112 STAINING OF BACTERIA 



Bunge's Method. 1. Prepare and fix cover-glasses as described 

 under the Loeffler method. 



2. Use the following mordant, which must be several days old : 



Concentrated watery solution of tannic acid . . ... 75 c.c. 

 5 per cent, watery solution of liquor ferri sesquichlorati . . 25 c.c. 

 Concentrated aqueous solution of fuchsin . . . .' . . 10 c.c. 



Place mordant in a beaker, float cover-glasses, prepared side down 

 on surface of mordant, heat over a small flame until fluid begins to 

 steam, but do not allow it to boil. 



3. Wash well in distilled water. 



4. Stain with carbol-gentian-violet solution, which is heated until 

 it steams on the cover-glass. 



5. Wash in water, dry, mount in Canada balsam. 



Carbol gentian violet is prepared like carbol-fuchsin, viz., take 



Gentian violet 1 gram 



Absolute alcohol 10 c.c. 



Aquae dest 100 c.c. 



Carbolic acid 5 c.c. 



Carbol fuchsin may be used instead of the carbol gentian violet. 



Pitfield's Method. Prepare the following two solutions, keep them 

 separate, and before use filter and mix. 



A. Saturated aqueous solution of alum 100 c.c. 



Saturated alcoholic solution gentian violet 10 c.c. 



B. Tannic acid 10 grams 



Aquae dest 100 c.c 



Mix in equal proportions before use. Cover film with staining 

 fluid, heat over a small flame until the stain boils, leave on for one 

 minute, wash well in water, dry, and mount in Canada balsam. 

 The following is not a real staining but a silver impregnation method : 

 Van Ermengem's Method of Silvering Flagella. 1. Prepare cover- 

 glasses as already indicated, and apply the following mordant, which 

 may be used hot for five minutes or cold for thirty minutes. Take 



20 per cent, watery solution of tannic acid ..... 60 c.c. 



2 per cent, watery solution of osmic acid . ... . 30 c.c. 



Glacial acetic acid . . . . . . .,,.-. .' . . 4 to 5 drops 



2. Wash in water. 



3. Wash in alcohol. 



4. Immerse for one to three seconds in \ to 1 per cent, watery 

 solution of nitrate of silver. 



5. W T ash for several seconds in the following solution : 



Gallic acid . . '. . . , . . . . .7 5 grams 



Tannic acid .... . . . . ... . . . . 3 grams 



Acetate of sodium . .... V . 10 grams 



Distilled water * : .V . . . . . 350 c.c. 



6. Immerse again in the \ to 1 per cent, watery solution of nitrate 

 of silver and move cover-glass, held in forceps, continually until 

 it assumes a black color. 



7. Wash well in water, dry, mount in Canada balsam. 



