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LABORATORY MANUAL OF

FOOD MICROBIOLOGY

FOR ETHIOPIAN HEALTH AND NUTRITION RESEARCH INSTITUTE

(FOOD MICROBIOLOGY LABORATORY)

UNIDO PROJECT

(YA/ETH/03/436/11-52)

DEC. 2003

DRAFTED BY

DR. CIIRA KIIYUKIA

(INIDO / FOOD ANALYSIS ­ MICROBIOLOGY)

TABLE OF CONTENTS INTRODUCTION ..................................................................................................... 4 MICROORGANISMS MORPHOLOGY AND STAINING............. 7

MICROSCOPY ..................................................................................................................... 7 STAINED PREPARATIONS ................................................................................................... 7 MAKING A SMEAR. ............................................................................................................ 8 A SIMPLE STAIN................................................................................................................. 8 A DIFFERENTIAL STAIN: GRAM'S STAINING METHOD ......................................................... 9 BACTERIAL MOTILITY ....................................................................................................... 9 ENDOSPORE STAINING (SCHAEFFER­FULTON OR WIRTZ­CONKLIN)................................. 10 FLAGELLA STAINING: WEST AND DIFCO'S SPOTTEST METHODS ...................................... 11

BASIC LABORATORY PROCEDURES AND CULTURE TECHNIQUES ......................................................................................................... 14

PREPARATION OF CULTURE MEDIA ................................................................................... 14 POURING A PLATE............................................................................................................ 14 STORAGE OF MEDIA ......................................................................................................... 14 STERILIZATION VS . DISINFECTION.................................................................................... 14 STERILIZATION OF EQUIPMENT AND MATERIALS .............................................................. 15 DISINFECTANTS ............................................................................................................... 15 INOCULATION AND OTHER ASEPTIC PROCEDURES ............................................................. 15 ESSENTIAL POINTS ........................................................................................................... 15 STREAK PLATE. ............................................................................................................... 17 POUR PLATE .................................................................................................................... 17 SPREAD PLATE................................................................................................................. 19 INCUBATION .................................................................................................................... 19 CLEARING UP ................................................................................................................... 20 PURE CULTURES ............................................................................................................... 20 MAINTAINING STOCK CULTURES ...................................................................................... 20 COTTON WOOL PLUGS ...................................................................................................... 21 ASEPTIC TRANSFER OF CULTURES AND STERILE SOLUTIONS .............................................. 21 TESTING SENSITIVITY TO ANTIBACTERIAL SUBSTANCES .................................................... 22

COMMON BIOCHEMICAL TESTS .......................................................... 24

1. INDOLE TEST................................................................................................................ 24 2. H2S PRODUCTION TEST:............................................................................................... 24 3. NITRATE REDUCTION TEST........................................................................................... 24 4. METHYL RED TEST ....................................................................................................... 24 5. VOGES- PROSKAUER'S TEST ........................................................................................ 24 6. UTILIZATION OF CITRATE AS THE SOLE SOURCE OF CARBON ......................................... 25 7. FERMENTATION OF SUGAR:.......................................................................................... 25 8. GELATIN LIQUEFACTION:............................................................................................. 25 9. ACTION ON LITMUS MILK :............................................................................................ 25 10. UTILIZATION OF URIC ACID AS THE SOLE CARBON SOURCE ......................................... 26 1

FOOD SAMPLING AND PREPARATION OF SAMPLE HOMOGENATE ..................................................................................................... 28

SAMPLE COLLECTION ....................................................................................................... 29 SAMPLE ANALYSIS .......................................................................................................... 31 CLASSIFICATION OF FOOD PRODUCTS FOR SAMPLING PURPOSES ....................................... 32 EQUIPMENT AND MATERIALS .......................................................................................... 34 RECEIPT OF SAMPLES ....................................................................................................... 34 THAWING ........................................................................................................................ 35 MIXING............................................................................................................................ 35 WEIGHING........................................................................................................................ 35 BLENDING AND DILUTING OF SAMPLES REQUIRING ENUMERATION OF MICROORGANISMS.. 35

ENUMERATION OF MICROORGANISMS IN FOODS .............. 37

A. DETERMINATION OF AEROBIC COLONY COUNT IN FOODS .................... 37 B. MOST PROBABLE NUMBER METHOD (MPN) ............................................... 41

CALCULATION OF MOST PROBABLE NUMBERS (MPN).................................................... 43 MPN TABLES .................................................................................................................. 45 C. ENUMERATION OF YEASTS AND MOULDS IN FOODS................................. 47 D. ENUMERATION OF COLIFORMS FAECAL COLIFORMS AND E. COLI IN FOODS USING THE MPN METHOD ........................................................................ 53

ISOLATION AND ENUMERATION OF PATHOGENIC MICROORGANISMS IN FOOD. ................................................................. 64

A. ISOLATION OF E. COLI 0157 IN FOODS ......................................................... 64 B. ENTEROCOCCUS .................................................................................................. 71 C. ISOLATION OF SALMONELLA FROM FOODS ................................................. 75 D. ENUMERATION OF STAPHYLOCOCCUS AUREAUS IN FOODS ................... 81 E. ISOLATION OF LISTERIA MONOCYTOGENS FROM ALL FOOD AND

ENVIRONMENTAL SAMPLES ................................................................................... 96 F. ISOLATION AND ENUMERATION OF BACILLUS CEREUS IN FOODS ...... 111 G. DETECTION OF CLOSTRIDIUM BOTULINUM IN HONEY AND SYRUPS... 121 H. ENUMERATION OF CLOSTRIDIUM PERFRIGENS IN FOODS ..................... 125

MICROBIOLOGY OF WATER.................................................................. 130

STANDARD QUALITATIVE ANALYSIS OF WATER ........................................................... 130 QUANTITITIVE ANALYSIS OF WATER.............................................................................. 133 PURPOSE........................................................................................................................ 133

HOWARD MOULD COUNT......................................................................... 136 EXAMINATION OF CANNED FOODS ................................................ 148 STANDARD OPERATING PROCEDURES (SOPS)..................... 161

QUALITY ASSURANCE IN MICROBIOLOGY LABORATORIES......................... 168

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INSTRUMENTAL MAINTENANCE, QUALITY CONTROL AND CALIBRATION

...................................................................................................................................... 169 LABORATORY AUDIT.............................................................................................. 185

MICROBIAL STANDARDS OF FOODS.............................................. 187 GUIDELINES FOR WRITING LAB REPORTS .............................. 198 REFERENCES AND SELECTED READINGS ................................ 201

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Introduction

The purpose of this manual is to provide the new food microbiology laboratory at the Ethiopian Health and Nutrition Research Institute with the standard methods for qualitative and quantitative detection of microorganisms in food and water. The manual contains detailed description of microbial enumeration, isolation and identification of pathogenic food-borne bacteria. Methods of estimating sanitary indicator microorganisms as well as enumeration of moulds and yeasts are documented. These methods have been adapted from methods recommended by the ICMSF, AOAC, FDA, APHA and Health Canada. The standard operating procedures and quality control guidelines relating to food sampling and methods of analysis are included. The manual is written in such a manner that it can be used for in-house training of new technicians. Description of equipment maintenance and calibration is detailed including quality control of media and internal laboratory audit. Isolation and identification of microbial food contaminants help to understand how infectious agents enter and spread through the food chain and therefore come up with procedures to prevent or minimize exposure of the consumer to such agents. There is the need to estimate the risk that foodborne pathogens pose to human health in a national and international context and to identify possible interventions to reduce or eliminate these risks. The standards, guidelines and recommendations adopted by international trade agreements, such as those administered by the WTO, are playing an increasingly important role in protecting the health of consumers. In the case of microbiological hazards, Codex has elaborated standards, guidelines and recommendations that describe processes and procedures for the safe preparation of food. The application of these standards, guidelines and recommendations is intended to prevent or eliminate hazards in foods or reduce them to acceptable levels. This requires an elaborate laboratory with equipment and personnel well trained to carry out the analysis. Most developing countries lack the resources to put up food microbiology laboratories and to man them adequately to international standards. The globalization of food trade and increasing problems worldwide with emerging and re-emerging foodborne diseases have increased the risk of crossborder transmission of infectious agents. Because of the global nature of food production, manufacturing, and marketing, infectious agents can be disseminated from the original point of processing and packaging to locations thousands of miles away. In this regard, developing countries are required to ensure that their sanitary and phytosanitary measures are based on an assessment, as appropriate to the circumstances, of the risks to human, animal or plant life or health, taking into account the risk assessment techniques developed by the relevant international organizations. The manual details microbiological risk assessment of various food categories, guidelines and recommendations related to food safety. There is a critical need for technical advice on risk assessment of microbiological hazards in foods to meet the needs of national governments, the food industry, the scientific community, trade organizations and international consumer groups. UNIDO, FAO and WHO have a direct role to play in assisting developing countries in matters related to food safety and should strengthen efforts to facilitate access to specific advice on microbiological risk assessment. This manual has been developed with the help of UNIDO inline with the above stated spirit.

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Microbial Food analysis 1: Reasons for microbial food analysis. · to meet certain set standards · to estimate the shelf-life of the product · to determine quality of the food · for public health purposes 2: The organisms to look for; i) Indicator organism(s); Definition: an indicator organism or group of organisms is one whose numbers in a product reflect the success or failure of "good manufacturing practices". Coliform group of microorganisms and Escherichia coli are commonly used as indicator organisms. ii) Index organism; Definition: an index organism is one whose presence implies the possible occurrence of a similar but pathogenic organism. E. coli is used an index organism a its presence nd indicates possible presence of pathogenic enterobacteriacea e.g. Salmonella sp. iii) Food poisoning organisms The are two types of food poisoning organisms · those which cause the decease by infection · those which produce toxin in food a)Those which cause infection must grow in food in large numbers and cause infection when consumed together with food. The most common microorganisms in this category includes Salmonella typimurium, enteropathogenic E. coli, Vibrio parahaemolyticus, Yersinia enterocolytica etc. b) Those which cause intoxication must grow in food in large numbers and produce enough toxin and when consumed together with food cause intoxication. The most common microorganisms in this category includes, Clostridium botulinum, Staphylococcus aureus and toxigenic fungi e.g. Aspergillus flavus. iv) Infectious microorganisms Definition: Organisms whose presence in small numbers in food or water and when consumed can cause infection. In this case the food acts as a vector but not necessarily as a growth medium.Infectious organisms can be transmitted by various ways including man to man and are said to be contagious. Organisms in this group includes; Vibrio cholerae O1, Salmonella typhi, Shigella sonnei, Bacillus anthracis, Hepatitis B virus etc. v) Spoilage organisms Definition: Spoilage organisms are the organisms whose growth in the food creates undesirable characteristics in that food. Any microorganism which is not intentionally added into food or intentionally allowed to grow in food so as to impart certain qualities in that food is considered a contaminant. Growth of the contaminant in that food will spoil the food making it unfit for human consumption. Some useful microorganisms e.g. lactic acid bacteria are considered as spoilage organisms when in beer, wine and fruit juices but not in milk.

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3: How to analyze i) Quantitative analysis · Serial decimal dilution · Aerobic plate count · Pour plate count · Total viable count · Most Probable Number (MPN) method · Yeast and Molds count ii) Qualitative analysis presence or absence of a specified microorganism e.g. · Salmonella sp. · E. coli · V. cholerae O1 4: Culture Methods · pre-enrichment broth · enrichment broth · selective enrichment · selective agar · Differential agar Biochemical tests · sugar fermentation · amino acid decarboxylation · gelatin liquefaction · lecithinase production Serology · agglutination · precipitin · coagulation Colony morphology · shape · colour · texture · size Cell shape by microscope · bacillus · coccus · streptococcus Gram stain characteristics · gram positive · gram negative Motility motile number of flagella arrangement of flagella non-motile

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Microorganisms morphology and staining

Microscopy Using the microscope The setting up of a microscope is a basic skill of microbiology yet it is rarely mastered. Only when it is done properly can the smaller end of the diversity of life be fully appreciated and its many uses in practical microbiology, from aiding in identification to checking for contamination, be successfully accomplished. The amount of magnification of which a microscope is capable is an important feature but it is the resolving power that determines the amount of detail that can be seen. Bacteria and yeast Yeast can be seen in unstained wet mounts at magnifications x100. Bacteria are much smaller and can be seen unstained at x400 but only if the microscope is properly set up and all that is of interest is whether or not they are motile. A magnification of x1000 and the use of an oil immersion objective lens for observing stained preparations are necessary for seeing their characteristic shapes and arrangements. The information gained, along with descriptions of colonies, is the starting point for identification of genera and species but further work involving physiology, biochemistry and molecular biology is then needed. . Moulds Mould mycelium and spores can be observed in unstained wet mounts at magnifications of x100 although direct observations of "mouldy" material through the lid of a Petri dish or specimen jar at lower magnifications with the plate microscope are also informative (but keep the lid on!). Routine identification of moulds is based entirely on the appearance of colonies to the naked eye and of the mycelium and spores in microscopical preparations. Stained preparations A "smear" of bacteria or yeast is made on a microscope slide, fixed, stained, dried and, without using a coverslip, examined with the aid of a microsope. Aseptic technique must be observed when taking samples of a culture for making a smear. A culture on agar medium is much preferable to a liquid culture for making a smear. A smear that is thin and even enables the shape and arrangement of cells to be clearly seen and ensures that the staining procedure is applied uniformly. There are two broad types of staining method: (1) a simple stain involves the application of one stain to show cell shape and arrangement and, sometimes, inclusions that do not stain, e.g. bacterial endospores; (2) a differential stain involves a sequence of several stains, sometimes with heating, and includes a stage which differentiates between either different parts of a cell, e.g. areas of fat storage, or different groups, e.g. between Gram-positive and Gram-negative bacteria. The reaction of bacteria to Gram's staining method is a consequence of differences in the chemical structure of the bacterial cell wall and is a key feature in their identification. Yeast cells can be stained by Gram's method but it is of no value in their identification. The basis of Gram's staining method is the ability or otherwise of a cell stained with crystal violet to retain the colour when

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treated with a differentiating agent, usually alcohol (although professionals sometimes use acetone). Bacteria that retain the violet/purple colour are called Gram-positive. Those that lose the colour, i.e. called Gram negative, are stained in the contrasting colour of a counterstain, usually pink/red. Making a smear. 1. Clean a plain microscope slide thoroughly using lens tissue. 2. Label a microscope slide with a marker pen to record the culture being used, date and initials; this is also a useful reminder of which side of the slide is being used. 3. Flame a wire loop to ensure that no culture accidentally remains from a previous operation. 4. Transfer one or two loopfuls of tap water on to the centre of the slide. 5. Flame loop and allow to cool. 6. Using aseptic technique, transfer a very small part of a single colony from a plate or slope of agar medium into the tap water. If the amount of culture on the loop is easily visible you have taken too much! 7. Make a suspension of the culture in the tap water on the slide and thoroughly but gently spread it evenly over an oval area of up to 2 cm length. 8. Flame the loop. If it is necessary to use a liquid culture or sample, the use of tap water to prepare the smear will probably be unnecessary and may result in a smear with too few cells. 9. Dry the suspension by warming gently over a Bunsen burner flame and then "fix" it by quickly passing it through the flame a few times. This is called a heat-fixed smear; it should be visible to the naked eye as a whitish area. Fixing is necessary to ensure that cells adhere to the slide and to minimise any post mortem changes before staining. A simple stain. 1. Put the slide with the fixed smear uppermost on a staining rack over a sink or staining tray. 2. Thoroughly cover the smear with stain and leave for, usually, 30 seconds. 3. Hold the slide with forceps (optional but avoids stained fingers), at a 45° angle over the sink. 4. Rinse off the stain with tap water. 5. Blot dry the smear with filter/fibre free blotting paper using firm pressure but not sideways movements that might remove the smear. 6. Examine under oil immersion. 7. When finished, dispose of slides into discard jar. Suitable stains include basic dyes (i.e. salts with the colour-bearing ion, the chromophore, being the cation) such as methylene blue, crystal violet and safranin. Staining solutions (relevant to procedures described below) Crystal violet solution: A. crystal violet 2.0g dissolved in absolute alcohol 100 ml B. ammonium oxalate 1.0g in distilled/deionised water 100ml Add 25 ml A to 100 ml B Lugol's iodine solution: iodine 1.0g, potassium iodide 2.0g distilled/deionised water 300 ml

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A differential stain: Gram's staining method Times of the staining periods depend on the formulation of the staining solutions which are not standard in all laboratories. Therefore, the times given here relate only to the solutions specified here. a. Put the slide with the fixed smear uppermost on a staining rack over a sink or staining tray. b. Thoroughly cover the smear with crystal violet solution and leave for 1 minute. c. Hold the slide with forceps (optional but avoids stained fingers), at a 45° angle over the sink. d. Pour off the stain, wash off any that remains (and any on the back of the slide) with iodine solution. e. Put the slide back on staining rack. f. Cover the smear with iodine solution and leave for 1 minute. Iodine solution acts as a "mordant" (a component of a staining procedure that helps the stain to adhere to the specimen), a crystal violet-iodine complex is formed and the smear looks black. g. Hold the slide with forceps at a 45° angle over the sink wash off the iodine solution with 95% (v/v) ethanol (not water); continue treating with alcohol until the washings are pale violet. h. Rinse immediately with tap water. i. Put the slide back on staining rack. j. Cover the smear with the counterstain, e.g. safranin solution, 0.5% w/v, for 30 seconds. k. Rinse off the stain with tap water. l. Blot dry the smear with filter/fibre free blotting paper using firm pressure but not sideways movements that might remove the smear. m. Examine under oil immersion. n. When finished, dispose of slides into discard jar. Always use a young culture because older cultures of Gram-positive bacteria tend to lose the ability t retain the crystal violet-iodine complex and appear to be Gram-negative; but some o bacteria are naturally only weakly Gram-positive. The amount of alcohol treatment (the differential stage) must be judged carefully because over-treatment washes the crystal violetiodine complex from Gram-positive bacteria and they will appear to be Gram-negative. Take care to make an even smear otherwise alcohol will continue to wash the violet/purple colour from thick parts of the smear while thin parts are being over-decolorised. At the end of the procedure, check that the labeling has not been washed off by the alcohol. Don't despair if the stained smear is not visible to the naked eye; this may happen with a Gram-negative reaction. Bacterial Motility 1. Hanging drop method of motility: - use the special microscope slide with a depression

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- a cover slip - a micropscope - immersion oil - actively growing bacterial culture Procedure Place one drop of the culture onto the cover slip Touch the corners of the cover slip with lanolin and invert it on the grooved microscope slide glass. Observe for motility using the high power lens. Motility is characterized by fast unidirectional movement as compared to the Brownian motion whereby the cells move round in one particular point. 2. Semi-solid agar method The agar medium is prepared with the agar content of 0.2%. The medium is put into test tubes. Inoculation is done by stabbing the medium at the center. The inoculated medium is incubated at appropriate temperature for 24 hr. motility is detected by observing turbidity at the line of inoculation. Endospore staining (Schaeffer­Fulton or Wirtz­Conklin) Materials 24-to 48 hours nutrient agar slant cultures of Bacillus megaterium (ATTC 12872) and Bacillus macerans (ATCC 8244), and old (more than 48 hours) thioglycollate cultures of Clostridium butyricum (ATCC 19398) and Bacillus circulars (ATCC 4513) Clean slides Microscope Immersion oil Wax pencil Inoculating loop Hot plate or boiling water bath with staining rack or loop 5 % malachite green solution Safranin Bibulous paper Paper toweling Lens paper Slide warmer Forceps Principle Bacteria in genera such as Bacillus and Clostridium produce quite a resistant structure capable for surviving of long periods in an unfavorable environment and then giving rise to a new bacterial cell. This structure is called an endospore since it develops within the bacterial cell. This location and size of endorspores vary with the species; thus, they are often of value in identifying bacteria. Endospores are spherical to elliptical in shape and may be either smaller or larger than the parent bacterial cell. Endospore position within the cell is characteristic and may be central, subs terminal, or terminal. Endospores do not stain easily but, once stained, they strongly resist decolorization. This property is the basis of the Schaeffer-Fulton or Wirtz-Conklin method of staining endospores. The

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endorspores are stained with malachite green. Heat is used to provide stain penetration. The rest of the cell is then decolirized and counterstained a light red with safranin. Procedure 1. With a wax pencil, place the names of the respective bacteria on the edge of four clean glass slides. 2. Aseptically transfer one species of bacterium with an inoculating oop to each of the l respective slides, air dry (or use a slide warmer), and heat-fix. 3. Place the slide to be stained on a hot plate or boiling water bath equipped with a staining loop or rack. Cover the smear with paper toweling that has been cut the same size as the microscope slide. 4. Soak the paper with the malachite green staining solution. Gently heat on the hot plate (just until the stain steams) for 5 to 6 minutes after the malachite green solution begins to steam. Replace the malachite green solution as it evaporates so that the paper remains saturated during heating. 5. Remove the paper using forceps, allow the slide to cool, and rinse the slide with water for 30 seconds. 6. Counterstain with safranin for 60 to 90 seconds 7. Rinse the slide with water for 30 seconds. 8. Blot dry with bibulous paper and examine under oil immersion. A coverslip is not necessary. The spores, both endospores and free spores, stain green; vegetative cells stain red.

Flagella staining: West and Difco's SpotTest Methods Materials Young, 18-hour tryptic soy agar slants of Alcaligenes faecalis (ATCC8750, peritrichously flagellated) and Pseudomonas fluorecens (ATCC 13525, polarly flagellated) Wax pencil Inoculating loop Acid-cleaned glass slides with frosted ends Clean distilled water Microscope Immersion oil Lens paper Boiling water bath (250 ml beaker with distilled water, rind stand, wire gauze pad, an Bunsen burner or hot plate) Pasteur pipettes with pipettor West stain Solution A

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Solution B Difco's SportTest Flagella stain

Principle Bacterial flagella are fine, threadlike organelles of locomotion. They are slender (about 10 to 30 nm in diameter) and can only be seen directly using the electron microscope. In order to observe them with the light microscope, the thickness of the flagella are increased by coating them with mordants like tannic acid and potassium alum, and staining them with basic fuchsin (Gray method) or crystal violet (Difco's method). Although flagella staining procedures are difficult to carryout, they often provide information about the presence and location of flagella, which is of great value in bacterial identification. Difco's SportTest flagella stain employs an alcoholic solution of crystal violet as the primary stain, and tannic acid and aluminum potassium sulfate as mordants. As the alcohol evaporates during the staining procedure, the crystal violet forms a precipitate around the flagella, thereby increasing their apparent size. Procedure 1. With a wax pencil, mark the left-hand corner of a clean glass slide with the name of the bacterium. 2. Aseptically transfer the bacterium with an inoculating loop from the turbid liquid at the bottom of the slant to 3 small drops of distilled water in the center of a clean slide that has been care fully wiped off with clean lens paper. Gently spread the diluted bacterial suspension over a 3cm area using the inoculating needle. 3. Let the slide air dry for 15 minutes 4. Cover the dry smear with solution A (the mordant) for 4 minutes 5. Rinse thoroughly with distilled water 6. Place a piece of paper toweling on the smear and soak it with solution B (the stain). Heat the slide in a boiling water bath for 5 minutes in an exhaust hood with the fan on. Add more stain to keep the slide from drying out. 7. Remove the toweling and rinse off excess solution B with distilled water. Flood the slide with distilled water and allow it to sit for 1 minute while more silver nitrate residue floats to the surface. 8. Then, rinse gently with water once more and carefully shake excess water off the slide. 9. Allow the slide to air dry at room temperature 10. Examine the slide with the oil immersion objective. The best specimens will probably be seen at the edge of the smear where bacteria are less dense.

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Procedure (Difco) 1. Draw a border around the clear portion of a frosted microscope slide with a wax pencil. 2. Place a drop of distilled water on the slide, approximately 1 cm from the frosted edge. 3. Gently touch a colony of the culture being tested with an inoculating loop and then lightly touch the drop of water without touching the slide. Do not mix. 4. Tilt the slide at a slight angle to allow the drop preparation to flow to the opposite end of the slide. 5. Let the slide air-dry at room temperature. Do not heat-fix. 6. Flood the slid with the contents of the Difco SportTest flagella stain ampule. 7. Allow the stain to remain on the slide for approximately 4 minutes. (Note: the staining time may need to be adjusted from 2 to 8 minutes depending on the age of the culture, the age of the stain, the temperature, and the depth of staining solution over the culture) 8. Carefully rinse the stain by adding water from a faucet or wash bottle to the slide while it remains on the staining rack. Do not tip slide before this is done. 9. After rinsing, gently tilt the slide to allow excess water to run off and let the slide air-dry at room temperature or place on a slide warmer. Examine the slide microscopically with the oil immersion objective. Begin examination at thinner areas of the preparation and move toward the center. Look for fields which contain several isolated bacteria, rather than fields which contain clumps of many bacteria. Bacteria and their flagella should stain purple

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Basic laboratory procedures and culture techniques

Media, Sterilization and Disinfectants

Media Preparation of culture media Re-hydrate powder according to manufacturer's instructions. Before sterilization, ensure ingredients are completely dissolved, using heat if necessary. Avoid wastage by preparing only sufficient for either immediate use (allowing extra for mistakes) or use in the near future. Normally allow 15-20 cm3 medium/ Petri dish. Dispense in volumes appropriate for sterilization in the autoclave/pressure cooker. Agar slopes are prepared in test tubes or Universal/McCartney bottles by allowing sterile molten cooled medium to solidify in a sloped position. Pouring a plate 1. Collect one bottle of sterile molten agar from the water bath. 2. Hold the bottle in the left hand; remove the lid with the little finger of the right hand. 3. Flame the neck of the bottle. 4. Lift the lid of the Petri dish slightly with the right hand and pour the sterile molten agar into the Petri dish and replace the lid. 5. Flame the neck of the bottle and replace the lid. 6. Gently rotate the dish to ensure that the medium covers the plate evenly. 7. Allow the plate to solidify. 8. Seal and incubate the plate in an inverted position. The base of the plate must be covered, agar must not touch the lid of the plate and the surface must be smooth with no bubbles. Storage of media Store stocks of prepared media at room temperature away from direct sunlight; a cupboard is ideal but an open shelf is satisfactory. Media in vessels closed by cotton wool plugs that are stored for future use will be subject to evaporation at room temperature; avoid wastage by using screw cap bottles. Re-melt stored agar media in boiling water bath, pressure cooker or microwave oven. Sterile agar plates can be pre-poured and stored in well-sealed plastic bags (media-containing base uppermost to avoid heavy condensation on lid). Sterilization vs. Disinfection Sterilization means the complete destruction of all the micro-organisms including spores, from an object or environment. It is usually achieved by heat or filtration but chemicals or radiation can be used. Disinfection is the destruction, inhibition or removal of microbes that may cause disease or other problems e.g. spoilage. It is usually achieved by the use of chemicals. Sterilization Use of the autoclave The principle of sterilization in an autoclave is that steam under pressure is used to produce a temperature of 121ºC which if held for 15 minutes all micro-organisms including bacterial endospores will be destroyed.

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Sterilization of equipment and materials Wire loop Heat to redness in Bunsen burner flame. Empty glassware and glass (not plastic!) pipettes and Petri dishes Either, hot air oven, wrapped in either grease proof paper or aluminum and held at 160ºC for 2 hours, allowing additional time for items to come to temperature (and cool down!). Note: plastic Petri dishes are supplied in already sterilized packs; packs of sterile plastic pipettes are also available but cost may be a consideration. Culture media and solutions - Autoclave/pressure cooker. Glass spreaders and metal forceps - Flaming in alcohol (70% industrial methylated spirit). Disinfectants Choice, preparation and use of disinfectants Specific disinfectants at specified working strengths are used for specific purposes. Commonly available disinfectants Hypochlorite (sodium chlorate I)

used in discard pots for pipettes and slides At 2500 ppm (0.25%, v/v) available chlorine Ethanol 70% (v/v) industrial methylated spirit When preparing working strength solutions from stock and dealing with powder form, wear eye protection and gloves to avoid irritant or harmful effects. Disinfectants for use at working strength should be freshly prepared from full strength stock or powder form. Use working strength hypochlorite on day of preparation. Inoculation and other aseptic procedures Essential points There are several essential precautions that must be taken during inoculation procedures to control the opportunities for the contamination of cultures, people or the environment. - Operations must not be started until all requirements are within immediate reach and must be completed as quickly as possible. - Vessels must be open for the minimum amount of time possible and while they are open all work must be done close to the Bunsen burner flame where air currents are drawn upwards. - On being opened, the neck of a test tube or bottle must be immediately warmed by flaming so that any air movement is outwards and the vessel held as near as possible to the horizontal. - During manipulations involving a Petri dish, exposure of the sterile inner surfaces to contamination from the air must be limited to the absolute minimum. - The parts of sterile pipettes that will be put into cultures or sterile vessels must not be touched or allowed to come in contact with other non-sterile surfaces, e.g. clothing, the surface of the working area, outside of test tubes/bottles. Using a wire loop Wire loops are sterilized using red heat in a Bunsen flame before and after use. They must be heated to red hot to make sure that any contaminating bacterial spores are destroyed. The handle of the wire loop is held close to the top, as you would a pen, at an angle that is almost vertical. This leaves the little finger free to take hold of the cotton wool plug/ screw cap of a test tube/bottle. Flaming procedure

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The flaming procedure is designed to heat the end of the loop gradually because after use it will contain culture, which may "splutter" on rapid heating with the possibility of releasing small particles of culture and aerosol formation. 1. Position the handle end of the wire in the light blue cone of the flame. This is the cool area of the flame. 2. Draw the rest of the wire upwards slowly up into the hottest region of the flame, (immediately above the light blue cone). 3. Hold there until it is red hot. 4. Ensure the full length of the wire receives adequate heating. 5. Allow to cool then use immediately. 6. Do not put the loop down or wave it around. 7. Re-sterilize the loop immediately after use. If a loop does not hold any liquid the loop has not made a complete circle. To correct the problem, first ensure that the loop has been sterilized and then reshape the loop with forceps. Do not use your fingers because of the possibility of puncturing the skin. Using a pipette Sterile graduated or dropping (Pasteur) pipettes are used to transfer cultures, sterile media and sterile solutions. 1. Remove the pipette from its container/ wrapper by the end that contains a cotton wool plug, taking care to touch no more than the amount necessary to take a firm hold. 2. Fit the teat. 3. Hold the pipette barrel as you would a pen but do not grasp the teat. The little finger is left free to take hold of the cotton wool plug/lid of a test tube/bottle and the thumb to control the teat. 4. Depress the teat cautiously and take up an amount of fluid that is adequate for the amount required but does not reach and wet the cotton wool plug. 5. Return any excess gently if a measured volume is required. The pipette tip must remain beneath the liquid surface while taking up liquid to avoid the introduction of air bubbles which may cause "spitting" and, conseque ntly, aerosol formation when liquid is expelled. 6. Immediately put the now contaminated pipette into a nearby discard pot of disinfectant. The teat must not be removed until the pipette is within the discard pot otherwise drops of culture will contaminate the working surface. A leaking pipette is caused by either a faulty or ill-fitting teat or fibres from the cotton wool plug between the teat and pipette. Flaming the neck of bottles and test tubes 1. Loosen the lid of the bottle so that it can be removed easily. 2. Lift the bottle/test tube with the left hand. 3. Remove the lid of the bottle/cotton wool plug with the little finger of the right hand. (Turn the bottle, not the lid.) 4. Do not put down the lid/cotton wool plug. 5. Flame the neck of the bottle/test tube by passing the neck forwards and back through a hot Bunsen flame. 6. Replace the lid on the bottle/cotton wool plug using the little finger. (Turn the bottle, not the lid.)

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Label tubes and bottles in a position that will not rub off during handling. Either marker pens or self-adhesive labels are suitable. Occasionally cotton wool plugs accidentally catch fire. Douse the flames by immediately covering with a dry cloth, not by blowing or soaking in water. Streak plate. The loop is used for preparing a streak plate. This involves the progressive dilution of an inoculum of bacteria or yeast over the surface of solidified agar medium in a Petri dish in such a way that colonies grow well separated from each other. The aim of the procedure is to obtain single isolated pure colonies. 1. Loosen the top of the bottle containing the inoculum. 2. Hold the loop in the right hand. 3. Flame the loop and allow to cool. 4. Lift the bottle/test tube containing the inoculum with the left hand. 5. Remove the lid/cotton wool plug of the bottle/test tube with the little finger of the left hand. 6. Flame the neck of the bottle/test tube. 7. Insert the loop into the culture broth and withdraw. At all times, hold the loop as still as possible. 8. Flame neck of the bottle/test tube. 9. Replace the lid/cotton wool plug on the bottle/test tube using the little finger. Place bottle/test tube on bench. 10. Partially lift the lid of the Petri dish containing the solid medium. 11. Hold the charged loop parallel with the surface of the agar; smear the inoculum backwards and forwards across a small area of the medium 12. Remove the loop and close the Petri dish. 13. Flame the loop and allow it to cool. Turn the dish through 90º anticlockwise. 14. With the cooled loop streak the plate from area A across the surface of the agar in three parallel lines. Make sure that a small amount of culture is carried over. 15. Remove the loop and close the Petri dish. 16. Flame the loop and allow to cool. Turn the dish through 90º anticlockwise again and streak from B across the surface of the agar in three parallel lines. 17. Remove the loop and close the Petri dish. 18. Flame the loop and allow to cool. Turn the dish through 90º anticlockwise and streak loop across the surface of the agar from C into the centre of the plate 19. Remove the loop and close the Petri dish. Flame the loop. 20. Seal and incubate the plate in an inverted position. Label the half of the dish that contains medium; use abbreviations and keep them to the edge of the plate so as not to interfere with the later observation of colonies. The same applies to the pour and spread plates described below. Either marker pens or self-adhesive labels are suitable. There are two approaches to making a streak plate: (1) with the base (containing medium) placed on the working surface, lift the lid vertically (i.e. still covering the base) the least amount that will allow access of the loop; (2) with the lid placed on the working surface, lift out the base, invert it and inoculate the upwards - facing agar surface. Pour plate A pour plate is one in which a small amount of inoculum from broth culture is added by pipette to a molten, cooled agar medium in a test tube or bottle, distributed evenly throughout the medium,

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thoroughly mixed and then poured into a Petri dish to solidify. Pour plates allow micro-organisms to grow both on the surface and within the medium. Most of the colonies grow within the medium and are small in size; the few that grow on the surface are of the same size and appearance as those on a streak plate. If the dilution and volume of the inoculum, usually 1 cm³, are known, the viable count of the sample i.e. the number of bacteria or clumps of bacteria, per cm³ can be determined. Pouring the pour plate 1. Roll the bottle gently between the hands to mix the culture and the medium thoroughly. Avoid making air bubbles. 2. Hold the bottle in the left hand; remove the lid with the little finger of the right hand. 3. Flame the neck of the bottle. 4. Lift the lid of the Petri dish slightly with the right hand and pour the mixture into the Petri dish and replace the lid. 5. Flame the neck of the bottle and replace the lid. 6. Gently rotate the dish to ensure that the medium covers the plate evenly. 7. Allow the plate to solidify. 8. Seal and incubate the plate in an inverted position. (The base of the plate must be covered, agar must not touch the lid of the plate and the surface must be smooth with no bubbles). Pouring the inoculated medium If pipettes are not available then a wire loop can be used. Several loopfuls of culture must be added to the cooled molten nutrient agar to ensure that there is enough inoculum present for growth. Using a spreader Sterile spreaders are used to distribute inoculum over the surface of already prepared agar plates. Wrapped glass spreaders may be sterilized in a hot air oven. They can also be sterilized by flaming with alcohol. It is advisable to use agar plates that have a well-dried surface so that the inoculum dries quickly. Dry the surface of agar plates by either incubating the plates for several hours, e.g. overnight, beforehand or put them in a hot air oven (ca 55-60ºC) for 30-60 minutes with the two halves separated and the inner surfaces directed downwards. Sterilization using alcohol 1. Dip the lower end of the spreader into a small volume of 70% alcohol contained in a vessel with a lid (either a screw cap or aluminium foil). 2. Pass quickly through a Bunsen burner flame to ignite the alcohol; the alcohol will burn and sterilize the glass. 3. Remove the spreader from the flame and allow the alcohol to burn off. 4. Do not put the spreader down on the bench. Flaming a glass spreader Ensure that the spreader is pointing downwards when and after igniting the alcohol to avoid burning yourself. Keep the alcohol beaker away from the Bunsen flame.

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Spread plate Spread plates, also known as lawn plates, should result in a culture spread evenly over the surface of the growth medium. This means that they can be used to test the sensitivity of bacteria to many antimicrobial substances, for example mouthwashes, garlic, disinfectants and antibiotics. The spread plate can be used for quantitative work (colony counts) if the inoculum is a measured volume, usually 0.1 cm3, of each of a dilution series, delivered by pipette. 1. Loosen the lid of the bottle containing the broth culture. 2. Hold a sterile pipette in the right hand and the bottle/test tube containing the broth culture in the left. 3. Remove the lid/plug of the bottle/test tube with the little finger of the right hand and flame the neck. 4. With the pipette, remove a small amount of broth. 5. Flame the neck of the bottle/test tube and replace the lid/plug. 6. With the left hand, partially lift the lid of the Petri dish containing the solid nutrient medium. 7. Place a few drops of culture onto the surface about 0.1 cm3 (ca 5 drops, enough to cover a 5 pence piece). 8. Replace the lid of the Petri dish. 9. Place the pipette in a discard jar. 10. Dip a glass spreader into alcohol, flame and allow the alcohol to burn off. 11. Lift the lid of the Petri dish to allow entry of spreader. 12. Place the spreader on the surface of the inoculated agar and, rotating the dish with the left hand move the spreader in a top-to-bottom or a side-to-side motion to spread the inoculum over the surface of the agar. Make sure the entire agar surface is covered. This operation must be carried out quickly to minimize the risk of contamination. 13. Replace the lid of the Petri dish. 14. Flame spreader using alcohol. 15. Let the inoculum dry. 16. Seal and incubate the plate in the inverted position. HINT Consider the calibrated drop method for colony counts of pure cultures of bacteria and yeast as a more economical method than the pour plate and spread plate. The procedure is as for the spread plate but fewer plates are needed because: (1) the inoculum is delivered as drops from a dropping pipette that is calibrated (by external diameter of the tip) to deliver drops of measured volume e.g. 0.02 cm³; (2) many drops (six or more) can be put on one plate. The method is not suitable for use with cultures that produce spreading growth including mixed cultures in many natural samples such as soil although yoghurt and cheese are among the exceptions. Incubation The lid and base of an agar plate should be taped together with 2-4 short strips of adhesive tape as a protection from accidental (or unauthorized!) opening during incubation. (Although tape is the preferred method Parafilm could be used as an alternative for sealing the plates.) Agar plates must be incubated with the medium-containing half (base) of the Petri dish uppermost otherwise condensation will occur on the lid and drip onto the culture. This might cause colonies to spread into each other and risk the spillage of the contaminated liquid.

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Water baths are used when accurately controlled temperatures are required, e.g. for enzyme reactions and growth temperature relationships, when temperature control of incubators is not sufficiently precise. They should be used with distilled or deionised water to prevent corrosion and emptied and dried for storage. Overlong incubation of mould cultures will result in massive formation of spores which readily escape, particularly from Petri dishes, and may cause contamination problems in the laboratory and be a health hazard. This can occur in an incubator, at room temperature and even in a refrigerator. Clearing up Working surfaces must be cleared after use. If cultures have been used the benches must be swabbed with disinfectant Discarded cultures, empty media tubes and all contaminated material must be placed in the appropriate labeled receptacles. Discard containers must be carefully and securely packed and never overloaded. Plastic Petri dishes must never be stacked above the lip of the discard container. Cultures and contaminated paper towels, gloves etc. must be autoclaved at 121ºC for 15 minutes before disposal. Slides, pipettes and Pasteur pipettes must be discarded in the appropriate containers of Hypochlorite (sodium chlorate 1). They must be soaked for at least 24 hours before disposal. Never discard sharp or broken items in a way which would endanger. After sterilization, all materials can be disposed of with normal waste. Care must be taken that glass is adequately packaged to prevent injury. Before leaving the laboratory, laboratory coats must be removed and hands washed with hot water and soap. Pure cultures The ability to keep pure cultures from becoming contaminated during inoculation and use is a key feature of GMLP. This skill is crucial for reasons of safety and for maintaining the scientific integrity of an investigation. Clearly, it is also vital skill to recognize when a culture has become contaminated. Maintaining stock cultures It may be convenient to maintain a stock of a pure culture instead of re-purchasing it when needed. Most of those considered suitable for use are also relatively easy to maintain by subculturing on the medium appropriate f growth but maintenance of stock cultures needs to be or well organized with attention to detail. Be prepared to transfer cultures four times a year to maintain viability. Cultures on streak plates are not suitable as stock cultures. Slope cultures in screw cap bottles are preferred because the screw cap reduces evaporation and drying out and cannot be accidentally knocked off (cf. a streak plate culture). Slope cultures are preferred to broth (i.e. liquid medium) cultures because the first sign of contamination is much more readily noticed on an agar surface. Two stock cultures should be prepared; one is the "working" stock for taking sub-cultures for classes, the other is the "permanent" stock which is opened only once for preparing the next two stock cultures. Incubate at an appropriate temperature until there is good growth. For growing strict aerobes it may be necessary to slightly loosen the cap for incubation (but close securely before storage) if there is insufficient air in the headspace. As soon as there is adequate growth, store the cultures at room temperature in either a cupboard or drawer. Keep on the lookout for contamination.

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Checking cultures for contamination Evidence for a culture being pure or otherwise is given by the appearance of colonies on a streak plates and of cells in a stained microscopical preparation. There should be uniformity of colony form and cell form (and consistency with the appearance of the original culture!). It is sensible to check purity on suspicion of contamination of the working stock culture from time to time and of the permanent stock when preparing new stock cultures. If a culture becomes contaminated, it is not advisable to try to remedy the situation by taking an inoculum from a single colony from a streak plate of the mixed culture because of the possibility of (1) not being able to distinguish between the colony forms of the contaminant and the original culture, and (2) culturing a variant of the original culture that does not behave as the original culture did. Instead, go back to the working (or permanent) stock cultures; that's what they are for! Cotton wool plugs Plugs made of non-absorbent cotton wool are used in test tubes and pipettes to prevent microorganisms from passing in or out and contaminating either the culture or the environment. The necessary movements of air in and gaseous products out are not prevented and the gaps between the cotton wool fibres are even wide enough for micro-organisms to pass through. However, this does not happen because micro-organisms (negatively charged) are "filtered" out by being attracted to and adsorbed on the oppositely charged cotton wool. The cotton wool must remain dry because this filtration property is lost if the cotton wool becomes moist ­ hence the use of nonabsorbent cotton wool. For use in test tubes a plug should be properly made to ensure that it can be held comfortably without being dropped and its shape and form are retained while being removed from and returned to a test tube several times. Aseptic technique cannot be maintained with poorly made plugs; working surfaces, floors and cultures may become contaminated and students may become understandably (but avoidably) frustrated and lose interest. Aseptic transfer of cultures and sterile solutions Regular practice is necessary to ensure that the manipulations involved in aseptic transfer of cultures and sterile Making a streak plate is a basic procedure that tests several skills and serves several purposes. During the inoculation procedure, the agar surface is protected from contamination by microorganisms that are carried in the air by keeping the time that the Petri dish is open to a minimum. There are two approaches: (1) with the base (containing medium) placed on the working surface, lift the lid vertically (i.e. still covering the base) the least amount that will allow access of the loop; (2) with the lid placed on the working surface, lift out the base, invert it and inoculate the upwards- facing agar surface. The second method is best reserved for older students working in a relatively dust and draught-free laboratory; it is the one used by professional microbiologists. The choice of loop or pipette for transfers between test tubes and screw cap bottles depends on whether they contain agar slopes, liquid media or sterile solutions. Although omitted from the table for simplicity, a straight wire may also be necessary for taking a small inoculum from liquid cultures for nutritional investigations. The wire loop is usually satisfactory for inoculating a tube or bottle from a separate colony on a plate but a straight wire is occasionally needed for dealing with very small colonies such as occur with pure cultures of some bacteria, e.g. species of Streptococcus and Lactobacillus, and on plates that are being used for isolating cultures from natural samples. Appropriate instruments for aseptic transfer procedures Microbial stock cultures for use in food microbiology are the equivalent of, say, solutions of chemicals or electrical circuits in other disciplines. The big difference, however, is that microbial

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cultures cannot be taken from a shelf and instantly be ready for use. It is necessary to begin to prepare cultures well in advance otherwise the outcome might not be as expected and the experience will be either diminished or lost. It is usual to grow moulds on the surface of an agar medium, allowing an incubation period of from several days to a week. The main points to observe are use of an adequate amount of inoculum, an appropriate culture medium and incubation temperature and, if it is necessary to grow a strictly aerobic organism in a single large volume of liquid culture and provision of adequate aeration. Moulds It is sometimes appropriate to prepare a mould inoculum as a spore suspension (particular care is necessary to prevent them from escaping into the air) but often the inoculum is a portion of the mycelium taken with a loop or straight wire with the end few millimetres bent at a right angle. When an agar plate with a mould inoculated at the centre is required, it is easy to inoculate accidentally other parts of the plate with tiny pieces of mould, usually spores, that fall off the loop or wire. This can be avoided by placing the Petri dish on the working surface lid down, lifting the base (containing medium) vertically above the lid and introducing the inoculum upwards onto the centre of the downwards-facing agar surface with a bent wire. Testing sensitivity to antibacterial substances The agar diffusion method is widely used in industry for testing the sensitivity of micro-organisms to antibiotics, antiseptics, toothpaste, mouthwashes, disinfectants, etc. The method involves preparing a pour or spread plate of a test micro-organism, adding small amount of test substance to either a well cut in the agar medium or (preferably) a paper disc which is then placed on the agar surface. After incubation, an inhibitory effect on the test organism is indicated by a clear zone (no growth) around the test substance; microbial growth is visible to the naked eye in areas of the plate that are unaffected. This is a straightforward activity that tests several practical skills and is relevant to other aspects of biology and to everyday life. In addition to using laboratory reagents, e.g. stains, and antibiotic discs, many preparations with antimicrobial activity are readily available in pharmacists and supermarkets. There is also opportunity to think of less obvious materials, e.g. plants and their products. Materials - Take one of the pour or spread plates prepared earlier in the day. - Sterile Filter paper discs, - Distilled/demineralised water (control) - Samples to be tested, 3 (e.g. mouthwashes, selected for a range of active ingredients) - Bunsen burner - Forceps - 70% (v/v) industrial methylated spirit in a small beaker covered in foil (CAUTION:flammable, should be kept covered away from flames) - Incubator at 25-30 °C (if available) Aseptic technique should be used throughout. 1. Mark and label four sections on the base of the Petri dish, for the three different samples and control (sterile water).

