Staining 189 



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. 



Fiocca* 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, 

 though somewhat cumbersome, and hence rarely employed 

 at the present time. Three solutions are used : 



(A)- 



Twenty per cent, aqueous solution of tannic 

 acid 3 .. 10 



Cold saturated aqueous solution of ferrous sul- 

 phate 5 



Alcoholic solution of fuchsin or methyl violet 



(B) One per cent, aqueous solution of caustic soda. 



(C) An aqueous solution of sulphuric acid of such strength 

 that i c.c. will exactly neutralize an equal quantity of 

 solution B. 



* "Centralbl. f. Bakt. u. Parasitenk.," July i, 1893, xiv, No. i. 

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



