DISSOLVING ENZYMES. 83 
hours. The use of formalin, however, is not a practical method of sterilizing broths preparatory 
to the study of the normal action of the enzym, since sterility is not assured by 2 or 3 hours subjec- 
tion to formalin and if a longer time elapses the activity of the enzym is affected. 
Bliss and Novi have shown that proteolytic enzyms have no effect upon fibrin, which has been 
acted upon a short time by formalin. This raised the query whether retardation in the cytolytic 
action is not due to the action of the formalin on the cell-wall of plant tissues rather than upon the 
enzym itself. Experiment proved that this was not the case. Sections immersed 24 hours or even 
a month in formalin or absolute alcohol were decomposed by the enzym solution as quickly as fresh 
sections. Bliss and Novi found that formalin inhibited certain enzyms (papain, trypsin, amylopsin) 
and not others (pepsin, malt diastase) and von Freudenreich discovered that formalin tends to 
lessen the action of galactase more promptly than it does that of pepsin and pancreatin. 
Phenol.—An addition of 0.3 per cent or 0.6 per cent of phenol, if the culture was well shaken, 
always produced sterility, and there was no apparent retardation of the activity of the enzym; 
o.1 per cent failed to sterilize, and 5 per cent totally inhibited the activity of the enzym. The phenol 
was allowed to act four days before the enzymic activity was tested. 
Thymol.—This was less effective than phenol, probably because of its slight solubility and slow 
diffusion in the broth. A small amount will kill the organism if the cultures are shaken thoroughly 
but even large amounts fail to sterilize in the absence of agitation. There is no evidence of any 
inhibition of the enzym action. 
Chloroform.—From to to 50 per cent of Powers & Weightman’s chloroform, “U.S. P. Standard,” 
added to cultures 7 to 9 days old failed to kill the organism in 3 days, but when the experiment 
was repeated 2 years later, using both Mallinckrodt’s “M. C. purified” chloroform and the 
U.S. P. grades both of this firm and of Powers & Weightman, sterility was secured in every case. 
The enzym action was not affected. 
Brown and Escombe report that the cytolytic enzym of barley is not appreciably affected by 
a saturated aqueous solution of chloroform. Smith found that many organisms are surprisingly 
resistant to chloroform and emphasizes the need of caution in its use. Potter did not obtain sterility 
in his cultures of the turnip white-rot organism with chloroform but Spieckermann succeeded in 
sterilizing the sap of vegetables invaded by his kale-rot organism by the use of it. There was no 
appreciable retardation of the cytolytic action with the exception of a possible gradual weakening 
after 15 days or more. Van Hall’s results with B. omnivorus are surprisingly at variance with these 
since he found the addition of even 0.5 per cent chloroform destroyed all trace of activity in bacterial 
juices in one quarter of an hour. 
A special series of these experiments was planned to demonstrate the comparative effect of these 
chemicals on the activity of the enzym. These comfirmed the evidence of previous trials and led 
the author to conclude that in the doses named neither chloroform, thymol, nor phenol caused any 
diminution of the cytolytic action; that formalin inhibited the enzymic activity; that filtration 
through porcelain reduced the enzym content. 
(4) Observations upon decaying vegetables have shown that cytolytic action goes on some 
distance in advance of the invasion of the organism. Experiments were carried on to test the diffusion 
of the enzym through some medium impenetrable to the bacteria. Small Petri dishes containing 
2 per cent beef broth agar about 3 mm. deep. were inoculated in the center with B. carotovorus. In 
3 days there was a good surface growth, about 1 cm. in diameter. A slice somewhat larger than this 
layer of agar, was then cut from the interior of a fresh turnip root and placed in a large sterile Petri 
dish. The layer of agar from the smaller dish was then placed upon the surface of the turnip, care 
being taken to avoid contamination. At the end of 24 hours the turnip showed an area immediately 
underlying the colony and somewhat larger than the same in which the tissues were slightly brown 
and softened exactly as though invaded by the organism. Bits of this rotten turnip tissue were 
transferred to each of three broth tubes but in none of these did growth develop, proving that this 
softening of the tissues was due solely to substances secreted by the bacteria which diffused through 
the layer of agar from the surface colony above. Microscopic examination showed isolation of the 
cells as a result of the solution of the middle lamella, a swelling of the residual walls, and granulation 
and plasmolysis of the cell-contents. There was no softening in the check. 
(5) By the use of strong alcohol a flocculent whitish precipitate is obtained from broth cultures. 
This includes the enzyms, various proteid matters and also the bodies of the bacteria. This dry 
precipitate can be preserved indefinitely, as the use of strong alcohol insures the elimination of the 
living organism, 25 per cent being fatal to B. carotovorus. 
It was found preferable not to pass the broth through a porcelain filter, as the filtered broth 
possesses less of the enzymic activity and, therefore, as repeated observations had shown that this 
enzym is entirely inactive on the celluloses proper, the culture broths were passed through filter 
paper. The succeeding steps were as follows: 
