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 (SCHAEFFER–FULTON OR WIRTZ–CONKLIN)................................. 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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UNIDO. developing countries are required to ensure that their sanitary and phytosanitary measures are based on an assessment. guidelines and recommendations adopted by international trade agreements. the scientific community. 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 manual is written in such a manner that it can be used for in-house training of new technicians. guidelines and recommendations that describe processes and procedures for the safe preparation of food. There is a critical need for technical advice on risk assessment of microbiological hazards in foods to meet the needs of national governments. Codex has elaborated standards. The manual details microbiological risk assessment of various food categories. In this regard. animal or plant life or health. of the risks to human. This manual has been developed with the help of UNIDO inline with the above stated spirit. isolation and identification of pathogenic food-borne bacteria. APHA and Health Canada. These methods have been adapted from methods recommended by the ICMSF. In the case of microbiological hazards. Description of equipment maintenance and calibration is detailed including quality control of media and internal laboratory audit. The application of these standards. This requires an elaborate laboratory with equipment and personnel well trained to carry out the analysis. as appropriate to the circumstances. such as those administered by the WTO. Because of the global nature of food production. taking into account the risk assessment techniques developed by the relevant international organizations. Most developing countries lack the resources to put up food microbiology laboratories and to man them adequately to international standards. AOAC. FDA. 4 . 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. The standards. guidelines and recommendations is intended to prevent or eliminate hazards in foods or reduce them to acceptable levels. 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. are playing an increasingly important role in protecting the health of consumers. Methods of estimating sanitary indicator microorganisms as well as enumeration of moulds and yeasts are documented. infectious agents can be disseminated from the original point of processing and packaging to locations thousands of miles away.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 standard operating procedures and quality control guidelines relating to food sampling and methods of analysis are included. The manual contains detailed description of microbial enumeration. and marketing. the food industry. guidelines and recommendations related to food safety. 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. manufacturing. trade organizations and international consumer groups.

Staphylococcus aureus and toxigenic fungi e. Yersinia enterocolytica etc. Coliform group of microorganisms and Escherichia coli are commonly used as indicator organisms. 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. 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. iv) Infectious microorganisms Definition: Organisms whose presence in small numbers in food or water and when consumed can cause infection. Definition: an index organism is one whose presence implies the possible occurrence of a similar but pathogenic organism. Hepatitis B virus etc. i) Indicator organism(s). The most common microorganisms in this category includes Salmonella typimurium. Some useful microorganisms e. ii) Index organism. Shigella sonnei. lactic acid bacteria are considered as spoilage organisms when in beer. wine and fruit juices but not in milk. In this case the food acts as a vector but not necessarily as a growth medium. Bacillus anthracis. Growth of the contaminant in that food will spoil the food making it unfit for human consumption.Microbial Food analysis 1: Reasons for microbial food analysis. Organisms in this group includes. Salmonella typhi.g. The most common microorganisms in this category includes. Definition: an indicator organism or group of organisms is one whose numbers in a product reflect the success or failure of "good manufacturing practices". 5 .Infectious organisms can be transmitted by various ways including man to man and are said to be contagious. b) Those which cause intoxication must grow in food in large numbers and produce enough toxin and when consumed together with food cause intoxication. v) Spoilage organisms Definition: Spoilage organisms are the organisms whose growth in the food creates undesirable characteristics in that food.g. coli. Vibrio cholerae O1. Vibrio parahaemolyticus. Salmonella sp. Clostridium botulinum. • 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. enteropathogenic E.g. Aspergillus flavus. coli is used an index organism a its presence nd indicates possible presence of pathogenic enterobacteriacea e. E.

• Salmonella sp.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. • E. 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 6 . coli • V.

Stained preparations A “smear” of bacteria or yeast is made on a microscope slide. Yeast cells can be stained by Gram’s method but it is of no value in their identification.g.g. stained. e. 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. bacterial endospores. 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. from aiding in identification to checking for contamination. without using a coverslip. areas of fat storage. between Gram-positive and Gram-negative bacteria. 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!). 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. be successfully accomplished. 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. examined with the aid of a microsope. or different groups. e. and includes a stage which differentiates between either different parts of a cell. A culture on agar medium is much preferable to a liquid culture for making a smear. along with descriptions of colonies. sometimes with heating.g. biochemistry and molecular biology is then needed. The basis of Gram’s staining method is the ability or otherwise of a cell stained with crystal violet to retain the colour when 7 . e. . (2) a differential stain involves a sequence of several stains. 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. The information gained. 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 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. dried and. is the starting point for identification of genera and species but further work involving physiology. 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. sometimes. inclusions that do not stain.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. fixed. Bacteria and yeast Yeast can be seen in unstained wet mounts at magnifications x100. Aseptic technique must be observed when taking samples of a culture for making a smear.

Flame loop and allow to cool. Hold the slide with forceps (optional but avoids stained fingers).0g dissolved in absolute alcohol 100 ml B. this is also a useful reminder of which side of the slide is being used.0g distilled/deionised water 300 ml 8 . being the cation) such as methylene blue. crystal violet and safranin. 1. Flame a wire loop to ensure that no culture accidentally remains from a previous operation.treated with a differentiating agent. transfer a very small part of a single colony from a plate or slope of agar medium into the tap water. Suitable stains include basic dyes (i.0g in distilled/deionised water 100ml Add 25 ml A to 100 ml B Lugol’s iodine solution: iodine 1. 5. 6. 3. usually pink/red. potassium iodide 2. When finished.e. 2.e. ammonium oxalate 1. 6. Staining solutions (relevant to procedures described below) Crystal violet solution: A. it should be visible to the naked eye as a whitish area. the use of tap water to prepare the smear will probably be unnecessary and may result in a smear with too few cells. This is called a heat-fixed smear. usually. Blot dry the smear with filter/fibre free blotting paper using firm pressure but not sideways movements that might remove the smear. i. 8. 7. Fixing is necessary to ensure that cells adhere to the slide and to minimise any post mortem changes before staining. crystal violet 2. salts with the colour-bearing ion. at a 45° angle over the sink. Examine under oil immersion. 9. Put the slide with the fixed smear uppermost on a staining rack over a sink or staining tray. 4. 4. dispose of slides into discard jar. Transfer one or two loopfuls of tap water on to the centre of the slide. Those that lose the colour. 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. Clean a plain microscope slide thoroughly using lens tissue. 5. If it is necessary to use a liquid culture or sample. Flame the loop. the chromophore. A simple stain. 2. Rinse off the stain with tap water. called Gram negative. Thoroughly cover the smear with stain and leave for. 30 seconds. Bacteria that retain the violet/purple colour are called Gram-positive. Using aseptic technique. are stained in the contrasting colour of a counterstain. date and initials. Label a microscope slide with a marker pen to record the culture being used. usually alcohol (although professionals sometimes use acetone).0g. 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. Making a smear. 3. If the amount of culture on the loop is easily visible you have taken too much! 7. 1.

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. Thoroughly cover the smear with crystal violet solution and leave for 1 minute. Therefore.5% w/v. a. j. but some o bacteria are naturally only weakly Gram-positive. g. wash off any that remains (and any on the back of the slide) with iodine solution. this may happen with a Gram-negative reaction. i. Hold the slide with forceps (optional but avoids stained fingers). at a 45° angle over the sink. l. Pour off the stain. for 30 seconds. Put the slide with the fixed smear uppermost on a staining rack over a sink or staining tray.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. Iodine solution acts as a “mordant” (a component of a staining procedure that helps the stain to adhere to the specimen). Put the slide back on staining rack. the times given here relate only to the solutions specified here. dispose of slides into discard jar. h. d. k. Bacterial Motility 1.g. f. Cover the smear with iodine solution and leave for 1 minute. continue treating with alcohol until the washings are pale violet. Blot dry the smear with filter/fibre free blotting paper using firm pressure but not sideways movements that might remove the smear. safranin solution. Rinse immediately with tap water. Hold the slide with forceps at a 45° angle over the sink wash off the iodine solution with 95% (v/v) ethanol (not water). check that the labeling has not been washed off by the alcohol. 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. 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.use the special microscope slide with a depression 9 . Examine under oil immersion. c. Put the slide back on staining rack. Don’t despair if the stained smear is not visible to the naked eye. Hanging drop method of motility: . e. Rinse off the stain with tap water. m. b. e. Cover the smear with the counterstain. When finished. At the end of the procedure. 0. n. a crystal violet-iodine complex is formed and the smear looks black.

Endospores do not stain easily but. Observe for motility using the high power lens. they strongly resist decolorization. The inoculated medium is incubated at appropriate temperature for 24 hr. Inoculation is done by stabbing the medium at the center. Endospore position within the cell is characteristic and may be central. This location and size of endorspores vary with the species. Semi-solid agar method The agar medium is prepared with the agar content of 0. 2.a cover slip .a micropscope . The medium is put into test tubes. motility is detected by observing turbidity at the line of inoculation.2%.immersion oil . Endospores are spherical to elliptical in shape and may be either smaller or larger than the parent bacterial cell. subs terminal. once stained. This property is the basis of the Schaeffer-Fulton or Wirtz-Conklin method of staining endospores. This structure is called an endospore since it develops within the bacterial cell. they are often of value in identifying bacteria. Motility is characterized by fast unidirectional movement as compared to the Brownian motion whereby the cells move round in one particular point. Endospore staining (Schaeffer–Fulton or Wirtz–Conklin) Materials 24-to 48 hours nutrient agar slant cultures of Bacillus megaterium (ATTC 12872) and Bacillus macerans (ATCC 8244).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.. 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. The 10 . thus. or terminal.

vegetative cells stain red. Gently heat on the hot plate (just until the stain steams) for 5 to 6 minutes after the malachite green solution begins to steam. The spores. and rinse the slide with water for 30 seconds. 2. air dry (or use a slide warmer). both endospores and free spores. 18-hour tryptic soy agar slants of Alcaligenes faecalis (ATCC8750. Blot dry with bibulous paper and examine under oil immersion. rind stand. Place the slide to be stained on a hot plate or boiling water bath equipped with a staining loop or rack. Flagella staining: West and Difco’s SpotTest Methods Materials Young. peritrichously flagellated) and Pseudomonas fluorecens (ATCC 13525. A coverslip is not necessary. place the names of the respective bacteria on the edge of four clean glass slides. Heat is used to provide stain penetration. allow the slide to cool. 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. Soak the paper with the malachite green staining solution. Aseptically transfer one species of bacterium with an inoculating oop to each of the l respective slides. Rinse the slide with water for 30 seconds. The rest of the cell is then decolirized and counterstained a light red with safranin. Replace the malachite green solution as it evaporates so that the paper remains saturated during heating. Remove the paper using forceps. Procedure 1. 3. 6. and heat-fix. Cover the smear with paper toweling that has been cut the same size as the microscope slide. 5. 8. wire gauze pad. 4.endorspores are stained with malachite green. With a wax pencil. Counterstain with safranin for 60 to 90 seconds 7. stain green. an Bunsen burner or hot plate) Pasteur pipettes with pipettor West stain Solution A 11 .

In order to observe them with the light microscope. which is of great value in bacterial identification. The best specimens will probably be seen at the edge of the smear where bacteria are less dense. 8. Flood the slide with distilled water and allow it to sit for 1 minute while more silver nitrate residue floats to the surface. Remove the toweling and rinse off excess solution B with distilled water. thereby increasing their apparent size. They are slender (about 10 to 30 nm in diameter) and can only be seen directly using the electron microscope. 3. Rinse thoroughly with distilled water 6. and tannic acid and aluminum potassium sulfate as mordants. With a wax pencil. Add more stain to keep the slide from drying out. the crystal violet forms a precipitate around the flagella. Difco’s SportTest flagella stain employs an alcoholic solution of crystal violet as the primary stain.Solution B Difco’s SportTest Flagella stain Principle Bacterial flagella are fine. 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. mark the left-hand corner of a clean glass slide with the name of the bacterium. 12 . Examine the slide with the oil immersion objective. Let the slide air dry for 15 minutes 4. the thickness of the flagella are increased by coating them with mordants like tannic acid and potassium alum. they often provide information about the presence and location of flagella. rinse gently with water once more and carefully shake excess water off the slide. threadlike organelles of locomotion. Allow the slide to air dry at room temperature 10. Then. 9. 2. Although flagella staining procedures are difficult to carryout. Cover the dry smear with solution A (the mordant) for 4 minutes 5. Place a piece of paper toweling on the smear and soak it with solution B (the stain). 7. and staining them with basic fuchsin (Gray method) or crystal violet (Difco’s method). Gently spread the diluted bacterial suspension over a 3cm area using the inoculating needle. Heat the slide in a boiling water bath for 5 minutes in an exhaust hood with the fan on. Procedure 1. As the alcohol evaporates during the staining procedure.

Look for fields which contain several isolated bacteria. the age of the stain. Begin examination at thinner areas of the preparation and move toward the center. approximately 1 cm from the frosted edge. 6. 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. Place a drop of distilled water on the slide. Do not tip slide before this is done. Tilt the slide at a slight angle to allow the drop preparation to flow to the opposite end of the slide. 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. 4. Carefully rinse the stain by adding water from a faucet or wash bottle to the slide while it remains on the staining rack. Examine the slide microscopically with the oil immersion objective. Flood the slid with the contents of the Difco SportTest flagella stain ampule. Let the slide air-dry at room temperature. and the depth of staining solution over the culture) 8. 3. Bacteria and their flagella should stain purple 13 . 7. 2. Draw a border around the clear portion of a frosted microscope slide with a wax pencil. (Note: the staining time may need to be adjusted from 2 to 8 minutes depending on the age of the culture. Allow the stain to remain on the slide for approximately 4 minutes. 5. the temperature. Do not mix. After rinsing. rather than fields which contain clumps of many bacteria.Procedure (Difco) 1. 9. Do not heat-fix.

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. Pouring a plate 1. 6. Re-melt stored agar media in boiling water bath. Disinfection Sterilization means the complete destruction of all the micro-organisms including spores. Sterilization and Disinfectants Media Preparation of culture media Re-hydrate powder according to manufacturer’s instructions. ensure ingredients are completely dissolved.Basic laboratory procedures and culture techniques Media. from an object or environment. Flame the neck of the bottle. inhibition or removal of microbes that may cause disease or other problems e. agar must not touch the lid of the plate and the surface must be smooth with no bubbles. 5. Seal and incubate the plate in an inverted position. using heat if necessary. Storage of media Store stocks of prepared media at room temperature away from direct sunlight. Media in vessels closed by cotton wool plugs that are stored for future use will be subject to evaporation at room temperature. 8. Disinfection is the destruction. Allow the plate to solidify.g. a cupboard is ideal but an open shelf is satisfactory. 3. It is usually achieved by heat or filtration but chemicals or radiation can be used. The base of the plate must be covered. Normally allow 15-20 cm3 medium/ Petri dish. Gently rotate the dish to ensure that the medium covers the plate evenly. Sterilization vs. 4. avoid wastage by using screw cap bottles. Before sterilization. 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. 2. 14 . Flame the neck of the bottle and replace the lid. Agar slopes are prepared in test tubes or Universal/McCartney bottles by allowing sterile molten cooled medium to solidify in a sloped position. spoilage. It is usually achieved by the use of chemicals. Collect one bottle of sterile molten agar from the water bath. Avoid wastage by preparing only sufficient for either immediate use (allowing extra for mistakes) or use in the near future. Hold the bottle in the left hand. Dispense in volumes appropriate for sterilization in the autoclave/pressure cooker. 7. Sterile agar plates can be pre-poured and stored in well-sealed plastic bags (media-containing base uppermost to avoid heavy condensation on lid). pressure cooker or microwave oven. remove the lid with the little finger of the right hand.

Flaming procedure 15 . Glass spreaders and metal forceps .25%. e. .g. allowing additional time for items to come to temperature (and cool down!).Sterilization of equipment and materials Wire loop Heat to redness in Bunsen burner flame.Flaming in alcohol (70% industrial methylated spirit).Autoclave/pressure cooker. . outside of test tubes/bottles. The handle of the wire loop is held close to the top. Using a wire loop Wire loops are sterilized using red heat in a Bunsen flame before and after use. clothing. wear eye protection and gloves to avoid irritant or harmful effects. hot air oven. people or the environment. . wrapped in either grease proof paper or aluminum and held at 160ºC for 2 hours. at an angle that is almost vertical. They must be heated to red hot to make sure that any contaminating bacterial spores are destroyed. Culture media and solutions . .Operations must not be started until all requirements are within immediate reach and must be completed as quickly as possible. v/v) available chlorine Ethanol 70% (v/v) industrial methylated spirit When preparing working strength solutions from stock and dealing with powder form. exposure of the sterile inner surfaces to contamination from the air must be limited to the absolute minimum. Disinfectants for use at working strength should be freshly prepared from full strength stock or powder form.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. Empty glassware and glass (not plastic!) pipettes and Petri dishes Either. the surface of the working area. as you would a pen. This leaves the little finger free to take hold of the cotton wool plug/ screw cap of a test tube/bottle.On being opened. 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. Disinfectants Choice. Use working strength hypochlorite on day of preparation. Note: plastic Petri dishes are supplied in already sterilized packs. . packs of sterile plastic pipettes are also available but cost may be a consideration. preparation and use of disinfectants Specific disinfectants at specified working strengths are used for specific purposes.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.During manipulations involving a Petri dish. 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. Commonly available disinfectants Hypochlorite (sodium chlorate I) used in discard pots for pipettes and slides At 2500 ppm (0.

Do not put down the lid/cotton wool plug. Return any excess gently if a measured volume is required. Fit the teat. 3. 4. 6. 5. Using a pipette Sterile graduated or dropping (Pasteur) pipettes are used to transfer cultures. 2. 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. Allow to cool then use immediately. Position the handle end of the wire in the light blue cone of the flame. 2. To correct the problem. first ensure that the loop has been sterilized and then reshape the loop with forceps.The flaming procedure is designed to heat the end of the loop gradually because after use it will contain culture. 4. 1. If a loop does not hold any liquid the loop has not made a complete circle. conseque ntly. 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. Remove the pipette from its container/ wrapper by the end that contains a cotton wool plug. 6. The teat must not be removed until the pipette is within the discard pot otherwise drops of culture will contaminate the working surface. Do not put the loop down or wave it around. (Turn the bottle. not the lid. Hold the pipette barrel as you would a pen but do not grasp the teat. Hold there until it is red hot. 3. Loosen the lid of the bottle so that it can be removed easily. Replace the lid on the bottle/cotton wool plug using the little finger. taking care to touch no more than the amount necessary to take a firm hold. 3. Remove the lid of the bottle/cotton wool plug with the little finger of the right hand. 7. 5. Ensure the full length of the wire receives adequate heating. Lift the bottle/test tube with the left hand. 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. Flaming the neck of bottles and test tubes 1. Immediately put the now contaminated pipette into a nearby discard pot of disinfectant. Draw the rest of the wire upwards slowly up into the hottest region of the flame. Flame the neck of the bottle/test tube by passing the neck forwards and back through a hot Bunsen flame. (immediately above the light blue cone). 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. sterile media and sterile solutions. This is the cool area of the flame.) 4. Re-sterilize the loop immediately after use. 2. not the lid. (Turn the bottle. Do not use your fingers because of the possibility of puncturing the skin. 5.) 16 . which may “splutter” on rapid heating with the possibility of releasing small particles of culture and aerosol formation. aerosol formation when liquid is expelled. 6. 1.

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

overnight. remove the lid with the little finger of the right hand. It is advisable to use agar plates that have a well-dried surface so that the inoculum dries quickly.g. 2. the alcohol will burn and sterilize the glass. Remove the spreader from the flame and allow the alcohol to burn off. Sterilization using alcohol 1. 4. are known. per cm³ can be determined. Several loopfuls of culture must be added to the cooled molten nutrient agar to ensure that there is enough inoculum present for growth. 3. If the dilution and volume of the inoculum. e. Avoid making air bubbles. 5. Wrapped glass spreaders may be sterilized in a hot air oven. Flaming a glass spreader Ensure that the spreader is pointing downwards when and after igniting the alcohol to avoid burning yourself. 18 . (The base of the plate must be covered. Gently rotate the dish to ensure that the medium covers the plate evenly. Seal and incubate the plate in an inverted position. Pouring the pour plate 1. 2. 4. Pouring the inoculated medium If pipettes are not available then a wire loop can be used. 7. Most of the colonies grow within the medium and are small in size. Flame the neck of the bottle. Allow the plate to solidify. Pass quickly through a Bunsen burner flame to ignite the alcohol. Using a spreader Sterile spreaders are used to distribute inoculum over the surface of already prepared agar plates. Do not put the spreader down on the bench. Roll the bottle gently between the hands to mix the culture and the medium thoroughly. 6. 3. They can also be sterilized by flaming with alcohol. the number of bacteria or clumps of bacteria. agar must not touch the lid of the plate and the surface must be smooth with no bubbles). Dry the surface of agar plates by either incubating the plates for several hours. the viable count of the sample i. 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). Pour plates allow micro-organisms to grow both on the surface and within the medium.thoroughly mixed and then poured into a Petri dish to solidify. Lift the lid of the Petri dish slightly with the right hand and pour the mixture into the Petri dish and replace the lid. 8. Hold the bottle in the left hand. Keep the alcohol beaker away from the Bunsen flame. 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. Flame the neck of the bottle and replace the lid. usually 1 cm³. the few that grow on the surface are of the same size and appearance as those on a streak plate.e.

Replace the lid of the Petri dish. usually 0. 4. for example mouthwashes. Replace the lid of the Petri dish. 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. This might cause colonies to spread into each other and risk the spillage of the contaminated liquid. delivered by pipette. The spread plate can be used for quantitative work (colony counts) if the inoculum is a measured volume. 0. Seal and incubate the plate in the inverted position. 6. Loosen the lid of the bottle containing the broth culture. partially lift the lid of the Petri dish containing the solid nutrient medium. 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. (Although tape is the preferred method Parafilm could be used as an alternative for sealing the plates. 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. 3. Make sure the entire agar surface is covered.g. 15. Place the pipette in a discard jar.1 cm3. 16. 11.Spread plate Spread plates. Remove the lid/plug of the bottle/test tube with the little finger of the right hand and flame the neck. 7. Place the spreader on the surface of the inoculated agar and. disinfectants and antibiotics. 12. remove a small amount of broth.1 cm3 (ca 5 drops. 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. (2) many drops (six or more) can be put on one plate. 1. flame and allow the alcohol to burn off. 14. This means that they can be used to test the sensitivity of bacteria to many antimicrobial substances. enough to cover a 5 pence piece).) 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. Flame spreader using alcohol. also known as lawn plates. 10. 2. 19 . Dip a glass spreader into alcohol. 9. Lift the lid of the Petri dish to allow entry of spreader. With the pipette. This operation must be carried out quickly to minimize the risk of contamination. Let the inoculum dry.02 cm³. Hold a sterile pipette in the right hand and the bottle/test tube containing the broth culture in the left. Flame the neck of the bottle/test tube and replace the lid/plug. 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. garlic. 8. should result in a culture spread evenly over the surface of the growth medium. 5. With the left hand. 13. Place a few drops of culture onto the surface about 0. of each of a dilution series.

store the cultures at room temperature in either a cupboard or drawer. Never discard sharp or broken items in a way which would endanger. Incubate at an appropriate temperature until there is good growth. all materials can be disposed of with normal waste. must be autoclaved at 121ºC for 15 minutes before disposal. laboratory coats must be removed and hands washed with hot water and soap. Overlong incubation of mould cultures will result in massive formation of spores which readily escape. e. empty media tubes and all contaminated material must be placed in the appropriate labeled receptacles. and may cause contamination problems in the laboratory and be a health hazard. This skill is crucial for reasons of safety and for maintaining the scientific integrity of an investigation. gloves etc. Two stock cultures should be prepared. liquid medium) cultures because the first sign of contamination is much more readily noticed on an agar surface. Discard containers must be carefully and securely packed and never overloaded. Pure cultures The ability to keep pure cultures from becoming contaminated during inoculation and use is a key feature of GMLP. They must be soaked for at least 24 hours before disposal. Care must be taken that glass is adequately packaged to prevent injury. Plastic Petri dishes must never be stacked above the lip of the discard container. Slope cultures are preferred to broth (i. Maintaining stock cultures It may be convenient to maintain a stock of a pure culture instead of re-purchasing it when needed.g. a streak plate culture). 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. Slides.e. Clearly. Be prepared to transfer cultures four times a year to maintain viability. at room temperature and even in a refrigerator. Keep on the lookout for contamination. Slope cultures in screw cap bottles are preferred because the screw cap reduces evaporation and drying out and cannot be accidentally knocked off (cf. it is also vital skill to recognize when a culture has become contaminated. They should be used with distilled or deionised water to prevent corrosion and emptied and dried for storage. 20 . the other is the “permanent” stock which is opened only once for preparing the next two stock cultures. particularly from Petri dishes. Clearing up Working surfaces must be cleared after use. 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. for enzyme reactions and growth temperature relationships.Water baths are used when accurately controlled temperatures are required. If cultures have been used the benches must be swabbed with disinfectant Discarded cultures. one is the “working” stock for taking sub-cultures for classes. pipettes and Pasteur pipettes must be discarded in the appropriate containers of Hypochlorite (sodium chlorate 1). Before leaving the laboratory. when temperature control of incubators is not sufficiently precise. Cultures on streak plates are not suitable as stock cultures. After sterilization. As soon as there is adequate growth. This can occur in an incubator. Cultures and contaminated paper towels.

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. still covering the base) the least amount that will allow access of the loop. a straight wire may also be necessary for taking a small inoculum from liquid cultures for nutritional investigations. 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. 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 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. 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. floors and cultures may become contaminated and students may become understandably (but avoidably) frustrated and lose interest. and on plates that are being used for isolating cultures from natural samples. There should be uniformity of colony form and cell form (and consistency with the appearance of the original culture!). 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. Aseptic technique cannot be maintained with poorly made plugs. If a culture becomes contaminated.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. The choice of loop or pipette for transfers between test tubes and screw cap bottles depends on whether they contain agar slopes.g.facing agar surface. lift the lid vertically (i. solutions of chemicals or electrical circuits in other disciplines. invert it and inoculate the upwards. it is the one used by professional microbiologists. Appropriate instruments for aseptic transfer procedures Microbial stock cultures for use in food microbiology are the equivalent of. The big difference. working surfaces. say. There are two approaches: (1) with the base (containing medium) placed on the working surface. Instead. However. The second method is best reserved for older students working in a relatively dust and draught-free laboratory. e. 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. species of Streptococcus and Lactobacillus. (2) with the lid placed on the working surface.e. 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. 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. and (2) culturing a variant of the original culture that does not behave as the original culture did. go back to the working (or permanent) stock cultures. liquid media or sterile solutions. 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. During the inoculation procedure. is that microbial 21 . Although omitted from the table for simplicity. lift out the base.

allowing an incubation period of from several days to a week.Sterile Filter paper discs. it is easy to inoculate accidentally other parts of the plate with tiny pieces of mould. 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. Mark and label four sections on the base of the Petri dish. toothpaste.70% (v/v) industrial methylated spirit in a small beaker covered in foil (CAUTION:flammable. This can be avoided by placing the Petri dish on the working surface lid down. 22 . 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. e. It is usual to grow moulds on the surface of an agar medium. e.g. The method involves preparing a pour or spread plate of a test micro-organism.Incubator at 25-30 °C (if available) Aseptic technique should be used throughout.Distilled/demineralised water (control) . and antibiotic discs. 1. usually spores. 3 (e. if it is necessary to grow a strictly aerobic organism in a single large volume of liquid culture and provision of adequate aeration. etc. mouthwashes. stains. 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.cultures cannot be taken from a shelf and instantly be ready for use. many preparations with antimicrobial activity are readily available in pharmacists and supermarkets. Materials .Samples to be tested. an inhibitory effect on the test organism is indicated by a clear zone (no growth) around the test substance. After incubation. . that fall off the loop or wire.Take one of the pour or spread plates prepared earlier in the day. The main points to observe are use of an adequate amount of inoculum. antiseptics.g. selected for a range of active ingredients) . mouthwashes. disinfectants. should be kept covered away from flames) . In addition to using laboratory reagents. . This is a straightforward activity that tests several practical skills and is relevant to other aspects of biology and to everyday life. for the three different samples and control (sterile water). 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. When an agar plate with a mould inoculated at the centre is required.Bunsen burner . Testing sensitivity to antibacterial substances The agar diffusion method is widely used in industry for testing the sensitivity of micro-organisms to antibiotics. an appropriate culture medium and incubation temperature and.g. There is also opportunity to think of less obvious materials.Forceps . microbial growth is visible to the naked eye in areas of the plate that are unaffected. plants and their products.

6. Using sterile forceps (flamed with alcohol and cooled) remove one filter paper disc. Incubation of the plate. 5. Invert the plate and incubate at 25-30°C or at room temperature for 48 hours. 4. 7. Consider what factors might be affecting the size of the zones of inhibition. Repeat for the remaining samples and the control (sterile water).2. Wash the forceps free of the sample. Remember to rinse and sterilize the forceps between each sample and to open the plate for the minimum possible time. Seal the lid to the base with tape. 23 . Measure and record the size of any zones of inhibition around the filter paper discs. 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. Examine the plate (without opening). 3.

10 drops) of the L-naphthol solution and 0. The H2S is usually tested for by demonstrating its ability to form black lead salt. Voges. 3. A distinct red colour is considered to be a positive test and yellow is negative. Pour ¼ th of the culture into a clean test tube. Method Inoculate a loop-full of culture in 2% peptone water. the blackening of lead acetate paper will take place. Add 0. Add about five drops of methyl red indicator solution.5. H2S production test: The activity of some bacteria on sulfur containing amino acids frequently results in the liberation of H2S. 2. Add solution B drop by drop. which causes the reduction of nitrate to nitrite. Method: Inoculate glucose phosphate broth with test culture and incubate at 37°C for 24 hr.5 ml (8. Then add 0. If H2S is produced.3% creatine. in the presence of which any acetylmethyl carbinol present becomes oxidized to diacetyl. The diacetyl will combine with creatine to give a red colour. Nitrate reduction test This is a test for the presence of the enzyme nitrate reductase. Method Inoculate 1% peptone water with one loop-full of culture and incubate at 37oC for 24 hrs.5 ml Ehrlich’s reagent. alkali is added. To the inoculated medium after incubation. 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.Common Biochemical Tests 1.1 ml of solution A and swirl. A red color developing within a few minutes indicates the presence of nitrite and hence the ability of the organism to reduce nitrate. Shake thoroughly and allow to stand for 5 to 24 . Method Inoculate glucose phosphate broth with test culture and incubate for 24 hr at 37 °C. 4. Method Inoculate a loop-full of culture in peptone nitrite water and incubate at 37°C for 24 hr.5 ml of the 40% KOH solution containing 0.Proskauer’s test This is a test for the production of acetylmethyl carbinol from glucose. Indole Test This test demonstrates the ability of certain bacteria to decompose the amino acid tryptophan to indole. Insert a lead acetate paper and incubate at 37°C for 24 hrs. Such that the pH of an old culture is sustained below a value of about 4. to test culture add 0. 5. A red colour indicates a positive reaction.

Gas. 9. a) Coagulation of the milk may occur as a result of proteolytic enzyme activity affecting the casein. Incubate at 37oC for 24 hrs. a) Acid production – shown by a change in the colour of the litmus to pink. although normally this is only visible if clotting has occurred. the colour of litmus remaining blue. The appearance of a pink to red colour indicates the presence of acetylmethyl carbinol. Observe the changes which have taken place. Incubate at 37 oC for 24 hrs. 25 . 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.30 minutes. Liquefaction is tested by removing the nutrient gelatin culture from the incubator and holding it at 4oC for 30 minutes before reading the results. 8. b) If sufficient acid is produced the milk will clot. Method: A stab culture of organisms to be tested is made using an inoculum from culture. 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. after 24 hr. Acid production is shown by change in the colour of Andrade’s indicator to pink. Fermentation of sugar: Most of bacteria will ferment a variety of sugars to form one or more acid end products. b) Hydrolysis of the casein as a result of proteolytic activity causes clearing and loss of opacity in the mix medium. Inoculate it at 37oC for 24 hr. Method Inoculate sugar medium with the test culture. accumulates in the Durham tube. Method: Inoculate a tube of litmus milk with a culture to be tested and incubate at 37 oC. 1. Gelatin liquefaction: Proteolytic organisms digest proteins and consequently may liquefy gelatin. Method Inoculate koser’s citrate medium with a wire needle. 7. if produced. usually referred to as peptonization. 2. Growth in he medium involving utilization of citrate as sole carbon source of carbon is shown by turbidity in the medium. 6. 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. 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). Proteolysis may also result in an alkaline reaction due to ammonia production.

1gm 95% ethanol 300 ml Distilled water top to 500 ml 4. 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. 380 ml 80 ml 26 . Incubate at 37oC for 24 hr. Methyl red indicator Methyl red 0. Griess. Reagents: Composition 1.3 gm 40% KOH 100 ml 6. growth in the medium is shown buy turbidity in the medium. Naphthol solution L-Naphthol 5 gm 95% ethanol top to 100 ml 5. 3. Method: Inoculate koser’s uric acid medium with a wire needle. Solution B: Dissolve 5 gm of á– naphthyl amine in 1 liter of 5N acetic acid. 4gm.Ilosvay’s reagents: Solution A: Dissolve 8 gm of sulphanilic acid in 1 liter of 5N acetic acid. HCL 2.10.5 % solution of acid fuchsin until the colour just becomes yellow. KOH– Creatine solution: Creatine 0. Ehrlich’s reagent p-dimethylaminobenzaldehyde 95% ethanol Conc. Andrades’s indicator: Add 1 N NaOH to a 0.

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. Medium 27 . coli E. Test. 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.OBSERVATIONS Regent (If Organism any) E.

Collect frozen samples in prechilled containers. at the time of sampling. unfrozen materials only) or plastic bottles are useful containers for line samples. take an additional sample as a temperature control. Do not use a felt pen on plastic because the ink might penetrate the container. Because interpretations about a large consignment of food are based on a relatively small sample of the lot. shall be taken. Use containers that are clean. Transport frozen or refrigerated products in approved insulated containers of rigid construction so that they will arrive at the laboratory unchanged. transfer representative portions to sterile containers under aseptic conditions. and of a size suitable for samples of the product. use sterile metal boxes. spatulas. Cool refrigerated samples. Identify each sample unit (defined later) with a properly marked strip of masking tape. unless stipulated in the method. avoid glass containers. submit samples to the laboratory in the original unopened containers. Keep frozen samples solidly frozen at all times. except shellfish and shell stock.Food Sampling and Preparation of Sample Homogenate The adequacy and condition of the sample or specimen received for examination are of primary importance. 2. Check the temperature of the control sample at the time of collection and on receipt at the laboratory. The composition and nature of each lot affects the homogeneity and uniformity of the total sample mass. 28 . Collection of samples 1. semisolid. and scissors in an autoclave or dry-heat oven. If products are in bulk or in containers too large for submission to the laboratory. If samples are improperly collected and mishandled or are not representative of the sampled lot. Whenever possible. Make a record for all samples of the times and dates of collection and of arrival at the laboratory. Place containers in a freezer long enough to chill them thoroughly. sterile. viscous. the laboratory results will be meaningless. The collector must determine the proper statistical sampling procedure. or packets with suitable closures. leak-proof. Unless otherwise specified. There can be no compromise in the use of sterile sampling equipment and the use of aseptic technique. Whenever possible. When collecting liquid samples. consisting of a specified number of sample units (usually five) drawn at random from each lot. established sampling procedures must be applied uniformly. which may break and contaminate the food product. Submit open and closed controls of sterile containers with the sample. forceps. refrigerated samples should not be analyzed more than 36 h after collection. Each sample unit shall consist of at least 100 ml or g. Sterile plastic bags (for dry. cans. Do not freeze refrigerated products. Containers such as plastic jars or metal cans that are leak-proof may be hermetically sealed. dry. For dry materials. 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. Deliver samples to the laboratory promptly with the original storage conditions maintained as nearly as possible. 3. Take care not to overfill bags or permit puncture by wire closure. or liquid. Collect original unopened container wherever possible. A sample. widemouthed. Dry or canned foods that are not perishable and are collected at ambient temperatures need not be refrigerated. 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. The number of units that comprise a representative sample from a designated lot of a food product must be statistically significant. according to whether the food is solid. Sterilize one-piece stainless steel spoons. bags. Use of a propane torch or dipping the instrument in alcohol and igniting is dangerous and may be inadequate for sterilizing equipment.

four 25 ml or g containers in each sample unit. to thaw during shipment Defination of Terms 1. that are usually frozen. Sample: The sample units taken per lot for analysis. Place the collected sample units in sterile containers. If the product is in bulk. 2) the possibility that the food may have undergone a step lethal to Salmonella during the manufacturing process or in the home. When there is no code identification. sampling plans for these organisms have received the attention of committees of national and international organizations. or be derived from this by multiplication and/or subtraction operations. Sample Unit: Usually a consumer size container of the product. during transport. and 3) the history of the food. or. 6. or a central portion (one-fifth) of the HGMF. Most Probable Number of Growth Units (MPNGU): On HGMF the Growth Unit (GU) is equivalent to the more familiar Colony Forming Unit (CFU). 7. 3. Employ aseptic techniques in collecting the sample units. 5.g. depending on the nature of the product. 1. the assignment to a sampling or food category depends on 1) the sensitivity of the consumer group (e. The MPNGU is derived from the HGMF score. and infants).. It may equal the HGMF count. 4. the infirm. The 29 . HGMF Count: Is the number obtained when counting either those HGMF grid-cells which contain colonies or those which do not. 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. as necessary. Salmonella species Sample collection Because of the continuing occurrence of Salmonella in foods. Counts may be made over the whole HGMF. and should consist of a minimum of 100 g (ml). 6. Generally. 2. A sample unit will consist of more than one container when the lot consists of containers smaller than 100 ml or g eg. while ensuring that the total number of sample units are not collected from one container.4. 5. several sample units can be collected from one container. the aged. HGMF Score: Is the total number of HGMF grid-cells containing colonies. Do not allow sample units. Analytical Unit: That amount of product withdrawn from the sample unit for analysis. at the same establishment and representing no more than one day's production. (b) the quantity of the same kind of product from one and the same manufacturer available for sampling at a fixed location. Lot: A batch or production unit which may be identified by the same code. 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. a lot may be considered as (a) that quantity of product produced under essentially the same conditions. unless stipulated in the method. Keep the sample unit refrigerated (0-4oC) or frozen. 7.

