organism. The number of them attached to each individual varies to a considerable extent with the species of the bacteria. Thus the individuals of some species have but one flagellum, while the individuals of other species may have few or many springing from all parts of the organism. The flagella are not rendered visible by the ordinary methods of staining, but special methods are necessary for their demonstration. These methods depend essentially upon the use of a mordant, which causes the flagella to take up the stain. The cover-glasses must be absolutely free from grease in these methods, so that the watery fluids may be spread evenly over them and not run into patches. The coverglasses may be prepared by warming them in concentrated sulphuric acid for a time, washing them in water, and keeping them in a mixture of equal parts of alcohol and strong ammonium hydroxid solution. When used they are to be dried on a cloth which has previously been soaked in ether and allowed to dry, in order that it may contain no trace of fat. Another way to treat the cover-glasses is to take them from alcohol, dry them with a clean cloth, and then heat them by means of the Cornet forceps in the Bunsen flame to burn off any fat or grease. The bacteria must be distributed upon the cover-glass well separated from one another in these methods. They should not be subjected to too much manipulation in doing this, for the flagella are readily broken up. The distribution of the bacteria upon the cover-glass is effected as follows: Upon each of three or four cover-glasses, free from grease, a small drop of water is placed by means of the platinum wire. A minute portion of the bacterial growth is then secured in the end of the platinum wire and gently mixed with a drop of water on one of the cover-glasses. Then a small portion of the suspension is transferred to the drop of water on the second cover-glass with the wire, and likewise spread on this cover-glass. In a similar manner the third cover-glass is prepared from the second. Another way is to place two drops of water on a coverglass-to draw the infected wire once through one of them across the surface, and then once through the other drop, thus making two streaks. This subjects the bacteria to less manipulation and gives a good distribution in places. The cover-glasses prepared as above indicated are to be allowed to dry in the air; "fixing" them in the flame is not necessary. As soon as they are thoroughly dry they are ready to be stained by any of the methods given below. The cultures used for the preparations should not be older than eighteen to twenty-four hours. Solid culture-media, such as agar-agar, should be employed. Löffler's Method.-Treat the preparation for about one minute with the freshly filtered mordant solution, which is— Aqueous solution of tannic acid (20 grams tannic acid to 100 c.c. water), Cold saturated solution of ferrous sulphate, violet or fuchsin, IO C.C.; 5 c.c.; I C.C. The cover-glass is to be covered with this while held with the Cornet forceps, as in ordinary methods of staining. The mordant, thus placed on the cover-glass, may be gently heated by holding the preparation high over the flame for a period of about one minute, but it must not be boiled. After this the preparation is to be washed in water, and then stained with a freshly prepared and filtered solution of aniline-gentian-violet or aniline-fuchsin, with gentle heating for thirty to sixty seconds. It is then again washed in water, and mounted in water or balsam for examination. In using this method, as well as the others, an important thing to avoid is overheating; in fact, the warming of the mordant is not really necessary. The mordant may be freshly mixed every time or kept indefinitely for use. The ferrous sulphate solution should always be freshly prepared, for it rapidly decomposes. The solution of tannic acid keeps well, however. The addition of varying quantities of acids or alkalies for different species of bacteria, as recommended by Löffler, is not necessary. Pitfield's Method.-Treat the preparation with a mixture. of equal parts of two solutions, which are as follows: Solution A.-Saturated aqueous solution of alum, Saturated alcoholic solution of gentian violet, Distilled water, Solution B.-Tannic acid, Distilled water, 5 c.c.; I C.C.; 5 c.c. I gram; IO C.C. These solutions are to be made up cold, and are to be freshly filtered before mixing. The mixture placed on the coverglass is to be gently heated, but not boiled, during about one minute, then washed in water and mounted. Precipitates occur with this method which mar the preparation, but do not necessarily obscure the flagella. If it is desired to avoid these, clearer but not so deeply stained preparations may be obtained by filtering the mixture of the two solutions before using, and then, after treatment with the filtered mixture, to stain for one minute in aniline-gentianviolet without boiling. Bunge's Method.