many Aspergillus species grow best at about 30C. However, different fungi grow over a wide range of temperatures; Aspergillus fumigatus grows well at 50C (Smith2) and Cladosporium herbarum will grow on meat at -6C 7,8. l Prepare positive control by inoculating Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404 and incubate at 22-25C for 5 days. l Prepare negative control by uninoculating medium. Identificationof Candida albicans 1. 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 28C. 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 reexamine. Interpretation Positive control Aspergillus niger ATCC 9642 Candida albicans ATCC 10231 Negative Control Uninoculated medium Expected Results White / yellow mycelium, black spores Good growth, cream coloured colonies No change

References 1. 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.

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Observation for chlamydospore production by Candida albicans and for the maintenance of fungal stock cultures
Introduction Corn 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 identification of this species. Prospero and Reyes1 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 greatly enhances the development of chlamydospores on the medium2,3,4,5,6. Mackenzie7 found that all 163 isolates of obtained from laboratories in the United Kingdom produced chlamydospores on Corn Meal Agar but Dawson8 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 species of Trichophyton e.g. Trichophyton rubrum 9. Material 1) 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). Equipment l Autoclave l Incubator l Laminar Air flow Procedure A 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 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 22C, 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 chlamydospores9. The addition of 0.001g % w/v Trypan blue to Corn Meal Agar provides a contrasting background for the observation of characteristic morphological features of yeast cultures10. 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 22C. Interpretation Positive Control Chlamydospore Production Candida albicans ATCC 10231 Negative Control Candida krusei ATCC 6258 Expected Results Good growth; white colonies and chlamydospores. Good growth; white / cream colonies, no chlamydospores.

References 1. 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.

Isolation of Nitrogen Fixing Bacteria from Soil


Introduction Nitrogen fixing bacteria are capable of taking gaseous nitrogen and combining it with hydrogen to make ammonia. The plant can use fixed nitrogen for growth. 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.

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Material l Jensen's Broth (AM50485) l Jensen's Medium (AM10486/AM50486) l Rhizobium leguminosarum ATCC 10004 l Rhixobium meliloti ATCC 9930 Equipment l Autoclave l Incubator l Laminar Air Flow Procedure l Prepare 20 ml sterile Jensens Broth tubes. l Prepare sterile Jensens Medium Plates. l Add aseptically 1-2 gms of soil sample into Jensens broth.

l l l l l

Incubate tubes for 7 days at 300 C. Check for growth and then transfer a loopful of enriched medium on Jensens Medium (Agar). Incubate plates for 7 days at 300 C. Observe characteristic colonies. Prepare positive control by inoculating Rhizobium leguminosarum ATCC 1004 and Rhixobium meliloti ATCC 9930 cultures and incubating at 300 C for 7 days.

Interpretation Positive control will give luxuriant growth. Reference 1. Vincent J. M. 1970, A Manual for Practical Study of Root Nodule Bacteria P194 IBP Hand Book, Backwell Scientific Publication Oxford.

Detection and Isolation of Phosphate Solubilizing Microoraganisms from soil


Introduction Both 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). Material l Pikovaskaya's Agar (AM508092/AM108092) l Distilled water l Aspergillus niger ATCC (16404) l Pseudomonas aeruginosa ATCC (27853) l Bacillus Subtilis ATCC (6633) Equipment l Incubator l Autoclave l Laminar Air Flow Procedure l 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. Incubate plates at 350C for 48 hours. Observe characteristic colonies and compare with growth response of positive control. For Positive control streak following cultures on Pikovaskaya's Agar and incubate at 350C for 48 hours. Aspergillus niger ATCC (16404) Pseudomonas aeruginosa ATCC (27853) Bacillus Subtilis ATCC (6633)
l l

Interpretation Positive control culture response Organism (ATCC) Aspergillus niger (16404) Pseudomonas aeruginosa (27853) Bacillus Subtilis (6633) Growth Luxuriant Luxuriant Good Phosphate Solubilization + + (+)

Key + = Clear zone surrounding the colony. (+) = Moderate clear zone surrounding the colony. References 1. 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.

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Microbiological Methods in Beverage Industry

Hand Book of Practicing Microbiologists

Detection and Enumeration of respiratory deficient yeast cells used in beverage


Introduction Respiratory 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. Material l Wort Agar (AM1111, AM5111) l TTC Solution 1% (AS0271) l Agar Powder (AB001) Equipment l Autoclave
l l

Incubator Oven

Procedure l Grow yeast cells on Wort Agar by spread plate technique or pour plate technique. l Prepare (100 ml) 1.5% sterile agar. 0 l 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. 0 l Incubate at 30 C for 1 hour. l Count white colonies and red colonies. Interpretation Normal respiratory sufficient colonies turn pink to red while respiratory deficient colonies remain white.

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


Introduction All 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. Material l Wort Agar (AM1111, AM5111) Equipment l Laminar Air Flow Unit
l l l

Autoclave Incubator Refrigerator

Procedure l Prepare 10 ml slants of Wort Agar Medium l 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.

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


Introduction All 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. To ensure the absence of unwanted microflora during manufacturing process at various stages product is monitored by performing sterility test by microfiltration technique. Material l WL Nutrient Agar (AM51092) l Raka- Ray Agar Base (AM10844)

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l l l l

WL Differential Agar (AM1109/ AM5109) Lysine Medium Base (AM10577/ AM50577) Lactobacillus MRS Agar (AM1051/ AM5051) 0.22 micron Filter paper.

l l l l

Equipment l Membrane holder l Autoclave l Laminar Air Flow l Incubator l Oil free vacuum pump. Procedure l Collect the samples in sterile bottles. l Conduct the experiment inside Laminar Air Flow unit. Table 1 Name of Media WL Nutrient Agar Incubation Temp. 300C

l l l

Arrange sterile membrane holder stand inside LAF unit. Moist the filter membrane by filtering sterile distilled water. Filter 100 ml sample through membrane with the aid of oil free vacuum pump. After filtration remove the membrane aseptically using sterile forcep and mount it on sterile agar plate. Use respective type of agar medium as per requirement. Incubate the plates at respective temperature and period as mentioned in table I. At the end of incubation period check for specific colonies.

Interpretation / Limit Agar plates should not exhibit typical standard colonies, Refer Table I for colonical characteristics.

Incubation Period 40-48 hours 1) 2) 3) 4) 1) 2) 3) 4)

Cultural Characteristcs S.cerevisiae-> E.coli -> L.Fermentum-> P.mirabilis-> E.coli-> L.fermentum-> P.mirabilis-> S.cerevisiae-> Good - Luxuraint Fair - Good Fair - Good Fair - Good Luxuriant Luxuriant Luxuriant Inhibited

WL Differential Agar

35 C

48 hours

Lysine Medium Base

25 C

7 Days

Lactobacillus MRS Agar

350C

18-24 hours

1) Pichia fermentans- > Luxuriant 2) L.plantarum-> Luxuriant 1) L.fermentum-> 2) E.coli-> Good Inhibited

Raka-Ray Agar Base

25-300C

4-7 days

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Medical Microbiology

Hand Book of Practicing Microbiologists

Introduction
The clinician uses the laboratory to assist in diagnosis and management of the patient due to following reasons.
l l l l l

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 laboratory l 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.

To confirm a clinical impression or establish a diagnosis. To rule out a diagnosis. To monitor therapy. To establish prognosis. 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

Blood Culture
INTRODUCTION Culture 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 situations 1. 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/MEDIAS l 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 formulaAM103011/AM503011) l Thioglycollate broth l Diphasic broth (Castaneda system)
l l l

Blood agar base (AM1014/AM5014), OR Blood agar base (Ready prepared media-250ml) (20500006) Modified Grams Stain Kit (20750020/20750021)

REQUIRED MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Sterile plating loops (10l). 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 EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens. 3. Incubator with air plus 5-10% CO2 at 370C20C. 4. Incubator with Nitrogen or Hydrogen plus 5-10% CO2 at 370C20C. 5. Autoclave. PROCEDURE a. Collection of sample: 1. Wear sterile gloves.

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2. Disinfect the venepuncture site on the patients 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% SPS 4. Cooked meat medium 5. Thioglycollate broth 6. 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% CO2 , at 37 C for 18-24 hours. l Incubate second plate anaerobically in nitrogen or hydrogen plus 5-10% CO2, at 370 C for 18-24 hours. 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). References 1. Basic Laboratory in Clinical Bacteriology, J.Vandepitte, K.Engbaek, P.Piot, C.C.Heuck, W.H.O. Geneva, 1991. 2. Diagnostic Microbiology, Bailey & Scott, 9th Ed., Mosby 1994, Ellen Jo Baron, Lance R. Peterson. 3. Practical Medical Microbiology, Mackie & McCartney, Vol 1, Microbial Infections, 13th Ed., Churchill Livingston 1978, Edited by J.P. Duguid, B.P. Marmion, R.H.A. Swain. 4. Practical Medical Microbiology, Mackie & McCartney, Vol. 2, 13th Ed., Churchill Livingston 1989, Edited by J.G. Collee, J.P. Duguid, A.G. Fraser, B.P.Marmion. 5. Handbook of Microbiological Media, Ronald M. Atlas, Lawrence C.Parks, 2nd Ed., 1997. 6. Data on file: Tulip Diagnostics (P) Ltd.

Upper respiratory tract infections, including throat, nose, ear and eye infections
INTRODUCTION The 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 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/MEDIAS Stuart transport medium (AM1094/AM5094) l Blood agar base (AM1014/AM5014 ) OR l Blood agar base (Ready prepared media-250ml) (20500006). l Stuart transport medium (AM1094/AM5094)

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l l l l l l

6mm disk containing 1 unit of benzyl penicillin 6mm disk containing 0.1 unit of bacitracin 6mm disk containing 50 unit nystatin 6mm disk containing 20g amphotericin 6mm disk containing 2 g of amoxycillin or ampicillin Candida identification kit (20794001)

containing 20g amphotericin on fourth blood agar plate. Place a 6mm disk containing 2g of amoxycillin or ampicillin on the well area of heated blood agar plate.

2. REQUIRED MATERIALS 1. Sterile hand gloves . 2. Face mask. 3. Sterile plating loops(10l). 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 EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens. 3. Incubator with air plus 5-10% CO2 at 370C20C. 4. Incubator with Nitrogen or Hydrogen plus 5-10% CO2 at 370C20C. 5. Autoclave. 4. PROCEDURE a. 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 refrigerator at 4 0C until testing or if it has to be transported it should be submitted in a tube of Stuart transport medium. b. Innoculation: Direct sensitivity method l 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

c. Incubation: i) Incubate one plate in air plus 5-10% CO2 , at 370 C for 18-24 hours. ii) Incubate second plate anaerobically in nitrogen or hydrogen plus 5-10% CO2 , at 370 C for 18-24 hours. 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 g 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. 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 & 20g amphotericin and later subjected to confirmatory test using commercially available identification kit). References 1. Basic Laboratory in Clinical Bacteriology, J.Vandepitte, K.Engbaek, P.Piot, C.C.Heuck, W.H.O. Geneva, 1991. 2. Diagnostic Microbiology, Bailey & Scott, 9th Ed., Mosby 1994, Ellen Jo Baron, Lance R. Peterson. 3. Practical Medical Microbiology, Mackie & McCartney, Vol 1, Microbial Infections, 13th Ed., Churchill Livingston 1978, Edited by J.P. Duguid, B.P. Marmion, R.H.A. Swain. 4. Practical Medical Microbiology, Mackie & McCartney, Vol. 2, 13th Ed., Churchill Livingston 1989, Edited by J.G. Collee, J.P. Duguid, A.G. Fraser, B.P.Marmion. 5. Handbook of Microbiological Media, Ronald M. Atlas, Lawrence C.Parks, 2nd Ed., 1997. 6. Data on file: Tulip Diagnostics (P) Ltd.

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Lower Respiratory Tract Infections


INTRODUCTION Unlike 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. SPECIMENS 1. Type of specimen 1. Sputum 2. Bronchial swabs and aspirates 3. Blood 2. Collection of specimen For 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. Sputum The 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 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 aspirates A 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 culture In all cases of suspected pneumonia a sample of blood should be taken for

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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 TUBERCULOSIS In 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 SPUTUM SUMMARY Acid Fast Bacilli staining is the first line microscopic procedure performed in detection of Mycobacterium tuberculosis. PRINCIPLE Carbol 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 REAGENTS 1. Mycostain (20307100) 2. Acid fast decolouriser (20308500) 3. Novachrom (20302125) REQUIRED MATERIALS 1. 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 EQUIPMENTS Biosafety hood with Bunsen burner, Microscope with oil immersion lens, Autoclave.

SPECIMEN COLLECTION REFER** PROCEDURE a. 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 decolouriser20308500) 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).

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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 No Acid Fast Bacilli 1-10 Acid Fast Bacilli >10 Acid Fast Bacilli Masses of Acid Fast Bacilli in several fields Report Negative Actual number + ++

2. DECONTAMINATION AND CONCENTRATION OF SPUTUM SUMMARY Before 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 REAGENTS Lyfectol (20301012) REQUIRED MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Sterile plating loops(10l). 4. Activated 2% glutaraldehyde solution. 5. Cedar wood oil. 6. Glass slide. 7. 5ml measuring cylinder. 8. 15-25ml universal container. REQUIRED EQUIPMENTS 1. 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. PRINCIPLE Proper 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** PROCEDURE The 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.

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: Number of Acid Fast Bacilli Observed No Acid Fast Bacilli 1-10 Acid Fast Bacilli >10 Acid Fast Bacilli Masses of Acid Fast Bacilli in several fields Report Negative Actual number + ++

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PREPARATION OF MUCOLYTIC REAGENT l 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 C. ****PROCESSING OF SPECIMEN l 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. CULTURING SUMMARY The optimal medium for tubercle bacilli are Lowenstein jensens solidified egg based medium with glycerol , Kirchner selective medium, Middle brook agar and broth with enriched supplement (for M.tuberculosis) or Lowenstein jensens 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 REAGENTS 1. 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 MATERIALS 1. Sterile hand gloves. 2. Face mask.

3. 4. 5. 6. 7. 8.

Sterile plating loops(10l). Activated 2% glutaraldehyde solution. Cedar wood oil. Glass slide. Micropippete (100l - 500l). Sterile micropipette tips.

REQUIRED EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens. 3. Vortex mixer, Incubator at 370.5C. 4. Autoclave. PRINCIPLE The 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) PROCEDURE l Bring the required media for inoculation to room temperature. l Label the medium slant appropriately. l Draw 10l 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 370.5C. l Observe for growth weekly till 8 weeks. INTERPRETATION OF RESULTS l 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

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sediment to the bottom. 4. SENSITIVITY TESTING SUMMARY Inadequate 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 REAGENTS 1. Sensicult primary (20305101) 2. Sensicult secondary (6 drugs) (20305201) 3. Sensicult secondary (10 drugs) (20305202) REQUIRED MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Sterile plating loops (10l). 4. Activated 2% glutaraldehyde solution. 5. Cedar wood oil. 6. Glass slide. 7. Micropippete (100l - 500l). 8. Sterile micropipette tips. REQUIRED EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens. 3. Vortex mixer. 4. Incubator at 370.5C. 5. Autoclave. PRINCIPLE Due 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. PROCEDURE INNOCULUM 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.

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. INNOCULATION l Bring the primary /secondary drug containing Lowenstein-jensen media panel for MTB sensitivity tests slants to room temperature . l Apply 100l from the seed culture-1 to each drug slant and 100l from the seed culture -2 to Lowenstein Jensen slant. l Close the cap tightly and incubate at 370.5C. l Observe for the growth till 8 weeks every alternate days. INTERPRETATION OF RESULTS As 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 TESTS SUMMARY Many 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.

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PROCEDURE I. CATALASE PRINCIPLE Most 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 7/68C ) method. Room Temperature Method Untreated catalase enzyme produced by Mycobacteria reduces hydrogen peroxide to water and oxygen. This is as bubbling of oxygen, which occurs following the addition of 50-100l of the Tween-Peroxide reagent to the 3-4 week old culture growth obtained on the solid plate or slant. Heat Stable (pH 7/68C ) method. Certain Mycobateria loose catalase activity when suspended in pH7.0 bufer and heated to 68C. 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 REAGENTS 1. Catalase Detection Kit (20403020). REQUIRED MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Activated 2% glutaraldehyde solution. 4. Micropippete (100l - 500l). 5. Sterile micropipette tips. 6. Test tube stand. 7. Screw cap test tubes (16x125mm). REQUIRED EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Water bath or constant temperature block heater. 3. Autoclave. SPECIMEN COLLECTION 3-4 weeks old cultures obtained from solid media only should be used for testing. TEST PROCEDURE i. PREPARATION OF 1ML TWEEN - PEROXIDE REAGENT Note: Tween - Peroxide reagent is to be prepared fresh, immediately

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 OBSERVATION Room Temperature Method: l Add 50-100l 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 7/68C ) 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 68C 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 7/68C) 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 RESULTS Room 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 7/68C ) method: l Formation of bubbles - Positive. l No bubbling until 20 minutes - Negative.

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II. TWEEN-80 HYDROLYSIS PRINCIPLE The commonly non-pathogenic, slow growing scotochromogens and nonchromogens 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 REAGENTS Tween 80 Hydrolysis Kit (20402100). REQUIRED MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Activated 2% glutaraldehyde solution. 4. Micropippete (100l - 500l). 5. Sterile micropipette tips. 6. Test tube stand. 7. Screw cap test tubes (16x125mm). REQUIRED EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Water bath /incubator at 372C. 3. Autoclave. SPECIMEN COLLECTION Slow growing cultures obtained from solid media can be inoculated on the Tween-80 substrate as soon as sufficient growth is obtained for transfer. TEST PROCEDURE i. PREPARATION OF TWEEN - HYDROLYSIS REAGENT l 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 100l of the concentrated mixed TWEEN-80 HYDROLYSIS reagent. l Slightly warm (50C) 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.

Allow the reagent to cool prior to testing.

ii. INNOCULATION l 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 35-37C. 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 RESULTS l 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 TEST PRINCIPLE The 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 Niacin + Cyanogen chloride g-carboxy-glutaconic aldehyde Cyanogen chloride g-carboxy-glutaconic aldehyde formation of Schiff base (Canary yellow colour)

REQUIRED REAGENTS Niacin drop test Kit (20401050). REQUIRED MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Activated 2% glutaraldehyde solution. 4. Micropippete (100l - 500l).

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5. 6. 7. 8. 9.

Sterile micropipette tips. Test tube stand. Screw cap test tubes(16x125mm). White background card or board,sterile. Sterile isotonic solution.

minutes. 8. Replace the caps and restore the NIACIN reagents at 2-8C immediately after use. INTERPRETATION OF RESULTS l 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 TEST PRINCIPLE PNB 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 REAGENTS 1. PNB Sensitivity Test Kit (20408006). 2. Mycocult Kit (20304006). 3. Mycocult PY (20314006). REQUIRED MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Activated 2% glutaraldehyde solution. 4. Micropippete (100l - 500l). 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 EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Water bath /incubator at 372C. 3. Vortex mixer. 4. Autoclave. SPECIMEN COLLECTION Three to four week old viable cultures on drug free solid media should be used for testing.

REQUIRED EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Centrifuge. 3. Autoclave. SPECIMEN COLLECTION Three 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 PROCEDURE l Allow the reagents from the kit to attain R.T.(20-25C) 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.(20-25C). 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

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TEST PROCEDURE IF THE PRIMARY ISOLATE IS SUSPECTED TO BE M.tuberculosis: 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 PNB slant L.J. control slant 1 L.J. control slant 2
l

l l

prior to inoculation. Retrieve one slant/vial from the PNB SENSIVITY TEST kit per isolate to be tested. Similarly, also retrieve 2 drug free L.J. control slants containing sodium pyruvate per isolate to be tested . 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. 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. Close all the caps tightly and incubate the three slants as follows: Temp. 370C 370C 250C Incubation specifications Internally illuminated incubator Internally illuminated incubator Dark incubator Examine at 3, 7, 14 and 21 days 3, 7, 14 and 21 days 3, 7, 14 and 21 days

Slants PNB slant L.J. control slant 1 L.J. control slant 2


l

When growth is evident on the drug free L.J. control slant incubated at 370C, examine all the three slants for pigmentation.

Temp. 370C 370C 250C

Incubation specifications Internally illuminated incubator Internally illuminated incubator Dark incubator

Examine at 3, 7, 14 and 21 days 3, 7, 14 and 21 days 3, 7, 14 and 21 days

INTERPRETATION OF RESULTS Members of the Mycobacterium tuberculosis complex can be identified on the following basis: 0 l Those that do not grow within three days at 37 C. 0 l 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 TEST PRINCIPLE TCH SENSITIVITY TEST is used to distinguish M.bovis from other nonchromogenic 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 REAGENTS TCH Sensitivity Test Kit (20404003). REQUIRED MATERIALS 1. Sterile hand gloves.

When growth is evident on the drug free L.J. control slant incubated at 370C, examine all the three slants for pigmentation.

TEST PROCEDURE IF THE PRIMARY ISOLATE IS SUSPECTED TO BE M.bovis: M.bovis: 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 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)

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2. 3. 4. 5. 6. 7. 8. 9. 10.

Face mask. Activated 2% glutaraldehyde solution. Micropippete (100l - 500l). Sterile micropipette tips. Test tube stand. Screw cap test tubes (16x125mm). Sterile 1ml glass bottles with glass beads. Mcfarland standard No. 1. Sterile isotonic solution.

INTERPRETATION OF RESULTS Slant Observations R1 No growth Growth R2 Growth No growth R3 No growth Growth Results M. bovis M. tuberculosis

REQUIRED EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Water bath /incubator at 372C. 3. Vortex mixer. 4. Autoclave. SPECIMEN COLLECTION Three 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 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 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 0 l Close all the caps tightly and incubate the slants at 37 C2 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:

VI. NITRATE REDUCTION KIT PRINCIPLE Some 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 REAGENTS Nitrate Reduction Kit (20405025). REQUIRED MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Activated 2% glutaraldehyde solution. 4. Micropippete (100l - 500l). 5. Sterile micropipette tips. 6. Test tube stand. 7. Screw cap test tubes (16x125mm).

