ROMANOWSKY METHODS OF STAINING. 149 



three ingredients are well mixed, add 5 c.c. of glycerin. Mix, then add 

 15 c.c. of alcohol; again mix, and finally add 30 c.c. of distilled water. 

 Keep the mixed stain about i week before using. The best fixatives 

 are heat and Whitney's modified Zenker. To use: Stain films from 

 2 to 5 minutes. Then wash and mount. The tri-acid stain is a good 

 tissue stain. The objections to the triacid stain are that it does not 

 stain malarial parasites or mast cells and that failure to obtain good 

 results is of frequent occurrence. 



Wright's Method. The stain is made by adding r gram of methylene 

 blue (Grubler) to 100 c.c. of a 1/2% solution of sodium bicarbonate in 

 water. This mixture is heated for i hour in an Arnold sterilizer. 

 When cool, add to the methylene-blue solution 500 c.c. of a i to 1000 

 eosin solution (yellow eosin, water soluble). Add the eosin solution 

 slowly, stirring constantly until the blue color is lost and the mixture 

 becomes purple with a yellow metallic luster on the surface and there 

 is formed a finely granular black precipitate. Collect this precipitate 

 on a filter-paper and when thoroughly dry (dry in the incubator at 

 38 C.) dissolve .3 gm. in 100 c.c. of pure methyl alcohol (acetone free). 

 This constitutes the stock solution. For use filter off 20 c.c. and add to 

 the filtrate 5 c.c. of methyl alcohol. 



A modification by Batch is very satisfactory. In this method in- 

 stead of polychroming the methylene blue with sodium bicarbonate 

 and heat, the method of Borrel is used. Dissolve i gm. of methylene 

 blue in 100 c.c. of distilled water. Next dissolve .5 gm. of silver 

 nitrate in 50 c.c. of distilled water. To the silver solution add a 2 to 

 5% caustic soda solution until the silver oxide is completely precipi- 

 tated. W r ash the precipitated silver oxide several times with distilled 

 water. This is best accomplished by pouring the wash-water on the 

 heavy black precipitate in the flask, agitating, then decanting and 

 again pouring on water. After removing all excess of alkali by 

 repeated washings, add the methylene-blue solution to. the precipitated 

 silver oxide in the flask. Allow to stand about 10 days, occasionally 

 shaking until a purplish color develops. The process may be hastened 

 in an incubator. When polychroming is complete, filter off and add 

 to the filtrate the i to 1000 eosin solution and proceed exactly as with 

 Wright's stain. 



