Staining 151 



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 10 



Cold saturated aqueous solution of ferrous sul- 

 phate 5 



Alcoholic solution of fuchsin or methyl violet 1 



(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. 



Some of the culture to be stained is mixed upon a cover- 

 glass with a drop of distilled water making a first dilution, 

 which is still too rich in bacteria to permit the flagella to 

 show well, so that it is recommended to prepare a second 

 by placing a small drop of distilled water, upon a cover 

 and taking a loopful from the first dilution to make the 

 second, and spreading it over the entire surface without 

 much rubbing or stirring. The film is allowed to dry, and 

 is then fixed by passing it three times through the flame. 

 When this is done with forceps there is some danger of the 

 preparation becoming too hot, so LofBer recommends that 



* "Centralbl. f Bakt. u. Parasitenk.," July 1, 1893, xiv, No. 1. 

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



