A. General Microbiological Guidance On Pre-warming Of Broths In All Qualitative Methods In The Compendium

Supplement to Appendix I
June 2005

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

Don_Warburton@hc-sc.gc.ca

Help on accessing alternative formats, such as Portable Document Format (PDF), MP3 and WAV files, can be obtained in the alternate format help section.

---

1. Application

The following information is offered as a supplement to all the qualitative (presence/absence) methods in The Compendium of Analytical Methods, and should be used with these methods unless otherwise specified. The purpose of this supplement is to provide additional information on pre-warming of broths.

2. Procedure

Preparation of Sample

Enrichment Procedures

General information is given in each method as to suitable pre-enrichment and enrichment broths, as appropriate to that particular organism and test. In addition to this information, the following instructions should be applied.

When compositing analytical units and for analytical sample units greater than 25 g, all initial enrichment broths should be warmed to approximately the temperature of incubation before proceeding with the dilution of sample with broth, with the exception of analysis that incubate at temperatures greater than 35°C, such as E. coli O157, Shigella spp., and Vibrio cholera. Broth for these analyses should be pre-warmed to approximately 35°C.

For secondary enrichment broths, as well as for analytical sample units of 25 g or less, it is acceptable for the temperature of the broth to be only increased to room temperature before proceeding with analysis.

Broth that has been pre-warmed, should not be warmed and then refrigerated repetitively. It is recommended that pre-warmed broth on the third cycle of pre-warming (i.e., has been previously pre-warmed and then refrigerated twice) be discarded at the end of the day if not required. Broth containing antibiotics or other supplements should only be pre-warmed once, then discarded if not required. Laboratories should validate their individual practices to determine if detrimental effects occur, if repetitively warming and refrigerating broth more than the recommended number of cycles.

Note: Holding of media at high temperature over an extended period of time may be detrimental depending on individual formulae, and this factor should be taken into account when choosing a standard operating procedure for pre-warming.

B. pH'ing Of Diluents And Enrichment Broths

Although mentioned in previous versions of some methods, adjusting the pH of the diluent plus food matrix before plating is not recommended for quantitative methods, except where stated by the manufacturer (for example, 3M requires this for Petrifilm). This will allow the product to be evaluated "as presented" to the consumer, preventing a false bias (positive or negative) that would result if the pH is adjusted before plating. This will be corrected in revised versions of these methods.

For qualitative methods the adjusting the pH of the enrichment broth plus food matrix is still required if mentioned in the method.

C. Use Of Commercial Reagents

Methyl-Red (MR) and Voges-Proskauer (VP) Tests

It is recognized that these standard microbial tests are available commercially as complete reagents. These are often more shelf-stable, user-friendly and reliable than reagents and solutions prepared in-house. Commercial reagents may be substituted for those listed in methods if the manufacturer's instructions for reconstitution and subsequent use are followed.

See Appendix I for other general Information on the use of commercial reagents.

D. Confirmation Of Presumptive Colonies In Qualitative Methods

The following may be followed for all qualitative methods:

If the colonies are well isolated on the selective agars: Pick a minimum of 5-10 typical colonies from each selective plate to purity plates (as specified in the original method). Concurrently pick the same colony to tubes and/or plates containing agars or biochemical agents (for example: motility, TSI, LIA, urea, and citrate slants).

If the colonies are NOT well isolated on the selective agars: Pick a minimum of 5-10 typical colonies from each selective plate to purity plates (as specified in the original method), streaking for separation. Incubate plates for 24-48 h or until growth is satisfactory. As needed, examine the plates for typical colonies and repurify if needed. Otherwise proceed with confirmation steps outlined in the method.