patients and observation of those by whom they are surrounded are naturally necessary.

Immunity and Protective Inoculation.

Plague-bac

teria retain their virulence until shortly before death. It is, therefore, impossible to immunize susceptible animals with old cultures (German Commission). Slightly susceptible animals can be readily immunized by increasing doses of living bacilli. Highly susceptible animals must, however, be treated carefully with dead cultures. For this purpose it is best to employ cultures that have been exposed for an hour to a temperature of 65° C. (149° F.). In subjecting a brown ape to protective inoculation the Commission employed a heated agar streak-culture. Seven days later the animal was immune to subcutaneous infection, but only after it had recovered from this was it protected against intraperitoneal inoculation. Filtered bouillon-cultures possess only slight immunizing properties.

Haffkine has employed his method of protective inoculation, which he first recommended for cholera, also in the treatment of plague. A fluid preparation, made from carefully devitalized plague-bacilli, is injected in doses of from 1⁄2 to 21⁄2 cu. cm., in accordance with the age of the individuals. Slight reaction follows, and the injection is repeated in a somewhat larger dose after the lapse of eight or ten days. The results obtained with Haffkine's method are not unfavorable, but a definite decision as to its value must be deferred for the present; at any rate, the protection conferred is not absolute.

A plague-serum is prepared in the Pasteur Institute for therapeutic purposes from the blood of highly immunized horses. In the observations of the German Commission, three cubic centimeters of this serum sufficed to protect brown apes against subsequent subcutaneous infection. As much as ten cubic centimeters, however, did not suffice to protect susceptible gray apes. The serum exhibits, also, an undoubtedly curative action (after twelve hours) in previously inoculated brown apes. With regard to its efficacy in cases of plague, nothing definite can as yet be stated.

DIPHTHERIA.

The diphtheria-bacillus was grown in pure culture in 1884 by Löffler.

The diphtheria-bacillus is a rather plump rod of varying size, from 1 to 6 long and from 0.5 to 1 μ thick. Its form is subject to great variations in different cultures. At times it appears as a small, wedge-shaped body; at other times as a rather long body, with a bulbous thickening at one extremitya so-called club; and at still other times as a double club, or a dumb-bell. Slender forms, occasionally curved slightly, are also observed, especially in membranes. Not rarely bifurcations are encountered, and upon the basis of this observation diphtheria

Fig. 51.-Bacillus diphtheriæ from a pure culture (Stengel).

bacilli have been placed in relation with streptothrices, and even with hyphomycetes. The diphtheria-bacillus is incapable of independent movement.

Spore-formation is wanting.

Staining Properties.- The diphtheria-bacillus is best stained with methylene-blue or dilute carbolfuchsin. Gentianviolet overstains and conceals the more delicate structural relations. The organism is stained by Gram's method. The small, blunt clubs stain equably, while the longer specimens exhibit unstained areas, so that the rods appear in places to consist of several segments. Flügge has described a method of double staining, devised by M. Neisser, which is considered to possess essential differential diagnostic significance. The preparation is placed for from three to five seconds in an acetic-acid solution of methylene-blue (methylene-blue 1, alcohol 20, distilled water 950, glacial acetic acid 50), rinsed in water, and counterstained for from three to five seconds in vesuvin (vesuvin 2,

dissolved in boiling distilled water 1000, and filtered). The bodies of the bacilli appear brown, and, as a rule, they contain two blue granules, which have at once been intensely stained by the first aniline dye, and have not yielded their color to the vesuvin subsequently employed. These are the so-called BabesErnst bodies (p. 19). The form, arrangement, and situation of these bodies are considered as characteristic of the diphtheriabacillus under the following conditions: The preparations must be made only from cultures that have grown upon Löffler's blood-serum (solidified at 100° C.-212° F.) at a temperature of 34° C. (93.2° F.) or 35° C. (95° F.), and never above 36° C. (96.8° F.). The cultures must not be less than nine

hours, and not more than from twenty to twenty-four hours, old. At one end, more frequently at both ends, of the brown rod a blue granule is then to be seen, and not rarely a third is visible in the middle. These granules are oval in shape, and possess a greater diameter than the bacillus itself, which, however, if the whole appearance is to be considered of diagnostic significance, must be distinctly visible in its entire length and form.

