sary, a small amount of normal salt solution (0.6 per cent.) may be added to dilute the material and facilitate the examination.

Staining Methods for Cover-glass Preparations.— Simple staining is used for the demonstration of bacteria in general, and also useful in gaining an idea of the character of the cellular elements in the preparation.

Löffler's alkaline methylene-blue solution is perhaps the best staining fluid to use for simple staining, for it does not stain so diffusely and intensely as do the other commonly used dyes, such as fuchsin and gentian violet, which may also be employed.

The cover-glass, covered with the staining fluid, should be warmed over the Bunsen flame, so that the fluid steams, for about fifteen seconds. Boiling should be avoided. The preparation is then washed in water for two or three seconds. and mounted.

Gram's Method of Staining.-I. Cover the preparation with aniline-gentian-violet solution for thirty seconds. 2. Wash in water for two or three seconds.

3. Cover the preparation with Gram's solution of iodin for thirty seconds.

4. Wash with 95 per cent. alcohol until the color ceases to come out of the preparation.

5. Wash in water for two or three seconds and mount.

Certain bacteria are stained by this method, while others are not. Bacteria when stained by it appear dark blue or black, while the nuclei of the cells are rather faintly stained. or not stained at all. The method is especially useful in the demonstration of bacteria which are stained by it when they are present in small numbers or when a few Gramstaining bacteria are mixed among numbers of bacteria. which do not stain by this method. It also has some value as a means of differentiating between bacteria which may be very much alike in size and shape.

In the following table the behavior of the more important pathogenic bacteria toward the method of Gram is indicated:

STAINED BY GRAM'S METHOD.

Staphylococcus pyogenes aureus.
Staphylococcus pyogenes albus.
Streptococcus pyogenes.

Pneumococcus.

Micrococcus tetragenus.

Bacillus diphtheria.

Bacillus tuberculosis.

Bacillus of anthrax.

Bacillus of tetanus.

Bacillus aerogenes capsulatus.

DECOLORIZED BY GRAM'S METHOD.
Gonococcus.

Diplococcus intra-cellularis meningi-
tidis.

Typhoid bacillus.

Bacillus coli communis.

Spirillum of Asiatic cholera.

Bacillus pyocyaneus.

Bacillus of influenza.

Bacillus of glanders.

Bacillus proteus.

Bacillus mucosus capsulatus.

Bacillus of malignant edema.

Stain for the Bacillus Tuberculosis (Gabbet's Method). -1. Stain in carbol-fuchsin solution for one minute, steaming the preparation, and adding more staining fluid as it evaporates. The surface of the cover-glass is to be kept completely covered with the staining fluid during the heating to avoid precipitation.

2. Wash in water for two or three seconds.

3. Cover with Gabbet's methylene-blue solution for about thirty seconds. The uncharged surface of the cover-glass should be cleansed at the same time from any dried stain by running over it a few drops of the same solution.

4. Wash in water thoroughly and mount.

In this way the bacillus tuberculosis is stained red, while other bacteria and the nuclei of cells are stained blue.

This method, as well as many others, depends upon the fact that the bacillus tuberculosis, when once thoroughly stained with an aniline dye, does not give up its stain in the presence of acids, as nearly all other bacteria do. The bacillus tuberculosis may therefore be identified even among a mixture of other bacteria by this property, taken in connection with its morphology, in most of the routine work of the pathological laboratory. Practically, the only other bacilli with which it may be confounded are the bacillus of leprosy and the smegma bacillus, both of which, when stained, resist the decolorizing action of acid. It may be differentiated from the smegma bacillus by the fact that it is not decolorized by alcohol (95 per cent.) after the usual treatment with

acid, while the smegma bacillus is decolorized under these circumstances. The preparation may be washed in alcohol for about thirty seconds.

As a rule, the differential test with alcohol need only be applied in the examination of urine and the material derived from about the external genitalia, especially in the case of females.

The differentiation from the bacillus of leprosy by certain quantitative differences in staining reactions has been attempted, but it is very unsatisfactory, and it is doubtful if there is as yet any reliable method of distinguishing between these two organisms, considered by themselves. The great rarity of leprosy in this country, however, renders confusion with the organism found in that disease very improbable.

