292 BIOLOGY OF PNEUMOCOCCUS 



non-polysaccharide since both the Molisch test and the test for reduc- 

 ing sugars after acid hydrolysis are negative. (3) The antigens so pre- 

 pared from Type I SSS produce active immunity in mice against both 

 Type I and Type II pneumococci. (4) The restoration of the biological 

 properties destroyed with hot NaOH by treatment with NH 4 OH shows 

 that the "acetyl" content is of no significance in determining the bio- 

 logical activity of the preparation studied, but conversely indicates that 

 this property is determined by a definite molecular configuration which 

 is readily altered by strong alkalis. 



Believing that the procedures hitherto employed in the prepara- 

 tion of pneumococcal polysaccharides might have disrupted the 

 molecular configuration of the carbohydrate molecule with a con- 

 sequent loss of essential radicals, Sevag (1934) 1257 applied gentler 

 measures for their isolation. Adopting the well-known action of 

 liquid air in disintegrating bacterial cells, Sevag first froze the 

 sediment from twelve-hour dextrose-serum broth cultures of a viru- 

 lent strain of Type I Pneumococcus and then subjected the detri- 

 tus to prolonged shaking in a mixture of water, chloroform, and 

 amyl alcohol. The protein coagulated by this treatment was re- 

 moved and the polysaccharide in the supernatant fluid was iso- 

 lated by precipitation with alcohol. After further purification with 

 chloroform, an alcohol-insoluble fraction was obtained which was 

 claimed to be protein-free, gave a strong Molisch reaction, and 

 contained between 6.60 and 6.72 per cent nitrogen, of which from 

 1.21 to 1.42 per cent was in the form of amino nitrogen. The spe- 

 cific rotation of the product was [a] D = + 218.3* to 219.1* 

 which after acid hydrolysis became +54.1 and showed a glucose 

 content of 23.98 per cent. The polysaccharide thus isolated, in a 

 dose of 0.0001 milligrams, protected mice against a thousand fatal 

 doses of virulent Type I Pneumococcus and in high dilutions gave 

 a high precipitation titer with homologous immune rabbit serum. 



Sevag entertained some doubts concerning the validity of Avery 

 and Goebel's claim that the capsular polysaccharide of Pneumo- 

 coccus Type I was an acetylated substance and queried whether 



•Typographical error in original figures gives +21.83 and 21.91. 



