Much practice is required in using either of the methods before one can judge accurately how long to leave sections in the staining reagents or decolourising agents, and the beginner must not be discouraged if at first he is unable to obtain good results although he follows the book directions most minutely.
Ehrlich method for tubercle bacilli.—Sections are stained for six to twenty-four hours in a one per cent. solution of gentian violet, methyl violet, methyl blue or fuchsine. They will stain more rapidly if the staining fluid be kept in an incubator at the body temperature. They should be removed from the staining fluid, and washed in distilled water, and then transferred (preferably on a glass section lifter) to a ten per cent. solution of nitric acid in distilled water until they are nearly decolourised. They should then be very thoroughly washed in distilled water. They may then be treated with some suitable contrast stain and mounted in Canada balsam.
Neelsen’s stain for tubercle bacilli.—Sections are placed in Ziehl’s carbol-fuchsine solution (p. [103]) which should be warmed for ten minutes to half-an-hour. They are then decolourised in a solution of sulphuric acid. Twenty-five per cent. is the strength originally recommended, but a ten per cent. solution does equally well and injures the section less. They are then very thoroughly washed in a large quantity of water, and afterwards may be treated with a contrast stain.
Gibbes’ double stain for tubercle bacilli.—
| (1) | Rosaniline hydrochlorate | 2 | grms. | 25 | grs. |
| Methyl blue | 1 | grm. | 12·5 | grs. |
Triturate in a glass mortar,
| (2) | Aniline oil | 3 | c.c. | 37·5 | grs. |
| Rectified spirit | 15 | c.c. | 3 1/2 | drms. |
Dissolve and add slowly to (1).
| (3) | Lastly add slowly to the mixture | ||||
| Distilled water | 15 | c.c. | 3 1/2 | drms. | |
Some of the solution is filtered into a watch glass and warmed. The sections are placed in it and left for some hours. They are then washed in methylated spirit till they are sufficiently decolourised, and then rapidly passed through absolute alcohol and oil of cloves and mounted in balsam and xylol. It is a very useful stain for examining the sputum for tubercle bacilli.