PREPARATION OF HEMOLYSIN 145 



animal from which the erythrocytes for injection are taken. The blood to 

 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 it 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 hemo- 

 globin. The fluid is carefully decanted, fresh saline added, the tube gently shaken, and 

 again centrifugalized. 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 begin- 

 ning of the experiment, the erythrocytes are obtained in the normal concentration, 

 just as in the blood, but completely free of serum. 



The washed, defibrinated blood can be injected subcuta- 

 Immuniza- neously, intravenously, or intraperitoneally. With the sub- 

 don, cutaneous and intraperitoneal methods in a rabbit, injections 



of from 5 to 20 c.cm. 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 in rabbits. 



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

 logical saline; o. 5 to i .o c.cm. 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 phe- 

 nomena. 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 variations 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,ithe animal can be allowed 

 to live; it will gradually lose its titer completely and will act apparently like a normal 



