OSWALD T. AVERY, COLIN M. MACLEOD, AND MACLYN MCCARTY 143 



chanically 30 to 60 minutes. The cells are separated by centrifugation, and the ex- 

 traction process is repeated 2 or 3 times. The desoxycholate extracts prepared in 

 this manner are clear and colorless. These extracts are combined and precipitated 

 by the addition of 3 to 4 volumes of absolute ethyl alcohol. The sodium desoxycho- 

 late being soluble in alcohol remains in the supernatant and is thus removed at this 

 step. The precipitate forms a fibrous mass which floats to the surface of the alcohol 

 and can be removed directly by lifting it out with a spatula. The excess alcohol is 

 drained from the precipitate which is then redissolved in about 50 cc. of saline. The 

 solution obtained is usually viscous, opalescent, and somewhat cloudy. 



Deproteinization and Removal of Capsular Polysaccharide. — The solution is then 

 deproteinized by the chloroform method described by Sevag (12). The procedure is 

 repeated 2 or 3 times until the solution becomes clear. After this preliminary treat- 

 ment the material is reprecipitated in 3 to 4 volumes of alcohol. The precipitate 

 obtained is dissolved in a larger volume of saline (150 cc.) to which is added 3 to 5 

 mg. of a purified preparation of the bacterial enzyme capable of hydrolyzing the 

 Type III capsular polysaccharide (13). The mixture is incubated at 37°C, and the 

 destruction of the capsular polysaccharide is determined by serological tests with 

 Type III antibody solution prepared by dissociation of immune precipitate accord- 

 ing to the method described by Liu and Wu (14). The advantages of using the 

 antibody solution for this purpose are that it does not react with other serologically 

 active substances in the extract and that it selectively detects the presence of the cap- 

 sular polysaccharide in dilutions as high as 1 : 6,000,000. The enzymatic breakdown 

 of the polysaccharide is usually complete within 4 to 6 hours, as evidenced by the loss 

 of serological reactivity. The digest is then precipitated in 3 to 4 volumes of ethyl 

 alcohol, and the precipitate is redissolved in 50 cc. of saline. Deproteinization by the 

 chloroform process is again used to remove the added enzyme protein and remaining 

 traces of pneumococcal protein. The procedure is repeated until no further film of 

 protein-chloroform gel is visible at the interface. 



Alcohol Fractionation. — Following deproteinization and enzymatic digestion of the 

 capsular polysaccharide, the material is repeatedly fractionated in ethyl alcohol as 

 follows. Absolute ethyl alcohol is added dropwise to the solution with constant 

 stirring. At a critical concentration varying from 0.8 to 1.0 volume of alcohol the 

 active material separates out in the form of fibrous strands that wind themselves 

 around the stirring rod. This precipitate is removed on the rod and washed in a 50 

 per cent mixture of alcohol and saline. Although the bulk of active material is re- 

 moved by fractionation at the critical concentration, a small but appreciable amount 

 remains in solution. However, upon increasing the concentration of alcohol to 3 

 volumes, the residual fraction is thrown down together with inert material in the form 

 of a flocculent precipitate. This flocculent precipitate is taken up in a small volume 

 of saline (5 to 10 cc.) and the solution again fractionated by the addition of 0.8 to 1.0 

 volume of alcohol. Additional fibrous material is obtained which is combined with 

 that recovered from the original solution. Alcoholic fractionation is repeated 4 to 5 

 times. The yield of fibrous material obtained by this method varies from 10 to 25 

 mg. per 75 liters of culture and represents the major portion of active material present 

 in the original crude extract. 



Effect of Temperature. — As a routine procedure all steps in purification were carried 



193 



