LABORATORY MANUAL OF FOOD MICROBIOLOGY FOR ETHIOPIAN HEALTH AND NUTRITION RESEARCH INSTITUTE (FOOD MICROBIOLOGY LABORATORY)

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 2 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 3 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