_Growth on egg-pork medium._—At a temperature of 20° to 25° C. the
cultures show a slight but distinct sour odor in from two to three
days. This odor, as before stated, closely resembles the odor of a
sour ham. Egg-pork cultures from three to five days old were given to
a trained meat inspector, who knew nothing whatever as to the contents
of the tubes, and he was asked to describe the odor; he described it as
that of a sour ham.
At one week the albumins of the medium are gelatinized or partly
coagulated and the odor is more pronounced. At ten days the albumins
are completely coagulated except at the surface, where there is
no apparent growth; the odor is more putrefactive in nature, and
the reaction of the medium is slightly acid. At three weeks the
coagulated albumin splits up into fragments and appears to undergo a
slow digestion, gas bubbles form in the lower portion of the culture,
and the odor becomes distinctly putrefactive in character. The slow
digestion of the albumin is probably due to a proteolytic enzyme
elaborated by the bacillus.
At the end of a week a dark zone usually appears at the surface of the
coagulated albumin and gradually darkens until it becomes almost black.
This zone is probably due to a pigment elaborated by the bacillus.
At ice-box temperature (8° to 10° C.) the same changes and the same
odor were noted, but were somewhat slower in developing.
_Glucose-pork-agar._—This medium was prepared from pork in the same
manner as beef-agar, and contained 1 per cent of glucose. The organism
grows readily on this medium and may be conveniently cultivated in deep
stab cultures. The medium was always thoroughly boiled and then rapidly
cooled in order to expel the inclosed air. The growth of the organism
was found to vary considerably with the reaction.
When the reaction was +1.5, deep stab cultures at three days (20° to
25° C.) showed a well-marked arborescent growth, appearing as delicate
filaments extending outward from the line of stab. The growth stopped
within one-fourth or one-half inch of the surface of the agar on
account of the presence of oxygen in the upper part of the culture
medium. As the growth extended toward the walls of the test tube the
agar became clouded, and there were sometimes gas bubbles in the depth
of the agar, but the gas formation was not extensive.
When the reaction of the agar is neutral or slightly alkaline,
extensive gas formation occurs and the agar is often much broken up.
The cultures developed a disagreeable, somewhat putrefactive odor,
but did not give the characteristic sour-ham odor obtained from the
egg-pork cultures.
The organism was also grown on anaerobic agar plates by Zinsser’s method,
which is said to give absolutely anaerobic conditions. The colonies on
agar have a cottony or woolly appearance at first, and spread slowly,
with slightly irregular margins.
In glucose-pork-agar to which azolitmin was added the azolitmin in the
lower portion of deep stab cultures was completely decolorized in five
days at room temperature (20° to 25° C).
In glucose-pork-agar containing neutral red the red color in the lower
portion of the tube was changed to yellow with the development of
fluorescence.
_Neutral gelatin._—Tubes of ordinary neutral gelatin without the
addition of glucose were inoculated and held at ice-box temperature
(8° to 10° C). At five days a delicate white growth appeared along
the line of stab in the lower portion of the tube. At seven days the
growth showed fine radial striæ, presenting an arborescent or tree-like
appearance, and extended halfway from the line of stab to the walls of
the test tube. At two weeks the growth had caused a delicate clouding
of the medium in the lower portion of the tube. At three weeks the
gelatin in the lower portion of the tube had become liquefied and the
growth had settled to the bottom as a white precipitate.
In gelatin containing glucose, gas bubbles are formed in the depth
of the medium through the splitting up of the glucose, and the
characteristic arborescent growth is obscured.
_Glucose-pork-bouillon._—This medium was prepared from pork instead
of beef and contained 1 per cent of glucose. The best results were
obtained when the reaction of the medium was neutral or slightly
alkaline.
Culture tubes, which had been previously boiled to expel the contained
air and then inoculated, were held in a Novy jar, in an atmosphere of
hydrogen at a temperature of 20° to 25° C. At three days the tubes
showed well-marked clouding. At one week the growth appeared as a
heavy, white, flocculent, cottony precipitate in the bottom of the
tubes with a slight flocculent precipitate above. When the culture was
removed from the jar and shaken, the heavy, flocculent precipitate at
the bottom of the tube broke up without much difficulty, giving rise to
a heavy uniform clouding with some small floating masses, which soon
settled to the bottom. On shaking the tube some evolution of gas in the
form of very fine bubbles was noticed.
In Smith fermentation tubes containing neutral glucose-pork-bouillon
the closed arm of the tube shows well-marked clouding with gas
formation at three days at room temperature (20° to 25° C). The
growth has a tufted, cottony appearance, and there are many filaments
and threads. The growth settles to the bottom of the closed arm as
a cottony, white precipitate (see Pl. IV). The organism splits the
glucose vigorously, and at 10 days the tubes show from 40 to 50
per cent of gas. The bouillon in the open arm of the tube remains
unclouded. The maximum gas production at room temperature is reached
in from 10 to 14 days, by which time the growth in the closed arm has
completely settled into the bend of the tube, leaving the bouillon
in the closed arm clear. The gas formula, as determined by Smith’s
method, was H/CO₂ = 5/1. The reaction of the bouillon becomes acid to
phenolphthalein.
The organism will grow on ordinary neutral bouillon without the
addition of glucose, and in Smith tubes containing this medium a small
amount of gas was formed, due to the splitting of the muscle sugar.
The bacillus also grows in a sugar-free broth—that is, a broth free
from muscle sugar—and from cultures grown in this medium a well-marked
indol test was obtained.
_Litmus-milk._—The organism was grown in litmus-milk in Smith
fermentation tubes at 20° to 25° C. At seven days the litmus in the
lower portion of the closed arm had assumed a brownish-buff color.
At two weeks the litmus in the closed arm had been reduced to a
brownish-buff color except at the top of the tube, where a pale, bluish
tinge remained, and the litmus in the open arm showed very slight
reddening as compared with a check tube. At three weeks the litmus in
the closed arm was entirely reduced to a light, brownish-buff color,
and the litmus in the open arm showed a slight but distinct reddening
as compared with the check. The reddening of the litmus in the open arm
was evidently due to the transfusion of acids formed by the growth of
the bacillus in the closed arm. After several weeks the milk is slowly
peptonized, probably as a result of enzyme action.