LAB. MANUAL 14 MANUAL OF METHODS T OF ANALYSIS OF FOODS F MICROBIOLOGICAL TESTING A R D FOOD SAFETY AND STANDARDS AUTHORITY OF INDIA MINISTRY OF HEALTH AND FAMILY WELFARE GOVERNMENT OF INDIA NEW DELHI 2012 MICROBIOLOGY OF FOODS 2012 MANUAL ON METHOD OF MICROBIOLOGICAL TESTING TABLE OF CONTENTS S.No. Page Title No. Chapter – 1: Microbiological Methods T 1. Aerobic Mesophilic Plate count 2. Aciduric Flat Sour Spore-formers 3. Bacillus cereus 4. Detection and Determination of Anaerobic Mes ophilic Spore F Formers in Foods (Clostridium perfringens ) 5. Detection and Determination of Coliforms, Faecal coliforms and E.coli in Foods and Beverages. 6. Direct Microscopic Count for Sauces, Tomato Puree an d Pastes A 7. Fermentation Test (Incubation test). 8. Rope Producing Spores in Foods 9. Detection and Confirmation of Salmonella species in F oods 10. Detection and Confirmation of Shigella species in Food s 11. Detection, DetermRination and Confirmation of Staphylo coccus aureus in Foods 12. Detection and Confirmation of Sulfide Spoilage Sp oreformers in Processed Foods 13. Detection and Determination of Thermophilic Fla t Sour Spore foDrmers in Foods 14. Detection and Confirmation of Pathogenic Vibrios in F oods Estimation of Yeasts and Moulds in Foods Detectioin and Confirmation of Listeria monocytogenes in Foods 15. Bacteriological Examination of water for Coliforms Bacteriological Examination of water for Detection, Determination and Confirmation of Escherichia coli Bacteriological Examination of water for Salmonella and Shigella Bacteriological Examination of water for Clostridium perfringens Bacteriological Examination of water for Bacillus cereus Bacteriological Examination of water for Pseudomonas aeruginosa 16. Chapter – 2: Culture Media’s 17. Chapter – 3: Equipment, Materials and Glassware’s 18. Chapter – 4: Biochemical Tests MICROBIOLOGY OF FOODS 2012 Microbiological Methods for Analysis of Foods, Water, Beverages and Adjuncts Chapter 1 T 1. Aerobic Mesophilic Plate count Indicates microbial counts for quality assessment of foods 1.2 Equipment: F Refer to Chapter 3 (Equipment, Materials & Glassware). 1.3 Medium: Plate count agar; o A Peptone water 0.1%, o (Chapter 2 for composition of medium) o R 1.4 Procedure: 1.4.1 Preparation of food homogenate Make a 1:10 dilution of the well mixed sample, by aseptically transferring sample to the desired volume of diluent. D Measure non-viscous liquid samples (i.e., viscosity not greater than milk) volumetrically and mix thoroughly with the appropriate volume of diluent (11 ml into 99 ml, or 10 ml into 90 ml or 50ml into 450 ml). Weigh viscous liquid sample and mix thoroughly with the appropriate volume of diluent (11 + 0.1g into 99ml; 10+ 0.1g into 90ml or 50+0.1g into 450ml). Weigh 50+0.1g of solid or semi-solid sample into a sterile blender jar or into a stomacher bag. Add 450 ml of diluent. Blend for 2 minutes at low speed (approximately 8000 rpm) or mix in the stomacher for 30-60 seconds. 1 MICROBIOLOGY OF FOODS 2012 Powdered samples may be weighed and directly mixed with the diluent. Shake vigorously (50 times through 30 cm arc). In most of the food samples particulate matter floats in the dilution water. In such cases allow the particles to settle for two to three minutes and then draw the diluent from that portion of dilution where food particles are T minimum and proceed. 