44 Neurological Technique 



PROCEDURE. 



Since it has been found that the tissues need not necessarily 

 be actually living, but need only be fresh to insure the best 

 action of the stain, two methods for bringing the stain in contact 

 with the nerve elements are now in common use: (i) by injec- 

 tion of the stain through the blood vessels into the fresh tissue ; 

 (2) by immersing small pieces of the fresh tissue in a limited 

 quantity of the methylen-blue solution. 



BY INJECTION. 



1. As soon as the animal is chloroformed, insert and tie a 

 canula in the main artery supplying the part containing the 

 nervous elements which it is required to stain, and with a syringe 

 or gravity apparatus inject the vessels full of the i per cent, 

 methylen-blue solution (B), taking care not to add pressure 

 sufificient to burst them. The part should assume a decidedly 

 blue color. If a syringe is used, leave it attached. Let the stain 

 diffuse through the walls of the blood vessels for an interval of 

 10 or 15 minutes, then inject more stain, and let the part remain 

 undisturbed for about 30 minutes. 



If a gravity apparatus is used, it may remain in connection 

 with the part for 30-40 minutes, thus keeping the blood vessels 

 distended with the stain solution. 



If the animal is small, it is best to inject the whole body 

 through the carotid artery or the abdominal aorta, using a 

 T-shaped canula if necessary. 



2. To develop. — After the part has remained subjected to the 

 undisturbed action of the stain for 30-40 minutes, small pieces 

 (2-3 mm. thick or less), containing the nerve elements desired, 

 are removed and exposed to the air on slides wet with physio- 

 logical salt solution. The pieces may be gently spread out with 

 teasing needles. Exposure to the air (probably due to ammonia) 

 seems to facilitate the selective staining of the nervous elements 

 by the methylen-blue. The tissues must remain exposed to the 

 air on the slide and, uncovered, must be examined under the 

 microscope every 2 or 3 minutes until the nerve-cells, axones, or 



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