IX. IDENTIFICATION OF MYCOPLASMAS
40 1
of the phenolphthalein diphosphate reagent. The pellet from 100ml of
uninoculated growth medium treated in the same way serves as a negative
control. After incubation at 37°C for 4 h, two drops of 5 N NaOH are
added. A positive reaction is indicated by the appearance of a red colour
in the test mixture.
3. Comments
The phosphatase test is based on the liberation of phenolphthalein and
its reaction with NaOH. The test on solid medium as recommended by
Aluotto et al. (1970) is used as the standard method at the FAO/WHO
Collaborating Centre for Animal Mycoplasmas. Whereas Aluotto et al.
perform the test after 3, 7 and 14 days’ incubation, using triplicate cultures,
one reading made after 7 days’ incubation is in our experience sufficient
for practical purposes. There is some indication that the plate test is working
less satisfactorily in some laboratories, false positive reactions frequently
being obtained. This may be explained by the presence in the test medium
of uninactivated phosphatase or perhaps by a spontaneous degradation of
sodium phenolphthalein diphosphate. Such difficulties may be overcome
by using the liquid medium test (2 above) which is slightly modified from
that of Burger et al. (1967) and Black (1973a). This method may have the
additional advantage of being more sensitive than the standard (plate)
method. A fluoregenic test allowing the detection of phosphatase in
mycoplasma cultures in 1 h has been described by Bradbury (1977).
F. Liquefaction of coqgulated serum
1. Test medium
medium, see Appendix, Section B, 5).
2. Test procedure
Two slopes are inoculated with a platinum loop, one from an undiluted
stock culture, the other from a dilution of 10-3. Readings are made at
frequent intervals during 14 days of incubation at 37°C. Liquefaction
becomes visible by the development of a shallow depressed area with a
moist base when growth is confluent, and of small pits when growth is
more scattered. I n the case of heavy liquefaction, fluid accumulates in the
angle between the base of the slope and the wall of the tube.
3. Comments
Other methods of demonstrating proteolytic activity, such as hydrolysis
of gelatin and digestion of casein have been used to characterise individual
strains or species (Freundt, 1958; Aluotto et al., 1970; Czekalowski et al.,
The substrate is horse serum coagulated as slopes in test tubes (Sd
14
40 1
of the phenolphthalein diphosphate reagent. The pellet from 100ml of
uninoculated growth medium treated in the same way serves as a negative
control. After incubation at 37°C for 4 h, two drops of 5 N NaOH are
added. A positive reaction is indicated by the appearance of a red colour
in the test mixture.
3. Comments
The phosphatase test is based on the liberation of phenolphthalein and
its reaction with NaOH. The test on solid medium as recommended by
Aluotto et al. (1970) is used as the standard method at the FAO/WHO
Collaborating Centre for Animal Mycoplasmas. Whereas Aluotto et al.
perform the test after 3, 7 and 14 days’ incubation, using triplicate cultures,
one reading made after 7 days’ incubation is in our experience sufficient
for practical purposes. There is some indication that the plate test is working
less satisfactorily in some laboratories, false positive reactions frequently
being obtained. This may be explained by the presence in the test medium
of uninactivated phosphatase or perhaps by a spontaneous degradation of
sodium phenolphthalein diphosphate. Such difficulties may be overcome
by using the liquid medium test (2 above) which is slightly modified from
that of Burger et al. (1967) and Black (1973a). This method may have the
additional advantage of being more sensitive than the standard (plate)
method. A fluoregenic test allowing the detection of phosphatase in
mycoplasma cultures in 1 h has been described by Bradbury (1977).
F. Liquefaction of coqgulated serum
1. Test medium
medium, see Appendix, Section B, 5).
2. Test procedure
Two slopes are inoculated with a platinum loop, one from an undiluted
stock culture, the other from a dilution of 10-3. Readings are made at
frequent intervals during 14 days of incubation at 37°C. Liquefaction
becomes visible by the development of a shallow depressed area with a
moist base when growth is confluent, and of small pits when growth is
more scattered. I n the case of heavy liquefaction, fluid accumulates in the
angle between the base of the slope and the wall of the tube.
3. Comments
Other methods of demonstrating proteolytic activity, such as hydrolysis
of gelatin and digestion of casein have been used to characterise individual
strains or species (Freundt, 1958; Aluotto et al., 1970; Czekalowski et al.,
The substrate is horse serum coagulated as slopes in test tubes (Sd
14