466 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



liver; the third is marked "A" for acetone-insoluble lipoids, and the 

 fourth is not marked at all or simply marked with the letters U S. C." 

 (serum control). 



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. Instead of the incubator a water-bath at 38 C. may be 

 employed for one hour (not less) for the primary and secondary periods 

 of incubation, or the primary period may be conducted in a refrig- 

 erator at 8 C. for four hours or over night. As shown by McNeal, the 

 latter method yields particularly delicate reactions. In the latter 

 method the secondary period of incubation is conducted in a water 

 bath at 38 C. for about an hour. At the end of primary incubation 

 the amboceptor and corpuscles are added to each tube except that 

 containing the corpuscle control. Each tube is shaken gently and re- 

 incubated for an hour or longer, depending upon the hemolysis of 

 the controls, when the readings are made. By making the read- 

 ings at this time the influence of an excess of hemolysin due to 

 the presence of natural antisheep hemolysin in the human sera is 

 avoided and lesser degrees of complement fixation detected, which may 

 become completely hemolyzed if the tubes are set aside over night. 



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 hemolyzed. The antigen tubes con- 

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



