July 21 , 1923 
Crowngall Organism and Its Host Tissue 
127 
used which showed the bacteria in mass about the puncture. In this 
case the differentiation was not so difficult because the bacteria were 
together in large numbers and the walls in these early stages had not 
undergone any change due to the bacterial action. But this gave no 
idea of the relation of small numbers of bacteria to the walls that took 
the stain more deeply. 
Since the usual paraffin method had yielded inconclusive results for 
the writer, as it had earlier for Smith {8, p. 233), it seemed evident that 
some variation in method must be employed if the organism were to be 
demonstrated in the tissue. At the suggestion of Doctor Eckerson a variety 
of microchemical tests were performed on free-hand sections of gall 
tissue in the hope that either the walls or protoplasm of the bacteria 
could be found to give a characteristic reaction. For the wall the 
chitosan reaction was tried, but it gave only unsatisfactory results. 
For protein, the biuret, Berlin blue, and xanthoprotein reactions and 
Millon’s reagent gave no better success, so far as demonstrating the 
bacteria was concerned. 
While these reactions were being tried, observations were made on 
free-hand sections of tomato galls that varied from one day to three weeks 
in age. These sections were mounted in water in the usual manner and 
examined under an oil immersion lens. In the middle lamella, especially 
of the eollenchymal cells, small bacteroid bodies were observed (PI. 4, A), 
which, however, varied considerably in size. At the same time, in cer¬ 
tain of the intercellular spaces, bacterium-like bodies were observed 
which were of rather uniform dimensions. These were associated with 
a yellowing of the adjacent cell walls. They were visible, just as bac¬ 
teria are in hanging-drop preparation, because of the differences in light 
refraction. Occasionally, also, as Smith observed (ri, p. if), bacteria 
could be seen in cut cells. However, the fact that they appeared inside 
the open cells was no proof that they were present in this position before 
the section was made. If they were in the intercellular spaces, one 
would expect some to escape as soon as the wall was cut, and then they 
might appear anywhere on the surface of the section. 
This possibility that the bacteria might wash out of the middle lamellae 
or intercellular spaces into the water during the sectioning made it 
seem advisable to cut the material dry. The actual practice, with a 
good sharp razor, proved to be much easier than anticipated. The sec¬ 
tions were mounted quickly in a small drop of water, in glycerin, or in 
lacto-phenol (equal parts of lactic acid, phenol, glycerin, and water). 
This last medium was the most convenient because it did not dry out 
like the water, nor did it contain so many annoying air bubbles as the 
glycerin. At the same time it proved to be an excellent preservative. - 
The large bacteroid granules observed in the middle lamellae especially 
between the eollenchymal cells were found in observations made in Feb¬ 
ruary, 1922, to be associated with the rapidly growing gall tissue but not 
with the more slowly growing uninoculated tissue (PI. 4, A, B). How¬ 
ever, when an examination was made in June, 1922, while the tomato 
stems were growing rapidly, similar granules were found in the normal 
as well as the gall tissue. Their presence seems to be associated with 
the rate of growth. Polarized light showed them not to be doubly 
refractive. They lost their identity quickly in 3 per cent hydrochloric 
acid upon the application of heat, and slowly in the lacto-phenol mount¬ 
ing fluid. They stained both with methylene blue and ruthenium red. 
