DISSOLVING ENZYMES. 81 
always gives a very strong iodoform reaction. The precipitate remaining in the flask is dissolved 
in part by acetic acid with evolution of gas; and with ammonium oxalate an abundant precipitate 
of oxalate of lime is obtained. It seems then that in the fermentation of the calcium metapectate, 
a portion of the lime is used to form carbonate of lime. The solution still contains some lime. When 
the solution was acidified with hydrochloric acid only the least trace of substance could be extracted 
with ether. No considerable acid fermentation, such as occurs in sugar solutions inoculated with 
the bacteria, takes place in the pectate flasks, since the liquid always remains neutral. 
Thus the part played by the enzym in the parasitism of this-organism is as follows: It makes 
possible a rapid penetration to the deeper plant tissues of the poison secreted by the bacteria; by 
the resulting death of the protoplasm the cell-sap which is rich in nutrient substances is made accessi- 
ble to the bacteria, insuring their more rapid multiplication, so that the plant is unable to bring 
its natural means of protection into play quickly enough. On the other hand, the middle lamella 
furnishes a suitable source of carbon to the organism, and finally the carbonate of lime liberated in 
the fermentation of the calcium pectate and perhaps also of the pectates themselves combines with 
the acids which are always produced by the bacteria in fluids containingsugar, and which would 
otherwise restrict the development of the organism. 
Further experiments were carried on as to the manner of the poisoning of the protoplasm of 
the plant cell by the bacteria. If sections of carrots immersed in the expressed juice of rotted potatoes 
or carrots to which no disinfectant has been added, are observed under the miscroscope, one sees in 
ten minutes a slow separation of the protoplasm from the cell-wall the former at the same time 
becoming plainly granular. This process takes place slowly. In 30 minutes the protoplasm is still 
further contracted and apparently dead; in 40 minutes it is rolled up into a ball, deformed, dark 
brown, dead, in the middle of the cell. The same phenomena occur with sections of potato, onions, 
and cattle beets (Futteriiben). 
If the juice from the decayed plants is boiled, its poisonous action seems to be completely lost. 
When sections of carrot are immersed in it nearly all the cells are living after 36 hours, as is shown 
by the production of plasmolysis with saltpeter solution. However, one finds that insuch sections 
some of the surface cells are dead and the protoplasm therein shows the same phenomena as that 
in those sections which have been immersed in the fresh juice. Furthermore, it appears that the 
poisonous substance diffuses through membranes only with the utmost difficulty, Thus filtrate 
from juice which has been passed through a bougie is usually at first not at all poisonous, and only 
slightly poisonous after the filtration of 400 cc., being similar in this respect to boiled juice. And 
the solution of the middle lamella and the action of the poison go hand in hand as can be easily 
observed under the microscope. The poisoning never precedes. If one uses sections which vary 
in thickness, then one sees very plainly that plasmolysis sets in at the same instant that the cells 
are separated from one another by the solution of the middle lamella. ‘The sound united cells and 
the diseased cells which are becoming separated are sharply differentiated. The poisonous action 
never extends beyond the decayed area, as is so often observed in fungous diseases. 
There is apparently no labile toxine present. No oxalic acid is present, at least not in the neutral 
to alkaline potato and carrot juice, and it was not detected in the acid fermentation produced by the 
bacteria in sugar, glycerin, and mannit solutions, a test which excludes free oxalic acid and various 
of its compounds. 
The poison also is precipitated from the juice of decayed potatoes, carrots, and onions, together 
with the slime and the pectine dissolving enzym, by the use of alcohol. If one dissolves the pre- 
cipitate in an equal amount of sterile water, one obtains a neutral solution which in the activity 
and manner of its poisoning differs in no way from the original juice. On boiling a granular pre- 
cipitate is obtained and the boiled solution still retains after many days a slight poisonous action 
on the protoplasm. : 
The most recent extensive contribution to the subject is by L. R. Jones (1910). A 
summary of this paper follows: 
A detailed study was first made of the enzym produced by Bacillus carotovorus, both living 
cultures and the enzym isolated from them being used in the experiments. Later, comparative 
studies were made with enzyms secreted by other soft-rot organisms, other classes of bacteria, fungi 
and germinating seeds. 
The same strain of the carrot-rot organism was used throughout. It is one isolated from decay- 
ing carrot tissues in 1899 and since* grown on artificial media (practically all of the time in beef 
broth), at room temperature 16° to 22° C. Jones believes that there has been a considerable decrease 
in pathogenicity accompanied by a corresponding decline in the amount of enzym production. 
*The studies here reported upon were carried on during the years 1901-1904. 