The number and weights of the sample units is correct. Samples should be preenriched at a 1:9 sample-to-broth ratio. Below are examples of situations that might confront the analyst. it may not be possible to fully conform to the sampling plan. The contents of each intact bag should be mixed to ensure homogeneity before the analytical units are withdrawn. 30 . Food Category I. 3 categories of foods are identified. as would be the case if the analyst were to treat the entire wheel as a single sample. These analytical units should be taken randomly from a wide variety of locations around the wheel. For example. . the analyst should only sample from the 10 intact bags. a single 10 lb wheel of cheese has arrived for testing. and infants. The number of sample units is incorrect. unless otherwise indicated in Chapter 5 or in the OMA. the analyst should still try to analyze as many analytical units as is required for the food of interest. thirty 25 g analytical units must be analyzed. . Food Category III. 1. The analytical units can be composited (fifteen 25 g units into a 375 g composite). Therefore. If Salmonella is present in a food.selection of a sampling plan depends mainly on the first 2 criteria cited. but the total weight of the sample unit(s) is greater than what would be necessary to perform the sample analysis.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. For the Salmonella sampling plan discussed here. and 15 analytical units for Category III foods. whether there was a past history of contamination. 30 analytical units for Category II foods. i. i. 3.5 g analytical units.e. Each sample should be mixed to ensure homogeneity before withdrawing a 25 g analytical unit. . the infirm. should be used to form the composite. but several of the sample units have been damaged and are unusable. 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.. In certain instances. ten 37. Since cheese is a Category II food. Nonetheless it is still important to ascertain whether or not Salmonella is present in the suspect food. In this case. 2. fifteen 1 lb bags of pasta have arrived for testing. For example. Since the analyst needs one 375 g composite. The number of sample units is correct. Individual 25 g analytical units may be combined into 375 g composites as described above unless otherwise indicated in Chapter 5 or the OMA. then the odds of detecting it will be enhanced if two 375 g composites are analyzed rather than a single 25 g analytical unit. from the remaining 10 intact bags.Foods that would not normally be subjected to a process lethal to Salmonella between the time of sampling and consumption. 60 analytical units for Category I foods. but 5 of the bags are torn and unusable. Food Category II.. The history of the food would be important in deciding whether to sample.Foods that would normally be subjected to a process lethal to Salmonella between the time of sampling and consumption.e.

For all samples positive 31 . A sample unit consists of a minimum of 100 g and is usually a consumer-size container of product. the entire amount of 375 g is analyzed for Salmonella. the total weight of the almonds had been less than 2 composites (750 g). 2 composite units. Food Category II. When using sample containers. Food Category II. then the analyst should analyze both a whole and a partial composite. 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. Sample analysis The laboratory will analyze each sample for the presence of Salmonella according to methods described in this manual. For each 375 g composite. Both composites. When a sample unit consists of more than one container.8 g) bag of almonds has arrived for testing. Category II foods require thirty 25 g analytical units (750g). In this case. but more than 1 composite. 60 sample units. Advise the laboratory in advance of perishable sample shipments.. four 25 g containers could constitute a sample unit). Take sample units at random to ensure that a sample is representative of the lot. the analytical units may be composited. one composite unit. the lot is rejected. in the above example. 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. The minimum number of composite units to be tested for each food category is as follows: Food Category I. Refrigerate perishable samples and samples supporting microbial growth. Submit all samples collected to the laboratory for analysis. If one or more composite units are positive for Salmonella. A lot will not be resampled unless the environmental control for Salmonella is positive. the analyst should analyze all of the almonds at a 1:9 sample-to-broth ratio (226. 30 sample units. Almonds are a Category II food. provided that the analytical control is negative for Salmonella. so it is impossible to analyze the amount of almonds required by the sampling plan.8g sample/2041 ml preenrichment medium). Food Category III. If. A sample unit will consist of more than one container when containers are smaller than 100 g (e. 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.g. An analytical control is required for each sample tested. should be preenriched at a 1:9 sample-to-broth ratio. The numbers of sample units to be collected in each food category are as follows: Food Category I. To reduce the analytical workload. an 8 oz (226. Food Category III. The maximum size of a composite unit is 375 g or 15 analytical units. Take a 25 g analytical unit at random from each 100 g sample unit. The analytical units comprising these composites should be taken randomly from a wide variety of locations in the lot of almonds. 15 sample units. aseptically mix the contents of each container before taking the 25 g analytical unit. The sampled lot is acceptable only if analyses of all composite units are negative for Salmonella. For example. There is less sample available than is necessary to form the required number of composites. 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. This sampling plan applies to the collection of finished products under surveillance and/or for determination of compliance for regulatory consideration. 4 composite units.4.

and other fish products. and nectars. dried 32 . pasteurized milk and raw fluid milk and fluid milk products for direct consumption. Examples are as follows: Industry Product Code 2 3 Milled grain products not cooked before consumption (bran and wheat germ) Bread. See Chapter 5 for information on further handling of these cultures. sodium caseinate. pasteurized and unpasteurized concentrated liquid milk products for direct consumption. and canned fruits and juices. Imports. unpasteurized eggs and egg products from unpasteurized eggs for consumption without further cooking 16 Canned and cured fish. and crustaceans for direct consumption 17 Meat and meat products. rolls. . These sampling plans apply to imported food products intended for human consumption. and icings 5 Breakfast cereals and other ready-to-eat breakfast foods 7 Pretzels.and cream-filled sweet goods. 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. vertebrates. frozen. shellfish. the infirm. poultry and poultry products. casein. crackers. and whey 12 Cheese and cheese products 13 Ice cream from pasteurized milk and related products that have been pasteurized. fresh and frozen raw shellfish and crustacean products for direct consumption. Listing does not necessarily mean that these products are probable sources of Salmonella. . dried milk and dried milk products for direct consumption. buns. and other snack foods 9 Butter and butter products. 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. Food Category II. determine the somatic group. smoked fish. chips.Foods that would not normally be subjected to a process lethal to Salmonella between the time of sampling and consumption. Food Category I. concentrates.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. and infants. and gelatin (flavored and unflavored bulk) 20-22 Fresh. custard.for Salmonella. 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. sugared breads.

fresh and frozen shellfish and crustaceans (except raw shellfish and crustaceans for direct consumption). Escherichiacoli (including enteropathogenic strains).. and edible seed products for direct consumption Vegetable juices. and butters Nuts. and squid) Vegetable protein products (simulated meats) normally cooked before consumption Fresh vegetables. gravies. salad dressing. stews. breads. edible seeds. total coliforms. pudding mixes. Bacilluscereus. Examples are as follows: Industry Product Code 2 3 4 16 Whole grain. Campylobacter spp. dried vegetables.. jellies. minerals. Yersinia spp. and rennet products that are cooked before consumption 18 24 26 35 2. frozen vegetables. other aquatic animals (including frog legs. and vegetables normally eaten raw Oils consumed directly without further processing. nut products. Aerobic plate counts. fecal coliforms. and honey Ready-to-eat sandwiches. vegetable sprouts. Vibrio spp. oil stock. cookies. milled grain products that are cooked before consumption (corn meal and all types of flour). sugars. and gelatin products Syrups. and Clostridium perfringens 33 . proteins. and vinegar Spices. and starch products for human use Prepared dry mixes for cakes. cured and processed vegetable products normally cooked before consumption Vegetable oils.. and vegetable shortening Dry dessert mixes.. 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. oleomargarine Dressings and condiments (including mayonnaise). Staphylococcus spp. preserves. vertebrates (except those eaten raw).23 24 26 27 28 29 30 31 33 34 35 36 37 38 39 54 fruits for direct consumption. flavors. Shigella spp.. marine snails. such as vitamins. and rolls Macaroni and noodle products Fresh and frozen fish. 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 sauces Soups Prepared salads Nutrient supplements. jams.

sharp-edge. be certain that twist wires did not puncture surrounding bags. Be certain that each sample is accompanied by a completed copy of the Collection Report and officially sealed with tape bearing the sample number. 1. Any crosscontamination resulting from one or more of above defects would invalidate the sample. b. with weights. Whether the sample is to be analyzed for regulatory purposes. 1000 ml.1 g Sterile beakers. scissors. 7. or for a bacteriological survey. Sample analysis. Butterfield's phosphate-buffered dilution water. Check sampling containers for gross physical defects. tablespoons. 250 ml. 2000 g capacity. If sample units were collected in plastic bottles. for investigation of a foodborne illness outbreak. Carefully inspect plastic bags and bottles for tears. low-form. stainless steel blades rotating at bottom of 4 lobe jar at 10. Suspended solids are reduced to fine pulp by action of blades and by lobular container. and tongue depressors (for sample handling) Receipt of samples The official food sample is collected by the FDA inspector or investigator. Sterile glass or metal high-speed blender jar. check bottles for fractures and loose lids. Equipment and materials 3. If plastic bags were used for sampling. spatulas.0 and 10. Collect the intact retail unit as the subsample even if it is larger than 8 oz. with cover. From any lot of food. Mechanical blender. it should be stored as described later. pinholes. meets these requirements. Analyze samples as indicated in current compliance programs.a. resistant to autoclaving for 60 min at 121°C 5. which swirls suspended solids into blades. forks. or equivalent.000-12. Waring blender. sensitivity of 0. 4. The term "high-speed blender" designates mixer with 4 canted. Condition of sampling container. As soon as the sample arrives at the laboratory. covered with aluminum foil Sterile graduated pipets.000 rpm or with equivalent shearing action. the analyst should note its general physical condition. Several types are available. Use blender that has several operating speeds or rheostat. Sample collection. strict adherence to the recommendations described here is essential. If the sample cannot be analyzed immediately. forceps. sterilized in bottles to yield final volume of 90 ± 1 ml 9. 6. collect ten 8-oz subsamples (or retail packages) at random. Balance. collecting official's 34 .0 ml 8. Labeling and records. and puncture marks. Sterile knives. and the collecting district should be notified. Do not break or cut larger retail packages to obtain an 8-oz subsample.

Add 450 ml Butterfield's phosphate-buffered dilution water to blender jar containing 50 g analytical unit and 35 . If necessary to temper a frozen sample to obtain an analytical portion. swab immediate work area with commercial germicidal agent. ne however. Mixing Various degrees of non-uniform distribution of microorganisms are to be expected in any food sample. When thawing a sample at elevated temperatures. agitate the sample continuously in thermostatically controlled water bath. Weighing Tare high-speed blender jar. do not thaw frozen samples before analysis. If contents of package are obviously not homogeneous (e. mix dried samples with sterile spoons or other utensils before withdrawing the analytical unit from a sample of 100 g or greater. Store nonperishable. Total volume in blender must completely cover blades.name. If rapid thawing is desired. however. Thawing Use aseptic technique when handling product.1 g) weigh unthawed food (if frozen) into jar. Blending and diluting of samples requiring enumeration of microorganisms All foods other than nut meat halves and larger pieces. Refrigerate unfrozen perishable samples at 0-4°C not longer than 36 h. If a sample fails to meet the above criteria and is therefore not analyzed. To ensure more even distribution. avoid transferring the sample to a second container for thawing. 25 g for Salmonella) may be recommended. Normally. determine whether the food has been sampled according to the most appropriate sampling plan. examine samples immediately upon receipt. Foods to be examined for Salmonella.g. Whenever possible. macerate entire contents of package and withdraw the analytical unit. Depending on the food and the type of analysis to be performed. or. shake liquid samples thoroughly and. weigh portion equivalent to one-half of sample and adjust amount of diluent or broth accordingly. and nut meal. clean immediate and surrounding work areas. notify the collecting district so that a valid sample can be obtained and the possibility of a recurrence reduced. depending on purpose of test. are sampled according to a statistically based sampling plan designed exclusively for use with this pathogen. if practical.g.. Other analytical unit sizes (e. store frozen samples at -20°C until examination. Most foods are collected under a specifically designed sampling plan in o of several ongoing compliance programs. Adherence to sampling plan. If possible. In addition. If analysis must be postponed. If entire sample weighs less than the required amount. a frozen dinner). 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. a sample can be thawed at 2-5°C within 18 h. Notification of collecting district. thaw it in the original container or in the container in which it was received in the laboratory. Before handling or analysis of sample. or low-moisture foods at room temperature until analysis. thaw the sample at less than 45°C for not more than 15 min. Use a 50 g analytical unit of liquid or dry food to determine aerobic plate count value and most probable number of coliforms. Preferably. then aseptically and accurately (± 0. preferably. Use analytical unit size and diluent volume recommended for appropriate Bacteriological Analytical Manual method being used. Storage. canned. depending on specific analysis to be performed. analyze each different food portion separately.

1 ml volumes. Make dilutions of original homogenate promptly. This results in a dilution of 10-1.0 ml. Nut meal. above) and shake vigorously 50 times through 30 cm arc to obtain 10-1 dilution. Add 90 ml of diluent (G-l. Not more than 15 min should elapse from the time sample is blended until all dilutions are in appropriate media. Aseptically weigh 10 g analytical unit into sterile screw-cap jar. Nut meat halves and larger pieces. Let stand 3-5 min and shake 5 times through 30 cm arc to resuspend just before making serial dilutions and inoculations. do not use pipet with capacity greater than 10 ml to deliver 1 ml volumes. Let stand 3-5 min and shake 5 times through 30 cm arc to resuspend just before making serial dilutions and inoculations. Prepare all decimal dilutions with 90 ml of sterile diluent plus 10 ml of previous dilution. For example. Aseptically weigh 50 g analytical unit into sterile screwcap jar. Add 50 ml diluent (G-l. unless otherwise specified. Do not deliver less than 10% of total volume of pipet. using pipets that deliver required volume accurately. 36 . for delivering 0. Shake all dilutions vigorously 25 times in 30 cm (1 ft) arc in 7 s. do not use pipet with capacity greater than 1.blend 2 min. above) and shake vigorously 50 times through 30 cm arc to obtain 100 dilution.

Thermophilic bacteria: an organism whose optimimum growth temperature is > 45oC. blender or equivalent 8) Incubator capable of maintaining the growth temperature required for the specific type of aerobic bacteria being enumerated (i. Analyze each sample unit individually.1%)(PW) 3) 2% sodium citrate (tempered to 450C) (for cheese samples only) 4) Sodium 2.20oC. and for thermophilic bacteria: 55oC) and 45oC waterbath 9) Colony counting device (optional) 4. Procedure Determine which type of aerobic bacteria are being enumerated.5 triphenyltetrazolium chloride (0. 37 . A portion of the product is mixed with a specified agar medium and incubated under specific conditions of time and temperature.1%) (optional) 5) 1N HCl and 1N NaOH 6) pH meter or paper capable of distinguishing to 0.Enumeration of microorganisms in foods A. Principle The Aerobic Colony Count (ACC) estimates the number of viable aerobic bacteria per g or mL of product. for mesophilic bacteria: 30 . 20 . Determination of Aerobic colony count in Foods 1. which has an upper limit for growth at ca.45oC.35oC. Application This method is applicable to the enumeration of viable aerobic bacteria (psychrophilic. 2.e.0 7) Stomacher. 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. and which has a lower limit of growth of 0oC or lower. Psychrophilic bacteria: an organism which grows optimally at or below 15oC. 1) Plate count agar (PC) 2) Peptone water diluent (0.0 to 8. Mesophilic bacteria: an organism whose optimim growth temperature lies within a range generally accepted as ca. 20oC. It is assumed that each viable aerobic bacterium will multiply under these conditions and give rise to a visible colony which can be counted.3 to 0. mesophilic and/or thermophilic bacteria) in foods.3. 3.5 pH units within a range of 5. for psychrophilic bacteria: 15 .

Sample units of frozen products shall be kept frozen. 4. keep the sample units refrigerated (0-5oC). 4.2. and remove an aliquot from below the liquid/foam interface. 4. 4. If a sample size other than 25 g or mL is used. 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.1. Preparation of Dilutions 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.3. 4.2. Preparation of Media 4.2. 4. agitate liquid or free flowing materials until the contents are homogeneous.6.1. Prepare sterile 0.3.1.2. Analyze sample units as soon as possible after receipt in the laboratory.3.2. a 10% solution (suspension) is represented by 10 g (mL) per 100 g (mL) of solution (suspension). 4.1. If a homogeneous suspension is to be obtained by shaking.e.3. Check the pH of the food suspension. NOTE: Volume in brackets indicates alternate procedure for marking dilutions. Sterilize.0 with sterile NaOH or HCl. such as 11 (10) g or mL into 99 (90) mL. Handling of Sample Units 4. 4. Clearly mark the duplicate Petri plates.1.4.3.3. the following shall apply: with the exception of shelfstable products.1% peptone water diluent. shake the dilution bottles 25 times through a 30 cm arc in approximately 7 sec.2.1. maintain the 1:10 sample to dilution ratio.4.3. During storage and transport. 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. as indicated in Table I. With foods that tend to foam. If the pH is outside the range of 5. Thaw frozen samples in a refrigerator or under time and temperature conditions which prevent microbial growth or death. the blending time should not exceed 2. 4. whereas a 1:10 dilution is based on 10 g (mL) of product (solute) plus 90 g (mL) of diluent (solvent).5-7. 4.2. 4. Clean surface of working area with a suitable disinfectant. Prepare plate count agar and dispense in appropriate quantities.3. If a homogeneous suspension is to be obtained by blending.3. i.2. obtain the analytical unit by taking a portion from several locations within the sample unit. To ensure a truly representative analytical unit.1.. 4. 38 .The test shall be carried out in accordance with the following instructions: 4.3.6.5 min in order to prevent over-heating. adjust the pH to 7.5. If the sample unit is a solid. use blender at low speed.

35oC. 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. In the case of products that tend to adhere to the bottom of the plates. Pipette 1 mL or 0.4.5. milk powder. The plates used to enumerate psychrophilic and thermophilic bacteria may be incubated up to 5 days. 4.6.3. Allow to solidify. select a representative portion of the plates free from spreaders. If plates contain colonies which spread.0 mL of sterile diluent previously placed in the Petri plate. Pour 12-15 mL of tempered agar into each plate. 4. select plates with 20-200 colonies (including pinpoint colonies).7. 4. 4. Prepare succeeding decimal dilutions as required.1 mL of the required dilutions to appropriately marked duplicate Petri plates.. add the inoculum to 1.1. etc.g. 4. 4. if the lab has verified their suitability.4.2.6.7. e. and mix by rotating and tilting. 4. and count the colonies in this area.6.4. Plating 4. Avoid crowding or excessive stacking of plates to permit rapid equilibration of plates with incubator temperature. Count colonies promptly after the incubation period. 4. using a separate sterile pipette for making each transfer.3. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present. Counting Colonies 4. count for the whole plate: 30 x 4 = 120 colonies. Food particles such as meat.4. 4.4.1.3. often interfere with the enumeration of the plates.3. If possible. for mesophilic bacteria: 30 . and for thermophilic bacteria: 55oC). 30 colonies counted on 1/4 of area of the plate. Plates should be poured not more than 15 min after preparation of dilutions.7 Differentiation of Colonies from Interfering Particles 4.4.6.1. 39 . Agitate each dilution bottle to resuspend material that may have settled out during preparation.8. Incubation Incubate plates in the inverted position for 48 h ± 4 h. Other combinations of time and temperature may be used. If counts do not fall within this range select plates that fall nearest to the 20-200 range.. Incubation temperature is dependent on the growth temperature requirements of the target organisms (for psychrophilic bacteria: 15 . 4.4.2. 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. if possible.20oC.

1 /10-1 = 10 4.. 4. and D is the respective dilution factor.8. where N is the number of colonies per g (mL) of product..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.5.1.850 as 2. for one millilitre and a set of duplicate plates (1 mL/plate) the value is < 0.. 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.5. Calculate the average count (arithmetic mean) of the duplicate plates4. Gently rock plates from side to side to cover the entire area with solution.5 This value is for a 10o dilution (Dilution Factor = 1). e. To compute the Aerobic Colony Count (ACC).1% 2. the numerical value of 0. (e. Pour off excessive solution and allow the plates to remain at room temperature for 3 hrs. triphenyltetrazolium chloride.8.900). The bacteria reduce the indicator to a formazan which colours the colonies red and aids in distinguishing the food particles.2 When reporting results (Table II) round-off the counts to two significant figures and record only the first two left hand digits.4.7. i. A is the average count per plate. The lowest possible average with one colony on one of the two duplicate plates is: 1 + 0 /2 = 0.2. the Dilution Factor. 40 .8. Alternatively. If the lowest dilution plated shows no colonies.e. use the formula: N = A x D. in an inverted position.3. 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. water etc.8. Table I Type of Food Liquids: milk. meats weigh into peptone water diluent all cheese spices Shellfish weigh into previously warmed (45oC) 2% sodium citrate (Na3C6H5O7.g.4.8. Recording Results 4.5 must be multiplied by the reciprocal of the dilution. Colonies cannot be picked for isolation after this method has been used. For other dilutions. 4.g. after incubation flood plates with 2 mL of 0. record 2.3.

440 counts lower than 20. Eisenhart and Wilson (1943). If. The sample is prepared in such a way that the bacteria are distributed randomly within it. here we will speak of these GUs or CFUs as individual bacteria. e. 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. and they do not repel each other.) at each dilution. e. De Man (1983) published a confidence interval method that was modified to make the tables below. the bacteria in the prepared sample in question can be found attached in chains that are not separated by the preparation and dilution.000 440..000 E 15. the numbers of inocula producing growth at each dilution.Table II Examples for Recording Results Examples of the average number of colonies Counts between 20-200..g. microbiologists use serial dilutions. In order to obtain estimates over a broad range of possible concentrations. The growth medium and conditions of incubation have been chosen so that every inoculum that contains even one viable organism will produce detectable growth. in the microbiologist's experience. will imply an estimate of the original. The "outcome". not clustered together. 41 . 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. incubating several tubes (or plates. however.000 E <500 B. and for those foods whose particulate matter may interfere with accurate colony counts. 144 Counts higher than 200. For simplicity.. The following assumptions are necessary to support the MPN method. Most Probable Number Method (MPN) The most probable number (MPN) is particularly useful for low concentrations of organisms (<100/g). 15 No count 0 Dilution 1:1000 Highest dilution 1:1000 Lowest dilution 1:1000 Lowest dilution 1:1000 Report as no. i. especially in milk and water. Only viable organisms are enumerated by the MPN determination.g. the MPN should be judged as an estimate of growth units (GUs) or colony-forming units (CFUs) instead of individual bacteria.. of bacteria per g (mL) 140. undiluted concentration of bacteria in the sample.e. The first accurate estimation of the number of viable bacteria by the MPN method was published by McCrady (1915). and Cochran (1950) published articles on the statistical foundations of the MPN.g. The bacteria are separate. e. Halvorson and Ziegler (1933). etc.

if positive results occur in higher unselected dilutions. illustrated by the table of examples below (with 5 tubes at each dilution). is 7 times larger. Case 1. refer to the 3 dilution table according to the following two cases. Select the 3 lowest dilutions (ex.20 4. a and b).01 g 0. 0. are very close approximations to those based on 4 or more dilutions. 0. When more than three dilutions are used in a decimal series of dilutions. If there were not enough higher dilutions tested to select three dilutions. 3. 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. then select the next lower dilutions (ex. No dilutions show all tubes positive. 0. however. the chance is at least 95 percent that the confidence interval associated with the eventual result will enclose the actual concentration. add those higher-dilution positive results to the results for the highest selected dilution (ex. In these cases. however. Table 1 Example 1. add those higher-dilution positive results to the results for the highest selected dilution (ex.7 Other compendia of methods require that no excluded lower dilutions may have any negative tubes. Before the tubes are inoculated. Until further research clarifies this situation. If there are still positive results in higher unselected dilutions. 0) occur too often to be random occurrences. report the extent to which such lower. 0) with MPN 4. d). f). This manual differs when the highest dilution that makes all tubes positive follows a lower dilution that has one or more negative tubes. c). The BAM selection method is based on FDA experience that for some organisms in some food matrices such outcomes as (2. Selecting Three Dilutions for Table Reference An MPN can be computed for any numbers of tubes at any numbers of dilutions. One or more dilutions show all tubes positive. 1. If there are positive results in higher unselected dilutions.001 g 0. analysts should continue to exclude dilutions lower than the highest dilution with all tubes positive.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. shift each selection to the next higher dilution (ex.0 g 0. e). 1. 5. 5.8/g.1 g 0.Confidence Intervals The 95 percent confidence intervals in the tables have the following meaning. Analysts working with 42 . The findings should. 33/g. 0) and (0. 1. Example b above would be read according to other compendia as (4. partially-negative dilutions have been excluded. Case 2. g). The BAM reading. 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. MPN values based on 3 decimal dilutions.

0 and 0.1 and 0. multiplying by 10 would make these inocula match the table inocula.. 1.1 g (ml) respectively are used.1 is positive (3) 5 tubes with 1 ml of 1:100 dilution of test material . 43/g. When higher dilutions are used. 0. 1. one would multiply the Table 1 MPN/g estimate. by 10 to arrive at 430/g. 5. 43 . multiply the MPN/g and confidence limits by whatever multiplier is required to make the inocula match the table inocula. plates overgrown by competing microflora at low dilutions). 0).1. If it is not desired to exclude the remaining tubes at or below the dilution of the excluded tubes. 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.0001 for 3 tubes each.g.3. Example: The following inoculated tubes give a positive reading: (1) 5 tubes with 10 ml of 1:10 dilution of test material . to correct for the actual amount of test material in the tubes. 0) as (4. and 0. 5.all 5 are positive (2) 5 tubes with 1 ml of 1:10 dilution of test material . If the results need to be expressed per g (ml).01. 1. They may also read outcomes such as (3. Conversion of Table Units The tables below apply directly to inocula 0. and also to obtain the MPN per g (ml) as is usually done for foods. rather than per 100 ml (g). the same procedure is followed.0 and 0. 0. Inconclusive Tubes In special cases where tubes or plates cannot be judged either positive or negative (e.001. 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 positive results from this 3 tube series were (3. 0). When different inocula are selected for table reference. 1.01 g (ml). if the inocula were 0. 5. 0. because they are not included in the tables. 1.none are positive The quantities in each of the five tubes of the three dilution series represent 1. For example. respectively of the test material. the MPN value is 330 ÷ 100 = 3. for which the values are given in the table. The entire dilutions at or below those in which exclusion occurs may be excluded. 0. The volume of diluent added to the tubes (and which accompanies the test material) is ignored when calculating the MPN. When other sized portions of the test material are placed in the tubes. 1 and 0. these tubes or plates should be excluded from the results.01. and 0. MPN values obtained from Table II must be multiplied by an appropriate number.materials with known limited complexity in research settings will want to use their professional judgement to read outcomes such as (4. Table I has been adapted from a conversion table prepared for the analysis of drinking waters where 10. the results will now have an unequal number of tubes at several dilutions. since only 1/10 of these amounts were actually used in the analysis.1 g (ml) in Table I. a reading of 5-1-0 gives a value of 33 when 10. According to Table I. 0. 0. 0) as too improbable to record.1 ml of the water under test are used as test portions. However. but the multiplier (dilution factor) is enlarged to relate the amount of test material actually present to the values given for 10.001 g. 1.

dilution with the smallest quantity of product). Example e. Select the highest dilution (last dilution. Table II. the number of such positive tubes should be added to those of the next lower dilution. 5. If only 3 dilutions are made. 1. Table II. Table II. use the dilution factor of the middle set of the three dilutions selected. If a positive tube occurs in the dilution higher than the 3 chosen to rule. 3. Example g. If more than 3 dilutions are employed. MPN/100 ml = No. Table II. use the results of only 3 consecutive dilutions. 2. To determine which consecutive dilutions to use.e. Example f. Examples c and d. For calculating the MPN. 4. Table II. use the first 3 dilutions. but none of the dilutions tested have all 5 tubes positive. If more than 3 dilutions are made. Refer to Table II and look up the value which corresponds to the number of positive tubes obtained. use the results for those 3 dilutions to compute the MPN. microorganisms x (Table I) dilution factor of middle set of tubes 44 . 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.Dilution factor = Reciprocal of the dilution of the analytical unit. refer to the combinations shown below: (See also Table III). i. in which all 5 tubes are positive and 2 subsequent higher dilutions. Examples a and b.

7 94 2 2 2 35 8.2 18 0 3 0 9.17 18 1 0 1 7.10 0.01.7 94 2 3 0 29 8.6 42 1 2 1 15 4.000 3 3 0 240 42 1.000 3 3 2 1100 180 4.5 42 2 0 0 9.5 42 1 3 0 16 4. lim.2 1.6 94 3 0 1 38 8.15 9.1.MPN Tables Table II. For 3 tubes each at 0.7 94 2 3 1 36 8.001 Low High 2 2 0 21 4.6 0 1 0 3.2 1.001 Low High 0 0 0 <3.3 20 1 1 1 11 3.5 42 2 2 1 28 8.000 3 3 1 460 90 2. 0.4 3.4 38 2 0 1 14 3. lim.100 3 3 3 >1100 420 -- 45 .6 38 1 2 0 11 3.7 110 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 1.5 0 0 1 3.9.15 11 0 1 1 6.7 42 2 1 1 20 4. Pos.7 94 Pos.4 1. and 0.6 38 1 1 0 7.5 42 2 1 0 15 3. 0.0 -.3 18 1 0 2 11 3.10 0.6 38 1 0 0 3.01 0.2 18 0 2 0 6.6 42 2 0 2 20 4.01 0.6 0. tubes MPN/g Conf. tubes MPN/g Conf.5 42 2 1 2 27 8.0 0.1 1.7 94 3 0 0 23 4. the MPNs per gram and 95 percent confidence intervals. 0.0 0.001 g inocula.2 1.

1.5 9.3 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 10.7 5.0.1 8. 1 and 0.1.2 10 12 15 17 8.Table III Most Probable Number (MPN) of Bacteria Per 100 g (ml) of Test Material Using 5 Tubes With 10.0.5 7.0.1 10 12 14 6.0.2 12 14 17 19 9.2 11 13 5.1.1.2 9 1.1 2 4 6 8 10 12 4 6.4 9.1 11 3.4 7.4 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 ml or g of Test Material 10.5 6.3 11 13 15 7.1 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 46 .3 9.5 7.0.8 9.1 12 14 16 6.8 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 10.1.1 <1.6 5.1.4 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 4.1 8.6 7.8 3.1 7.8 3.8 9.6 5.8 1.1 000 001 002 003 004 005 010 011 012 013 014 015 020 021 022 023 024 025 030 031 032 033 034 035 040 041 042 043 044 045 050 051 052 053 054 055 MPN 10.4 11 13 15 17 9.0.

Enumeration of yeasts and moulds in foods (not specified below) These agars are suitable for foods where the a is above 0. Colonies appearing on the medium are then counted and/or examined. Xerophilic: Moulds capable of growing at reduced water activity (aw).. containing (a) adequate nutrients for growth of most yeasts and moulds and (b) antibiotics for inhibition of most bacteria.3.5) 3. Such media are now recognized as inferior to antibiotic supplemented media that are formulated to suppress bacterial colony development. Osmophilic: Yeasts preferring reduced aw for growth. 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.95. 3. The method described here is a "general purpose" method and may not be suitable for detection of yeasts and moulds adapted to certain foods. Note: If the analyst uses any variations of the media listed here (either product that is commercially available or made from scratch). Defination of terms 3. Precautions Some yeasts and moulds can be infectious or can cause allergic responses. plates should be held in incubators. 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. therefore. not in an open room. 2. 4. it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency. enhance resuscitation of injured fungi. foods of very low water activity. e. A medium.) (7. Plate lids should generally only be removed for procedures such as the preparation of a slide for microscopic examination. and reference 7. Principle In the past. Ideally. W vegetables. meat and dairy). Cultures should never be smelled. is inoculated with a given quantity of the product or with scrapings from equipment or the manufacturing environment.2.1.3 for the formula of individual media. acidified media were used to enumerate yeasts and moulds in foods. Scrapings: Suspected yeast and mould material scraped from food plant equipment and the manufacturing environment. it is important to be fairly cautious when working with fungi. 47 . It is incubated at 22-25oC for 3-5 days. such as fresh foods (fruit. Flamed needles should be cooled before making transfers to avoid dispersal of conidia and other cells. (Yeasts preferring reduced aw are also sometimes referred to as xerophilic.C. and minimize precipitation of food particles. Enumeration of yeasts and moulds in foods 1.g. 3.

Procedure Each sample unit shall be analyzed individually. Sample units of frozen products shall be kept frozen. nuts. the following shall apply: with the exception of shelfstable products. keep the sample units refrigerated (0-5oC). 48 .1%) (PW) 9) 2% sodium citrate tempered to 45oC (diluent for high fat foods. 5.3 to 0. Thaw frozen samples in a refrigerator or under time and temperature conditions which prevent microbial growth or death. blender or equivalent 13) pH meter or paper capable of distinguishing to 0.3. fruit concentrates. jellies.5 pH units within a range of 5.0 to 8. see 6. Handling of Sample Units and Scrapings 5. 55oC waterbath (and 45oC waterbath if sodium citrateistobeused).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. 5. such as cheese) (optional) 10) 1N HCl and 1N NaOH 11) Gram stain solutions 12) Stomacher.1) 7) Malt extract agar containing 50% (w/w) sucrose Other: 8) Peptone water (0. and spices 5) Dichloran-glycerol DG 18 agar (DG-18) Enumeration of xerophilic yeasts and moulds in jams.0 14) Light microscope 15) Colony counting device (optional) 16) Incubator (darkened) capable of maintaining 22 to 25oC.1.1. flours.1. and dried fruits 6) 20% sucrose (diluent additive for osmophiles.1. During storage and transport. The test shall be carried out in accordance with the following instructions: 5.2.

