26 Neurological Technique 



This gives a solution of i part of toluidin blue to 3,000 of water. In 

 some instances it has been found necessary to use a still weaker solution. 



PROCEDURE. 



1. To fix, place pieces of fresh nervous tissue (4-10 mm. 

 thick) in 30-40 volumes of the 5 per cent, nitric add solution 

 (A) for 24 hours. 



During this period the temperature of the fluid should not 

 rise above 20° C. There have been trials in which even a lower 

 temperature (15° C.) was thought to produce better results for 

 neurofibrillae. In hot weather a weaker solution of the acid (3 

 per cent.) is recommended. 



It is essential that the fixing fluid act equally upon the 

 tissue from all sides. To insure this, it is best to lay the pieces 

 on a layer of filter paper and also to turn them over from time 

 to time. 



If it is only desired to demonstrate the neurofibrillae, the spinal 

 cord is suggested as containing cells rich in fibrillse. The fresh 

 spinal cord of the dog is good material. 



At the end of the period of fixation the pieces should be of 

 a light yellow color. 



2. To harden and wash, transfer direct from the nitric acid 

 solution to 20-30 volumes of 96 per cent, alcohol (B) for 12-24 

 hours. 



3. Development with ammonia. — Replace 96 per cent, alcohol 

 with an equal amount of the atnmoniated alcohol (C) for 12-24 

 hours. 



Here also the temperature must not exceed 20° C. 



The pieces become a dark yellow. If a black-brown color 

 is produced, the indication is that nitrates have been formed in 

 the tissue in excess. Either the nitric solution was too strong 

 or the temperature too high during the action of the acid or of 

 the ammonia. 



4. To wash, transfer the tissue again to 96 per cent, alcohol 

 for 6-12 hours. 



5. Development with hydrochloric acid. — Transfer to 20—30 

 volumes of the alcoholic hydrochloric acid solution (D) and, as 



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