numbers and frequently in the pus-cells. These bacilli frequently occur in pairs, and resemble pairs of cocci. Their ends may be more deeply stained than the central portions. For the staining of cover-glass preparations of the sputum Pfeiffer recommends that a very dilute carbol-fuchsin solution be applied for five to ten minutes. The cover-glass preparation is to be made from a distinctly purulent portion of the sputum. Staining with Löffler's methylene-blue solution. also gives good results.

The bacillus of influenza may be cultivated from the sputum by breaking up a small portion of a distinctly purulent character in 1 or 2 c.c. of bouillon, and then spreading a platinum loopful of the suspension over the surface of a blood-agar-agar slant, which is then placed in the incubator. After eighteen to twenty-four hours the characteristic colonies may be visible with the aid of a hand-lens. These should not grow in ordinary media unless blood or hemoglobin be present, and should have the morphology of the bacillus of influenza.

Examination of Sputum for Tubercle Bacilli.— The morning sputum should be taken for examination. Select one of the dense, grayish-white particles, and with the aid of small pointed forceps or the platinum wire rub it over the surface of a cover-glass, breaking it up as much as possible. The material should be spread in a very thin layer. The preparation is next to be "fixed" in the ordinary way described for cover-glass preparations (see p. 89), and is then to be treated as follows:

1. Stain in carbol-fuchsin solution, steaming for one minute over the Bunsen flame, with the staining solution thoroughly covering all the surface of the cover-glass. None of the surface of the cover-glass should be allowed to become dry by evaporation, as this causes a precipitate to form, but more of the staining fluid should be added. from time to time to keep it completely covered as evaporation occurs.

2. Wash in water.

3. Cover with Gabbet's solution for twenty seconds. The

solution should also be applied to the uncharged slide of the cover-glass to remove any dried stain which may have collected thereon.

4. Wash thoroughly in water.

5. Mount in water or balsam. Water is to be preferred, for the reason that the apparent size of the bacilli is larger when examined in water.

FIG. 74.-Tubercle bacilli in sputum (carbol-fuchsin and methylene-blue).

Another method is as follows:

1. Stain in carbol-fuchsin solution or aniline-fuchsin solution in the manner above indicated.

2. Wash in water.

3. Wash in 95 per cent. alcohol for a few seconds.

4. Decolorize in a solution composed of

Water,
Alcohol,

Concentrated sulphuric acid,

until the preparation has a faint pink color.

5. Wash thoroughly in water.

6. Stain for thirty seconds with Löffler's methylene-blue solution without heating.

7. Wash in water and mount.

By both of these methods the tubercle bacilli are stained red, while other bacteria and the nuclei of cells are stained blue (Fig. 74). There are other methods for staining tubercle bacilli, but these fulfil all the requirements of practical work.

Tubercle bacilli, when present in sputum in very small numbers, may sometimes be demonstrated by methods of sedimentation. A good means of sedimentation consists in heating the sputum in a test-tube either with boiling water or in the steam sterilizer for fifteen minutes. The heat coagulates the cells and albuminous constituents, which sink to the bottom, carrying with them the bacilli. The supernatant liquid may then be poured off and the sediment examined as above.

Another method of treating the sputum when only a small number of bacilli may be present is as follows: Place 10-15 c.c. of the sputum in a wide-mouthed bottle of 100 c.c. capacity. Add 10 c.c. of water and 6 c.c. of liquefied carbolic-acid crystals. Close the flask with a rubber cork and shake for one minute. After shaking, fill the bottle with water and shake again. Then pour the contents of the bottle into a sedimenting glass, let stand for twelve to twenty-four hours, and examine the sediment. The cover-glass preparations of the sediment before staining are to be washed in ether or chloroform, and then in alcohol, or they may be washed in a mixture of alcohol and ether, equal parts.

Tubercle Bacilli in Urine.-The sediment of the urine should be examined. This may be rapidly thrown down by the centrifuge.

