222 bulletin: museum of comparative zoology. 



ear adhering to it by the eighth nerve, was removed. By careful 

 dissection it is possible, when desired, to remove the entire ear intact. 

 The material was then transferred to 12 % formalin containing 1% 

 nitric acid, for the decalcification of the otoliths, which cannot readily 

 be removed by dissection without injury to the sensory areas. It has 

 been found that 24 hours is an adequate time for decalcification. The 

 carbon dioxide evolved acts as a float for the ear sac and causes it to 

 rise to the surface of the fluid. The gas must therefore be removed, 

 by gentle pressure, through a suitable opening made at some non- 

 sensory portion in the wall of the ear sac. This opening also serves to 

 admit to the interior of the ear the impregnating and reducing fluids 

 used later, thus insuring the contact of the inner ends of the sense cells 

 with these fluids. After decalcification it is necessary to rinse the 

 material in flowing water for several hours, to remove all traces of 

 nitric acid. 



The material is now transferred to a 2 % solution of silver nitrate, 

 in which it is allowed to remain for about 24 hours, after which it is 

 removed and immersed in an ammoniacal solution of silver oxide, 

 commonly known as Bielschowsky's fluid, for a period varying with the 

 kind of material that is being used. This is followed by a rapid rins- 

 ing, to remove the excess of the fluid from the surface, after which the 

 material is transferred to a 20% solution of formalin. After 12 hours 

 or more in this fluid the material is rapidly dehydrated, cleared, im- 

 bedded in paraffin, sectioned, and mounted in balsam, in the usual way. 



An essential feature of this method of silver impregnation is that 

 the nerve paths become occupied by a finely divided precipitate of 

 metallic silver, set free from the silver compound by the reducing 

 action of the aldehyde. In this it differs from the Golgi method, in 

 which there is present more or less of silver chromate, a crystalline 

 product which is probably responsible to a large degree for the notori- 

 ously capricious results obtained by that method. It differs from the 

 somewhat analogous reduction method devised by Ramon y Cajal 

 (:04^) in three rather important particulars: (1) ammoniacal solution 

 of silver oxide is used for impregnation, in addition to the silver nitrate 

 solution common to both methods; (2) the reducing agent employed 

 is formaldehyde, instead of the reagents commonly used in developing 

 photographic plates; and (3) all the processes are carried on at room 

 temperature, rather than at the higher temperatures required by Ramon 

 y Cajal's method. 



Bielschowsky's fluid is pre})ared as follows: To a suitable quantity 

 of a 2% solution of silver nitrate a few drops of a 40% solution of 



