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 rapijera). 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 , 23C). 

 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 lamellae. 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 



