168 CROWN-GALL OF PLANTS. 



something else, e. g., cell detritus. The fact that the organism as it 

 occurs in the gall comes up slowly on agar poured plates, but grows 

 as promptly as other bacteria in the same medium when transferred 

 from cultures, may be coupled with the fact that irregularly shaped 

 involution forms are common in this species when it is exposed to 

 certain unfavorable conditions, e. g., cold or sodium chloride. If 

 such involution forms were the common form also in the plant, it 

 might explain many of our failures. Such club-shaped and branched 

 forms occur abundantly in some of the nitrogen root nodules of 

 Leguminosae. 



If this were true, namelv, that there is a pretty nearly even balance 

 between the growth of the bacteria in the tumor tissue and the destruc- 

 tion of the same, then we might have the tumor rapidly proliferating 

 as the result of the stimulus of enzymes, toxins, acids, alkalies, or 

 other substances, dissolved out of the dead bacteria, and not in the 

 tumor at any given time very many bacteria demonstrable by 

 means of stains, because inactive and partially disorganized bacteria 

 are proverbially difficult to stain." Might not some phenomena of 

 the kind mentioned be present in malignant animal tumors and 

 thus complicate the determination of their etiology? We know in 

 case of the crown-galls, even when we can not stain the bacteria in 

 the tissues, that they are there, because b}'' selecting small tumors 

 no part of which has yet passed into a necrotic condition we can 

 obtain therefrom cultures of the gall-producing organism, and have 

 done it over and over again. All that it is necessary to do is to 

 scrape thoroughly and wash the unfissured surface of the gall, then 

 soak it in some germicide long enough to sterilize the surface (an 

 hour or less in 1:1,000 mercuric chloride water usually suffices), dry 

 it, and dig into the depths of the tumor (or in case of hard tumors 

 into superficial rapid-growing portions) with sterile instruments, 

 remove and crush some of this interior portion in sterile bouillon, and 

 make poured plates in -f 15 nutrient agar. We are further led to the 

 belief that living bacteria are not numerous in the galls by the fact 

 that even when the melted cooled agar for plates is inoculated rather 

 copiously, what one would ordinarily call very copiously (say, 1,000 

 or 10,000 or even 100,000 times too much) if he were dealing with 

 other diseases, the colonies developing on the plates (which under 

 the conditions mentioned are sometimes absolutely free from in- 

 truders) are not very numerous. Third, even this procedure will 

 frequently fail to yield any colonies, unless one also takes the added 

 precaution of allowing the living bacteria present in the partially 



a Since this was written we have obtained numerous involution forms in agar and bouillon by adding 

 wealc acetic acid. These facts, couiiled with the knowledge derived from the flask analyses, viz, that 

 acetic acid is formed from sugar by this organism, makes it very probable that both acetic acid and involu 

 tion forms occur iu the tumor. 

 213 



