large quantities of virulent spores (Koch), but has proved very successful practically in anthrax regions (Pasteur). Hankin has tried to produce an immunity in animals with the metabolic products of anthrax without positive results; the serum from sheep in which a very high active immunity has been produced possesses immunizing action only for sheep and not for rabbits (Sobernheim). The bactericidal power of the serum in vitro is not increased over the serum from normal animals. The serum does not cause agglutination. Sobernheim considers the injection of a mixture of immune sheep serum (16 c.c.) and attenuated bacilli (loopful) to be the most certain method of conferring a more lasting vaccination protection (Sobernheim, Z. H. xxv, 301; and XXXI, 89). Emmerich and Löw have observed very noticeable curative and immunizing results in rabbits from pyocyanase (p. 110).

Special Methods for Demonstration and Differential Diagnosis.-If the question, as is usual, is one of diagnosis in an infected man or animal, very often a good preparation of smeared blood stained by Gram's method gives valuable results. For the differential diagnosis ordinary agar plates are especially to be prepared, which, after seventeen to twenty-four hours in the incubator at 37°, present spores within threads. Also observations as to motility are useful, as also are sugar-agar shake cultures. The Differential Diagnosis between the Varieties which most Come in Question may be Represented as Follows:

The result is always obtained with absolute certainty in thirty-six hours.

For the differential diagnosis between symptomatic anthrax and malignant edema, see page 314.

It may be difficult to differentiate an anthrax bacillus from soil from the closely related spore-forming varieties. If a virulent form is in question, then the inoculation with a sample of earth into several guinea-pigs will often decide the question. The dead animals will be examined as described above. Thus it is possible that one animal dies of anthrax, others of malignant edema, tetanus, etc., the causes of which were all present as spores in the sample of soil. Non-virulent forms of anthrax, isolated from soil, are only recognizable by comparison with known anthrax, whereby the five species of the table (p. 314) are to be excluded.

The following are described as closely related to anthrax: B. pseudanthracis Burri. According to Hartleb and Stutzer, it is widely distributed in American meat powder. The cultures isolated from different samples were not exactly identical. The cultures are motile, especially when grown in bouillon. Also it is important that in bouillon there is first diffuse turbidity, then a clearing up, with the formation of a precipitate and pellicle. All other characteristics are deceptively like those of anthrax. Its virulence for mice and guinea-pigs is slight. Compare C. B. L. II, 81, where also is a description of cultures of B. pseudanthracis II and III, which are still a little further removed from anthrax.

B. anthracoides Hüppe and Wood (from soil), which we obtained from Král and carefully studied. We found, indeed, the agar cultures macroscopically very much like anthrax, but microscopically they resemble B. subtilis; also upon gelatin the similarity to Bac. subtilis when magnified 60 times was much greater than to Bac. anthracis. In young colonies loop-like projections extend out, reminding one of Bact. vulgare. When magnified 1000 times, sluggish motion was unmistakable.

B. anthraci similis McFarland (C. B. XXIV, 556). It was once found upon a laboratory plate, and is entirely non-pathogenic. Perhaps it was true anthrax.

Bacillus mycoides. Flügge.

(Plates 37 and 38, I-IV.)

Synonym.-Root bacillus.

Microscopic Appearance.-Rather large rods, scarcely at all rounded at the ends, 1.6-3.6 μ long and 0.8 μ thick. Sometimes arranged in threads (38, 1). Forms oval spores.

Motility. We have seen no cultures which exhibited active motility. Usually all the individuals are quiet except a few, which are only detected in motion after more prolonged observation. Also in the stained preparation the impression is given of only a few being provided with flagella. Formerly we believed we had observed entirely non-motile cultures, but can not now demonstrate this. Here belongs the non-motile Bacillus radicosus Zimm. Staining Properties.-Stains by Gram's method. Requirements as to Nutrient Media. Slight; also grows without oxygen but scantily.

Gelatin Plate.-(a) Natural size: In earliest stage the colonies consist of a scarcely perceptible circle of little. hairs (37, VI). After one or two days the gelatin is liquefied a little, while the colonies have become distinctly larger. The hairy circle ramifies more and more, and thicker branches are formed, especially at the center, which, toward the periphery, are replaced by irregular, fine, root-like branches (37, Ix).

