III. Animal Experiments.

(A) Infection.

1. Subcutaneous Inoculation.-After the skin in some part has been washed with 1: 1000 corrosive sublimate solution, a shallow incision is made with scissors, and inoculating material is introduced beneath the skin by means of a stout platinum wire with a loop. Mice are usually inoculated above the root of the tail, they being simply held by the tip of the tail and allowed to hang into a glass which is covered in great part by a piece of board. Guinea-pigs and rabbits are inoculated on the side of the thorax.

2. Subcutaneous injection is usually carried out with Koch's rubber-ball injection syringe or with Strohschein's syringe. A fold of skin is picked up upon some part of the body, and the needle introduced in the direction of the fold. If several cubic centimeters are to be injected, it may be simply done as follows: Upon a graduated pipet is fastened a short piece of rubber tubing provided with an injection needle, and the whole sterilized. The pipet is sucked full, and the fluid forced out with the mouth or a rubber bulb.

3. Intraperitoneal injection is made by perforating the abdominal wall at a single thrust with a sterile hollow needle; then, cautiously advancing the needle, the fluid is injected.

Regarding infection by feeding, inhalation, etc., consult more extensive works on technic.

(B) Observation.

Mice may be kept in sterile glass vessels provided with cotton and closed with wire gauze. Larger animals must be kept in sterilized cages or stalls.

(C) Autopsy and Disposition of the Body.

Autopsies must be made at once after death; at least, the animal must be placed on ice after death. The animal is placed upon a board on its back and nailed or tied by its four legs. The abdomen and chest are thoroughly moistened with sublimate solution and then the abdominal cavity first opened with a sterile knife. The abdominal walls are separated, and from the spleen, liver, and kidneys, some blood (or tissue juice) is obtained with a sterile platinum loop and smeared at once upon previously prepared agar plates. The organs are carefully cut out, avoiding contact with the intestines, and placed in absolute alcohol for further examination. Then the thorax is opened with scissors, and blood removed from the heart and also the lungs. These organs are also placed in alcohol. Before each operation the instruments must be carefully heated to a glow or thoroughly burned. It is better to have numerous sterilized instruments ready. The hands must be perfectly clean.

In interpreting the findings at the autopsy it is to be remembered that often very soon (sometimes during the death agony) micro-organ

the organs from the intestine. If living bacteria are abdominal cavity or trachea of cadavers, they can cuni in the organs after a time (C. B. XXIII, 418). spy the body is best burned. If this is not practicas wrapped in coverings wet with sublimate and buried er Jeep, and quicklime filled in about it.

APPENDIX V.

so Guide to the Recognition of Bacteria.

(Illustrated with an example.)

case is one of eczematous conjunctivitis in which a of the bacteria occurring in diseased eyes are Purulent or serous material removed from the al sic or edge of the lid with a platinum loop is

Microscopic Examination (Smear upon Slide or Cover-glass).

Stained with fuchsin, we see:

Covi, especially diplococci in heaps, usually disbiscuit-shaped," many times within cells (percocci).

Covi, single or united in irregular clusters (probably AV Pyogenes).

Sort chains, of two or three links, of lance-shaped se with capsules (probably Streptoc. lanceolat.).

is larger or smaller, often very irregular in form, in segments, ends rounded or pointed, often of of cocci (true diphtheria, pseudodiphtheria, or Maciilus).

is regular, rather thick, but small (perhaps coli

gods, often in pairs, quite large, the ends not rounded hough at the time without spores, a bacillus

r).

(b) Gram's stain: All the organisms in the preparation are stained except the biscuit-shaped cocci and the small, plump regular bacteria. The loss of color speaks in favor of the cocci being gonococci, and the rods Bact. coli. (c) Stain for tubercle bacilli with carbol fuchsin: In the differentiation the preparation is completely decolorized with sulphuric acid. After counter-staining with methylene-blue only organisms which are stained blue are seen. Thus, in our case the tubercle bacillus and those resembling it are excluded.

If, as sometimes occurs, no micro-organisms can be seen in the fuchsin preparation, then a preparation is also stained by Gram's method because the stained cocci and bacilli are more readily seen after the mucus and coagulum have been decolorized. In any case if the examination of the slide is negative, the plate method is always employed.

