266 BIOLOGY OF PNEUMOCOCCUS 



grown in broth containing rabbits' blood and killed by the addi- 

 tion of formalin. He obtained an immune rabbit serum by treat- 

 ing the animals intravenously with repeated injections of the au- 

 tolysate and of the killed hormone broth-rabbit blood cultures and 

 this serum was designated "Anti-A" serum. In addition, to other 

 rabbits Enders gave injections of formalinized saline suspensions 

 of agar-grown pneumococci. When the Anti-A serum was tested 

 against SSS* no precipitate was formed, but with an autolysate 

 from smooth Type I Pneumococcus the serum was definitely pre- 

 cipitating, and the reaction was type-specific. Furthermore, both 

 the Type I autolysate and the supernatant fluid from the ace- 

 tic acid precipitation of the autolysate gave precipitates with 

 Anti-A serum but none with antipneumococcic serum prepared 

 from rough strains. 



By the method devised by Ward, Enders then added to his 

 "normal" serum specific carbohydrate until precipitation was com- 

 plete. The centrifuged supernatant fluid no longer gave any visible 

 precipitation with SSS but, when the homologous autolysate was 

 added, an abundant, flocculent precipitate was observed. Inasmuch 

 as the SSS had apparently exhausted its specific antibody from 

 the immune serum, the appearance of a precipitate on the addition 

 of autolysate indicated to Enders the presence in the autolysate of 

 a precipitinogen other than the specific polysaccharide and, ac- 

 cordingly, to this hypothetical substance he gave the name, "A 

 Substance." 



There are two other characters of the A substance of Enders 

 which should be mentioned. One was instability when exposed to 

 heat. Boiled for one-half hour at pH 9 its effectiveness as a pre- 

 cipitating antigen was reduced at least one thousandfold, while the 

 soluble specific substance under similar treatment remained un- 

 changed. When, however, the reaction was adjusted to pH 4 with 

 10 per cent acetic acid, a solution containing the A substance 



* For convenience, the soluble specific substance or type-specific polysaccha- 

 rides of Pneumococcus will hereafter be frequently referred to as SSS. 



