liquefying tendency of the micro-organisms. Various types of gelatin cultures are shown in the accompanying diagrams, and it is rather important that the student should familiarize himself with the terms by which these different growths are described, in order that uniformity of description may be maintained. Gelatin cultures may not be kept in the incubating oven, as the medium liquefies at such temperatures. On the other hand, it must not be kept where the temperature is too low, else the bacterial growth may be retarded. The temperature of a comfortably heated room, not subject to excessive variations, such as are caused by steam heat and the burning of gas, etc., is about the most appropriate. Like the colonies, the cultures must be carefully examined from day to day, as it not infrequently happens that a growth

2

Fig. 61.-Types of streak cultures: 1, Filiform (B. coli); 2, echinulate (Bact. acidi-lactici); 3, beaded (Str. pyogenes); 4, effuse (B. vulgaris); 5, arborescent (B. mycoides) (Frost).

which shows no signs of liquefaction to-day may begin to liquefy to-morrow or a week hence, or even as late as two weeks hence.

The Agar-agar Culture.-Different operations vary in the exact method adopted in transplanting to agar-agar. In most cases, a simple stroke is made from the bottom of the tube in which the agar-agar has been obliquely solidified, and where it is fresh and moist, to the upper part, where it is thin and dry. In addition to this, it is advisable to make a puncture from the center of the oblique surface to the bottom of the tube. This enables us to tell whether the bacteria can grow as readily below the surface as above. Some workers always make a zigzag stroke upon the surface of the agar-agar. This does not seem to have any particular

advantage except in cases where it is desired to scatter the transplanted organisms as much as possible, in order that the bacteria will grow, or in order that a large bacterial mass may be secured.

Cultures upon Potato.-These are made by simply stroking the surface of the culture medium, the opacity of the potato making it impracticable to puncture it.

Cultures in Fluid Media.-Here, as has already been stated, transplantation consists in simply stirring in the bacteria so as to distribute them fairly well throughout the medium.

men.

Adhesion Preparations.-Sometimes it is desirable to preserve an entire colony as a permanent microscopic speciTo do this a perfectly clean cover-glass, not too large in size, is momentarily warmed, then carefully laid upon the surface of the gelatin or agar-agar containing the colonies. Sufficient pressure is applied to the surface of the glass to exclude bubbles, but not to destroy the integrity of the colony. The cover is gently raised by one edge, and if successful the whole colony or a number of colonies, as the case may be, will be found adhering to it. It is treated exactly as any other cover-glass preparation-dried, fixed, stained, mounted, and kept as a permanent specimen. It is called an adhesion preparation—“Klatschpräparat.”

Special Methods of Securing Pure Cultures.-Pure cultures from single colonies may also be secured by a very simple manipulation suggested by Banti.* The inoculation is made into the water of condensation at the bottom of an agar-agar tube, without touching the surface. The tube is then inclined so that the water flows over the agar, after which it is stood away in the vertical position. Colonies will grow where bacteria have been floated upon the agar-agar, and may be picked up later in the same manner as from a plate.

When the bacterium to be isolated (gonococcus, etc.) will not grow upon the media capable of alternate solidification and liquefaction, the blood-serum, potato, or other medium may be repeatedly stroked with the platinum wire dipped in the material to be investigated. Where the first strokes were made, confluent impure cultures occur; but as the wire became freer of organisms by repeated contact with the medium, the colonies become scattered and can be studied and transplanted.

*"Centralbl. f. Bakt. u. Parasitenk.," 1895, xvii, No. 16.

In some cases pure cultures may be most satisfactorily secured by animal inoculation. For example, when the tubercle bacillus is to be isolated from milk or urine which contains bacteria that would outgrow the slow-developing tubercle bacillus, it is necessary to inject the fluid into the abdominal cavity of a guinea-pig, await the development of tuberculosis in the animal, and then seek to secure pure cultures of the bacillus from the unmixed infectious material in the softened lymphatic glands.

In many cases, when it is desired to isolate Micrococcus tetragenus, the pneumococcus, and others, it is easier to inoculate the animal most susceptible to the infection and recover it from the blood or organs, than to plate it out and search for the colony among many others similar to it.

The growth upon agar-agar is in many ways less characteristic than in gelatin, but as the medium does not liquefy except at a high temperature (100° C.), it has that advantage. The colorless or almost colorless condition of the preparation also aids in the detection of chromogenesis.

Sometimes the growth is colored; at times the production of soluble pigment colors the agar-agar as well as the growth; sometimes the bacterial mass has one color and the agar-agar another. The growth may be filamentous, or simply a smooth, shining band. Occasionally the bacterium does not. grow upon agar-agar unless glycerin be added (tubercle bacillus); sometimes it will not grow even then (gonococcus).

Still less characteristic are the growths upon potato. Most bacteria produce smooth, shining, irregularly extending growths, that may show characteristic colors.

In milk and litmus milk one should observe change in color from the occurrence of acid or alkali production, coagulation, gelatinization, and digestion of the coagulum.

Blood-serum is liquefied by some bacteria, but the majority of organisms have no characteristic reaction upon it. A few, as the bacillus of diphtheria, are, however, characterized by rapid development at given temperatures.

While most of the saprophytic bacteria grow well at the temperature of a well-warmed room, the important pathogenic forms must be kept at the temperature of the body either to permit growth or to secure typical development. To do this satisfactorily an incubating oven or thermostat becomes a necessity. Various forms, of wood and metal, are in the market, one being shown in the illustration (Fig. 62).

The growth in gelatin is generally so far removed from the walls of the tube (a central puncture nearly always being

made in the culture-medium, in order that the growth be symmetric) that it is impossible to make a microscopic ex

amination of it with any power beyond that given by a hand-lens.

MICROSCOPIC STUDY OF CULTURES.

Some attention has been given to the preparation of microtome sections of the gelatin growth, which can be done if the glass be warmed just sufficiently to permit the gelatin containing the growth to be removed and placed in Müller's fluid (bichromate of potassium 2-2.5, sulphate of sodium 1, water 100), where it is hardened. When quite firm it is washed in water, passed through alcohols ascending in strength from 50 to 100 per cent., embedded in celloidin, cut wet, and stained like a section of tissue.

A ready method of doing this has been suggested by Winkler, who bores a hole in a block of paraffin with the smallest size cork-borer, soaks the block in bichlorid solution for an hour, pours liquid gelatin into the cavity, allows it to solidify, inoculates it by the customary puncture of the platinum wire, allows it to develop sufficiently, and when ready cuts the sections under alcohol, subsequently staining them with much diluted carbol-fuchsin.

Neat museum specimens of plate and puncture cultures in gelatin can be made by simultaneously killing the microorganisms and permanently fixing the gelatin with formaldehyd, which can either be sprayed upon the gelatin or applied in dilute solution. As gelatin fixed in formaldehyd cannot subsequently be liquefied, such preparations will last indefinitely.

Standardizing Freshly Isolated Cultures.-This is a matter of some importance, as in bringing bacteria into the new environment of artificial cultivation their biologic peculiarities are temporarily altered, and it takes some time for them to recover themselves. While the appearances of the freshly isolated organism should be carefully noted, too much stress should not be laid upon them, and before beginning the systematic study of any new organism it should be made to grow for several successive generations upon two or three of the most important culture media. Its saprophytic existence being thus established, the characteristics manifested become the permanent peculiarities of the species.

* "Fortschritte der Medicin," Bd. xi, 1893, No. 22.