SOP: Enumeration of Total Aerobic Bacteria in Food Products and Food Ingredients Using 3M Petrifilm Aerobic Count Plates

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

This method enumerates aerobic bacteria in food products and food ingredients; it may also be used for environmental sampling (laboratory procedure MFLP-41). It can be used to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act and/or other relevant federal regulations.

Petrifilm plates are a ready-to-use product developed by the 3M Company, St. Paul, MN. The media are coated onto films, so traditional media preparation is unnecessary. Aerobic bacteria are enumerated on Petrifilm Aerobic Count (AC) plates, which contain dry medium and a cold-water-soluble gelling agent. A 1 mL sample is added directly to the plate. Plastic spreading devices are provided with the plates; the concave side spreads the sample over the growth area — pressure applied to the spreader, placed on the overlay film, spreads the sample over 20 cm². The gelling agent is allowed to solidify, and the plates are then incubated and counted. Petrifilm AC plates contain modified Standard Methods nutrients, a cold-water-soluble gelling agent, and 2,3,5-triphenyltetrazolium chloride (TTC) indicator; reduction of the TTC turns the colonies red.

2. Apparatus required

• Stomacher, blender or equivalent
• Incubator capable of maintaining 35°C
• pH meter or pH paper capable of distinguishing 0.3–0.5 pH units within the range 6.0–7.5
• Colony counter and/or magnified illuminator (optional)

3. Media and reagents

• Petrifilm Aerobic Count plates 6400/6406 (3M Canada Inc.)
• A. Petrifilm AC plates, stored under refrigeration
• B. Plastic spreading device
• C. Product Instructions and Interpretation Guide (available from 3M)
• Diluents: Butterfield’s phosphate buffer (IDF phosphate buffer, 0.0425 g/L of KH₂PO₄ adjusted to pH 7.2), peptone salt diluent (ISO method 6887), buffered peptone water (ISO method 6579), 0.1% peptone water, saline solution (0.85 to 0.90%), bisulfite-free Letheen broth, or distilled water. For the analysis of cheese, where citrate buffer is specified, use warmed (40–45°C) Butterfield’s phosphate buffer.
• Buffered peptone water (BPW): peptone — 10.0 g; sodium chloride (NaCl) — 5.0 g; disodium phosphate — 3.5 g; potassium dihydrogen phosphate — 1.5 g; distilled water — 1.0 L. Final pH 7.2 ± 0.2.
• To prepare (BPW): dissolve the ingredients in 1.0 L of distilled water ↓ dispense into appropriate glassware ↓ autoclave at 121 °C for 15 min.
• 1N NaOH or 1N HCl

Note: Do not use diluents containing citrate, bisulfite or thiosulfate with the Petrifilm plate method.

4. Preparation of sample

• Except for shelf-stable foods, keep sample units refrigerated or frozen in the laboratory before analysis, depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions that prevent microbial growth or death.
• Analyse sample units as soon as possible after their receipt in the laboratory.
• Agitate liquids or free-flowing materials until homogeneous, to ensure a representative analytical unit. For solid foods, take a portion from several locations within the sample unit.
• Prepare a 1 in 10 (1:9) dilution in an appropriate diluent; stomach or blend as required for thorough mixing.
• Adjust the pH of the diluted sample to 6.6–7.2 with 1N NaOH or 1N HCl.

5. Procedure

Have sterile diluent ready

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Clean the surface of the working area with a suitable disinfectant

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Place the Petrifilm plate on a flat, level surface

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Lift the top film and carefully inoculate 1 mL of sample or diluted sample onto the centre of the bottom film, using a pipette, pipettor or equivalent device held perpendicular to the film

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Release the top film and allow it to drop onto the sample; do not roll the top film down

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Distribute the sample evenly with downward pressure on the centre of the plastic spreader, concave side down; do not slide the spreader across the film. Leave the plate undisturbed for at least one minute to let the gel solidify. Discard and re-prepare any plate showing bubbles or a non-circular inoculation area, as they may be enumerated inaccurately

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Return unused plates to the foil pouch and seal it to exclude air; store the resealed pouch in a cool, dry place. Use plates within one month of opening. Avoid exposing plates to temperatures above 25°C and/or relative humidity greater than 50%, which can affect plate performance. Do not use plates showing discoloration. Respect the expiration date that is part of the lot number on each package