II. Plate Cultures.

In examining an animal body for micro-organisms the nature of which we do not know, we employ "the best nutrient medium": i. e., serum or ascitic fluid-agar, and, as a substitute, glycerin-agar. 1 1

The usual plate method consists in placing the material to be examined in liquefied gelatin or agar in various dilutions and pouring it out into double dishes. This is not especially suitable for the examination of materials which contain relatively few germs. In our case we prefer to pour the nutrient medium into plates, and, after it is solidified, to carefully make several streaks over the surface with a platinum loop which carries the pus or mucus, etc., to be examined. The double dishes are then turned upside down (so the agar will not dry so rapidly) and placed in the incubator.

After forty-eight hours there appear upon the plate:

1. Moist, white, yellow, and orange, roundish, slightly elevated colonies, 2 which, when magnified sixty times, are

1 On the contrary, many varieties from soil, water, etc., grow only upon nutrient media poor in nutrient substances, like the ordinary nutrient media (see p. 200).

2 The colonies here described are always such as lie on the surface of the medium.

finely granular. In stained preparation, magnified a thousand times, they are micrococci (probably Micrococcus pyogenes albus, citreus, and aureus). The examination must be carried further, as indicated on page 163.

If there are only one or two colonies, especially yellow ones, one may often recognize it as a contamination of the plate by germs in the air, most often sarcina. The edges of the sarcina colonies, when magnified sixty times, are coarsely granular or jagged. When magnified one thousand times, packets of micrococci are seen.

2. Most minute, scarcely perceptible colonies, not elevated, half a millimeter in diameter. When magnified sixty times, extremely delicate, transparent, very delicately punctate. The edges practically smooth (recalling gonococcus, Streptococcus lanceolatus, and Streptococcus pyogenes). In the last, one often observes upon ascites-agar that chains of cocci grow out from the edge of the colony in the form of the finest curling threads! When the stained preparation is examined under a magnification of 1000, the examination is to be continued according to page 163, if cocci; page 134, if streptococci; page 195, if bacilli.

In order to render still more secure the diagnosis founded upon morphologic and biologic peculiarities, several such small colonies are taken up with a platinum loop and introduced beneath the skin of a mouse, or this may be done by employing more abundant infecting material (bouillon culture). If we are dealing with a Streptococcus lanceolatus, we find in the blood and organs characteristic forms of this variety with capsules. Smears from the blood and organs are to be examined for the characteristic organisms (Strept. pyogenes, Strept. lanceolatus), and also new smear inoculations made upon nutrient media.

3. Tiny white to yellowish-white points, rather dense, and just visible with certainty after twenty-four to forty-eight hours. If the plates are kept longer, there is usually only a slight increase in size, up to about 0.5 mm., and then, with very few exceptions, they become no larger. They are, however, always distinguished from those named before by the tougher consistency. When magnified sixty times, the border is ragged, often as if gnawed away, and

splintery (see Plate 59, 1) and of a yellowish color. Magnified 1000 times: Stained in segments, highly polymorphous, short, long, thick, thin, clubbed, pointed, also with the form of cocci (apparently diphtheria or pseudodiphtheria or xerosis bacilli). In the pseudodiphtheria bacillus the border is often coarsely granular, similar to sarcinæ. The further examination is conducted according to page 384.

4. Larger, moist, sometimes slimy, luxuriant colonies, somewhat elevated, whitish to gray, with transmitted light somewhat iridescent. When magnified sixty times, the edge is smooth. Microscopic preparation magnified. 1000 times: Small, plump or more slender rods, perhaps also isolated short chains. Not stained by Gram's method. Belongs to the group of non-sporulating bacteria. Perhaps or probably the Bact. coli or a closely related variety. It is to be further studied regarding motility, gas-formation, indol, coagulation of milk, according to page 169, etc. When transferred to gelatin plates, the colon group presents, upon slight magnification, the characteristic, wavy, smooth-edged, transparent colonies with intersecting lines.

5. Macroscopic: Similar to the colonies described under 4, but never slimy; grayish-white, often gray. When magnified sixty times, the border is matted or curly. Microscopic preparations magnified 1000 times show sturdy bacilli, of equal length, the ends not rounded, staining by Gram's method, often united in chains (very probably sporulating organisms of the subtilis, anthrax, and mesentericus group). To be further studied according to page 304.

6. Essentially the same as under 5, but the periphery is exceedingly delicate and transparent. There is neither observed formation of curls nor irregular breaking up of the periphery into a felty structure. The microscopic preparation magnified 1000 times shows bacilli similar to those described under 5, but usually arranged in pairs (probably Bacterium duplex).

It may here be again stated for the beginner that the diagnosis, especially the separation of the bacteria into separate groups, may be much facilitated by paying attention to the periphery of the colonies. In the follow