FLAGELLA STAINING 115 



platinum wire as quickly as possible. The film thus made should 

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



Age of the culture. A twenty -four hours' culture does quite well 

 (a younger one is perhaps better, but flagella can be shown for a 

 week or fortnight or more). 

 I. The mordant : 



Osmic acid, 2 per cent. . . .1 part 

 Tannic acid, 20 per cent, watery solution 3 or 4 parts 

 II. Silver solution : Silver nitrate . . 1 per cent. 



III. Gallic acid, 2 per cent, solution . . 1 part 

 Ammonia fort. ... .1 part 



To be mixed before using and to be used immediately. 



To stain. Place the mordant on the film for one or two minutes 

 or less (time unimportant). 



1. Wash in tap- water thoroughly. 



2. Shake off as much water as possible. 



3. Place a few drops of silver nitrate on the slide for a few seconds 

 or longer. 



4. Shake off all excess. 



5. Allow one drop of the ammonia-gallic solution to fall on the 

 middle of the slide from a small pipette. A wave spreads away 

 from the centre to each end of the slide. As soon as the film is 

 seen standing out clearly and black in the centre (in a few seconds), 

 wash off in tap-water. 



6. Add again a drop or two of the silver solution and allow it to 

 act for half a minute or thereabouts. 



7. Wash in tap-water, blot, and dry over the flame. 



8. It is best not to mount in balsam or in cedar-wood oil, as the 

 preparations rapidly fade in these. 



If done with any care, the film should now appear black and 

 distinct to the naked eye with no precipitate, and the flagella will 

 be found to be stained distinctly and intensely with hardly any 

 ground substance, or at least insufficient to interfere with a clear 

 view of them. 



(6) 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 