-The mordant used consists ofLiquor ferri sesquichloridi diluted with distilled water I: 20, Saturated aqueous solution of tannic acid, I part; 3 parts. This mixture improves by keeping, and is to be filtered for use. It gives good results after standing in a loosely stoppered bottle for two or three weeks, when it has acquired a red-brown color and a scum on the surface. It may then be said to be "ripe." The fresh mordant artificially ripened by the addition of the peroxid of hydrogen gives the best results. This is done as follows: Filter the above mixture into a test-tube and add peroxid of hydrogen (3 per cent. solution), drop by drop, until a redbrown color appears. Then shake and filter immediately through a double filter on the cover-glass preparations. Allow the mordant to act one minute on this; then wash in water, dry off with filter-paper, and stain the preparation with carbol-gentian-violet, gently warming, but not boiling. Then wash in water and mount. The artificially ripened mordant is good only for a few minutes. The amount of peroxid necessary will be about 0.5 c.c. per 5 c.c. of mordant. Capsules, in addition to flagella, may also be demonstrated in the case of certain bacteria by treating the preparation, before applying the mordant and stain, with 5 to 10 per cent. acetic acid, and afterward decolorizing with 1 per cent. acetic acid. The procedure for the demonstration of both capsules and flagella may be summarized as follows: 1. 5 to 10 per cent. acetic-acid solution, thirty to sixty. seconds. 2. Wash in water and dry with filter-paper. 3. Apply mordant one minute, not heating or with gentle heat. 4. Wash in water and dry with filter-paper. 5. Stain with carbol-gentian-violet, warming gently for one minute. 6. Wash in water and decolorize in I per cent. acetic acid solution thirty to sixty seconds. 7. Wash in water and mount. Bunge recommends that the preparations be made from gelatin cultures two days old, rather than from cultures grown in the thermostat on agar-agar. Van Ermengem's Method.-Make a very dilute suspension in 0.6 per cent. sodium chlorid solution of an eighteenhour agar-agar culture of the organism, and place a drop of the suspension in the center of a thoroughly clean coverglass. Allow the drop to dry in the air, and then "fix" the preparation by passing the cover-glass rapidly three times through the flame. To the "fixed" preparation apply the following mordant for half an hour at room-temperature or for five minutes at 50° to 60° C. : Osmic acid (2 per cent. solution), I; 2. To this four or five drops of glacial acetic acid may be added. Next wash in water and in alcohol (95 per cent.), and then immerse for a few seconds in a 0.25 to 0.5 per cent, solution of silver nitrate. From this, without washing, transfer to a small dish containing the "reinforcing bath," which is of the following composition : In this the preparation is washed for a few seconds, and is then again immersed in the solution of silver nitrate (0.25 to 0.5 per cent.). The preparation is moved about in this solution until the solution begins to acquire a brown or black color, when it is removed, washed thoroughly in water, dried, and mounted in balsam. 2. METHODS OF OBTAINING PURE CULTURES. When it is desired to obtain a pure culture of bacteria, a colony or a portion of a colony of the organism is secured on the end of the sterile platinum wire, and transferred by this means to the culture-medium in another test-tube. The bacteria thus sown in the fresh culture-medium multiply there, and produce a growth visible to the naked eye which exhibits appearances more or less characteristic of the species. This growth, if the medium be a solid one, will usually be in the form of confluent colonies; if the medium be a fluid one, the growth may appear as a sediment with or without clouding of the liquid, or it may manifest other peculiarities according to the species to which the organism. belongs. If other bacteria are present in the culture from which it is desired to obtain material for a pure culture, it is important that the material should be taken from a colony. of the organisms which is well separated from other colonies. -i. e. that the colony should be a so-called "discrete" one. In transplanting, the culture-tube containing the colony |