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culture when not under examination should be kept in an incubator at 20°-25° (68°-77° F.). If it is desired to follow the multiplication of one particular cell it must be carefully marked, or the slide must be fixed on the microscope, and the latter placed in an incubator or other suitably warm place. (For mode of propagation of yeast by budding, see Klöcker, p. 191.)

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FIG. 24. Multiplication of top yeast: I., 2, 7 P.M.; II., 27, 8 A.M.; III., 9 A.M.; IV., 10 a.m.; V., 12 noon; VI., 3 P.M.; VII., 8 P.M.; VIII., 28, 8 A.M.; IX., 10 A.M.; X., 11 A.M.; XI., 1 P.M.; XII., 2o, 8 P.M.; XIII., ; XIV., , 12 o'clock. (After Mitscherlich.)

A Study of Some of the Well-recognised Races of Yeasts and Torula.-When the student is familiar with the appearance and ordinary mode of reproduction of brewer's yeast, he should then proceed to study some of the well-recognised races and species of yeasts and torula. Inoculations

VI

from pure cultures of these should be obtainable in the laboratory in which the student works, and should at first be grown in tubes of malt wort. The general appearance of the organisms should be studied under the microscope and also their modes of development in drop culture, careful drawings being made at all times. A selection from the following list of organisms is recommended :Saccharomyces cerevisiæ. Top fermentation race.

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Saaz and Logos; low fermentation races. (See Klöcker, p. 220.)

Pastorianus I., II., and III. (See Klöcker, pp. 254-256.)

ellipsoideus I. and II. (See Klöcker, pp. 257259.)

Marxianus. (See Klöcker, p. 261.)

anomalus. (See Klöcker, p. 262.)

Schizo-saccharomyces Pombe. (See Klöcker, p. 269.)

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octosporus. (See Klöcker, p. 270.)
Saccharomyces (?) apiculatus. (See Klöcker, p. 294.)
Mycoderma vini. (See Klöcker, p. 297.)
Torula. (See Klöcker, p. 289.)

A Study of Some of the more Common Species of Moulds.-The close relationship of the saccharomycetes to the moulds, and the important fermentation changes induced by many species of moulds, renders it most desirable for the student to study some of the more important types of these organisms.

Inoculations from pure cultures of the moulds should be made in malt wort and on wort gelatin, and the modes of growth of the organisms studied.

At the same time drop cultures should be made with spores or mycelia of the moulds, and the different stages in the growth and development of the organisms carefully followed under the microscope. Drawings should be made throughout these studies.

A selection from the following moulds is recommended :

Mucor racemosus. (See Klöcker, p. 179.)

Mucor (Amylomyces) Rouxii. (See Klöcker, p. 181.)
Penicillium glaucum. (See Klöcker, p. 278.)

Aspergillus glaucus. (See Klöcker, p. 274.)
Monilia candida. (See Klöcker, p. 298.)
Oidium lactis. (See Klöcker, p. 303.)
Dematium pullulans. (See Klöcker, p. 305.)

Preparation of Spore Cultures of the Saccharo

mycetes.

Requirements:

A pure culture of yeast twenty-four hours old.
Sterilised gypsum blocks in covered glass dishes. (See
Klöcker, p. 64.)

Sterilised water.

Sterilised pipettes. (These may be made by drawing out thin glass tubing in a gas flame.)

In order to induce sporulation in yeast, the culture employed must be young and vigorous (see Klöcker, p. 122).

Inoculate a small flask or test-tube of sterile wort from a pure culture of top fermentation yeast, and keep it in an incubator at 25° (77° F.) for twenty-four hours, in which time a sufficient deposit

of freshly formed yeast for the purpose of the experiment will probably be found.

Sterilise the gypsum blocks and dishes in the hot-air steriliser for an hour at 110° to 115° (230° to

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239 F.). The temperature must not rise above 120° (248° F.) or the blocks may be spoiled by dehydration of the gypsum.

The small pipettes should be wrapped separately in filter-paper and sterilised at about 150° (302° F.).

To prepare the spore culture, pour away the supernatant fermenting liquid from the yeast culture, and take out a small quantity of the sedimentary yeast with a pipette and spread it in a thin layer on a gypsum block. Pour water from the sterilised water-holder into the dish in which the gypsum block is placed until it is about two-thirds filled. During these manipulations uncover the dish for as short a time as possible. Place the covered dish in an incubator at 25° (77° F.). Examine the culture after twenty-four hours under the microscope for the formation of spores, removing for this purpose a little of the yeast with a sterilised needle and mounting it in water on a glass slide (see

Klöcker, p. 122). If spores are not found, examine at intervals of six or eight hours, and note the time when spore formation is first observed.

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FIG. 26.-Saccharomyces cerevisia I., Hansen. First stages of development of the spores. 1000. (After Hansen.)

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FIG. 27.-Saccharomycetes which form ascospores. 1. Sacch. cerevisia I. 2. Sacch. Pastorianus I. 3. Sacch. Pastorianus II. 4. Sacch. Pastorianus III. 5. Sacch. ellipsoideus I. 6. Sacch. ellipsoideus II. a, Cells with septa; b, cells with more than normal number of spores; c, cells distinctly beginning to sporulate. About 5o. (After Hansen.)

Make spore cultures of the various species of yeasts which are being studied, and note the different times at which spore formation is first observed (see Klöcker on spore formation, pp. 202 et seq.).

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