30 STAINING METHODS. 



three hundred partis) ; then washed in alcohol, dried, and, if for per- 

 manent preservation, mounted in balsam. 



METHODS OF STAINING THE TUBERCLE BACILLUS. Numerous 

 methods of staining the tubercle bacillus in sputum dried upon a 

 cover glass have been proposed, but we shall only give here two or 

 three of the most approved methods, either one of which may be 

 relied upon for satisfactory results if carefully followed. 



1. The Ehrlich-Weigert Method. Place in a watch glass a little 

 of the aniline-methyl-violet solution (No. 2); float upon the surface 

 of this the cover glass with the dried film downward ; heat over a 

 small flame until it begins to steam, then allow it to stand for from 

 two to five minutes ; decolorize in a tray containing one part of nitric 

 acid to three parts of water the cover glass, held in forceps, is gently 

 moved about in the decolorizing solution for a few seconds. It is 

 then washed off in sixty-per-<5ent alcohol to remove the remaining 

 blue color this usually takes but a second or two and then in water. 

 For a contrast stain a saturated aqueous solution of vesuvin may be 

 used, a few drops being left upon the cover glass for five minutes. 

 The stained preparation is then washed, dried, and mounted in 

 balsam. 



2. The Ziehl-Neelson Method. Float the cover glass upon the 

 carbol-f uchsin solution (No. 3) ; heat gently until steam commences 

 to rise from three to five minutes' time will usually be sufficient ; 

 wash off in water, and decolorize in nitric or sulphuric acid, twenty- 

 five-per-cent solution, then in sixty-per-cent alcohol for a very short 

 time to remove remaining color from albuminous background; wash 

 well in water and mount in Canada balsam. 



3. Friedldnder's Method. Spread and dry the sputum upon 

 the slide ; fix by passing the slide three times through the flame of 

 an alcohol lamp or Bunsen burner ; place upon the dried film three or 

 four drops of carbol-fuchsin (No. 3); heat gently over a flame until 

 steam is given off ; wash in a dish of distilled water ; drain off excess 

 of water, and add a few drops of the following decolorizing solution : 



Acid, nitric, pure, . . . . . 5 cc. 



Alcohol (eighty per cent), . . . .to 100 cc. 



- usually the preparation will be decolorized in about half a minute ; 

 wash in water ; add a few drops of an aqueous solution of methylene 

 blue as a contrast stain ; allow the stain to act for about five minutes, 

 without heating ; wash again in water, dry, and mount in balsam, 

 or for a temporary mount use a drop of cedar oil. 



4. Gabbett's Method. This is a slight modification only of a 

 very useful method recommended by B. Frankel in 1884. The con- 

 trast stain is added to the decolorizing solution. After staining with 



