be avoided. These can, however, when formed, be expelled by a cautious tapping on the cover glass. Yeast cells and moulds are usually examined in the unstained condition.

Water-mounted preparations can be kept for some time if the cover glass is sealed round the edge to the glass slip so that no evaporation can take place. A very suitable medium for this purpose is a solution of common sealing wax in spirit, or a melted mixture of vaseline and beeswax. If it is wished to make really durable preparations, Hantsch's solution (see p. 92) is added drop by drop to an ordinary preparation in water so that after some time the only liquid remaining in the preparation is glycerine, the alcohol and water having evaporated. The gradual addition is necessary so that the form of the cells may not be altered too much by the water-absorbing property of glycerine. Afterwards the edge of the cover glass is sealed either with the above-mentioned sealing wax solution or with asphalt lac. This method is especially suitable for preparations of yeast cells and moulds. For the preparation of stained bacteria specimens see below. If the latter are to be made permanent they are mostly mounted in Canada balsam. In permanent specimens the cells always lose their natural form to some extent.

Removal of Grease from Cover Glasses.-In preparing a microscopical specimen which is to be fixed, hardened and stained, it is necessary to use perfectly clean cover glasses. To obtain these it is not sufficient to clean them in the ordinary way, but means must be employed to remove the thin layer of grease which always adheres to the glass. The cover glass is first laid in some strong mineral acid (hydrochloric or sulphuric), then washed with water and boiled in a soda solution; it is again washed with distilled water, dried, washed in absolute alcohol and again dried.

Fixing and Staining of Yeast Cells. After the cover glass is carefully cleaned in this manner a drop of the culture is spread over it in as thin a layer as possible, and the cover glass left under a glass bell until the drop has completely dried up. If the culture is present in a solid substratum, a little of it is distributed in a water drop and the mixture spread on the cover glass. As regards the staining of a yeast cell preparation, e.g., with an aniline dye, the preparation, thoroughly dried in air by the above method, is taken up by means of a pair of forceps with the prepared surface upwards and drawn through a small gas fame three times with uniform speed describing a vertical circle with a diameter of about one-third of a metre, the three motions occupying about three seconds. The specimen is thus fixed and hardened. A little of the staining solution is now put on the cover glass, allowed to act for some minutes and then washed off with distilled water. The clean side of the cover glass is next dried with filter paper, and the specimen is then ready for examination.

The distinguishing of dead cells from living ones has been assiduously carried on in most brewery laboratories since the microscope came into general use. But the value of the indications given by the reagents employed for this purpose has been very much overestimated. The question seems to deserve proper investigation. According to Wehmer, a half per cent. methylene blue solution will stain the dead cells indigo blue, while the living cells remain

colourless.

Staining of Yeast Spores. Ziehl's carbol fuchsine. solution (see page 92) is used for colouring the spores of yeast cells. As soon as the preparation has been fixed in the above-described manner, it is laid in a small crucible or watch glass with carbol fuchsine, heated for a short time to boiling, and then washed with water, afterwards

with dilute acid (5 per cent.), and then again with water. The spores are then usually coloured red; the rest is colourless. Sometimes other coloured bodies appear besides spores, and it may also happen that some single spores remain colourless.

Staining of the Yeast Cell Nucleus.-The detection of the cell nucleus is no easy matter. Janssens and Leblanc recommend a modification of Moeller's method, viz., the following: A few drops of a solution of iodine in potassium iodide (1 part potassium iodide, 100 parts of water, iodine to saturation) are placed on a glass slip; and a little of the yeast in question is stirred in. A drop of the mixture is then spread on a well-cleaned cover glass. Immediately after the mixture has dried, the cover glass is put into the iodine solution and allowed to lie for twenty-four hours. The specimen is now hardened; the cover glass is taken out of the iodine solution, placed first in water, then in 33 per cent. alcohol, next in 80 per cent., and, finally, in 95 per cent. alcohol. Before proceeding to stain, the yellow colour of the cells must be completely removed. In case this is not effected by the 80 per cent. alcohol, an aqueous solution of potassium iodide (1 to 3 per cent.), or ether may be used. The specimen ought to lie at least forty-eight hours in the 95 per cent. alcohol; a longer soaking is not harmful, but is unnecessary. Carbol fuchsine is used for staining, the cover glass being warmed in a little of this liquid contained in a watch glass. The cover glass is then washed several times with water, and, finally, with very dilute sulphuric acid.

Heidenhain's method may also be adopted, the procedure being as follows: The fixed specimen is laid for four hours in a solution of 2.5 grams of iron alum in 100 c.c. of distilled water; it is then placed for twelve to eighteen hours in a solution of 0.5 gram of haematoxylin in 100 c.c.

of distilled water. Lastly it is decolorised in the usual

manner.

Fixing and Staining of Bacteria.-A preparation of bacteria is stained and fixed in the same way as a yeast preparation. A drop of an alcoholic aniline dye solution is then allowed to act for some minutes, after which washing with distilled water takes place.

A special method of staining is described by Chr. Gram, which has found extensive application, chiefly because it is used as a method of diagnosing certain species of bacteria. According to this method the fixed specimen is stained from one to three minutes in a hot saturated solution of gentian violet in aniline water, and then immersed for one to three minutes or longer in a solution of iodine in potassium iodide. A precipitate is thus formed which is only deposited on the bacteria. The preparation is then washed with absolute alcohol until every trace of the colouring matter has been removed.

Staining of Bacteria Spores. Bacteria spores are stained by boiling the fixed specimen for a long time in carbol fuchsine (in some cases for an hour, during which the evaporated liquid is constantly renewed); it is then washed in alcohol.

Aujeszky has recently communicated a simpler method for staining spores. A little of the culture containing the spores is spread on a cover glass, and while the smear is drying in the air, a half per cent. hydrochloric acid solution is warmed over a Bunsen flame in a porcelain dish until bubbles begin to appear. When this point is reached the Bunsen is removed, and the cover glass, now dried, but not fixed, is laid for three to four minutes in the liquid. The preparation is afterwards washed with water, dried, fixed and treated with Ziehl's carbol fuchsine, then held in forceps over the Bunsen flame and heated until it fumes. As soon

as the staining solution begins to fume, the preparation is drawn out of the flame for some seconds, this heating being twice repeated. The preparation is then allowed to cool one to two minutes more, after which decolorising with 4 to 5 per cent. sulphuric acid follows. The latter process should not be carried too far in case the spores again become colourless.

Finally, we shall describe the method given by Alex. Klein, who found that spores easily become stained without any previous treatment if the dye is allowed to act on them in the moist state. The process is the following: Preparation of an emulsion of the spore-containing material in 0·7 per cent. salt solution (in a watch glass) and addition of an equal quantity of filtered carbol fuchsine solution, then gentle heating, steam being given off at the surface for six minutes, dust being kept off by covering with a second watch glass. The preparations are then spread out and allowed to dry in air and fixed by passing twice through the flame. Decolorising is effected by 1 per cent. sulphuric acid acting for one to two seconds, and lastly the preparation is washed with water.

Staining of Flagella according to Löffler.-Staining assumes a special significance when the question is one of detecting the motile organs, the flagella, of bacteria. There are several ways of doing this, one of the most used being that described by Löffler. After the preparation has been carefully fixed it is treated with a mordant, which consists of 2 parts of a 20 per cent. solution of tannin, some drops of an aqueous saturated solution of ferrous sulphate and 1 part of logwood extract (1 to 8).

Some drops of this mixture are placed on the cover glass and warmed directly over the flame until steam begins to form. Afterwards the cover glass is washed with water and stained with carbol fuchsine or with Löffler's solution,