

Staining 167 



Holler* finds it advantageous to prepare the films, before 

 staining, by immersion in chloroform for two minutes, fol- 

 lowing this by immersion in 5 per cent, chromic acid solu- 

 tion for one-half to two minutes. 



Anjeszky f recommends the following method of staining 

 spores, which is said always to give good results even with 

 anthrax bacilli: A cover-glass is thinly spread with the 

 spore-containing fluid and dried. While it is drying, some 

 0.5 per cent, hydrochloric acid is warmed in a porcelain 

 dish over a Bunsen flame until it steams well and bubbles 

 begin to form. When the solution is hot and the smear 

 dry, the cover-glass is dropped upon the fluid, which is 

 allowed to act upon the unfixed smear for three or four 

 minutes. The cover is removed, washed with water, dried, 

 and fixed for the first time, then stained with Ziehl's carbol- 

 fuchsin solution, which is warmed twice until fumes arise. 

 The preparation is allowed to cool, decolorized with a 4-5 

 per cent, sulphuric acid solution, and counterstained for a 

 minute or two with malachite green or methylene-blue. 

 The whole procedure should not take longer than eight to 

 ten minutes. 



FioccaJ suggests the following rapid method: " About 

 20 c.c. of a 10 per cent, aqueous solution of ammonium are 

 poured into a watch-glass, and 10 to 20 drops of a saturated 

 solution of gentian violet, fuchsin, methyl blue, or safranin 

 added. The solution is warmed until vapor begins to rise, 

 then is ready for use. A very thinly spread cover-glass, 

 carefully dried and fixed, is immersed for three to five 

 minutes (sometimes ten to twenty minutes), washed in 

 water, washed momentarily in a 20 per cent, solution of 

 nitric or sulphuric acid, washed again in water, then counter- 

 stained with an aqueous solution of vesuvin, chrysoidin, 

 methyl blue, malachite green, or safranin, according to the 

 color of the preceding stain. This whole process is said to 

 take only from eight to ten minutes, and to give remarkably 

 clear and beautiful pictures." 



Method of Staining Flagella. This is somewhat more 

 difficult than the staining of the bacteria or their spores. 



Loffler's Method.^ This is the original and best method, 



* " Centralbl. f. Bakt. u. Parasitenk.," Bd. x, p. 273. 

 t Ibid., Feb. 27, 1898, xxm, No. 8, p. 329. 

 t Ibid., July i, 1893, xiv, No. i. 

 Ibid., 1890, Bd. vii, p. 625. 



