Plate count method APHA 2001 for total coliforms 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) and  the  Standard Methods for the Examination of Dairy Products (Davidson  et al., 2004).

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

Coliform count

•  Sterile, empty 20 × 100 mm Petri dishes

•  Violet Red Bile (VRB) Agar

•  Brilliant Green Bile Broth (BGB)

•  Laboratory incubator set to 35 ± 1°C (32 ± 1°C for milk and dairy products)

2 - Procedure

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

a)  Preparation of the samples and inoculation.

For preparation of the samples and serial dilutions (10⁻¹, 10⁻² and serial subsequent) follow the procedures described in Chapter 2. Select three appropriate dilutions of the sample and inoculate in  Violet Red Bile (VRB) 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.

For processed foods expected to contain sub-lethally damaged or stressed coliforms (unable to growth and form typical colonies on VRB), include a recovery step: pour plate in a non-selective agar (e.g.  Trypticase Soy Agar, TSA), allow 2 ± 0.5 h of recovery at room temperature, and then overlay the TSA layer with VRB.

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

Note a.1)   The  Standard Methods for the Examination of Dairy Products (Davidson  et al., 2004) recommends to overlay the TSA layer with fortified VRB (VRB containing double strength of bile salts, neutral red and crystal violet).

b)  Incubation and colony counting. Incubate the plates in an inverted position at 35 ± 1°C/18–24 h (32 ± 1°C/24 ± 2 h for milk and dairy products).

Select plates with 15–150 colonies and count only the typical coliforms colonies on the VRB medium: red-purple, 0.5 mm or greater in diameter, sur-rounded by a reddish halo characteristic of the precipitation of the bile salts.

c) Confirmation: Select five suspicious colonies and inoculate each colony into a tube of  Brilliant Green Bile Broth (BGB). Incubate BGB tubes at 35 ± 0.5°C/24 ± 2 h (32 ± 1°C for milk and dairy products) and examine for gas production. Consider confirmed cultures exhibiting gas without surface pellicle. If gas and pellicle are observed, perform Gram stain and oxidase test. Consider confirmed the cultures of Gram negative rods, oxidase negative.

d)  Calculation of the results. Calculate number of total coliforms/g of sample based on percentage of colonies tested that are confirmed as total coliforms. Example: The presumptive count obtained with 10⁻⁴ dilution of sample was 65 and four of five colonies tested were confirmed (80%). The number of coliforms cells/g of food is 65  × 0.8  × 104  = 520,000 CFU/g = 5.2 × 105 CFU/g.

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 .

International Organization for Standardization (2005) ISO 7251:2005.  Microbiology of food and animal stuffs –  Horizontal method for the detection and enumeration of presumptive Escherichia coli – Most probable number technique. Geneva, ISO.

Hunt, M.E. & Rice, E.W. (2005) Microbiological examination. In: Eaton, A.D., Clesceri, L.S., Rice, E.W. & Greenberg, A.E. (eds). Standard Methods for the Examination of Water & Wastewater. 21^st edition. Washington, American Public Health Association (APHA), American Water Works Association (AWWA) & Water Environment Federation (WEF). Part 9000, pp. 9.1–9.169.

Davidson, P.M., Roth, L.A. & Gambrel-Lenarz, S.A. (2004) Coliform and other indicator bacteria. In: Wehr, H.M. & Frank, J.F (eds). Standard Methods for the Examination of Dairy Products. 17th edition. Washington, American Public Health Association. Chapter 7, pp. 187–226.

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 Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 8, pp. 69–82.