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2. Using sterile forceps (flamed with alcohol and cooled) remove one filter paper disc. Dip into the first test sample, drain on the side of the container and place firmly onto the appropriate section of the seeded agar plate. 3. Wash the forceps free of the sample. 4. Repeat for the remaining samples and the control (sterile water). Remember to rinse and sterilize the forceps between each sample and to open the plate for the minimum possible time. 5. Seal the lid to the base with tape. Incubation of the plate. 6. Invert the plate and incubate at 25-30°C or at room temperature for 48 hours. 7. Examine the plate (without opening). Measure and record the size of any zones of inhibition around the filter paper discs. Consider what factors might be affecting the size of the zones of inhibition.

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Common Biochemical Tests

1. Indole Test This test demonstrates the ability of certain bacteria to decompose the amino acid tryptophan to indole. Method Inoculate 1% peptone water with one loop-full of culture and incubate at 37oC for 24 hrs. Then add 0.5 ml Ehrlich's reagent. A red colour indicates a positive reaction. 2. H2S production test: The activity of some bacteria on sulfur containing amino acids frequently results in the liberation of H2S. The H2S is usually tested for by demonstrating its ability to form black lead salt. Method Inoculate a loop-full of culture in 2% peptone water. Insert a lead acetate paper and incubate at 37°C for 24 hrs. If H2S is produced, the blackening of lead acetate paper will take place. 3. Nitrate reduction test This is a test for the presence of the enzyme nitrate reductase, which causes the reduction of nitrate to nitrite. Method Inoculate a loop-full of culture in peptone nitrite water and incubate at 37°C for 24 hr. to test culture add 0.1 ml of solution A and swirl. Add solution B drop by drop. A red color developing within a few minutes indicates the presence of nitrite and hence the ability of the organism to reduce nitrate. 4. Methyl red test The methyl red test is employed to detect the production of sufficient acid during fermentation of glucose and the maintenance of acid condition. Such that the pH of an old culture is sustained below a value of about 4.5. Method: Inoculate glucose phosphate broth with test culture and incubate at 37°C for 24 hr. Add about five drops of methyl red indicator solution. A distinct red colour is considered to be a positive test and yellow is negative. 5. Voges- Proskauer's test This is a test for the production of acetylmethyl carbinol from glucose. To the inoculated medium after incubation, alkali is added, in the presence of which any acetylmethyl carbinol present becomes oxidized to diacetyl. The diacetyl will combine with creatine to give a red colour. Method Inoculate glucose phosphate broth with test culture and incubate for 24 hr at 37 °C. Pour ¼ th of the culture into a clean test tube. Add 0.5 ml (8- 10 drops) of the L-naphthol solution and 0.5 ml of the 40% KOH solution containing 0.3% creatine. Shake thoroughly and allow to stand for 5 to

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30 minutes. The appearance of a pink to red colour indicates the presence of acetylmethyl carbinol. 6. Utilization of citrate as the sole source of carbon This is a test for the ability of an organism to utilize citrate as the sole carbon and energy source for growth. Method Inoculate koser's citrate medium with a wire needle. Incubate at 37oC for 24 hrs. Growth in he medium involving utilization of citrate as sole carbon source of carbon is shown by turbidity in the medium. 7. Fermentation of sugar: Most of bacteria will ferment a variety of sugars to form one or more acid end products. Method Inoculate sugar medium with the test culture. Inoculate it at 37oC for 24 hr. Acid production is shown by change in the colour of Andrade's indicator to pink. Gas, if produced, accumulates in the Durham tube. 8. Gelatin liquefaction: Proteolytic organisms digest proteins and consequently may liquefy gelatin. Liquefaction of gelatin is a routinely used index of proteolytic activity useful in differentiating certain microorganism but a positive result may take many days to develop. Method: A stab culture of organisms to be tested is made using an inoculum from culture. Incubate at 37 oC for 24 hrs. Liquefaction is tested by removing the nutrient gelatin culture from the incubator and holding it at 4oC for 30 minutes before reading the results. 9. Action on litmus milk: The end results of the action of bacteria on milk depend on whether the organism attacks the carbohydrates and the protein of the skim milk. 1. a) Acid production ­ shown by a change in the colour of the litmus to pink. b) If sufficient acid is produced the milk will clot. This is known as acid clot (AC) c) Reduction of the litmus and loss of colour may occur (R) d) Gas may also be produced and can be seen as gas bubbles in the medium (G), although normally this is only visible if clotting has occurred. 2. a) Coagulation of the milk may occur as a result of proteolytic enzyme activity affecting the casein, the colour of litmus remaining blue. b) Hydrolysis of the casein as a result of proteolytic activity causes clearing and loss of opacity in the mix medium, usually referred to as peptonization. Proteolysis may also result in an alkaline reaction due to ammonia production. Method: Inoculate a tube of litmus milk with a culture to be tested and incubate at 37 oC. Observe the changes which have taken place, after 24 hr.

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10. Utilization of uric acid as the sole carbon source This is a test for the ability of an organism to utilize uric acid as the sole source of nitrogen for growth. Method: Inoculate koser's uric acid medium with a wire needle. Incubate at 37oC for 24 hr. growth in the medium is shown buy turbidity in the medium. Reagents: Composition 1. Ehrlich's reagent p-dimethylaminobenzaldehyde 95% ethanol Conc. HCL 2. Griess- Ilosvay's reagents: Solution A: Dissolve 8 gm of sulphanilic acid in 1 liter of 5N acetic acid. Solution B: Dissolve 5 gm of á­ naphthyl amine in 1 liter of 5N acetic acid. 3. Methyl red indicator Methyl red 0.1gm 95% ethanol 300 ml Distilled water top to 500 ml 4. Naphthol solution L-Naphthol 5 gm 95% ethanol top to 100 ml 5. KOH­ Creatine solution: Creatine 0.3 gm 40% KOH 100 ml 6. Andrades's indicator: Add 1 N NaOH to a 0.5 % solution of acid fuchsin until the colour just becomes yellow.

4gm. 380 ml 80 ml

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OBSERVATIONS Regent (If Organism any) E. coli E. aerogenenes 1 Indole test 1% peptone Ehrlich's +ve -ve water reagents 2 Methyl red test Glucose Methyl red phosphate soln +ve -ve broth 3 Voges-proskaner's test Glucose KOH and á­ phosphate naphthol ­ve +ve broth soln. 4 Utilization of citrate Koser's citrate -ve +ve 5 Nitrate reduction test Peptone nitrate L-naphthyl water amine and +ve +ve sulfanilic acid 6 H2S production test 2% peptone Lead acetate ­ve ­ve water paper 7 Utilization of uric acid Koser's uric ­ve +ve acid 8 Liquefaction of gelatin Nutrient gelatin ­ve ­ve 9 Action on litmus milk Litmus milk Acidic Acidic 10 Fermentation of urea Urea broth ­ve ­ve 11 Fermentation of sugars Glucose + + Lactose + + Maltose + + Sucrose + + Mannitol + + Xylose + + No. Test. Medium

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Food Sampling and Preparation of Sample Homogenate

The adequacy and condition of the sample or specimen received for examination are of primary importance. If samples are improperly collected and mishandled or are not representative of the sampled lot, the laboratory results will be meaningless. Because interpretations about a large consignment of food are based on a relatively small sample of the lot, established sampling procedures must be applied uniformly. A representative sample is essential when pathogens or toxins are sparsely distributed within the food or when disposal of a food shipment depends on the demonstrated bacterial content in relation to a legal standard. The number of units that comprise a representative sample from a designated lot of a food product must be statistically significant. The composition and nature of each lot affects the homogeneity and uniformity of the total sample mass. The collector must determine the proper statistical sampling procedure, according to whether the food is solid, semisolid, viscous, or liquid, at the time of sampling. Whenever possible, submit samples to the laboratory in the original unopened containers. If products are in bulk or in containers too large for submission to the laboratory, transfer representative portions to sterile containers under aseptic conditions. There can be no compromise in the use of sterile sampling equipment and the use of aseptic technique. Sterilize one-piece stainless steel spoons, forceps, spatulas, and scissors in an autoclave or dry-heat oven. Use of a propane torch or dipping the instrument in alcohol and igniting is dangerous and may be inadequate for sterilizing equipment. Use containers that are clean, dry, leak-proof, widemouthed, sterile, and of a size suitable for samples of the product. Containers such as plastic jars or metal cans that are leak-proof may be hermetically sealed. Whenever possible, avoid glass containers, which may break and contaminate the food product. For dry materials, use sterile metal boxes, cans, bags, or packets with suitable closures. Sterile plastic bags (for dry, unfrozen materials only) or plastic bottles are useful containers for line samples. Take care not to overfill bags or permit puncture by wire closure. Identify each sample unit (defined later) with a properly marked strip of masking tape. Do not use a felt pen on plastic because the ink might penetrate the container. Submit open and closed controls of sterile containers with the sample. Deliver samples to the laboratory promptly with the original storage conditions maintained as nearly as possible. When collecting liquid samples, take an additional sample as a temperature control. Check the temperature of the control sample at the time of collection and on receipt at the laboratory. Make a record for all samples of the times and dates of collection and of arrival at the laboratory. Dry or canned foods that are not perishable and are collected at ambient temperatures need not be refrigerated. Transport frozen or refrigerated products in approved insulated containers of rigid construction so that they will arrive at the laboratory unchanged. Collect frozen samples in prechilled containers. Place containers in a freezer long enough to chill them thoroughly. Keep frozen samples solidly frozen at all times. Cool refrigerated samples, except shellfish and shell stock, in ice at 0-4°C and transport them in a sample chest with suitable refrigerant capable of maintaining the sample at 0-4°C until arrival at the laboratory. Do not freeze refrigerated products. Unless otherwise specified, refrigerated samples should not be analyzed more than 36 h after collection. Collection of samples 1. A sample, consisting of a specified number of sample units (usually five) drawn at random from each lot, shall be taken. 2. Each sample unit shall consist of at least 100 ml or g, unless stipulated in the method. 3. Collect original unopened container wherever possible.

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4. If the product is in bulk, several sample units can be collected from one container, while ensuring that the total number of sample units are not collected from one container. More than one sample unit may also be collected from large institutional or bulk containers when the total number of sample units required exceeds the number of containers in the lot. Place the collected sample units in sterile containers. A sample unit will consist of more than one container when the lot consists of containers smaller than 100 ml or g eg. four 25 ml or g containers in each sample unit. 5. Employ aseptic techniques in collecting the sample units. 6. Keep the sample unit refrigerated (0-4oC) or frozen, depending on the nature of the product, during transport. 7. Do not allow sample units, that are usually frozen, to thaw during shipment Defination of Terms 1. 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 kind of product from one and the same manufacturer available for sampling at a fixed location. 2. Sample: The sample units taken per lot for analysis. 3. Sample Unit: Usually a consumer size container of the product, and should consist of a minimum of 100 g (ml), unless stipulated in the method. 4. Analytical Unit: That amount of product withdrawn from the sample unit for analysis. 5. HGMF Count: Is the number obtained when counting either those HGMF grid-cells which contain colonies or those which do not. Counts may be made over the whole HGMF, or a central portion (one-fifth) of the HGMF. 6. HGMF Score: Is the total number of HGMF grid-cells containing colonies. It may equal the HGMF count, or be derived from this by multiplication and/or subtraction operations, as necessary. 7. Most Probable Number of Growth Units (MPNGU): On HGMF the Growth Unit (GU) is equivalent to the more familiar Colony Forming Unit (CFU). The MPNGU is derived from the HGMF score. 1. Salmonella species Sample collection Because of the continuing occurrence of Salmonella in foods, sampling plans for these organisms have received the attention of committees of national and international organizations. Each of these committees has recommended varying the number of samples from a particular lot of food according to the sampling category to which a food is assigned. Generally, the assignment to a sampling or food category depends on 1) the sensitivity of the consumer group (e.g., the aged, the infirm, and infants); 2) the possibility that the food may have undergone a step lethal to Salmonella during the manufacturing process or in the home; and 3) the history of the food. The 29

selection of a sampling plan depends mainly on the first 2 criteria cited. The history of the food would be important in deciding whether to sample, i.e., whether there was a past history of contamination. For the Salmonella sampling plan discussed here, 3 categories of foods are identified. Food Category I. - Foods that would not normally be subjected to a process lethal to Salmonella between the time of sampling and consumption and are intended for consumption by the aged, the infirm, and infants. Food Category II. - Foods that would not normally be subjected to a process lethal to Salmonella between the time of sampling and consumption. Food Category III. - Foods that would normally be subjected to a process lethal to Salmonella between the time of sampling and consumption. In certain instances, it may not be possible to fully conform to the sampling plan. Nonetheless it is still important to ascertain whether or not Salmonella is present in the suspect food. Therefore, the analyst should still try to analyze as many analytical units as is required for the food of interest, i.e., 60 analytical units for Category I foods, 30 analytical units for Category II foods, and 15 analytical units for Category III foods. Individual 25 g analytical units may be combined into 375 g composites as described above unless otherwise indicated in Chapter 5 or the OMA. Below are examples of situations that might confront the analyst. 1. The number and weights of the sample units is correct. Each sample should be mixed to ensure homogeneity before withdrawing a 25 g analytical unit. The analytical units can be composited (fifteen 25 g units into a 375 g composite), unless otherwise indicated in Chapter 5 or in the OMA. Samples should be preenriched at a 1:9 sample-to-broth ratio. 2. The number of sample units is correct, but several of the sample units have been damaged and are unusable. For example, fifteen 1 lb bags of pasta have arrived for testing, but 5 of the bags are torn and unusable. In this case, the analyst should only sample from the 10 intact bags. The contents of each intact bag should be mixed to ensure homogeneity before the analytical units are withdrawn. Since the analyst needs one 375 g composite, ten 37.5 g analytical units, from the remaining 10 intact bags, should be used to form the composite. The composite should be combined with its preenrichment medium at a 1:9 sample-to-broth ratio (375 g sample/3375 ml preenrichment) as directed in Chapter 5 or the OMA. 3. The number of sample units is incorrect, but the total weight of the sample unit(s) is greater than what would be necessary to perform the sample analysis. For example, a single 10 lb wheel of cheese has arrived for testing. Since cheese is a Category II food, thirty 25 g analytical units must be analyzed. These analytical units should be taken randomly from a wide variety of locations around the wheel. If Salmonella is present in a food, then the odds of detecting it will be enhanced if two 375 g composites are analyzed rather than a single 25 g analytical unit, as would be the case if the analyst were to treat the entire wheel as a single sample.

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4. There is less sample available than is necessary to form the required number of composites. For example, an 8 oz (226.8 g) bag of almonds has arrived for testing. Almonds are a Category II food. Category II foods require thirty 25 g analytical units (750g), so it is impossible to analyze the amount of almonds required by the sampling plan. In this case, the analyst should analyze all of the almonds at a 1:9 sample-to-broth ratio (226.8g sample/2041 ml preenrichment medium). If, in the above example, the total weight of the almonds had been less than 2 composites (750 g), but more than 1 composite, then the analyst should analyze both a whole and a partial composite. The analytical units comprising these composites should be taken randomly from a wide variety of locations in the lot of almonds. Both composites, should be preenriched at a 1:9 sample-to-broth ratio. This sampling plan applies to the collection of finished products under surveillance and/or for determination of compliance for regulatory consideration. It also applies to the collection of factory samples of raw materials in identifiable lots of processed units and/or finished products where regulatory action is possible. It does not apply to the collection of in-line process sample units at various stages of manufacture since those samples do not necessarily represent the entire lot of food under production. A sample unit consists of a minimum of 100 g and is usually a consumer-size container of product. Take sample units at random to ensure that a sample is representative of the lot. When using sample containers, submit a control consisting of one empty sample container that has been exposed to the same conditions as those under which the sample was collected. Collect more than one sample unit from large institutional or bulk containers when the number of sample units required exceeds the number of containers in the lot. A sample unit will consist of more than one container when containers are smaller than 100 g (e.g., four 25 g containers could constitute a sample unit). The numbers of sample units to be collected in each food category are as follows: Food Category I, 60 sample units; Food Category II, 30 sample units; Food Category III, 15 sample units. Submit all samples collected to the laboratory for analysis. Advise the laboratory in advance of perishable sample shipments. Sample analysis The laboratory will analyze each sample for the presence of Salmonella according to methods described in this manual. Take a 25 g analytical unit at random from each 100 g sample unit. When a sample unit consists of more than one container, aseptically mix the contents of each container before taking the 25 g analytical unit. To reduce the analytical workload, the analytical units may be composited. The maximum size of a composite unit is 375 g or 15 analytical units. The minimum number of composite units to be tested for each food category is as follows: Food Category I, 4 composite units; Food Category II, 2 composite units; Food Category III, one composite unit. For each 375 g composite, the entire amount of 375 g is analyzed for Salmonella. Refrigerate perishable samples and samples supporting microbial growth. An analytical control is required for each sample tested. The sampled lot is acceptable only if analyses of all composite units are negative for Salmonella. If one or more composite units are positive for Salmonella, the lot is rejected, provided that the analytical control is negative for Salmonella. A lot will not be resampled unless the environmental control for Salmonella is positive. For all samples positive

31

for Salmonella, determine the somatic group. See Chapter 5 for information on further handling of these cultures. Recommendations for regulatory action may be based on the identification of the Salmonella somatic group and will not require definitive serotyping before initiation of regulatory action. Imports. These sampling plans apply to imported food products intended for human consumption. Classification of food products for sampling purposes Foods that have been classified into the 3 categories described above for regulatory sampling are listed in the categories according to the Industry Product Code sequence and nomenclature. Listing does not necessarily mean that these products are probable sources of Salmonella. Food Category I. - Foods that would not normally be subjected to a process lethal to Salmonella between the time of sampling and consumption and are intended for consumption by the aged, the infirm, and infants. Food Category II. - Foods that would not normally be subjected to a process lethal to Salmonella between the time of sampling and consumption. Examples are as follows:

Industry Product Code 2 3 Milled grain products not cooked before consumption (bran and wheat germ) Bread, rolls, buns, sugared breads, crackers, custard- and cream-filled sweet goods, and icings 5 Breakfast cereals and other ready-to-eat breakfast foods 7 Pretzels, chips, and other snack foods 9 Butter and butter products, pasteurized milk and raw fluid milk and fluid milk products for direct consumption, pasteurized and unpasteurized concentrated liquid milk products for direct consumption, dried milk and dried milk products for direct consumption, casein, sodium caseinate, and whey 12 Cheese and cheese products 13 Ice cream from pasteurized milk and related products that have been pasteurized, raw ice cream mix and related unpasteurized products for direct consumption 14 Pasteurized and unpasteurized imitation dairy products for direct consumption 15 Pasteurized eggs and egg products from pasteurized eggs, unpasteurized eggs and egg products from unpasteurized eggs for consumption without further cooking 16 Canned and cured fish, vertebrates, and other fish products; fresh and frozen raw shellfish and crustacean products for direct consumption; smoked fish, shellfish, and crustaceans for direct consumption 17 Meat and meat products, poultry and poultry products, and gelatin (flavored and unflavored bulk) 20-22 Fresh, frozen, and canned fruits and juices, concentrates, and nectars; dried

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23 24 26 27 28 29 30 31 33 34 35 36 37 38 39 54

fruits for direct consumption; jams, jellies, preserves, and butters Nuts, nut products, edible seeds, and edible seed products for direct consumption Vegetable juices, vegetable sprouts, and vegetables normally eaten raw Oils consumed directly without further processing; oleomargarine Dressings and condiments (including mayonnaise), salad dressing, and vinegar Spices, flavors, and extracts Soft drinks and water Beverage bases Coffee and tea Candy (with and without chocolate; with and without nuts) and chewing gum Chocolate and cocoa products Pudding mixes not cooked before consumption, and gelatin products Syrups, sugars, and honey Ready-to-eat sandwiches, stews, gravies, and sauces Soups Prepared salads Nutrient supplements, such as vitamins, minerals, proteins, and dried inactive yeast

Food Category III: Foods that would normally be subjected to a process lethal to Salmonella between the time of sampling and consumption. Examples are as follows: Industry Product Code 2 3 4 16 Whole grain, milled grain products that are cooked before consumption (corn meal and all types of flour), and starch products for human use Prepared dry mixes for cakes, cookies, breads, and rolls Macaroni and noodle products Fresh and frozen fish; vertebrates (except those eaten raw); fresh and frozen shellfish and crustaceans (except raw shellfish and crustaceans for direct consumption); other aquatic animals (including frog legs, marine snails, and squid) Vegetable protein products (simulated meats) normally cooked before consumption Fresh vegetables, frozen vegetables, dried vegetables, cured and processed vegetable products normally cooked before consumption Vegetable oils, oil stock, and vegetable shortening Dry dessert mixes, pudding mixes, and rennet products that are cooked before consumption

18 24 26 35

2. Aerobic plate counts, total coliforms, fecal coliforms, Escherichiacoli (including enteropathogenic strains), Staphylococcus spp., Vibrio spp., Shigella spp., Campylobacter spp., Yersinia spp., Bacilluscereus, and Clostridium perfringens 33

a. Sample collection. From any lot of food, collect ten 8-oz subsamples (or retail packages) at random. Do not break or cut larger retail packages to obtain an 8-oz subsample. Collect the intact retail unit as the subsample even if it is larger than 8 oz. b. Sample analysis. Analyze samples as indicated in current compliance programs.

Equipment and materials 3. Mechanical blender. Several types are available. Use blender that has several operating speeds or rheostat. The term "high-speed blender" designates mixer with 4 canted, sharp-edge, stainless steel blades rotating at bottom of 4 lobe jar at 10,000-12,000 rpm or with equivalent shearing action. Suspended solids are reduced to fine pulp by action of blades and by lobular container, which swirls suspended solids into blades. Waring blender, or equivalent, meets these requirements. 4. Sterile glass or metal high-speed blender jar, 1000 ml, with cover, resistant to autoclaving for 60 min at 121°C 5. 6. 7. Balance, with weights; 2000 g capacity, sensitivity of 0.1 g Sterile beakers, 250 ml, low-form, covered with aluminum foil Sterile graduated pipets, 1.0 and 10.0 ml

8. Butterfield's phosphate-buffered dilution water, sterilized in bottles to yield final volume of 90 ± 1 ml 9. Sterile knives, forks, spatulas, forceps, scissors, tablespoons, and tongue depressors (for sample handling)

Receipt of samples The official food sample is collected by the FDA inspector or investigator. As soon as the sample arrives at the laboratory, the analyst should note its general physical condition. If the sample cannot be analyzed immediately, it should be stored as described later. Whether the sample is to be analyzed for regulatory purposes, for investigation of a foodborne illness outbreak, or for a bacteriological survey, strict adherence to the recommendations described here is essential. Condition of sampling container. Check sampling containers for gross physical defects. Carefully inspect plastic bags and bottles for tears, pinholes, and puncture marks. If sample units were collected in plastic bottles, check bottles for fractures and loose lids. If plastic bags were used for sampling, be certain that twist wires did not puncture surrounding bags. Any crosscontamination resulting from one or more of above defects would invalidate the sample, and the collecting district should be notified. Labeling and records. Be certain that each sample is accompanied by a completed copy of the Collection Report and officially sealed with tape bearing the sample number, collecting official's

34

name, and date. Assign each sample unit an individual unit number and analyze as a discrete unit unless the sample is composited as described previously in this chapter. Adherence to sampling plan. Most foods are collected under a specifically designed sampling plan in o of several ongoing compliance programs. Foods to be examined for Salmonella, ne however, are sampled according to a statistically based sampling plan designed exclusively for use with this pathogen. Depending on the food and the type of analysis to be performed, determine whether the food has been sampled according to the most appropriate sampling plan. Storage. If possible, examine samples immediately upon receipt. If analysis must be postponed, however, store frozen samples at -20°C until examination. Refrigerate unfrozen perishable samples at 0-4°C not longer than 36 h. Store nonperishable, canned, or low-moisture foods at room temperature until analysis. Notification of collecting district. If a sample fails to meet the above criteria and is therefore not analyzed, notify the collecting district so that a valid sample can be obtained and the possibility of a recurrence reduced. Thawing Use aseptic technique when handling product. Before handling or analysis of sample, clean immediate and surrounding work areas. In addition, swab immediate work area with commercial germicidal agent. Preferably, do not thaw frozen samples before analysis. If necessary to temper a frozen sample to obtain an analytical portion, thaw it in the original container or in the container in which it was received in the laboratory. Whenever possible, avoid transferring the sample to a second container for thawing. Normally, a sample can be thawed at 2-5°C within 18 h. If rapid thawing is desired, thaw the sample at less than 45°C for not more than 15 min. When thawing a sample at elevated temperatures, agitate the sample continuously in thermostatically controlled water bath. Mixing Various degrees of non-uniform distribution of microorganisms are to be expected in any food sample. To ensure more even distribution, shake liquid samples thoroughly and, if practical, mix dried samples with sterile spoons or other utensils before withdrawing the analytical unit from a sample of 100 g or greater. Use a 50 g analytical unit of liquid or dry food to determine aerobic plate count value and most probable number of coliforms. Other analytical unit sizes (e.g., 25 g for Salmonella) may be recommended, depending on specific analysis to be performed. Use analytical unit size and diluent volume recommended for appropriate Bacteriological Analytical Manual method being used. If contents of package are obviously not homogeneous (e.g., a frozen dinner), macerate entire contents of package and withdraw the analytical unit, or, preferably, analyze each different food portion separately, depending on purpose of test. Weighing Tare high-speed blender jar; then aseptically and accurately (± 0.1 g) weigh unthawed food (if frozen) into jar. If entire sample weighs less than the required amount, weigh portion equivalent to one-half of sample and adjust amount of diluent or broth accordingly. Total volume in blender must completely cover blades. Blending and diluting of samples requiring enumeration of microorganisms All foods other than nut meat halves and larger pieces, and nut meal. Add 450 ml Butterfield's phosphate-buffered dilution water to blender jar containing 50 g analytical unit and

35

blend 2 min. This results in a dilution of 10-1. Make dilutions of original homogenate promptly, using pipets that deliver required volume accurately. Do not deliver less than 10% of total volume of pipet. For example, do not use pipet with capacity greater than 10 ml to deliver 1 ml volumes; for delivering 0.1 ml volumes, do not use pipet with capacity greater than 1.0 ml. Prepare all decimal dilutions with 90 ml of sterile diluent plus 10 ml of previous dilution, unless otherwise specified. Shake all dilutions vigorously 25 times in 30 cm (1 ft) arc in 7 s. Not more than 15 min should elapse from the time sample is blended until all dilutions are in appropriate media. Nut meat halves and larger pieces. Aseptically weigh 50 g analytical unit into sterile screwcap jar. Add 50 ml diluent (G-l, above) and shake vigorously 50 times through 30 cm arc to obtain 100 dilution. Let stand 3-5 min and shake 5 times through 30 cm arc to resuspend just before making serial dilutions and inoculations. Nut meal. Aseptically weigh 10 g analytical unit into sterile screw-cap jar. Add 90 ml of diluent (G-l, above) and shake vigorously 50 times through 30 cm arc to obtain 10-1 dilution. Let stand 3-5 min and shake 5 times through 30 cm arc to resuspend just before making serial dilutions and inoculations.

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Enumeration of microorganisms in foods

A. Determination of Aerobic colony count in Foods

1. Application This method is applicable to the enumeration of viable aerobic bacteria (psychrophilic, mesophilic and/or thermophilic bacteria) in foods. 2. Principle The Aerobic Colony Count (ACC) estimates the number of viable aerobic bacteria per g or mL of product. A portion of the product is mixed with a specified agar medium and incubated under specific conditions of time and temperature. It is assumed that each viable aerobic bacterium will multiply under these conditions and give rise to a visible colony which can be counted. Psychrophilic bacteria: an organism which grows optimally at or below 15oC, which has an upper limit for growth at ca. 20oC, and which has a lower limit of growth of 0oC or lower. Mesophilic bacteria: an organism whose optimim growth temperature lies within a range generally accepted as ca. 20 - 45oC. Thermophilic bacteria: an organism whose optimimum growth temperature is > 45oC.

3. Materials and special equipment The following media and reagents (1-4) are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. 1) Plate count agar (PC) 2) Peptone water diluent (0.1%)(PW) 3) 2% sodium citrate (tempered to 450C) (for cheese samples only) 4) Sodium 2,3,5 triphenyltetrazolium chloride (0.1%) (optional) 5) 1N HCl and 1N NaOH 6) pH meter or paper capable of distinguishing to 0.3 to 0.5 pH units within a range of 5.0 to 8.0 7) Stomacher, blender or equivalent 8) Incubator capable of maintaining the growth temperature required for the specific type of aerobic bacteria being enumerated (i.e. for psychrophilic bacteria: 15 - 20oC, for mesophilic bacteria: 30 - 35oC, and for thermophilic bacteria: 55oC) and 45oC waterbath 9) Colony counting device (optional) 4. Procedure Determine which type of aerobic bacteria are being enumerated. Analyze each sample unit individually.

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The test shall be carried out in accordance with the following instructions: 4.1. Handling of Sample Units 4.1.1. During storage and transport, the following shall apply: with the exception of shelfstable products, keep the sample units refrigerated (0-5oC). Sample units of frozen products shall be kept frozen. 4.1.2. Thaw frozen samples in a refrigerator or under time and temperature conditions which prevent microbial growth or death. 4.1.3. Analyze sample units as soon as possible after receipt in the laboratory. 4.2. Preparation of Media 4.2.1. Prepare plate count agar and dispense in appropriate quantities. Sterilize. 4.2.2. Temper prepared melted agar in a waterbath to 45oC ensuring that the water level is 1 cm above the level of the medium in the bottles. 4.2.3. Clean surface of working area with a suitable disinfectant. 4.2.4. Clearly mark the duplicate Petri plates. 4.3. Preparation of Dilutions 4.3.1. Prepare sterile 0.1% peptone water diluent. 4.3.2. To ensure a truly representative analytical unit, agitate liquid or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit. 4.3.3. Prepare a 1:10 dilution of the food by aseptically blending 25 g or mL (the analytical unit) into 225 mL of the required diluent, as indicated in Table I. If a sample size other than 25 g or mL is used, maintain the 1:10 sample to dilution ratio, such as 11 (10) g or mL into 99 (90) mL. NOTE: Volume in brackets indicates alternate procedure for marking dilutions. 4.3.4. If a homogeneous suspension is to be obtained by blending, the blending time should not exceed 2.5 min in order to prevent over-heating. With foods that tend to foam, use blender at low speed, and remove an aliquot from below the liquid/foam interface. If a homogeneous suspension is to be obtained by shaking, shake the dilution bottles 25 times through a 30 cm arc in approximately 7 sec. 4.3.5. In some instances it may be advantageous to prepare the initial dilution on a percent basis to obtain a more accurate test material weight than is attained by the dilution ratio method; i.e., a 10% solution (suspension) is represented by 10 g (mL) per 100 g (mL) of solution (suspension), whereas a 1:10 dilution is based on 10 g (mL) of product (solute) plus 90 g (mL) of diluent (solvent). 4.3.6. Check the pH of the food suspension. If the pH is outside the range of 5.5-7.6, adjust the pH to 7.0 with sterile NaOH or HCl. 38

4.3.7. Prepare succeeding decimal dilutions as required, using a separate sterile pipette for making each transfer. 4.3.8. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present. 4.4. Plating 4.4.1. Agitate each dilution bottle to resuspend material that may have settled out during preparation. 4.4.2. Pipette 1 mL or 0.1 mL of the required dilutions to appropriately marked duplicate Petri plates. 4.4.3. In the case of products that tend to adhere to the bottom of the plates, add the inoculum to 1.0 mL of sterile diluent previously placed in the Petri plate. 4.4.4. Pour 12-15 mL of tempered agar into each plate, and mix by rotating and tilting. Allow to solidify. Plates should be poured not more than 15 min after preparation of dilutions. 4.5. Incubation Incubate plates in the inverted position for 48 h ± 4 h. Incubation temperature is dependent on the growth temperature requirements of the target organisms (for psychrophilic bacteria: 15 - 20oC, for mesophilic bacteria: 30 - 35oC, and for thermophilic bacteria: 55oC). The plates used to enumerate psychrophilic and thermophilic bacteria may be incubated up to 5 days. Other combinations of time and temperature may be used, if the lab has verified their suitability. Avoid crowding or excessive stacking of plates to permit rapid equilibration of plates with incubator temperature. 4.6. Counting Colonies 4.6.1. Count colonies promptly after the incubation period. 4.6.2. If possible, select plates with 20-200 colonies (including pinpoint colonies). If counts do not fall within this range select plates that fall nearest to the 20-200 range. 4.6.3. If plates contain colonies which spread, select a representative portion of the plates free from spreaders, if possible, and count the colonies in this area. The total count of the entire plate is estimated by multiplying the count for the representative area counted by the reciprocal of the fraction of the plate counted; e.g., 30 colonies counted on 1/4 of area of the plate; count for the whole plate: 30 x 4 = 120 colonies. 4.7 Differentiation of Colonies from Interfering Particles 4.7.1. Food particles such as meat, milk powder, etc., often interfere with the enumeration of the plates. This can be eliminated by making one extra plate of each dilution containing interfering particles and holding it under refrigeration as a control for comparison during counting.

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4.7.2. Alternatively, after incubation flood plates with 2 mL of 0.1% 2,3,5, triphenyltetrazolium chloride. Gently rock plates from side to side to cover the entire area with solution. Pour off excessive solution and allow the plates to remain at room temperature for 3 hrs. in an inverted position. The bacteria reduce the indicator to a formazan which colours the colonies red and aids in distinguishing the food particles. Colonies cannot be picked for isolation after this method has been used. 4.8. Recording Results 4.8.1. Calculate the average count (arithmetic mean) of the duplicate plates4.8.2 When reporting results (Table II) 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). 4.8.3. If the lowest dilution plated shows no colonies, the recorded value will be the lowest average obtainable with given volume plated onto a given set of replicate plates preceeded by a "less than" (<) sign, e.g., for one millilitre and a set of duplicate plates (1 mL/plate) the value is < 0.5. The lowest possible average with one colony on one of the two duplicate plates is: 1 + 0 /2 = 0.5

This value is for a 10o dilution (Dilution Factor = 1). For other dilutions, the numerical value of 0.5 must be multiplied by the reciprocal of the dilution; i.e., the Dilution Factor, 1 /10-1 = 10

4.8.4. To compute the Aerobic Colony Count (ACC), use the formula: N = A x D, where N is the number of colonies per g (mL) of product, A is the average count per plate, and D is the respective dilution factor. Table I Type of Food Liquids: milk, water etc. Preparation* pipette directly into Petri dishes and/or into peptone water diluent weigh into peptone water diluent Treatment shake shake shake stomach or blend stomach or blend shake stomach or blend

viscous lipids Solids: water soluble weigh into peptone water diluent solids powder, meats weigh into peptone water diluent all cheese spices Shellfish

weigh into previously warmed (45oC) 2% sodium citrate (Na3C6H5O7.2H2O) weigh into diluent weigh into peptone water diluent

*Sample may be weighed into a stomacher or blender jar with the diluent added prior to mixing. 40

Table II Examples for Recording Results Examples of the average number of colonies Counts between 20-200, e.g., 144 Counts higher than 200, e.g., 440 counts lower than 20, e.g., 15 No count 0 Dilution 1:1000 Highest dilution 1:1000 Lowest dilution 1:1000 Lowest dilution 1:1000 Report as no. of bacteria per g (mL) 140,000 440,000 E 15,000 E <500

B. Most Probable Number Method (MPN)

The most probable number (MPN) is particularly useful for low concentrations of organisms (<100/g), especially in milk and water, and for those foods whose particulate matter may interfere with accurate colony counts. Only viable organisms are enumerated by the MPN determination. If, in the microbiologist's experience, the bacteria in the prepared sample in question can be found attached in chains that are not separated by the preparation and dilution, the MPN should be judged as an estimate of growth units (GUs) or colony-forming units (CFUs) instead of individual bacteria. For simplicity, however, here we will speak of these GUs or CFUs as individual bacteria. The following assumptions are necessary to support the MPN method. The sample is prepared in such a way that the bacteria are distributed randomly within it. The bacteria are separate, not clustered together, and they do not repel each other. The growth medium and conditions of incubation have been chosen so that every inoculum that contains even one viable organism will produce detectable growth. The essence of the MPN method is the dilution of a sample to such a degree that inocula will sometimes but not always contain viable organisms. The "outcome", i.e., the numbers of inocula producing growth at each dilution, will imply an estimate of the original, undiluted concentration of bacteria in the sample. In order to obtain estimates over a broad range of possible concentrations, microbiologists use serial dilutions, incubating several tubes (or plates, etc.) at each dilution. The first accurate estimation of the number of viable bacteria by the MPN method was published by McCrady (1915). Halvorson and Ziegler (1933), Eisenhart and Wilson (1943), and Cochran (1950) published articles on the statistical foundations of the MPN. Woodward (1957) recommended that MPN tables should omit those combinations of positive tubes (high for low concentrations and low for high concentrations) that are so improbable that they raise concerns about laboratory error or contamination. De Man (1983) published a confidence interval method that was modified to make the tables below.

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Confidence Intervals The 95 percent confidence intervals in the tables have the following meaning. Before the tubes are inoculated, the chance is at least 95 percent that the confidence interval associated with the eventual result will enclose the actual concentration. Selecting Three Dilutions for Table Reference An MPN can be computed for any numbers of tubes at any numbers of dilutions. MPN values based on 3 decimal dilutions, however, are very close approximations to those based on 4 or more dilutions. When more than three dilutions are used in a decimal series of dilutions, refer to the 3 dilution table according to the following two cases, illustrated by the table of examples below (with 5 tubes at each dilution). Case 1. One or more dilutions show all tubes positive. Select the highest dilution that gives positive results in all tubes (even if a lower dilution gives negative results) and the next two higher dilutions (ex. a and b); if positive results occur in higher unselected dilutions, shift each selection to the next higher dilution (ex. c). If there are still positive results in higher unselected dilutions, add those higher-dilution positive results to the results for the highest selected dilution (ex. d). If there were not enough higher dilutions tested to select three dilutions, then select the next lower dilutions (ex. e). Case 2. No dilutions show all tubes positive. Select the 3 lowest dilutions (ex. f). If there are positive results in higher unselected dilutions, add those higher-dilution positive results to the results for the highest selected dilution (ex. g). Table 1 Example 1.0 g 0.1 g 0.01 g 0.001 g 0.0001 g a 5 5 1 0 0 b 4 5 1 0 0 c 5 4 4 1 0 d 5 4 4 0 1 e 5 5 5 5 2 f 0 0 1 0 0 g 4 4 1 1 0 Combination of Positives 5-1-0 5-1-0 4-4-1 4-4-1 5-5-2 0-0-1 4-4-2 MPN/g 33 33 40 40 5400 0.20 4.7

Other compendia of methods require that no excluded lower dilutions may have any negative tubes. This manual differs when the highest dilution that makes all tubes positive follows a lower dilution that has one or more negative tubes. Example b above would be read according to other compendia as (4, 5, 1, 0, 0) with MPN 4.8/g. The BAM reading, 33/g, is 7 times larger. The BAM selection method is based on FDA experience that for some organisms in some food matrices such outcomes as (2, 5, 1, 0, 0) and (0, 3, 1, 0, 0) occur too often to be random occurrences. In these cases, it appears that some factor (a competing organism or adverse set of compounds) is present at the lowest dilutions in such concentrations that it can reduce the detection of the target microbes. Until further research clarifies this situation, analysts should continue to exclude dilutions lower than the highest dilution with all tubes positive. The findings should, however, report the extent to which such lower, partially-negative dilutions have been excluded. Analysts working with

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materials with known limited complexity in research settings will want to use their professional judgement to read outcomes such as (4, 5, 1, 0, 0) as (4, 5, 1, 0, 0). They may also read outcomes such as (3, 5, 1, 0, 0) as too improbable to record, because they are not included in the tables. Inconclusive Tubes In special cases where tubes or plates cannot be judged either positive or negative (e.g., plates overgrown by competing microflora at low dilutions), these tubes or plates should be excluded from the results. The entire dilutions at or below those in which exclusion occurs may be excluded. If it is not desired to exclude the remaining tubes at or below the dilution of the excluded tubes, the results will now have an unequal number of tubes at several dilutions. Conversion of Table Units The tables below apply directly to inocula 0.1, 0.01, and 0.001 g. When different inocula are selected for table reference, multiply the MPN/g and confidence limits by whatever multiplier is required to make the inocula match the table inocula. For example, if the inocula were 0.01, 0.001, and 0.0001 for 3 tubes each, multiplying by 10 would make these inocula match the table inocula. If the positive results from this 3 tube series were (3, 1, 0), one would multiply the Table 1 MPN/g estimate, 43/g, by 10 to arrive at 430/g. Calculation of Most Probable Numbers (MPN) Table I 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 I 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. When other sized portions of the test material are placed in the tubes, MPN values obtained from Table II 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 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 I, 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 II must be multiplied by 10 giving 33 x 10 = 330 organisms per 100 g (ml) of test material. If the results need 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 I.

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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 III). 1. If only 3 dilutions are made, use the results for those 3 dilutions to compute the MPN. Examples a and b, Table II. 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 subsequent higher dilutions. Examples c and d, Table II. 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, Table II. 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, Table II. 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, Table II.

Refer to Table II and look up the value which corresponds to the number of positive tubes obtained. MPN/100 ml = No. microorganisms x (Table I) dilution factor of middle set of tubes

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MPN Tables Table II. For 3 tubes each at 0.1, 0.01, and 0.001 g inocula, the MPNs per gram and 95 percent confidence intervals. Pos. tubes MPN/g Conf. lim. 0.10 0.01 0.001 Low High 0 0 0 <3.0 -- 9.5 0 0 1 3.0 0.15 9.6 0 1 0 3.0 0.15 11 0 1 1 6.1 1.2 18 0 2 0 6.2 1.2 18 0 3 0 9.4 3.6 38 1 0 0 3.6 0.17 18 1 0 1 7.2 1.3 18 1 0 2 11 3.6 38 1 1 0 7.4 1.3 20 1 1 1 11 3.6 38 1 2 0 11 3.6 42 1 2 1 15 4.5 42 1 3 0 16 4.5 42 2 0 0 9.2 1.4 38 2 0 1 14 3.6 42 2 0 2 20 4.5 42 2 1 0 15 3.7 42 2 1 1 20 4.5 42 2 1 2 27 8.7 94 Pos. tubes MPN/g Conf. lim. 0.10 0.01 0.001 Low High 2 2 0 21 4.5 42 2 2 1 28 8.7 94 2 2 2 35 8.7 94 2 3 0 29 8.7 94 2 3 1 36 8.7 94 3 0 0 23 4.6 94 3 0 1 38 8.7 110 3 0 2 64 17 180 3 1 0 43 9 180 3 1 1 75 17 200 3 1 2 120 37 420 3 1 3 160 40 420 3 2 0 93 18 420 3 2 1 150 37 420 3 2 2 210 40 430 3 2 3 290 90 1,000 3 3 0 240 42 1,000 3 3 1 460 90 2,000 3 3 2 1100 180 4,100 3 3 3 >1100 420 --

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Table III

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

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 10;1;0,1 <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 19 100 101 102 103 104 105 110 111 112 113 114 115 120 121 122 123 124 125 130 131 132 133 134 135 140 141 142 143 144 145 150 151 152 153 154 155 MPN 10;1;0,1 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 24 200 201 202 203 204 205 210 211 212 213 214 215 220 221 222 223 224 225 230 231 232 233 234 235 240 241 242 243 244 245 250 251 252 253 254 255 MPN 10;1;0,1 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 32 300 301 302 303 304 305 310 311 312 313 314 315 320 321 322 323 324 325 330 331 332 333 334 335 340 341 342 343 344 345 350 351 352 353 354 355 MPN 10;1;0,1 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 45 400 401 402 403 404 405 410 411 412 413 414 415 420 421 422 423 424 425 430 431 432 433 434 435 440 441 442 443 444 445 450 451 452 453 454 455 MPN 10;1;0,1 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 81 500 501 502 503 504 505 510 511 512 513 514 515 520 521 522 523 524 525 530 531 532 533 534 535 540 541 542 543 544 545 550 551 552 553 554 555 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

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C. Enumeration of yeasts and moulds in foods

1. Application This method is applicable to the enumeration of viable yeasts and moulds in foods and food ingredients It may also be used to confirm the viability of apparent yeast and mould material scraped from food plant equipment and the manufacturing environment. 2. Principle In the past, acidified media were used to enumerate yeasts and moulds in foods. Such media are now recognized as inferior to antibiotic supplemented media that are formulated to suppress bacterial colony development, enhance resuscitation of injured fungi, and minimize precipitation of food particles. A medium, containing (a) adequate nutrients for growth of most yeasts and moulds and (b) antibiotics for inhibition of most bacteria, is inoculated with a given quantity of the product or with scrapings from equipment or the manufacturing environment. It is incubated at 22-25oC for 3-5 days. Colonies appearing on the medium are then counted and/or examined. The method described here is a "general purpose" method and may not be suitable for detection of yeasts and moulds adapted to certain foods, e.g., foods of very low water activity. 3. Defination of terms 3.1. Scrapings: Suspected yeast and mould material scraped from food plant equipment and the manufacturing environment. 3.2. Xerophilic: Moulds capable of growing at reduced water activity (aw). (Yeasts preferring reduced aw are also sometimes referred to as xerophilic.) (7.5) 3.3. Osmophilic: Yeasts preferring reduced aw for growth. Precautions Some yeasts and moulds can be infectious or can cause allergic responses, therefore, it is important to be fairly cautious when working with fungi. Ideally, plates should be held in incubators, not in an open room. Plate lids should generally only be removed for procedures such as the preparation of a slide for microscopic examination. Flamed needles should be cooled before making transfers to avoid dispersal of conidia and other cells. Cultures should never be smelled. 4. Materials and special equipment The following media and reagents (1-8) are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. and reference 7.3 for the formula of individual media. Note: If the analyst uses any variations of the media listed here (either product that is commercially available or made from scratch), it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency. Enumeration of yeasts and moulds in foods (not specified below) These agars are suitable for foods where the a is above 0.95, such as fresh foods (fruit, W vegetables, meat and dairy). 47

1) Dichloran rose bengal chloramphenicol agar (DRBC) 2) Plate count agar with chloramphenicol (PCA-C) 3) Potato dextrose agar with chloramphenicol (PDA-C) 4) Potato dextrose salt agar with chloramphenicol (PDSA-C) (for analysis of 'spreader' moulds) Enumeration of xerophilic yeasts and moulds in grains, flours, nuts, and spices 5) Dichloran-glycerol DG 18 agar (DG-18) Enumeration of xerophilic yeasts and moulds in jams, jellies, fruit concentrates, and dried fruits 6) 20% sucrose (diluent additive for osmophiles, see 6.3.1) 7) Malt extract agar containing 50% (w/w) sucrose Other: 8) Peptone water (0.1%) (PW) 9) 2% sodium citrate tempered to 45oC (diluent for high fat foods, such as cheese) (optional) 10) 1N HCl and 1N NaOH 11) Gram stain solutions 12) Stomacher, blender or equivalent 13) pH meter or paper capable of distinguishing to 0.3 to 0.5 pH units within a range of 5.0 to 8.0 14) Light microscope 15) Colony counting device (optional) 16) Incubator (darkened) capable of maintaining 22 to 25oC, 55oC waterbath (and 45oC waterbath if sodium citrateistobeused). 5. Procedure Each sample unit shall be analyzed individually. The test shall be carried out in accordance with the following instructions: 5.1. Handling of Sample Units and Scrapings 5.1.1. During storage and transport, the following shall apply: with the exception of shelfstable products, keep the sample units refrigerated (0-5oC). Sample units of frozen products shall be kept frozen. 5.1.2. Thaw frozen samples in a refrigerator or under time and temperature conditions which prevent microbial growth or death.