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8. Mcfarland Standard No. 10. 9. Sterile 1ml screw capped glass vial with glass beads. 10. Sterile isotonic solution. REQUIRED EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Water bath /incubator at 372C. 3. Vortexing mixer. 4. Autoclave. SPECIMEN COLLECTION AND PREPARATION 1. 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 PROCEDURE Method I l In a clean sterile test tube aseptically pipette out 0.5ml of nitrate substrate reagent (R1 reagent). l Aseptically emulsify 300l to 400l 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 372C 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. OR Put one drop (10l -25l) 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 II l In a clean sterile test tube aseptically pipette out 0.5ml of nitrate substrate reagent (R1 reagent). l Aseptically emulsify 100l of MTB culture suspension matching Mcfarland standard 10 in 0.5ml of nitrate substrate reagent (R1

l l l

l l

reagent) Shake by hand to mix and incubate at 372C for 16-24 hours. Retrieve the required number of nitrite detection strips. Dip the Nitrite detection strip into the incubated test substrate for the solution to be just absorbed on the reaction pad. OR Put one drop (10l -25l) of the incubated test substrate for the solution to be just absorbed on the reaction pad. Observe for colour change from colourless to pink/red/violet at 30-60 seconds. 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.

INTERPRETATION OF RESULTS Colour change observed Result Directly with nitrite With Nitrite detection strip detection strip on addition of zinc dust Pale pink or deep --Nitrate positive strain red or violet No colour change No colour change Nitrate positive strain No colour change Pale pink or deep red Nitrate negative strain or violet VII. BIOCHEMICAL TEST SUMMARY Tuberculosis 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-MTB PRINCIPLE Adenosine 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. ADA Adenosine+H2O --------> Ammonia + Inosine

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Alkaline Ammonia+Phenol+Hypochlorite --------> Blue Indophenol Complex Medium REQUIRED REAGENTS ADA-MTB Test Kit (20306015). REQUIRED MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Activated 2% glutaraldehyde solution. 4. Micropippete (500l - 1000l) and (10l - 500l). 5. Sterile micropipette tips. 6. Test tube stand. 7. Test tubes. REQUIRED EQUIPMENTS 1. Water bath /incubator at 372C. 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 PROCEDURE l Reagent Preparation 1. Reagent L1(Buffer reagent), L2 (Adenosine Reagent) and Standard are ready to use. 2. Adenosine Reagent (L2) may from crystals at 2-80C. Dissolve the same by gently warming (370C - 500C) 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 stable for at least 6 months when stored at 2-80C in tightly closed bottles.

TESTING 1) 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 370C. 3) Pipette into clean dry test tubes labeled Blank (B), Standard (S), Sample Blank (SB) and Test (T) as follows Addition Sequence Buffer Reagent Adenosine Reagent Deionised water Standard Sample B (ml) 0.20 0.20 S (ml) 0.20 0.20 SB (ml) 0.20 T (ml) 0.20 0.20

4) Mix well and incubate at 370C 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 1.00 Reagent 5) Mix well and incubate at 370C 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 Total ADA activity in U/L Abs.T-Abs.SB = ------------------------ x 50 Abs.S-Abs.B

8) Linearity The 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 Normal <30 U/L Suspect 30U/L to 40 U/L Strong Suspect >40U/L to 60 U/L Positive >60U/L CSF Normal <10U/L Positive >10U/L It is recommended that each laboratory establish its own normal range representing its patient population. Serum, Plasma, Pleural, Pericardial & Ascitic Fluids

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Examination for common infections Summary All 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: Summary There 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/MEDIAS Solution of dithiothreitol (Sputolysin/Calbiochem). REQUIRED MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Activated 2% glutaraldehyde solution. REQUIRED EQUIPMENTS 1. Vortex mixer. 2. Incubator at 370C20C. 3. Machine that tilts to and fro. PROCEDURE l 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 or l Preferably, mix gently and continuously on a machine that tilts to and fro placed in an incubator for 30 min at 370C. l Take the homogenized sputum for microscopy, and culture. 3) Microscopy REQUIRED REAGENTS/MEDIAS Modified grams stain kit (20750020 / 20750021).

REQUIRED MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Sterile plating loops(10l). 4. Activated 2% glutaraldehyde solution. 5. Cedar wood oil. 6. Glass slide. REQUIRED EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens. 3. Autoclave. PROCEDURE 1. Gram Stain l 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. Observation l 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. Interpretation l 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 -lactamase resistant penicillin, such as flucloxacillin, is urgently required. 2. Wet film l Place approximately 100l 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.

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Observation l Note the presence of sporing heads. Interpretation l 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) Culture SUMMARY A semi-quantitative method of culture is recommended, so that the presence of a potential pathogen in only small numbers, e.g. Less than 106/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/MEDIAS l Blood agar base (AM1014/AM5014). OR l Blood agar base (Ready prepared media - 250ml) (20500006). l Fildes digest agar. l Sabouraud dextrose agar (AM1014/AM5014). OR l Blood agar base (Ready prepared media-250ml) (20600006). l Malt extract agar (AM1087/AM5087). l Nutrient broth (AM1077/AM5077). OR l Nutrient broth (Ready prepared media-5ml) (20500006). l Peptone Water (AM1079/AM5079). OR l Peptone Water (Ready prepared media-5ml) (20572006). l 50 mg optochin disk. l 1 unit benzyl pencillin disk. l 2 g 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 MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Sterile plating loops(10l).

4. 5. 6. 7.

Activated 2% glutaraldehyde solution. Glass spreader. 70% Isopropyl alcohol. Sterile forceps.

REQUIRED EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Autoclave. PROCEDURE 1. Dilute the 1-in-2 homogenized sputum a further 1 in 100 in Sterile Nutrient Broth/Peptone water 2. Inoculate a 0.005 ml loopful of the dilution on to each culture plate. Blood Agar Heated blood (chocolate) agar Fildes digest agar Sabouraud dextrose agar OR Malt extract agar 3. The inoculum should be spread confluently over half of the plate and streaked out over the other half 4. Place 50 g 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 g amoxycillin disk onto confluently seeded area of Fildes digest agar. INCUBATION 1. Incubate one set of Blood Agar, Heated blood (chocolate) agar, Fildes digest agar at 370C for 18-24 hrs in humid air plus 5-10% CO2, and reincubate if the colonies are then still small and indistinct. 2. 2nd set of each agar should be incubated at 370C for 2-4 days anaerobically. 3. Incubate one set of Sabouraud dextrose agar OR Malt extract agar at 35370C for 2 days. 4. And 2nd set of Sabouraud dextrose agar OR Malt extract agar at 280C for 10 days. OBSERVATION Observe for growth and zone of inhibition. INTERPRETATION 1. The growth on the whole area of the plate of 25 or more colonies of the same potential pathogen will then indicate that 106 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.

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3. Sensitive to the respective antibiotics if zone of inhibition observed. 4. Resistant to the respective antibiotics if no zone of inhibition observed. 4. 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.

5. 6. 7. 8. 9. 10. 11.

References 1. Basic Laboratory in Clinical Bacteriology, J.Vandepitte, K.Engbaek, P.Piot, C.C.Heuck, W.H.O. Geneva, 1991. 2. Diagnostic Microbiology, Bailey & Scott, 9th Ed., Mosby 1994, Ellen Jo Baron, Lance R. Peterson. 3. Practical Medical Microbiology, Mackie & McCartney, Vol 1, Microbial

Infections, 13th Ed., Churchill Livingston 1978, Edited by J.P. Duguid, B.P. Marmion, R.H.A. Swain. Practical Medical Microbiology, Mackie & McCartney, Vol. 2, 13th Ed., Churchill Livingston 1989, Edited by J.G. Collee, J.P. Duguid, A.G. Fraser, B.P.Marmion. Handbook of Microbiological Media, Ronald M. Atlas, Lawrence C.Parks, 2nd Ed., 1997. Tuberculosis; A Clinical HandBook, 1st Edition 1995, Edited by L.I. Lutwick. Clinical Diagnosis & Management by Laboratory Methods, Todd, Sanford & Davidsohn, 17th Edition 1998, Edited by john Bernard Henry. Tuberculosis Case Finding and Chemotherapy, K, Toman, World Health Organisation, Geneva, 1979. Bombay Hospital Journal ; Drug Resistance in Tuberculosis; by Lina Deodhar et al. April 1999. Laboratory methods for clinical and public health-Mycobacteriology, U.S. Dept of health, education and welfare. Data on file: Tulip Diagnostics (P) Ltd.

Wound, Skin & Deep Sepsis


SUMMARY Wound 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. REQUIRED REAGENTS/MEDIAS l Blood agar base (AM1014/AM5014). OR l Blood agar base (Ready prepared media-250ml) (20500006). l Modified grams stain kit (20750020 / 20750021). l Stuart transport medium (AM1094/AM5094). l Mycostain kit and Acidfast decolorizer (20307100 / 20308500). l Immunofluorescent stain. l Nutrient broth (AM1077/AM5077). OR l Nutrient broth (Ready prepared media-5ml) (20500006). l Peptone Water (AM1079/AM5079). OR l Peptone Water (Ready prepared media-5ml) (20572006). l MacConkey Agar (AM1061/AM5061). OR l MacConkey Agar (Ready prepared media-250ml) (20550006/10550006). l CLED Agar (AM1026/AM5026). OR l CLED Agar (Ready prepared media-250ml) (20520006/10520006). l PNPG Blood Agar (0.43 gm p-nitrophenyl glycerol or blood agar containing 2-3 times extra agar). l Gram negative bacteria identification kit (20791001). l Staph identification kit (20792001). l Strept identification kit (20793001).

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Cooked meat medium (AM1030/AM5030) (BIS formula AM103011/AM503011)

REQUIRED MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Sterile plating loops (10l). 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 EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Microscope. 3. Autoclave. PROCEDURE a. Collection of sample: l Wear sterile gloves. l 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. l Replace the swab in its tube with care not to soil the rim. l It should be taken for testing within a hour or should be placed in a refrigerator at 4 0 C until testing or if it has to be transported it should be submitted in a tube of Stuart transport medium l Collect pus or exudate in a small screw capped bottle, a firmly stopperred tube or syringe or a sealed capillary tube. l 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. l Note: Pathogens grow in directly seed broth and in subcultures from it. l 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. l 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. l Homogenize in a tissue grinder with a little sterile Nutrient Broth/Peptone water broth.

Fluid Aspirates Aspirates 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 dialysis Many 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 Examination The 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 Examination Note the appearance of a specimen of pus or exudate on initial examination. Interpretation l 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 strawcoloured 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. Microscopy 1. Gram Stain l 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.

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And examine with oil-immersion.

Observation l 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 Gramnegative bacteria. Interpretation l 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 Gramnegative 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 film l Place approximately 100l 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. Observation l Carefully search for the presence of fungi, motile bacteria and uric acid crystals. Interpretation l 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.)

3. Ziehl-Neelsen method l 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. Culture 1) Inoculate the specimen on the following media l Blood Agar l MacConkey Agar or CLED Agar l Cooked Meat Broth l 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 g gentamicin disk on one plate. l 5 g metronidazole and 50 g neomycin on another blood agar plate. 3) Incubate l One plate with One unit benzyl penicillin and 10 g gentamicin aerobically air plus 5-10% CO2 at 370C + 20C, l Second plate with 5 g metronidazole and 50 g neomycin anaerobically in nitrogen / hydrogen plus 5-10% CO2 at 370C + 20C, 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.

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Observation Media Incubation Aerobically Blood Agar Anaerobically MaConkey Agar with sodium taurocholate Aerobically 1) Growth/No growth 2) Growth with or without lysis of RBC's Showing zones of inhibition or no zones of inhibition towards respective antibiotics disks Growth 1) Growth/No growth 2) Growth with or without lysis of RBC's Sensitivity Showing zones of inhibition or no zones of inhibition towards respective antibiotics disks

1) Growth/No growth Showing zones of inhibition or no zones of inhibition 2) Growth-Pink colonies or colourless towards respective antibiotics disks colonies 1) Growth/No growth 2) Growth-Pink colonies or Blue colourless colonies Growth/No growth

CLED Agar

Aerobically

Cooked Meat Broth PNPG Blood agar with 2-3 times the usual concentration of Agar

Aerobically

Aerobically

Growth/No growth

Identification The obtained cultures should be purified and subjected to following identification test. 1) Coagulase 2) Lance field grouping 3) Biochemical identification for Staph, Strept and Gram negative coliforms with commercially available biochemical identification test kit. Interpretation and Reporting A 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

mixed faecal and skin bacteria present', without giving identities or antibiotic sensitivities. References 1. Basic Laboratory in Clinical Bacteriology, J.Vandepitte, K.Engbaek, P.Piot, C.C.Heuck, W.H.O. Geneva, 1991. 2. Diagnostic Microbiology, Bailey & Scott, 9th Ed., Mosby 1994, Ellen Jo Baron, Lance R. Peterson. 3. Practical Medical Microbiology, Mackie & McCartney, Vol 1, Microbial Infections, 13th Ed., Churchill Livingston 1978, Edited by J.P. Duguid, B.P. Marmion, R.H.A. Swain. 4. Practical Medical Microbiology, Mackie & McCartney, Vol. 2, 13th Ed., Churchill Livingston 1989, Edited by J.G. Collee, J.P. Duguid, A.G. Fraser, B.P.Marmion. 5. Handbook of Microbiological Media, Ronald M. Atlas, Lawrence C.Parks, 2nd Ed., 1997. 6. Data on file: Tulip Diagnostics (P) Ltd.

Genital Tract Infections


Introduction The 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 infections, e.g gonorrhoea, syphilis and chlamydial infection, are common to bothsexes, there are usually differences in the presenting symptoms and the sites and the sites and methods of collection of specimens in these infections. Moreover,

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some other infections, e.g. Vaginitis and uterine sepsis, are confined to women. Genital infections in Women Summary These include urethritis, vaginitis, vaginosis, genital ulceration, cervicitis, uterine sepsis, salpingitis, ophoritis and the condition recognized as pelvic in inflammatory disease. REQUIRED REAGENTS/MEDIAS l Blood agar base (AM1014/AM5014). OR l 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). OR l 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 g metronidazole disk. l 50 g neomycin disk. l PPLO broth. l Horse serum. l Urea. l Phenol red indicator. REQUIRED MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Sterile plating loops (10l). 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 EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Microscope.

3. Autoclave. 4. Incubator with air plus 5% CO2 at 370C + 20C. 5. Anaerobic Incubator CO2 at 370C + 20C. PROCEDURE Collection 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. Microscopy 1. Gram Stain l Make a smear of the swab exudates on a clean scratch free glass slide l Stain by commercially available modified grams stain kit. Follow manufacturers instruction. l And examine with oil-immersion. Observation and Interpretation l Gram positive yeast cells- Candidosis l Gram positive hyphae- Pseudomycelium l Small Gram negative or Gram variable bacilli of diptheroid morphologyanaerobic vaginosis. l Gram negative diplococci from endocervical swab- gonococci infection. (Note:- Positive smears must be confirmed by culture). 2. Wet film. l Place approximately 100l 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.

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Observation and Interpretation Observe 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. Culture 1) Inoculate the specimen on the following media l Blood Agar 2) Place following antibiotics disks on to blood agar plate prior to incubation. l 5 g metronidazole and 50 g neomycin. 3) Incubate 0 0 l One plate in a humid aerobically air plus 5% CO2 at 37 C + 2 C . 0 0 l Second plate anaerobically with CO2 at 37 C + 2 C. Observation l 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.) Interpretation l 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 humid aerobically air plus 5% CO2 at 370C + 20C . (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 modified New York City selective medium for incubation at 35- 370C 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 20g 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

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 trachomatis l 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 Procedure 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. 1 l Express the exudate from the swab into 0.9 ml medium to obtain a 10 , dilution. 6 l Prepare further dilutions up to 10 .
l

Incubation 0 l Incubate at 37 C and examine for a colour change each day up to 7 days. Observation l With colour changes resulting from ureaplasma growth the medium remains clear. l Any turbidity is a sign of bacterial contamination which invalidates the result.

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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 men Summary The infections in men are mostly caused by the same organisms as in women, but are seldom asymptomatic. Collection of Specimens:1. Direct collection l 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

l l

examination. The examination of a chancre requires the careful collection of exudate and its preparation for darkground microscopy. 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. References 1. Basic Laboratory in Clinical Bacteriology, J.Vandepitte, K.Engbaek, P.Piot, C.C.Heuck, W.H.O. Geneva, 1991. 2. Diagnostic Microbiology, Bailey & Scott, 9th Ed., Mosby 1994, Ellen Jo Baron, Lance R. Peterson. 3. Practical Medical Microbiology, Mackie & McCartney, Vol 1, Microbial Infections, 13th Ed., Churchill Livingston 1978, Edited by J.P. Duguid, B.P. Marmion, R.H.A. Swain. 4. Practical Medical Microbiology, Mackie & McCartney, Vol. 2, 13th Ed., Churchill Livingston 1989, Edited by J.G. Collee, J.P. Duguid, A.G. Fraser, B.P.Marmion. 5. Handbook of Microbiological Media, Ronald M. Atlas, Lawrence C.Parks, 2nd Ed., 1997. 6. Data on file: Tulip Diagnostics (P) Ltd.

Gastrointestinal Infections
SUMMARY The 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. 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 REAGENTS l 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. or l 0.1% Peptone Water.

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l l l l l l l l l l l l l l l l l l

Nutrient agar (20570006/10570006 /AM1074/AM5074). or Urea agar. MacConkey agar (AM1061/AM5061). Blood agar (20500006 /AM1014/AM5014). 6% NaCl nutrient agar (AM1074, AM5074). Mannitol salt agar (AM1069, AM5069). Poly-myxin B. Selective medium of Holbrook and Anderson. Modified Grams Stain Kit (20750020/20750021). TCBS agar (AM1095/AM5095). Alkaline peptone water (AM1910/AM5910) . Oxidase reagent (20690040). Peptone water with phenol red and glucose (AM1080/AM5080). Peptone water with phenol red and sucrose (AM1080/AM5080). Alkaline peptone water (AM1910/AM5910). CLED-(AM1026/AM5026 and 20520006,10520006). Tellurite taurocholate gelatin agar. 1% sodium dimethyl-p-phenylenediamine monohydrochloride.

Faeces l 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 faeces 1) 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) Culturing Preparation of faecal suspension Dilute the portion of a solid specimen (0.3 gms) in 3 ml of Phosphate buffered saline or 0.1% Peptone Water. FOR CULTURING OF CAMPYLOBACTER l 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, incubate for 24 hours at 42-430C, and then subculture on to a plate of Campylobacter selective medium.) Incubation 0 l Incubate for 48 hours at 42-43 C under microaerophilic conditions 56% O2 , 7-10% CO2 in H2 or N2 . FOR CULTURING OF SALMONELLA AND SHIGELLA l 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 Broth l 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,

REQUIRED MATERIALS 1. 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 (10l). 5. Activated 2% glutaraldehyde solution. 6. Cedar wood oil. 7. Glass slide. REQUIRED EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens. 3. Autoclave. 4. Incubator at 37C2C. Collection of specimen l 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.

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Incubation 0 l Incubate aerobically for 18-24 h at 37 C. Observation l 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 BACTERIA INTRODUCTION If 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 PERFRINGENS Note: 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 AUREUS Plate 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 Incubate aerobically for 18-24 hrs at 370C 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 CEREUS Note:- 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 Incubation Incubate aerobically for 18-24 hrs at 370C. Observation Look for large rough pale colonies on MacConkey agar and blue colonies surrounded by a precipitate on Holbrook and Anderson's medium . Identification Identify them by their appearance in a Gram film with commercially available modified grams stain kit. FOR VIBRO PARAHAEMOLYTICUS Note:- Particularly in outbreaks attribute to raw sea fish and shellfish, plate a few loopfuls of a 1 in 10 suspension of faeces on 1) Thiosulphate citrate bile sucrose (TCBS) agar (AM1095/AM5095) and incubate aerobically for 18-24 hrs at 370C. 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 250C, then subculture on to a TCBS plate and incubate for 18-24 hrs at 370C. Observation Inspect the plates for large (2-5 mm) blue or green, (non-sucrose-fermenting) colonies. Identification Demonstrate 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.

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l l l l

Peptone water with phenol red and sucrose Alkaline peptone water with 8% NaCl Alkaline peptone water without 8% NaCl CLED

polyvalent sera for enterotoxigenic serotypes of E.coli. MICROSCOPY FOR PROTOZOA, CYSTS AND OVA Introduction Usually 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. Procedure l 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 enterocolitis Summary When 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. Procedure Make 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 PATHOGENS Introduction Culture of faeces for other intestinal pathogens should be attempted when the circumstances suggest they may be present. For Clostridium difficile Always examine for this organism in cases in which pseudomembranous colotis is suspected. The faeces may be tested directly for cytotoxin. For Vibro cholerae Introduction This 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. Procedure 1) Culture the faces in alkaline peptone water on TCBS agar and on tellurite taurocholate gelatin agar.

