of the vessels with the culture media. Sterilise with steam in the usual manner, noting that wort gelatin should be heated for only sufficient time to ensure perfect sterilisation, as prolonged heating affects its setting property.

Sterile Water. A supply of sterile water is frequently required during work with micro-organisms. A holder containing a supply of sterile water should be prepared. For description see Klöcker, p. 67.

A. THE SACCHAROMYCETES AND LOWER FUNGI.

Study of the Morphology of a Yeast Cell.-A sample of ordinary matured brewer's yeast is required. Place a small drop of water on a microscope slide and add to it a minute drop of yeast, so that the appearance of the water drop is slightly milky. Cover with a clean cover-glass and examine under the microscope with a 4-inch objective.

Observe that the yeast cells are spherical or slightly elliptical in shape. Like most living cells, they consist of protoplasm, or living matter, enclosed by a membrane-the cell wall.

To render the cell wall easily visible, remove the slide from the microscope and press the centre of the cover-glass gently, in order to burst some of the yeast cells. Examine again under the microscope and the walls of the burst cells will now be visible as very thin transparent membranes.

Examine another preparation of yeast in water,

and note that the contents of the cells are not homogeneous, but that one or two round, clear spaces of considerable size are visible in each cell which have the appearance of being of less density than the protoplasm. These spaces are called vacuoles, and are filled with cell sap.

Examine the protoplasm carefully and it will be noticed that a number of dense particles lie embedded in it, giving it a mottled appearance. These particles, or granules, are composed of fatty matter enclosed in a layer of albuminous material. The granules situated in the protoplasm are without motion, but granules may be frequently observed within the vacuoles which possess a Brownian movement indicating the fluid condition of the cell sap in which they are floating. (See Technical Mycology, Lafar, vol. ii., p. 145, concerning the Anatomy of the Yeast Cell".)

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The protoplasm of the yeast cell, like that of all living cells, is composed of a central body— the nucleus surrounded by cytoplasm; but the nucleus of the yeast cell is only rendered visible by certain staining processes which are somewhat difficult of execution (Klöcker, p. 89).

Dry, stain and mount a preparation of the yeast in the usual bacteriological manner (Klöcker, p. 88).

Having now studied the general anatomy of the mature yeast cell, the student should proceed to

observe the changes which take place in the appearance of the cell during the various stages of its growth.

Add a little of the mature yeast already examined to some hopped malt wort in a small flask, and place the flask in an incubator kept at a temperature of about 25°. Proceed to examine the yeast cells from time to time under the microscope, at first at intervals of one hour, and afterwards at longer intervals. Note the changes which rapidly take place in the general appearance of the cells. The granular appearance of the protoplasm disappears and the cells become very transparent; the walls of the cells also appear to become thinner.

Note also the changes in appearance of the vacuoles. Observe that the first signs of reproduction of the yeast cell by budding is evidenced after two or three hours by a slight protrusion on one side of the cell. The bud gradually enlarges until a full-sized cell is formed from the mother cell; but this method of reproduction will be followed better when employing the drop culture method described below. Observe as fermentation draws to a close that the protoplasm of the yeast cells becomes more granulated, and the cells return to the original appearance of the mature yeast with which the present study was commenced.

Determine the average size of the yeast cells by means of the micrometer eye-piece of the micro

scope, and express in micromillimeters (1 μ or micromillimeter equals 001 mm.)

Growth of Yeast in a "Drop Culture ".-Requirements for the preparation of a "drop culture". Thin glass rods in a tin box with a glass cover.

A glass plate and glass bell jar or beaker to act as a

cover.

Microscope slides and Böttcher's moist chambers (see
Klöcker, p. 69), or hollow-ground slides.

Microscope cover-glasses in small Petri dish (for
cleaning cover-glasses, see Klöcker, p. 87).
Freudenreich flasks of sterilised wort.

Vaseline.
Forceps.

FIG. 28.-Moist Chamber.

Sterilise the box containing glass rods, and the Petri dish containing cover-glasses, in the hot air steriliser. Sterilise the glass plate and cover with a Bunsen flame, and afterwards sterilise the microscope slides and moist chambers in the same manner, holding them in the flame with the forceps. Place the slides and moist chambers after sterilisation on the glass plate underneath the cover.

Preparation of a Drop Culture.-In special work the preparation of a drop culture is conducted in a sterile chamber (see Klöcker, p. 21), but this precaution is not necessary in the ordinary course of a student's work, and therefore reference to this part of the process is omitted here.

Take a glass slide from below the cover with the forceps, and lay it on the top of a Petri dish which has been sterilised by being passed through a Bunsen flame. Place three drops of sterile wort from a Freudenreich flask side by side on the glass slide by means of a sterile glass rod. A trace of the yeast to be examined is now added by means of the glass rod to one of the drops of water and the whole well mixed.

A little of the first drop

is then added to the second by means of a second sterile rod. After mixing, a little of the second drop is added to the third by means of another sterile rod. A small drop of the final mixture is now placed in the centre of a cover-glass, and the glass is then rapidly inverted and fixed on the ring of a moist chamber, the edge of which has been previously smeared with vaseline. A small drop of wort from the Freudenreich flask should be placed on the bottom of the moist chamber previous to the fixing of the cover-glass, in order to keep the air of the chamber moist.

Examine the drop culture under the microscope, which must be in an upright position. The yeast cells present should be so few in number that not more than one or two are visible in each field of view. If the cells are too numerous, another drop culture must be made with further dilution of the culture.

Study the cells from time to time as they multiply by budding, and make drawings. The