[Vor. 7 
264 ANNALS OF THE MISSOURI BOTANICAL GARDEN 
portant factor in causing the increasing inhibitory action up 
to 96 hours. Subculture 4, with the same added nutrients as 
Subculture 3 but sterilized and reinoculated, did not attain the 
growth of Subculture 3, probably because of the small inocula- 
tion. However, the fact that the sterilized subculture did not 
surpass the unsterilized would indicate that in the parent cul- 
ture or in the other subcultures the inhibition was not due to 
a substance which could be killed by sterilizing. 
Some investigators have reported that the inhibitory action 
disappeared on standing and that a good growth was attained 
upon reinoculation, although the acidity was unaltered. To 
check this with Bacillus coli, 3 to 5 days after the cultures re- 
ported in table m1 became sterile, Subcultures 1, 3, and 4 were 
mixed together and divided into three equal 200-cc. portions, 
designated Cultures A, B, and C. Culture A was unchanged; 
Culture B was sterilized 15 minutes at 120? C.; and Culture C 
was readjusted to Pu 7.3. All were inoculated from the same 
culture of Bacillus coli with approximately 275,000 bacteria 
per ec. The growth and hydrogen ion concentration changes 
are recorded in table rv and fig. 4. Where unaltered, the hy- 
drogen ion concentration in Cultures A and B is Py 5.1 at inocu- 
lation, progressing to Py 4.9 in a short time. Death of the 
bacteria occurs shortly, with very little difference between the 
sterilized and unsterilized cultures. In Culture C, unsterilized 
but with acidity corrected to Py 7.3, growth and formation 
of acid occur similar to that in a new culture. Normal growth 
when the acidity was adjusted to neutral and no growth when it 
was not, both in the sterilized and unsterilized cultures, would indi- 
cate that no thermolabile substance which disappears on stand- 
ing was present and that the hydrogen ion concentration of 
the medium was the important inhibitory factor. 
The combined results expressed in the four tables might be 
summarized as follows: Inhibition to the point of death occurred 
only in dextrose bouillon in conjunction with acid formation, 
and not in plain bouillon with alkali formation. A slight in- 
hibitory action was found in dextrose bouillon, increasing with 
the age of the culture up to 96 hours, which was not attributable 
to acid but which probably was due to a diminution of the 
nutrients in the medium. No indication was found of an in- 
