and Laboratory Methods. 



1359 



Sea water (or normal salt), - - . - 50 c. c. 

 Cone. c. p. glycerin, - - - - - - 50 c. c. 



Cone, ammonia, .-.-.. i drop. 



Add more glycerin, and finally mount in glycerin containing just enough of 

 the ammonium picrate to color it slightly. Specimens put up in this way have 

 kept their color now for over three years, no noticeable change having taken place 

 during that time. 



The above method was frequently modified. I did not find, as some later 

 writers have done, that better results were obtained by a bath of longer duration 

 in ammonium picrate. A short bath of from y^ to \ minute suffices for sensory 

 nerves and peripheral sense organs, and somewhat longer for deeper lying nerves. 

 I am inclined to take Apathy's view regarding the successful staining of the 

 nerve fibers. He believes that he gets a true stain and not an impregnation. 

 In a few very successful cases I have succeeded in following to the nerve cell a 

 bundle of fibers, which I believe to correspond to primitive neurofibrils. 



More frequently, however, an impregnation probably takes place, the whole 

 interfibrillar space taking the blue. This can best be shown by following the 

 successive changes which take place in the tunicate nerve fiber after death, or 

 during the diffuse staining which takes place in the later stages of every suc- 

 cessful nerve staining with methylen blue. The successful stain of a fiber shows 

 an almost unbroken wavy blue line, or series of interlacing fibrils, around which 

 can be seen very faintly the sheath. This fiber has almost no knobs or granula- 

 tions in its course except at the true ending. At a point of branching a triangular 

 blue area, probably caused by the stretching of the sheath, can be seen. As the 



tissue dies, however, a change 

 takes place. The nerve fiber 

 begins to bead, at first hardly 

 noticably, but later these head- 

 ings become so large as to dis- 

 tort the whole fiber and com- 

 pletely change its appearance. 

 Ultimately all trace of the fiber 

 disappears except a line of 

 irregularly placed blue globules. 

 (See figure.) 



This same beading of the 

 fiber is frequently induced by 

 fixation with ammonium picrate, 

 and it is only in rare cases 

 when we have the fiber fixed so that it shows the individual fibrils. Frequently 

 there appears to be a vacuolization of the nerve. Large vacuoles appear, at 

 one side of which there is a heavier deposit of blue. This is, perhaps, a 

 pathological condition induced by the injection of the methylen blue. 



Fixation for imbedding and sectioning. — Parker's sublimate and alcohol method 

 was tried with no success. Bethe's ammonium molybdate method (Arch. f. Mik. 

 Anat. xliv. '94) yielded poor results. His later method (Anat. Anz. xii. '96) 



Four stages in the degeneration of a nerve fibre : 

 I. A successful impregnation. 

 II. Ten minutes later; beading commencing. 



III. Twenty minutes later; much beading. 



IV. Two hours later. 



