of an animal dead of anthrax into a drop of nourishing material, fluid or solid, on the centre of a clean cover-glass, the edges of which have been prepared as just mentioned, and fasten this on the above slide so that the specimen faces the concave pit : expose this so prepared specimen to a constant temperature, either by placing it in the incubator and examining it with the microscope from hour to hour, or on the warm stage (Stricker, Ranvier) used in histological work for directly observing the influence of temperature on the various cells and tissues; or, place it simply on the stage of the microscope and expose the whole (i.e. microscope and all) in a suitable warm chamber (after Klebs), but so that the chamber allows light to pass by means of a small window to the mirror of the microscope, while the eyepiece is so arranged as to project through a hole in the

A

B

A

FIG. 7.-A GLASS CELL, FOR OBSERVING UNDER THE MICROSCOPE THE PROGRESS OF GROWTH OF MICRO-ORGANISMS.

The upper figure shows the cell in perspective; the lower figure in profile or cross section.

A. Glass slide.

B. Cover-glass.

C. Glass ring forming the wall of the chamber.

P. Drop of nourishing material in which the micro-organisms grow.

upper wall of the chamber. The plan which I generally follow is with slight modifications that of Koch.

A glass cell (Fig. 7) is made by cementing a glass ring, 3— inch in diameter and about - inch high, on to an ordinary glass slip. The chamber of this cell is well cleaned with absolute alcohol. A thin cover-glass, square or round, about one inch in breadth, is well heated by holding it for a few seconds over the flame of a gas-burner or spirit-lamp. On the upper edge of the above glass ring is placed with a camels' hair brush a thin layer of clean olive oil; a droplet of water is deposited on the bottom of the cell in order to keep this

afterwards well supplied with moisture; a drop of the sterile nourishing material (broth, aqueous humour, hydrocele fluid, blood serum, liquefied gelatine mixture, liquefied Agar-Agar mixture, &c.) is then deposited by means of a capillary pipette on to the centre of the cover-glass; then the point of a capillary pipette or needle containing the material it is desired to sow is rapidly plunged into the drop of the nourishing material (or if this is solidified is deposited in lines or points on the drop of nourishing material), the cover-glass inverted and placed on to the glass ring the layer of olive oil keeps the edges of the cover-glass air-tight on the glass ring. This cell is then placed into the incubator and exposed there to the desired temperature. Microscopic examination is carried out from time to time to watch the progress made. This can be done with high powers, since the growth is taking place on the lower surface of the cover-glass.

Although contamination with air-organisms is not excluded, still it is possible by making several specimens at the same time and operating rapidly, to obtain pure cultures. This glass cell can be also watched on a warm stage, or in a Klebs' warm chamber.

M. Nachet of Paris has designed a glass cell, in which the drop of nourishing material is deposited on to the bottom of the cell, the glass slip being here replaced by a very thin glass; but then there is a peculiar arrangement in the microscope, by which the lower surface of the glass cell, i.e. the one nearest to the growth, is directly subjected to microscopic observation.

After what has been said above about inoculation of solid and fluid nourishing media with solid matter, it is not necessary to dwell specially on the method of inoculation with earth or similar substances.

3. Examination of Water for Micro-organisms. Most water contains bacteria of some kind, as has been shown by direct experiment by Burdon Sanderson. If any sample of water is to be examined for micro-organisms, particularly bacterial forms, it is allowed to stand for a few hours, till most of the particulate matter is settled, and then with a capillary pipette a little of the fluid and sediment is drawn out and used for (a) microscopic specimens to be examined fresh; (b) microscopic specimens prepared after the Weigert-Koch method, i.e. by spreading out on a cover-glass a thin layer, drying it, staining it with suitable aniline dyes, e.g. Spiller's purple, gentian violet,

