COMPLEMENT FIXATION 



175 



bacterial emulsions and extracts. Therefore, it is necessary to be sure that the 

 amount of bacterial emulsion used in the test is not " anti-complementary," but 

 yet in sufficient concentration to operate well. The emulsion is made from a 

 twenty-four-hour slant agar culture (see page 81 for preparation) and may be 

 killed by heat or formalin. The preliminary titration may be set up as follows : 



Each tube should be made up to a volume of 2.0 c.c. with salt solution before 

 primary incubation. If convenient the erythrocytes may be sensitized by the 

 previous addition of amboceptor. In the protocol C.H. indicates complete 

 hemolysis, P.H. partial hemolysis and no hemolysis. 



The Test. The results given indicate that 0.5 c.c. bacterial emulsion is defi- 

 nitely anti-complementary, but the 0.3 c.c. has no such influence. The last tube 

 excludes hemolytic activity on the part of the emulsion. In order to be abso- 

 lutely sure that the final test will not be misleading through the anti-comple- 

 mentary action of the bacterial emulsion it is advisable to use the next smaller 

 amount than the titration shows to be free of anti-complementary activity, which 

 in this case is 0.2 c.c. This being the case 2.0 c.c. bacterial emulsion may be 

 diluted with 3.0 c.c. salt solution, whereupon 0.5 c.c. of the dilution will contain 

 0.2 c.c. original emulsion. The complement-fixation test may then be set up 

 as follows : 



The first incubation permits of fixation of the complement by the bacteria 

 and their specific immune serum and the second determines whether or not 

 complement is free to act upon the sensitized erythrocytes. For sensitization of 

 erythrocytes the hemolytic immune serum should be diluted so that 0.5 c.c. 

 contains two units hemolytic amboceptor, then added to an equal volume 5 per 

 cent, erythrocyte suspension. In the above test the immune serum is diluted, so 

 that 2.5 c.c. contain ten units amboceptor; it is then added to 2.5 c.c. 5 per cent, 

 erythrocyte suspension and the mixture allowed to remain at room temperature 

 for one hour. The protocol given above shows, reading from below upward, 

 that the hemolytic immune serum used for sensitization is not of itself hemolytic, 

 that the complement is in sufficient concentration for hemolysis, that neither the 

 bacterial emulsion nor the typhoid immune serum is anti-complementary in the 

 amounts used. In the first tube the bacterial emulsion, specific anti-bacterial 

 serum and complement interact so that the complement is not free to combine with 

 the sensitized erythrocytes, whereas tube 2 shows that normal rabbit serum will 

 not fix complement. 



Specific Character of the Test. In order to elaborate the test and to show 

 its specificity it is well also to titrate an emulsion of some other organism, for 

 example, colon bacilli for anti-complementary activity at the same time the 

 typhoid emulsion is titrated and in the same manner. If this shows anti-comple- 



