IX. IDENTIFICATION OF MYCOPLASMAS
405
3. Comments and suggestions fGr modi’jications
As mentioned already, the growth inhibition test is used very extensively
for identification of mycoplasmas. The test has the advantage of being easy
to perfoim and of exhibiting a high degree of specificity. As it has the
disadvantage, on the other hand, of being rather insensitive a variety of
modifications have been developed in attempts to increase the sensitivity.
In this respect the agar well method is definitely superior to the disc
technique. Inhibition zones may be increased still further by refilling the
wells at appropriate intervals during incubation (Black, 1973b). A further
improvement in sensitivity may be obtained by using suboptimal growth
conditions for the test organism, i.e. by lowering the incubation temperature
from 37°C to about 27-30°C and/or by reducing the concentration of
horse serum in the growth medium from 15-20y0 to 2% and omitting the
yeast extract (Freundt et al., 1973b). All these manoeuvres have the effect
of altering the ratio of the reactants, so that, within a given period of time,
increased amounts of antiserum react against a smaller number of mycoplasma cells proliferating at a reduced rate. The sensitivity of the test may
also be increased by the use of discs impregnated with antisera concentrated
with a dry polyacrylamide gel (Windsor and Trigwell, 1976).
It may also be mentioned at this point that the difficulties encountered
with the growth inhibition test because of its low sensitivity are more apt to
occur with some species than with others, and also that strains within one
species may vary. Such variations are not easily understood, but they may
be attributed to antigenic differences. This appears to be the case with, for
example, M . hominis. The rather frequent failure, experienced by several
workers, of antisera produced against the type strain (PG21) of this species
to inhibit the growth of heterotypic strains may be compensated for by the
use of combined type-specific antisera (Lin et al., 1975). Antisera against
three strains of M . hominis that differed serologically by agglutination and a
complement-dependent mycoplasmacidal test, although cross-reacting by
growth inhibition, proved in the study of Lin and collaborators to inhibit a
much wider spectrum of field strains than did each of the individual sera.
Apart from antigenic properties, the result of the G I test may also depend
on other factors such as membrane stability and the possible existence of
surface structures external to the cell membrane.
The choice of the G I procedure to be used for routine identification
depends on the given circumstances, such as the species involved and the
potency of the antisera available. I n general the best results are obtained
with the agar well method and the use of suboptimal growth conditions. The
agar well method is somewhat more laborious than the disc test, which has
the additional advantage of allowing long-term storage of the serumimpregnated discs at 4°C.
405
3. Comments and suggestions fGr modi’jications
As mentioned already, the growth inhibition test is used very extensively
for identification of mycoplasmas. The test has the advantage of being easy
to perfoim and of exhibiting a high degree of specificity. As it has the
disadvantage, on the other hand, of being rather insensitive a variety of
modifications have been developed in attempts to increase the sensitivity.
In this respect the agar well method is definitely superior to the disc
technique. Inhibition zones may be increased still further by refilling the
wells at appropriate intervals during incubation (Black, 1973b). A further
improvement in sensitivity may be obtained by using suboptimal growth
conditions for the test organism, i.e. by lowering the incubation temperature
from 37°C to about 27-30°C and/or by reducing the concentration of
horse serum in the growth medium from 15-20y0 to 2% and omitting the
yeast extract (Freundt et al., 1973b). All these manoeuvres have the effect
of altering the ratio of the reactants, so that, within a given period of time,
increased amounts of antiserum react against a smaller number of mycoplasma cells proliferating at a reduced rate. The sensitivity of the test may
also be increased by the use of discs impregnated with antisera concentrated
with a dry polyacrylamide gel (Windsor and Trigwell, 1976).
It may also be mentioned at this point that the difficulties encountered
with the growth inhibition test because of its low sensitivity are more apt to
occur with some species than with others, and also that strains within one
species may vary. Such variations are not easily understood, but they may
be attributed to antigenic differences. This appears to be the case with, for
example, M . hominis. The rather frequent failure, experienced by several
workers, of antisera produced against the type strain (PG21) of this species
to inhibit the growth of heterotypic strains may be compensated for by the
use of combined type-specific antisera (Lin et al., 1975). Antisera against
three strains of M . hominis that differed serologically by agglutination and a
complement-dependent mycoplasmacidal test, although cross-reacting by
growth inhibition, proved in the study of Lin and collaborators to inhibit a
much wider spectrum of field strains than did each of the individual sera.
Apart from antigenic properties, the result of the G I test may also depend
on other factors such as membrane stability and the possible existence of
surface structures external to the cell membrane.
The choice of the G I procedure to be used for routine identification
depends on the given circumstances, such as the species involved and the
potency of the antisera available. I n general the best results are obtained
with the agar well method and the use of suboptimal growth conditions. The
agar well method is somewhat more laborious than the disc test, which has
the additional advantage of allowing long-term storage of the serumimpregnated discs at 4°C.