Fish Microbiology and Quality Assurance (2+2)

Part C: Completed test for the confirmation of E. coli

The positive result obtained in the previous test is confirmed by subjecting the suspected colonies that developed on EMB agar to biochemical tests. The indole test, methyl red (MR) test, Voges –Proskauer (VP) test and citrate utilization tests differentiate E. coli from other coliform bacteria. E. coli is positive for indole and MR test while, negative for VP and citrate utilization test. The colonies showing typical reaction as E. coli are referred back to MPN tubes and MPN numbers expressed by referring to MPN table.

Indole test:
Methyl red test:
Voges- Proskauer test:
Citrate utilization test:

Biochemical reactions of E. coli

Tests	Reactions	Results
Indole	Formation of red ring	Positive
M.R.	Magenta red	Positive
V.P.	Pink colour	Negative
Citrate utilization	No growth (green colour)	Negative

Media composition

Lauryl Sulphate Tryptose Broth (LSTB)

Tryptone Lactose Sodium chloride Dipotassium hydrogen phosphate Potassium Dihydrogen phosphate Sodium lauryl sulphate Distilled water pH

- 20.0 g - 5.0 g - 5.0 g - 2.75 g - 2.75 g - 0.1 g - 1000 ml - 6.8 ± 0.2

Sterilize at 110⁰C for 10 minutes.

E. coli (EC) Broth

Tryptone Lactose Bile salt Mixture Dipotassium phosphate Monopotassium phosphate Sodium chloride Distilled water pH

- 20.0 g - 5.0 g - 1.5 g - 4.0 g - 1.5 g - 5.0 g - 1000.0 ml - 6.9 ± 0.2

Sterilize at 110⁰ C for 10 minutes.

Eosine Methylene Blue (EMB) Agar

Peptone Lactose Sucrose Dipotassium phosphate Agar Distilled water Eosine Y Methylene Blue pH

- 10.0 g - 5.0 g - 5.0 g - 2.0 g - 13.5 g - 1000.0 ml - 0.4 g - 0.065 g - 7.2 ± 0.2

Sterilize at 121⁰C for 15 minutes. Cool, shake and then pour into plates.