FLAGELLUM STAINING 153 



bath consisting of equal parts of the following solutions mixed just before use and 

 filtered : 



SOLUTION A. 



Orcein, - 1 gram. 



Absolute alcohol, - - 50 c.c. 



Distilled water, - - 40 



SOLUTION B. 



Tannin, - 8 grams. 



Distilled water, - - 40 c.c. 



Use heat to dissolve the tannin. 



In staining the flagella of the Vibrio cholerce asiaticce add 1 c.c. of a saturated 

 solution of alum for each 10 c.c. of mordant. 



2. Washing. Wash in water. Dry. 



3. Staining. Flood the film with aniline-gentian-violet and heat until steam 

 just rises from the film for 15-30 seconds. 



4. Mounting. Wash. Dry. Mount in balsam. 



(ix) Gemelli's method. 



1. Immerse the films in the following solution for 10-20 minutes. 



Potassium permanganate, - - 0'25 gram. 



Distilled water, - - 100 c.c. 



2. Wash in distilled water. 



3. Stain for 15-30 minutes in the following mixture : 



0'75 per cent, aqueous solution of calcium chloride, - - 20 c.c. 

 1 per cent, aqueous solution of neutral-red, - - 1 



4. Wash in water. Dry in the air. Mount in balsam. 



(x) Sclavo's method. 



Sclavo's method fails to stain the flagella of some micro-organisms especi- 

 ally the flagella of the cholera vibrio. The author's experience has been 

 that it is equally unsuited for the flagella of the colon bacillus. 



1. Flood the film with a large drop of the mordant, viz. : 



50 per cent, alcohol, ..... . 100 c.c. 



Tannin, 1 gram. 



Leave for 1 minute and then wash in water. 



2. Treat for 1 minute with the following solution on the film : 



Phospho-tungstic acid, ------- 5 grams. 



Water, - - - 100 c.c. 



3. Wash quickly in water. 



4. Stain for 3 or 5 minutes with a drop of aniline-gentian-violet heating the 

 stain until steam rises gently from the film. 



5. Wash and examine in water. Dry. Mount hi balsam. 



| (xi) Stephens' method.] 

 (Method recommended.) 



[The method worked out by J. W. W. Stephens is a modification of van 

 Ermengem's (p. 149) and depends upon the use of very strong ammonia as 

 the reducing agent. With ordinary care a satisfactory result can be absolutely 

 relied upon. 



[To clean the slides. Rub the slides with a clean cloth, place them on a piece of 

 clean wire gauze and heat with a smokeless flame for some minutes (by this means 

 grease is completely removed). Leave the slides until cool. 



[To prepare the film. Rub a little of the culture in a small drop of tap-water in 

 a watch-glass. Transfer a drop with a very small platinum loop to a minute drop 

 of water on the slide. Mix. Spread with the loop as quickly as possible. The 

 film should dry immediately if only a small drop of water has been used. A 