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5.1.3. Analyze the sample units as soon as possible after receipt at the laboratory. 5.2. Preparation of Medium 5.2.1. Prepare the appropriate media for the analysis being carried out (see Section 5). NOTE: DRBC agar should not be exposed to light, since photo-degradation of rose bengal produces compounds that are toxic to fungi. 5.2.2. Temper melted agar in a 55oC waterbath, ensuring that the water level is 1 cm above the level of the medium in the bottles. 5.2.3. Clean surface of working area with a suitable disinfectant. 5.2.4. Mark clearly the duplicate petri plates identifying sample, sample unit, dilution and date of inoculation. 5.3. Preparation of Dilutions 5.3.1. Prepare 0.1% peptone water as diluent. An appropriate solute, such as 20% sucrose, should be added to the diluent when enumerating osmophiles in foods such as syrups and fruit juice concentrates. In addition, a 2% solution of sodium citrate, pre-warmed to 45°C, can be used as diluent for high-fat foods such as cheese. 5.3.2. To ensure a representative analytical portion, agitate liquid or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit. 5.3.3. Some degree of soaking may be beneficial for the recovery of yeasts and moulds from dried or intermediate-moisture foods. Soaking may allow for the repair of sub-lethally damaged cells (resuscitation). Rehydrate dried foods for 1 h with an equal amount of distilled water or peptone water and store at room temperature. 5.3.4. Prepare a 1:10 dilution of the food by aseptically blending 25 g or mL (the analytical unit) into 225 mL of the required diluent, as indicated in Table I. If a sample size other than 25 g or mL is used, maintain the 1:10 sample to dilution ratio, such as 11 (10) g or mL into 99 (90) mL. NOTE: Weight or volume in brackets indicates alternate procedure for making dilutions. 5.3.5. Stomach, blend or shake according to the type of food as indicated in Table 1. Blend or stomach for the minimum time required to produce a homogeneous suspension. To prevent over-heating, blending time should not exceed 2.5 min. With foods that tend to foam, use blender at low speed and remove aliquot from below liquid/foam interface. 5.3.6. Verify the pH of the suspension. If the pH is not between 5.5 and 7.5, adjust the pH to 7.0 with a sterile solution of 1N NaOH or 1N HCl. 5.3.7. If the 1:10 dilution is prepared in a dilution bottle, it should be mixed by shaking the bottle 25 times through a 30 cm arc in approximately 7 sec.

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5.3.8. Prepare succeeding decimal dilutions as required, using a separate sterile pipette for making each transfer. 5.3.9. Because mould propagules may settle out within a few minutes, it is important to shake all dilutions (as in 5.3.7) immediately prior to making transfers to ensure uniform distribution of the microorganisms present. 5.4. Plating 5.4.1 Agitate each dilution bottle to resuspend material that may have settled out during preparation. 5.4.2 Moulds should be enumerated by a surface spread-plate technique rather than with pour plates. This technique provides maximal exposure of the cells to atmospheric oxygen and avoids heat stress from molten agar. Agar spread plates should be dried overnight before being inoculated. Spread 0.1 mL onto duplicate plates (see Section 5 for appropriate plating media) 5.4.3 For determination of viability of suspected yeast and mould material from food plant equipment and the manufacturing environment, aseptically tease the scrapings apart and distribute the pieces over the surface of solidified medium. 5.5. Incubation Incubate plates undisturbed in an upright position at 22 to 25oC for 3-5 days. Incubate plates in the dark. Normally, count colonies on plates after 5 days. Examine on the third day and if mould colonies are numerous, count them and then count again on the fifth day, if possible. Handle the plates as little as possible when counting on day 3 so spores will not be dislodged, which may result in secondary growth 5.6. Counting Colonies and Examining Growth 5.6.1. Count colonies, distinguishing, if required, yeast colonies from mould colonies, according to their colonial morphology. Microscopic examination with crystal violet stained smears may be necessary to distinguish yeast colonies from some bacterial colonies that may look like yeast. 5.6.2. If possible, select plates with 10-150 colonies. Determine the identity of pin-point colonies microscopically. If counts do not fall within this range, select plates that fall nearest to the 10-150 range. If the mycoflora consists primarily of moulds, the lower population range is selected; if primarily yeast colonies, the upper limit is counted. Alternatively, 5.6.3. If plates contain colonies which spread, select a representative portion of the plates free from spreaders, if possible, and count colonies in this area. The total count of the whole plate is estimated by multiplying the count for the representative area by the reciprocal of the fraction of the plate counted, e.g., 30 colonies counted on 1/4 of the area of the plate; count for the whole plate: 30 x 4 = 120 colonies. Results are expressed as an estimated count.

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5.6.4. Wet mounts and gram stains of several diverse types of cells per sample should be examined to confirm that bacteria are not present. Yeast cells and asexual mould spores are generally gram-positive, whereas mould mycelia are gram-negative. 5.7. Differentiation of Colonies from Interfering Particles 5.7.1. Food particles such as meat, milk powder, etc., often interfere with the enumeration of colonies. This can be eliminated by making one extra plate of each dilution containing interfering particles, and holding it under refrigeration as a control for comparison during counting. 5.8. Recording Results 5.8.1. Calculate the average count (arithmetic mean) of the duplicate plates, following the examples in Table II: Standard Methods for the Examination of Dairy Products. 5.8.2. Avoid creating erroneous ideas of precision and accuracy when computing counts (Table II). Round-off counts to two significant figures and record only the first two left hand digits. 5.8.3. If the lowest dilution plated shows no colonies, the recorded value will be the lowest average obtainable with a given volume plated onto a given set of replicate plates preceded by a "less than" (<) sign, e.g., for 1 mL and a set of duplicate plates (1 mL/plate), the value is <0.5. (The lowest possible average with one colony on one of the two duplicate plates is: 1+0/2 = 0.5). This value is for a 100 dilution (Dilution Factor = 1). For other dilutions, the numerical value of 0.5 must be multiplied by the reciprocal of the dilution, i.e., the Dilution Factor. E.g. 1/10-1 = 10. 5.8.4. To compute the yeast and mould count, use the formula: N = A x D, where N is the number of colonies per g (mL) of product, A is the average count per plate, and D is the respective dilution factor

TABLE I Preparation of Initial Dilution Type of food Preparation* Liquids milk, water, juice, etc. pipette directly into peptone water diluent Viscous liquids Weigh into peptone water diluent Solids Water soluble solids Weigh into peptone water diluent Powder, meats Weigh into peptone water diluent all cheese Weigh into previously warmed 45oC 2% aqueous sodium citrate (NA3C6H5O7-2H2O) Spices Weigh into peptone water diluent shellfish, fish products Weigh into peptone water diluent

Treatment shake shake shake stomach or blend stomach or blend shake stomach or blend

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Sample may be added into an empty stomacher bag, blender jar or dilution bottle and the diluent added prior to mixing. TABLE II Examples for Recording Results Examples of the average number of Dilution Report as no. of yeasts and moulds per colonies g (mL) count between 10-150, e.g., 144 1:1000 140,000 counts higher than 150, e.g., 440 Highest dilution440,000 E* 1:1000 counts lower than 15, e.g., 10 Lowest dilution10,000 E 1:1000 no count Lowest dilution<500 1:1000 * E is the estimated count

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D. Enumeration of coliforms faecal coliforms and E. coli in foods using

the MPN method

1. Application The Most Probable Number (MPN) method is applicable to the enumeration of coliforms, faecal coliforms and aerogenic Escherichia coli in foods, food ingredients and water, including contact water from food manufacturing plants. Note: This method is not intended to be used to isolate and enumerate E. coli serotypes associated with human illness, particularly the enterohemorrhagic serotype O157:H7. Many of the pathogenic serotypes do not give a positive faecal coliform reaction and therefore would not be detected and recovered by this method. 2. Description 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. The method has been shown to produce satisfactory results with naturally-contaminated foods and water for the detection of coliforms, faecal coliforms and aerogenic E. coli. 3. Principle The terms "coliform" and "faecal coliform" have no taxonomic validity and, therefore, are only meaningful when expressed in terms of the analytical test parameters of medium, time and temperature of incubation. Coliforms, faecal coliforms, and E. coli are considered "indicator organisms." The presence of "indicator organisms" in foods processed for safety may indicate one of the following possibilities: 1. inadequate processing and/or post-processing contamination; and/or 2. microbial growth. The presence of faecal coliforms and E. coli may indicate faecal contamination; however, it must be understood that these microorganisms can survive and multiply in a variety of non-intestinal environments, including the processing plant. When assessing the presence of "indicator organisms" in a sample, one must assess the results against the tolerance limits specified by government standards or guidelines, health agencies, or a laboratory's inhouse specifications, keeping in mind that established standards and guidelines are specifically linked to the method used to develop these standards. As indicated in section 1, the presence of coliforms, faecal coliforms and aerogenic E. coli in food and 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;

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gas production at 44.5 or 45o C from EC broth is used as confirmation of faecal coliform presence; and appearance of typical nucleated, dark-centred colonies with or without metallic sheen when positive EC broths are streaked onto L -EMB agar are indicative of E. coli. The typical colonies on L -EMB agar must be confirmed by further biochemical tests to prove the presence of E. coli.

4. Materials and special equipment The media listed below (1 to 8) are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. Note: If the analyst uses any variations of the media listed here (either product that is commercially available or made from scratch), it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency. 1) Peptone Water (0.1% and 0.5%) 2) Aqueous Sodium Citrate (2.0%), tempered to 40-45oC 3) Lauryl Sulfate Tryptose (LST) broth 4) Brilliant Green Lactose 2% Bile (BGLB) broth 5) Escherichia coli (EC) broth or EC broth with MUG (4-methylumbelliferyl-ß-D-glucuronide) 6) Levine's Eosin Methylene Blue (L-EMB) agar or Endo agar 7) MacConkey agar 8) Nutrient Agar (NA) or other non-selective agar 9) Covered water baths, with circulating system to maintain temperature of 44.5oC and 45oC. Water level should be above the medium in immersed tubes. 10) Thermometer, calibrated and traceable 11) Incubator, 35oC. 12) Stomacher, blender or equivalent. 13) Control cultures (use ATCC cultures or equivalent): positive control(s): E. coli that is known to produce gas at 44.5 / 45o C and is capable of fermenting lactose to produce typical reactions on L-EMB agar; if using EC-MUG, a strain that is known to produce ß-glucuronidase EMB / IMViC negative control: Enterobacter aerogenes or an equivalent gram negative rod that does not produce "positive" reactions on EMB and is indole-negative, methyl red-negative, 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 (LST, BGLB 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. 54

14) pH meter capable of distinguishing to 0.1 pH units within the range of pH 5.0 to 8.0 or pH paper capable of distinguishing from 0.3 to 0.5 pH units, within the same range. 15) Supplies needed for confirmation (commercially available): The following supplies may be needed for confirmation; use A or B (see 7.9). The choice of further identification schemes (7.9.5) may require alternate media A. IMViC media and reagents: a. Tryptone (or tryptophane) broth Indole reagents (available commercially) b. Buffered Glucose broth Voges-Proskauer test reagents (available commercially) Methyl red solution c. Simmon's Citrate (SC) agar B. Rapid Identification Kits or Systems (such as API, Vitek or equivalent) 5. Procedure Each sample unit may be analyzed individually or the analytical units may be combined where requirements of the applicable sampling plan can be met. Carry out the test in accordance with the following instructions: 5.1. Handling of Sample Units 5.1.1. In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated (0-5oC) or frozen, depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death. 5.1.2. Analyze sample units as soon as possible after their receipt in the laboratory. Shellfish must be analyzed within 24 hours of collection. 5.2. Preparation for Analysis 5.2.1. Have ready sterile peptone water. 5.2.2. Clean the surface of the working area with a suitable disinfectant. 5.2.3. Arrange LST broth tubes in rows of five and mark them identifying the sample unit and the dilution to be inoculated (Table II). 5.3. Preparation of Sample, Initial Set-up and Reporting- Raw and Processed Shellfish 5.3.1. For all shellfish, always use 0.5% peptone water for all dilutions. 5.3.2. Include only live animals in the sample for unfrozen shellfish. Select 10 or more animals to obtain a minimum of 200 g of meat and liquor. 5.3.3. Scrape off all extraneous growth and loose material from the shell and scrub the shellfish (including the crevices at the juncture of the shells) with a sterile stiff brush under running water of potable quality. Do no use faucets equipped with aerators. Drain shellfish in a clean container or on clean towels.

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5.3.4. Disinfect hands (soap and water, rinse with potable water then rinse with 70% alcohol) or gloves (dipped in iodophore solution or other suitable disinfectant then rinsed with potable water) prior to shucking shellfish. Alternatively, use disposable gloves disinfected with 70% alcohol. A protective mail glove may be worn under the disposable glove to prevent accidental injury. Using a sterile shucking knife, open the shellfish through the bill, not hinge, and collect meats and liquor into a sterile container. 5.3.5. Weigh at least 200 g of shellfish and liquor into a tared blender jar and add an equal amount of 0.5% peptone water. Blend for 1 - 2 minutes. Blended homogenate represents a 1 in 2 dilution. 5.3.6. To obtain a 1 in 10 dilution, add 20 g of the homogenate to 80 g of peptone water and shake. Shake dilutions 25 times through a 1 -foot (30 cm) arc in approximately 7 seconds. 5.3.7. Prepare succeeding decimal dilutions as required using a separate sterile pipette for making each transfer. 5.3.8. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present. 5.3.9. Immediately (i.e., within 2 minutes after blending) prepare the dilutions from the ground sample and then proceed to inoculate into tubes. Inoculate each of separate sets of five tubes of LST broth with each dilution to be tested, according to the scheme in (Table II) as follows: Inoculate shellfish samples into LST: 10 mL of a 1 in 10 dilution into each of 5 tubes of double strength LST, 1 mL of 1 in 10 dilution into each of 5 tubes of single strength LST, and 1 mL of 1 in 100 dilution to each of 5 tubes of single strength LST. 5.3.10. Follow incubation of LST and confirmation steps for coliforms, faecal coliforms and E. coli as required, and record results as MPN per 100 g of shellfish. 5.4. Preparation of Sample, Initial Set-up and Reporting - Water 5.4.1. Inoculate each of separate sets of five tubes of LST broth with each dilution to be tested, according to the scheme in (Table II), as follows. Inoculate each of the five tubes of 10 mL double strength LST broth (first row) with 10 mL of the undiluted water sample. Inoculate each of the five tubes of 10 mL single strength LST broth (second row) with 1 mL undiluted water. Inoculate each of the five tubes of 10 mL single strength LST broth (third row) with 0.1 mL of undiluted water. 5.4.2. Follow incubation of LST and confirmation steps for coliforms, faecal coliforms and E. coli as required, and record results as MPN per 100 mL of water 5.5. Preparation of Sample, Initial Set-up and Reporting - All other commodities 5.5.1. To ensure a truly representative analytical unit, agitate liquids or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit. To reduce the workload, the

56

analytical units may be combined for analysis. It is recommended that a composite contain not more than 500 g. 5.5.2. Prepare a 1 in 10 dilution of the food by aseptically blending 11 (10) g or mL (the analytical unit) into 99 (90) mL of the required diluent, as indicated in Tables I and II. If five sub-samples are composited for analysis, aseptically blend 50 g or mL into 450 mL of the required diluent. For fish products an alternative method may be used. Weigh 100 g fish products and add 300 mL of 0.1% peptone water. Blend for 2 minutes. Blended homogenate represents a 1 in 4 dilution. Weigh 40 g of homogenate into 60 mL of 0.1 % peptone to obtain a 1 in 10 dilution. Pipette into LST as in 5.3.9 and express results as MPN/100g. 5.5.3. With products that require blending, blend or stomach for the minimum time required to produce a homogeneous suspension and to avoid overheating, blending time should not exceed 2.5 min. When blending foods that tend to foam, use blender at low speed and remove aliquot from below liquid/foam interface. 5.5.4. Check pH of the food suspension. If the pH is outside the range of 5.5-7.5, adjust pH to 7.0 with sterile 1N NaOH or 1N HCl. 5.5.5. Allow the food homogenate (1 in 10 dilution) of dry foods to stand at room temperature for 15 min. In all other instances, continue the analysis without this delay. 5.5.6. Prepare succeeding decimal dilutions as required using a separate sterile pipette for making each transfer. Shake dilutions 25 times through a 1-foot (30 cm) arc in approximately 7 seconds. 5.5.7. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present. 5.5.8. Inoculate each of separate sets of five tubes of LST broth with each dilution to be tested, according to the scheme in (Table II) as follows. 5.5.9. Inoculate each of the five tubes of 10 mL single strength LST broth (first row) with 1 mL of the10 -1 dilution. Inoculate each of the five tubes of succeeding rows of single strength LST with 1 mL additional dilutions. 5.5.10. Follow incubation of LST and confirmation steps for coliforms, faecal coliforms and E. coli as required. Compute MPN per g (mL) of food (per 100 g of shellfish or fish products or per 100mL of water) convert the number of gas-positive tubes to MPN values. 5.6. Incubation of LST 5.6.1. In order to verify growth conditions in the elevated temperature water baths, inoculate one LST broth tube with the MPN broths positive control and one LST 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. 5.6.2. Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials. 57

5.6.3. Incubate the inoculated LST broth tubes at 35oC for 24 ± 2 h. 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. 5.6.4. Incubate gas-negative tubes (except raw shellfish and fish products) for an additional 24 ± 2 h, examine, record the number of additional gas-positive tubes, add to the result obtained in 5.6.3 and begin the confirmed coliform, faecal coliform and E. coli tests for the additional gas-positive tubes, as required. 5.6.5. The absence of gas in all of the tubes at the end of 48 ± 4 h (24 ± 2 h for raw shellfish and fish products) of incubation constitutes a negative presumptive test. 5.7. Confirmation Steps for Determination of Coliforms 5.7.1. Use BGLB broth dispensed in 10 mL volumes in tubes containing gas vials. 5.7.2. Shake or rotate the positive LST broth tubes to mix the contents and transfer one loopful from each tube to a tube of BGLB broth (avoid transferring pellicle). Sterile wood applicator sticks or other appropriate transfer devices may be used for making the transfers. 5.7.3. Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials. 5.7.4. Incubate the inoculated BGLB broth tubes at 35oC for 24 ± 2 h. Examine for gas formation (gas bubble or effervescence) and record results. 5.7.5. Incubate gas-negative tubes for an additional 24 ± 2 h, re-examine, record the numbers of additional gas-positive tubes and add to the result obtained in 5.7.4. 5.7.6. Formation of gas during 48 ± 4 h incubation constitutes a positive confirmed test. 5.7.7. Compute the MPN of Confirmed Coliforms per g (mL) of food (per 100 g of shellfish or fish products or per 100 mL of water) convert the number of gas-positive tubes to MPN values. 5.8. Confirmation Steps for Determination of Faecal Coliforms 5.8.1. Use EC broth (with or without MUG), dispensed in 10 mL volumes in tubes containing gas vials. 5.8.2. Shake or rotate the positive LST broth tubes (obtained in 5.6) 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. This transfer should be made simultaneously with 5.7 above 5.8.3. Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials. 5.8.4. Incubate the inoculated EC broth tubes in a water bath at 45oC for 24 ± 2 h (for shellfish and fish products analysis incubate at 44.5oC). Maintain the water level in the bath at least 1 cm above the level of the medium in the tubes.

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5.8.5. Examine for gas production (gas bubble or effervescence), record results, and begin on the same day E. coli identification for all gas-positive tubes (5.9). 5.8.6. Incubate gas-negative tubes (except raw shellfish and fish products) for an additional 24 ± 2 h, examine, record the number of additional gas-positive tubes, add to the results obtained in 5.8.5 and begin the E. coli identification for the additional gas-positive tubes. 5.8.7. The absence of gas in all of the tubes at the end of 48 ± 4 h (24 ± 2 h for raw shellfish and fish products) of incubation constitutes a negative presumptive test. 5.8.8. Formation of gas during 48 ± 4 h (24 ± 2 h for raw shellfish and fish products) incubation constitutes a positive faecal coliform test. 5.8.9. Tubes containing EC-MUG broth should also be examined under UV light (366 nm) for glucuronidase activity. Blue-green fluorescence indicates a positive presumptive E. coli test; these tubes may be used for further testing described in 5.9 to confirm presence of E. coli. Precautions: Follow safety precautions in the manufacturer's instructions when using the UV light. Negative controls of the EC-MUG broth should be also examined under the UV light to ensure that the tubes do not fluoresce. 5.8.10. Compute faecal coliform MPN per g (mL) of food (per 100 g of shellfish and fish products or per 100 mL of water) convert the number of gas-positive tubes to MPN values. 5.9. Confirmation Steps for Identification of E. coli 5.9.1. Gently shake each gas-positive EC broth tube or each fluorescing EC- MUG broth tube (5.8.5 and 5.8.6) and streak a loopful of the culture onto a L-EMB or Endo agar plate. 5.9.2. Incubate the plates at 35oC for 18 to 24 h, and examine for typical non-mucoid, nucleated, dark-centred colonies with or without a metallic sheen which are indicative of E. coli. Note: It is up to the Laboratory Supervisor to determine which dilutions and sets of presumptive (gas- positive) MPN tubes are to be confirmed (and, subsequently, the number of colonies picked per plate) to adequately determine the final and confirmed MPN count. 5.9.3. 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 4oC, to be taken to non-selective media if the initial colony does not confirm as E. coli. Incubate at 35oC for 18-24 h. Use these cultures for further confirmation. 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. Note: Confirmation can be done by either completing the GIMViC tests (5.9.4) or by the use of a rapid identification kit (7.9.5).

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5.9.4. GIMViC From the streaked plates (NA, EMB or MacConkey), transfer inoculum into a separate tube of each of EC broth (G medium) 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 44.5o C or 45.0oC (G) Incubate inoculated tubes of G medium (EC broth) in a water bath at 44.5o C or 45.0oC for 24 ± 2 h. Examine for gas production. If no gas has been produced, incubate for an additional 24 ± 2 h and re-examine. Record results. Indole (I) Incubate inoculated tubes of Tryptone or tryptophane broth at 35oC for 24 ± 2 h. Add indole reagent (commercially available) to each tube following manufacturer's instructions. A dark red colour in the alcohol 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 35oC for 48 ± 2 h. 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 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 35oC for 48 ± 2 h and observe for growth. Visible growth (positive reaction) is usually accompanied by a change of colour from green to deep blue Interpretation The characteristic GIMViC reaction pattern for E. coli is given in Table III. If necessary, commonly occurring coliforms may be differentiated by using the data in Table IV. 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 60

considered to be absent from the tube of primary EC broth from which the isolates originated. 5.9.5. Rapid Identification Kits Rapid identification kits may be used to identify E. coli. Follow manufacturer's instructions. 5.9.6. Calculation of MPNs Compute the MPN of E. coli per g(mL) of food (per 100 g of shellfish and fish products or per 100 mL of water) based on the number of tubes found to contain isolates that produce GIMViC reaction patterns characteristic of E. coli as given above or confirmed by rapid identification kits as E. coli.

TABLE I Preparation of the Initial Dilution Type of Food Preparation* Product Liquids: milk, water, etc. pipette directly into LST and/or into peptone water diluent weigh into peptone water diluent viscous liquids Solids: Water soluble weigh into peptone water diluent solids Powder, meats weigh into peptone water diluent all cheese spices shellfish, products Treatment

shake shake shake or or

blend stomach weigh into previously warmed (40-45oC) 2% aqueous sodium blend citrate (Na3C6H5O7.2H2O) stomach weigh into peptone water diluent shake fish weigh into peptone water diluent blend stomach

or

* Sample may be added into an empty stomacher bag, blender jar or dilution bottle and the diluent added prior to mixing.

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TABLE II Marking and Inoculating Scheme Tube Marking* WATER 1 Dilution Volume of dilution inoculated into LST Amount of product broth tubes represented per tube

101 10 mL of undiluted water into 10 mL 10 mL double strength medium 0 0 undil. 10 1 mL of undiluted water into 10 mL1 mL single strength medium -1 -1 undil. 10 0.1 mL of undiluted water into 10 mL 0.1 mL single strength medium RAW SHELLFISH (Optional for Fish Products) 0 undil. 100 10 mL of 10-1 dilution of solids into 10 1 g mL of double strength medium -1 -1 1 in 10 10 1 mL of 10-1 dilution into 10 mL single 0.1 g strength medium -2 -2 1 in 100 10 1 mL of 10-2 dilution into 10 mL single 0.01 g strength medium ALL OTHER COMMODITIES 0 undil. 100 1 mL of undiluted liquids into 10 mL1 mL single strength medium 0 0 undil. 10 10 mL of 10-1 dilution of solids into 10 1 g mL of double strength medium -1 -1 1 in 10 10 1 mL of 10-1 dilution into 10 mL single 0.1 g or mL strength medium -2 -2 1 in 100 10 1 mL of 10-2 dilution into 10 mL single 0.01 g or mL strength medium -3 -3 1 in 1000 10 1 mL of 10-3 dilution into 10 mL single 0.001 g or mL strength medium -4 -4 1 in 10 1 mL of 10-4 dilution into 10 mL single 0.0001 g or mL 10000 strength medium Further dilutions of the food may be inoculated in the same manner, into single strength medium, depending on the anticipated level of contamination of the food. For inoculation of initial dilution of shellfish see Section 5.3 undil. * Other marking schemes may be used.

Table III GIMViC Pattern for E. coli Biotypes Gas at 44.5 - 45oC Type I G Type II + (Anaerogenic) Indole I + Methyl Red M + + Voges-Proskauer V Citrate C -

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TABLE IV** Differentiation of Commonly Occurring Coliforms Gas in EC broth at Indole 44.5 - 45oC test Escherichia coli Type I (typical) + Type II (anaerogenic) Intermediates Type I Type II Enterobacter aerogenes Type I Type II Enterobacter cloacae Irregular Type I Type II + Type VI + Irregular Other types Reactions variable + Methyl red Proskauer + + test Vogestest Growth citrate on

+ + + -

+ + + + -

-* -* + + + +

+ + + + + +

* Weak positive reactions are occasionally found.

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Isolation and Enumeration of pathogenic microorganisms in food.

A. Isolation of E. coli 0157 in foods

1. Application This method is applicable to the isolation of viable Escherichia coli O157 in foods. 2. Description The method has been shown to produce satisfactory results with artificially-contaminated meats (including beef, veal and pork), vegetables, dairy products, spices and environmental samples. This method can be used successfully for the detection of E. coli O157 in other foods, food ingredients and environmental samples. 3. Principle The sample is enriched in a selective broth and plated on selective agars. Presumptive positives are determined within 48 h. Confirmatory biochemical and serological tests are performed on purified colonies. 4. Materials and special equipment Broths and agars (base media and supplements are commercially available) 1) Modified Tryptic Soy Broth with Novobiocin (mTSB-n) 2) Enterohemorrhagic E. coli (EHEC) Enrichment Broth (EEB) 3) Modified Hemorrhagic Coli Agar (mHC) with Tellurite and Cefsulodin 4) Modified Sorbitol MacConkey agar (TCCSMAC) with Tellurite, Cefixime, and Cefsulodin 5) Purple broth base with cellobiose Other necessary supplies and equipment 6) 0.5% K2SO4 (needed for some spices and foods containing large amounts of spices) 7) 1N HCl and 1N NaOH 8) pH meter or paper capable of distinguishing 0.3 to 0.5 pH units within a range of 6.0 to 7.5 9) Stomacher, blender, or equivalent 10) Control cultures (use ATTC cultures or equivalent) positive control: E. coli O157 (H7 or other serovars) negative control: E. coli (NOT an O157) 11) Incubators capable of maintaining 35 and 42°C Confirmation media and reagents (commercially available) 12) Trypticase Soy Agar with Yeast Extract (TSA-YE) 13) Rapid Identifcation kits 14) Latex Agglutination kits

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Optional (commercially available) 15) IMVIC Reagents (see MFHPB-19) 16) urea agar slants 17) MUG (4-methylumbelliferyl-$-D-glucuronide) 18) BCIG (5-bromo-4-chloro-3-indolyl-$-D-glucuronide) (cyclohexylammonium) salt 19) O157 and H7 antisera 5. Procedure Each sample unit may be analyzed individually or the analytical units may be combined. Carry out the test in accordance with the following instructions: 5.1. Handling of Sample Units 5.1.1. In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated (0-5oC) or frozen, depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death. 5.1.2. Analyze sample units as soon as possible after their receipt in the laboratory. 5.2. Preparation for Analysis 5.2.1 Have ready sterile mTSB-n (and/or EHEC enrichment broth (EEB)). 5.2.2 Clean the surface of the working area with a suitable disinfectant. 5.3. Preparation of Sample 5.3.1. To ensure a truly representative analytical unit agitate liquids or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit. To reduce the workload, the analytical units may be combined for analysis. It is recommended that a composite contain not more than five analytical units. Note: When analyzing larger volumes, the enrichment broth should be prewarmed to 35oC. 5.3.2. Prepare a 1:10 dilution of the food by aseptically adding 25 g or mL (the analytical unit) into 225 mL of the enrichment broth mTSB-n (and EEB if applicable). Stomach or blend. A second primary enrichment broth started directly in EEB should be done when there is a high bacterial load of competing organisms in the sample. Note: Some spices, such as onion and garlic powder are antimicrobial in nature. When analysing spices or products containing large amounts of spices, add 0.5% K2SO4 to mTSB-n before autoclaving. Garlic especially affects E. coli O157, therefore garlic or garlic containing products need to be diluted 1:100. Other spices also may need to be analyzed using larger dilutions either -Na or -CHX

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5.3.4. After the addition of the sample to the broth adjust the pH of the mixture, if necessary, to 6.0 to 7.0 with 1N NaOH or 1N HCl. 5.3.5. A positive and a negative control should be set up at the same time. 5.3.6. Incubate the enrichment mixture and controls for 22-24 h at 42oC. 5.3.7. Either screen the enrichment broth for E. coli O157 by using rapid kits or proceed to 7.5. Note: When using the rapid kits, the incubation temperature (5.3.6) may be adjusted to the manufacturer's instructions. Retain enrichment broths (5.3.7) under refrigeration temperatures as all rapid kits that show positive reactions must be confirmed culturally following this method. 5.4. Secondary Enrichment in EEB Note: This step must be used when a rapid kit has identified the presence of E. coli O157 but it was not isolated when the primary enrichment broths, mTSB-n and/or EEB, were plated onto the selective agars. E. coli O157 may be difficult to isolate from some samples with high ACC levels. After agitation of the enrichment broth, transfer 1 mL of the mTSB-n and/or EEB to 9 mL of EEB. Incubate 18-24 h at 35oC. Plate 0.1 mL of 10-4 to 10-6 dilutions (made in 0.1% peptone water) from the enrichment broths onto the selective agars, as below. Follow confirmation steps for typical colonies.

5.5. Selective Isolation 5.5.1 Plate dilutions of 10-4 to 10-6 from each enrichment broth onto mHC and TCCSMAC agar plates. Incubate for 18-24 h at 42oC. Due to the increased selectivity of TCCSMAC, the counts may be one log less than on mHC. 5.5.2 On the mHC agar, typical E. coli O157:H7 colonies appear blue. On TCCSMAC, typical E. coli O157:H7 colonies appear colorless, bear the tint of the medium or are gray to pink with smokey centers. Other serovars of E. coli, including O157 (not H7) will be yellow on mHC and red on TCCSMAC. Some of these sorbitol positive colonies may be pathogenic also. 5.6. Confirmation Steps 5.6.1. If the suspect colonies are well isolated, confirm that they are sorbitol negative and cellobiose negative (5.6.2) using the same isolate for all tests. Rapid identification kits may be used (5.6.6). If not well isolated, streak suspect colonies onto mHC and/or TCCSMAC and/or TSA-YE for purity, and then continue, as below. 5.6.2. Draw a grid on mHC and TCCSMAC. Inoculate 5-10 typical and isolated colonies onto the grid cells of mHC agar and TCCSMAC plates. Incubate at 42°C for 18-24 h. Inoculate the same isolates into tubes of purple broth base containing 1% cellobiose. Incubate at 35°C for 18-24 h.

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5.6.3. Continue confirmation of colonies that are sorbitol negative and cellobiose negative (no acid production). See Table 1 for typical biochemical and serological reactions. Rapid identification kits may also be used. Repick from the original plates (5.5.1) if the cellobiose reactions are positive, i.e. show acid production (yellow reaction), until a total of 10 colonies have been screened. Secondary enrichment may be necessary. (See 5.4.) 5.6.4. Use the purified colonies (5.6.1) and continue confirmation steps. Unless stipulated, incubate all tests at 35oC for 22-24 h. 5.6.5. Do a Gram stain. 5.6.6. Use rapid identification kits following manufacturers' instructions. 5.6.7. Confirm isolates as an O157 using latex agglutination kits. Optional Confirmation Steps: See Table 1 for typical reactions. 5.6.8. Streak suspect colonies onto Phenol red sorbitol agar with MUG (PSRA), and/or Sorbitol MacConkey agar with BCIG. Incubate plates at 35oC for 22-24 h. 5.6.9. Inoculate IMViC tests and Urea slants). Incubate at 35oC. 5.6.10 Complete serological testing using O157 antisera and H7 antisera. Follow manufacturer's instructions. The isolate must be "resuscitated" in M broth or on motility agar several times (at least three times). 6. Preparation of media When steam sterilization is used, it is essential to allow sufficient time for the load to reach the required temperature before the actual sterilizing period commences. This varies with the nature and size of the load. Thus, proper exposure times should be followed to ensure sterilization of solutions in flasks and heat stable culture media. Refer to the sterilizer manual. 6.1. EHEC Enrichment broth (EEB) mTSB without novobiocin (9.2) Vancomycin Cefsulodin Cefixime Distilled water 33 g 8 mg 10 mg 0.05 mg 1.0 L

Dissolve, and adjust pH to 7.4 - 7.6. Autoclave for 15 min at 121oC. Cool to 50oC and just before use add the filter sterilized antibiotics.

6.2. Modified TSB with Novobiocin (mTSB-n)

Tryptic Soy Broth or Tryptone Soya Broth Bile Salts No. 3 Dipotassium phosphate Distilled water

30g 1.5 g 1.5 g 1.0 L

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Dissolve, adjust pH to 7.4-7.6, heat to boiling, dispense in 225 mL to 1 L amounts. Autoclave for 15 min at 121oC. After cooling store at 4oC.

Novobiocin Solution (100 mg/mL) Novobiocin (sodium salt) Deionized water 100 mg 1.0 mL

Dissolve novobiocin. Filter sterilize, using 0.2 µm filter and s yringe. May be stored several months in a dark bottle at 4oC. Add 0.2 mL solution per 1 L of mTSB before use.

6.3. Modified HC Agar (mHC) Tryptone Bile Salts No. 3 Sodium chloride Sorbitol 4-methylumbelliferyl-ß-Dglucuronide (MUG) (optional) 1.6% Bromocresol purple 0.1% K Tellurite 1% Cefsulodin Agar Distilled water 20.0 g 1.12 g 5.0 g 20.0 g 0.10 g 0.94 mL 2.5 mL 1.0 mL 15.0 g 1.0 L

Heat to boiling with stirring to dissolve completely. Autoclave 15 min at 121oC and dispense. Final pH should be 7.4 ± 0.2. Temper to 50°C. Filter sterilize cefsulodin and add aseptically to the agar before dispensing. Store prepared plates at 4oC for two weeks. Note: Cefsulodin is not needed if the enrichment broth EEB is used. 6.4. Modified Sorbitol MacConkey Agar (TCCSMAC) Sorbitol MacConkey Agar 0.1% K Tellurite (50 mg/mL) 1% Cefsulodin Cefixime (1.0 mg/mL) Distilled water 60 µ L 1.0 mL 60 µL 1.0 L

Prepare Sorbitol MacConkey Agar according to manufacturers' instructions. Add the tellurite and heat to dissolve completely. Autoclave 15 min at 121oC and dispense. Final pH should be 7.2 ± 0.2. Temper to 50oC. Filter sterilize antibiotics and add aseptically to the agar before dispensing. Store prepared plates at 4oC for two weeks. Note: Cefsulodin is not needed if the enrichment broth EEB is used. 6.5. Nutrient Agar Follow manufacturers' instructions.

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6.6. Phenol Red Sorbitol Agar with MUG Adjust pH to 6.8 to 6.9 and autoclave 15 min at 121oC. Cool and dispense. Store at 4oC. When using the MUG supplement, follow manufacturers' instructions. Phenol Red Broth Base Agar D-sorbitol 4-methylumbelliferyl-ß-D-glucuronide (MUG) or MUG supplement 6.7. Purple broth base Proteose peptone Beef extract Sodium chloride Bromcresol purple Distilled water 10 g 1g 5g 0.02 g 1.0 L 20 g 20 g 0.005%

Heat to boiling to dissolve completely. Final pH should be 6.8 ± 0.2 at 25oC. Add 10 g of the cellobiose. Stir. Dispense 10 mL into tubes and autoclave at 121oC for 15 min. 6.8. Sorbitol MacConkey Agar with BCIG Sorbitol MacConkey Agar (BCIG) 5-bromo-4-chloro-3-indolyl-$-D-glucuronide-Na (or CHX) salt Distilled water 0.1 g 1L

Prepare Sorbitol MacConkey agar according to manufacturers' instructions and add BCIG-Na salt. Autoclave 15 min at 121oC, cool and dispense. Store at 4oC. Note: If using BCIG-CHX, it must be in solution, as follows: dissolve 0.1 g BCIG in 2.5 mL 95% ethanol and 0.5 mL of 1N NaOH. Heat slightly to dissolve and add to 1 L Sorbitol MacConkey agar. 6.9. Tryptic Soy Agar - Yeast Extract (TSA-YE) TSA Yeast extract 40 g 6g

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Distilled water

1.0 L

Autoclave for 15 min at 121oC, cool and dispense in Petri plates. 6.10. Urea Agar Slants

Table 1. Characteristics of E. coli O157:H7 E. coli ReactionsA Negative O157:H7 Other or non-E. coli Reactions Positive - (No Color) - (Yellow) + (Red) + (Blue or Growth) + (Yellow) + (Brilliant Pink) + (Pigment) + (Fluorescence) + (Coloured) - (Negative) - (Negative) - (Negative)

Gram Stain IMViCs Indole Methyl Red Vogues-Proskauer Citrate Cellobiose Urea slants Pigment Production on Nutrient Agar MUG Reaction BCIG Reaction Latex Agglutination O157 H7

A B

+ (Red) + (Red) - (No Color) - (Green or No Growth) - (Purple) - (Pale) - (No Pigment) - (No fluorescenceb) - (Pale) + (Positive) + (Positive) + (Positive)

+ (Positive Reactions); - (Negative Reactions) E. coli O157:H16 and H45 fluoresce when MUG is present.

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B. Enterococcus

1. Identifying characteristics

· · · · · · · · · · · ·

Gram positive Cocci shape Nonmotile Occur in pairs or short chains Cells are one micrometer in diameter Predominately inhabit human intestines Faculative anaerobes (prefer anaerobic) Complex and variable nutritional requirements Resistant to many Gram positive antibiotics Perform simple fermentation Mechanism of pathogenicity unknown Used as indicators of fecal pollution in the purification of water and dried and frozen foods Members of genus streptoccous Belong to Lancefield's serologic group D Streptococcus Catalase negative Can grow in 6.5% NaCl Can grow at a pH range of 9.6 to 4.6 Can grow at temperatures ranging from 10 to 45°C Optimunm growth at 37°C Sensitive to chlorination

· · · · · · · ·

2. Taxonomic description The enterococcus group is a subgroup of the fecal streptococci that includes at least five species: S. faecalis, S. faecium, S. durans, S. gallinarum, and S. avium. The enterococci are differentiated from other streptococci by their ability to grow at high pH (9.6 at 10), high temperature (45°C) and in high salt concentrations (6.5% sodium chloride). The enterococcus are generally resistant to many Gram positive antibiotics such as the tetracyclines, aminoglycosides, sulfonamides, some penicillins, and lincosamides. E. faecalis and E. faecium are the most frequent species found in humans. E. faecalis is the only enterococcus species that has been genetically characterized. Its genome is 3 mb in length. The two genetic mechanisms first

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discovered in the enterococci were conjugative transposons and sex pheromone plasmids. Some strains require vitamin B and amino acids for growth. Selected differential physiological characteristics for species of the enterococci.

E.faecalis E. faecium E. durans E. bovis E.equinus

Hemolysis Growth at10 °C Growth at 45°C Growth at 50°C Growth at pH 9.6 Growth at 6.5% NaCl Growth at 40% bile Resists 60°C for 30 min NH3 from arginine Gelatin liquefied Tolerates 0.04% Pot. tellurite Acid from Glycerol Acid from Mannitol Acid from Sorbitol Acid from L-arabinose Acid from Lactose Acid from Sucrose Acid from Raffinose

-/+ + + + + +/+ + + -/+ + + + + + + -

+ + + + +/+ + + + + + +/-

+/+ + +/+/+ +/+ + -

+ + -/+ -/+ +/+ + +

+ + + -

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Acid from Melibiose Acid from Melezitose Starch hydrolyzed Tetrazolium reduced at pH 6.0

+ +

+ -

-

+ + +/-

-

3. Isolation and ecology Most procedures employ presumptive media followed by confirmatory tests. Primary selective agents can be azide, tellurite, bile, neonycin, Tween 80, taurocholate, selenite, NaCl, alcohol, phenylethyl, and thallium. For isolation, the Association of Food and Drug Officials of the United States recommends KF agar medium. This is selective differential agar that contains sodium azide, that inhibits catalase positive organisms, and tetrazolium chloride which produces a red color in the colonies. Ethyl violet azide (EVA) broth can be used as a confirmation. Fecal enterococci from water can be isolated, cultivated, and enumerated in this broth. Growth of fecal enterococci in EVA results in turbidity and a purple sediment in the bottom of liquid cultures. There is also a tyrosine decarboxylase activity procedure and a mentagan test that works well. Enterococci are able to grow in the presence of bile and hydrolyze the esculin; the liberated diphydroxycourmarin complexes with ferric citrate present in the media to form a dark brown/black soluble compound. The picture on the left shows the differential reaction that identifies the enterococci on bile esculin agar. Enterococci occur naturally in soil and can be readily isolated from most plant roots as well. They are also found routinely in frozen seafood, cheese, dried whole egg powder, raw and pasteurized milk, frozen fruits, fruit juices, and vegetables. Occasionally they are used as starter cultures for making hard cheese. Some strains produce high levels of the amines tyramine and histamine. Tyramine may be involved n causing migraines. They are capable of producing extracellular i proteinases and peptidases to hydrolyse large peptides and transport them into the cell to convert them to amino acids. Due to diet, E. faecalis dominates the guts of humans in the United States and England. In India and Japan, E. faecalis and E. faecium are equally found in the intestines. They get into food through vegetation, processing equipment, processing environments, or fecal contamination. Symptoms are similar to B. cereus and C. perfringens. Symptoms include nausea, vomiting, and diarrhea, but are milder than those caused by other food borne illnesses. The picture at left shows hemolysis on blood agar by S. pyogenes, a group A streptococcus. Blood agar is often used as a diagnostic test for the enterocococci, especially when isolations are made from food or clinical samples. Two of the five enterococcal species (faecalis and durans) will usually produce hemolysis on blood agar (see above table). 4. Public health significance The enterococci are used as a bacterial indicator for determining the extent of fecal contamination in foods and in recreational surface waters. Water quality guidelines based on enterococcal density have been proposed for recreational waters. The guideline is 33 enterococci/100 mL for 73

recreational fresh waters. For marine waters, the guideline is 35 enterococci/100 mL. The guidelines are based on the geometric mean of at least five samples per 30-d period during the swimming season. There are two types of selection methods. The membrane filter technique is used for samples of fresh and saline waters; however, it is unsuitable for highly turbid waters. The multiple-tube technique is also applicable to fresh and marine waters, but is primarily used for raw and chlorinated wastewater. For the presumptive test procedure of the multiple-tube technique, a series of azide dextrose broth tubes are inoculated and incubated. If not turbid, tubes are reincubated. Tubes showing turbidity are streaked onto Pfizer selective enterococcus (PSE) agar. Brownish-black colonies with brown halos confirm the presence of fecal streptococci. These colonies are transferred to a tube of brain-heart infusion broth containing 6.5% NaCl. Growth indicates colonies of the enterococcus group. In the membrane filter technique, the sample is filtered, the filter containing the colonies are transferred to an agar medium which is incubated. The filter is transferred to EIA medium containing esculin and ferric acid as selective agents. Pink to red enterococci colonies develop a black or reddish-brown precipitate. A well isolated colony from brain-heart infusion agar is then transferred onto a brain-heart infusion broth tube and incubated. After growth, a sample of the culture is transferred to bile esculin agar, brain-heart infusion broth, and brain-heart infusion broth with 6.5% NaCl. Growth at 45°C in 6.5% NaCl indicates presence of enterococcus group. For clinical or food samples, additional tests that may be conducted include bile solubility (above left picture; the tube on the far left is positive), and antibiotic sensitivity (the above right picture shows the antibiotic disk assay for bacitracin).

