134 BACTERIOLYSINS AND HEMOLYSINS. 



consists therefore in allowing varying quantities of hemolysin with a constant 

 amount of complement to act upon a constant quantity of red blood cells. The 

 simplest method of accomplishing this is first to destroy the complement by in- 

 activation of the hemolytic serum, then to make the desired dilutions, and finally 

 to add to all, the same amount of normal serum as complement. The normal 

 serum of an animal of the same species as that which provided the immune 

 serum can under no circumstances serve as complement. On the contrary, 

 foreign sera are much more suitable; and guinea-pig's serum is especially 

 recommended as complement, when immune rabbit's serum is used. Not 

 every complement serves equally well for any immune serum. 



A very good hemolytic system which is almost exclusively used for the 

 complement fixation reaction, is sheep's blood as antigen, rabbit's immune 

 hemolysin as amboceptor and normal guinea-pig's serum as complement. 

 The preparation of these ingredients should be carried out as follows : 



i. Sheep's Blood. This should be defibrinated and washed. Washing 

 is necessary because fresh sheep's blood contains complement; and if the 

 blood is a few days old, washing is even more important. 



Although serum which is not fresh does not contain sufficient active 

 Comple- complement to cause the danger of superfluous complement, it never- 

 mentoids. theless contains substances which interfere with hemolysis. Probably 



the existence of "complementoids" is the disturbing factor. It must 

 be assumed that complement is composed of two biologically different parts, as is the case 

 with toxins and ferments. One is the haptophore group, which has affinity for the com- 

 plementophile group of the amboceptor and is the more stable of the two. The other 

 corresponds to the energy group of the toxins (toxophore element) and of the ferments. 

 Just as- after the destruction of the toxophore group there remain only innocuous toxoids 

 whose single perceptible activity consists in their ability to neutralize antitoxins, so also, 

 after the destruction of the weakly resistant energy elements of the complement, there 

 remain complementoids which lack the ability to activate a bacteriolytic or hemolytic 

 amboceptor, although by virtue of their uninjured haptophore groups they bind the 

 complementophile groups of the amboceptors. In this way they usurp the place of 

 whatever active complement may still be present, rendering the latter inactive, and as a 

 result hemolysis is absent or incomplete. 



Following the technique of Ehrlich and Morgenroth, a 5 per cent, sus- 

 pension of washed red blood corpuscles is employed to test a hemolysin. A 

 pipette, closed at the top by pressure of the index finger is thrust to the bot- 

 tom of the washed erythrocytes contained in the centrifuge tube; a definite 

 amount, for instance i c.c. is withdrawn and allowed to flow into a graduate. 

 For diluting purposes (in this case up to 20 c.c.) only isotonic or weakly 

 hypertonic NaCL solutions may be used. If water, hypotonic or strongly 

 hypertonic salt solutions are employed, the red blood cells disintegrate. 

 This is not a true biological hemolysis, but depends upon physical basis. 

 0.85 per cent, saline is most suitable for the majority of erythrocytes (man, 

 rabbit, guinea-pig, ox, sheep). When instead of an isotonic salt solution, 

 an isotonic sugar solution is made, the red cells are retained in their proper 