1 Reports of the Medical Officer of the Privy Council, 1870.

methyl blue, or magenta, washing with water, then spirit, then distilled water, then drying, and finally mounting it in Canadabalsam solution. (c) Test-tubes containing sterile nourishing material (broth, Agar-Agar mixture, gelatine mixture, Cohn's or Pasteur's fluid) are inoculated in the manner described previously, i.e. by piercing the cotton-wool plug with the pointed end of the capillary pipette. These test-tubes are then exposed in the incubator, and after one or two days or more, a sample is withdrawn with a capillary pipette, and used for microscopic examination. As a rule, after a day or two of incubation we can already distinguish with the unaided eye whether there are any organisms present, the nourishing fluid either being uniformly turbid this is generally the case or there being a growth at the bottom of the fluid. But of course the microscopic examination only shows what kind of organisms are present. New cultivations are made from this one, if any are required. (d) A good plan of recognising easily that there are present various kinds of organisms in such cultures is one similar to that recommended by Professor Angus Smith.1 Sterile gelatine broth or gelatine only, contained in sterile test-tubes plugged with sterile cotton-wool, is liquefied, but of course not heated to more than about 35° -40° C., then inoculated with the water (to be tested), by means of the capillary pipette; after inoculation the gelatine is mixed by shaking the test-tube slightly. In this way the organisms present in the water are distributed in the gelatine. Then the gelatine is allowed to set and is kept in this solid state. The organisms being distributed in the gelatine, after some days' growth are noticeable as clusters which gradually increase in extent and are distributed in various parts of the medium. The various species, owing to difference of growth, form clusters differing in aspect, size, and arrangement.

4. Examination of Air.-The simplest plan to test for the presence of organisms in the air is to draw out the cotton-wool plug of several test-tubes or flasks containing the sterile nourishing material, or, if this be boiled, potato, paste, or gelatine (see p. 14), to expose their surface, and to leave it thus for variable periods, froni a few seconds to several minutes. Then replace everything and expose the material to incubation, or keep it only at the ordinary temperature of the room. other method is to collect the particles present in the air on glasses moistened with pure glycerine (Maddox), and then to make microscopic specimens or inoculate tubes with this glycerine.

1 Sanitary Record, p. 344, 1883.

An

A method which is very useful is the one recommended by Cohn and Miflet.1 The principle of it is, that by means of an aspirator, an air-pump of any kind—e.g. a Sprengel pump, or simply the fall of water-air of a particular locality is drawn into one, two, or more Wolff's bottles (each with the ordinary two bent glass tubes), connected with one another by short pieces of india-rubber tubing, and containing the sterile material in which the organisms are required to grow. All bottles and tubes being of course sterile, the plugging of the tubes after the air has passed is done with sterile cotton-wool. Any quantity of air for any length of time can thus be passed through a series of such bottles, the one that receives the air first being of course most contaminated.

The bottles are after the experiment placed in the incubator if required, the outer end of their tubes being plugged with cotton-wool.

Miquel 2 has carefully described many ingenious methods for the study of air-organisms

Zeitschr. f. Biol. d. PA. iii. 1, p. 119.

2 Les Organismes vivants de l'Atmosphère, Paris, 1883.

A

CHAPTER VI.

MORPHOLOGY OF BACTERIA.

BACTERIA are minute organisms not containing chlorophyll, and multiplying by fission-hence the term schizomycetes (v. Nägeli). They are composed of a kind of protoplasm, the mycoprotein of Nencki, and are invested with a membrane, which is composed chiefly of cellulose and a certain amount of mycoprotein (Nencki).

Their contents are transparent and clear, but sometimes contain minute bright granules of sulphur (Beggiatoa). Owing to the cellulose membrane they resist the action of acids and alkalies. Many species of bacteria—micrococcus, bacterium, spirillum are able by rapid multiplication to form colonies; the individuals are then embedded in a hyaline gelatinous matrix produced by them, this is also mycoprotein. Some species are possessed of one or two straight or slightly spiral cilia or flagella, and thereby they are capable of locomotion, darting through, or spinning round, in the fluid in which they are suspended. Such is the case with many kinds of bacteria, bacilli, and spirilla.

Bacteria grow best when left undisturbed; movement of the vessel in which they grow is not advantageous. Light and electricity do not appear to have a decided influence, since most of them grow well in the light. According to Cohn and Mendelssohn,1 strong electric currents have a noxious influence on the growth of micrococci.

Some bacteria require free access of oxygen, and are called aerobic (Pasteur); others grow without free oxygen, and are anaerobic (Pasteur). All require for their growth certain nourishing materials containing carbon and nitrogen. Water

Cohn's Beitr. z. Biol. d. Pf. Bd. iii. 1.