bacilli and the age of the culture. An evidence of attenuation is found in a greater lengthening and thinning of the microorganism, and of virulence by the presence of short, thick bacilli. A considerable variation in size is recognized in bacilli taken from the sputum, which, as a rule, are of greater length than those observed from culture growth. Differences also exist in the contour or shape of the microorganisms, in their disposition with reference to one another, and in their specific staining reaction. A notched, beaded, or clubbed appearance, with a tendency toward flexion, is suggestive of an old attenuated bacillus. A fragmentary or broken-down rod also is strongly indicative of degenerative change. On the other hand, the aggregation of bacilli in clumps, as opposed to an isolated or scattered disposition, is somewhat characteristic of active virulence. The same is true of their ability to take rapidly a deep primary stain. An essential property of the tubercle bacillus is its resistance to decolorization by acids. On account of this attribute it is easy to recognize the bacillus in the sputum after the removal of the primary stain from the cellular elements and associated bacteria. The subsequent blue contrast stain of these portions of the specimen renders the continuous red color of the bacilli especially pronounced. An important preliminary to the staining process is the selection from the midst. of the tuberculous sputa of small cheesy particles often found in the more dependent portions. Tubercle bacilli are much more frequent in these caseous deposits than elsewhere in the expectoration. When the bacilli are particularly scanty, it is sometimes advisable to dilute 100 c.c. of sputum with 200 c.c. of water to which have been added about 8 drops of a 10 per cent. solution of sodium hydroxid. A homogeneous solution is obtained by boiling, and the centrifuged sediment is examined for tubercle bacilli. A thin smear of this portion of the sputum should be made either upon the cover-glass or the slide. Several methods of staining are employed, notably the procedures of Gabbet, Ehrlich, the Weigert-Ehrlich, the Ziehl-Neelsen, and the Pappenheim. The principle employed in each instance is the production of a deep, primary red stain with carbol-fuchsin and the subsequent decolorization of all portions of the specimen except the tubercle bacilli, which later are made more conspicuous by the contrast stain of the cellular elements. The carbolfuchsin solution is composed of one part of fuchsin, 100 parts of a 5 per cent. solution of phenol, and 10 parts of absolute alcohol. The decolorization is secured by a dilute solution of sulphuric, nitric, or hydrochloric acid. The smears are allowed to dry in the air upon the cover-glass and later are fixed by being passed quickly three times through the flame of an alcohol lamp or Bunsen burner. The specimen is then ready for staining with the carbol-fuchsin solution. It was formerly the practice to immerse the cover-glass in this solution for twenty-four hours, but by the use of heat this process may be shortened to a few minutes. convenient and rapid way of staining consists of placing a few drops. of the carbol-fuchsin solution upon the cover-glass and holding this for a few minutes over the flame. The fluid should be allowed to simmer or steam, but must not be brought to the boiling-point. At the end of one minute the specimen is washed with water and decolorized by adding a few drops of 25 per cent. solution of sulphuric acid. The decolorization should be repeated once or twice until the film upon the cover-glass when held to the light appears colorless and opaque. The process is made more complete by the addition of a few drops of 98 per

A

cent. alcohol. After washing, the specimen is stained with methyleneblue for about thirty seconds, although no harm results, as a rule, if this is permitted to remain for a considerably longer period. After washing again with water and drying the specimen usually is mounted in Canada balsam.

The retention of the primary stain by the microorganism is of exceeding value in the differentiation of the true from the pseudotubercle bacillus. There remain, however, a considerable number of acid-fast bacilli which closely resemble the tubercle bacillus in other respects. For this reason the appellation "pseudotubercle bacillus' is usually restricted to bacteria which simulate the appearance of tubercle bacilli and do not yield to acid decolorization. Among the varieties which do not surrender their primary stain upon the employment of acids the smegma bacillus is the most important, as the presence of this microorganism may lead to unfortunate errors of diagnosis involving to a vast degree the well-being of the patient. In cases susceptible of reasonable doubt a staining in accordance with the ordinary methods must lead to inevitable confusion in the interpretation of results. It is found that the smegma bacillus holds its color after a prolonged exposure to absolute alcohol, but yields its stain upon boiling in alcohol. Aronson has shown that the tubercle bacillus contains wax and fatty acids, on account of which combination the microorganism is acid fast. The smegma bacillus, on the other hand, has fat and sebaceous matter in place of wax. This bacillus may be robbed of its fat by boiling in alcohol, which has slight effect upon the wax contained in the tubercle bacillus. Bienstock and Gottstein were able to treat other bacilli with fats, as butter, lanolin, paraffin, and wax, and make them acid fast. By the addition of these substances to several culture-media they were able also to give a degree of acid-fastness to bacilli which otherwise yielded to acid decolorization. Various procedures are employed for the differential staining of the true and the smegma bacillus, all based upon the application of the influence of alcohol. The use of inorganic acids is found of no value. Pappenheim has devised a method which produces a ready decolorization of the smegma bacillus by fluorescein-alcohol. After the production of the primary stain the preparations are immersed several times in Pappenheim's solution, which consists of one part of corallin in 100 parts of absolute alcohol, to which methylene-blue is added to saturation and diluted with 20 parts of glycerin (Simon). As a practical matter, error in the differentiation of the bacilli may take place principally in connection with genito-urinary tuberculosis. It is now the custom of clinicians and surgeons to insist upon catheterized specimens of urine before submission to bacteriologists for examination. This plan obviates to a degree the danger of confusing the tubercle bacillus with the smegma microorganism.

