S.O.P.4: DIGITONIN SENSITIVITY TEST FOR THE DETECTION OF ACHOLEPLASMA CONTAMINATION.

Mycoplasmas require sterols in the growth media for incorporation into their cytoplasmic membranes. Cholesterol in particular plays a key role in the synthesis and maintenance of the integrity of mycoplasma cell membranes. Cholesterol is normally supplied by animal serum which is added to mycoplasma growth media. Acholeplasmas are able to synthesise sterols on their own and they can therefore grow in media without added sterol sources such as animal serum. Digitonin induces lysis of mycoplasma cells via the formation of digitonide-cholesterol precipitation complexes which lead to increased permeability of mycoplasma cell membranes. This increased leakiness of the membrane eventually causes mycoplasma death. Thus growth of mycoplasmas colonies on agar is inhibited by digitonin while Acholeplasmas are unaffected. This differential characteristic can therefore be used to detect any Acholeplasma contamination in CBPP vaccines.

I. Equipment

• Class II laminar flow cabinet
• Universal (30 ml) containers
• Sterile pipettes, 5 and 10 ml
• Pipetters range 5 to 30 μl ; 40 to 200 μl and tips
• Incubator, set at 37± 0.5°C
• 60 × 15 mm Petri dishes
• 18 gauge needles
• Sterile thumb forceps
• Humidified box

II. Materials

• Digitonin discs (SOP 1)
• Mycoplasma agar (SOP 1)
• CBPP Vaccine

III. Test procedure

1. Reconstitute a vial of CBPP vaccine using a volume of cold, sterile distilled water that is equal to the filling volume of the vaccine vial. Keep the vaccine at 4°C

2. Dry the previously prepared mycoplasma agar plate (with lid on) by keeping it in a 37°C for 2 hours.

3. Taking the reconstituted vaccine suspension as neat, prepare ten-fold dilutions using sterile distilled water, PBS or counting medium base so as to give a mycoplasma concentration of about 10⁶ (1 million). In most CBPP vaccines tested at PANVAC this would be dilution 10^-3. Using 150 μl of the appropriately diluted vaccine, flood the plate of the agar medium uniformly.

4. Place the disc of digitonin on the inoculated agar plate and press lightly on to the agar surface.

5. With the lid on allow the agar plate to dry by keeping it in a laminar air flow cabinet for 1 hour.

6. Place the agar plate upside down in a moist chamber and incubate it at 37°C.

7. Examine the agar plate on day 3, day 6 and finally on day 10 under a 16x objective of an inverted microscope for zone and extent of inhibition of mycoplasma growth all around the digitonin discs. Record the observations.

IV. Interpretation of the observations

The vaccine passes the test for digitonin sensitivity if a zone of mycoplasma growth inhibition measuring at least 5 mm from the edge of the digitonin disc is observed. If there are breakthrough colonies within the inhibition zone Acholeplasma and/or bacterial contamination can be suspected and the vaccine fails the test for digitonin sensitivity. The breakthrough colonies should be cloned and identified.