150 FLAGELLUM STAINING 



The following reagents are required : 



Fuchsin ^ ink. 



25 per cent, aqueous solution of tannin, - - 10 c.c. 



Cold saturated solution of ferrous sulphate, - 5 



Saturated alcoholic solution of fuchsin, - * 1 



Alkaline solution. 



Alcoholic soda, - 1 gram. 



Distilled water, - - 100 c.c. 



Acid solution. 



Pure sulphuric acid, 1 gram. 



Distilled water, - 100 c.c. 



Staining solution. 



Aniline water, - 100 c.c. 



1 per cent, solution of soda, - 1 



Gentian-violet or fuchsin, 4 to 5 grams. 



Shake. Leave for a few hours in a bottle. Filter. 



Technique. 1. Mordanting. Drop on to a film prepared as above (see general 

 rules) a large drop of the fuchsin ink containing a few drops of the acid or alkaline 

 solution. The amount of the latter depends upon the species of organism under 

 examination. Heat the film over the pilot flame of a Bunsen until steam just 

 begins to rise and continue the heating for 30-50 seconds : be careful not to boil 

 the liquid. This stage of the method is very tricky and is liable to failure. 



The amount of the acid or alkaline solution to be added to 16 c.c. of fuchsin ink 

 has been determined by experiment. The following table shows the amounts 

 necessary for the principal ciliated bacteria. 



Mordant alone without acid or alkali, - - Spirillum concentricum. 



Mordant + -1 drop of acid solution, - - V. cholerse asiaticse. 



+6 drops - B. pyocyaneus. 



+ 18-20 - Micrococcus agilis. 



+20 - B. chauvsei. 



+ 20-30 drops of alkaline solution, - - B. febris entericae. 



+ 28-30 - B. subtilis. 



+26-28 ,, ,, - B. maligni OBdematis. 



. (from 20 drops of acid solution "\ T> . f , , .,, 



" + { to 15 drops of alkaline solution, } 



2. Washing. Wash in water, then in absolute alcohol. 



3. Staining. Place a drop of the staining solution on the film, heat until steam 

 begins to rise gently, and let the hot stain act for about a minute. 



4. Mounting. Wash in a large volume of water and examine in water. If 

 satisfactory, dry and mount in balsam. 



(iii) Remy and Sugg's method. 



This method, which is a modification of Loeffler's, is designed to avoid the 

 formation of granular precipitates. The fuchsin solution is used cold, and 

 after staining the film is treated with iodine. 



Instead of Loeffler's the following stain is used : 



Staining solution. 



Phenylamine water, 1 .- - 20 c.c. 



Alcoholic solution of gentian-violet, - 1 drop. 



Distilled water, ... 5 c.c. 



Mix the gentian-violet with the water and then add the phenylamine water. 



Technique. 1. The mordanting process is the same as in Lceffler's method but 

 the solution is left on the film for 15-30 minutes and is not heated. 



2. Pour off the mordant and replace it at once with a drop of Gram's solution. 



3. Wash in water, then in absolute alcohol. 



4. Place the film in a watch-glass filled with the stain and leave it for half an 

 hour, preferably in the warm (37C.) incubator. 



5. Wash in water. Examine in water. Dry. Mount in balsam. 



1 Prepare in a similar manner to aniline oil water (p. 139). 