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 GASTROENTERITIS Most 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 COLI Summary There 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. Procedure 1) Plate a loopful of faecal suspension on a blood agar plate and MacConkey plate and incubate 18-24 h at 370C. 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 Summary Strains 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'. Procedure 1. Plate a loopful of faecal suspension on a blood agar plate and MacConkey plate and incubate 18-24 h at 370C. 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
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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 hydrophila Procedure l 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 . Observation l 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 shigelloides Inoculate on to MacConkey and DCA media. Observation Growth on MacConkey and DCA media, usually as pale, but sometimes pink colonies. Note: Distinguished from those of Shigella and Escherichia coli by their oxidasepositive reaction. For Yersinia enterocolitica Procedure 1) 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 the culture for up to 6 weeks at 40C. 2) Inoculate a loopful of the faecal suspension, and at weekly intervals

thereafter loopfuls of the selenite broth culture, on to plates of DCA or Yersinina selective agar base with Yersinina selective supplement. Incubation Incubate for 24 hrs at 320C. Observation Inspect 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 0 l It is motile at 22 C. 0 l Hydrolyses urea at 35 C. Reporting Antibiotic 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. References 1. Basic Laboratory in Clinical Bacteriology, J.Vandepitte, K.Engbaek, P.Piot, C.C.Heuck, W.H.O. Geneva, 1991. 2. Diagnostic Microbiology, Bailey & Scott, 9th Ed., Mosby 1994, Ellen Jo Baron, Lance R. Peterson. 3. Practical Medical Microbiology, Mackie & McCartney, Vol 1, Microbial Infections, 13th Ed., Churchill Livingston 1978, Edited by J.P. Duguid, B.P. Marmion, R.H.A. Swain. 4. Practical Medical Microbiology, Mackie & McCartney, Vol. 2, 13th Ed., Churchill Livingston 1989, Edited by J.G. Collee, J.P. Duguid, A.G. Fraser, B.P.Marmion. 5. Handbook of Microbiological Media, Ronald M. Atlas, Lawrence C.Parks, 2nd Ed., 1997. 6. Data on file: Tulip Diagnostics (P) Ltd.

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Urinary Tract Infections


Summary Samples 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 REAGENTS l 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) disk l Nalidixic acid (30 mg) disk l Ciprofloxacin (5 mg) disk l Nitrofurantin (50 mg) disk l Trimethoprim (2.5 mg) disk l Amoxcycillin (20 mg) + clavulante (10 mg) Amoxyclav (30 mg) disk l Cefaroxime (30 mg) disk l Gentamicin (10 mg) disk l Mcfarland 0.5 standard (20701040) l Modified Gram Stain Kit. REQUIRED MATERIALS 1. Sterile hand gloves. 2. Face mask. 3. Sterile plating loops(10l). 4. Activated 2% glutaraldehyde solution. 5. Cedar wood oil. 6. Glass slide. 7. Glass spreader. 8. Micropipette and tips (100l - 1000l). REQUIRED EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens. 3. Incubator at 370C+ 20C. 4. Autoclave. PROCEDURE COLLECTION OF SAMPLE Procedure 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 BACTERIA The pathogenic organisms can be identified as per the colour chart provided with the EASYBACT kit and the table mentioned below: Proteus Green Regular Blue/Grey Klebsiella Dull Large Mucous Green/Grey Candida White Regular Pink Pseudomonas Colourless Convex Blue/Green Streptococci Pink Regular Pink/Red Staphylococci Pink Regular Pink/Red

E. coli Colonies Medium Pink Regular 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.

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(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 TEST 2 Methods: 1) Direct Sensitivity 2) Indirect Sensitivity 1) Direct Sensitivity This 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 0 l Incubate the plates at 37 C+ 2 C for 18-24 hours. Observation Observe for zone of inhibition. Interpretation Interpret as resistant/sensitive. 2) Indirect Sensitivity This 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 l on each plate Muller Hinton Agar and spread it evenly with a sterile glass spreader l Dry the surface.

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) 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. For patients in hospital place following antibiotic disk. 1) Cefaroxime (30 mg) 2) Gentamicin (10 mg) Incubate the plates at 370C+ 20C for 18-24 hours.

Observations Observe for zone of inhibition. Interpretation Interpret 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'. References 1. Basic Laboratory in Clinical Bacteriology, J.Vandepitte, K.Engbaek, P.Piot, C.C.Heuck, W.H.O. Geneva, 1991. 2. Diagnostic Microbiology, Bailey & Scott, 9th Ed., Mosby 1994, Ellen Jo Baron, Lance R. Peterson. 3. Practical Medical Microbiology, Mackie & McCartney, Vol 1, Microbial Infections, 13th Ed., Churchill Livingston 1978, Edited by J.P. Duguid, B.P. Marmion, R.H.A. Swain. 4. Practical Medical Microbiology, Mackie & McCartney, Vol. 2, 13th Ed., Churchill Livingston 1989, Edited by J.G. Collee, J.P. Duguid, A.G. Fraser, B.P.Marmion. 5. Handbook of Microbiological Media, Ronald M. Atlas, Lawrence C.Parks, 2nd Ed., 1997. 6. Detection, Prevention and Management of urinary Tract Infections, C.M. Kunin, 4th Edition, 1987. 7. Data on file: Tulip Diagnostics (P) Ltd.

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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. 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. REQUIRED REAGENTS 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. Blood agar-20500006 (AM1014/AM5014). 7. Cooked meat broth. 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 MATERIALS 1. Sterile hand gloves. 2. Fresh sterile screw-capped containers. 3. Face mask. 4. Sterile plating loops(10l). 5. Activated 2% glutaraldehyde solution. 6. Cedar wood oil. 7. Glass slide. 8. Modified Fuchs-Rosenthal slide chamber. 9. Cover slip. REQUIRED EQUIPMENTS 1. Biosafety hood with Bunsen burner. 2. Microscope with oil immersion lens. 3. Autoclave. 4. Incubator with microaerophilic condition at 42C-43C. 5. Incubator at 37C2C. PROCEDURE COLLECTION OF SPECIMENS CSF The 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 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.) l The patient should lie on his side with his back overhanging the edge of a firm couch or bed. l His head, flexed forwards, should be on the same level as his sacrum, and his knees should be drawn up. l 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. l Anesthetize by the intra dermal and subcutaneous injection of a little local anesthetic. l 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. l Withdraw the stylet, check for first drop of CSF appear within a few seconds. l If no fluid appears replace and push the needle a little further on. l If the needle strikes bone withdraw a short distance and pressed in again in a different direction. l 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

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l l

l l

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 0 incubator at 37 C.

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 CHAMBER Introduction 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 mm3 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 thus the count per mm3 . Dilution CSF 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 ml Propan 1, 2 diol 2.5 ml Crystal violet 1% in water 1.5 ml Distilled water 100 ml Procedure 1) 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

Blood culture Blood for cultures should be collected at the same time as the CSF, if possible before antibiotics are given. LABORATORY EXAMINATION OF CSF Visual Examination Examined with the naked eye for the presence of turbidity and any sign of contamination with blood from the puncture wound. Interpretation l Normal CSF -If clear and colourless like water. l Previous cerebral haemorrhage -If yellow color . l Infected CSF - If turbid and blood contaminated . If turbid and blood contaminated, the specimen should be further examined for the following: l Cell count. l Gram film. l Culture. l Glucose and protein contents. l Presence of haemophilus, meningococcal or pneumococcal antigens. Cell count Microscopy l 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 l Normal CSF - 0-5 leucocytes/mm , mainly lymphocytes. 3 (Note: In neonates up to 30/mm , mainly polymorphs. ) 3 l Purulent meningitis -100-3000 leucocytes/ mm mostly polymorphs. 3 l Aseptic meningitis-10-500 leucocytes/mm , mostly lymphocytes, though polymorphs may predominate in the earliest acute stage of the illness. 3 l Tuberculous meningitis - 100-500 leucocytes/mm , mostly lymphocytes.

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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 specimens, multiply by the dilution factor to get the count per mm3. 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 CSF Centrifuge 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. Note When 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. Observation A 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. Note The 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 CSF Procedure Immediately after centrifugation of the CSF and the removal of some of the deposit

for the Gram film, Seed the remainder of the deposit, on to following culture media, e.g l A plate of blood agar. l A plate of heated-blood ('chocolate') agar for incubation in humid air plus 510 % CO2. l A tube of cooked meat broth. Note 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% CO2. Incubation Incubate 18-24 hrs at 370C+ 20C. Observation l 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 370C+ 20C. Biochemical Tests Test the supernatant from the Centrifuged CSF for its content of glucose and protein with commercially available glucose and protein detection kits. Interpretation l 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 Antigens A 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.

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Medical Microbiology

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Viral meningitis In the presence of aseptic meningitis isolate the virus from l the CSF, l a throat swab or a specimen of faeces, l and examine paired sera for viral antibodies. Note: Keep CSF at -700C until inoculation into cell cultures, Submit throat swab in viral transport medium for culturing of mumps virus. Faeces may be cultured for echo, coxsackie and polio virus.

Microscopy Observe the CSF under dark-ground illumination for motile leptospira. Culture Innoculate the CSF or Blood into Korthoff's or stuarts or Ellinghausen & McCullough's medium. Incubation and observation Incubate at 28C-30C and examine periodically up to 6 weeks before discarding it as negative. References 1. Basic Laboratory in Clinical Bacteriology, J.Vandepitte, K.Engbaek, P.Piot, C.C.Heuck, W.H.O. Geneva, 1991. 2. Diagnostic Microbiology, Bailey & Scott, 9th Ed., Mosby 1994, Ellen Jo Baron, Lance R. Peterson. 3. Practical Medical Microbiology, Mackie & McCartney, Vol 1, Microbial Infections, 13th Ed., Churchill Livingston 1978, Edited by J.P. Duguid, B.P. Marmion, R.H.A. Swain. 4. Practical Medical Microbiology, Mackie & McCartney, Vol. 2, 13th Ed., Churchill Livingston 1989, Edited by J.G. Collee, J.P. Duguid, A.G. Fraser, B.P.Marmion. 5. Handbook of Microbiological Media, Ronald M. Atlas, Lawrence C.Parks, 2nd Ed., 1997. 6. Data on file: Tulip Diagnostics (P) Ltd.

l l l

Tuberculous meningitis In suspect of tuberculous infection l Prepare a smear of centrifuged deposit of the CSF and examine in a ZiehlNeelsen 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 meninggitis In 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.

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 infection for detail procedure) 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. 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.

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References 1. Basic Laboratory in Clinical Bacteriology, J.Vandepitte, K.Engbaek, P.Piot, C.C.Heuck, W.H.O. Geneva, 1991. 2. Diagnostic Microbiology, Bailey & Scott, 9th Ed., Mosby 1994, Ellen Jo Baron, Lance R. Peterson. 3. Practical Medical Microbiology, Mackie & McCartney, Vol 1, Microbial Infections, 13th Ed., Churchill Livingston 1978, Edited by J.P. Duguid, B.P.

Marmion, R.H.A. Swain. 4. Practical Medical Microbiology, Mackie & McCartney, Vol. 2, 13th Ed., Churchill Livingston 1989, Edited by J.G. Collee, J.P. Duguid, A.G. Fraser, B.P.Marmion. 5. Handbook of Microbiological Media, Ronald M. Atlas, Lawrence C.Parks, 2nd Ed., 1997. 6. Data on file: Tulip Diagnostics (P) Ltd.

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Quantitative Analysis of culture media using Ecometric Method

Hand Book of Practicing Microbiologists

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. Requirements 1) 1 l nichrome loop 2) 5 ml vials of non selective medium 3) Soyabean Casein Digest Medium (AM1092/AM5092) 4) Tryptone Glucose Extract Broth (AM1102/AM5102) 5) Brain Heart infusion Broth (AM1017/AM5017) Procedure l 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 4 hours at 37o C + 2 o C for bacteria and 25 o C - 30 o 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:

Figure I l Charge 1l 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). Calculation l Refer table1 and note down values of absolute growth index end points for test and control l Calculate RGI using formula Relative growth Index = AGI test x 100 AGI control Table1 Absolute Growth Index (AGI) A1=5 A2=25 A3=45 A4=65 A5=85 B1=10 B2=30 B3=50 B4=70 B5=90 C1=15 C2=35 C3=55 C4=75 C5=95 D1=20 D2=40 D3=60 D4=80 D5=100

Limits l For efficiency activity RGI should be Minimum 70% l For inhibition activity RGI should be 0% References 1. 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 Standards-3rd 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.

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Vibrio

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l l

Bacillus Cereus Agar Base (AM1009/AM5009)

Baird Parker Agar Base (AM1011/AM5011)

BP Sulpha Supplement* (AS005)

Egg Yolk Emulsion* (A010)

Egg Yolk Tellurite Emulsion* (AS011)

Potassium Tellurite 3.5%* (AS023)


l l l l l l l l l l

Bile Esculin Agar (AM1012/AM5012)

Bismuth Sulphite Agar (AM1013/AM5013)

Blood Agar Base (AM1014/AM5014)

Brain Heart Infusion Agar (AM1016/AM5016)

Brain Heart Infusion Broth (AM1017/AM5017)

Brilliant Green Agar, Modified (AM1018/AM5018)

Sulpha Supplement* (AS027)


l l l

Brilliant Green Bile Broth 2% (AM1020/AM5020)

Chloramphenicol Yeast Glucose Agar* (AM1024/AM5024)

Colombia Blood Agar Base (AM1029/AM5029)

Staph-Strepto Supplement* (AS025)

Strepto Supplement* (AS026)


l l l l l l l l l

Cooked Meat Medium (AM1030/AM5030)

DNase Test Agar Base (AM10381)

Deoxycholate Citrate Agar (AM1031/AM5031)

Dextrose Tryptone Agar (AM1036/AM5036)

Dextrose Tryptone Broth (AM1037/AM5037)

Differential Reinforced Clostridial Broth (AM1038/AM5038)

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EC Broth (AM1039/AM5039)

Acid uric Flat S and h our SpoTe ermophilic r formers Bacillus c ereus Campylo bacter

Iron Sulp hite spore formers Listeria Mesophili (Aerobic) c Sporeformers Mesophi (Anaeroblic Sporeformers ic) Osmophil ic organis ms Proteolyti c microorg anisms Psychrotr ophic Org anisms Salmone lla Shigella

S treptococ cus/Entero coccus Total Plat e Count

Yeasts an d Moulds Yersinia e nterocolit i ca

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Vibrio
l l l l

Clostridiu m Coliforms

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Acid uric Flat S and our SpoThermophilic reformers Bacillus c ereus Campylo bacter

PRODUCTS
l l l l l l l l l l l l l

EMB Agar,Levine (AM1040/AM5040)

Fluid Lactose Medium (AM1042/AM5042)

Fluid Sabouraud Medium (AM1043/AM5043)

Fluid Selenite Cystine Medium (AM1044/AM5044)

Fluid Thioglycollate Medium (AM1045/AM5045)

Heart Infusion Agar (AM10481/AM50481)

Heart Infusion Broth (AM10482/AM50482)

Iron Sulphite Agar (AM10483/AM50483)

Karmali Campylobacter Agar Base (AM1049/AM5049)

Campylobacter Selective Supplement with Hemin (Karmali), Modified* (AS007)


l l l l l l l

Kligler Iron Agar (AM1050/AM5050)

Lactobacilli MRS Agar (AM1051/AM5051)

Lactose Broth (AM1052/AM5052)

Lauryl Tryptose Broth (AM1053/AM5053)

Listeria Identification Agar Base, PALCAM (AM1055/AM5055)

Listeria Selective Supplement *(AS018)


l l l

MacConkey Agar with Crystal Violet, NaCl and 0.15% Bile Salts (AM1058/AM5058)

MacConkey Broth with Neutral Red (AM1064/AM5064)

l l l l l

MacConkey Broth Purple with Bromocresol Purple (AM1063/AM5063)

Malonate Broth Ewing Modified (AM1065/AM5065)

Malt Agar (AM1066/AM5066)


l

Malt Extract Agar (AM1067/AM5067)

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l

Malt Extract Broth (AM1068/AM5068)

Hand Book of Practicing Microbiologists

Mannitol Motility Test Medium (AM10681/AM50681)

Iron Sulp hite spore formers Listeria Mesophi (Aerobic)lic Sporeformers Mesophil (Anaerob ic Sporeformers ic) Osmophi lic organis ms Proteolyt ic microo rganisms Psychrotr ophic Org anisms Salmone lla Shigella

S treptococ cus/Entero coccus Total Plat e Count Yeasts an d Moulds Yersinia e nterocolit ica

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Clostridiu m Coliforms

Staphyloc occus

S treptococ cus/Entero coccus Total Plate Count

Vibrio

Acid uric Flat S and our SpoThermophilic reformers Bacillus c ereus Campylo bacter

PRODUCTS
l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l

Mannitol Salt Agar (AM1069/AM5069)

MR-VP Medium (AM1071/AM5071)

Nutrient Agar (AM1074/AM5074)

Nutrient Agar pH 6.8 (AM1075/AM5075)

Nutrient Broth (AM1077/AM5077)

Plate Count Agar (AM1081/AM5081)

Potato Dextrose Agar (AM1082/AM5082)

Potato Dextrose Broth (AM1083/AM5083)

Reinforced Clostridial Agar (AM1085/AM5085)

Sabouraud Dextrose Agar (AM1087/AM5087)

Sabouraud Dextrose Broth (AM1088/AM5088)

Selenite F Broth (AM1089/AM5089)

Simmons Citrate Agar (AM1090/AM5090)

Skim Milk Agar (AM10901/AM50901)

Soyabean Casein Digest Agar (AM1091/AM5091)

Soyabean Casein Digest Medium (AM1092/AM5092)

SS Agar (AM1093/AM5093)

TCBS Agar (AM1095/AM5095)

Tetrathionate Broth Base, Hajna (AM1096/AM5096)

Triple Sugar Iron (TSI) Agar (AM1099/AM5099)

Tryptone Glucose Extract Agar (AM1101/AM5101)

Tryptone Glucose Extract Broth (AM1102/AM5102

Tryptone Phosphate Broth (AM1103/AM5103)

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Tryptone Water (AM1104/AM5104)

Iron Sulp hite spore formers Listeria Mesophil (Aerobic) ic Sporeformers Mesophil (Anaerob ic Sporeformers ic) Osmophili c organis ms Proteolyt ic microo rganisms Psychrotro phic Orga nisms Salmone lla Shigella

Yeasts an d Moulds Yersinia e nterocolit ica

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S treptococ cus/Entero coccus Total Plat e Count Vibrio


l l

PRODUCTS
l l l l

Urea Broth Base (AM1106/AM5106)

Urea 40%* (AS028)


l l

Violet Red Bile Agar (AM1107/AM5107)

Vogel Johnson Agar Base (AM1108/AM5108)

Potassium Tellurite 1%* (AS022)


l l l

XLD Agar (AM1112/AM5112)

Yeast Extract Agar (AM1113/AM5113)

Yeast Malt Agar (AM1114/AM5114)

Yersinia Selective Agar Base (AM1116/TM116005)

Yersinia Selective Supplement *(AS029)

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* - Store between 2-80C.

Acid uric Flat S and our SpoThermophilic reformers Bacillus c ereus Campylo bacter

Iron Sulp hite spore formers Listeria Mesophili (Aerobic) c Sporeformers Mesophi (Anaeroblic Sporeformers ic) Osmophi lic organis ms Proteolyt ic microo rganisms Psychrotr ophic Org anisms Salmone lla Shigella

Yeasts an d Moulds Yersinia e nterocolit ica


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uld

c activity

Proteoly ti

lla/Shig ella

occus

Campylo bacter

Coliform s

Lactoba cilli

Salmon e

Staphyl oc

Li s t eri monoca ytogene s

Strepto Enteroccoccus/ occus

Yeast & Mo

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Anaero Microo bic (Includirganisms ng Clos tridia) Bacill (and us Species t beareosher spore r ) Stand Count ard Plate
l l l l l l l l l l l l l l l l l l l l l

PRODUCTS

Azide Blood Agar Base (AM1006/AM5006)

Bacillus Cereus Agar Base (AM1009/5009)

Polymyxin B Selective Supplement* (AS021)

Egg Yolk Supplement* (AS010)

Baird Parker Agar Base (AM1011/AM5011)

B P Sulpha Supplement* (AS005)

Egg Yolk Emulsion* (A010)

Egg Yolk Tellurite Emulsion* (AS011)

Potassium Tellurite 3.5%* (AS023)

Bile Esculin Agar (AM1012/AM5012)

Bismuth Sulphite Agar (AM1013/AM5013)

Brain Heart Infusion Agar (AM1016/AM5016)

Brain Heart Infusion Broth (AM1017/AM5017)

Brilliant Green Agar Modified (AM1018/AM5018)

Sulpha Supplement* (AS027)

Brilliant Green Bile Broth 2% (AM1020/AM5020)

Chloramphenicol Yeast Glucose Agar (AM1024/AM5024)

Cooked Meat Medium (AM1030/AM5030)

Differential Reinforced Clostridial Broth (AM1038/AM5038)

EC Broth (AM1039/AM5039)

EMB Agar, Levine (AM1040/AM5040)

Endo Agar (AM1040/AM5040)

Fluid Lactose Medium (AM1042/AM5042)

Fluid Selenite Cystine Medium (AM1044/AM5044)

Iron Sulphite Agar (AM10483/AM50483)

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Yersinia enteroc olitica

128

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Dairy Analysis

APPLICATIONS
/Shigell a ul d
Application

Proteoly ti

Salmon ella

Coliform s

Lactoba cilli

Li s t eri monoca ytogene s

Staphyl ococcus

Yeast & Mo
l l l l l l

Campyl obacter

Stand Count ard Plate

PRODUCTS
l l l

Lactose Broth (AM1052/AM5052)

Lauryl Tryptose Broth (AM1053/AM5053)

Listeria Identification Agar Base, PALCAM, (AM1055/AM5055)

Listeria Selective Supplement* (AS018)


l l

MacConkey Agar with Crystal Violet, NaCl and 0.15%Bile Salts (AM1059/AM5059)
l l l

MacConkey Broth with Bromocresol Purple (AM1063/AM5063)

Malonate Broth, Ewing Modified (AM1065/AM5065)

Malt Agar (AM1066/AM5066)

Malt Extract Agar (AM1067/AM5067)


l l l l l l l

Mannitol Salt Agar (AM1069/AM5069)

MR-VP Medium (AM1071/AM5071)

Nutrient Agar (AM1074/AM5074)

Nutrient Broth (AM1077/AM5077)

Plate Count Agar (AM1081/AM5081)

Potato Dextrose Agar (AM1082/AM5082)

Potato Dextrose Broth (AM1083/AM5083)


l

Preston Selective Supplement (Campylobacter Selective Supplement IV, Modified) (AS231)


l l

Reinforced Clostridial Agar (AM1085/AM5085)

Anaero Microo bic (Includirganisms ng Clos tridia) Bacill (and us Species beareother spore rs)

Simmons Citrate Agar (AM1090/5090)

l l l l l l l l l

Skim Milk Agar (AM10901/AM50901)