1.4.2 Dilution: F If the count is expected to be more than 2.5 x103 per ml or g, prepare decimal dilutions as follows. Shake each dilution 25 times in 30 cm arc. For A each dilution use fresh sterile pipette. Alternately use auto pipette. Pipette 1 ml of food homogenate into a tube containing 9 ml of the diluent. From the first dilution transfer 1ml to second dilution tube containing 9ml of the R diluent. Repeat using a third, fourth or more tubes until the desired dilution is obtained. D 1.4.3 Pour plating: Label all petriplates with the sample number, dilution, date and any other desired information. Pipette 1ml of the food homogenate and of such dilutions which have been selected for plating into a petri dish in duplicate. Pour into each petri dish 10 to 12ml of the molten PCA (cooled to 42-45oC) within 15 min from the time of preparation of original dilution. Mix the media and dilutions by swirling gently clockwise, anti-clockwise, to and fro thrice and taking care that the contents do not touch the lid. Allow to set. 2 MICROBIOLOGY OF FOODS 2012 1.5 Incubation: Incubate the prepared dishes, inverted at 35oC for 48+2 hours. (Or the desired temperature as per food regulation e.g. in case of packaged drinking water). T 1.6 Counting Colonies: Following incubation count all colonies on dishes containing 30-300 colonies and record the results per dilution counteFd. 1.7 Calculation A In dishes which contain 30-300 colonies count the actual number in both plates of a dilution and as per the formula given below: ∑ C N= R (N1+0.1N2) D ∑ C is the sum of colonies counted on all the dishes retained N1 is the no. of dishes retained in the first dilution D N2 is the no of dishes retained in the second dilution D is the dilution factor corresponding to first dilution E.g. At the first dilution retained (10-2):165 & 218 colonies At the second dilution retained (10-3) 15 & 24 N= 165+218+15+24 = 422 = 19182 [2 + (0.1x2) x 10x-2] 0.022 Rounding the result to first two digits gives 19000 CFU. 3 MICROBIOLOGY OF FOODS 2012 1.8 Expression of Result Aerobic (Mesophilic) Plate Count = 19000 CFU/g or 1.9x104 CFU/g or If plates from all dilutions have no colonies and inhibitory substances have not been detected, the result is expressed as less than 1 x 101 CFU per g or T ml. If plates from the lowest dilutions contain less than 30 colonies, record the actual number and calculate as above but express Fresults as CFU per g or ml. Note:- This method, as all other methods, has some limitations. Microbial A cells often occur as clumps, clusters, chains or pairs in foods, and may not be well distributed irrespective of the mixing and dilution of the sample. Moreover the single agar medium used, the conditions of incubation, R aeration etc., are not conducive to the growth of various populations of bacteria that may be present in a food sample. For statistDical reasons alone, in 95% of cases the confidence limits of this test vary from ± 12% to ± 37%. In practice even greater variation may be found specially among results obtained by different microbiologists. (Corvell and Morsettle, J. Sci. Fd. Agric., 1969, vol. 20 p 573) References: 1. Official Methods of Analysis of AOAC International (1995). 16th Edition. Edited by Patricia Cuniff. Published by AOAC International. Virginia. USA. Test 17.2.01 p.3-4. 4 MICROBIOLOGY OF FOODS 2012 2. Compendium of Methods for the Microbiological Examination of Foods. (1992) Carl Vanderzant and Don F. Splittstoesser Eds. Washington D.C. p. 75-87 3. Bacteriological Analytical Manual (1992) 6th Edn. Arlington, V.A. Association of Official Analytical Chemists for FDAT, Washington, D.C. p. 17-21. 4. Microbiology- General guidance for the enumeration of F Microorganisms-Colony count technique at 35oC (first revision) IS5402-2002, ISO4833:1991. Bureau of Indian Standards, Manak Bhavan, 9 Bhadur Shah Zafar Marg, New Delhi110002. A 2. To Determine and Confirm Aciduric Flat Sour Spore Formers in Foods. R The organism of this group is Bacillus coagulans. It is responsible for spoilage of canned products. 