SOP: Enumeration of Yeast and Mould in Food Products and Food Ingredients Using 3M Petrifilm Yeast and Mold Count Plates

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

Ready-to-use Petrifilm plates from 3M Company (St. Paul, MN) replace traditional medium preparation and save labour and time. Yeasts and moulds are enumerated on the Petrifilm Yeast and Mould Count (YM) plates, which carry dry medium and a cold-water-soluble gelling agent. A 1 mL sample goes directly onto the plate; pressure applied to the plastic spreading device on the overlay film spreads it over the 30 cm² growth area. After the gel solidifies, the plates are incubated and counted. The plates also contain 5-bromo-4-chloro-3-indolyl phosphate (BCIP), an indicator of phosphatase enzyme activity, which enhances visualization of the colonies. The method has been studied extensively against conventional methods (see References).

2. Apparatus required

• Stomacher, blender or equivalent
• Bench or incubator maintaining 20–25 °C
• Colony counter and/or magnified illuminator

3. Media and reagents

• Petrifilm Yeast and Mould Count plates 6407/6417 (3M Canada Inc.); the plates contain nutrients supplemented with antibiotics, the cold-water-soluble gelling agent and BCIP. Store at or below 8 °C. Each set comes with a plastic spreading device (use the concave side to spread the sample over the growth area) and a package insert including instructions for use; an Interpretation Guide is available on request.
• Diluents: Butterfield’s phosphate buffer, 0.1% peptone water and distilled water. Do not use citrate buffer or sodium thiosulfate with the Petrifilm plate method; where citrate buffer is indicated in a standard, substitute it with warmed (40–45 °C) Butterfield’s phosphate buffer.

4. Sample handling and preparation

• Before analysis, keep samples refrigerated (2–8 °C) or frozen, depending on the nature of the product — except shelf-stable foods. Thaw frozen samples in a refrigerator, or under time and temperature conditions that prevent microbial growth or death.
• Analyse samples as soon as possible after their receipt in the laboratory.
• Have sterile diluent ready and disinfect the surface of the work area.
• Place the Petrifilm plate on a flat surface and mark identifying sample information on the film.
• To make the analytical unit representative: agitate liquids until the contents are homogeneous; for solid foods, take representative portions from several locations within the sample. Prepare a 1:10 dilution in the appropriate diluent and mix thoroughly.

5. Procedure

Lift the top film and carefully inoculate 1 mL of the sample (or diluted sample) onto the centre of the bottom film, using a pipette or a plate-loop continuous pipetting syringe

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Drop the top film down onto the sample

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Distribute the sample evenly by pressing downward on the centre of the plastic spreader, concave side down; do not slide the spreader across the film; leave the plate undisturbed for a minimum of 1 minute so the gel can solidify

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Return unused plates to the foil pouch; seal the pouch by folding and taping the open end; store the resealed pouch in a cool dry place and use the plates within one month after opening the pouch; do not use plates showing orange or brown discolouration (the expiration date and lot number are noted on each package and the lot number also appears on each individual test film)

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Incubate the plates in a horizontal position, clear side up, in stacks not exceeding 20 units; follow current standards for incubation temperature; observe the plates for growth at both 3 and 5 days

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Count the plates promptly after the incubation period; if counting at once is impossible, store the plates in the freezer (avoid this as a routine practice)

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Count with a standard colony counter; a magnifier-illuminator may also be used

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If a plate holds more than 150 colonies, estimate: count the colonies in one or more representative squares, take the average per square and multiply by 30 (see Calculation)

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When counting duplicate plates of consecutive dilutions, compute the mean number of colonies for each dilution before determining the average count

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To isolate a colony for further identification, lift the top film and pick the colony from the gel

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Read the colony types: yeast colonies are blue-green or off-white and form small defined colonies; mould colonies are larger and more diffuse, usually blue, but they may take on their natural pigmentation (black, yellow, green and so on); high numbers of yeast colonies may turn the entire growth area blue, and high numbers of mould colonies may turn it blue, black, yellow and so on — when this happens, further dilution of the sample is required

6. Calculation

For plates with more than 150 colonies:

Estimated total colonies per plate = average colonies per representative square × 30

Where,

• average colonies per representative square = the mean count in one or more representative squares of the growth area
• 30 = the approximate growth area of the plate in cm²

For duplicate plates of consecutive dilutions, the mean number of colonies for each dilution is computed before the average count is determined. Calculate the number of colonies per mL or g of sample from the counts obtained on plates at dilution levels that give a statistically significant result.

7. Precautions

• Store Petrifilm plates at or below 8 °C; do not use plates with orange or brown discolouration.
• Use opened plates within one month of opening the pouch and keep resealed pouches in a cool dry place; exposure to temperatures above 24 °C and/or humidity above 50% RH can affect plate performance.
• Never use citrate buffer or sodium thiosulfate with this method; substitute warmed (40–45 °C) Butterfield’s phosphate buffer where a standard calls for citrate buffer.
• Incubate plates in a horizontal position, clear side up, in stacks of no more than 20 units; count at both 3 and 5 days and count promptly after incubation.
• Where 35 °C is recommended, the incubator may be at 35 ± 1.0 °C; 30 or 25 °C may be ± 1.0 °C; at higher recommended temperatures such as 43 or 45.5 °C, the incubator or water bath must be held within 0.5 °C.
• If the entire growth area turns blue, black or yellow from high colony numbers, dilute the sample further and repeat.

References

Health Canada, Compendium of Analytical Methods, Volume 2 — HPB Method MFHPB-32, March 2003: “Enumeration of Yeast and Mould in Food Products and Food Ingredients Using 3M Petrifilm Yeast and Mold Count Plates” (replaces MFHPB-32, April 1997).

• AOAC. 2000. Official Method 997.02. Official Methods of Analysis of AOAC International. 17th Ed. AOAC International, Gaithersburg, MD, USA.
• Betts, G.D., Betts, R.P. and Taylor, R. 1994. Evaluation of 3M Petrifilm for Aerobic Plate Count, Yeast and Mould Count and Escherichia coli Count. Campden Food & Drink Research Association, Chipping Campden, Gloucestershire, UK.
• Beuchat, L.R., Nail, B.V., Brackett, R.E. and Fox, T.L. 1991. Comparison of the Petrifilm Yeast and Mould Culture Film Method to Conventional Methods for Enumerating Yeasts and Moulds in Foods. J. Food Protection. 54:443-447.
• Beuchat, L.R., Nail, B.V., Brackett, R.E. and Fox, T.L. 1990. Evaluation of a Culture Film (Petrifilm YM) Method for Enumerating Yeasts and Moulds in Selected Dairy and High-Acid Foods. J. Food Protection. 53:864, 869-874.
• Knight, M.T., Newman, M.C., Benzinger, Jr. and Agin, J.R. 1994. Comparison of the Petrifilm Dry Rehydratable Film and Conventional Culture Methods for the Enumeration of Yeasts and Moulds in Foods. AOAC International Meeting and Exposition, Abstract 23-012.
• Knight, M.T., Newman, M.C., Benzinger, Jr., Neufang, K., Agin, J.R., McAllister, J.S. and Ramos, M. 1997. Comparison of the Petrifilm Dry Rehydratable Film and Conventional Culture Methods for the Enumeration of Yeasts and Moulds in Foods: Collaborative Study. J. AOAC International. 80:806-823.
• Vleamynck, G.M. 1994. Comparison of Petrifilm and Plate Count Methods for Enumerating Moulds and Yeasts in Cheese and Yogurt. J. Food Protection. 57:913-914.