FLAGELLA STAINING 131 



THE MORDANT 



A. Ferric chloride (1-20 aqueous solution) . 1 part 

 Saturated aqueous solution of tannic acid . 3 parts 



This solution improves with age and should be filtered before 

 use. 



B. Anilin oil 1 part 



95 per cent, alcohol .... 4parts 



Eight drops of. A are placed on the film and one drop of B is 

 immediately added. The mixture is allowed to act for two minutes 

 and is then washed off with water, and the water is drained off 

 and absorbed by touching the edge of cover-glass or slide with 

 filter-paper. The film is then flooded with the stain, which may 

 be carbol-fuchsin, ariilin gentian violet or Loffler's methylene 

 blue. The staining solution to be preferred is prepared as follows, 

 and keeps well : 



Loffler's methylene blue . . . .30 c.c. 



Solution B of mordant ..... 3 c.c. 



The stain is allowed to act for two to three minutes, washed 

 thoroughly with water and allowed to dry. The mordanting and 

 staining are done at room temperature, and distilled water should 

 be used throughout. 1 



This is a modification of Loffler's original flagella stain, and we 

 have found it work well at the King's College Laboratory. 



(b) Pitfield's method. Two solutions are freshly prepared : 



A. Saturated aqueous solution of alum . . 10 c.c. 

 Saturated alcoholic solution of gentian violet 1 c.c. 



B. Tannic acid ...... 1 grm. 



Distilled water . . . . .10 c.c. 



The solutions should be made with cold water, filtered, and 

 preserved in separate bottles. For use equal quantities are mixed 

 together. The specimens are flooded with the mixture and held 

 over the flame until it nearly boils : they are then laid aside, with 

 the hot stain on them, for one minute, and are finally washed 

 in water. After washing, the preparations are flooded with 

 anilin gentian violet for one second, washed in water, dried and 

 mounted. 



1 Journ. of Bacteriology, v, 1920, p. 181. 



92 