The lepra bacillus is of but slight interest to practitioners in this country. It presents, however, several points of resemblance to the tubercle bacillus, particularly in form and staining reaction. Cornet has referred to the disposition of the lepra bacilli, as pointed out by Bâbes, this feature distinguishing them from the tubercle bacillus. The former are found by preference within the cell and grouped very closely together. The latter are usually without the cells, but if intracellular, do not arrange themselves in such dense masses as to obscure the nucleus. He also calls attention to the comparative rapidity with which the lepra

bacillus takes the primary stain and its ability to do this even without the exhibition of heat, a quality not possessed by the tubercle bacillus. According to James, there are now thirty microorganisms more or less resembling the tubercle bacillus in form, and almost equally resistant. to acids. A close analogy has been suggested between these varieties and the tubercle bacillus, the essential difference pertaining to evolutionary changes of adaptability in accordance with a varying environment. These microorganisms are not found, as a rule, within the body, though occasionally they do occur in the sputum, feces, or secretions from nose and pharynx and within the tonsillar crypts. They are distributed, to some extent, in the vegetable kingdom and in the excrement and secretions of animals accustomed to herbivorous diet. The most important of these microorganisms is the timothy-grass bacillus. Others have been found in milk, butter, manure, and earth subjected to fertilization. The grass bacillus, unlike the other varieties mentioned, will grow at the same temperature as the tubercle bacillus, and is thought to be equally resistant to external influences. Its discoverer, Moeller, claims to have been able to produce the same lesions with this agent as with the tubercle bacillus, but finds cultural differences and a much diminished virulence.

The cultural characteristics of true tubercle bacilli consist of their very slow growth and the necessary maintenance of a high temperature, approximating that of the blood. Cultures may be obtained from tuberculous tissue and infected sputum. The usual method is to inoculate a small portion of the sputum or infective material into a healthy guinea-pig. After about three weeks the animal is killed, and, under strict aseptic precautions, the tubercles are dissected from the tissues and transplanted into culture-media. Colonies may also be produced by the direct implantation of tuberculous material into the various culture-media; this method, however, being quite difficult. This is done by thorough rubbing of the surface of the media with an infected platinum loop. The medium may be solid or liquid, and consist of blood-serum, bouillon, glycerin-agar, or glycerin bouillon. After a period of two or three weeks in the incubator at a temperature of 37° C'., there is found upon the surface of the culture-medium a growth resembling an irregular cauliflower excrescence, yellowish white in color, and of a peculiarly dry or crumbly appearance. When cultivated in bouillon, a small scale from a solid culture-medium is transplanted to the liquid in such a manner that it floats upon the surface. By means of Hesse's Nährboden spread in Petri dishes the time may be shortened from ten days, which was required by the older methods, to four or five days, the fresh sputum being deposited upon the medium by drawing a portion of the slimy pus over its surface. Successive transplantation to new media results in more profuse vegetation and a more luxuriant flora. Some observers attribute a diminished virulence to cultures of rapid and luxuriant growth, and an increased pathogenic power to those difficult of cultivation and of scant growth. This is the opinion of Lartigau, as the result of experimental inoculation. The cultural vitality also of the tubercle bacillus is supposed to diminish with the rapidity. of growth. Temperature greatly in excess of that of the blood is extremely detrimental to its vital resistance. Its growth is destroyed at 42° C., and the bacillus quickly becomes extinct at 70°C. A distinctly inhibitory influence upon its development is ascribed to sunlight, especially if

Tubercle bacilli from specimen of sputum. Bacilli red, other organisms and tissues blue. The so-called attenuated variety of tubercle bacilli. Note the elongated rods, some of which are notched and beaded. Note also faintness and irregularity of stain.