For staining preparations with gentian-violet Crookshank employs the following useful method:—Place four or five drops of pure aniline in a test-tube, add distilled water to three-quarters full, close mouth with thumb, shake thoroughly. Filter the emulsion twice, pour filtrate into watch-glass. To the perfectly clear aniline water thus obtained, add, drop by drop, a concentrated alcoholic solution of gentian-violet till precipitation commences. Cover-glasses must be left in this solution ten minutes, transferred to iodine-potassic-iodide until the film becomes uniformly brown, then rinsed in alcohol. The decolourisation may be hastened by dipping the cover-glass in clove oil and returning to alcohol. Again immerse cover-glass in clove oil, dry by gently pressing between two layers of filter-paper, and mount in Canada balsam.
Double-staining of cover-glass preparations.—They can be treated by Ehrlich’s method for staining tubercular sputum, or by Neelsen’s modification, or by staining with eosin after treatment by the method of Gram.
Ehrlich’s Method is as follows: Five parts of aniline oil are shaken up with one hundred parts of distilled water, and the emulsion filtered through moistened filter-paper. A saturated alcoholic solution of fuchsine, methyl-violet, or gentian-violet, is added to filtrate in watch-glass, drop by drop, until precipitation commences. Cover-glass preparations are floated in this mixture for fifteen minutes to half an hour, then washed for a few seconds in dilute nitric acid (one part of nitric acid to two of water), then rinsed in distilled water.
Neelsen’s Solution and Methylene Blue.—Ziehl suggested the use of carbolic acid as a substitute for aniline blue. Neelsen recommended a solution of carbolic acid, absolute alcohol and fuchsine. (See Appendix.)
Gram’s Solution and Eosin.—After using Gram’s method as above and decolourising in alcohol, the cover-glass is placed in a weak solution of eosin for two or three minutes, washed in alcohol, immersed in clove oil, dried, and mounted in balsam.
Staining of Spores.—The cover-glass preparation must be heated to 210° C. for half an hour, or passed about twelve times through the flame of a Bunsen burner, or exposed to the action of strong sulphuric acid for several seconds, then a few drops of a watery solution of aniline dye applied in the usual way. To double-stain spore-bearing bacilli the cover-glass preparation must be floated from twenty minutes to an hour on Ehrlich’s fuchsine-aniline-water, or on the Ziehl-Neelsen solution. The stain must be heated until steam arises.
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.