4i6 RESEARCH IN PROTOZOOLOGY 



bacterial action during extraction. In the actual test, the super- 

 natant, either filtered or centrifuged or both, is used. 



Although both lipoidal and aqueous solvents give rise to reactive 

 test antigens, the reactive principles in each case may be different 

 components of the original parasitic material. The great drawback 

 to lipoidal solvents resides in the tendency of the resultant ex- 

 tract to react with syphilitic serums. This is not surprising in 

 view of the remarkable reactivity of syphilitic serums in the Was- 

 sermann reaction where the test antigen is made up of a lipoidal 

 extract of various normal organs. It may, however, be ruled out 

 by dilution, i.e., by ascertaining to what extent syphilitic serums 

 react with a given test antigen and thereafter testing with higher 

 dilutions of the test antigen. 



B. Materials required. Since the complement fixation test is a 

 delicate measure of whether or not complement is fixed, each 

 of the ingredients has to be carefully titrated as far as possible. 

 Furthermore, as the strength of the experimental antiserum and 

 (until standardized) of the test antigen is unknown, it is ex- 

 tremely important to have a definite balance between complement, 

 hemolytic antiserum and red blood cells, otherwise the test might 

 be invalidated. For this reason, a standard has been arbitrarily 

 accepted by most investigators and is as follows : The titre of the 

 hemolytic antiserum is the smallest amount of undiluted, specific 

 serum necessary to hemolyse completely one cubic centimeter of 

 a five per cent suspension of blood corpuscles (one unit) in the 

 presence of two units of complement. Two units of complement is 

 generally about 0.08 of a cubic centimeter of fresh, normal, un- 

 diluted guinea-pig serum, but many investigators, following the 

 earlier procedure, use o.i cubic centimeter. In some techniques, 

 half, quarter or fifth units are frequently successfully employed 

 for economy of materials. The latter are used in the following 

 protocols which are patterned after the technique used by. Prof. 

 C. G. Bull of the Johns Hopkins University and are taken from a 

 book by the present author (1929). Thus, 0.5 of a cubic centimeter 

 of a two per cent suspension of corpuscles is one-fifth unit and 

 0.2 of a cubic centimeter of ten per cent guinea-pig serum (com- 

 plement) is one-fifth of the older arbitrary dose of o.i of a cubic 

 centimeter of undiluted serum. 



In all cases control tubes are an essential accompaniment of 

 every test to insure against one or another of the reagents, by 



