Mannitol Egg Yolk Polymixin (MYP) agar is prepared and sterilized.
Aseptically transfer the MYP agar into a petriplate.
Divide the agar plate into three equal parts.
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.
Repeat the same procedure for the other Bacillus culture in Nutrient broth. Inoculate the culture on a segment of MYP agar.
Inoculate uninoculated broth (control) on one side of MYP agar.
Incubate the plates at (35 ± 1°c) for 24-48 hours.
After 24-48 hours examine the colour change in the medium and opacity zones around colonies.(use transmitted light to observe the halo)
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
Egg yolk agar plate was prepared, sterilized and aseptically transferred to a sterile petriplate.
The plate was divided into two halves.
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.
Allow to absorb and dry and mark the side of the plate inoculated with antitoxin.
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.
Inoculate the control organisms on the same plate in the similar procedure.
Incubate anaerobically in a gas pak jar immediately after streaking and transfer into the incubator maintained at 35-37o C for 24-48 hours.
Examine the plate for the opalescent halo surrounding the inoculum and disappearance of halo zones in the antitoxin side of the plate.
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.
Differences Encountered in a Real Laboratory:
In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab:
Always wear gloves, and lab coat.
Tie your hair properly to prevent any contamination from the microbial culture.
When you enter the lab switch on the exhaust fans.
Switch on the lights of the laminar air flow and blower. Prepare your work space (Laminar Air Flow Cabinet) or lab bench by wiping down the area with disinfectant.
Properly adjust the flame of the Bunsen burner. The proper flame is a small blue cone; it is not a large plume, nor is it orange.
Always label all tubes and plates with:
1. The name of the organism
2. The type of media
3. Your initials
4. The date.
While flaming the inoculation loop be sure that each segment of metal glows orange/red-hot before you move the next segment into the flame.
Once you have flamed your loop, do not lay it down, blow on it, touch it with your fingers, or touch it to any surface other than your inoculums. If you do touch the tip to another surface or blow on it, you will have to re-flame the loop before you proceed to your experiment.
Allow your loop to cool before you try to pick up your organism to avoid killing the inoculum.
When removing the caps from tubes, always keep the caps in your hand. Never set them on the table, as they could pick up contaminants.
Always handle open tubes at an angle near to the flame of the burner; never let them point directly up, since airborne or other environmental organisms could fall into the tube and cause contamination.
Open the lid of the plate sufficiently (45 degrees) to introduce an inoculation loop and only for the time it takes to obtain inoculums.
Rotate the plate counter clockwise 90 degrees and cross the prior streaks to pick up some bacteria and spread them into the next quadrant (Repeat in all the four quadrants).
Streak gently; does not gouge the agar.
As soon as the inoculation is completed, flame your loop or needle. Never place a contaminated tool on your workbench.
Turn the inoculated petriplate upside down while keeping it in the incubator.Do not expose the methylene blue indicator too much into the air. Close the anaerobic chamber tightly.
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