STAINING 749 



III. Decolorizing Solution: 



1 per cent, aqueous solution of oxalic acid 50 c.c. 



1 per cent, aqueous solution of potassium sulphate 50 c.c. 



This last solution should be freshly prepared by mixing the two 

 stock solutions just prior to use. 



1. Stain sections (six to twenty-four hours) until black. 



2. Wash well in water. A few drops of lithium carbonate solution 

 added to the water may improve the color, which should become a 

 deep blue-black. 



3. Differentiate until the gray matter becomes brown (one-quarter 

 to two minutes). 



4. Rinse in water. 



5. Decolorize until the white matter becomes a steel blue, the gray 

 matter a light brown (one-quarter to one minute), watching each 

 section with care. 



6. Wash thoroughly in several changes of water, or in running water. 



7. If desired, counter-stain with alum carmin, and wash in water. 



8. Dehydrate, clear, and mount. 



Methylene Blue, for non-medullated nerve fibers (Intravitam 

 Method) : 



I. Stain : 



Methylene blue (Griibler's f rectif. nach 



Ehrlich') 0.1 grm. 



Distilled water 100 c.c. 



Dissolve with heat, cool, and filter. 



II. Fixing Solution (Bethe's) : 



Ammonium molybdate 1 grm. 



Distilled water 20 c.c. 



Hydrochloric acid, C. P 1 drop. 



The solution should be freshly made and kept at or near C. 



1. The method is only applicable to living tissues, by injecting the 

 blood-vessels with the stain, or by partially immersing in the staining 

 fluid small pieces of tissue, freshly removed from the living animal. 



2. After ten to thirty minutes, rinse in normal saline solution, and 

 place in the cold fixing solution for two to six hours, according to the 

 size of the pieces. The tissue should be kept cold. 



3. Wash well in distilled water. 



