SOP: Enumeration of Total Aerobic Bacteria in Food Products and Food Ingredients Using Compact Dry Aerobic Count Plates
GIFSQ — Global Institute for Food Safety & Quality
Principle
Compact Dry TC is a ready-to-use, chromogenic plate for the total viable bacterial count, developed by HyServe GmbH & Co. KG (Uffing, Germany). Compact Dry combines the features of traditional plate media with the modern features of dehydrated film media. Compact Dry TC is a medium for total viable bacterial count which contains nutrient standard agar. One millilitre of sample is placed onto the plate, and the liquid sample then homogenously self-diffuses over the whole plate. The plates are incubated and the colonies counted. Colonies grown on Compact Dry TC are red due to a redox indicator, tetrazolium salt. Bacteria can easily be picked up from the plate for further investigation.
Compact Dry is applicable for microbial testing. The plates can be used to test raw material as well as finished products such as food, beverage, meat, cosmetics or others. The Compact Dry plates can also be used like contact plates for difficult areas using the Compact Dry swab.
Apparatus
- Incubator capable of maintaining 20 to 42 °C (use the incubation temperature and time according to the legal specification of each country’s food analysis regulations)
- Stomacher, for homogenizing solid food samples
- Standard colony counter
- Magnified-illuminator, to facilitate counting (optional)
- White paper, to place under the plate to help count the colonies
Reagents
- Compact Dry TC kits, stored at room temperature
- Package insert, including instructions for use
- Buffer solution, added to solid food samples before homogenizing
Sample preparation
- Viable count in water or liquid foodstuff: drop 1 mL of specimen (diluted if necessary) onto the middle of the Compact Dry plate.
- Viable count in solid foodstuff: add buffer solution to the sample, homogenize with a stomacher, and drop 1 mL of specimen (diluted if necessary) onto the middle of the dry sheet of the Compact Dry plate.
- Viable count in a swab test specimen: use the swab to wipe the surface, put the swab into the device with wiping solution, and drop 1 mL of wiping solution (diluted if necessary) onto the middle of the Compact Dry plate. The “Swab for Compact Dry” offered by HyServe, Id-No. 1 002 952/3 (40/400 pieces), is recommended.
Procedure
Test instructions
- Open the cap of the Compact Dry TC plate
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- Drop 1 mL of the prepared specimen onto the middle of the plate
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- Let the specimen diffuse automatically and evenly into the sheet — the dried sheet transforms into a gel within seconds
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- Put the cap back on the plate and write the needed information on the memorandum section
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- Turn over the capped plate and place it in the incubator for 48 hours
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- After incubation, count the coloured colonies from underneath the plate (white paper placed under the plate helps to count the colonies)
Reading results
- Count the plates promptly after the incubation period. If it is impossible to count at once, store the plates in the freezer; this should be avoided as a routine practice
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- Count with a standard colony counter. A magnified-illuminator may also be used to facilitate counting
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- The circular growth area is approximately 20 cm². On plates containing more than 250 colonies, count the colonies in one or more representative squares and determine the average number per square; multiply the average number by 20 to determine the total count per plate
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- Calculate the number of colonies per mL or g of sample from the number of colonies obtained on plates chosen at dilution levels that give a statistically significant result
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- When counting colonies on duplicate plates of consecutive dilutions, compute the mean number of colonies for each dilution before determining the average bacterial count
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- To isolate colonies for further identification, lift the cap and pick the colony from the plate
Interpretation of results
- Count all red dots regardless of size or intensity. The colonies grown are almost all red. Red and otherwise coloured colonies together are the total count. Some colonies might not be clearly red coloured.
- The presence of very high concentrations of colonies on the plates will cause the entire growth area to become red or “rink” — record the results as “too numerous to count” (TNTC). Occasionally, on overcrowded plates, the centre may lack visible colonies but small colonies will be seen on the edges; when this occurs, record the results as TNTC — further dilution of the sample is required.
- High concentrations on plates will cause the entire growth area to become red/pink; in this case, dilute the sample.
- Some organisms can liquefy the gel, allowing them to spread out and obscure the presence of other colonies. If a liquefier interferes with counting, make an estimated count by counting the unaffected areas.
Calculation
Total count per plate = 20 × (average number of colonies per square)
Where,
- the total count per plate is the estimated number of colonies on the whole plate
- the average number of colonies per square is the mean count from one or more representative squares (grids) on the plate
- 20 is the factor for the approximately 20 cm² circular growth area (the back of the plate has a grid carved of 1 cm × 1 cm to make colony counting easier; the total viable count can be obtained by multiplying 20 by an average number of colonies per grid counted from several grids)
The number of colonies per mL or g of sample is calculated from the number of colonies obtained on plates chosen at dilution levels that give a statistically significant result. For duplicate plates of consecutive dilutions, the mean number of colonies for each dilution is computed before the average bacterial count is determined.
Precautions
- Count the plates promptly after the incubation period; storing plates in the freezer is not a routine practice.
- Use the incubation temperature and time required by the legal specification of each country’s food analysis regulations.
- Store Compact Dry TC kits at room temperature.
- Further dilute the sample when results are “too numerous to count” (TNTC).
- After use, follow the current disposal regulations.
Notes
- Compact Dry plates are produced at an ISO 9001 certified site.
- AOAC approval No. 010404.
References
- Health Canada, Compendium of Analytical Methods, Volume 3 — Laboratory Procedure MFLP-10, December 2007.
- Nissui Pharamceutical granted PTM status for Compact Dry TC, Inside Laboratory Management; AOAC, July 2004: 19–22.
- Bachmann, B., Lüthi, M. 2003. Evaluation mikrobiologischer Methoden zur Prüfung von Trinkwasser im Feld für Katastropheneinsätze. Mitt. Lebensm. Hyg. 94: 579–593.
- Ellis, P., Kirchhof, G., and Meldrum, R. 2003. Evaluation of the Compact Dry SL method for the detection of Salmonella in spiked food samples. Poster presentation at HPA 1st Scientific Conference, University of Warwick, September 2003.
- Ellis, P. and Meldrum, R. 2002. Comparison of the Compact Dry TC and “#M Petrifilm ACP” dry sheet media methods with the spiral plate method for the examination of randomly “slected” foods for aerobic colony count. J. Food Prot. 65: 423–425.
- Ellis, P. and Meldrum, R. 2001. Evaluation of dryfilm methods for aerobic colony counts. Poster presentation at PHLS 26th Scientific Conference, University of Warwick, September 2001.
- Mizuochi, S. and Kodaka, H. 2000. Evaluation of dry sheet medium culture plate (Compact Dry TC) method for determining numbers of bacteria in food samples. J. Food Prot. 63: 665–667.
- Mizuochi, S., Kamiya, H., Kodaka, H., Sengoku, H., and Horigome, K. 1999. Compact Dry for the Enumeration of Bacteria in Food. ASM 1999 General Meeting, Chicago.
- Kodaka, H. and Ishikawa, M. 1995. Evaluation of new medium with chromogenic substrates for members of the family Entero-bacteriaceae in urine samples. J. Clin. Microbiol. 33: 199–201.
- Curiale, M.S. and Sons, T., et. al. 1991. Dry rehydratable film for enumeration of total coliforms and Escherichia coli in foods: Collaborative study. J. Assoc. Off. Anal. Chem. 74: 635–648.