444 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



cent, suspension and used in doses of 1 c.c.; antisheep amboceptor is 

 adjusted to the complement and corpuscles by a method of titration, 

 as given with the first method, and used in a dose equal to 1J/2 hemo- 

 lytic units. 



The Test. Seven test-tubes are arranged in a row and marked 

 with the patient's name; in each of the first six tubes the dose of 

 antigen is placed. The following amounts of diluted serum (1 : 10) are 

 then added: 



10 = 0.01 c.c. serum. 



10 = 0.02 c.c. serum. 



10 = 0.04 c.c. serum. 



10 = 0.06 c.c. serum. 



10 = 0.08 c.c. serum. 



10 = 0.1 c.c. serum. 



10 = 0.1 c.c. serum (control). 



The seventh tube contains no antigen, and is the serum control 

 with the maximum dose employed. 



In testing cerebrospinal fluid the following doses may be used 

 (undiluted): 0.1 c.c., 0.2 c.c., 0.4 c.c., 0.6 c.c., 0.8 c.c., 1 c.c., and 

 1 c.c. for the seventh or control tube. 



To each tube 1 c.c. of the diluted complement (1 : 20) is now added, 

 and sufficient salt solution to bring the total volume in each up to 3 c.c. 



Controls. Unless one is examining a large number of serums and 

 is sure that the antigen is satisfactory, known positive and negative 

 serum controls may be included in the series. As a general rule, 

 however, they should not be omitted. 



The hemolytic system control receives at this time 1 c.c. of the 

 complement dilution and 2 c.c. of salt solution. The antigen control 

 receives the dose employed plus 1 c.c. of complement and sufficient 

 salt solution to bring the total volume up to 3 c.c. The corpuscle 

 control receives 1 c.c. of the suspension and 3 c.c. of salt solution, and 

 the tube is plugged with cotton to show that it is finished. 



All tubes are shaken gently and incubated for one hour at 37 C., 

 at the end of which time 1J^ units of amboceptor and 1 c.c. of the 

 corpuscle suspension are added to all except the corpuscle control. 

 Each tube is shaken and all are reincubated for an hour or an hour 

 and a half, the length of time depending upon the hemolysis of the 

 serum controls when all are placed in the refrigerator at a low tem- 

 perature overnight, readings being made the next morning. 



Reading the Results. As is usual, the controls are examined first. 