5 and 7. If the pH is not between 5.5 min.2.7. can be used as diluent for high-fat foods such as cheese. Temper melted agar in a 55oC waterbath.5. 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. such as 20% sucrose. adjust the pH to 7.5.2. 5. since photo-degradation of rose bengal produces compounds that are toxic to fungi.3.3.3.4. Preparation of Dilutions 5.0 with a sterile solution of 1N NaOH or 1N HCl. Soaking may allow for the repair of sub-lethally damaged cells (resuscitation).2. should be added to the diluent when enumerating osmophiles in foods such as syrups and fruit juice concentrates. 5. Stomach. Prepare the appropriate media for the analysis being carried out (see Section 5). Rehydrate dried foods for 1 h with an equal amount of distilled water or peptone water and store at room temperature. 5.2.1. 5. NOTE: DRBC agar should not be exposed to light. as indicated in Table I. An appropriate solute. To prevent over-heating. agitate liquid or free flowing materials until the contents are homogeneous. obtain the analytical unit by taking a portion from several locations within the sample unit. a 2% solution of sodium citrate.1% peptone water as diluent.6. 5.3.1. If a sample size other than 25 g or mL is used. If the 1:10 dilution is prepared in a dilution bottle.4. Verify the pH of the suspension.3.2.2. Mark clearly the duplicate petri plates identifying sample. To ensure a representative analytical portion.2. 5. Prepare 0. pre-warmed to 45°C. it should be mixed by shaking the bottle 25 times through a 30 cm arc in approximately 7 sec. such as 11 (10) g or mL into 99 (90) mL. 5. ensuring that the water level is 1 cm above the level of the medium in the bottles.3. 5.3. If the sample unit is a solid. With foods that tend to foam. Analyze the sample units as soon as possible after receipt at the laboratory. maintain the 1:10 sample to dilution ratio. Preparation of Medium 5. In addition. NOTE: Weight or volume in brackets indicates alternate procedure for making dilutions.1. use blender at low speed and remove aliquot from below liquid/foam interface. blending time should not exceed 2.3. Blend or stomach for the minimum time required to produce a homogeneous suspension. sample unit.3.3. Clean surface of working area with a suitable disinfectant. Some degree of soaking may be beneficial for the recovery of yeasts and moulds from dried or intermediate-moisture foods. 5.5.3. 49 . 5. 5. dilution and date of inoculation. blend or shake according to the type of food as indicated in Table 1.

6. Counting Colonies and Examining Growth 5.8.5.4.6. 5.9. e.2. If counts do not fall within this range. select a representative portion of the plates free from spreaders. Determine the identity of pin-point colonies microscopically. count colonies on plates after 5 days.3.1.g. distinguishing. it is important to shake all dilutions (as in 5.3 For determination of viability of suspected yeast and mould material from food plant equipment and the manufacturing environment.4. 5.3. if required. select plates with 10-150 colonies. aseptically tease the scrapings apart and distribute the pieces over the surface of solidified medium. if possible. 5.4. Spread 0. the lower population range is selected.. if possible.6. which may result in secondary growth 5. If plates contain colonies which spread.1 Agitate each dilution bottle to resuspend material that may have settled out during preparation. Microscopic examination with crystal violet stained smears may be necessary to distinguish yeast colonies from some bacterial colonies that may look like yeast. using a separate sterile pipette for making each transfer. 5. Agar spread plates should be dried overnight before being inoculated. 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. yeast colonies from mould colonies.3. count for the whole plate: 30 x 4 = 120 colonies.3. 50 .1 mL onto duplicate plates (see Section 5 for appropriate plating media) 5.7) immediately prior to making transfers to ensure uniform distribution of the microorganisms present. Because mould propagules may settle out within a few minutes. according to their colonial morphology. 5. Prepare succeeding decimal dilutions as required. Incubate plates in the dark. 30 colonies counted on 1/4 of the area of the plate. the upper limit is counted. count them and then count again on the fifth day. Normally. Examine on the third day and if mould colonies are numerous. Handle the plates as little as possible when counting on day 3 so spores will not be dislodged. Alternatively. This technique provides maximal exposure of the cells to atmospheric oxygen and avoids heat stress from molten agar.2 Moulds should be enumerated by a surface spread-plate technique rather than with pour plates. 5.6.5. Plating 5. select plates that fall nearest to the 10-150 range. If the mycoflora consists primarily of moulds. If possible. and count colonies in this area. if primarily yeast colonies. Count colonies. Incubation Incubate plates undisturbed in an upright position at 22 to 25oC for 3-5 days.4. Results are expressed as an estimated count.

g.8. often interfere with the enumeration of colonies.1.e.3. where N is the number of colonies per g (mL) of product. milk powder.4. Avoid creating erroneous ideas of precision and accuracy when computing counts (Table II). i.8. etc. 1/10-1 = 10. the Dilution Factor. pipette directly into peptone water diluent Viscous liquids Weigh into peptone water diluent Solids Water soluble solids Weigh into peptone water diluent Powder. Yeast cells and asexual mould spores are generally gram-positive. use the formula: N = A x D. Recording Results 5. 5.8. following the examples in Table II: Standard Methods for the Examination of Dairy Products. 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. (The lowest possible average with one colony on one of the two duplicate plates is: 1+0/2 = 0. A is the average count per plate.2. If the lowest dilution plated shows no colonies. Calculate the average count (arithmetic mean) of the duplicate plates.1. For other dilutions. This value is for a 100 dilution (Dilution Factor = 1). Round-off counts to two significant figures and record only the first two left hand digits. e. 5. for 1 mL and a set of duplicate plates (1 mL/plate).. juice. E.8. the numerical value of 0. 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. To compute the yeast and mould count. the value is <0. and D is the respective dilution factor TABLE I Preparation of Initial Dilution Type of food Preparation* Liquids milk.g..5 must be multiplied by the reciprocal of the dilution. Differentiation of Colonies from Interfering Particles 5.6.. whereas mould mycelia are gram-negative. water.5.8. This can be eliminated by making one extra plate of each dilution containing interfering particles.7. fish products Weigh into peptone water diluent Treatment shake shake shake stomach or blend stomach or blend shake stomach or blend 51 . and holding it under refrigeration as a control for comparison during counting. 5.4. 5. Wet mounts and gram stains of several diverse types of cells per sample should be examined to confirm that bacteria are not present.7.5. etc.5). Food particles such as meat. 5.

e.g.000 counts higher than 150.000 E* 1:1000 counts lower than 15. TABLE II Examples for Recording Results Examples of the average number of Dilution Report as no.g. e. blender jar or dilution bottle and the diluent added prior to mixing.Sample may be added into an empty stomacher bag.g... 10 Lowest dilution10.. of yeasts and moulds per colonies g (mL) count between 10-150. 440 Highest dilution440. e. 144 1:1000 140.000 E 1:1000 no count Lowest dilution<500 1:1000 * E is the estimated count 52 .

coli is present. As indicated in section 1. including the processing plant. i. The method is progressive. or a laboratory’s inhouse specifications. health agencies. coli serotypes associated with human illness. Many of the pathogenic serotypes do not give a positive faecal coliform reaction and therefore would not be detected and recovered by this method. food ingredients and water. 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. time and temperature of incubation. to extinction. then determining if these tubes also contain faecal coliforms. coli in food and water may be determined by means of the MPN procedure. Note: This method is not intended to be used to isolate and enumerate E. coli. this method involves serially diluting out the target organisms in the sample. and then confirming whether E. 53 . Briefly. first determining the presence of coliforms in the tubes. the most probable number present can be estimated from a standard statistical MPN table.” The presence of “indicator organisms” in foods processed for safety may indicate one of the following possibilities: 1. Enumeration of coliforms faecal coliforms and E. the presence of coliforms. 3. gas production from BGLB broth is considered confirmation of coliform presence. faecal coliforms and aerogenic Escherichia coli in foods. particularly the enterohemorrhagic serotype O157:H7. 2. The probable level of the target organisms is then statistically estimated from an MPN table.. Based on the number of tubes indicating the presence / absence of the three groups of organisms. however. including contact water from food manufacturing plants.e. The presence of faecal coliforms and E. coli in foods using the MPN method 1. therefore. are only meaningful when expressed in terms of the analytical test parameters of medium. Application The Most Probable Number (MPN) method is applicable to the enumeration of coliforms.D. The method has been shown to produce satisfactory results with naturally-contaminated foods and water for the detection of coliforms. faecal coliforms. one must assess the results against the tolerance limits specified by government standards or guidelines. keeping in mind that established standards and guidelines are specifically linked to the method used to develop these standards. faecal coliforms and aerogenic E. coli may indicate faecal contamination. faecal coliforms and aerogenic E. in 5-replicate aliquots. coli are considered “indicator organisms. When assessing the presence of “indicator organisms” in a sample. Gas production is used as an indication of ability to ferment lactose from LST broth (presumptive coliform test). and/or 2. microbial growth. it must be understood that these microorganisms can survive and multiply in a variety of non-intestinal environments. Principle The terms “coliform” and “faecal coliform” have no taxonomic validity and. Coliforms. inadequate processing and/or post-processing contamination. and E.

12) Stomacher.5 / 45o C and is capable of fermenting lactose to produce typical reactions on L-EMB agar. 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. aerogenes or an equivalent culture for EMB agar and IMViC tests. BGLB and EC broths) by producing a small gas bubble. dark-centred colonies with or without metallic sheen when positive EC broths are streaked onto L -EMB agar are indicative of E. VogesProskauer-positive. and appearance of typical nucleated. 35oC. it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency. 10) Thermometer. 4. aerogenes will give false-positive reactions in the MPN broths (LST. calibrated and traceable 11) Incubator. Note: If the analyst uses any variations of the media listed here (either product that is commercially available or made from scratch). 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.gas production at 44. 1) Peptone Water (0. The typical colonies on L -EMB agar must be confirmed by further biochemical tests to prove the presence of E. if using EC-MUG. berta or an equivalent culture for these broths and E. and citrate positive.0%). 13) Control cultures (use ATCC cultures or equivalent): positive control(s): E. coli. coli that is known to produce gas at 44. 54 .5 or 45o C from EC broth is used as confirmation of faecal coliform presence.5oC and 45oC. Water level should be above the medium in immersed tubes. 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. with circulating system to maintain temperature of 44. Therefore. methyl red-negative. 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. use S.5%) 2) Aqueous Sodium Citrate (2. coli.1% and 0. blender or equivalent.

depending on the nature of the product.1.3. Analyze sample units as soon as possible after their receipt in the laboratory. Rapid Identification Kits or Systems (such as API.2. Thaw frozen samples in a refrigerator. 5.1 pH units within the range of pH 5. Select 10 or more animals to obtain a minimum of 200 g of meat and liquor. Have ready sterile peptone water.0 to 8. Shellfish must be analyzed within 24 hours of collection. except for shelf-stable foods. 5. Tryptone (or tryptophane) broth Indole reagents (available commercially) b. within the same range.1.Raw and Processed Shellfish 5.9. 5. 5. 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.3 to 0. 5. Preparation of Sample.1. 55 . Initial Set-up and Reporting. keep sample units refrigerated (0-5oC) or frozen.2.1.5) may require alternate media A.3. Simmon's Citrate (SC) agar B. Do no use faucets equipped with aerators. 5. Drain shellfish in a clean container or on clean towels.5 pH units. 15) Supplies needed for confirmation (commercially available): The following supplies may be needed for confirmation.0 or pH paper capable of distinguishing from 0. Vitek or equivalent) 5.3.2.3. Carry out the test in accordance with the following instructions: 5. Handling of Sample Units 5. use A or B (see 7. In the laboratory prior to analysis.5% peptone water for all dilutions.14) pH meter capable of distinguishing to 0. For all shellfish. Include only live animals in the sample for unfrozen shellfish. Arrange LST broth tubes in rows of five and mark them identifying the sample unit and the dilution to be inoculated (Table II). Preparation for Analysis 5. IMViC media and reagents: a. Clean the surface of the working area with a suitable disinfectant.2. always use 0.9). Buffered Glucose broth Voges-Proskauer test reagents (available commercially) Methyl red solution c. or under time and temperature conditions which prevent microbial growth or death. 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.1.2.2.1.2.3.3. 5. The choice of further identification schemes (7.

Immediately (i.3.10.5% peptone water.6. as follows. add 20 g of the homogenate to 80 g of peptone water and shake. Inoculate each of the five tubes of 10 mL double strength LST broth (first row) with 10 mL of the undiluted water sample. 5. and record results as MPN per 100 g of shellfish.e.8. Using a sterile shucking knife. 1 mL of 1 in 10 dilution into each of 5 tubes of single strength LST. 5.9. 5. Follow incubation of LST and confirmation steps for coliforms.. Inoculate each of separate sets of five tubes of LST broth with each dilution to be tested.1 mL of undiluted water.4. Disinfect hands (soap and water.3. and record results as MPN per 100 mL of water 5.3. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present. To ensure a truly representative analytical unit.7. 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.5. 5. Initial Set-up and Reporting . open the shellfish through the bill. within 2 minutes after blending) prepare the dilutions from the ground sample and then proceed to inoculate into tubes. 5. Preparation of Sample.3.4. Shake dilutions 25 times through a 1 -foot (30 cm) arc in approximately 7 seconds. agitate liquids or free flowing materials until the contents are homogeneous.1.2. Alternatively.5.4.1. not hinge.3. Follow incubation of LST and confirmation steps for coliforms.4. A protective mail glove may be worn under the disposable glove to prevent accidental injury. Preparation of Sample. and 1 mL of 1 in 100 dilution to each of 5 tubes of single strength LST. the 56 . obtain the analytical unit by taking a portion from several locations within the sample unit.5. 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. To reduce the workload. Initial Set-up and Reporting . and collect meats and liquor into a sterile container. Inoculate each of separate sets of five tubes of LST broth with each dilution to be tested. Inoculate each of the five tubes of 10 mL single strength LST broth (second row) with 1 mL undiluted water. If the sample unit is a solid. faecal coliforms and E. according to the scheme in (Table II). 5. Blended homogenate represents a 1 in 2 dilution. coli as required.3.2 minutes.All other commodities 5. coli as required. 5. faecal coliforms and E.Water 5. Prepare succeeding decimal dilutions as required using a separate sterile pipette for making each transfer. 5.3. Weigh at least 200 g of shellfish and liquor into a tared blender jar and add an equal amount of 0. Blend for 1 . To obtain a 1 in 10 dilution.5. Inoculate each of the five tubes of 10 mL single strength LST broth (third row) with 0. use disposable gloves disinfected with 70% alcohol.

In all other instances. Pipette into LST as in 5. 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.5. Weigh 100 g fish products and add 300 mL of 0.10.5.5. Blend for 2 minutes.5-7.5. 5. Set up an uninoculated tube of medium corresponding to each step in the procedure as a media control. faecal coliforms and E. Incubation of LST 5. coli as required. Mix inoculum and medium by gently shaking or rotating the tubes.5.9 and express results as MPN/100g.5. Weigh 40 g of homogenate into 60 mL of 0. In order to verify growth conditions in the elevated temperature water baths.2. Follow incubation of LST and confirmation steps for coliforms. 57 . Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present. For fish products an alternative method may be used. Check pH of the food suspension.2.3.4. blend or stomach for the minimum time required to produce a homogeneous suspension and to avoid overheating. Inoculate each of the five tubes of 10 mL single strength LST broth (first row) with 1 mL of the10 -1 dilution. If the pH is outside the range of 5. With products that require blending. 5.6. blending time should not exceed 2.5 min. 5. Allow the food homogenate (1 in 10 dilution) of dry foods to stand at room temperature for 15 min. 5.5. When blending foods that tend to foam. but avoid entrapping air in the gas vials.3.analytical units may be combined for analysis.9.5. If five sub-samples are composited for analysis.1% peptone water. 5. 5.8. adjust pH to 7.6. 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. use blender at low speed and remove aliquot from below liquid/foam interface. 5.6. Inoculate each of separate sets of five tubes of LST broth with each dilution to be tested. for each bath used.5. as indicated in Tables I and II. Blended homogenate represents a 1 in 4 dilution.1 % peptone to obtain a 1 in 10 dilution. 5.7. aseptically blend 50 g or mL into 450 mL of the required diluent.1.5. 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. according to the scheme in (Table II) as follows. Inoculate each of the five tubes of succeeding rows of single strength LST with 1 mL additional dilutions.5.0 with sterile 1N NaOH or 1N HCl. Transfer into all media used at different stages of the procedure. 5. 5. It is recommended that a composite contain not more than 500 g. continue the analysis without this delay. inoculate one LST broth tube with the MPN broths positive control and one LST broth tube with the MPN negative control.6. 5.

5.6. record the numbers of additional gas-positive tubes and add to the result obtained in 5.5. record results. examine. as required.4. Formation of gas during 48 ± 4 h incubation constitutes a positive confirmed test. record the number of additional gas-positive tubes.4. 5. and begin the confirmed coliform. 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.5.8.5.2. 5. but avoid entrapping air in the gas vials.2.3. Examine for gas formation (gas bubble or effervescence) and record results.4.7. Mix inoculum and medium by gently shaking or rotating the tubes. 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.7.5oC). Mix inoculum and medium by gently shaking or rotating the tubes. 5.7. coli tests for all gas-positive tubes.6. 5. 58 . 5. Sterile wood applicator sticks or other appropriate transfer devices may be used for making the transfers. dispensed in 10 mL volumes in tubes containing gas vials. Incubate gas-negative tubes (except raw shellfish and fish products) for an additional 24 ± 2 h. and E. as required.3 and begin the confirmed coliform.4. Maintain the water level in the bath at least 1 cm above the level of the medium in the tubes.7. Use EC broth (with or without MUG).6. 5.7. Shake or rotate the positive LST broth tubes (obtained in 5.6.7. coli tests for the additional gas-positive tubes. This transfer should be made simultaneously with 5. 5. Examine for gas formation (gas formation may be either a gas bubble or effervescence). 5. Use BGLB broth dispensed in 10 mL volumes in tubes containing gas vials. 5. add to the result obtained in 5.7. but avoid entrapping air in the gas vials. Incubate the inoculated LST broth tubes at 35oC for 24 ± 2 h. Confirmation Steps for Determination of Faecal Coliforms 5.6. Incubate the inoculated EC broth tubes in a water bath at 45oC for 24 ± 2 h (for shellfish and fish products analysis incubate at 44.7.7. faecal coliform and E.8. 5. faecal coliform.3.1.8. Confirmation Steps for Determination of Coliforms 5.7.7 above 5.8.1.8.6) to mix the contents and transfer one loopful from each tube to a tube of EC broth (avoid transferring pellicles). re-examine. Incubate the inoculated BGLB broth tubes at 35oC for 24 ± 2 h.3. Incubate gas-negative tubes for an additional 24 ± 2 h. Sterile wood applicator sticks or other appropriate transfer devices may be used for making the transfers. 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). 5.

8. coli identification for the additional gas-positive tubes. Note: It is up to the Laboratory Supervisor to determine which dilutions and sets of presumptive (gas.8. add to the results obtained in 5.9. pick two typical colonies and re-streak onto EMB to obtain discrete colonies. 5. coli 5. pick one typical colony and streak onto a non-selective agar such as NA (EMB or MacConkey can also be used).8. 5. 5.9.2. 59 . 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.1.8. 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). record the number of additional gas-positive tubes.3. Note: Confirmation can be done by either completing the GIMViC tests (5.7. subsequently.5. 5.8. coli test. coli identification for all gas-positive tubes (5.9 to confirm presence of E.5 and begin the E. 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. examine.5 and 5.9. 5. Circle one other typical colony on EMB before storing the plates at 4oC. the number of colonies picked per plate) to adequately determine the final and confirmed MPN count.8. Examine for gas production (gas bubble or effervescence). record results.10.5). 5.9).9. nucleated.8. Formation of gas during 48 ± 4 h (24 ± 2 h for raw shellfish and fish products) incubation constitutes a positive faecal coliform test. coli. Use these cultures for further confirmation. If the colonies are not well isolated on L-EMB or Endo agar plates. and examine for typical non-mucoid. to be taken to non-selective media if the initial colony does not confirm as E.6.positive) MPN tubes are to be confirmed (and. Incubate the plates at 35oC for 18 to 24 h. Precautions: Follow safety precautions in the manufacturer’s instructions when using the UV light. Incubate gas-negative tubes (except raw shellfish and fish products) for an additional 24 ± 2 h.8. 5. Confirmation Steps for Identification of E. Negative controls of the EC-MUG broth should be also examined under the UV light to ensure that the tubes do not fluoresce.5.9. Incubate at 35oC for 18-24 h.4) or by the use of a rapid identification kit (7. Incubate as above and use these cultures for further confirmation. these tubes may be used for further testing described in 5. dark-centred colonies with or without a metallic sheen which are indicative of E. If the colonies are well isolated on L -EMB or Endo agar plates.MUG broth tube (5. coli.9. coli.9. Blue-green fluorescence indicates a positive presumptive E. Gently shake each gas-positive EC broth tube or each fluorescing EC. Tubes containing EC-MUG broth should also be examined under UV light (366 nm) for glucuronidase activity.8. Refrigerate the second EMB plate in case it is needed at a later point. and begin on the same day E.6) and streak a loopful of the culture onto a L-EMB or Endo agar plate.8.

The test is VP-positive if an eosin pink colour develops after 5-10 minutes. Inoculate one tube of each of the GIMViC media for each of the isolates to be identified. and negative if a yellow colour develops. Alternatively. "I" -medium is Tryptone broth. If both isolates fail to produce IMViC reaction patterns characteristic of E. A yellow colour would be considered negative. use a straight needle and apply a light inoculum. Add indole reagent (commercially available) to each tube following manufacturer’s instructions. IMViC tests may be done using any commercially available testing system. Collectively they are referred to as the GIMViC media.5o C or 45. If no gas has been produced. Record results. test the second isolate for its GIMViC reaction pattern. Gas Production at 44.5. A dark red colour in the alcohol layer indicates a positive test. coli are obtained with GIMViC tests. 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. "M". Inoculate IMViC positive and negative controls into each of the IMViC media and MPN positive and negative controls into secondary EC broth.4. If necessary. Indole (I) Incubate inoculated tubes of Tryptone or tryptophane broth at 35oC for 24 ± 2 h. However. coli. EMB or MacConkey). coli is given in Table III. the other isolate need not be further tested. Examine for gas production. then E. transfer inoculum into a separate tube of each of EC broth (G medium) and the IMViC media.9. if the first isolate gives a non-characteristic IMViC pattern. GIMViC From the streaked plates (NA. and "C"-medium is Simmon's Citrate agar.and "V"-medium is Buffered Glucose broth. incubate for an additional 24 ± 2 h and re-examine. prepare fresh plates or slants prior to inoculating the GIMViC media. If GIMViC tests are not carried out within 96 h of inoculating the non-selective agar. where the "G"-medium is the secondary EC broth. An orange colour probably indicates the presence of skatole and may be reported as a ± reaction.0oC (G) Incubate inoculated tubes of G medium (EC broth) in a water bath at 44. coli is 60 . commonly occurring coliforms may be differentiated by using the data in Table IV.0oC for 24 ± 2 h. If characteristic reactions for E. Incubate the slants at 35oC for 48 ± 2 h and observe for growth. Repeat confirmation steps.5o C or 45. 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. Methyl-Red (MR) and Voges-Proskauer (VP) Tests (MVi) Inoculate 2 tubes of Buffered Glucose broth and incubate at 35oC for 48 ± 2 h. Simmon's Citrate Test (C) In inoculating the slants of SC agar. The MR test is positive if a red colour develops. Use MR and VP reagents (commercially available) following manufacturer’s instructions.

Rapid Identification Kits Rapid identification kits may be used to identify E. 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.6. blender jar or dilution bottle and the diluent added prior to mixing. 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.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. Calculation of MPNs Compute the MPN of E. coli. coli. coli as given above or confirmed by rapid identification kits as E.9. 5. water. 61 . products Treatment shake shake shake or or blend stomach weigh into previously warmed (40-45oC) 2% aqueous sodium blend citrate (Na3C6H5O7. meats weigh into peptone water diluent all cheese spices shellfish. 5. etc.5.considered to be absent from the tube of primary EC broth from which the isolates originated.9. TABLE I Preparation of the Initial Dilution Type of Food Preparation* Product Liquids: milk. Follow manufacturer’s instructions.

1 g strength medium -2 -2 1 in 100 10 1 mL of 10-2 dilution into 10 mL single 0.5 . 100 1 mL of undiluted liquids into 10 mL1 mL single strength medium 0 0 undil. depending on the anticipated level of contamination of the food.1 g or mL strength medium -2 -2 1 in 100 10 1 mL of 10-2 dilution into 10 mL single 0. into single strength medium.45oC Type I G Type II + (Anaerogenic) Indole I + Methyl Red M + + Voges-Proskauer V Citrate C - 62 . 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 0.3 undil.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 0.01 g or mL strength medium -3 -3 1 in 1000 10 1 mL of 10-3 dilution into 10 mL single 0. coli Biotypes Gas at 44. 10 1 mL of undiluted water into 10 mL1 mL single strength medium -1 -1 undil. * Other marking schemes may be used.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. Table III GIMViC Pattern for E. For inoculation of initial dilution of shellfish see Section 5.001 g or mL strength medium -4 -4 1 in 10 1 mL of 10-4 dilution into 10 mL single 0.01 g strength medium ALL OTHER COMMODITIES 0 undil.0001 g or mL 10000 strength medium Further dilutions of the food may be inoculated in the same manner.1 mL of undiluted water into 10 mL 0. 10 0.

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. 63 .TABLE IV** Differentiation of Commonly Occurring Coliforms Gas in EC broth at Indole 44.5 .

blender. Description The method has been shown to produce satisfactory results with artificially-contaminated meats (including beef.5 9) Stomacher. spices and environmental samples. 4. Principle The sample is enriched in a selective broth and plated on selective agars. Presumptive positives are determined within 48 h. This method can be used successfully for the detection of 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 64 .Isolation and Enumeration of pathogenic microorganisms in food. vegetables. and Cefsulodin 5) Purple broth base with cellobiose Other necessary supplies and equipment 6) 0. 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. food ingredients and environmental samples. coli (EHEC) Enrichment Broth (EEB) 3) Modified Hemorrhagic Coli Agar (mHC) with Tellurite and Cefsulodin 4) Modified Sorbitol MacConkey agar (TCCSMAC) with Tellurite.5 pH units within a range of 6. coli O157 (H7 or other serovars) negative control: E.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. Cefixime. coli 0157 in foods 1. coli O157 in other foods. veal and pork). Confirmatory biochemical and serological tests are performed on purified colonies.0 to 7. or equivalent 10) Control cultures (use ATTC cultures or equivalent) positive control: E. 2. dairy products. A. Application This method is applicable to the isolation of viable Escherichia coli O157 in foods.3 to 0. Isolation of E.

To reduce the workload. Other spices also may need to be analyzed using larger dilutions either -Na or -CHX 65 . such as onion and garlic powder are antimicrobial in nature. keep sample units refrigerated (0-5oC) or frozen. add 0. 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). coli O157. In the laboratory prior to analysis.1. 5.5% K2SO4 to mTSB-n before autoclaving. It is recommended that a composite contain not more than five analytical units. 5. 5.2. 5. the analytical units may be combined for analysis. depending on the nature of the product.2.1 Have ready sterile mTSB-n (and/or EHEC enrichment broth (EEB)). or under time and temperature conditions which prevent microbial growth or death.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. Garlic especially affects E.2 Clean the surface of the working area with a suitable disinfectant. 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. Thaw frozen samples in a refrigerator. Preparation of Sample 5. Procedure Each sample unit may be analyzed individually or the analytical units may be combined.1. To ensure a truly representative analytical unit agitate liquids or free flowing materials until the contents are homogeneous. Carry out the test in accordance with the following instructions: 5.2.2. 5. obtain the analytical unit by taking a portion from several locations within the sample unit. Handling of Sample Units 5. If the sample unit is a solid.3. the enrichment broth should be prewarmed to 35oC.3. except for shelf-stable foods.1.1. therefore garlic or garlic containing products need to be diluted 1:100. Stomach or blend.1. Analyze sample units as soon as possible after their receipt in the laboratory. Note: Some spices. Note: When analyzing larger volumes. Preparation for Analysis 5. When analysing spices or products containing large amounts of spices.3.2.

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

67

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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coli O157:H7 E.Distilled water 1.(No Color) .(No Pigment) .(Negative Reactions) E.(Pale) + (Positive) + (Positive) + (Positive) + (Positive Reactions).(Negative) .(Yellow) + (Red) + (Blue or Growth) + (Yellow) + (Brilliant Pink) + (Pigment) + (Fluorescence) + (Coloured) . Urea Agar Slants Table 1. 6. coli O157:H16 and H45 fluoresce when MUG is present.(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) . cool and dispense in Petri plates. 70 .(Negative) . coli ReactionsA Negative O157:H7 Other or non-E. coli Reactions Positive .(Pale) .(No fluorescenceb) . Characteristics of E.(No Color) .(Green or No Growth) . .(Purple) .0 L Autoclave for 15 min at 121oC.10.

some penicillins. The enterococci are differentiated from other streptococci by their ability to grow at high pH (9. and S. gallinarum.B. S. Its genome is 3 mb in length. and lincosamides. The two genetic mechanisms first 71 .5% sodium chloride). E. aminoglycosides. faecalis and E.6 Can grow at temperatures ranging from 10 to 45°C Optimunm growth at 37°C Sensitive to chlorination • • • • • • • • 2. E. durans. faecalis. faecium are the most frequent species found in humans. avium. faecium.6 to 4. Enterococcus 1. S.5% NaCl Can grow at a pH range of 9.6 at 10). high temperature (45°C) and in high salt concentrations (6. faecalis is the only enterococcus species that has been genetically characterized. Taxonomic description The enterococcus group is a subgroup of the fecal streptococci that includes at least five species: S. The enterococcus are generally resistant to many Gram positive antibiotics such as the tetracyclines. 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. sulfonamides. S.

Some strains require vitamin B and amino acids for growth. Selected differential physiological characteristics for species of the enterococci. E. tellurite Acid from Glycerol Acid from Mannitol Acid from Sorbitol Acid from L-arabinose Acid from Lactose Acid from Sucrose Acid from Raffinose -/+ + + + + +/+ + + -/+ + + + + + + - + + + + +/+ + + + + + +/- +/+ + +/+/+ +/+ + - + + -/+ -/+ +/+ + + + + + - 72 .5% NaCl Growth at 40% bile Resists 60°C for 30 min NH3 from arginine Gelatin liquefied Tolerates 0.faecalis E. durans E.6 Growth at 6.04% Pot. faecium E.discovered in the enterococci were conjugative transposons and sex pheromone plasmids.equinus Hemolysis Growth at10 °C Growth at 45°C Growth at 50°C Growth at pH 9. bovis E.

a group A streptococcus. The picture on the left shows the differential reaction that identifies the enterococci on bile esculin agar. faecalis dominates the guts of humans in the United States and England. fruit juices. Water quality guidelines based on enterococcal density have been proposed for recreational waters. 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. or fecal contamination. neonycin. Tyramine may be involved n causing migraines. 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. selenite. especially when isolations are made from food or clinical samples. The picture at left shows hemolysis on blood agar by S. Symptoms include nausea. and diarrhea. phenylethyl. Two of the five enterococcal species (faecalis and durans) will usually produce hemolysis on blood agar (see above table). raw and pasteurized milk. NaCl. Isolation and ecology Most procedures employ presumptive media followed by confirmatory tests. bile. For isolation. dried whole egg powder. They are also found routinely in frozen seafood. Ethyl violet azide (EVA) broth can be used as a confirmation. cheese. Fecal enterococci from water can be isolated. processing equipment. Enterococci occur naturally in soil and can be readily isolated from most plant roots as well. frozen fruits. faecalis and E. that inhibits catalase positive organisms. perfringens. This is selective differential agar that contains sodium azide. alcohol. In India and Japan. and thallium. E. the Association of Food and Drug Officials of the United States recommends KF agar medium. pyogenes. They get into food through vegetation. There is also a tyrosine decarboxylase activity procedure and a mentagan test that works well. taurocholate. vomiting. cereus and C. and enumerated in this broth.Acid from Melibiose Acid from Melezitose Starch hydrolyzed Tetrazolium reduced at pH 6. Enterococci are able to grow in the presence of bile and hydrolyze the esculin. Growth of fecal enterococci in EVA results in turbidity and a purple sediment in the bottom of liquid cultures. but are milder than those caused by other food borne illnesses. and tetrazolium chloride which produces a red color in the colonies. The guideline is 33 enterococci/100 mL for 73 . Due to diet.0 + + + - - + + +/- - 3. and vegetables. the liberated diphydroxycourmarin complexes with ferric citrate present in the media to form a dark brown/black soluble compound. 4. Primary selective agents can be azide. Blood agar is often used as a diagnostic test for the enterocococci. processing environments. E. faecium are equally found in the intestines. Occasionally they are used as starter cultures for making hard cheese. tellurite. cultivated. Some strains produce high levels of the amines tyramine and histamine. Tween 80. Symptoms are similar to B.

74 . A well isolated colony from brain-heart infusion agar is then transferred onto a brain-heart infusion broth tube and incubated. and antibiotic sensitivity (the above right picture shows the antibiotic disk assay for bacitracin). If not turbid. In the membrane filter technique. the tube on the far left is positive). the guideline is 35 enterococci/100 mL. additional tests that may be conducted include bile solubility (above left picture. Brownish-black colonies with brown halos confirm the presence of fecal streptococci. however. The membrane filter technique is used for samples of fresh and saline waters.recreational fresh waters.5% NaCl indicates presence of enterococcus group. For clinical or food samples. After growth. Growth at 45°C in 6. but is primarily used for raw and chlorinated wastewater. For the presumptive test procedure of the multiple-tube technique. These colonies are transferred to a tube of brain-heart infusion broth containing 6. For marine waters. There are two types of selection methods. a series of azide dextrose broth tubes are inoculated and incubated. The filter is transferred to EIA medium containing esculin and ferric acid as selective agents.5% NaCl. it is unsuitable for highly turbid waters. a sample of the culture is transferred to bile esculin agar.5% NaCl. Tubes showing turbidity are streaked onto Pfizer selective enterococcus (PSE) agar. The guidelines are based on the geometric mean of at least five samples per 30-d period during the swimming season. and brain-heart infusion broth with 6. brain-heart infusion broth. The multiple-tube technique is also applicable to fresh and marine waters. the sample is filtered. tubes are reincubated. Growth indicates colonies of the enterococcus group. Pink to red enterococci colonies develop a black or reddish-brown precipitate. the filter containing the colonies are transferred to an agar medium which is incubated.

Biochemical Screening Isolates are screened using determinant biochemical reactions. 3. desiccation. preservatives. Tryptone) Soy Broth. 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. cultures should be sent to a reference typing centre for complete serotyping. Non-Selective Enrichment (Preenrichment). 2 Collection of samples Sampling Food control efforts frequently target processes and products presenting significant human health risks. Each food category carries an appropriately stringent sampling plan to determine the acceptability of the food product. 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. The International Commission on Microbiological Specifications for Foods (ICMSF) has categorized foods according to the degree of hazard associated with product use. Materials And Special Equipment 1) Nutrient Broth (NB).C. 75 . Serological Identification Polyvalent and/or somatic grouping antisera are used to support the tentative identification of isolates as members of Salmonella spp. 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. Isolation of Salmonella from foods 1. 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. freezing. 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. 2) Trypticase (Tryptic. For confirmation.

17) Physiological Saline. 4) Buffered Peptone Water (BPW). analyze these samples as soon as possible preferably within 24 h of receipt. 14) Urea Agar (Christensen's). including those that are received in a partially thawed condition. 5) Skim Milk Medium. Dried and shelf stable foods may be stored at room temperature. 6) Tetrathionate Brilliant Green Broth (TBG). 76 . 13) Lysine Iron Agar (LIA). If sample units have been abused in transit. 12) Triple Sugar Iron Agar (TSI). b. Refrigerate all other foods. stomacher or other homogenizing device. Frozen Foods: Sample units that show no signs of thawing upon receipt may be stored in the freezer at -10oC to -20oC. 15) Commercial biochemical test kits. 18) Blender. c. store samples under time and temperature conditions that will prevent the growth or death of native microflora. Thaw frozen samples at room temperature within 60 min. 10) MacConkey Agar. 8) Bismuth Sulfite Agar (BS). resampling of the lot should be carried out. 19) Incubator or water bath capable of maintaining 35±0. 7) Selenite Cystine Broth (SC). a. 9) Brilliant Green Sulfa Agar (BGS). If necessary. 4. 16) Polyvalent and single grouping somatic (O) and flagellar (H) antisera. Procedure Handling of Sample Units Analyze samples as soon as possible. SS agar 11) Nutrient Agar.5oC and 43±0.3) Brilliant Green Water. if this is not possible. thaw the samples at refrigerator (4 to 10oC) temperature.5oC.