Soyabean Casein Digest Agar (AM1091/AM5091)

Soyabean Casein Digest Medium (AM1092/AM5092)

SS Agar (AM1093/AM5093)

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Tetrathionate Broth Base, Hajna (AM1096/AM5096)

Hand Book of Practicing Microbiologists

Tomato Juice Agar (AM1097/AM5097)

Strepto Enteroccoccus/ occus

Yersinia enteroc olitica

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Dairy Analysis

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Application

Coliform s

ic activit

Campylo bacter

Proteoly t

Staphylo coccus

Lactoba cilli

Stand Count ard Plate

Microxpress
el l a Anaero Microo bic r (Includinganisms g Clostr idia) Bacill (and us Species beareother spore rs)
l l l l l l l l l l l l

PRODUCTS

Tomato Juice Agar, Special (AM1098/AM5098)

Triple Sugar Iron (TSI) Agar (AM1099/AM5099)

Tryptone Glucose Extract Agar (AM1101/AM5101)

Tryptone Phosphate Broth (AM1103/AM5103)

Tryptone Water (AM1104/AM5104)

Urea Agar Base, Christensen (AM1105/AM5105)

Urea 40%* (AS028)


l

Urea Broth Base (AM1106/AM5106)

Urea 40%* (AS028)


l l

Violet Red Bile Agar Base (AM1107/AM5107)

Vogel Johnson Agar Base (AM1108/AM5108)

Potassium Tellurite 1%* (AS022)


l l

XLD Agar (AM1112/AM5112)

Yersinia Selective Agar Base (AM1116/TM116005)

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Yersinia Selective Supplement* (AS029)

Liste ri monoca ytogene s

Salmon ella/Shi g

Strept Enteroococcus/ c oc c us

Yeast & Mould

Yersinia enteroc olitica

130

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APPLICATIONS
Microbial Limit Test
s Pseudo mona e ting ay
Application

Salmon ella

Staphylo coccus

Sterility Tes

Vitamin Ass
l l l l

PRODUCTS
l l l l l

Alternative Thioglycollate Medium (AM1900/AM5900)

Antibiotic Assay Medium A (No. 1, Seed Agar) (AM1002/AM5002)

Antibiotic Assay Medium C (No. 3, Assay Broth) (AM1003/AM5003)

Antibiotic Assay Medium No 11 (Neomycin, Erythromycin Assay Agar) (AM1004/AM5004)

Antifungal Assay Agar (AM10041/AM50041)

B12 Assay Agar using E.coli Mutant Culture (AM1007/AM5007 )

B12 Assay Medium (AM10071)

B12 Maintenance Medium for E.coli Mutant (AM1008/AM5008)


l

Baird Parker Agar Base (AM1011/AM5011)

B P Sulpha Supplement* (AS005)

Egg Yolk Emulsion* (AS010)

Egg Yolk Tellurite Emulsion* (AS011)

Potassium Tellurite 3.5%* (AS023)


l l

Bismuth Sulphite Agar (AM1013/AM5013)

Brilliant Green Agar Modified (AM1018/AM5018)

Sulpha Supplement* (AS027)


l

Cetrimide Agar Base (AM1022/AM5022)

Nalidixic Selective Supplement* (AS020)


l l l l l l

Deoxycholate Citrate Agar (AM1031/AM5031)

EMB Agar, Levine (AM1040/AM5040)

Fluid Lactose Medium (AM1042/AM5042)

Fluid Selenite Cystine Medium (AM1044/AM5044)

Fluid Thioglycollate Medium (AM1045/AM5045)

Glucose Yeast Extract Agar (AM1048/AM5048)


l l

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

Lactose Broth (AM1052/AM5052)

Hand Book of Practicing Microbiologists

MacConkey Agar with Crystal Violet,NaCl and 0.15% Bile Salts (AM1059/AM5059)

Antib Mediaiotic Assay

E. coli

To tal Ae Microbiarobic l Count Yeast Count and Mould

Lactoba cillus Co unt

Microxpress

Pharmaceutical Analysis

131

onas

l l ae

Sterility Testing

Pseudo m

Salmon e

Staphyl ococcus

Microxpress
Antib Mediaiotic Assay
l l l l l l l l l l l l l l l l l

PRODUCTS

MacConkey Broth with Bromocresol Purple (AM1063/AM5063)

Mannitol Salt Agar (AM1069/AM5069)

Nutrient Broth (AM1077/AM5077)

Peptone Water (AM1079/AM5079)

Potato Dextrose Agar (AM1082/AM5082)

Sabouraud Chloramphenicol Agar (AM1086/AM5086)

Sabouraud Dextrose Agar (AM1087/AM5087)

Selenite F Broth (AM1089/AM5089)

Soyabean Casein Digest Agar (AM1091/AM5091)

Soyabean Casein Digest Medium (AM1092/AM5092)

Tetrathionate Broth Base, Hajna (AM1096/AM5096)

Triple Sugar Iron (TSI) Agar (AM1099/AM5099)

Urea Broth Base (AM1105)

Urea 40%* (AS028)


l

Vogel Johnson Agar Base (AM1108/AM5108)

Potassium Tellurite 1%*(AS022)


l

XLD Agar (AM1112/AM5112)

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

* - Store between 2-80C.


Hand Book of Practicing Microbiologists

E. coli

To tal A Microbierobic al Coun t Yeast Count and Mould

Vitamin Assay

Lactoba cillus Co unt

132

APPLICATIONS
Application

Pharmaceutical Analysis

Microbial Limit Test

APPLICATIONS
Application

Water and Wastewater Analysis


Qualitative Tests Quantitative Tests
ter

Campylo bac

Clostridia

Klebsiella

Vibrio
l

PRODUCTS
l l l l

Aspargine Proline Broth (AM1005/AM5005)

Azide Blood Agar Base (AM1006/AM5006)

Baird Parker Agar Base (AM1011/AM5011)

BP Sulpha Supplement* (AS005)

Egg Yolk Emulsion* (A010)

EY Tellurite Enrichment* (AS011)

Potassium Tellurite 3.5%* (AS023)


l l l l l l l

Beef Extract Agar (AM10111/AM50111)

Bile Esculin Agar Base (AM1012/AM5012)

Bismuth Sulphite Agar (AM1013/AM5013)

Brain Heart Infusion Agar (AM1016/AM5016)

Brain Heart Infusion Broth (AM1017/AM5017)

Brilliant Green Agar Modified (AM1018/AM5018)

Sulpha Supplement* (AS027)


l l l

Brilliant Green Bile Broth 2% (AM1020/AM5020)

Cetrimide Agar Base (AM1022/AM5022)

Nalidixic Selective Supplement* (AS020)


l

Cetrimide Broth (AM1023/AM5023)

Corn Meal Agar (AM10301/AM50301)


l l l l l l l

Deoxycholate Citrate Agar (AM1031/AM5031)

Differential Reinforced Clostridial Broth (AM1038/AM5038)

EC Broth (AM1039/AM5039)

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

EMB Agar, Levine (AM1040/AM5040)

Hand Book of Practicing Microbiologists

Endo Agar (AM1041/AM5041)

MPN Pre sumptive Test MPN Con firmed Te st MPN Com pleted Te st Fecal Co liforms Different Nonfecaliation of Fecal & Coliform Coliphag e Detecti on Fecal Stre ptococci

Legionella Species Pseudom onas Pre-E nri Selectivechment/ Enrichme nt Selective Isolation Identifica Biochemi tion & cal Tests MPN Pre sumptive MPN Con firmed Staphyloc occus Total Plate Count

Yeasts an d Moulds Yersinia E nterocolit ica

Microxpress
Total coliform Salmonella/Shigella

133

Campylo bac

Clostridia

Klebsiella

Vibrio

PRODUCTS
l l l l l l l l

Fluid Lactose Medium (AM1042/AM5042)

Fluid Selenite Cystine Medium (AM1044/AM5044)

Kligler Iron Agar (AM1050/AM5050)

Lactose Broth (AM1052/AM5052)

Lauryl Tryptose Broth (AM1053/AM5053)

Legionella Agar Base (AM1054/AM5054)

Legionella Growth Supplement* (AS016)


l l l l l l l l l l l l l l l l l l l l l l

Legionella Selective Supplement* (AS017)

MacConkey Agar with Crystal Violet, NaCl and with 0.15% Bile Salts (AM1059/AM5059) MacConkey Agar without Crystal Violet and with 0.15% Bile Salts(AM1060/AM5060) MacConkey Agar without Crystal Violet, NaCl and with 0.5% Sodium Taurocholate (AM1061/AM5061)

MacConkey Broth with Neutral Red (AM1064/AM5064)

MacConkey Broth Purple W/Bromocresol Purple (AM1063/AM5063) Malonate Broth Ewing, Modified (AM1065/AM5065)

Malt Agar (AM1066/5066)

Malt Extract Agar (AM1067/5067)

Mannitol Motility Test Medium (AM10681/AM50681)

Mannitol Salt Agar (AM1069/AM5069)

MR-VP Medium (AM1071/AM5071)

Nutrient Agar pH 6.8 (AM1075/AM5075)

Nutrient Broth (AM1077/AM5077)

Plate Count Agar (AM1081/AM5081)

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

Hand Book of Practicing Microbiologists

Preston Agar Base (AM10831/AM50831)

MPN Pre sumptive Test MPN Con firmed Te st MPN Com pleted Te st Fecal Co liforms Different Nonfecaliation of Fecal & Coliform Coliphag e Detecti on Fecal Str eptococc i

Legionella Species Pseudom onas Pre-E nri Selectivechment/ Enrichme nt Selective Isolation Identifica ti Biochemic on & al Tests MPN Pre sumptive MPN Con firmed Staphyloc occus Total Plate Count

Yeasts an d Moulds Yersinia E nterocolit ic a

Microxpress
Total coliform Qualitative Tests Quantitative Tests Salmonella/Shigella
ter

134

APPLICATIONS
Application

Water and Wastewater Analysis

APPLICATIONS
Application

Water and Wastewater Analysis


Qualitative Tests
a Vibrio
l l l l l l l l l l l l l l l

Total coliform Quantitative Tests

Salmonella/Shigella

Campylo bac Clostridia

Klebsiella

PRODUCTS

Preston Selective Supplement (Campylobacter Selective Supplement IV, Modified) (AS0231)


l

Pseudomonas Agar Base (AM1084/AM5084)

Cetrinix Supplement* (AS008)


l l l l l l l l l l

CFC Supplement* (AS009)

Reinforced Clostridial Agar (AM1085/AM5085)

Selenite F Broth (AM1089/AM5089)

Simmions Citrate Agar (AM1090/AM5090)

Soyabean Casein Digest Agar (AM1091/AM5091)

Soyabean Casein Digest Medium (AM1092/AM5092)

SS Agar (AM1093/AM5093)

TCBS Agar (AM1095/AM5095)

Tetrathionate Broth Base, Hajna (AM1096/AM5096)

Triple Sugar Iron Agar (AM1099/AM5099)

Tryptone Glucose Extract Agar (AM1101/AM5101)

Tryptone Water (AM1104/AM5104)

Urea Agar Base, Christensen (AM1105/AM5105)

Urea 40%* (AS028)


l

Urea Broth Base (AM1106/AM5106)

Urea 40%* (AS028)


l l l

Violet Red Bile Agar (AM1107/AM5107)

XLD Agar (AM1112/AM5112)

Yeast Malt Agar (AM1114/AM5114)

Yeast Malt Broth (AM1115/AM5115)

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

Yersinia Selective Agar Base (AM1116/TM5116)

Hand Book of Practicing Microbiologists

Yersinia Selective Supplement* (AS029)

* - Store between 2-80C.

MPN Pre sumptive Test MPN Con firmed Te st MPN Com pleted Te st Fecal Co liforms Differenti Nonfecal ation of Fecal & Coliform Coliphag e Detecti on Fecal Str eptococc i

Legionell a Species Pseudom onas Pre-E nri Selectivechment/ Enrichme nt Selective Isolation Identifica ti Biochemic on & al Tests MPN Pre sumptive MPN Con firmed Staphyloc occus Total Plat e Count Yeasts an d Moulds Yersinia E nterocolit ic

Microxpress
ter

135

Brucella

Coliforms

Listeria

Pseudom onas

Salmone lla/Shigella

Staphyloc occi

Mycobac

Anaerobe s Clostridia (Including )

PRODUCTS
l l l

Azide Blood Agar Base (AM1006/AM5006)

Baird Parker Agar Base (AM1011/AM5011)

BP Sulpha Supplement* (AS005)

Egg Yolk Emulsion*(A010)

Egg Yolk Tellurite Emulsion* (AS011)

Potassium Tellurite 3.5%* (AS023)


l l l l

Bismuth Sulphite Agar (AM1013/AM5013)

Brain Heart Infusion Agar (AM1016/AM5016)

Brain Heart Infusion Broth (AM1017/AM5017)

Brilliant Green Agar Modified (AM1018/AM5018)

Sulpha Supplement* (AS027)


l

Cetrimide Agar Base (AM1022/AM5022)

Nalidixic Selective Supplement* (AS022)


l l l

Columbia Blood Agar Base (AM1029/AM5029)

Brucella Selective Supplement* (AS006)

Staph-Strepto Supplement* (AS025)

Strepto Supplement* (AS026)


l l l l l l

Cooked Meat Medium (AM1030/AM5030)

Corn Meal Agar (AM10301/AM50301)

DNase Test Agar Base (AM10381)

EMB Agar, Levine (AM1040/5040)

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

Hand Book of Practicing Microbiologists

Endo Agar (AM1041/AM5041)

General P urpose C ulture Media

Streptoco cci

Vibrio

Yeast & M oulds

Microxpress
terium

136

APPLICATIONS
Application

Veterinary Testing

APPLICATIONS
Application

Veterinary Testing
Mycobac terium Yeast & M oulds
l l l l l l l l l l l l l

Brucella

Coliforms

Listeria

Pseudom onas

Salmone lla/Shigella

Streptoco cci Vibrio

Anaerobe s Clostridia (Including )

PRODUCTS
l l l l l

Listeria Identification Agar Base PALCAM (AM1055/AM5055)

Fluid Selenite Cystine Medium (AM1044/AM5044)

Fluid Thioglycollate Medium (AM1045/AM5045)

Heart Infusion Agar (AM10481/AM50481)

Listeria Selective Supplement* (AS018)


l l l l

Liver Infusion Agar (AM10551/AM50551)

Liver Infusion Broth (AM10552/AM50552)

Lowenstein Jensen Medium Base (AM1057/AM5057)

MacConkey Agar Base (AM1058/AM5058)

Malt Agar (AM1066/AM5066)

Malt Extract Agar (AM1067/AM5067)

Mannitol Salt Agar (AM1069/AM5069)

MR-VP Medium (AM1071/AM5071)

Potato Dextrose Agar (AM1082/AM5082)

Pseudomonas Agar Base (AM1084/AM5084)

Cetrinix Supplement* (AS008)

CFC Supplement* (AS009)


l

Reinforced Clostridial Agar (AM1085/AM5085)

Sabouraud Dextrose Agar (AM1087/AM5087)

Sabouraud Dextrose Broth (AM1088/AM5088)

Selenite F Broth (AM1089/AM5089)

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

Hand Book of Practicing Microbiologists

Simmons Citrate Agar (AM1090/AM5090)

General P urpose C ulture Media

Staphyloc occi

Microxpress

137

Coliforms

Listeria

Mycobac

Pseudom onas

Staphyloc occi

Streptoco cci

Vibrio

Brucella

Anaerobe s Clostridia (Including )

PRODUCTS
l l l l l l l l l l

Soyabean Casein Digest Agar (AM1091/AM5091)

Soyabean Casein Digest Medium (AM1092/AM5092)

SS Agar (AM1093/AM5093)

TCBS Agar (AM1095/AM5095)

Tetrathionate Broth Base, Hajna (AM1096/AM5096)

Triple Sugar Iron (TSI) Agar (AM1099/AM5099)

Tryptose Agar (AM11041/AM51041)

Urea Agar Base, Christensen (AM1105/AM5105)

Urea 40%* (AS028)


l l

Violet Red Bile Agar (AM1107/AM5107)

Vogel Johnson Agar Base (AM1108/AM5108)

Potassium Tellurite 1%* (AS022)


l l l

XLD Agar (AM1112/AM5112)

Yeast Malt Agar (AM1114/AM5114)

Yeast Malt Broth (AM1116/AM5116)

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

* - Store between 2-8 C.


Hand Book of Practicing Microbiologists

General P urpose C ulture Media

Salmone lla/Shigel la

Yeast & M

Microxpress
terium oulds

138

APPLICATIONS
Application

Veterinary Testing

APPLICATIONS
Fermentation Process Brewery Analysis

Products for Brewery and Fermentation Analysis


i Agar Me dia Liquid M edia Coliform s Lactoba cill

Application

Microxpress PRODUCTS
l l l l l l l l l l l

Fluid Lactose Medium (AM1042/AM5042)

Lactobacillus MRS Agar (AM1051/AM5051)

MacConkey Agar with Crystal Violet, NaCl and 0.15%Bile Salts (AM1059/AM5059)

MacConkey Broth Purple with Bromocresol Purple (AM1063/AM5063)

Malt Agar (AM1066/AM5066)

WL Differential Agar (AM1109/AM5109)

WL Nutrient Broth (AM1110/AM5110)

Wort Agar (AM1111/AM5111)

Yeast Malt Agar (AM1114/AM5114)

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

* - Store between 2-8 C.

Hand Book of Practicing Microbiologists

139

Bactero i de s Bordete lla

E. coli

Haemop hilis Klebsie lla

Legione lla Listeria

Mycoba cteria Mycopla s ma Neisseri a Pseudo m on a s

Proteus Salmon ella

Shigella Staphyl ococcus Streptoc oc c u s

Vibrio

Microxpress
Brucella Ca mp Selec ylobacte tive Isola r tion Clostrid ia Coryneb acterium
C C C/I/S C S S C/N C/S C/N E S N E S C S N N N N N N N N N N N N S S
0

PRODUCTS

Anaerobic Agar (AM1000/AM5000)

Azide Blood Agar Base (AM1006/AM5006)

Bacteroides Bile Esculin Agar (AM1010/AM5010)

Bacteroides Selective Supplement* (AS001)

Baird Parker Agar Base (AM1011/AM5011)

B P Sulpha Supplement* (AS005)

Egg Yolk Emulsion* (AS010)

Egg Yolk Tellurite Emulsion* (AS011)

Potassium Tellurite 3.5%* (AS023)

Bismuth Sulphite Agar (AM1013/AM5013)

Blood Agar Base (AM1014/AM5014)

Bordet Gengou Agar Base (AM1015/AM5015)

Bordetella Selective Supplement* (AS004)

Brain Heart Infusion Broth (AM1017/AM5017)

Briliiant Green Agar Modified (AM1018/AM5018)

Sulpha Supplement* (AS027)

Brilliant Green Bile Agar (AM1019/AM5019)

Brilliant Green Bile Broth 2% (AM1020/AM5020)

Cetrimide Agar Base (AM1022/AM5022)

Nalidixic Selective Supplement* (AS020)

Cetrimide Broth (AM1023/AM5023)

Chloramphenicol Yeast Glucose Agar (AM1024/AM5024)


N N

CLED Agar with Andrade indicator (AM1026/AM5026)

CLED Agar with Bromothymol Blue (AM1027/AM5027)

Coagulase Mannitol Agar Base (AM1028/AM50258)

Columbia Blood Agar Base (AM1029/AM5029)

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

Hand Book of Practicing Microbiologists

C - Cultivation, E - Enrichment, I - Identification, N - Non-Selective and S - Selective Isolation. * - Store between 2-8 C.

Yeast & Moulds Yersinia enteroc olitica

140

APPLICATIONS
Application

Products for Medical and Research Institutes

APPLICATIONS
Application

E. co il

Proteus Salmon ella Shigella

Staphylo c oc c u s Streptoc occus Vib ior


E I C/S C/S

Legione ll a Listeria

PRODUCTS

Brucella Selective Supplement* (AS006) Campylobacter Selective Supplement with Hemin (Karmali) Modified* (AS007)

Staph-Strepto Supplement* (AS025)


E/C S C S C/S N N N C/S N E E N N E S

Strepto Supplement* (AS026)

Cooked Meat Medium (AM1030/AM5030)

Deoxycholate Citrate Agar (AM1031/AM5031)

Differential Reinforced Clostridial Broth (AM1038/AM5038)

EC Broth (AM1039/AM5039)

EMB Agar (AM10391/AM50391)

EMB Agar, Levine (AM1040/AM5040)

EM Broth (AM10401/AM50401)

Endo Agar (AM1041/AM5041)

Fluid Lactose Medium (AM1042/AM5042)

Fluid Sabouraud Medium (AM1043/AM5043)


E C C/S C E

Fluid Selenite Cystine Medium (AM1044/AM5044)

Fluid Thioglycollate Medium (AM1045/AM5045)

G.C Agar Base (AM1046/AM5046)

G.C Supplement* (AS012)


I E S I I I

Haemoglobin Solution* (AS014)

Kligler Iron Agar (AM1050/AM5050)

Lauryl Tryptose Broth (AM1053/AM5053)

Legionella Agar Base (AM1054/AM5054)

Legionella Growth Supplement* (AS016)


S
0

Legionella Selective Supplement* (AS017)

Listeria Identification Agar Base,PALCAM (AM1055/AM5055)

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

Hand Book of Practicing Microbiologists

C - Cultivation, E - Enrichment, I - Identification, N - Non-Selective and S - Selective Isolation. * - Store between 2-8 C.

