New YorK AGRICULTURAL EXPERIMENT STATION. ae 
remarkably sensitive to desiccation as shown in our earlier 
work (1900 :528). It seems to us not unlikely that the weaken- 
ing observed at the New York Station may have resulted from 
the intermittent desiccation incident to its culture on agar. 
Exact estimates as to relative ability in enzym production 
Shown now and formerly are even more difficult to make than 
are those as to pathogenicity. Our first isolations of enzym 
were made in 1901. We have compared some of the preserved 
samples of these earlier enzyms with those isolated in 1903. ° 
Those of 1901 were certainly more active than those of 1903. 
This difference might, however, possibly be accounted for by 
variations in medium and vigor of development as shown later 
in this article. 
In our judgment, along with the loss of pathogenicity there 
has been a corresponding decline in enzym-producing power, 
but both of these have been so gradual as to be hard to estimate 
quantitatively. 
The first thing undertaken in the course of these investiga- 
tions was to determine whether the softening of the tissues 
associated with the invasion of this bacillus was certainly 
due to an enzym, and whether, if so, the enzym was separable 
from the organism. Five methods were tried with the object 
of securing such enzym, if it existed, apart from the organism, 
viz., (1) heat, (2) filtration, (8) germicides, (4) diffusion 
through agar, (5) precipitation by alcohol. 
The first three methods involved, in all cases alike, the fol- 
lowing procedures: The cultivation of the organism in beef 
broth for periods varying from three to fourteen days; the 
treatment of such cultures by the methods under trial; the 
determination of the sterility in the broth so treated; in case 
sterility was secured, the testing of the cytolytic activity of 
this sterilized broth by immersion in it of sterile blocks cut 
from fresh uncooked carrot or turnip roots, or from potato 
tubers or of cotyledon of immature pea; finally, tests to 
determine the continued sterility of the broth during this 
last trial period. In all cases where chemicals were used 
control trials were made to be sure that the chemical itself 
was not the cause of the changes observed. 