375 g or mL).24 h. This eventuality is more frequently encountered in situations involving consumer complaints or food poisoning investigations. whole chicken) may not readily thaw at refrigerator temperatures. If the sample unit received for analysis is less than the recommended analytical unit. For products that do not require blending. For greater expediency. A positive Salmonella a a negative medium control should be set up in parallel with the test nd samples.g.0. Use aseptic techniques and sterile equipment at all stages of analysis.0 . analyze the entire amount and record the weight used. enclose the frozen sample in a heavy-walled paper bag and thaw overnight at room temperature. 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. adjust with 1N NaOH or 1N HCl. the analytical unit shall then consist of equal portions from each of the containers. If not possible or practical.g. Excessive blending could result in physical damage that would adversely affect the viability of endogenous microflora.NOTE: a) Large samples (e. If a sample unit consists of more than one container.5oC for 18 . b) Appropriate containers should ensure that the drippings from the product do not contaminate the laboratory environment. 77 . Containment during the handling of powdered products is critical if cross-contamination of the work environment is to be avoided. This technique maintains the product surface cold during the thawing process. If the pH of the preenrichment mixture lies outside the range of 6. disperse the analytical unit into the appropriate preenrichment broth. Blending of samples should be limited to the minimum time required to produce a homogeneous suspension. aseptically mix the contents of the containers prior to withdrawal of the analytical unit.7. Incubate the preenrichment mixture and the positive and negative controls at 35±0. 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. up to 15 x 25 g (mL) analytical units may be composited into a single test sample (e. Non-selective Enrichment (Preenrichment) Compositing of Analytical Units To reduce the workload. 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.

5oC for 24±2 h. proceed with serological testing.0 mL of the preenrichment culture into each of 9 mL of selenite cystine (SC) and tetrathionate brilliant green (TBG) broths.Selective Enrichment With a sterile pipette. If serological testing is not to be performed within 72 h. Store the agar slants at refrigerator (4 to 10oC) temperature. NOTE: Erroneous biochemical results may be obtained if tubes are not loosely capped during incubation. The absence of suspect colonies on the plates indicates that the analytical or composite test samples did not contain Salmonella spp. A heavy growth of non-salmonellae may also mask the presence of Salmonella on this medium. inoculate suspect colonies into the biochemical media or in commercial diagnostic kits that would yield equivalent results. lactose-positive biotypes will occur as pink colonies. If none of the isolates from a particular analytical unit are suggestive of Salmonella. Selective Plating Streak replicate loopsful of each selective enrichment culture onto BS and BGS agar to obtain well isolated colonies. BS agar can retard the growth of Salmonella serovars other than S. The enrichment c ultures may be streaked onto additional plating media for the isolation of Salmonella. typhi unless poured plates are refrigerated (4 to 10oC) for 24 h prior to streaking. b. and as black colonies on BS agar with or without a metallic sheen. Purification Streak suspect colonies onto MacConkey agar for purification.5oC. and showing a gradual H2S.5oC and 43±0. inoculate suspect isolates into nutrient agar slants and incubate at 35±0. Typical Salmonella usually occur as pink to fuchsia colonies surrounded by red medium on BGS agar. Lactose-and/or sucrose-fermenting Salmonella strains develop a coliform-like (greenish) appearance on BGS agar. respectively. If colonies suggestive of Salmonella have not developed on BS plates. Commercially available diagnostic kits may be used to obtain detailed biochemical profiles of bacterial isolates. Nutrient 78 . Examine incubated plates for colonies suggestive of Salmonella. However. incubate for an additional 24±2 h. Incubate the biochemical media for 18-24 h at 35±0. the analytical unit is considered to be free of salmonellae.dependent blackening of the surrounding medium with increasing incubation time.5oC for 24±2 h. NOTE: a. If the presence of Salmonella is suspected.5oC for 24±2 h. transfer 1. Incubate plates at 35±0.5oC. Incubate plates at 35±0. Typical Salmonella colonies are lactose-negative and will appear as colourless colonies on this medium. Biochemical Screening With a sterile needle. Incubate SC and TBG broths for 24±2 h at 35±0.

Prepare fresh agar slants for this purpose. C(negative control) and T (test culture). and two drops to the area marked C-.with a sterile needle or loop. 79 . prepare a heavy suspension of a known Salmonella culture in the area marked C+. Add one drop of physiological saline to each of the areas marked T and C+.C. Salmonella belonging to uncommon serogroups may be missed. Mark the following sections on an agglutination plate: C-(negative control) and T (test culture).D. or E. The inoculum should be withdrawn from the slope portion of agar slants. The serological test for a given culture is invalidated if the negative control shows agglutination (autoagglutination). Nevertheless. Such misleading reactions can be resolved through further testing with somatic grouping and flagellar antisera. Mix the culture-saline-antiserum suspensions in T and C+ and the saline-culture mixture in C. Prepare somatic polyvalent antisera as directed by the manufacturer. Remove sufficient culture material from a triple sugar iron. Salmonella cultures usually agglutinate within 1 min. - - - 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. Serological Identification Testing with somatic polyvalent antiserum Mark the following sections on an agglutination plate: C+ (positive control). it is important to recognize that unless a complete set of grouping antisera is available. False positive reactions from microorganisms that are closely related to Salmonella may occur.agar slants that have been stored for more than 72 h should n be used for serological ot testing. 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. Hold the slide against a dark background and observe for agglutination. lysine iron or nutrient agar slant to prepare a heavy suspension in the test area (T) and in the negative control (C-) area. For the positive control. add one drop to each of the areas marked T and C+. Tilt the slide preparation back and forth for 1 min.

If the serological test is positive. add one drop to each of the areas marked T and C+. Hold the slide against a dark background and observe for agglutination. If the culture-saline-antiserum mixture does not agglutinate.with a sterile needle or loop. 80 . The inoculum should be withdrawn from the slope portion of the agar slants. the culture should be sent to a Salmonella typing centre for complete serotyping. Tilt the slide preparation back and forth for 1 min. Salmonella cultures usually agglutinate within 1 min. Mix the culture-saline-antiserum suspensions in T and C+ and the s aline. Remove sufficient culture material from a triple sugar iron. 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. and two drops to the area marked C-. The serological test for a given culture is invalidated if the negative control shows agglutination (autoagglutination). prepare C+ (positive control) Add one drop of physiological saline to each of the areas marked T and C+. lysine iron or nutrient agar slant to prepare a heavy suspension in the test area and in the negative control area. Salmonella isolates agglutinable with somatic antisera should be further identified by testing with polyvalent H antiserum. repeat the procedure with another somatic group antiserum.culture mixture in C.If a Salmonella control culture is available for each somatic group tested. Prepare somatic group antiserum as directed by the manufacturer.

or they may also indicate a lack of adherence to Good Hygienic Practices. From the results of these tests. e. and/or produce a heat-stable nuclease (TNase). the number of S. e.0 8) Stomacher.g. food ingredients and environmental samples. aureus coagulase negative. use the following or equivalent strains. This method determines the presence of S. e. aureus by plating known quantities of (dilutions of) a food sample onto a selective agar. ATCC 14990. either Blood agar (BA). presumptive staphylococcal colonies are selected. 3. 4) A non-selective agar.0 to 8. vegetables. This ability to produce enterotoxins. This method can be used successfully for the detection of Staphylococcus aureus in other foods. ATCC 7700 11) Crystal violet stain 12) Coagulase (Rabbit) Plasma. Positive Controls: S. and artificially-contaminated foods. with few exceptions. cereals and dairy products. poultry.D. The numbers present may indicate a potential for the presence of enterotoxin. 33501 Negative Controls: Escherichia coli. ATCC 23509 Pseudomonas aeruginosa. ATCC 27154. aureus coagulase positive. aureus per g or mL of the food is calculated. or Trypticase Soy agar (TSA) 5) TSA slants 6) Peptone Water diluent 7) pH meter or paper capable of distinguishing to 0.g. Application This method is applicable to the enumeration of Staphylococcus aureus in foods. Description The method has been shown to produce satisfactory results with naturally-contaminated meats.g. 25923 S.1 pH units within the range of pH 5. 2. Follow manufacturer's instructions for reconstitution 81 . Enumeration of Staphylococcus aureaus in Foods 1. 4. and subjected to confirmatory tests. After incubation. Principle Certain staphylococci produce enterotoxins which cause food poisoning. is limited to those strains that are coagulase-positive. fish. blender or equivalent 9) Vortex mixer or equivalent 10) Control strains. Nutrient agar (NA).g. Materials and special equipment 1) Baird-Parker (BP) agar base 2) Egg Yolk Tellurite emulsion 3) Brain Heart Infusion (BHI) broth. e.

Thaw frozen samples in a refrigerator.1. Procedure: Each sample unit shall be analyzed individually.5% glucose. Handling of Sample Units 5.13) Incubator capable of maintaining 35oC. Anaerobic utilization of glucose (Phenol red carbohydrate broth with 0. to ensure a representative analytical unit. Preparation of sample 5. or 82 . Latex Agglutination kits D.1 Have sterile peptone water prepared (may be stored under refrigeration for up to 12 weeks).2 Analyze the sample units as soon as possible after receipt at the laboratory.2.5% mannitol. 5. 5. sterile paraffin oil) F. Sample units of frozen products shall be kept frozen. 5. lysostaphin solution) H.1.2.1. The test shall be carried out in accordance with the following instructions: 5.2. Accuprobe B. keep the sample units refrigerated (0-5 C). Preparation for Analysis 5. sterile paraffin oil) G. Rapid ID kits E.3. Lysostaphin sensitivity (phosphate saline buffer. 5. Combine portions from several locations within each solid sample unit. the following shall apply: w the exception of shelfith o stable products.2 Clean the surface of the working area with a suitable disinfectant. Anaerobic utilization of mannitol (Phenol red carbohydrate broth with 0. 14) Waterbath capable of maintaining 50-55oC 15) Supplies needed for confirmation: (The following supplies may be needed for confirmation) A.3. Enterotoxin assay C. or under time and temperature conditions which prevent microbial growth or death.1 During storage and transport. TNase 5.1.

agar plates. Agitate each dilution to resuspend material that may have settled during preparation. Retain the plates in an upright position until the inoculum has been absorbed by the medium (approximately 10 minutes on properly dried plates).3.3.4. If counts of fewer than 1. the analysis should be continued as soon as possible.4.5. With foods that tend to foam. blending time should not exceed 2. 5. Plating 5. In all other instances. If the inoculum is not readily absorbed by the medium. spread 0.7. aureus 5.4. thoroughly mix each sample unit by shaking the container. 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.000 S.P.1.4. 5. 5. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present. If the pH is outside the range of 5. shake the dilution bottle 25 times through a 30 cm arc in approximately 7sec. see Table I). The liquid should not be spread right to the edge of the plate.000 S.e.6. use blender at low speed and remove aliquot from below liquid/foam interface. 5. Routinely (i. 5. If the sample unit is a liquid or a free-flowing solid (powder).2 mL of the undiluted analytical unit may be spread onto duplicate B.4.5-7.3.2b. 83 . Blend/stomach for the minimum time required to produce a homogeneous suspension. Shake. 0.1. the plates may be placed in an upright position in an incubator for up to one hour.4. Plating should be carried out within 15 min of preparing the dilutions.9. stomach or blend according to the type of food as indicated in Table I.2.6. 5.3.1. adjust pH to 7.1.0 with sterile NaOH or HCl.1. 5.5 If the 1:10 dilution is to be mixed by shaking.4. Check pH of the food suspension.5. since this causes confluent growth at the plate-agar interface which is difficult to count.3. agar plates. to avoid overheating. If the sample units are liquid. 5.4. NOTE: Weight or volume in brackets indicates alternate procedure for making dilutions.2a. 5.1.P. 5.3. spread 0. 5. agar plates. for counts higher than 1.5 min.1. using a separate sterile pipette for making each transfer. Prepare succeeding decimal dilutions in peptone water as required.8.2 mL of each dilution to be used onto duplicate B. aureus per g or mL of the food).4 mL of the 1:10 dilution evenly over the surface of each of five B.P.3. 5.3.3.1.4. aureus per g of a solid food are expected. 5. The food homogenate (1:10 dilution) of dry foods should stand at room temperature for 15 min.4 Enumeration of Presumptive S.3.

Type 4.2.4.4. If the number of presumptive staphylococcal colonies on some of the five plates is < 20.3. Plates should be observed at 24-30 h for possible overgrowth.4. If the number of all presumptive staphylococcal colonies per plate is between 20 and 200.4. Add the results and report as the total presumptive count per g of food.4.4. but on others is 20. 5. Count colonies immediately after the incubation period.4. Each colony type may show grey-white margins around the colonies and/or opaque zones (double halos). above. Convex. 5.5.2.04 g of food) (A/2).4. entire. proceed as in 5.4.1. count separately the colonies of each type and compute the respective average presumptive count per plate (per 0. Count the colonies of each type and record separately.4. and record as the respective presumptive count per g of food (C).4.4. 5. This is the count of one of the four types per 2 mL (0.4.1.1. Such colonies usually belong to the genus Bacillus.2.3. shiny black without well defined clear zones.4. 5.2.1. Counting of five plates of the 1:10 dilution (solid food only) 5. 5. If the number of all presumptive staphylococcal colonies per plate is fewer than 20.4 mL.4.3.4. Incubation 5. Multiply each count by 25 and record as the respective presumptive count per g of food (C). 5. add separately the counts for each type from all five plates and record as the respective presumptive count. Convex.2 g of food). Observe the following four types of presumptive staphylococcal colonies: Type 1. Counting Colonies and Recording Results 5. shiny black surrounded by clear zones extending into the opaque medium. 5. Grey colonies similar to type 1.4. and report as the total presumptive count per g of food.2. Multiply each count by 5. Type 3. Add the results.3. Counting of duplicate plates (any dilution) 84 . select two plates at random. Grey colonies similar to type 2. presumptive colonies may be counted at this time but the count should be verified at 48 ± 2 h. Type 2. which is equivalent to 0. Invert the plates and incubate at 35oC for 48 ± 2 h. 5.2. Black mucoid colonies larger than 2 mm in diameter and swarmers should not be counted.3. entire.5. Avoid excessive crowding or stacking of plates in order to permit rapid equilibration of plates with incubator temperature.3.4. but add together to give the total presumptive count.

5. the counts on all four plates should be used to arrive at the average count.4. 5.5.4.6. then the isolate is considered S.4.5.5 x the dilution factor for duplicate plates.4.6. no further confirmation is required. Inasmuch as the four different types of colonies are to be counted separately and it is quite possible that individual counts may be < 20. Run controls (positive and negative cultures as well as media controls) simultaneously when performing all confirmation tests. perform at least two of the following confirmation tests. the counts are to be averaged as shown below.4. If no presumptive staphylococcal colonies are obtained. If plates from more than one dilution are used. this average itself becomes an estimated value. Select plates containing 20-200 presumptive staphylococcal colonies per plate consisting of the combined counts of all types.5.5. When an estimated count contributes to an average count. or < 2. and record as presumptive count per g or mL of food for each type (C). multiply by five and by the appropriate dilution factor.4.4. see Table II.4. Confirmatory Tests For confirmation of S. estimated counts may be made on plates giving presumptive counts outside this range. 5.1. aureus. Compute the average presumptive count per plate for each type (A/2). record presumptive counts as < 5 per g or mL for the five plates of the 1:10 dilution. although the combined counts are within range. it is important that lysostaphin sensitivity 85 . 3) Latex agglutination kits. perform the coagulase test (following manufacturer’s instructions) as an initial step. 5. 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.5. Report results as estimated counts when results are outside the range of 20-200. aureus is available. 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. 5. 5.2. If no plate containing 20-200 presumptive S.5. estimates and true values would have to be combined in order to arrive at an average value. Add the results and report as the total presumptive count per g or mL of food.3. 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.1.3. ( 1 /(Dilution1) + 1 / Dilution2) 5. 2) Staphylococcal enterotoxin assays. 4) Rapid ID kits or5) at least one of the additional confirmatory tests listed in section 5. If the coagulase reaction is 3+ or less. If two of these tests are positive.5. 1) Accuprobe method. 5.aureus. aureus.5.

5.5.1. pick five colonies of that type at random.1.1.7. Incubate the inoculated BHI broth tubes at 35oC for 18-24 h and observe for growth.1. Selection of Colonies 5.5. such as BA.3.5.5 mL certified coagulase plasma. utilizes glucose and mannitol anaerobically.5. pick all colonies of that type.5. epidermidis.1. 5.and anaerobic utilization of glucose are not the only two tests carried out since these do not distinguish between S. 5. 5. Incubate at 35oC for 24 ± 2 h. and submit them to the subsequent tests as required. 5.1.1.8. Inoculate a culture of Staphylococcus aureus known to produce coagulase and TNase.5.5. If an isolate is not pure. 5. 5.2.5. Do not shake tubes during incubation. When the total count per type for all plates of a dilution is equal to or greater than five colonies. 5. aureus and S.. If the isolates are composed of cocci only.5. Make a smear from the growth of each isolate on the non-selective medium and stain with a simple stain (e. Additional Confirmatory Tests 86 .3 Distinct clotting as shown in Fig.2 Incubate tubes at 35oC and examine after 1 h and after 4 h.2. 5.3.2. 2 is considered a positive coagulase reaction. Retain BHI broth cultures. Coagulase Test 5.5.1.4. 5. Use uninoculated medium from the same batch of BHI broth as a negative control. into BHI broth to serve as a positive control. From the replicate plates counted.1 Transfer 0.2.1. 5. 5.2 mL of each BHI broth culture into sterile 13 x 100 mm tubes containing 0.5.5.g.9.2 above and repeat colony isolation steps above.1. transfer inoculum from each into a separate tube of BHI broth.6. Inoculate the controls along with the test cultures. 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.5.5. Negative tubes should be incubated overnight at room temperature and rechecked. NA or TSA to obtain discrete colonies.1.5. 5.5.2.5. Observe microscopically for the presence of cocci. and is lysostaphin-sensitive. Transfer a representative colony from one of the non-selective media to a TSA slant. Mix thoroughly.1. Streak each colony picked onto a non-selective medium. choose another colony at step 5. crystal violet).

2.4.5. 5.3. Lysostaphin sensitivity Inoculate culture to be tested into 0. the isolate is considered to be S.2. Same as for glucose utilization except that the source of carbohydrate is mannitol.1.3. Add 0. it is essential that the load be sufficiently pre-heated before the actual sterilization period commences.3.1 mL of phosphate saline buffer to serve as a negative control.At least one of the following additional tests may be done. Colour change indicating an acid reaction is a positive test for S. aureus usually gives a positive reaction but some strains do not ferment mannitol. colonies tested (G) Same for types 2.5. Overlay with sterile paraffin oil and incubate at 35oC for 1824 h. aureus per g or mL of food (N T ). S. aureus types 1.5% glucose. aureus (P) / x g or mL (N1) 1(C) No. aureus type 1 per presumptive count type = S. 3 and 4 (N T = N1 + N2 + N3 + N4) No. Hence.1 Baird-Parker (BP) Medium 87 . record the total number of S. Total No. of colonies confirmed as No. Inoculate culture to be tested into a tube of carbohydrate fermentation medium containing 0.5. Anaerobic utilization of glucose. aureus per g or mL equals the sum of No. proper exposure times should be followed to ensure sterilization of flask solutions and heat stable culture media. aureus.5. keeping in mind that Anaerobic utilization of glucose and Lysostaphin sensitivity must not be the only two tests performed. Thermonuclease Test Perform the test for the presence of thermostable nuclease (TNase). 5.3.3. If the turbidity clears in the tube containing cells plus lysostaphin. See Table II 6. If clearing has not occurred in 2 h. S. Preparation of media For steam sterilization. Anaerobic utilization of mannitol.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. If two of the three additional confirmatory tests are positive. the test is positive for S. S. the test is negative.5. This varies considerably with the nature and size of the load. On the basis of the confirmatory tests for each of the four types of cultures. and there is no clearing in the control tube. particularly when prepared in large volumes (Refer to your sterilizer manual). aureus. 5.1 mL of lysostaphin solution to the original tube to give a concentration of 25 mg lysostaphin per mL of cell suspension. 5. Incubate both tubes at 35oC for up to 2 h. aureus. 6.5. 5. S. 3 and 4.3.

If commercial egg yolk emulsion is used. If commercial EY-Tellurite Enrichment is used. 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). do not use non-opaque plates.1. Egg yolk emulsion containing tellurite is also commercially available. Egg yolk emulsion (50%). The pH should be 7. b. The surface of the agar should be dried before inoculation. infusion from Proteose peptone Dextrose 200 g 250 g 10 g 2g 88 . Egg yolk emulsion is commercially available and usually contains about 50% egg yolk. 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 50 mL to 950 mL of the tempered (50-55oC) basal medium. Adjust the total volume of the complete medium to 1000 mL. It is therefore advisable to store the plates at room temperature overnight before inoculation.2.1 Preparation of complete medium.2 Brain Heart Infusion (BHI) Broth Calf brain.5% egg yolk. Use as per manufacturer’s instructions. 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. Cool to 50-55oC in a water bath. It has been observed that freshly prepared BP medium may be toxic to injured cells. Sterilize at 121oC (15 lb pressure) for 15 min. c. c. b. Use as per manufacturer’s instructions. Poured plates may be stored in the refrigerator for up to 4 days. a. Soak fresh clean eggs in 70% alcohol for 15 min. The medium should be opaque. (EY-Tellurite Enrichment). 6.a. make certain to add the equivalent of 2. 6. Add above ingredients to 950 mL of distilled water and heat to boiling to dissolve the medium completely. Mix thoroughly but gently and dispense into petri plates. infusion from Beef heart. Filter-sterilized 1% tellurite solution.

Sodium chloride Sodium Phosphate (Na2HPO4) pH 7.(Trypticase Blood agar base) Trypticase Beef extract Sodium chloride (NaCl) Agar pH 7. This medium is commercially available.5 g Dissolve ingredients in 1. Reconstitute as directed by the manufacturers.4 Coagulase Plasma Certified rabbit plasma containing EDTA is commercially available. Dispense and sterilize at 121oC. Mix thoroughly but avoid incorporation of air bubbles.5 Lysostaphin Solution Dissolve lysostaphin in phosphate saline buffer (0:02 M.4 ± 0. Cool to 45-50oC and add aseptically 5% sterile defibrinated blood. Heat to boiling to dissolve ingredients. Heat to boiling to dissolve ingredients. the plasma solution is cold enough to delay clotting for 10-15 min.000 mL of distilled water. Dispense. Sterilize at 121oC.000 mL of distilled water. It is not satisfactory for use if gross contamination occurs. pH 7.3-7.1 5g 2. The solidified. This medium is commercially available.4) to obtain a concentration of 50 mg lysostaphin per mL. 6. 6.0 g of Bacto peptone or equivalent in 1.1 Suspend ingredients in 1000 mL of distilled water. After being kept in the refrigerator. If dehydrated product is not available.6 Nutrient Agar (NA) Beef extract 3 g Peptone 5g Agar 15 g pH 6. complete medium cannot be reliquified. Peptone is commercially available.8 ± 0. 89 . use fresh rabbit plasma collected aseptically in containers with EDTA (1 mL of a 15% solution of the potassium salt per 100 mL blood). The reconstituted plasma may be kept in the refrigerator for five days without loss of potency. 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.1 10 g 3g 5g 15 g Suspend ingredients in 1000 mL distilled water. This delay can be prevented by warming the plasma solution to 35oC before use. Distribute as required (tubes or flasks) and sterilize at 121oC.3 Blood Agar .7 Peptone Water Dissolve 1. Dispense 99 mL into dilution bottles and sterilize at 121oC. 6. 6. 6.2 ± 0.

Autoclave for 15 min at 118oC and allow to cool.3-7. Stock solution 1 Distilled water Approximate pH = 8. final pH.2 Stock solution 2 Distilled water Approximate pH = 5.9 Phosphate Saline Buffer (pH 7. aureus.02 m) Prepare stock solutions of 0.6 50 mL 450 mL 10.0 mL By means of a pH meter. Final pH. and dispense 2 mL portions in 13 x 100 mm test tubes containing inverted fermentation tubes.3-7.8 Phenol Red Carbohydrate Broth Trypticase or proteose peptone no.4 g 85.2.5 mL portions in 13 x 100 mm test tubes containing inverted 6 x 50 mm fermentation tubes. in 800 mL of water with heat and occasional agitation. Aseptically add 0. Dissolve carbohydrate in 200 mL of water and sterilize by passing the solution through a bacteria retaining filter. Autoclave for 10 min at 118oC. 0.02 M phosphate saline buffer for use in the lysostaphin susceptibility test on S.4. e.4 ± 0.2 6.4 by adding approximately 65 mL of diluted solution 2.5 mL of sterile filtrate to each tube of sterilized broth after cooling to 45oC. 7. These stock solutions are for preparation of the 0.2 mL of 0.02 M phosphate saline (0. titer the diluted solution 1 to a pH of 7. Dispense 2.g. omitting carbohydrate.0 mL 90.6 g 85. The resulting solution will be 0.5% salt (NaCl) solutions.018 g 1000 mL Dissolve 5 g of glucose or mannitol in this basal broth.3 ± 0. dissolve the ingredients. 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. Alternatively. Shake gently to mix.25% solution of phenol red) Distilled water 10 g 5g 1g 0.0 g 1000 mL Make 1:10 dilutions of aliquots of each stock solution to obtain 0.and di-sodium phosphate in 8. 7.0 g 1000 mL 28.85%) buffers.2 M mono.6. 90 .02 M phosphate saline buffer. 3 Sodium chloride Beef extract (optional) Phenol red (or 7.

Addition of 0. This medium is commercially available TABLE I Preparation of the Initial Dilution Type of Food Product Preparation Liquids: milk. Dispense and sterilize at 121oC.QC: Do not titer 0. 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 91 .000 mL of distilled water. 6.10 Trypticase Soy (TSA) Agar Trypticase peptone Phytone peptone Sodium chloride (NaCl) Agar pH 7.1 15 g 5g 5g 15 g Suspend ingredients in 1.3-7.3.02 M strength.2.2 M phosphate buffer to pH 7.3 ± 0. 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. Heat with frequent agitation and boil for 1 min or until solution is accomplished. water etc. The pH of the medium should be 7. This results in a drop in pH of approximately 0.25.4 and then dilute to 0.85% salt after pH adjustment also results in a drop of approximately 0.

"C" C = Types per g or mL Duplicate Plates "A" 1/2AxD*5** "N" N = (P/G)xC Fewer than 5 (e.000 and N2 = 100 and N3 = 0. of Isolates Total No. N2. aureus Count per g or mL of Food Total No.500 3. of Colonies of No. if N1 = 1. of S. Report total number of Staphylococcus aureus per g or mL of food to two significant figures 92 . All (4) 2 1.000 + 100 = 1. of No. Likewise. one of the four Types from one of the four the four Types on Tested "G" aureus "P" per g or mL.000 500 4) More than 5 (e. aureus Colonies of one of Isolates Confirmed as S. 5(5) 4 4. 0. N3 and N4 for each colony type to obtain total number of S. of No. when 5 plates of the 1:10 dilution are counted.2 mL per plate. Divide by 2 since "A" represents the total count of one of the four types on two duplicate plates.100 per g * Dilution factor = 100 ** For duplicate plates.g. and N4 = 0 NT = 1.g. divide by 5.600 18) Calculate N1. aureus.TABLE II Example of Computing S. (N T ) per g or mL NT = N1 + N2 + N3 + N4 e.g.

FIGURE 1 Flow Diagram for Confirmation Process FIGURE 2 Coagulase test reaction Tube number Intensity of reaction NEGATIVE POSITIVE (degree of clotting) 93 .

aureus reduces tellurite to form grey-black shiny colonies and then produces clear zones around the colonies by proteolytic action. saprophyticus produce both clear zones and opaque haloes but experienced workers can distinguish these from S. The selective agents glycine. most strains of Staph. Irregular and may produce clearing. no clearing. Not all strains of S. to protect damaged cells and aid their recovery2 and egg yolk emulsion as a diagnostic agent. Dry the surface of agar plates for a minimal period of time prior to use. Egg yolk emulsion makes the medium yellow and opaque. Egg yolk reaction negative strains of S. Growth Characteristics: Microorganism S. 94 . aureus. aureus Growth Good Colony Morphology Grey-black shiny convex 1-1.2 and improved its reliability in isolating S.5mm diameter (18 hours) up to 3mm (48 hours) narrow white entire margin surrounded by zone of clearing 2-5mm. This clear zone with typical grey-black colony is diagnostic for S. aureus. No clearing. Yeasts Technique Variable Variable 1. Not shiny black and seldom produces clearing. aureus may occur in some foods. epidermidis Variable S. Colonies typical of S. On further incubation. aureus produce both reactions. S. aureus form opaque haloes around the colonies. aureus by the longer incubation time required-5. Wide opaque zones may be produced in 24hrs. Baird-Parker added sodium pyruvate. lithium and tellurite have been carefully balanced to suppress the growth of most bacteria present in foods. It is now widely recommended by national and international bodies for the isolation of S. without inhibiting S. Brown-black with no clearing.Description (E-Y-T Emulsion) : Baird-Parker' developed this medium from the tellurite-glycine formulation of Zebovitz et al. saprophyticus Variable Bacillus sp. White. S. Variable Variable Proteus sp. Variable Escherichia coli Micrococcus sp. and this is probably the action of a lipase. aureus. especially cheese. aureus from foods. Some strains of S. Large brown-black Very small in shades of brown and black. aureus but without an egg yolk reaction should also be tested for coagulase production . Dark brown matt with occasional clearing after 48hrs.

Report S.1 ml aliquots of food dilutions made up in buffered peptone water on the agar surface until it is dry. 3. Re-incubate negative cultures for a further 24 hours. Results Incubate the dishes for 48 hours and select those with 20-200 colonies. Incubate the inverted dishes at 35'C. With a glass spatula or spreader (spread O. aureus.2. Count the S. aureus results per gram of food. aureuslike colonies and test them for coagulase reaction. Up to 0.5 ml may be used on larger dishes (24 cm). 95 . Examine after 24 hours and look for typical colonies of S.

E.). Isolation of Listeria monocytogens from all food and environmental samples 1. Novel chromogenic and other isolation agars may be used in conjunction with the above media. uses cycloheximide as a selective agent. Listeria innocua. poultry. However. have already produced alternative supplements for their media. then in a screening broth. Oxford. Application The method is applicable to the detection of viable Listeria monocytogenes in foods (seafood. which can be substituted in the media. dairy products. red meat. Listeria ivanovii. 8) Control cultures (use ATCC strains or equivalent) Positive controls: Listeria monocytogenes. (Staphylococcus aureus and Rhodococcus equi . Some media suppliers. and as a result cycloheximide will be unavailable shortly. 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. monocytogenes cells will multiply under these conditions and give rise to visible colonies which can be identified . etc. it is up to the users of this method to ensure that their in-house validation data meets their criteria. blender or equivalent. The organization holding the patent on this antibiotic is no longer producing it. monocytogenes in the product. Principle This method determines the presence of viable L. It is assumed that viable L. such as Oxoid. Environmental samples can also be analysed using this method. 2. 3. vegetables.optional) 9) Stomacher. Data may be obtained from the manufacturer and should be kept on file. plated onto a specified agar medium and one additional plating medium. vortex mixer 10) Microscope 96 . and then incubated under specified conditions of time and temperature. A portion of the product is enriched first in a primary broth.

maltose. depending on the nature of the product. Confirmation Media and Reagents 12) Tryptose broth and agar (TA) 13) Trypticase soy broth and agar.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.1. Thaw frozen samples in a refrigerator.5oC. Maintain a ratio of 1 part sample material to 9 parts sterile enrichment broth. where higher temperatures are recommended.1 In the laboratory prior to analysis. with 0.1) Optional 17) Rapid identification kits.1. Micro-ID Listeria (Organon Teknika Corp. Where 35oC is recommended in the text of the method.0oC. the incubator may be at 35 +/-1. 4.5oC due to potential lethality of higher temperatures on the microorganism being isolated. except for shelf-stable foods. such as 43 or 45. Information regarding Listeria distribution can be obtained by analyzing each analytical unit separately. Similarly.optional) 15) Motility test medium 16) Carbohydrate fermentation agars or broths (mannitol.2 Analyze sample units as soon as possible after their receipt in the laboratory. keep sample units refrigerated (0-5oC) or frozen.dextrose. 97 . Note: these biochemicals may be done via rapid identification kits (see 6. However.). or under time and temperature conditions which prevent microbial growth or death. Procedure Each sample unit may be analyzed individually or the analytical units may be composited according to the sampling scheme in Table 4. -methyl-D-mannoside 21) Beta-lysine discs (Remel) 22) Listeria monocytogenes antisera 4.) or the Listeria AccuprobeTM Test (Gen-Probe. MFLP-88) or equivalent 18) Gram stain solutions 19) 3% hydrogen peroxide (catalase) 20) Biochemicals . it is imperative that the incubators or water baths be maintained within 0. rhamnose and xylose). such as the Vitek or API Listeria (Bio Mérieux Vitek.6% yeast extract (TSB-YE and TSA-YE) 14) Horse blood agar and (sheep blood agar . lower temperatures of 30 or 25oC may be +/. Carry out the test in accordance with the following instructions: 4.1. esculin. Handling of Sample Units 4.0oC.1. Inc.8.

6.6. or PALCAM agar (PAL). 98 .2 Streak MFB onto plates if positive. stomach or vortex as required for thorough mixing. Preparation of Sample To ensure a representative analytical unit. dark brown or dark green. modified Oxford agar (MOX). one of the analytical composites described in Table 4 is added to a sufficient amount of LEB.4. 4.2 Clean the surface of the working area with disinfectant. those inoculated from LEB at 24 and 48 h onto two different plating media (streaking LEB is optional but preferable for obtaining all listeriae). Maintain a ratio of 1 part sample material to 9 parts LEB. striking the plate bottom at a 45o angle.1 Have sterile Listeria enrichment broth (LEB) ready. Incubate LEB culture for 48 h at 30oC. Streak positive MFB. Reading the MFB at 26 h can substantially reduce the plating done at 48 h. Selective Enrichment 4. 4.Examine LPM plates for suspect colonies using beamed white light powerful enough to illuminate the plate well. the colonies can look smooth with a blue tinge around the perimeter. Blend. reincubate another 24 h and streak if positive.2. A positive broth has darkened and may be black. Alternatively.2. Incubate LPM plates at 30oC for 24-48 h and OXA. or transferred to a sterile Erlenmeyer flask. HELPFUL HINT: Vortex the MFB at 20 to 24 h. 4.1. 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.3.5).5. Place environmental swabs in 10 mL portions of LEB in test tubes. add 50 g to 450 mL of LEB. Incubate 24-26 h at 35oC.5. For composite samples.5. agitate liquids or free flowing materials until the contents are homogeneous.4. A negative MFB has the straw colour of a newly made broth.6. Use Oxford agar (OXA) and one of the following: lithium chloride-phenylethanolmoxalactam medium (LPM).6. Other media may be used along with the two selective agars (see 4. LEB culture may be incubated in the stomacher bag or test tube.1. If the sample unit is a solid. Alternately. If negative. and inoculate 10 mL of modified Fraser Broth (MFB) with 0.1 mL of the LEB culture. Isolation Procedure 4. an even blue-grey iridescent sheen can be observed.2. then reincubate for an additional 2 to 6 h before reading reaction. Proceed with Step 4. obtain the analytical unit by taking a portion from several locations within the sample unit. LPM . When growth becomes near confluent. mix the LEB culture by swirling or vortexing. MOX and PAL plates at 35oC for 24-48 h. 4. 4. 4. 4. Preparation for Analysis 4.6. 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.2. At 24 and 48 h.

