MICROSCOPIC STUDY AND STAINING 105 



Decolorize with thirty-three per cent nitric acid one-half to one 

 minute. 



Treat with sixty per cent alcohol, until no color can be seen to come 

 off. 



Counterstain with aqueous methylene-blue. 



Rinse in water, dry, and mount. 



Ziehl-Neelson Method.' — Thin smears are made upon cover- 

 slips or slides. 



Fix by heat. 



Stain in carbol-fuchsin solution as given on page 97. The slide 

 or cover-slip may be flooded with the stain, and this gently heated with 

 the flame until it steams, or else the cover-slip may be inverted upon 

 the surface of the staining fluid, in a porcelain dish or watch-glass, and 

 this heated until it steams. This is continued for three to five min- 

 utes. Decolorize with either five per cent nitric acid, five per cent 

 sulphuric acid, or one per cent hydrochloric acid for three to five 

 seconds. The treatment with the acid is continued until subsequent 

 washing with water will give only a faint pink color to the preparation. 



Wash with ninety per cent alcohol until no further color can be re- 

 moved. If, after prolonged washing with alcohol, a red color still re- 

 mains in very thick places upon the smear, while the thin areas appear 

 entirely decolorized, this may be disregarded. 



Wash in water and counterstain in aqueous methylene-blue for 

 one-half to one minute. 



Rinse n water, dry, and mount. 



By this method the tubercle bacilli are colored red, other bacteria 

 and cellular elements which may be present are stained blue. 



Gabbet's Method.^ — Gabbet has devised a rapid method in which 

 the decolorization and counterstaining are accomplished by one solu- 

 tion. The specimen is prepared and stained with carbol-fuchsin as in 

 the preceding method. It is then immersed for one minute directly in 

 the following solution: 



Methylene-blue 2 gms. 



Sulphuric acid 25 per cent (sp. gr. 1018) 100 c.c. 



Then rinse in water, dry, and mount. 



This method, while rapid and very convenient, is not so reliable as 

 the Ziehl-Neelson method. 



1 Ziehl, Deut. med. Woch., 1882; Neelson, Deut. med. Woch., 1883. 



2 Gahbet, Lancet, 1887. 



