152 FLAGELLUM STAINING 



Pour these solutions into a well plugged flask : heat the mixture to 100 C. in a 

 water bath, thoroughly mixing the ingredients by shaking and continue to heat 

 until the liquid is pale in colour. The solution must be kept in an hermetically 

 sealed bottle. 



1. Mordanting. Flood the cover-glass with a few drops of the mordant for 2 or 

 3 minutes at 25 C. or 10 minutes at 15 C. Wash in water. Dry. 



2. Staining. Stain in the following solution for a few seconds, heating the 

 solution until steam just begins to rise : 



Fuchsin, 0'25 gram. 



Absolute alcohol, - - 10 c.c. 



Carbolic acid crystals, - 5 grams. 



Distilled water, - - 100 c.c. 



3. Mounting. Wash in water. Dry. Mount in balsam. 



(vii) Fitfield's method. Benignetti and Gino's method. 



In Pitfield's method the mordant and the stain are combined in one 

 solution, thus : 



Saturated aqueous solution of alum, - 10 c.c. 



10 per cent, solution of tannin in water, - - 10 



Saturated alcoholic solution of gentian-violet, - 2 



Benignetti and Gino obtain very satisfactory results with the following 

 very simple method which is a modification of the above. 



The combined mordanting and staining solution is prepared thus : 



A. Zinc sulphate, 

 Tannin, 

 Distilled water, 



B. Solution A, - 



Saturated aqueous solution of alum, 

 Saturated alcoholic solution of gentian-violet, 



1 gram. 

 10 grams. 

 100 



5 c.c. 

 5 

 3 , 



Technique. Fix the film with heat, and when cool flood it with a large 

 drop of solution B, and heat it over the pjrot light of a Bunsen until steam 

 just begins to rise. Wash in water. Dry. Mount in balsam. 



[R. Muir's modification of Pitfield's method. 



[Prepare : 



A. Filtered 10 per cent, aqueous solution of tannin, - 10 c.c. 



Saturated aqueous solution of perchloride of mercury, 5 



Saturated aqueous solution of alum, 5 



Carbol-fuchsin, - 5 



[Mix thoroughly. Allow the precipitate to settle. Decant off the clear 

 supernatant fluid. The mordant will keep for about a fortnight. 



B. Saturated aqueous solution of alum, - 10 c.c. 



Saturated alcoholic solution of gentian- violet, - 2 



The stain does not keep. 



[1. Prepare and fix a film as above (p. 149). 



[2. Flood the preparation with the mordant and heat until steam just 

 begins to rise. Let the solution act for 1 minute. 



[3. Wash thoroughly in running water. Blot and dry over the flame. 



[4. Flood the film with the stain, heat as before for 1 minute. 



[5. Wash well in water. 



[6. Blot. Dry. Mount in balsam. ] 



(viii) Bowhffl's method. 



This method is troublesome and offers no special advantages. 



1. Mordanting. Place the film for 10 minutes at a temperature of 40-50 C. hi a 



