OSWALD T. AVERY, COLIN M. MACLEOD, AND MACLYN MCCARTY 139 



1. Nutrient Broth. — Beef heart infusion broth containing 1 per cent neopeptone 

 with no added dextrose and adjusted to an initial pH of 7.6-7.8 is used as the basic 

 medium. Individual lots of broth show marked and unpredictable variations in the 

 property of supporting transformation. It has been found, however, that charcoal 

 adsorption, according to the method described by MacLeod and Mirick (10) for 

 removal of sulfonamide inhibitors, eliminates to a large extent these variations; conse- 

 quently this procedure is used as routine in the preparation of consistently effective 

 broth for titrating the transforming activity of extracts. 



2. Serum or Serous Fluid. — In the first successful experiments on the induction of 

 transformation in vitro, Dawson and Sia (5) found that it was essential to add serum 

 to the medium. Anti-R pneumococcal rabbit serum was used because of the observa- 

 tion that reversion of an R pneumococcus to the homologous S form can be induced 

 by growth in a medium containing anti-R serum. Alloway (6) later found that as- 

 citic or chest fluid and normal swine serum, all of which contain R antibodies, are 

 capable of replacing antipneumococcal rabbit serum in the reaction system. Some 

 form of serum is essential, and to our knowledge transformation in vitro has never 

 been effected in the absence of serum or serous fluid. 



In the present study human pleural or ascitic fluid has been used almost exclusively. 

 It became apparent, however, that the effectiveness of different lots of serum varied 

 and that the differences observed were not necessarily dependent upon the content 

 of R antibodies, since many sera of high titer were found to be incapable of support- 

 ing transformation. This fact suggested that factors other than R antibodies are 

 involved. 



It has been found that sera from various animal species, irrespective of their 

 immune properties, contain an enzyme capable of destroying the transforming prin- 

 ciple in potent extracts. The nature of this enzyme and the specific substrate on 

 which it acts will be referred to later in this paper. This enzyme is inactivated by 

 heating the serum at 60°-65°C, and sera heated at temperatures known to destroy 

 the enzyme are often rendered effective in the transforming system. Further an- 

 alysis has shown that certain sera in which R antibodies are present and in which the 

 enzyme has been inactivated may nevertheless fail to support transformation. This 

 fact suggests that still another factor in the serum is essential. The content of this 

 factor varies in different sera, and at present its identity is unknown. 



There are at present no criteria which can be used as a guide in the selection of 

 suitable sera or serous fluids except that of actually testing their capacity to support 

 transformation. Fortunately, the requisite properties are stable and remain unim- 

 paired over long periods of time; and sera that have been stored in the refrigerator 

 for many months have been found on retesting to have lost little or none of their 

 original effectiveness in supporting transformation. 



The recognition of these various factors in serum and their role in the reaction 

 system has greatly facilitated the standardization of the cultural conditions 

 required for obtaining consistent and reproducible results. 



3. The R Strain (R36A). — The unencapsulated R strain used in the present 

 study was derived from a virulent "S" culture of Pneumococcus Type II. 

 It will be recalled that irrespective of type derivation all "R" variants of 

 Pneumococcus are characterized by the lack of capsule formation and the 



