636 APPENDIX: LABORATORY METHODS 



If the medium is allowed to stand for a time, the thioglycollic acid 

 becomes oxidized, and therefore a sterile solution of the acid should be 

 available and an amount sufficient to give a concentration of 0.01 per 

 cent should be added immediately before use. To obtain a culture of 

 standard density a small inoculum from blood broth is transferred to a 

 tube of the medium and incubated overnight. This is then subcultured 

 into fresh medium at 37° and incubated for 5 to 6 hours. 



Pneumococcal Protein (Avery and Morgan 54 ) 



Concentrated suspensions of organisms in 0.002 N NaOH are sub- 

 jected to repeated freezing and thawing to disrupt the cell bodies. This 

 thick solution of dissolved organisms is then diluted with salt solution 

 to one-tenth the volume of original culture fluid and passed through a 

 Berkefeld V filter. To this filtered solution N acetic acid is added 

 slowly and the mixture carefully shaken. Flocculation of the protein 

 occurs promptly and completely at a reaction faintly acid to litmus. The 

 precipitated protein is separated by centrifugation, washed several 

 times in distilled water, and redissolved in salt solution by adding 0.1 

 N NaOH until the solution is faintly alkaline to litmus. Solutions of 

 pneumococcal protein freshly prepared by this method exhibit the usual 

 qualitative color reactions for substances of this nature: positive biuret, 

 Hopkins-Cole, Millon, xanthoproteic, and Molisch reactions. The hy- 

 drolyzed protein gives the purine reaction with Fehling's solution. The 

 protein solutions are standardized on the basis of their nitrogen content. 



Somatic Carbohydrate or C Fraction (Tillett, Goebel, and 

 Avery 1410 ) 



The bacterial cells from 3 liters of broth culture of an R strain de- 

 rived from Type II Pneumococcus were collected by centrifugation. 

 The bacteria were resuspended in 50 cc. of salt solution and were re- 

 peatedly frozen and thawed to break up the bacterial bodies. To this 

 solution of bacteria 0.5 cc. N acetic acid was added, and the mixture 

 was then heated for 10 minutes in a boiling water-bath. The tube was 

 cooled and the coagulated protein was separated from the clear super- 

 natant liquid which contained the C substance. In this manner the non- 

 coagulable material from 30 to 40 liters of culture was collected. The 

 combined supernatant extract, after neutralization, was finally filtered 

 through a Berkefeld candle and then concentrated in vacuo to 50 cc. 

 The concentrates from 36 liters of bacteria obtained as described above 



