Understand the principles and role of the Corynebacterium diphtheriae virulence test.

Operation method

Agar plate virulence test

Principle

The toxin and the antitoxin meet by diffusion within the agar, and when in the right proportions they form specific precipitation lines visible to the naked eye. This test replaces the animal intradermal test, and has the advantage of simplicity, without the need for animals, and is basically the same as the results of the animal test.

Move

Take 11 ml of phosphate protein agar and dissolve it by heating, leave it to cool down to 50 °C and pour it into a sterilized 7.5 cm diameter flat dish.

After solidification, put it into a 37 ℃ incubator for about one hour to dry the surface of the medium.

Tweezers take a small circle of filter paper sheet immersed in diphtheria antitoxin (note that the remaining liquid on the filter paper should be allowed to drain away when it is taken out), and then place the round filter paper sheet containing antitoxin in the center of the plate.

At a distance of 6-8mm from the edge of the small circle filter paper, inoculate several strains of diphtheria rod-shaped bacillus cultures to be examined, and the inoculation area is about equal to the size of the small circle filter paper (it is better to inoculate more bacteria).

Incubate at 37 ℃ for 24-48h, and a white precipitation line will appear between the small round filter paper and the culture in positive cases.

Common Problems

If the strain to be examined is virulent, a clear white precipitation line appears between it and the filter paper sheet containing diphtheria antitoxin; if there is a positive control, this precipitation line often coincides with that of the positive control strain.

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