130 Journal of Agricultural Research voi. xxv, No. $ 
They apparently diminished the toxic action of the mercuric chlorid 
sufficiently to permit the crowngall colonies to grow around them. So, 
occasionally, in the clear zones surrounding the treated gall tissue, a 
contaminator appeared, surrounded by a small zone of colonies of the 
crowngall organism. 
The presence of a large number of causal organisms in crowngall 
tissue was demonstrated by dissecting out and grinding in white sand 
and water, under aseptic conditions, a 2 mm. cube of young gall tissue 
which was produced by inoculation. Dilution plates were poured of 
the suspension secured. Counts and computations indicated that this 
block of gall tissue contained approximately 50,000 bacteria capable 
of producing colonies. Another similar cube was found to contain 
about 30,000. Under different conditions a piece of gall tissue^ c. mm. 
in volume (1X2X0.125 mm.) was dissected out under sterile conditions 
and crushed in a Petri dish. Over 1,100 colonies of the gall organism 
developed, with practically no contamination. Doubtless many more 
would have appeared if the plate had not been so crowded. Other similar 
pieces have produced colonies ranging in number from 18 to 11,600. 
When these figures are multiplied by 10, one secures numbers consistent 
with those indicated by the microscopic examinations. 
Although the crowngall bacteria have been found in larger numbers 
than has commonly been supposed, a very wide range of variation has 
been observed. The number of bacteria secured in culture seems to 
depend on the number of intercellular pockets of bacteria that are 
broken open so as to release the organisms. Since the number of these 
pockets varies with the age of the gall, the portion of the gall selected 
for isolation, the conditions of growth, etc., considerable differences 
are to be expected in the numbers of bacteria that appear in plates or 
sections. 
Since it was discovered that mercuric chlorid exerted this inhibiting 
influence, its use has been abandoned in routine isolations for the crown¬ 
gall organism. A procedure like the following has been found success¬ 
ful. A 2 to 3 mm. cube of young gall tissue is dissected out under asep¬ 
tic conditions. This is dropped into 10 cc. of sterile distilled water and 
crushed. Then dilution plates are poured from the suspension. 
A further check on the accuracy of the interpretation of the previ¬ 
ously described intercellular bodies as the crowngall bacteria was made 
by observing their multiplication from free hand sections of gall tissue, 
subsequently by isolating them from such preparations, and by estab¬ 
lishing their identity with the usual cultural and inoculation methods. 
This was accomplished in the following manner. 
Free-hand sections of crowngall tissue were cut under aseptic con¬ 
ditions and mounted in drops of melted nutrient dextrose agar on thin 
cover slips. After the agar had solidified, each cover slip was fixed 
with vaseline on a van Tieghem cell as in the preparation of a hanging- 
drop. When the bacteria developed, observations made at about eight- 
hour intervals showed that the colonies were unusually definitely local¬ 
ized on account of the solid medium. No difficulty was experienced in 
examining the preparation even with an oil-immersion lens. Colonies 
were observed to grow consistently from the previously described inter¬ 
cellular bacterial pockets. When the examination was completed, usu¬ 
ally when the preparations were two or three days old, the sections in 
agar were crushed in separate tubes of sterile distilled water, from which 
dilution plates were poured. These plates have commonly yielded prac- 
