SEROLOGICAL METHODS IN THE STUDY OF PROTOZOA 419 



for a given i :io dilution of the glycerinated antiserum will hold 

 for about a week.) 



Where the serums from a large number of healthy guinea-pigs 

 are pooled, the unit of complement can be taken as a definite 

 amount (in fifth units, 0.2 of a cubic centimeter of a i :io dilu- 

 tion). In practice, however, where complement is obtained from 

 one or two animals, it is necessary to rule out individual variations 

 by a preliminary titration. (See Table 2.) In the example given, 

 since complete hemolysis occurs through Tube 4 the unit of com- 

 plement is 0.1 of a cubic centimeter of a i :io dilution. In the 

 actual fixation tests two units are used, or according to the titration 



TABLE 2 

 PROTOCOL FOR THE TITRATION OF COMPLEMENT 



in Table 2, 0.2 of a cubic centimeter of i :io. A much lower 

 dilution is not considered desirable. This titration has to be re- 

 peated for every batch of serum, or at least every other day when 

 tests are run regularly. 



The next step before performing the actual fixation test is to 

 ascertain to what extent the test antigen will non-specifically fix 

 complement. A protocol for this titration is given in Table 3, 

 and in the example given, since partial hemolysis occurs in Tube 

 6, the anticomplementary dose is 0.6 of a cubic centimeter of a 

 1 :20 dilution, and half of it can be considered a safe dilution for 

 the actual test. Sometimes, it may happen that an experimental 

 test antigen will be hemolytic, a condition which would be indicated 

 if hemolysis occurred in Control Tube 10. In that event, the above 

 protocol should be repeated leaving out the complement alto- 

 gether. The subsequent dilution for the actual test would then 

 have to be higher than the one that caused hemolysis of the cells 

 and higher than the anticomplementary dilution. (Such titrations, 



