82 BACTERIA IN RELATION TO PLANT DISEASES. 



Five methods were used for the isolation of the enzym: (i) 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 at55C. (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 destntctans 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 



