FLAGELLUM STAINING 151 



(iv) Nicolle's and Morax's method. 

 (Method recommended.) 



This method, a simplification of Loeffler's, does away with the use of the 

 acid and alkaline solutions and is a satisfactory stain for the flagella of all 

 motile organisms. Carbol-fuchsin is used instead of Loeffler's stain. 



1. Mordanting. Place a large drop of fuchsin ink (without any addition 

 of acid or alkali) on the film. Heat for 10 seconds over the pilot flame of a 

 Bunsen. 



When steam begins to rise, pour off the solution, tilt the cover-glass and 

 run a gentle stream of water from a wash-bottle on to its upper angle so as 

 to wash the film well without washing the organisms away. 



Repeat the mordanting and washing two or three times. Wipe the under 

 surface of the cover-glass and the teeth of the forceps each time after 

 washing : otherwise when the mordant is poured on again the solution will 

 run under the cover-glass and along the forceps. 



2. Staining. Put a drop of carbol-fuchsin on the film and heat once or 

 twice until steam has been rising for 15 seconds. 



3. Mounting. Wash and examine in water. If the preparation be satis- 

 factory, dry and mount in balsam. 



Bunge's and de Rossi's methods. These methods differ slightly from that of 

 Nicolle and Morax but have no advantage over the latter. 



Bunge's mordant. 



Saturated aqueous solution of tannin, - 3 parts. 



1 in 20 aqueous solution of perchloride of iron, - 1 part. 



To ten parts of this mixture add one part of a saturated aqueous solution of 

 fuchsin. The mordant must be exposed to the air for a few weeks before use ; 

 while so exposed it acquires a brownish -red colour. 



Filter a few drops of the above solution on to the film and leave it for 5 minutes : 

 wash in water and dry : stain with carbol-fuchsin as in Nicolle and Morax's 

 method : wash, dry and mount inbalsam. 



De Rossi treats the film for 10 minutes with the following solution : 



Tannin, 5 grams. 



O'l per cent, aqueous solution of potash, - - 100 c.c. 



Wash in water. Dry. Stain with carbol-fuchsin as in Nicolle's and Morax's 

 method. Wash. Dry. Mount. 



(v) Trenkmann's method. 



This method gives satisfactory results but it takes too long to be of use 

 in routine work. 



1. Leave the film in the following solution for 6-8 hours : 



Tannin, 2 grams. 



Distilled water, - - - 100 c.c. 



Pure hydrochloric acid, - 4 drops. 



2. Wash in water. Treat the film for 1 hour in a watch-glass containing a 

 saturated solution of metallic iodine in distilled water. 



3. Wash in water. Stain in aniline-gentian-violet for half an hour. 



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



(vi) Cerrito's method. 



This method requires a good deal of care, because the mordant frequently 

 gives rise to troublesome deposits. 



The mordant consists of the following somewhat complex mixture : 

 25 per cent, aqueous solution of tannin in ether, - 20 c.c. 



5 per cent, aqueous solution of pure iron-alum, - 10 ,, 



Saturated solution of fuchsin in 90 per cent, alcohol, - 1 