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C. Isolation of Salmonella from foods

1. Principle The procedure consists of six distinct stages. The initial handling of the food and the non-selective enrichment stage (preenrichment) vary according to the type of food examined. Non-Selective Enrichment (Preenrichment). The test sample is initially inoculated into a non-inhibitory liquid medium to favour the repair and growth of stressed or sublethally-injured salmonellae arising from exposure to heat, freezing, desiccation, preservatives, high osmotic pressure or wide temperature fluctuations Selective Enrichment Replicate portions of each preenrichment culture are inoculated into two enrichment media to favor the proliferation of salmonellae through a selective repression or inhibition of the growth of competing microorganims. Selective Plating Enrichment cultures are streaked onto selective differential agars for the isolation of salmonellae Purification Presumptive Salmonella isolates are purified on MacConkey agar plates or SS agar plates. Biochemical Screening Isolates are screened using determinant biochemical reactions. Serological Identification Polyvalent and/or somatic grouping antisera are used to support the tentative identification of isolates as members of Salmonella spp. For confirmation, cultures should be sent to a reference typing centre for complete serotyping. 2 Collection of samples Sampling Food control efforts frequently target processes and products presenting significant human health risks. The International Commission on Microbiological Specifications for Foods (ICMSF) has categorized foods according to the degree of hazard associated with product use. Each food category carries an appropriately stringent sampling plan to determine the acceptability of the food product. The choice of sampling plan may require some subjective judgement based on the number and kinds of factors that contribute to the degree of hazard. 3. Materials And Special Equipment 1) Nutrient Broth (NB). 2) Trypticase (Tryptic, Tryptone) Soy Broth.

75

3) Brilliant Green Water. 4) Buffered Peptone Water (BPW). 5) Skim Milk Medium. 6) Tetrathionate Brilliant Green Broth (TBG). 7) Selenite Cystine Broth (SC). 8) Bismuth Sulfite Agar (BS). 9) Brilliant Green Sulfa Agar (BGS). 10) MacConkey Agar, SS agar 11) Nutrient Agar. 12) Triple Sugar Iron Agar (TSI). 13) Lysine Iron Agar (LIA). 14) Urea Agar (Christensen's). 15) Commercial biochemical test kits. 16) Polyvalent and single grouping somatic (O) and flagellar (H) antisera. 17) Physiological Saline. 18) Blender, stomacher or other homogenizing device. 19) Incubator or water bath capable of maintaining 35±0.5oC and 43±0.5oC. 4. Procedure Handling of Sample Units Analyze samples as soon as possible. If necessary, store samples under time and temperature conditions that will prevent the growth or death of native microflora. If sample units have been abused in transit, resampling of the lot should be carried out. a. Frozen Foods: Sample units that show no signs of thawing upon receipt may be stored in the freezer at -10oC to -20oC. b. Dried and shelf stable foods may be stored at room temperature. c. Refrigerate all other foods, including those that are received in a partially thawed condition; analyze these samples as soon as possible preferably within 24 h of receipt. Thaw frozen samples at room temperature within 60 min; if this is not possible, thaw the samples at refrigerator (4 to 10oC) temperature.

76

NOTE: a) Large samples (e.g. whole chicken) may not readily thaw at refrigerator temperatures. For greater expediency, enclose the frozen sample in a heavy-walled paper bag and thaw overnight at room temperature. This technique maintains the product surface cold during the thawing process. b) Appropriate containers should ensure that the drippings from the product do not contaminate the laboratory environment. If the sample unit received for analysis is less than the recommended analytical unit, analyze the entire amount and record the weight used. Blending of samples should be limited to the minimum time required to produce a homogeneous suspension. Excessive blending could result in physical damage that would adversely affect the viability of endogenous microflora. For products that do not require blending, disperse the analytical unit into the appropriate preenrichment broth. Use aseptic techniques and sterile equipment at all stages of analysis. Containment during the handling of powdered products is critical if cross-contamination of the work environment is to be avoided. Non-selective Enrichment (Preenrichment) Compositing of Analytical Units To reduce the workload, up to 15 x 25 g (mL) analytical units may be composited into a single test sample (e.g. 375 g or mL). If a sample unit consists of more than one container, aseptically mix the contents of the containers prior to withdrawal of the analytical unit. If not possible or practical, the analytical unit shall then consist of equal portions from each of the containers. Sample Analysis The required analytical unit is dispersed into a suitable non-selective enrichment broth Nutrient broth (NB) and buffered peptone water (BPW) are equally reliable and can be used interchangeably as general purpose preenrichment. If the pH of the preenrichment mixture lies outside the range of 6.0 - 7.0, adjust with 1N NaOH or 1N HCl. NOTE: If the sample unit consists of a container with little food material, thoroughly rinse the interior of the container with a suitable preenrichment broth medium and incubate the rinse in a sterile flask. This eventuality is more frequently encountered in situations involving consumer complaints or food poisoning investigations. A positive Salmonella a a negative medium control should be set up in parallel with the test nd samples. Incubate the preenrichment mixture and the positive and negative controls at 35±0.5oC for 18 - 24 h. NOTE: The negative medium control should not show any evidence of growth after incubation whereas the absence of growth in the positive control would invalidate test results.

77

Selective Enrichment With a sterile pipette, transfer 1.0 mL of the preenrichment culture into each of 9 mL of selenite cystine (SC) and tetrathionate brilliant green (TBG) broths. Incubate SC and TBG broths for 24±2 h at 35±0.5oC and 43±0.5oC, respectively. Selective Plating Streak replicate loopsful of each selective enrichment culture onto BS and BGS agar to obtain well isolated colonies. The enrichment c ultures may be streaked onto additional plating media for the isolation of Salmonella. Incubate plates at 35±0.5oC for 24±2 h. If colonies suggestive of Salmonella have not developed on BS plates, incubate for an additional 24±2 h. Examine incubated plates for colonies suggestive of Salmonella. Typical Salmonella usually occur as pink to fuchsia colonies surrounded by red medium on BGS agar, and as black colonies on BS agar with or without a metallic sheen, and showing a gradual H2S- dependent blackening of the surrounding medium with increasing incubation time. NOTE: a. Lactose-and/or sucrose-fermenting Salmonella strains develop a coliform-like (greenish) appearance on BGS agar. A heavy growth of non-salmonellae may also mask the presence of Salmonella on this medium. b. BS agar can retard the growth of Salmonella serovars other than S. typhi unless poured plates are refrigerated (4 to 10oC) for 24 h prior to streaking. The absence of suspect colonies on the plates indicates that the analytical or composite test samples did not contain Salmonella spp. Purification Streak suspect colonies onto MacConkey agar for purification. Incubate plates at 35±0.5oC for 24±2 h. Typical Salmonella colonies are lactose-negative and will appear as colourless colonies on this medium. However, lactose-positive biotypes will occur as pink colonies. Biochemical Screening With a sterile needle, inoculate suspect colonies into the biochemical media or in commercial diagnostic kits that would yield equivalent results. Incubate the biochemical media for 18-24 h at 35±0.5oC. NOTE: Erroneous biochemical results may be obtained if tubes are not loosely capped during incubation. Commercially available diagnostic kits may be used to obtain detailed biochemical profiles of bacterial isolates. If none of the isolates from a particular analytical unit are suggestive of Salmonella, the analytical unit is considered to be free of salmonellae. If the presence of Salmonella is suspected, proceed with serological testing. If serological testing is not to be performed within 72 h, inoculate suspect isolates into nutrient agar slants and incubate at 35±0.5oC for 24±2 h. Store the agar slants at refrigerator (4 to 10oC) temperature. Nutrient

78

agar slants that have been stored for more than 72 h should n be used for serological ot testing. Prepare fresh agar slants for this purpose. Serological Identification Testing with somatic polyvalent antiserum Mark the following sections on an agglutination plate: C+ (positive control), C(negative control) and T (test culture). Add one drop of physiological saline to each of the areas marked T and C+, and two drops to the area marked C-. Remove sufficient culture material from a triple sugar iron, lysine iron or nutrient agar slant to prepare a heavy suspension in the test area (T) and in the negative control (C-) area. The inoculum should be withdrawn from the slope portion of agar slants. For the positive control, prepare a heavy suspension of a known Salmonella culture in the area marked C+. Prepare somatic polyvalent antisera as directed by the manufacturer; add one drop to each of the areas marked T and C+. Mix the culture-saline-antiserum suspensions in T and C+ and the saline-culture mixture in C- with a sterile needle or loop. Tilt the slide preparation back and forth for 1 min. Hold the slide against a dark background and observe for agglutination. Salmonella cultures usually agglutinate within 1 min. False positive reactions from microorganisms that are closely related to Salmonella may occur. Such misleading reactions can be resolved through further testing with somatic grouping and flagellar antisera. The serological test for a given culture is invalidated if the negative control shows agglutination (autoagglutination).

-

-

-

Testing with Somatic Grouping Antisera It is advantageous to test presumptive Salmonella cultures with somatic grouping antisera whenever possible. Many foodborne Salmonella belong to somatic groups B,C,D, or E. Nevertheless, it is important to recognize that unless a complete set of grouping antisera is available, Salmonella belonging to uncommon serogroups may be missed. NOTE: It should be stressed that any non-agglutinating culture possessing the biochemical reactions suggestive of Salmonella should be sent to a reference typing centre for identification. Mark the following sections on an agglutination plate: C-(negative control) and T (test culture).

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If a Salmonella control culture is available for each somatic group tested, prepare C+ (positive control) Add one drop of physiological saline to each of the areas marked T and C+, and two drops to the area marked C-. Remove sufficient culture material from a triple sugar iron, lysine iron or nutrient agar slant to prepare a heavy suspension in the test area and in the negative control area. The inoculum should be withdrawn from the slope portion of the agar slants. Prepare somatic group antiserum as directed by the manufacturer; add one drop to each of the areas marked T and C+. Mix the culture-saline-antiserum suspensions in T and C+ and the s aline- culture mixture in C- with a sterile needle or loop. Tilt the slide preparation back and forth for 1 min. Hold the slide against a dark background and observe for agglutination. Salmonella cultures usually agglutinate within 1 min. If the culture-saline-antiserum mixture does not agglutinate, repeat the procedure with another somatic group antiserum. If the serological test is positive, the culture should be sent to a Salmonella typing centre for complete serotyping. The serological test for a given culture is invalidated if the negative control shows agglutination (autoagglutination). A biochemically suspect Salmonella isolate (Table IV) that fails to yield any positive serological reaction should be sent to a reference typing centre for identification. Testing with Flagellar (H) Antisera In instances where the services of a reference typing centre are not available, Salmonella isolates agglutinable with somatic antisera should be further identified by testing with polyvalent H antiserum.

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D. Enumeration of Staphylococcus aureaus in Foods

1. Application This method is applicable to the enumeration of Staphylococcus aureus in foods. 2. Description The method has been shown to produce satisfactory results with naturally-contaminated meats, fish, poultry, vegetables, cereals and dairy products, and artificially-contaminated foods. This method can be used successfully for the detection of Staphylococcus aureus in other foods, food ingredients and environmental samples. 3. Principle Certain staphylococci produce enterotoxins which cause food poisoning. This ability to produce enterotoxins, with few exceptions, is limited to those strains that are coagulase-positive, and/or produce a heat-stable nuclease (TNase). This method determines the presence of S. aureus by plating known quantities of (dilutions of) a food sample onto a selective agar. After incubation, presumptive staphylococcal colonies are selected, and subjected to confirmatory tests. From the results of these tests, the number of S. aureus per g or mL of the food is calculated. The numbers present may indicate a potential for the presence of enterotoxin, or they may also indicate a lack of adherence to Good Hygienic Practices. 4. Materials and special equipment 1) Baird-Parker (BP) agar base 2) Egg Yolk Tellurite emulsion 3) Brain Heart Infusion (BHI) broth. 4) A non-selective agar; either Blood agar (BA), Nutrient agar (NA), or Trypticase Soy agar (TSA) 5) TSA slants 6) Peptone Water diluent 7) pH meter or paper capable of distinguishing to 0.1 pH units within the range of pH 5.0 to 8.0 8) Stomacher, blender or equivalent 9) Vortex mixer or equivalent 10) Control strains; use the following or equivalent strains. Positive Controls: S. aureus coagulase positive, e.g. ATCC 27154, 25923 S. aureus coagulase negative, e.g. ATCC 14990, 33501 Negative Controls: Escherichia coli, e.g. ATCC 23509 Pseudomonas aeruginosa, e.g. ATCC 7700 11) Crystal violet stain 12) Coagulase (Rabbit) Plasma. Follow manufacturer's instructions for reconstitution

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13) Incubator capable of maintaining 35oC. 14) Waterbath capable of maintaining 50-55oC 15) Supplies needed for confirmation: (The following supplies may be needed for confirmation) A. Accuprobe B. Enterotoxin assay C. Latex Agglutination kits D. Rapid ID kits E. Anaerobic utilization of glucose (Phenol red carbohydrate broth with 0.5% glucose, sterile paraffin oil) F. Anaerobic utilization of mannitol (Phenol red carbohydrate broth with 0.5% mannitol, sterile paraffin oil) G. Lysostaphin sensitivity (phosphate saline buffer, lysostaphin solution) H. TNase 5. Procedure: Each sample unit shall be analyzed individually. The test shall be carried out in accordance with the following instructions: 5.1. Handling of Sample Units 5.1.1 During storage and transport, the following shall apply: w the exception of shelfith o stable products, keep the sample units refrigerated (0-5 C). Sample units of frozen products shall be kept frozen. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death. 5.1.2 Analyze the sample units as soon as possible after receipt at the laboratory. 5.2. Preparation for Analysis 5.2.1 Have sterile peptone water prepared (may be stored under refrigeration for up to 12 weeks). 5.2.2 Clean the surface of the working area with a suitable disinfectant. 5.3. Preparation of sample 5.3.1. Combine portions from several locations within each solid sample unit, to ensure a representative analytical unit, or

82

5.3.2. If the sample unit is a liquid or a free-flowing solid (powder), thoroughly mix each sample unit by shaking the container. 5.3.3. Prepare a 1:10 dilution of the food by adding aseptically 11(10) g or mL (the analytical unit) to 99(90) mL of diluent (peptone water, see Table I). Shake, stomach or blend according to the type of food as indicated in Table I. NOTE: Weight or volume in brackets indicates alternate procedure for making dilutions. 5.3.4. Blend/stomach for the minimum time required to produce a homogeneous suspension; to avoid overheating, blending time should not exceed 2.5 min. With foods that tend to foam, use blender at low speed and remove aliquot from below liquid/foam interface. 5.3.5 If the 1:10 dilution is to be mixed by shaking, shake the dilution bottle 25 times through a 30 cm arc in approximately 7sec. 5.3.6. Check pH of the food suspension. If the pH is outside the range of 5.5-7.6, adjust pH to 7.0 with sterile NaOH or HCl. 5.3.7. The food homogenate (1:10 dilution) of dry foods should stand at room temperature for 15 min. In all other instances, the analysis should be continued as soon as possible. 5.3.8. Prepare succeeding decimal dilutions in peptone water as required, using a separate sterile pipette for making each transfer. 5.3.9. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present. 5.4 Enumeration of Presumptive S. aureus 5.4.1. Plating 5.4.1.1. Agitate each dilution to resuspend material that may have settled during preparation. Plating should be carried out within 15 min of preparing the dilutions. 5.4.1.2a. If counts of fewer than 1,000 S. aureus per g of a solid food are expected, spread 0.4 mL of the 1:10 dilution evenly over the surface of each of five B.P. agar plates. 5.4.1.2b. If the sample units are liquid, 0.2 mL of the undiluted analytical unit may be spread onto duplicate B.P. agar plates. 5.4.1.3. Routinely (i.e. for counts higher than 1,000 S. aureus per g or mL of the food), spread 0.2 mL of each dilution to be used onto duplicate B.P. agar plates. 5.4.1.4. The liquid should not be spread right to the edge of the plate, since this causes confluent growth at the plate-agar interface which is difficult to count. 5.4.1.5. Retain the plates in an upright position until the inoculum has been absorbed by the medium (approximately 10 minutes on properly dried plates). If the inoculum is not readily absorbed by the medium, the plates may be placed in an upright position in an incubator for up to one hour. 83

5.4.2. Incubation 5.4.2.1. Invert the plates and incubate at 35oC for 48 ± 2 h. Plates should be observed at 24-30 h for possible overgrowth; presumptive colonies may be counted at this time but the count should be verified at 48 ± 2 h. 5.4.2.2. Avoid excessive crowding or stacking of plates in order to permit rapid equilibration of plates with incubator temperature. 5.4.3. Counting Colonies and Recording Results 5.4.3.1. Observe the following four types of presumptive staphylococcal colonies: Type 1. Convex, entire, shiny black surrounded by clear zones extending into the opaque medium. Type 2. Convex, entire, shiny black without well defined clear zones. Type 3. Grey colonies similar to type 1. Type 4. Grey colonies similar to type 2. Each colony type may show grey-white margins around the colonies and/or opaque zones (double halos). Black mucoid colonies larger than 2 mm in diameter and swarmers should not be counted. Such colonies usually belong to the genus Bacillus. 5.4.3.2. Count the colonies of each type and record separately, but add together to give the total presumptive count. 5.4.3.3. Count colonies immediately after the incubation period. 5.4.4. Counting of five plates of the 1:10 dilution (solid food only) 5.4.4.1. If the number of all presumptive staphylococcal colonies per plate is fewer than 20, add separately the counts for each type from all five plates and record as the respective presumptive count. This is the count of one of the four types per 2 mL (0.2 g of food). Multiply each count by 5, and record as the respective presumptive count per g of food (C). Add the results, and report as the total presumptive count per g of food. 5.4.4.2. If the number of all presumptive staphylococcal colonies per plate is between 20 and 200, select two plates at random, count separately the colonies of each type and compute the respective average presumptive count per plate (per 0.4 mL; which is equivalent to 0.04 g of food) (A/2). Multiply each count by 25 and record as the respective presumptive count per g of food (C). Add the results and report as the total presumptive count per g of food. 5.4.4.3. If the number of presumptive staphylococcal colonies on some of the five plates is < 20, but on others is 20, proceed as in 5.4.4.1. above. 5.4.5. Counting of duplicate plates (any dilution) 84

5.4.5.1. Select plates containing 20-200 presumptive staphylococcal colonies per plate consisting of the combined counts of all types. 5.4.5.2. Compute the average presumptive count per plate for each type (A/2), multiply by five and by the appropriate dilution factor, and record as presumptive count per g or mL of food for each type (C). Add the results and report as the total presumptive count per g or mL of food. 5.4.5.3. If plates from more than one dilution are used, the counts are to be averaged as shown below. 5.4.5.4. If no plate containing 20-200 presumptive S. aureus is available, estimated counts may be made on plates giving presumptive counts outside this range. Report results as estimated counts when results are outside the range of 20-200. 5.4.5.5. When an estimated count contributes to an average count, this average itself becomes an estimated value. 5.4.6. Averaging of counts over two dilutions If plates from two consecutive decimal dilutions contain counts within the range of 20-200 presumptive staphylococcal colonies per plate, the counts on all four plates should be used to arrive at the average count. Inasmuch as the four different types of colonies are to be counted separately and it is quite possible that individual counts may be < 20, although the combined counts are within range, estimates and true values would have to be combined in order to arrive at an average value. This can be avoided by using the following formula: Average colony count/g or mL = Total number of colonies counted / Volume used per dilution For an example of counting colonies, see Table II. 5.4.6.1. If no presumptive staphylococcal colonies are obtained, record presumptive counts as < 5 per g or mL for the five plates of the 1:10 dilution, or < 2.5 x the dilution factor for duplicate plates. ( 1 /(Dilution1) + 1 / Dilution2)

5.5. Confirmatory Tests For confirmation of S. aureus, perform the coagulase test (following manufacturer's instructions) as an initial step. A firm clot which does not move when the tube is tipped on its side (4+ coagulase reaction) is considered a positive test for S. aureus; no further confirmation is required. Run controls (positive and negative cultures as well as media controls) simultaneously when performing all confirmation tests. If the coagulase reaction is 3+ or less, perform at least two of the following confirmation tests. If two of these tests are positive, then the isolate is considered S.aureus. 1) Accuprobe method, 2) Staphylococcal enterotoxin assays, 3) Latex agglutination kits, 4) Rapid ID kits or5) at least one of the additional confirmatory tests listed in section 5.5.3, it is important that lysostaphin sensitivity

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and anaerobic utilization of glucose are not the only two tests carried out since these do not distinguish between S. aureus and S. epidermidis. 5.5.1. Selection of Colonies 5.5.1.1. From the replicate plates counted, a number of each colony type observed is selected as follows to check for culture purity: When the total count per type for all the plates of a dilution is less than five, pick all colonies of that type. 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. 5.5.1.2. Streak each colony picked onto a non-selective medium, such as BA, NA or TSA to obtain discrete colonies. 5.5.1.3. Incubate at 35oC for 24 ± 2 h. 5.5.1.4. 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. 5.5.1.5. If the isolates are composed of cocci only, transfer inoculum from each into a separate tube of BHI broth. If an isolate is not pure, choose another colony at step 5.5.1.2 above and repeat colony isolation steps above. 5.5.1.6. Incubate the inoculated BHI broth tubes at 35oC for 18-24 h and observe for growth. 5.5.1.7. Retain BHI broth cultures. 5.5.1.8. Transfer a representative colony from one of the non-selective media to a TSA slant. 5.5.1.9. Inoculate a culture of Staphylococcus aureus known to produce coagulase and TNase, utilizes glucose and mannitol anaerobically, and is lysostaphin-sensitive, into BHI broth to serve as a positive control. Use uninoculated medium from the same batch of BHI broth as a negative control. Inoculate the controls along with the test cultures, and submit them to the subsequent tests as required. 5.5.2. Coagulase Test 5.5.2.1 Transfer 0.2 mL of each BHI broth culture into sterile 13 x 100 mm tubes containing 0.5 mL certified coagulase plasma. Mix thoroughly. 5.5.2.2 Incubate tubes at 35oC and examine after 1 h and after 4 h. Do not shake tubes during incubation. Negative tubes should be incubated overnight at room temperature and rechecked. 5.5.2.3 Distinct clotting as shown in Fig. 2 is considered a positive coagulase reaction. 5.5.3. Additional Confirmatory Tests 86

At least one of the following additional tests may be done; keeping in mind that Anaerobic utilization of glucose and Lysostaphin sensitivity must not be the only two tests performed. 5.5.3.1. Anaerobic utilization of glucose. Inoculate culture to be tested into a tube of carbohydrate fermentation medium containing 0.5% glucose. Overlay with sterile paraffin oil and incubate at 35oC for 1824 h. Colour change indicating an acid reaction is a positive test for S. aureus. 5.5.3.2. Anaerobic utilization of mannitol. Same as for glucose utilization except that the source of carbohydrate is mannitol. S. aureus usually gives a positive reaction but some strains do not ferment mannitol. 5.5.3.3. Lysostaphin sensitivity Inoculate culture to be tested into 0.2 mL of phosphate saline buffer and emulsify. Transfer one half of the suspended cells to another tube (13 x 100 mm) and mix with 0.1 mL of phosphate saline buffer to serve as a negative control. Add 0.1 mL of lysostaphin solution to the original tube to give a concentration of 25 mg lysostaphin per mL of cell suspension. Incubate both tubes at 35oC for up to 2 h. If the turbidity clears in the tube containing cells plus lysostaphin, and there is no clearing in the control tube, the test is positive for S. aureus. If clearing has not occurred in 2 h, the test is negative. 5.5.3.4. Thermonuclease Test Perform the test for the presence of thermostable nuclease (TNase). 5.5.3.5. If two of the three additional confirmatory tests are positive, the isolate is considered to be S. aureus. On the basis of the confirmatory tests for each of the four types of cultures, record the total number of S. aureus per g or mL of food (N T ). Total No. S. aureus per g or mL equals the sum of No. S. aureus types 1, 2, 3 and 4 (N T = N1 + N2 + N3 + N4) No. of colonies confirmed as No. S. aureus type 1 per presumptive count type = S. aureus (P) / x g or mL (N1) 1(C) No. colonies tested (G) Same for types 2, 3 and 4. See Table II

6. Preparation of media For steam sterilization, it is essential that the load be sufficiently pre-heated before the actual sterilization period commences. This varies considerably with the nature and size of the load. Hence, proper exposure times should be followed to ensure sterilization of flask solutions and heat stable culture media, particularly when prepared in large volumes (Refer to your sterilizer manual). 6.1 Baird-Parker (BP) Medium 87

a. Basal medium Tryptone Beef extract Yeast extract Glycine Lithium chloride Sodium pyruvate Agar

10 g 5g 1g 12 g 5g 10 g 20 g

The basal medium is commercially available. Add above ingredients to 950 mL of distilled water and heat to boiling to dissolve the medium completely. Sterilize at 121oC (15 lb pressure) for 15 min. Cool to 50-55oC in a water bath. The pH should be 7.2. b. Filter-sterilized 1% tellurite solution. c. Egg yolk emulsion (50%). Soak fresh clean eggs in 70% alcohol for 15 min. Separate egg yolks aseptically and mix with an equal amount of physiological saline for about 5 min on a magnetic stirrer at low speed (do not heat). Egg yolk emulsion is commercially available and usually contains about 50% egg yolk. Use as per manufacturer's instructions. Egg yolk emulsion containing tellurite is also commercially available. (EY-Tellurite Enrichment). Use as per manufacturer's instructions. 6.1.1 Preparation of complete medium. a. Add aseptically 10 mL of the prewarmed (50-55oC) tellurite solution and 50 mL of the prewarmed (50-55oC) egg yolk emulsion to 950 mL of the tempered (50-55oC) basal medium. Mix thoroughly but gently and dispense into petri plates. b. If commercial egg yolk emulsion is used, make certain to add the equivalent of 2.5% egg yolk. Adjust the total volume of the complete medium to 1000 mL. c. If commercial EY-Tellurite Enrichment is used, add 50 mL to 950 mL of the tempered (50-55oC) basal medium. The surface of the agar should be dried before inoculation. It has been observed that freshly prepared BP medium may be toxic to injured cells. It is therefore advisable to store the plates at room temperature overnight before inoculation. Poured plates may be stored in the refrigerator for up to 4 days. The medium should be opaque; do not use non-opaque plates.

6.2 Brain Heart Infusion (BHI) Broth Calf brain, infusion from Beef heart, infusion from Proteose peptone Dextrose 200 g 250 g 10 g 2g 88

Sodium chloride Sodium Phosphate (Na2HPO4) pH 7.4 ± 0.1

5g 2.5 g

Dissolve ingredients in 1,000 mL of distilled water. Distribute as required (tubes or flasks) and sterilize at 121oC. This medium is commercially available. 6.3 Blood Agar - (Trypticase Blood agar base) Trypticase Beef extract Sodium chloride (NaCl) Agar pH 7.2 ± 0.1 10 g 3g 5g 15 g

Suspend ingredients in 1000 mL distilled water. Heat to boiling to dissolve ingredients. Sterilize at 121oC. Cool to 45-50oC and add aseptically 5% sterile defibrinated blood. Mix thoroughly but avoid incorporation of air bubbles. Dispense. The solidified, complete medium cannot be reliquified. 6.4 Coagulase Plasma Certified rabbit plasma containing EDTA is commercially available. Reconstitute as directed by the manufacturers. The reconstituted plasma may be kept in the refrigerator for five days without loss of potency. It is not satisfactory for use if gross contamination occurs. After being kept in the refrigerator, the plasma solution is cold enough to delay clotting for 10-15 min. This delay can be prevented by warming the plasma solution to 35oC before use. If dehydrated product is not available, use fresh rabbit plasma collected aseptically in containers with EDTA (1 mL of a 15% solution of the potassium salt per 100 mL blood). Dilute plasma 1:2 or 1:3 with sterile distilled water and test each batch with coagulase-positive and coagulasenegative strains of staphylococci before putting it into routine use. 6.5 Lysostaphin Solution Dissolve lysostaphin in phosphate saline buffer (0:02 M; pH 7.3-7.4) to obtain a concentration of 50 mg lysostaphin per mL. 6.6 Nutrient Agar (NA) Beef extract 3 g Peptone 5g Agar 15 g pH 6.8 ± 0.1 Suspend ingredients in 1000 mL of distilled water. Heat to boiling to dissolve ingredients. Dispense and sterilize at 121oC. This medium is commercially available. 6.7 Peptone Water Dissolve 1.0 g of Bacto peptone or equivalent in 1,000 mL of distilled water. Dispense 99 mL into dilution bottles and sterilize at 121oC. Peptone is commercially available.

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6.8 Phenol Red Carbohydrate Broth Trypticase or proteose peptone no. 3 Sodium chloride Beef extract (optional) Phenol red (or 7.2 mL of 0.25% solution of phenol red) Distilled water 10 g 5g 1g 0.018 g 1000 mL

Dissolve 5 g of glucose or mannitol in this basal broth. Dispense 2.5 mL portions in 13 x 100 mm test tubes containing inverted 6 x 50 mm fermentation tubes. Autoclave for 10 min at 118oC; final pH, 7.3 ± 0.2. Alternatively, dissolve the ingredients, omitting carbohydrate, in 800 mL of water with heat and occasional agitation; and dispense 2 mL portions in 13 x 100 mm test tubes containing inverted fermentation tubes. Autoclave for 15 min at 118oC and allow to cool. Dissolve carbohydrate in 200 mL of water and sterilize by passing the solution through a bacteria retaining filter. Aseptically add 0.5 mL of sterile filtrate to each tube of sterilized broth after cooling to 45oC. Shake gently to mix. Final pH, 7.4 ± 0.2 6.9 Phosphate Saline Buffer (pH 7.3-7.4, 0.02 m) Prepare stock solutions of 0.2 M mono- and di-sodium phosphate in 8.5% salt (NaCl) solutions. These stock solutions are for preparation of the 0.02 M phosphate saline buffer. Stock Solution 1 Na2HPO4 (Anhydrous Reagent Grade) NaCl (Reagent Grade) Distilled water to make Stock Solution 2 NaH2PO4H2O (Reagent Grade) NaCl (Reagent Grade) Distilled water to make 27.6 g 85.0 g 1000 mL 28.4 g 85.0 g 1000 mL

Make 1:10 dilutions of aliquots of each stock solution to obtain 0.02 M phosphate saline (0.85%) buffers; e.g. Stock solution 1 Distilled water Approximate pH = 8.2 Stock solution 2 Distilled water Approximate pH = 5.6 50 mL 450 mL 10.0 mL 90.0 mL

By means of a pH meter, titer the diluted solution 1 to a pH of 7.3-7.4 by adding approximately 65 mL of diluted solution 2. The resulting solution will be 0.02 M phosphate saline buffer for use in the lysostaphin susceptibility test on S. aureus.

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QC: Do not titer 0.2 M phosphate buffer to pH 7.3-7.4 and then dilute to 0.02 M strength. This results in a drop in pH of approximately 0.25. Addition of 0.85% salt after pH adjustment also results in a drop of approximately 0.2. 6.10 Trypticase Soy (TSA) Agar Trypticase peptone Phytone peptone Sodium chloride (NaCl) Agar pH 7.3 ± 0.1 15 g 5g 5g 15 g

Suspend ingredients in 1,000 mL of distilled water. Heat with frequent agitation and boil for 1 min or until solution is accomplished. The pH of the medium should be 7.3. Dispense and sterilize at 121oC. This medium is commercially available TABLE I Preparation of the Initial Dilution Type of Food Product Preparation Liquids: milk, water etc. pipette directly into Petri dishes and/or into peptone water diluent viscous liquids weigh into peptone water diluent Solids: water soluble solids weigh into peptone water diluent powder, meats weigh into peptone water diluent Spices weigh into peptone water diluent Shellfish weigh into peptone water diluent Treatment shake shake shake blend shake blend

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TABLE II Example of Computing S. aureus Count per g or mL of Food Total No. of No. of No. of Isolates Total No. of Colonies of No. of S. aureus Colonies of one of Isolates Confirmed as S. one of the four Types from one of the four the four Types on Tested "G" aureus "P" per g or mL. "C" C = Types per g or mL Duplicate Plates "A" 1/2AxD*5** "N" N = (P/G)xC Fewer than 5 (e.g. All (4) 2 1,000 500 4) More than 5 (e.g. 5(5) 4 4,500 3,600 18) Calculate N1, N2, N3 and N4 for each colony type to obtain total number of S. aureus. (N T ) per g or mL NT = N1 + N2 + N3 + N4 e.g. if N1 = 1,000 and N2 = 100 and N3 = 0, and N4 = 0 NT = 1,000 + 100 = 1,100 per g * Dilution factor = 100 ** For duplicate plates, 0.2 mL per plate. Divide by 2 since "A" represents the total count of one of the four types on two duplicate plates. Likewise, when 5 plates of the 1:10 dilution are counted, divide by 5. Report total number of Staphylococcus aureus per g or mL of food to two significant figures

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FIGURE 1 Flow Diagram for Confirmation Process

FIGURE 2 Coagulase test reaction Tube number

Intensity of reaction NEGATIVE POSITIVE (degree of clotting) 93

Description (E-Y-T Emulsion) : Baird-Parker' developed this medium from the tellurite-glycine formulation of Zebovitz et al.2 and improved its reliability in isolating S. aureus from foods. Baird-Parker added sodium pyruvate, to protect damaged cells and aid their recovery2 and egg yolk emulsion as a diagnostic agent. It is now widely recommended by national and international bodies for the isolation of S. aureus. The selective agents glycine, lithium and tellurite have been carefully balanced to suppress the growth of most bacteria present in foods, without inhibiting S. aureus. Egg yolk emulsion makes the medium yellow and opaque. S. aureus reduces tellurite to form grey-black shiny colonies and then produces clear zones around the colonies by proteolytic action. This clear zone with typical grey-black colony is diagnostic for S. aureus. On further incubation, most strains of Staph. aureus form opaque haloes around the colonies. and this is probably the action of a lipase. Not all strains of S. aureus produce both reactions. Some strains of S. saprophyticus produce both clear zones and opaque haloes but experienced workers can distinguish these from S. aureus by the longer incubation time required-5. Colonies typical of S. aureus but without an egg yolk reaction should also be tested for coagulase production . Egg yolk reaction negative strains of S. aureus may occur in some foods, especially cheese. Growth Characteristics: Microorganism S. aureus Growth Good Colony Morphology Grey-black shiny convex 1-1.5mm diameter (18 hours) up to 3mm (48 hours) narrow white entire margin surrounded by zone of clearing 2-5mm. Not shiny black and seldom produces clearing. Irregular and may produce clearing. Wide opaque zones may be produced in 24hrs. Dark brown matt with occasional clearing after 48hrs. Large brown-black Very small in shades of brown and black. No clearing. Brown-black with no clearing. White, no clearing.

S. epidermidis

Variable

S. saprophyticus

Variable

Bacillus sp.

Variable

Escherichia coli Micrococcus sp.

Variable Variable

Proteus sp. Yeasts Technique

Variable Variable

1. Dry the surface of agar plates for a minimal period of time prior to use.

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2. With a glass spatula or spreader (spread O.1 ml aliquots of food dilutions made up in buffered peptone water on the agar surface until it is dry. Up to 0.5 ml may be used on larger dishes (24 cm). 3. Incubate the inverted dishes at 35'C. Examine after 24 hours and look for typical colonies of S. aureus. Re-incubate negative cultures for a further 24 hours. Results Incubate the dishes for 48 hours and select those with 20-200 colonies. Count the S. aureuslike colonies and test them for coagulase reaction. Report S. aureus results per gram of food.

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E. Isolation of Listeria monocytogens from all food and environmental samples

1. Application The method is applicable to the detection of viable Listeria monocytogenes in foods (seafood, dairy products, red meat, poultry, vegetables, etc.). Environmental samples can also be analysed using this method. 2. Principle This method determines the presence of viable L. monocytogenes in the product. A portion of the product is enriched first in a primary broth, then in a screening broth, plated onto a specified agar medium and one additional plating medium, and then incubated under specified conditions of time and temperature. It is assumed that viable L. monocytogenes cells will multiply under these conditions and give rise to visible colonies which can be identified . Novel chromogenic and other isolation agars may be used in conjunction with the above media. 3. Material and special equipment Listeria broths and agars (base media and supplements are commercially available) 1) Listeria enrichment broth (LEB) 2) Modified Fraser broth (MFB) 3) Oxford agar (OXA) 4) Lithium chloride-phenylethanol-moxalactam medium (LPM) 5) Modified Oxford agar (MOX) 6) PALCAM agar (PAL) 7) Chromogenic media (follow manufacturer's instructions for preparation and use) NOTE: The Listeria isolation agar, Oxford, uses cycloheximide as a selective agent. The organization holding the patent on this antibiotic is no longer producing it, and as a result cycloheximide will be unavailable shortly. Some media suppliers, such as Oxoid, have already produced alternative supplements for their media, which can be substituted in the media. However, it is up to the users of this method to ensure that their in-house validation data meets their criteria. Data may be obtained from the manufacturer and should be kept on file. 8) Control cultures (use ATCC strains or equivalent) Positive controls: Listeria monocytogenes, Listeria ivanovii, Listeria innocua, (Staphylococcus aureus and Rhodococcus equi - optional) 9) Stomacher, blender or equivalent, vortex mixer 10) Microscope

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11) Incubators capable of maintaining 30oC and 35oC NOTE: It is the responsibility of each laboratory to ensure that the temperature of the incubators or water baths are maintained at the recommended temperatures. Where 35oC is recommended in the text of the method, the incubator may be at 35 +/-1.0oC. Similarly, lower temperatures of 30 or 25oC may be +/- 1.0oC. However, where higher temperatures are recommended, such as 43 or 45.5oC, it is imperative that the incubators or water baths be maintained within 0.5oC due to potential lethality of higher temperatures on the microorganism being isolated. Confirmation Media and Reagents 12) Tryptose broth and agar (TA) 13) Trypticase soy broth and agar, with 0.6% yeast extract (TSB-YE and TSA-YE) 14) Horse blood agar and (sheep blood agar - optional) 15) Motility test medium 16) Carbohydrate fermentation agars or broths (mannitol, rhamnose and xylose). Note: these biochemicals may be done via rapid identification kits (see 6.8.1) Optional 17) Rapid identification kits, such as the Vitek or API Listeria (Bio Mérieux Vitek, Inc.), Micro-ID Listeria (Organon Teknika Corp.) or the Listeria AccuprobeTM Test (Gen-Probe; MFLP-88) or equivalent 18) Gram stain solutions 19) 3% hydrogen peroxide (catalase) 20) Biochemicals - dextrose, esculin, maltose, -methyl-D-mannoside 21) Beta-lysine discs (Remel) 22) Listeria monocytogenes antisera 4. Procedure Each sample unit may be analyzed individually or the analytical units may be composited according to the sampling scheme in Table 4. Maintain a ratio of 1 part sample material to 9 parts sterile enrichment broth. Information regarding Listeria distribution can be obtained by analyzing each analytical unit separately. Carry out the test in accordance with the following instructions: 4.1. Handling of Sample Units 4.1.1 In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated (0-5oC) or frozen, depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death. 4.1.2 Analyze sample units as soon as possible after their receipt in the laboratory.

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4.2. Preparation for Analysis 4.2.1 Have sterile Listeria enrichment broth (LEB) ready. 4.2.2 Clean the surface of the working area with disinfectant. 4.3. Preparation of Sample To ensure a representative analytical unit, agitate liquids or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit. 4.4. Enrichment Procedure (see Figure 1) Add the environmental sponge to 100 mL of LEB or composite up to 10 sponges with 100 mL LEB for each sponge. Add 25 g or mL of the food (the analytical unit) to 225 mL of LEB in a blender jar or stomacher bag. Alternately, add 50 g to 450 mL of LEB. For composite samples, one of the analytical composites described in Table 4 is added to a sufficient amount of LEB. Maintain a ratio of 1 part sample material to 9 parts LEB. Place environmental swabs in 10 mL portions of LEB in test tubes. Blend, stomach or vortex as required for thorough mixing. LEB culture may be incubated in the stomacher bag or test tube, or transferred to a sterile Erlenmeyer flask. Incubate LEB culture for 48 h at 30oC. 4.5. Selective Enrichment 4.5.1. At 24 and 48 h, mix the LEB culture by swirling or vortexing, and inoculate 10 mL of modified Fraser Broth (MFB) with 0.1 mL of the LEB culture. Incubate 24-26 h at 35oC. HELPFUL HINT: Vortex the MFB at 20 to 24 h, then reincubate for an additional 2 to 6 h before reading reaction. Reading the MFB at 26 h can substantially reduce the plating done at 48 h. 4.5.2 Streak MFB onto plates if positive. A positive broth has darkened and may be black, dark brown or dark green. A negative MFB has the straw colour of a newly made broth. If negative, reincubate another 24 h and streak if positive. Proceed with Step 4.6. 4.6. Isolation Procedure 4.6.1. Streak positive MFB; those inoculated from LEB at 24 and 48 h onto two different plating media (streaking LEB is optional but preferable for obtaining all listeriae). Use Oxford agar (OXA) and one of the following: lithium chloride-phenylethanolmoxalactam medium (LPM), modified Oxford agar (MOX), or PALCAM agar (PAL). Incubate LPM plates at 30oC for 24-48 h and OXA, MOX and PAL plates at 35oC for 24-48 h. Other media may be used along with the two selective agars (see 4.6.5). 4.6.2. LPM - Examine LPM plates for suspect colonies using beamed white light powerful enough to illuminate the plate well, striking the plate bottom at a 45o angle. Under optimum transillumination the more isolated and larger (48 h old) Listeria colonies appear as whitish piles of crushed glass often showing mosaic-like internal structures occasionally having bluegrey iridescence that tends to sparkle. Alternatively, the colonies can look smooth with a blue tinge around the perimeter. When growth becomes near confluent, an even blue-grey iridescent sheen can be observed. 98

4.6.3. OXA and MOX agars - L. monocytogenes forms 1 mm diameter black colonies surrounded by black haloes after 24 h. At 48 h colonies are 2-3 mm in diameter, black with a black halo and sunken centre. The colonies can also appear brown-black or green-black. Other Listeria species show a similar appearance. When examined before 24 h, growth of Listeria spp. is sometimes apparent but without the characteristic blackening. Some strains of this genus, other than L. monocytogenes, are inhibited on this medium when incubated at 35oC. 4.6.4. PAL agar - L. monocytogenes forms 2 mm grey-green colonies with a black sunken centre and a black halo on a cherry-red background. Some Enterococcus and Staphylococcus strains form grey colonies with a brown-green halo or yellow colonies with a yellow halo. 4.6.5. Chromogenic agar - novel chromogenic and other isolation agars may be used, but in conjunction with the plating media above. Follow manufacturer's instructions for preparation and use. 4.7. Identification Procedure - Confirmation 4.7.1. If the colonies are well isolated on the selective agars: Pick a minimum of 5 typical colonies from each selective plate to horse blood agar (as in 4.7.2). If the colonies are NOT well isolated on the selective agars: Pick a minimum of 5 typical colonies from each selective plate to Tryptose agar (TA) or Trypticase soy agar with 0.6% yeast extract (TSA-YE), streaking for separation. Incubate plates at 30oC for 24-48 h or until growth is satisfactory. Examine the plates for typical colonies using the light arrangement already described in 4.6.2. HELPFUL HINT: Listeria confirmation and speciation of L. monocytogenes can be accomplished by using motility, hemolysis and 3 carbohydrate agars (mannitol, rhamnose and xylose). Other biochemical tests are optional. Rapid identification kits may be helpful to reinforce confirmation of these results and differentiate the different Listeria species (see 4.8.1). 4.7.2. Hemolysis: On horse blood agar plates, draw a grid of 20-25 spaces on the plate bottom. Pick typical colonies from the selective agars (if colonies are well isolated) or from the TA or TSA-YE plates (if streaked for purity) and inoculate the horse blood agars by stabbing one culture per grid. Stab blood agar plates, motility agar and carbohydrate plates1 concurrently from the same colony. Ensure that each colony is placed in the same position on all grid plates. Always stab positive and negative controls (L. monocytogenes, L. ivanovii and L. innocua). Incubate for 24 h at 35oC. 1Note: Carbohydrate plates may be replaced by rapid identifcation kits (see 4.8.1). Examine blood agar plates containing culture stabs by transillumination using a bright light (holding the plate so that the light shines through from the back of the plate). L. monocytogenes produces a slight cleared zone around the stab; L. innocua shows no zone of hemolysis, whereas L. ivanovii produces a well-defined zone of clearing around the stab. 4.7.3. Motility:

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Agar: Stab motility test medium from selective agars, TA or TSA-YE. (Do blood agar and carbohydrates concurrently (see 4.7.2)). Incubate for up to 48 h at room temperature. Observe daily. ONLY Listeria cells give typical umbrella growth pattern. and/or Wet mount: Pick at least one typical colony from each selective agar, TA or TSA-YE. Inoculate TSB-YE broths and incubate overnight at 30oC. Transfer a loopful of the overnight cultures to a fresh TSB-YE and incubate at 25oC for 6 hours. Put a drop of each 6 hour culture onto a glass slide and examine for typical Listeria motility using the oil immersion objective or phase contrast microscope. Listeria appears as slim, short rods with tumbling motility. Always compare to a known Listeria culture. Cocci, large rods, or rods with rapid swimming motility are not Listeria. 4.7.4. Carbohydrate Utilisation Plates On carbohydrate (mannitol, rhamnose and xylose) agar plates, draw a grid of 20-25 spaces on the plate bottom. Pick typical colonies from the selective agars, TA or TSA-YE plates and inoculate agars by stabbing one culture per grid. Ensure that each colony is placed in the same position on all grid plates. Always stab positive and negative controls (L. ivanovii, L. monocytogenes and L. grayi). See Table 1 for guidance. Incubate for 24 h at 35oC. and/or Broths From TSB-YE culture, inoculate the following carbohydrates set up as 0.5% solutions in purple carbohydrate broth: dextrose, esculin, maltose, mannitol, rhamnose, methyl-D-mannoside and xylose. Incubate 7 days at 35oC. Examine daily. Listeria spp. produce acid with no gas, or no reaction. Consult Table 1 for the carbohydrate reactions of the Listeria spp. All species should be positive for dextrose, esculin, and maltose. All Listeria spp. except L. grayi and L. murrayi should be mannitol-negative 4.8 Identification Procedure - Optional Tests 4.8.1 Rapid Identification Kits Rapid identification kits, such as the Vitek or API Listeria (Bio Mérieux Vitek, Inc.), MicroID Listeria (Organon Teknika Corp.) or the Listeria AccuprobeTM Test (Gen-Probe) or equivalent. Follow manufacturer's instructions for use. 4.8.2 Catalase Test a typical colony for catalase. Transfer a colony onto a clean glass slide and add one drop of 3% hydrogen peroxide. Development of bubbles is indicative of a positive reaction. Listeria cells are catalase-positive. Avoid picking test colonies from agars containing blood as they can produce a false positive result. 4.8.3 Gram stain Listeria is a small Gram-positive rod.

