138 Report oF DEPARTMENT OF BACTERIOLOGY OF THE 
STUDIES WHEREIN THE ENZYM WAS ISOLATED BY 
vy’ HEATING. 
The thermal death point of the carrot-rot bacillus was care- 
fully determined in connection with our earlier studies. It 
was found that recently inoculated thin-walled-tube cultures 
immersed for ten minutes at 51° C., or slightly under this, are 
rendered sterile.® 
Previous studies (cf. Green, 1901: 98) upon cytolytic en- 
zyms have shown that when in solution they are destroyed 
by heating to a temperature of 60°—65° C. It seemed probable, 
assuming that we are dealing with the same or a similar 
enzym, that there might be an intermediate temperature where 
the organism would be destroyed and the enzym left in the 
solution. This matter was tested by cultivating the organism 
in beef broth, heating these cultures to sterilize, making trans- 
fers to prove sterility and inserting bits of sterile carrot or 
other fresh vegetable tissues to determine enzym action. 
The details of a single experiment will suffice to make clear 
the methods and aid in interpreting the results. 
A series of six 10 ¢. c. beef broth cultures seven days old 
were immerséd for ten minutes in a water bath of which the 
temperature was held at 55° C. These tubes were of thin 
glass about 15 x 1.5 cm. in size, immersed in the water three- 
fourths of their depth. The original reaction of the broth 
was +1.5%. To make sure of continued sterility transfers 
were made from the tubes soon after leaving the bath and 
again at the close of the experiment. Immediately after 
heating, a small block of living carrot tissue, cut from the 
interior of the root with proper precautions to insure sterility, 
was inserted into each tube. Like blocks of tissue were put 
into control tubes of each of two kinds, first, sterile uninocu- 
lated broth, and second, living broth cultures of the same age 
as the ones heated. The result was that the tissues in sterile 
uninoculated broth remained unsoftened, those in the living 
cultures rapidly softened and were fully decomposed in three 
* The above trials were first made in 1899 and repeated with like results 
in 1901. A careful repetition in 1903, using the same methods, showed a 
thermal death point fully one degree lower. This is doubtless the result 
of long cultivation in the laboratory. It is simply a matter of biological 
interest, which does not in any way affect the methods or results of the 
enzym studies discussed above. | | 
