Tested October 1971
Disc Growth Inhibition Test:
Antiserum disc: Filter-paper disc were impregnated with 0.02 mL antiserum, dried overnight at 37°C, and stored at 4°C
Test plate: M. pneumoniae and other mycoplasma species were inoculated onto plates and dried; the antiserum-impregnated discs were placed in the inoculation zone
Time and temperature: 4 days at 27°C
Result: 8 mm zone of inhibition; specific for M. pneumoniae
Metabolic Inhibition Test:
Test preparation: Serial two-fold dilutions of antiserum (heat-treated at 56°C for 30 minutes) were mixed with M. pneumoniae and other mycoplasma species and added to wells of a microtiter plate
Temperature: 37°C
Reading: Endpoint was recorded as the highest antiserum dilution which prevented tetrazolium reduction
Titer: 1:4096; specific for M. pneumoniae
Indirect Hemagglutination Test:
Antigen: Supernatant fluid from an ultrasonicated M. pneumoniae suspension
Red blood cells: Tanned sheep red blood cells (1% suspension) and sensitized at pH 7.2 with M. pneumoniae
Test preparation: Serial two-fold dilutions of antiserum (heat-treated at 56°C for 30 minutes) were mixed with washed, packed M. pneumoniae-sensitized red blood cells (1 mL per mL of antiserum)
Time and temperature: 30 minutes at 4°C
Titer: 1:65536
Complement Fixation Test:
Antigen: Phenol treated
Red blood cells: Sensitized at pH 7.2 with antibody to sheep red blood cells
Test preparation: Antiserum/antigen mixtures were incubated overnight at 4°C before the addition of sensitized sheep red blood cells. Two units of complement and two units of hemolysin were added.
Time and temperature: 30 minutes at 37°C
Reading: The titer was defined as the highest antiserum dilution at which 75% of the sheep erythrocytes were not hemolyzed.
Titer: 1:128
Purity tested October 1971
Bacterial Sterility: Negative