Appendix A - Definitions of Terms: Applicable to All Methods in the Compendium of Analytical Methods

Volumes 1-3
November 2006

Microbiological Methods Committee
Microbiology Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Health Products and Food Branch,
Health Canada
Postal Locator: 2204A1
Ottawa, Ontario K1A 0L2

E-mail: Don_Warburton@hc-sc.gc.ca

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1. Application

The following information is applicable to all methods that detect bacteria in food, food ingredients and environmental samples and should be used with these methods. The purpose of this revised Appendix is to provide additional information and replaces Appendix A, dated January 1993.

2. Definition of Terms

• 2.1. Lot: A batch or production unit which may be identified by the same code. When there is no code identification, a lot may be considered as (a) that quantity of product produced under essentially the same conditions, at the same establishment and representing no more than one day's production; or, (b) the quantity of the same kind of product from one and the same manufacturer available for sampling at a fixed location.
• 2.2. Sample: The total number of sample units taken per lot for analysis. The number of sample units taken is usually between 5 and 60 drawn at random from each lot.
• 2.3. Sample Unit: Usually a consumer size container of the product, and should consist of a minimum of 100 g (mL), unless stipulated in the method, or requested in the specific commodity sampling plan. Each sample unit may consist of more than one container in order to meet the size specifications of the sampling plan.
• 2.4. Analytical Unit: That amount of product withdrawn from each of the sample units (that constitute the sample) for analysis. The analytical units may be tested individually, or composited as directed in specific methods or sampling plans.
• 2.5. Plate or Direct Counts (CFU/g or CFU/mL): Plate count methods provide a direct count of living organisms expressed in CFU/mL or CFU/g (colony forming unit (CFU)) and estimate of the number of viable microorganisms in food according to the medium employed and the time and temperature of incubation. Each colony represents the minimum number of separable cells on the surface of or in semi-solid agar medium which gives rise to a visible colony of progeny on the order of tens of millions of cells in number. Each colony that appears on the agar plates can arise from a single cell, pairs, chains and clusters of cells.
• 2.6. Most Probable Number (MPN): See MFHPB-19. MPN values are estimates (statistical in nature), while plate counts are direct counts of living organisms expressed in CFU/mL. MPN values are, however, particularly useful when low concentrations of organisms (<100/g) are encountered in such materials as milk, food, water and soil where particulate matter of the matrix may interfere with obtaining accurate colony counts. Serial dilutions of the sample are prepared and inoculated into a series of 3 or 5 tubes of liquid media. Tubes that show a positive reaction, i.e. growth with gas formation, are usually inoculated into another series of selective or differential liquid media and/or plated onto selective agars. Numbers of organisms in the original sample are determined by the use of standard MPN tables.
• 2.7 Hydrophobic Grid Membrane Filter Method (HGMF): The growth in a HGMF grid-cell is equivalent to the more familiar Colony Forming Unit (CFU). The Most Probable Number of Growth Units (MPNGU) is derived from the HGMF score by an equation (refer to Appendix C). HGMF methods yield counts that are as high as, and more precise than, the Most Probable Number method. A single dilution gives an accurate count over a wide range of contamination levels. Counting precision may be better than on conventional plates or membrane filters because the HGMF reduces the effect of individual visual acuity on the count.
• 2.8 Dilutions: Most methods state to prepare a 1:10 dilution for most analysis (refer to Appendix H). Some methods state to prepare a 1 in 9 dilution or a 1/10 dilution. Regardless of the format, this refers to diluting 1 part of the compound or food matrix in question in 9 parts of diluent for a total of 10 parts. The dilution can be on a volume and/or weight basis.
• 2.9 Temperature of Incubation: Most Compendium methods state the following: "NOTE: It is the responsibility of each laboratory to ensure that the temperature of the incubators or waterbaths are maintained at the recommended temperatures. Where 35°C is recommended in the text of the method, the incubator may be 35 +/-1.0°C. Similarly, lower temperatures of 30 or 25°C may be +/- 1.0°C. However, where higher temperatures are recommended, such as 43 or 45.5°C, it is imperative that the incubators or waterbaths be maintained within 0.5°C due to potential lethality of higher temperatures on the microorganism being isolated."

  To further clarify this recommendation, temperatures greater than 37°C should be maintained within 0.5°C.

• 2.10 Presumptive positive: A presumptive positive result is defined as any positive signal obtained from a validated indirect method, which give only an indication, a probability or reasonable grounds for belief that the targeted microorganism is present based on the detection of characteristics other than the viability of the microorganism (e.g., DNA amplification, surface antigen detection, etc.).
• 2.11 Confirmed positive: A presumptive positive result is confirmed positive when the presence and the viability of the targeted microorganism has been assessed by a "direct" culture method, usually the standard cultural method. Therefore a confirmed positive result indicates that a viable organism has been isolated and identified as the organism of concern.
• 2.12 Pathogen: An organism capable of producing disease in a host.
• 2.13 Plasmid: An extrachromosomal genetic element found in bacteria, not essential for growth. Usually contains genetic information for resistance to an antimicrobial agent or for degradation of additional substrates.
• 2.14 Injury: Reversible phenotypic response of bacteria exposed to sublethal stressors.