with the staining fluid, which is poured upon it from a dropping-bottle.' It may then be heated over the flame of the Bunsen burner, washed in water, and submitted to any further manipulation which may be necessary while still in the grasp of the forceps. When the staining is completed the preparation is next to be prepared for microscopic examination. This is done by placing the cover-glass, with as much water as will adhere to it, charged side downward, on a "slide," and then removing all remaining water, except a thin film of water between the slide and cover-glass, by gentle pressure with several thicknesses of filter-paper. The preparation is then ready for examination with an oil-immersion lens. The presence of this film of water is very essential for a satisfactory examination with the microscope, and its evaporation may be compensated for by a drop of water placed at the edge of the cover-glass. The preparation may also be mounted in balsam after carefully drying it first between filter-paper and then holding it in the fingers over the Bunsen flame, but the examination in water mount is better, because the apparent size of the bacteria is greater in this than when mounted in balsam.

It is of the greatest importance that cover-glass preparations be made from all exudates or acute inflammatory conditions of organs or tissues, so that the results of cultures can be controlled and some idea formed of the number and character of the bacteria present. They are of especial use as enabling one sometimes to recognize the presence of an organism which does not grow in the culture for some reason, and whose presence might escape notice, while their importance in many cases in the identification of the pneumococcus and the bacillus tuberculosis must be apparent. In certain cases it is best to examine the material in its natural state and without drying and staining. This is especially true of suspected infections with actinomyces and the amebæ coli. In these cases the material should be spread over a cover-glass, and this placed, while the material is still

1 The form of drop-bottle known by dealers in bacteriological supplies as the "T. K. patent," with flat stopper, is the best.

moist, charged surface downward, on a "slide," and then examined with various powers of the microscope. If necessary, 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.

Pappenheim's pyronin and methyl-green mixture may be used as above described in place of Löffler's methyleneblue solution. This mixture is composed of:

Saturated aqueous solution of methyl-green, 3-4 parts;

Bacteria are stained brilliant red and the nuclei of cells are stained blue or purple. The mixture is said to keep several weeks.

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 1 The formula for staining fluids will be found in Part III. (see Index).

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:

Special staining methods for cover-glass preparations will be found in the section on Special Bacteriology.

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, and in certain instances special media are to be used, as will be pointed out in the following pages. 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 testtube 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. In making cultures from clinical material, the platinum loop may be used for fluids.

The "platinum wire" or "loop" consists of a piece of platinum wire of about 22 gauge, 21⁄2 to 3 inches long, fixed in the end of a small glass or metal rod 8 or 10 inches long. It is often of great convenience to have two of these instruments, one with the wire curled into a simple loop about 1 to 2 mm. in diameter at the free end, and the other a straight wire with the free extremity hammered flat into a very small spatula. The latter is of great utility in picking up minute portions of bacterial colonies.

Both this instrument and the stiffer wire, above mentioned, should be heated to a red heat in a flame immediately before using, in order to destroy any bacteria that may be upon them.

If the material is on a "swab," the surface of the bloodserum or other media may be conveniently inoculated directly by gently rubbing the swab over it. In this case it is usually best to make a dilution or two by means of the platinum wire, as described below, especially if there be a large amount of material on the swab or if the cover-glass examination

has shown that a large number of bacteria are present. In any case it is important that the infected material be spread over all of the surface of the medium, and not in the form of one or two narrow streaks.

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 culture, a sufficiently small number of colonies in the second. tube, so that they may be discrete-i. e. separated from one