IV. ISOLATION OF COMPONENTS 639 



acetate solution and 5 cc. of glacial acetic acid, and reprecipitated free 

 from copper, after which it was dissolved in 100 cc. of water and the 

 solution again tested for glycogen. If this is present the copper precipi- 

 tation is repeated. The SI was finally precipitated with redistilled alco- 

 hol in the presence of a small amount of sodium acetate, washed with 

 redistilled alcohol, filtered, and dried. Yield: 0.9 gram of the neutral 

 sodium salt of SI. 



b. Preparation of the specific polysaccharide of Type II Pneumococ- 

 cus. 10 liters of a 4-day culture of Type II Pneumococcus were treated 

 in the same way as for Type I. No differences were observed up to the 

 point at which the copper acetate precipitation was made. However, 

 only one-third of the SII was thrown down as the copper salt. This 

 fraction, which was free from glycogen, was reprecipitated as the cop- 

 per salt and isolated separately. 



The copper-soluble fraction was freed from copper salts by several 

 precipitations with alcohol in the presence of acetic acid and sodium 

 acetate and was redissolved in a small volume of water. Even this con- 

 centrated solution failed to react with copper acetate. As glycogen was 

 present in this fraction it was removed by adjusting the pH to 6.5 and 

 adding a small amount of saliva. After a few minutes the iodine test was 

 negative. To remove protein impurities added in the saliva, 5 grams of 

 sodium acetate and 2.5 cc. of acetic acid were added, and the solution 

 (volume 100 cc.) was repeatedly shaken with chloroform and a little 

 butyl alcohol until an emulsion layer was no longer formed. Since the 

 polysaccharide solution still contained nitrogen it was adjusted to con- 

 tain 5 grams of sodium acetate per 100 cc. and was chilled and precipi- 

 tated with 5 volumes of glacial acetic acid. The precipitate was centri- 

 fuged in the cold, taken up in 50 cc. of 5 per cent sodium acetate 

 solution, and again precipitated with 5 volumes of acetic acid. This was 

 followed by two precipitations with alcohol from 5 per cent sodium 

 acetate solution and one precipitation with redistilled alcohol, after 

 which the polysaccharide was washed with redistilled alcohol, filtered, 

 and dried. One cc. of the broth contained 0.59 mg. or a total of 0.590 

 gm. of SII in the 10 liters used. Recovery, 78 per cent. 



c. Preparation of the specific polysaccharide of Type III Pneumo- 

 coccus. The culture filtrate was concentrated in vacuo to one-tenth its 

 original volume. After three precipitations with 1.5 volumes of alcohol, 

 part of the protein contained in the material was removed by 40 per 

 cent saturation with sodium sulfate at 37°. The SHI was then precipi- 

 tated by completely saturating the solution with sodium sulfate. After 



