BACTERIOLYSINS AND H^MOLYSINS 123 



.4 of a cc. of antigen, to the second .3, then .2, .1, 

 and .05. Next o.i cc. complement is added to each 

 tube. These tubes are known as the anti-comple- 

 mentary control. They are placed in the water bath 

 at 37 C. for half an hour and the sensitized sheep 

 cells added. The tube which shows the least inhibi- 

 tion of haemolysis contains the anti-complementary 

 dose of the antigen ; not more than J of this amount 

 may be used in the test proper and it is well to use 

 even less than this if possible. The exact amount to 

 be used is determined by adding varied amounts of 

 the antigen to known positive sera. If at least six 

 strongly positive sera are pooled, the use of a small 

 amount of this mixture will give a fairly constant 

 fixation unit. Six tubes are required; in each is 

 placed .01 cc. of the pooled sera. To the first is 

 added .25 then .2, .12, .1, .05, .025 cc. of antigen and 

 .1 cc. of complement to each. The amounts of fluid 

 in the tubes are then equalized by adding the neces- 

 sary amount of normal saline. They are placed 

 in the water bath at 37 C. for half an hour and 

 the sensitized sheep cells added. With a good 

 antigen there should be no haemolysis in any of the 

 tubes or, at least, only a faint trace in the tube 

 containing .025 of antigen. In this case four times 

 .025 or .1 is the proper amount to use in the 

 test proper. The difference in the amount of 

 antigen between the anti-complementary unit and 

 the fixing unit is the range of that antigen and the 



