FLAGELLUM STAINING 149 



and its action on these is uncertain. The best results are obtained with Vibrio 

 cholerce asiaticce, V. finkler-prior, V. metchnikowi. The method fails altogether 

 to stain the flagella of many organisms 'such as B. febris entericce, B. coli communis, 

 B. subtilis, etc. 



2. The staining of flagella in dried preparations. 

 General rules. 



1. Take a small quantity of a young agar culture and make a perfectly 

 homogeneous, very faintly opalescent emulsion in a watch-glass containing 

 ordinary water, or preferably distilled water. 



2. Fix an absolutely clean cover-glass in a pair of Cornet's forceps, pass it 

 through the flame, and when cool place a drop of the emulsion on it with a 

 pipette. Unless the cover-glass be perfectly clean the liquid will not spread 

 uniformly. 



3. By tilting the cover-glass run the liquid over the surface, then let the 

 excess gravitate to one corner and aspirate it with a pipette. 



4. Dry the film in the air away from dust and, without fixing, stain by one 

 of the methods described below. 



By following the above instructions, a dilution is obtained such that each field 

 of the microscope contains only a few organisms, which is an essential condition 

 for good results. By this method, also, the mucoid substances which agglomerate 

 organisms in cultures and form precipitates on the cover-glass (thus obscuring the 

 details) are as far as possible excluded. 



(i) Van Ermengem's method. 

 (Method recommended.) 



This method is based on the reduction of nitrate of silver in the flagella and 

 gives very beautiful preparations. It is generally used in preference to any 

 other method. [Vide also method (xi) p. 153.] 



1. Place the film for 1 minute at 50 C. (or 30 minutes at room temperature) 

 in the following bath which must be freshly prepared : 



2 per cent, aqueous solution of osmic acid, 8 c.c. 



10 per cent, aqueous solution of tannin, - - 16 



Glacial acetic acid, 1 drop. 



2. Wash in water, then in absolute alcohol. 



3. Treat the film with silver for 1 or 2 minutes. 



Crystals of silver nitrate, 1 gram. 



Distilled water, - - 200 c.c. 



4. Without washing, transfer the film for 1 minute to the reducing 



solution. 



Gallic acid, - 5 grams. 



Tannin, 3 



Fused sodium acetate, - 10 



Distilled water, - - 350 c.c. 



5. Without washing, put the preparation into the silver bath again and 

 keep the liquid moving over the film until the latter assumes a black tint. 



6. Wash. Dry. Mount in balsam. 



(ii) Lceffler's method. 



The method devised by Lceffler, for a long time the classical method of 

 staining flagella, requires a good deal of practice and gives only mediocre 

 results. The films are often covered with an abundant precipitate which 

 obscures the flagella and renders their detection difficult. 



