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.
MORPHOLOGY.
The organism is a large bacillus having an average size of 4 to 8 μ in length by 0.5 to 0.7 μ in thickness, but there are many longer forms measuring from 10 to 20 μ in length. It develops in long, irregular chains or filaments, which at times show a slightly spiral form.
Fig. 5.—Ham-souring bacillus (Bacillus putrefaciens) grown on egg-pork medium, showing tendency to form chains. Partly developed and fully developed spores are shown at ends of rods; also free spores. (Pen-and-ink drawing made with camera lucida from preparation stained by Gram’s method.× 640.)
The individual organisms show at times a widely open, slightly spiral form, which was more apparent in hanging-drop preparations made from bouillon cultures, where the organisms had been comparatively undisturbed. This appearance was also noted at times in the stained sections of soured muscular tissue, where the organisms were stained in place. The organism possesses no motility. It stains with the ordinary aniline dyes and by Gram’s method.