Journal of Applied Microscopy. 



65 



general use, both are worthy of consid- 

 eration: 



1. Injecting the methylen blue into 

 the blood vessels, as first suggested by 

 Ehrlich. 



2. Adding a few drops of the methy- 

 len blue solution to the living tissues, as 

 first suggested by Dogiel and especially 

 developed by him. 



Method of injecting the solution: 



A.— Solution used. In the past few 

 years I have employed a one per cent, 

 solution of methylen blue (Methylenblau, 

 rectiflciert nach Ehrlich. Gruebler) in 

 normal salt. This solution is prepared 

 by mixing in a flask one gram of methy- 

 len blue with one hundred cubic centi- 

 meters of normal salt solution, and heat- 

 ing over a flame until the solution 

 becomes hot. It is then allowed to cool; 

 when filtered it is ready for use. 



B. — Injection of the stain. I have in 

 the past few years obtained the best 

 results by killing the animal with chloro- 

 form, and as soon as dead, exposing the 

 main artery going to the part in which 

 it is desired to stain the nerves. If 

 desirous of studying the nerve endings 

 or ganglia in the tongue, in the eye or 

 other parts of the head, I have exposed 

 one of the carotid arteries; if the nerve 

 endings in the extremities, the femoral 

 or brachial; if in the viscera, usually the 

 aorta, other times the main artery going 

 to the organ to be studied. A canula is 

 then tied into the artery and the methy- 

 len blue solution injected. The amount 

 of the solution injected varies of course 

 with the extent of the vascular area 

 injected. I always inject a sufilcient 

 quantity to give a light blue color to the 

 part in which the nerves are to be 

 studied. As soon as the blue color 

 appears, the injection of the methylen 

 blue is interrupted. If too much stain is 

 injected, structures other than nerves 

 are stained so deeply that it becomes 

 difficult to see the nerve fibers. After 

 the injection, the parts to be studied 

 remain undisturbed for about one hour, 

 after which time they are exposed, and 

 small, or at least thin pieces are removed 

 to a slide moistened in normal salt, and 

 exposed to the air; and here the good 

 sense of the observer must tell him to 

 what extent the tissues to be studied 

 must be divided in order to obtain the 

 right kind of small pieces, always bear- 

 ing in mind that it is necessary to bring 

 the nerve fibers which it is desired to 

 stain as freely as possible in contact 

 with the air, for it has long been known 

 that the methylen blue forms with the 

 nerve fibers (if that expression may be 

 allowed) a leuco-base, which is oxydized 

 in the presence of the air into the blue 

 stain desired. I may briefly indicate 



what is meant, in this connection, by 

 small pieces. If. for example, it is 

 desired to obtain a preparation showing 

 the nerve-endings in the epithelium and 

 mucosa of the mucous membrane lining 

 the mouth, side of the tongue for in- 

 stance, it is only necessary to separate 

 the mucous membrane from the underly- 

 ing tissue and transfer it to a slide, epi- 

 thelial side downward if the plexus in the 

 mucosa is desired, mucosa-side down if 

 the ultimate endings in the epithelium 

 are sought for. If it is desired to obtain 

 motor endings in striated muscle, a thin 

 muscle like one of the eye muscles may 

 be selected, or a larger muscle may be 

 divided into ribbon-like pieces. If it is 

 desired to stain the endings in and about 

 the taste-buds, it is convenient to cut 

 out with a double knife, from the 

 foliate papilla, a thin strip about two 

 millimeters in thickness. The same 

 method may be used for nerve endings in 

 the skin. Small ganglia may be at once 

 removed to a slide,larger ganglia are best 

 divided into halves with a sharp knife. 

 It is of course understood that in each 

 instance the parts to be studied were 

 injected with methylen blue, as above 

 directed, before they were removed to the 

 slide. On the slide, the tissues remain 

 until the nerve fibers are satisfactorily 

 stained, that is, until the blue color has 

 developed in the nerve fibers (axis cylin- 

 ders) and their endings. This may take 

 five, ten or fifteen minutes; usually if at 

 the end of fifteen minutes the nerves are 

 not stained, longer waiting does not avail 

 much. After the tissues are removed to 

 the slide, they are to be examined every 

 two or three minutes to see what prog- 

 ress has been made. While such exami- 

 nations are being made — and I refer here 

 of course to a microscopical examination 

 — it is best not to cover the preparation 

 with a cover-glass. With a little care 

 the observer is usually able to see under 

 a low power whether the nerve fibers 

 are stained or not, so that it is not nec- 

 essary to use a cover-glass. If it 

 becomes necessary to use a high power 

 and consequently a cover-glass, this 

 should remain on the tissue only long 

 enough to satisfy the observer whether 

 the proper degree of staining has been 

 attained. If longer examination of the 

 fresh tissue is desired one must be pre- 

 pared to sacrifice the preparations, as 

 they usually bleach in a few minutes 

 when covered with a cover-glass. As 

 soon as the nerve fibers seem to be 

 stained, the tisues are to be placed at 

 once in one or the other of the following 

 fixatives. The selection of the fixative 

 depends on the results sought for. If it 

 is desired to gain a preparation giving 

 the general course of nerves, the forma- 

 tion of plexuses, the relation of efferent 



