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E. A. FREUNDT ET AL.
some antisera may occasionally exhibit species non-specific inhibitory activity. For example, antisera produced against strains of the bovine
“serogroup 7” (Leach, 1967) have been observerved in various laboratories
to inhibit the growth of M. bovigenitalium, although neither biochemical nor
other serological data suggest a relationship on the species level between
these two taxa (Ern0 and Jurmanovi, 1973). Mention may also be made of
the cross-reactions as yet unexplained, which are occasionally seen between
different species of the genus Acholeplasma.
A major disadvantage of the G I test is its low degree of sensitivity.
Another inconvenience, that may or may not be associated with the
relative lack of sensitivity of this test, is the difficulty sometimes experienced
in obtaining satisfactory inhibition of growth on solid medium, even with
antisera that may prove highly potent in the metabolism inhibition test.
This phenomenon tends to be more pronounced with some species than
with others. It is encountered particularly frequently within the genus
Acholeplasma, but may be seen also with rapidly growing strains of the
genus Mycoplasma, such as M . mycoides subsp. Capri. As described in
more detail later, several modifications have been developed with the
purpose of enhancing the sensitivity of the G I test. The following parameters have a decisive influence on the test results: the size of the inoculum
of mycoplasmas, medium composition, incubation temperature, amount of
antiserum, and the relative proportions of the different classes of immunoglobulin. As to the size of the inoculum, the density of colonies produced
by an inoculum containing 104-105 colony forming units per ml (CFUlml)
is about optimal. A lower density of colonies will detract from the accuracy
of measuring the size of the inhibitory zone or even make the reading of the
test unreliable. A higher density will diminish the zone of inhibition.
Variations in the medium composition, incubation temperature, and the
amount of antiserum used are made in order to create optimal conditions
for the antigen-antibody reaction, by allowing a larger amount of antibody
to react with a smaller number of mycoplasma cells within a given period of
time.
Very little is known about the involvement of different classes of immunoglobulins in growth inhibition, but some observations suggest an
association of the inhibitory effect with IgM and IgG, though perhaps to a
greater extent with the latter.
(b) Metabolism inhibition. The M I test utilises the ability of antibody to
inhibit metabolic activities such as glucose fermentation (Taylor-Robinson
et al., 1966), arginine catabolism (Purcell et al., 1966a), hydrolysis of urea
(Purcell et al., 1966b) or reduction of 2,3,5-triphenyl tetrazolium chloride
(Senterfit and Jensen, 1966).
E. A. FREUNDT ET AL.
some antisera may occasionally exhibit species non-specific inhibitory activity. For example, antisera produced against strains of the bovine
“serogroup 7” (Leach, 1967) have been observerved in various laboratories
to inhibit the growth of M. bovigenitalium, although neither biochemical nor
other serological data suggest a relationship on the species level between
these two taxa (Ern0 and Jurmanovi, 1973). Mention may also be made of
the cross-reactions as yet unexplained, which are occasionally seen between
different species of the genus Acholeplasma.
A major disadvantage of the G I test is its low degree of sensitivity.
Another inconvenience, that may or may not be associated with the
relative lack of sensitivity of this test, is the difficulty sometimes experienced
in obtaining satisfactory inhibition of growth on solid medium, even with
antisera that may prove highly potent in the metabolism inhibition test.
This phenomenon tends to be more pronounced with some species than
with others. It is encountered particularly frequently within the genus
Acholeplasma, but may be seen also with rapidly growing strains of the
genus Mycoplasma, such as M . mycoides subsp. Capri. As described in
more detail later, several modifications have been developed with the
purpose of enhancing the sensitivity of the G I test. The following parameters have a decisive influence on the test results: the size of the inoculum
of mycoplasmas, medium composition, incubation temperature, amount of
antiserum, and the relative proportions of the different classes of immunoglobulin. As to the size of the inoculum, the density of colonies produced
by an inoculum containing 104-105 colony forming units per ml (CFUlml)
is about optimal. A lower density of colonies will detract from the accuracy
of measuring the size of the inhibitory zone or even make the reading of the
test unreliable. A higher density will diminish the zone of inhibition.
Variations in the medium composition, incubation temperature, and the
amount of antiserum used are made in order to create optimal conditions
for the antigen-antibody reaction, by allowing a larger amount of antibody
to react with a smaller number of mycoplasma cells within a given period of
time.
Very little is known about the involvement of different classes of immunoglobulins in growth inhibition, but some observations suggest an
association of the inhibitory effect with IgM and IgG, though perhaps to a
greater extent with the latter.
(b) Metabolism inhibition. The M I test utilises the ability of antibody to
inhibit metabolic activities such as glucose fermentation (Taylor-Robinson
et al., 1966), arginine catabolism (Purcell et al., 1966a), hydrolysis of urea
(Purcell et al., 1966b) or reduction of 2,3,5-triphenyl tetrazolium chloride
(Senterfit and Jensen, 1966).