Rapid Method for Enumeration of E. coli Biotype I

This method is modified by Anderson and Parker.

Rapid Method for Enumeration of E. coli Biotype I

  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.

About Author

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Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He is doing his Ph.D. at the Central Department of Microbiology, Tribhuvan University, Kathmandu, Nepal. He was awarded the DAAD Research Grant to conduct part of his Ph.D. research work for two years (2019-2021) at Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. Sagar is interested in research on actinobacteria, myxobacteria, and natural products. He is the Research Head of the Department of Natural Products, Kathmandu Research Institute for Biological Sciences (KRIBS), Lalitpur, Nepal. Sagar has more than ten years of experience in blogging, content writing, and SEO. Sagar was awarded the SfAM Communications Award 2015: Professional Communicator Category from the Society for Applied Microbiology (Now: Applied Microbiology International), Cambridge, United Kingdom (UK). Sagar is also the ASM Young Ambassador to Nepal for the American Society for Microbiology since 2023 onwards.

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