588 
Journal of Agricultural Research 
Vol. XXIV, Na 7 
by such mechanical agencies as jarring or rapid addition of water. It 
was observed that when such a point was reached in a few of the pre¬ 
liminary experiments, germination was very much lower than in the 
later experiments when puddling was avoided by very careful handling 
of the soil. It is obvious, then, that the condition of the soil containing 8o 
per cent of its moisture-holding capacity was not far removed from that 
of puddled soil. Among the several factors that may have been re¬ 
sponsible for reduced germination in wet soil, that of absence of suffi¬ 
cient oxygen appears the most probable. For this reason experiments 
were carried on to determine whether the spores germinate in the absence 
of oxygen. 
THE INFLUENCE OF OXYGEN ON SPORE GERMINATION 
In the search for a method whereby oxygen could be excluded from a 
spore suspension which could be kept under observation, many devices 
were tried before a satisfactory 
technique was finally adopted. The 
apparatus used is shown in Figure 3. 
Oxygen-free air was forced through 
an Engelmann cell (fig. 3, C) where 
spores were suspended in beef broth 
(Ph 6.4), as in the germination ex¬ 
periments previously described. 
The beef broth had been freed from 
Fig, 3.—Apparatus for excluding oxyg«a from a 
spore suspension; A, reservoir of oxygen-free 
water; B, bottle containing pyrogallic add; C, 
Engelmann cell; and D, bottle of water. 
oxygen by heating at 100® C. and 
kept in an air-tight receptacle with 
a solution of pyrogallic acid. The 
oxygen was removed froih the air in 
the air-tight bottle (fig. 3, B) by 
introducing a liter of alkaline pyro- 
gallic acid and shaking it within the 
bottle for 20 minutes. Normal 
pressure was restored after shaking 
by siphoning boiled water from the 
reservoir (fig. 3, A) into the oxygen-free bottle. The tube from the 
reservoir to the bottle was then tightly clamped, the reservoir covered 
with a quarter of an inch of kerosene to exclude oxygen, and the latter 
raised to the position shown in the figure. 
The Engelmann cell with its spore suspension was then attached to the 
outlet tube of the bottle, and a flow of water allowed to pass through the 
siphon into the bottle of oxygen-free air whose connection with the Engel¬ 
mann cell was then opened. The oxygen-free air thus forced through the 
Engelmann cell was allowed to bubble through water in a small bottle 
placed at a much lower level (fig. 3, D). 
^ By regulating the inflow of water from the reservoir, the oxygen-free 
air was forced rapidly through the cell for at least 40 minutes, and then 
inore slowly for the remainder of the 24-hour period, except during the 
time when the air passing through the cell was collected for analysis in a 
Hempel pipette by the replacement of mercury. During each experi¬ 
ment four or five pipettes of gas were collected and the percentage of 
oxygen determined over phosphorus.® This anatlysis was made in order 
* The writer is indebted to Dr. A. S. Eoevenhart and the Department of Pharmacology, University of 
Wisconsin, for the use of the oxgen-analysis apparatus. 
