FLAGELLA STAINS. 



191 



Fischer's Flagella Stain. 



This is a slight modification of Dr. Corner's, 

 which is itself a modification of Loeffler's. 



(1) Mordant: 



Dry tannin 2 



Water 20 



Solution ferrous sulphate (1:2).... 4 

 Concentrated alcoholic solution basic 



fuchsin i 



Filter. This mordant will keep for several 

 weeks. Drop it on the cover and heat over a 

 gentle flame until steam rises ; then continue one- 

 half minute longer without boiling. Wash. 

 Treatment with alcohol is unnecessary. 



(2) Stain : Place on the cover a few drops of 

 concentrated watery solution of basic fuchsin. 

 Heat slowly, so that steam rises after about i 

 minute. Then expose for one-half minute 

 longer, so that the stain boils up once or twice. 

 Wash, dry, and mount. Fischer says tannin ab- 

 sorbs moisture readily, and advises keeping it 

 in a desiccator. 



Bunge's Flagella Stain. 



(1) Mordant: 



Tannin 20, water 100 30 



Liquor ferri sesquichlorati i 



Water 20 



Saturated watery solution basic 



fuchsin 5 



This must ripen some weeks by exposure to 

 the air in a flask loosely plugged with cotton. 



(2) Stain : Carbol-fuchsin. 



Expose to the filtered mordant 5 minutes, 

 using heat if necessary. Wash and stain. 



Van Ermengem's Flagella Stain. 

 This is made as follows : 



(1) Mordant: 



Osmic acid (2 per cent water solu- 

 tion) 50 



Tannin (10 to 25 per cent in water) .. 100 

 Four drops of glacial acetic acid may be added 

 to this. 



(2) Silver bath: 0.25 to 0.5 per cent nitrate of 

 silver dissolved in distilled water in a very clean 

 bottle. 



(3) Reducing and strengthening bath : 



Gallic acid 5 



Tannin 3 



Fused sodium acetate (some books 



say fused potassium acetate) 10 



Distilled water 3SO 



The flamed cover-glass (it may be unflamed) 

 is first covered with the mordant for one-half 



hour, or if in a thermostat at 50 C. for 5 or 10 

 minutes. The mordant is then carefully re- 

 moved by thorough washing in water, alcohol 

 (some say absolute alcohol), and water. The 

 cover (film side up) is now put into the silver 

 bath (a few cubic centimeters in a small, per- 

 fectly clean beaker or watch-glass) for a few 

 seconds, during which it is gently agitated. 

 Without rinsing, it is put next into a few cubic 

 centimeters of the reducing solution and gently 

 agitated until the fluid begins to blacken. It is 

 then washed in water and examined. If not 

 stained deeply enough the cover is returned to 

 the silver bath, then once more passed through 

 the reducing bath. It is finally dried and 

 mounted in balsam. All the dishes must be 

 scrupulously clean. The fluids must not be con- 

 taminated by the fingers nor by dipping iron or 

 steel instruments into them. 



Kuntze has suggested some improvements. 

 (See Centralb. f. Bakt, I Abt., Bd. XXXII, 

 1902, pp. 555-560.) 



PMeld's Flagella Stain. 



1 i ) Mordant : 



Tannic acid, 10 per cent aq. sol 10 



Corrosive sublimate, sat. aq. sol 5 



Alum, sat. aq. sol 5 



Carbol-fuchsin 5 



Heat on cover until steam rises; keep at this 

 temperature i minute; then wash, dry, and 

 stain. 



(2) Stain : 



Alum, sat. (cold) aq. sol 10 



Gentian violet, sat. ale. sol 2 



Kendall, in Journ. Applied Micr., Vol. V, 

 1902, p. 1836, says this has proved a very satis- 

 factory stain to himself and his associates. 



Another formula is given as follows : 



(1) Saturated (cold) aqueous solution of 



alum 10 



Saturated alcoholic solution of gentian 

 violet i 



(2) Tannic acid (tannin) i 



Distilled water 10 



A mixture of these two fluids is put on the 

 flamed cover, which is held over the flame and 

 gently heated until nearly ready to boil. The 

 cover is then put aside for i minute, after which 

 it is washed in water, dried, and mounted in bal- 

 sam. If the mixed mordant-stain is filtered be- 

 fore using, it is best to stain a second time for a 

 moment in anilin-water gentian violet. 