Chromogenic agar .8. Follow manufacturer’s instructions for preparation and use. Identification Procedure . hemolysis and 3 carbohydrate agars (mannitol.7. Incubate plates at 30oC for 24-48 h or until growth is satisfactory. 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. monocytogenes.2. 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.Confirmation 4. 4.2).6. 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 0.1). innocua shows no zone of hemolysis. L. Examine the plates for typical colonies using the light arrangement already described in 4. are inhibited on this medium when incubated at 35oC. ivanovii produces a well-defined zone of clearing around the stab. 4. Some Enterococcus and Staphylococcus strains form grey colonies with a brown-green halo or yellow colonies with a yellow halo.7. innocua). draw a grid of 20-25 spaces on the plate bottom. monocytogenes.L. Other biochemical tests are optional. 4. black with a black halo and sunken centre.3.3. Incubate for 24 h at 35oC.6.4.6. monocytogenes forms 1 mm diameter black colonies surrounded by black haloes after 24 h.4. monocytogenes forms 2 mm grey-green colonies with a black sunken centre and a black halo on a cherry-red background. When examined before 24 h. 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).6% yeast extract (TSA-YE). 1Note: Carbohydrate plates may be replaced by rapid identifcation kits (see 4. is sometimes apparent but without the characteristic blackening.1). Some strains of this genus. OXA and MOX agars . L. Rapid identification kits may be helpful to reinforce confirmation of these results and differentiate the different Listeria species (see 4. Always stab positive and negative controls (L. monocytogenes produces a slight cleared zone around the stab. rhamnose and xylose). At 48 h colonies are 2-3 mm in diameter. motility agar and carbohydrate plates1 concurrently from the same colony. Motility: 99 . 4.7.novel chromogenic and other isolation agars may be used. 4. monocytogenes can be accomplished by using motility. growth of Listeria spp.7.5. PAL agar .L. ivanovii and L. Stab blood agar plates. other than L. whereas L.1. Ensure that each colony is placed in the same position on all grid plates. HELPFUL HINT: Listeria confirmation and speciation of L. Hemolysis: On horse blood agar plates.2. The colonies can also appear brown-black or green-black. but in conjunction with the plating media above.7. Other Listeria species show a similar appearance.6. L. streaking for separation.8.

Listeria cells are catalase-positive. produce acid with no gas.Optional Tests 4. L. Development of bubbles is indicative of a positive reaction. maltose.7. Ensure that each colony is placed in the same position on all grid plates. rhamnose and xylose) agar plates.7. esculin. TA or TSA-YE. 4.5% solutions in purple carbohydrate broth: dextrose. Consult Table 1 for the carbohydrate reactions of the Listeria spp. Carbohydrate Utilisation Plates On carbohydrate (mannitol. Incubate for 24 h at 35oC. short rods with tumbling motility. or rods with rapid swimming motility are not Listeria. All species should be positive for dextrose. such as the Vitek or API Listeria (Bio Mérieux Vitek. and/or Wet mount: Pick at least one typical colony from each selective agar.2)). Listeria spp. methyl-D-mannoside and xylose. (Do blood agar and carbohydrates concurrently (see 4. murrayi should be mannitol-negative 4.2 Catalase Test a typical colony for catalase. Inc. Always compare to a known Listeria culture.4. inoculate the following carbohydrates set up as 0. Incubate for up to 48 h at room temperature.). draw a grid of 20-25 spaces on the plate bottom.1 Rapid Identification Kits Rapid identification kits. Listeria appears as slim. mannitol. or no reaction. esculin. TA or TSA-YE plates and inoculate agars by stabbing one culture per grid. 4.8 Identification Procedure . All Listeria spp. ivanovii. 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. Pick typical colonies from the selective agars. except L.Agar: Stab motility test medium from selective agars. Cocci.8. Transfer a colony onto a clean glass slide and add one drop of 3% hydrogen peroxide.3 Gram stain Listeria is a small Gram-positive rod. Follow manufacturer’s instructions for use. Transfer a loopful of the overnight cultures to a fresh TSB-YE and incubate at 25oC for 6 hours. MicroID Listeria (Organon Teknika Corp. grayi). grayi and L. monocytogenes and L. ONLY Listeria cells give typical umbrella growth pattern. 4. Avoid picking test colonies from agars containing blood as they can produce a false positive result.) or the Listeria AccuprobeTM Test (Gen-Probe) or equivalent. Incubate 7 days at 35oC. rhamnose.8. Observe daily. and/or Broths From TSB-YE culture. 100 . Inoculate TSB-YE broths and incubate overnight at 30oC.8. TA or TSA-YE. large rods. and maltose. Examine daily. Always stab positive and negative controls (L. See Table 1 for guidance.

All biochemical. but is rhamnose-positive. 4. being careful not to touch the disc with the inoculum. seeligeri by the CAMP test. Gram-positive motile rods that are catalase-positive. L. ivanovii. 101 .5 Serology Follow manufacture's instructions provided with the antisera. seeligeri from L. ivanovii hemolysis is enhanced near the Rhodococcus streak.4. All species give +/+ reactions in MR-VP broth. 4. They utilize dextrose. ivanovii from L. esculin. L. ivanovii are strongly hemolytic and form a clear beta hemolytic line along the entire streak. L. 4. monocytogenes. innocua can only be differentiated from L. After 18-24 h incubation at 35oC.8.8. monocytogenes by its lack of hemolysis on blood agar plates and negative reaction in the CAMP test.2 An alternative and convenient CAMP test may be performed using the S. urea-negative. 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.8. L. L. and pathogenicity data are summarized in the tables below. seeligeri shows enhanced hemolysis only at the Staphylococcus streak and L. seeligeri is enhanced in the vicinity of the Staphylococcus streak. seeligeri unless the CAMP test is run. and a weakly-hemolytic L.8. L.negative may be confused with a weakly-hemolytic L. with some species also using mannitol.4 CAMP test For the CAMP test. welshimeri. Of the three.1 The hemolysis of L.4. examine the plates for hemolysis in the zone of the vertical streaks. are small. a very sharp CAMP reaction between L. Separate the vertical streaks so that test strains may be streaked horizontally between them without touching the vertical streaks. In this test. and maltose. Complete all data collection before making species determinations. only L. aureus factor in commercially prepared sterile beta-lysine discs. 4. monocytogenes cannot utilize xylose. monocytogenes and L. The test can differentiate L. murrayi are the only two species which utilize mannitol. serological. grayi and L. L. and produce an acid slant and butt in TSI without production of H2S. seeligeri (weak) produce hemolysis in horse or sheep blood agar and are also positive in the CAMP test. murrayi is the only species which can reduce NO3. ivanovii can be differentiated from L. seeligeri cultures and the disc can be observed. while L. seeligeri. where L. monocytogenes or L. streak fresh isolates of beta-hemolytic Staphylococcus aureus and Rhodococcus equi vertically on a sheep blood agar plate. and xylose with production of acid. After 24-48 h incubation at 35oC. The other Listeria species are CAMP test negative. and L.to NO2-. rhamnose. equi streak. welshimeri that is rhamnose.4. L.9 Interpretation of Results for Speciation Listeria spp. ivanovii shows enhanced hemolysis in the area of the R.

welshimeri + d + L. ivanovii + +d + + L. murrayi + + d - d + d + -f + d + + + + + + + + + + + + + + + + + + + + - + + + + - 102 . + aureus) CAMP-test (R. grayi + + L. genes b innocua Gram stain + + c Beta-Hemolysis + Mannitol L-Rhamnose + d D-Xylose e CAMP-test (S. 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.Table 1 Characteristics differentiating the species of the genus Listeriaa Characteristics L. monocyto.L. seeligeri + + + + L.

Not all strains of L. 4c.a. A very wide zone or multiple zones of hemolysis are usually exhibited by L. monocytogenes exhibit beta-hemolysis . un* + * un = undefined. equi + serotype 103 . did not give a positive reaction. 1/2c.negative. ivanovii strains. -: > or equal to 90% negative. 3c. innocua L. 7 L. 6b. e. monocytogenes 1/2a. seeligeri 1/2b. monocytogenes L. (-) . the type strain. welshimeri 6a. 6a. d. 6b L. 4b(x). Hemolytic Activity andMouse Virulence for Listeria Species species hemolysis of horse blood mouse (7%) stab virulence L.the type strain ATCC 15313 is nonhemolytic on horse. 4d.positive. Table 2 Serology. c. innocua 4ab. 4c. Of 30 strains. sheep and bovine blood. 4b. ivanovii L. ATCC 15313. 1 gave a positive reaction. Of 10 strains tested. aureus + + R.89% of strains are positive b. welshimeri L. +: > or equal to 90% positive. 4e. 1/2b. 6b. 3b. un* L. 4a. 4d. ivanovii 5 + + L. + + 4ab. 3a. seeligeri hemolytic reaction S. d: 11 . Standard symbols: (+) . Table 3 Camp Test Reactions of Listeria Species species L.

Foods not supporting growth of LM include the following: (a) pH 5. modified random from atmosphere (MAP) sandwiches. salted fish.porting 5 sample units growth of LM with refrigerated (100 g or mL shelf -life >10d (e. monocytogenes (LM) Food Product Sampling 1. hard cheese.92 regardless of pH (d) frozen foods The pH and aw determinations should be done on 3 out of 5 analytical units.Table 4 The Sampling Scheme for Ready-to-eat (RTE) Foods 1 being Analyzed for L. 5x10 g analytical 4 DIRECT 4 units are analyzed PLATING separately. All other RTE foods sup. monocytogenes" 2 3 At present. packaged salads.5 and aw <0.0-5. this product is not commonly found in the Canadian marketplace. RTE foods causally linked to 5 sample units listeriosis (e. cooked seafood. this list presently (100 g or mL includes soft cheese.g. 2. 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. 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. ice cream. dry salami.g. random from jellied pork tongue2 ) each lot. The food is presumed to support the growth of L. packaged lot. For a definition of RTE foods. monocytogenes has been found in the environment of the finished product area. For Category 3 foods. salads. 4 5 The designated analytical unit is taken from each sample unit. refrigerated sauces) 3. if GMP is inadequate and L. 5x5 g analytical units4 ENRICHMENT are either analyzed ONLY separately or composited.0 regardless of aw (c) aw 0.95 (b) pH <5. 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. each lot. Where enrichment is ENRICHMENT necessary5 5x5 g analytical units 4 are analyzed separately or composited. or where examination of Good Manufacturing Practice 104 . each) taken at coleslaw mix with shelf life > 10d. please see the latest version of the field compliance guide entitled "RTE foods contaminated with L. liver pâté.g cooked seafood. vacuumeach) taken at packaged meats.

At 24 and 48 h transfer 0. or rapid identification kits. mannitol.(GMP) status is not possible. Figure 1 A Flow Diagram Showing the Isolation Procedure Blend or stomach in LEB broth. Streak positiveMFB onto selective agar plates. both MFLP-74 (Enumeration of Listeria monocytogenes in Food) and MFHPB-30 may be used as appropriate. Incubate plates for 24-48h Confirmation Testsmotility.Record reactions for all tubes.1 mL of theLEB into MFB. Incubate 24-48 h at 35oC. rhamnose biochemicals.hemolysis. as required. and xylose. Incubate at 30oC for 48 h. Reincubate negative MFB for an additional 24 h. Streak LEB (optional but preferable) onto plates. other 105 .

2.5% solution and autoclave at 115oC for 15 min. Add 1 mL carbohydrate solution to 9 mL broth base to yield a final concentration of 0. Commercial plates may also be used.5% carbohydrate in broth. Rehydrate and sterilize as recommended by the manufacturer.1 Blood Agar Prepare blood agar plates as soon as possible after receiving fresh blood. Autoclave at 121oC for 15 min. Sterilize the base at 121oC for 15 minutes. 5. 5. as 5% solutions and filter sterilize.5. A 5% solution of esculin at room temperature is a gel that cannot be pipetted. Sheep blood plates for the CAMP test are prepared and stored in a similar way. then pour thick plates. Basal Medium: Purple broth base Bromcresol purple (1.1 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.2. Temper. Plates can be stored for at least 2-3 weeks at 4oC.2 Carbohydrate Fermentation Broth and Agar 5. MEDIA 5. Carbohydrate Solution: Filter sterilize 100 mL each of 20% aqueous solutions of rhamnose. Prepare all carbohydrates.2 Carbohydrate Fermentation Agar a. add 50 mL of the sterile carbohydrate solution. using blood agar base or preferably Trypticase Soy agar with 7% defibrinated horse blood.6% aqueous) Agar Distilled water b. Plates stored at 4oC can last for 1 month. 16 g 1 mL 16 g 950 mL 106 . except esculin. Agar and blood should both be at 45-50oC before combining and pouring plates. mannitol and xylose. Longer storage times must be validated by the individual lab. Add esculin directly into base broth to make a 0.

0 Filter sterilize. 0. add 1. The basal medium cannot be made in advance and reheated.2% aqueous acriflavin solution. Moxalactam solution: 2 mL Moxalactam (ammonium or sodium salt) Potassium phosphate buffer. 5. Sterilize at 121oC for 15 minutes. On the day of use. b. Store at 4oC.0 g 1000 mL b.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.4 Lithium Chloride-Phenylethanol-Moxalactam Medium (LPM ) a. Basal Medium: Phenylethanol agar Glycine anhydride Lithium chloride Distilled water 35.1 M. 1g 100 mL 107 . Basal Medium: Proteose peptone Tryptose Lab lemco powder (Oxoid) Yeast extract NaCl KH2PO4 Na2HPO4 Esculin 1 Nalidixic acid (2% solution in 0. Cool to 45oC-50oC and add 2 mL of moxalactam solution. Store at 4oC for 2 months.3 Listeria Enrichment Broth (LEB) a. cool at once after removal from the sterilizer. LEB broth is available commercially as UVM 1 formulation.5 g 10.5.1M NaOH) Distilled water 1 5g 5g 5g 5g 20 g 1. Acriflavin Solution: Filter sterilize 25 mL of 1. Do not overheat.0 mL of acriflavin solution to 1000 mL of basal medium. pH 6.0 g 5. Store the solution frozen in 2 mL aliquots Sterilize the basal medium at 121oC for 15 min. Pour 12-15 mL in each petri dish and store at 4oC.

Dispense aseptically in 10 mL amounts in presterilized 16 x 150 mm test tubes.35 g 12 g 1g 3g 1 mL 1000 mL Rehydrate with constant stirring with a magnetic mixer and adjust pH to 7. Sterilize at 121oC for 15 minutes. 5. add 1. Dispense 10 mL portions of basal medium in 16 x 150 mm test tubes.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.2 if necessary. Store at 4oC for 2 months. Cool at once after sterilization and store at 4oC. Just prior to use. a.0 mL of each stock solution to each 100 mL bottle and mix. mix again. Stock Solutions: Acriflavin (0. Autoclave at 121oC for 10 minutes. cool at once after removal from the sterilizer.1 mL of each stock solution to each tube before use.0% in distilled water) Filter sterilize the solutions.5 g/L 15 g/L 1 mL 1000 mL 5g 5g 5g 5g 20 g 1. see b. Add 0.6 Modified Oxford Agar (MOX) MOX agar is a slight modification of the Listeria selective agar (Oxford Formulation).) Distilled water 39-44 g/L 2 g/L 1 g/L 0. Do not overheat. OR: Dispense 100 mL portions of the basal medium in screw capped bottles and sterilize at 121oC for 15 min. 108 .25% in distilled water) Ferric ammonium citrate (5. and cool rapidly to 46oC in a water bath. Basal Medium: Columbia blood agar base (depending of the brand) Agar Esculin Ferric ammonium citrate Lithium chloride Colistin (1% solution. Store at 4oC.

0 g 0.5 g 15.0 1g 100 mL Filter sterilize. pH 6.02 g 0. or manufacturer's supplements. 5. 0.0 g 0. Dispense 6 mL portions into 16 x 125 mm screw-capped tubes. Cool to 50oC.005 g 1000 mL Suspend the ingredients in the water. 5. Supplements: Cefotetan Fosfomycin 0.01 g Add the cefotetan and fosfomycin. c.1 M.0 1g 100 mL Colistin solution is not sterilized.002 g 0.7 Motility Test Medium Rehydrate and sterilize according to manufacture's instructions. methane sulfonate Potassium phosphate buffer. mix well.0 g 1. b. Add 2 mL of moxalactam solution to the basal medium. Basal Medium: Columbia blood agar base Esculin Ferric ammonium citrate Lithium chloride Cycloheximide Colistin Acriflavin Distilled or deionised water 39. Store the solution at -20oC in 2 mL aliquots. Store at -20oC in small aliquots (3-5 mL).4 g 0. Sterilize by autoclaving at 121oC for 15 min. mix and pour the plates.1 M. Bring to a boil to dissolve completely. or 3 mL portions in 13 x 100 mm screw-capped tubes. pH 6. 109 .b. Both the basal medium and supplements are available commercially.8 Oxford Agar (OXA) a. and pour 12 mL per plate. Moxalactam Solution: Moxalactam (ammonium or sodium salt) Potassium phosphate buffer. Colistin Solution: Colistin. 0.

110 .12 Trypticase Soy Agar with 0. add the manufacturer's supplements containing polymxin-B-sulphate. ceftazidime and acriflavine aseptically.08 g 1000 mL Suspend the ingredients in the water and adjust pH if necessary to 7.1. 5. make generous slants.6% Yeast Extract (TSB-YE) Trypticase soy broth 30.0 g 10.0 g Distilled water 1000 mL Autoclave at 121oC for 15 min and dispense into 16 x 100 mm tubes.9 Palcam Agar (PAL) a. autoclave at 121oC for 15 min and prepare slants. and then pour the plates.0 g 0.5. 5. 5.0 g 5.0 g 1000 mL Autoclave at 121oC for 15 min.0 g 5. Cool to 50oC.0 g Distilled water 1000 mL Dispense into screw-capped tubes.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 0.6% Yeast Extract (TSA-YE) Trypticase soy agar 40.0 g Yeast extract 6. Basal Medium: Peptone Starch Sodium chloride Agar Mannitol Ferric ammonium citrate Esculin Dextrose Lithium chloride Phenol red Distilled or deionized water 23.0 g 13. Bring to a boil to dissolve completely.2±0.5 g 0.11 Trypticase Soy Broth with 0.0 g 1.5 g 15.8 g 0. Sterilize by autoclaving at 121oC for 15 min.0 g 1. For agar.0 g Yeast extract 6.0 g 15.

Application This method is applicable to the isolation.5% aqueous solution. anthracis is non-motile and non-hemolytic.3% in 70% ethanol. Consider the source of the sample when identifying the isolates as B. Description The method has been shown to produce satisfactory results with naturally-contaminated meats. When B. 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. This method determines the presence of B. Safranin. Sudan Black B. thuringiensis are likely to occur naturally in food products. vegetables. cereus per g or mL of the food is calculated. cereus colonies are selected and subjected to confirmatory testing. 3. mycoides characteristically produces rhizoid colonies on agar media and B. 1) Peptone Water diluent (PW) 2) Citrate solution. See Section 6 for the formula of individual media. 0. identification and enumeration of Bacillus cereus (with limitations as described in the method) in foods. However. presumptive B. the number of B. cereus.F. cereus by plating known quantities of (dilutions of) a food sample onto a selective agar. 2%. sufficient enterotoxin may be produced resulting in foodborne illness. Principle Bacillus cereus is widely distributed in nature and is commonly found in a variety of foods.5% aqueous solution OR TB Carbol-fuchsin ZN stain (Difco) [protein toxin crystals] 111 . 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. cereus and B. 0. NOTE: B. 0. Xylol 9) Basic fuchsin. cereus is not easily distinguished from other closely related organisms in the B. B. 4. Only B. After incubation. Isolation and Enumeration of Bacillus cereus in foods 1. cereus are variable in expression of motility and hemolysis and further testing may be necessary to identify the isolates. dairy products. 5% aqueous solution. cereus Group. From the results obtained.1) or TSA-MnSO4 agar (optional) 8) Staining solutions (optional): Malachite Green. cereus grows to high numbers in a food (> 106/g). cereals and dried foods. atypical strains of B.

ATCC or equivalent 14) Blender. 112 . No obvious morphological differentiation between some strains of B.2. If the sample unit is a liquid or a free-flowing solid (powder). 5. thoroughly mix each sample unit by shaking the container.1. Shake. keep the sample units refrigerated (0-5°C) or frozen depending on the nature of the product. 10) Methanol [protein toxin crystals] 11) BC Motility Medium 12) Rapid identification kits (optional) 13) Control cultures. 5. or under time and temperature conditions which prevent microbial growth or death.Note: Both Basic Fuchsin and TB Carbol-fuchsin ZN stains are toxic and possibly carcinogenic.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). Preparation of Dilutions 5. cereus and B. anthracis will occur. The food homogenate (1:10 dilution) of dry foods should stand at room temperature for 15 min. Use appropriate safety precautions.1. 5.1. The test shall be carried out in accordance with the following instructions: SAFETY NOTE: PEMBA media supports the growth of B.1. In all other instances. 5. stomacher or equivalent 15) Microscope 16) Incubators capable of maintaining 30 and 35°C 5. Thaw frozen samples in a refrigerator.2.2. It is recommended that commercially-available products be purchased. the analysis should be continued as soon as possible. Procedure Each sample unit shall be analyzed individually.2. During transport.1.4.2. 5. Note: Weight or volume in brackets indicates alternate procedure for making dilutions. blend or stomach according to the type of food as indicated in Table 1. Analyze the sample units as soon as possible after receipt at the laboratory. Handling of Sample Units 5. with the exception of shelf-stable products.2. combine portions from several locations within each solid sample unit. anthracis. 5. To ensure a representative analytical unit from a solid sample.2. Take suitable precautions.

cereus per g are expected: spread 0. 5. Retain the plates in an upright position until the inoculum has been absorbed by the medium (approximately 10 minutes on properly dried plates). (ii) Routinely.3.000 B. cereus 5.2. If the inoculum is not readily absorbed by the medium.3. cereus.2. or if counts higher than 1. Dry PEMBA plates in a bio-hood or laminar flow hood immediately before using.2. the plates may be placed in an upright position in an incubator for up to 1 h.6.1. Count the number of presumptive B. Liquid sampes: If the sample units are liquid.2 mL of each dilution on each of duplicate PEMBA plates 5. Enumeration of Presumptive B. 5. With foods that tend to foam. shake the dilution bottle 25 times through a 30 cm arc in approximately 7 sec. Plating should be carried out within 15 min of preparing the dilutions.2.7.5. Plating 5.2 mL of the undiluted analytical unit may be spread on each of duplicate PEMBA plates.3. 5.2.3. use blender at low speed and remove aliquot from below liquid/foam interface.3). 5.1. Mix for the minimum time required to produce a homogeneous suspension to avoid overheating.3.2.8. Shake all dilutions (as in 5. 5. Avoid excessive crowding or stacking of plates in order to permit rapid equilibration of plates with incubator temperature.3. 113 . Prepare succeeding decimal dilutions as required. Invert the plates and incubate at 35°C for 24 ± 2 h. Re-incubate the plates at room temperature for an additional 24 h and re-examine. If the 1:10 dilution is to be mixed by shaking. 5.3.2 mL of the 1:10 dilution evenly over the surface of one of each of ten selective agar plates (PEMBA).2.4.2.2.3.3. 5. blending or stomaching time should not exceed 2 min. Examine the plates for presumptive B.1. NOTE: The liquid should not be spread right to the edge of the plate. 5. Incubation 5.6) immediately prior to making transfers to ensure uniform distribution of the microorganisms present.2. using a separate sterile pipette for making each transfer.1. 5.5.3.1. Agitate each dilution to resuspend material that may have settled during preparation.3.1.2.1.3. 0. Solid foods (i) If fewer than 1. 5. cereus per g are expected: spread 0.000 B. cereus colonies present (Sec. since this causes confluent growth at the plate-agar interface which is difficult to count.3.

3.2(b. multiply by five and by the appropriate dilution factor. Counting of Duplicate Plates (Any Dilution) 5. and report as the total presumptive count per g of food. 5. cereus.3.3.3. and record as the respective presumptive count per g of food (C). 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.3. due to overgrowth of the colonies. When re-examined at 48 h.3. cereus colonies on some of the ten plates is < 20. Multiply each count by 50 and record as the respective presumptive count per g of food (C). cereus colonies on PEMBA: Type 1: Uneven margins. If the number of presumptive B. In this case. 2 to 5 mm in diameter. cereus colonies per plate is fewer than 20.3.3. whiter and more raised when compared to B. add separately the counts for each type from all ten plates and record as the respective presumptive count. Colonies of B.3. cereus colonies per plate consisting of the combined counts of the two types. It may appear that there are fewer colonies at 48 h.3. Add the results and report as the total presumptive count per g or mL of food. 5.3. This is the count of one of the two types per 2 mL.) (A) If the number of all presumptive B.3. look for colonies that were not present at 24 h and add to the 24 h count.2 mL) (A/2).3.) If the number of all presumptive B. 5.3. count separately the colonies of each type and compute the respective average presumptive count per plate (per 0.2(a. Multiply the count by 5. Add the results.3. 5. 5.3. Counting the Ten Plates of the 1:10 Dilution (Solid Food Only) 5. An alternate counting range of 10100 or 10-150 may be used. Counting Colonies and Recording Results Note: On PEMBA B. select two plates at random. and record as presumptive count per g or mL of food for each type (C). but on others is > 20. Add the results and report as the total presumptive count per g of food. 5.2. Select plates containing 20-200 presumptive B.Helpful Hint: Circle presumptive colonies at 24 h.2(a) above.3.1.2(c). anthracis may appear to be smaller.3. proceed as in 5. as these ranges are recommended in other standard methods due to the spreading nature of Bacillus colonies. anthracis (and some strains of B.3. fimbriate or slightly rhizoidal.3(b). Look for the following 2 types of presumptive B. cereus colonies is greater than 20 per plate but the total count of the two types does not exceed 200. 114 .2 g of food) (B).3. cereus) have very little or no zone of egg yolk precipitate. Count colonies immediately after the incubation period. Compute the average presumptive count per plate for each type (A/2). Type 2: Colonies similar to type 1 but with no surrounding halo of precipitation. 5.3(a).3.(0. the count at 24 h is more accurate.

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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Store in tightly stoppered container. shake blend* shake blend* TABLE II Example of Computing B.g. of No. of No. 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. of B. 0.5 g 20 mL 80 mL Dissolve 0. All (4) 2 1.3. cereus / B. of No. Note: Fuchsin stain is toxic and possibly carcinogenic. 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. (N T ) per g or mL N T = N1 + N2 119 .000 500 4) More than 5(e. thuringiensis Count per g or mL of Food Total No. Filter solution if necessary thru fine paper to remove excess dye particles. 5(5) 4 4. of Isolates Total No.5% Basic Fuchsin Stain (8. cereus. 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.500 3.600 18) Calculate N1 and N2 for each colony type to obtain total number of B.2H 2O) solution Spices weigh into peptone water diluent Powders.6.5 g basic fuchsin in 20 mL of alcohol and dilute to 100 mL with water.g.3) basic fuchsin alcohol distilled water 0. meat and other weigh into peptone water diluent solids * A stomacher may also be used to provide the initial blend.

2(b)).4.000 + 100 = 1.5.2 mL per plate (5. If the two of the ten plates of the 1:10 dilution are counted (5.000 and N2 = 100 NT = 1. 0. If the ten plates of the dilution are counted (5.100/g * Dilution factor = 100 ** For duplicate plates. N.B.g.3. C=Bx10x0. C=1/2Ax10x5 120 .5).e.2(a)).3. Divide by 2 since "A" represents the total count of one of the two types on two duplicate plates.3.3. if N1 = 1. where B is the total count of one of the two types on all ten plates. Report total number of Bacillus cereus / Bacillus thuringiensis per g or mL of food to two significant figures.

7) Sterile dH2O. 6) Sterile beakers. 2) Millipore membrane filters (MF) HAWP04700. 12) Centrifuge. 2. 8) Sterile 1% Tween 80. 4) 27G 1/2" needles. and (b) the toxin cannot be neutralized by Type A or B antisera. cultivation of the membrane in a liquid medium. analysis of the culture for toxin. The procedure is therefore geared towards the detection of these 2 types.1. 15) Anaerobic jars or anaerobic chamber. Of the common human types of C. Filtration Equipment 121 . 9) 150 mL screw capped dilution bottles. 13) Laminar flow cabinet. 5) Botulinal antitoxins. 2.G. 17) 0.45 µm filter with Luer lock. 18) Gelatin phosphate buffer. and identification of toxins with specific botulinal antisera. Detection of Clostridium botulinum in honey and syrups 1. Materials and Special equipment 1) Millipore sterilfil holders XXII04710. 3) 1 cc tuberculin syringes. only types A and B are commonly involved in infant botulism. 19) White mice (approx 20 g). Principle The procedure involves the removal of botulinal spores from the liquid portion of honey or syrups by membrane filtration. 16) Paraffin oil. A rare human type (F) may be considered as the possible source of toxin if (a) injected mice show the typical signs of botulism. 14) TPGYB medium. botulinum. 11) Water bath set to 65oC. 10) 300 mL centrifuge bottles.

recorder number 5602.000 xg for 20 min. water through each MF.1. 3.3. For honeys. In the analysis of honey. Incubate at 35oC for 7 days under anaerobic conditions. but the direction of optimum flow bears no relation to their orientation in the packages. cold dist. These are retailed in boxes containing 4 packages of 25 filters each. transfer the 125 mL suspensions to 300 mL centrifuge bottles. Two or more units may be linked. Examine at least one filter each of the remaining 3 packages in the same way to ascertain proper orientation. Keep the sediment temporarily at 4oC and filter. Recommended needle: 27G 1/2. 3. M2R 3T4 416) 667-2701 Wellcome Laboratories. also B-D 2. and centrifuge at 15. Add 100 mL of sterile distilled water with 1% Tween 80 and stir until the solution is homogeneous. heated to 65oC. to a manifold which is connected to a vacuum pump.2 Syringes and Needles Recommended syringe: 1 cc tuberculin. These are placed on 1-litre suction flasks. Cap loosely to prevent pressure build-up. Connaught Laboratories. and filter through a membrane filter (MF). 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. North York. Millipore membrane filters (MF) HAWP 04700. Preparation of diluted samples Weigh 25 g of honey (or syrup) into a sterile foil-covered beaker. Kent.2. Filter 100 mL of diluted honey (20% w:v). Botulinal antitoxins Trivalent (A. transfer the 125 mL suspensions to 150 mL screw-capped dilution bottles. Transfer the MF in a laminar flow cabinet into 110 mL of TPGYB medium.B. After filtration rinse dilution bottle and funnel with about 5 mL of sterile. 1755 Steeles Ave. Filter the supernate through a membrane filter. 122 . Hold in a water bath at 65oC for 30 min.1. Check the bottles daily. filtration and incubation For syrups. England. Procedure 3. Note: Flow rate and volume of filterable material depend on the direction in which the MF are placed in the filter units.2. in parallel. carefully add the sediment from the centrifugation to the dilution bottle containing TPGYB medium and the filter. hold in a water bath at 65oC for 30 min. Millipore Sterifil holders XXII 04710. Becton-Dickinson.E) antiserum. Bechenham. Maintain the orientation with the higher flow rates for the remaining 25 MF in the first package.1.1. 2. When a new box is opened. Spore activation. through both and record the flow rates. Monovalent (A and B) antisera. 2. Ontario.2. West.

botulinum type F may be the source of toxin. B. ii) Trivalent (A. weak or paralysed limbs) and none of the antisera has a protective effect. Keep at 65oC for 30 min. Centrifuge at 20.4. Inject intraperitoneally two mice (about 24 g) each with 0.7. Confirmation of botulinal toxin Select all samples causing death in 1/2 or 2/2 mice. 3.5 mL of diluted filtrate and observe for 4 days. inject 2 more mice.B) antiserum.15 mL of botulinal antiserum (Appendix B. select the bottles with signs of growth and withdraw about 20 mL of culture. if possible within 24 h after the first injection. Observe for 4 days. B. Notes: i) Dilution of filtrate is required to prevent anaphylactic shock from the high protein content of the medium. botulinum in cereals Weigh 25 g of cereal directly into 600 mL of TPGYB medium tempered to 65oC. botulinum type B is confirmed if mice are protected with trivalent A. E and monovalent A antisera. and keep the mixtures at ambient temperature for 45 min. Notes: i) If there are signs of botulism prior to death (ruffled fur. ship the remaining filtrate to the Botulism Reference Service for identification and store the original culture at 4oC for future reference. In that case.3. laboured abdominal breathing.45 µm membrane filter (Millipore) fitted on the syringe. Samples are considered positive for toxin if 2/2 or at least 2/4 mice are killed. 4): trivalent A. transfer the unfiltered portion to a second filter unit. Store the unused portions of diluted and undiluted filtrate at 4oC. 3. Place 1.55 mL of each filtrate/antiserum mixtures and 0. and transfer both filters to a single bottle of TPGYB medium. E) antiserum is used in lieu of divalent (A.6.2 in case of clogged filters In the rare event that the MF filter becomes clogged before the filtration of 125 mL is completed. Add 0. C. ii) 95% of the mice killed by botulinal toxin in TPGYB medium will be dead or near death after 24 h 3. If a sample kills only 1/2 mice. Rinse. Detection of C. E and monovalent B antisera. B. Modifications of 3. Mix. monovalent B to the third. to 1 h. monovalent A to the second. Preparation of culture filtrate After 7 days of incubation. B. 123 . none to the fourth. Clostridium botulinum type A is confirmed if mice are protected with trivalent A. 3. Incubate anaerobically at 35oC for 7 days. C. transfer the rinse water to the second unit and complete the filtration. Take about 10 mL of supernate up in a disposable syringe and sterilize by filtration through a Millex HA 0. E to the first.5 mL each of diluted filtrate in four 10 x 75 mm test tubes.5 mL of filtrate without antiserum.5.000 x g for 20 min and decant the supernate.3. Rinse the funnel of the first unit with water. Inject two mice each with 0. Detection of toxin Dilute 4 mL of sterile filtrate with 4 mL of gelatin phosphate buffer.

Gelatin phosphate buffer Gelatin Disodium hydrogen phosphate (Na2HPO4) Distilled water 2g 10 g 1L 1L 10 g Adjust pH to 6. 4. though rarely.2. CAUTION: immunization does not assure protection of the eye from botulinal toxin. and splashes may result in blindness. botulinum contain high levels of toxin and should be h andled only by experienced personnel after immunization with botulinal toxoid. 4.3.1a. botulinum of types A to E. Disposable gloves should be worn and pipetting by mouth is to be avoided. heatresistant container which should be placed in the autoclave by the investigator.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.6 with N HCl. Goggles must be worn whenever an accidental splash may be expected. cotton or tissue paper is collected in autoclave bags for hazardous waste and are autoclaved. CAUTION: the toxoid supplied by CDC protects only against C. or by placing the dilution bottles in boiling water. Autoclave at 121oC and 15 lb pressure for 15 min POTENTIAL HAZARDS TO THE INVESTIGATOR Liquid cultures of C. Disposable material such as gloves. for 10 min. 124 . Used glassware and other supplies in contact with toxin are placed in a sturdy. 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. involved in food-borne or infant botulism. deaerate prior to use by steaming at 100oC in the autoclave for 10 min. Tween 80 diluent Tween 80 (polyethylene sorbitan monooleate) Distilled water Filter-sterilize. Preparation of media 4. If accidental spills occur. the toxin may be inactivated with saturated or dry sodium bicarbonate. about 6 cm deep.4. not against type F which may be.

the AnaeroGenTM anaerobic atmosphere generation system (Oxoid) or an anaerobic incubator capable of maintaining 35oC. they usually contain relatively low levels of toxin.1%) 6) Lactose gelatin (LG) 7) Stomacher. The number of confirmed Clostridium perfringens is calculated from the ratio of presumptive colonies confirmed to presumptive colonies tested. A portion of the product is mixed and incubated with a selective medium by the pour plate technique. Application This method is applicable to the enumeration of viable Clostridium perfringens in foods. such as. 4. such as anhydrous CaSO4). A minimum of five of these colonies are subjected to confirmatory tests. 3. Principle The procedure estimates the number of viable Clostridium perfringens per g or mL of food.3 to 0. 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.5 pH units within a range of 5. 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. blender or equivalent 8) pH meter or paper capable of distinguishing to within 0.0 to 8.Incriminated foods (excepting sealed products) and clinical specimens may be analyzed by experienced personnel without the need for immunization. anaerobic jars (with a venting system or disposable H2/CO2 gas generator envelopes and a desiccant. Description This method has been shown to produce satisfactory results with naturally-contaminated meat and poultry products. Typical black colonies are counted as presumptive Clostridium perfringens. 2.0 9) 1N HCl and 1N NaOH 10) A system capable of generating anaerobic conditions. See also Appendix G of Volume 2 and reference 8. H.1 for the formula of individual media. Enumeration of Clostridium perfrigens in foods 1. 125 . if toxic.

except for shelf-stable foods. where higher temperatures are recommended. 5.3.5oC. Thaw frozen samples in a refrigerator. use blender at low speed and remove aliquot from below liquid/foam interface.1. However.2.3. lower temperatures of 30 or 25oC may be +/. 5. Carry out the test in accordance with the following instructions: 5.3.0oC. 5. 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. Preparation for Analysis 5. blending time should not exceed 2.3. 15) Colony counting device 5. Handling of Samples Units 5. With foods that tend to foam. 5. keep sample units refrigerated (0-5oC) or frozen.1.2.0oC.3. or under time and temperature conditions which prevent microbial growth or death. to avoid overheating. 126 . the incubator may be 35 +/-1. Similarly. depending on the nature of the product. If a sample size other than 25 g or mL is used. If the sample unit is a solid.1% peptone water diluent or other required diluent (Table 1). Blend for the minimum time required to produce a homogeneous suspension.2 Analyze sample units as soon as possible after their receipt in the laboratory. Procedure Each sample unit should be analyzed individually.1. To ensure a truly representative analytical unit agitate liquids or free flowing materials until the contents are homogeneous. 5. Preparation of Sample 5. as indicated in Table I. maintain the 1:10 sample to dilution ratio.2.1. such as 11 (10) g or mL into 99 (90) mL. Prepare a 1:10 dilution of the food by aseptically shaking.2. such as 43 or 45. Where 35oC is recommended in the text of the method.2 Clean the surface of the working area with a suitable disinfectant.1 Have ready 0. stomaching or blending 25 g or mL (the analytical unit) into 225 mL of the required diluent.5oC due to potential lethality of higher temperatures on the microorganism being isolated.5 min. NOTE: Weight or volume in brackets indicates alternate procedure for making dilutions. obtain the analytical unit by taking a portion from several locations within the sample unit.1 In the laboratory prior to analysis. it is imperative that the incubators or waterbaths be maintained within 0.1. 5.11) 5% C02.

