84 BACTERIA IN RELATION TO PLANT DISEASES. 



"To the filtrate was added enough 95 per cent alcohol to render it alcoholic to the desired degree, 

 usually 80 per cent. The precipitate allowed to settle, the supernatant alcohol syphoned off, the 

 precipitate collected on filter paper, washed with either absolute or 95 per cent alcohol and quickly 

 dried, partially in a current of warm air, then in a dessicator over sulphuric acid. The dried precipitate, 

 which is gray and somewhat brittle, was then powdered before redissolving in water. It is of course 

 important to secure quick drying to avoid the possibility of alteration as a result of bacterial growth 

 or of chemical changes in the precipitate. The drying must also be done at so low a temperature 

 as to preclude danger of injury from heat to the sensitive enzym." 



Tests of different per cents of alcohol showed that 80 per cent secured practically all of the enzym 

 and that the latter was in a state of the highest activity. Re-precipitation proved to be of little ad van- 

 tage. The activity of the enzym increased with the strength of the solution, but not proportionally. 

 After several trials the author came to the conclusion that, with the precipitate used, it required on an 

 average, 25 minutes for a i per cent solution of the precipitate to secure as complete enzymic action 

 as was secured in 15 minutes in the 5 per cent solution, and in 10 minutes in the 10 per cent solution. 



In all the work reported here 5 per cent solutions have been used unless otherwise stated. 



Comparative tests showed that no loss of cytolytic activity occurs through precipitation and 

 re-solution. On the other hand, the 5 per cent solution of the precipitate rotted the vegetable 

 sections in less than one-third the time required by the living cultures (5 per cent solution of the 

 precipitate, however, contains about 15 times as much of the enzym as the broth cultures). 



Jones found the relation of cultural conditions to enzym production to be as follows : 



The Medium. The vigor of growth of the organism varies widely with the medium, age and 

 temperature. Various experiments were undertaken to determine the relation of these factors to 

 enzym formation, some previous investigators having concluded that enzym production is in some 

 cases a starvation phenomenon. 



The media used were (i) Dunham's peptone solution = a very weak growth; (2) the same + 2 

 per cent cane sugar = twice as dense growth as in the simple Dunham's solution; (3) neutral beef 

 broth = good growth; (4) the same +2 per cent cane sugar = more rapid growth than in plain 

 broth; (5) cooked carrot broths (a) those in which equal weights of pieces of fresh carrot roots 

 and water were cooked and sterilized together by discontinuous sterilization, and (b) the same in 

 which after the first cooking in the steamer the cell-wall substances were removed by crushing the 

 roots and filtering through several thicknesses of paper. This filtrate was then sterilized in the steamer 

 by the fractional process. Both of these have, except in certain cases, proved to be the most favorable 

 cooked media for this organism; (6) living vegetables this class of media has given the greatest 

 and most active enzym product. Beef broth yields on the average about 0.25 per cent of dry pre- 

 cipitate while expressed juice from decayed turnip after filtration through paper, has yielded over 

 0.5 per cent precipitate, as per centaqueous solution of which caused the complete rotting of the razor 

 section of turnip in 10 minutes, whereas a like solution of the precipitate from a beef broth culture 

 required nearly 2 hours. Thus not only was twice as much precipitate obtained from the decayed 

 living vegetable but the solution was 12 times as active. 



When sections were immersed directly in the living cultures, i. e., in the juice from decaying 

 turnip, and in beef broth cultures, those in the former were rotted 2 or 3 times as quickly as those in 

 the latter. The presence or absence of cell-wall substances, the effect of which was tried in the unfil- 

 tered and filtered vegetable broths, makes no difference in the enzym production. The cooked vege- 

 table media, however, were not uniformly satisfactory. In some cases there was excellent growth, in 

 others very little. The enzymic development was directly proportional to the amount of growth. 

 Jones used beef broth cultures largely in his comparative studies because more reliance could be 

 placed upon the uniformity of growth in this medium. 



The addition of 2 per cent sucrose causes a more vigorous growth of the organism especially in 

 the earlier stages. More precipitate and more enzymic activity were developed in the sugar broth 

 than in the plain broth. In conclusion, then, the author notes that the amount of enzym developed 

 seems to be directly proportional to the rate and vigor of growth; that the presence of cell-wall 

 substances have no appreciable effect on the amount of enzym developed; and that in beef broth 

 cultures the addition of sugar favors growth and increases the enzym production. Enzymic produc- 

 tion in this case at least is not a starvation phenomenon. The experiments with filtered and unfil- 

 tered vegetable broths indicate that the organism makes little or no use, for nutrition purposes, 

 of the cell-wall substance which it dissolves. 



The A ge of the Culture. The enzym content in carrot broth cultures increased with the age of 

 the latter. Cultures were grown at 20 to 22 C. and tested at the end of 1.5, 3, 5, 7, and 9 days. 

 Between 1.5 days and 5 days there was a rapid increase in the enzymic action (scarcely distinguishable 

 in 1.5 days, but very pronounced in 5 days). From the fifth to the ninth day it continued to 

 increase but at a slower rate. Beef broth cultures tested at the end of 4, 6, 9, and 18 days gave 



