Enumeration of Escherichia coli in Foods by the Hydrophobic Grid-Membrane Filter Method (HGMF) Method

4. DEFINITION OF TERMS

See Appendix A of Volume 2.

5. COLLECTION OF SAMPLES

See Appendix B of Volume 2.

6. MATERIALS AND SPECIAL EQUIPMENT

The media listed below (items 3-5) are commercially available and are to be prepared and sterilized according to the manufacture's instructions.

1) HGMF (1600 grid-cell, 0.45 µm pore size). (Available as ISO-GRID filters from Oxoid Ltd., Nepean, Ont.)

2) Membrane filter forceps (Millipore Corp., Bedford, MA.)

3) Peptone water (0.1%) (PW) or Peptone/Tween 80 diluent (PT)

4) Nutrient agar (NA) plates (if a resuscitation step is required)

5) Tryptone bile agar (TBA) plates

6) Enzyme solutions (Appendix E of Volume 2; 9.7) as required for some food products

7) Stomacher/or equivalent

8) Whatman No. 1 or equivalent filter papers, 7 cm dia

9) Indole reagent

10) Spreadfilter with funnel plus pipet-tip prefilters (Filtaflex Ltd., P.O. Box 1224, Almonte, Ont.), or ISO-GRID filtration unit (Oxoid, Nepean, Ont.)

11) HGMF Linecounter for manual counting (Available from Oxoid Ltd., Nepean, Ont.)(optional)

12) An incubator capable of maintaining 44.5⁰C

Note: It is the responsibility of each laboratory to ensure that the temperature of the incubators or water baths are maintained at the recommended temperatures. Where 35EC is recommended in the text of the method, the incubator may be at 35 +/-1.0E C. Similarly, lower temperatures of 30 or 25 may be +/- 1.0EC. However, where higher temperatures are recommended, such as 43 or 45.5EC, it is imperative that the incubators or water baths be maintained within 0.5EC due to potential lethality of higher temperatures on the microorganism being isolated.