Practical Technique- Cultivation of Clostridia From Soil

Clostridia are obligatory anaerobic, gram-positive, spore bearing rods, unlike spores of genus Bacillus, species of genus Clostridium produce spores with prominent and elaborate bulging, moreover spores are round or oval and occur at the terminal end; hence they have drumstick or racket shaped appearances.

Clostridia are commonly found in soil, manure, sewage polluted lands, and in intestinal tract of man and animals. They require complex organic media such as cooked meat medium and blood-glucose-infusion agar or broth. Majority of Clostridia are harmless and helpful saprophytes. Many of them produce enzymes, chemicals and are of great value in industrial fermentations. Some important species are as follows.

·         Clostridium tetani: produces a potent endotoxin (neurotoxin), responsible for tetanus (lockjaw).

·         Clostridium perfringens: produces a potent exotoxin (enterotoxin), responsible for food poisoning.

·         Clostridium perfringens, Clostridium novyi, Clostridium septicum and Clostridium histolyticum: cause gas gangrene (rapid invasion and liquefictive necrosis of muscle with profuse gas formation).

·         Clostridium botulinum: produces a potent endotoxin (neurotoxic), responsible for botulism (food-borne intoxication).

·         Clostridium acetobutylicum: used for industrial production of butanol, acetone and ethanol.

·         Clostridium pasteurianum: is nitrogen fixing bacterium.

Principle: 

Selective isolation of Clostridia from soil can be achieved by;

1.      Heat treatment of the soil suspension: heat shock destroys the sensitive vegetative non-spore forming bacteria. However, Clostridia can survive heat shock because they form endospores.

2.      Providing anaerobic environments: among the surviving spore formers, only anaerobic organisms (Clostridia) will be able to grow.

Requirements

1.      Thioglycollate broth and Robertson’s bullock’s heart cooked medium (RCM).

2.      Sterile distilled water tubes (10ml) and sterile pipettes (10ml and 1ml).

3.      Sterile paraffin oil.

4.      Soil sample from sewage polluted lands or horse manure.

Procedure

1.      Suspend 1g of soil manure in 10ml of sterile distilled water.

2.      Mix contents of the tube well and hold the tube in water bath at 90°C for 10 minutes, so as to destroy vegetative cells.

3.      After heat shock, place the tube aside and leave it undisturbed to allow particulate matter to settle down.

4.      Place tubes of thioglycollate broth and RCM medium in boiling water bath for 10 minutes to expel out oxygen present in tubes.

5.      Remove media tubes from water bath immediately cool to 50°C.

6.      With the help of 1ml sterile pipette inoculate 0.5-1ml of soil supernatant deep at the bottom of the medium into thioglycollate and RCM medium.

7.      With the help of 10ml sterile pipette, overlay sterile paraffin oil up to about 2-3 cm above the medium, (it is specifically recommended for medium which are filled in ordinary tubes rather than screw-capped tubes)

8.      Incubate broth media at Room temperature for 5-7 days.

9.      After incubation observe changes occurring in media.

10.  Withdraw a loopful of suspension from bottom of the tube and prepare smear. Stain the smear by Gram’s staining.

11.  Examine the slide under microscope and search for Gram-positive rods, having bulging terminal or sub terminal spores; i.e., racket shaped or drumstick shaped cells.

12.  Presence of such cells indicates the growth of Clostridium in an anaerobic medium.

Interpretations

·         Thioglycollate broth:

Change in colour of litmus or methylene blue to colourless state indicates the anaerobic conditions of the medium, and the development of turbidity in the medium indicates the growth of organisms.

Gram’s staining showing the presence of gram-positive racket shaped cells indicates the growth of Clostridia.

·         Robertson’s cooked meat medium:

Following changes are observed in medium.

1.      The colour of meat changes pink to black.

2.      Putrefaction of the meat pieces, which generates foul smell.

Gram’s staining showing presence of gram-positive racket shaped cells which indicate growth of Clostridia.


Did you read ? 
Maintenance of Sterility in Animal Tissue Culture.
Practical Technique- Fluorescence Staining for Mycoplasma