Fish Microbiology and Quality Assurance (2+2)

5. Isolation and identification of fecal streptococci from fish and fishery products

Streptococci are Gram-positive, non-motile, non-sporeforming, facultatively anaerobic and catalase-negative cocci. They are important human pathogens and grow only in media containing fermentable carbohydrates or enriched with blood or serum. They form circular, transluscent to opaque , pinpoint colonies on solid media. The primary habitats of the faecal streptococci are the intestinal tract of human beings, animals and birds and their presence in foods including seafoods indicates faecal contamination of the foods similar to E. coli. Contamination of foods with this organisms takes place through theeuse of contaminated water and unclean workers. Since they are resistant to low and high temperature processing treatments, they are considered as the good indicators of faecal contamination in processed food products.

Materials required

• K.F. (Kenner Faecal) Agar
• 1% 2,3,5-Triphenyl Tetrazolium Chloride (TTC)
• Physilogical Saline
• Blood Agar
• Glucose-Phosphate Medium
• Sorbitol
• Trehalose
• Lactose
• Maltose
• Mannitol
• Dextrin
• Hydrogen peroxide
• Nitrate broth
• Gelatin agar

Procedure

About 25 g of fish sample is taken aseptically and homogenized with 225 ml of physiological saline. 0.1 ml of appropriate dilution of homogenized sample is spread plated onto KF agar plates and incubated at 37±1⁰C for 48 h. The colonies of faecal streptococci on KF agar appearing as red pinpoint colonies counted and results are expressed as number of colony forming units per g of the sample.

Calculation

Faecal Streptococci per g =(Number of colonies x dilution factor x10)/Weight of sample

Typical colonies may be isolated, purified, subcultured onto TSA plates and further subjected to the following biochemical reactions for confirmation. The typical reactions characteristic of faecal streptococci are as follows;

 

Tests	Reactions	Results
Catalase V.P Gelatinase Haemolysis (Blood agar) Nitrate Sorbitol Trehalose Lactose Maltose Dextrin Mannitol	No effervescence No change in colour Not liquefied Clear zone Not reduced Fermentative Fermentative Fermentative Fermentative Fermentative Fermentative	Negative Negative Negative Positive Negative Positive Positive Positive Positive Positive Positive

 

Media Composition
Kenner Faecal (KF) Streptococcal Agar

Protease peptone Yeast extract Sodium chloride Sodium glycerophosphate Maltose Latcose Sodium azide Sodium carbonate Agar Distilled water Bromocresol purple pH

- 10.0 g - 10.0 g - 5.0 g - 10.0 g - 20.0 g - 1.0 g - 0.4 g - 0.63 g - 20.0 g - 1000.0 ml - 0.015 g - 7.2 ± 0.2

Sterilize at 121^oC for 15 minutes. Cool to 50^oC and add 10 ml of 1% of 2,3,5 – Triphenyl tetrazolium chloride (TTC)and pour onto sterile plates.