132 BACTERIOLYSINS AND HEMOLYSINS. 



be injected, can be employed in just the condition in which it flows from the 

 vein. Nevertheless it is as a rule defibrinated, to prevent coagulation. 

 The simplest and most practical way of doing this is to place some glass 

 beads into a bottle or Erlenmeyer flask and then sterilize by dry heat. The 

 blood coming from the vein is allowed to flow into one of these flasks and 

 then it is repeatedly shaken for several minutes. This suffices to defibrinate 

 the blood and thus prevent coagulation. 



The production of hemolysins depends entirely upon the red blood 

 corpuscles. The presence of the serum is not only superfluous, but even 

 harmful, as experience has shown that dangerous reactions may follow 

 the injection of foreign serum. 



Before injecting, therefore, the erythrocytes are washed. For this 

 Washing of purpose a few cubic centimeters of defibrinated blood are poured into 

 Red Blood a centrifuge tube and the level of the fluid marked on the tube. An 

 Corpuscles, equal or double this amount of 0.85 per cent, saline is added, and the 

 tube rapidly centrifugalized. The erythrocytes fall to the bottom, while 

 the upper layers of the tube consist of diluted serum more or less tinged with hemoglobin. 

 The fluid is carefully decanted, fresh saline added, the tube shaken, and again centrifu- 

 galized. If this is done two to three times the erythrocytes can be freed of the last 

 traces of serum; finally, by adding saline up to the mark made at the beginning of the 

 experiment the erythrocytes are obtained in the normal concentration, just as in the 

 blood, but completely free of serum. 



The injection of the washed, defibrinated blood, can be af- 

 Immunization. fected subcutaneously, intravenously, or intraperitoneally. 



With the subcutaneous and intraperitoneal methods in a rab- 

 bit, injections of from 5 to 20 c.c. are necessary at intervals of five to six days. 

 Far larger quantities should be given to bigger animals, like goats and 

 sheep. Subcutaneous injections often cause infiltrations and occasionally 

 abscesses. The author therefore uses the intravenous method exclusively 

 in rabbits. 



A suspension of washed blood corpuscles is diluted four to five times with physiological 

 saline; 0.5 to i.o c.c. of this fluid is slowly injected into the ear vein every five to six 

 days. Three injections are almost always sufficient for procuring a good serum. The 

 animals sustain the first two injections with ease, but the third and following ones are 

 not altogether without danger. This is supposed to be akin to anaphylactic phenomena. 

 It is therefore advisable to immunize several animals simultaneously, so that in case one 

 dies there is another to replace it. Furthermore there are such marked individual varia- 

 tions in the ability to produce hemolysins that it is best to have several animals to choose 

 from. Beginning on the sixth day after the third injection, blood should be withdrawn 

 for the determination of the hemolytic strength and this process repeated daily until 

 the titer has reached a satisfactory height and then the animal should be bled. If only 

 a small amount of hemolysin is needed, the animal can be allowed to live; it will gradually 

 lose its titer completely and will act apparently like a normal 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 



