and the operator ought not to consider himself efficient until he can keep flasks containing yeast water or meat extracts sterile after repeated manipulations.

Experiments with Solid Culture Media. If a portion of a growth is to be inoculated from a solid culture medium into a flask containing nutrient solution or on to new solid medium, then metal wires, such as platinum, or glass rods are used. Before use they are sterilised in the flame and placed in a flamed tin box, which is covered with a glass plate likewise flamed. They are left in the box until quite cool. This is very important, as one might otherwise easily kill the growth to be introduced. Immediately before use they are once again drawn quickly through the flame. When glass rods are to be used it will be better in many cases to take one drawn out into a long thin point, especially when small specks of growth (e.g., from a moist chamber) or a fine mycelium, which clings easily to inoculation needles when these are used, has to be introduced, as the point of the rod can be broken off and left in the infected culture liquid. The infection is thus performed more surely and more quickly than when a metal wire has to be rubbed against the sides of the flask in order to leave particles of growth in the liquid. Sometimes small pieces of platinum wire are used for the same purpose, being manipulated by a pair of forceps.

Infection of a solid culture medium takes place either on or below its surface. A surface culture is laid on most easily as a streak made with a metal wire or glass rod; if the culture to be introduced is contained in a liquid, a pipette may be used, by means of which we then sow a drop on the surface of the solid culture medium; or we may use a metal loop. On the other hand, if the culture is to be sown in the substratum, this may be performed by aid of a metal wire (an inoculation needle), which with the

adherent growth is thrust into the medium; a so-called "stab" culture is thus obtained. Development of the organism then takes place on and below the surface. Cultures in the body of the substratum may also be obtained by mixing the growth with liquefied nutrient gelatine.

Plate cultures, which are employed in the production of pure cultures, are described on page 103.

Cultures of Anaërobic Organisms. If it is wished to start a culture of an anaerobic organism on a solid medium, this can be done by covering the medium with a plate of mica (Koch), which is pressed on, the medium having a perfectly level surface. A glass plate can be used in place of the mica, or melted gelatine is poured on to the infected medium (Hesse). Of other methods that of H. Buchner may be mentioned. According to the latter the organism under examination is placed in a small test tube with nutrient gelatine, and this put into a second larger test tube in which there is an alkaline solution of pyrogallic acid (1 vol. of an almost saturated solution of pyrogallic acid mixed with 10 vols. of potash solution [1 + 1]); the outer test tube is kept well closed with a tight plug. The alkaline solution of pyrogallic acid absorbs all the oxygen so that the culture in the inner open test-tube grows in an oxygen-free atmosphere.

The process may also be carried out by displacing the atmospheric air by an indifferent gas, e.g., by hydrogen. The method described by Fränkel consists in the use of ordinary wide test tubes fitted with double-bored bungs through which two glass tubes pass, one reaching almost to the bottom, the other ending just below the bung. This culture tube after being filled with the gelatine is sterilised, inoculated, and then has a current of hydrogen passed through it. After all the air has been driven out, the glass tubes are closed by fusing.

Suppression of Bacteria in Yeast Growths.—If it is required to encourage the development of a bacteriainfected culture of an alcoholic yeast, the method usually adopted is to cultivate the impure growth in an acid nutrient medium. Nearly all bacteria are killed in this way, or are to a great extent hindered in their development, so that the alcoholic yeasts in the mixture preponderate. But it is not always possible to use such an acid medium, as the organism to be cultivated may also be influenced adversely. In such a case the cultures can be exposed to the action of light, as bacteria can withstand the action of light only to a small degree. This was proved several years ago by Downes and Blunt. Experiments in this direction have also been made at the Carlsberg laboratory, and have shown that spore cultures of saccharomycetes on gypsum blocks can be kept free from bacteria if exposed to light.

3.-Preparation of Pure Cultures.

The methods for preparing pure cultures described in the following pages are only those of practical importance and application.

PURE CULTURES FOR INVESTIGATIONS IN MORPHOLOGY AND DEVELOPMENT.-The preparation of pure cultures for investigating morphology and development was begun very early, the development of the single cell being observed under the microscope. This was perhaps first done by Ehrenberg (1821); later, Mitscherlich studied the budding of the yeast cell in the same manner. The process of microscopical examination as a means of studying morphology and development was largely employed in investigating fungi belonging to the most widely separated divisions of the system. The technique was brought to a high degree of perfection, especially by Brefeld (1875).

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Brefeld's Glass Slip Cultures.-The essential point in this method is the direct microscopical observation of all stages of development. Brefeld takes a fruit carrier with spores, or a small mass of spores, from the mould in question, e.g., a Mucor, with the aid of a fine needle and distributes them in a drop of sterile water; he continues diluting until only one or two spores are present in any drop, and one of these drops is then placed on a glass slip. He then adds nutrient solution, the position of the mould spore is marked, and, to prevent evaporation, the glass slip culture is kept under a moist bell-jar when not under observation. If the investigation is prolonged, the culture medium is replaced by gelatine to prevent evaporation. These Brefeld glass slip cultures are quite open, which is of some importance in the investigation of the higher fungi as they have then sufficient room for growing; the culture, however, is in this way exposed to infection from the air. In order to mitigate this danger, a paper shield is placed on the microscope. But even when foreign germs, e.g., a Penicillium spore, find their way into the culture, it is of little importance; no error can be made, as the culture is under microscopical observation the whole time. This technique is excellent in its own sphere.

Brefeld in his investigations on Bacillus subtilis used moist chambers, viz., those of v. Recklinghausen. The latter are only suitable for morphological work and have not found a place in this book, all the more so as they have been recently replaced by a better design. (See the moist chambers mentioned on page 68.)

PURE MASS CULTURES.-If the pure culture, on the contrary, is to lead to an absolutely pure mass culture, other considerations arise than when the former is designed only for morphological and developmental purposes. In the latter case it is of no importance if a foreign organism is present along with those being cultivated, for the whole

investigation is carried out on the stage of the microscope under continued observation. It is quite otherwise when the physiological experiment is carried on with flasks containing mass cultures; another technique is here necessary.

These pure culture methods are divided into two groups, one comprising those based on the principle of dilution, whilst in the second the physiological behaviour of the species forms the basis. It is only by use of the dilution method, and that in its most developed form, that an undoubted pure culture is obtained. Such a culture is prepared by sowing out a single cell in a sterile culture medium and by further cultivation in such a way that no foreign organisms are able to force their way in.

I. The Dilution Methods.-Dilution methods may be again divided into two groups, according as the dilution takes place in nutrient liquids or solids. In the dilution of nutrient liquids the method is to count the number of germs in a certain unit of volume, and then to dilute with a calculated quantity of the liquid until there is one cell per unit of volume. This method was used by Lister (1878, pure cultures of a lactic acid bacterium), Nägeli, Fitz and Hansen (1882). The method was brought to its greatest perfection by Hansen.

Hansen's Dilution Method. The dilution method as used by Hansen's predecessors was quite uncertain. It was never really known whether those flasks in which a growth was developing contained a pure culture or not, i.e., whether the seeding consisted of one or several cells. The counting method in use was not exact; but even with exact counting the seeding might consist, in certain flasks, of more than one cell. Therefore, Hansen added to the method two elements, by means of which it gained in certainty, viz.: (1) an indication to decide whether the infected flask has received one cell or several; and (2) an