442 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



To each of the four tubes 0.1 or 0.2 c.c. of the patient's serum is 

 added, or 0.8 c.c. of cerebrospinal fluid. 



To each tube 1 c.c. of the diluted complement (1 : 20) and suffi- 

 cient salt solution to bring the total volume in each up to 3 c.c. are 

 now added. 



Controls. A known positive and negative serum should be included, 

 unless one is performing a large number of tests with reliable antigens 

 every week, in which case, among many serums, a few at least are 

 likely to be positive. Under these circumstances these controls may 

 be omitted; as a general rule, however, they should be included. 



To the hemolytic system control tube 1 c.c. of complement dilu- 

 tion and 2 c.c. of salt solution are now added. Three antigen control 

 tubes are set up for each antigen with the dose employed, plus 1 c.c. 

 of complement dilution and sufficient salt solution to make the total 

 volume about 3 c.c. The corpuscle control receives 1 c.c. of the sus- 

 pension plus 3 c.c. of salt solution. 



All the tubes are shaken gently and placed in the incubator for an 

 hour at 37 C. At the end of this time the amboceptor and corpuscles 

 are added to each tube except that containing the corpuscle control. 

 Each tube is shaken gently and reincubated for an hour or longer, 

 depending upon the hemolysis of the serum controls. A preliminary 

 reading is now made, and the tubes set aside in the refrigerator at low 

 temperature until the corpuscles have settled. As a general rule, the 

 two readings coincide quite closely. Occasionally, with serums of 

 vigorously treated cases of syphilis, at the preliminary reading the 

 antigen tubes will show very slight inhibition or delay in hemoly is, 

 whereas if allowed to settle overnight this may not be noticeable. 



Reading the Results. The readings are made in the same manner 

 as described in the first method, the controls always being inspected 

 first. The hemolytic, antigen, and serum controls and known nega- 

 tive serum tubes should all be hemoly zed. The antigen tubes con- 

 taining the positive syphilitic serum should not be hemolyzed. Re- 

 sults with the unknown serums are dependent upon whether or not 

 the serums are luetic, and if they are, upon the quantity of syphilis 

 antibody present. 



With strongly positive serums there is complete inhibition of 

 hemolysis with all three antigens. With serums of long-standing or 

 treated cases of syphilis containing smaller amounts of antibody the 

 reaction with the cholesterinized extract is usually strongly positive, 

 whereas with the other two antigens the degree of inhibition of hemol- 