Welch's Method of Staining the Capsule of the Pneumococcus.-This method depends upon the fact that acetic acid precipitates the mucin-like substance of which the capsule is composed, and that the precipitated material of the capsule is not soluble in a 2 per cent. solution of sodium chlorid. Therefore, any necessary washing of the preparation is to be done in the solution of sodium chlorid, and the mount is to be made in the same solution or in balsam.

The method is as follows:

1. Cover the preparation with glacial acetic acid for a few seconds.

2. Drain off and replace (without washing in water) with aniline-gentian-violet solution. The staining solution is to be repeatedly added to the surface of the cover-glass until all of the acid is replaced.

3. Wash in 2 per cent. solution of sodium chlorid and mount in the same.

Curry's Method of Staining the Capsule of the Pneumococcus..-I. Cover the preparation with glacial acetic acid for a few seconds.

2. Wash off the acetic acid with a 1 per cent. solution of potassium hydroxid.

3. Stain with aniline-gentian-violet for one minute, without previously washing off the hydroxid.

4. Wash quickly in water.

5. Dry thoroughly and mount.

If the specimen is stained too deeply, the capsule may be slightly decolorized by washing for a few seconds in a 0.5 per cent. solution of acetic acid and then drying thoroughly. It is important that the specimen be quite dry when mounted, as the xylol tends to decolorize the capsules when moisture is present.

This method has been in use in the Pathological Laboratory of the Boston City Hospital, where it was originated, and has been found to work very satisfactorily.

The capsule may also be demonstrated by Gram's method (see page 90) if the decolorization with alcohol be not pushed too far. In this case the capsule will be outlined around the deeply stained organism as a broad clear zone bounded by a dark line.

Examination by Cultures.-The demonstration of the presence of bacteria in a tissue or exudate by means of cultures consists in bringing a small amount of the material to be examined in contact with some solid nutrient substance in which the bacteria will thrive. On this the bacteria by multiplication form masses or colonies visible to the naked eye, and present appearances which enable a practised eye in many cases to recognize the species of the bacteria of which they are composed. Of the solid culture-media described in the preceding section, the coagulated blood-serum is distinctly the best to use for the demonstration of the presence of bacteria in routine pathological work, because certain of the most important pathogenic bacteria grow better upon it than upon agar-agar or similar media. The other media have important uses in the study of the bacteria after their isolation from the tissues, as is pointed out elsewhere. The blood-serum medium here described has been found entirely suitable for the isolation of the bacillus tuberculosis from tubercular lesions, which proves its efficiency as a culture-medium.

Method of Preparing Cultures on Blood-serum.-The preparation of cultures on the coagulated blood-serum consists in distributing over the surface of the medium in a test

tube as much of the tissue or other material as will adhere to the end of a piece of stiff platinum wire hammered flat at the end. The wire is fixed in the end of a glass or metal rod, and should be about 8 cm. long. It should have a rounded spatula-like extremity, and should be thick enough not to bend easily.

It is, of course, essential that the material brought in contact with the culture-medium should be free from bacteria not originally present in it, or that it be not contaminated with bacteria from outside sources. Therefore, in taking material from the interior of organs and tissues the surface is first sterilized by searing it with a hot knife, such as an ordinary case-knife, which has been heated in the Bunsen flame, and then, through a small incision made with another hot knife in this seared or sterilized area, the material from the interior is collected on the end of the platinum wire, which has also been previously heated in the Bunsen flame to sterilize it, and then cooled either by plunging it in the water of condensation of the culture-tube for a few seconds or by moving it about in the interior of the tissue.

In the case of exudations on free surfaces, however, this searing is impossible, and therefore care should be exercised at the autopsy not to contaminate any such exudate by handling before the material for culture has been obtained with the platinum wire. The material thus secured is then transferred by means of the platinum wire to the surface of a blood-serum culture-tube, and the infected wire gently rubbed over all of the surface of the culture-medium, avoiding, however, the breaking of the surface. It is important that the material be well distributed over the nutrient surface. If the material is suspected of containing a large number of bacteria, as in the case of suppurations or acute inflammatory lesions, a second tube should be inoculated from the first one by touching the platinum wire, previously sterilized and cooled, to the infected surface of the first tube, and then gently rubbing the infected wire over the surface of the second tube. This operation is called "diluting." The object of this is to obtain, after the development of the cul