OSWALD T. AVERY, COLIN M. M.AcLEOD~ AND ~T.ACLYlq 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
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 r61e 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