S.O.P.8: BIOCHEMICAL TESTS FOR THE IDENTIFICATION OF M. MYCOIDES SUBSP. MYCOIDES

I The Test for Fermentation of Glucose

1. Reconstitute a vial of vaccine in volume of cold, sterile distilled water equivalent to the filling volume of the vial.

2. Inoculate the vaccine suspension into tubes of glucose medium (see SOP 1) at a ratio of 1 part vaccine suspension to 9 parts medium. (At PANVAC 2 bijoux bottles each containing 2.25 ml of medium are inoculated with 250 μl of vaccine suspension).

3. A positive control (e.g. M. bovirhinis) and the vaccine reconstitution fluid as the negative control should be included in the test.

4. Incubate the tubes at 37 °C for a maximum of 10 days.

5. A positive reaction is indicated by change of medium colour from pink to yellow. A similar colour change should occur in the positive control tube. There should be no colour change in the negative control bottles.

6. The test is valid if no colour change occurs in the negative control tubes and the colour of medium changes from pink to yellow in the positive control tubes.

II The Test for Arginine Hydrolysis

1. Using the reconstituted vaccine suspension 2 tubes of arginine medium (SOP 1) are inoculated as in the test for glucose fermentation.

2. The positive control in this test is a tube inoculated with a culture of M. arginini while the negative control tube is inoculated with the vaccine reconstitution fluid.

3. The tubes are incubated aerobically at 37°C for 10 days.

4. A positive reaction is indicated by change of medium colour to deep purplish-red. The test is valid if the colour of positive control medium changes to deep purple and no colour change occurs in the negative control medium tubes.

III The Test for Tetrazolium Reduction

A. Broth Culture Method

1. Inoculate 4 tubes of tetrazolium reduction medium (SOP 1) as in the test for glucose fermentation. Two tubes of positive control medium are inoculated with a culture of M. bovirhinis while two negative control tubes are inoculated with the fluid used to reconstitute the vaccine.

2. Incubate one pair of the tubes aerobically and the other half anaerobically at 37°C for 10 days.

4. A positive tetrazolium reduction test is indicated by the development of a brick red coloration in the test medium.

5. The test is valid if the colour of the positive control medium develops a brick-red colouration and no colour change occurs in the negative control tubes.

B. Agar Plate Method

In this procedure an overlay of agar containing the tetrazolium salt is poured over Mycoplasma colonies on agar and the plate examined for colour change typical of tetrazolium reduction.

1. Prepare a culture of the vaccine under test on two mycoplasma agar plates as was described for the test of digitonin sensitivity (SOP 4). Incubate one plate aerobically and the other anaerobically at 37°C for 6–10 days.

2. Prepare 1% w/v suspension of agar pH 7.2 in distilled water. Melt the agar by autoclaving at 110°C for 10 minutes. Equilibrate the molten agar to 50°C in a water bath. Prepare 0.2% w/v solution of 2,3,5-triphenyl tetrazolium chloride in distilled water. Sterilize the solution by filtration through a membrane filter of pore size 0.2 μm then equilibrate it to 50°C.

3. Mix equal volumes of the molten agar and the tetrazolium salt solution. Add 4 ml of this mixture on to the surface of each of the agar plates containing the mycoplasma colonies being tested.

4. Incubate the plate aerobically at 37°C. Examine the plate after 1, 2 and 3 hours post incubation. A positive tetrazolium reduction test is indicated by the appearance of a brick-red colouration around the mycoplasma colonies. The test is valid if a brick-red colouration is observed in the positive control plate and the negative control plate should not show such colour change.

IV. The test for Phosphatase activity

1. Reconstitute the content of the vaccine vial using a volume of cold, sterile distilled water equivalent to the vial filling volume.

2. Inoculate a plate of phosphatase agar medium (SOP 1) with 0.2 ml of the reconstituted vaccine suspension. The positive control in this test is a plate inoculated with M. mycoides subsp. mycoides (LC) while the negative control is a medium plate inoculated with the sterile distilled water used for reconstitution.

3. Incubate inoculated plates in a moist chamber at 37°C for 5–10 days.

4. Examine the plates using an inverted microscope (16x) for the appearance of mycoplasma colonies. When the colonies are clearly visible (usually on day 6 of incubation), pour 1 ml of 40% solution of NaOH or KOH onto each plate.

5. A positive phosphatase activity is indicated by the appearance of a pink-red colouration around the mycoplasma colonies within 30 seconds upon the addition of the alkaline solution. The test is valid if the positive control plate shows an appearance of red colour around the mycoplasma colonies and there should be no colour change in the negative control plate.

V. The test for liquefaction of coagulated serum

1. Reconstitute the vaccine as described for the test of phosphatase activity. Prepare a culture using the vaccine suspension by inoculating 0.25 ml into a bijoux bottle containing 2.25 ml of complete Counting or Gourlay medium (SOP 1). Incubate the bottles at 37°C for 2–3 days.

2. By means of a sterile bacteriological loop streak the vaccine broth culture along the surface of the serum slant of the test medium (SOP 1). A medium tube similarly inoculated with a culture of M. mycoides subsp.mycoides (LC) serves as the positive control. The negative control is a medium tube streaked with Counting or Gourlay medium.

3. Incubate inoculated tubes at 37°C for 14 days. Examine test medium tubes on day 5, day 10 and finally on day 14.

4. A positive proteolysis reaction is indicated by the appearance of gutters along the streak lines with the medium becoming transparent and/or the collection of fluid at the bottom of the slant. The test is valid if the positive control tube shows a positive reaction and the negative control tube shows no evidence of serum proteolysis.

CHARACTERISTICS OF M. mycoides subsp. mycoides

NB: v = Variable +/- = positive or negative