ROMANO WSKY STAINING METHODS 183 



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 

 precipitated. 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 nitrate 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 1/2% solution of sodium carbonate. This is kept at 

 65 C. 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. 



Method of staining: 



1. Make films and air dry. 



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

 (to fix). 



3. Add water to the stain on the cover-glass or slide, drop by drop, until a 

 yellow metallic scum begins to form. It is advisable to add the drops of water 

 rapidly in order to eliminate precipitates on the stained film. Practically, we may 

 add i drop of water for every drop of stain used. 



4. Wash thoroughly in water until the film has a pinkish tint. 



5. Dry with filter-paper and mount. 



Red cells are stained orange to pink; nuclei, shades of violet; eosinophile granules, 

 red; neutrophile granules, yellow to lilac; blood platelets, purplish; malarial parasites, 

 blue; chromatin, metallic-red to rose-pink 



Giemsa's Modification of the Romanowsky Method. This is one of 

 the most perfect of the modifications. The objection is that greater 

 time in staining films is required than with the Wright or Leishman 

 method and the stain is very expensive. 



Take of Azur II eosin 0.3 gram. Azur II 0.08 gram. 



Dissolve this amount of dry powder in 25 c.c. of glycerine at 60 C. 

 Then add 25 c.c. of methyl -alcohol at the same temperature. Allow 

 the glycerine methyl-alcohol solution to stand overnight and then filter. 

 This is the stock stain. To use: Dilute i c.c. with 10 to 15 c.c. of dis- 



