Plate count method APHA 2001 for Enterobacteriaceae in foods

Method of the American Public Health Association (APHA), as described in  the 4th Edition of the Compendium of Methods for the Microbiological Examination of Foods (Kornacki and Johnson, 2001).

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

Enumeration (pour plate)

•  Sterile, empty 20 × 100-mm Petri dishes

•  Culture medium: Violet Red Bile Glucose (VRBG) Agar

•  Laboratory incubator set to 35 ± 1°C

2 - Procedure

A general flowchart for the enumeration of Enterobacteriaceae in foods using the plate count method APHA 2001 is shown in Figure 1.

a)  Preparation of the samples and serial dilutions.

b) Inoculation. Select three appropriate dilutions of

the sample and inoculate in  Violet Red Bile Glucose (VRBG) Agar. Use the pour plate technique and, after complete solidification of the medium, cover the surface with a 5–8 ml thick layer of the same medium.

c) Incubation. Incubate the plates in an inverted position at 35 ± 1°C/18–24 h.

d) Counting of the colonies and calculating the results. Select plates with 15–150 colonies and count only the typical  Enterobacteriaceae colonies on the VRBG medium: red-purple, 0.5 mm or greater in diameter, surrounded by a reddish halo characteristic of the precipitation of the bile salts.

On crowded plates the colonies may remain small and fail to reach 0.5 mm. Determine the number of CFU/g or ml by multiplying the number of typical colonies by the inverse of the dilution.

Figure 1   Scheme of analysis for the enumeration of Enterobacteriaceae in foods using the plate count method APHA 2001 (Kornacki and Johnson, 2001).

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 .

Kornacki, J.L. & Johnson, J.L. (2001) Enterobacteriaceae, coliforms, and Escherichia coli as quality and safety indicators. In: Downes, F.P. & Ito, K. (eds).  Compendium of Methods for the Microbio-logical Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 8, pp. 69–82.