176 



1. Nutrie?n Broth.— Beef heart infu- 

 sion broth containing 1 per cent neo- 

 peptone 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 unpre- 

 dictable variations in the property of 

 supporting transformation. It has been 

 found, however, that charcoal adsorp- 

 tion, according to the method de- 

 scribed by MacLeod and Mirick (10) 

 for removal of sulfonamide inhibitors, 

 eliminates to a large extent these varia- 

 tions; consequently this procedure is 

 used as routine in the preparation of 

 consistently effective broth for titrat- 

 ing the transforming activity of ex- 

 tracts. 



2. Serum or Serous Fluid.— \n the 

 first successful experiments on the in- 

 duction 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 observation 

 that reversion of an R pneumococcus 

 to the homologous S form can be in- 

 duced by growth in a medium con- 

 taining anti-R serum. Alloway (6) 

 later found that ascitic or chest fluid 

 and normal swine serum, all of which 

 contain R antibodies, are capable of 

 replacing antipneumococcal rabbit se- 

 rum in the reaction system. Some form 

 of serum is essential, and to our knowl- 

 edge 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, how- 

 ever, that the effectiveness of different 

 lots of serum varied and that the dif- 

 ferences observed were not necessarily 

 dependent upon the content of R anti- 

 bodies, since many sera of high titer 

 were found to be incapable of support- 

 ing transformation. This fact sug- 



AVERY, MACLEOD, MCCARTY 



gested that factors other than R anti- 

 bodies 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 principle in potent ex- 

 tracts. 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 de- 

 stroy the enzyme are often rendered 

 effective in the transforming system. 

 Further analysis has shown that certain 

 sera in which R antibodies are present 

 and in which the enzyme has been in- 

 activated may nevertheless fail to sup- 

 port 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 transforma- 

 tion. Fortunately, the requisite prop- 

 erties are stable and remain unimpaired 

 over long periods of time; and sera that 

 have been stored in the refrigerator for 

 many months have been found on re- 

 testing to have lost little or none of 

 their original effectiveness in support- 

 ing transformation. 



The recognition of these various 

 factors in serum and their role in the 

 reaction system has greatly facilitated 

 the standardization of the cultural con- 

 ditions required for obtaining consist- 

 ent and reproducible results. 



3. The R Strain (R36A).-i:\\t un- 

 encapsulated R strain used in the pres- 

 ent study was derived from a virulent 

 "S" culture of Pneumococcus Type 

 II. It will be recalled that irrespective 



