276 BIOLOGY OF PNEUMOCOCCUS 



cose. In other words there is present in this fraction a complex 

 carbohydrate."* 



Felton 417 amplified the study of the acid-soluble and acid-insolu- 

 ble fractions of Pneumococcus on which he had already published 

 a preliminary note. 416 The material was prepared by dehydrating 

 broth-grown pneumococci with acetone, followed by dessication in 

 vacuo over calcium chloride. Prepared by this method the organ- 

 isms retained their antigenicity at least through the period of the 

 study. Watery suspensions of the dried pneumococci were treated 

 first with enough sodium hydroxide to make the concentration of 

 the alkali one-tenth normal, and then after the suspensions were 

 allowed to stand at room temperature for one-half hour, an equal 

 volume of various mineral and organic acids was added. In this way 

 it was possible, as Heidelberger and Avery, and Wadsworth and 

 Brown had found, to separate the pneumococcal material into an 

 acid-soluble and an acid-insoluble fraction. The former possessed 

 most of the immunizing activity of the cell, and the immunity pro- 

 duced by injecting this fraction into white mice proved to be 

 .largely type-specific. The latter fraction, probably containing 

 some intact cells, also possessed a small amount of the immunizing 

 substance and evoked in mice a heterologous immunity. Precipita- 

 tion of the acid-soluble fraction with ethyl alcohol or acetone 

 yielded at least 90 per cent of the immunizing substance. 



Felton's experiments, therefore, confirmed the work of Schie- 

 mann and Caspar, 1228 Saito and Ulrich, 1214 Wadsworth and 

 Brown, 1466 and Zozaya and Clark 1590 in that active immunity can 

 be produced in white mice by a fraction smaller than the intact 

 pneumococcal cell. The precipitating action of the acid-insoluble 



* In another part of the same paper, Felton stated that in his experience the 

 Molisch test failed in the presence of other organic substances to indicate the 

 presence of polysaccharide which on hydrolysis gave a glucose content of from 

 0.2 to 0.5 per cent, and emphasized the fact that the biuret test for protein is 

 notably insensitive, giving a positive test with concentrations of the majority of 

 proteins in a dilution no higher than 1 to 10,000. When one considers the ex- 

 traordinary immunological activity of bacterial polysaccharides and proteins, it 

 need scarcely be said that less reliance should be placed on these or similar 

 tests as criteria for determining the precise chemical nature of these antigens. 



