Pleuro-Pneumonia of Cattle 185 



it was distinctly noticeable that in the former tubes the charac- 

 teristic opalescence was more marked, but there was no recog- 

 nisable sedimentation at the bottom of the culture tubes. It is 

 not considered that the increased opalescence in the cultures 

 growing in the media containing the serum of Experimental Calf 

 I was produced by a more vigorous growth of the organisms in 

 those tubes, but in all probability it was due to a partial agglu- 

 tination of the organisms in the culture fluid, and the intercep- 

 tion of rays of light in their passage through the culture fluid 

 .by the slight agglomerations of partly agglutinated organisms 

 floating in the culture media. The experiment was adequately 

 -controlled by incubating at the same time tubes of the same media 

 which had not been inoculated with the primary culture. These 

 ^control tubes maintained an unaltered appearance throughout. 



Complement Fixation. 



To test the complement fixation reaction in contagious pleuro- 

 pneumonia, the following ingredients for the test were prepared 

 :as required. 



(i) Haemolytic Amboceptor. — The serum of a rabbit immun- 

 ised by repeated injections of the washed red blood corpuscles 

 of a sheep. This rabbit's serum was inactivated by heating in 

 -a water bath at 56°C. for 30 minutes, and for the tests was em- 

 -ployed in a dilution of 1 in 1000 with saline solution. The exact 

 dilution and the amount for the test was established at the com- 

 mencement of the test by one or more titration experiments. The 

 titre of this Haemolytic Amboceptor was never less than 1 in 

 1000 when used in any of the complement fixation tests. 



(2) Corpuscle Suspension. — A 5 per cent, suspension in saline 

 -solution of the washed red blood cells of a sheep. 



(3) Complement. — Fresh guinea-pig's serum, diluted 1 in 10 

 with saline solution, the minimum haemolytic dose (M.H.D.) 



^being established by titration. 



(4) Saline Solution. — 0.9 per cent, sodium chloride solution, 

 filtered, and sterilised. 



(5) Test Sera. — Obtained from Bovines. 



In the earlier complement fixation tests, which were carried 

 on over a period of several months, the test sera were diluted 

 with equal parts of 1 per cent, carbolic acid in saline solution 

 for preservation, and were kept in the ice chest for use as re- 

 quired. They were further diluted with physiological saline solu- 



