MICROSCOPIC STUDY AND STAINING 131 



Decolorize with anilin oil until no more color comes off. 

 This both decolorizes and dehydrates. 

 Treat with xylol. Mount in balsam. 



METHOD FOR GRAM POSITIVE AND GRAM NEGATIVE ORGANISMS IN TISSUES, 

 RECOMMENDED BY PAPPENHEIMER. 



Fixation: Zenker preferred. Formalin or alcohol. 

 Paraffin sections, 5 mu or less. 

 Staining : 



1. Stirling's Gentian violet 5 minutes. 



2. Gram's iodin 1 minute. 



3. Aniline oil or aniline oil xylol. Decolorize to pale violet. 



4. 100% ale. Few seconds only. 



5. Distilled water. 



6. Aqueous saffranin. l /2% 30 seconds. 



7. Distilled water. 



8. Blot. 100% ale. Few seconds only. 



9. Clear in xylol. 



GRAM-WEIGERT METHOD (for paraffin sections). Preferably Zenker's 

 fixation : 



1. Stain sections lightly in alum-hematoxylin. 



2. Wash in running water. 



3. Four per cent aqueous solution soluble in water, five minutes to one- 

 half hour. 



4. Wash in water. 



5. Aniline methyl-violet, one-half to one hour. 



6. Wash off with water. 



7. LugoFs solution, one to two minutes. 



8. Wash off with water. 



9. Blot with filter-paper and dehydrate and clear in several changes of 

 aniline and xylol, equal parts, or in aniline oil alone. 



10. Wash off with xylol. 



11. Mount in xylol-colophonium. 



MALLORY'S EOSIN AND METHYLENE-BLUE STAIN. For paraffin sections 

 after Zenker's fixation only: 



1. Stain paraffin sections in a 5 per cent aqueous solution of eosin for 

 twenty minutes or longer. Sometimes it is advisable to get a deeper eosin 

 stain by placing the sections in the paraffin oven for fifteen to twenty minutes. 



2. Wash in water to get rid of excess of eosin. 



3. Stain in Unna's alkaline methylene-blue solution, diluted ^4 or 5 with 

 water, for ten or fifteen minutes. 



4. Wash in water. 



5. Differentiate and dehydrate in a cUsh of 95 per cent alcohol, to which 



