Staining of Flagella.
Koch first stained flagella by floating the cover-glass on a watery solution of hæmatoxylin, transferring them to a five per cent. solution of chromic acid, or to Müller’s fluid, by which they obtained a brownish-black coloration.
Löffler’s Method.—Add together aqueous solutions of ferrous-sulphate and tannin (twenty per cent.) until the mixture turns a violet-black colour, then add three or four cc. of a one-in-eight aqueous solution of logwood; a few drops of carbolic acid may be added before transferring to a stoppered bottle; that is the mordant. The dye consists of 1 cc. of a one per cent. solution of caustic soda, added to 100 cc. of aniline water, in which four or five grammes of either methyl-violet, methylene blue, or fuchsine, are dissolved. A cover-glass preparation is made in the usual way, then the film is covered with mordant, and cover-glass held over flame until steam rises, the mordant is then washed off with distilled water. The stain is filtered and a few drops allowed to fall on film, after a few minutes the cover-glass is again warmed until steam rises. The stain is then washed off with distilled water, and the preparation is ready to be mounted for examination.
As Löffler’s process is somewhat complicated, a modification has been said to afford more satisfactory results. A specimen is taken from a recent gelatine culture and diluted with water. A little of the fluid is then transferred to a warm cover-glass by means of a pipette and allowed to dry, after which a drop of the following mordant is applied:—Aqueous solution of tannin (twenty per cent.), ten cc.; cold saturated solution of ferrous sulphate, five cc.; saturated solution of fuchsine in absolute alcohol, one cc. The cover is next heated gently for a short time until vapours are given off, then washed carefully. This process is repeated two or three times, and the specimen washed after each application. Subsequently, staining is effected by means of Ziehl’s fuchsine solution, the cover is afterwards warmed once or twice for about fifteen seconds, then washed, and the specimen examined in water to ascertain if the colour is sufficiently intense. If satisfactory, the preparation may then be dried and finally mounted in Canada balsam or dammar.
Preservation of Preparations.—After examining a cover-glass preparation with an oil-immersion objective the cedar oil must be carefully wiped off, and the slide set aside for the Canada balsam to set. At a convenient time these preparations should be sealed with a ring of Hollis’s glue.