IX. IDENTIFICATION OF MYCOPLASMAS
417
The optimal sensitising concentration (OSC) of the haemolytic serum is
in that dilution which gives most lysis with the highest dilution of complement. I n the example shown in Table V, it is 1 :200 and the haemolytic
serum should be used at that dilution in subsequent titrations. One unit
(HC50) of complement is contained in that dilution which gives 50% lysis
(reading 2) at the OSC of the haemolytic serum. I n the titration shown in
Table V, the HC50 is in the 1 : 114 dilution. I n the test proper 3 HC50 are
used, so the working dilution of complement is 1 : 38.
Since haemolytic serum is stable for long periods, a full chessboard
titration need not be done frequently. Once the OSC is established, a
simple “line” titration of complement can be carried out with that dilution
of haemolysin.
(f) Production of antigen. Mycoplasma antigens for C F tests usually consist
of washed suspensions of whole organisms. The practice at the Lister
Institute has been to grow the organisms in 500 ml of medium containing
10-20% serum. Cultures are usually incubated for 6-7 days, because the
anticomplementary activity of many antigens is reduced by this extended
incubation period. This may be due to the loss of complement-inactivating
enzymes as the organisms age. After harvesting, the mycoplasmas are
washed twice in 0.85% saline, resuspended in 5-10 ml saline containing
1 : 10 000 merthiolate (Eli Lilly and Co. Ltd, Basingstoke, England) and
stored at 4°C. Antigens thus prepared retain their activity for many
months.
The problem of anticomplementary activity is a real one. Most preparations are anticomplementary at low dilutions, but the optimal antigen
dilution is usually found to be in dilutions higher than those which are
anticomplementary. If an antigen proves to be too anticomplementary to
use, it must be discarded and a fresh culture initiated as there is little one
can do to “improve” an antigen. Absorption with complement has, for
example, never been effective in our hands. Extending the incubation
period still further (15-24 days) has effectively overcome the anticomplementary activity of A. Zaidlawii antigens. T h e use of 1 4 % PPLO
serum fraction (Difco) instead of whole serum may also reduce the anticomplementary activity of the resulting antigen. For species which possess
lipid haptens, for example, M. pneumoniae and M . fermentans, the lipid
extracted with chloroform-methanol (2 : 1) is usually less anticomplementary than whole cell suspensions. Heating may also reduce the anticompleurentary activity. For example, the C F antigen used for the diagnosis of
contagious bovine pleuropneumonia is an aqueous suspension of M .
mycoides subsp. mycoides boiled for 10 min in a brine bath and then made
isotonic with sodium chloride (Campbell, 1938). It is noteworthy that
417
The optimal sensitising concentration (OSC) of the haemolytic serum is
in that dilution which gives most lysis with the highest dilution of complement. I n the example shown in Table V, it is 1 :200 and the haemolytic
serum should be used at that dilution in subsequent titrations. One unit
(HC50) of complement is contained in that dilution which gives 50% lysis
(reading 2) at the OSC of the haemolytic serum. I n the titration shown in
Table V, the HC50 is in the 1 : 114 dilution. I n the test proper 3 HC50 are
used, so the working dilution of complement is 1 : 38.
Since haemolytic serum is stable for long periods, a full chessboard
titration need not be done frequently. Once the OSC is established, a
simple “line” titration of complement can be carried out with that dilution
of haemolysin.
(f) Production of antigen. Mycoplasma antigens for C F tests usually consist
of washed suspensions of whole organisms. The practice at the Lister
Institute has been to grow the organisms in 500 ml of medium containing
10-20% serum. Cultures are usually incubated for 6-7 days, because the
anticomplementary activity of many antigens is reduced by this extended
incubation period. This may be due to the loss of complement-inactivating
enzymes as the organisms age. After harvesting, the mycoplasmas are
washed twice in 0.85% saline, resuspended in 5-10 ml saline containing
1 : 10 000 merthiolate (Eli Lilly and Co. Ltd, Basingstoke, England) and
stored at 4°C. Antigens thus prepared retain their activity for many
months.
The problem of anticomplementary activity is a real one. Most preparations are anticomplementary at low dilutions, but the optimal antigen
dilution is usually found to be in dilutions higher than those which are
anticomplementary. If an antigen proves to be too anticomplementary to
use, it must be discarded and a fresh culture initiated as there is little one
can do to “improve” an antigen. Absorption with complement has, for
example, never been effective in our hands. Extending the incubation
period still further (15-24 days) has effectively overcome the anticomplementary activity of A. Zaidlawii antigens. T h e use of 1 4 % PPLO
serum fraction (Difco) instead of whole serum may also reduce the anticomplementary activity of the resulting antigen. For species which possess
lipid haptens, for example, M. pneumoniae and M . fermentans, the lipid
extracted with chloroform-methanol (2 : 1) is usually less anticomplementary than whole cell suspensions. Heating may also reduce the anticompleurentary activity. For example, the C F antigen used for the diagnosis of
contagious bovine pleuropneumonia is an aqueous suspension of M .
mycoides subsp. mycoides boiled for 10 min in a brine bath and then made
isotonic with sodium chloride (Campbell, 1938). It is noteworthy that