MS 21.0 



METAL STAINS 



533 



which the sunhght was reflected. Though 

 this is possibly ratlier an excessive view, 

 tliere is no doubt that the state of the 

 weather has more effect on the technique 

 than any other single factor. It would 

 perhaps be simplest to suggest that the 

 specimen be exposed to bright daylight, 

 that is, some intermediate condition be- 

 tween the dingy darkness of a winter day 

 and bright, direct sunhght. It should re- 

 main in the acetic acid for from one to two 

 days. The period of time is, of course, de- 

 pendent upon the degree of illumination 

 to which it is exposed. 



If the technique is successful the entire 

 muscle will change to a dull purple color. 

 The word c?mM should be emphasized. Dai'k 

 purple indicates too great a degree of re- 

 duction, and there will be no differentia- 

 tion of the nervous tissues. A hght mauve 

 color indicates that the material has not 

 been sufficiently reduced, and it is doubt- 

 ful that it is worth exposing it further to 

 dayhght, since prolonged exposure to in- 

 ferior illumination does not have the same 

 effect as a correct length of exposure to 

 the proper illumination. 



The best way of determining whether 

 or not the impregnation has been success- 

 ful is to examine the muscle under a 

 binocular microscope with the strongest 

 possible illumination from beneath. If the 

 beginnings of the divisions of the nerve 

 witliin the muscle can be seen, the 

 preparation is satisfactory; if, however, 

 the material is so dark that the nerve 

 cannot be followed beyond the point of 

 its entry into the muscle, it is doubtful 

 that it is worth proceeding further. 



Successful impregnations should be re- 

 moved from the acetic acid and placed in 

 the formic acid for 48 hours in the dark. 

 Then take a straight upright tube of about 

 one inch in diameter by four inches long, 

 and place an inch of pure glycerol in the 

 bottom; with a pipet, very carefully place 

 about a two-inch layer of 20 % formic acid 

 on the top of the glycerol, being careful to 

 mix them as little as possible. The prepa- 

 ration is now transferred to the upper 

 layer of formic acid, and will naturally 

 float at the interphase between the formic 

 acid and the glycerol. After about 24 hours 



the muscle will have sunk through the 

 glycerol, but it should not be removed 

 until it shows no further streams of 

 formic acid rising from it through the 

 glycerol. The formic acid is then carefully 

 pipetted from the top of the tube, and a 

 fresh portion of glycerol is added. There 

 will probably have been a considerable 

 mixture of formic acid and glycerol by 

 diffusion, thus the muscle will again float 

 at the interphase between this diluted 

 glycerol and the fresh glycerol which has 

 been added. As soon as the muscle has 

 again sunk, thus demonstrating that it is 

 completelj'' impregnated, the partially 

 diluted glycerol should be pipetted from 

 the surface and the muscle transferred to 

 a watch glass or stender dish full of fresh 

 glycerol. 



This is an excellent time at which to 

 issue the muscle and nerve to a class, and 

 there are manj^ methods which may sub- 

 sequently be followed to secure prepara- 

 tions showing the nerve endings. The sim- 

 plest for class purposes is to divide the 

 muscle into a series of freehand sections 

 taken longitudinally, each section being 

 about J^ o-niillimeter thick. If each stu- 

 dent is provided with one of these sections, 

 he can then examine it under the micro- 

 scope, and will usually without further 

 trouble be able to see the fine terminations 

 of the nerves. To make better and more 

 permanent preparations, it is desirable to 

 take these thin sections and to tease them 

 with needles so as to separate the individ- 

 ual fibers, following the process under a 

 binocular dissecting microscope. Fibers 

 which look as though they might show 

 endings, are then transferred to a slide and 

 mounted permanently in glj'cerol jelly. 

 In place of glycerol jelly one may also 

 employ any of the gum-arabic-glycerol 

 media, or probably, though the author has 

 never tried it, some of the polj^vinyl-alco- 

 hol media. 



These preparations are not very perma- 

 nent^unless thej'' are preserved in the dark, 

 and no method seems to be known by 

 which permanency can be given to them. 

 Under no circumstances can they be 

 mounted in anything save an aqueous 

 medium. 



