Fish Microbiology and Quality Assurance

4. Isolation and identification of E. coli from fish and fishery products by MPN and membrane filter methods

Coliforms are aerobic and facultatively anaerobic Gram-negative, non-sporing rods that ferment lactose in MacConkey broth or Brilliant green Lactose Bile salt broth at 37 ± 1⁰C within 48 h. They exhibit general morphological and biochemical similarities and are grouped together in the family of Enterobacteriaceae. The predominant aerobic bacterial flora of the large intestine of human beings and animals is composed of non-spore forming, Gram-negative bacteria. Members of the coliforms, including faecal coliforms are referred to as indicator organisms, since their presence may indicate the potential presence of pathogens in foods. E. coli is highly motile. An elevated temperature is used to differentiate coliforms of faecal and non-faecal origin. Faecal coliforms ferment lactose at 44.5 ± 0.5⁰C within 24 h, whereas non-faecal coliforms cannot do so. Faecal coliforms normally grow in the gastro-intestinal tract of humans and other warm-blooded animals. They include members of the genera Escherichia, Klebsiella and Enterobacter. E. coli is normally present in faecal contaminated water and foods. They can not withstand the low and high temperature processing treatments. The maximum allowable number of E. coli in raw fish and fishery products is 20/g and it should be absent in cooked fishery products. Faecal coliforms in fish processing water should be less than 1/100ml by MPN technique.

Enumeration of total coliforms, faecal coliforms and E. coli is done by Most Probabale Number (MPN) technique. MPN method makes use of a statistical technique for estimating the probable number of bacteria present per specified unit of material. In this method, specified volumes from at least three serial decimal dilutions of the sample are inoculated to the tubes containing specific media. The number of positive tubes in each set is referred to MacCrady’s Tables from which the most probable number of bacteria per unit sample (MPN index) can be calculated with a known degree of certainty. Three serial decimal dilutions are generally used and 5 tubes of medium are inoculated from each dilution. Usually the first three dilutions prepared are used but an expected high count may necessitate using higher dilutions.

Three sequential steps are generally followed for the detection of coliform bacteria in water or any other food sample. These include presumptive test for total coloforms, confirmed test for faecal coliforms, and completed test for confirming coliforms of faecal origin (E. coli).

Materials required

1. Lauryl Sulphate Tryptose Broth (LSTB)
2. E.C. Broth
3. Eosine Methylene Blue (EMB) agar
4. Glucose
5. Citrate medium
6. Tryptone water
7. Kovac’s reagent
8. 0.04% Methyl red solution
9. 6% α - Napthol
10. Potassium hydroxide solution
11. MacConkey broth
12. Physiological saline