the neck of the tube should be heated in the Bunsen flame until the cotton begins to brown before inserting the cork, which should also be charred in the Bunsen flame before insertion. The tubes may also be sealed with wax or paraffin or covered with small rubber caps.

Cultures may also be made from other organs containing tubercular lesions, but the lymphatic glands seem to offer the best examples of large tubercular foci, and therefore are preferred for the purpose in view.

The tubercular lesions in human tissues are not ordinarily favorable for the isolation of the bacillus, on account of the frequent presence of other bacteria in them and because of the small number of tubercle bacilli usually present in tissue otherwise suitable.

We have found the firmly coagulated, opaque blood-serum medium above mentioned to be quite adequate for the cultivation of the bacillus from its lesions, both in man and guinea-pigs.

A special culture-medium for the cultivation of the bacillus tuberculosis is that recommended by Lubinski. Its composition and mode of preparation are as follows-to make one liter of medium :

Take i kilo of potato in small pieces, and, after thoroughly washing, cook in the steam sterilizer for three or four hours with 1500 c.c. of water. Of the mass thus formed take 1000 c.c., and add to it 500 grams of finely chopped beef, and allow it to stand in the cold for twenty-four hours, then boil the mixture for a few minutes and filter. Add to the filtrate 1 to 1.5 per cent. agar-agar, and boil until the agar-agar is thoroughly dissolved. This will require say half an hour's boiling. Next add 1 per cent. pepton and 0.5 per cent. sodium chlorid. When these are thoroughly in solution, and after about five minutes' continued boiling, the mixture is to be neutralized with sodium hydrate solution, boiling meanwhile. When brought to the proper reaction it is to be filtered, and to the filtrate add 4 per cent. glycerin. The medium is then to be run into test-tubes and sterilized as in the case of agar-agar. The loss by evaporation in boiling is to be made up with water.

A bouillon may be also made by leaving out the agar-agar.

Another special culture-medium is that recommended by Capaldi. This consists in mixing with fluid agar-agar in tubes, at a temperature of 45° C., three or four large loopsful of the yelk of a fresh egg to each tube. When the portion of the yelk taken is thoroughly mixed with the fluid agar, the tube is placed on its

side, and its contents allowed to become solid by cooling so as to form a "slant." In securing the yelk aseptic precautions must be used. The egg must be quite fresh. By suitable manipulations the yelk is to be freed from the "white" and placed in a small, clean dish. Then the yelk-membrane is to be seared with a hot knife and an opening made in it. Through this opening the yelk is obtained with the loop. Before using the culture-tubes so prepared, they should be tested as to their sterility by placing in the incubator for twenty-four hours.

Occurrence. In tubercular lesions generally and in the sputum of pulmonary phthisis, in the urine in many cases of genito-urinary tuberculosis, and in the feces in intestinal tuberculosis. The tuberculosis of cattle is generally considered to be due to this organism, while the tuberculosis of birds is probably due to a different variety.

Does not multiply outside of the body except in cultures. May occur on the surface of objects contaminated with the excreta of tuberculous individuals or in the dust of places inhabited by such individuals.

1

Spirillum of Asiatic Cholera (Comma Bacillus) 1 (see also Clinical Bacteriology).-Morphology (Figs. 50, 51). -In fresh cultures the organism appears usually as a slightly curved rod somewhat shorter than the tubercle bacillus, but much thicker. The curving of the rod varies, being very marked in some individuals and absent in others. Sometimes two rods are joined end to end with their convexity pointing in opposite directions, or moderately long, undulating threads may be found. It seems probable that the curved rods represent the segments of a spirillum, and hence the name of the organism.

In cultures some days old degenerated and atypical forms are found (involution forms). The organism is motile, and a single flagellum is attached to the end of the rod.

It is not stained by Gram's method.

