126 
Journal of Agricultural Research 
Vol. XXV, No. 3 
beginning it appeared to be the simple and natural suggestion from this 
evidence that the bacterial invasion not only started in the intercellular 
spaces but possibly also continued therein in a manner comparable to 
that of various other bacterial pathogens. 
With this question in mind a search was instituted with the hope of 
detecting the bacteria in situ. In the beginning it was realized that the 
organism might be located only in certain portions of the gall and might 
also be more apparent at one time in the stages of development than at 
another. Consequently, in this search the efforts were not confined to 
any portion of the stem taken at one time, but were directed to whole 
sections of stems with galls in different stages of development. 
Material was killed in such fixatives as chrom-acetic, Flemming’s, 
Camoy’s, picro-formal, formal-alcohol, and Benda’s. Paraffin sections 
were then cut and stained with carbol-fuchsin, methylene blue, gentian- 
violet, Haidenhain’s iron-alum-haematoxylin, safranin, rose Bengal, 
Giemsa, neutral red, orange G., and light green, either alone or in combi¬ 
nation. No satisfactory demonstration of the organism in the tumor 
tissue was secured. The organisms could not be distinguished, except 
as Smith states (n, p. 17), relatively near the entrance of the needle. 
However, it was noted that the bacteria could be traced from the puncture 
out through the intercellular spaces for a short distance (PI. 3, C), and 
could also be located in many of the intercellular spaces above or below 
the puncture. This was quite easy in the cortex and pith where the 
intercellular spaces were large. Search was of course persistently made 
for evidence of the occurrence of bacteria within the cells, but they were 
detected only in those cells which were close enough to the puncture to 
be subject to injury. In such cases the organisms were present in 
sufficient quantities to occupy a major portion of the cells (PL 3, D). 
It appears, therefore, that if entrance into the cells is provided for the 
bacteria, they collect there in considerable numbers. It seems quite 
improbable, however, that cells showing such abundant invasion should 
survive. 
Efforts were made to locate the bacteria with gold chlorid (7, p. 127) 
and with Gram’s stain, employing amyl alcohol as used by Smith (rr, 
p. 18-19), but these also gave unsatisfactory results. Such combina¬ 
tions as ruthenium red and methylene blue, used so successfully by 
Jones (j, p. 332) were equally ineffective. 
While studying paraffin sections the writer noticed that certain of 
the walls bounding intercellular spaces took the stain much more 
deeply than others. This was found to be true of the spaces that had 
been filled with liquid after the puncture and to which the bacteria 
had gained access (PI. 3, A). The bacteria seemed to have exerted an 
action on the walls which made them stain more deeply. This influence 
was noticed to extend a short distance into the middle lamella (PI. 3, B), 
but its exact range has not been determined. This seemed to be in 
accordance with the hypothesis that the bacteria were located in the 
intercellular spaces and possibly to some extent in the middle lamellae. 
If this were true, a satisfactory explanation would be given alike for the 
different types of staining shown by different cell walls, and for the 
development of galls from the region water-soaked by the puncture. 
So the problem became one of trying to differentiate between the cell 
wall, which had been made more sensitive to stain by the action of the 
bacteria and the organisms themselves. For this purpose sections were 
