148 BACTERIOLYSINS AND HEMOLYSINS 



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

 bottom of the washed erythrocytes contained in the centrifuge tube; a 

 definite amount, for instance i c.cm., is withdrawn and allowed to flow 

 into a graduate. For diluting purposes (in this case up to 20 c.cm.) 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 de- 

 pends 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 form, but the addition of hemolysin 

 and complement produces no hemolysis. The presence of salt is indis- 

 pensable for hemolysis as well as agglutination 



Undiluted, unwashed, defibrinated blood if removed sterile can be 

 kept several days in the ice-box. The "Frigo" apparatus is unsuited 

 for this purpose, because the thawing of the frozen blood breaks the 

 capsule of the red blood corpuscle. The deterioration of the preserved 

 blood is recognized by the large hemoglobin content of the serum or the 

 violet color of the blood. 



Occasionally blood left in an ice-box becomes dark. This is due to the lack of 

 oxygen. When the 5 per cent, suspension is made and thoroughly shaken, the red color 

 returns. Such blood of course is perfectly serviceable. 



Still, it is best not to keep blood longer than four days. Blood older than that, even 

 if apparently unchanged, possesses a lowered resistance and can give a far higher' titer 

 in hemolysin tests than fresh blood. 



2 The rabbit's hemolysin must have been inactivated for one-half 

 hour at 56 C. Dilutions are made with physiological saline. 



3. Guinea-pig's complement is obtained by bleeding to death a healthy 

 normal animal. 



The blood is allowed to flow directly into a centrifuge tube and then to clot; the clear 

 serum is obtained by centrifugalization. For titration of hemolysin it is best to use a 

 constant dose of complement as i c.cm. of a i/io dilution. Complement can be kept 

 for twenty-four hours in the ice-box. When older than this it suffers a distinct decrease 

 in efficiency as complementoid is produced. (See above.) In the "Frigo," comple- 

 ment may be kept for weeks. Stern, however, does not recommend complement pre- 

 served in "Frigo" for use in complement fixation tests, as its affinity for amboceptor 

 is noticeably decreased. 



One c.cm. of each of the three reagents (each so diluted with saline that 

 the desired dose is contained within i c.cm.) is mixed and 2 c.cm. of 0.85 

 salt solution is added to make the total volume up to 5 c.cm. 



The following controls are absolutely necessary. 



