160 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS 



diately. If centrifuge tubes are used, an amount of blood not more 

 than one-third the capacity of the tube (about 5 c.c.) is collected and 

 twice the volume of sterile salt solution is added to it. The corpuscles 

 are sedimented, the supernatant solution is pipetted off, fresh salt 

 solution is poured in, and the corpuscles resuspended by careful 

 stirring with a clean glass rod. This process is repeated five times, 

 each time discarding the washings. The last time the volume occu- 

 pied by the erythrocytes is read off on the graduations of the tube 

 and they are suspended in a volume of salt solution twenty times that 

 occupied by the erythrocytes. This makes a 5 per cent, suspension. 

 Erythrocytes are obtained by centrifugalization from the citrated 

 blood in precisely the same manner. This suspension of red blood 

 cells, kept in a cool, dark place, may be used for two days, but not 

 longer. Beyond that time the cells deteriorate and hemolyze with 

 abnormal readiness, thus vitiating the value of the test. 



(c) Standardization of Hemolytic System. It is very important to 

 know with exactness the amount of hemolytic serum (inactivated, 

 of course) which will effect complete hemolysis of 1 c.c. of a 5 per 

 cent, suspension of sheep erythrocytes in the presence of a constant 

 amount of complement. The determination of this factor gives the 

 hemolytic titer of the hemolytic serum. It is readily determined 

 by adding to a series of tubes, 0.1 c.c. of fresh guinea-pig serum (com- 

 plement), 1 c.c. of erythrocyte suspension, and varying amounts of 

 the inactivated hemolytic serum. The smallest amount of hemolysin 

 which will effect hemolysis under the conditions stated is the hemolytic 

 titer orunit. Thus, the following tubes incubated at 37 C. for one 

 hour showed : 



Result. 

 Complete hemolysis. 



Partial hemolysis. 

 No hemolysis. 



0.0025 of this serum is one unit; the hemolytic titer is 0.0025 c.c., in other words. 

 It is customary to use two units in the actual test, consequently 0.005 c.c. would be 

 the amount used. 



Complement control. 



2 Erythrocyte control. 