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4.8.4 CAMP test For the CAMP test, streak fresh isolates of beta-hemolytic Staphylococcus aureus and Rhodococcus equi vertically on a sheep blood agar plate. Separate the vertical streaks so that test strains may be streaked horizontally between them without touching the vertical streaks. After 24-48 h incubation at 35oC, examine the plates for hemolysis in the zone of the vertical streaks. 4.8.4.1 The hemolysis of L. monocytogenes and L. seeligeri is enhanced in the vicinity of the Staphylococcus streak; while L. ivanovii hemolysis is enhanced near the Rhodococcus streak. The other Listeria species are CAMP test negative. The test can differentiate L. ivanovii from L. seeligeri, and a weakly-hemolytic L. seeligeri from L. welshimeri. 4.8.4.2 An alternative and convenient CAMP test may be performed using the S. aureus factor in commercially prepared sterile beta-lysine discs. In this test, a betalysine disc is placed in the center of the sheep blood plate and 4-5 Listeria cultures are streaked as radiating lines from the disc, being careful not to touch the disc with the inoculum. After 18-24 h incubation at 35oC, a very sharp CAMP reaction between L. monocytogenes or L. seeligeri cultures and the disc can be observed. L. ivanovii are strongly hemolytic and form a clear beta hemolytic line along the entire streak. 4.8.5 Serology Follow manufacture's instructions provided with the antisera. 4.9 Interpretation of Results for Speciation Listeria spp. are small, Gram-positive motile rods that are catalase-positive, urea-negative, and produce an acid slant and butt in TSI without production of H2S. They utilize dextrose, esculin, and maltose, with some species also using mannitol, rhamnose, and xylose with production of acid. All species give +/+ reactions in MR-VP broth. L. grayi and L. murrayi are the only two species which utilize mannitol. L. murrayi is the only species which can reduce NO3- to NO2-. L. monocytogenes, L. ivanovii, and L. seeligeri (weak) produce hemolysis in horse or sheep blood agar and are also positive in the CAMP test. Of the three, only L. monocytogenes cannot utilize xylose, but is rhamnose-positive. L. ivanovii can be differentiated from L. seeligeri by the CAMP test, where L. seeligeri shows enhanced hemolysis only at the Staphylococcus streak and L. ivanovii shows enhanced hemolysis in the area of the R. equi streak. L. innocua can only be differentiated from L. monocytogenes by its lack of hemolysis on blood agar plates and negative reaction in the CAMP test. L. welshimeri that is rhamnose- negative may be confused with a weakly-hemolytic L. seeligeri unless the CAMP test is run. All biochemical, serological, and pathogenicity data are summarized in the tables below. Complete all data collection before making species determinations.

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Table 1 Characteristics differentiating the species of the genus Listeriaa Characteristics L. monocyto- L. genes b innocua Gram stain + + c Beta-Hemolysis + Mannitol L-Rhamnose + d D-Xylose e CAMP-test (S. + aureus) CAMP-test (R. Equi) Acid production from: L-Arabinose Dextrin d Galactose d Glycogen Lactose d + D-Lyxose Melezitose d d Melibiose alpha-Methyl-D+ + glucoside alpha-Methyl-D+ + mannoside Sorbitol d Soluble starch Sucrose d Voges-Proskauer + + Hydrolysis of: Cellulose Hippurate + + Starch d d Lecithinase d d Phosphatase + + Reduction NO3 to NO2 Pathogenicity for + mice L. seeligeri + + + + L. welshimeri + d + L. ivanovii + +d + + L. grayi + + L. murrayi + + d -

d + d + -f + d + + + + +

+ + + + +

+ + + + +

+

+

+ + + -

+ + + + -

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a. Standard symbols: (+) - positive; (-) - negative; +: > or equal to 90% positive; -: > or equal to 90% negative; d: 11 - 89% of strains are positive b. Not all strains of L. monocytogenes exhibit beta-hemolysis - the type strain ATCC 15313 is nonhemolytic on horse, sheep and bovine blood. c. A very wide zone or multiple zones of hemolysis are usually exhibited by L. ivanovii strains. d. Of 30 strains, ATCC 15313, the type strain, did not give a positive reaction. e. Of 10 strains tested, 1 gave a positive reaction.

Table 2 Serology, Hemolytic Activity andMouse Virulence for Listeria Species species hemolysis of horse blood mouse (7%) stab virulence L. monocytogenes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, + + 4ab, 4b, 4b(x), 4c, 4d, 4e, 7 L. ivanovii 5 + + L. innocua 4ab, 6a, 6b, un* L. welshimeri 6a, 6b L. seeligeri 1/2b, 4c, 4d, 6b, un* + * un = undefined. Table 3 Camp Test Reactions of Listeria Species species L. monocytogenes L. ivanovii L. innocua L. welshimeri L. seeligeri hemolytic reaction S. aureus + + R. equi + serotype

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Table 4 The Sampling Scheme for Ready-to-eat (RTE) Foods 1 being Analyzed for L. monocytogenes (LM) Food Product Sampling 1. RTE foods causally linked to 5 sample units listeriosis (e.g. this list presently (100 g or mL includes soft cheese, liver pâté, each) taken at coleslaw mix with shelf life > 10d, random from jellied pork tongue2 ) each lot. 2. All other RTE foods sup- porting 5 sample units growth of LM with refrigerated (100 g or mL shelf -life >10d (e.g. vacuumeach) taken at packaged meats, modified random from atmosphere (MAP) sandwiches, each lot. cooked seafood, packaged salads, refrigerated sauces) 3. RTE foods supporting growth of 5 sample units LM with refrigerated shelf-life (100 g or mL 10d and all foods not 3 each) taken at supporting growth random from the (e.g cooked seafood, packaged lot. salads, ice cream, hard cheese, dry salami, salted fish, breakfast and other cereal products)

1

Analysis Type of analysis 5x10 g or 2x25 g ENRICHMENT analytical units4 are ONLY either analyzed separately or composited. 5x5 g analytical units4 ENRICHMENT are either analyzed ONLY separately or composited.

5x10 g analytical 4 DIRECT 4 units are analyzed PLATING separately. Where enrichment is ENRICHMENT necessary5 5x5 g analytical units 4 are analyzed separately or composited.

For a definition of RTE foods, please see the latest version of the field compliance guide entitled "RTE foods contaminated with L. monocytogenes"

2 3

At present, this product is not commonly found in the Canadian marketplace. Foods not supporting growth of LM include the following: (a) pH 5.0-5.5 and aw <0.95 (b) pH <5.0 regardless of aw (c) aw 0.92 regardless of pH (d) frozen foods

The pH and aw determinations should be done on 3 out of 5 analytical units. The food is presumed to support the growth of L. monocytogenes if any one of the analytical units fall into the range of pH and aw values which are thought to support the growth of the organism.

4 5

The designated analytical unit is taken from each sample unit.

For Category 3 foods, if GMP is inadequate and L. monocytogenes has been found in the environment of the finished product area, or where examination of Good Manufacturing Practice 104

(GMP) status is not possible, both MFLP-74 (Enumeration of Listeria monocytogenes in Food) and MFHPB-30 may be used as appropriate.

Figure 1 A Flow Diagram Showing the Isolation Procedure Blend or stomach in LEB broth. Incubate at 30oC for 48 h.

At 24 and 48 h transfer 0.1 mL of theLEB into MFB. Incubate 24-48 h at 35oC.Record reactions for all tubes. Streak LEB (optional but preferable) onto plates.

Streak positiveMFB onto selective agar plates. Reincubate negative MFB for an additional 24 h. Incubate plates for 24-48h

Confirmation Testsmotility,hemolysis, mannitol, rhamnose biochemicals, or rapid identification kits, as required.

and

xylose;

other

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5. MEDIA 5.1 Blood Agar Prepare blood agar plates as soon as possible after receiving fresh blood, using blood agar base or preferably Trypticase Soy agar with 7% defibrinated horse blood. Rehydrate and sterilize as recommended by the manufacturer. Agar and blood should both be at 45-50oC before combining and pouring plates. Commercial plates may also be used. Plates stored at 4oC can last for 1 month. Sheep blood plates for the CAMP test are prepared and stored in a similar way. 5.2 Carbohydrate Fermentation Broth and Agar 5.2.1 Carbohydrate Fermentation Broth Purple broth base Distilled water 16 g 900 mL

Dispense 9 mL portions in 16 x 125 mm tubes each containing a Durham tube. Autoclave at 121oC for 15 min. Prepare all carbohydrates, except esculin, as 5% solutions and filter sterilize. Add 1 mL carbohydrate solution to 9 mL broth base to yield a final concentration of 0.5% carbohydrate in broth. Add esculin directly into base broth to make a 0.5% solution and autoclave at 115oC for 15 min. A 5% solution of esculin at room temperature is a gel that cannot be pipetted. 5.2.2 Carbohydrate Fermentation Agar a. Basal Medium: Purple broth base Bromcresol purple (1.6% aqueous) Agar Distilled water b. Carbohydrate Solution: Filter sterilize 100 mL each of 20% aqueous solutions of rhamnose, mannitol and xylose. Sterilize the base at 121oC for 15 minutes. Temper, add 50 mL of the sterile carbohydrate solution, then pour thick plates. Plates can be stored for at least 2-3 weeks at 4oC. Longer storage times must be validated by the individual lab. 16 g 1 mL 16 g 950 mL

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5.3 Listeria Enrichment Broth (LEB) a. Basal Medium: Proteose peptone Tryptose Lab lemco powder (Oxoid) Yeast extract NaCl KH2PO4 Na2HPO4 Esculin 1 Nalidixic acid (2% solution in 0.1M NaOH) Distilled water

1

5g 5g 5g 5g 20 g 1.35 g 12 g 1g 1 mL 1000 mL

NOTE: The amount of nalidixic acid given here is 1/2 the amount given in the original formula.

Sterilize at 121oC for 15 minutes. Do not overheat; cool at once after removal from the sterilizer. Store at 4oC. LEB broth is available commercially as UVM 1 formulation. b. Acriflavin Solution: Filter sterilize 25 mL of 1.2% aqueous acriflavin solution. Store at 4oC for 2 months. On the day of use, add 1.0 mL of acriflavin solution to 1000 mL of basal medium. 5.4 Lithium Chloride-Phenylethanol-Moxalactam Medium (LPM ) a. Basal Medium: Phenylethanol agar Glycine anhydride Lithium chloride Distilled water 35.5 g 10.0 g 5.0 g 1000 mL

b. Moxalactam solution: 2 mL Moxalactam (ammonium or sodium salt) Potassium phosphate buffer, 0.1 M, pH 6.0 Filter sterilize. Store the solution frozen in 2 mL aliquots Sterilize the basal medium at 121oC for 15 min. Cool to 45oC-50oC and add 2 mL of moxalactam solution. Pour 12-15 mL in each petri dish and store at 4oC. The basal medium cannot be made in advance and reheated. 1g 100 mL

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5.5 Modified Fraser Broth (MFB) Proteose peptone Tryptose Lab lemco powder (Oxoid) Yeast extract NaCl KH2PO4 Na2HPO4 Esculin Lithium chloride Nalidixic acid (2% solution in distilled water) Distilled water b. Stock Solutions: Acriflavin (0.25% in distilled water) Ferric ammonium citrate (5.0% in distilled water) Filter sterilize the solutions. Store at 4oC for 2 months. Dispense 10 mL portions of basal medium in 16 x 150 mm test tubes. Sterilize at 121oC for 15 minutes. Do not overheat; cool at once after removal from the sterilizer. Store at 4oC. Add 0.1 mL of each stock solution to each tube before use. OR: Dispense 100 mL portions of the basal medium in screw capped bottles and sterilize at 121oC for 15 min. Cool at once after sterilization and store at 4oC. Just prior to use, add 1.0 mL of each stock solution to each 100 mL bottle and mix. Dispense aseptically in 10 mL amounts in presterilized 16 x 150 mm test tubes. 5.6 Modified Oxford Agar (MOX) MOX agar is a slight modification of the Listeria selective agar (Oxford Formulation). a. Basal Medium: Columbia blood agar base (depending of the brand) Agar Esculin Ferric ammonium citrate Lithium chloride Colistin (1% solution; see b.) Distilled water 39-44 g/L 2 g/L 1 g/L 0.5 g/L 15 g/L 1 mL 1000 mL 5g 5g 5g 5g 20 g 1.35 g 12 g 1g 3g 1 mL 1000 mL

Rehydrate with constant stirring with a magnetic mixer and adjust pH to 7.2 if necessary. Autoclave at 121oC for 10 minutes, mix again, and cool rapidly to 46oC in a water bath.

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b. Colistin Solution: Colistin, methane sulfonate Potassium phosphate buffer, 0.1 M, pH 6.0 1g 100 mL

Colistin solution is not sterilized. Store at -20oC in small aliquots (3-5 mL). c. Moxalactam Solution: Moxalactam (ammonium or sodium salt) Potassium phosphate buffer, 0.1 M, pH 6.0 1g 100 mL

Filter sterilize. Store the solution at -20oC in 2 mL aliquots. Add 2 mL of moxalactam solution to the basal medium, mix well, and pour 12 mL per plate. Both the basal medium and supplements are available commercially. 5.7 Motility Test Medium Rehydrate and sterilize according to manufacture's instructions. Dispense 6 mL portions into 16 x 125 mm screw-capped tubes, or 3 mL portions in 13 x 100 mm screw-capped tubes. 5.8 Oxford Agar (OXA) a. Basal Medium: Columbia blood agar base Esculin Ferric ammonium citrate Lithium chloride Cycloheximide Colistin Acriflavin Distilled or deionised water 39.0 g 1.0 g 0.5 g 15.0 g 0.4 g 0.02 g 0.005 g 1000 mL

Suspend the ingredients in the water. Bring to a boil to dissolve completely. Sterilize by autoclaving at 121oC for 15 min. Cool to 50oC. b. Supplements: Cefotetan Fosfomycin 0.002 g 0.01 g

Add the cefotetan and fosfomycin, or manufacturer's supplements, mix and pour the plates.

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5.9 Palcam Agar (PAL) a. Basal Medium: Peptone Starch Sodium chloride Agar Mannitol Ferric ammonium citrate Esculin Dextrose Lithium chloride Phenol red Distilled or deionized water 23.0 g 1.0 g 5.0 g 13.0 g 10.0 g 0.5 g 0.8 g 0.5 g 15.0 g 0.08 g 1000 mL

Suspend the ingredients in the water and adjust pH if necessary to 7.2±0.1. Bring to a boil to dissolve completely. Sterilize by autoclaving at 121oC for 15 min. Cool to 50oC, add the manufacturer's supplements containing polymxin-B-sulphate, ceftazidime and acriflavine aseptically, and then pour the plates. 5.10 Tryptose Broth and Agar for Confirmation Tests and Serology Tryptose Sodium chloride Dextrose Agar (leave out of broth formula) Distilled water 20.0 g 5.0 g 1.0 g 15.0 g 1000 mL

Autoclave at 121oC for 15 min. For agar, make generous slants. 5.11 Trypticase Soy Broth with 0.6% Yeast Extract (TSB-YE) Trypticase soy broth 30.0 g Yeast extract 6.0 g Distilled water 1000 mL Autoclave at 121oC for 15 min and dispense into 16 x 100 mm tubes.

5.12 Trypticase Soy Agar with 0.6% Yeast Extract (TSA-YE) Trypticase soy agar 40.0 g Yeast extract 6.0 g Distilled water 1000 mL Dispense into screw-capped tubes, autoclave at 121oC for 15 min and prepare slants.

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F. Isolation and Enumeration of Bacillus cereus in foods

1. Application This method is applicable to the isolation, identification and enumeration of Bacillus cereus (with limitations as described in the method) in foods. 2. Description The method has been shown to produce satisfactory results with naturally-contaminated meats, vegetables, dairy products, cereals and dried foods. 3. Principle Bacillus cereus is widely distributed in nature and is commonly found in a variety of foods. When B. cereus grows to high numbers in a food (> 106/g), sufficient enterotoxin may be produced resulting in foodborne illness. This method determines the presence of B. cereus by plating known quantities of (dilutions of) a food sample onto a selective agar. After incubation, presumptive B. cereus colonies are selected and subjected to confirmatory testing. From the results obtained, the number of B. cereus per g or mL of the food is calculated. NOTE: B. cereus is not easily distinguished from other closely related organisms in the B. cereus Group. B. mycoides characteristically produces rhizoid colonies on agar media and B. anthracis is non-motile and non-hemolytic. However, atypical strains of B. cereus are variable in expression of motility and hemolysis and further testing may be necessary to identify the isolates. Consider the source of the sample when identifying the isolates as B. cereus. Only B. cereus and B. thuringiensis are likely to occur naturally in food products. 4. Materials and special equipment The following media and reagents (1-5) are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See Section 6 for the formula of individual media. 1) Peptone Water diluent (PW) 2) Citrate solution, 2%, warmed to 45°C (for cheese) 3) Trypticase Soy Broth (TSB) 4) Nutrient Agar plates 5) Polymyxin Pyruvate Egg Yolk Mannitol Bromthymol Blue Agar (PEMBA Medium) 6) Blood Agar plates (TSB agar with 5% sheep blood) 7) Sporulation broth (9.1) or TSA-MnSO4 agar (optional) 8) Staining solutions (optional): Malachite Green, 5% aqueous solution; Safranin, 0.5% aqueous solution; Sudan Black B, 0.3% in 70% ethanol; Xylol 9) Basic fuchsin, 0.5% aqueous solution OR TB Carbol-fuchsin ZN stain (Difco) [protein toxin crystals]

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Note: Both Basic Fuchsin and TB Carbol-fuchsin ZN stains are toxic and possibly carcinogenic. Use appropriate safety precautions. It is recommended that commercially-available products be purchased. 10) Methanol [protein toxin crystals] 11) BC Motility Medium 12) Rapid identification kits (optional) 13) Control cultures, ATCC or equivalent 14) Blender, stomacher or equivalent 15) Microscope 16) Incubators capable of maintaining 30 and 35°C 5. Procedure Each sample unit shall be analyzed individually. The test shall be carried out in accordance with the following instructions: SAFETY NOTE: PEMBA media supports the growth of B. anthracis. No obvious morphological differentiation between some strains of B. cereus and B. anthracis will occur. Take suitable precautions. 5.1. Handling of Sample Units 5.1.1. During transport, with the exception of shelf-stable products, keep the sample units refrigerated (0-5°C) or frozen depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death. 5.1.2. Analyze the sample units as soon as possible after receipt at the laboratory. 5.2. Preparation of Dilutions 5.2.1. To ensure a representative analytical unit from a solid sample, combine portions from several locations within each solid sample unit. 5.2.2. If the sample unit is a liquid or a free-flowing solid (powder), thoroughly mix each sample unit by shaking the container. 5.2.3 Prepare a 1:10 dilution of the food by adding aseptically 11 (10) g or mL (the analytical unit) to 99 (90) mL of diluent (Table 1). Shake, blend or stomach according to the type of food as indicated in Table 1. Note: Weight or volume in brackets indicates alternate procedure for making dilutions. 5.2.4. The food homogenate (1:10 dilution) of dry foods should stand at room temperature for 15 min. In all other instances, the analysis should be continued as soon as possible.

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5.2.5. Mix for the minimum time required to produce a homogeneous suspension to avoid overheating; blending or stomaching time should not exceed 2 min. With foods that tend to foam, use blender at low speed and remove aliquot from below liquid/foam interface. 5.2.6. If the 1:10 dilution is to be mixed by shaking, shake the dilution bottle 25 times through a 30 cm arc in approximately 7 sec. 5.2.7. Prepare succeeding decimal dilutions as required, using a separate sterile pipette for making each transfer. 5.2.8. Shake all dilutions (as in 5.2.6) immediately prior to making transfers to ensure uniform distribution of the microorganisms present. 5.3. Enumeration of Presumptive B. cereus 5.3.1. Plating 5.3.1.1. Dry PEMBA plates in a bio-hood or laminar flow hood immediately before using. Agitate each dilution to resuspend material that may have settled during preparation. Plating should be carried out within 15 min of preparing the dilutions. 5.3.1.2. Solid foods (i) If fewer than 1,000 B. cereus per g are expected: spread 0.2 mL of the 1:10 dilution evenly over the surface of one of each of ten selective agar plates (PEMBA). (ii) Routinely, or if counts higher than 1,000 B. cereus per g are expected: spread 0.2 mL of each dilution on each of duplicate PEMBA plates 5.3.1.3. Liquid sampes: If the sample units are liquid, 0.2 mL of the undiluted analytical unit may be spread on each of duplicate PEMBA plates. NOTE: The liquid should not be spread right to the edge of the plate, since this causes confluent growth at the plate-agar interface which is difficult to count. 5.3.1.4. Retain the plates in an upright position until the inoculum has been absorbed by the medium (approximately 10 minutes on properly dried plates). If the inoculum is not readily absorbed by the medium, the plates may be placed in an upright position in an incubator for up to 1 h. 5.3.2. Incubation 5.3.2.1. Invert the plates and incubate at 35°C for 24 ± 2 h. 5.3.2.2. Avoid excessive crowding or stacking of plates in order to permit rapid equilibration of plates with incubator temperature. 5.3.2.3. Examine the plates for presumptive B. cereus. Count the number of presumptive B. cereus colonies present (Sec. 5.3.3). Re-incubate the plates at room temperature for an additional 24 h and re-examine.

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Helpful Hint: Circle presumptive colonies at 24 h. When re-examined at 48 h, look for colonies that were not present at 24 h and add to the 24 h count. It may appear that there are fewer colonies at 48 h, due to overgrowth of the colonies. In this case, the count at 24 h is more accurate. 5.3.3. Counting Colonies and Recording Results Note: On PEMBA B. anthracis (and some strains of B. cereus) have very little or no zone of egg yolk precipitate. Colonies of B. anthracis may appear to be smaller, whiter and more raised when compared to B. cereus. 5.3.3.1. Count colonies immediately after the incubation period. Look for the following 2 types of presumptive B. cereus colonies on PEMBA: Type 1: Uneven margins, fimbriate or slightly rhizoidal, 2 to 5 mm in diameter, turquoise to peacock blue (intensity variable) in color with flat ground glass surface and surrounded by a grey to turquoise halo of dense precipitate (egg yolk reaction) which may become peacock blue after 48 h incubation. Type 2: Colonies similar to type 1 but with no surrounding halo of precipitation. 5.3.3.2. Counting the Ten Plates of the 1:10 Dilution (Solid Food Only) 5.3.3.2(a.) (A) If the number of all presumptive B. cereus colonies per plate is fewer than 20, add separately the counts for each type from all ten plates and record as the respective presumptive count. This is the count of one of the two types per 2 mL.(0.2 g of food) (B). Multiply the count by 5, and record as the respective presumptive count per g of food (C). Add the results, and report as the total presumptive count per g of food. 5.3.3.2(b.) If the number of all presumptive B. cereus colonies is greater than 20 per plate but the total count of the two types does not exceed 200, select two plates at random, count separately the colonies of each type and compute the respective average presumptive count per plate (per 0.2 mL) (A/2). Multiply each count by 50 and record as the respective presumptive count per g of food (C). Add the results and report as the total presumptive count per g of food. 5.3.3.2(c). If the number of presumptive B. cereus colonies on some of the ten plates is < 20, but on others is > 20, proceed as in 5.3.3.2(a) above. 5.3.3.3. Counting of Duplicate Plates (Any Dilution) 5.3.3.3(a). Select plates containing 20-200 presumptive B. cereus colonies per plate consisting of the combined counts of the two types. An alternate counting range of 10100 or 10-150 may be used, as these ranges are recommended in other standard methods due to the spreading nature of Bacillus colonies. 5.3.3.3(b). Compute the average presumptive count per plate for each type (A/2), multiply by five and by the appropriate dilution factor, and record as presumptive count per g or mL of food for each type (C). Add the results and report as the total presumptive count per g or mL of food.

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5.3.3.3(c). If plates from more than one dilution are used, the counts are to be averaged as shown below (Sec. 5.3.3.4) 5.3.3.3(d). If no plates containing 20-200 presumptive B. cereus are available, estimated counts may be made on plates giving presumptive counts outside this range. Report results as estimated counts when results are outside the range of 20-200. 5.3.3.3(e). When an estimated count contributes to an average count, this average itself becomes an estimated value. 5.3.3.4. Averaging of Counts Over Two Dilutions 5.3.3.4(a). If plates from two consecutive decimal dilutions contain counts within the range of 20-200 presumptive B. cereus colonies per plate, the counts on all four plates should be used to arrive at the average count. Inasmuch as the two different types are to be counted separately and it is quite possible that individual counts may be < 20, although the combined counts are within range, estimates and true values would have to be combined in order to arrive at an average value. This can be avoided by using the following formula:

Average colony Total number of colonies counted / = count/g or mL Volume used per dilution (1/dilution1 + 1/dilution2) For an example of counting colonies see Table II. 5.3.3.4(b) If no presumptive B. cereus colonies are obtained, record presumptive counts as < 5 per g or mL for the ten plates of the 1:10 dilution, or < 2.5 x the dilution factor for duplicate plates. 5.4. Confirmation 5.4.1. Selection of Colonies 5.4.1.1. From the plates counted, a number of each colony type observed is selected as follows: a) When the total count per type for all the plates of a dilution is less than five, pick all colonies of that type. b) 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. 5.4.2. Screening for B. cereus / B. thuringiensis It is recommended that suspect colonies be streaked onto non-selective agar (Nutrient or Blood agar) for purity. Inoculate 5 mL of Trypticase-soy broth (TSB) with suspect colonies, as well as appropriate controls, and incubate for 18 h at 30/C. 5.4.2.1. Motility

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Inoculate BC motility medium (BCMM) by stabbing down the center of the tube with a 3 mm loopful of a 24 h culture suspension. Incubate tubes for 18 to 24 h at 30oC and examine for type of growth along the stab line. Most strains of B. cereus and B. thuringiensis are motile by peritrichous flagella, and produce diffuse growth out into the medium away from the stab. B. anthracis and all but a few strains of B. mycoides are non-motile. NOTE: A few strains of B. cereus are non-motile. 5.4.2.2. Rhizoid growth Inoculate a pre-dried nutrient agar plate by touching the medium surface near the center with 2 mm loopful of culture. Let the inoculum be absorbed, and incubate the plate in an upright position for 24 to 48 h at 30oC. Check the plate for rhizoid growth characterized by root or hairlike structures which may extend several cm from the point of inoculation. This type of growth is typical for B. mycoides species. B. cereus strains produce rough irregular colonies that should not be confused with rhizoid growth. 5.4.2.3. Hemolytic activity After incubation of broth, divide a blood agar plate into 6 to 8 equal segments. Label each segment and inoculate one or more segments near the center by gently touching the agar surface with a loopful of incubated broth. Let inoculum be absorbed, and incubate plates for 24 h at 30oC. Check plates for hemolytic activity. B. cereus is usually strongly beta hemolytic. B. thuringiensis and B. mycoides are often weakly beta hemolytic with production of complete hemolysis only underneath the colonies. B. anthracis is usually non-hemolytic. Aging cultures may demonstrate weak gamma hemolysis. Take proper precautions if a non-hemolytic colony is isolated. Note: This is a subjective test which may not differentiate B. cereus from B. thuringiensis or B. mycoides, but the detection of beta hemolysis will rule out B. anthracis. 5.4.2.4. Use of a rapid identification system such as VITEK or API may be useful to confirm that the isolate is B. cereus or B. thuringiensis. Systems such as Vitek will not differentiate these two species, even though a good identification is made by the system of B. cereus or B. thuringiensis. Note: Some labs have trouble differentiating colour reactions with API 50CH. BioMerieux recommends that API 50CH be used in conjunction with API CHB/E. In addition, the first 12 tests in API 20E may aid in identification. Check with your BioMerieux representative. 5.4.2.5. Isolates that are motile, do not exhibit rhizoid growth and are hemolytic have a high probability of being B. cereus or B. thuringiensis. Strongly hemolytic strains are likely B. cereus. To confirm the presence of B. cereus, the following test for protein toxin crystals will differentiate B. cereus from B. thuringiensis.

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5.4.2.6. Protein toxin crystals Inoculate nutrient agar slants with 3 mm loopfuls of 24 h TSB culture suspensions. Incubate slants 24 h at 30/C and then at room temperature 2-3 days. Prepare smears with sterile distilled water. Air-dry and lightly heat-fix. Place slide on staining rack and flood with methanol. Let stand 30 s, pour off methanol, and allow slide to air-dry. Return slide to staining rack and flood completely with 0.5% Basic fuchsin or TB carbolfuchsin ZN stain (Difco). Heat slide gently from below until steam is seen. Wait 1-2 min and repeat this step. Let stand 30 s, pour off stain, and rinse slide thoroughly with clean tap water. Dry slide without blotting and examine under oil immersion for presence of free spores and darkly stained tetragonal (diamond- shaped) toxin crystals. Crystals are usually smaller than spores. Toxin crystals are usually abundant in a 3- to 4-day-old culture of B. thuringiensis but cannot be detected by the staining technique until lysis of the sporangium has occurred. Therefore, unless free spores can be seen, cultures should be held at room temperature for a few more days and re-examined for toxin crystals. B. thuringiensis usually produces protein toxin crystals that can be detected by the staining technique either as free crystals or parasporal inclusion bodies within the exosporium. B. cereus and other members of the B. cereus group do not produce protein toxin crystals. 5.4.2.7. Confirm with staining procedure as outlined below if necessary. It is recommended that a sporulation step be included before following this procedure. 5.4.3. Sporulation Procedure (Optional) 5.4.3.1. Inoculate a prepared flask of sporulation broth with one isolated presumptive B. cereus colony from PEMBA.. Place on a stir plate (without heat), loosen the cap and stir moderately at room temperature for five days. Stain as outlined in 5.4.4. 5.4.3.2. Alternately, streak presumptive colony onto TSA-MnSO4 agar. Incubate at room temperature for 2-3 days. Stain as outlined in 5.4.4. 5.4.4. Staining Procedure (Optional) 5.4.4.1. Prepare smears on glass microscope slides from t e centre of colonies h selected. 5.4.4.2. Air dry the smears and fix with minimal flaming. 5.4.4.3. Place the slides on a staining rack and flood with 5% w/v Malachite Green. 5.4.4.4. Heat slides with a gentle flame until vapour can be seen to rise. Continue for 3 min taking care not to boil the staining solution on the slides. 5.4.4.5. Wash slides well with cold tap water; blot dry. 5.4.4.6. Flood slides with 0.3% w/v Sudan Black B in 70% ethanol. Allow to sit for 15 minutes. 5.4.4.7. Wash slides well with cold water; blot dry. 5.4.4.8. Flood slides with xylol for 5 seconds.

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Note: Follow suitable safety precautions when using xylol. 5.4.4.9. Wash slides with cold tap water; blot dry. 5.4.4.10. Flood slides with 0.5% aqueous Safranin for 30 seconds. 5.4.4.11. Wash slides with cold tap water and allow to dry in an upright position. 5.4.4.12. Vegetative cells of B. cereus stain red and generally have a characteristic `boxcar' appearance 4-5 : long and 1.0-1.5 : wide with square ends and rounded corners usually appearing as chains. Spores stain pale to mid-green and lipid globules are black. Vegetative cells displaying: i) central or paracentral spores not obviously swelling the sporangium and ii) lipid globules, confirm the isolates as B. cereus Group. 5.4.5. Calculations and Reporting (See also Table 2) On the basis of the confirmatory tests for each of the two types of cultures, record the total number of B. cereus per g or mL of food (N). Total number of B. cereus per g or mL equals the sum of the number of B. cereus types 1 and 2 (N T =N1+N2). No. B.cereus/ = type 1 per g or mL(N) No. of colonies confirmed presumptive count as B. cereus(P)/ X type 1 (C) No. colonies tested (G)

6. Preparation of Media 6.1. Sporulation Broth Glucose Yeast extract Manganese sulphate (MnSO4) Distilled water 50.0 g 30.0 g 3.0 g 1.0 L

Add ingredients to 1L of distilled water and bring to a boil to dissolve. Dispense 100 mL into 500 mL erlenmeyer flasks. Autoclave at 121/C for 15 minutes. 6.2. BC Motility Agar (8.3) Trypticase Yeast extract Dextrose Na2HPO4 Agar Distilled water 10.0 g 2.5 g 5.0 g 2.5 g 3.0 g 1L

Heat to dissolve and dispense into tubes (2 mL into 13 X 100 mm tubes is suggested). Autoclave 10 minutes at 121°C. Final pH 7.4 ± 0.2. For best results store at room temperature for 2 to 4 days before use to prevent growth along the side of the medium.

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6.3. 0.5% Basic Fuchsin Stain (8.3) basic fuchsin alcohol distilled water 0.5 g 20 mL 80 mL

Dissolve 0.5 g basic fuchsin in 20 mL of alcohol and dilute to 100 mL with water. Filter solution if necessary thru fine paper to remove excess dye particles. Store in tightly stoppered container. Note: Fuchsin stain is toxic and possibly carcinogenic. Use appropriate safety precautions

TABLE 1 Preparation for the Initial Dilution Type of Food Product Liquids Preparation pipette directly into petri plate and/or peptone water diluent weigh into peptone water diluent Treatment shake blend*

Viscous and non-miscible liquids Solids Water soluble solids weigh into peptone water diluent Cheese weigh into previously warmed (45°C) sterile 2% sodium citrate (Na3C6H5O7.2H 2O) solution Spices weigh into peptone water diluent Powders, meat and other weigh into peptone water diluent solids * A stomacher may also be used to provide the initial blend.

shake blend* shake blend*

TABLE II Example of Computing B. cereus / B. thuringiensis Count per g or mL of Food Total No. of No. of No. of Isolates Total No. of No. of B. cereus from one Colonies of one of Isolates Confirmed as Colonies of one of of the two Types per g or the two Types on Tested "G" B. cereus "P" the two Types per g mL "N" N= (P/G)xC Duplicate Plates or mL "C" C= "A" 1/2AxD*x 5** Fewer than 5(e.g. All (4) 2 1,000 500 4) More than 5(e.g. 5(5) 4 4,500 3,600 18) Calculate N1 and N2 for each colony type to obtain total number of B. cereus. (N T ) per g or mL N T = N1 + N2

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e.g. if N1 = 1,000 and N2 = 100 NT = 1,000 + 100 = 1,100/g * Dilution factor = 100 ** For duplicate plates, 0.2 mL per plate (5.4.5). Divide by 2 since "A" represents the total count of one of the two types on two duplicate plates. Report total number of Bacillus cereus / Bacillus thuringiensis per g or mL of food to two significant figures. N.B. If the ten plates of the dilution are counted (5.3.3.2(a)); C=Bx10x0.5, where B is the total count of one of the two types on all ten plates. If the two of the ten plates of the 1:10 dilution are counted (5.3.3.2(b)); C=1/2Ax10x5

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G. Detection of Clostridium botulinum in honey and syrups

1. Principle The procedure involves the removal of botulinal spores from the liquid portion of honey or syrups by membrane filtration, cultivation of the membrane in a liquid medium, analysis of the culture for toxin, and identification of toxins with specific botulinal antisera. Of the common human types of C. botulinum, only types A and B are commonly involved in infant botulism. The procedure is therefore geared towards the detection of these 2 types. A rare human type (F) may be considered as the possible source of toxin if (a) injected mice show the typical signs of botulism, and (b) the toxin cannot be neutralized by Type A or B antisera. 2. Materials and Special equipment 1) Millipore sterilfil holders XXII04710. 2) Millipore membrane filters (MF) HAWP04700. 3) 1 cc tuberculin syringes. 4) 27G 1/2" needles. 5) Botulinal antitoxins. 6) Sterile beakers. 7) Sterile dH2O. 8) Sterile 1% Tween 80. 9) 150 mL screw capped dilution bottles. 10) 300 mL centrifuge bottles. 11) Water bath set to 65oC. 12) Centrifuge. 13) Laminar flow cabinet. 14) TPGYB medium. 15) Anaerobic jars or anaerobic chamber. 16) Paraffin oil. 17) 0.45 µm filter with Luer lock. 18) Gelatin phosphate buffer. 19) White mice (approx 20 g). 2.1. Filtration Equipment

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2.1.1. Millipore Sterifil holders XXII 04710. These are placed on 1-litre suction flasks. Two or more units may be linked, in parallel, to a manifold which is connected to a vacuum pump. 2.1.2. Millipore membrane filters (MF) HAWP 04700. These are retailed in boxes containing 4 packages of 25 filters each. Note: Flow rate and volume of filterable material depend on the direction in which the MF are placed in the filter units, but the direction of optimum flow bears no relation to their orientation in the packages. When a new box is opened, take 2 filters from a package and place them (in succession or in parallel) in filter units (a) in the same orientation as in the package (keeping the top side up) and (b) with top and bottom sides reversed. Filter 100 mL of diluted honey (20% w:v), heated to 65oC, through both and record the flow rates. Maintain the orientation with the higher flow rates for the remaining 25 MF in the first package. Examine at least one filter each of the remaining 3 packages in the same way to ascertain proper orientation. 2.2 Syringes and Needles Recommended syringe: 1 cc tuberculin, recorder number 5602, Becton-Dickinson. Recommended needle: 27G 1/2; also B-D 2.3. Botulinal antitoxins Trivalent (A,B,E) antiserum; Connaught Laboratories, 1755 Steeles Ave. West, North York, Ontario, M2R 3T4 416) 667-2701 Wellcome Laboratories, Bechenham, Kent, England.

Monovalent (A and B) antisera; 3. Procedure

3.1. Preparation of diluted samples Weigh 25 g of honey (or syrup) into a sterile foil-covered beaker. Add 100 mL of sterile distilled water with 1% Tween 80 and stir until the solution is homogeneous. 3.2. Spore activation, filtration and incubation For syrups, transfer the 125 mL suspensions to 150 mL screw-capped dilution bottles, hold in a water bath at 65oC for 30 min. and filter through a membrane filter (MF). For honeys, transfer the 125 mL suspensions to 300 mL centrifuge bottles. Hold in a water bath at 65oC for 30 min. and centrifuge at 15,000 xg for 20 min. Filter the supernate through a membrane filter. Keep the sediment temporarily at 4oC and filter. After filtration rinse dilution bottle and funnel with about 5 mL of sterile, cold dist. water through each MF. Transfer the MF in a laminar flow cabinet into 110 mL of TPGYB medium. In the analysis of honey, carefully add the sediment from the centrifugation to the dilution bottle containing TPGYB medium and the filter. Incubate at 35oC for 7 days under anaerobic conditions. Check the bottles daily. Cap loosely to prevent pressure build-up. 122

3.3. Modifications of 3.2 in case of clogged filters In the rare event that the MF filter becomes clogged before the filtration of 125 mL is completed, transfer the unfiltered portion to a second filter unit. Rinse the funnel of the first unit with water, transfer the rinse water to the second unit and complete the filtration. Rinse, and transfer both filters to a single bottle of TPGYB medium. 3.4. Detection of C. botulinum in cereals Weigh 25 g of cereal directly into 600 mL of TPGYB medium tempered to 65oC. Keep at 65oC for 30 min. Incubate anaerobically at 35oC for 7 days. 3.5. Preparation of culture filtrate After 7 days of incubation, select the bottles with signs of growth and withdraw about 20 mL of culture. Centrifuge at 20,000 x g for 20 min and decant the supernate. Take about 10 mL of supernate up in a disposable syringe and sterilize by filtration through a Millex HA 0.45 µm membrane filter (Millipore) fitted on the syringe. 3.6. Detection of toxin Dilute 4 mL of sterile filtrate with 4 mL of gelatin phosphate buffer. Inject intraperitoneally two mice (about 24 g) each with 0.5 mL of diluted filtrate and observe for 4 days. Store the unused portions of diluted and undiluted filtrate at 4oC. Notes: i) Dilution of filtrate is required to prevent anaphylactic shock from the high protein content of the medium. ii) 95% of the mice killed by botulinal toxin in TPGYB medium will be dead or near death after 24 h 3.7. Confirmation of botulinal toxin Select all samples causing death in 1/2 or 2/2 mice. Place 1.5 mL each of diluted filtrate in four 10 x 75 mm test tubes. Add 0.15 mL of botulinal antiserum (Appendix B, 4): trivalent A, B, E to the first, monovalent A to the second, monovalent B to the third, none to the fourth. Mix, and keep the mixtures at ambient temperature for 45 min. to 1 h. Inject two mice each with 0.55 mL of each filtrate/antiserum mixtures and 0.5 mL of filtrate without antiserum. Observe for 4 days. If a sample kills only 1/2 mice, inject 2 more mice, if possible within 24 h after the first injection. Samples are considered positive for toxin if 2/2 or at least 2/4 mice are killed. Clostridium botulinum type A is confirmed if mice are protected with trivalent A, B, E and monovalent A antisera; C. botulinum type B is confirmed if mice are protected with trivalent A, B, E and monovalent B antisera. Notes: i) If there are signs of botulism prior to death (ruffled fur, laboured abdominal breathing, weak or paralysed limbs) and none of the antisera has a protective effect, C. botulinum type F may be the source of toxin. In that case, ship the remaining filtrate to the Botulism Reference Service for identification and store the original culture at 4oC for future reference. ii) Trivalent (A, B, E) antiserum is used in lieu of divalent (A,B) antiserum.

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4. Preparation of media 4.1a.Trypticase-Peptone-Glucose-Yeast Extract-Beef Extract (TPGYB) Medium Trypticase (BBL)* Peptone (Difco) Dextrose (Difco) Yeast extract (Difco) Beef extract (Difco) Sodium thioglycollate Distilled water 50 g 5g 4g 20 g 10 g 1g 1L

* May be substituted with special peptone L72 (Oxoid) Note: If the medium is not used on the day of autoclaving, deaerate prior to use by steaming at 100oC in the autoclave for 10 min, or by placing the dilution bottles in boiling water, about 6 cm deep, for 10 min. 4.2. Tween 80 diluent Tween 80 (polyethylene sorbitan monooleate) Distilled water Filter-sterilize. 4.3. Gelatin phosphate buffer Gelatin Disodium hydrogen phosphate (Na2HPO4) Distilled water 2g 10 g 1L 1L 10 g

Adjust pH to 6.6 with N HCl. Autoclave at 121oC and 15 lb pressure for 15 min POTENTIAL HAZARDS TO THE INVESTIGATOR Liquid cultures of C. botulinum contain high levels of toxin and should be h andled only by experienced personnel after immunization with botulinal toxoid. CAUTION: the toxoid supplied by CDC protects only against C. botulinum of types A to E, not against type F which may be, though rarely, involved in food-borne or infant botulism. Contaminated sealed products (canned or vacuum-packaged) may be under pressure and must be opened in a fume hood or safety cabinet for protection from aerosols. Goggles must be worn whenever an accidental splash may be expected. CAUTION: immunization does not assure protection of the eye from botulinal toxin, and splashes may result in blindness. Disposable gloves should be worn and pipetting by mouth is to be avoided. Used glassware and other supplies in contact with toxin are placed in a sturdy, heatresistant container which should be placed in the autoclave by the investigator. Disposable material such as gloves, cotton or tissue paper is collected in autoclave bags for hazardous waste and are autoclaved. If accidental spills occur, the toxin may be inactivated with saturated or dry sodium bicarbonate.

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Incriminated foods (excepting sealed products) and clinical specimens may be analyzed by experienced personnel without the need for immunization; if toxic, they usually contain relatively low levels of toxin.

H. Enumeration of Clostridium perfrigens in foods

1. Application This method is applicable to the enumeration of viable Clostridium perfringens in foods. 2. Description This method has been shown to produce satisfactory results with naturally-contaminated meat and poultry products. 3. Principle The procedure estimates the number of viable Clostridium perfringens per g or mL of food. A portion of the product is mixed and incubated with a selective medium by the pour plate technique. Typical black colonies are counted as presumptive Clostridium perfringens. A minimum of five of these colonies are subjected to confirmatory tests. The number of confirmed Clostridium perfringens is calculated from the ratio of presumptive colonies confirmed to presumptive colonies tested. 4. Material and special equipment The following media and reagents (1-3) are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 2 and reference 8.1 for the formula of individual media. 1) Sulfite cycloserine agar (SC) (originally designated as Egg yolk free tryptose sulfite cycloserine agar 2) Nitrate-motility (NM) agar 3) Nitrate reagents 4) 2% sodium citrate (tempered to 45oC) (may be used for cheese samples) 5) Peptone water diluent (PW) (0.1%) 6) Lactose gelatin (LG) 7) Stomacher, blender or equivalent 8) pH meter or paper capable of distinguishing to within 0.3 to 0.5 pH units within a range of 5.0 to 8.0 9) 1N HCl and 1N NaOH 10) A system capable of generating anaerobic conditions, such as, anaerobic jars (with a venting system or disposable H2/CO2 gas generator envelopes and a desiccant, such as anhydrous CaSO4); the AnaeroGenTM anaerobic atmosphere generation system (Oxoid) or an anaerobic incubator capable of maintaining 35oC.

