ESTIMATION OF BACTERICIDAL SUBSTANCES 09 



tendered highly immune against the organism in question by vaccina- 

 tion (which see). In such a case a much larger quantity of bacteria 

 may be injected (1 oese) with impunity, and it will be observed 

 that notwithstanding the large dose no bacteria will be found in the 

 peritoneal cavity at the expiration of one hour (Pfeiffer's experiment). 

 The same result will be obtained if instead of using a normal 

 guinea-pig and injecting some immune serum together with the 

 bacteria, these are introduced by themselves into a previously immu- 

 nized animal. 



The reason why bacteriolysis will be so much more extensive 

 in the presence of immune serum is the fact that as a result of 

 infection (vaccination, immunization) the amboceptor content of 

 the blood serum is materially increased. The complement which is 

 necessary for the experiment is normally present in the living animal. 

 In its absence, of course, bacteriolysis could not take place, and 

 as complement readily becomes inactive outside of the body, after 

 standing even for a relatively short time at body or room temperature, 

 it is essential if the experiment is conducted in vitro that only per- 

 fectly fresh serum be used. Otherwise bacteriolysis will not occur, 

 even though the serum may be rich in natural amboceptors. 



Quantitative Estimation of Bactericidal Substances. The quanti- 

 tative estimation of the content in bactericidal substances of a given 

 serum is most conveniently carried out by starting with a suspen- 

 sion, of known number, of the organism to be examined, and inocu- 

 lating tubes containing known amounts of serum, after which these 

 are incubated for a certain period of time and plates are prepared 

 in which the number of surviving organisms is finally determined 

 by a direct count. As serum is in itself an admirable culture medium 

 for most organisms it is, of course, essential to reduce this factor 

 as much as possible in the experiment. To this end one can either 

 determine the total number of bacteria Which is completely killed 

 by a given amount of serum in a given length of time, or one can 

 determine the extreme degree of dilution in which a given serum will 

 still exercise a bactericidal effect, or one may determine the 

 maximal bactericidal effect, which is observed after different 

 intervals of time. The general arrangement of such a test is apparent 

 from the following example, which is taken from Wright, and which 

 represents the titration of a given serum against cholera vibrios on 

 the one hand and typhoid bacilli on the other: 



