386
E. A. FREUNDT ET AL.
antigen, Chanock and co-workers used agar-grown colonies transferred to
microscope glass slides. This technique, which was further modified by
Lind (1970) and Rosendal and Black (1972), is relatively laborious and
time-consuming. An approach to a rapid laboratory diagnosis of M .
pneumoniae pneumonia was subsequently made by Hers (1963) using
immunofluorescence to identify M. pneumoniae associated with respiratory
tract cells excreted in the sputum of patients suffering from primary
atypical pneumonia. This method apparently never came into common use.
A major advance in the utilisation of the immunofluorescence technique
for the identification of mycoplasmas was made in 1967 by Del Giudice
et al., who developed the epi-immunofluorescence test. This allowed the
application of the fluorescent antibody staining method to mycoplasma
colonies grown on solid medium and the direct examination of the stained
colonies using a microscope with an attachment for incident illumination.
Whereas the direct fluorescent antibody staining technique was employed
by Del Giudice et al., definite advantages were demonstrated by Rosendal
and Black (1972) in using the indirect staining technique.
As is the case with the GI and M I tests the antigens reacting with
antibody in the immunofluorescence test are mainly associated with the
cell membrane. The testing of living unfixed colonies in situ on the agar
surface, i.e. the avoidance of any manipulation with the mycoplasma cells
prior to antibody-labelling, implies that the native structure of the antigens
involved in the reaction is maintained.
The specificity of the epi-immunofluorescence test is equal to that of
the G I test and its sensitivity is much higher. T h e epi-immunofluorescence
test has the additional advantage over the G I test of producing a quantitative result, since sera can be titrated and endpoints of 1 : 1000 or more are
usually obtained. Since, moreover, the epi-immunofluorescence test is easy
to standardise, a high priority must obviously be given to this test for the
routine identification of mycoplasmas. The only real drawback of the test is
the tendency of strains of some species to autofluoresce.
It should further be pointed out that the unexpected cross-reactions
between different species of the genus Acholeplasma that were previously
mentioned when discussing the G I test may also be seen with the immunofluorescence test. Collectively, these observations suggest that species
differentiation within the genus Acholeplasma is perhaps less well-founded
and not as clear-cut as in the genus Mycoplasma.
(d) Direct agglutination. Agglutination of mycoplasma cells by antiserum
was among the first tests used for serological classification. Although in the
hands of experienced investigators (Klieneberger, 1938; Edward, 1950;
Edward and Fitzgerald, 1951, 1954; Nicol and Edward, 1953; Edward and
E. A. FREUNDT ET AL.
antigen, Chanock and co-workers used agar-grown colonies transferred to
microscope glass slides. This technique, which was further modified by
Lind (1970) and Rosendal and Black (1972), is relatively laborious and
time-consuming. An approach to a rapid laboratory diagnosis of M .
pneumoniae pneumonia was subsequently made by Hers (1963) using
immunofluorescence to identify M. pneumoniae associated with respiratory
tract cells excreted in the sputum of patients suffering from primary
atypical pneumonia. This method apparently never came into common use.
A major advance in the utilisation of the immunofluorescence technique
for the identification of mycoplasmas was made in 1967 by Del Giudice
et al., who developed the epi-immunofluorescence test. This allowed the
application of the fluorescent antibody staining method to mycoplasma
colonies grown on solid medium and the direct examination of the stained
colonies using a microscope with an attachment for incident illumination.
Whereas the direct fluorescent antibody staining technique was employed
by Del Giudice et al., definite advantages were demonstrated by Rosendal
and Black (1972) in using the indirect staining technique.
As is the case with the GI and M I tests the antigens reacting with
antibody in the immunofluorescence test are mainly associated with the
cell membrane. The testing of living unfixed colonies in situ on the agar
surface, i.e. the avoidance of any manipulation with the mycoplasma cells
prior to antibody-labelling, implies that the native structure of the antigens
involved in the reaction is maintained.
The specificity of the epi-immunofluorescence test is equal to that of
the G I test and its sensitivity is much higher. T h e epi-immunofluorescence
test has the additional advantage over the G I test of producing a quantitative result, since sera can be titrated and endpoints of 1 : 1000 or more are
usually obtained. Since, moreover, the epi-immunofluorescence test is easy
to standardise, a high priority must obviously be given to this test for the
routine identification of mycoplasmas. The only real drawback of the test is
the tendency of strains of some species to autofluoresce.
It should further be pointed out that the unexpected cross-reactions
between different species of the genus Acholeplasma that were previously
mentioned when discussing the G I test may also be seen with the immunofluorescence test. Collectively, these observations suggest that species
differentiation within the genus Acholeplasma is perhaps less well-founded
and not as clear-cut as in the genus Mycoplasma.
(d) Direct agglutination. Agglutination of mycoplasma cells by antiserum
was among the first tests used for serological classification. Although in the
hands of experienced investigators (Klieneberger, 1938; Edward, 1950;
Edward and Fitzgerald, 1951, 1954; Nicol and Edward, 1953; Edward and