41 2
E. A. FHEUNDT ET AL.
Conjugated normal rabbit serum is used in the direct, and non-conjugated
normal rabbit serum in the indirect method. The less experienced investigator is also advised to include positive 'controls in each test, i.e. to
test each antiserum against a homologous reference strain.
4. Comments
The epi-IMF test, especially when using unfixed agar-grown colonies as
antigen, has several obvious advantages and has in fact contributed very
significantly to making I M F a practical and easy tool for rapid identification
of mycoplasmas. I n the hands of an experienced investigator the epi-IMF
test is as easy to perform as the G I test, highly reliable, and may lead to a
specific diagnosis within a few hours. In contrast to the G I test the reading
is not affected by the inoculum size, as long as the colonies are small and
well separated. The use of colonies grown on less complex media (ssumfree or reduced concentration of serum) is advantageous. Moreover, the
test can be carried out on a mixed culture and may even help to detect the
occurrence of a mixture of organisms in a culture or a single colony (Del
Giudice et al., 1967; Al-Aubaidi and Fabricant, 1971).
Although both the direct and the indirect versions of the I M F test have
been described above, the indirect method would seem for several reasons
to be superior to the direct method: (1) only one conjugate is needed, (2)
the non-specific background fluorescence that may interfere with reading
and interpretation of the test is less pronounced, and (3) it has been shown
to be more sensitive, yielding two to eight-fold higher titres than the
direct method (Rosendal and Black, 1972; Black and Krogsgaard- Jensen,
1974). Recently, a promising new method, the indirect immunoperoxidase
test, has been introduced and compared with the indirect I M F test (PolakVogelzang et al., 1978).
E. Direct agglutination
Although the authors have very little personal experience with direct
agglutination as applied to mycoplasmas it is felt that the basic techniques
of this very simple method should be briefly described. T h e description that
follows of the tube agglutination test is based essentially on the procedure
used by Edward (1950).
1. Tube agglutination
(a) Preparation of antigen. A culture in 200-1000 ml of liquid medium is
harvested at the end of the logarithmic growth phase in a refrigerated angle
centrifuge for 1 h at approximately 10 000 rev/min (12 000 x g). The
deposit is washed once by suspending in saline and recentrifuging. For
E. A. FHEUNDT ET AL.
Conjugated normal rabbit serum is used in the direct, and non-conjugated
normal rabbit serum in the indirect method. The less experienced investigator is also advised to include positive 'controls in each test, i.e. to
test each antiserum against a homologous reference strain.
4. Comments
The epi-IMF test, especially when using unfixed agar-grown colonies as
antigen, has several obvious advantages and has in fact contributed very
significantly to making I M F a practical and easy tool for rapid identification
of mycoplasmas. I n the hands of an experienced investigator the epi-IMF
test is as easy to perform as the G I test, highly reliable, and may lead to a
specific diagnosis within a few hours. In contrast to the G I test the reading
is not affected by the inoculum size, as long as the colonies are small and
well separated. The use of colonies grown on less complex media (ssumfree or reduced concentration of serum) is advantageous. Moreover, the
test can be carried out on a mixed culture and may even help to detect the
occurrence of a mixture of organisms in a culture or a single colony (Del
Giudice et al., 1967; Al-Aubaidi and Fabricant, 1971).
Although both the direct and the indirect versions of the I M F test have
been described above, the indirect method would seem for several reasons
to be superior to the direct method: (1) only one conjugate is needed, (2)
the non-specific background fluorescence that may interfere with reading
and interpretation of the test is less pronounced, and (3) it has been shown
to be more sensitive, yielding two to eight-fold higher titres than the
direct method (Rosendal and Black, 1972; Black and Krogsgaard- Jensen,
1974). Recently, a promising new method, the indirect immunoperoxidase
test, has been introduced and compared with the indirect I M F test (PolakVogelzang et al., 1978).
E. Direct agglutination
Although the authors have very little personal experience with direct
agglutination as applied to mycoplasmas it is felt that the basic techniques
of this very simple method should be briefly described. T h e description that
follows of the tube agglutination test is based essentially on the procedure
used by Edward (1950).
1. Tube agglutination
(a) Preparation of antigen. A culture in 200-1000 ml of liquid medium is
harvested at the end of the logarithmic growth phase in a refrigerated angle
centrifuge for 1 h at approximately 10 000 rev/min (12 000 x g). The
deposit is washed once by suspending in saline and recentrifuging. For