Materials Required:

1. For Bacillus Species Identification:

1. Two Nutrient broth tubes subcultured with the suspected Bacillus colonies.
2. Inoculating loop.
3. Incubators, (35 ± 1°c).

Cultures:

1. Bacillus cereus.
2. Bacillus subtilis.

Medium Used:

1. Mannitol-egg yolk-Polymyxin (MYP) agar

 

2. For differentiating Clostridium species: Nagler's reaction

1. Thioglycolate broth with inoculated Clostridia species to be differentiated.
2. Clostridium perfringens Type A antitoxin.
3. Inoculating loop.
4. Hockey stick spreader.
5. Incubator.

Medium used

1. AnaeroGRO™ Egg Yolk Agar.

Cultures:

1. Positive control: Clostridium perfringens
2. Negative control: Clostridium sporogenes

Equipment used:

1. Anaerobic gas pak system

 

Procedure:

For Bacillus Species identification:

1. Mannitol Egg Yolk Polymixin (MYP) agar is prepared and sterilized.
2. Aseptically transfer the MYP agar into a petriplate.
3. Divide the agar plate into three equal parts.
4. Transfer a loopful of culture from the first nutrient broth of the suspected Bacillus culture. Inoculate a segment on the MYP agar surface with a very visible circular amount of organism. Continuosly streak on the medium to obtain isolated colonies.
5. Repeat the same procedure for the other Bacillus culture in Nutrient broth. Inoculate the culture on a segment of MYP agar.
6. Inoculate uninoculated broth (control) on one side of MYP agar.
7. Incubate the plates at (35 ± 1°c) for 24-48 hours.
8. After 24-48 hours examine the colour change in the medium and opacity zones around colonies.(use transmitted light to observe the halo)

 

Result:

Positive test: Bacillus colonies in the first plate appear pink red without mannitol fermentation and exhibit clear zones of opacity confirming lecithinase activity.

Negative test: Colonies in the second plate ferment mannitol, appear yellow with absence of opacity zones.

Control: No color change.

Gram positive rods with large zones of opacity that are lecithinase positive belong to Bacillus cereus group was confirmed in the first half of the plate and the second half of the plate confirmed the presence of lecithinase negative , mannitol fermenting Bacillus subtilis group.

 

For differentiating Clostridium species: Nagler's reaction

1. Egg yolk agar plate was prepared, sterilized and aseptically transferred to a sterile petriplate.
2. The plate was divided into two halves.
3. To one half of the plate add 60 µl of Clostridium perfringens type A antitoxin and spread evenly with the aid of a hockey spreader.
4. Allow to absorb and dry and mark the side of the plate inoculated with antitoxin.
5. Take a loopful of test organism from the Thioglycolate broth and streak it as a straight line from the antitoxin free half across to the antitoxin side of the plate.
6. Inoculate the control organisms on the same plate in the similar procedure.
7. Incubate anaerobically in a gas pak jar immediately after streaking and transfer into the incubator maintained at 35-37o C for 24-48 hours.
8. Examine the plate for the opalescent halo surrounding the inoculum and disappearance of halo zones in the antitoxin side of the plate.

 

Result:

Positive result: Disappearance or marked reduction of opacity in the antitoxin half of the plate due to lecithin neutralization by the antitoxin.

Negative result: No disappearance of opacity zones in the antitoxin half of the plate.