212 MICROMETRY AND BLOOD PREPARATIONS 



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

 mixed stain about one week before using. The best fixatives are heat and Whitney's 

 modified Zenker. To use, stain films from two to five minutes; then wash and 

 mount. The triacid 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 i gram of methylene blue (Grub- 

 ler) to 100 c.c. of a %% solution of sodium bicarbonate in water. This mixture is 

 heated for one hour in an Arnold sterilizer. The flask containing the alkaline methyl- 

 ene-blue solution should be of such size and shape that the depth of the fluid does 

 not exceed 2% inches. 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 38C.) dissolve 0.3 gram in 100 c.c. of pure 

 methyl alcohol (acetone free). Wright lately has recommended using o.i in 60 c.c. 

 methyl alcohol. 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 instead of poly- 

 chroming the methylene blue with sodium bicarbonate and heat, the method of 

 Borrel is used. Dissolve i gram of methylene blue in 100 c.c. of distilled water. 

 Next dissolve 0.5 gram 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 pre- 

 cipitated. Wash 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 ten days, occasionally 

 shaking until a purplish color develops. The process may be hastened in an incu- 

 bator. 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. 



In Leishman's method the polychroming is accomplished by adding i gram of 

 methylene blue to 100 c.c. of a V^% solution of sodium carbonate. This is kept at 

 65C. for twelve hours and allowed to stand at room temperature for ten days 

 before the eosin solution is added. The succeeding steps are as for Wright's stain. 



In all Romanowsky methods distilled water should be used. If not 

 obtainable, the best substitute is rain-water collected in the open and 

 not from a roof. 



If the yellow color in water in a test-tube to which has been added a small pinch of 

 haematoxylin does not change to blue in from one to five minutes it is too acid and 

 should be treated with a i % sod. carb. sol. until it does show blue. Alkaline waters 

 are less easy to correct. 



Method of staining: 



1. Make films and air dry. 



2. Cover dry film preparation with the methyl-alcohol stain for one minute (to 

 fix). 



