ture-medium to fill them to a depth of about 2 cm., but not much more than this, for with a greater quantity the preparation of a thin layer is difficult.

In carrying out this method it is best to first make a depression in a block of ice with a test-tube filled with hot water. In this depression the culture-tube containing the fluid is placed in a nearly horizontal position, and rotated rapidly with the fingers until the medium is properly distributed and solid, care being exercised not to permit the medium to come in contact with the cotton stopper. The even distribution of the medium over the inner surface of the tube is best obtained by manipulating the tube while the medium is still fluid, in such a way as to moisten its inner surface up to about 1 cm. of the cotton stopper before proceeding to rotate it on the ice.

Gelatin is the best culture-medium to use in this method. Agaragar can be used, but has the great disadvantage of readily slipping down toward the bottom of the tube if the tube is placed upright. It is necessary, therefore, to keep the tubes on their sides. The method has little or no advantage over the method of Petri.

The Determination of the Motility of Bacteria.— This is done by observing the individual organisms, un

FIG. 24.-The "hanging drop" seen from above and in profile.

stained, in a drop of bouillon or similar fluid under the oilimmersion lens. For this purpose a so-called “hanging drop" is prepared, for which a special form of slide known as a "hollow slide" is necessary. The hollow slide is a slide having a shallow circular concavity, about 1 cm. in diameter, ground out in its center (Fig. 24).

In preparing a hanging drop the procedure is as follows: A small drop of a bouillon culture or of the water of condensation of a blood-serum or agar-agar slant is placed in the center of a cover-glass by means of the platinum wire.

The cover-glass is then placed, drop downward, over the circular depression in the hollow slide. To hold the coverglass in its place and to prevent evaporation of the fluid in which the organisms are suspended, a little vaselin is painted around the margin of the depression before placing the cover-glass in position. The hanging drop thus prepared is then examined by focusing upon it with the oil-immersion lens, a small aperture of the iris diaphragm of the condenser being used to render the bacteria visible by refraction. To facilitate focusing, the edge of the drop should be brought into the center of the field of the low-power objective, and then the oil-immersion put in place and focused upon it, the edge of the drop being more readily seen as a sharp line, owing to refraction, than the organisms. Great care is necessary to avoid breaking the cover-glass in the effort to bring the bacteria into view. Hanging drops may also be prepared from suspensions of bacteria grown on solid media, by mixing a portion of the growth with a small quantity of bouillon.

In the study of spore-formation the hanging drop is of great utility. Here the slide and cover-glass must be carefully sterilized before using, the cavity between the cover-glass and the slide well sealed with vaselin, and other precautions taken to prevent contamination of the drop with other bacteria. The preparations may be placed in the incubator or on a "warm stage and the process of spore-formation followed.

3. THE INOCULATION OF ANIMALS.

The animals ordinarily used in the laboratory are guineapigs, rabbits, and mice. The instruments, etc. used in the inoculation of animals should be sterilized beforehand, but strict surgical asepsis is not necessary as a rule.

Guinea-pigs are in most instances inoculated either subcutaneously or into the peritoneum.

Subcutaneous inoculation is effected either by injection with a hypodermic syringe (see page 71) or by the introduction of the material to be inoculated through a small incision in the skin. The best point for subcutaneous inoculation is the tissue of the anterior abdominal wall.

In inoculating, the animal is to be held abdomen uppermost by an assistant, who grasps the neck and fore quarters with one hand and the hind quarters with the other. If the skin is to be incised, the hair about the point of inoculation is to be cut short with a pair of scissors and the skin cleansed with soap and water. An incision is then to be made about 8 or 10 mm. long through the skin, including the subcutaneous tissue, and the superficial tissues separated from the muscle for a distance of 10 or 15 mm. toward one side of the wound by inserting the points of scissors or other instrument, so as to form a "pocket" beneath the skin. In this "pocket" the material for inoculation is introduced, either on the platinum wire (see page 72) or by means of small forceps.

If pieces of tissue are used, it may be well in some cases to close the wound by one or two sutures in order to prevent the extrusion of the material after the release of the animal.

