Rapid Method for Enumeration of E. coli Biotype I

This method is modified by Anderson and Parker.

1. Prepare serial tenfold dilutions in peptone water of food.
2. Aseptically transfer sterile cellulose acetate membranes to the surface or dried glutamate agar.
3. Transfer duplicate 1.0 mL aliquots of each dilution to cellulose acetate membranes, using a sterile glass Drigalski spreader, distribute the fluid over the entire membrane (except the periphery).
4. Incubate plates 4 h at 35°C (facilitate resuscitation).
5. Transfer each membrane to tryptone bile agar plate and incubate for 18 h at 44°C.
6. Remove lids of plates.
7. Add 3 mL Kovacs reagent into each lid.
8. Remove membrane from agar and immerse in reagent for 5 min.
9. Remove membrane and drain excess reagent.
10. Dry the membranes under a UV lamp.
11. Count pink-stained colonies within 30 min.
12. Calculate the number of type I E. coli by selecting the dilution giving an average of
    20–50 pink colonies and both membranes and multiply total by the dilution factor.