1914] 
COOLEY—SCLEROTINIA CINEREA 811 
іп the agar just below the fungous filaments, thus indicating 
that the cellulose is being hydrolyzed. With increasing age of 
the fungus, this clear and almost transparent area gradually 
enlarges downward, although the fungus shows little or no cor- 
responding penetration. At the expiration of three weeks or a 
month, there is а very distinct, clear, and nearly transparent 
zone in the medium below the region occupied by the fungous 
mycelium. Since one could see very distinctly how far the 
fungous filaments had penetrated into the substrate, it was 
very evident that the cyto-hydrolytic enzyme had diffused 
beyond the limits of the mycelium. 
The method employed in this investigation for the demonstra- 
tion of cellulase was the same as that used by Kellerman in his 
recent work (31) and was utilized to demonstrate the fact that 
the cyto-hydrolytic enzyme secreted by this fungus penetrates 
the substrate considerably beyond the limits of the filaments 
themselves. Tubes containing cellulose agar, in which the 
fungus had been growing for four weeks, were disinfected exter- 
nally by washing with a bichloride of mercury solution, and cut 
off at à point about 12 mm. below the clear portion of the me- 
dium. Тһе cotton plug was then flamed and pushed into the 
tube with a glass rod until the agar was partially shoved out 
of the eut end of the tube. Тһе clear portion of the agar was 
then cut into disks about 12 mm. in thickness, which were laid 
on plates poured with nutrient cellulose agar, great care, of 
course, being exercised throughout the operation to maintain 
aseptic conditions. The plates so prepared were then placed 
in an incubator at 25°C. where they remained for two weeks, 
at the expiration of which time the cellulose was very distinctly 
hydrolyzed in a ring about the sterile slices of agar. Micro- 
scopic examination confirmed the macroscopic observation that 
these agar disks were free from any infection. 
As might be expected, the activity of the secretion of the 
enzyme cellulase is influenced by temperature, a fact which is 
well illustrated by the following experiment: Tubes containing 
cellulose agar inoculated with the brown-rot fungus were kept 
at temperatures of 10-12, 16-20, and 24-26°С. respectively, and 
at the end of twelve days the following results were noted: In 
the cultures maintained at 10-12°C. no apparent growth or 
4 
