l88 BACTERIOLYSIS METHODS OF RESEARCH 



Here 0-0125 c>c - f ^he immune serum contained sufficient 

 immune body to sensitize fully i c.c. of a 5 per cent, emulsion of 

 corpuscles i.e., a given volume of serum will sensitize 1-25 of its 

 own volume of corpuscles. 



The determination of the amount of complement is made by an 

 inversion of this method. Thus Gay, who has made numerous 

 investigations as to the amount of complement present in human 

 serum, proceeds as follows : The sensitizing serum is derived from 

 a rabbit which has been injected with ox corpuscles. This is 

 heated, and the amount necessary for complete sensitization of a 

 definite amount of ox corpuscles is determined; thus in his 

 experiment 0-7 c.c. saturated 7 c.c. of a 5 per cent, emulsion. A 

 series of tubes, each containing i c.c. of a 5 per cent, emulsion of 

 fully-sensitized corpuscles, is prepared, and varying doses of the 

 serum to be tested are added ; the amount, which is small, is pre- 

 pared by dilution with normal saline to such an extent that the 

 actual bulk added is 0*1 c.c. The subsequent treatment is as 

 above. Gay and Ayer find that on the average about -$ c.c. has 

 to be added to bring about complete haemolysis, the limits being 

 i 1 ^ and g^ c.c. 



Quantitative researches on the bacteriolytic action of the serum 

 are very much more difficult. The actual determination of the 

 amount of bactericidal action is by no means easy, and the results 

 obtained are of very little importance, since the serum may be 

 very deficient in complement, and deviation may occur. The 

 method which has been chiefly employed is that of plating out 

 after the bacteria and serum have been allowed to act together at 

 incubator temperature for a given period. The method is briefly 

 as follows : The emulsion of bacteria must be of constant strength. 

 As a rule, it is sufficient to take a twenty-four-hour broth culture, 

 and to dilute it to the same degree in all experiments ; or the 

 same loop may be employed throughout, or some one or other of 

 the counting methods which have been described may be used. 

 The emulsions should be dilute, so that all the bacteria may 

 be killed. - Klien recommends i : 8,000 of a twenty-four-hour 

 broth culture in the case of B. typhosus. Lastly, normal saline 

 solution is better than broth as a diluting agent, since it diminishes 

 the chance of error owing to the multiplication of bacteria during 

 the somewhat lengthy process of preparing the dilutions. 



The actual process is as follows : Measured small amounts of 

 the serum to be tested are placed in a series of tubes, a uniform 



