IX. IDENTIFICATION OF MYCOPLASMAS
407
(a) Antigens. As mentioned above, the antigen is a live mycoplasma culture
in liquid medium. For production of the antigen, the organism is preferably
grown in the test medium. Adaptation to this medium may be achieved by
subculturing two or three times. If the strain to be tested is not growing
well in medium B or SU (Appendix, Sections A, 1 and 3), another medium
must be selected on the basis of growth studies. For most strains, media
can be selected that will provide suitable conditions for performance of the
test. Difficulties are more likely to occur with glucose- and argininenegative strains, as the reduction of tetrazolium may be weak and inconsistent with some strains of species that are generally described as being
tetrazolium positive. I n such cases the problem may be overcome by
supplementing the test medium with sodium thioglycollate (0.1 %) which
reduces the redox potential and thereby potentiates the reduction caused
by the mycoplasmas.
A batch of the appropriate medium is inoculated with mycoplasmas,
incubated to the end of the log phase of growth and frozen as 1 ml aliquots
at - 70°C.
(b) Titration of antigen. Titration of the antigen is carried out to determine
the number of colour changing units (CCU) as defined below.
(1) 1 ml volumes of ten-fold serial dilutions (10-1-10-6) are prepared in
tubes using test medium as diluent.
(2) Two drops (0.05 ml) of each antigen dilution, and six drops (0.15 ml)
of test medium are mixed in the wells of a microtitre tray. Eight drops
(0.2ml) of test medium are placed in each of four wells and serve as
medium controls. Endpoint controls consist of four wells each containing
eight drops of test medium with the pH adjusted to the endpoint required
in the test. This control is not included in tetrazolium reduction tests.
(3) The plate is sealed with tape to avoid evaporation and incubated at
37°C or as otherwise required.
(4) The test is read daily over the reading mirror, and colour changes
noted. When the number of wells showing a change is stable, the endpoint
is determined. The highest dilution of antigen that causes a colour change
is regarded as containing 1 CCUjO.05 ml. From this is calculated the
dilution required to make an antigen preparation containing 100-1000
CCUjO.05 ml, the dose to be used in the test proper. At the same time one
obtains an idea of the length of the incubation period required for the test
proper. The stored antigen will maintain its titre for a considerable period
of time, and hence it is usually unnecessary to repeat the antigen titration
when performing the test proper. The frozen antigen should not, however,
be thawed more than once.
(c) Test medium. This consists of a suitable growth medium supplemented
407
(a) Antigens. As mentioned above, the antigen is a live mycoplasma culture
in liquid medium. For production of the antigen, the organism is preferably
grown in the test medium. Adaptation to this medium may be achieved by
subculturing two or three times. If the strain to be tested is not growing
well in medium B or SU (Appendix, Sections A, 1 and 3), another medium
must be selected on the basis of growth studies. For most strains, media
can be selected that will provide suitable conditions for performance of the
test. Difficulties are more likely to occur with glucose- and argininenegative strains, as the reduction of tetrazolium may be weak and inconsistent with some strains of species that are generally described as being
tetrazolium positive. I n such cases the problem may be overcome by
supplementing the test medium with sodium thioglycollate (0.1 %) which
reduces the redox potential and thereby potentiates the reduction caused
by the mycoplasmas.
A batch of the appropriate medium is inoculated with mycoplasmas,
incubated to the end of the log phase of growth and frozen as 1 ml aliquots
at - 70°C.
(b) Titration of antigen. Titration of the antigen is carried out to determine
the number of colour changing units (CCU) as defined below.
(1) 1 ml volumes of ten-fold serial dilutions (10-1-10-6) are prepared in
tubes using test medium as diluent.
(2) Two drops (0.05 ml) of each antigen dilution, and six drops (0.15 ml)
of test medium are mixed in the wells of a microtitre tray. Eight drops
(0.2ml) of test medium are placed in each of four wells and serve as
medium controls. Endpoint controls consist of four wells each containing
eight drops of test medium with the pH adjusted to the endpoint required
in the test. This control is not included in tetrazolium reduction tests.
(3) The plate is sealed with tape to avoid evaporation and incubated at
37°C or as otherwise required.
(4) The test is read daily over the reading mirror, and colour changes
noted. When the number of wells showing a change is stable, the endpoint
is determined. The highest dilution of antigen that causes a colour change
is regarded as containing 1 CCUjO.05 ml. From this is calculated the
dilution required to make an antigen preparation containing 100-1000
CCUjO.05 ml, the dose to be used in the test proper. At the same time one
obtains an idea of the length of the incubation period required for the test
proper. The stored antigen will maintain its titre for a considerable period
of time, and hence it is usually unnecessary to repeat the antigen titration
when performing the test proper. The frozen antigen should not, however,
be thawed more than once.
(c) Test medium. This consists of a suitable growth medium supplemented