Check pH of the food suspension.1 After 20 h of incubation. The presumptive count N (as number of colonies per g (mL)) is N=A x D. count or estimate the number and record the results with the letter E. 5..5.5. If a stomacher is used. If the 1:10 dilution in a dilution bottle is to be mixed by shaking.5. Incubate plates anaerobically in an upright position at 35oC for 20 h. 5. 5. the AnaeroGenTM anaerobic atmosphere generation system (Oxoid) may be used.4.5-7. 5.4. 5. 5. If the 127 . In all other instances. the bottom of the jars should be covered with anhydrous CaSO4 or another suitable desiccant. Presumptive Clostridium perfringens count 5.3 Count presumptive colonies and average the count of duplicate plates.3. 5.3.7. Prepare succeeding decimal dilutions as required.3. Inversion of the plates may result in agar displacement by gas.2 Select plates containing 20-200 black colonies.1. about 1-2.6.5. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present. where A is the average presumptive count from duplicate plates. Pinpoint black colonies are not to be counted. using a separate sterile pipette for making each transfer. If the pH is outside the range of 5.4. If envelopes are used.8.0 with sterile 1N NaOH or 1N HCl.3. 1. adjust pH to 7. Alternately. to indicate a lower degree of accuracy.3. 5. For a large number of plates an anaerobic incubator is preferable. the analysis should be continued as soon as possible 5.4. Longer incubation may result in excess blackening along the bottom rim of the plates.5 mm in diameter.9. e. Pipette 1 mL of the required dilutions into each of duplicate sterile Petri plates. 10% H2 and 85% N2. 5.5. Pour into each plate approximately 20 mL of sulfite cycloserine (SC) agar and mix by gentle rotation. The food homogenate (1:10 dilution) of dry foods should stand at room temperature for 15 min. If the lowest number of colonies per plate exceeds 150.3. Small numbers of plates may be incubated in anaerobic jars.8 x 106 E. macerate for 1 min. 5. Anaerobic incubators and jars require three evacuations and replacements with a mixture of 5% CO2.2.5.4. check the indicators to ascertain anaerobiosis (without anaerobiosis the analysis is discontinued). shake the bottle 25 times through a 30 cm arc in approximately 7 sec. either with disposable H2/CO2 gas generator envelopes or with a venting system. and D the dilution factor.3.g. Each jar and incubator must contain an anaerobic indicator.5. Plating and incubation 5.

0 x 106. 5. record the count as <0.6.2. 128 . In parallel. >2. To each NM tube with a distinct line of non-motile growth along the stab.4. e. Production of a red color at the top indicates reduction of nitrate to nitrite (a positive test). 5.6.6. of colonies confirmed / No.5 x D.7. 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). reduce nitrate. inoculate the same colonies deep into lactose gelatin (LG). API An-ident or Vitek Anaerobic cards.g. If no presumptive colonies are found. slightly more intense than the blanks. record the count as <0.g.. add 0. record the result with the letter E. where D is the dilution factor. 5. rapid identification kits may be used. 5.4 mL of nitrate reagent. reduced nitrate to nitrite and is lactose fermentation positive. If growth is limited to the lower part of the tube and little or no colour develops. Place the LG tubes in ice water for 10 min or in the refrigerator (4oC) for one h. are lactose positive and liquefy gelatin within 48 h are confirmed Clostridium perfringens. record the minimum number estimable with a > sign.3. 5. 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. e. as well as a color change from red to yellow which is indicative of lactose fermentation (lactose positive). Examine LG tubes for gas production. Anaerobic incubation is not required. Isolates that are non-motile.number is too high to be estimated. If the highest number of colonies per plate is below 15. Close the tubes tightly and incubate at 35-37oC for 24 h. Incubate two uninoculated NM and LG tubes as controls. Confirmatory tests 5.2 x 103 E.6.5.20.6.5 x D. 1. re-incubate the LG tube for another 24 h. If no liquefaction has occurred within 24 h of incubation but the organism is non-motile. such as the API 20A.6. 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. of colonies tested If no colonies are confirmed. should be counted as negative. Faint color reactions. aspirate and discard the upper part of the medium in the tube and again add the nitrate reagent to the remainder.6. 5. In addition to the tests mentioned.6.1.6. 5.. Add a small quantity of zinc dust to a negative culture.

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. 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. Viscous and nonmiscible liquids Solids: water soluble solids powder. water etc. blender jar or dilution bottle and the diluent added prior to mixing. 129 .TABLE I Preparation of the Initial Dilution Type of Food Product Liquids: milk.

and paratyphoid fever. their detection is facilitated by use of this medium. each composed of three tubes of the specified medium. Tubes of this lactose medium are inoculated with 10-ml. bile salt used to suppress the growing of organisms other than coliform bacteria. Because these bacteria are capable of using lactose as a carbon source (the other enteric organisms are not). As a result. Measured aliquots of the water to be tested are added to a lactose fermentation broth containing an inverted gas vial (Durham tubes). These pathogens are responsible for intestinal infections such as bacillary dysentery. With increasing industrialization. water is examined to detect Escherichia coli. and 0. 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. To determine the presence of coliform bacteria in a water sample. Since Escherichia coli is always present in the human intestine. The tests are performed sequentially on each sample under analysis. 1-ml. Standard Qualitative Analysis of Water The three basic tests to detect coliform bacterial water are presumptive. The tubes in each group are then inoculated with the designated volume of the water sample as described under "procedure". Development of gas in any of the tubes is presumptive evidence of the presence of coliform bacteria in the sample. 2. the greater the sensitivity of the test. Analysis of water samples on a routine basis would not be possible if each pathogen required detection. As with milk or water. Polluted waters contain vast amounts of organic matter that serves as excellent nutritional sources for the growth and multiplication of microorganisms. the medium also contains a surface tension depressant. confirmed. In addition to lactose. Therefore. 130 . typhoid fever. 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). its presence in water alerts public health officials to the possible presence of other human or animal intestinal pathogens. the bacterium that indicates fecal pollution. Principle The presumptive test is specific for detection of coliform bacteria. cholera. The MPN is estimated by determining the number of tubes in each group that show gas following the incubation period.To obtain some index as to the possible number of organisms present in the sample under analysis. non sporeforming bacilli that ferment lactose with the production of acid and gas that is detectable following a 24-hour incubation period at 37ºC. water has become a formidable factor in disease transmission. . They detect the presence of coliform bacteria (indicators of fecal contamination) the gram-negative.1-ml aliquots of the water sample. The greater the number of tubes per group. water sources available for consumption and recreation have been adulterated with industrial as well as animal and human wastes. and completed. Both qualitative and quantitative methods are used to determine the sanitary condition of water.Microbiology of water Introduction The importance of potable (drinking) water supplies cannot be overemphasized. Presumtive coliform test Determination of the most probable number of coliform Purpose 1. The series consists of at least three groups.

Negative: There is no gas in the tube in the series in 48 hours. transfer 1 ml of water to the three tubes. sterile 10-ml pipettes. Equipment Bunsen burner. mechanical pipetting device. label the water source and volume of sample inoculated. Exercise care in handling sewage waste water sample because enteric pathogens may be present. 2. c. Confirmation of these results is necessary. Media lactose broth . Incubate all tubes for 48 hours at 37 degrees centigrade. 3. b. since positive presumptive tests may be the result of organisms of non-coliform origin that are not recognized as indicators of fecal pollution. 8. for each tube. coli colonies and the surrounding medium pink. 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. in a test-tube rack. producing dark centers and a green metallic sheen. 5. Positive: 10 percent or more of gas appears in a tube in 24 hours. Examine all tubes after 24 and 48 hours of incubation. and glassware marking pencil. Repeat steps 2 through 5 for the tap and pond water sample. Procedure 1. EMB forms a complex that precipitates out onto the coliform colonies. Mix sewage plant water sample by shaking thoroughly. Flame bottle and then using a 10-ml pipette. In the present of an acid environment. sterile 1 -ml pipettes. pond. a total of nine tubes per series. Principle The presence of a positive or doubtful presumptive test immediately suggests that the water sample is non-portable. 4. Flame bottle and then using a 1-ml pipette. transfer 10-ml aliquots to the three tubes. the major indicator of fecal pollution. which forms a dark pink complex that turns the E. 7. Endo agar is a nutrient medium containing the dye fuchsin. Record your results in the chart as: a. which inhibits the growth of gram-positive organisms. This reaction is characteristic for Escherichia coli. 131 . Confirmed coliform test Purpose To confirm the presence of coliform bacteria in a water sample for which the presumptive test was positive. and tap.Materials Cultures Water samples from sewage plant. Doubtful: Gas develops in a tube after 48 hours. Eosin-methylene blue agar contains the dye methylene blue. Set up three separate series consisting of three groups.

Label the covers of the three EMB plates and three endo agar plates with the source of the water sample (sewage. . Using a positive 24-hour lactose broth culture from the sewage water series from thepresumptive test. 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. 132 .Materials Cultures One 24-hour-old positive broth culture from each of the three series from the presumptive test. pond. 3. if necessary. glassware marking pencil. Media Nutrient agar slants and lactose broth. Incubate all plate cultures in an inverted position for 24 hours at 37 degree centigrade. 4. Completed coliform test Purpose To confirm the presence of coliform bacteria in a water sample.Repeat Step 2 using the positive lactose broth cultures from the pond and tap water series to inoculate the remaining plates. and tap). to confirm a suspicious but doubtful result of the previous test. and inoculating loop. Media Eosin-methylene blue agar plates and endo agar plates. Following inoculation and incubation. 2. Equipment Bunsen burner. Materials Cultures One 24-hour coliform-positive EMB or endo agar culture from each of the three series of the confirmed test. streak the surface of one EMB and one endo agar plate.coli. or. Principle The completed test is the final analysis of the water sample. 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. Procedure 1. and indicative of a positive completed test. It is used to examine the coliform colonies that appeared on the EMB or endo agar plates used in the confirmed test. 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.

the results are readily reproducible.5ºC. <0. particulate matter clogs the pores and inhibits passage of the specific volume of water. glassware and marking pencil. 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. >4 indicates human pollution. Principle Bacteria-tight membrane filters capable of retaining microorganisms larger than 0. Equipment Bunsen burner. microscope. and safranin. 3. (2) larger volumes of sample can be processed.Reagents Crystal violet. Inoculate one lactose broth and one nutrient agar slant from the same isolated E. Quantititive analysis of water Purpose To determine the quality of water samples using the membrane filter method. Streaking onto EMB and Endo agar from the positive tubes can follow as above. the types of fecal pollution. 95 percent ethyl alcohol. A water sample is passed through a sterile membrane filter that is housed in a special filter apparatus contained in a suction flask. staining tray. if any. blotting paper. 2. lens paper. Also. the number of colonies present on the filter is counted with the aid of a microscope. and (3) because of the high accuracy of this method. Incubate all tubes for 24 hours at 37 degrees centigrade. coli colony obtained from an EMB or an endo plate from each of the experimental water samples. Label each tube with the source of its water sample. inoculating loop. Following filtration. Positive results are characterized by the growth and production white precipitate. indicative of pollution from other animal origins. 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. The ratio of the fecal coliforms to fecal streptococci per millimeter of sample is interpreted as follows: Between 4 indicates human and animal pollution. multiple-tube method of water analysis: (1) Results are available in a shorter period of time. Following incubation. 133 . These filters offer several advantages over the conventional. are established by means of a fecal coliform count. A disadvantage involves the processing of turbid specimens that contain large quantities of suspended materials.7 indicates poultry and livestock pollution. Gram's iodine. Procedure 1. differential liquid medium. A total count of coliform bacteria determines the potability of the water sources. the filter disc that contains the trapped microorganisms is aseptically transferred to a sterile petri dish containing an absorbent pad saturated with a selective. indicative of human pollution and a fecal streptococcal count.45 micrometer are frequently used for analysis of water.

c. 4. Using a sterile forceps dipped in 95 percent alcohol and flamed. 15 sterile millipore membrane filters and absorbent pads. Arrange the 15 petri dishes into three sets of five plates. 10-1. on the sintered glass platform. Attach a rubber hose from the side-arm on the vacuum source. 10-3. Label the four 90-ml water blanks with the source of the water samples and dilution (10-1. 134 . Procedure The following instructions are for analysis of one of the provided water samples. With sterile forceps. place a sterile membrane filter disc. 2. forceps. and 10-4 dilutions. 10-3. 10-2. b. using the four 90-ml water blanks to effect the 10-1. 12 10-ml pipetting device. add a sterile pad to all Petri dishes. d. Using the filter clamp. 5. small beaker of 95 percent alcohol. place 20ml of the dilution into the funnel and start the vacuum. Two ml of K-F broth to each pad in the plates labeled FSC. Unwrap and carefully place the funnel section of the apparatus on top of the filter disc.broth. wash the inner surface of the funnel with 20 ml of sterile water.Materials Cultures Water samples collected upstream (labeled U) and down stream (labeled D) from an outlet of a sewage treatment plant. 1-litre suction flask. c. and glassware marking pencil. dissecting microscope. and 10-4). When the entire sample has been filtered. and one 300-ml flask of sterile water. 3. grid side up. Different samples may be assigned to individual groups. 6. For total coliform count (TCC) and dilutions (undiluted. Equipment Sterile Millipore membrane apparatus (base. Using the highest sample dilution (10-4) and a pipette. one 20-ml tube of EC . funnel. c. b. For fecal streptococcal count (FSC) and dilutions as in Step 3a. a. Media one 20-ml tube of lactose broth. Two ml of EC. For fecal coliform count (FCC) and dilutions as in Step 3a. one 20-ml tube of K-F broth. 15 sterile 50-mm Petri dishes. and 10-4). 10-2. 10-3. 10-2. b. Label each set as follows: a.broth to each pad in the plates labeled FCC. 1. and clamp). Two ml of lactose broth to each pad in the plates labeled TCC. Aseptically assemble the sterile paper-wrapped membrane filter unit as follows: a. aseptically perform a 10-fold serial dilution of the assigned undiluted water sample. Using 10-ml pipettes. Unwrap and insert the sintered glass filter base into the neck of a 1liter side-arm suction flask. secure the funnel to the filter base. four 90-ml sterile water blanks.

FCC plates sealed with waterproof tape and placed in a weighted watertight plastic bag. adding the filter discs to the 10-4 dilution plates labeled FCC and FSC. unclamp the filter assembly. FSC: Count colonies on K-F agar that are pink to red (performed on a disc showing 20 to 100 of these colonies).7. Remove the filter discs from the petri dishes and allow to dry on absorbent paper for 1 hour. Incubate the plates in an inverted position as follows a. gram’s characteristics and production of gas from lactose. TCC: Count colonies on M-endo agar that present a golden metallic sheen (performed on a disc showing 20 to 80 colonies). 10-2. Repeat Steps 8 through 10. TCC and FSC plates for 24 hours at 37 degrees centigrade. FCC: Count colonies on M-FC agar that are blue (performed on a disc showing 20 to 60 of these colonies). reassemble the filtration apparatus. Check for the typical colonies as explained earlier. Colony Count x dilution factor ml of sample used x 100 3. 9. a. 135 .5 degrees centigrade water bath for 24 hours. b. 2. Carry out the biochemical test below: indole production methyl red voges/proskeur citrate utilization In addition check for the motility. and repeat Steps 7 through 9 twice. Isolation of Escherichia coli Carry out the coliform test up to fecal coliforms. 11. The EC media is streaked onto the EMB agar. b. which is then submerged in 44. 10-4). and with sterile forcepts remove the membrane filter and place it on the medium saturated pad in the Petri dish labeled TCC. 10. For each of the three counts. using 20ml of the 10-3. Examine all filter discs under a dissecting microscope and perform colony counts on each set of discs as follows. determine the number of fecal organisms present in 100 ml of the water sample. Aseptically place a new membrane on the platform. c. and 10-1 dilutions and the undiluted samples. Disconnect the vacuum. using the following formula. Observation and results 1.

Howard Mould Count 1. 2. If the blade is not of this size it may be ground down to the designated width and to a flat surface.7. f. Compound microscope.6.1. Application This method shall be used for the determination of mould filaments in canned tomatoes. mark the blade l0. puree. b.5. 2.8. tomato pulp and tomato catsup.1). mechanical stage.4.1. condenser with iris diaphragm.1.S. The ocular must be equipped with a micrometer disk cross-ruled in sixths of ocular diaphragm opening (Preparation of Microscope. 136 .382 mm (Preparation of Microscope. 2. source of illumination. Diagram IIa or IIb and cover glass. standard sieve no. mm.0 mm from the tip to give a working area of 50 sq.1. tomato paste. pulp. Spatula with a 5.1. 2.9. Howard mould counting chamber or cell of the type with specifications as outlined in Part 6.5 x oculars.2. The 10 x objective must be calibrated with the ocular to give a field diameter of 1. 2. Dissecting needle.1. 2.3. Spoon or other suitable utensil (for puree. either binocular or monocular equipped with: a. 2. 2 (for canned tomatoes). U. section 2. 2. two objectives . With a glass pencil.0 mm flat blade. The purpose of recommending this spatula is to standardize the quantity of product transferred from the sample to the Howard cell.1. g. Apparatus 2. c.2. Bunsen burner. Procedure The examination shall be carried out in accordance with the following instructions. Distilled water. paste and catsup).1. Wide mouth bottles with screw caps or other suitable containers (for canned tomatoes.10. 2.1.2). 2.1. paste and catsup). d. pulp. section 2. Lint-free clean towel or cloth for drying Howard cell and cover glass. tomato juice and vegetable juice.1.2.1. 8 x . e.12.a 10 x (16 mm) for counting and a 20 x (8mm) for confirmation. and in tomato puree. 2.

Locate and focus a mould filament with the microscope.382 mm on stage. To make the grid.11.3.382 mm as in step 2. 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 1.1.14 mm) that will coincide with 1. 2. Use a coloured filter if necessary to increase contrast of filaments. c. If not. Coarse filter paper or celluwipe (for puree. 2. 2. Calibrate the 10 x objective with the ocular to give a field of view diameter of 1. Examine each sample unit immediately after it is prepared. adjust the height of the condenser. remove disk and change lines as necessary. 2.382 mm scribed on a Howard cell. raise the height of the ocular(s) until the diameter coincides with 1. Mark width on micrometer disk. etch grid on micrometer disk with very fine lines making certain grid is centred on the disk.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 or make an accessory drop-in ocular diaphragm with aperture accurately cut to necessary size. b. place disk in ocular and check that width coincides. c. b.2.3. If there is any delay. pulp and paste).2. 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. b. If the field diameter is less than 1. Once the proper width has been determined. Sampling.2 Preparation of Microscope 2.2.1. the sample unit should be thoroughly shaken again prior to examination.12. 137 . Equip microscope with a micrometer disk cross-ruled in sixths of ocular diaphragm opening as follows: a.2.382 mm as follows: a. 2. Preparation of Sample Units 2. (Part 6. calculate the width of grid (10 . pulp and paste).1.12. Focus the light source into the condenser. Each sample unit shall be analyzed separately. Diagram I).2.2. Refractometer (for puree. Obtain or make a micrometer disk of suitable diameter to fit into ocular (approximately 21 mm) and 1 mm thick.1.3. If the field diameter is greater. Using the 10 x objective and ocular(s) in the range of 8 .1. Establish adequate light source for examination as follows: a.2.382 mm use lower power ocular(s).3. Each sample shall consist of six sample units of one container each as outlined in section 2. 2.

pass the flame of a Bunsen burner lightly over the surface to disperse the foam. Preparation of Howard Mould Count Cell.4. Tomato Puree. c.3. transfer 100 ml to a wide mouth bottle. (ii) Open container. (iii) Proceed as in step 2. a hazy image will be formed which is hard to centre and read. a. 138 . Removal of the pulp from tomato mixture does not affect the refractive index as it is based only on the soluble solids. Preparation of Howard Mould Count Cell. Canned Tomatoes (i) Before opening.3.3.a. (v) Measure refractive index as in step (ii) and correct if necessary. Drain liquid from canned tomatoes through a no.3. (ii) Open container. (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. shake container (sample unit) 60 times in 30 sec through a 30 cm arc. shake container (sample unit) 60 times in 30 sec through a 30 cm arc. (iv) Repeat procedure for remaining five sample units. (iv) Continue as in step 2.2.3. (vi) Proceed as in step 2.4. secure lid and repeat mixing. (iv) Mix sample unit as in step (i). If the pulp is not removed.3454 at 20oC or 1. (ii) Transfer a small portion onto a coarse filter paper or celluwipe and measure the refractive index of the filtrate. d.3448 -1. 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. b.3442 -1. If considerable foam is produced. add required amount of distilled water. 2 sieve into a suitable clean receptacle. Tomato Catsup (i) Open container (sample unit) and mix 60 times in 30 sec with a spoon or other suitable instrument.3448 at 25oC. (vii) Repeat procedure for remaining five sample units.3. Tomato Juice and Vegetable Juice (i) Before opening. (iii) Transfer liquid to a wide mouth bottle and screw lid on securely.

using a dissecting needle to facilitate the transfer. only the sample. Determine adequate cleanliness of slide by placing cover glass in position and pressing it firmly against the shoulders. 2. so that the material spreads i evenly over the entire surface of the disk. secure lid and shake 60 times in 30 sec through a 30 cm arc.4.3. Prepare glass slide using technique (a) or (b) as follows a. (iv) Lower the cover glass slightly until it almost touches the test material on the disk. 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.3. for in doing so. These rings resemble a rainbow in colour and when properly formed are broken arcs of concentric circles. Clean spatula and dissecting needle. Inclined Cover Glass Technique (i) Remove cover from Howard cell.3. Preparation of Howard Mould Count Cell. flame and cool. dry with a lint free cloth and pass lightly over a Bunsen flame. b.1. Do not allow the spatula or needle to touch the central disk.3. thus ruining the mount. On the other hand.3. (v) Repeat procedure for remaining five sample units.(ii) Transfer a measured well mixed representative portion to a wide mouth bottle.4. otherwise the test material will not spread evenly over the disk. 2. then. do not lower too gently. rinse in distilled water. (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.4. If Newton's rings are not formed re-wash slide and cover glass. Clean Howard cell and cover glass making certain central area of cell is clean.2.3. (iii) Dilute contents of bottle with an equal volume of distilled water. the slide is considered sufficiently clean. 2. If Newton's rings appear between each shoulder and the cover glass. 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. a portion of the sample may splash over onto one or both of the shoulders. (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. 2. (iv) Proceed as in step 2. lower it rapidly but gently nto place.4.4.3. and remain after pressure has been released. (v) Do not lower the cover glass too rapidly. Preparation of Howard Mould Count Cell (1) 2. Parallel Cover Glass Technique 139 . Rinse with distilled water.4.4.

5. Then move the mechanical stage vertically to the next alternate row and examine 5 more alternate fields in reverse horizontal direction. Microscopical Examination 2. only the sample.6. 2. d. Do no allow the spatula or needle to touch the central disk. Absence of air bubbles.5.3.382 mm (1.5 sq.3. and consequently moulds. (iv)While maintaining contact with the test sample. Absence of Newton's rings.4. when cover glass is put in place. Uneven distribution of material. d. 2. so that the test portion spreads evenly over the entire surface of the disk. then.5. Otherwise. Repeat this process until 25 fields have been examined. 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. using a dissecting needle to facilitate the transfer. Sufficient material to fill area used for counting. (ii)Dip spatula into well mixed sample up to 10 mm line and transfer a sample portion onto the approximate centre of the disk. If a field with an air bubble is encountered. The recognized procedure for examining a mount is to examine alternate fields in alternate rows throughout the entire area of the mount. without stopping lower it rapidly but gently until it just touches the shoulders of the cell.2). (iii)Hold the cover glass parallel to the surface of the central disk and lower it slowly until it just touches the sample portion. Numerous air bubbles.3.(i)Remove cover from Howard cell.3. b. alternately raise and lower the cover glass very slightly 2 or 3 times.4. Even distribution of material on slide. b. may be more abundant at the centre of the mount. mm) as outlined in Preparation of the Microscope (section 2. Newton's rings visible.1. 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.5. Discard any mount showing: a. Ensure sample portion is taken from a thoroughly mixed sample.3. To accomplish this. move to another field unless 140 . Liquid which has been drawn across the moat and between the cover glass and shoulder. c. Ensure the slide is characterized by: a. Use higher magnification (180 250 x) only for confirmation of mould. c. 2.2. examine alternate fields horizontally across the slide preparation until 5 fields have been examined. insoluble material. 2.

6. (ii) Recognize the difference between various mould filaments and plant remnants such as tracheal tube thickenings. 2. boil in low count tomato juice to simulate actual conditions and examine microscopically.3. Count field as positive when the aggregate length of < 3 of the longest filaments present exceeds 1/6 diameter of field. 2. Calculation and Recording Results 2.6. 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 141 . pieces of cell wall.4.5.3. A clump or mass of mould has the same value as a single filament (Part 6. Calculate proportion of positive fields from results of examination of all observed fields for each sample unit. Observe each field noting presence or absence of mould filaments as characterized in Part 6.2. (iii) It is not necessary to classify types of mould. lint or fabric segments. 2. Diagram III. Otherwise never move the slide purposely to exclude or include mould filaments.1.6.mould is seen at first glance. These filaments may be separate or attached to each other. Diagram IV). Diagram III.3. only to positively identify mould filaments as characterized in Part 6.5. If not familiar with the diverse forms of mould.3.3. examine known moulds as follows: (i) Remove mouldy areas from fresh tomatoes infected with various types of mould. because the field will contain insufficient sample.3. 2.

shall be applied in determining whether the tested lot of the product complies with Section B. 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.3544 1. The tolerance as specified hereafter and representing the maximum incidence of positive fields in tomato puree.0 158. 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.474 14.2.145 1.1. The tolerance as specified hereafter and representing the maximum incidence of positive fields in canned tomatoes.3610 1.2 144.2 147.171 2.11. 142 .2 232.3593 1.3494 1.3577 1.024 2.5 29.5 73. Dilution Factor Amt.0 187.440 1.876 2. tomato juice or vegetable juice.017 of the Food and Drug Regulations.0 58.2 132. shall be applied in determining whether the tested lot of the product complies with the Food and Drug Regulations. tomato paste.3462 1. 3. of Water to be Added to 100 ml Total Volume of Diluted Sample of Sample Unit Unit 1.3560 1.4 3.292 1. tomato pulp or tomato catsup.4 114.322 2.3478 1.5 173.730 1. Interpretation 3.585 1.6 102.4 117.2 44.3511 1.2 247.6 202.5 129.4 217.0 87.TABLE I DILUTION OF PUREE (PULP) FOR MOULD COUNT AT 20oC (2) Actual Index Refr.3527 1.

DIAGRAM I MICROMETER DISK A: Length of grid that coincides with 1.382 mm and cross ruled in sixths 143 .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.

1. 15 X 20 mm G: Moat 144 .HOWARD MOULD COUNTING CHAMBER DIAGRAM 11a E F G A: Calibration circle.382 mm apart F: Rectangle.382 mm diameter B: Area of liquid for mould count C: Cover glass D: Cover glass E: Two engraved parallel lines spaced 1.

occasionally encountered. mm.[1. mm.15 cu..382/2]2 X 3.5 X 0.1416 = 1. 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). area of microscopic field1. 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). parallel walls of even intensity with one end blunt and the other end rounded 145 .5 sq.1 = 0..

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 146 .F: Occasionally encountered.

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 147 .

Underprocessed and leaking cans are of major concern and both pose potential health hazards. toxin. the hazard is obvious. do not produce gas and therefore cause no abnormal appearance of the can. excessive contamination of the product for which normally adequate processes are insufficient. Overfilling. When the can contains a spoiled product and no viable microorganisms. to soft swell. Underprocessing may be caused by undercooking. or controls. to springer. However. A viable mixed microflora of bacterial rods and cocci is indicative of leakage. 148 . with consequent lengthening of the heat penetration time. buckling. if Clostridium botulinum (spores. Naturally. cans may progress from normal to flipper. 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. but when it occurs it must be investigated properly. denting. they cause spoilage of the product.Examination of Canned Foods The incidence of spoilage in canned foods is low. or closing while cool may also be responsible. such as drained weight and product formulation. Microbial spoilage and hydrogen. 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. Some microorganisms that grow in canned foods. It must be determined that the can is intact (commercially acceptable seams and no microleaks) and that other factors that may lead to underprocessing. unless proved otherwise. Contaminated cooling water sometimes leaks to the interior through pinholes or poor seams and introduces bacteria that cause spoilage. are the principal causes of swelling. to hard swell. However. before a decision can be made regarding the potential health hazard of a low-acid canned food. punctures. sporeforming rods should be considered underprocessed. or rough handling. spoilage may have occurred before processing or the microorganisms causing the spoilage may have died during storage. gauges. spoilage is not the only cause of abnormal cans. Grampositive. which may usually be confirmed by can examination. High summer temperatures and high altitudes may also increase the degree of swelling. retort operations that are faulty because of inaccurate or improperly functioning thermometers. Leakage occurs from can defects. nevertheless. produced by the interaction of acids in the food product with the metals of the can. Intact cans that contain only mesophilic. certain basic information is necessary. accidental bypassing of the retort operation altogether. During spoilage. or. changes in formulation or handling of the product that result in a more viscous product or tighter packing in the container. have been evaluated. Swollen cans often indicate a spoiled product. or both) is found. Spoilage is usually caused by growth of microorganisms following leakage or underprocessing. sometimes. however.

moderate or severe etching slight. when brought down sharply on its end on a flat surface.a can that normally appears flat. When sufficient pressure is applied to this end. flat surface. A hard swell will generally "buckle" before the can bursts. Springer . 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. On special occasions these procedures may not yield all the required information. Unspoiled cans may be examined bacteriologically to determine the presence of viable but dormant organisms. Useful descriptive terms for canned food analysis. it remains in this condition even when the can is brought down sharply on its end on a solid. Flipper . 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 . culturing 4-6 cans may be adequate. The procedure is the same as that 149 .a can with one end permanently bulged. When pressure is applied to this end.a can bulged at both ends. Bursting usually occurs at the double seam over the side seam lap. and additional tests must be devised to collect the necessary data. but the other end will flip out. or in the middle of the side seam. and so tightly that no indentation can be made with thumb pressure. but in some cases it may be necessary to culture 10-50 cans before the cause of spoilage can be determined. but not so tightly that the ends cannot be pushed in somewhat with thumb pressure. Soft swell . it flips in again and the can appears flat. moderate or severe blackening slight. one end flips out. The number of cans examined bacteriologically should be large enough to give reliable results.a can with both ends concave.a can bulged at both ends. When the cause of spoilage is clear-cut. Hard swell .Table 1. it will flip in.

cotton-plugged. With marking pen. sterile 8. Nutrient agar (NA) (M112) 7. crystal violet (R16). Liver-veal agar (without egg yolk) (LVA) (M83) 5. potentiometer 3. sterile 6. all sterile 5. sterile and nonsterile 10. brush. Serological pipets. Incubators. Soap. sterile 7. 4% Iodine in 70% ethanol (R18) C. Equipment and materials 1. Media and reagents 1. Indelible ink marking pen 11. and coverslips 4. Acid broth (M4) 6. Sabouraud's dextrose agar (SAB) (M133) 9. thermostatically controlled at 30. and towels. 35. Can opener. Bromcresol purple (BCP) dextrose broth (M27) 2. Diamond point pen for marking cans 12. Separate all cans by code 150 . and 55°C 2. Malt extract broth (M94) 4. 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. bacteriological can opener. Can preparation Remove labels. sterile 9. Test tubes. Chopped liver broth (M38) or cooked meat medium (CMM) (M42) 3. slides. Nontapered pipets. A. Petri dishes. cotton-plugged (8 mm tubing). Methylene blue stain (R45). or Gram stain (R32) 8.used for spoiled foods except that the number of cans examined and the quantity of material subcultured must be increased. Examination pans (Pyrex or enamel baking pans) B. water. pH meter. Microscope. and can punch.

. condition. e. Make opening in sterilized end of can large enough to permit removal of sample. test for headspace gas. When abnormal can or one becoming increasingly swollen is found. After 14 days remove flat and flipper cans from incubator and test at least 6. Immediately turn tube upright and pour in a small amount of lime water. or use can-puncturing press to capture some escaping gas in a syringe. using a qualitative test or the gasliquid chromatography method described below. Before observing cans for classification. After incubation. a. evidence of leakage.Flat and flipper cans. When can becomes a hard swell or when swelling no longer progresses. and perform remaining steps of canned food examination. cover can with sterile towel or invert sterile funnel over can. botulinum organisms or Gram-positive rods. Examine at frequent intervals for 14 days. if available. DO NOT FLAME. When swelling occurs. Wipe off iodine mixture with 151 . when reserve portion must be held. warm water. For a qualitative test. and all identifying marks on label. Chill hard swells in refrigerator before opening. Badly swollen cans may spray out a portion of the contents. dents. Observe cans for progressive swelling at frequent intervals for 14 days. or use separate presterilized can openers. Place cans (excluding those held in reserve) in incubator at 35°C. Flipper and flat cans. Scrub entire uncoded end and adjacent sides of can using abrasive cleanser. Hard swells. which may be toxic. pinholes or rusting. buckling or other abnormality. and a brush. examine for preformed toxin of C. Immediately analyze springers. NOTE: Cans must be at room temperature for classification. or abrasive pad. botulinum if microscopic examination shows typical C. Retain examples of each. and a brush. Classify each can according to the descriptive terms in Table 1. Swollen cans. A slight explosion indicates presence of hydrogen. Scrub entire uncoded end and adjacent sides of can using abrasive cleanser. one for each can. cold water. make sure cans are at room temperature. At the time a swollen can is punctured. above. Open can in an environment that is as aseptic as possible. Sanitize can end to be opened with 4% iodine in 70% ethanol for 30 min and wipe off with sterile towel. Flip mouth of tube to flame of Bunsen burner. 1. follow directions in l a. b. Place remaining flat and flipper cans (excluding those held in reserve) in incubator at 35°C. Use of vertical laminar flow hood is recommended. Gently shake cans to mix contents before sanitizing. Flood end of can with iodine-ethanol solution and let stand at least 15 min. and springers . soft swells. and a representative number (at least 6. Sterilize can opener by flaming until it is almost red. steel wool.Sampling can contents a. 2. make note of it. product. Take some precaution to guard against this hazard. if available. Examination of can and contents Classification of cans. Opening the can.numbers and record size of container. swells. culture sampled contents. code. bring cans back to room temperature before classifying them. b. A white precipitate indicates presence of CO2. Rinse and dry with clean sterile towel. if available) of flat and flipper cans. hold mouth of sterile test tube at puncture site to capture some escaping gas.g. (It is not necessary to analyze all normal cans. Rinse and dry with clean sterile towel.) Do not incubate cans at temperatures above 35°C. steel wool. or abrasive pad. D.

