PRESERVATION OF HEMOLYSINS. 133 



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 

 ThePreser- best to bleed the animal to death. For the preservation of 



vation of hemolysins the author recommends the following procedure 



Hemolysins. which he has found very trustworthy. One to 3 c.c. of serum 



obtained sterile, are poured into sterile tubes, which are 



closed with absorbent cotton. The tubes are placed into a water bath at 



56 C. for one-half hour to inactivate the serum and are then covered 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 dissolve 

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

 nearly related animals, and that it has only the effect of a normal serum upon 

 the erythrocytes of other animals. The quantitative estimation 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 serum and 

 the mixtures placed into the thermostat. Results like the following will be 

 obtained. 



Antigen blood. 



Hemolytic serum of immune rabbit, j Result after 2 hours. 



i c.c. of 5% sheep's blood | i c.c. of active serum, i to 10 Hemolysis. 



i c.c. of 5% sheep's blood j i c.c. of active serum, i to 20 Incomplete hemolysis. 



i c.c. of 5% sheep's blood i i c.c. of active serum, i to 50 Incomplete hemolysis. 



i c.c. of 5% sheep's blood i c.c. of active serum, i to 100 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 

 dilutions decreases therefore not only the amount of hemolysin, the quanti- 

 tative estimation of which is the object of the experiment, but also the 

 complement. Inasmuch as the latter was not at first increased, a point is 

 soon reached where there is no complement at all in the diluted fluid; as a 

 result hemolysis cannot occur, for only the combination of hemolysin + 

 sufficient complement, can exhibit any hemolytic action. Correct titration 



