38 STAINING METHODS 



7. Counterstain with methylene blue one-half minute. 



8. Dehydrate in alcohol. Clear in xylol and mount. (May simply dry speci- 

 men with filter-paper.) 



Rosenow's Capsule Stain. Make a very thin smear of the pathological material 

 and when nearly dry cover the preparation for ten to twenty seconds with 10% 

 tannic acid solution. Wash in water and blot. Stain with aniline gentian violet 

 by gently steaming for one-half to one minute. Wash in water. Apply Gram's 

 iodine solution for one-half to one minute. Decolorize in 95% alcohol and then stain 

 with alcoholic solution of eosin. Wash in water, dry and mount. 



Flagella Staining. Inoculate a tube of sterile water (gently) in 

 upper part, with just enough of an eighteen to twenty-four-hour-old 

 agar culture to produce faint turbidity. Incubate for two hours at 37 

 C. From the upper part of culture take a loopful and deposit it on a 

 cover-glass. Dry in thermostat for one to five hours or over night. 

 Use perfectly clean cover-glasses. To stain by 



Muir's Modified Pitfield Method. 



i. Flood specimen with mordant. Steam gently one minute. 



Mordant. Tannic acid (10% aqueous solution), 10 c.c. 



Sat. aq. sol. mercuric chloride, 5 c.c. 



Sat. aq. sol. alum, 5 c.c. 



Carbol fuchsin, 5 c.c. 



Allow precipitate to settle or centrifuge. Keeps only one week. 



2. Wash well in water for two minutes. 



3. Dry carefully preferably in incubator. 



4. Pour on stain. Steam gently one minute. 



Stain. Sat. aq. sol. alum, 10 c.c. 



Sal. ale. sol. gentian violet, 2 c.c. 



(May use carbol fuchsin instead of gentian violet.) 

 Stain only keeps two days. 



5. Wash well in water. Dry and mount. 



Zettnow's Flagella Staining Method. 



Solution I. Dissolve 2 grams of tartar emetic in 40 c.c. water. 



Solution II. Dissolve 10 grams tannin in 200 c.c. water. To the 200 c.c. 

 solution II, warmed to 50 or 60 C., add 30 c.c. of the tartar emetic solution. 

 The turbidity of the mordant should entirely clear up on heating. The mordant 

 should keep for months when a small crystal of thymol is added to it. 



Next dissolve i gram silver sulphate in 250 c.c. distilled water. Of this solution 

 take 50 c.c. and add to it drop by drop ethylamine (this comes in a 33% solution) 

 until the yellowish-brown precipitate which forms at first is entirely dissolved and 

 the fluid is entirely clear. It requires only a few drops. The bacterial preparations 

 prepared as described above are floated in a little mordant contained in a Petri dish 



