30 HUBER. [Vol. XVI. 



been reached by me ; namely, that better results were obtained 

 when the hydrogen peroxide was omitted from Bethe's formula. 

 It may be stated that it has always been my plan to expose 

 the ganglia, or other tissues to be stained, to the air, either in 

 the animal or on the slide, until all the elements which it was 

 thought would stain were clearly seen under the microscope ; 

 or, in other words, until the leukobase, the tetramethyldiamido- 

 thiodiphenylamin, which is formed by the living tissues when 

 methylene blue comes in contact with them, is again oxi- 

 dized, as it is in the presence of air. Hydrogen peroxide, 

 or, as used in the formula, hypermolybddnsauren ammonium, 

 would in such cases be unnecessary, and does, to some extent 

 at least, as Meyer suggests, have a bleaching effect. I wish 

 further to call attention to other modifications which Bethe 

 (3) himself has suggested. He has tried to obviate the neces- 

 sity of placing the tissues into an " ice-cool mixture," as his 

 earlier method requires, by changing the unstable methylene 

 blue stain, as found in fresh tissues, into one insoluble in water. 

 To this end he places the fresh tissue in a saturated aqueous 

 solution of ammonium picrate (suggested by Smirnow and 

 Dogiel), and, at the expiration of fifteen to twenty minutes, 

 transfers them to a solution prepared after one of several form- 

 ulas, one of which is the following : 



Ammonium molybdate i grm. 



Aqua dest 20 c.cm. 



Hydrochloric acid i gtt. 



Tissues two to three millimeters in thickness remain in this 

 second solution three-fourths to one hour. 



My experience with this modification of Bethe's earlier method 

 has not been extended ; as far as it goes, I have been led to 

 conclude that the prefixation in ammonium picrate is open to 

 a number of objections. 



In the first place, picrate of ammonium seems, even in the 

 short time the tissues are exposed to it, to act more or less as 

 a macerating fluid, which is obviously an objection where the 

 relation of end fibrillae to cells is to be investigated. Secondly, 

 tissues prefixed in the picrate solution do not stain so readily 

 as when the molybdate alone is used. 



