Fish Microbiology and Quality Assurance (2+2)

10. Isolation and identification of Clostridia in fish and fishery products

Among Clostridia, Clostridium botulinum is important pathogen involved in intoxication type of food poisoning. These are widely distributed in soils and sediments of oceans and lakes. These anaerobic, spore forming, Gram-positive, rod shaped bacteria heat resistant, catalase-negative bacteria produce a potent proteinaceous toxin. Botulism is the type of food poisoning resulting from the consumption of foods contaminated with this toxin. Among seven recognized antigenic types (A, B, C, D, E, F and G), toxins of types C and D are non-proteolytic while others produce protein toxin. Clostridium botulinum type E is associated with the aquatic environments and hence involved in cases of botulism involving fish and other seafoods.

The isolation of Clostridium botulinum requires enrichment to facilitate their growth over other competing organisms and plating on selective agar medium.

Materials required

1. Chopped liver broth or cooked meat medium (CMM)
2. Trypticase peptone glucose yeast extract broth (TPGYB)
3. Trypticase peptone glucose yeast extract agar (TPGYA)
4. Gas-Pak system

Procedure

About 2-5 g of sample is aseptically transferred directly into 100 ml of enrichment broth (CMM/TPGYB) and incubated at 28 ± 2⁰C for 5 – 6 days. This enriched culture contacting both vegetative cells and endospore of C. botulinum are treated with alcohol or heat to kill vegetative cell. Two ml each of enrichment culture sediment is transferred to two sterile test tubes. Equal volume of filter sterilized absolute ethyl alcohol is added to 1 tube and held at room temperature for 1 h with occasional shaking to kill all vegetataive cells. The other tube is heated at 80⁰C for 10 min to destroy vegetataive cells. After pre-treatment, enrichment cultures are streaked onto pre-dried TSGYA plates of and incubated for 48 – 72h at 30⁰ C under anaerobic conditions of Gas-Pak system.
Typical Clostridium botulinum colonies on TSGYA appear raised or flat and commonly show some spreading and exhibit surface irridescence (pearly layer) covering both the zone of precipitation and the halo of clearing around the colony as seen by reflected light. These colonies can be picked and further checked for the toxin production by growing in tubes of CMM or TPGY broth at 30⁰ C for 3 days. Presence of toxin is detected by centrifuging a portion of the culture at 10000xg for 15min at 4⁰C and using the supernatant for toxicity test by mouse bioassay.

Media composition

Cooked Meat Medium (CMM)

Beef heart : 500.0 g
Tryptone : 10.0 g
Proteose peptone : 20.0 g
Yeast extract : 5.0 g
Sodium chloride : 5.0 g
D-Glucose : 4.0 g
Sodium thioglycollate : 1.0 g
Soluble starch : 2.0 g
Distilled water : 1000.0 ml
Sodium hydroxide (1N) : 25.0 ml
pH : 7.4

Sterilize at 121⁰C for 15 minutes.

Trypticase-Peptone Glucose Yeast extract Broth (TPGYB)

Tryptone : 50.0 g
Bacto-peptone : 5.0 g
Yeast extract : 20.0 g
Dexttrose : 4.0 g
Sodium thioglycollate : 1.0 g
Distilled water : 1000 ml
Final pH : 7.0 ± 0.1

Sterilize at 121⁰C for 15 min. For the growth of C. botulinum type E, aseptically add 1 ml of trypsin solution per 15 ml broth. Trypsin solution is prepared by dissolving 15 g trypsin (1:250) in 100 ml water and sterilized by filtering through 0.45 µm Millipore membrane filter.

Tryptone Soytone Glucose Yeast extract Agar (TSGYA)

Tryptone : 50.0 g
Soytone : 5.0 g
Yeast extract : 20.0 g
Dextrose : 50.0 g
Sodium bicarbonate : 1.0 g
Agar : 20.0 g
Distilled water : 900 ml
Egg yolk emulsion (50%) : 100ml
pH : -7.2 ± 0.1

Sterilize at 121⁰C for 15 min. Dextrose and egg yolk emulsion (50%) are added to the sterile cooled medium. 50 % sterile glucose solution is prepared by filtering through 0.2µm Millipore filter.