OZENA AND DISTEMPER 505 



COMPLEMENT FIXATION REACTIONS WITH B. BRONCHISEPTICUS 

 AND PEREZ' BACILLUS 



Complement fixation tests were made with the same antisera 

 as used for the agglutination reactions. 



Technic. The volume of all the complement fixation tests 

 and all titrations was 4.5 cc. The method in general was that 

 given by Kolmer in "Infection, Immunity and Specific Therapy." 



For the hemolytic system, sheep corpuscles in 5 per cent solu- 

 tion (the blood was defibrinated with beads, measured, washed 

 five times in salt solution, diluted back to original volume and 

 5 cc. of this used in 100 cc. salt solution), rabbit amboceptor, and 

 guinea-pig complement in a 1 in 20 dilution (serum from at least 

 two pigs being mixed) were used. The amboceptor was titrated 

 daily with each complement and corpuscle suspension and that 

 amount which showed just complete hemolysis in one hour at 

 37°C. was taken as the unit. In the tests one and a half times 

 the unit was used. 



Each antigen was titrated for its anticomplementary and its 

 antigenic unit. In the tests, one-half to one-fourth the anti- 

 complementary unit was used. 



Each antiserum was tested to determine the smallest amount 

 which, with its homologous antigen, completely inhibited hemoly- 

 sis. 



The test. Antigen plus serum plus complement was incubated 

 one hour at 37°C. then amboceptor and cells were added and 

 incubated from one to one and one-half hour depending on the 

 antigen controls. The tests were then placed in the ice-chest 

 and read the following morning. 



Antigens. B. bronchisepticus and Perez' Bacillus were trans- 

 planted daily for several days, then planted on plain agar in 

 whiskey flasks; incubated for twenty-four hours at 37°C.; the 

 growths washed off in sterile distilled water (about 50 cc. per 

 flask); shaken in a mechanical shaker for forty-eight hours; 

 brought up in a water bath to 56°C. and incubated at that 

 temperature over night; the antigens were then filtered through 

 asbestos and to nine parts of clear filtrate one part of 8.5 per 



