418 PATHOGENIC MICROORGANISMS 



accomplished by beginning with an injection of about 1 c.c. of broth 

 culture heated for ten minutes at 60° in order to kill the bacilli. After 

 five or six days, a second injection of a larger dose of dead bacilli is 

 administered; at similar intervals, gradually increasing doses of dead 

 bacilli are given and finally considerable quantities of a living and fully 

 virulent culture may be injected without serious consequences to the 

 animal. While this method is convenient and usually successful, it 

 is also possible to obtain satisfactory immunization by beginning with 

 very small doses of living microorganisms, according to the early 

 method of Chantemesse and Widal,' and others. 



Such active immunization, successfully carried out upon rabbits and 

 guinea-pigs, within a short time after the discovery of the typhoid bacil- 

 lus, was believed to depend upon the development of antitoxic sub- 

 stances in immunized animals. This point of view, however, was not 

 long tenable, and was definitely disproven by the investigations of Pfeif- 

 fer and Kolle '^ in 1896. These investigators, as well as a large number of 

 others working subsequently, have shown satisfactorily that there are 

 present in the blood serum of typhoid-immune animals and human 

 beings, bacteriolytic, bactericidal, and agglutinating substances, and to 

 a lesser extent, precipitating and opsonic bodies. 



Bactericidal and Bacteriolytic Substances. — The bacteriolytic sub- 

 stances in typhoid-immune serum may be demonstrated either by the 

 intraperitoneal technique of Pfeiffer or in vitro. In the former experi- 

 ment a small quantity of a fresh culture of typhoid bacilli is mixed 

 with the diluted immune serum and the emulsion injected into the 

 peritoneal cavity of a guinea-pig. Removal of peritoneal exudate with 

 a capillary pipette and examination in the hanging drop will reveal, 

 within a short time, a swelling and granulation of the bacteria — the 

 so-called Pfeiffer phenomenon. The test in vitro, as recommended by 

 Stem and Korte,^ may be carried out by adding definite quantities of 

 a fresh agar culture of typhoid bacilli to progressively increasing dilu- 

 tions of inactivated immune serum together with definite quantities of 

 complement in the form of fresh normal rabbit or guinea-pig serum. 

 At the end of several hours' incubation at 37.5° C. definite quantities 

 of the fluid from the various tubes are inoculated into melted agar 

 and plates are poured to determine the bactericidal action. Carefu] 

 colony counting in these plates and comparison with proper controls 



' Chantemesse and Widal, Ann. de I'inst. Pasteur, 1892. 



2 Pfeiffer und KolU, Zeit. f. Hyg., xxi, 1896. 



3 Stem und Korte, Berl. klin. Woch., x., 1904. 



