IV. ISOLATION OF COMPONENTS 637 



were again acidified with 0.5 cc. of N acetic acid and reheated for 15 

 minutes at 100°. A small amount of coagulated protein was thus sepa- 

 rated and removed by centrifugation. The clear solution was now 

 treated with 5 volumes of alcohol and after standing overnight in the 

 ice-box, a precipitate settled out which contained all of the so-called C 

 substance, together with certain nitrogenous impurities. This precipi- 

 tate, when redissolved in saline solution, was found to give a specific 

 precipitin test with antipneumococcic horse serums of Types I, II, and 

 III. The solution of the C fraction was found to contain, however, ma- 

 terial which gave a positive biuret test, but this impurity was precipi- 

 tated from solution by making the mixture alkaline with sodium hydrox- 

 ide, without loss of the serologically reactive substance. The alkaline 

 solution of the C fraction, now at a volume of 50 cc, was reprecipitated 

 by the addition of 5 volumes of alcohol. The material was centrifuged, 

 redissolved in 40 cc. of water, and again precipitated from faintly acid 

 solution by alcohol. This procedure was repeated altogether four times. 

 The carbohydrate recovered from the final alcoholic precipitation was 

 dissolved in 15 cc. of water and was cooled to 0°. To the solution were 

 added 2 cc. of hydrochloric acid (sp. gr. 1.09). A small amount of in- 

 soluble inactive material separated from the solution and was centri- 

 fuged off. The clear acid solution was now precipitated with five vol- 

 umes of redistilled alcohol. After standing at 0° for 4 hours the C 

 substance was separated by centrifugation. It was redissolved in 10 cc. 

 of water and then reprecipitated with alcohol and acid. The final prod- 

 uct was washed free from chlorides with 85 per cent alcohol, and was 

 washed finally with redistilled alcohol and ether. The yield was about 

 65 milligrams from 36 liters of broth cultures. 



Capsular Polysaccharide (Heidelberger, Kendall, and Scherp 627 ) 



a. Preparation of the specific polysaccharide of Type I Pneumococ- 

 cus. 10 liters of phosphate meat-infusion broth containing 0.3 per cent 

 of added glucose were seeded with a highly virulent Type I pneumococ- 

 cus.* After 72 hours at 37°, 1 per cent of phenol was added and the 

 culture allowed to stand overnight. It was then centrifuged in a Shar- 

 pies centrifuge and the effluent concentrated to 1 liter under reduced 

 pressure, keeping the temperature below 35°. 100 grams of crystalline 

 sodium acetate were dissolved in the concentrated broth and 1,250 cc. 



* Recent mouse passage is necessary for good yields of the polysaccharide. 

 After a period of 3 weeks between the last mouse passage and the preparation 

 of the polysaccharide the yield was reduced 80 per cent. 



