180 



activity have shovv^n that not more than 

 10 to 15 per cent of the active material 

 is lost in the washing, a loss which is small 

 in comparison to the amount of inert sub- 

 stances which are removed by this pro- 

 cedure. 



After the final washing, the cells are 

 extracted in 150 cc. of sahne containing 

 sodium desoxycholate in final concentra- 

 tion of 0.5 per cent by shaking the mix- 

 ture mechanically 30 to 60 minutes. The 

 cells are separated by centrifugation, and 

 the extraction process is repeated 2 or 3 

 times. The desoxycholate extracts pre- 

 pared in this manner are clear and color- 

 less. These extracts are combined and 

 precipitated by the addition of 3 to 4 

 volumes of absolute ethyl alcohol. The 

 sodium desoxycholate 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 re- 

 moved directly bv lifting it out with a 

 spatula. The excess alcohol is drained 

 from the precipitate which is then re- 

 dissolved in about 50 cc. of saline. The 

 solution obtained is usually viscous, 

 opalescent, and somewhat cloudy. 



Deprotei7iizatio7i a?id Re?fioval of Cap- 

 sular 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 treatment the material is re- 

 precipitated 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 capa- 

 ble of hydrolyzing the Type 111 capsular 

 polysaccharide (13). The mixture is in- 

 cubated at 37°C., and the destruction of 

 the capsular polysaccharide is determined 

 by serological tests with Type III anti- 

 body solution prepared by dissociation of 

 immune precipitate according 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 selec- 

 tively detects the presence of the capsular 



AVERY, MACLEOD, MCCARTY 



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 re- 

 dissolved in 50 cc. of sahne. Deprotein- 

 ization by the chloroform process is 

 again used to remove the added enzyme 

 protein and remaining traces of pneumo- 

 coccal protein. The procedure is repeated 

 until no further film of protein-chloro- 

 form gel is visible at the interface. 



Alcohol Fractionation. — Following de- 

 proteinization 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 con- 

 stant 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 them- 

 selves around the stirring rod. This pre- 

 cipitate is removed on the rod and 

 washed in a 50 per cent mixture of al- 

 cohol and saline. Although the bulk of 

 active material is removed by fractiona- 

 tion at the critical concentration, a small 

 but appreciable amount remains in solu- 

 tion. However, upon increasing the con- 

 centration of alcohol to 3 volumes, the 

 residual fraction is thrown down together 

 with inert material in the form of a floc- 

 culent precipitate. This flocculent pre- 

 cipitate 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 re- 

 peated 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 ac- 

 tive material present in the original crude 

 extract. 



Effect of Temperature. — As a routine 

 procedure all steps in purification were 

 carried out at room temperature unless 

 specifically stated otherwise. Because of 

 the theoretical advantage of working at 