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11) 5% C02, 10% H2 and 85% N2 (if anaerobic incubator or jars with venting system are used) 12) Anaerobic indicator 13) Aerobic incubator capable of maintaining 35o 14) 45oC waterbath (if sodium citrate is to be used) NOTE: It is the responsibility of each laboratory to ensure that the temperature of the incubators or waterbaths is maintained at the recommended temperatures. Where 35oC is recommended in the text of the method, the incubator may be 35 +/-1.0oC. Similarly, lower temperatures of 30 or 25oC may be +/- 1.0oC. However, where higher temperatures are recommended, such as 43 or 45.5oC, it is imperative that the incubators or waterbaths be maintained within 0.5oC due to potential lethality of higher temperatures on the microorganism being isolated. 15) Colony counting device 5. Procedure Each sample unit should be analyzed individually. Carry out the test in accordance with the following instructions: 5.1. Handling of Samples Units 5.1.1 In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated (0-5oC) or frozen, depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death. 5.1.2 Analyze sample units as soon as possible after their receipt in the laboratory. 5.2. Preparation for Analysis 5.2.1 Have ready 0.1% peptone water diluent or other required diluent (Table 1). 5.2.2 Clean the surface of the working area with a suitable disinfectant. 5.3. Preparation of Sample 5.3.1. To ensure a truly representative analytical unit agitate liquids or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit. 5.3.2. Prepare a 1:10 dilution of the food by aseptically shaking, stomaching or blending 25 g or mL (the analytical unit) into 225 mL of the required diluent, as indicated in Table I. If a sample size other than 25 g or mL is used, maintain the 1:10 sample to dilution ratio, such as 11 (10) g or mL into 99 (90) mL. NOTE: Weight or volume in brackets indicates alternate procedure for making dilutions. 5.3.3. Blend for the minimum time required to produce a homogeneous suspension; to avoid overheating, blending time should not exceed 2.5 min. With foods that tend to foam, use blender at low speed and remove aliquot from below liquid/foam interface.

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5.3.4. If the 1:10 dilution in a dilution bottle is to be mixed by shaking, shake the bottle 25 times through a 30 cm arc in approximately 7 sec. 5.3.5. If a stomacher is used, macerate for 1 min. 5.3.6. Check pH of the food suspension. If the pH is outside the range of 5.5-7.5, adjust pH to 7.0 with sterile 1N NaOH or 1N HCl. 5.3.7. The food homogenate (1:10 dilution) of dry foods should stand at room temperature for 15 min. In all other instances, the analysis should be continued as soon as possible 5.3.8. Prepare succeeding decimal dilutions as required, using a separate sterile pipette for making each transfer. 5.3.9. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present. 5.4. Plating and incubation 5.4.1. Pipette 1 mL of the required dilutions into each of duplicate sterile Petri plates. 5.4.2. Pour into each plate approximately 20 mL of sulfite cycloserine (SC) agar and mix by gentle rotation. 5.4.3. Incubate plates anaerobically in an upright position at 35oC for 20 h. Longer incubation may result in excess blackening along the bottom rim of the plates. Inversion of the plates may result in agar displacement by gas. Small numbers of plates may be incubated in anaerobic jars, either with disposable H2/CO2 gas generator envelopes or with a venting system. If envelopes are used, the bottom of the jars should be covered with anhydrous CaSO4 or another suitable desiccant. Alternately, the AnaeroGenTM anaerobic atmosphere generation system (Oxoid) may be used. For a large number of plates an anaerobic incubator is preferable. Anaerobic incubators and jars require three evacuations and replacements with a mixture of 5% CO2, 10% H2 and 85% N2. Each jar and incubator must contain an anaerobic indicator. 5.5. Presumptive Clostridium perfringens count 5.5.1 After 20 h of incubation, check the indicators to ascertain anaerobiosis (without anaerobiosis the analysis is discontinued). 5.5.2 Select plates containing 20-200 black colonies, about 1-2.5 mm in diameter. Pinpoint black colonies are not to be counted. 5.5.3 Count presumptive colonies and average the count of duplicate plates. The presumptive count N (as number of colonies per g (mL)) is N=A x D, where A is the average presumptive count from duplicate plates, and D the dilution factor. If the lowest number of colonies per plate exceeds 150, count or estimate the number and record the results with the letter E, e.g., 1.8 x 106 E, to indicate a lower degree of accuracy. If the

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number is too high to be estimated, record the minimum number estimable with a > sign, e.g., >2.0 x 106. If the highest number of colonies per plate is below 15, record the result with the letter E, e.g., 1.2 x 103 E. If no presumptive colonies are found, record the count as <0.5 x D. 5.6. Confirmatory tests 5.6.1. Randomly select a minimum of five presumptive colonies from the appropriate plates (or all presumptive colonies if less than five are encountered). Stab-inoculate with a plain needle (or a needle with a minute loop) each of the selected colonies into nitrate-motility (NM) agar. In parallel, inoculate the same colonies deep into lactose gelatin (LG). 5.6.2. Close the tubes tightly and incubate at 35-37oC for 24 h. Incubate two uninoculated NM and LG tubes as controls. Anaerobic incubation is not required. 5.6.3. To each NM tube with a distinct line of non-motile growth along the stab, add 0.20.4 mL of nitrate reagent. Production of a red color at the top indicates reduction of nitrate to nitrite (a positive test). Faint color reactions, slightly more intense than the blanks, should be counted as negative. Add a small quantity of zinc dust to a negative culture. The development of a red color is indicative of a negative test and the absence of a color change is positive (no nitrate remains having been reduced by the culture beyond the nitrite stage). If growth is limited to the lower part of the tube and little or no colour develops, aspirate and discard the upper part of the medium in the tube and again add the nitrate reagent to the remainder. 5.6.4. Examine LG tubes for gas production, as well as a color change from red to yellow which is indicative of lactose fermentation (lactose positive). 5.6.5. Place the LG tubes in ice water for 10 min or in the refrigerator (4oC) for one h. If no liquefaction has occurred within 24 h of incubation but the organism is non-motile, reduced nitrate to nitrite and is lactose fermentation positive, re-incubate the LG tube for another 24 h. Isolates that are non-motile, reduce nitrate, are lactose positive and liquefy gelatin within 48 h are confirmed Clostridium perfringens. 5.6.6. In addition to the tests mentioned, rapid identification kits may be used, such as the API 20A, API An-ident or Vitek Anaerobic cards. 5.6.7. Calculate the confirmed Clostridium perfringens count from the presumptive count and the relative number of confirmed colonies: Confirmed count/g(mL) = Presumptive count/g(mL) X No. of colonies confirmed / No. of colonies tested

If no colonies are confirmed, record the count as <0.5 x D, where D is the dilution factor.

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TABLE I Preparation of the Initial Dilution Type of Food Product Liquids: milk, water etc. Viscous and nonmiscible liquids Solids: water soluble solids powder, meats all cheese spices Shellfish Preparation* pipette directly into Petri plates and/or peptone water diluent weigh into peptone water diluent Treatment shake shake

weigh into peptone water diluent weigh into peptone water diluent weigh into previously warmed (45oC) 2% aqueous sodium citrate (Na3C6H5O7.2H2O) weigh into peptone water diluent weigh into peptone water diluent

shake stomach or blend stomach or blend shake stomach or blend

* Sample may be added into an empty sterile stomacher bag, blender jar or dilution bottle and the diluent added prior to mixing.

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Microbiology of water

Introduction The importance of potable (drinking) water supplies cannot be overemphasized. With increasing industrialization, water sources available for consumption and recreation have been adulterated with industrial as well as animal and human wastes. As a result, water has become a formidable factor in disease transmission. Polluted waters contain vast amounts of organic matter that serves as excellent nutritional sources for the growth and multiplication of microorganisms. As with milk or water, the presence and number of coliform bacteria and other enteric organisms in water is indicative of fecal contamination and may suggest the presence of pathogens. These pathogens are responsible for intestinal infections such as bacillary dysentery, typhoid fever, cholera, and paratyphoid fever. Analysis of water samples on a routine basis would not be possible if each pathogen required detection. Therefore, water is examined to detect Escherichia coli, the bacterium that indicates fecal pollution. Since Escherichia coli is always present in the human intestine, its presence in water alerts public health officials to the possible presence of other human or animal intestinal pathogens. Both qualitative and quantitative methods are used to determine the sanitary condition of water. Standard Qualitative Analysis of Water The three basic tests to detect coliform bacterial water are presumptive, confirmed, and completed. The tests are performed sequentially on each sample under analysis. They detect the presence of coliform bacteria (indicators of fecal contamination) the gram-negative, non sporeforming bacilli that ferment lactose with the production of acid and gas that is detectable following a 24-hour incubation period at 37ºC. Presumtive coliform test Determination of the most probable number of coliform Purpose 1. To determine the presence of coliform bacteria in a water sample. 2. .To obtain some index as to the possible number of organisms present in the sample under analysis. Principle The presumptive test is specific for detection of coliform bacteria. Measured aliquots of the water to be tested are added to a lactose fermentation broth containing an inverted gas vial (Durham tubes). Because these bacteria are capable of using lactose as a carbon source (the other enteric organisms are not), their detection is facilitated by use of this medium. In addition to lactose, the medium also contains a surface tension depressant, bile salt used to suppress the growing of organisms other than coliform bacteria. Tubes of this lactose medium are inoculated with 10-ml, 1-ml, and 0.1-ml aliquots of the water sample. The series consists of at least three groups, each composed of three tubes of the specified medium. The tubes in each group are then inoculated with the designated volume of the water sample as described under "procedure". The greater the number of tubes per group, the greater the sensitivity of the test. Development of gas in any of the tubes is presumptive evidence of the presence of coliform bacteria in the sample. The presumptive test also enables the microbiologist to obtain some idea of the number of organisms present by means of the most probable number test (MPN). The MPN is estimated by determining the number of tubes in each group that show gas following the incubation period.

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Materials Cultures Water samples from sewage plant, pond, and tap. Media lactose broth . Equipment Bunsen burner, sterile 10-ml pipettes, sterile 1 -ml pipettes, mechanical pipetting device, and glassware marking pencil. Procedure 1. Set up three separate series consisting of three groups, a total of nine tubes per series, in a test-tube rack; for each tube, label the water source and volume of sample inoculated. 2. Mix sewage plant water sample by shaking thoroughly. Exercise care in handling sewage waste water sample because enteric pathogens may be present. 3. Flame bottle and then using a 10-ml pipette, transfer 10-ml aliquots to the three tubes. 4. Flame bottle and then using a 1-ml pipette, transfer 1 ml of water to the three tubes. 5. Repeat steps 2 through 5 for the tap and pond water sample. 7. Incubate all tubes for 48 hours at 37 degrees centigrade. 8. Examine all tubes after 24 and 48 hours of incubation. Record your results in the chart as: a. Positive: 10 percent or more of gas appears in a tube in 24 hours. b. Doubtful: Gas develops in a tube after 48 hours. c. Negative: There is no gas in the tube in the series in 48 hours. Confirmed coliform test Purpose To confirm the presence of coliform bacteria in a water sample for which the presumptive test was positive. Principle The presence of a positive or doubtful presumptive test immediately suggests that the water sample is non-portable. Confirmation of these results is necessary, since positive presumptive tests may be the result of organisms of non-coliform origin that are not recognized as indicators of fecal pollution. The confirmed test requires that selective and differential media such as eosinmethylene blue (EMB) or endo agar be streaked from a positive lactose broth tube obtained from the presumptive test. Eosin-methylene blue agar contains the dye methylene blue, which inhibits the growth of gram-positive organisms. In the present of an acid environment, EMB forms a complex that precipitates out onto the coliform colonies, producing dark centers and a green metallic sheen. This reaction is characteristic for Escherichia coli, the major indicator of fecal pollution. Endo agar is a nutrient medium containing the dye fuchsin, which forms a dark pink complex that turns the E. coli colonies and the surrounding medium pink.

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Materials Cultures One 24-hour-old positive broth culture from each of the three series from the presumptive test. Media Eosin-methylene blue agar plates and endo agar plates. Equipment Bunsen burner, glassware marking pencil, and inoculating loop. Procedure 1. Label the covers of the three EMB plates and three endo agar plates with the source of the water sample (sewage, pond, and tap). 2. Using a positive 24-hour lactose broth culture from the sewage water series from thepresumptive test, streak the surface of one EMB and one endo agar plate. 3. .Repeat Step 2 using the positive lactose broth cultures from the pond and tap water series to inoculate the remaining plates. 4. Incubate all plate cultures in an inverted position for 24 hours at 37 degree centigrade. N/B: The confirmed test can also be done using the brilliant green lactose bile broth (BGLB) by transferring one loop of the positive test tubes of presumptive test into the BGLB broth arranged as in the presumptive test and containing the Durham tubes.

Completed coliform test Purpose To confirm the presence of coliform bacteria in a water sample, or, if necessary, to confirm a suspicious but doubtful result of the previous test. Principle The completed test is the final analysis of the water sample. It is used to examine the coliform colonies that appeared on the EMB or endo agar plates used in the confirmed test. An isolated colony is picked from the confirmatory test plate and inoculated into a tube of lactose broth and streak on a nutrient agar slant to perform a Gram stain. Following inoculation and incubation, tubes showing acid and gas in the lactose broth and the presence of gram-negative bacilli on microscopic examination are further confirmation of the presence E.coli, and indicative of a positive completed test.

Materials Cultures One 24-hour coliform-positive EMB or endo agar culture from each of the three series of the confirmed test. Media Nutrient agar slants and lactose broth.

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Reagents Crystal violet, Gram's iodine, 95 percent ethyl alcohol, and safranin. Equipment Bunsen burner, staining tray, inoculating loop, lens paper, blotting paper, microscope, glassware and marking pencil. Procedure 1. Label each tube with the source of its water sample. 2. Inoculate one lactose broth and one nutrient agar slant from the same isolated E. coli colony obtained from an EMB or an endo plate from each of the experimental water samples. 3. Incubate all tubes for 24 hours at 37 degrees centigrade. N/B Completed coliform test can also be carried out using the EC-broth in test tubes and inoculated with loopful of the positive tubes from the BGLB broth and incubated at 44.5ºC. Positive results are characterized by the growth and production white precipitate. Streaking onto EMB and Endo agar from the positive tubes can follow as above.

Quantititive analysis of water Purpose To determine the quality of water samples using the membrane filter method. Principle Bacteria-tight membrane filters capable of retaining microorganisms larger than 0.45 micrometer are frequently used for analysis of water. These filters offer several advantages over the conventional, multiple-tube method of water analysis: (1) Results are available in a shorter period of time, (2) larger volumes of sample can be processed, and (3) because of the high accuracy of this method, the results are readily reproducible. A disadvantage involves the processing of turbid specimens that contain large quantities of suspended materials; particulate matter clogs the pores and inhibits passage of the specific volume of water. A water sample is passed through a sterile membrane filter that is housed in a special filter apparatus contained in a suction flask. Following filtration, the filter disc that contains the trapped microorganisms is aseptically transferred to a sterile petri dish containing an absorbent pad saturated with a selective, differential liquid medium. Following incubation, the number of colonies present on the filter is counted with the aid of a microscope. This experiment is used to analyze a series of dilutions of water samples collected upstream and downstream from an outlet of a sewage treatment plant. A total count of coliform bacteria determines the potability of the water sources. Also, the types of fecal pollution, if any, are established by means of a fecal coliform count, indicative of human pollution and a fecal streptococcal count, indicative of pollution from other animal origins. The ratio of the fecal coliforms to fecal streptococci per millimeter of sample is interpreted as follows: Between 4 indicates human and animal pollution; >4 indicates human pollution; <0.7 indicates poultry and livestock pollution.

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Materials Cultures Water samples collected upstream (labeled U) and down stream (labeled D) from an outlet of a sewage treatment plant.

Media one 20-ml tube of lactose broth, one 20-ml tube of EC - broth, one 20-ml tube of K-F broth, four 90-ml sterile water blanks, and one 300-ml flask of sterile water. Equipment Sterile Millipore membrane apparatus (base, funnel, and clamp), 1-litre suction flask, 15 sterile millipore membrane filters and absorbent pads, 15 sterile 50-mm Petri dishes, 12 10-ml pipetting device, small beaker of 95 percent alcohol, forceps, dissecting microscope, and glassware marking pencil. Procedure The following instructions are for analysis of one of the provided water samples. Different samples may be assigned to individual groups. 1. Label the four 90-ml water blanks with the source of the water samples and dilution (10-1, 10-2, 10-3, and 10-4). 2. Using 10-ml pipettes, aseptically perform a 10-fold serial dilution of the assigned undiluted water sample, using the four 90-ml water blanks to effect the 10-1, 10-2, 10-3, and 10-4 dilutions. 3. Arrange the 15 petri dishes into three sets of five plates. Label each set as follows: a. For total coliform count (TCC) and dilutions (undiluted, 10-1, 10-2, 10-3, and 10-4). b. For fecal coliform count (FCC) and dilutions as in Step 3a. c. For fecal streptococcal count (FSC) and dilutions as in Step 3a. 4. Using a sterile forceps dipped in 95 percent alcohol and flamed, add a sterile pad to all Petri dishes. a. Two ml of lactose broth to each pad in the plates labeled TCC. b. Two ml of EC- broth to each pad in the plates labeled FCC. c. Two ml of K-F broth to each pad in the plates labeled FSC. 5. Aseptically assemble the sterile paper-wrapped membrane filter unit as follows: a. Unwrap and insert the sintered glass filter base into the neck of a 1liter side-arm suction flask. b. With sterile forceps, place a sterile membrane filter disc, grid side up, on the sintered glass platform. c. Unwrap and carefully place the funnel section of the apparatus on top of the filter disc. Using the filter clamp, secure the funnel to the filter base. d. Attach a rubber hose from the side-arm on the vacuum source. 6. Using the highest sample dilution (10-4) and a pipette, place 20ml of the dilution into the funnel and start the vacuum. When the entire sample has been filtered, wash the inner surface of the funnel with 20 ml of sterile water.

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7.

9.

10. 11.

Disconnect the vacuum, unclamp the filter assembly, and with sterile forcepts remove the membrane filter and place it on the medium saturated pad in the Petri dish labeled TCC, 10-4). Aseptically place a new membrane on the platform, reassemble the filtration apparatus, and repeat Steps 7 through 9 twice, adding the filter discs to the 10-4 dilution plates labeled FCC and FSC. Repeat Steps 8 through 10, using 20ml of the 10-3, 10-2, and 10-1 dilutions and the undiluted samples. Incubate the plates in an inverted position as follows a. TCC and FSC plates for 24 hours at 37 degrees centigrade. b. FCC plates sealed with waterproof tape and placed in a weighted watertight plastic bag, which is then submerged in 44.5 degrees centigrade water bath for 24 hours.

Observation and results 1. 2. Remove the filter discs from the petri dishes and allow to dry on absorbent paper for 1 hour. Examine all filter discs under a dissecting microscope and perform colony counts on each set of discs as follows. a. TCC: Count colonies on M-endo agar that present a golden metallic sheen (performed on a disc showing 20 to 80 colonies). b. FCC: Count colonies on M-FC agar that are blue (performed on a disc showing 20 to 60 of these colonies). c. FSC: Count colonies on K-F agar that are pink to red (performed on a disc showing 20 to 100 of these colonies). For each of the three counts, determine the number of fecal organisms present in 100 ml of the water sample, using the following formula. Colony Count x dilution factor ml of sample used x 100

3.

Isolation of Escherichia coli Carry out the coliform test up to fecal coliforms. The EC media is streaked onto the EMB agar. Check for the typical colonies as explained earlier. Carry out the biochemical test below: indole production methyl red voges/proskeur citrate utilization In addition check for the motility, gram's characteristics and production of gas from lactose.

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Howard Mould Count

1. Application 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. 2. Procedure The examination shall be carried out in accordance with the following instructions. 2.1. Apparatus 2.1.1. Compound microscope, either binocular or monocular equipped with: a. mechanical stage. b. condenser with iris diaphragm. c. source of illumination. d. two objectives - a 10 x (16 mm) for counting and a 20 x (8mm) for confirmation. e. 8 x - 12.5 x oculars. f. The 10 x objective must be calibrated with the ocular to give a field diameter of 1.382 mm (Preparation of Microscope, section 2.2.1). g. The ocular must be equipped with a micrometer disk cross-ruled in sixths of ocular diaphragm opening (Preparation of Microscope, section 2.2.2). 2.1.2. Howard mould counting chamber or cell of the type with specifications as outlined in Part 6, Diagram IIa or IIb and cover glass. 2.1.3. Distilled water. 2.1.4. Lint-free clean towel or cloth for drying Howard cell and cover glass. 2.1.5. Bunsen burner. 2.1.6. 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. 2.1.7. Dissecting needle. 2.1.8. U.S. standard sieve no. 2 (for canned tomatoes). 2.1.9. Wide mouth bottles with screw caps or other suitable containers (for canned tomatoes, puree, pulp, paste and catsup). 2.1.10. Spoon or other suitable utensil (for puree, pulp, paste and catsup).

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2.1.11. Refractometer (for puree, pulp and paste). 2.1.12. Coarse filter paper or celluwipe (for puree, pulp and paste). 2.2 Preparation of Microscope 2.2.1. Calibrate the 10 x objective with the ocular to give a field of view diameter of 1.382 mm as follows: a. Using the 10 x objective and ocular(s) in the range of 8 - 12.5 x measure the diameter of field of view with a stage micrometer or with the two parallel lines or circle measuring 1.382 mm scribed on a Howard cell. b. If the field diameter is less than 1.382 mm use lower power ocular(s). c. If the field diameter is greater, raise the height of the ocular(s) until the diameter coincides with 1.382 mm or make an accessory drop-in ocular diaphragm with aperture accurately cut to necessary size. 2.2.2. 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 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 2.2.1, (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. 2.2.3. 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. 2.3. Preparation of Sample Units 2.3.1. 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. 2.3.2. 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.

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2.3.3. 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 2.3.4, Preparation of Howard Mould Count Cell. (iv) Repeat procedure for remaining five sample units. 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 step 2.3.3.a. 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 as in step 2.3.4, Preparation of Howard Mould Count Cell. (vii) Repeat procedure for remaining five sample units. d. Tomato Catsup (i) Open container (sample unit) and mix 60 times in 30 sec with a spoon or other suitable instrument.

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(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 as in step 2.3.4, Preparation of Howard Mould Count Cell. (v) Repeat procedure for remaining five sample units. 2.3.4. Preparation of Howard Mould Count Cell (1) 2.3.4.1. Clean Howard cell and cover glass making certain central area of cell is clean. Rinse with distilled water, dry with a lint free cloth and pass lightly over a Bunsen flame. 2.3.4.2. 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. 2.3.4.3. Clean spatula and dissecting needle, rinse in distilled water, flame and cool. 2.3.4.4. Prepare glass slide using technique (a) or (b) as follows a. 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 nto place, so that the material spreads i 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. b. Parallel Cover Glass Technique

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(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. 2.3.4.5. 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. 2.3.4.6. 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. 2.3.5. Microscopical Examination 2.3.5.1. 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 2.2). Use higher magnification (180 250 x) only for confirmation of mould. 2.3.5.2. 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 140

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. 2.3.5.3. 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) It is not necessary to classify types of mould, only to positively identify mould filaments as characterized in Part 6, Diagram III. 2.3.5.4. 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). 2.3.6. Calculation and Recording Results 2.3.6.1. Calculate proportion of positive fields from results of examination of all observed fields for each sample unit. 2.3.6.2. Report results as a percentage of fields containing mould filaments individually for each sample unit: Number of positive fields/Number x 100 = % positive fields per sample unit 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

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TABLE I DILUTION OF PUREE (PULP) FOR MOULD COUNT AT 20oC (2)

Actual Index Refr. Dilution Factor Amt. of Water to be Added to 100 ml Total Volume of Diluted Sample of Sample Unit Unit

1.3462 1.3478 1.3494 1.3511 1.3527 1.3544 1.3560 1.3577 1.3593 1.3610

1.145 1.292 1.440 1.585 1.730 1.876 2.024 2.171 2.322 2.474

14.5 29.2 44.0 58.5 73.0 87.6 102.4 117.2 132.2 147.4

114.5 129.2 144.0 158.5 173.0 187.6 202.4 217.2 232.2 247.4

3. Interpretation 3.1. The 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 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. 3.2. 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.

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DIAGRAM I

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

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HOWARD MOULD COUNTING CHAMBER DIAGRAM 11a

E F G

A: Calibration circle, 1.382 mm diameter B: Area of liquid for mould count C: Cover glass D: Cover glass E: Two engraved parallel lines spaced 1.382 mm apart F: Rectangle, 15 X 20 mm G: Moat

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[1.382/2]2 X 3.1416 = 1.5 sq. mm., area of microscopic field1.5 X 0.1 = 0.15 cu. mm., volume of material in microscopic field 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

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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

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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

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Examination of Canned Foods

The incidence of spoilage in canned foods is low, but when it occurs it must be investigated properly. Swollen cans often indicate a spoiled product. During spoilage, cans may progress from normal to flipper, to springer, to soft swell, to hard swell. However, spoilage is not the only cause of abnormal cans. Overfilling, buckling, denting, or closing while cool may also be responsible. Microbial spoilage and hydrogen, produced by the interaction of acids in the food product with the metals of the can, are the principal causes of swelling. High summer temperatures and high altitudes may also increase the degree of swelling. Some microorganisms that grow in canned foods, however, do not produce gas and therefore cause no abnormal appearance of the can; nevertheless, they cause spoilage of the product. Spoilage is usually caused by growth of microorganisms following leakage or underprocessing. Leakage occurs from can defects, punctures, or rough handling. Contaminated cooling water sometimes leaks to the interior through pinholes or poor seams and introduces bacteria that cause spoilage. A viable mixed microflora of bacterial rods and cocci is indicative of leakage, which may usually be confirmed by can examination. Underprocessing may be caused by undercooking; retort operations that are faulty because of inaccurate or improperly functioning thermometers, gauges, or controls; excessive contamination of the product for which normally adequate processes are insufficient; changes in formulation or handling of the product that result in a more viscous product or tighter packing in the container, with consequent lengthening of the heat penetration time; or, sometimes, accidental bypassing of the retort operation altogether. When the can contains a spoiled product and no viable microorganisms, spoilage may have occurred before processing or the microorganisms causing the spoilage may have died during storage. Underprocessed and leaking cans are of major concern and both pose potential health hazards. However, before a decision can be made regarding the potential health hazard of a low-acid canned food, certain basic information is necessary. Naturally, if Clostridium botulinum (spores, toxin, or both) is found, the hazard is obvious. Intact cans that contain only mesophilic, Grampositive, sporeforming rods should be considered underprocessed, unless proved otherwise. It must be determined that the can is intact (commercially acceptable seams and no microleaks) and that other factors that may lead to underprocessing, such as drained weight and product formulation, have been evaluated. The preferred type of tool for can content examination is a bacteriological can opener consisting of a puncturing device at the end of a metal rod mounted with a sliding triangular blade that is held in place by a set screw. The advantage over other types of openers is that it does no damage to the double seam and therefore will not interfere with subsequent seam examination of the can.

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Table 1. Useful descriptive terms for canned food analysis. Exterior can condition leaker dented rusted buckled paneled bulge Micro-leak test packer seam side panel side seam cut code pinhole Internal can condition normal peeling slight, moderate or severe etching slight, moderate or severe blackening slight, moderate or severe rusting mechanical damage Product odor Product liquor putrid cloudy acidic clear butyric foreign metallic frothy sour cheesy fermented musty sweet fecal sulfur off-odor Pigment Consistency darkened slimy light fluid changed viscous ropy

Solid product Liquid product digested cloudy softened clear curdled foreign uncooked frothy overcooked

Flat - a can with both ends concave; it remains in this condition even when the can is brought down sharply on its end on a solid, flat surface. Flipper - a can that normally appears flat; when brought down sharply on its end on a flat surface, one end flips out. When pressure is applied to this end, it flips in again and the can appears flat. Springer - a can with one end permanently bulged. When sufficient pressure is applied to this end, it will flip in, but the other end will flip out. Soft swell - a can bulged at both ends, but not so tightly that the ends cannot be pushed in somewhat with thumb pressure. Hard swell - a can bulged at both ends, and so tightly that no indentation can be made with thumb pressure. A hard swell will generally "buckle" before the can bursts. Bursting usually occurs at the double seam over the side seam lap, or in the middle of the side seam. The number of cans examined bacteriologically should be large enough to give reliable results. When the cause of spoilage is clear-cut, culturing 4-6 cans may be adequate, but in some cases it may be necessary to culture 10-50 cans before the cause of spoilage can be determined. On special occasions these procedures may not yield all the required information, and additional tests must be devised to collect the necessary data. Unspoiled cans may be examined bacteriologically to determine the presence of viable but dormant organisms. The procedure is the same as that

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used for spoiled foods except that the number of cans examined and the quantity of material subcultured must be increased. A. Equipment and materials 1. Incubators, thermostatically controlled at 30, 35, and 55°C 2. pH meter, potentiometer 3. Microscope, slides, and coverslips 4. Can opener, bacteriological can opener, and can punch, all sterile 5. Petri dishes, sterile 6. Test tubes, sterile 7. Serological pipets, cotton-plugged, sterile 8. Nontapered pipets, cotton-plugged (8 mm tubing), sterile 9. Soap, water, brush, and towels, sterile and nonsterile 10. Indelible ink marking pen 11. Diamond point pen for marking cans 12. Examination pans (Pyrex or enamel baking pans) B. Media and reagents 1. Bromcresol purple (BCP) dextrose broth (M27) 2. Chopped liver broth (M38) or cooked meat medium (CMM) (M42) 3. Malt extract broth (M94) 4. Liver-veal agar (without egg yolk) (LVA) (M83) 5. Acid broth (M4) 6. Nutrient agar (NA) (M112) 7. Methylene blue stain (R45), crystal violet (R16), or Gram stain (R32) 8. Sabouraud's dextrose agar (SAB) (M133) 9. 4% Iodine in 70% ethanol (R18) C. Can preparation Remove labels. With marking pen, transfer subnumbers to side of can to aid in correlating findings with code. Mark labels so that they may be replaced in their original position on the can to help locate defects indicated by stains on label. Separate all cans by code

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numbers and record size of container, code, product, condition, evidence of leakage, pinholes or rusting, dents, buckling or other abnormality, and all identifying marks on label. Classify each can according to the descriptive terms in Table 1. Before observing cans for classification, make sure cans are at room temperature. D. Examination of can and contents Classification of cans. NOTE: Cans must be at room temperature for classification. 1.Sampling can contents a. Swollen cans. Immediately analyze springers, swells, and a representative number (at least 6, if available) of flat and flipper cans. Retain examples of each, if available, when reserve portion must be held. Place remaining flat and flipper cans (excluding those held in reserve) in incubator at 35°C. Examine at frequent intervals for 14 days. When abnormal can or one becoming increasingly swollen is found, make note of it. When can becomes a hard swell or when swelling no longer progresses, culture sampled contents, examine for preformed toxin of C. botulinum if microscopic examination shows typical C. botulinum organisms or Gram-positive rods, and perform remaining steps of canned food examination. b.Flat and flipper cans. Place cans (excluding those held in reserve) in incubator at 35°C. Observe cans for progressive swelling at frequent intervals for 14 days. When swelling occurs, follow directions in l a, above. After 14 days remove flat and flipper cans from incubator and test at least 6, if available. (It is not necessary to analyze all normal cans.) Do not incubate cans at temperatures above 35°C. After incubation, bring cans back to room temperature before classifying them. 2. Opening the can. Open can in an environment that is as aseptic as possible. Use of vertical laminar flow hood is recommended. a. Hard swells, soft swells, and springers . Chill hard swells in refrigerator before opening. Scrub entire uncoded end and adjacent sides of can using abrasive cleanser, cold water, and a brush, steel wool, or abrasive pad. Rinse and dry with clean sterile towel. Sanitize can end to be opened with 4% iodine in 70% ethanol for 30 min and wipe off with sterile towel. DO NOT FLAME. Badly swollen cans may spray out a portion of the contents, which may be toxic. Take some precaution to guard against this hazard, e.g., cover can with sterile towel or invert sterile funnel over can. Sterilize can opener by flaming until it is almost red, or use separate presterilized can openers, one for each can. At the time a swollen can is punctured, test for headspace gas, using a qualitative test or the gasliquid chromatography method described below. For a qualitative test, hold mouth of sterile test tube at puncture site to capture some escaping gas, or use can-puncturing press to capture some escaping gas in a syringe. Flip mouth of tube to flame of Bunsen burner. A slight explosion indicates presence of hydrogen. Immediately turn tube upright and pour in a small amount of lime water. A white precipitate indicates presence of CO2. Make opening in sterilized end of can large enough to permit removal of sample. b. Flipper and flat cans. Scrub entire uncoded end and adjacent sides of can using abrasive cleanser, warm water, and a brush, steel wool, or abrasive pad. Rinse and dry with clean sterile towel. Gently shake cans to mix contents before sanitizing. Flood end of can with iodine-ethanol solution and let stand at least 15 min. Wipe off iodine mixture with

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clean sterile towel. Ensure sterility of can end by flaming with burner in a hood until iodineethanol solution is burned off, end of can becomes discolored from flame, and heat causes metal to expand. Be careful not to inhale iodine fumes while burning off can end. Sterilize can opener by flaming until it is almost red, or use separate presterilized can openers for each can. Make opening in sterilized end of can large enough to permit removal of sample. 3. Removal of material for testing. Remove large enough portions from center of can to inoculate required culture media. Use sterile pipets, either regular or wide-mouthed. Transfer solid pieces with sterile spatulas or other sterile devices. Always use safety devices for pipetting. After removal of inocula, aseptically transfer at least 30 ml or, if less is available, all remaining contents of cans to sterile closed containers, and refrigerate at about 4°C. Use this material for repeat examination if needed and for possible toxicity tests. This is the reserve sample. Unless circumstances dictate otherwise, analyze normal cans submitted with sample organoleptically and physically (see 5-b, below), including pH determination and seam teardown and evaluation. Simply and completely describe product appearance, consistency, and odor on worksheet. If analyst is not familiar with decomposition odors of canned food, another analyst, preferably one familiar with decomposition odors, should confirm this organoleptic evaluation. In describing the product in the can, include such things as low liquid level (state how low), evidence of compaction, if apparent, and any other characteristics that do not appear normal. Describe internal and external condition of can, including evidence of leakage, etching, corrosion, etc. 4. Physical examination. Perform net weight determinations on a representative number of cans examined (normal and abnormal). Determine drained weight, vacuum, and headspace on a representative number of normal-appearing and abnormal cans (1). Examine metal container integrity of a representative number of normal cans and all abnormal cans that are not too badly buckled for this purpose (see Chapter 22). CAUTION: Always use care when handling the product, even apparently normal cans, because botulinal toxin may be present. 5. Cultural examination of low-acid food (pH greater than 4.6). If there is any question as to product pH range, determine pH of a representative number of normal cans before proceeding. From each container, inoculate 4 tubes of chopped liver broth or cooked meat medium previously heated to 100°C (boiling) and rapidly cooled to room temperature; also inoculate 4 tubes of bromcresol purple dextrose broth. Inoculate each tube with 1-2 ml of product liquid or product-water mixture, or 1-2 g of solid material. Incubate as in Table 2. Table 2. Incubation times for various media for examination of low acid foods (pH > 4.6). Medium Chopped liver (cooked meat) Chopped liver (cooked meat) Bromcresol purple dextrose broth Bromcresol purple dextrose broth No. of tubes Temp. (°C) 2 35 2 55 2 55 2 35 Time of incubation (h) 96-120 24-72 24-48 96-120

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After culturing and removing reserve sample, test material from cans (other than those classified as flat) for preformed toxins of C. botulinum when appropriate.. a. Microscopic examination. Prepare direct smears from contents of each can after culturing. Dry, fix, and stain with methylene blue, crystal violet, or Gram stain. If product is oily, add xylene to a warm, fixed film, using a dropper; rinse and stain. If product washes off slide during preparation, examine contents as wet mount or hanging drop, or prepare suspension of test material in drop of chopped liver broth before drying. Check liver broth before use to be sure no bacteria are present to contribute to the smear. Examine under microscope; record types of bacteria seen and estimate total number per field. b. Physical and organoleptic examination of can contents. After removing reserve sample from can, determine pH of remainder, using pH meter. DO NOT USE pH PAPER. Pour contents of cans into examination pans. Examine for odor, color, consistency, texture, and overall quality. DO NOT TASTE THE PRODUCT. Examine can lining for blackening, detinning, and pitting. Table 3.Schematic diagram of culture procedure for low-acid canned foods

a

LVA, liver-veal agar; NA, nutrient agar; CMM, cooked meat medium; BCP, bromcresol purple dextrose broth. Table 4. Incubation of acid broth and malt extract broth used for acid foods (pH 4.6) Medium Acid broth Acid broth Malt extract broth No. of tubes 2 2 2 Temp. (°C) 55 30 30 Time of incubation (h) 48 96 96

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Table 5. Pure culture scheme for acid foods (pH 4.6).

a

NA, nutrient agar; SAB, Sabouraud's dextrose agar. E. Cultural findings in cooked meat medium (CMM) and bromcresol purple dextrose broth (BCP) Check incubated medium for growth at frequent intervals up to maximum time of incubation (Table 2). If there is no growth in either medium, report and discard. At time growth is noted streak 2 plates of liver-veal agar (without egg yolk) or nutrient agar from each positive tube. Incubate one plate aerobically and one anaerobically, as in schematic diagram (Table 3). Reincubate CMM at 35°C for maximum of 5 days for use in future toxin studies. Pick representatives of all morphologically different types of colonies into CMM and incubate for appropriate time, i.e., when growth is sufficient for subculture. Dispel oxygen from CMM broths to be used for anaerobes but not from those to be used for aerobes. After obtaining pure isolates, store cultures to maintain viability. 1. If mixed microflora is found only in BCP, report morphological types. If rods are included among mixed microflora in CMM, test CMM for toxin, as described in Chapter 17. If Gram-positive or Gram-variable rods typical of either Bacillus or Clostridium organisms are found in the absence of other morphological types, search to determine whether spores are present. In some cases, old vegetative cells may appear to be Gram-negative and should be treated as if they are Gram-positive.

Table 6. Classification of food products according to acidity Low acid--pH greater than 4.6 Meats Seafoods Milk Meat and vegetable Mixtures and "specialties" Acid pH 4.6 and below Tomatoes Pears Pineapple Other fruit 154

Spaghetti Soups Vegetables Asparagus Beets Pumpkin Green beans Corn Lima beans

Sauerkraut Pickles Berries Citrus Rhubarb

Table 7. Spoilage microorganisms that cause high and low acidity in various vegetables and fruits Spoilage type Thermophilic Flat-sour Thermophilic(a) Sulfide spoilage(a) Mesophilic Putrefactive anaerobes(a) Butyric anaerobes Aciduric flat-sour(a) Lactobacilli Yeasts Molds

a

pH groups >5.3 >4.8 >5.3 >4.8 >4.0 >4.2 4.5-3.7 <3.7 <3.7

Examples Corn, peas Spinach, corn Corn, peas Corn, asparagus Tomatoes, peas Tomato juice Fruits Fruits Fruits

The responsible organisms are bacterial sporeformers.

Group of organisms Flat-sour

Table 8. Spoilage manifestations in low-acid products Classification Manifestations Can flat Product Possible loss of vacuum on storage Appearance not usually altered; pH markedly lowered, sour; may have slightly abnormal odor; sometimes cloudy liquor May burst Fermented, sour, cheesy or butyric odor H2S gas absorbed by product Usually blackened; rotten egg odor May burst

Thermophilic anaerobe Sulfide spoilage Putrefactive

Can swells Product Can flat Product Can swells

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anaerobe Aerobic sporeformers

Product Can flat or swollen

May be partially digested; pH slightly above normal; typical putrid odor Usually no swelling, except in cured meats when nitrate and sugar present; coagulated evaporated milk, black beets

Table 9. Spoilage manifestations in acid products Type of organism Classification Manifestation Bacillus thermoacidurans (flat, sour Can flat Little change in vacuum tomato juice) Product Slight pH change; off-odor Butyric anaerobes (tomatoes and Can swells May burst tomato juice) Product Fermented, butyric odor Nonsporeformers (mostly lactic Can swells Usually burst, but swelling may be types) arrested Product Acid odor

Can Product appearance Odor Normal, sour or putrid, but generally consistent from can to can PH Usually fairly constant Microscopic Cultures show sporeforming rods only and cultural Growth at 35 and/or 55°C. May be characteristic on special growth media, e.g., acid agar for tomato juice.

Table 10. Laboratory diagnosis of bacterial spoilage Underprocessed Leakage Flat or swelled; seams generally normal Swelled; may show normal defects(a) Sloppy or fermented Frothy fermentation; viscous Sour, fecal; generally varying from can to can Wide variation Mixed cultures, generally rods and cocci; only at usual temperatures

If product misses retort completely, rods, cocci,yeast or molds, or any combination of these may be present. History Spoilage usually confined to certain Spoilage scattered portions of pack In acid products, diagnosis may be less clearly defined; similar organisms may be involved in understerilization and leakage. a Leakage may be due not to can defects but to other factors, such as contamination of cooling water or rough handling, e.g., can unscramblers, rough conveyor system.

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Table 11. pH range of a few selected commercially canned foods Food pH range Food pH range Apples, juice Apples, whole Asparagus, green Beans Baked Green Lima Soy Beans with pork Beef, corned, hash Beets, whole Blackberries Blueberries Boysenberries Bread White Date and nut Broccoli Carrot juice Carrots, chopped Cheese Parmesan Roquefort Cherry juice Chicken Chicken with noodles Chop suey Cider Clams Cod fish Corn Cream style On-the-cob Whole grain Brine-packed Vacuum-packed Crab apples, spiced Cranberry Juice 3.3 - 3.5 3.4 - 3.5 5.0 - 5.8 4.8 - 5.5 4.9 - 5.5 5.4 - 6.3 6.0 - 6.6 5.1 - 5.8 5.5 - 6.0 4.9 - 5.8 3.0 - 4.2 3.2 - 3.6 3.0 - 3.3 5.0 - 6.0 5.1 - 5.6 5.2 - 6.0 5.2 - 5.8 5.3 - 5.6 5.2 - 5.3 4.7 - 4.8 3.4 - 3.6 6.2 - 6.4 6.2 - 6.7 5.4 - 5.6 2.9 - 3.3 5.9 - 7.1 6.0 - 6.1 5.9 - 6.5 6.1 - 6.8 Jam, fruit Jellies, fruit Lemon juice Lemons Lime juice Loganberries Mackerel Milk Cow, whole Evaporated Molasses Mushroom Olives, ripe Orange juice Oysters Peaches Pears (Bartlett) Peas Pickles Dill Sour Sweet Pimento Pineapple Crushed Juice Sliced Plums Potato salad Potatoes Mashed White, whole Prune juice Pumpkin Raspberries Rhubarb Salmon Sardines Sauerkraut 3.5 - 4.0 3.0 - 3.5 2.2 - 2.6 2.2 - 2.4 2.2 - 2.4 2.7 - 3.5 5.9 - 6.2 6.4 - 6.8 5.9 - 6.3 5.0 - 5.4 6.0 - 6.5 5.9 - 7.3 3.0 - 4.0 6.3 - 6.7 3.4 - 4.2 3.8 - 4.6 5.6 - 6.5 2.6 - 3.8 3.0 - 3.5 2.5 - 3.0 4.3 - 4.9 3.2 - 4.0 3.4 - 3.7 3.5 - 4.1 2.8 - 3.0 3.9 - 4.6 5.1 5.4 - 5.9 3.7 - 4.3 5.2 - 5.5 2.9 - 3.7 2.9 - 3.3 6.1 - 6.5 5.7 - 6.6 3.1 - 3.7 157

6.0 - 6.4 3.3 - 3.7 2.5 - 2.7

Sauce Currant juice Dates Duck Figs Frankfurters Fruit cocktail Gooseberries Grapefruit Juice Pulp Sections Grapes Ham, spiced Hominy, lye Huckleberries Strawberries Sweet potatoes Tomato juice Tomatoes Tuna Turnip greens Vegetable juice Vegetables, mixed Vinegar Youngberries

2.3 3.0 6.2 - 6.4 6.0 - 6.1 4.9 - 5.0 6.2 - 6.2 3.6 - 4.0 2.8 - 3.1 2.9 - 3.4 3.4 3.0 - 3.5 3.5 - 4.5 6.0 - 6.3 6.9 - 7.9 2.8 - 2.9 3.0 - 3.9 5.3 - 5.6 3.9 - 4.4 4.1 - 4.4 5.9 - 6.1 5.4 - 5.6 3.9 - 4.3 5.4 - 5.6 2.4 - 3.4 3.0 - 3.7

Juice Shrimp Soups Bean Beef broth Chicken noodle Clam chowder Duck Mushroom Noodle Oyster Pea Spinach Squash Tomato Turtle Vegetable Miscellaneous products Beers Ginger ale Human Blood plasma Duodenal contents Feces Gastric contents Milk Saliva Spinal fluid Urine Magnesia, milk of Water Distilled, CO2 Mineral Sea Wine

3.3 - 3.4 6.8 - 7.0 5.7 - 5.8 6.0 - 6.2 5.5 - 6.5 5.6 - 5.9 5.0 - 5.7 6.3 - 6.7 5.6 - 5.8 6.5 - 6.9 5.7 - 6.2 4.8 - 5.8 5.0 - 5.8 4.2 - 5.2 5.2 - 5.3 4.7 - 5.6 4.0 - 5.0 2.0 - 4.0 7.3 - 7.5 4.8 - 8.2 4.6 - 8.4 1.0 - 3.0 6.6 - 7.6 6.0 - 7.6 7.3 - 7.5 4.8 - 8.4 10.0 -10.5 6.8 - 7.0 6.2 - 9.4 8.0 - 8.4 2.3 - 3.8

2. If no toxin is present, send pure cultures for evaluation of heat resistance to Cincinnati District Office, FDA, 1141 Central Parkway, Cincinnati, OH 45202, if cultures meet the following criteria:

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§

Cultures come from intact cans that are free of leaks and have commercially acceptable seams. (Can seams of both ends of can must be measured; visual examination alone is not sufficient.) Two or more tubes are positive and contain similar morphological types.

