gelatine is liquefied at 30° C. (the agar-agar at 40°), and is then mixed with the equally hot litmus solution. The latter should only be so strong that its action as a reagent is just distinguishable.

These substrata

Potatoes are

Other Solid Media.-Bread might be mentioned as one of those solid media which are sometimes employed. It is made into a paste with a little water and sterilised by steam; rice may be sterilised in the same way. Manure (with or without addition of water) is sterilised several times with an interval of one or two days. are employed, e.g., in cultivating moulds. frequently used for the culture of bacteria. are cleaned well with a brush and laid for some minutes in a 10 per cent. solution of sublimate and afterwards in a 01 per cent. sublimate solution for half an hour to fifteen hours. Finally they are well washed with water and then sterilised for two hours in steam and cut up.

The potatoes

1.—Microscopical Investigation of Micro-Organisms. Preparation Making.-A preparation of a micro-organism is made as a rule by putting it in a drop of liquid or in Canada balsam, etc., on a glass slip and laying a cover glass on it. Water is often used (best when distilled and sterile so as to exclude outside organisms from the preparation); a little of the growth to be investigated is taken out with a platinum wire or similar instrument and stirred in the water. If it is in a culture liquid, a sample can be taken out with a small glass rod and placed direct on the glass slip without addition of water. The needles and rods used for taking out samples must of course be sterilised beforehand, when the cultures are to be preserved pure; all the usual precautions must be observed. When the cover glass is laid on the drop the enclosing of air bubbles in the liquid is to

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 flame 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