METHODS FOR CONDUCTING THE SYPHILIS REACTION 473 



0.5 c.c. of a 2J per cent, suspension of cells: 0.1 c.c., 0.15 c.c., 0.2 c.c., 

 0.25 c.c., 0.3 c.c., and 0.4 c.c. In the antigenic titration the following 

 amounts of antigen diluted 1 : 20 are placed in a series of ten test-tubes: 

 0.001 c.c., 0.005 c.c., 0.008 c.c., 0.01 c.c., 0.05 c.c., 0.08 c.c., 0.1 c.c., 

 0.2 c.c., 0.3 c.c., and 0.4 c.c.; 0.1 c.c. of fresh inactivated syphilitic serum 

 preferably from several persons mixed, plus 2 units of complement 

 and sufficient salt solution to make 3 c.c., are added to each tube and 

 incubated in a water-bath at 38 C. for one hour, when 1 unit of hemol- 

 ysin and 0.5 c.c. of corpuscles are added, tubes shaken, and re-incubated 

 for an hour. The reading may be made at once or after the corpuscles 

 have settled; the unit is the smallest amount of antigen yielding complete 

 inhibition of hemolysis. 



In the anticomplementary titration the extract is diluted 1 : 20 and 

 the following amounts placed in a series of ten test-tubes with 0.1 c.c. 

 of fresh inactivated normal serum and 2 units of complement: 0.1, 

 0.2, 0.3, 0.5, 0.8, 1.0, 1.2, 1.5, and 2.0 c.c. The titration is conducted 

 in the same manner, and the anticomplementary unit is the smallest amount 

 of extract producing inhibition of hemolysis. 



Hemolytic and serum controls on both the syphilitic and normal 

 serum should be included and show complete hemolysis. 



A satisfactory extract is one in which the antigenic unit is at least 

 ten times less than the anticomplementary unit, and in conducting the 

 Wassermann reaction 2 units of antigen are employed as the dose. 



5. Fluid to be Tested. Serum is heated at 56 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 1 c.c. 



6. The Test. For each serum eight test-tubes are arranged in a row. 

 One is labeled with the patient's name and all are numbered. Into 

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

 six tubes are placed the dose of antigen and 2, 3, 4, 5, 6, and 8 units of 

 complement respectively. The last two tubes are the serum controls, 

 to determine the amount of complement fixed by serum alone, and re- 

 ceive 1 and 2 units of complement respectively. Sufficient salt solution 

 is added to each tube to bring the total volume up to 3 c.c., and all are 

 incubated in the water-bath at 38 C. for an hour. One unit of hemolysin 

 and 0.5 c.c. of corpuscle suspension are now added to each tube and re- 

 incubated in the water-bath for an hour, when the readings are made. 

 Sharper readings may be made after the corpuscles have settled either by 

 centrifuging or standing the tubes in the refrigerator overnight. 



A human hemolytic system may be employed if a sufficiently potent 



