146 Report OF DEPARTMENT OF BACTERIOLOGY OF THB 
quired twice as long; after twenty-four hours it required four 
times as long. 
2. Actions on the organism :—Formalin to make 0.2% solu- 
tion was added to broth cultures, thoroughly shaken; trans- 
fers at the end of each two, three, six, and nine hours showed 
erowth. There was, however, progressive retardation. Thus 
the control was clouded in less than twenty-four hours; that 
made at the end of two hours showed clouding first on the 
third day; that at the end of three hours first clouded on the 
fifth day; six hours, on the seventh day; nine hours, on the 
twelfth day. After clouding appeared growth progressed with 
normal rapidity. 
These results show sufficient variation between the different 
trials to forbid sweeping generalizations. They agree, how- 
ever, with each other and with Spieckermann’s results in show- 
ing that the action on the organism is more rapid than on 
the enzym. There was no appreciable retardation of the en- 
zym action until after a period varying from three to nine 
hours, or in.one case twenty-four hours, whereas there was 
marked inhibition in growth of the organisms after two or 
three hours. These results were of such a nature, however, 
as to discourage us from looking to the use of formalin as a 
practical method of sterilizing broths preparatory to the study 
of the normal action of the enzym. If used within two or 
three hours as Spieckermann directs, sterility is not insured ; 
if a longer time elapses, the activity of the enzym will be 
reduced quantitatively at least and conceivably affected quali- 
tatively. :; 
Bliss and Novy (1899: 52) have shown that fibrin, which 
has been acted upon for a short time by formalin, resists 
thereafter the digestive action of proteolytic enzyms. These 
observations raised the question as to whether the retardation 
in the cytolytic action already noted might be in any degree 
the result of the action of the formalin on the wall of the 
vegetable tissues rather than upon the enzym itself. In order 
to determine this, razor sections of turnip and radish were 
immersed twenty-four hours in full strength formalin, then 
washed out in water, and the rapidity of action of enzym 
solution on these compared with that on freshly cut sections 
