184 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 



Bacteria which retain the initial stain Gram-positive bacteria 

 are colored dark purple or blue; those which fail to retain the initial 

 stain Gram-negative bacteria are brown, or bright pink if fuchsin 

 is used as a counterstain. 



2. Stains for Acid-fast Bacteria. Ziehl-Neelsen Method. 1 (a) Stain 

 dried and fixed smear with carbol fuchsin, as described on page 180. 



(6) Wash thoroughly with water. 



(c) Decolorize with acid alcohol 2 until no more color can be washed 

 out. 



(d) Wash with water. 



(e) Counterstain lightly with Loffler's alkaline methylene blue. 

 (/) Wash, dry, and mount. 



Acid-fast bacilli and spores red; all others blue. 



3. Frankel-Gabbet Method. 3 (a) Stain with carbol fuchsin as in 

 the Ziehl-Neelsen method and wash in water. 



(b) Decolorize and counterstain simultaneously with the following 

 solution: 



Methylene blue , 2 grams 



Water 75 c.c. 



Sulphuric acid 25 c.c. 



The counterstain is allowed to act for one minute. 



(c) Wash, dry, and mount. 



4. Polychrome Stains. Polychrome stains are of special value for 

 the examination of exudates, body fluids or tissues in which the his- 

 tological relations of bacteria are to be investigated. These stains, 

 or modifications of them, are also useful in the study of treponemata, 

 spirochetes, and protozoa. 



Wright's Stain* Preparation. "To a 0.5 per cent, aqueous 

 solution of sodium bicarbonate add methylene blue (B.X., or 'medi- 

 cinally pure') in the proportion of 1 gm. of the dye to each 100 c.c. 

 of the solution. Heat the mixture in a steam sterilizer at 100 C. for 

 one full hour, counting the time after the sterilizer has become thor- 

 oughly heated. The mixture is to be contained in a flask, or flasks, 

 of such size and shape that it forms a layer not more than 6 cm. deep. 

 After heating the mixture is allowed to cool, placing the flask in cold 

 water if desired, and is then filtered to remove the precipitate which 



1 Ziehl, Deutsch. med. Wchnschr., 1882, 451; Neelsen, Fort. d. med., 1885, 200. 



2 Ninety per cent, alcohol containing 3 per cent, by volume of hydrochloric acid. 



3 Frankel, Berl. klin. Wchnschr., 1884; Gabbet, Lancet, 1887. 



4 Mallory and Wright, Pathological Technic, 1915, 6th ed., p. 382. 



