BIOLOGICAL AND MORPHOLOGICAL CHARACTERISTICS OF THE HAM-SOURING BACILLUS.

CONDITIONS FAVORABLE TO GROWTH.

The most favorable medium for the growth of the organism was found to be the modified egg-meat mixture of Rettger, which has been previously described. In this medium the organism develops rapidly at a temperature of 20° to 25° C., giving rise to the characteristic sour-meat odor. Like the bacillus described by Klein, it also grows readily on pork-agar and pork-bouillon containing glucose, but differs from Klein’s bacillus in that it will grow, though less luxuriantly, on ordinary nutrient media—agar, gelatin, and bouillon—without the addition of glucose.

The optimum temperature for growth is 20° to 25° C. The organism does not grow at incubator temperature (37.5° C.). At ice-box temperature (8° to 10° C.) it develops readily, although the growth is less rapid than at 20° to 25° C. That the organism will develop at even lower temperatures was shown in the inoculation experiments with hams, where it developed and multiplied extensively in the bodies of the hams at the temperature of the pickling cellars, which are held usually at 34° to 36° F.(1° to 2° C.).

The organism develops best in a neutral or slightly alkaline medium.

GROWTH ON DIFFERENT CULTURE MEDIA.

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.

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.

SPORE FORMATION.

The organism develops large, terminal spores, which are at first oval, but when fully developed are perfectly round and measure from 1.5 to 2 μ in diameter.

Spores develop rapidly in the egg-pork medium at 20° to 25° C., fully developed spores being noted in from five to seven days. At ice-box temperature (8° to 10° C.) partly developed spores were noted in the egg-pork medium at 10 days and fully developed spores at 2 weeks.

Occasional spores were noted in old agar and gelatin cultures, but abundant spore formation was seen only in the egg-pork medium. No spores were noted in bouillon cultures, even at 10 weeks.

RESISTANCE TO HEAT AND CHEMICAL AGENTS.

In its vegetative form the bacillus is killed at 55° C. in 10 minutes. The spores survive a temperature of 80° C. for 20 minutes, but are killed at 100° C. in 10 minutes.

When sodium chlorid and potassium nitrate were added to glucose-pork broth in varying amounts, it was found that 3 per cent of sodium chlorid or 3 per cent of potassium nitrate was sufficient to inhibit completely the growth of the bacillus at room temperature (20° to 25° C.).

While the growth of the bacillus was inhibited by sodium chlorid and potassium nitrate as just stated, it was found that very much stronger solutions of the two salts failed to destroy the bacillus. Thus it was found that the bacillus or its spores retained their vitality after an exposure of 30 days in a solution containing 23 per cent of sodium chlorid and 6 per cent of potassium nitrate.

GAS PRODUCTION.

The organism splits glucose, but not lactose or saccharose. That it possesses the power of splitting muscle sugar was shown by the formation of gas in Smith fermentation tubes containing ordinary neutral bouillon without the addition of any sugar.

The formation of gas in glucose bouillon varies considerably with the reaction of the medium. The largest amount of gas was formed when the broth was neutral or slightly alkaline. When the reaction of the broth was distinctly acid or distinctly alkaline the amount of gas was diminished. The gas which is formed in bouillon cultures consists chiefly of hydrogen and carbon dioxide. In order to collect a sufficient amount of the gas for analysis, two large fermentation tubes capable of holding 150 cubic centimeters each were constructed. These tubes were filled with pork-bouillon and inoculated with the bacillus. After 20 days at room temperature (20° to 25° C.) the gas was collected and the carbon dioxide and hydrogen determined, with the following result:

Cubic centimeters.
Total amount of gas collected37.7
Carbon dioxide, by absorption with NaOH6.2
Hydrogen, by difference31.5

This analysis gives an approximate gas formula of H/CO2= 5/1, which agrees with the gas formula as determined in the small fermentation tubes by Smith’s method.

In hams which had undergone spontaneous souring and in hams which had been artificially soured by inoculation, hydrogen-sulphid was often noted when the sour portions of the meat were tested with lead-acetate paper, but no distinct odor of the gas could be obtained. Hydrogen sulphid was also noted in egg-pork cultures of the bacillus.

ACID PRODUCTION.

In glucose-bouillon, butyric and lactic acids are formed and the reaction of the medium becomes distinctly acid. Butyric and lactic acids were also noted in the egg-pork cultures.