Unless circumstances dictate otherwise. Make opening in sterilized end of can large enough to permit removal of sample. and headspace on a representative number of normal-appearing and abnormal cans (1). if apparent. Medium Chopped liver (cooked meat) Chopped liver (cooked meat) Bromcresol purple dextrose broth Bromcresol purple dextrose broth No. if less is available. Cultural examination of low-acid food (pH greater than 4. Removal of material for testing. Incubate as in Table 2. consistency. even apparently normal cans. Simply and completely describe product appearance. aseptically transfer at least 30 ml or. or 1-2 g of solid material. or use separate presterilized can openers for each can. Remove large enough portions from center of can to inoculate required culture media. determine pH of a representative number of normal cans before proceeding. of tubes Temp.6). After removal of inocula. Ensure sterility of can end by flaming with burner in a hood until iodineethanol solution is burned off. If analyst is not familiar with decomposition odors of canned food. analyze normal cans submitted with sample organoleptically and physically (see 5-b.clean sterile towel. below). Incubation times for various media for examination of low acid foods (pH > 4. Use this material for repeat examination if needed and for possible toxicity tests. etc. preferably one familiar with decomposition odors. 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). Be careful not to inhale iodine fumes while burning off can end. should confirm this organoleptic evaluation. another analyst. include such things as low liquid level (state how low). Determine drained weight. Use sterile pipets. Physical examination. either regular or wide-mouthed. vacuum. including evidence of leakage. Sterilize can opener by flaming until it is almost red. In describing the product in the can. end of can becomes discolored from flame. Inoculate each tube with 1-2 ml of product liquid or product-water mixture. evidence of compaction. corrosion. 5. because botulinal toxin may be present. all remaining contents of cans to sterile closed containers. CAUTION: Always use care when handling the product. and any other characteristics that do not appear normal. Perform net weight determinations on a representative number of cans examined (normal and abnormal). etching. From each container. including pH determination and seam teardown and evaluation. If there is any question as to product pH range. and heat causes metal to expand. 3. inoculate 4 tubes of chopped liver broth or cooked meat medium previously heated to 100°C (boiling) and rapidly cooled to room temperature.6). Transfer solid pieces with sterile spatulas or other sterile devices. and odor on worksheet. and refrigerate at about 4°C. 4. Describe internal and external condition of can. Always use safety devices for pipetting. This is the reserve sample. also inoculate 4 tubes of bromcresol purple dextrose broth. Table 2. (°C) 2 35 2 55 2 55 2 35 Time of incubation (h) 96-120 24-72 24-48 96-120 152 .

examine contents as wet mount or hanging drop. consistency. b. and stain with methylene blue. a. Prepare direct smears from contents of each can after culturing. liver-veal agar.. test material from cans (other than those classified as flat) for preformed toxins of C. DO NOT USE pH PAPER. DO NOT TASTE THE PRODUCT. record types of bacteria seen and estimate total number per field. or prepare suspension of test material in drop of chopped liver broth before drying. determine pH of remainder. Examine can lining for blackening. If product washes off slide during preparation.Schematic diagram of culture procedure for low-acid canned foods a LVA. fixed film. and pitting. Check liver broth before use to be sure no bacteria are present to contribute to the smear. cooked meat medium. and overall quality. crystal violet.After culturing and removing reserve sample. fix. Examine under microscope. rinse and stain. botulinum when appropriate. Table 4. Physical and organoleptic examination of can contents. texture. detinning. using pH meter. color. using a dropper. Examine for odor. After removing reserve sample from can. Incubation of acid broth and malt extract broth used for acid foods (pH 4.6) Medium Acid broth Acid broth Malt extract broth No. Dry. or Gram stain. If product is oily. (°C) 55 30 30 Time of incubation (h) 48 96 96 153 . Pour contents of cans into examination pans. NA. of tubes 2 2 2 Temp. bromcresol purple dextrose broth. Table 3. BCP. CMM. add xylene to a warm. Microscopic examination. nutrient agar.

If there is no growth in either medium. 1. In some cases. SAB. report and discard. If Gram-positive or Gram-variable rods typical of either Bacillus or Clostridium organisms are found in the absence of other morphological types. store cultures to maintain viability. Classification of food products according to acidity Low acid--pH greater than 4.e. old vegetative cells may appear to be Gram-negative and should be treated as if they are Gram-positive. Pick representatives of all morphologically different types of colonies into CMM and incubate for appropriate time.Table 5. Table 6. Reincubate CMM at 35°C for maximum of 5 days for use in future toxin studies. After obtaining pure isolates. 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). as in schematic diagram (Table 3). report morphological types. nutrient agar. search to determine whether spores are present. i. Dispel oxygen from CMM broths to be used for anaerobes but not from those to be used for aerobes. E. a NA.6 Meats Seafoods Milk Meat and vegetable Mixtures and "specialties" Acid pH 4. 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..6 and below Tomatoes Pears Pineapple Other fruit 154 . Pure culture scheme for acid foods (pH 4. when growth is sufficient for subculture.6). If mixed microflora is found only in BCP. test CMM for toxin. Sabouraud's dextrose agar. If rods are included among mixed microflora in CMM. as described in Chapter 17.

0 >4. Spoilage manifestations in low-acid products Classification Manifestations Can flat Product Possible loss of vacuum on storage Appearance not usually altered. rotten egg odor May burst Thermophilic anaerobe Sulfide spoilage Putrefactive Can swells Product Can flat Product Can swells 155 .3 >4.7 <3.7 <3.8 >5. sour. cheesy or butyric odor H2S gas absorbed by product Usually blackened. sour. may have slightly abnormal odor.8 >4. peas Tomato juice Fruits Fruits Fruits The responsible organisms are bacterial sporeformers.2 4. pH markedly lowered. corn Corn. sometimes cloudy liquor May burst Fermented.Spaghetti Soups Vegetables Asparagus Beets Pumpkin Green beans Corn Lima beans Sauerkraut Pickles Berries Citrus Rhubarb Table 7.3 >4.5-3. peas Spinach. Group of organisms Flat-sour Table 8. 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 >5.7 Examples Corn. asparagus Tomatoes. peas Corn.

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. diagnosis may be less clearly defined. rough conveyor system. off-odor Butyric anaerobes (tomatoes and Can swells May burst tomato juice) Product Fermented.. e. e. History Spoilage usually confined to certain Spoilage scattered portions of pack In acid products. typical putrid odor Usually no swelling. Spoilage manifestations in acid products Type of organism Classification Manifestation Bacillus thermoacidurans (flat. except in cured meats when nitrate and sugar present. May be characteristic on special growth media. rods.g. or any combination of these may be present. 156 .g. coagulated evaporated milk. such as contamination of cooling water or rough handling. may show normal defects(a) Sloppy or fermented Frothy fermentation. fecal. similar organisms may be involved in understerilization and leakage. generally varying from can to can Wide variation Mixed cultures. seams generally normal Swelled.yeast or molds.anaerobe Aerobic sporeformers Product Can flat or swollen May be partially digested. pH slightly above normal. generally rods and cocci. Laboratory diagnosis of bacterial spoilage Underprocessed Leakage Flat or swelled. sour or putrid. black beets Table 9. butyric odor Nonsporeformers (mostly lactic Can swells Usually burst. but swelling may be types) arrested Product Acid odor Can Product appearance Odor Normal. cocci.. viscous Sour. a Leakage may be due not to can defects but to other factors. Table 10. acid agar for tomato juice. only at usual temperatures If product misses retort completely. sour Can flat Little change in vacuum tomato juice) Product Slight pH change. can unscramblers.

4. juice Apples.2 . fruit Jellies.6.4.2 .7.9 .2 3.4.0 3.5 .5 .3 5.5 2. whole Asparagus.1 5.3.6.9 3.3 .4 .6.Table 11.5.6.3 5.0 .1 .3.0 4. ripe Orange juice Oysters Peaches Pears (Bartlett) Peas Pickles Dill Sour Sweet Pimento Pineapple Crushed Juice Sliced Plums Potato salad Potatoes Mashed White.5.7 2.7 .0 5.6 6.5 .3 .7 .3.1 .6.6.0 5.4 .9 .5 5.3.4 2.1 6.7.0 . whole Blackberries Blueberries Boysenberries Bread White Date and nut Broccoli Carrot juice Carrots.9 .6.5 2.0 6.3.2 .5.2.6.0 .6 5.0 .3.5.2 .3 3.6.5 5.5 4.3 5.1 2.4.5.6 3.6.8 .3 4.9 .9 .4.7 157 6.4 6.9 .3.6.7 3.0 .4 3.6.2 6.4 .5 6.6.8 .8 5.8 .3. green Beans Baked Green Lima Soy Beans with pork Beef.8 3.3 .6 3. whole Prune juice Pumpkin Raspberries Rhubarb Salmon Sardines Sauerkraut 3.4.6 5.3 6.4 .3 .9 .3 .6.5.8 4. 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.9 .8 Jam.3.5 5.5 .5.0 3.2 .9 .3.4 .8 5. pH range of a few selected commercially canned foods Food pH range Food pH range Apples.6.1 .6 2.3.3.0 3.0 . fruit Lemon juice Lemons Lime juice Loganberries Mackerel Milk Cow.6 5.2.1 .5 2.2 .9 3.5 .4.5.5 5.4 .7 .5.6 5.4 .5.5.4.4 2.3 6.3.4.6 5.2 .1 5.3.7 5.9 .0 .2 .3.6 2.6.3 5.0 .2 .5 2.0 .4.2.6 .5 5.0 .2 3.8 3.9 .7 .4 6.8 5.6 .2 . whole Evaporated Molasses Mushroom Olives.5.6.7 2. spiced Cranberry Juice 3.5 3. corned.4.2.5.1 .6.7 3.0 4.3.7 .0 .3.4 .0 . hash Beets.8 3.6.2 .

3.0 .3 .3.3 .8.6 3.9 .4.6 7.1 .1 2.9 5.1 5.9 5. Cincinnati.4.7 .2.6 . CO2 Mineral Sea Wine 3.8. If no toxin is present.8 4.6.8 .7.0 .7.2 .4 3.0 6. send pure cultures for evaluation of heat resistance to Cincinnati District Office.0 .6 6.7 .7 .8 6.4 5. 1141 Central Parkway.7.4.0 .4 3.5.9 .3 .2 .2 .0 .5. OH 45202.7 5.3 .0 6.4 .2 .6 .6.5 6.3 4.4 8.0 7.4 1.3.5.1 4.2 4.7.0 .3 3.7.0 2.6.3 .2 4.5.4.8 .6 .8 6.3.3.5.0 2.3.0 .9 .3.4.5.8 . if cultures meet the following criteria: 158 . mixed Vinegar Youngberries 2.6 .6.8 .5 6.2 5.9 .6.0 .9 5.3 5.5 3.4 10.5 4.5.5.0 5.6 4.5.6.0 .6.4 6.6 .4 .4 4.5 5.5 .5. milk of Water Distilled.7.5.4 6.7 6. FDA.6 3.6.2 .5 .5.6.9.0 .0 .9 .4 3.0 6.6 2.2 3.Sauce Currant juice Dates Duck Figs Frankfurters Fruit cocktail Gooseberries Grapefruit Juice Pulp Sections Grapes Ham. lye Huckleberries Strawberries Sweet potatoes Tomato juice Tomatoes Tuna Turnip greens Vegetable juice Vegetables.5 4.9 3.6.3 .0 -10.8 .5.8.5 .9 .7.8.8 5.2 5. spiced Hominy.0 .3.5.9 2.8 2.0 6.8 .4 2.4.8 .3 6.0 .4 .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.

a spoilage characteristic similar to that resulting from leakage. Spoilage encountered in products such as tomatoes. and clostridial forms may be seen on microscopic examination. thermobutylicum may be indicated by gas in cooked meat at 55°C and a cheesy odor. Incubation at 55°C will not cause a change in the appearance of the can. Spoilage by mesophilic organisms such as Bacillus thermoacidurans or B. Spoilage in acid products is usually caused by nonsporeforming lactobacilli and yeasts.§ Cultures come from intact cans that are free of leaks and have commercially acceptable seams. spores. but which grow and cause spoilage when the product is subjected to elevated temperatures (50-55°C). figs. a sporeforming anaerobe which produces gas and a butyric acid odor. A mixed microflora of viable bacterial rods and cocci usually indicates leakage. visual examination alone is not sufficient. Pure cultures may be isolated as shown in Table 5. with or without lowered pH. perfringens may be indicated in cooked meat at 35°C by gas and a putrid odor. or C. which are flat-sour types. Examination of acid foods (pH 4. botulinum. Spoilage by thermophilic anaerobes such as C. Spoilage of this type is an exception to the general rule that products below pH 4. sporogenes. due to aerobic.) Two or more tubes are positive and contain similar morphological types. but leakage at some time in 5. make smears and stain. 4. requiring recall of all cans bearing the same code. § 3. 159 . stearothermophilus are thermophiles responsible for flat-sour decomposition in acid and low-acid foods.6 and below) by cultivation. (Can seams of both ends of can must be measured. Many canned foods contain thermophiles which do not grow under normal storage conditions. since viable botulinal spores in canned foods indicate a potential public health hazard. thermosaccolyticum is a thermophilic anaerobe which causes swelling of the can and a cheesy odor of the product. and pineapples is occasionally caused by C. or Record presence or absence of growth in each tube. rods. Can examination may not substantiate the bacteriological findings. mesophilic. in cans with satisfactory seams and no microleaks indicates underprocessing if their heat resistance is equal to or less than that of C. Spoilage by C. Presence or absence of growth in this case must be determined by subculturing. No definitive conclusions may be drawn from inspection of cultures in broth if the food produced an initial turbidity on inoculation. Cans of spoiled tomatoes and tomato juice remain flat but the products have an off-odor. and from those that show evidence of growth. C. Report types of organisms seen. Cans which bypass the retort without heat processing usually are contaminated with nonsporeformers as well as sporeformers. thermoacidurans and B. The presence of only sporeforming bacteria. Always test supernatants of such cultures for botulinal toxin even if no toxin was found in the product itself. and thermophilic sporeformers. and incubate as in Table 4. may be indicated by acid production in BCP tubes at 35 and/or 55°C in high-acid or low-acid canned foods. which grow at 35°C. botulinum. From each can.6 are immune to spoilage by sporeformers. 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. C. pasteurianum. stearothermophilus. 6. respectively. B. coagulans and/or thermophilic organisms such as B. but the product has an off-odor with or without a lowered pH. pears.

7. such as tomato paste. If can remains flat. aseptically inoculate growing organism into another normal can. A mixed microflora in the product. mincemeats. molasses. and incubate 14 days at 35°C. the cans may have missed the retort altogether. The proportion of hydrogen varies with the length and condition of storage. This results from bacterial growth in the product before canning. in which there are large numbers of bacteria visible but no growth in the cultures. open it aseptically and subculture as previously described. If any swelling of container or product changes occur. If no evidence of microbial growth can be found in swelled cans. and appearance. This is particularly true of concentrated products containing sugar and some acid. Chemical breakdown of the product may result in evolution of carbon dioxide. The reaction is accelerated at elevated temperatures. solder the hole closed. Alternatively. 160 . as shown by direct smear. If a culture of the same organism is recovered and the product is normal. 9. in which case a high rate of swells would also be expected.the past must be presumed. 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. Thermophilic anaerobes produce gas. The product may be abnormal in pH. To confirm. and since cells disintegrate rapidly after growth. odor. it is possible to confuse thermophilic spoilage with hydrogen swells. consider the product commercially sterile since the organism does not grow under normal conditions of storage and distribution. may indicate precanning spoilage. 8. the organism was probably not in the o riginal sample. and highly sugared fruits. the swelling may be due to development of hydrogen by chemical action of contents on container interiors.

For example. Personnel Personnel should have the education. 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. 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. the manufacturer's instructions must be followed exactly. SOP’s should include limitations of each test and a list of precautions. Similarly. Employees must be fully aware of their QA responsibilities and the adverse consequences that will arise from failure to carry out their duties carefully. The SOP’s should also indicate how a test should be read and what should be looked for in a positive and negative test. Care should be taken to ensure that the photographs do not fade. 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). for pre-prepared kits such as the mini-kits used for biochemical tests. Mistakes can arise from using an incorrect viewing angle or the wrong method of inoculation. Management should ensure that a safe. SOP's should also include the names of personnel to be contacted if out-of-control procedures are found. experience and motivation necessary to perform their jobs and the requirements of a quality assurance program. Modifications to standard methods should not be used unless comparative studies have shown that the modified methods are as reliable as reference methods.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. efficiently designed facility. Colour photographs can be used to assist personnel in reading tests. Possible interferences should be described. supervision and workload direction is as important as selection of appropriate personnel. Facilities 161 . the potential presence of natural inhibitory substances should be noted and methods for diluting out or neutralizing these substances must be included. sufficient supplies and equipment are available. Management of laboratory personnel through motivation. however neither should be used as substitutes for SOP's.

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:

162

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

163

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

164

-

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

165

faecalis – + – + – Optochin disc S. Coagulase Indole S. coli E. coli Voges Proskauer Bacitracin disc E.aeruginosa E.aeruginosa E. aerogenes Oxidase P. coli The testing should be done each time a new batch of working solution is prepared. aureus E.Table 3: QC for commonly-used tests Procedure/result Catalase Test Control organism S. aerogenes Methyl red E. viridans Oxidase disc P. aureus Streptococcus spp. coli E. pneumoniae S.. 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. 166 . aerogenes E.

influenzae type b) Antigen control Negative control serum Positive control serum Pneumococci Haemolytic streptococci H. Test 1. Course-curriculum of such trainings should focus on the issues highlighted above (SOPs). 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.Quality control procedures used for the detection of antigen or antibodies by various test methods : Table 4. The control or referral laboratories should organise EQAS in some commonly used tests. 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. 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. Capsular Quellung reaction (Omni serum.: ___________________ 167 . Direct agglutination (Widal test) 4. 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. Latex agglutination test Positive control serum (ASO) 3.

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. laboratory staff would be well advised to adopt a TQM approach of continuous improvement to their work. Appropriate procedures and controls over procurement of laboratory materials Use of appropriate analytical procedures Statistical quality control Audits of the quality system. The application of good laboratory practice ensures accuracy and confidence in test results. These elements should be encompassed in a quality manual. N/B Ideally. It also prevents cross-contamination of samples and protects personnel against infection and other laboratory hazards. 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 168 . Although the accuracy required depends on the how the information is to be used. storage and delivery. 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. Good record keeping activities also help manage and encourage staff proficiency. Standardization of methods will also reduce variation between results produced by one laboratory and between results produced by associate laboratories. handling.

the sampling plan should also be included. It should encourage and support staff to carry out the laboratory's quality policy. If possible. It may also involve rewarding groups or individuals or if necessary disciplining staff who do not meet the laboratory's expectations. the samples must be stored to maintain their original condition and then tested as soon as possible. Therefore. To sterilize clean. i. Autoclaves are standard items in all microbiology laboratories. Management is responsible for providing resources and ensuring that these resources are suitable for the functioning of the laboratory. It is also responsible for ensuring that the quality system functions as planned. Autoclave Uses of autoclave in microbiology 1. Records should include for each cycle. For example. This will involve auditing and verification of the laboratory practices. target organisms may increase or decrease in number during sample handling. it is important that the laboratory insist that the original condition of the sample and its container be maintained. They should be equipped with accurate and calibrated pressure and temperature gauges. man-power and effort and can have a demoralizing effect on staff. 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. calibrated and regularly tested. Preventative maintenance must be carried out on a regular basis. date and time of collection. Temperature sensors and temperature controllers in incubators. To sterilize media 3. There should also be adequate documentation stating the sample's source. wrapped instruments and containers 2. analysis requirements and required storage conditions.e. Preferably a temperature recorder should also be used to keep a record of each sterilization cycle. with saturated steam 121oC is equivalent to 15 PSI. Equipment such as balances. changes in the microbial content and change in the chemical make-up.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 is responsible for designating responsibilities and authorities of persons involved in assuring the quality of output from the laboratory. This section presents guidelines for maintenance and standardization of selected instruments. To render microbiologically contaminated materials biologically safe (Sterile) before they are discarded. It may be necessary for a single technician to have responsibility of the sample. before a sample is accepted for analysis. Instrumental Maintenance. Does not affect radioactivity or chemical toxicity of autoclaved materials. Analysis of a mishandled sample is a waste of time . Microbiological Sample management Samples which are normally encountered in the Microbiology laboratory are typically subject to change. water-baths and hot air sterilizers should similarly be accurate. 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. Due to mishandling. Upon receipt of the sample. (1) temperature and time settings (2) materials in 169 . thermometers and pH meters should be calibrated to meet national standards. This must be documented and staff should be trained in how to carry out preventative maintenance.

. test the same total volume of 170 . For sterilization of liquid volumes of greater than 0. This is best done by removing the air by vacuum. Autoclave tape while a useful indicator that autoclaving has been undertaken does not show whether the sterilization cycle has been completed.0 34 44 2.0 43 53 4. Another factor which will affect the efficiency of sterilization will be the presence of air in the autoclave. Place biological indicators into each type of item that maybe autoclaved 1. vary both the total volume of liquid and the number of containers being autoclaved (i. Place spore strips into center of wrapped packs. The presence of air will result in a temperatures which are less than those above for a given pressure. 2. Modern autoclaves automatically exhaust the air before reaching the sterilization temperature. A. For verification of sterilization of liquid. 3. large volumes of media will require longer holding times than small volumes. Calibrate the autoclave with the maximum permissible load. 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. Although the standard temperature. The proper functioning of the autoclave should be ascertained using biological indicators such as spore ampoules or strips of Bacillus stearothermophilus. pressure and holding time used for sterilizing is 121oC at 15 psi for 15 minutes.e.0 37 47 3.0 52 62 Thus for a four litre container of medium.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 following holding times are recommended: Liquid Volume per container (l) Time required to reach 121oC (min) Total holding time (min) 0.5 l. 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.5 19 29 1. this is not suitable for heat-sensitive sugars such as glucose and sucrose. These sugars should be autoclaved at 110oC for 30 minutes. a holding time of 62 minutes instead of 15 minutes must be used. The air should thus be removed during when the heating stage of the autoclave cycle. Because heat transfer through liquids is not instantaneous. Physical measurements of temperature and pressure should also be used. Drop biological indicator ampoules into flasks of medium (tie ampoule with string to facilitate removal). The standard operating procedures used for the operation of an autoclave should take into account the properties of media being sterilized.

Because they require no oxygen. Clean up any spills immediately Incinerator burner Uses in the microbiology laboratory. Be sure that the unit does not generate any smoke or persistent odor suggestive of burning rubber.liquid but distribute it among few containers for one load and among numerous containers for another load). 1. 2. Plug the incinerator into a grounded electrical circuit. Removing particulate matter. Incinerators are also useful for working in laminar-flow biosafety cabinets. In the heated condition. Concentrating etiologic agents and cells. Inspect for small cracks in the ceramic casing and residue buildup during both cool and heated conditions. replace the heater element. Observe recording chart for proper time and temperature. The absence of an open flame provides safer alternative to the Bunsen burner. B. Air Incubators 1. Check each load for proper balance. 3. B. intensely yellow orange fissures. Routine quality control Visually inspect the heater element daily to determine if the heater element core is worn. Centrifuge Uses in the microbiology laboratory. Routine use 1. Electric incinerator burners are particularly useful in laboratories not equipped with gas or flame sterilizing burners. a smoother centrifugation will result in better separation. Separating components of mixed suspensions on the basis of densities. The manufacturer prior to shipment should perform initial calibration. both for even distribution and for weight to within 0. C. Initial calibration A. Cracks do not inhibit the sterilization ability if the unit. Calibration of centrifuge rotation speed. particularly for today’s solid-state circuit instruments should be done only by skilled personnel. they work well in anaerobic chambers. Instrument setup 171 . 2. Look for a red glow inside the heater element. but they create an electrical safety hazard. 2. Incubator Uses in the microbiology laboratory The incubator is used to maintain a constant and appropriate temperature for the growth of microorganisms. 3. Initial calibration 1. cracks can be seen as small. Incubate biological indicator to verify sterilization. Well-balanced loads prevent wear on the drive train and motor bearings. Additionally. A.5g. Check that appropriate temperature is maintained during operation. 3. If defects are notes.

Adjust the regulating thermostat knob until the dial thermometer reaches 35°C. e. Check the temperature in four other spots (top front. d. c. 3.5 h. Setting the safety thermostat (Example: setting for 35°C incubator) a. When the temperature reaches the desired safety temperature (usually 2. Check the product manual for instructions d.5 h. and put some tape or a sign near the door handle so that the door will not be opened during the calibration. Do not use these areas for sensitive cultures. Verify that the power source is compatible with the incubator power system before plugging the system into the electric outlet. After 10 min with the door closed. Calibration of the internal thermometer a. If there is a safety thermostat. Allow the incubator to run overnight and recheck parameters before using. Regulating the humidity a. tighten the temperature control knob (or put a tape over the control) to prevent accidental turning of the control knob. 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. and allow the incubator to equilibrate for 0. c. Close the door of the incubator.0°C above the actual desired maximum temperature). b. d. If both pilot lights go on at the same time the safety knob is set too low and must be reset. turn the safety thermostat down very slowly until the safety pilot light just goes on.a. b. if the humidity is less than 40%. b. bottom front and bottom back corners) with the NBS thermometer so that any hot or cold spots will be known. 172 . Allow the incubator to equilibrate for 0. 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. 4. b. If there is no safety thermostat. skip to step 3 below. b. c. and then turn the regulating thermostat control knob down until both the safety pilot light and the regulating pilot light go off. Turn both the regulating thermostat control knob and the satiety thermostat knob to the highest available setting and monitor the temperature. allow the temperature to remain at 37°C for 0. Read the humidity after 1 h. d. the safety pilot light should go off during normal operation. After the temperature has stabilized at 35°C. Install shelves at the desired levels. compare the temperature on the NBS thermometer with that on the thermometer in the incubator. c. 5. adjusting the safety thermostat knob as needed to get the temperature to stay at the selected value (usually 37°C). Setting the regulating thermostat a. 2. If there is a discrepancy. Turn OFF-ON switch to ON so that the heating element (an the blower if applicable) will go on. c. Lay a NBS thermometer on the back of the shelf nearest the center of the cabinet. top back. it is usually possible to remove the face of the dial thermometer and adjust the reading.5 h. Place a hygrometer (humidity measuring device) on the central shelf of the incubator. place a pan of water (containing antifungal agent) with a large surface area ( 150 cm2) on the shelf of the incubator.

Microaerophillic atmosphere primarily for Campylobacter species. etc. Haemophilus species. Holding and transporting pure cultures of anaerobic or microaerophilic organisms under appropriate atmosphere. Anaerobic atmosphere primarily for strict anaerobes. Open the field diaphragm all the way. fungi. Koehler illumination is a precise way of aligning the light pathway onto the specimen plane to evenly illuminate the field of view. Rejuvenate catalysts after each use by heating the basket of pellets (rendering them less inactive). Place a cleaned stained specimen (cells or bacteria. anaerobic atmosphere sufficient to change the indicator is achieved and QC organisms can grow. b. 4. store heated catalyst baskets in a very dry place. Before opening an anaerobic jar. subject identity is not critical. Carbon dioxide enriched atmosphere primarily for Neisseria species. but best to have distinct form for ease of focus) slide right side up in the stage slide holder. parasites and host cells in various stained and unstained preparations. such as a desiccator jar. This procedure ensures the highest resolution for the optical system. If an anaerobic atmosphere was not maintained the anaerobic culture result is invalid 4. Discard all strips that are not white when packet is opened. Initial aligning procedures A. microbroth dilution plates. Primary plate incubation (all atmospheres) a. 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. 3. Routine quality control A. Incubation of other cultures that require special atmosphere: test strips. Check the indicator strip has not turned color inside the sealed packet. Initial calibration: Anaerobic and Microaerophilic jars Test the jar with generator system and indicator QC organisms to verify that seal is air tight. 2. 1. 1. certain microaerophillic streptococci and other capnophiles 2. Maintenance of specimens (such as tissues) under anaerobic atmosphere. c.Anaerobic Jars Uses in the microbiology laboratory. tubers. 173 . record the QC checks in a label attached to each lid. 3. 1. Check jar and lid for cracks 2. observe the indicator strip to be sure that an anaerobic atmosphere was maintained during incubation. enabling visualization of as much detail as possible. Anaerobic jars: each time they are used Since the lid is the component most likely to fail.

Other objective magnifications (50× oil or 63 × oil immersion lenses) may also be used. the disk is usually calibrated as a line divided into 50 Units. 6. using he course and fine adjustment knobs.and 0. swing it out of the light path. 18. If the condenser has an auxiliary lens.1. 5. 4. usually a stage micrometer with a scale of 0. Otherwise.3. 16. 11. Do not disturb the condenser height. Do not open any further. Look through the fixed eyepiece.01-mm divisions 3. If the diaphragm image is not centered. if available. 15.) Adjust fine focus knob so that the specimen is in sharp focus. to compensate for any dioptic discrepancy (different ability to focus) between each eye. Immersion oil. Rack the condenser all the way up. 13. bacteria. B. 174 . (Rule of the thumb is to remove the eyepiece.mm divisions. Rotate the nosepiece to the 10× objective position (8× to 20× objective is acceptable). and adjust the aperture to two-thirds open. 10. 14. then center it by gently turning the centering screws located on the condenser. Materials A. Open the diaphragm until the image of the diaphragm just goes out of the field of view. Open the condenser diaphragm all the way. Use the focusing eyepiece. 12. you will not be able to image the diaphragm.1. Control contrast by adjusting the condenser diaphragm.and 0. one of which is size. Set the optimal contrast by gradually closing the aperture diaphragm. and 100× objectives. Use the adjustable eyepiece to focus the image sharply. Lower the condenser slightly until the diaphragm edge is as sharp as possible. comfortable light intensity. 8. 40×. protozoa and other parasites depend on several factors. Calibration of microscope with an ocular micrometer I. Principle The identification of molds. 17. look down the tube at the back focal plane of the objective. Ocular micrometer disk (line divided into 50U) 2. 4. Equipment 1. Stage micrometer with a scale of 0. 7. Lens paper B. II. 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. Gradually close the field diaphragm until you see a multisided polygon (image of diaphragm) around the field of the specimen. Binocular microscope with 10×. Most microscopes will not need further alignment for higher magnification. Adjust the binoculars to your inter papillary distance. (A magenta color may be seen. The ocular disk division must be compared with a known calibrated scale. Turn on the light source and adjust it to a low.01. 9. and focus on the specimen. Depending on the objective magnification used the divisions in the disk represent different measurements. Adjust both eyepieces to equal height. Supplies 1.

D. D. The second set of lines should be as far to the right of the 0 lines as possible.1. Some may prefer 5×. Makes sure you understand the divisions on the scale before proceeding. Unscrew the eye lens of a 10× ocular. Oculars should be 10×. Single 10× ocular to be used to calibrate all laboratory microscopes (to be used when any organism is being measured) III. Use lens paper to handle the disk.0 µm (factor) Oil immersion (100×) 0.2.05 mm/ 62 U × 1000µm/ 1 mm = 0. 3. and focus on the scale. When these two 0 lines are lined up. You should be able to distinguish the difference between the 0. the thin ocular line should be lined up in the center or at the corresponding edge of the broad stage micrometer line. keep al surfaces free of dust or lint.and 0.8 µm (factor) 175 .0 µm (factor) High dry power (40×) 0. Latex or polystyrene beads of a standardized diameter can be used to check the calculations and measurements beads of 10µm and 90µm are recommended. count the number of 0.mm divisions between the 0 lines and the second set of superimposed lines.8 mm/ 100 U × 1000µm/ 1 mm = 8. F. twice a year is recommended. Then. Calculate the factors as follows. Place the calibrate micrometer on the stage. Look to the right of the 0 line for another set of lines superimposed on each other. do not move the stage micrometer any further. Thus when the second set of superimposed line is found. Calculate the portion of a millimeter that is measured by a single ocular unit. C. Recalibrate the microscope a minimum of once each year. Examples: Stage reading (mm)/ ocular reading × 1000µm/ 1 mm = ocular units (µm) Low power (10×) 0. G. Quality control A. on the stage micrometer. whereas the ocular line will remain the same size. Record all measurements in QC records. The thin ocular 0 line should be lined up in the center or at one edge of the broad stage micrometer 0 line. ×400. If the scope receives heavy use. B. B.1. ×1. ad place the micrometer disk (engraved side down) within the ocular. 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.5 µm) is used to check the calibrations of the three magnifications (×100. E. however the distance varies with the objectives being used. H.1mm/ 50 U × 1000µm/ 1 mm = 2. Often the measurement of RBCs (approximately 7. Count the number of ocular divisions between the 0 lines and the point where the second set of lines is superimposed. the 0 line of the stage micrometer will increase in size.01 mm divisions. IV.000). Procedure A. When the high dry and oil immersion objectives are used. however smaller magnification may make final identification more difficult. C.

depending on the objective magnification. The final multiplication factors will be on as good as your visual comparison of the ocular 0 and stage micrometer 0 lines.6 µm. then.0 µm (for that objective). C. V. Once the number of ocular lines per width and length of the organism is measured. the size for the object measured should be the same (or very close). Hot air oven In microbiology laboratory the oven is used to dry glassware. Example: If a protozoan cyst measures 27 ocular units with the oil immersion objective.8 µm (for that objective). 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. However. VI. This single ocular can be inserted when measurements are taken. B. a factor will be generated (1 ocular unit = certain number of micrometers. then multiply the measurements by the factor 2. Each microscope used to measure organisms must be calibrated as a unit. Initial calibration A. then multiply the measurements by the factor 0. C. the high dry objective (40×) factor should be approximately 2. The low-power objective (10×) factor should be approximately 10 times the oil immersion objective (100×) factor. this particular ocular containing the ocular micrometer disk must also have been used as the ocular during microscope calibration. 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. sterilize metal and certain hightemperature stable glass objects and to reactivate palladium-covered alumina catalysts used in anaerobic chambers and jars. Connect the power cord to the grounded outlet. The original oculars and objectives that were used to calibrate the microscope must also be used when an organism is measured. After each objective has been calibrated.Example: If a helminth egg measures 15 ocular units by 7 ocular units with the high dry objective. VIII. Limitations of the procedure A. B. Procedure noted A.5 times more than the oil immersion objective (100×) factor. VII. For each objective magnification. If standardized latex or polystyrene beads or an RBC is measured with various objectives. The cyst then measures 21. The objective containing the ocular micrometer can be stored until needed. regardless of the objective magnification. the oculars containing the disk and/or these can not be interchanged with corresponding objectives or oculars on another microscope B. Results A. Comparison of these measurements with reference measurements in various books and manuals should confirm the organism identification. The egg the measure 30 by 14 µm and is probably Clonorchis sinensis. As a rule of thumb. B. Reporting results A. 176 .

