TITRATION OF HEMOLYSIN 



149 



1. A test showing that hemolysin in strong dosage but without com- 

 plement is ineffective ; 



2. A test indicating that without hemolysin complement in the 

 dosage used is ineffective; 



3. A test which shows that the NaCl solution is iso tonic. 



The three reagents must be thoroughly mixed by careful shaking of 

 the tubes which are then placed in 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.cm. of complement under observation for one-half hour, or it is 

 i : 500 with o. i complement under observation for two hours. The time in 

 which hemolysins work is very different. While many hemolysins of the 

 same titer act in a few moments, others require two hours. The author 

 has made it a rule to read the result after two hours' observation, but he 

 notes the progress of the reaction every one-half hour in order to 

 determine whether it is a slowly or rapidly acting hemolysin. 



The following chart demonstrates the titration of a hemolysin as a preliminary 

 experiment to the complement fixation method. 



Determining the end reaction is a source of difficulty for the beginner. 

 Between the extreme "o" i.e., entire absence of hemolysis, where the 

 appearance of the tube corresponds to that of control III representing a 

 suspension of red blood cells diluted with isotonic saline, and the other 

 extreme "complete/' i.e., complete hemolysis, where every trace of cor- 

 puscular elements has disappeared and a fluid looking like dilute red-wine 



