MICROBIOLOGICAL METHODS. Chapter 17, p. 32 ‘AOAC OFFICIAL METHODS OF ANALYSIS (2005) ‘Table 996.020. Repeatability standard deviations of the ‘modified Petrifilm HSC plate and the original Petrifim HSCC plate methods Modified Petnfim —Originl Petiim Food HSC plates, HSC plato 5, autor 0.1996 0.1912 Buttermik 0.1288 oat Cheese 0.1073 0.1000 Chocolate rik 0.1264 0.1000 Cream 0.103 0.1489 Frozen yogurt 0.1148 0.1338 lea cream 0.1408 0.1276 ‘Sour cream 0.1516 0.1443 ‘Sweetened condensed mik 0.1170 0.1231 Whole ei 0.0883 0.1050 area can be considered to contain ca 60 em’. To isolate colonies for further identification, lift top film and pick colony from gel. Reference: J. AOAC Int. 80, 505(1997), 173.04 AOAC Official Method 991.14 Coliform and Escherichia coll Counts in Foods Dry Rehydratable Film {Potritim™ E. collColtform Count Plate™ and, Potriflm™ Coliform Count Plate") Methods First Action 1994 Final Action 1994 ‘See Table 991.14 for the results of the interlaboratory study supporting acceptance ofthe method. A. Principle ‘See 989.104 (see 173.03). 1B. Apparatus and Reagent ‘See 989.10B(b)-(P) (see 17.3.03). E. coli count plaies—Plates axe silat to coliform count plates, 989.10B(b) (sce 173.03) with adtion of S-bromo-4-chlor-3indoly-B.0- sluewronide, an indica of gucurnidase activity. This allows colifrmas and E. col to be read on the same plate. Peiflm E. col! Count Pats (Microbiology Products, 3M Center, Blig 75-SW-485, Se Paul, MN'SS144, USA) meet these specifications. CC. Proparation of Test Suspension ‘See 966.23B (see 17.2.01) and 986.33C (see 17.3.02). D. Analysis (a) Coliform count—Place dry-film . coli count plate, B, of coliform count plate, 989.10B(}) (see 17.3.03), on flat surface, Li {op film and inoculate | mL test suspension onto center of film base, Carefully place top film down onto inoculum. Distribute test suspension over prescribed growth area with downward pressure on center of plastic spreader device (flat side down). Leave plate ‘undisturbed 1 min to permit gelto solidify. Incubate plates 24:2hat 35° 1°C, {© 2008 AOAG INTERNATIONAL In incubator, place plates in horizontal position, clear side up in stacks not exceeding 20 units. Count plates promptly after incubation period, After incubation is complete, plates may be stored frozen (<15°C) up to 7 days. This should be avoided as routine practice. Use standard colony counter for counting purposes. Magnifierilluminator may also be used to facilitate counting Coliforms appear as red colonies that have one or more gas bubbles associated (within one colony diameter) wit them, Count al colonies in countable range (15-150 colonies). Red colonies without gas ‘bubbles are not counted as coliform organisms. (b) E. coli count —Use E. coli count plate and proceed as in (a), Incubate an additional 24+ 2h (48 24h otal. E, col colonies appear asblue colonies associated with gas bubbles; other coliforms appear as red colonies with gas Reference: JAOAC 74, 635(199)). Revised: March 2002 173.040 AOAC Official Method 2000.15 Rapid Enumeration of Coliforms in Foods First Action 2000 Final Action 2005 (Applicable to determination of confirmed coliforms at 14 and 24 in foods. Not applicable to hash brown potatoes.) ‘See Tables 2000.15 A and B forthe results ofthe interlaboratory study supporting acceptance of the method A. Principle ‘Method uses bacterial culture plates of dry medium and cold-water-soluble gel. Undiluted or diluted test suspensions are ‘Added to plates at a volume of 1.0 mL. per plate. Pressure, when applied to plastic spreader placed on overlay film, spreads the suspension evenly over a 20 cm? growth area. Gelling agent is allowed to solidify, and plates are incubated and then counted. A serological pipet or automatic pipet can be used for suspension addition for bacterial count analysis, B. Apparatus and Reagents (a) Petrifiim Rapid Coliform Count (RCC) plates —Plates (evailable from 3M Microbiology Products, St. Paul, MN 55144, USA) contain modified violet red bile (VRB) nutrients, cold: ‘water-soluble gelling agent, tetrazolium indicator dye, and pH indicator. (b) Plastic spreader—Provided with Petrifilm plates, has & ‘evessed side and a smooth flat side, designed to spread suspension evenly over plate growth ara. (©) Pipets—1.0 and 10.0 mL serological pipets with 0.1 mL ‘graduations. Pipets must accurately deliver required volume. Do not use<10%of their total volume. Forexample,to deliver ! ml, do not use pipet >10 mL; to deliver 0.1 mL, do not use pipet >1 mL, (Calibrated 3M™ Electronic Pippetor, or equivalent, may beusedt0 deliver 10 ml.) (@) Colony counter—Quedec Dark-Field Colony Counter (VWR International; www.vwrsp.com), oF equivalent. (©) Sterile sodium hydroxide solution.—IM. Dissolve 40 [NaOH in IL water. Autoclave 15 min at 121°C.