Bactero ides Bordete lla Brucella Ca mp Sele ylobac ctive Iso ter lation Clostrid ia Coryneb acterium

Haemop hilis Klebsie l la

Mycoba cteria M y c o pl a sma Neisseri a Pseudo monas

Yeast & Moulds Yersinia enteroc olitica

Microxpress

Products for Medical and Research Institutes

141

E. co il

Mycoba cteria Mycopla s ma

Proteus Salmon ella Shigella

Staphylo c o c c us Streptoc occus

Vib ior

Microxpress
Bactero ides Bordete lla Brucella Ca mp Selec ylobacte tive Isola r tion Clostrid ia Coryneb acterium
C/N

PRODUCTS

Listeria Selective Supplement* (AS008)

Loeffler Medium Base (AM1056/AM5056)

Horse Serum* (AS015)


C/N

Lowenstein Jensen Medium Base (AM1057/AM5057)


N N N I I N N N N N N N N N

Gruft Mycobacterial Supplement* (AS013)

Mac Conkey Agar with Crystal Violet, NaCl and 0.15% Bile Salts (AM1059/AM5059)

MacConkey Agar without Crystal Violet, NaCl and with 0.5% Sodium Tauracholate (AM1061/AM5061)

MacConkey Broth Purple with Bromocresol Purple (AM1063/AM5063)

Malonate Broth Ewing Modified (AM1065/AM5065)

Malt Agar (AM1066/AM5066)

Haemop hilis Klebsie l la

Legione lla Listeria

Neisseri a Pseudo monas

C C C S I I C/S I

Malt Extract Agar (AM1067/AM5067)

Malt Extract Broth (AM1068/AM5068)

Mannitol Salt Agar (AM1069/AM5069)

MR-VP Medium (AM1070/AM5070)

Mycoplasma Agar Base (PPLO Agar) (AM1073/AM5073)

Mycoplasma Enrichment Supplement* (AS019)


C/N C/N S S

Horse Serum* (AS015)

Potato Dextrose Agar (AM1082/AM5082)

Potato Dextrose Broth (AM1083/AM5083)

Preston Agar Base (AM10831/AM50831)

Pseudomonas Agar Base (AM1084/AM5084)

Cetrinix Supplement* (AS008)


C

CFC Supplement* (AS009)

Reinforced Clostridial Agar (AM1085/AM5085)

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

Hand Book of Practicing Microbiologists

C - Cultivation, E - Enrichment, I - Identification, N - Non-Selective and S - Selective Isolation. * - Store between 2-80C.

Yeast & Moulds Yersinia enteroc olitica

142

APPLICATIONS
Application

Products for Medical and Research Institutes

APPLICATIONS
Application

E. coli

Haemop hilis Klebsie lla Legione lla Listeria

Mycoba cteria Mycopla s ma Neisseri a Pseudo monas Proteus Salmon ella Shigella

Staphylo c oc c u s Streptoc occus Vibrio


S C/N C/N I S C/N C/N C/S C S

Bactero ides Bordete lla

PRODUCTS

Sabouraud Chloramphenicol Agar (AM1086/AM5086)

Sabouraud Dextrose Agar (AM1087/AM5087)

Sabouraud Dextrose Broth (AM1088/AM5088)


E I S S I I I

Selenite F Broth (AM1089/AM5089)

Simmons Citrate Agar (AM1090/AM5090)

SS Agar (AM1093/AM5093)

TCBS Agar (AM1095/AM5095)


E I I I I

Tetrathionate Broth Base, Hajna (AM1096/AM5096)

Triple Sugar Iron (TSI) Agar (AM1099/AM5099)

Tryptic Digest Broth (AM1100/AM5100)


E I I I I I

Tryptone Phosphate Broth (AM1103/AM5103)

Tryptone Water (AM1104/AM5104)

Urea Agar Base, Christensen (AM1105/AM5105)

Urea 40%* (AS028)


I I I I

Urea Broth Base (AM1106/AM5106)

Urea 40%* (AS028)


N

Violet Red Bile Agar (AM1107/AM5107)

Vogel Johnson Agar Base (AM1108/AM5108)

Potassium Tellurite 1%* (AS022)


S S

XLD Agar (AM1112/AM5112)

Yeast Malt Agar (AM1114/AM5114)

Yeast Malt Broth (AM1115/AM5115)

Yersinia Selective Agar Base (AM1116/AM5116)

Yersinia Selective Supplement* (AS029)

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

Hand Book of Practicing Microbiologists

C - Cultivation, E - Enrichment, I - Identification, N - Non-Selective and S - Selective Isolation. * - Store between 2-80C.

Brucella Ca mp Selec ylobac tive Isolter ation Clostrid ia Coryneb acterium

Yeast & Moulds Yersinia enteroc olitica

Microxpress

Products for Medical and Research Institutes

143

ation

Haemolys is Test

Citrate Uti lization

Deoxyribo nuclease Test

Esculin H ydrolysis

Indole Pro duction

Lecithinas e Test

Malonate

Carbon U tiliz

M edia CarbohWithout ydrates

Media CarbohWith One ydrate

PRODUCTS
l l

Andrade Peptone Water(AM1001/AM5001)

Baird Parker Agar Base(AM1011/AM5011)

BP Sulpha Supplement* (AS005)

Egg Yolk Emulsion* (AS010)

Egg Yolk Tellurite Emulsion* (AS011)

Potassium Tellurite 3.5%* (AS023)


l l l l l l l l l

Bile Esculin Agar (AM1012/AM5012)

Blood Agar Base (AM1014/AM5014)

Brain Heart Infusion Agar (AM1016/AM5016)

Bromothymol Blue Lactose Agar (AM1021/AM5021)

Christensen Citrate Agar(AM1025/AM5025)

Columbia Blood Agar Base (AM1029/AM5029)

DNase Test Agar Base (AM10381)

Heart Infusion Agar (AM10481/AM50481)

Staph-Strepto Supplement* (AS025)

Strepto Supplement* (AS026)


l l l l l l l l l

Kligler Iron Agar (AM1050/AM5050)

Malonate Broth, Ewing Modified (AM1065/AM5065)

MR-VP Medium (AM1071/AM5071)

Peptone Water (AM1079/AM5079)

Peptone Water with Phenol Red (AM1080/AM5080)

Simmons Citrate Agar (AM1090/AM5090)

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

Hand Book of Practicing Microbiologists

* - Store between 2-8 C.

Media W One Carbith More Than ohydrate

Hydrog Producten Sulphide ion

Methyl Re d Test

Urease Te st

Microxpress
Carbohydrate Fermentation

Utilization

144

APPLICATIONS
Application

Products for Biochemical Tests

APPLICATIONS
Carbohydrate Fermentation
Application

Products for Biochemical Tests


ation ation uclease T est drolysis Carbon U tiliz Citrate Ut iliz Deoxyribo n Esculin H y Haemolys is Test Indole Pro duction Lecithinas e Test Malonate Utilization Methyl Re d Test
l l

M edia CarbohWithout ydrates

Media CarbohWith One ydrate

Media W One Carbith More Than ohydrate

PRODUCTS
l l l l

Soyabean Casein Digest Agar (AM1091/AM5091)

Triple Sugar Iron (TSI) Agar (AM1099/AM5099)

Tryptone Water (AM1104/AM5104)

Urea Agar Base, Christensen (AM1105/AM5105)

Urea 40%* (AS028)

Urea Broth Base (AM1106/AM5106)

Urea 40%* (AS028)

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

* - Store between 2-80C.

Hydrogen Productio Sulphide n

Urease Te s

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145

Bacillus

Pseudom onas

Staphyloc occi

Yeast & M oulds

PRODUCTS
l l

Anaerobic Agar (AM1000/AM5000)

Baird Parker Agar Base (AM1011/AM5011)

BP Sulpha Supplement* (AS005)

Egg Yolk Emulsion* (AS010)

Egg Yolk Tellurite Emulsion* (AS011)

Potassium Tellurite 3.5%* (AS023)


l l l

Brilliant Green Bile Broth 2%(AM1020/AM5020)

Bromothymol Blue Lactose Agar (AM1021/5021)

Cetrimide Agar Base (AM1022/AM5022)

Nalidixic Selective Supplement* (AS020)


l l l l l l l l l l l l l l l l

Cetrimide Broth (AM1023/5023)

CLED Agar with Andrade Indicator (AM1026/AM5026)

CLED Agar with Bromothymol Blue (AM1027/AM5027)

Cooked Meat Medium (AM1030/AM5030)

Differential Reinforced Clostridial Broth (AM1038/AM5038)

DNase Test Agar Base (AM10381)

EMB Agar, Levine (AM1040/AM5040)

Endo Agar (AM1041/AM5041)

Fluid Lactose Medium (AM1042/AM5042)

Fluid Thioglycollate Medium (AM1045/AM5045)

MacConkey Agar with Crystal Violet, NaCl and 0.15% Bile Salts (AM1059/AM5059)

MacConkey Agar without Crystal Violet and with 0.15% Bile Salts (AM1060/AM5060)

Malonate Broth, Ewing Modified (AM1065/AM5065)

Malt Agar (AM1066/AM5066)

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

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Malt Extract Agar (AM1067/AM5067)

Anaerobe s Clostridiu (including m)

Bacterial Counts

Enteroba cteriacea e

Sterility T est

Yeast & M ould Cou nts

146

APPLICATIONS
Application

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Cosmetic Analysis

APPLICATIONS
Application

Bacterial Counts

Enteroba cteriacea e

Pseudom onas

Sterility T est

Bacillus

PRODUCTS
l l l l l l l l

Malt Extract Broth (AM1068/AM5068)

Mannitol Salt Agar (AM1069/AM5069)

MR-VP Medium (AM1070/AM5070)

Nutrient Agar (AM1074/AM5074)

Nutrient Broth (AM1077/AM5077)

Plate Count Agar (AM1081/AM5081)

Potato Dextrose Agar (AM1082/AM5082)

Pseudomonas Agar Base (AM1084/AM5084)

Cetrinix Supplement* (AS008)

CFC Supplement* (AS009)


l l l l l l l l l l

Reinforced Clostridial Agar (AM1085/AM5085)

Sabouraud Dextrose Agar (AM1087/AM5087)

Sabouraud Dextrose Broth (AM1088/AM5088)

Soyabean Casein Digest Agar (AM1091/AM5091)

Soyabean Casein Digest Medium (AM1092/AM5092)

Triple Sugar Iron (TSI) Agar (AM1099/AM5099)

Vogel Johnson Agar Base (AM1108/AM5108)

Potassium Tellurite 1%* (AS022)


l

Yeast Malt Agar (AM1114/AM5114)

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

Hand Book of Practicing Microbiologists

* - Store between 2-8 C.

Anaerobe s Clostridiu (including m)

Staphyloc occi

Yeast & M oulds

Yeast & M ould Cou nts

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CAT. NO.

DESCRIPTION

CAT. NO. AM1003 AM5003

DESCRIPTION Antibiotic Assay Medium C (No 3) (Assay Broth) A medium for determining antibiotic potency by microbiological assay techniques as per USP/IP. 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. 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. Antibiotic Assay Medium No 11(Neomycin, Erythromycin Assay Agar) A medium for determining antibiotic potency by microbiological assay techniques as per USP. Antibiotic Assay Medium G (No.19) A medium for determining antibiotic potency by microbiological assay techniques as per USP/IP. Antibiotic Assay Medium No.32 A medium for determining antibiotic potency by microbiological assay techniques as per USP. Antifungal Assay Agar A medium recommended for assaying the antifungal activity. Artificial Sea Water A medium for cultivation of marine organisms. Ashbys Glucose Agar A medium for cultivation of Azotobacter species by using glucose as carbon source. Ashbys Mannitol Agar A medium for cultivation of Azotobacter species by using mannitol as carbon source. Asparagine Proline Broth A medium for cultivation of Pseudomonas aeruginosa by membrane filtrationtechnique.

DEHYDRATED CULTURE MEDIA AM5908 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.

AM50031

AM50032 AM1910 AM5910 AM5911 Alkaline Peptone Water A medium for enrichment of Vibrio species. Alkaline Peptone Water ISO A medium for enrichment of Vibrio species in compliance with ISO specification ISO / DIS 8914 : 1990. Alkaline Peptone Water BIS A medium for enrichment of Vibrio species in compliance with BIS specification IS 5887 (Part 5) 1976, reaffirmed 1986. Alternative Thioglycollate Medium A medium recommended for sterility testing of certain biological products, which may be turbid or viscous. Anaerobic Agar A medium recommended for cultivation of anaerobic microorganisms. Andrade Peptone Water A basal medium to which various carbohydrates may be added to study fermentation reactions, particularly of members of the Enterobacteriaceae. Andrade Peptone water BIS A 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) Antibiotic Assay Medium A (No 1) (Seed Agar) A medium for determining antibiotic potency by microbiological assay techniques as per USP/IP. AM1004 AM5004

AM1912 AM5912

AM500411

AM1900 AM5900

AM500412

AM1000 AM5000

AM10041 AM50041 AM50042

AM1001 AM5001

AM50043

AM10011

AM50044

AM1002 AM5002

AM1005 AM5005

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

148

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CAT. NO. AM1006 AM5006

DESCRIPTION Azide Blood Agar Base A medium for selective isolation and cultivation of Staphylococcus and Streptococcus species and when supplemented with blood for hemolytic reactions. Azide Dextrose Broth A selective medium for detection and cultivation of Streptococci in water, sewage, milk and other materials. B12 Assay Agar* (using E. coli mutant culture) A medium for microbiological assay of vitamin B12 by the cup plate or disc plate method. B12 Assay Medium* (using E. coli mutant and Lactobacillus leichmannii culture) A medium for determining vitamin B12 concentration by the microbiological assay technique. B12 Maintenance Media* (for E.coli Mutant) A medium for the propagation, cultivation and maintenance of E. coli mutant 113-3 D, ATCC 11105, which is the test organism in vitamin B12 assay. Bacillus Cereus Agar Base A selective medium for isolation and enumeration of Bacillus cereus. Polymixin B Selective Supplement* AS021 Egg Yolk Emulsion* AS010 Bacteroides Bile Esculin Agar A medium for selective isolation, cultivation and presumptive identification of Bacteroides fragilis group. Bacteroides Selective Supplement* AS001 Baird Parker Agar Base A medium with added supplements for selective isolation and enumeration of coagulase positive Staphylococci from clinical and non-clinical specimens. Egg Yolk Tellurite Emulsion* AS011 Egg Yolk Emulsion* AS010 Potassium Tellurite 3.5%* AS023 B P Sulpha Supplement*AS005

CAT. NO. AM101111 AM501111

DESCRIPTION Baird Parker Agar Base IP 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* AS010 Potassium Tellurite 1%* AS022 Baird Parker Agar Base USP 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* AS010 Potassium Tellurite 1%* AS022 Baird Parker Agar Base (Agar medium O) EP 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* AS010 Potassium Tellurite 1%* AS022 Baird Parker Agar Base (Agar medium O) BP 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* AS010 Potassium Tellurite 1%* AS022 Baird Parker Agar Base BIS 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* AS011 Egg Yolk Emulsion* AS010 Potassium Tellurite 3.5%* AS023 Beef Extract Agar A general purpose nutrient medium, which supports the growth of not particularly fastidious bacteria. Bile Esculin Agar A differential medium for isolation and presumptive identification of Group D Streptococci / Enterococci from foods.

AM50061

AM101112 AM501112

AM1007

AM10071

AM101113 AM501113

AM1008

AM101114 AM501114

AM1009 AM5009

AM1010 AM5010

AM101115 AM501115

AM1011 AM5011

AM10111 AM50111

AM1012 AM5012

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM501211

DESCRIPTION Bile Esculin Agar ISO A differential medium for isolation and presumptive identification of Yersinia enterocolitica in compliance with ISO specification ISO 10273, 1994. Bile Esculin Azide Agar A medium for selective isolation and presumptive identification of faecal Streptococci. Bile Salt Agar A medium used for the isolation and enumeration of enteric bacilli. Bile Salt Agar BIS A medium used for the isolation and enumeration of bile tolerant enteric bacilli in compliance with BIS specification IS:5887 (Part5) 1976 reaffirmed 1986. Bismuth Sulphite Agar A highly selective medium for isolation of Salmonella species, particularly S.typhi from clinical and non-clinical specimens. Bismuth Sulphite Agar IP (Twin Pack) A highly selective medium for isolation of Salmonella species, particularly S.typhi from sewage and other specimens in compliance with IP. Bismuth Sulphite Agar Medium USP A highly selective medium for isolation of Salmonella species, particularly S.typhi from sewage and other specimens in compliance with USP. Blood Agar Base 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. Bordet Gengou Agar Base A medium for the detection and isolation of Bordetella pertussis and Bordetella parapertussis from clinical specimens. Bordetella Selective Supplement* AS004 Brain Heart Infusion Agar A general-purpose medium for cultivation of a wide variety of

CAT. NO.

DESCRIPTION microorganisms including bacteria, yeasts and moulds.

AM1017 AM5017

AM501212

Brain Heart Infusion Broth 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. Brilliant Green Agar, Modified A medium for selective isolation of Salmonella other than S. typhi from clinical and non-clinical samples. Sulpha Supplement* AS027 Brilliant Green Agar, Modified IP A medium for selective isolation of Salmonella other than S. typhi from foods, dairy products and other samples in compliance with IP. Brilliant Green Agar, Modified USP A medium for selective isolation of Salmonella other than S. typhi from foods, dairy products and other samples in compliance with USP. Brilliant Green Agar, Modified ( Agar Medium L) EP A medium for selective isolation of Salmonella other than S. typhi from foods, dairy products and other samples in compliance with EP. Brilliant Green Agar, Modified ( Agar Medium L) BP A medium for selective isolation of Salmonella other than S. typhi from foods, dairy products and other samples in compliance with BP. Brilliant Green Bile Agar A medium for isolating, differentiating and enumerating coliform bacteria. Brilliant Green Bile Broth 2% 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. Bromothymol Blue Lactose Agar A medium for detection and isolation of pathogenic Staphylococci.

AM10121 AM50121 AM10122 AM50122

AM1018 AM5018

AM10181 AM50181

AM1013 AM5013

AM10182 AM50182

AM10131 AM50131

AM10183 AM50183

AM10132 AM50132

AM10184 AM50184

AM1014 AM5014

AM1019 AM5019

AM1015 AM5015

AM1020 AM5020

AM1016 AM5016

AM1021 AM5021

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM10211 AM50211

DESCRIPTION Buffered Peptone Water A medium used for pre-enrichment of injured Salmonella species from clinical and non- clinical specimens. Buffered Peptone Water BIS 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. Buffered Peptone Water ISO A medium used for pre-enrichment of injured Salmonella species from clinical and non- clinical specimens in compliance with ISO specification 657 9: 2002. Buffered Peptone Water with NaCl A medium used for pre-enrichment of injured Salmonella species from clinical and non- clinical specimens . Buffered Peptone Water with NaCl IP A medium used for pre-enrichment of injured Salmonella species from clinical and non- clinical specimens in compliance with IP. Buffered Peptone Water with NaCl EP A medium used for pre-enrichment of injured Salmonella species from clinical and non- clinical specimens in compliance with EP. Buffered Peptone Water with NaCl BP A medium used for pre-enrichment of injured Salmonella species from clinical and non- clinical specimens in compliance with BP. Campylobacter Agar Base A medium for selective isolation of Campylobacter species from clinical and non-clinical samples. Campylobacter Supplement-I (Blaser-Wang)* AS0061 Campylobacter Supplement-III (Skirrow)* AS0071 Candida Medium A medium for selective isolation and cultivation of Candida species. Carbohydrate Consumption Broth Base A medium for cultivation and differentiation of Listeria species. Cetrimide Agar Base A selective medium for isolation of Pseudomonas aeruginosa from

CAT. NO.

DESCRIPTION clinical and non-clinical specimens. Nalidixic Selective Supplement* AS020

AM10212 AM50212

AM10221 AM50221

Cetrimide Agar Base IP A selective medium for isolation of Pseudomonas aeruginosa from clinical and non-clinical specimens in compliance with IP. Cetrimide Agar Base Medium USP A selective medium for isolation of Pseudomonas aeruginosa from clinical and non-clinical specimens in compliance with USP. Cetrimide Agar Base ( Agar Medium N) EP A selective medium for isolation of Pseudomonas aeruginosa from clinical and non-clinical specimens in compliance with EP. Cetrimide Agar Base ( Agar Medium N) BP A selective medium for isolation of Pseudomonas aeruginosa from clinical and non-clinical specimens in compliance with BP. Cetrimide Broth A medium for selective cultivation of Pseudomonas aeruginosa. Chapman Stone Agar A medium for selective isolation of Staphylococci from clinical and non-clinincal specimens. Chloramphenicol Yeast Glucose Agar* A selective medium for isolation and enumeration of yeasts and moulds in milk and milk products. Christensen Citrate Agar A medium for differentiation of enteric pathogens and coliforms on the basis of citrate utilization. Chromogenic Coliform Agar* A chromogenic medium with sodium lauryl sulphate recommended for simultaneous detection of Escherichia coli and total coliforms in water and food samples. Chromogenic E. coli Agar* A chromogenic medium for detection and enumeration of Escherichia coli in foods without further confirmation on membrane filter or by indole reagent.

AM10222 AM50222

AM10213 AM50213

AM10223 AM50223

AM50214

AM10224 AM50224

AM50215

AM1023 AM5023 AM50231

AM50216

AM50217

AM1024 AM5024

AM50218

AM1025 AM5025

AM10251 AM50251

AM50219

AM50220

AM10252 AM50252

AM1022 AM5022

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

Microxpress

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CAT. NO. AM10253 AM50253

DESCRIPTION Chromogenic Enterococci Broth* A chromogenic medium for identification and differentiation of Enterococci from water samples. Chromogenic UTI Agar* A chromogenic differential medium for presumptive identification of microorganisms mainly causing urinary tract infections. C.L.E.D Agar with Andrade Indicator A medium for isolation, enumeration and presumptive identification of microorganisms from urine, giving good colonial differentiation. C.L.E.D Agar with Bromothymol Blue A medium for isolation, enumeration and presumptive identification of microoganisms from urine. Coagulase Mannitol Agar Base A medium with added plasma for isolation and differentiation of Staphylococci from clinical specimens or for classifying pure cultures. Columbia Blood Agar Base 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* AS006 Staph-Strepto Supplement* AS025 Strepto Supplement* AS026 Columbia Agar Base (Medium Q) EP A medium for detection of Clostridium perfringens from pharmaceutical products in compliance with EP. Columbia Agar Base (Medium Q) BP A medium for detection of Clostridium perfringens from pharmaceutical products in compliance with BP. Cooked Meat Medium A medium for cultivation and maintenance of aerobes and anaerobes, especially Clostridium species. Cooked Meat Medium BIS A medium for cultivation and maintenance of aerobes and

CAT. NO.