Intraperitoneal inoculation may be performed essentially as above indicated. If the inoculation be by incision, the opening into the peritoneal cavity should be as small as possible, and the wound should be firmly closed with silk sutures in order to prevent extrusion of the intestines.

In inoculating with the hypodermic syringe the needle should not be pushed in too far or the intestines may be wounded. The needle is best introduced a little to one side of, or slightly below, the umbilicus.

Rabbits. These animals may be inoculated both subcutaneously and intraperitoneally, essentially as described for guinea-pigs.

In lifting or in carrying rabbits from one place to another the animals are to be grasped by the ears. During the operation of inoculating, the assistant grasps the ears with one hand and the hind legs with the other, while the body of the animal rests upon the table, abdomen uppermost. Rabbits held for a few seconds in this position usually become perfectly quiet, and often do not show any evidence of pain during the operation.

Intravenous inoculation is usually done on rabbits, because of the ease with which the needle of a hypodermic syringe may be introduced into the long and prominent marginal vein of the ear. In inoculating in this manner the tip of the ear is held by the thumb and fingers of the left hand, while the right manipulates the syringe, the needle of which is pushed through the skin of the external surface of the ear into the vein which runs along the outer margin of the ear (Fig. 25).

FIG. 25.-Method of making an intravenous injection into a rabbit. Observe that the needle enters the posterior vein from the hairy surface (McFarland).

By the exercise of care and gentleness the animal may be thus inoculated without being held by an assistant, especially if the fur between the ears be stroked for a short time just before the introduction of the needle. In some cases it may be necessary to anesthetize the animal on account of violent struggling. (See below.)

Injection of bacteria into the mesenteric veins by means of the hypodermic syringe, after laparotomy, may be performed both on rabbits and on guinea-pigs. This is to be done under anesthesia. Ether is very satisfactory for this purpose. Guinea-pigs bear it well, but it is to be used with caution on rabbits. With the latter animals death is liable to occur if the ether is "pushed" after

complete anesthesia is established. Rabbits once thoroughly anesthetized seem to remain so for a considerable time without additional ether being necessary. The incision for this form of inoculation should be in the lower half of the abdominal wall in the median line, for in this region the coils of the small intestine are most numerous. The length of the incision should be about 2 cm. Several loops of intestine are brought out through the wound, and a mesenteric vein of the proper size to admit the needle of the syringe (see page 71) sought for. When found the needle is to be introduced and held firmly in position while an assistant carefully presses inward the piston of the syringe. After the injection of the material the needle is withdrawn, the punctured vein picked up with the artery-forceps, and the vessel tied on both sides of the puncture with silk thread. The loops of the intestine are then replaced and the wound closed in two layers, one consisting of the muscles and peritoneum, the other of the skin. The so-called "button-hole stitch" with silk thread is very well fitted for the closing of the wound.

Little or no aseptic precautions are necessary to obtain primary union in the wound. Before the operation, however, the hair of the region should be cut off close and the skin cleansed with soap and water.

This form of inoculation may be useful in studying the local effects of bacteria upon liver-tissue, for large numbers of them will be lodged in the capillaries of the liver, and microscopical sections of any part of the organ will contain them, so that any local lesion produced by them may be subjected to observation after variable intervals of time.

Mice are usually inoculated subcutaneously at the root of the tail. The animal, manipulated by means of chemists' crucible tongs or a similar instrument grasping his tail, is to be persuaded to crawl into a cylinder of wire gauze, about 8 to 10 cm. long and about 3 cm. in diameter, which is fixed. on a small board. The cylinder is open at both ends, and when the mouse has crawled into it—a thing which he will readily do the end near his tail is bent inward so as to prevent him from backing out of it, while an ordinary small screw-clamp is adjusted firmly to his tail to prevent his escaping through the other end. The animal is thus secured and ready for the operation of inoculation. A more complete form of this apparatus, with a fixed clamp for the animal's tail, is shown in Fig. 26.

In making the inoculation the mouse is pulled backward by the tail until his rump is exposed in the end of the cylin