AVERY, MACLEOD, MCCARTY 



being large, glistening, mucoid colo- 

 nies typical of Pncumococcus Type 

 III. Figs. 1 and 2 illustrate these differ- 

 ences in colony form. 



179 



A typical protocol of a titration of 

 the transforming activity of a highly 

 purified preparation is given in Table 

 IV. 



Figs. 1 and 2. (1) Colonies of the R variant (R36A) derived from Pneumococcus 

 type II. Plated on blood agar from a culture grown in serum broth in the absence of 

 the transforming substance. X3.5. (2) Colonies on blood agar of the same cells after 

 induction of transformation during growth in the same medium with the addition 

 of active glistening, mucoid colonies shown are characteristic of Pneumococcus Type 

 III and readily distinguishable from the small, rough colonies of the parent R strain 

 illustrated in Fig. 1. X3.5. (The photograph was 7nade by Mr. Joseph B. Haiilenbeek.) 



Preparative Methods 



Source Material. — In the present inves- 

 tigation a stock laboratory strain of Pneu- 

 mococcus Type III (A66) has been used 

 as source material for obtaining the active 

 principle. A4ass cultures of these organ- 

 isms are grown in 50 to 75 liter lots of 

 plain beef heart infusion broth. After 16 

 to 18 hours' incubation at 37°C. the bac- 

 terial cells are collected in a steam-driven 

 sterilizable Sharpies centrifuge. The cen- 

 trifuge is equipped with cooling coils im- 

 mersed in ice water so that the culture 

 fluid is thoroughly chilled before flowing 

 into the machine. This procedure retards 

 autolysis during the course of centrifuga- 

 tion. The sedimented bacteria are re- 

 moved 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 thor- 



oughly emulsified. The glass vessel con- 

 taining 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 in- 

 activates the intracellular enzyme known 

 to destroy the transforming principle. 



Extraction of Heat-Killed Cells. — Al- 

 though 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. "Jhe chief value of the 

 washing process is to remove a large ex- 

 cess of capsular polysaccharide together 

 with much of the protein, ribonucleic 

 acid, and somatic "C" polysaccharide. 

 Quantitative titrations of transforming 



