The growth inhibition test (GIT) stems from the fact that high titre antiserum added into mycoplasma growth medium will inhibit the growth of the homologous mycoplasma species against which the antiserum was produced.
There are several methods of performing GIT; all differing on the mode of application or presentation of the antiserum. Thus antiserum may be impregnated into a paper disc and then the disc is placed on the surface of inoculated solid medium or it may be added directly into wells in the growth medium. It may also be added directly on to the inoculated surface of growth medium as a drop.
Whichever procedure is selected, it is important to validate it before its adoption. At PANVAC the method used and the one that is reproducible and is thus recommended is the agar-well method and this is the procedure described here.
I. Equipment:
II. Materials
CBPP Vaccine (previously titrated : SOP 2)
III. Procedure
Reconstitute a vial of pretitrated vaccine batch using 10 ml of cold, sterile distilled water, PBS or base counting medium.
Prepare ten-fold dilution series of the reconstituted vaccine suspension in the diluent used for reconstitution so as to obtain approximately 106 CCU per ml of suspension.
Lift the Petri dish cover slightly then place 150 μl of the diluted vaccine suspension at one end of agar plate. Tilt the plate so that the inoculum spreads evenly over the whole agar surface.
With the cover on, allow the plate to dry in a laminar air flow cabinet for 30 minutes.
Using a sterile 5 mm diameter metal or glass tube cut out a well from the inoculated agar medium. The cut out piece is removed by gentle suction or carefuly scooped out by use of a sterile 18-gauge needle.
Add 50μl of antiserum into the well and leave the plate covered in the laminar air flow cabinet for 2 to 3 hours to allow the serum to diffuse into the agar medium.
Place the agar plate upside down in a humidified chamber e.g. plastic sandwich box and incubate at 37°C.
Using an inverted microscope (16x) or a binocular dissection microscope (20x), examine the plate on day 3, day 6 and finally on day 10 post incubation for the appearance of mycoplasma colonies and the development and extent of a zone of growth inhibition around the antiserum well.
Measure the diameter of the inhibition zone in millimetres starting from the edge of the well on day 6, and finally on day 10 post-inoculation.
Record the observations.
IV. Interpretation of the result
A zone of inhibition measuring at least 2 mm should be observed. Inhibition of mycoplasma growth can be total (i.e. complete absence of mycoplasma colonies around the antiserum well), or nearly total (presence of break-through mycoplasma colonies within the inhibition zone) or it may be partial (relative decrease of colony numbers and size in the inhibition zone). Experience at PANVAC has shown that the degree of inhibition depends on the vaccine seed strain of mycoplasma used for preparation of the vaccine. For instance, quite often T1SR produces break-through colonies while T1/44 rarely does so. Where such break through colonies occur, they should be coloned and identified. If they are confirmed to be M. mycoides subsp. mycoides the vaccine preparation passes the test of identity in the growth inhibition test.
