MEDICAL AND BIOLOGICAL STAINING TECHNIQUES 



Results: 



Granules, dark red. Body of cells, pale blue. Other bacteria, 

 pale. 



PYRONIN - ALPHANAPHTHOL (Graham) 

 For oxidase granules in blood smears 



Solutions required: 



A. a-Naphthol, pure . . . . i gm. 



Alcohol 40% . . . . . . 100 ml. 



Hydrogen peroxide 20 vols. . . 0-2 ml. 



Note: This solution deteriorates in four or five 

 days when the hydrogen peroxide has been added, 

 and it is, therefore, better not to add the hydrogen 

 peroxide until the solution is required for immediate 

 use. 



B. Pyronin . . . . . . . . o-i gm. 



Alcohol 40% . . . . . . 96 ml. 



Aniline oil . . . . . . • • 4 nil. 



C. Methylene blue 0-5% aqueous. 



Technique: 



1. Freshly spread air-dried blood smears are fixed in 10% 

 formalin for two minutes ; then washed well in distilled water. 



2. Stain for five minutes in Solution A (as above) ; then wash 

 for fifteen minutes in running water. 



3. Stain for two minutes in Solution B (as above) ; then wash in 

 distilled water. 



4. Stain for one half to one minute in Solution C (as above) ; 

 then wash with water ; blot and dry thoroughly ; mount in balsam. 



Results: 



Neutrophile granules, which give the oxidase reaction, are 

 stained purple to red; while eosinophile granules are lighter red, 

 larger and more refractile. Basophile granules are stained a deep 

 purple; cell nuclei, blue; cytoplasm, pale blue; erythrocytes 

 appear greenish yellow to pink. 



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