SPECIAL STAINING METHODS 663 



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

 grey 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. 



Methylen Blue for Nerve Tissues (A, Intravitam Method): 



I. Stain : 



Methylen blue (Griibler's "rectif. nach 



Ehrlich") 0.1 grm. 



Distilled water 100 cc. 



Dissolve with heat, cool, and filter. 



II. Fixing Solution (Bethe's) : 



Ammonium molybdate 1 grm. 



Distilled water 20 cc. 



Hydrochloric acid, 0. 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. 



4. Dehydrate quickly in 95 per cent, and absolute alcohol, kept 

 at or near C. 



5. Embed in paraffin. At a convenient time, cut and mount. 

 The stain is rather unstable, but may be kept fairly well if 



mounted in glycerin or in neutral balsam. 



Methylen Blue for Nerve Tissues (B, Nissl's Method): 



I. Stain: 



Methylen blue (Griibler's "B pat."). . . 3.75 grm. 



Venetian soap (white Castile) 1.75 grm. 



Distilled water 1000 cc. 



II. Differentiating Solution: 



Anilin oil (pure) 10 cc. 



95 per cent, alcohol 90 cc. 



