therefore, require a considerable amount of nitrogenous food during its growth.

The Nature of the Food Requirements of the Yeast Cell.-Before commencing this study it is necessary for the student to learn the use of the hæmacytometer for the purpose of counting yeast

cells.

1. Use of the Homacytometer.-The form of hæmacytometer recommended is the one known as Thoma's (Fig. 20).

FIG. 20.-Figure of Thoma's Hæmacytometer.

A is a thick glass microscope slide on which a square of glass (a) 0.2 mm. thick with a circular hole in the middle is cemented. A circular glass (c) 0.1 mm. thick is cemented centrally in this hole, leaving an annular space (d) between (a) and (c). In the middle of (c) two sets of equi-distant parallel lines are etched, cutting each other at right angles. There is thus formed a large square with a side of 1 mm. subdivided into 400 small squares each

having a side of 0.05 mm. If a drop of liquid is placed on the square and enclosed by the cover glass (), the depth of the layer of liquid thus formed is 0.1 mm. As the large square has an area of 1 square mm. and the column of liquid above it is 0.1 mm. in depth, the volume of the liquid prism above the large square is thus 0.1 cubic mm.; and as the large square is subdivided into 400 small squares, the prism of liquid above each small square is oo, or 00025, of a cubic mm.

If now a liquid placed in the cell contains yeast cells, these after standing a short time will settle on the surface of (c), and the cells in the prism of liquid over the large etched square will settle on the square. If the cells resting on the large square are then counted under the microscope the number found will represent the number contained in 0.1 cubic mm. of the original liquid. The same remarks apply also to the small squares, each of which represents Too of a cubic mm. of liquid.

The above description indicates very briefly the nature of Thoma's hæmacytometer and the manner in which it is used; for fuller information the student is referred to Klöcker's Fermentation Organisms, p. 126. But it should be understood that when the student is learning the use of the hæmacytometer and other special operations connected with fermentation work referred to later on, he must work under the direction of a competent

instructor, for personal guidance in the details of such work is necessary for all but the most experienced students.

Experiment.

Weigh out 1 grm. of pressed yeast in a small beaker, and mix it with a little water to a thin liquid. Transfer the liquid yeast and the washings of the beaker to a 100 c.c. flask, and dilute with water to a volume of 100 c.c.

Shake the flask violently to disseminate the yeast cells evenly throughout the liquid. If this cannot be done sufficiently well owing to the limited capacity of the 100 c.c. flask, transfer the liquid to a dry flask of larger capacity. Immediately after shaking the flask, transfer 50 c.c. of the liquid by means of a pipette into another flask and dilute with 50 c.c. of water. Again shake violently and remove as rapidly as possible a small drop with a capillary glass tube or pointed glass rod to the hæmacytometer, and cover it at once with the glass (6). Note that the drop taken must be of sufficient size to touch the lower surface of the glass (b), but must not be so large as to run into the annular space (d). If the size of the drop does not conform to these conditions, it must be wiped off, and the experiment repeated. When a satisfactory drop is obtained, transfer the hæmacytometer to the stage of a microscope placed in an upright position, and allow a few minutes for the yeast cells to settle. Then proceed to count the cells under a 4-inch objective. It will usually be

found convenient to count the number of cells in a perpendicular column of twenty of the smaller squares; but before commencing to do so it is necessary of course to decide with regard to those cells which may happen to touch the lines bounding the column of squares. If all those touching the lines bounding the left hand and top of the column are counted, those touching the right hand and bottom lines must be omitted; either this or a reverse method must be adopted, or a correct number will not be obtained. The cells in at least five columns of the smaller squares, (100 squares), must be counted in order to obtain a fair average. After making one count of the cells, the hæmacytometer should be cleaned and the cells counted in a fresh drop. If the numbers obtained agree closely, the determinations may be regarded as satisfactory; if not, further countings of other preparations must be made until close agreement is obtained.

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The average number of cells obtained for a small square represents the number present in 4000 or 00025 of a cubic mm. of the liquid. Calculate the number of cells present in the original 1 grm. of pressed yeast taken, bearing in mind that the original preparation was diluted to one-half its volume before counting.

In order to assist in realising the vast number of cells obtained, calculate the distance a single row of the cells would cover if placed in a straight

line with the cells just touching each other, assuming that the average diameter of a cell is 6p. A μ, or micro millimeter, equals 1000 or 001 of a millimeter, and is the unit employed in microscopic

measurement.

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2. The Nature of the Food Requirements of the Yeast Cell. Three flasks, of similar shape, with a capacity of about 250 c.c. are required for this study.

Introduce into flask (a) 100 c.c. of a 10 per cent. solution of pure cane-sugar in distilled water.

Introduce into flask (b) 100 c.c. of a 10 per cent. solution of cane-sugar in Pasteur's nutritive mineral solution. This solution is prepared by dissolving the following constituents in 1000 c.c. of water :—

Introduce into flask (c) 100 c.c. of a 10 per cent. solution of cane-sugar in yeast water.

Weigh out 5 grms. of pressed yeast, and after mixing it thoroughly with a little water, dilute to 100 c.c. with more water, and agitate the mixture violently. Now add 5 c.c. of the diluted yeast to each of the flasks (a), (b) and (c), so that the contained solutions are yeasted with an equal number of cells in an equal volume of liquid. Close the flasks with cotton-wool stoppers, and place them in an incubator at a temperature of about 20°. At