142 TRANSFORMATION OF PNEUMOCOCCAL TYPES 



The anti-R properties of the serum in the medium cause the R cells to 

 agglutinate during growth, and clumps of the agglutinated cells settle to the 

 bottom of the tube leaving a clear supernatant. When transformation occurs, 

 the encapsulated S cells, not being affected by these antibodies, grow diffusely 

 throughout the medium. On the other hand, in the absence of transformation 

 the supernatant remains clear, and only sedimented growth of R organisms 

 occurs. This difference in the character of growth makes it possible by inspec- 

 tion alone to distinguish tentatively between positive and negative results. 

 As routine all the cultures are plated on blood agar for confirmation and further 

 bacteriological identification. Since the extracts used in the present study 

 were derived from Pneumococcus Type III, the differentiation between the 

 colonies of the original R organism and those of the transformed S cells is 

 especially striking, the latter being large, glistening, mucoid colonies typical of 

 Pneumococcus Type III. Figs. 1 and 2 illustrate these differences in colony 

 form. 



A typical protocol of a titration of the transforming activity of a highly 

 purified preparation is given in Table IV. 



Preparative Methods 



Source Material. — In the present investigation a stock laboratory strain of Pneu- 

 mococcus Type III (A66) has been used as source material for obtaining the active 

 principle. Mass cultures of these organisms are grown in 50 to 75 liter lots of plain 

 beef heart infusion broth. After 16 to 18 hours' incubation at 37°C. the bacterial 

 cells are collected in a steam-driven sterilizable Sharpies centrifuge. The centrifuge 

 is equipped with cooling coils immersed in ice water so that the culture fluid is thor- 

 oughly chilled before flowing into the machine. This procedure retards autolysis 

 during the course of centrifugation. The sedimented bacteria are removed from the 

 collecting cylinder and resuspended in approximately 150 cc. of chilled saline (0.85 

 per cent NaCl), and care is taken that all clumps are thoroughly emulsified. The 

 glass vessel containing the thick, creamy suspension of cells is immersed in a water 

 bath, and the temperature of the suspension rapidly raised to 65°C. During the 

 heating process the material is constantly stirred, and the temperature maintained 

 at 65°C. for 30 minutes. Heating at this temperature inactivates the intracellular 

 enzyme known to destroy the transforming principle. 



Extraction of Heat-Killed Cells. — Although various procedures have been used, 

 only that which has been found most satisfactory will be described here. The heat- 

 killed cells are washed with saline 3 times. The chief value of the washing process 

 is to remove a large excess of capsular polysaccharide together with much of the pro- 

 tein, ribonucleic acid, and somatic "C" polysaccharide. Quantitative titrations of 

 transforming activity have shown 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 substances which are removed by this procedure. 



After the final washing, the cells are extracted in 150 cc. of saline containing sodium 

 desoxycholate in final concentration of 0.5 per cent by shaking the mixture me- 



192 



