THE NERVOUS TISSUES. 183 



tissues through the blood-vessels ; (2) adding a few drops of the stain to 

 small pieces of perfectly fresh tissues removed from the body. The solu- 

 tion used for injecting tne tissues is prepared as follows : i gm. of methyl- 

 ene-blue 1 is mixed in a small flask with 100 c.c. of normal salt solution 

 and heated over a flame until the solution becomes hot. It is then allowed 

 to cool ; when filtered, it is ready for use. A cannula is tied into the 

 main artery of the part in which it is desired to stain the nerve elements, 

 and sufficient of the foregoing methylene-blue solution injected to give 

 the part a decidedly blue color. After the injection the part to be 

 studied remains undisturbed for about one-half hour, after which time 

 small, or at least thin, pieces of the tissue to be studied are removed to a 

 slide moistened in normal salt solution, and exposed to the air. The 

 tissues remain on the slide until the nerve-cells, nerve-fibers, or nerve- 

 endings seem satisfactorily stained. After placing the tissues on the slide, 

 they are examined under the microscope (without covering with a cover- 

 glass) every two or three minutes, until such examination shows blue 

 color in the neuraxes of the nerve-fibers and their terminations, or in the 

 nerve-cells, if there be any in the tissues examined. Care should be 

 taken not to miss the time when the staining has reached its full develop- 

 ment, as the blue color usually fades again and only inferior preparations 

 are obtained. 



Tissues thus stained may be fixed by one of two methods (or modifi- 

 cations of these methods), the selection of the method depending some- 

 what on the results desired. If it is desired to gain preparations giving 

 the general course of nerves, the formation of nerve-plexuses, the relations 

 of afferent and efferent nerves to the nerve-cells in ganglia, or the gen- 

 eral arrangement of the terminal branches of nerve-fibers in nerve end- 

 organs, the tissues are placed in a saturated aqueous solution of ammo- 

 nium picrate (Dogiel) in which the blue color of the tissues is in a short 

 time changed to a purplish color. In this solution the tissues remain for 

 from twelve to twenty-four hours, and are then transferred to a mixture 

 consisting of equal parts of a saturated aqueous solution of ammonium 

 picrate and glycerin, in which they remain another twenty -four hours ; 

 they may, however, without detriment remain in the mixture several 

 days. The tissues are then mounted in this ammonium picrate-glycerin 

 mixture. 



If, on the other hand, it is desired to section tissues stained intra 

 I'itam in methylene-blue, the following method, slightly modified from 

 that given by Bethe, is suggested. The following fixative is prepared : 

 Ammonium molybdate, i gm.; distilled water, 10 c.c.; hydrochloric 

 acid, i drop. The solution is prepared by grinding the ammonium 

 molybdate to a fine powder, removing it to a flask, and adding the 

 required quantity of water. The flask is now heated until the ammonium 

 molybdate is entirely dissolved, when the hydrochloric acid is added. 

 Before using this fixative it is necessary to cool it to 2-5 C. It is, there- 

 fore, well to prepare it before the injection is made, and surround it with 

 an ice mixture. In this fixative the tissues remain for from twelve to 

 twenty-four hours. After the first six to eight hours it is not necessary to 

 keep the fixative below ordinary room -temperature. After fixation the 

 tissues are washed for an hour in distilled water. They are then hard- 

 ened and dehydrated in absolute alcohol. It is advisable to hasten this 

 step as much as possible, though not at the risk of imperfect dehydration. 



1 Methylenblau, rectificiert nach Ehrlich, Griibler, Leipzig. 