A series of Smith fermentation tubes containing 10 c. c. each of glucose-pork broth medium was inoculated with the bacillus and held at room temperature (20° to 25° C.). These cultures were titrated against [N/40]NaOH, with phenolphthalein as an indicator at intervals of two days up to nineteen days, and then at two-week intervals up to sixty-one days. Three of the cultures were titrated each time so as to give a fair average of the acidity of the cultures, and an uninoculated check tube was also titrated each time to see if there was any change in the reaction of the medium. The results of the titrations are shown in the following table:

Acidity determinations in glucose-pork broth cultures.

Age of culture
(days).		Culture
A.		Culture
B.		Culture
C.		Average.Medium.Acidity of
culture.
Per cent.
20.0380.0300.0400.0360.0090.027
4.105.100.102.102.009.093
6.106.110.109.108.009.099
8.124.115.117.119.009.110
10.128.130.126.128.009.119
12.129.120.129.126.009.117
19.126.125.125.125.009.116
33.125.123.125.124.009.115
47.122.120.121.121.009.112
61.121.116.119.118.009.109

From the above table it will be seen that the maximum acidity was reached at ten days, after which there was a gradual reduction in the acidity, due probably to the formation of ammonia compounds.

PATHOGENIC PROPERTIES.

Rabbits, guinea pigs, and white mice were inoculated and fed with cultures of the bacillus without effect, from which it would appear that the bacillus possesses no pathogenic or disease-producing properties.

NATURE OF THE BACILLUS.

The bacillus is essentially a saprogenic bacterium with zymogenic properties. A preliminary study of the chemical changes which take place in sour hams shows that these changes are of a putrefactive nature. Hams which had undergone spontaneous souring were compared with hams which had been artificially soured by inoculation, and the chemical changes were found to be identical. A chemical study was also made of the changes taking place in egg-pork cultures of the bacillus at different stages of growth, and these changes were found to be of a putrefactive nature and similar in character to the changes which occur in sour hams. Among the putrefactive products formed by the growth of the bacillus in the egg-pork medium were indol, skatol, volatile fatty acids, skatol-carbonic acid, and hydrogen sulphid.[4]

[4] The tests for the putrefactive products formed by the growth of the bacillus in the egg-pork medium were made by P. Castleman, of the Biochemic Division, who also determined the percentage composition of the gas formed by the growth of the bacillus in the glucose-pork-bouillon medium.

Bul. 132, Bureau of Animal Industry, U. S. Dept. of Agriculture. Plate IV.

Glucose Bouillon Culture in Smith Fermentation Tube at Four Days. Culture Grown at Room Temperature (20° to 25° C.). Growth Confined Entirely to Closed Arm, with Gas Collecting at Top.

A more extended study is now being carried on in the Biochemic Division of the chemical changes which take place in hams during the process of souring, together with a further study of the chemical changes which result from the growth of the bacillus in the egg-pork medium. The results of this investigation will be given in a later paper.

The bacillus described in this paper belongs to the class of putrefactive anaerobes, which are widely distributed in nature in dust, soil, and excrementitious matters. This group of bacteria contains both pathogenic and nonpathogenic forms. The former have received considerable attention, but the latter have never been thoroughly cleared up. The bacillus isolated from sour hams belongs in the latter category, being possessed of no pathogenic or disease-producing properties. It occurs in the dust and dirt of the packing house and finds its way into the hams in the various manipulations to which the hams are subjected.

The bacillus described in this paper does not seem to correspond with any forms heretofore described. It differs from Klei bacillus (Bacillus fœdans) in the following important particulars:(1) It forms large terminal spores, whereas Klein’s bacillus formed no spores;(2) it will grow at a temperature of 34° F., while Klei bacillus did not grow below 50° F.;(3) it produces an acid reaction in culture media, while Klei bacillus gave a distinctly alkaline reaction;(4) it will grow on the ordinary nutrient media—gelatin, agar, and broth—without the addition of glucose, while Klein’s bacillus did not;(5) it peptonizes the casein in milk, whereas Klein’s bacillus had no action on milk; (6) it liquefies gelatin more rapidly, causing complete liquefaction after three weeks at 8° to 10° C., whereas Klein’s bacillus caused only partial liquefaction after eight weeks at 20° C.;(7) it can be conveyed

from turbid broth cultures to new culture material by means of the platinum loop, whereas Klein’s bacillus could not be thus conveyed.

For the bacillus described in the present paper the following name is proposed:Bacillus putrefaciens.