In

(b) Magnified fifty times: Colorless, more or less winding threads, interlacing in a most extraordinary manner. the center the colony is sometimes felted and opaque. The branchings are only apparent, since always two closely lying threads diverge from each other at the point of apparent branching.

Gelatin Stab.-It is characterized by delicate little hairs of quite uniform length growing outward, parallel 1 to each other, along the stab canal (37, 1). The liquefaction of the gelatin begins in the form of a saucer and then

1 In more advanced stages the hairs are often directed upward (37, II). The zone of liquefaction is usually clear or slightly cloudy.

becomes cylindric. Upon the surface of the liquefied medium there is a thick white film, reminding one of a covering of asbestos. If this falls to the bottom of the liquid, a new one at once forms, so that cultures may be found having many such films.

Agar Plate.-(a) Natural size: At first the colonies are very like those in the gelatin plate, but more sturdy (37, VII). The further growth is absolutely irregular, there being found, as well, colonies with a dense center and very distinct main branches, and also those with a delicate central portion, and about this a growth in the form of a circle (37, v).

(b) Magnified fifty times: Exactly like the colonies in the gelatin plate. Plate 38, 1, represents a colony with an open central part. Plate 38, IV, represents a part of the same magnified 150 times.

Agar Stab. Stab: Parallel brush-shaped outgrowths, usually of unequal length, delicate gray, but a little denser than in the gelatin stab (37, IV). Surface growth: Exactly like the colonies on the agar plate; light gray, moist, shining (37, v).

Agar Streak.-Grayish-white, moist, shining growth, with root-like outgrowths, showing most extraordinary abundant branchings. In a short time it covers the entire surface (37, III).

Potato Culture.-Extremely like the potato culture of the Bac. subtilis. It is white, when older yellowish, a little elevated, crummy, dull, provided with a delicate, insignificant fringe at the periphery (38, 11).

Chemical Activities. See page 307. There is also no formation of H,S.

Distribution.-Very common in soil.

Bacillus subtilis.

F. Cohn. (Beiträge, Bd. i,
H. ii, 175.)

(Plates 39 and 40.)

Common Name.-Hay bacillus.

Microscopic Appearance. -Short (1.3-3), rather thick (0.8-1.2 μ), sturdy rods with rounded ends, often

united in long strings, and not infrequently the separation into individual rods is indistinct, so that long threads occur (40, v).

Spores. When air is admitted, oval spores are readily formed, which germinate at right angles to the long diameter. (Compare p. 27.)

Motility. The short forms are very actively motile because of long, abundant, peritrichous flagella. The chains of bacilli still show flagella, even when they are no longer motile (40, VI, IX).

Staining Properties.-Stain by Gram's method.

Requirements as to Nutrient Media and Oxygen. -Grows upon the most various nutrient media at room and incubator temperatures, but, when oxygen is excluded it grows poorly and without sporulation. Growth is rapid.

Gelatin Plate.-(a) Natural size: In a short time the colonies sink into a saucer-shaped depression. The content of the liquefied zone is grayish-white. At the center lies the whitish, ragged colony (40, III). A later stage is shown in 40, IV.

(b) Magnified sixty times: At first the colonies are roundish, even-edged, crumbly, yellowish, sometimes surrounded by a delicate row of hairs (40, II, i). Later, especially in the case of the superficially located colonies, the periphery becomes wavy, and with advancing liquefaction of the gelatin breaks up into innumerable tangled locks, which surround the colony. The central part is still held firmly together, being granular and yellowish to brownish until after four to five days, when it also becomes completely disintegrated (40, II, e).

Gelatin Stab.-The surface growth is whitish-gray, and, after thirty-six hours, sinks into the gelatin in the form of a saucer. The gray liquefied content of the saucer contains whitish bunches in suspension (39, 1). The liquefaction progresses in a cylindric form, the contents being grayish-white, and cloudy, especially below. Upon the surface is a thick white scum, firmly attached to the wall of the tube (39, II).

Agar Plate. (a) Natural size: Small, irregular, shining, grayish-white colonies (39, vIII).