472 MICROBIOLOGY 



The titration proper is carried out as follows. Eleven 

 test tubes and three for controls are used. To each tube 1 cc. 

 of salt solution is added, then 1 cc. of the complement of the 

 previously determined smallest quantity established by the 

 titration done before. To each tube add 1 cc. of the different 

 dilutions (1-10, 1-30, 1-50, etc.) of the extract as obtained in 

 Table IV. The tubes are shaken, placed in a rack and the rack 

 placed in an incubator at 37 C. for one hour.* The tubes 

 are then removed and 1 cc. of a double quantity of the pre- 

 viously titrated amboceptor is added to each tube, and finally 

 1 cc. of a 5% suspension of washed sheep blood corpuscles. 

 Three control tubes are used. The first serves for a control to 

 show that the complement alone (without the amboceptor) 

 .does not produce hemolysis, the second to show that the ambo- 

 'ceptor alone does not produce hemolysis and the third that the 

 ;salt alone does not produce hemolysis. In the first control 

 lube 3 cc. of salt solution, 1 cc. of complement and 1 cc. of 

 blood corpuscles are added. The second tube contains 3 cc. 

 of salt solution, 1 cc. of amboceptor and 1 cc. of blood cor- 

 puscles, while the third contains 4 cc. of salt solution and 1 

 cc. of suspension of sheep blood corpuscles. The following 

 table (Table V) gives a summary of the foregoing method. 



Each tube is shaken and all the tubes placed in the incu- 

 bator at 37 C. for two hours. t The results are then read. 

 The tube in which hemolysis is no longer prevented represents 

 the titre of the extract. In the glanders test, however, one-half 

 that quantity is used. For example, if the results of the titra- 

 tion should be that the first tube which does not prevent 

 hemolysis contains a dilution of 1-80 (tube 5, Table V), then 

 a dilution of 1-160 of the extract is used for the glanders test. 

 .Antigen which possesses, too much anti-complementary sub- 

 stance should not be used ; that is, where hemolysis is prevented 

 in as high dilutions as 1-300, 1-400, 1-500, etc. 



5. Suspected serum. The suspected glandered horse is 

 bled usually through the jugular and about 20 cc. of the blood 



* May place in water bath at 37 C. for y 2 hour. 

 t Or water bath at 37 C. for one hour. 