Colonies on Gelatin Plates (Fig. 52).-After twenty-four to forty-eight hours at a temperature of 20° to 22° C. the largest colonies will appear as masses of indefinite granular material lying in circular areas of liquefied gelatin in which

Koch: Deutsche med. Wochenschr., 1884 and 1885.

granular shreds are scattered. Within the next twenty-four hours the areas of liquefaction increase, and the colonies ap

FIG. 50.-Spirillum of Asiatic cholera, from a bouillon culture three weeks old, showing long and degenerate forms; X 1000 (Fränkel and Pfeiffer).

pear under the low power as a dense granular mass surrounded by an area of liquefaction through which can be seen

granular prolongations of the colony, usually extending irregularly between the periphery and the central mass" (Ab

bott), while the margin of the liquefied area is marked by delicate radiating filaments closely packed together.

The colonies on agar-agar plates are not characteristic. Growth is rapid.

Gelatin Stab.-Growth all along the line of inoculation. with liquefaction at the surface in funnel form after fortyeight hours. The liquefaction proceeds in such a manner that the liquefied area has a smaller diameter at the surface than immediately beneath, and, owing to the fact that the liquefied gelatin does not fill the cavity, a space is left between the surface of the medium and the surface of the liquefied gelatin so that the appearance of an air-bubble is pro

FIG. 52.-Developmental stages of colonies of the spirillum of Asiatic cholera at 20° to 22° C. on gelatin; X about 75 diameters (Abbott): a, after sixteen to eighteen hours; b, after twenty-four to twenty-six hours; c, after thirty-eight to forty hours; d, after forty-eight to fifty hours; e, after sixty-four to seventy hours.

duced. Along the deeper portions of the line of inoculation the liquefaction is slow.

Bouillon.-Diffusely clouded. A thin pellicle forms on the surface after a time.

Litmus-milk.-Turned red and coagulated.

Indol-production.-In cultures in Dunham's pepton solution or in the pepton solution of Koch (2 per cent. pepton and I per cent. sodium chlorid) a rose-color is produced by the addition of sulphuric acid alone. (Concentrated c. p. acid is to be employed, as in the test for indol-production by the bacillus coli communis.) The production of the rose-color without the addition of the sodium nitrite shows that nitrites as well as indol are formed by the growth of the organism

in the pepton solution. The reaction can be obtained in cultures which have been but eight hours in the incubator.

Potato.-Thin, dry, grayish-white growth which does not spread over the surface.

Pathogenesis. The pathogenic effects of the cholera spirillum are best shown by the inoculation of guinea-pigs. There are two methods of inoculation, as follows:

1. The Method of Pfeiffer.-Scrape from the surface of a fresh agar-agar culture as much of the growth as will adhere to a platinum wire bent into the form of a small loop. Suspend this amount of material in 1 c.c. of bouillon, and inject the suspension into the peritoneal cavity of a guinea-pig by means of a hypodermic syringe. With virulent cultures, this inoculation soon produces a fall in the temperature of the animal, which continues and becomes more marked, death occurring in from twelve to twenty-four hours. At the autopsy of the animal a clear fluid will be found in the peritoneal cavity and in the thorax.

2. The Method of Koch.-This depends upon the fact that the animal may be infected through the alimentary canal, provided the acidity of the gastric juice be neutralized, this acidity being destructive to the cholera spirillum.

A soft catheter is passed into the stomach of the animal through the mouth, and through this 5 c.c. of a 5 per cent. solution of sodium carbonate is injected. After ten or fifteen minutes 10 c.c. of a bouillon culture of the organism are injected through the catheter, and immediately afterward the animal receives subcutaneously I c.c. of the tincture of opium for every 200 grams of its body-weight. The object of this opium administration is to stop peristalsis, so that the organisms may be longer in contact with a given area of the mucous membrane of the intestine. The result of the inoculation first appears after about twenty-four hours. The animal then has no appetite and is listless. Later, paralysis of the hinder extremities appears, respiration is prolonged and weak, the heart-beats become feeble, and the body-temperature may become subnormal. Death usually occurs after the animal has been a few hours in this condition.