Enumeration of Coliforms, Faecal Coliforms and of E. Coli in Foods using the MPN Method

6. MATERIALS AND SPECIAL EQUIPMENT

The media listed below (1 to 8) are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 3 and reference 8.8 for the formulae of individual media.

Note: If the analyst uses any variations of the media listed here (either product that is commercially available or made from scratch), it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency.

1) Peptone Water (0.1% and 0.5%)

2) Aqueous Sodium Citrate (2.0%), tempered to 40-45^oC

3) Lauryl Sulfate Tryptose (LST) broth

4) Brilliant Green Lactose 2% Bile (BGLB) broth

5) Escherichia coli (EC) broth or EC broth with MUG (4-methylumbelliferyl-ß-D-glucuronide)

6) Levine's Eosin Methylene Blue (L-EMB) agar or Endo agar

7) MacConkey agar

8) Nutrient Agar (NA) or other non-selective agar

9) Covered water baths, with circulating system to maintain temperature of 44.5^oC and 45^oC. Water level should be above the medium in immersed tubes.

10) Thermometer, calibrated and traceable

11) Incubator, 35^oC.

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 35^oC is recommended in the text of the method, the incubator may be at 35 +/-1.0^o C (except for shellfish, where the incubators must be at 35 ± 0.5^oC). Similarly, lower temperatures of 30 or 25 may be +/- 1.0^oC. However, where higher temperatures are recommended, such as 43 or 44.5^oC, it is imperative that the incubators be maintained within 0.5^oC and water baths be maintained within 0.2^oC due to potential lethality of higher temperatures on the microorganism being isolated.

12) Stomacher, blender or equivalent.

13) Control cultures (use ATCC cultures or equivalent): positive control(s): E. coli that is known to produce gas at 44.5 / 45^o C and is capable of fermenting lactose to produce typical reactions on L-EMB agar; if using EC-MUG, a strain that is known to produce ß-glucuronidase EMB / IMViC negative control: Enterobacter aerogenes or an equivalent gram negative rod that does not produce "positive" reactions on EMB and is indole-negative, methyl red-negative, Voges-Proskauer-positive, and citrate positive. MPN broths negative control: Salmonella berta or an equivalent gram negative rod that is gas-negative in MPN broths and in the secondary EC broth