DESCRIPTION anaerobes, especially Clostridium species in compliance with BIS specification IS: 5887 ( Part2) 1976.

AM10254 AM50254

AM10301 AM50301 AM10302 AM50302

Corn Meal Agar A general purpose medium for cultivation of fungi. Czapek Dox Agar A semisynthetic medium for general cultivation of fungi, yeasts and soil bacteria. Deoxycholate Agar 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. Deoxycholate Citrate Agar A selective medium for isolation of enteric pathogens particularly Salmonella and Shigella species. Deoxycholate Citrate Agar IP A selective medium for isolation of enteric pathogens particularly Salmonella and Shigella species in compliance with IP. Deoxycholate Citrate Agar ( Agar Medium J) EP A selective medium for isolation of enteric pathogens in compliance with EP. Deoxycholate Citrate Agar ( Agar Medium J) BP A selective medium for isolation of enteric pathogens in compliance with BP. Dextrose Agar A medium used with or without blood for the cultivation of wide variety of microorganisms. Dextrose Broth A medium for the cultivation of fastidious microorganisms and for detecting gas production from enteric bacilli. Dextrose Peptone Agar A general-purpose medium for the cultivation of a wide variety of microorganisms.

AM1026 AM5026

AM10303 AM50303

AM1027 AM5027

AM1031 AM5031

AM1028 AM5028

AM10311 AM50311

AM1029 AM5029

AM10312 AM50312

AM50313

AM50291

AM1032 AM5032

AM50292

AM1033 AM5033

AM1030 AM5030

AM1034 AM5034

AM103011 AM503011

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

152

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CAT. NO. AM1035 AM5035

DESCRIPTION Dextrose Peptone Broth A general-purpose medium for the cultivation of a wide variety of microorganisms. Dextrose Tryptone Agar A medium for cultivation and enumeration of mesophilic and thermophilic aerobic microorganisms associated with food spoilage. Dextrose Tryptone Broth A medium for enrichment of mesophilic and thermophilic organisms associated with food spoilage. 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. 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. Differential Reinforced Clostridial Broth A medium for cultivation of Clostridia from water.

CAT. NO. AM503911

DESCRIPTION E. C. Broth ISO A medium recommended for selective enumeration of presumptive Escherichia coli by MPN technique in compliance with ISO specification ISO 7251 : 1993. EC D Agar A medium for selective isolation of coliforms, specially Escherichia coli in water and food by membrane filter technique. E.E.Broth , Mossel A medium for selective enrichment of Enterobacteriaceae in bacteriological examination of foods. E.E.Broth , Mossel (Enrichment Broth Medium E ) EP A medium for selective enrichment of Enterobacteriaceae in bacteriological examination of foods in accordance with EP. E.E.Broth , Mossel (Enrichment Broth Medium E ) BP A medium for selective enrichment of Enterobacteriaceae in bacteriological examination of foods in accordance with BP. E.E.Broth , Mossel with Oxgall A medium for selective enrichment of Enterobacteriaceae in bacteriological examination of foods. Eijkman Lactose Broth A medium for detection and differentiation of Escherichia coli from other coliforms on the basis of their ability to librate gas from lactose. EMB Agar A slightly selective and differential medium recommended for isolation, cultivation and differentiation of gram-negative enteric bacilli from clinical and non-clinical specimens. E M B Agar, Levine A slightly selective and differential medium for isolation, enumeration and differentiation of members of Enterobacteriaceae. E M B Agar, Levine IP (Levine Eosin-Methylene Blue Agar Medium) A slightly selective and differential medium for isolation, enumeration and differentiation of members of Enterobacteriaceae in compliance with IP.

AM1036 AM5036

AM503912

AM1037 AM5037

AM103913 AM503913

AM50371

AM503914

AM503915 AM50372

AM503916

AM1038 AM5038 AM503811

AM503917 Differential Reinforced Clostridial Broth Base ISO A medium for cultivation of Clostridia from water, in compliance with ISO specification ISO 6461- 1: 1986. DNase Test Agar Base A differential medium for detection of deoxyribonuclease activity to aid in the identification of bacteria and fungi isolated from clinical specimens especially Staphylococci. Double Sugar Agar, Russell A medium for differentiation of gram- negative enteric bacilli on the basis of their ability to ferment dextrose and lactose with or without gas formation. E. C. Broth A medium for detection of coliform bacteria at 350 C and E. coli at an elevated temperature of 44.5 or 45.50C. AM10391 AM50391

AM10381

AM50382

AM1040 AM5040

AM1039 AM5039

AM104011 AM504011

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM104012 AM504012

DESCRIPTION E M B Agar, Levine USP A slightly selective and differential medium for isolation, enumeration and differentiation of members of Enterobacteriaceae in compliance with USP. E M B Agar, Levine BIS A slightly selective and differential medium for isolation, enumeration and differentiation of members of Enterobacteriaceae in compliance with BIS specification IS:5401(1969) and IS 5887 ( Part 1) 1976. EMB Broth A slightly selective and differential medium recommended for isolation, cultivation and differentiation of gram-negative enteric bacilli from clinical and non-clinical specimens. Endo Agar A differential and slightly selective medium for detection of coliforms and other enteric microorganisms. Fluid Casein Digest Soya Lecithin Medium ( Twin Pack) IP A medium for detection of microbes on sanitized surfaces in compliance with IP. Fluid Casein Digest Soya Lecithin Medium ( Twin Pack) USP. A medium for detection of microbes on sanitized surfaces in compliance with USP. Fluid Lactose Medium A medium for detection of coliforms and the study of lactose fermentation by common bacteria. Fluid Lactose Medium IP A medium for detection of coliforms and the study of lactose fermentation by common bacteria in compliance with IP. Fluid Sabouraud Medium A liquid medium for sterility testing of moulds and lower bacteria in pharmaceutical preparations. Fluid Selenite Cystine Medium (Twin pack) A selective enrichment medium for isolation of Salmonellae from faeces, foods, pharmaceutical articles, water and other materials of sanitary importance.

CAT. NO. AM10441 AM50441

DESCRIPTION Fluid Selenite Cystine Medium IP (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 IP. Fluid Selenite Cystine Medium USP (Twin pack) A selective enrichment medium for isolation of Salmonellae from faeces, foods, pharmaceutical articles, water and other materials of sanitary importance incompliance with USP. 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. Fluid Thioglycollate Medium A medium for sterility testing of biologicals and cultivation of aerobes, anaerobes and microaerophiles. Fluid Thioglycollate Medium IP A medium for sterility testing of biologicals and cultivation of aerobes, anaerobes and microaerophiles in compliance with IP. Fluid Thioglycollate Medium USP A medium for sterility testing of biologicals and cultivation of aerobes, anaerobes and microaerophiles in compliance with USP. Fluid Thioglycollate Medium EP A medium for sterility testing of biologicals and cultivation of aerobes, anaerobes and microaerophiles in compliance with EP. Fluid Thioglycollate Medium BP A medium for sterility testing of biologicals and cultivation of aerobes, anaerobes and microaerophiles in compliance withBP. Fraser Broth Base A 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* AS0112 Fraser Supplement* AS0114

AM104013 AM504013

AM10442 AM50442

AM50443 AM10401 AM50401

AM1041 AM5041

AM1045 AM5045

AM10411 AM50411

AM10451 AM50451

AM10412 AM50412

AM10452 AM50452

AM1042 AM5042

AM10453 AM50453

AM10421 AM50421

AM10454 AM50454

AM1043 AM5043

AM50455

AM1044 AM5044

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM50456

DESCRIPTION Fraser Broth Base ISO A 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* AS0113 Fraser Supplement* AS0114 Fraser Secondary Enrichment Broth Base A medium for the isolation, cultivation and enrichment of Listeria monocytogenes from foods, environmental specimens and animal feeds. Fraser Enrichment Supplement* AS0111 Fraser Selective Supplement* AS0112 G.C.Agar Base A medium with various additives to isolate and cultivate Gonococci and other fastidious organisms. G.C.Supplement* AS012 Haemoglobin Powder Soluble* AS014 Glucose Agar A medium for determining the fermentation reaction of presumptive Enterobacteriaceae. Glucose Broth A medium in glucose fermentation studies where pH indicator is not desired. Glucose Salt Teepol Broth (Twin Pack) A medium for enrichment of Vibrio parahaemoliticus and marine isolates in compliance with BIS specification IS: 5887 (Part 5) 1976 reaffirmed 1986. Glucose Yeast Extract Agar A medium for enumeration of Lactobacilli in pharmaceutical preparations. Heart Infusion Agar A general purpose medium used in the cultivation of a wide range of microorganisms from a variety of clinical and non-clinical specimens.

CAT. NO. AM10482 AM50482 AM104821 AM504821

DESCRIPTION Heart Infusion Broth A medium used for cultivating fastidious microorganisms. Hektoen Enteric Agar A selective medium for detection and isolation of pathogenic intestinal bacteria including Salmonella and Shigella from clinical and non-clinical specimens. Hottinger Broth A medium for cultivation of less fastidious microorganisms and determination of indole production. Hoyle Medium Base A selective medium for isolation and differentiation of Corynebacterium diptheriae. Potassium Tellurite 3.5% * AS023 Inactivator Broth (Twin Pack) A medium for detection and isolation of bacterial contamination from clean surfaces and accidently contaminated raw material samples of pharmaceutical formulations. Iron Sulphite Agar A medium for the detection of thermophilic anaerobic organisms causing sulphide spoilage in foods. IUT Medium Base A medium for cultivation of Mycobacterium tuberculosis. Jensen s Broth A medium for detection and cultivation of nitrogen fixing bacteria. Jensens Medium A medium for detection and cultivation of nitrogen fixing bacteria. Karmali Campylobacter Agar Base 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 Kings Medium A Base A medium for isolation, cultivation and pigment production of Pseudomonas species.

AM50457

AM104822

AM104823 AM504823

AM1046 AM5046

AM104824 AM504824

AM50461

AM10483 AM50483

AM1047 AM5047

AM10484 AM50484 AM50485

AM10471 AM50471

AM10486 AM50486 AM1049 AM5049

AM1048 AM5048

AM10481 AM50481

AM50491

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM50492

DESCRIPTION Kings Medium B Base A medium for isolation, cultivation and pigment production of Pseudomonas species. Kirchner Medium Base, Modified A medium for cultivation of Mycobacterium tuberculosis. Horse Serum** AS 015 Kligler Iron Agar 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. Kligler Iron Agar ISO A 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. Lactobacillus MRS Agar* A medium for isolation and cultivation of all Lactobacillus species. Lactobacillus MRS Agar ISO* A medium recommended by ISO committee for isolation and enumeration of lactic acid bacteria from meat and meat products. Lactose Broth A medium for the detection of coliforms, as a pre-enrichment broth for Salmonellae and in the study of lactose fermentation in general. Lactose Broth IP A medium for the detection of coliforms in water, food and dairy products in compliance with IP. Lactose Broth (Broth Medium D) EP A medium for the detection of coliforms in water, food and dairy products in compliance with EP. Lactose Broth (Broth Medium D) BP A medium for the detection of coliforms in water, food and dairy products in compliance with BP.

CAT. NO. AM10524 AM50524

DESCRIPTION Lactose Broth BIS A medium for the detection of coliforms in water, food and dairy products in compliance with BIS specification IS: 5041 -1969. Lactose Sulphite Broth Base A medium recommended for detection and enumeration of Clostridium perfringens in pharmaceutical products. Lactose Sulphite Broth Base (Medium R) EP A medium recommended for detection and enumeration of Clostridium perfringens in pharmaceutical products in compliance with EP. Lactose Sulphite Broth Base ( Broth Medium R) BP A medium recommended for detection and enumeration of Clostridium perfringens in pharmaceutical products in compliance with BP. Lauryl Tryptose Broth A medium for detection of coliform organisms in materials of sanitary importance. Lecithin Agar* A medium for detection of bacterial contamination of surfaces in unprotected and protected areas. Lees Agar A medium for differentiation and enumeration of yoghurt starter bacteria. Lees Multi-differential Agar A medium for the cultivation, enumeration and identification of brewery bacteria. Legionella Agar Base A medium with added supplements for cultivation of Legionella species. Legionella Growth Supplement* AS016 Legionella Selective Supplement* AS017 Listeria Identification Agar Base (PALCAM) A medium with added supplement for selective isolation and identification of Listeria species. Listeria Selective Supplement (PALCAM)* AS018

AM50493

AM50525

AM1050 AM5050

AM50526

AM50501

AM50527

AM1053 AM5053

AM1051 AM5051 AM50511

AM10531 AM50531

AM50532 AM1052 AM5052

AM50533

AM10521 AM50521

AM1054 AM5054

AM10522 AM50522

AM10523 AM50523

AM1055 AM5055

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM105511 AM505511

DESCRIPTION Listeria Identification Broth Base (PALCAM) A medium with added supplement for selective isolation and identification of Listeria species. Listeria Selective Supplement (PALCAM)* AS018 Listeria Oxford Medium Base A medium for isolation of Listeria species from pathological specimens. Oxford Listeria Supplement* AS0201 Listeria Moxalactam Supplement* AS0171 Liver Infusion Agar A medium for the cultivation of Brucella and other pathogenic bacteria. Liver Infusion Broth A medium for the cultivation of highly fastidious microorganisms, particularly Brucella species and anaerobes. Loeffler Medium Base A medium with added horse serum for cultivation of Corynebacterium diphtheriae from clinical specimens and in pure cultures. Horse Serum** AS015 Lowenstein Jensen Medium Base A medium supplemented with eggs for cultivation and isolation of Mycobacterium species, especially M. tuberculosis. Gruft Mycobacterium Supplement* AS013 Egg Yolk Emulsion* AS010 Luria Agar A medium recommended for the cultivation and maintenance of recombinant strains of Escherichia coli and for routine cultivation of not particularly fastidious microorganisms. Luria Broth A medium recommended for the cultivation and maintenance of recombinant strains of Escherichia coli and for routine cultivation of not particularly fastidious microorganisms. Luria Bertani Agar, Miller A medium recommended for the cultivation and maintenance of Escherichia coli in molecular biology procedures and for routine

CAT. NO.

DESCRIPTION cultivation of not particularly fastidious microorganisms.

AM50574

AM105512 AM505512

Luria Bertani Broth, Miller A medium recommended for the cultivation and maintenance of Escherichia coli in molecular biology procedures and for routine cultivation of not particularly fastidious microorganisms. LPM Agar Base A medium for isolation and cultivation of Listeria monocytogenes from food and dairy products. Moxalactam Supplement* AS0182 Lysine Iron Agar A medium for differentiation of enteric organisms specially Salmonella species, based on their ability to decarboxylate or deaminate lysine and production of hydrogen sulphide. Lysine Medium Base A medium for isolation and enumeration of wild yeasts in pitching yeasts. Potassium Lactate 50% ( 10ml/Vial)* AS0211 MacConkey Agar Base A medium for studying carbohydrate fermentation reactions of coliforms by adding the desired carbohydrate. MacConkey Agar Base Medium USP A medium for studying carbohydrate fermentation reactions of coliforms by adding the desired carbohydrate in compliance with USP. MacConkey Agar with crystal violet, NaCl, and 0.15% Bile Salts A slightly selective and differential medium for the detection of coliforms and other enteric pathogens. MacConkey Agar with crystal violet, NaCl, and 0.15% Bile Salts IP A slightly selective and differential medium for the detection of coliforms and other enteric pathogens in compliance with IP. MacConkey Agar with crystal violet, NaCl, and 0.15% Bile Salts USP A slightly selective and differential medium for the detection of coliforms and other enteric pathogens in compliance with USP.

AM10575

AM10551 AM50551

AM10576 AM50576

AM10552 AM50552

AM10577 AM50577

AM1056 AM5056

AM1058 AM5058

AM1057 AM5057

AM10581 AM50581

AM10571 AM50571

AM1059 AM5059

AM50591 AM10572 AM50572

AM50592

AM50573

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM50593

DESCRIPTION MacConkey Agar with crystal violet, NaCl, and 0.15% Bile Salts (Agar Medium H) EP A slightly selective and differential medium for the detection of coliforms and other enteric pathogens in compliance with EP. MacConkey Agar with crystal violet, NaCl, and 0.15% Bile Salts (Agar Medium H) BP A slightly selective and differential medium for the detection of coliforms and other enteric pathogens in compliance with BP. MacConkey Agar without crystal violet and with 0.15% Bile Salts A medium for selective isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacteria. MacConkey Agar without crystal violet and with 0.5% Bile Salts A medium for isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacteria. MacConkey Agar without crystal violet, NaCl, and with 0.5% Sodium Taurocholate A medium for cultivation and differentiation of enteric bacteria and potentially pathogenic gram-positive organisms while restricting swarming of Proteus species. MacConkey Broth Double Strength with Neutral Red A medium for primary isolation of coliforms from large samples such as water and waste water. MacConkey Broth Double Strength with Neutral Red BIS 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 54011969. MacConkey Broth Purple with BCP 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. MacConkey Broth Purple with BCP IP 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.

CAT. NO. AM10632 AM50632

DESCRIPTION MacConkey Broth Purple with BCP(Broth Medium G) EP 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. MacConkey Broth Purple with BCP (Broth Medium G) BP 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. MacConkey Broth Purple with BCP BIS 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. MacConkey Broth Purple with BCP ISO 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. MacConkey Broth with Neutral Red A standard medium for primary isolation as well as presumptive identification of coli-aerogenes group in food and water. MacConkey Broth with Neutral Red BIS A standard medium for enrichment and enumeration of coliforms in compliance with BIS specification IS: 5887 (Part 1 and Part 2) 1976. Malonate Broth Ewing Modified A medium for differentiation of Enterobacteriaceae on the basis of malonate utilization. Malt Agar A medium for isolating and cultivating yeasts and moulds from food and dairy products and carrying stock cultures of the same. Malt Extract Agar A medium for enumeration, cultivation and isolation of yeasts and moulds.

AM50594

AM10633 AM50633

AM1060 AM5060

AM10634 AM50634

AM50601

AM1061 AM5061

AM10635 AM50635

AM1062 AM5062

AM1064 AM5064

AM10621 AM50621

AM10641 AM50641

AM1065 AM5065

AM1063 AM5063

AM1066 AM5066

AM10631 AM50631

AM1067 AM5067

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM1068 AM5068

DESCRIPTION Malt Extract Broth A medium for detection of yeasts, moulds and aciduric microorganisms. Mannitol Motility Test Medium A semisolid medium suitable for determining motility and mannitol fermentation.

CAT. NO.

DESCRIPTION Rosolic Acid* AS0232

AM506924

AM10681 AM50681

M-FC Broth Base A medium for detection and enumeration of faecal coliforms using membrane filter technique at higher temperature (44.5 0C) Rosolic Acid * AS0232 M-(HPC) Heterotrophic Plate Count Agar Base A medium for enumeration of heterotrophic microorganisms from water samples using membrane filter technique. M-(HPC) Heterotrophic Plate Count Broth Base A medium for enumeration of heterotrophic microorganisms from water samples using membrane filter technique. Middlebrook 7H9 Agar Base A medium for isolation, cultivation and sensitivity testing of Mycobacterium tuberculosis. Middlebrook OADC Growth Supplement* AS0181 Modified Rappaport Vassiliadis Medium A medium for selective enrichment of Salmonella from environmental and food specimens. Modified Rappaport Vassiliadis Medium for Water Testing BIS A medium for selective enrichment of Salmonella from water, environmental and food specimens in compliance with BIS specification IS 15187:2002. Modified Rappaport Vassiliadis Medium for Water Testing ISO A 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. Modified Teepol Broth( Twin Pack) ISO A medium for selective isolation and identification of enteric lactose fermenting bacteria in compliance with ISO specification ISO 9308-1: 1990. Modified Tergitol 7 Agar Base ISO A 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

AM506925 AM1069 AM5069 Mannitol Salt Agar A selective medium for isolation and identification of Staphylococcus aureus from clinical and non-clinical specimens. AM506926 AM10693 AM50693 Mannitol Salt Agar IP A selective medium for isolation and identification of Staphylococcus aureus from clinical and non-clinical specimens in compliance with IP. Mannitol Salt Agar Medium USP A selective medium for isolation and identification of Staphylococcus aureus from clinical and non-clinical specimens in compliance with USP. Mannitol Salt Broth A selective medium for isolation and identification of Staphylococcus aureus from clinical and non-clinical specimens. Marine Agar 2216 ( Zobell Marine Agar) A medium for isolation and enumeration of heterotrophic marine bacteria. AM506930 AM10692 AM50692 AM106921 AM506921 Marine Broth2216 ( Zobell Marine Broth) A medium for cultivation of heterotrophic marine bacteria. M-Endo Agar LES A medium for enumeration of coliforms in water using a two step membrane filter technique. M-Endo Broth A medium for estimation of coliforms in water using a membrane filter technique. M-FC Agar Base A medium for detection and enumeration of faecal coliforms using membrane filter technique at higher temperature (44.5 0C)

AM506927

AM10694 AM50694

AM506928

AM10695 AM50695

AM506929

AM 10691 AM50691

AM506931

AM106922 AM506922

AM506932

AM506923

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM506933

DESCRIPTION Motility Test Medium A medium for detection of bacterial motility.

CAT. NO.

DESCRIPTION with ISO specification ISO/DIS 7932,1993.

