PRESERVATION OF HEMOLYSINS. 135 



form, but the addition of hemolysin and complement produces no hemolysis. 

 The presence of salt is indispensable 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 be inactivated for one-half hour at 56 

 C. if it is not kept ready for use in an inactivated state. 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 hemolysin titration it is best to use a 

 constant dose of complement as i c.c. 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," complement 

 may be kept for weeks. Stern, however, does not recommend complement preserved 

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

 decreased. 



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

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

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



The following controls are absolutely necessary. 



1. A test tube showing that hemolysin without complement in strong 

 dosage is ineffective; 



2. A test tube indicating that complement without hemolysin in the 

 dosage used is ineffective; 



3. A test tube which shows that the NaCL solution is isotonic. 



The three reagents must be thoroughly mixed by careful shaking of 

 the tubes which are then placed into the thermostat at 37 C. and hemolysis 

 watched for. The duration of the observation is a matter of personal 

 preference. Only the length of time must always be mentioned. One 

 must say, for instance, that the titer of this hemolysin is i :8oo, using o.i c.c. 

 of complement under observation for one-half hour, or it is 1 11500 with o.i 

 complement under observation for two hours. The time in which hemo- 

 lysins work is very different. While many hemolysins of the same titer act 