§

3. Examination of acid foods (pH 4.6 and below) by cultivation. From each can, inoculate 4 tubes of acid broth and 2 tubes of malt extract broth with 1-2 ml or 1-2 g of product, using the same procedures as f low-acid foods, and incubate as in Table 4. or Record presence or absence of growth in each tube, and from those that show evidence of growth, make smears and stain. Report types of organisms seen. Pure cultures may be isolated as shown in Table 5. 4. The presence of only sporeforming bacteria, which grow at 35°C, in cans with satisfactory seams and no microleaks indicates underprocessing if their heat resistance is equal to or less than that of C. botulinum. Spoilage by thermophilic anaerobes such as C. thermobutylicum may be indicated by gas in cooked meat at 55°C and a cheesy odor. Spoilage by C. botulinum, C. sporogenes, or C. perfringens may be indicated in cooked meat at 35°C by gas and a putrid odor; rods, spores, and clostridial forms may be seen on microscopic examination. Always test supernatants of such cultures for botulinal toxin even if no toxin was found in the product itself, since viable botulinal spores in canned foods indicate a potential public health hazard, requiring recall of all cans bearing the same code. Spoilage by mesophilic organisms such as Bacillus thermoacidurans or B. coagulans and/or thermophilic organisms such as B. stearothermophilus, which are flat-sour types, may be indicated by acid production in BCP tubes at 35 and/or 55°C in high-acid or low-acid canned foods. No definitive conclusions may be drawn from inspection of cultures in broth if the food produced an initial turbidity on inoculation. Presence or absence of growth in this case must be determined by subculturing. Spoilage in acid products is usually caused by nonsporeforming lactobacilli and yeasts. Cans of spoiled tomatoes and tomato juice remain flat but the products have an off-odor, with or without lowered pH, due to aerobic, mesophilic, and thermophilic sporeformers. Spoilage of this type is an exception to the general rule that products below pH 4.6 are immune to spoilage by sporeformers. Many canned foods contain thermophiles which do not grow under normal storage conditions, but which grow and cause spoilage when the product is subjected to elevated temperatures (50-55°C). B. thermoacidurans and B. stearothermophilus are thermophiles responsible for flat-sour decomposition in acid and low-acid foods, respectively. Incubation at 55°C will not cause a change in the appearance of the can, but the product has an off-odor with or without a lowered pH. Spoilage encountered in products such as tomatoes, pears, figs, and pineapples is occasionally caused by C. pasteurianum, a sporeforming anaerobe which produces gas and a butyric acid odor. C. thermosaccolyticum is a thermophilic anaerobe which causes swelling of the can and a cheesy odor of the product. Cans which bypass the retort without heat processing usually are contaminated with nonsporeformers as well as sporeformers, a spoilage characteristic similar to that resulting from leakage. A mixed microflora of viable bacterial rods and cocci usually indicates leakage. Can examination may not substantiate the bacteriological findings, but leakage at some time in

5.

6.

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the past must be presumed. Alternatively, the cans may have missed the retort altogether, in which case a high rate of swells would also be expected. 7. A mixed microflora in the product, as shown by direct smear, in which there are large numbers of bacteria visible but no growth in the cultures, may indicate precanning spoilage. This results from bacterial growth in the product before canning. The product may be abnormal in pH, odor, and appearance. If no evidence of microbial growth can be found in swelled cans, the swelling may be due to development of hydrogen by chemical action of contents on container interiors. The proportion of hydrogen varies with the length and condition of storage. Thermophilic anaerobes produce gas, and since cells disintegrate rapidly after growth, it is possible to confuse thermophilic spoilage with hydrogen swells. Chemical breakdown of the product may result in evolution of carbon dioxide. This is particularly true of concentrated products containing sugar and some acid, such as tomato paste, molasses, mincemeats, and highly sugared fruits. The reaction is accelerated at elevated temperatures. Any organisms isolated from normal cans that have obvious vacuum and normal product but no organisms in the direct smear should be suspected as being a laboratory contaminant. To confirm, aseptically inoculate growing organism into another normal can, solder the hole closed, and incubate 14 days at 35°C. If any swelling of container or product changes occur, the organism was probably not in the o riginal sample. If can remains flat, open it aseptically and subculture as previously described. If a culture of the same organism is recovered and the product is normal, consider the product commercially sterile since the organism does not grow under normal conditions of storage and distribution.

8.

9.

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Standard Operating Procedures (SOPs)

Standard procedures should describe

· · · · · · ·

procedures for the acceptance or rejection of samples methodology to be followed appropriate control procedures quality assurance procedures procedures for cleaning, sterilizing and calibrating equipment procedures for preparing media and reagents procedures for handling and disposing of contaminated materials

Analytical methods should be based on a standard method such as those published by the International Commission on Microbiological Specifications for Foods (ICMSF) or the Association of Official Analytical Chemists (AOAC). Modifications to standard methods should not be used unless comparative studies have shown that the modified methods are as reliable as reference methods. Similarly, for pre-prepared kits such as the mini-kits used for biochemical tests, the manufacturer's instructions must be followed exactly. Mistakes can arise from using an incorrect viewing angle or the wrong method of inoculation. SOP's should include limitations of each test and a list of precautions. Possible interferences should be described. For example, the potential presence of natural inhibitory substances should be noted and methods for diluting out or neutralizing these substances must be included. SOP's should also include the names of personnel to be contacted if out-of-control procedures are found. The SOP's should also indicate how a test should be read and what should be looked for in a positive and negative test. Colour photographs can be used to assist personnel in reading tests. Care should be taken to ensure that the photographs do not fade. Instructions on how tests should be read should also be included on results sheets. SOP's can be c onstructed from manufacturer's brochures and manuals, however neither should be used as substitutes for SOP's. Personnel Personnel should have the education, experience and motivation necessary to perform their jobs and the requirements of a quality assurance program. Management of laboratory personnel through motivation, supervision and workload direction is as important as selection of appropriate personnel. Training should be ongoing and should aim to ensure that workers know the exact duties they are to perform so as to obtain results of the highest quality. Employees must be fully aware of their QA responsibilities and the adverse consequences that will arise from failure to carry out their duties carefully. Management should ensure that a safe, efficiently designed facility, sufficient supplies and equipment are available. Facilities

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Laboratory facilities should be designed with the safety of workers being of utmost importance. It should also be designed to provide for the convenience of the workers and operations. It should be adequately equipped to carry out all objectives of the laboratory. Ventilation, temperature, laboratory and bench space, storage (including refrigeration) facilities, sinks, electricity outlets should be considered in the design of the laboratory. Laboratory grade water should be available. Distilled water is suitable for most microbiological work. Laboratory water should be tested for its toxicity or stimulatory effects on microorganisms. Housekeeping A routine cleaning and disinfection schedule for the laboratory should be established, monitored and documented. All laboratory benches should be disinfected before and after each use. Laboratory materials should be stored after use and unneeded and outdated materials should be discharged according to an appropriate and documented procedure. Where necessary, hygroscopic chemicals such dry-form media, should be stored in dry cabinets or in appropriately desiccated containers. Dust should not be allowed to build up and attention should be paid to hard-to-clean areas.

Quality Assurance

Quality Assurance (QA) is a wide ranging concept covering all matters that individually or collectively influence the quality of a product. It denotes a system for continuously improving reliability, efficiency and utilization of products and services. In the context of quality assurance two important definitions need to be clearly understood: i. Internal Quality Control (IQC): which denotes a set of procedures undertaken by the staff of health facility (medical, paramedical workers as well as laboratorians) for continuously and concurrently assessing laboratory work so that quality results are produced by the laboratory. External Quality Assessment (EQA): is a system of objectively assessing the laboratory performance by an outside agency. This assessment is retrospective and periodic but is aimed at improving the IQC.

ii.

IQC and EQA are complementary in ensuring the reliability of the procedures and the results. What is the objective of QA? QA programmes are required for the following reasons: To generate reliable, reproducible results. To establish inter-laboratory comparability in laboratory testing To establish the credibility of the laboratory among scientists and the public at large. Motivating the staff for further improvement. Prevention of legal complications which may follow poor quality results. Factors affecting the quality It is commonly believed that the quality of laboratory results solely depends upon the laboratory undertaking this analysis. However, there are many pre-analytical and post-analytical factors which influence the quality of the end results to a very significant extent. Some of the important factors influencing quality are listed here:

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i.

Specimen: This is the single most important factor. Selection of the right sample, collection in a right manner, adequate quantity, proper transportation to the laboratory, and processing of the sample before testing, are crucial factors. Record the history of the sample i.e. how it was collected, by whom, when and for what reason. Personnel: The quality of the laboratory results generated is directly proportional to the training, commitment and motivation of the technical staff. The appropriate technician should handle the analysis he/she is competent in i.e. bacterial, moulds and yeast. Environmental factors: Inadequate lighting, workspace or ventilation or unsafe working conditions may influence the laboratory results. Fans should not be used in the laboratory. No direct wind from outside should be allowed in the laboratory. Analytical factors: The quality of reagents, chemicals, glassware, stains, culture media, use of standard procedures and reliable equipment all influence laboratory results. Failure to examine a sufficient number of microscope fields can lead to false negative results. Proper labeling of reagents and media all the time to avoid mistakes in the mix up.

ii.

iii.

iv.

Post analytical factors: Transcription errors, incomplete reports, and improper interpretation can adversely influence the laboratory results. Every step in the analysis should be recorded in the worksheet available for every single analysis. Requirements of Internal Quality Control (IQC) Comprehensive: Cover all steps from collection of sample to reporting. Regular and continuous monitoring. Economical: Should be cost-effective and within the provided budget. SOPMs should be periodically reviewed and revised and religiously followed in the laboratories.

Maintenance of equipment

Good quality equipment is absolutely essential to generate quality results. Care of the equipment purchased is also crucial. The quality control steps for some of the commonly-used equipment at the intermediate/peripheral laboratory level is depicted in Table 1. Table 1: Suggested maintenance of commonly-used equipment Equipment Autoclave Maintenance Instructions Clean and change water monthly Adjust water level before each run Record time, temperature and pressure for each run Inspect gasket in the lid weekly Technical inspection every six months Clean inside walls once in a month Record temperature at the start of each working day Technical maintenance every six months

Incubator

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Hot air oven

Clean the inside at least once a month Record time and temperature with every run Technical inspection every six months Wipe lenses with lens paper at the end of each day's work Protect the microscope from dust, vibrations and moisture Place a shallow plate containing dry blue silica gel in a box to absorb moisture Check alignment of the condenser once a month Technical inspection once in a year Keep the balance and weights clean and dry Always use a container or weighing paper, do not put material directly on the pan Prevent the balance from drafts of air Place at least 10 inches away from the wall Clean and defrost at least every two months Record temperature daily Technical service at least once a year Check water level daily Check temperature before and during use Clean monthly Technical inspection once in six months Check the flow of air Clean with antiseptic solution after every use Wipe inner walls with antiseptic solution weekly Balance well the samples before putting them in the centrifuge. Check brushes and bearings every six months Discard chipped glassware Ensure these are free of detergents Do not store sterile glassware for more than three weeks before it is used.

Microscope

Balance

Refrigerator

Water Bath

Clean Bench (Laminar Flow) Centrifuge

Glassware

Performance tests on culture media - Culture media may be prepared from the individual ingredients or may be prepared from dehydrated powders available commercially. The important points in QC of media are listed below - Do not over-stock the media. Store the required quantities only which can be used in 6-12 months. - Store the media away from moisture by securing the caps of all the containers tightly. - Store in a dark, cool and well-ventilated place as per the manufacturer's instructions

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Keep a record of the receipt, and opening of the media container. Discard all dehydrated media that are either darkened or caked. Rotate the stock of media, following the principle of "first in, first out". For preparation of media adhere strictly to the manufacturer's instructions. Prepared media should be protected from sunlight and heat. Sterility testing, performance testing and pH test of the prepared media should be done as listed in Table-2.

Table 2: Performance tests on commonly-used media Medium Blood Agar Incubation 24h Control Organism S. aureus S. pneumoniae Chocolate agar MacConkey agar With crystal violet 24h 24h H. influenzae E. coli P. mirabilis E. faecalis E. coli K .pneumoniae E. coli ATCC 25922 Expected Result Growth and beta-haemolysis Growth and alphahaemolysis Growth Red colonies Colourless colonies (no swarming) No growth Positive/negative Negative/positive Acceptable zone sizes

Methyl red/VogesProskauer Mueller-Hinton Peptone water (indole) Simmons citrate (incubate with loose screwcap) Thiosulfate citrate bile salt (TCBS) agar

48h 24h 24h 48h

P. aeruginosa ATCC 27853 Acceptable zone sizes E. coli Positive K. pneumoniae E. coli K . pneumoniae Negative No growth Growth, blue colour

24h

Vibrio spp. (non agglutinable Yellow colonies

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Table 3: QC for commonly-used tests Procedure/result Catalase Test Control organism S. aureus Streptococcus spp. Coagulase Indole S. aureus E. coli E. aerogenes Methyl red E. coli E.. aerogenes Oxidase P.aeruginosa E. coli Voges Proskauer Bacitracin disc E. aerogenes E. coli + ­ + + ­ + ­ + ­ + ­ Expected reaction Bubbling reaction No bubbling Clot formation in 4 hours Red ring at surface Yellow ring at surface Instant red colour No colour change Purple colour in 20 seconds No colour in 20 seconds Red colour No colour change Zone of inhibition Zone of inhibition Zone of inhibition No zone of inhibition Purple colour in 30 seconds No change in colour

Streptococcus group A + E. faecalis ­ + ­ + ­

Optochin disc

S. pneumoniae S. viridans

Oxidase disc

P.aeruginosa E. coli

The testing should be done each time a new batch of working solution is prepared.

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Quality control procedures used for the detection of antigen or antibodies by various test methods : Table 4. Test 1. Flocculation test (RPR) Control procedures required Expected results No clumping Clumping of graded activity Clumping of graded activity No clumping Clumping No clumping No clumping Clumping Capsular swelling No reaction Capsular swelling No reaction

Nonreactive serum control Weakly reactive serum control Reactive serum control Negative control serum 2. Latex agglutination test Positive control serum (ASO) 3. Direct agglutination (Widal test) 4. Capsular Quellung reaction (Omni serum, H.influenzae type b) Antigen control Negative control serum Positive control serum Pneumococci Haemolytic streptococci H.influenzae type b Acinetobacter anitratum

In service training of staff Periodic updating of the skills and knowledge of the laboratory technicians is essential for maintaining quality. Course-curriculum of such trainings should focus on the issues highlighted above (SOPs). Participation in external quality assessment Participation in EQAS reassures about the correctness of the results generated by the laboratory and finds out whether IQC is in place or not. The control or referral laboratories should organise EQAS in some commonly used tests. Standard Operating Procedure Record Prepared By: ________________________________________Date: ___/___/___ Print Name:______________________________ Reviewed By: _______________________________________ Date: ___/___/___ Print Name:______________________________ Technical Staff _______________________________________ Date: ___/___/___ Print Name:_____________________________ QA Officer _______________________________________ Date: ___/___/___ Print Name:_____________________________ Laboratory Director Date Issued: ___/___/___ Withdrawn By: ______________________________________ Date: ___/___/___ Controlled Copy No.: ___________________

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Quality assurance in microbiology laboratories

The objective of laboratory quality assurance is to verify the accuracy and precision of information obtained from analyses and to ensure that data obtained from analyses are suitable for decision making. The application of good laboratory practice ensures accuracy and confidence in test results. It also prevents cross-contamination of samples and protects personnel against infection and other laboratory hazards. Standardization of methods will also reduce variation between results produced by one laboratory and between results produced by associate laboratories. Good record keeping activities also help manage and encourage staff proficiency. Although the accuracy required depends on the how the information is to be used, laboratory staff would be well advised to adopt a TQM approach of continuous improvement to their work. The elements of the laboratory quality assurance programme A laboratory quality assurance system consists of the following elements:

· · · · · · · · · · · · · · ·

A Quality Policy Quality planning Appropriate record keeping Control of quality documents Standard operating procedures Designation of responsibility and authority of personnel Training Instrument calibration Equipment maintenance Validation of data including the use of appropriate reagent and culture controls Appropriate sample identification, handling, storage and delivery. Appropriate procedures and controls over procurement of laboratory materials Use of appropriate analytical procedures Statistical quality control Audits of the quality system.

These elements should be encompassed in a quality manual. N/B Ideally, both the construction and the implementation of the quality manual should be a quality effort involving management, designated quality assurance personal and other laboratory staff. Management responsibility

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Management is responsible for establishing the system which will seek initially to ensure that an appropriate standard of quality is met and then drive the laboratory to improve on that standard. It should encourage and support staff to carry out the laboratory's quality policy. It is also responsible for ensuring that the quality system functions as planned. This will involve auditing and verification of the laboratory practices. It may also involve rewarding groups or individuals or if necessary disciplining staff who do not meet the laboratory's expectations. Management is responsible for providing resources and ensuring that these resources are suitable for the functioning of the laboratory. It is responsible for designating responsibilities and authorities of persons involved in assuring the quality of output from the laboratory. Microbiological Sample management Samples which are normally encountered in the Microbiology laboratory are typically subject to change; i.e. changes in the microbial content and change in the chemical make-up. Due to mishandling, target organisms may increase or decrease in number during sample handling. Analysis of a mishandled sample is a waste of time , man-power and effort and can have a demoralizing effect on staff. Therefore, before a sample is accepted for analysis, it is important that the laboratory insist that the original condition of the sample and its container be maintained. There should also be adequate documentation stating the sample's source, date and time of collection, analysis requirements and required storage conditions. If possible, the sampling plan should also be included. Upon receipt of the sample, the samples must be stored to maintain their original condition and then tested as soon as possible. It may be necessary for a single technician to have responsibility of the sample.

Instrumental Maintenance, quality control and calibration

Every instrument or piece of equipment used in the performance of diagnostic tests or for reagent preparation and handling in the microbiology laboratory must function properly and perform according to established standards to ensure the reliability of diagnostic test results. Equipment such as balances, thermometers and pH meters should be calibrated to meet national standards. Temperature sensors and temperature controllers in incubators, water-baths and hot air sterilizers should similarly be accurate, calibrated and regularly tested. Preventative maintenance must be carried out on a regular basis. This must be documented and staff should be trained in how to carry out preventative maintenance. This section presents guidelines for maintenance and standardization of selected instruments. Autoclave Uses of autoclave in microbiology 1. To sterilize clean, wrapped instruments and containers 2. To sterilize media 3. To render microbiologically contaminated materials biologically safe (Sterile) before they are discarded. Does not affect radioactivity or chemical toxicity of autoclaved materials. Autoclaves are standard items in all microbiology laboratories, They should be equipped with accurate and calibrated pressure and temperature gauges. A simple test (although not conclusive) test of the status of the pressure and temperature gauges is to determine if the correlation between the two variable holds. For example, with saturated steam 121oC is equivalent to 15 PSI. Preferably a temperature recorder should also be used to keep a record of each sterilization cycle. Records should include for each cycle, (1) temperature and time settings (2) materials in 169

the chamber (3) pressure and temperature readings once the autoclave has reached the sterilizing region of the cycle and (4) date and time that the sterilizing cycle has started and finished. The standard operating procedures used for the operation of an autoclave should take into account the properties of media being sterilized. Although the standard temperature, pressure and holding time used for sterilizing is 121oC at 15 psi for 15 minutes, this is not suitable for heat-sensitive sugars such as glucose and sucrose. These sugars should be autoclaved at 110oC for 30 minutes. Because heat transfer through liquids is not instantaneous, large volumes of media will require longer holding times than small volumes. For sterilization of liquid volumes of greater than 0.5 l, the following holding times are recommended: Liquid Volume per container (l) Time required to reach 121oC (min) Total holding time (min) 0.5 19 29 1.0 34 44 2.0 37 47 3.0 43 53 4.0 52 62 Thus for a four litre container of medium, a holding time of 62 minutes instead of 15 minutes must be used. Another factor which will affect the efficiency of sterilization will be the presence of air in the autoclave. The typical pressure-temperature relationship often used in microbiology laboratories which states the following: Pressure (psi) Temperature oC 5 109 10 115 15 121 20 126 applies in a system where only water (liquid) and its vapour are present in equillibrium. The presence of air will result in a temperatures which are less than those above for a given pressure. The air should thus be removed during when the heating stage of the autoclave cycle. This is best done by removing the air by vacuum. If this is not possible then an alternative (but less satisfactory) is to open the exhaust valve while the autoclave is being heated to reach the set temperature. Modern autoclaves automatically exhaust the air before reaching the sterilization temperature. The proper functioning of the autoclave should be ascertained using biological indicators such as spore ampoules or strips of Bacillus stearothermophilus. Physical measurements of temperature and pressure should also be used. Autoclave tape while a useful indicator that autoclaving has been undertaken does not show whether the sterilization cycle has been completed. A. Calibrate the autoclave with the maximum permissible load. Place biological indicators into each type of item that maybe autoclaved 1. Drop biological indicator ampoules into flasks of medium (tie ampoule with string to facilitate removal). 2. Place spore strips into center of wrapped packs. 3. For verification of sterilization of liquid, vary both the total volume of liquid and the number of containers being autoclaved (i.e., test the same total volume of

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liquid but distribute it among few containers for one load and among numerous containers for another load). B. Observe recording chart for proper time and temperature. C. Incubate biological indicator to verify sterilization. Centrifuge Uses in the microbiology laboratory. 1. Concentrating etiologic agents and cells. 2. Removing particulate matter. 3. Separating components of mixed suspensions on the basis of densities. The manufacturer prior to shipment should perform initial calibration. Calibration of centrifuge rotation speed, particularly for today's solid-state circuit instruments should be done only by skilled personnel. Routine use 1. Check each load for proper balance, both for even distribution and for weight to within 0.5g. Well-balanced loads prevent wear on the drive train and motor bearings. Additionally, a smoother centrifugation will result in better separation. 2. Check that appropriate temperature is maintained during operation. 3. Clean up any spills immediately Incinerator burner Uses in the microbiology laboratory. Electric incinerator burners are particularly useful in laboratories not equipped with gas or flame sterilizing burners. Incinerators are also useful for working in laminar-flow biosafety cabinets. Because they require no oxygen, they work well in anaerobic chambers. The absence of an open flame provides safer alternative to the Bunsen burner. A. Initial calibration 1. Plug the incinerator into a grounded electrical circuit. 2. Look for a red glow inside the heater element. 3. Be sure that the unit does not generate any smoke or persistent odor suggestive of burning rubber, B. Routine quality control Visually inspect the heater element daily to determine if the heater element core is worn. Inspect for small cracks in the ceramic casing and residue buildup during both cool and heated conditions. In the heated condition, cracks can be seen as small, intensely yellow orange fissures. If defects are notes, replace the heater element. Cracks do not inhibit the sterilization ability if the unit, but they create an electrical safety hazard. Incubator Uses in the microbiology laboratory The incubator is used to maintain a constant and appropriate temperature for the growth of microorganisms. Initial calibration A. Air Incubators 1. Instrument setup

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a. Adjust t e leveling feet with a wrench if necessary so that the unit is level (bubble h centered in the carpenters level) and steady. b. Install shelves at the desired levels. c. Verify that the power source is compatible with the incubator power system before plugging the system into the electric outlet. d. Turn OFF-ON switch to ON so that the heating element (an the blower if applicable) will go on. 2. Setting the safety thermostat (Example: setting for 35°C incubator) a. Close the door of the incubator, and put some tape or a sign near the door handle so that the door will not be opened during the calibration. If there is no safety thermostat, skip to step 3 below. b. Turn both the regulating thermostat control knob and the satiety thermostat knob to the highest available setting and monitor the temperature. c. When the temperature reaches the desired safety temperature (usually 2.0°C above the actual desired maximum temperature), turn the safety thermostat down very slowly until the safety pilot light just goes on. d. Allow the incubator to equilibrate for 0.5 h, adjusting the safety thermostat knob as needed to get the temperature to stay at the selected value (usually 37°C). e. When the temperature has stabilized at 37°C, tighten the safety thermostat control knob lock (or place tape over the control) to prevent accidental changes in the setting. 3. Setting the regulating thermostat a. If there is a safety thermostat, allow the temperature to remain at 37°C for 0.5 h, and then turn the regulating thermostat control knob down until both the safety pilot light and the regulating pilot light go off. b. Adjust the regulating thermostat knob until the dial thermometer reaches 35°C, and allow the incubator to equilibrate for 0.5 h. the safety pilot light should go off during normal operation. c. After the temperature has stabilized at 35°C, tighten the temperature control knob (or put a tape over the control) to prevent accidental turning of the control knob. d. If both pilot lights go on at the same time the safety knob is set too low and must be reset. 4. Calibration of the internal thermometer a. Lay a NBS thermometer on the back of the shelf nearest the center of the cabinet. b. After 10 min with the door closed, compare the temperature on the NBS thermometer with that on the thermometer in the incubator. c. If there is a discrepancy, it is usually possible to remove the face of the dial thermometer and adjust the reading. Check the product manual for instructions d. Check the temperature in four other spots (top front, top back, bottom front and bottom back corners) with the NBS thermometer so that any hot or cold spots will be known. Do not use these areas for sensitive cultures. 5. Regulating the humidity a. Place a hygrometer (humidity measuring device) on the central shelf of the incubator. b. Read the humidity after 1 h. if the humidity is less than 40%; place a pan of water (containing antifungal agent) with a large surface area ( 150 cm2) on the shelf of the incubator. c. Allow the incubator to run overnight and recheck parameters before using.

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Anaerobic Jars Uses in the microbiology laboratory. 1. Primary plate incubation (all atmospheres) a. Microaerophillic atmosphere primarily for Campylobacter species. b. Anaerobic atmosphere primarily for strict anaerobes. c. Carbon dioxide enriched atmosphere primarily for Neisseria species, Haemophilus species, certain microaerophillic streptococci and other capnophiles 2. Maintenance of specimens (such as tissues) under anaerobic atmosphere. 3. Holding and transporting pure cultures of anaerobic or microaerophilic organisms under appropriate atmosphere. 4. Incubation of other cultures that require special atmosphere: test strips, tubers, microbroth dilution plates, etc. Initial calibration: Anaerobic and Microaerophilic jars Test the jar with generator system and indicator QC organisms to verify that seal is air tight, anaerobic atmosphere sufficient to change the indicator is achieved and QC organisms can grow. Routine quality control A. Anaerobic jars: each time they are used Since the lid is the component most likely to fail, record the QC checks in a label attached to each lid. 1. Check jar and lid for cracks 2. Check the indicator strip has not turned color inside the sealed packet. Discard all strips that are not white when packet is opened. 3. Before opening an anaerobic jar, observe the indicator strip to be sure that an anaerobic atmosphere was maintained during incubation. If an anaerobic atmosphere was not maintained the anaerobic culture result is invalid 4. Rejuvenate catalysts after each use by heating the basket of pellets (rendering them less inactive), store heated catalyst baskets in a very dry place, such as a desiccator jar. An extra gallon jar with several inches of calcium carbonate desiccant in the bottom and the lid tightly screwed on is acceptable Microscope Uses in the microbiology laboratory In microbiology laboratory microscopes are used in observation and description of the microscopic morphology of bacteria, fungi, parasites and host cells in various stained and unstained preparations. Initial aligning procedures A. Koehler illumination is a precise way of aligning the light pathway onto the specimen plane to evenly illuminate the field of view. This procedure ensures the highest resolution for the optical system, enabling visualization of as much detail as possible. 1. Place a cleaned stained specimen (cells or bacteria; subject identity is not critical, but best to have distinct form for ease of focus) slide right side up in the stage slide holder. 2. Open the field diaphragm all the way.

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3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

18.

Open the condenser diaphragm all the way. If the condenser has an auxiliary lens, swing it out of the light path. Otherwise, you will not be able to image the diaphragm. Rack the condenser all the way up. Rotate the nosepiece to the 10× objective position (8× to 20× objective is acceptable). Turn on the light source and adjust it to a low, comfortable light intensity. Adjust the binoculars to your inter papillary distance. Use the focusing eyepiece, if available, to compensate for any dioptic discrepancy (different ability to focus) between each eye. Adjust both eyepieces to equal height. Look through the fixed eyepiece, and focus on the specimen, using he course and fine adjustment knobs. Use the adjustable eyepiece to focus the image sharply. Gradually close the field diaphragm until you see a multisided polygon (image of diaphragm) around the field of the specimen. Lower the condenser slightly until the diaphragm edge is as sharp as possible. (A magenta color may be seen.) Adjust fine focus knob so that the specimen is in sharp focus. If the diaphragm image is not centered, then center it by gently turning the centering screws located on the condenser. Open the diaphragm until the image of the diaphragm just goes out of the field of view. Do not open any further. Do not disturb the condenser height. Set the optimal contrast by gradually closing the aperture diaphragm. (Rule of the thumb is to remove the eyepiece, look down the tube at the back focal plane of the objective, and adjust the aperture to two-thirds open. Control contrast by adjusting the condenser diaphragm. Most microscopes will not need further alignment for higher magnification.

B. Calibration of microscope with an ocular micrometer I. Principle The identification of molds, bacteria, protozoa and other parasites depend on several factors, one of which is size. Any laboratory doing diagnostic work in microbiology and parasitology should have a calibrated microscope available for precise measurements. Measurements are made with a micrometer disk that is placed in the ocular of the microscope; the disk is usually calibrated as a line divided into 50 Units. Depending on the objective magnification used the divisions in the disk represent different measurements. The ocular disk division must be compared with a known calibrated scale, usually a stage micrometer with a scale of 0.1- and 0.01- mm divisions. II. Materials A. Supplies 1. Ocular micrometer disk (line divided into 50U) 2. Stage micrometer with a scale of 0.1- and 0.01-mm divisions 3. Immersion oil. 4. Lens paper B. Equipment 1. Binocular microscope with 10×, 40×, and 100× objectives. Other objective magnifications (50× oil or 63 × oil immersion lenses) may also be used.

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2. Oculars should be 10×. Some may prefer 5×; however smaller magnification may make final identification more difficult. 3. Single 10× ocular to be used to calibrate all laboratory microscopes (to be used when any organism is being measured) III. Quality control A. Recalibrate the microscope a minimum of once each year. If the scope receives heavy use, twice a year is recommended. B. Often the measurement of RBCs (approximately 7.5 µm) is used to check the calibrations of the three magnifications (×100, ×400, ×1,000). C. Latex or polystyrene beads of a standardized diameter can be used to check the calculations and measurements beads of 10µm and 90µm are recommended. D. Record all measurements in QC records. IV. Procedure A. Unscrew the eye lens of a 10× ocular, ad place the micrometer disk (engraved side down) within the ocular. Use lens paper to handle the disk; keep al surfaces free of dust or lint. B. Place the calibrate micrometer on the stage, and focus on the scale. You should be able to distinguish the difference between the 0.1- and 0.01 mm divisions. Makes sure you understand the divisions on the scale before proceeding. C. Adjust the stage micrometer so that the "0" line on the ocular micrometer is exactly lined up on the top of the 0 line on the stage micrometer. D. When these two 0 lines are lined up, do not move the stage micrometer any further. Look to the right of the 0 line for another set of lines superimposed on each other. The second set of lines should be as far to the right of the 0 lines as possible; however the distance varies with the objectives being used. E. Count the number of ocular divisions between the 0 lines and the point where the second set of lines is superimposed. Then, on the stage micrometer, count the number of 0.1- mm divisions between the 0 lines and the second set of superimposed lines. F. Calculate the portion of a millimeter that is measured by a single ocular unit. G. When the high dry and oil immersion objectives are used, the 0 line of the stage micrometer will increase in size, whereas the ocular line will remain the same size. The thin ocular 0 line should be lined up in the center or at one edge of the broad stage micrometer 0 line. Thus when the second set of superimposed line is found, the thin ocular line should be lined up in the center or at the corresponding edge of the broad stage micrometer line. H. Calculate the factors as follows. Examples: Stage reading (mm)/ ocular reading × 1000µm/ 1 mm = ocular units (µm) Low power (10×) 0.8 mm/ 100 U × 1000µm/ 1 mm = 8.0 µm (factor) High dry power (40×) 0.1mm/ 50 U × 1000µm/ 1 mm = 2.0 µm (factor) Oil immersion (100×) 0.05 mm/ 62 U × 1000µm/ 1 mm = 0.8 µm (factor)

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Example: If a helminth egg measures 15 ocular units by 7 ocular units with the high dry objective, then multiply the measurements by the factor 2.0 µm (for that objective). The egg the measure 30 by 14 µm and is probably Clonorchis sinensis. Example: If a protozoan cyst measures 27 ocular units with the oil immersion objective, then multiply the measurements by the factor 0.8 µm (for that objective). The cyst then measures 21.6 µm. V. Results A. For each objective magnification, a factor will be generated (1 ocular unit = certain number of micrometers. B. If standardized latex or polystyrene beads or an RBC is measured with various objectives, the size for the object measured should be the same (or very close), regardless of the objective magnification. VI. Reporting results A. Post the multiplication factor for each objective either on the base of the microscope or on a nearby wall or bulletin board for easy reference. B. Once the number of ocular lines per width and length of the organism is measured, then, depending on the objective magnification, the factor (1 ocular unit = certain number of millimeters) can be applied to the number of lines to obtain the width and length of the organism. C. Comparison of these measurements with reference measurements in various books and manuals should confirm the organism identification. VII. Procedure noted A. The final multiplication factors will be on as good as your visual comparison of the ocular 0 and stage micrometer 0 lines. B. As a rule of thumb, the high dry objective (40×) factor should be approximately 2.5 times more than the oil immersion objective (100×) factor. The low-power objective (10×) factor should be approximately 10 times the oil immersion objective (100×) factor. VIII. Limitations of the procedure A. After each objective has been calibrated, the oculars containing the disk and/or these can not be interchanged with corresponding objectives or oculars on another microscope B. Each microscope used to measure organisms must be calibrated as a unit. The original oculars and objectives that were used to calibrate the microscope must also be used when an organism is measured. C. The objective containing the ocular micrometer can be stored until needed. This single ocular can be inserted when measurements are taken. However, this particular ocular containing the ocular micrometer disk must also have been used as the ocular during microscope calibration. Hot air oven In microbiology laboratory the oven is used to dry glassware, sterilize metal and certain hightemperature stable glass objects and to reactivate palladium-covered alumina catalysts used in anaerobic chambers and jars. Initial calibration A. Connect the power cord to the grounded outlet.

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B. Place a dial gauge or mercury bulb thermometer that has been previously calibrated with an NBS thermometer on a shelf in the middle of the oven. C. Switch the oven on, and adjust it to the desired temperature. D. Check the thermometer every 15 min until the desired temperature is reached. E. Readjust the temperature gauge if the temperature increases more than 5°C above the desired set temperature. F. Allow the temperature to stabilize for at least 2 h before using the oven for the first time. pH meter Uses of pH meter in the microbiology laboratory. 1. Precise measurement and adjustment of the pH of solutions. a. Antibiotic activity and solubility vary with pH of diluents. b. Bacteria, fungi and viruses often require specific pH ranges of media or transport solution to remain viable or to multiply. c. Reagents used for testing metabolic reactions must be prepared to a specific pH fro the reaction to work correctly and be correctly interpreted Routine quality control procedure A. Each time, even if more than one use occurs in the same day. 1. Calibrate the pH meter with the standard calibration buffer at pH 7.00 and the standard whose pH is nearest to the expected pH of the liquid you plan to test. If your expected solution us pH 8.2, for example, calibrate with pH 10.00 standard. 2. Read and record the result of pH standard calibration buffer opposite the pH of the standard that you read in step 1. For example given above, test the pH 4.00 standard calibration buffer. Results must be within ± 0.10-pH unit of the value of the standard. 3. Read and record the pH of the QC-certified buffer whose pH is closest to that of the expected pH of the sample. For example given above, use QC- certified buffer pH 8.00. Value must be within ±0.05 pH unit of the pH of the QC- certified buffer. Refrigerators and freezers Uses in the microbiology laboratory Low temperatures are required for storage and preservation if stock cultures, reagents and media. Refrigeration or freezing may inhibit the growth of contaminants, slow reactions that would otherwise inactivate reagents and delay evaporation. The use of refrigerators and freezer must be carefully controlled. A refrigerator which does not cool to the correct temperature will cause sample degradation and loss of culture viability. They should be routinely sanitized. They should be arranged in a tidy manner such that sample or culture access is n hampered. A record of materials entering and being removed from the ot refrigerator should be maintained. All samples must be appropriately identified with the sample's identity, date of placement in refrigerator, contact person and if possible the use-by date. Mislabeled and unlabelled samples can lead to many analysis errors. Appropriate containers should be used to hold samples and cultures. Spillage of cultures in the refrigerator or onto the floor is very dangerous and can arise from failure to use the proper holding containers, overfilling containers and from cluttered refrigerator. Cultures should be maintained in the refrigerator on slopes and not on agar plates. Agar plates, no matter how well sealed will tend come apart,

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leading to a potential safety hazard. The formation of condensate on the lid of the Petri dish can also lead to the contamination of the plate. Initial calibration A. Preparing thermometers 1.Number each individual thermometer, and calibrate each one against the NBS thermometer 2.Use waterproof colored felt-tip pen or bands of colored tape to mark of the area ±10°C of the working temperature of the unit in which the thermometer will be use 3.Immerse thermometers for refrigerators in 50% glycerol, polyethylene glycol (antifreeze), or alcohol in an empty blood bank pheresis bag or in a bottle stoppered with a single hole cork or rubber stopper. 4.Immerse thermometers for freezers in alcohol or polyethylene glycol in plastic bag or bottle. Be certain to use containers that will withstand sub zero temperatures. 5.If the built in thermometer will be used exclusively, place and NBS-calibrated thermometer into the unit and verify the accuracy of the internal thermometer. If necessary, adjust the thermometers to read identically (consult the product manual), or note on the internal thermometer the correction factor needed to match the NBS measurements. Calibration of such internal thermometers must be performed at the same intervals as for any thermometer QC protocol (every 6 months for thermometers with intervals of 1 °C and every month for those with smaller divisions). B. Placing thermometers 1. Place thermometer where it can be viewed easily is not exposed to trauma and will not be subjected to air drafts that will distort the reading as soon as the door is opened. 2. At least one thermometer should be placed into each interior chamber or a refrigerator or freezer; very-large-volume units (more than 60 ft3 [ca. 13 m3]) may be better served with additional thermometers. For a large unit, we recommend that one thermometer be placed in the top left rear and on be placed in the bottom right front. Before placing a new unit in service, place several (three or more) thermometers in diverse areas, close to the door, allow the temperature to equilibrate for 1 h, and check for consistent temperature readings. If warm spots are found, it is prudent to place the single thermometer there or to use more than one thermometer. C. Setting the temperature 1. Set refrigerator temperature to 6°C by using regulating dial inside the unit. 2. Set standard freezer temperature to -20°C by using external dial or gauge. 3. Set ultra low freezers to appropriate temperatures (common temperatures are ­40, -70, -80 and -100°C) 4. Allow the temperature to equilibrate for at least 1 h after the compressor stops running for the first time after startup, and then check the internal thermometers. Adjust the gauges, and re-equilibrate the temperatures until it is within 0.5 °C of the desired temperature. If variation among all internal thermometers are >4°C, call your manufacturer for advise (and service) 5. Monitor the temperature of the unit for stability several times a day for 1 to 2 days before placing materials inside. Laminar flow cabinets (Clean bench) Uses in microbiology laboratories

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1. To contain infections aerosols generated by processing of samples or cultures. Examples include the following. a. Grinding or mincing of tissue b. Preparing direct smears and wet mounts c. Plating of specimens. d. Identification procedures for molds 2. To protect samples or materials from external contamination a. Performing procedures with sterile fluids b. Preparation of media or reagents solutions aseptically. N/B The maintenance of laminar flow hoods should include checking of the ultraviolet light source, the HEPA filter and the air compressor rate. All will need routine replacement or routine maintenance. Challenge tests should be used to test for their effectiveness the UV-light and filter. The hood should not be positioned near doorways, air-conditioning vents or near where there is heavy human traffic. They should not be used as a storage area and should not be left on when not in use. Bunsen burners should not be used in the hood as the column of air arising from the burner can disturb the laminar air-flow patterns. For the similar reasons, media bottles and other large containers should be removed once they are no longer required.

Installation and certification A. Every biological safety cabinet must be tested in certified for adequate functioning after it is in place in the laboratory, since testing that occurs elsewhere will not ensure that the cabinet will function properly under the conditions actually found in the laboratory 1. Specified airflow velocities must be met but not exceeded. 2. The cabinet and filters must be free of air leaks 3. Intake and exhaust airflow rates must be balanced. 4. Field test should include the following. a. Determination of the work are velocity profile b. Measurement of the face velocity (airflow velocity through the front opening) c. Determination of HEPA filter integrity d. Determination of air flow patterns (smoke test) e. Checks on lighting intensity, temperature, vibration and noise levels. 5. Ask that the cabinet function be checked while equipment used in or around the cabinet is running to ascertain if the cabinet function is impaired. B. Certification must be done by specially trained personnel. Previously there have been no standards for certification if personnel who certify biological cabinets. Cabinet users had to (and still should) know enough about the operation of a cabinet to ensure that the person certifying the cabinets was adequately trained and familiar with that particular type of cabinet. C. Cabinets must be recertified before being operate after the have been moved. For long distance moves, especially when nonmicrobiology personnel are involved, cabinets should be decontaminated prior to the move. 1. Ensure that the cabinet was nit damaged during the move 2. Ensure that the placement if the cabinet does not interfere with proper functioning of the cabinet. D. Cabinets must be recertified whenever HEPA filters are changed or repaired E. Cabinets must be recertified at least annually. Institutional or regulatory guideline may call for more frequent certifications.

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1. The CAP commission of Laboratory Accreditation mandates annual certification 2. Medicare and Clinical Laboratory Improvement Act regulations call for a minimum air intake of 50 lfpm through the front opening 3. JCAHO makes no specific recommendations. Routine Quality control. A. Each time the safety cabinet is turned on or at least once per day 1. Routine disinfections While the cabinet is running but before you begin work, wipe down the cabinet surfaces, including the back and sidewalls, with disinfectant. If bleach is used as the disinfectant rinse all metal surfaces to prevent corrosion of the metal. 2. Check blower function. a. Listen for the blower b. Observe that airflow check strips are drawn in onward the workspace of the cabinet 3.Record gauge reading (if gauge is provided). 4.Cover working surface with absorbent covering (laboratory diaper pr paper toweling) (optional). Use of absorbent coverings minimizes splatters and makes clean up easier. B. Each week Clean UV lights (if present) by wiping with 70% ethanol. C. Each month 1. Once each month (or with heavy use, more frequently), remove the vent covers and clean the gutter with disinfectant. (If this area is provides with a drain valve, disinfectant can be poured into the area and allowed to stand for 20 min before draining. Flush with several liters of water) 2. Measure UV light output (if UV light is provided; optional). D. Each year Have the cabinet recertified. E. As needed. 1. Clean up small spills. Replace absorbent covering if contaminated. 2. Remove and replace discard containers Thermometers Precise temperature measurements can be made only with instruments whose accuracy has been verified. This can be accomplished by comparing routine thermometers used in the laboratory against the SRM thermometers or NBS-calibrated thermometers. A. Liquid-in-glass thermometers 1. Prepare an ice bath using shaves ice and small amount of water to form a tightly packed slush. Use only enough water to allow good contact with thermometer. Ice must not float. Remove excess water. Avoid handling the ice so as not to contaminate it. 2. Rinse the bulb of the standard thermometer with distilled water before inserting it into the slush bath. 3. Take the ice point reading of the standard thermometer. Gently tap thermometer to ensure that the mercury does not stick to the capillary wall. Wait at least 5 min before

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4.

5.

6.

7.

making determination. Liquid-in-glass thermometers do not respond instantaneously; they needs time to adjust and stabilize. Compare the ice point of the standard with the report of calibration that is received with the standard, they should agree, Note: if the manufacturer has included a correction factor with the standard thermometer, then always add or subtract the correction factor from the reading of the standard to measure the actual temperature in the liquid being measured. Place the standard thermometer and the test thermometers upright in the rack in the heated liquid bath. Adjust the temperature to 25°C and read on the standard thermometer. Read the temperatures indicated on all test thermometers. Take readings at 25, 30, and 37°C (or whichever temperatures the SRM thermometer has been calibrated for). Thermometers that fall within the tolerance range (as provided by the manufacturer) are acceptable for routine use. Log all results, serial numbers, ranges and tolerances of test thermometers and standard thermometers on the QC forms.

Water Baths Uses in the microbiology laboratory Water bath are used in the microbiology laboratory for incubating at or maintaining a constant temperature. They are ideal for application requiring accurate temperature control. One application specific to microbiology laboratory is equilibrating melted agar to a working temperature for pouring agar plates. Boiling water baths are used to melt agar. Initial calibration A. Fill the bath with demineralized water per manufacturer's recommendations, making allowance for displacement by samples to be immersed. B. Connect the power cord to a grounded outlet. C. Construct a calibration table for reference dial setting versus temperature. Note: Keep the water bath covered during use to prevent evaporation and conserve heat. 1. Set the reference dial to a number midway between the highest and the lowest settings (usual range is 0­9) 2. Place an NBS-calibrated thermometer deep enough into the liquid to obtain accurate measurements. 3. When the light goes off, indicating heating is complete, record the dial setting and the corresponding temperature. 4. Continue to raise the dial setting 1 U at a time and record the temperature each time the light goes off. 5. In this way construct a reference table of dial settings versus water bath temperature. Routine Quality control A. Daily 1. Check the water in the bath for bacterial or algae growth. Visual inspection is sufficient. If a problem exists empty and clean the tank and add an antimicrobial agent in the water. 2. Inspect the bath for build up of mineral deposits. Wash the bath and scour with cleaning supplied if a problem exists. 3. Check for leaks

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4. Check the temperature with a thermometer that has been calibrated with a NBS thermometer. Record the temperature on the QC sheet B. Monthly (for baths filled with water) 1. Drain and clean the bath. Baths used infrequently or those filled with mineral oil maybe cleaned less frequently. a. Used a siphon tube to drain a) Completely immerse siphon in the water b) Cover one end of the tube, lift that end from the bath, and then lower it below the water level in the bath before uncovering the closed end. 2. Clean the bath with mild, soapy water and a damp cloth. Do not use bleaches or cleaners containing abrasives or chlorine on stainless steel baths. 3. Refill the bath with demineralized, distilled, or deionized water or mineral oil. C. Semiannually Clean the unit as needed with a scouring compound or tartaric acid to remove scale build up or corrosion caused by impurities in water. Pipetters/ calibrated Loops Uses in the microbiology laboratory Pipetters are used to dilute sera, set up quantitative cultures, prepare inocula for antimicrobial tests, add ingredients to media and reagents and add exact amounts of reagents or specimen during a test procedure. Pipetters are used because of their excellent accuracy and precision and because they expeditiously dispense small volumes repeatedly. Quantitative loops are commonly used to setup quantitative cultures and prepare inocula for antimicrobial tests. Quantitative loops are less accurate than pipetters yet are excellent way to setup a semi quantitative culture or dilution. Quantitative loops are used when 20% error is acceptable. Calibration methods for volume dispensing instruments.

