82 BACTERIA IN RELATION TO PLANT DISEASES. 
Five methods were used for the isolation of the enzym: (1) heat, (2) filtration, (3) germicides, 
(4) diffusion through agar, (5) precipitation by alcohol. 
(1) Cultures 7 days old were immersed for 10 minutes in the water bath at 55° C. (51° C. thermal 
death point of the organism). Blocks of living carrot tissues were cut under sterile conditions and 
put, some into the heated tubes, some into living beef-broth cultures, and some into sterile unin- 
oculated broth. 
Result.—Tissues rapidly softened and fully decomposed in 3 or 4 days in the living cultures; 
a similar softening and complete decomposition in 10 days in the heated tubes; no softening in the 
sterile, uninoculated broth. Similar results were obtained with turnip root and cotyledons of imma- 
ture peas. ~ Heating at 54° to 62°C. inhibited the action of the enzym, and temperatures above 
63° C. (also at 62° and 63° C. with one exception) checked it entirely. 
(2) A sterile solution was obtained without difficulty by means of the Chamberland filter. 
Broth cultures, varying in age from 7 to 14 days, were used. In all cases the middle lamella in sterile 
blocks of fresh roots of carrot and turnip, potato tubers, and the cotyledons of young peas immersed 
in the filtrate was dissolved as in the presence of the living organisms. In one experiment a piece 
of carrot about 3 mm. in diameter was perceptibly softened in 24 hours and softened throughout 
in 3 days, while potato blocks of the same size showed the first signs of softening in 5 days. 
Comparisons were made of the enzym activity of filtered sterile broth, unfiltered broth cultures 
and cultures of broths sterilized with chemicals. In one experiment thin razor sections from roots 
of carrot and turnip were immersed in (a) culture broth sterilized by filtration, (b) culture broth 
sterilized by a 20 per cent addition of chloroform, (c) solutions of alcoholic precipitate from culture 
broth, The solutions (b) and (c) acted about alike, whereas (a) required at least twice as long to 
disintegrate the tissues. 
Similar trials made using razor sections of turnip, showed that the enzym-action in sterile 
broths like (b) and (c) was practically the same as in living cultures. Some of the experiments indicate 
that possibly four-fifths of the enzym was lost by filtration through the porcelain. 
All these experiments show that passage through Chamberland filters removes a large proportion 
of the enzym-content of the broth. The reason for this has not been determined. Freudenreich 
found that although all the bougies used by him retained considerable of the nitrogenous matter 
a new filter retained much less than the same one after it had been used several times. Later he 
found that the enzym galactase was removed from milk passed through these filters. 
Potter found that the enzym produced by Pseudomonas destructans passed through a Cham- 
berland filter and Laurent found that similar bacterial enzyms were not removed by filtration through 
porcelain. Spieckermann, on the other hand, obtained contrary results, the culture broths which he 
had filtered through a Reichel porcelain filter having not the least enzymic action. Van Hall found 
that the juice from potato decayed by Bacillus subtilis, when passed through the porcelain filter was 
still capable of rapidly destroying potato tissue. The juice from iris invaded by Bacillus omnivorus 
when passed through a porcelain filter retained its enzymic activity but in a less degree. In other 
trials he found that filtered broths lost all enzymic activity. Jones is at a loss to reconcile some of 
these results with his own, except by attributing the discrepancy to differences in the filters. 
(3) Trial was made of the addition of formalin, phenol, thymol, and chloroform, respectively, 
to beef broth cultures. 
Formalin.—Both the organism and the enzym are extremely sensitive to this chemical. However 
it is possible to use an amount which will sterilize the broth and leave the enzym active. The follow- 
ing conclusions have been reached: 
One-tenth per cent of formalin sterilizes a beef-broth culture of B. carotovorus, one to ten days 
old, provided that the tube is thoroughly shaken. Otherwise more formalin, 0.2 per cent or more, 
may be necessary. Enzym action is completely inhibited by 0.6 per cent of formalin and even 0.3 
per cent retards to a marked degree, while there was perceptible retardation from 0.06 per cent 
formalin, although this amount was too small to insure sterilization. In all the above trials several 
days elapsed between the addition of the formalin and the trial of enzymic activity. 
Similar results were obtained when tests were made to determine the effect of formalin in solu- 
tions of the enzym obtained from broth cultures by precipitation with alcohol. 
Spieckermann reports that a 0.2 per cent solution of formalin sterilized the cultures of the 
soft-rot organism of cabbage with which he was working without inhibiting the action of the cytolytic 
enzym, at least for several hours. Jones undertook to learn the relative rate of action of formalin 
upon both B. carotovorus and its enzym. Broth cultures to which 0.2 per cent of formalin had been 
added were shaken thoroughly and tested at frequent intervals both for viability and enzym-action. 
The results though varying considerably, agreed in showing that the action on the organism is more 
rapid than on the enzym. There was no appreciable effect on the enzym action for from 3 to 9 hours, 
or, in one case, 24 hours, whereas there was marked inhibition to growth of the organism after 2 or 3 
