first placed in a solution which contained in 200 c.c. of water 1 c.c. of a concentrated solution of methylene blue; to this was added 2 c.c. of a 10 p.c. solution of potash. The preparation remained in the solution 20-24 hours (or with warming to 40° only half an hour), afterwards it was placed in a concentrated solution of vesuvin for 15-20 minutes in the case of sections, and for but 1-2 minutes in the case of dried sputum. In this way the tubercle bacilli were coloured blue, the rest brown.

Two years later Koch1 published a more complete and detailed account of his researches on the etiology of phthisis. Koch recommended in this treatise a modification of Ehrlich's staining method, which consists in placing the preparation in a solution composed of 100 c.c. aniline water, 11 c.c. alcoholic solution of methyl violet or fuschin, and 10 c.c. absolute alcohol. The preparation remains in this solution at least twelve hours, but the time can be shortened by warming; it is then transferred to dilute nitric acid (1:3), then washed for a few minutes with absolute alcohol (covering glass preparations only a few seconds) and finally coloured by soaking for a few minutes in aqueous solution of vesuvin or methyleneblue.

(345) Characteristics of the Tubercle Bacilli.

Koch 2 described the general characteristics of the micro-organisms as follows:-"They always appear in the form of rods the length of which is equal to that of half a red blood corpuscle (about 0015mm. 0035mm.). Though the length of the bacilli may vary somewhat, the diameter is constant so long as the same staining method is employed; the alkaline methylene blue process causes them to appear more slender than when stained with the process of Ehrlich. The bacilli are commonly not quite straight rods but there are found among them slight curves and often also slight twistings, in the longest examples, these may even suggest the notion of the commencement of a spiral. The bacilli are separated from other bacilli which come nearest to them in size and appearance by this tendency to curve."

See Plates II. and III. Plate II. a represents a giant cell containing numerous bacilli arranged in a radiating form × 700

1 Mittheilungen d. Kaiser. Gesundheitsamte. 2 Band.

2 Mittheilungen aus dem Kaiserlich Gesundheitsamte. Berlin, 1884.

(after Koch); b represents a few colonies derived from the artificial culture represented in Plate III. b x 700. Plate III. a is the naked eye appearance of an artificial culture of the tubercle bacilli on coagulated blood serum; b is a portion of the same culture × 80 (after Koch).

The tubercle bacilli are frequently beaded; they may occur singly, or in pairs, or in bundles. The bacilli are always rounded at the ends (see Fig. 51). They are non-motile, do not liquefy the culture medium, and form spores both within the body and in artificial cultures. With regard to the latter Crookshank remarks that "the tubercle bacillus, when examined by such powers as Powell and Lealands in

FIG. 51.

Hom. imm. is frequently seen to consist of a very delicate sheath, holding together a number of deeply stained granules, for the most part round or cylindrical, with irregular contour and differing considerably in size, while the light interspaces are seen to vary in form according to the shape of the granules. In some preparations more distinct and clearly ovoid granules may be observed which are sometimes terminal. It is not impossible that these ovoid granules are spores which in their behaviour to staining reagents thus form an exception to the general rule. But there can be little doubt that a tubercle bacillus consists for the most part of a very delicate sheath, with protoplasmic contents which have a great tendency to be broken up or coagulated into little segments or roundish granules, owing, possibly, to the treatment they are subjected to in making a microscopical examination. This, however, does not always occur, for the bacilli at times are not beaded, but are stained in their entirety."

(346) Artificial Cultivation of Tubercle Bacilli.

Koch found the most suitable medium in which to cultivate the bacilli was blood serum coagulated at a temperature of 65°; it remains transparent if this heat is not exceeded. He sterilises it by heating for five alternate days for one hour each day,

1 Manual of Bacteriology. 2nd edition.