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This method is modified by Anderson and Parker.
- Prepare serial tenfold dilutions in peptone water of food.
- Aseptically transfer sterile cellulose acetate membranes to the surface or dried glutamate agar.
- 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).
- Incubate plates 4 h at 35°C (facilitate resuscitation).
- Transfer each membrane to tryptone bile agar plate and incubate for 18 h at 44°C.
- Remove lids of plates.
- Add 3 mL Kovacs reagent into each lid.
- Remove membrane from agar and immerse in reagent for 5 min.
- Remove membrane and drain excess reagent.
- Dry the membranes under a UV lamp.
- Count pink-stained colonies within 30 min.
- 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.