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Supplement to All Methods in the Compendium: General Microbiological Guidance
Shelf-life of Media
The shelf-life of some media is included in compendial methods and in Appendix G. It is recognized that commercial media may use shelf-stable components that can enhance the shelf-life of the reconstituted product. It is also recognized that chemicals and ingredients from different sources may alter the recommended shelf-life. It is up to the analyst or lab supervisor to verify and set appropriate shelf-lives for media used in their laboratory.
Comparable Media
It is recognized that problems with suppliers, manufacturers and shipments, and short-term emergencies, may force the need for substitution of one medium for another. For example, blood agar plates made with horse blood may be substituted for those made with sheep blood. In such cases, it is the responsibility of the lab supervisor to instill suitable controls to ensure the analysis is reliable or complete.
It is also recognized that other media (for example, chromogenic media) may be more efficient and suitable for certain foods or conditions. These media may be available for some time before they become incorporated into compendial methods. Novel media must be used in conjunction with the recommended media in validation studies.
Use of Culture Controls
Negative and positive controls are recommended in most compendial methods. When controls are not listed in the compendial methods, it is recommended that suitable strains be used to ensure the reliability of each analysis. Where specific strains are recommended, it is recognized that these may not be available to all laboratories and that equivalent strains can be substituted by the lab supervisor to meet the requirements of the method.
Use of Commercial Reagents, Identification Kits and Tests
It is recognized that standard microbial tests, such as indole and oxidase tests, are available commercially as complete reagents, kits and tests. These are often more shelf-stable, user-friendly and reliable than reagents and solutions prepared in-house. It is also recognized that rapid identification kits may incorporate many of the confirmation steps listed in a method. Commercial reagents, identification kits and tests may be substituted for those listed in methods when equivalency is shown.
7. Procedure
Preparation of Sample
Weighing of Analytical Unit
General information is given in each method, as well as in Appendices F and H. In addition, the analyst can tare either a blender jar or Stomacher bag (when using the appropriate holder) on the balance prior to weighing the analytical unit. The analyst can then remove the analytical unit from the sample (following the instructions in the method and/or Appendices F and H) and place it into the tared blender or Stomacher bag. The analyst can then add a suitable amount of diluent to obtain the 1:10 dilution, prior to macerating. Weight measurement is considered a more reliable means of obtaining the initial dilution.
Limits of Detection and Use of Alternate Dilutions
The usual first dilution recommended in compendial methods is 1:10. In order to meet clients' needs, it is recognized that it may be necessary to increase the sensitivity of a method and alter it's "Limit of Detection" (LOD). This may be accomplished by: 1. increasing the amount analysed (for example, doing MPNs using 100, 10, and 1 g instead of the usual 10, 1, and 0.1 g); 2. decreasing the first dilution to 1:5; 3. increasing the amount spread onto an agar plate (for example, plating 0.2 mL instead 0.1 mL); and 4. increasing the number of plates being used with the first dilution (for example, spreading 0.1 mL on 5 agar plates instead of the usual duplicate (2) agar plates . These and other suitable means of increasing or decreasing the LOD may be used if they are validated properly in the laboratory.
After the initial 1:10 dilution, further dilutions may be done using 9, 10, 90 or 99 mL aliquots (or similar volumes) of diluent.
