IX. IDENTIFICATION OF MYCOPLASMAS
41 1
may be read by means of an ordinary fluorescence microscope, using transmitted ultraviolet light, as well as by the epi-immunofluorescence method.
When applying the indirect immunofluorescence method to unfixed agar
colonies, the blocks with colonies are incubated with a drop of appropriately diluted antiserum for 30 min at 20°C in a moist chamber. The blocks
are washed twice for 10 min with PBS, p H 7.2, in test-tubes on the test-tube
rotator. A drop of appropriately diluted fluorescein-conjugated antirabbit
globulin is placed on each block, and the blocks are incubated for another
30 min at 20°C. After washing twice in PBS the blocks are mounted on
slides and examined by the epi-immunofluorescence method. A technical
modification of the epi-IMF test was recently described by Mdler (1979).
It has the advantage, inter alia, that the agar blocks are held in the same
position during the whole procedure and do not have to be moved from
slides to test tubes and back again.
Indirect immunofluorescence of hot water-fixed colonies is carried out
in essentially the same way, but here again washing is performed in a slide
carrier, the final wash being in distilled water.
(a) Working dilution of conjugates and antisera. The determination of
appropriate working dilutions for the conjugates and antisera to be used in
the test is a prerequisite for the correct procedure.
For the direct immunofluorescence test, fluorescein-conjugated antiserum is tested as described above in two-fold serial dilutions between,
for example, 1 : 10 and 1 : 320 against the homologous strain. Thereby the
optimal dilution, as defined by the highest dilution giving maximal fluorescence, is determined. It is advisable, however, to choose a somewhat lower
working dilution, since, to obtain a positive reaction, freshly isolated
strains of a species quite often require a higher concentration of antibody
than that needed for the type strain used in the production of the antiserum.
For the indirect immunofluorescence test, the working dilution of the
antirabbit globulin is determined on the basis of a chessboard titration
against a potent rabbit antiserum. Thereafter, the titration of other rabbit
antisera may be carried out solely against this dilution of conjugate to
determine the optimal dilution of each serum. Here again, in order to avoid
false negative results, it is advisable to select a working dilution lower than
the optimal for the identification of unknown strains.
The diluted conjugated antirabbit globulin should not be stored for
more than 1 week before use, whereas the diluted rabbit antisera are usable
for at least 1 month, even if subjected to repeated thawing and freezing.
(b) Controls. For each strain being tested, it is necessary to compare the test
slide with a negative control in order to detect non-specific binding.
41 1
may be read by means of an ordinary fluorescence microscope, using transmitted ultraviolet light, as well as by the epi-immunofluorescence method.
When applying the indirect immunofluorescence method to unfixed agar
colonies, the blocks with colonies are incubated with a drop of appropriately diluted antiserum for 30 min at 20°C in a moist chamber. The blocks
are washed twice for 10 min with PBS, p H 7.2, in test-tubes on the test-tube
rotator. A drop of appropriately diluted fluorescein-conjugated antirabbit
globulin is placed on each block, and the blocks are incubated for another
30 min at 20°C. After washing twice in PBS the blocks are mounted on
slides and examined by the epi-immunofluorescence method. A technical
modification of the epi-IMF test was recently described by Mdler (1979).
It has the advantage, inter alia, that the agar blocks are held in the same
position during the whole procedure and do not have to be moved from
slides to test tubes and back again.
Indirect immunofluorescence of hot water-fixed colonies is carried out
in essentially the same way, but here again washing is performed in a slide
carrier, the final wash being in distilled water.
(a) Working dilution of conjugates and antisera. The determination of
appropriate working dilutions for the conjugates and antisera to be used in
the test is a prerequisite for the correct procedure.
For the direct immunofluorescence test, fluorescein-conjugated antiserum is tested as described above in two-fold serial dilutions between,
for example, 1 : 10 and 1 : 320 against the homologous strain. Thereby the
optimal dilution, as defined by the highest dilution giving maximal fluorescence, is determined. It is advisable, however, to choose a somewhat lower
working dilution, since, to obtain a positive reaction, freshly isolated
strains of a species quite often require a higher concentration of antibody
than that needed for the type strain used in the production of the antiserum.
For the indirect immunofluorescence test, the working dilution of the
antirabbit globulin is determined on the basis of a chessboard titration
against a potent rabbit antiserum. Thereafter, the titration of other rabbit
antisera may be carried out solely against this dilution of conjugate to
determine the optimal dilution of each serum. Here again, in order to avoid
false negative results, it is advisable to select a working dilution lower than
the optimal for the identification of unknown strains.
The diluted conjugated antirabbit globulin should not be stored for
more than 1 week before use, whereas the diluted rabbit antisera are usable
for at least 1 month, even if subjected to repeated thawing and freezing.
(b) Controls. For each strain being tested, it is necessary to compare the test
slide with a negative control in order to detect non-specific binding.