Plate count method APHA 2001 for yeasts and molds in foods

Method of the American Public Health Association (APHA), of the 4th Edition of the  Compendium of Methods for the Microbiological Examination of Foods (Beuchat and Cousin, 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 (spread plate)

•  Dichloran Rose Bengal Chloramphenicol (DRBC) Agar plates (for foods with water activity >0.95

•  Dichloran 18% Glycerol (DG18) Agar plates (for foods with water activity ≤0.95

•  Laboratory incubator set to 22–25°C

2- Procedure

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

The procedure described below does not present these details, as they are supposed to be known to the analyst.

a)  Preparation of the samples and serial dilutions: For foods with intermediate moisture levels, keep the sample soaked in the diluent for a certain period of time, to soften the product and facilitate the release of the microorganisms present.

b) Inoculation: Select three adequate dilutions of the sample and inoculate (spread plating) 0.1 ml of each dilution on previously prepared and dried plates, containing one of the following culture media:

•   Dicloran Rose Bengal Chloramphenicol agar (DRBC), for foods with water activity higher than 0.95.

•   Dicloran Glycerol 18 (DG-18), for foods with water activity lower than or equal to 0.95. In the case of analysis of raw materials intended for use in formulating products with water activity greater than 0.95, DRBC Agar should be used.

Spread the inoculum with a glass or plastic spreaders (Drigalski), from the plates inoculated with the greatest dilution to the plates inoculated with the smallest dilution, until all excess liquid is absorbed. If the estimated counts of the sample are smaller than 100/g or ml, inoculate 1 ml of the first dilution, dividing the volume over four plates, three with 0.3 ml and one with 0.1 ml.

 Note b.1)   In the analysis of thickeners (gums, pectin, cellu-lose) the initial dilution must be greater than 1:10, because of the high viscosity of these products. If the estimated counts are low, 1 g of the sample may be plated directly onto the plate, spreading the material over the entire surface (Gray and Pinkas, 2001).

Note b.2)  Before use, store the plates with DRBC or DG18 in the refrigerator, protected from light, to avoid photo degradation of Rose Bengal, which can be accompanied by the formation of compounds that inhibit the growth of yeasts and molds.

c) Incubation:  Wait until the plates dry (at least 15 minutes) and incubate at 22–25°C for five days, without inverting the plates, in stacks of no more than three plates, in the dark. It is advisable to not count the colonies before five days, since moving the plates about may result in secondary growth (due to displacement of spores), invalidating the final count.

 Note c.1)   The International Commission on Food Mycology (ICFM) recommends incubation at 25°C/5 days as a standard condition (Hocking  et al.,  2006). Pitt and Hocking (2009) recommend 30°C/5 days for products stored at ambient temperature in tropical regions and 22ºC/5 days in temperate climate regions such as Europe.

d) Counting the colonies and calculating the results: For counting the colonies and calculating the results, select plates with 15 to 150 colonies with the aid of a magnifying glass fitted onto a colony counter.

On the selected plate, count separately the colonies with a filamentous, cotton-like or pulverulent (i.e. powdery) appearance, which are characteristic of molds, and record the result.

On the same plate, count the remaining colonies, which may be yeasts or bacteria that are eventually capable of growth under the conditions of

Figure 1   Scheme of analysis for enumeration of yeasts and molds in foods using the plate count method APHA 2001 (Beuchat and Cousin, 2001).

the test. Select at least five of these colonies and verify the morphology of the cells under the micro-scope, observing whether the culture is made up of yeasts, bacteria or a mixture of both. For that purpose, a wet mount can be prepared or gram staining can be done, Consider as confirmed all the colonies that present yeasts or mixtures of yeasts and bacteria. Determine the number of yeast colonies on the plate in function of the confirmed percentage. For example, out of 30 colonies counted, five were subjected to confirmation, and three were confirmed as yeasts (60%).

So, the number of yeast colonies on the plate is 30 × 0.6 = 18.

To calculate the number of CFU/g or ml of molds, multiply the number of typical mold colonies by ten and by the inverse of the dilution. To calculate the number of CFU/g or ml of yeasts, multiply the number of colonies confirmed as yeasts by ten and by the inverse of the dilution. To calculate the total number of molds and yeasts, add the number of mold colonies to the number of colonies confirmed as yeasts and multiply by ten and the inverse of the dilution.

Example

Dilution: 10^–2 (inoculated: 0.1ml). Total number of typical mold colonies on the plate: 30. Number of presumptive yeast colonies on the plate: 40, five subjected to confirmation, four confirmed (80%). Total number of yeast colonies on the plate: 40 × 0.8 = 32. Molds count in CFU/g or ml: 30 × 10² × 10 = 3.0 × 10⁴. Yeasts count in CFU/g or ml: 32  × 10²  × 10  = 3.2 × 104. Molds and yeasts count in CFU/g or ml: (30 + 32) × 10² × 10 = 6.2 × 10⁴.

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 .

Beuchat, L.R. & Cousin, M.A. (2001) Yeasts and molds. 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 20, pp. 209–215.

Gray, R.J.H. & Pinkas, J.M. (2001) Gums and spices. In: Downes, F.P. & Ito, K. (eds).  Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 52, pp. 533–540.

Pitt, J.I. & Hocking, A.D. (eds) (2009) Fungi and Food Spoilage. 3rd edition. London, Springer.