EXPERIMENTAL PRODUCTION OF PARASITES. 49 
Cultures of B. subtilis were always similar in appearance when made from the decaying spots, 
but a peculiar variation developed in cultures after a time. Streak cultures on malt-agar often 
showed in 2 or 3 days transparent outgrowths, contrasting strongly with the milky-white or yellow- 
ish streak. A microscopic examination showed these transparent outgrowths to contain no spores, 
though spores were present in great numbers in the streak itself. By transfers pure cultures of this 
“‘asporogenous variety”’ were easily obtained and remained free of spores when further cultivated. 
Yet the ability to form spores was not, he thinks, entirely lost, for now and then atavistic forms 
arose which were spore-producing. Such forms were too rare to be detected by the microscope, but 
when a rather old culture was scraped from the agar, covered with water, heated to boiling and then 
poured over malt-agar or sterilized potato there frequently appeared one or more colonies which 
belonged to the original spore-forming variety. [It is not stated whether this was direct from the 
translucent part of a spore-bearing streak; a pure culture from a colony of the non-sporogenous 
form; or a subculture from one of the outgrowths, and this is important in judging whether he had 
two organisms on the start or only one.] 
; Inoculations were made with pure cultures of both forms (sporogenous and non-sporogenous) 
as follows: 
The parts of the plants to be used were scrubbed with soap under the tap, then pared, and kept 
for a few minutes in 2 per cent solution of corrosive sublimate. Finally they were washed in sterile 
water. Freshly cut slices of this material were then placed in Petri dishes and streaks were made 
from pure cultures. Results showed that both sorts were equally active. At 30° C., potatoes, Jeru- 
salem artichokes, early turnips (Mairiibe), celery, and carrots were badly decayed, and kohlrabi 
hazel nut and chestnut slightly attacked; while all of these, except the chestnut, were badly 
decayed at 37° C. Many more varieties of plants succumbed than had done when earth was strewed 
on the section. This indicated to him that the result of infection depends on the number of bacteria 
in the material used for inoculation. No results were obtained from inoculation on any of these 
plants when kept at 23° C. 
Three months later repeated attempts to inoculate from these same pure cultures, kept during 
that time on artificial media, gave negative results, except in case of the potato which the sporo- 
genous form still rotted readily and the non-sporogenous form slightly. Virulence was, therefore, 
greatly reduced by culture for 3 months on artificial media. This virulence was restored to both 
forms by a single passage through the potato. 
Inoculations were then made on whole tubers of potato, Jerusalem artichoke, and the Mairiibe 
(B. rapa rapifera). ‘The surface was cleaned and freed from surface bacteria, as before mentioned, 
and the bacteria (sporogenous form) introduced through a small wound. The tubers were then 
placed in sterile glass vessels, in the thermostat and keptat constant temperatures (37°, 30°, 23°C.). 
At 37 C. it required only 4 or 5 days to complete the decay of each species. At 30° it required 10 
to 12 days, while at 23° no result was obtained. 
The decay is a characteristic soft rot, the progress of which may be easily followed. In the 
cells between the healthy and the decayed parts, the protoplasm contracts and becomes granular. 
At the same time the cells separate with the disintegration of the middle lamella. This is due, not 
to the presence of bacteria, they having not penetrated so far, but to their secretions. B. subtilis 
is not able to digest cellulose nor to penetrate into the cells, hence it is found only in the intercellular 
spaces. As destruction progresses the granular protoplasm disappears almost entirely while the 
starch granules, which remain intact longest, gradually lose their sharp outlines, mass together 
and slowly dissolve. In streaks on nutrient agar containing 0.5 starch, tests with iodine after 3 or 4 
days growth showed absence of the blue color in the vicinity of the streak. 
A characteristic browning or blackening accompanies this process in potatoes and Jerusalem 
artichokes. This is probably due not to the bacteria directly, but to the oxidation of an enzyme 
(tyrosinase), which the bacteria have not destroyed. 
The decayed parts also gave off a rather characteristic odor, chiefly of trimethylamin and 
ammonia. The reaction was always alkaline. 
In comment on the above it may be said if one formulated the following hypothesis, 
viz., van Hall was working ‘with mixed cultures, a non-sporogenous parasite, and a spore- 
bearing saprophyte related to or identical with Bacillus subtilis, I do not see how it could 
be combated with any degree of certainty by means of any statements in his paper, since 
he nowhere speaks of beginning any of his successful inoculations with descendants of a 
single well-identified spore. 
To demonstrate more clearly the presence of a virus secreted by the bacteria, potatoes decayed 
by B. subtilis were crushed and their juice filtered through a porcelain filter. One drop of this 
