viously 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 cover-glass 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 off. A good way is to make a dilute suspension of the bacteria in distilled water, and place one or two loopfuls of this on the cover-glass, not spreading with the loop, but making the suspension flow over the surface by inclining the cover-glass. 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, and are then to be heated for a few seconds over a flame while held between the fingers. They are then 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 cover-glass 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 others, an important thing to avoid is overheating. 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 as Modified by J. Blackburn Smith.— The mordant is prepared as follows: A saturated solution of mercuric chlorid, made by boiling, is poured while still hot into a bottle in which crystals of ammonia alum have been placed in quantity more than sufficient to saturate the fluid. The bottle is then well shaken and allowed to cool. To 10 c.c. of this fluid 10 c.c. of a freshly made 10 per cent. solution of tannic acid are added and 5 c.c. of carbol fuchsin solution. After mixing, filter. This mordant will keep. In staining, the mordant is filtered on to the cover-glass preparation, which is heated until steam is given off during about three minutes. Boiling is to be avoided. The preparation is then washed in distilled water and is stained in a mixture of I c.c. of a saturated alcoholic solution of gentian violet and 10 c.c. of a saturated solution of ammonia alum. This mixture is filtered on to the preparation. Bowhill's Method.-Stain the preparation in the following solution for ten to fifteen minutes, slightly warming: Saturated alcoholic solution of orcein, This mixture is to be filtered before using. alcoholic solution of orcein should be at old. 15 c.c. IO C.C. 30 c.c. The saturated least ten days. G. H. Neuman's Method.—Make this mixture: Add to this, after the ingredients are dissolved, 0.5 gram of night blue, dissolved in 20 c.c. of absolute alcohol, mixing thoroughly. Filter off the precipitate that forms. The filtrate is the staining fluid. It will keep only a few days and should be filtered immediately before use. 1. Stain the preparation (which has been dried at roomtemperature) for two minutes or more, changing the fluid two or three times. 2. Wash thoroughly in water. 3. Stain with a saturated aqueous solution of gentianviolet for two minutes, in order to stain the bodies of the bacteria. 4. Wash in water and mount. Williams' Method.—This is a modification of van Ermengem's method along the lines of the modification of Hinterberger and others. It has been adopted by Dr. Hugh Williams after a large experience with various. methods in the Laboratory of the Massachusetts General Hospital. The method is capable of giving black bacteria and flagella, with little or no precipitate. The method is as follows: 1. Cover the cover-glass with a mordant consisting of Alumnol,' I per cent. solution, 5 c.c.; 5 c.c.; 15 c.c. Shake the mixture, and add three drops of glacial acetic acid, and again shake. 2. Apply the mordant less than one minute without heating. Wash thoroughly in water. 3. Cover the preparation, during about one minute, with a I Farbwerke vorm. Meister Lucius u. Brüning, Höchst a. M., Germany. per cent. solution of silver nitrate to which sufficient ammonium. hydroxid has been added to keep the silver in solution. 4. Wash in water. 5. Wash with 0.6 per cent. solution of sodium chlorid. 6. Flood the preparation with a 30 per cent. solution of ammonium hydroxid, and immediately wash in water. 7. Apply a few drops of Ortol photographic developer. The directions for making up this developer come with the Ortol. 8. Wash in water. 9. Cover with a I per cent. solution of gold chlorid during a few seconds. 10. Wash in water, and apply Ortol developer for a few seconds. 11. Wash in water, and cover with a 1 per cent. solution of mercuric chlorid for a few seconds. 12. Wash in water. 13. Apply Ortol developer for a few seconds. 14. Wash in water, and repeat the application of chlorid of gold, the washing, and the application of the developer two or more times. Between the various applications of the chlorid of gold the preparation should be inspected with a high, dry lens to determine the progress of the staining. This is readily done by placing the cover-glass, charged side upward, on a slide. In this way the process of impregnation with gold may be controlled; for the flagella, if stained, may be easily seen with the high-power dry lens. The preparation is very conveniently held during the process in cover-glass forceps. The washing is best done in a small stream of water from a faucet. The various solutions are conveniently applied from dropping-bottles, see p. 93. It will be seen that the process consists essentially in the impregnation of the flagella with silver, followed by intensification, in the photographic sense, with mercury and gold. The object of the application of the sodium chlorid and ammonia is to remove the excess of silver compounds which adhere to the surface of the cover-glass in spite of washing. This excess of silver compounds is chiefly respon sible for the precipitates which appear on the preparation after the intensification. In spite of the application of the sodium chlorid and ammonia solutions, some precipitate will occur if the intensification is pushed too far. On this account it is advisable to observe the progress of the intensification under the microscope as above indicated. Although this method may appear complicated, in practice it requires but a few minutes to stain a preparation. Methods of Staining the Capsules of Bacteria. Welch's Method. This method depends upon the fact that acetic acid precipitates the mucin-like substance of which the capsule is composed, and that the precipitated material of the capsule is not soluble in a 2 per cent. solution of sodium chlorid. Therefore, any necessary washing of the preparation is to be done in the solution of sodium chlorid, and the mount is to be made in the same solution or in balsam. The method is as follows: 1. Cover the preparation with glacial acetic acid for a few seconds. 2. Drain off and replace (without washing in water) with aniline-gentian-violet solution. The staining solution is to be repeatedly added to the surface of the cover-glass until all of the acid is replaced. 3. Wash in a 2 per cent. solution of sodium chlorid and mount in the same. W. H. Smith's Method. This has been found particularly useful in demonstrating the pneumococcus in the sputum. The sputum or other material should be fresh. The cover-glasses should be spread as thinly as possible and fixed by passing three times through the flame in the usual manner. 1. Stain in aniline-gentian-violet solution for a few seconds, gently warming until the staining fluid steams. 2. Wash in water. 3. Cover with Gram's solution of iodin for thirty seconds. 4. Wash with 95 per cent. alcohol until the color ceases to come out. 5. Wash with ether for a few seconds. (To remove fat.) 6. Wash in absolute alcohol for a few seconds. |