2.1Equipment: D Refer to Chapter 3 (Equipment, Material and Glassware) 2.2 Culture Media: Dextrose tryptone agar (with bromocresol purple) 2.3 Procedure: Weighed samples or dilutions of the sample are taken in a test tube and heat shocked at 88oC for 5 min. The sample tubes are immediately cooled and one ml of the heat shocked sample or decimal volume is transferred to petri plates. 18 to 20 ml of melted bromocresol purple agar is added. After mixing the plates are incubated at 55oC for 48h. 5 MICROBIOLOGY OF FOODS 2012 Surface colonies on dextrose tryptone agar will appear slightly moist, usually slightly convex and pale yellow. Subsurface colonies on this medium are compact with fluffy edges. Colonies are surrounded by a yellow zone. Suspected colonies are counted and expressed as number per g of the sample. T 2.4 Calculation Average plate count x dilution factor = XF 2.5 Expression of Result Aciduric flat sour spore formers = X/g A References: 1. Official Methods of Analysis of AOAC International (1995). 16th R Edition. Edited by Patricia Cuniff. Published by AOAC International. Virginia. USA. Test. 17.6.03., p.44. 2. Compendium of Methods for the Microbiological Examination of FooDds. (1992) Carl Vanderzant and Don F. Splittstoesser Eds. Washington.D.C.p.291-295. 3. Detection and Determination of Bacillus cereus in Foods, and Beverages. 3.1 Equipment: Refer to Chapter 3 3.2 Culture media and reagents Mannitol-egg yolk-polymyxin (MYP) agar o Trypticase-soy-polymyxin broth o Phenol red dextrose broth o 6 MICROBIOLOGY OF FOODS 2012 Nitrate broth o Nutrient agar slants and plates o Nutrient agar with L-tyrosine o Nutrient broth with lysozyme o Modified Voges- Proskauer medium (VP) o T Motility medium o Nitrate test reagents o Voges- Proskauer test reagents F o 3.3 Procedure A 3.4 Preparation of food homogenate Prepare as directed in 1.4.1 R 3.4.1 Dilution Prepare decimal dilutions by pouring 1ml in 9 ml of dilution water. 3.4.A Most Probable Number Method D This procedure is suitable for the examination of foods which are expected to contain fewer than 1000 B.cereus per g. i. Inoculate each of three tubes of trypticase-soy-polymyxin broth with 1 ml food homogenate and its dilutions. ii. Incubate at 30oC for 48 hours. iii. Examine for dense growth typical of B.cereus iv. Vortex-mix and using a 3mm loop transfer one loopful from each growth positive tube to dried MYP medium plates. Streak to obtain isolated colonies. v. Incubate at 30oC for 48 hours 7 MICROBIOLOGY OF FOODS 2012 vi. Pick one or more eosin pink (mannitol fermentation positive) colonies surrounded by precipitate zone (due to lecithinase activity) from each plate and transfer to nutrient agar slants for confirmation tests. vii. The confirmed B.cereus count is determined using the MPN Table 4 T of Test No. 10 for coliform count. On the basis of the number of tubes at each dilution in which B.cereus was detected and reported as MPN of B.cereus per gram. F 3.4.B Plate Count Techniques A This procedure is suitable for the examination of foods expected to contain more than 1000 B. cereus per gram. R Inoculate duplicate MYP agar plates with the homogenate and each dilution of homogenate by spreading 0.1 ml evenly on to each plate in duplicate with sterile bent glass streaking rods (hockey sticks). Incubate plates 24 hDours at 30oC. 3.4.B.I Counting Colonies The number of eosin pink colonies surrounded by lecithinase zone are counted. If reactions are not clear, incubate plates for added 24 hours before counting. Plates must ideally have 15-150 colonies. Five or more colonies of presumptive B.cereus are picked from plates and transferred to nutrient agar slants for confirmation (3.5). 8