It is further said to be characteristic of the diphtheria-bacillus that the individual bacteria are arranged side by side like palisades. As the most distinctive feature, M. Neisser considers the appearance of impression-preparations from a serum-plate six hours old that has developed at a temperature of from 34° C. (93.2° F.) to 36° C. (96.8° F.). In these there are

visible" moderate-sized free masses in which the slender, rather long, slightly curved bacilli lie in characteristic irregular arrangement-an appearance that is to some degree represented by placing the extended fingers of one hand in varying combinations over or by the side of those of the other."

Cultural Properties.-The diphtheria-bacillus thrives only at temperatures between 20° C. (68° F.) and 40° C. (104° F.) upon all slightly alkaline culture-media. Its temperature-optimum is that of the body.

Upon gelatin-plates round, whitish colonies form that remain small. Microscopically these appear yellowish brown and granular, with an irregular border.

In gelatin stab-cultures similar small, whitish, spherical colonies, not exceeding a certain size, form along the line of inoculation. At a temperature of 24° C. (75.2° F.) superficial growth takes place, with indications of nail-culture. The gela

tin is not liquefied.

Upon agar, and better upon glycerin-agar plates, after from twenty-four to forty-eight hours, small, grayish-white, glistening colonies form that often exhibit microscopically concentric lamination; with low powers of the microscope they appear peculiarly granular, with an irregular border.

In agar streak-cultures, after twenty-four hours, small, translucent, slightly raised colonies appear. Further growth is inconsiderable, and scarcely extends beyond the line of inoculation.

In agar stab-cultures the colonies develop along the line of inoculation, and there is slight growth upon the surface.

Upon Löffler's serum, after twenty-four hours, fairly large, whitish, opaque colonies of firm consistence appear. In the succeeding days these colonies increase but little in size. Löffler's blood-serum (p. 84) constitutes, all in all, the best culture-medium for diphtheria-bacilli. It is, therefore, always employed for purposes of differential diagnosis.

In bouillon, after twenty-four hours some precipitate has formed, after two days slight turbidity, which increases to the fifth day, then to grow less, until finally the fluid overlying the crumbling, flocculent precipitate is completely clear. Not rarely a thin, fragile coating appears upon the surface of the bouillon.

A delicate coating forms upon the surface of potato rendered alkaline.

Milk constitutes a favorable nutritive medium, but is not coagulated.

In boiled and unboiled egg, both white as well as yolk, the diphtheria-bacillus grows well; upon coagulated egg-albumin it not rarely exhibits branching.

In slightly alkaline Löffler's meat-infusion bouillon the diph

theria-bacillus generates acid. The increase in acidity is distinctly appreciable within twenty-four hours; it augments from the second day, then to subside. Later, after two or three weeks, the bouillon becomes again alkaline. On addition of litmus to the culture-medium these variations can be distinctly followed by the changes in color.

Tenacity of the Diphtheria-bacilli.—A solution of mercuric chlorid, 1:1000, destroys cultures in a thick layer within twenty seconds; and five per cent. potassium permanganate, five per cent. aqueous solution of carbolic acid, three per cent. solution of carbolic acid in thirty per cent. alcohol, four per cent. solution of kresol in forty per cent. alcohol, within the same time. Five per cent. potassium chlorate is still ineffective after sixty seconds (Löffler). Pure lemon-juice likewise destroys the bacilli speedily. They are destroyed by exposure for ten minutes to a temperature of 60° C. (140° F.), although in a somewhat thicker layer they withstand drying for months. In the form of dust, however, they rapidly die. They withstand cold well, although in the refrigerator the diphtheriabacilli rapidly lose their property of generating toxins. Abel found diphtheria-bacilli on building-blocks with which a child suffering from diphtheria had played six months previously. Diphtheria-bacilli have been found, further, upon soiled bed-linen, on the rim of a drinking-glass, in the hair and on the shoes of nurses, etc. In gelatincultures they may survive, according to Löffler, for three hundred and thirty-one days. Diphtheric membranes dried and preserved in the dark yield cultures even after the lapse of months.

Pathogenic Properties of the Diphtheria-bacillus for Animals.-Diphtheria does not naturally occur in animals. So-called spontaneous diphtheria of fowl, of pigeons, etc., are etiologically different diseases.

Diphtheria-bacilli give rise to the formation of true diphtheric pseudomembrane, with multiplication of the bacilli, in the previously injured vagina or conjunctiva of guineapigs, in the trachea of guinea-pigs and rabbits after tracheotomy. Most birds, especially pigeons and chickens, then young dogs, rabbits, and especially guinea-pigs, are susceptible to the diphtheria-bacillus. Subcutaneous introduction of the bacilli is first followed by purely local alterations: more or less extensive hemorrhagic edema of the subcuta