PRACTICAL APPLICATIONS 399 



TABLE 11. TITRATION OF HEMOLYSIN 



The following controls should be set up at the same time : 



1. 1 c.c. of corpuscles in 1 c.c. of amboceptor dilution. This tube 



should show no hemolysis, as the serum has been inactivated 

 and is too highly diluted for complement activity, even though 

 native complement were present. 



2. 1 c.c. of corpuscles in 1 c.c. of complement dilution. This tube 



may show a trace of hemolysis, due to the presence of a small 

 amount of natural amboceptor for the corpuscles used. As a 

 general rule, guinea-pig serum is free from natural antisheep 

 amboceptor, or the amount is so small under these conditions 

 that it is not necessary to remove it. 



. 3. 1 c.c. of corpuscles in 3 c.c. of salt solution. This tube should show 

 no hemolysis, and serves to show that the diluent was isotonic. 

 In the foregoing titration it is found that 0.25 c.c. of 1 : 1000 dilution 

 of amboceptor is the unit, or the titer is 1 : 4000. This method is less 

 difficult than preparing a series of dilutions of amboceptor as a 1 : 100, 

 1 : 200, 1 : 500, 1 : 1000, 1 : 2000, etc., using a cubic centimeter of each 

 dilution. The method requires accurate pipets and careful work, but 

 yields uniform and satisfactory results (Fig. 106). 



The rabbit may now be bled under anesthesia. The serum is sep- 

 arated and inactivated and again titrated, as the final titration, for some 

 unknown reason, is likely to be a little lower than in the primary tests. 

 When suitably preserved, a hemolytic serum will maintain its activ- 

 ity for long periods of time; it should always, however, be titrated before 

 complement-fixation tests are undertaken. 



