66 



Journal of Applied Microscopy. 



and afferent nerves to the ganglion cells 

 of ganglia, the arrangement of the ter- 

 minal fibrils in free endings and in end 

 organs, the tissues are to be placed in a 

 saturated aqueous solution of ammo- 

 nium picrate, suggested by Dogiel. In 

 this solution the bluish colored tissues 

 in a short time assume a purplish color. 

 In it they remain for twelve to twenty- 

 four hours; are then transferred to a 

 mixture of equal parts of the saturated 

 aqueous solution of ammonium picrate 

 and glycerine (Dogiel), in which they 

 remain twelve to twenty-four hours; 

 they may, however, remain several days 

 without detriment. The tissues are to 

 be mounted in the same solution. If the 

 pieces are too large for the mounting, 

 they may be teased, or small pieces may 

 be snipped off with sharp scissors or 

 they may be pressed out between slide 

 and cover-glass or perhaps better 

 between two slides, the top slide being 

 replaced by a cover-glass. If, on the 

 other hand, it is desired to work out 

 some of the finer details, in which case 

 it is often desirable to section the tissues, 

 the following method, slightly modified 

 from that given by Bethe, may be most 

 highly recommended for fixing the 

 stain : 



Ammonium molybdate, one gram. 



Distilled water, ten cubic centimeters. 



Hydrochloric acid, one drop. 



The solution is prepared by grinding 

 the ammonium molybdate to a fine pow- 

 der, 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 is added. Before using 

 this fixative it is necessary to cool it to 

 two degrees to five degrees Fahrenheit, 

 above zero. It is therefore well to pre- 

 pare it before the injection is made, and 

 surround it with an ice mixture. In this 

 fixative the tissues remain for 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 hardened and 

 dehydrated in absolute alcohol. It is 

 advisable to hasten this step as much as 

 possible, though not at the risk of imper- 

 fect dehydration. The tissues are then 

 transferred to xylol and embedded In 

 paraffin, sectioned and fixed to the slide 

 or cover glass with albumen fixative, 

 and may be double stained in alum car- 

 mine or alum cochineal. After staining 

 in either of these stains, the sections are 

 thoroughly dehydrated, cleared in oil of 

 bergamot. The oil is washed off with 

 xylol and the sections are mounted in 

 Canada balsam. 



Local Application of the Methylen 

 Blue. — In staining the nerve fibers with 

 methylen blue by means of local appli- 

 cation of the stain to the tissues, the 

 following method has given me the best 

 results. The method here given is much 

 the same as that so successfully used 

 by Dogiel. The tissues to be studied are 

 divided into small pieces, much as above 

 suggested, and are spread out on slides 

 or in Petri dishes; the slides and dishes 

 having been previously moistened with 

 normal salt. 



Solution used. — I usually use a one- 

 twentieth per cent, solution of the 

 methlen blue in normal salt solution. Of 

 this, two, three or four drops are placed 

 on each piece of tissue to be stained, the 

 stain being constantly renewed as soon 

 as it is noticed that that previously ap- 

 plied is beginning to evaporate. It is 

 essential that not enough stain is added 

 at any one time, to completely cover 

 the tissue. It is, for instance, impossible 

 to obtain a stain of the nerve fibers In 

 a tissue by immersing it in the above 

 solution. Here, as in the previous 

 method, is is essential that the air have 

 access to the tissue while it is being 

 stained. The length of time required to 

 stain the nerve fibers by this method 

 varies greatly. Sometimes they are 

 stained in half an hour, again it may 

 require two and one-half hours; on an 

 average, about one hour. It is therefore 

 necessary to examine the tissues from 

 time to time to see whether the nei-ves 

 are stained, the same precautions about 

 covering them with a cover glass being 

 observed. As soon as it is found that 

 the nerve fibers are stained, the tissues 

 are fixed in ammonium picrate or am- 

 monium molybdate, as above described. 

 It seems hardly necessary to add that 

 when it is stated that the stain colors 

 the nerve fibers, it is of course under- 

 stood, as all who may have used this 

 method know, that reference is had to 

 the axis cylinder (neuraxis) and its 

 branches. The dentrites of the neurons 

 and the cell bodies of the neurons are 

 also stained, if ganglia come within the 

 field of observation. Before closing, a 

 few general remarks about the method 

 may not be out of place. 



It must always be borne in mind that 

 even an extended acquaintance with this 

 method does not prevent one from hav- 

 ing failures now and then. Why this 

 is the case, I am unable to say. 



It must ever be borne in mind that 

 methylen blue, when used after either 

 of the above methods, does not stain 

 only nerve fibers and their endings, but 

 also other tissue elements. Indeed, It 

 may be said that it Is almost impossible 

 to obtain a preparation where only nerve 

 fibers are stained. This is very often 