In urine smegma bacilli may be mistaken for tubercle. bacilli, especially in the urine of females. The smegma bacillus resembles the tubercle bacillus very closely in form, and, like the latter, it retains its stain in the presence of acids. It differs from the tubercle bacillus, however, in that it is decolorized by alcohol. Therefore in staining for tubercle bacilli in the urine alcohol should be used in decolorizing. The procedure is as follows:

1. Make a cover-glass preparation from the sediment.

2. Stain with carbol-fuchsin solution for one minute, as described in the case of sputum.

3. Wash in water.

4. Decolorize in 20 per cent. sulphuric acid until the preparation has a pink color.

5. Wash thoroughly in water.

6. Wash in alcohol (95 per cent.) thirty seconds.

7. Wash in water.

8. Stain with Löffler's methylene-blue solution twenty seconds, without heat.

9. Wash in water and mount.

Surgical Tuberculosis.-The demonstration of the tuberculous nature of material removed at operations may be made by the histological examination, by the demonstration of the tubercle bacilli on cover-glass preparations, as in sputum, and by the results of the inoculation of guinea-pigs with the material. Cultures are not ordinarily practicable.

The examination of cover-glass preparations is commonly of little value, owing to the small number of bacilli usually present.

The histological examination may be often made very satisfactorily by frozen sections of the tissues. If possible, however, regularly hardened and imbedded tissues, cut in fairly thin sections and stained with hematoxylin and eosin, are preferable.

The inoculation of guinea-pigs is to be made subcutaneously in the abdominal wall, either with a hypodermic syringe if the material is fluid, or, if it be in the form of tissue, by inserting a small piece beneath the skin. Material obtained on a swab may also be used for inoculation by introducing the infected swab beneath the skin and moving it back and forth a few times. If tubercle bacilli are present in the material, the animal will show enlargement of the inguinal lymphatic glands in about three weeks and will usually die of miliary tuberculosis in the course of six to ten weeks. If necessary, the glands in the inguinal region may be examined. histologically after three weeks for the presence of tubercular lesions, or examined by cover-glass preparations for tu

bercle bacilli. The discharges from sinuses, etc. may also be tested for the presence of tubercle bacilli as above indicated, the material being obtained on a "swab."

Cultures from the Blood during Life.-With a sterilized hypodermic syringe secure a cubic centimeter of blood from one of the large veins at the flexure of the elbow. Mix this immediately with one or two tubes of fluid agar-agar which has been kept at hand at a temperature of about 40° C. and form a “slant," or make a Petri plate of the mixture. (For keeping the agar-agar tubes at the desired temperature an ordinary cup or similar vessel filled with water of the proper temperature will suffice.)

When the "slant" or "plate" has become solid it is to be placed in the incubator for development.

It may be mentioned that this mixture of blood and agaragar is a good medium for the growth of the gonococcus, and that this organism has been cultivated from the blood in gonorrheal endocarditis by this means. Before puncturing the vein the skin over it should be thoroughly cleansed with alcohol and ether, but no antiseptics, such as carbolic acid or corrosive sublimate, should be used. The syringe should be thoroughly sterilized by steam for half an hour, and should be brought to the patient in a sterilized test-tube, from which it should be removed only when it is to be used.

Intraperitoneal inoculation of mice may also be made with to I c.c. of the blood. This may give rise to streptococcus or pneumococcus septicemia.

Gonorrhea.-Cover-glass examination of the pus shows cocci in the form of paired hemispheres, mainly inside puscells (Fig. 75). For the identification of these cocci as gonococci their morphology and position inside the leucocytes are not alone sufficient, for essentially the same appearances may be sometimes seen in pus infected with staphylococci or streptococci.

A positive diagnosis of infection with the gonococcus is not justified until it has been determined that the cocci are decolorized by Gram's method, for the ordinary pyogenic cocci are stained by this method.