IX. IDENTIFICATION OF MYCOPLASMAS
397
IV. BIOCHEMICAL METHODS IN IDENTIFICATION
A. Sensitivity to digitonin
1. Test medium
This is a conventional agar medium containing 10-20% serum and
supporting good growth of the test strain. I t has been shown that all
Acholeplasma species are inhibited by 1.5% digitonin when grown on
either serum-free medium or on medium containing bovine serum fraction
instead of whole serum. For the distinction between Mycoplasma and
Acholeplasma species it is essential, therefore, that the digitonin test is
consistently carried out on medium enriched with serum (Freundt et al.,
1973a).
2. Reagent
Sterile filter paper discs, about 6.0 mm in diameter, of the type used in
antibiotic sensitivity tests, are impregnated with 0.02 ml of a 1.5% (w/v)
ethanolic solution of digitonin (E. Merck A.G., Darmstadt, West Germany)
dried overnight at 37°C and stored at 4°C until use. T o dissolve digitonin
in ethanol, heating for 20-30 min in a 50-56°C water bath is required.
3. Test procedure
The test is performed on agar medium which should be free from
surface moisture before inoculation. By means of a calibrated platinum
loop, a 0.01 ml drop from a liquid stock culture (Section 111) containing
approximately 105 CFU/ml is allowed to run down the surface of a slightly
tilted plate (running drop technique). When the fluid of the inoculum has
been absorbed, a digitonin disc is placed in the centre of the inoculated
area. The agar plates are incubated under optimal growth conditions for the
test and a reading is made when growth is visible under a low-power
microscope. The width of the zone of inhibition is measured in mm
(Ern0 and Stipkovits, 1973; Freundt et al., 1973a).
Each batch of digitonin discs should be tested against known positive
and negative control strains.
B. Urease activity
1. Reagent
(MnC12.4Hz0) is the test reagent.
2. Test procedure
An agar block, about 1 cm square, is cut from a plate culture and placed
with the colony side upward on a slide. One or two drops of the ureaA solution containing 1% urea and 0.8% manganous chloride
397
IV. BIOCHEMICAL METHODS IN IDENTIFICATION
A. Sensitivity to digitonin
1. Test medium
This is a conventional agar medium containing 10-20% serum and
supporting good growth of the test strain. I t has been shown that all
Acholeplasma species are inhibited by 1.5% digitonin when grown on
either serum-free medium or on medium containing bovine serum fraction
instead of whole serum. For the distinction between Mycoplasma and
Acholeplasma species it is essential, therefore, that the digitonin test is
consistently carried out on medium enriched with serum (Freundt et al.,
1973a).
2. Reagent
Sterile filter paper discs, about 6.0 mm in diameter, of the type used in
antibiotic sensitivity tests, are impregnated with 0.02 ml of a 1.5% (w/v)
ethanolic solution of digitonin (E. Merck A.G., Darmstadt, West Germany)
dried overnight at 37°C and stored at 4°C until use. T o dissolve digitonin
in ethanol, heating for 20-30 min in a 50-56°C water bath is required.
3. Test procedure
The test is performed on agar medium which should be free from
surface moisture before inoculation. By means of a calibrated platinum
loop, a 0.01 ml drop from a liquid stock culture (Section 111) containing
approximately 105 CFU/ml is allowed to run down the surface of a slightly
tilted plate (running drop technique). When the fluid of the inoculum has
been absorbed, a digitonin disc is placed in the centre of the inoculated
area. The agar plates are incubated under optimal growth conditions for the
test and a reading is made when growth is visible under a low-power
microscope. The width of the zone of inhibition is measured in mm
(Ern0 and Stipkovits, 1973; Freundt et al., 1973a).
Each batch of digitonin discs should be tested against known positive
and negative control strains.
B. Urease activity
1. Reagent
(MnC12.4Hz0) is the test reagent.
2. Test procedure
An agar block, about 1 cm square, is cut from a plate culture and placed
with the colony side upward on a slide. One or two drops of the ureaA solution containing 1% urea and 0.8% manganous chloride