144 TRANSFORMATION OF PNEUMOCOCCAL TYPES 



out at room temperature unless specifically stated otherwise. Because of the theo- 

 retical advantage of working at low temperature in the preparation of biologically 

 active material, the purification of one lot (preparation 44) was carried out in the cold. 

 In this instance all the above procedures with the exception of desoxycholate ex- 

 extraction and enzyme treatment were conducted in a cold room maintained at 0-4°C. 

 This preparation proved to have significantly higher activity than did material simi- 

 larly prepared at room temperature. 



Desoxycholate extraction of the heat-killed cells at low temperature is less efficient 

 and yields smaller amounts of the active fraction. It has been demonstrated that 

 higher temperatures facilitate extraction of the active principle, although activity is 

 best preserved at low temperatures. 



Analysis of Purified Transforming Material 



General Properties. — Saline solutions containing 0.5 to 1.0 mg. per cc. of the 

 purified substance are colorless and clear in diffuse light. However, in strong 

 transmitted light the solution is not entirely clear and when stirred exhibits a 

 silky sheen. Solutions at these concentrations are highly viscous. 



Purified material dissolved in physiological salt solution and stored at 2^i C. 

 retains its activity in undiminished titer for at least 3 months. However, when 

 dissolved in distilled water, it rapidly decreases in activity and becomes com- 

 pletely inert within a few days. Saline solutions stored in the frozen state in a 

 CO2 ice box (— 70°C.) retain full potency for several months. Similarly, 

 material precipitated from saline solution by alcohol and stored under the 

 supernatant remains active over a long period of time. Partially purified 

 material can be preserved by drying from the frozen state in the lyophile 

 apparatus. However, when the same procedure is used for the preservation 

 of the highly purified substance, it is found that the material undergoes changes 

 resulting in decrease in solubility and loss of activity. 



The activity of the transforming principle in crude extracts withstands 

 heating for 30 to 60 minutes at 65°C. Highly purified preparations of active 

 material are less stable, and some loss of activity occurs at this temperature. 

 A quantitative study of the effect of heating purified material at higher tem- 

 peratures has not as yet been made. Alloway (6), using crude extracts pre- 

 pared from Type III pneumococcal cells, found that occasionally activity could 

 still be demonstrated after 10 minutes' exposure in the water bath to tem- 

 peratures as high as 90°C. 



The procedures mentioned above were carried out with solutions adjusted 

 to neutral reaction, since it has been shown that hydrogen ion concentrations 

 in the acid range result in progressive loss of activity. Inactivation occurs 

 rapidly at pH 5 and below. 



Qualitative Chemical Tests. — The purified material in concentrated solution 

 gives negative biuret and Millon tests. These tests have been done directly 

 on dry material with negative results. The Dische diphenylamine reaction 



194 