AM50733 AM506934 Motility Test Medium ( Edwards and Ewing) BIS A medium for testing of motility of enteric bacteria in compliance with BIS specifications IS: 5887 (Part 1 and Part5) 1976 reaffirmed, 1986. M R -V P Medium A medium for the differentiation of coli-aerogenes group by means of the Methyl Red and Voges Proskauer reactions. M R -V P Medium BIS 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. M R -V P Medium ISO 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. Mueller Hinton Agar A medium for antimicrobial disk diffusion susceptibility testing of common, rapidly growing bacteria by the Bauer-Kirby method. Mueller Hinton Broth A medium for determining the antimicrobial susceptibility of bacteria by the tube dilution method. Mycoplasma Agar Base (PPLO Agar) A medium when supplemented with nutritive enrichments for isolation and cultivation of Mycoplasma species. Mycoplasma Enrichment Supplement* AS019 Horse Serum** AS 015 Nitrate Broth BIS 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. Nitrate Broth ISO A medium for detection of nitrate reduction by bacteria. Also recommended for the enumeration of Bacillus cereus in compliance AM1076 AM5076

Nitrofurantoin Broth Base A medium for enrichment and isolation of Pseudomonas species. Nutrient Agar 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. Nutrient Agar IP A general purpose culture medium for the cultivation of microorganisms in compliance with IP. Nutrient Agar 1.5% ISO A 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. Nutrient Agar pH 6.8 A medium for the cultivation of a wide variety of bacteria and for the enumeration of microorganisms in water, sewage, faeces and other materials. Nutrient Agar with 1% Peptone A general purpose medium for the examination of water and dairy products. Nutrient Broth 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. Nutrient Broth with 1% Peptone A general purpose medium for the examination of water and dairy products. Nutrient Broth Medium IP A general purpose medium for aerobes in compliance with IP. Orange Serum Agar A medium for isolation, cultivation and enumeration of acid tolerant microorganisms present in citrus products.

AM1074 AM5074

AM1070 AM5070

AM10741 AM50741

AM10701 AM50701

AM50742

AM10702 AM50702

AM1075 AM5075

AM1071 AM5071

AM1072 AM5072

AM1073 AM5073

AM1077 AM5077

AM1078 AM5078

AM10731 AM50731

AM10781 AM50781 AM50782

AM10732 AM50732

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

160

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CAT. NO. AM50783

DESCRIPTION Orange Serum Broth A medium for cultivation of acid tolerant microorganisms present in citrus products. Pantothenate Assay Medium A medium for microbiological assay of Pantothenic acid or its salts using Lacttobacillus plantarum test. Pantothenate Assay Medium, AOAC A medium recommended by AOAC for microbiological assay of Pantothenic acid or its salts using Lacttobacillus plantarum ATCC 8014. Pantothenate Culture Agar A medium for culturing Lacttobacillus plantarum ATCC 8014. Pantothenate Inoculum Broth A medium used in preparation of inoculum for pantothenate assay. Peptone Water A non-selective medium for cultivating non-fastidious organisms and a base for carbohydrate fermentation media. Peptone Water BIS 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. Peptone Water with Phenol Red A non-selective medium for cultivating non-fastidious organisms and a base for carbohydrate fermentation media. Peptone Water with Phenol Red ISO A medium for studying fermentation ability of Yersinia enterocolitica, in compliance with ISO specification ISO /DIS 10273 : 1994. Phenol Red Dextrose Broth A medium for dextrose fermentation studies of microorganisms. Phenol Red Lactose Broth A medium for lactose fermentation studies of microorganisms.

CAT. NO. AM50804

DESCRIPTION Phenol Red Lactose Broth ISO A medium for lactose fermentation studies of coliforms in compliance with ISO specification ISO 9308 -1: 1990. Phenol Red Maltose Broth A medium for maltose fermentation studies of microorganisms. Phenol Red Mannitol Broth A medium for mannitol fermentation studies of microorganisms. Phenol Red Sorbitol Broth A medium for sorbitol fermentation studies of microorganisms. Phenol Red Sucrose Broth A medium for sucrose fermentation studies of microorganisms. Phenol Red Xylose Broth A medium for xylose fermentation studies of microorganisms. Phenylalanine Agar A medium for differentiation of Proteus and Providencia from other Enterobacteriaceae on the basis of deamination of phenylalanine. Pikovskayas Agar A medium for detection of phosphate solubilizing soil microorganisms. Plate Count Agar (Standard Methods Agar) A medium for obtaining microbial plate counts from milk and dairy products, foods, water and other materials of sanitary importance. Plate Count Agar BIS 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. PNY Medium A medium for cultivation and isolation of Lactobacillus species. Potato Dextrose Agar A medium for the cultivation and enumeration of yeasts and moulds from dairy and food products.

AM10784

AM50805

AM50806 AM10785

AM50807

AM10786

AM50808

AM10787

AM50809

AM1079 AM5079

AM108091 AM508091

AM10791 AM50791

AM108092 AM508092

AM1081 AM5081

AM1080 AM5080

AM10811 AM50811

AM50801

AM10812 AM50812 AM1082 AM5082

AM50802

AM50803

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM10821 AM50821

DESCRIPTION Potato Dextrose Agar USP A medium for the cultivation and enumeration of yeasts and moulds from dairy and food products in compliance with USP. Potato Dextrose Broth A medium for the cultivation and enumeration of yeasts and moulds from dairy and food products. Preston Agar Base 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 Pseudomonas Agar Base A medium with added supplements for the selective isolation of Pseudomonas species. CFC Supplement* AS009 Cetrinix Supplement* AS008 Pseudomonas Agar ( For Fluorescein) A medium for enhancement of fluorescein production by Pseudomonas species. Pseudomonas Agar ( For Fluorescein ) IP A medium for enhancement of fluorescein production by Pseudomonas species in compliance with IP. Pseudomonas Agar ( For Fluorescein ) USP A medium for enhancement of fluorescein production by Pseudomonas species in compliance with USP. Pseudomonas Agar ( For Pyocyanin ) A medium for enhancement of pyocyanin production by Pseudomonas species. Pseudomonas Agar ( For Pyocyanin ) IP A medium for enhancement of pyocyanin production by Pseudomonas species in compliance with IP. Pseudomonas Agar ( For Pyocyanin ) USP A medium for enhancement of pyocyanin production by Pseudomonas species in compliance with USP.

CAT. NO. AM108417 AM508417

DESCRIPTION Pseudomonas Isolation Agar Base A medium for selective isolation and identification of Pseudomonas aeruginosa from clinical and non-clinical specimens R-2A Agar A medium for enumeration of heterotrophic bacteria in treated potable water. R-2A Agar (Agar Medium S) EP A medium for enumeration of heterotrophic bacteria in treated potable water in compliance with EP. R-2A Agar (Agar Medium S) BP A medium for enumeration of heterotrophic bacteria in treated potable water in compliance with BP. 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 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 Reddys Differential Agar, Modified (Lactic Streak Agar) A medium for the qualitative and quantitative differentiation of lactic Streptococci. Reinforced Clostridial Agar A medium for the cultivation and enumeration of Clostridia and other anaerobes. Reinforced Clostridial Broth USP A medium for cultivation and enumeration of Clostridia and other anaerobes in compliance with USP. Reinforced Clostridial Broth ( Medium P) EP A medium for cultivation and enumeration of Clostridia and other anaerobes in compliance with EP.

AM1083 AM5083

AM10841 AM50841

AM10831 AM50831

AM50842

AM50843

AM1084 AM5084

AM10844

AM108411 AM508411

AM10845

AM108412 AM508412

AM50846

AM108413 AM508413

AM1085 AM5085

AM108414 AM508414

AM10851 AM50851

AM108415 AM508415

AM50852

AM108416 AM508416

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM50853

DESCRIPTION Reinforced Clostridial Broth ( Medium P ) BP A medium for cultivation and enumeration of Clostridia and other anaerobes in compliance with BP. Rogosa SL Agar A medium for cultivation of oral, vaginal and faecal Lactobacilli.

CAT. NO.

DESCRIPTION and aciduric bacteria in compliance with USP.

AM1088 AM5088

AM50854

Sabouraud Dextrose Broth A general-purpose medium for the cultivation of yeasts, moulds and aciduric bacteria. Sabouraud Dextrose Broth USP A general-purpose medium for the cultivation of yeasts, moulds and aciduric bacteria in compliance with USP. Sabouraud Dextrose Broth EP A general-purpose medium for the cultivation of yeasts, moulds and aciduric bacteria in compliance with EP. Sabouraud Glucose Agar with Antibiotics IP A medium for selective cultivation of yeasts and moulds in compliance with IP. Sabouraud Glucose Agar with Antibiotics (Agar Medium C) EP A medium for selective cultivation of yeasts and moulds in compliance with EP. Sabouraud Glucose Agar with Antibiotics (Agar Medium C) BP A medium for selective cultivation of yeasts and moulds in compliance with BP. Selenite F Broth (Twin pack) An enrichment medium for the isolation of Salmonella species from faeces, urine, water, foods and other materials of sanitary importance. Selenite F Broth IP (Twin pack) An enrichment medium for the isolation of Salmonella species from faeces, urine, water, foods and other materials of sanitary importance in compliance with IP. SIM Medium A medium for determination of H2S production, indole formation and motility of enteric bacteria. Simmons Citrate Agar A medium for the differentiation of gram-negative bacteria on the basis of citrate utilization.

AM50881 AM50855 Rogosa SL Broth A medium for cultivation of oral, vaginal and faecal Lactobacilli. Rose Bengal Agar Base A medium for selective isolation and enumeration of yeasts and moulds from environmental materials and food stuffs. Chloramphenicol Selective Supplement* AS00911 Rose Bengal Chloramphenicol Agar* A medium selective isolation and enumeration of yeasts and moulds from environmental materials and food stuffs . AM50884 AM1086 AM5086 AM50861 Sabouraud Chloramphenicol Agar* A medium for selective cultivation of yeasts and moulds. Sabouraud Chloramphenicol Agar*IP A medium for selective cultivation of yeasts and moulds in compliance with IP. Sabouraud Chloramphenicol Agar*EP A medium for selective cultivation of yeasts and moulds in compliance with EP. Sabouraud Chloramphenicol Agar*BP A medium for selective cultivation of yeasts and moulds in compliance with BP. Sabouraud Dextrose Agar A general-purpose medium for the cultivation of yeasts, moulds and aciduric bacteria. Sabouraud Dextrose Agar IP A general-purpose medium for the cultivation of yeasts, moulds and aciduric bacteria in compliance with IP. Sabouraud Dextrose Agar USP A general-purpose medium for the cultivation of yeasts, moulds AM50885 AM50882

AM50856

AM50883 AM10857 AM50857

AM50862

AM1089 AM5089

AM50863

AM10891 AM50891

AM1087 AM5087

AM50892

AM10871 AM50871

AM1090 AM5090

AM10872 AM50872

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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Hand Book of Practicing Microbiologists

CAT. NO. AM109011 AM509011

DESCRIPTION Simmons Citrate Agar BIS 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. Skim Milk Agar A medium for cultivation and enumeration of microorganisms in milk and dairy products. Soyabean Casein Digest Agar (Antibiotic Assay Medium No 36) (Tryptone Soya Agar) A general-purpose medium for the isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms. Soyabean Casein Digest Agar (Casein Soyabean Digest Agar) IP A general-purpose medium for the isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms in compliance with IP. Soyabean Casein Digest Agar Medium USP A general-purpose medium for the isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms in compliance with USP. Soyabean Casein Digest Agar (Agar Medium B) (Casein Soyabean Digest Agar) EP A general-purpose medium for the isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms in compliance with EP. Soyabean Casein Digest Agar (Agar Medium B) (Casein Soyabean 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. Soyabean Casein Digest Medium (Antibiotic Assay Medium No 37) (Tryptone Soya Broth) A general-purpose medium for the isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms. Soyabean Casein Digest Medium IP A general-purpose medium for the isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms in compliance with IP.

CAT. NO. AM10922 AM50922

DESCRIPTION Soyabean Casein Digest Medium USP A general-purpose medium for the isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms in compliance with USP.
Soyabean Casein Digest Medium (Broth Medium A) (Casein Soyabean Digest Broth)EP A general-purpose medium for the isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms in compliance with EP. Soyabean Casein Digest Medium (Broth Medium A) (Casein Soya Bean 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.

AM10901 AM50901

AM50923

AM1091 AM5091

AM50924

AM10911 AM50911

AM50925

Soyabean Casein Digest Medium, Sterile Powder

AM50925-5K A medium for evaluation of sterility in manufacturing process.

AM10912 AM50912

AM1093 AM5093

SS Agar A differential and selective medium for isolation of Salmonella and some Shigella species from clinical and non-clinical specimens. SS Agar, Modified A differential and selective medium for isolation of Salmonella and many Shigella species from clinical and non-clinical specimens. Staphylococcus Agar No. 110 (Gelatin Mannitol Salt Agar) A medium for selective isolation and differentiation of pathogenic Staphylococci. Streptococcus Selection Agar A medium for selective isolation and enumeration of Streptococci species . Streptococcus Selection Broth A medium for selective isolation and cultivation of Streptococci species . Stuart Transport Medium A medium for collecting, transporting and preserving microbiological specimens, particularly Neisseria species and other fastidious organisms.

AM10913 AM50913

AM50931

AM10932 AM50932

AM10914 AM50914

AM50933

AM1092 AM5092

AM50934

AM10921 AM50921

AM1094 AM5094

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM50941

DESCRIPTION TB Broth Base A medium for cultivation of Mycobacterium tuberculosis. TCBS Agar A selective medium for isolating and cultivating vibrios causing cholera and food poisoning from clinical and non-clinical specimens. Teepol Broth (Twin Pack ) A medium for selective isolation and identification of lactose fermenting enteric bacteria. Tergitol-7 Agar Base A selective medium for the detection of coliform bacteria and for early detection of E.coli in water analysis. TTC Solution 1%* AS0271 Tergitol-7 Agar Base BIS 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 Tergitol-7 Broth A selective medium for the detection of coliform bacteria and for early detection of E.coli in water analysis. Tetrathionate Brilliant Green Bile Broth A medium for isolation and identification of Salmonella. Tetrathionate Bile Brilliant green Broth IP A medium for isolation and identification of Salmonella in compliance with IP . Tetrathionate Bile Brilliant green Broth (Broth Medium I) EP A medium for isolation and identification of Salmonella in compliance with EP. Tetrathionate Bile Brilliant green Broth (Broth Medium I) BP A medium for isolation and identification of Salmonella in compliance with BP. Tetrathionate Broth Base, Hajna A medium for selective enrichment of Salmonella, particularly in

CAT. NO.

DESCRIPTION food and dairy products prior to isolation.

AM1095 AM5095

AM10961 AM50961

Tetrathionae Broth Base IP A medium for selective enrichment of Salmonella in compliance with IP. Tetrathionate Broth Base Medium USP A medium for selective enrichment of Salmonella in compliance with USP. Tinsdale Agar Base A medium for primary isolation and identification of Corynebacterium diptheriae. Diptheria Virulence Supplement (Part A & B)*AS0091 Tomato Juice Agar* A medium for cultivation and enumeration of Lactobacillus species. Tomato Juice Agar, Special* A medium for cultivation and enumeration of acidophilic microorganisms from clinical and non-clinical specimens. Triple Sugar Iron Agar A medium for identification of gram negative enteric bacilli on the basis of dextrose, lactose and sucrose fermentation and hydrogen sulphide production. Triple Sugar Iron Agar IP 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. Triple Sugar Iron Agar USP 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. Triple Sugar Iron Agar (Agar Medium M) EP 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.

AM10962 AM50962

AM509511

AM50963

AM10951 AM50951

AM1097 AM5097 AM1098 AM5098

AM10952 AM50952

AM50953

AM1099 AM5099

AM50954

AM10991 AM50991

AM50955

AM50956

AM10992 AM50992

AM50957

AM10993 AM50993

AM1096 AM5096

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM10994 AM50994

DESCRIPTION Triple Sugar Iron Agar BIS 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. Triple Sugar Iron Agar ISO 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 ISO specifications ISO/DIS 6579:1993. Tryptic Digest Broth (Fields Tryptic Digest Broth) A medium for cultivation of fastidious microorganisms. Tryptone Agar A general purpose medium for growth of non fastidious microorganisms. Tryptone Glucose Beef Extract Agar (TGB Agar) A medium for enumeration of bacteria in water, air, milk and dairy products. Tryptone Glucose Extract Agar A medium for enumeration of bacteria in water, air, milk and dairy products. Tryptone Glucose Extract Broth A general purpose enrichment medium for a wide variety of microorganisms. Tryptone Phosphate Broth A medium for enrichment of enteropathogenic E. coli. Tryptone Soya Agar with Lecithin and Tween 80* 80* ( 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. Tryptone Soya Yeast Extract Agar ISO A medium for confirmation of Listeria in Henrys light, in compliance with ISO specifications ISO 10560: 1993.

CAT. NO. AM51033

DESCRIPTION Tryptone Soya Yeast Extract Broth ISO A medium for confirmation of Listeria in Henrys light, in compliance with ISO specifications ISO 10560: 1993. Tryptone Water A medium for detection of indole production especially by coliforms. Tryptone Water without Sodium Chloride A medium for detection of Vibrio cholerae and Vibrrio parahaemolyticus in compliance with BIS specifications IS: 5887 (Part 5) 1976 reaffirmed 1986. Tryptose Agar A medium for cultivation of Brucella species and other fastidious microorganisms. Universal Beer Agar (UB Agar) A medium for cultivation of microrganisms related to brewing industry. Urea Agar Base, Christensen A medium with added urea for detection of urease production, particularly by the genus Proteus. Urea 40%* AS028 Urea Agar Base, Christensen BIS 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 Urea Agar Base, Christensen ISO A 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 Urea Broth Base A medium with added urea for the detection of urease production, to differentiate Proteus species from Salmonella and Shigella species. Urea 40%* AS028

AM1104 AM5104

AM50995

AM110411 AM510411

AM1100 AM5100 AM11001 AM51001

AM11041 AM51041

AM11042

AM11002 AM51002

AM1105 AM5105

AM1101 AM5101

AM1102 AM5102

AM11051 AM51051

AM1103 AM5103 AM11031 AM51031

AM51052

AM51032

AM1106 AM5106

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM51061

DESCRIPTION Urea Broth IP* A medium with added urea for the detection of urease production, to differentiate Proteus species from Salmonella and Shigella species in compliance with IP.

CAT. NO.

DESCRIPTION Staphylococcus aureus from clinical and non-clinical specimens in compliance with USP. Potassium Tellurite 1%* AS022

AM51083 AM1107 AM5107 AM11071 AM51071 Violet Red Bile Agar A selective medium for the detection and enumeration of coliforms . Violet Red Bile Agar BIS A selective medium for the detection and enumeration of coliforms in compliance with BIS specification IS : 5401- 1969. Violet Red Bile Broth A medium for detection and enumeration of coliforms from water and food. Violet Red Bile Glucose Agar. A medium for enumeration of Enterobacteriaceae. Violet Red Bile Glucose Agar without Lactose ISO A medium for enumeration of Enterobacteriaceae in compliance with ISO specification ISO 7402 : 1993 Violet Red Bile Glucose Agar with Lactose (Agar Medium F) EP A medium for enumeration of Enterobacteriaceae in compliance with EP. Violet Red Bile Glucose Agar with Lactose (Agar Medium F) BP A medium for enumeration of Enterobacteriaceae in compliance with BP. Vogel Johnson Agar Base w/o Tellurite A medium with the addition of potassium tellurite for isolation of Staphylococcus aureus from clinical and non-clinical specimens. Potassium Tellurite 1%* AS022 Vogel Johnson Agar Base w/o Tellurite IP 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 Vogel Johnson Agar Base w/o Tellurite USP A medium with the addition of potassium tellurite for isolation of AM1109 AM5109

Wilson Blair Agar Base A medium base for isolation and differentiation of Salmonella serotype Typhi. W.L. Differential Agar A medium for selective isolation and enumeration of bacteria encountered in breweries and industrial fermentation. W.L. Differential Broth A medium for selective isolation and enumeration of bacteria encountered in breweries and industrial fermentation. WL Nutrient Agar A medium for enumeration and cultivation of yeasts, moulds and bacteria encountered in brewing and fermentation processes. W.L. Nutrient Broth A medium for cultivating yeasts, moulds and bacteria encountered in brewing and fermentation processes. Wort Agar A medium for cultivation and enumeration of yeasts. X.L.D. Agar A moderately selective medium for isolation and differentiation of Salmonella and Shigella. X.L.D. Agar IP A moderately selective medium for isolation and differentiation of Salmonella andShigella.in compliance with IP. X.L.D. Agar Medium USP A moderately selective medium for isolation and differentiation of Salmonella and Shigella.in compliance with USP. X.L.D. Agar ( Agar Medium K) EP A moderately selective medium for isolation and differentiation of Salmonella and Shigella in compliance with EP.

AM51072

AM51091

AM51073

AM51092

AM51074

AM1110 AM5110

AM51075

AM1111 AM5111 AM1112 AM5112

AM51076

AM1108 AM5108

AM51121

AM51122 AM11081 AM51081

AM51123

AM11082 AM51082

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

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CAT. NO. AM51124

DESCRIPTION X.L.D. Agar (Agar Medium K) BP A moderately selective medium for isolation and differentiation of Salmonella and Shigella in compliance with BP. X.L.D. Agar, Modified ISO A moderately selective medium for isolation and differentiation of Salmonella and Shigella.in compliance with ISO specification ISO 6579:2002. Yeast and Mould Broth A medium for isolation and cultivation of yeasts and moulds. Yeast Extract Agar A highly nutritive medium for cultivation of a wide variety of bacteria. Yeast Malt Agar A medium for isolation and cultivation of yeasts, moulds and aciduric bacteria. Yeast Malt Broth A medium for isolation and cultivation of yeasts, moulds and aciduric bacteria. Yeast Mannitol Agar with 1.5% Agar A medium for cultivation, Isolation and enumeration of soil microorganisms like Rhizobium species. Yeast Mannitol Agar with Congo Red A medium for cultivation of soil microorganisms like Rhizobium species. Yeast Mannitol Broth A medium for cultivation of Rhizobium species. Yersinia Selective Agar Base A medium with added supplements for isolation and enumeration of Yersinia enterocolitica from clinical and non-clinical specimens. Yersinia Selective Supplement* AS029

CAT. NO.

