Petrifilm™  AOAC official methods 990.12 - 989.10 - 986.33 for  aerobic mesophilic bacteria in foods

Methods of the AOAC International, as described in Revision 3 of the 18th Edition of the  Official Methods of Analysis of  AOAC International (Horwitz and Latimer, 2010), applied to milk (986.33), dairy products (989.10) and foods in general (990.12).

 Petrifilm™ (3M Company) is a modified version of the Colony Forming Units (CFU) plate count method, consisting of two sterile, dry rehydratable films impregnated with culture medium and cold-water-soluble gelling agents. Inoculation is carried out on the surface of the plating surface (bottom film), which, after inoculation is covered with the top film. The inoculum is spread evenly using a plastic spreader and applying gentle manual pressure. After solidification of the gel, the plates are incubated for the development of colonies. The medium is the Standard Plate Count Agar (PCA) but supplemented with 2,3,5-triphenyltetrazolim chloride, an indicator which, when reduced, gives the colonies a deep red color (to facilitate counting).

1 - Material required for analysis

Preparation of the sample and serial dilutions

•  Diluent: 0.1% Peptone Water (PW) or Butterfield’s Phosphate Buffer

•  Dilution tubes containing 9 ml 0.1% Peptone Water (PW) or Butterfield’s Phosphate Buffer

•  Observation: to check on special cases in which either the type or volume of diluent vary as a function of the sample to be examined.

Total plate count

•  Petrifilm™ Aerobic Count plates (3M Microbiology Products)

•  Laboratory incubator set to 35 ± 1°C

•  Laboratory incubator set to 32 ± 1°C (for milk and dairy products)

2 - Procedure

a)  Preparation of the samples and serial dilutions.

b) Inoculation. Place the Petrifilm plates on a flat surface. Select three adequate dilutions of the sample for inoculation. Inoculate 1 ml of each dilution on separate  Petrifilm™AC Total Count Plates (3M Company), lifting the top film and dispensing the inoculum at the center of the circular area of the bottom film. Lower the top film covering the inoculum, place the spreader (which comes with the Petrifilm kits) with the recessed side down on the center of the plate and press gently to spread the inoculum evenly over the plate growth area.

c) Incubation. Wait one minute for the gel to solidify and incubate at 35  ± 1°C/48  ± 2h, in stacks of no more than 20 films, without inverting them. In the case of milk and dairy products, incubate at 32 ± 1°C/48 ± 3h.

d)  Counting the colonies and calculating the results. The circular growth area of the Petri-film plates are about 20 cm² in size and counting should be performed on the Petrifilms exhibiting 30 to 300 colonies (25 to 250 for dairy products). Count all the red colonies, irrespective of size or color intensity. Calculate the number of CFU per gram or milliliter of sample by multiplying the number of colonies by the inverse of the dilution. To determine the number of colonies on Petrifilm plates with more than 250 colonies, count the number of colonies in one or more representative 1 cm² square area(s), calculate the average per cm² and multiply by 20.

Detection limit: 1 CFU/ml for liquid samples or 10 CFU/g for solid samples.

References

Silva, N.D .; Taniwaki, M.H. ; Junqueira, V.C.A.;  Silveira, N.F.A. , Nasdcimento , M.D.D. and Gomes ,R.A.R .(2013) . Microbiological examination methods of food and water a laboratory Manual. Institute of Food Technology – ITAL, Campinas, SP, Brazil .

Horwitz, W. & Latimer, G.W. (eds) (2010) Official Methods of Anal-ysis of AOAC International. 18th edition, revision 3. Gaithersburg, Maryland, AOAC International.