IX. IDENTIFICATION OF MYCOPLASMAS
423
1972). Alternatively, antisera can be absorbed with lyophilised medium
at a rate of 10 mg ml-l (Ross and Karmon, 1970).
(d) Production of antigen. Cultures in the late log-phase are harvested by
centrifugation at 4°C and washed three times in PBS. If the last wash is
carried out in a known volume and samples are taken before centrifugation
for protein estimation (Lowry et al., 1951), the protein content of the final
pellets can be determined. Pellets are drained well and stored frozen at or
below -30°C.
For D I D tests, the mycoplasmas need to be disrupted or lysed. The
methods usually employed are: alternate cycles of freezing and thawing,
ultrasonic treatment, or lysis with detergents. For freezing and thawing.
pellets are resuspended in deionised water to an appropriate protein
concentration (5-10 mg protein ml-I), frozen rapidly in a mixture of dry
ice and acetone, and thawed in luke-warm water. This is carried out
10-20 times. The details of ultrasonic treatment depend on the instrument
available. With a Branson Sonifier S-75 operating at 20 kc s-1, mycoplasmas
suspended in PBS are treated for five periods of 1 min, each period being
separated by a cooling interval of 1 min. Tubes are kept in ice and water
throughout.
Most of the precipitin bands produced by frozen-thawed or ultrasonically treated suspensions are due to cytoplasmic antigens (Hollingdale
and Lemcke, 1970; Lemcke, 1973). T o release additional antigens which are
bound to the membrane and thus to obtain a more complete precipitin
pattern, it is necessary to use a detergent. We have found that the non-ionic
detergent Triton X-100 is best for this purpose because it does not give
non-specific precipitates with rabbit serum as do sodium dodecyl sulphate
and sodium deoxycholate. Triton X-100 is added to suspensions in PBS
at the rate of 5 mg mg-1 of mycoplasma protein and incubated for 15 min
at 37°C.
Antigens prepared by freezing and thawing, ultrasonic treatment or
lysis with Triton X-100 are usually satisfactory at 5-10 mg protein rnl-l,
but it is advisable to first test an antigen against its homologous antiserum
over a range of concentrations (e.g. 1.25, 2.5, 5-0 and 10 mg ml-1) to see
at which concentration the lines are sharpest and best resolved. The
choice of concentration is inevitably somewhat arbitrary since each
“antigen” is, in fact, a complex mixture of antigens and the concentration
selected cannot be optimal for all the components.
3. Test procedure
T h e slides or cover glasses are wiped free of acetone, set out on a levelling
table (Shandon Southern Instruments Ltd) and the metal rings centred on
423
1972). Alternatively, antisera can be absorbed with lyophilised medium
at a rate of 10 mg ml-l (Ross and Karmon, 1970).
(d) Production of antigen. Cultures in the late log-phase are harvested by
centrifugation at 4°C and washed three times in PBS. If the last wash is
carried out in a known volume and samples are taken before centrifugation
for protein estimation (Lowry et al., 1951), the protein content of the final
pellets can be determined. Pellets are drained well and stored frozen at or
below -30°C.
For D I D tests, the mycoplasmas need to be disrupted or lysed. The
methods usually employed are: alternate cycles of freezing and thawing,
ultrasonic treatment, or lysis with detergents. For freezing and thawing.
pellets are resuspended in deionised water to an appropriate protein
concentration (5-10 mg protein ml-I), frozen rapidly in a mixture of dry
ice and acetone, and thawed in luke-warm water. This is carried out
10-20 times. The details of ultrasonic treatment depend on the instrument
available. With a Branson Sonifier S-75 operating at 20 kc s-1, mycoplasmas
suspended in PBS are treated for five periods of 1 min, each period being
separated by a cooling interval of 1 min. Tubes are kept in ice and water
throughout.
Most of the precipitin bands produced by frozen-thawed or ultrasonically treated suspensions are due to cytoplasmic antigens (Hollingdale
and Lemcke, 1970; Lemcke, 1973). T o release additional antigens which are
bound to the membrane and thus to obtain a more complete precipitin
pattern, it is necessary to use a detergent. We have found that the non-ionic
detergent Triton X-100 is best for this purpose because it does not give
non-specific precipitates with rabbit serum as do sodium dodecyl sulphate
and sodium deoxycholate. Triton X-100 is added to suspensions in PBS
at the rate of 5 mg mg-1 of mycoplasma protein and incubated for 15 min
at 37°C.
Antigens prepared by freezing and thawing, ultrasonic treatment or
lysis with Triton X-100 are usually satisfactory at 5-10 mg protein rnl-l,
but it is advisable to first test an antigen against its homologous antiserum
over a range of concentrations (e.g. 1.25, 2.5, 5-0 and 10 mg ml-1) to see
at which concentration the lines are sharpest and best resolved. The
choice of concentration is inevitably somewhat arbitrary since each
“antigen” is, in fact, a complex mixture of antigens and the concentration
selected cannot be optimal for all the components.
3. Test procedure
T h e slides or cover glasses are wiped free of acetone, set out on a levelling
table (Shandon Southern Instruments Ltd) and the metal rings centred on