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Incubate plates at 35 ± 1°C in a horizontal position, clear side up, in stacks of no more than 20 units, for up to 48 ± 3 hours

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Count plates promptly after the incubation period. If plates cannot be counted within 1 hour of removal from the incubator, they may be stored in a sealable container, frozen at temperatures of −15°C or below, for no longer than one week — this should be avoided as routine practice

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Count the colonies; a colony counter and/or magnified illuminator can facilitate counting. The circular growth area is approximately 20 cm²: on plates containing more than 250 colonies, estimate by counting the colonies in one or more representative squares, finding the average number per square, and multiplying that average by 20 (one square = 1 cm²) to obtain the total count per plate

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Calculate the number of colonies per mL or g of sample from plates at dilution levels giving a statistically significant result (25 to 250 colonies). If no plate falls within that range, use plates containing between 1 and 25 colonies

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When counting duplicate plates of consecutive dilutions, use only plates with counts in the statistically significant range to determine the average

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

6. Interpretation of results

• Count all red dots as colonies, regardless of size or intensity.
• Very high colony concentrations will turn the entire growth area red or pink — record the result as “too numerous to count” (TNTC). Occasionally, on overcrowded plates, the centre lacks visible colonies while many small colonies appear at the edges; record this as TNTC as well and dilute the sample further.
• Some organisms liquefy the gel, spreading out and obscuring other colonies. If a liquefier interferes with counting, make an estimated count from the unaffected squares only; do not count colonies within the liquefied area.
• Distinguish colonies from food particles, which may appear opaque on the Petrifilm plates.

7. Precautions

• The laboratory Supervisor must ensure that the analysis is carried out in accordance with ISO/IEC 17025:2005 (or the latest version): General Requirements for the Competence of Testing and Calibration Laboratories.
• The laboratory is responsible for ensuring equivalency if any variations of the listed media formulations are used (commercial product or made from scratch); equivalency data is forwarded to the Editor of the Compendium of Analytical Methods for consideration of modification of the method.
• Where 35°C is recommended, the incubator may be at 35 ± 1.0°C; lower temperatures of 30 or 25°C may be ± 1.0°C. Where higher temperatures are recommended, such as 43 or 45.5°C, the incubators or water baths must be maintained within 0.5°C, because of the potential lethality of higher temperatures to the microorganism being isolated.

References

Health Canada, Compendium of Analytical Methods, Volume 2 — HPB Method MFHPB-33, April 2015: “Enumeration of Total Aerobic Bacteria in Food Products and Food Ingredients Using 3M Petrifilm Aerobic Count Plates.” This revised method replaces MFHPB-33, dated February 2001.

• Baylis, C. L., C. de W. Blackburn, and S.B. Petitt. 1994. Evaluation of Petrifilm Plates for the Enumeration of Total Aerobic Flora, Coliforms, and E. coli, in Foods. Leatherhead Food RA.
• Betts, G. D., R.P. Betts and R. Taylor. 1994. Technical Memorandum N. 703: 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.
• Curiale, M. S., T. Sons, J.S. McAllister, B. Halsey and T.L. Fox. 1990. Dry Rehydratable Film for Enumeration of Total Aerobic Bacteria in Foods: Collaborative Study. Journal of the Association of Official Analytical Chemists. 73: 242-248.
• Curiale, M. S., P. Fahey, T.L. Fox and J.S. McAllister. 1989. Dry Rehydratable Films for Enumeration of Coliforms and Aerobic Bacteria in Dairy Products: Collaborative Study. Journal of the Association of Official Analytical Chemists. 72: 312-318.
• Ginn, R. E., V.S. Packard and R.L. Fox. 1986. Enumeration of Total Bacteria and Coliforms in Milk by Dry Rehydratable Film Methods: Collaborative Study. Journal of the Association of Official Analytical Chemists. 69: 527-531.
• Bureau of Microbial Hazards, Health Canada. 2014. Determination of the aerobic colony count in foods (MFHPB-18). In: Volume 2.
• 3M Petrifilm Aerobic Count Plate — Interpretation Guide.