448 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



method. This is diluted 1 : 7 with normal salt solution to secure the 

 maximum turbidity, and is used in a constant dose of 0.6 c.c. 



A cholesterinized alcholic extract of human heart, acetone-insolu- 

 ble lipoids, or plain alcoholic extract of syphilitic liver may be used in 

 four times their titrated antigenic dose, with satisfactory results. 



4. Fluid to be Tested. Serum is heated at 55 C. for half an 

 hour and used in dose of 0.1 c.c. Cerebrospinal fluid should be fresh, 

 and is used unheated in dose of 0.8 or 1 c.c. 



5. The Test. Two series of tubes are required. 



Series A : These tubes should contain the dose of antigen employed 

 with one, two and three hemolytic doses of complement respectively, 

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

 These are the antigen controls to determine how much complement 

 may be fixed by antigen alone. Usually but one or at most a portion 

 of two doses are fixed. 



Series B: For each serum 12 test-tubes are arranged in a row. 

 They are labeled with the initial of the patient's name and numbered. 

 Into each is placed 0.1 c.c. of the patient's serurn. Into each of the 

 first nine tubes are placed the dose of antigen and 2, 4, 6, 8, 10, 12, 15, 

 18, 20 hemolytic doses of complement respectively. The last three 

 tubes are the serum controls, to determine the amount of complement 

 fixed by serum alone, and receive 1, 2 and 3 hemolytic units of com- 

 plement respectively. Sufficient salt solution is added to each tube 

 to bring the total volume up to 3 c.c., and all are incubated for an 

 hour at 37 C. 



Controls. Series "A" contains the antigen controls, and these 

 suffice for making any number of tests with the same complement and 

 sensitized corpuscles. 



(2) Each serum is controlled in the last three tubes of series B. 



(3) A known positive syphilitic serum may be included and tested 

 in the usual manner. 



(4) A known negative normal serum may be included, but it is 

 not necessary to use more than 2 to 4 and 6 hemolytic units of com- 

 plement instead of running out to 18 or 20 units, as is done with an 

 unknown serum or with cerebrospinal fluid. 



(5) A hemolytic control is set up with one dose of complement and 

 sufficient salt solution to make 3 c.c. 



(6) A corpuscle control may be included, containing 1 c.c. of 

 sensitized corpuscles and 3 c.c. of salt solution. This tube is plugged 

 with cotton to indicate that it is finished. It controls the tonicity of 

 the salt solution and should show no hemolysis. 



