146 BACTERIOLYSINS AND HEMOLYSINS 



animal. Nevertheless, an essential difference exists. For if the animal previously 

 immunized is again injected, hemolysins reappear after a short incubation period, 

 whereas in a normal animal a prolonged immunization is necessary. Hemolysins, 

 therefore, exist to a certain extent in a preformed state in the cells of an immunized 

 animal. If a stimulus to immunization occurs, the hemolytic substances are thrown 

 off into the circulation, while in a normal animal the formation of hemolysins by the 

 cells must first take place. 



If a great amount of hemolysin of the same titer is needed, it is 

 The Preser- best to bleed the animal to death. For the preservation of 

 vation of hemolysins the author recommends the following procedure 

 Hemolysins. w hich he has found very trustworthy. One to 3 c.cm. of 

 serum obtained sterile are poured into sterile tubes, which 

 are closed with non-absorbent cotton. The tubes are placed into a water 

 bath at 56 C. for one-half hour to inactivate the serum and are then cov- 

 ered with sterile rubber caps. (These are sterilized by placing them in a 

 i per cent, sublimate solution for forty-eight hours.) 



An immune hemolysin must answer both qualitative and quantitative 

 determinations; qualitative, whereby is proven that the serum can hem- 

 olyze only the red blood cells which serve as antigen or to a slight degree 

 those of related animals, and that it has only the effect of a normal 

 serum upon the erythrocytes of other animals. The quantitative estima- 

 tion supplies the only means for the absolute differentiation between a 

 normal and an immune serum. In complement fixation where hemolysis 

 bears an active part, it is the quantitative use of the hemolysin which 

 decides the result of the reaction. The immune serum must therefore 

 be "titrated." 



If fresh active hemolytic immune serum is used, a constant quantity of 

 blood serving as antigen is mixed with decreasing quantities of the immune 

 serum and the mixtures placed in the thermostat. Results like the follow- 

 ing will be obtained. 



Antigen blood 



Hemolytic serum of immune 

 rabbit 



Result after 2 hours 



i c.cm. of 5% sheep's blood ! i c.cm. of active serum, i to 10 



i c.cm. of 5% sheep's blood i c.cm. of active serum, i to 20 



i c.cm. of 5% sheep's blood, 

 i c.cm. of 5% sheep's blood. 



i c.cm. of active serum, i to 50 

 i c.cm. of active serum, i to 100 



Hemolysis. 



Incomplete hemolysis. 

 Incomplete hemolysis. 

 No hemolysis. 



On the basis of this experiment the titer of the hemolytic serum for 

 sheep's blood would lie between i/io and 1/20. But this is incorrect, as 

 it was pointed out previously that by immunization only the amboceptors 

 are increased and the complement remains unchanged. Each of the above 