Cultures should be maintained in the refrigerator on slopes and not on agar plates. Read and record the pH of the QC-certified buffer whose pH is closest to that of the expected pH of the sample. 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. date of placement in refrigerator. D. use QC. E. Switch the oven on. A record of materials entering and being removed from the ot refrigerator should be maintained.certified buffer.10-pH unit of the value of the standard. Antibiotic activity and solubility vary with pH of diluents. They should be arranged in a tidy manner such that sample or culture access is n hampered. pH meter Uses of pH meter in the microbiology laboratory. Refrigeration or freezing may inhibit the growth of contaminants. test the pH 4.00. 2.05 pH unit of the pH of the QC. For example given above. Each time. Value must be within ±0. Allow the temperature to stabilize for at least 2 h before using the oven for the first time. 1. F. Read and record the result of pH standard calibration buffer opposite the pH of the standard that you read in step 1. Appropriate containers should be used to hold samples and cultures. b. A refrigerator which does not cool to the correct temperature will cause sample degradation and loss of culture viability. All samples must be appropriately identified with the sample's identity.B. slow reactions that would otherwise inactivate reagents and delay evaporation. 177 .certified buffer pH 8. 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.00 standard calibration buffer. for example. Bacteria. even if more than one use occurs in the same day. Spillage of cultures in the refrigerator or onto the floor is very dangerous and can arise from failure to use the proper holding containers. Results must be within ± 0. a. Refrigerators and freezers Uses in the microbiology laboratory Low temperatures are required for storage and preservation if stock cultures. c. Mislabeled and unlabelled samples can lead to many analysis errors. Agar plates. 1. They should be routinely sanitized. reagents and media. Check the thermometer every 15 min until the desired temperature is reached. no matter how well sealed will tend come apart. For example given above.00 standard. Readjust the temperature gauge if the temperature increases more than 5°C above the desired set temperature. 3. C. calibrate with pH 10. and adjust it to the desired temperature. The use of refrigerators and freezer must be carefully controlled. 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. If your expected solution us pH 8. Precise measurement and adjustment of the pH of solutions. overfilling containers and from cluttered refrigerator. fungi and viruses often require specific pH ranges of media or transport solution to remain viable or to multiply. contact person and if possible the use-by date.2.

Adjust the gauges. 2.Number each individual thermometer. and then check the internal thermometers. For a large unit. Monitor the temperature of the unit for stability several times a day for 1 to 2 days before placing materials inside. very-large-volume units (more than 60 ft3 [ca. or note on the internal thermometer the correction factor needed to match the NBS measurements. 13 m3]) may be better served with additional thermometers. If warm spots are found. The formation of condensate on the lid of the Petri dish can also lead to the contamination of the plate. Be certain to use containers that will withstand sub zero temperatures. 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. Setting the temperature 1. and re-equilibrate the temperatures until it is within 0. polyethylene glycol (antifreeze). call your manufacturer for advise (and service) 5. and check for consistent temperature readings.If the built in thermometer will be used exclusively. 5. we recommend that one thermometer be placed in the top left rear and on be placed in the bottom right front.5 °C of the desired temperature.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. close to the door. Set standard freezer temperature to -20°C by using external dial or gauge.Immerse thermometers for refrigerators in 50% glycerol. allow the temperature to equilibrate for 1 h. C. If necessary. Allow the temperature to equilibrate for at least 1 h after the compressor stops running for the first time after startup. 2. adjust the thermometers to read identically (consult the product manual). it is prudent to place the single thermometer there or to use more than one thermometer. Placing thermometers 1. -70. Before placing a new unit in service. Laminar flow cabinets (Clean bench) Uses in microbiology laboratories 178 . Preparing thermometers 1. place several (three or more) thermometers in diverse areas. and calibrate each one against the NBS thermometer 2. Initial calibration A. At least one thermometer should be placed into each interior chamber or a refrigerator or freezer.Immerse thermometers for freezers in alcohol or polyethylene glycol in plastic bag or bottle. If variation among all internal thermometers are >4°C. Set ultra low freezers to appropriate temperatures (common temperatures are –40. place and NBS-calibrated thermometer into the unit and verify the accuracy of the internal thermometer. or alcohol in an empty blood bank pheresis bag or in a bottle stoppered with a single hole cork or rubber stopper. 3.leading to a potential safety hazard. B. 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). Set refrigerator temperature to 6°C by using regulating dial inside the unit. -80 and -100°C) 4. 4.

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. 1. temperature. Determination of the work are velocity profile b. Grinding or mincing of tissue b. Checks on lighting intensity. C. Previously there have been no standards for certification if personnel who certify biological cabinets. For the similar reasons. vibration and noise levels. Intake and exhaust airflow rates must be balanced. 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. Identification procedures for molds 2. 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. cabinets should be decontaminated prior to the move. N/B The maintenance of laminar flow hoods should include checking of the ultraviolet light source. a. Cabinets must be recertified at least annually. 2. To protect samples or materials from external contamination a. d. Plating of specimens. air-conditioning vents or near where there is heavy human traffic. 4. Installation and certification A.1. 5. Measurement of the face velocity (airflow velocity through the front opening) c. Every biological safety cabinet must be tested in certified for adequate functioning after it is in place in the laboratory. The cabinet and filters must be free of air leaks 3. Performing procedures with sterile fluids b. since testing that occurs elsewhere will not ensure that the cabinet will function properly under the conditions actually found in the laboratory 1. a. Certification must be done by specially trained personnel. B. D. To contain infections aerosols generated by processing of samples or cultures. Determination of air flow patterns (smoke test) e. Preparation of media or reagents solutions aseptically. For long distance moves. Challenge tests should be used to test for their effectiveness the UV-light and filter. Examples include the following. Preparing direct smears and wet mounts c. the HEPA filter and the air compressor rate. media bottles and other large containers should be removed once they are no longer required. Determination of HEPA filter integrity d. Cabinets must be recertified before being operate after the have been moved. Cabinets must be recertified whenever HEPA filters are changed or repaired E. Field test should include the following. 179 . Institutional or regulatory guideline may call for more frequent certifications. All will need routine replacement or routine maintenance. Specified airflow velocities must be met but not exceeded. especially when nonmicrobiology personnel are involved. They should not be used as a storage area and should not be left on when not in use. The hood should not be positioned near doorways. 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.

Medicare and Clinical Laboratory Improvement Act regulations call for a minimum air intake of 50 lfpm through the front opening 3. 2. Remove excess water. Check blower function. Each time the safety cabinet is turned on or at least once per day 1. Avoid handling the ice so as not to contaminate it. Ice must not float.Record gauge reading (if gauge is provided). E. B. Use of absorbent coverings minimizes splatters and makes clean up easier. This can be accomplished by comparing routine thermometers used in the laboratory against the SRM thermometers or NBS-calibrated thermometers. Each month 1. Rinse the bulb of the standard thermometer with distilled water before inserting it into the slush bath. disinfectant can be poured into the area and allowed to stand for 20 min before draining. optional). remove the vent covers and clean the gutter with disinfectant. Replace absorbent covering if contaminated. Measure UV light output (if UV light is provided. Take the ice point reading of the standard thermometer. Routine disinfections While the cabinet is running but before you begin work. D. wipe down the cabinet surfaces. Remove and replace discard containers Thermometers Precise temperature measurements can be made only with instruments whose accuracy has been verified. Once each month (or with heavy use. Routine Quality control. Flush with several liters of water) 2. Wait at least 5 min before 180 . The CAP commission of Laboratory Accreditation mandates annual certification 2. A. (If this area is provides with a drain valve. JCAHO makes no specific recommendations. with disinfectant.1. Liquid-in-glass thermometers 1. Each week Clean UV lights (if present) by wiping with 70% ethanol. including the back and sidewalls. If bleach is used as the disinfectant rinse all metal surfaces to prevent corrosion of the metal. more frequently).Cover working surface with absorbent covering (laboratory diaper pr paper toweling) (optional). Use only enough water to allow good contact with thermometer. 2. Gently tap thermometer to ensure that the mercury does not stick to the capillary wall. 4. Prepare an ice bath using shaves ice and small amount of water to form a tightly packed slush. 1. As needed. A. 3. Clean up small spills. 2. Observe that airflow check strips are drawn in onward the workspace of the cabinet 3. Listen for the blower b. C. a. Each year Have the cabinet recertified.

Continue to raise the dial setting 1 U at a time and record the temperature each time the light goes off. If a problem exists empty and clean the tank and add an antimicrobial agent in the water. making allowance for displacement by samples to be immersed. When the light goes off. Read the temperatures indicated on all test thermometers. serial numbers. 3. B. Inspect the bath for build up of mineral deposits. Daily 1. Routine Quality control A. Place the standard thermometer and the test thermometers upright in the rack in the heated liquid bath. then always add or subtract the correction factor from the reading of the standard to measure the actual temperature in the liquid being measured.4. Boiling water baths are used to melt agar. Note: Keep the water bath covered during use to prevent evaporation and conserve heat. record the dial setting and the corresponding temperature. 5. Set the reference dial to a number midway between the highest and the lowest settings (usual range is 0–9) 2. making determination. Fill the bath with demineralized water per manufacturer’s recommendations. indicating heating is complete. they should agree. 1. Place an NBS-calibrated thermometer deep enough into the liquid to obtain accurate measurements. 3. They are ideal for application requiring accurate temperature control. Note: if the manufacturer has included a correction factor with the standard thermometer. Thermometers that fall within the tolerance range (as provided by the manufacturer) are acceptable for routine use. Connect the power cord to a grounded outlet. One application specific to microbiology laboratory is equilibrating melted agar to a working temperature for pouring agar plates. In this way construct a reference table of dial settings versus water bath temperature. 30. 4. Check for leaks 181 . Water Baths Uses in the microbiology laboratory Water bath are used in the microbiology laboratory for incubating at or maintaining a constant temperature. Adjust the temperature to 25°C and read on the standard thermometer. Check the water in the bath for bacterial or algae growth. Log all results. C. 5. 2. Visual inspection is sufficient. Liquid-in-glass thermometers do not respond instantaneously. they needs time to adjust and stabilize. Initial calibration A. Construct a calibration table for reference dial setting versus temperature. 7. and 37°C (or whichever temperatures the SRM thermometer has been calibrated for). 6. Compare the ice point of the standard with the report of calibration that is received with the standard. ranges and tolerances of test thermometers and standard thermometers on the QC forms. Take readings at 25. Wash the bath and scour with cleaning supplied if a problem exists.

01 ml Colorimetric Quantitative loops Loop vol between 0. prepare inocula for antimicrobial tests. Changing pipetter tips during calibration procedure Use the same pipetter tip for all deliveries during the calibration procedure. Baths used infrequently or those filled with mineral oil maybe cleaned less frequently. a. Pipetters/ calibrated Loops Uses in the microbiology laboratory Pipetters are used to dilute sera. Clean the bath with mild. whether the pipetter is used for repetitive dispensing of several aliquots of the same liquid (e. 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. Pipetters are used because of their excellent accuracy and precision and because they expeditiously dispense small volumes repeatedly. Do not use bleaches or cleaners containing abrasives or chlorine on stainless steel baths.001 ml Special precautions and Environmental concerns A. 2. 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. C. and then lower it below the water level in the bath before uncovering the closed end. Drain and clean the bath.. soapy water and a damp cloth. Calibration methods for volume dispensing instruments.g. distilled.01 and 0.002 ml Spectrophotometric Pipetters Vol dispensed must be >0. 3. Record the temperature on the QC sheet B. Refill the bath with demineralized.4. set up quantitative cultures. Quantitative loops are commonly used to setup quantitative cultures and prepare inocula for antimicrobial tests. add ingredients to media and reagents and add exact amounts of reagents or specimen during a test procedure. or deionized water or mineral oil. Quantitative loops are less accurate than pipetters yet are excellent way to setup a semi quantitative culture or dilution. Check the temperature with a thermometer that has been calibrated with a NBS thermometer. Quantitative loops are used when 20% error is acceptable. reagents) or for 182 . lift that end from the bath. Used a siphon tube to drain a) Completely immerse siphon in the water b) Cover one end of the tube. buffers. Monthly (for baths filled with water) 1. Special precautions for pipetter calibration 1.

B.3 Periodic toxicity testing should be undertaken with washed glassware and disposable glassware and plasticware which may be sterilized by ethylene oxide gas. d. the specifications of laboratory glassware and plasticware should be established and followed. 183 . 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. a different tip must be used for each different liquid. Washing may also leave toxic residues on glassware. Temperatures of pipetter to be calibrated. If the pipetter is normally used for repetitive dispensing of several aliquots of the same liquid. Keep temperature stable through out the procedure 2. Complete weighing steps quickly. If the pipetter is normally used for transferring single aliquots of different liquids. Note: during actual pipetting for routine use. This reduces evaporation and limits build up of static electricity b.g. Prepare aqueous test liquid from NCCLS type I or II water. Temperature control b. The extent to which this is undertaken is dependent on how the glassware or plasticware is used. Toxicity testing should be performed annually. Maintain relative humidity at 45 to 75%. Environmental concerns for pipetter calibration 1. prerinse pipetter tip at the beginning. test liquid (water). Glassware made from soft glass can cause problems due to leaching of components from the surface of the glass. c. prerinsing may not be necessary.5°C). a. before dispensing the first aliquot. and other equipment should be identical (± 0. Prerinsing improves uniformity and precision by providing identical contact surfaces for all aliquots. 2. Use water with no visible bubbles.5 and blue above pH 7. b. Glassware should be made from high quality borosilicate glass. serum). Temperature should be as close as possible to the temperature at which the pipetter is used.5 to 7.3 and yellow below pH 6. Many detergents have a high affinity for glassware and some are bacteriostatic. The calibration of pipettes and other volumetric and graduated glassware should be checked and the calibration verified. Use a lid on the weighing vessel to decrease evaporation.transferring single aliquots of different liquids (e. Detergents should be completely removed from glassware. Prerinse by aspirating an aliquot of the liquid into the tip and then dispensing it back into the original container or discarding it. Air bubbles alter measured volume. Reusable glassware and plastic ware should be sterilized and washed using appropriate methods. Laboratory glassware and plastic ware Ideally. Miscellaneous environmental concerns a. c. which prevents impurities from affecting water density. Prerinsing pipetter tips “Prerinsing” is the precoating of the inside of the tip with the liquid being dispensed. d. room air. These precautions obviate the need for and evaporation factor in the calculations.

use of tap water or using water from a malfunctioning still or water deionizer. Keep no more than 6 months' to a years supply on hand. 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. This problem can be overcome by swabbing the surface of the agar plate and then incubating the swabs in a nonselective medium. Common errors include • • Incorrect weighing of dried materials Use of dry materials that have deteriorated as a result of exposure to heat. Failure to pre-melt agar before sterilization can lead to uneven gel strength through an agar medium. The absence of growth indicates that the plates are sterile. Look for changes in flow properties and in colour. • • • • • • • 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. Use older stocks first and do not exceed supplier's expiration date. moisture or oxidation Incorrect measurement of water volumes (eg. Media and reagents Microbiological media and test reagents should be tested for their performance. Use of contaminated containers and glassware Incomplete mixing or solubilization of ingredients during preparation. The temperature should be that normally used for these media. using the markings on beakers as volume guides). then discard it.The sterility of supplies and equipment must be tested. Neither of these may however be present if the medium is designed to inhibit various bacteria. Similarly the sterility of tubes and pipettes can be ascertained with sterile broth medium. The plates should be inspected for turbidity or colony formation. A similar technique can be used with liquid media. This should be done for pre-prepared media and media formulated in the laboratory. Quality control procedures 184 . If an any item in question. 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.

stains.. This includes media.what do we do? . Spectrophotometers. 185 . identification and analysis of microorganisms.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. Control strains should be used to validate procedures and test media and reagents. laboratory manuals. control organisms or slides. Volumetric dispensers should be tested with each run.Can we improve what we do? . Request forms. biohazard cabinets and microscopes should be tested yearly. oxidase and catalase tests should be tested weekly. Control strains can be purchased from local distributors or directly from culture collections in the USA. date of sampling.Are we doing what we should be doing? . safety policies. For example. The number of subcultures permitted for each strain again should be in accordance with the standard method. The strain used should comply with the standard method being followed. biochemical test reagents and equipment such incubators and autoclaves. The intervals between QC tests varies with the critical importance of the material or method and the likelihood that QC will be a problem. Continual passaging of microbial strains on agar media can lead to a loss or change in a critical characteristic. are they easy to use? Are all relevant details provided by the user. The chief technician will examine the following areas.what should we do? . Each lot or batch of materials should undergo quality control. 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. time of sampling and temperature of sample at the time of sampling. Gram stains should tested on a weekly basis. Audit is a means of assessing whether one is achieving one's stated objectives. Europe or Australia. isolation. while agglutinating sera is tested monthly or quarterly. As discussed above equipment and glassware should also undergo quality control tests. Laboratory Audit Audit is an essential part of the quality assurance program of a laboratory. Incubators and anaerobic jars should be tested on a daily basis. record sheets and trained personnel. equipment maintenance etc. It may include policies on the induction and training of new staff. The strains should be well maintained. A quality assurance programme covers all aspects of the service provided. As with quality assurance. there should be a procedure manual. For example. There are five key questions in the audit process: . The ATCC has one such collection. defined QC criteria.Quality control should be performed on all materials and equipment which are critical to the detection. staff development.

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. Every single accident in the laboratory should be recorded and improvements made if necessary. 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. The equipment and apparatus should be checked to make sure that they area functioning as required. The use of dangerous substances should be audited. To guide them in the types of tests to carry out on a sample. Safety policies and procedures.Sample. Functioning of Equipment. 186 . Every laboratory should have a comprehensive safety policy. Sufficient use of staff.

sell or advertise any article in such a manner that it is likely to be mistaken for such food. microbiological guidelines and injurious extraneous material guidelines. Since guidelines are not part of the Regulations. Standards and guidelines can be applied only when the appropriate method of analysis (or equivalent) is used. misleading or deceptive or is likely to create an erroneous impression regarding its character. (b) is unfit for human consumption. Frequently. When there is no code identification. 4. prepare.Microbial Standards of Foods Quoting selected sections of the American FDA act may help in understanding and appreciating this section. The Act defines "unsanitary conditions" as "such conditions or circumstances as might contaminate a food. however. preserved. at the same establishment and representing no more than one 187 . prepared. 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). rotten. sell or advertise any food in a manner that is false. package. package. No person shall sell an article of food that (a) has in or upon it any poisonous or harmful substance. 3. storing. putrid. as additional data become available. process. filth or render the same injurious to health". processing. No person shall label. (d) is adulterated. a lot may be considered as (a) that quantity of product produced under essentially the same conditions.g. merit or safety. packaged or stored under unsanitary conditions. etc). or (e) was manufactured. Guidelines A given guideline may embody the same limiting criteria that would be employed in a standard. preserve. package or store for sale any f ood under unsanitary conditions. 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. glass in jam. quantity. (c) consists in whole or in part of any filthy. splinters. composition. Where a standard has been prescribed for a food. value. transporting and handling of food that could lead to an injury (e. if necessary. no person shall label. disgusting. unless the article complies with the prescribed standard. treat. 2. No person shall manufacture. decomposed or diseased animal or vegetable substance. they can be readily modified. drug or cosmetic with dirt. Sections of the Food and Drugs Act 1. There are two distinct groups of guidelines related to health and safety. The latter includes foreign matter associated with objectionable conditions or practices in manufacturing.

Hazards can be identified from relevant data sources. “M” separates sample units of marginally acceptable quality from those of defective quality. the lot becomes unacceptable. c: The maximum allowable number of marginally acceptable sample units. imminent spoilage or gross insanitation. government agencies. usually per g or mL. and studies on analogous microorganisms and situations. moisture content or water activity (aw). investigations of the characteristics of microorganisms. that indicate a (potential) health or injury hazard. Predictive microbiology can be a useful tool. Hazard Identification The purpose of hazard identification is to identify the microorganisms or the microbial toxins of concern with food. are influenced by processing and packaging. including the temperature of storage. from databases such as those in the food industry. 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. usually per g or mL. When this number is exceeded. or death of microorganisms. growth. Hazard identification will predominately be a qualitative process. the interaction between microorganisms and their environment through the food chain from primary production up to and including consumption. Several factors 188 . and relevant international organizations and through solicitation of opinions of experts. hazard characterization and risk characterization. The “m” values listed in the following tables are based on levels achievable under GMP. This is the sample. It might be based on the potential extent of food contamination by a particular agent or its toxins. and the likelihood of their occurrence in foods at the time of consumption. and on dietary information. RISK ASSESSMENT Risk assessment is composed of four elements: hazard identification. Information on hazards can be obtained from scientific literature. the storage environment. 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. Exposure assessment estimates the level of microbiological pathogens or microbiological toxins. m: The numerical value of “m” represents acceptable concentrations of microorganisms or amounts of extraneous material. the presence of antimicrobial substances. or (b) the quantity of the same variety of product from one and the same manufacturer available for sampling at a fixed location. exposure assessment. including pathogens in foods. epidemiological studies and surveillance. A value determined for any one sample unit of a sample that is greater than that of “M” renders the pertaining lot unacceptable. Exposure Assessment Exposure assessment includes an assessment of the extent of actual or anticipated human exposure. M: the numerical value of “M” represents unacceptable concentrations of microorganisms or amounts of extraneous material.day's production. pH. laboratory animal studies. The presence. survival. and competing microflora. “c” is the acceptance number of a plan. Relevant information includes data in areas such as: clinical studies.

In relation to the microorganism the following are important: microorganisms are capable of replicating. immune status and previous exposure history. e. access to and use of medical care. Note When products characterized with a Health 2 risk are associated with illness in an outbreak. In relation to the host the following may be important: genetic factors. microorganisms can be spread through secondary and tertiary transmission.g.need to be considered in hazard characterization. A health hazard has not been identified. Follow-up action should ensure that the cause has been determined and appropriate corrective action has been taken to correct the problem. They are related to both the microorganism. the hazard identified must be present in sufficient numbers to present a risk to human health. 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 Risk Characterization represent the integration of the hazard identification. Sanitation The problem identified is an indication of a breakdown in hygienic practice. progressive steps should be taken to bring about compliance. population characteristics such as population immunity. in 189 . Risk characterization depends on available data and expert judgments. even 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. A review of the manufacturer’s GMP/HACCP is appropriate where M or c/m values are exceeded. Health 2 The health hazard identified represents a situation that could cause temporary. hazard characterization. the virulence and infectivity of microorganisms can change depending on their interaction with the host and the environment. the onset of clinical symptoms can be substantially delayed following exposure. 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. low doses of some microorganisms can cause a severe effects. increased susceptibility due to breakdowns of physiological barriers. consideration should be given to modification of the risk characterization to Health 1 with appropriate action to the consumer level. If c/m values are exceeded. Appropriate action should be taken to limit further distribution if the M value is exceeded. Appropriate action should be taken to limit further distribution of the product. action at the consumer level if the product has been distributed). status. genetic material can be transferred between microorganisms leading to transfer of characteristics such as antibiotic resistance and virulence factors. In some situations. the attributes of a food may alter the microbial pathogenicity. Repeated violations should be investigated. adverse health consequences. concurrent infections. and persistence of the organism in the population. and the human host. initially review GMP/HACCP. The probability of serious adverse consequences is considered remote. high fat content of a food vehicle. 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.g. microorganisms can persist in certain individuals leading to continued excretion and continued risk of spread of infection. not life-threatening.

coli S. cancer patients. 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. TABLE 1a. the elderly or immunocompromised individuals (e.8/100mL 10 0 0 104 10/100mL 10/100mL - Mineral or Spring Water Water in ACC Coliforms Sanitation Sealed Sanitation Containers Pre. 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.g. transplant recipients. consideration should be given to modification of the risk characterization to Health 1 with appropriate action to the consumer level. etc. AIDS patients. 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. Foods for which there is a Microbiological Standard. Therefore.).packaged Coliforms Sanitation Ice Egg Products Salmonella Health 2 190 . where products are contaminated with Health 2 risks but are directed to sensitive populations such as children less than five years of age.sensitive populations.

Smoked Fish in Hermetically Sealed Commercial Sterility.TABLE 2.8 2 103 1 <1. 9% salt Health 1 ContainersB (NaCl). Foods for which Microbiological Guidelines have been established. Freezing. Health 1 Low Acid Foods in Hermetically Sealed Commercial Sterility. coli Health 2E Powdered Infant Salmonella Health 1 Formula (if M S. aureus Health 2 exceeded Health 1. 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.8 101 5 2 3x104 106 5 5 10 20 10 10 10 2 <1.8 00 1 101 1 102 1 102 102 104 101 0 102 104 103 191 . Refrigeration at < Health 1 ContainersA 4/C. if c exceeded Bacillus cereus Health 2 Clostridium Health 2 Health 2) perfringens 5 2 2X103 104 5 2 <1. Foods for which there are Standards other than Microbiological FOOD CATEGORY NATURE OF CONCERN Dairy products made from pasteurized milk Phosphatase Test. STANDARD TABLE 3a.

Raw Campylobacter Fermented coli or C. 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. coli Health 2 5 1 101 Fermented S. aureus Health 2 5 2 102 Salmonella Health 2 5 0 0 Heat Treated E. coli fermented S. Foods for which Microbiological Guidelines have been established.5x102 Sausage NonE.TABLE 3b. coli Health 2 5 1 <1. aureus Health 2 5 1 5x101 Sausage Raw E. 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. 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.Yersinia fermented Sausage enterocolitica A E. Foods for which Microbiological Guidelines have been established. coli Health 2 5 1 102 Fermented 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. aureus Health 2 5 1 2.8 S. coli Health 2C 5 2 <1. coliB Products 192 . jejuniA Sausage and Non.8 Yeast and Sanitation 5 2 5x102 mould S. coli O157 Deboned ACC Poultry E.

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. Health 2 coliA Y.g. Foods for which Microbiological Guidelines have been established. coli Health 2 S.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 - 193 .Products (Precooked) S. ACC Sanitation Sauce.C. Soup) Heat ColiformsB Sanitation and Serve Yeast and Moulds Sanitation E. jejuni or C. aureus Health 2 5 1 102 104 Salmonella Health 2 C. 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. aureus Health 2 C. enterocoliticaA Health 2 Dry Mixes (Gravy. aureus Health 2 Salmonella Health 2 Yersinia Health 2 enterocolitica Spices (Readyto. 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. coli Health 2 S. coli Health 2 S. Alfalfa and E. perfringens Health 2 eat only) B. cereus Health 2 E.

S. perfrigens Health 2 g/day) C b) Powdered Protein. ACC Sanitation Meal Replacements. coli Health 2 and Dietary Salmonella Health 1 Supplements S.8 0 101 102 102 105 103 104 105 105 104 101 102 104 103 Health Foods ACC Sanitation a) Raw Organ Derived E. aureus Health 2 capsules or powders. cereus C. 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. 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. cereus Health 2 C.TABLE 3e. 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. E. Health 2 consumed at <10 B. coli Health 2 Products and Herbal Salmonella Health 1 Products (in tablets. if for infant postprocessing formula then Health 1 194 . 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. Foods for which Microbiological Guidelines have been established. Foods for which Microbiological Guidelines have been established. perfringens Health 2 TABLE 3f. Foods for which Microbiological Guidelines have been established. aureus Health 2 B. coli O157:H7 E.

Mushroom water activityC > 0. botulinum and the 195 .92 Vegetable or water activity: Products packed OR Health 1 Mushroom MFLP-66.coli trimming or O157:H7 carcasses found Generic E. coli Health 2 positive for E.85E but <0. coli Sanitation 5 0 0 >100 Raw ground beef Positive for Health 2 derived from E. coli >100 cfu/g or level not determined Raw ground beef Negative for E. coli O157:H7 O157:H7 Generic E.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.0 or not controlled OR iv) If product has not Health 1 received a thermal processF sufficient to kill spores of proteolytic C.92 and pH >5. in OilA ii) If pHD is the sole Products in Oil commercial sterility barrier and <4.coli B 1 if generic E. and Health 1 Prepared MFHPB-03.6. 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.coli O157:H7 Generic E.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. coli Becomes Health 5 0 0 100 1 if generic E. coli Becomes Health 5 0 0 100 positive for E.

aureus count is < 10 cfu/g can be used for further processing. There is a Health 2 concern if the level of S.aureus level is >10 cfu/g but <104 196 .botulinumG but is not stored at refrigeration temperature. botulinum has been addressed. botulinum (eg 10 minutes at 90/C or equivalent) but is not labelled and stored refrigerated. 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.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. Containers with an S.g. low brine. aureus. If the S.aureus in a container or lots is >104 cfu/g and implicated product should be destroyed. cloudy brine etc. 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. 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. aureus Examine for Health 2 Enterotoxin GUIDANCE OF There is a sanitation concern for product which is visually defective e. 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. musty. off smell. For example every container has received a pasteurization process sufficient to inactivate the nonproteolytic spores of C. 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.

There is a Health 2 concern if S. 197 . The implicated container or lot is not acceptable for further processing and should be destroyed.aureus enterotoxin is detected in a container or a lot.examine for enterotoxin.

When possible.) 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. The details of the protocol do not need to be reproduced in the text but an appropriate reference should be cited.e. 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 should be sequentially numbered. 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. Isolation of MudJ insertion mutants. Introduction: The Introduction should tell the reader (i) what is the experimental question the paper will address. Not all results deserve a separate table or figure.” If necessary to interpret the figure. For example. 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. not a numerical series of steps as in an experimental protocol). (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. Tables and Figures: All tables and figures should be put into a contextual framework in the corresponding text. Write the opening sentence in bold font for emphasis. Your report should focus on what worked.g. Figures should be sequentially numbered. 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. “Figure 1. Bacterial strains and plasmids used in this study. but what you actually did). etc). the results should be summarized in accompanying text (Note that when referring to a particular table or figure. “Table 1. (ii) what is the background biology that makes this an interesting question (e.. what are the properties of the organisms used. Tables and figures should typically summarize results. Each figure should have a title (shown below the table) that describes the point of the table. specific descriptions about what a result 198 . For example.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.” If necessary to interpret the table. Tables and figures should present information in a format that is easily evaluated by the reader. As a rule of thumb. Each table should have a title (shown above the table) that describes the point of the table. the results should provide some way of evaluating the reproducibility or statistical significance of any numbers presented. not simply present large amounts of raw data. It is not appropriate to indicate volumes of solutions added – instead indicate the relevant information about the experiment such as final concentrations used. etc. 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. they should be capitalized: Table 1. not things that didn’t work (unless they didn’t work for reasons that are interesting and provide biological insights). Any changes from the protocol provided in handouts should be described. However. Figure 6. specific descriptions about what a result represents or how the results were obtained can be described in a legend below the table.) Any results that include multiple data points that are critical for the reader to evaluate the experiment should be shown in tables or figures.

Genetic Analysis of Pathogenic Bacteria. G. Nomenclature. a space should be left between numbers and the accompanying unit.g. The complete genome sequence of the marine Roseophage SIO1 shares homology with nonmarine phages. NJ. Results described in your paper should be described in past tense(you’ve done these experiments. do not use an abbreviation unless a term is used at least three times in the manuscript. 2000. L..) Methods in Molecular Biotechnology: Protocols in Bioremediation. not mls). Wolven. min. Segall. Abbreviations. 1997. Define all other abbreviations the first time they are used. Totowa. Breitbart. In D. The enumeration of hydrocarbon-degrading bacteria. Discussion: Do not simply restate the results — explain your interpretations of the results. M. Oceanography 45: 408-418. However. Use correct bacterial nomenclature. Use standard abbreviations (hr. 99109. what experiments and what are the predicted results)? References: Give complete references to the sources for any fact. then subsequently use the abbreviation [e. using the format for different sources shown below: Books: Maloy. and future tense. Limnol. Sheehan (ed. abbreviations should not be written in the plural form (e. S. idea. V. Even when credit is given. Cold Spring Harbor Laboratory Press. List the references alphabetically at the end of your report. the tables and figures may be integrated into the paper if you are using a page layout program. Azam. Humana Press. sec. Taylor. but your results are not yet accepted “facts”). or opinion not your own which was cited in the report. F. and F. Tables and figures should not be directly copied from another source without credit.. NY. Steward. pp. In general. and R. Results from published papers should be described in the present tense (based upon the assumption that published results are“facts”). Stewart. the table or figure should be redrawn. Ampicillin resistant (Amp)]. 1996. V.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. Format and proofreading: Certain general rules are commonly followed in scientific writing. and B.. Published papers: Rohwer. With two exceptions (the degree symbol and percent symbol). etc) instead of writing complete words. Past. F. As a general rule. present. How did your results compare with the expected results? Are there other experiments that could be done to confirm your results (if so. Some common abbreviations that do not require definition are shown on the attached table. Book chapters: Rice. if they are integrated into the paper make sure that there is not a page break in the middle of a table or figure. 1 ml or 5 ml.g. A. Hemmingsen. Only experiments that you plan to do in the future should be described in the future tense.Seguritan. 199 .

Always spellcheck your paper and carefully proofread your paper before submission. It is common to use a pronoun such as “it” or “they” to refer to a concept from the previous sentence. not with anthropomorphic or possessive terms (e. Most text should be written in the third person to avoid sounding like an autobiographical account penned by a narcissistic author.” Proofreading...g. they should be arranged so that it is explicit which word they modify.. It is better to error on the side of redundancy by repeating the concept in subsequent sentences. It is OK to use first person in scientific writing. However.Third vs first person. etc) should be described in third person. it is better to say“It is possible to .”. Avoid double parentheses.. Parentheses. say “the chromosomal att site”). Empty phrases. Likewise. proteins. “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). Don’t make the reader guess what you mean. If several expressions modify the same word. read your paper to yourself as if you were reading it out loud to ensure that the wording and sentence construction is not clumsy.” (shorten to simply “To . but it should be used sparingly reserve the use of first person for things that you want to emphasize that “you” uniquely did (i. In short.. However. In addition. the title of a table of results does not benefit from the preface “Results of . Writing that uses the impersonal pronoun “one” often seems noncommittal and dry.. For example.e. instead of saying “its att site”.. Specify...”. inanimate objects (like genes. For example.” (delete).. 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). don’t use more words than you need to make your point. “In order to . In addition to checking for errors and typos..”).” than to say “One could . Avoid using phrases that do not contribute to understanding. This is OK as long as there is only one concept that “it” or “they” means. than to take the chance of confusing the reader. 200 .. the following phrases could be shortened (or completely deleted) without altering the meaning of a sentence: “the fact that . not things that many others have done as well).

8. Bacteriol. Chapter 3. Arlington. 13. 5. 12. C. 15th ed.D. Association of Official Analytical Chemists. Standard Methods for the Examination of Water and Waste Water.C. VA. AOAC. Washington. 1992. 2. Arlington. Laboratory Procedures for the Examination of Seawater and Shellfish. AOAC. The Howard Mold Count Method as Applied to Tomato Products. Methods for recovering injured "classical" enteric pathogenic bacteria (Salmonella. Boca Raton. Baird-Parker. 1997.S. American Public Health Association.M. Andrews.) Injured Index and Pathogenic Bacteria.C. Ray (ed. Eighth Edition. Clesceri. (eds. 25:12-19. American Public Health Association. F. Third edition. Twentieth Edition. Downes and K.C. 16th ed. 9. Arlington. Vanderzant and D. Research Division. A. A. Compendium of Methods for the Microbiological Examination of Foods Fourth edition. American Public Health Association Inc. VA. 1989. AOAC. 1995. Md. Association of Official Analytical Chemists (AOAC). Gaithersburg. 10. Washington. 11. Appl. NJ. The craft of scientific writing. 1992. American Public Health Association. Sixteenth edition.E.. CRC Press. Eaton. Alley. 15. 1999. APHA. 2001. 1962. 3 rd edition. pp. Shigella. Vol. AOAC INTERNATIONAL. 4. American Public Health Association (APHA).C. Greenberg and A. 14. Chapter 6. FDA Bacteriological Analytical Manual. 1996. 7. Illinois. Ito (editors). J.F. American Public Health Association (APHA).. W.References and selected readings 1. 1990. Splittstoesser (eds. Atlas. 201 . [and accompanying web site: http://filebox. pp:32-34. Official Methods of Analysis. Second edition. 1957. Association of Official Analytical Chemists (AOAC) International. In: B. 2001. Official Methods of Analysis. Parks (editor). VA. American Public Health Association (APHA). Maywood. Washington. 6. Bacteriological Analytical Manual (Online) Chapter 14.edu/eng/mech/writing/] 3. 1995. DC.C. L. USDA. 5th ed. and enteropathogenic Escherichia coli) from foods.P. 16th. 1985. CRC Press Inc. Handbook of Microbiological Media. FL. D.American Public Health Association. Washington.C. Prentice Hall. D. Compendium of Methods for the Microbiological Examination of Foods. American Can Company.vt.). An improved diagnostic and selective medium for isolating coagulase positive staphylococci. Bacillus cereus. I.H. M. D. Official Methods of Analysis of AOAC International. American Can Company. Washington. 55-113. 1998. Chapt. D. Standard Methods for the Examination of Dairy Products. Center for Food Safety and Applied Nutrition.). American Public Health Association. Inc. 17. Lenore. R. AOAC International.AOAC INTERNATIONAL.

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