Method Gravimetric Instrument Pipetters (recommended method) Basis of system 1 ml of water = 1 g (adjusted for temperature and pressure) Absorbance of potassium dichromate used to create calibration curve Absorbance of Evans blue dye used to create calibration curve Limitation Vol dispensed must be >0.002 ml

Spectrophotometric

Pipetters

Vol dispensed must be >0.01 ml

Colorimetric

Quantitative loops

Loop vol between 0.01 and 0.001 ml

Special precautions and Environmental concerns A. Special precautions for pipetter calibration 1. Changing pipetter tips during calibration procedure Use the same pipetter tip for all deliveries during the calibration procedure, whether the pipetter is used for repetitive dispensing of several aliquots of the same liquid (e.g., buffers, reagents) or for 182

transferring single aliquots of different liquids (e.g. serum). Note: during actual pipetting for routine use, a different tip must be used for each different liquid. 2. Prerinsing pipetter tips "Prerinsing" is the precoating of the inside of the tip with the liquid being dispensed. Prerinse by aspirating an aliquot of the liquid into the tip and then dispensing it back into the original container or discarding it. Prerinsing improves uniformity and precision by providing identical contact surfaces for all aliquots. a. If the pipetter is normally used for repetitive dispensing of several aliquots of the same liquid, prerinse pipetter tip at the beginning, before dispensing the first aliquot. b. If the pipetter is normally used for transferring single aliquots of different liquids, prerinsing may not be necessary. B. Environmental concerns for pipetter calibration 1. Temperature control b. Temperatures of pipetter to be calibrated, room air, test liquid (water), and other equipment should be identical (± 0.5°C). c. Temperature should be as close as possible to the temperature at which the pipetter is used. d. Keep temperature stable through out the procedure 2. Miscellaneous environmental concerns a. Maintain relative humidity at 45 to 75%. This reduces evaporation and limits build up of static electricity b. Prepare aqueous test liquid from NCCLS type I or II water, which prevents impurities from affecting water density. c. Use water with no visible bubbles. Air bubbles alter measured volume. d. Complete weighing steps quickly. Use a lid on the weighing vessel to decrease evaporation. These precautions obviate the need for and evaporation factor in the calculations. Laboratory glassware and plastic ware Ideally, the specifications of laboratory glassware and plasticware should be established and followed. The calibration of pipettes and other volumetric and graduated glassware should be checked and the calibration verified. The extent to which this is undertaken is dependent on how the glassware or plasticware is used. Glassware should be made from high quality borosilicate glass. Glassware made from soft glass can cause problems due to leaching of components from the surface of the glass. Reusable glassware and plastic ware should be sterilized and washed using appropriate methods. Detergents should be completely removed from glassware. Many detergents have a high affinity for glassware and some are bacteriostatic. A drop of bromothymol blue pH indicator is useful in determining if the cleaning agent has been completely removed. Bromothymol blue is blue-green between pH 6.5 to 7.3 and yellow below pH 6.5 and blue above pH 7.3 Periodic toxicity testing should be undertaken with washed glassware and disposable glassware and plasticware which may be sterilized by ethylene oxide gas. Washing may also leave toxic residues on glassware. Toxicity testing should be performed annually.

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The sterility of supplies and equipment must be tested. Sterility tests of petri dishes can be performed by pouring a non-selective medium onto dish and incubating the solidified plate both anaerobically and aerobically. The absence of growth indicates that the plates are sterile. Similarly the sterility of tubes and pipettes can be ascertained with sterile broth medium. Media and reagents Microbiological media and test reagents should be tested for their performance. This should be done for pre-prepared media and media formulated in the laboratory. Common errors include

· ·

Incorrect weighing of dried materials Use of dry materials that have deteriorated as a result of exposure to heat, moisture or oxidation Incorrect measurement of water volumes (eg. using the markings on beakers as volume guides), use of tap water or using water from a malfunctioning still or water deionizer. Use of contaminated containers and glassware Incomplete mixing or solubilization of ingredients during preparation. Failure to pre-melt agar before sterilization can lead to uneven gel strength through an agar medium. Overheating of media during preparation and sterilization Improper determination of pH Failure to perform quality control on finished media Failure to perform quality control on dehydrated media Store media in tightly capped bottles or tightly closed plastic liners in a cool dry place protected from light. Keep no more than 6 months' to a years supply on hand. Use older stocks first and do not exceed supplier's expiration date. Look for changes in flow properties and in colour. If an any item in question, then discard it.

·

· ·

· · · ·

General guidelines for the storage of dehydrated media includes:

·

·

·

Sterility testing of prepared media Sterility testing can be performed by incubating placing selected plates or tubed media prior to use or along with inoculated media. The temperature should be that normally used for these media. The plates should be inspected for turbidity or colony formation. Neither of these may however be present if the medium is designed to inhibit various bacteria. This problem can be overcome by swabbing the surface of the agar plate and then incubating the swabs in a nonselective medium. A similar technique can be used with liquid media. Quality control procedures

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Quality control should be performed on all materials and equipment which are critical to the detection, isolation, identification and analysis of microorganisms. This includes media, stains, biochemical test reagents and equipment such incubators and autoclaves. As with quality assurance, there should be a procedure manual, control organisms or slides, defined QC criteria, record sheets and trained personnel. The intervals between QC tests varies with the critical importance of the material or method and the likelihood that QC will be a problem. For example, oxidase and catalase tests should be tested weekly, while agglutinating sera is tested monthly or quarterly. Gram stains should tested on a weekly basis. Each lot or batch of materials should undergo quality control. As discussed above equipment and glassware should also undergo quality control tests. Volumetric dispensers should be tested with each run. Incubators and anaerobic jars should be tested on a daily basis. Spectrophotometers, biohazard cabinets and microscopes should be tested yearly. Control strains should be used to validate procedures and test media and reagents. The strain used should comply with the standard method being followed. The strains should be well maintained. The number of subcultures permitted for each strain again should be in accordance with the standard method. Continual passaging of microbial strains on agar media can lead to a loss or change in a critical characteristic. Control strains can be purchased from local distributors or directly from culture collections in the USA, Europe or Australia. The ATCC has one such collection.

Laboratory Audit

Audit is an essential part of the quality assurance program of a laboratory. A quality assurance programme covers all aspects of the service provided. It may include policies on the induction and training of new staff, staff development, laboratory manuals, safety policies, equipment maintenance etc. Audit is a means of assessing whether one is achieving one's stated objectives. There are five key questions in the audit process: - what should we do? - what do we do? - Are we doing what we should be doing? - Can we improve what we do? - Have we improved? All technicians are required to participate in laboratory audit which is defined as the systematic critical analysis of the quality of food microbiological analysis.. Laboratory audit is concerned primarily with the everyday aspects of the work of the department and is a means of providing feedback to both the users of the laboratory and its staff. The chief technician will examine the following areas. Request forms; are they easy to use? Are all relevant details provided by the user. For example, date of sampling, time of sampling and temperature of sample at the time of sampling.

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Sample; is the right sample received at the right time? Are the appropriate tests selected by the laboratory staff? The laboratory staff should use the laboratory MANUAL. To guide them in the types of tests to carry out on a sample. Safety policies and procedures. Every laboratory should have a comprehensive safety policy. Every single accident in the laboratory should be recorded and improvements made if necessary. The use of dangerous substances should be audited. Sufficient use of staff. Do senior staff perform duties that should or could be delegated to others. Efficient use of staff would be a much more important consideration in a small laboratory than a larger one. Functioning of Equipment. The equipment and apparatus should be checked to make sure that they area functioning as required.

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Microbial Standards of Foods

Quoting selected sections of the American FDA act may help in understanding and appreciating this section. Standards and guidelines can be applied only when the appropriate method of analysis (or equivalent) is used.

Sections of the Food and Drugs Act 1. No person shall sell an article of food that (a) has in or upon it any poisonous or harmful substance; (b) is unfit for human consumption; (c) consists in whole or in part of any filthy, putrid, disgusting, rotten, decomposed or diseased animal or vegetable substance; (d) is adulterated; or (e) was manufactured, prepared, preserved, packaged or stored under unsanitary conditions. 2. No person shall label, package, treat, process, sell or advertise any food in a manner that is false, misleading or deceptive or is likely to create an erroneous impression regarding its character, value, quantity, composition, merit or safety. 3. Where a standard has been prescribed for a food, no person shall label, package, sell or advertise any article in such a manner that it is likely to be mistaken for such food, unless the article complies with the prescribed standard. 4. No person shall manufacture, prepare, preserve, package or store for sale any f ood under unsanitary conditions. The Act defines "unsanitary conditions" as "such conditions or circumstances as might contaminate a food, drug or cosmetic with dirt, filth or render the same injurious to health". Guidelines A given guideline may embody the same limiting criteria that would be employed in a standard. Frequently, however, they are based on fewer data than those used in developing a standard but they serve as useful indicators of levels achievable using Good Manufacturing Practices (GMPs). Since guidelines are not part of the Regulations, they can be readily modified, if necessary, as additional data become available. There are two distinct groups of guidelines related to health and safety; microbiological guidelines and injurious extraneous material guidelines. The latter includes foreign matter associated with objectionable conditions or practices in manufacturing, processing, storing, transporting and handling of food that could lead to an injury (e.g. glass in jam, splinters, etc). Sampling plans The symbols used in the plans and their definitions are as follows: 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

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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 or amounts of extraneous material, usually per g or mL. The "m" values listed in the following tables are based on levels achievable under GMP. M: the numerical value of "M" represents unacceptable concentrations of microorganisms or amounts of extraneous material, 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.

RISK ASSESSMENT Risk assessment is composed of four elements: hazard identification, exposure assessment, hazard characterization and risk characterization. Hazard Identification The purpose of hazard identification is to identify the microorganisms or the microbial toxins of concern with food. Hazard identification will predominately be a qualitative process. Hazards can be identified from relevant data sources. Information on hazards can be obtained from scientific literature, from databases such as those in the food industry, government agencies, and relevant international organizations and through solicitation of opinions of experts. Relevant information includes data in areas such as: clinical studies, epidemiological studies and surveillance, laboratory animal studies, investigations of the characteristics of microorganisms, the interaction between microorganisms and their environment through the food chain from primary production up to and including consumption, and studies on analogous microorganisms and situations. Exposure Assessment Exposure assessment includes an assessment of the extent of actual or anticipated human exposure. It might be based on the potential extent of food contamination by a particular agent or its toxins, and on dietary information. Exposure assessment estimates the level of microbiological pathogens or microbiological toxins, and the likelihood of their occurrence in foods at the time of consumption. The presence, growth, survival, or death of microorganisms, including pathogens in foods, are influenced by processing and packaging, the storage environment, including the temperature of storage, pH, moisture content or water activity (aw), the presence of antimicrobial substances, and competing microflora. Predictive microbiology can be a useful tool. Hazard Characterization This step provides a qualitative or quantitative description of the severity and duration of adverse effects that may result from the ingestion of a microorganism or its toxin in food. Several factors 188

need to be considered in hazard characterization. They are related to both the microorganism, and the human host. In relation to the microorganism the following are important: microorganisms are capable of replicating; the virulence and infectivity of microorganisms can change depending on their interaction with the host and the environment; genetic material can be transferred between microorganisms leading to transfer of characteristics such as antibiotic resistance and virulence factors; microorganisms can be spread through secondary and tertiary transmission; the onset of clinical symptoms can be substantially delayed following exposure; microorganisms can persist in certain individuals leading to continued excretion and continued risk of spread of infection; low doses of some microorganisms can cause a severe effects; the attributes of a food may alter the microbial pathogenicity, e.g. high fat content of a food vehicle. In relation to the host the following may be important: genetic factors; increased susceptibility due to breakdowns of physiological barriers; status, concurrent infections, immune status and previous exposure history; population characteristics such as population immunity, access to and use of medical care, and persistence of the organism in the population. Risk Characterization Risk Characterization represent the integration of the hazard identification, hazard characterization, and exposure assessment determination to obtain a risk estimate. Risk characterization brings together all of the qualitative or quantitative information of the previous steps to provide a sound estimate of risk for a given population. Risk characterization depends on available data and expert judgments. The following categories have been used to characterize the health risks: Health 1 The health hazard identified represents a situation that could cause serious adverse health consequences or death. Appropriate action should be taken against the product to limit or prevent exposure in the population to the product. Such action should ensure that the product is no longer sold and the population does not consume what they have at home (e.g. action at the consumer level if the product has been distributed). Follow-up action should ensure that the cause has been determined and appropriate corrective action has been taken to correct the problem. Health 2 The health hazard identified represents a situation that could cause temporary, not life-threatening, adverse health consequences. The probability of serious adverse consequences is considered remote. Appropriate action should be taken to limit further distribution of the product. In some situations, the hazard identified must be present in sufficient numbers to present a risk to human health. Appropriate action should be taken to limit further distribution if the M value is exceeded. Repeated violations should be investigated. If c/m values are exceeded, progressive steps should be taken to bring about compliance, initially review GMP/HACCP. Sanitation The problem identified is an indication of a breakdown in hygienic practice. A review of the manufacturer's GMP/HACCP is appropriate where M or c/m values are exceeded. A health hazard has not been identified. Note When products characterized with a Health 2 risk are associated with illness in an outbreak, consideration should be given to modification of the risk characterization to Health 1 with appropriate action to the consumer level. It should also be noted that some health risk are typically characterized as Health 2 for the general population but may cause more severe adverse health consequences, even death, in

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sensitive populations. Therefore, where products are contaminated with Health 2 risks but are directed to sensitive populations such as children less than five years of age, the elderly or immunocompromised individuals (e.g. AIDS patients, transplant recipients, cancer patients, etc.), consideration should be given to modification of the risk characterization to Health 1 with appropriate action to the consumer level. TABLE 1a. Foods for which there is a Microbiological Standard. FOOD STANDARD CATEGORY NATURE OF CONCER N Salmonella Health 2 Salmonella Health 2 Salmonella Health 2 Aerobic colony Sanitation count (ACC) ACC Sanitation Escherichia coli Staphylococcus aureus E. coli S. aureus Coliforms ACC Coliform ACC Coliforms SAMPLING PARAMETERS n c m M

Chocolate Cocoa Milk Powder Flavoured Milks Milk for Manufacture Cheese from Pasteurized Milk Cheese from Unpasteurized Milk Cottage Cheese Ice Cream Ice Milk

10 10 20 5 5

0 0 0 2 0 2 2 2 2 1 2 1 2 1

0 0 0 5x104 2x106 102 102

106 2x103 104

Health 25 Health 2 5 Health 2 Health 2 Sanitation Sanitation Sanitation Sanitation Sanitation 5 5 5 5 5 5 5

5 X 102 2x103 103 104 101 105 101 105 101 103 106 103 106 103

TABLE 1b. Foods for which there is a Microbiological Standard FOOD CATEGORY STANDARD NATURE SAMPLING PARAMETERS OF CONCER n c m M N Coliforms Sanitation 10 1 0/100 mL 10/100mL 5 2 102 10 <1.8/100mL 1 10 1 <1.8/100mL 10 0 0 104 10/100mL 10/100mL -

Mineral or Spring Water Water in ACC Coliforms Sanitation Sealed Sanitation Containers Pre- packaged Coliforms Sanitation Ice Egg Products Salmonella Health 2

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TABLE 2. Foods for which there are Standards other than Microbiological FOOD CATEGORY NATURE OF CONCERN Dairy products made from pasteurized milk Phosphatase Test. Health 1 Low Acid Foods in Hermetically Sealed Commercial Sterility, Refrigeration at < Health 1 ContainersA 4/C. Smoked Fish in Hermetically Sealed Commercial Sterility, Freezing, 9% salt Health 1 ContainersB (NaCl). STANDARD

TABLE 3a. Foods for which Microbiological Guidelines have been established. FOOD CATEGORY Cocoa GUIDELINE NATURE SAMPLING OF PARAMETERS CONCERN n c m M 5 2 105 106

ACC Sanitation includes aerobic spore formers Yeast and Sanitation Moulds Coliforms Sanitation Chocolate ACC Sanitation includes aerobic spore formers Coliforms Sanitation Instant Infant ACC Sanitation Cereal and E. coli Health 2E Powdered Infant Salmonella Health 1 Formula (if M S. aureus Health 2 exceeded Health 1; if c exceeded Bacillus cereus Health 2 Clostridium Health 2 Health 2) perfringens

5 2 2X103 104 5 2 <1.8 101 5 2 3x104 106

5 5 10 20 10 10 10

2 <1.8 2 103 1 <1.8 00 1 101 1 102 1 102

102 104 101 0 102 104 103

191

TABLE 3b. Foods for which Microbiological Guidelines have been established. FOOD GUIDELINE NATURE SAMPLING CATEGORY OF PARAMETERS CONCERN n c m Fresh and Dry ACC Sanitation 5 2 5x104 Pasta Yeast and Sanitation 5 2 2x103 moulds E. coli Health 2C 5 2 <1.8 S. aureus Health 2 5 2 5x102 Salmonella Health 2D 5 0 0 Bakery ACC Sanitation 5 2 5x104 Products Coliforms Sanitation 5 2 5x101 E. coli Health 2 5 1 <1.8 Yeast and Sanitation 5 2 5x102 mould S. aureus Health 2 5 2 102 Salmonella Health 2 5 0 0 Heat Treated E. coli Health 2 5 1 101 Fermented S. aureus Health 2 5 1 5x101 Sausage Raw E. coli Health 2 5 1 102 Fermented S. aureus Health 2 5 1 2.5x102 Sausage NonE. coli fermented S. aureus Ready-to-eat Sausage Health 2 Health 2 5 5 2 102 2 102

M 106 104 103 104 -106 104 103 104 104 -103 104 103 104 103 104

TABLE 3c. Foods for which Microbiological Guidelines have been established. FOOD CATEGORY GUIDELINE NATURE SAMPLING OF PARAMETERS CONCERN n c m M Health 2 Health 2 Health 2 Health 1 Sanitation Health 2 5 00 5 00 5 00 5 00 5 3 104 5 2 101 ----106 103

Heat Treated Salmonella Sausage, Raw Campylobacter Fermented coli or C. jejuniA Sausage and Non- Yersinia fermented Sausage enterocolitica A E. coli O157 Deboned ACC Poultry E. coliB Products

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Products (Precooked)

S. aureus

Health 2

5 1 102

104

Salmonella Health 2 C. jejuni or C. Health 2 coliA Y. enterocoliticaA Health 2 Dry Mixes (Gravy, ACC Sanitation Sauce, Soup) Heat ColiformsB Sanitation and Serve Yeast and Moulds Sanitation E. coli Health 2 S. aureus Health 2 C. perfringens Health 2 Salmonella Health 2

5 00 5 00 5 5 5 5 5 5 5 5 0 3 3 3 2 2 2 0 0 104 101 5x102 101 102 102 0

106 103 104 103 104 103 -

TABLE 3d. Foods for which Microbiological Guidelines have been established. FOOD CATEGORY GUIDELINE NATURE SAMPLING OF PARAMETERS CONCERN n c m M 5 2 105 5 5 5 5 5 5 5 5 5 5 5 2 2 0 0 2 2 2 2 0 2 0 102 102 0 0 107 103 104 -

Soybean Products Psychrotrophic Sanitation (Ready-to-eat) bacteria E. coli Health 2 S. aureus Health 2 Salmonella Health 2 Yersinia Health 2 enterocolitica Spices (Readyto- C. perfringens Health 2 eat only) B. cereus Health 2 E. coli Health 2 S. aureus Health 2 Salmonella Health 2 Yeast and Mould Sanitation Bottled Water Pseudomonas Health 2 aeruginosa Aeromonas Health 2 hydrophila Sprouted Seeds Fecal Coliforms Sanitation (e.g. Alfalfa and E.coli Health 2 Bean Sprouts) Salmonella Health 2

104 106 104 106 102 103 102 104 0 2 10 104 0/100 mL -

5 0 0/100 mL 5 2 103 5 2 102 500 105 103 -

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TABLE 3e. Foods for which Microbiological Guidelines have been established. FOOD CATEGORY GUIDELINE NATURE SAMPLING OF PARAMETERS CONCERN n c m M 5 5 5 5 5 5 5 5 5 5 5 5 3 1 0 2 1 2 2 1 0 2 1 2 104 101 0 102 104 104 103 <1.8 0 101 102 102 105 103 104 105 105 104 101 102 104 103

Health Foods ACC Sanitation a) Raw Organ Derived E. coli Health 2 Products and Herbal Salmonella Health 1 Products (in tablets, S. aureus Health 2 capsules or powders, Health 2 consumed at <10 B. cereus C. perfrigens Health 2 g/day) C b) Powdered Protein, ACC Sanitation Meal Replacements, E. coli Health 2 and Dietary Salmonella Health 1 Supplements S. aureus Health 2 B. cereus Health 2 C. perfringens Health 2

TABLE 3f. Foods for which Microbiological Guidelines have been established. FOOD CATEGORY Raw Oyster GUIDELINE NATURE SAMPLING OF PARAMETERS CONCERN n c m M

Unpasteurized apple juice

Vibrio parahaemolyticus At harvest At consumer level E. coli O157:H7 E. coli

Health 2 Health 1 Health 1 Health 2

30 5 5 5

15 1 0 2

101 102 102 104 0 100 1000

TABLE 3g. Foods for which Microbiological Guidelines have been established. FOOD GUIDELINE NATURE OF CATEGORY CONCERN Low-acid foods processed If heat process alone is to achieve F0 < 3 to commercial sterility commercial sterility F0=3 must be achieved Health 1 If a retorted product is not commercially Health 2 sterile but heat process is known to be above F0 = 3 If the product is judged to be Health 1 underprocessed F0 <3 If contamination is assessed at Health 2; if for infant postprocessing formula then Health 1

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TABLE 4 Raw Foods for which there is a proposed guideline FOOD CATEGORY GUIDELINE NATURE OF SAMPLING CONCERN PARAMETERS n c m M Ground beef found E. coli Health 2 positive for E. coli O157:H7 O157:H7 Generic E. coli Becomes Health 5 0 0 100 1 if generic E. coli >100 cfu/g or level not determined Raw ground beef Negative for E.coli O157:H7 Generic E. coli Sanitation 5 0 0 >100 Raw ground beef Positive for Health 2 derived from E.coli trimming or O157:H7 carcasses found Generic E. coli Becomes Health 5 0 0 100 positive for E.coli B 1 if generic E. O157:H7 at coli >100 cfu/g Processor or level not determined

TABLE 5 Foods for which there is a Guideline other than Microbiological FOOD CATEGORY METHOD OR PROPOSED EQUIVALENT GUIDELINE GUIDANCE NATURE OF CONCERN

Commercially for pH: Plant and I) If pHC >4.6, and Health 1 Prepared MFHPB-03, Mushroom water activityC > 0.92 Vegetable or water activity: Products packed OR Health 1 Mushroom MFLP-66, in OilA ii) If pHD is the sole Products in Oil commercial sterility barrier and <4.6 but and/or thermal there is no control over processing: the growth of yeast and MFHPB-01 mould OR Health 2 iii) If aw >0.85E but <0.92 and pH >5.0 or not controlled OR iv) If product has not Health 1 received a thermal processF sufficient to kill spores of proteolytic C. botulinum and the 195

Home Prepared Garlicin-oil Products

thermal process is the Health 1 sole barrier OR v) If the product relies on the thermal process as the sole barrier and only is sufficient to kill nonproteolytic spores of C.botulinumG but is not stored at refrigeration temperature. It's Your Health Product stored at room Health 1 Garlic-in-oilB temperature or stored refrigerated but has a use by date of >10 days from the date of manufacturing.

For example a thermal process where every container received a minimal heat treatment to render the product commercially sterile. Documentation could include time-temperature profile charts and thermal process calculations to verify the Fo delivered for the worst case situation. Results from inoculated pack challenge studies or predictive modelling results can also be used as additional evidence that the concerns over proteolytic strains of C. botulinum has been addressed. For example every container has received a pasteurization process sufficient to inactivate the nonproteolytic spores of C. botulinum (eg 10 minutes at 90/C or equivalent) but is not labelled and stored refrigerated.

TABLE 6 Raw Foods for which there is a Guideline FOOD GUIDELINE CATEGORY Brined Mushrooms for Imported Further Processing Mushroom March 1998 NATURE CONCERN Brined Visual inspection by lot Sanitation Protocol or container Determine % salt as Sanitation specified in protocol Determine the level of Health 2 S. aureus Examine for Health 2 Enterotoxin GUIDANCE OF

There is a sanitation concern for product which is visually defective e.g. low brine, off smell, musty, cloudy brine etc. and for containers which have a salt concentration <15 % or lots with a Q value <16% (average salt concentration) as the salt concentration could be low enough to permit the growth of S. aureus. There is a Health 2 concern if the level of S.aureus in a container or lots is >104 cfu/g and implicated product should be destroyed. Containers with an S. aureus count is < 10 cfu/g can be used for further processing. If the S.aureus level is >10 cfu/g but <104 196

examine for enterotoxin. There is a Health 2 concern if S.aureus enterotoxin is detected in a container or a lot. The implicated container or lot is not acceptable for further processing and should be destroyed.

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Guidelines for Writing Lab reports

Name and Date: Your name and the date should be shown on the top of the first page of the lab report. Introduction: The Introduction should tell the reader (i) what is the experimental question the paper will address, (ii) what is the background biology that makes this an interesting question (e.g. what are the properties of the organisms used, etc), and (iii) a very brief overview of the approach used to answer the experimental question Materials and Methods: The Materials and Methods section should succinctly describe what was actually done in a narrative format (i.e., not a numerical series of steps as in an experimental protocol). It should include sufficient description of the techniques used that it is clear to the reader what experiments were done and how (not what was written in the protocol, but what you actually did). The details of the protocol do not need to be reproduced in the text but an appropriate reference should be cited. Any changes from the protocol provided in handouts should be described. It is not appropriate to indicate volumes of solutions added ­ instead indicate the relevant information about the experiment such as final concentrations used, etc Results: Begin each paragraph with an opening sentence that tells the reader what property is being tested in the experiments described in that paragraph and why. Write the opening sentence in bold font for emphasis. (Sometimes a complete sentence is used and sometimes a short phrase is used ­ either style is OK but the style should be used consistently throughout the report.) Any results that include multiple data points that are critical for the reader to evaluate the experiment should be shown in tables or figures. However, the results should be summarized in accompanying text (Note that when referring to a particular table or figure, they should be capitalized: Table 1, Figure 6, etc.) The text of the Results section should be succinct but should provide the reader with a summary of the results of each table or figure. Not all results deserve a separate table or figure. As a rule of thumb, if there are only a few numerical results or a very simple conclusion describe the results in the text instead of in a table or figure. Your report should focus on what worked, not things that didn't work (unless they didn't work for reasons that are interesting and provide biological insights). Tables and Figures: All tables and figures should be put into a contextual framework in the corresponding text. Tables and figures should present information in a format that is easily evaluated by the reader. A good rule of thumb is that it should be possible to figure out the meaning of a Table or Figure without referring to the text. Tables and figures should typically summarize results, not simply present large amounts of raw data. When possible, the results should provide some way of evaluating the reproducibility or statistical significance of any numbers presented. Tables should be sequentially numbered. Each table should have a title (shown above the table) that describes the point of the table. For example, "Table 1. Bacterial strains and plasmids used in this study." If necessary to interpret the table, specific descriptions about what a result represents or how the results were obtained can be described in a legend below the table. Figures should be sequentially numbered. Each figure should have a title (shown below the table) that describes the point of the table. For example, "Figure 1. Isolation of MudJ insertion mutants." If necessary to interpret the figure, specific descriptions about what a result

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represents or how the results were obtained can be described immediately following the title. Tables and figures may be printed on separate pages that follow the Reference section Alternatively, the tables and figures may be integrated into the paper if you are using a page layout program. However, if they are integrated into the paper make sure that there is not a page break in the middle of a table or figure. Tables and figures should not be directly copied from another source without credit. Even when credit is given, the table or figure should be redrawn. Discussion: Do not simply restate the results -- explain your interpretations of the results. How did your results compare with the expected results? Are there other experiments that could be done to confirm your results (if so, what experiments and what are the predicted results)? References: Give complete references to the sources for any fact, idea, or opinion not your own which was cited in the report. List the references alphabetically at the end of your report, using the format for different sources shown below: Books: Maloy, S., V. Stewart, and R. Taylor. 1996. Genetic Analysis of Pathogenic Bacteria. Cold Spring Harbor Laboratory Press, NY. Book chapters: Rice, L., and B. Hemmingsen. 1997. The enumeration of hydrocarbon-degrading bacteria. In D. Sheehan (ed.) Methods in Molecular Biotechnology: Protocols in Bioremediation, pp. 99109. Humana Press, Totowa, NJ. Published papers: Rohwer, F., A. Segall, G. Steward, V.Seguritan, F. Wolven, M. Breitbart, and F. Azam, 2000. The complete genome sequence of the marine Roseophage SIO1 shares homology with nonmarine phages. Limnol. Oceanography 45: 408-418.

Format and proofreading: Certain general rules are commonly followed in scientific writing. Nomenclature. Use correct bacterial nomenclature. Abbreviations. Use standard abbreviations (hr, min, sec, etc) instead of writing complete words. Some common abbreviations that do not require definition are shown on the attached table. Define all other abbreviations the first time they are used, then subsequently use the abbreviation [e.g. Ampicillin resistant (Amp)]. As a general rule, do not use an abbreviation unless a term is used at least three times in the manuscript. With two exceptions (the degree symbol and percent symbol), a space should be left between numbers and the accompanying unit. In general, abbreviations should not be written in the plural form (e.g. 1 ml or 5 ml, not mls). Past, present, and future tense. Results described in your paper should be described in past tense(you've done these experiments, but your results are not yet accepted "facts"). Results from published papers should be described in the present tense (based upon the assumption that published results are"facts"). Only experiments that you plan to do in the future should be described in the future tense.

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Third vs first person. It is OK to use first person in scientific writing, but it should be used sparingly reserve the use of first person for things that you want to emphasize that "you" uniquely did (i.e. not things that many others have done as well). Most text should be written in the third person to avoid sounding like an autobiographical account penned by a narcissistic author. However, it is better to say"It is possible to .." than to say "One could ...". Writing that uses the impersonal pronoun "one" often seems noncommittal and dry. In addition, inanimate objects (like genes, proteins, etc) should be described in third person, not with anthropomorphic or possessive terms (e.g., instead of saying "its att site", say "the chromosomal att site"). Empty phrases. Avoid using phrases that do not contribute to understanding. For example, the following phrases could be shortened (or completely deleted) without altering the meaning of a sentence: "the fact that ..." (delete); "In order to ..." (shorten to simply "To ..."). Likewise, the title of a table of results does not benefit from the preface "Results of ...". In short, don't use more words than you need to make your point. Specify. If several expressions modify the same word, they should be arranged so that it is explicit which word they modify. It is common to use a pronoun such as "it" or "they" to refer to a concept from the previous sentence. This is OK as long as there is only one concept that "it" or "they" means. However, if there are more than one concepts it is easy for the reader to get confused about what the pronoun is meant to specify (even if you know which one you mean). It is better to error on the side of redundancy by repeating the concept in subsequent sentences, than to take the chance of confusing the reader. Don't make the reader guess what you mean. Parentheses. Avoid double parentheses. For example, "Three gene products catalyze reactions in the pathway for proline biosynthesis (Figure 1) (3)" could be reworded to say "Figure 1 shows the three reactions of the pathway for proline biosynthesis (3)." Proofreading. Always spellcheck your paper and carefully proofread your paper before submission. In addition to checking for errors and typos, read your paper to yourself as if you were reading it out loud to ensure that the wording and sentence construction is not clumsy.

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References and selected readings

1. American Public Health Association (APHA). 1992. Compendium of Methods for the Microbiological Examination of Foods. Third edition. C. Vanderzant and D.F. Splittstoesser (eds.). American Public Health Association Inc., Washington, D.C. 2. Alley, M. 1996. The craft of scientific writing, 3 rd edition. Prentice Hall, NJ. [and accompanying web site: http://filebox.vt.edu/eng/mech/writing/] 3. American Can Company. 1957. The Howard Mold Count Method as Applied to Tomato Products. American Can Company, Research Division, Maywood, Illinois. 4. American Public Health Association (APHA). 1992. Standard Methods for the Examination of Dairy Products. Chapter 6. Sixteenth edition, American Public Health Association, Washington, D.C. 5. American Public Health Association (APHA). 2001. Compendium of Methods for the Microbiological Examination of Foods Fourth edition. F.P. Downes and K. Ito (editors), American Public Health Association, Washington, D.C. 6.American Public Health Association. 1985. Laboratory Procedures for the Examination of Seawater and Shellfish, 5th ed. APHA, Washington, DC. 7. American Public Health Association. 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. 8. Andrews, W.H. 1989. Methods for recovering injured "classical" enteric pathogenic bacteria (Salmonella, Shigella, and enteropathogenic Escherichia coli) from foods. Chapter 3. In: B. Ray (ed.) Injured Index and Pathogenic Bacteria, CRC Press, Boca Raton, FL. pp. 55-113. 9.AOAC INTERNATIONAL. 1995. Official Methods of Analysis, 16th ed. AOAC INTERNATIONAL, Arlington, VA. 10. AOAC. 2001. Bacteriological Analytical Manual (Online) Chapter 14, Bacillus cereus. USDA, Center for Food Safety and Applied Nutrition. 11. Association of Official Analytical Chemists (AOAC) International. 1995. FDA Bacteriological Analytical Manual, Eighth Edition. AOAC International, Arlington, VA. 12. Association of Official Analytical Chemists (AOAC). 1999. Official Methods of Analysis of AOAC International. 16th. AOAC. Gaithersburg, Md. Vol. I, Chapt. 17. pp:32-34. 13. Association of Official Analytical Chemists. 1990. Official Methods of Analysis, 15th ed. AOAC, Arlington, VA. 14. Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition. L.C. Parks (editor). CRC Press Inc. 15. Baird-Parker, A.C. 1962. An improved diagnostic and selective medium for isolating coagulase positive staphylococci. J. Appl. Bacteriol. 25:12-19.

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16. Baird-Parker, A.C. and E. Davenport. 1965. The effect of recovery medium on the isolation of Staphylococcus aureus after heat treatment and after storage of frozen or dried cells. J. Appl. Bacteriol. 28:390-402. 17. Banks, J.G. and R.G. Board. 1987. Some factors influencing the recovery of yeasts and moulds from chilled foods. Intl. J. Food Microbiol. 4:197. 18. Beuchat, L.R. and A.D. Hocking. 1990. Some considerations when analysing foods for the presence of xerophilic fungi. J. Food Prot. 53:984-989. 19. Bhatia Rajesh & lchhpujani R.L.: Quality Assurance in Microbiology CBS Publishers and Distributors, New Delhi, 1995. 20. Cochran, W. G. 1950. Estimation of bacterial densities by means of the "Most Probable Number." Biometrics 6:105-116. 21. Collins-Thompson, D.L., A. Hurst and B. Aris. 1974. Comparison of selective media for the enumeration of sublethally heated food-poisoning strains of Staphylococcus aureus. Can. J. Microbiol. 20:1072-1075. 22. Continental Can Company. 1968. Mold Counting of Tomato Products. Continental Can Company Inc., Research and Development, Chicago, Illinois. 23. Crisley, F.D., J.T. Peeler and R. Angelotti. 1965. Comparative evaluation of five selective and differential media for the detection and enumeration of coagulase-positive staphylococci in foods. Appl. Microbiol. 13:140-156. 24. D'Aoust, J.-Y. 1977. Effect of storage conditions on the performance of bismuth sulfite agar. J. Clin. Microbiol. 5: 122-124. 25. D'Aoust, J.-Y. 1984. Effective enrichment-plating conditions for detection of Salmonella in foods. J. Food Prot. 47:588-590. 26. D'Aoust, J.-Y. 1989. Salmonella. Chapter 9. In: M.P. Doyle (ed.). Foodborne Bacterial Pathogens, Marcel Dekker Inc., New York, NY. pp. 327-445. 27. D'Aoust, J.-Y., 1981. Update on preenrichment and selective enrichment conditions for detection of Salmonella in foods. J. Food Prot. 44:369-374. 28. D'Aoust, J.-Y., A.M. Sewell and P. Greco. 1993. Detection of Salmonella in dry foods using refrigerated preenrichment and enrichment broth cultures: interlaboratory study. J. of AOAC Int. 76:814-821. 29. D'Aoust, J.-Y., C. Maishment, D.M. Burgener, D.R. Conley, A. Loit, M. Milling and U. Purvis. 1980. Detection of Salmonella in refrigerated preenrichment and enrichment broth cultures. J. Food Prot. 43:343-345. 30. D'Aoust, J.-Y., H.J. Beckers, M. Boothroyd, A. Mates, C.R. McKee, A.B. Moran, P. Sado, G.E. Spain, W.H. Sperber, P. Vassiliadis, D.E. Wagner, and C. Wiberg. 1983. ICMSF Methods Studies. XIV. Comparative study on recovery of Salmonella from refrigerated preenrichment and enrichment broth cultures. J. Food Prot. 46:391-399.

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31. D'Aoust, J.-Y., A.M. Sewell and D.W. Warburton. 1992. A comparison of standard cultural methods for the detection of foodborne Salmonella. Int. J. Food Microbiol. 16: 41-50. 32. D'Aoust. J.-Y., A.M. Sewell and C. McDonald. 1995. Recovery of Salmonella spp. from refrigerated preenrichment cultures of dry food composites. J. AOAC Int. 78: 1322-1324. 33. Day, R. 1995. Scientific English: A guide for scientists and other professionals, 2 nd edition. Orynx Press 34. Day, R. 1998. How to write and publish a scientific paper, 5 th edition. Orynx Press. 35. de Man, J. C. 1983. MPN tables, corrected. Eur. J. Appl. Biotechnol. 17:301-305. 36. Deak, T., J. Chen, D.A. Golden, M.S. Tapia, J. Tornai-Lehoczki, B.C. Viljoen, M.T. Wyder and L.R. Beuchat. 2001. Comparison of dichloran 18% (DG18) agar with general purpose mycological m edia for enumerating food spoilage yeasts. Int. J. Food Microbiol. 67:49-53. 37. Eisenhart, C., and P. W. Wilson. 1943. Statistical methods and control in bacteriology. Bacteriol. Rev. 7:57-137. 38. Flowers, R. S., Gecan, J.S. and Pusch, D.J. 1992. Laboratory Quality Assurance. In Vanderzant, C. and Splittstoesser, D.F. (ed). Compendium of Methods for the Microbiological Examination of Foods. American Public Health Association. 39. Food and Agriculture Organization of the United Nations. 1991. Manual of Food Quality Control 12. Quality Assurance in the Food Control Microbiology Laboratory. Rome. Italy 40. Food and Drug Administration. 1978. EDRO Data Codes Manual. Product Codes: Human Foods. FDA, Rockville, MD. 41. Food and Drug Administration. 1989. Laboratory Procedures Manual. FDA, Rockville, MD. 42. Food and Drug Administration. 1993. Investigations Operations Manual. FDA, Rockville, MD. 43. Garthright, W. E. 1993. Bias in the logarithm of microbial density estimates from serial dilutions. Biom. J.35: 3, 299-314. 44. Garthright, W. G. and Blodgett, R.J. 2003, FDA's preferred MPN methods for Standard, large or unusualtests, with a spreadsheet. Food Microbiology 20:439-445. 45. Goben, G., and J. Swan. 1990. The science of scientific writing. Am. Scientist 78: 550558. [Available online at http://www.research.att.com/~andreas/sci.html] 46. Gould, W.A. 1974. Tomato Production, Processing and Quality Evaluation. AVI Publishing Co., Inc., Westport, Connecticut. 47. Haldane, J.B.S. 1939. Sampling errors in the determination of bacterial or virus density by the dilution method. J. Hygiene. 39:289-293.

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48. Halvorson, H. O., and N. R. Ziegler. 1933. Application of statistics to problems in bacteriology. J. Bacteriol. 25:101-121; 26:331-339; 26:559-567. 49. Harmon, S.M. 1982. New Method for Differentiating Members of the Bacillus cereus Group: Collaborative Study. J. Assoc. Off. Anal. Chem. 65:1134-1139. 50. Hauschild, A.H.W. 1975. Criteria and procedures for implicating Clostridium perfringens in food-borne outbreaks. Can. J. Public Health 66:388-392. 51. Hauschild, A.H.W. and R. Hilsheimer. 1974. Enumeration of food-borne Clostridium perfringens in egg yolk-free tryptose-sulfite-cycloserine agar. Appl. Microbiol. 27:521526. 52. Hauschild, A.H.W., R.J. Gilbert, S.M. Harmon, M.F. O'Keeffe and R. Vahlefield. 1977. ICMSF methods studies. VIII. Comparative study for the enumeration of Clostridium perfringens in foods. Can. J. Microbiol. 23:884-892. 53. Health Canada. 2003 . Compendium of Analytical Methods . http://www.hcsc.gc.ca/food-aliment/mh-dm/mhe-dme/compendium/e_index.html 54. Hitchins, A.D. 1995. Listeria monocytogenes. Chapter 10. Revised 1998. Bacteriological Analytical Manual (BAM). AOAC International. Gaithersburg, MD. 55. Holbrook, R. and Anderson, J.M., 1980. An improved selective and diagnostic medium for the isolation and enumeration of Bacillus cereus in foods. Can. J. Microbiol. 26: 753759. 56. Horwitz, W. (ed.) 1980. Official Methods of Analysis of the Association of Official Analytical Chemists. (44.096), Thirteenth edition. AOAC., Washington, D.C. 57. Howaritz PJ, Kowanitz JH: Laboratory quality assurance (McGraw Hill Book Companry, New York (1987). 58. International Commission on Microbiological Specifications for Foods (ICMSF). 1978. Microorganisms in foods. Their significance and methods of enumeration. Second edition. p. 157-159, 318-319. University of Toronto Press, Toronto. 59. International Commission on Microbiological Specifications for Foods. 1986. Microorganisms in Foods. 2. Sampling for Microbiological Analysis:Principles and Specific Applications, 2nd ed. University of Toronto Press, Toronto, Ontario, Canada. 60. Jarvis, B. 1973. Comparison of an improved rose bengal-chlortetracycline agar with other media for the selective isolation and enumeration of moulds and yeasts in foods. J. Appl. Bacteriol. 36: 723-727. 61. Jarvis, B. and A.P. Williams. 1987. Methods for detecting fungi in foods and beverages. In: Beuchat, L.R. (ed.), Food and beverage mycology. Second edition. p. 599-636. Avi Publishing Co., Westport, Connecticut. 62. Koskitalo, L.D. and M.E. Milling. 1969. Lack of correlation between egg yolk reaction in staphylococci medium 110 supplemented with egg yolk and coagulase activity of staphylococci isolated from cheddar cheese. Can. J. Microbiol. 15:132-133.

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63. Landry, W.I., J.E. Gilchrist, S. McLaughlin, and J.T. Peeler 1988. Analysis of abnormal canned foods. AOAC Abstracts 64. Lobban, C. and M. Schefter. 2002. Successful Lab reports: A Manual for Science Students. Cambridge University Press 65. Lovett, J. 1987. Listeria isolation. Chapter 29. Bacteriological Analytical Manual (BAM). AOAC. Arlington, Virginia. 66. Lovett, J. and A.D. Hitchins. 1988. Listeria isolation. Chapter 29. Revised October 13, 1988. Bacteriological Analytical Manual (BAM). AOAC. Arlington, Virginia. 67. McClain, D. and W.H. Lee. 1988. Development of USDA-FSIS Method for Isolation of Listeria Monocytogenes from Raw Meat and Poultry. JAOAC. 71(3):660-664. 68. McClain, D. and W.H. Lee. 1989. FSIS Method for the Isolation and Identification of Listeria Monocytogenes From Processed Meat and Poultry Products. USDA-FSIS Laboratory Communication No. 57. Revised May 24, 1989. 69. McCrady, M. H. 1915. The numerical interpretation of fermentation-tube results. J. Infect. Dis. 17:183-212. 70. McGuire, O.E. 1964. Wood Applicators for the Confirmatory Test in Bacteriological Analysis of Water. Public Health Reports. 79: 812-814. 71. McMillan, V. 1988. Writing papers in the biological sciences. Bedford Books, NY. 72. Mislivec, P.B., L.R. Beuchat and M.A. Cousin. 1992. Yeasts and Moulds. In: Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C. p. 237-249. 73. Mossel, D.A.A., C.L Vega, and H.M.C. Put. 1975. Further studies on the suitability of various media containing antibacterial antibiotics for the enumeration of moulds in food and food environments. J. Appl. Bacteriol. 39: 15-22American Public Health Association. 1984. Recommended Procedures for the Examination of Sea Water and Shellfish; Fourth Edition. A.E. Greenberg and D.A. Hunt (eds.). American Public Health Association Inc., Washington, D.C. 74. National Academy of Sciences. 1969. An Evaluation of the Salmonella Problem. National Academy of Sciences, Washington, DC. 75. Peeler, J. T., G. A. Houghtby, and A. P. Rainosek. 1992. The most probable number technique, Compendium of Methods for the Microbiological Examination of Foods, 3rd Ed., 105-120. 76. Powers, E.M. and T.G. Latt. 1977. Simplified 48-Hour IMViC Test: an Agar Plate Method. Appl. Environ. Microbiol. 34: 274-279. 77. Rayman, M.K., C.E. Park, J. Philpott and E.C.D. Todd. 1975. Reassessment of the coagulase and thermostable nuclease tests as means of identifying Staphylococcus aureus. Appl. Microbiol. 29:451-454. 78. Report of the Forty-Fifth Session of the Executive Committee of the Codex Alimentarius Commission. Joint FAO/WHO Food Standards Programme. Rome, 3-5 June 1998.

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