DESCRIPTION

MEDIA BASES AB001 Agar Powder For use as a solidifying agent in microbiological culture media. AB002 Beef Extract Powder A nutritious extract used in the preparation of a variety of culture media for the cultivation of a wide variety of microorganisms. Brain Heart Infusion Powder A nutritious ingredient used in the preparation of a variety of culture media for the cultivation of a wide variety of fastidious microorganisms. 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. Fish Peptone A nutritious ingredient used in culture media for the cultivation of a variety of bacteria and fungi. Gelatin Peptone For use in culture media, especially for non-fastidious microorganisms. Also used in fermentation studies. Heart Infusion Powder A nutritious ingredient used in preparation of culture media for cultivation of fastidious microorganisms. Liver Extract Powder A nutritious extract used in the preparation of culture media for cultivation of fastidious bacteria. Malt Extract Powder An ideal ingredient used in the preparation of culture media for the cultivation of yeasts and moulds. Meat Extract Powder A nutritious extract used as a ingredient in the preparation of culture media for cultivation of a wide variety of fastidious microorganisms.

AM51125

AB003 AM51126

AM1113 AM5113

AB004

AM1114 AM5114

AB005

AM1115 AM5115

AB0051

AM51151

AB006

AM51152

AB007

AM51153

AB008

AM1116 AM5116

AB009

Store Dehydrated Media below 300C, * Store all Supplements and Media* between 2-80C, ** Store below -200C.

Store Media Bases below 300C.

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CAT. NO. AB010

DESCRIPTION Mycological Peptone (Peptone M) A nutritious ingredient used in the preparation of culture media for cultivation of yeasts and moulds. Peptone, Bacteriological A nutritious ingredient used in the preparation of culture media for the cultivation of a wide variety of bacteria and fungi. Peptone Special A nutritious enzymatic preparation used in the preparation of culture media for cultivation of a wide variety of fastidious microorganisms. Peptonized Milk A refined enzymatic digest of milk solids used in the preparation of culture media suitable for the cultivation of lactobacilli, yeasts and moulds. Proteose Peptone A nutritious ingredient used in the preparation of culture media employed for cultivation of a wide variety of microorganisms and in producing bacterial toxins. Soya Peptone Papaic digest of soyabean meal, used in the preparation of culture media for the cultivation of many fastidious microorganisms, including fungi. Tryptose An enzymatic hydrolysate of protein that can replace meat infusion used in the preparation of culture media for the cultivation of many fastidious microorganisms. Veg Peptone Veg 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. Yeast Extract Powder A nutritious extract used in the preparation of culture media for the cultivation of a wide variety of microorganisms.

CAT. NO.

DESCRIPTION

AB011

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. Bile Salts No.3* A selective agent used in microbiological culture media to inhibit gram-positive microorganisms. Bordetella Selective Supplement* An antibiotic supplement recommended for selective isolation of Bordetella pertussis. Bordet Gengou Agar Base AM1015/5015 BP Sulpha Supplement* An antibiotic supplement recommended for use in Baird Parker Agar Base to suppress the growth of Proteus species. Baird Parker Agar Base AM1011/5011 Brucella Selective Supplement, Modified* An antibiotic supplement recommended for selective isolation of Brucella species in milk. Columbia Blood Agar Base AM1029/5029 Campylobacter selective supplement (Blaser - Wang)* An antibiotic supplement recommended for selective isolation of Campylobacter species. Campylobacter Agar Base AM 50218 Campylobacter Selective Supplement with Hemin (Karmali), Modified* An antibiotic supplement recommended for the isolation of thermotolerant Campylobacter species. Karmali Campylobacter Agar Base AM1049/5049 Campylobacter supplement (Skirrow)* An antibiotic supplement recommended for selective isolation of Campylobacter species. Campylobacter Agar Base AM 50218

AB012

AS003

AB0121

AS004

AB013

AS005

AB014

AS006

AB015

AS0061

AB0151

AS007

AB016

AS0071

Store Media Bases below 300C.

* Store all Supplements, Agents and Enrichments between 2-80C, ** Store below -200C. Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits

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CAT. NO. AS008

DESCRIPTION Cetrinix Supplement* An antibiotic supplement recommended for the selective isolation of Pseudomonas species. Pseudomonas Agar Base AM1084/5084 CFC Supplement* An antibiotic supplement recommended for the selective isolation of Pseudomonas species. Pseudomonas Agar Base AM1084/5084 Chloramphenicol Selective Supplement* An antibiotic supplement recommended for yeasts and moulds. Rose Bengal Agar Base AM50856 Diptheria Virulence Supplement (Part A & Part B)* A selective supplement recommended for the isolation and presumptive identification of Corynebacterium diptheriae. Tinsdale Agar Base AM50693 Egg Yolk Emulsion (100 ml/vial)* A sterile, stabilized emulsion of egg yolk recommended for use in culture media. Bacillus Cereus Agar Base AM1009/5009 Baird Parker Agar Base AM1011/5011 Baird Parker Agar Base IP AM101111/501111 Baird Parker Agar Base USP AM101112/501112 Baird Parker Agar Base EP AM101113/501114 Baird Parker Agar Base BP AM101114/501114 Baird Parker Agar Base BIS AM101115/501115 Mannitol Salt Agar Base AM1069/5069 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 AM1011/5011 Baird Parker Agar Base BIS AM101115/501115 Fraser Enrichment Supplement* An 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

CAT. NO. AS0112

DESCRIPTION Fraser Selective Supplement* An 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 Fraser Broth Base AM 50455 Fraser Selective Supplement ISO* An 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 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 50455 Fraser Broth Base ISO AM 50456 G.C. Supplement* An enrichment and antibiotic supplement recommended for the selective isolation of pathogenic Neisseria. G.C.Agar Base AM1046/5046 Gruft Mycobacterium Supplement* An enrichment and antibiotic supplement recommended for the selective cultivation of mycobacteria. Lowenstein Jensen Medium Base AM1057/5057 Haemoglobin Powder Soluble (100 gms/Vial)* An enrichment supplement whose 2% w/v solution is autoclavable. GC Agar Base AM1046/5046 Horse Serum** An enrichment supplement recommended for isolation and cultivation of Mycoplasma, Trichomonas, Streptococcus species and C.diphtheriae. Mycoplasma Agar Base AM1073/5073 Loeffler Medium Base AM1056/5056 Kirchner Medium Base, Modified AM50493

AS009

AS0113

AS00911

AS0091

AS0114

AS010

AS012

AS013

AS011

AS014

AS0111

AS015

* Store all Supplements, Agents and Enrichments between 2-80C, ** Store below -200C.

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CAT. NO. AS0151

DESCRIPTION Lactic Supplement* A supplement for selective isolation of lactic acid bacteria in beer and brewing procedures. Raka Ray Agar Base (Lactic Acid Bacteria Selective Agar Base) AM10844 Raka Ray No. 3 Broth Base (Lactic Acid Bacteria Selective Broth Base) AM10845 Legionella Growth Supplement* An enrichment supplement used for enhancing growth of Legionella species. Legionella Agar Base AM1054/5054 Legionella Selective Supplement* An antibiotic supplement recommended for the selective isolation of Legionella species. Legionella Agar Base AM1054/5054 Listeria Moxalactam Supplement* An antibiotic supplement recommended for the isolation of Listeria monocytogenes from mixed flora. Listeria Oxford Medium Base AM105512/505512 Listeria Selective Supplement* An antibiotic supplement recommended for the selective isolation and identification of Listeria monocytogenes. Listeria Identification Agar Base (PALCAM) AM1055/5055 Listeria Identification Broth Base (PALCAM) AM105511/505511 Middlebrook OADC Growth Supplement* An enrichment supplement recommended for cultivation of Mycobacteria . Middlebrook 7H9 Agar Base A. AM506927 Moxalactam Supplement* An antibiotic supplement recommeded for selective isolation and cultivation of LPM Agar Base AM 10575

CAT. NO. AS020

DESCRIPTION Nalidixic Selective Supplement* An antibiotic supplement recommended for the selective isolation of Pseudomonas aeruginosa. Cetrimide Agar Base AM1022/5022 Oxford Listeria Supplement* An antimicrobial supplement recommended for selective isolation of Listeria species. Listeria Oxford Medium Base AM105512/505512 Polymixin B Selective Supplement* An antibiotic supplement recommended for the selective isolation of B. cereus. Bacillus Cereus Agar Base AM1009/5009 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 Potassium Tellurite 1% (10ml /vl)* A filter sterilized supplement recommended for the selective isolation of Staphylococci. Baird Parker Agar Base IP AM101111/501111 Baird Parker Agar Base USP AM101112/501112 Baird Parker Agar Base EP AM101113/501114 Baird Parker Agar Base BP AM101114/501114 Vogel Johnson Agar Base W/O Tellurite AM1108/5108 Vogel Johnson Agar Base W/O Tellurite IP AM11081/51081 Vogel Johnson Agar Base W/O Tellurite USP AM11082/51082 Potassium Tellurite 3.5% (10ml/vl)* A filter sterilized supplement recommended for the selective isolation of Staphylococci. Baird Parker Agar Base AM1011/5011 Baird Parker Agar Base BIS AM 101115/501115 Hoyle Medium Base AM 104823/504823 Preston Selective Supplement* (Campylobacter Selective Supplement IV, Modified) A supplement recommended for the selective isolation of Campylobacter species. Preston Agar Base AM 10831/50831

AS0201

AS016

AS021

AS017

AS0211

AS0171

AS022

AS018

AS0181

AS023

AS0182

AS0231 AS019 Mycoplasma Enrichment Supplement* An enrichment and antibiotic supplement recommended for the selective isolation of Mycoplasma species. Mycoplasma Agar Base AM1073/5073

* Store all Supplements, Agents and Enrichments between 2-80C, ** Store below -200C.

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CAT. NO. AS0232

DESCRIPTION Rosolic Acid (0.1 gms per vial)* A supplement recommended for selective isolation of coliform bacteria. M-FC Agar Base AM506923 MFC Broth Base AM506924 Sodium Deoxycholate* A sodium salt of deoxycholic acid, used in culture media to inhibit gram-positive microorganisms. Staph-Strepto Supplement* An antibiotic supplement recommended for the selective isolation of Streptococcus species. Columbia Blood Agar Base AM1029/5029 Strepto Supplement* An antibiotic supplement recommended for the selective isolation of Streptococcus species. Columbia Blood Agar Base AM1029/5029 Sulpha Supplement* An antibiotic supplement recommended for the selective isolation of Salmonella species. Brilliant Green Agar Modified AM1018/5018 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 506932 Tergitol 7 Agar Base AM10951/50951 Tergitol 7 Agar BaseBIS AM10952/50952 Urea 40% (5ml /vl)* A filter sterilized supplement for detection of urease activity. Urea Agar Base, Christensen AM1105/5105 Urea Agar Base, Christensen BIS AM11051/51051 Urea Agar Base, Christensen ISO AM51052 Urea Broth Base AM1106/5106 Yersinia Selective Supplement* An antibiotic supplement recommended for the selective isolation of Yersinia Enterocolitica. Yersinia Selective Agar Base AM1116/5116

CAT. NO.

DESCRIPTION

READY TO USE WATER QUALITY TESTING KITS 20409001 PA coliform Test Kit* For the detection of presence or absence of coliform bacteria from water samples. 20410001 Rapid Coliform Test Kit** For rapid detection of E.coli and coliforms from water samples on the basis of enzyme substrate reaction. Rapid Enterococci Test Kit** For rapid identification and differentiation of enterococci from water samples. Rapid H2S Test Kit* For simultaneous detection of Salmonella, Vibrio species, Citrobacter and E.coli from water samples.

AS024

AS025

20420001

20430010 AS026

AS027

PRESUMPTIVE IDENTIFICATION TESTS 20440010 PYR Test Kit** PYR Test to differentiate between group A Streptococci and Enterococci. 20450010 Presumptive Identification Test Kit for E.coli** Test for fluorescence detection of E.coli on the basis of enzyme substrate reaction. Test Kit for E.coli** Test for presumptive identification of E.coli on the basis of enzyme substrate reaction and Indole test. Test Kit for Esculin Hydrolysis** Test for detection of Esculin Hydrolysis. Test Kit for MR-VP** Test for detection of acid and acetoin production and for differentiation of Enterobacteriaceae. Test Kit for Phenylalanine Deamination** Test for differentiation of Proteus, Providencia from enteric bacilli.

AS0271

20460010

20470010

AS028

20480010

20490010

AS029

READY PREPARED MEDIA 20501006 Alkaline Peptone Water A medium for enrichment of microorganisms.
* Store at R.T., ** Store betweeb 2-80C.

* Store all Supplements, Agents and Enrichments between 2-80C, ** Store below -200C.

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CAT. NO. 20502006

DESCRIPTION BHI Broth A highly nutritious general purpose liquid medium for cultivation of variety of fastidious and non-fastidious microorganisms, including aerobic and anaerobic bacteria. Bile Broth A medium for cultivation of members of the Enyerobacteriaceae. Blood Agar Base** A non-selective general-purpose medium to which blood may be added for use in isolation and cultivation fastidious organisms and detecting hemolytic activity. Brain Heart Infusion Agar** A general-purpose medium for cultivation of a wide variety of microorganisms including bacteria, yeasts and moulds. C.L.E.D Agar with Andrade Indicator** A medium for isolation, enumeration and presumptive identification of microorganisms from urine, giving good colonial differentiation. Columbia Blood Agar Base** A basal medium for preparation of blood and chocolate agar and for various selective and identification media in isolating and cultivating fastidious microorganisms. E M B Agar, Levine** A slightly selective and differential medium for isolation, enumeration and differentiation of members of Enterobacteriaceae. Glucose Broth A medium used for cultivation and fermentation studies of microorganisms. Glucose Phosphate Broth A medium for differentiation of coli- aerogenes group by means of MethylRed and Voges Proskauer reactions. Hartley Broth A medium for cultivation of wide variety of bacteria from blood specially fastidious Streptococci and Corynebacterium diptheriae.

CAT. NO. 20540006 10540006

DESCRIPTION Mac Conkey Agar with Crystal violet, NaCL, and 0.15% Bile Salts** A slightly selective and differential medium for the detection of coliforms and other enteric pathogens. Mac Conkey Agar without crystal violet, NaCL, and with 0.5% Sodium Taurocholate** A medium for cultivation and differentiation of enteric bacteria and potentially pathogenic gram-positive organisms while restricting swarming of Proteus species. Mueller Hinton Agar** A medium for antimicrobial susceptibility testing of common, rapidly growing microorganisms by the Bauer-Kirby method. Nutrient Agar** 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 Nutrient Broth A general purpose medium for cultivation of bacteria. Peptone Water A non selective medium for cultivating non- fastidious organisms. Plate Count Agar(Standard Methods Agar)** A medium for obtaining microbial plate counts from milk and dairy products, foods, water and other materials of sanitary importance. Potato Dextrose Agar** A medium for cultivation and enumeration of yeasts and moulds from dairy and other food products. Sabouraud Dextrose Agar** A general-purpose medium for cultivation of yeasts, moulds and aciduric bacteria. Selenite Broth An enrichment medium for the isolation of Salmonella species from faeces, urine, water, foods and other material of sanitary importance. Simmons Citrate Agar** A medium for differentiation of gram-positive bacteria on the basis of citrate utilization.

20550006 20503006 10550006

20500006 10500006

20560006 10560006

20510006 10510006

20570006 10570006

20520006 10520006

20571006

20521006 10521006

20572006

20580006 10580006

20530006 10530006

20590006 10590006

20531006

20600006 10600006

20532006

20601006

20533006

20610006 10610006

* Store at R.T., ** Store betweeb 2-80C.

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CAT. NO. 20620006 10620006

DESCRIPTION Soyabean Casein Digest Agar (Antibiotic Assay Medium No 36) (Tryptone Soya Agar)** A general purpose medium for isolation and cultivation of a wide variety of fastidious and non-fastidious microorganisms. 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. SS Agar** A differential and selective medium for isolation of Salmonella and some Shigella species from clinical and non-clinical specimens. Triple Sugar Iron Agar** A medium for identification of gram negative enteric bacilli on the basis of dextrose, lactose and sucrose fermentation and hydrogen sulphide production. Violet Red Bile Agar** A selective medium for the detection and enumeration of coliforms. X.L.D Agar** A moderately selective medium for isolation and differentiation of Salmonella and Shigella.

CAT. NO. 20105300 20105600

DESCRIPTION Mueller Hinton Agar* A medium for antimicrobial susceptibility testing of common and rapidly growing microorganisms by the disk diffusion technique. Nutrient Agar* 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. Sabouraud Dextrose Agar* A medium for cultivation of yeasts, moulds and aciduric bacteria. SS Agar* A medium for differential and selective isolation of Salmonella and some Shigella species from pathological specimens and suspected foodstuffs.

20621006

20101300 20101600

20103300 20622006 10622006

20106300

20630006 10630006

20640006 10640006 20650006 10650006

MYCOBACTERIOLOGY 20308500 Acid Fast Decolorizer* Pre-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-MTB** For the determination of adenosine deaminase activity in serum, plasma & biological fluids. Catalase Detection Kit** For differentiation of isoniazide resistant strains of M. tuberculosis and M. gastri from genus mycobacterium based on catalase activity. Combicult** Combipack of solid and liquid medium for mycobacterium tuberculosis isolation. Lyfectol** Mucolytic, disinfectant, specimen pretreatment and buffering system for AFB staining and culture. Mycocult** Ready to use L J. solid medium for Mycobacterium tuberculosis isolation.

BLOOD CULTURING SYSTEMS 20660700 BHI-Supplemented with 0.05% SPS** 20660200 For qualitative detection of microorganisms in blood. 20661700 20662700 20670700 20670200 INSTAPREP 20104300 20104600 Glucose Broth - Supplemented with 0.05% SPS** For qualitative detection of microorganisms in blood.

20403020

20303001 Soyabean Casein Digest Broth- Supplemented with 0.05% SPS** For qualitative detection of microorganisms in blood. 20301012 CLED Agar* A medium for isolation and differentiation of urinary pathogens on the basis of lactose fermentation. 20304006 20102300 20102600 MacConkey Agar* A medium for selective isolation and differentiation of coliforms and other enteric pathogens.

* Store at R.T., ** Store betweeb 2-80C.

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Hand Book of Practicing Microbiologists

CAT. NO. 20314006

DESCRIPTION Mycocult PY** Ready to use L.J solid medium with sodium pyruvate for isolation of Mycobacterium bovis.

CAT. NO.

DESCRIPTION M.tuberculosis with a nitrate reductase assay using proportion method.

20404003 20307100 Mycostain** Acid fast stain set for screening of M.tuberculosis and M.leprae. 20402100 20406003 Mycovue NRA** LJ slants with nitrate substrate for detection of nitrate reduction by M. tuberculosis. Niacin Drop Test** For differentiation of M. tuberculosis from M.bovis based on niacin production. Nitrate Reduction Kit** For determination of nitrate reduction in cultures. Novachrom** Rapid two step cold AFB stain. 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. Sensicult Primary** Primary drug containing L-J media panel for MTB sensitivity tests. 20720025 20305201 Sensicult Secondary** (6 drugs) Secondary drug containing L-J media panel for MTB sensitivity tests. 20730040 20305202 Sensicult Secondary** (10 drugs) Secondary drug containing L-J media panel for MTB sensitivity tests. 20740040 20407001 Sensivue Primary** Drug susceptibility (primary anti-tubercular drugs) test for M.tuberculosis with a nitrate reductase assay using proportion method. Sensivue Secondary** Drug susceptibility (secondary anti-tubercular drugs) test for 20700040

TCH Sensitivity Test** For differentiation of M. tuberculosis from M. bovis. Tween 80 Hydrolysis** Biochemical test for differentiation of saprophytic species of mycobacteria.

20401050

ANALYTICAL REAGENTS AND INDICATORS* 20680020 Barritt Reagent A, Barritt Reagent B, Creatine* For 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. Kovacs' Reagent (Indole)* For Indole Test, particularly useful in the identification of E.coli. McFarland Standard No. 0.5 Turbidity standard, used in antimicrobial susceptibility testing. Methyl Red Indicator* To detect the ability of an organism to produce and maintain stable acid end products formed from glucose fermentation. Nitrite Detection Kit* For differentiating and identifying various types of bacteria by their ability to reduce nitrate. PYR Reagent* For detection of pyroglutamate aminopeptidase activity in group A streptococci and enterococci. TDA Reagent* For phenylalanine deamination reaction in differentiating Proteus from other members of Enterobacteriaceae.

20405025

20302125

20408006

20701040

20710040

20305101

20407101

READYMADE STAINING SOLUTIONS 20750020 Modified Gram's Stain Kit (containing A, B, and C)* 20750021 To differentiate between gram-negative and gram-positive bacteria.

* Store at R.T., ** Store betweeb 2-80C.

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CAT. NO.

DESCRIPTION Stains for Fungi Lactophenol Cotton Blue* For wet mount and staining of fungi Lactophenol Picric Acid* For wet mount and staining of fungi Picric Acid* For staining of fungi

CAT. NO.

DESCRIPTION contains sterile broth for Urease detection and 11 different carbohydrates-Melibiose, Lactose, Maltose, Sucrose, Raffinose, Galactose, Trehalose, Cellobiose, Inositol, Xylose, Dulcitol).

20760040 20770040 20780040

20795001

BIOCHEMICAL IDENTIFICATION KITS 20790001 Enterobacteriaceae Identification Test Kit** A 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 Kit** A 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, H2S production, Citrate utilization and 5 different carbohydrates-Glucose, Adonitol, Arabinose, Lactose, Sorbitol). Reagents supplied with the kit: TDA Reagent, Nitrite Detection Strip, Zinc Dust. Staph Identification Kit** A 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. Strep Identification Kit** A 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. Candida Identification Kit** A panel of 12 tests for identification of Candida species (Kit

Neisseria Identification Kit** A 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. E.coli Identification Kit** A 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. Salmonella Identification Kit** A panel of 12 tests for the identification of Salmonella species (Kit contains sterile broth for MR test, Voges Proskauer test, Citrate utilization, Urease detection, H2S production, ONPG utilization, 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. Listeria Identification Kit** A 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.

20796001

20797001

20792001

20798001

20793001

20794001

URINE CULTURE 20201012 Easybact** Chromogenic, differential, semi-quantitative bacteriuria collection and screening system.

* Store at R.T., ** Store betweeb 2-80C.

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