along the line of inoculation and spreads through the medium as a cloudiness extending to within a few millimeters of the surface. The employment of glucose culture-media not older than a week or so seems to be important for success in cultivating this organism.

In the vegetative form the organism is sluggishly motile. It has numerous flagella. It is stained by Gram's method. Glucose-bouillon.-Growth appears first, after twenty-four to forty-eight hours, as a diffuse cloudiness. Later the fluid becomes clear, and a grayish sediment collects at the bottom of the tube. Only a small amount of gas is produced.

Occurrence.-Found in the soil, and often in the feces of herbivorous animals. In cases of tetanus the bacillus is to be found only in the wound or at the point of inoculation. It does not invade the blood-current.

The bacillus of tetanus acts by the production of a “ toxin" or "toxalbumin." This is also produced in cultures. It may be demonstrated in the bacteria-free filtrate of bouillon cultures some days or weeks old. A very few drops of this fluid will give rise to fatal tetanus in a mouse.

Method of Isolation.-Tetanus bacilli will grow in aërobic culture if other bacteria are growing with them. Since tetanus wounds usually contain other bacteria, all that is necessary to obtain an impure culture of the tetanus bacillus is to inoculate an ordinary blood-serum culture-tube (see page 79) with material from the wound. After several days or a week in the incubator, if tetanus bacilli are present they can be recognized by cover-glass preparations from the growth in the tube by their morphology and sporeformation (see Fig. 88). There will also be a peculiar, stinking odor about the culture. The isolation of the tetanus bacillus is now to be proceeded with as follows: Mix a loopful of the mixed growth on blood-serum with a tube of sterile bouillon, and heat in a water-bath for at least fifteen minutes at 80° C., then make anaerobic cultures from this (see Anaërobic Methods, page 121), taking several loopfuls for inoculation.

If other spore-bearing bacilli are present in the mixed culture in the blood-serum tube, it will be necessary to use some form of anaërobic culture on a solid medium in order to obtain separate colonies of the tetanus bacillus for further cultures.

The bacillus may be isolated from wounds and from the

FIG. 88. Spore-bearing tetanus bacilli in an impure culture on blood-serum from a case of tetanus. In the bacillus on the extreme left the beginning of spore-formation is shown (Wright and Brown).

soil by inoculation of mice subcutaneously, and proceeding as above described with material from the seat of inoculation. Bacillus Aërogenes Capsulatus.'-Will not grow in the presence of oxygen.

Morphology-Bacilli of about the thickness of the anthrax bacillus, variable in length, but usually 3 to 6μ long. Ends rounded or square cut. Occurs singly, in pairs, in clumps, and sometimes in short chains, less frequently in threads and long chains.

May show unstained spots or deeply staining granules in the protoplasm. Capsules may be frequently demonstrated in the specimens from the tissues, and sometimes in agaragar cultures.

Colonies in anaërobic cultures are grayish to brownishwhite, with a central darker spot by transmitted light. In time they may attain a diameter of 2 to 3 mm. or more. Colonies in the depths are spherical or oval, sometimes presenting knob-like or feathery projections.

Effects on Animal Tissues.—Not pathogenic for rabbits.

1 Welch and Flexner: Journal of Experim. Medicine, vol. i. No. 1, 1896.

If a rabbit that has received 0.5 to 1 c.c. of a bouillon culture injected into the ear-vein be killed immediately afterward and the body kept for twenty-four hours at a temperature of 18° to 20° C., or for four to six hours at a temperature of 30° to 35° C., the vessels and organs will be found to contain a great quantity of gas and large numbers of the bacilli. The organism multiplies post-mortem in the blood of the animal and produces the gas. This effect upon the tissues of the dead animal is characteristic of the bacillus.

The subcutaneous inoculation of guinea-pigs with young cultures may produce fatal gas phlegmons. The hemorrhagic fluid from the dead animal is virulent for other guineapigs, and may be virulent for rabbits.

Gas-production is marked in agar-agar and gelatin cultures containing glucose. The gas produced burns with a blue flame and is odorless.

Gelatin is liquefied slowly and to a limited extent.

Glucose Bouillon.-Diffusely clouded at first, later becoming clearer, with an abundant whitish, more or less viscid sediment.

Milk.-Coagulated, the clot being firm, retracted, and furrowed with the marks of gas-bubbles.

Potato-Growth thin, moist, and grayish-white, or it may not be visible.

The bacillus is stained by Gram's method. It is not motile. Spore-formation has been observed by E. K. Dunham.

The vitality of the organism depends upon the character of the culture-medium and the mode of cultivation. It survives longer when cultivated by Buchner's method (see page 124) than when cultivated under hydrogen. Cultures on glucose media are shorter lived than those on plain media.

Occurrence.-Occurs at autopsies in which gas-bubbles are present in the larger vessels, accompanied by the formation. of numerous small cavities in the liver containing gas. It has been found also in emphysematous phlegmons, in puerperal sepsis, in peritonitis, and in other conditions.

Bacillus of Malignant Edema.'-This bacillus will not grow in the presence of oxygen.

Morphology.-Rather large bacilli, sometimes growing into threads (Fig. 89), but occurring frequently in pairs, in which the proximal ends are square while the distal ends are rounded. Forms oval spores in the middle of the rod, which may give the rod a spindle or oval shape.

The colonies in anaerobic glucose-gelatin cultures appear as spheres of cloudy liquefied gelatin marked by delicate radiating streaks. Gas-bubbles are formed in the medium (Fig. 90).

FIG. 89.-Bacillus of malignant edema from the edema fluid of a guinea-pig inoculated with garden-earth; X 1000 (Fränkel and Pfeiffer).

In glucose-agar the colonies appear as hazy points made up of interlacing filaments and resembling very much the colonies of the tetanus bacillus.

Pathogenesis.-Subcutaneous inoculation of mice, guineapigs, and rabbits is followed by death in from sixteen to forty-eight hours, depending upon the animal, mice being most susceptible. The typical lesions are extensive subcutaneous edema containing gas-bubbles and more or less blood, and enlargement of the spleen. The bacilli are found

1 Liborius: Zeitschrift f. Hygiene u. Infectionskrankheiten, Bd. 1, 1886.

in the edema, in the viscera, and on the serous surfaces of the organs, but not in the blood of the heart if the examination be made immediately after death, except sometimes in mice. The organism is not capable of multiplying in the living blood, owing to the presence of oxygen. In inoculating subcutaneously a deep pocket should be made in the skin, and the material for inoculation introduced into the tissue as far away from the opening as possible. This is to

prevent the access of too much oxygen to the organism.

Slightly motile. Flagella may be demonstrated by special staining methods.

The bacilli in tissues are stained by Gram's method, but in cultures most of them are decolorized by it, probably because of rapid degenerative changes in them.

Growth in anaerobic agar-agar and bouillon culture is good, but not characteristic.

Occurrence.-Widely distributed in the soil and in putrefying substances. Only a very few cases are on record of infection in man by this bacillus.

The Micro-organism of Actinomycosis.'-The proper name of this micro-organism is "Actinomyces bovis." It belongs to of filamentous branching micro-organisms which are regarded as occupying an inter

the

group

mediate position between the bacteria, on one hand, and the moulds or hyphomycetes on the other.

The organism appears in the pus from subacute or chronic

"The Biology of the Micro-organism of Actinomycosis," by James Homer Wright, Journal of Medical Research, new series, vol. viii., p. 349.