Method VII 47 



The piece of tissue is wet in the physiological salt solution, 

 is then frozen and sectioned on the freezing microtome,' and the 

 sections are transferred to the slide, and there treated with the 

 dilute stain and fixed according to the procedure given above 

 for the application of the stain by immersion. The fixation must, 

 of course, be done on the slide. 



PARAFFIN SECTIONS. 



When, on the other hand, it is found necessary to make a 

 more detailed study of the relations of the elements stained 

 intra vitam than is possible from the thicker mounts according to 

 the above directions, a method has been devised by which the 

 tissues may be imbedded in parafifin, and by this means sections 

 obtained as thin as desired. A method for imbedding which 

 gives uniform success, however, has not yet been devised, and 

 it must be remembered that, in the necessary manipulation for 

 imbedding, frequent failures are produced upon material which 

 the preliminary examination may show to be excellently stained. 



Preliminary to imbedding in parafifin, Bethe's fixing fluid 

 (C, 2) is prepared. Pulverize the ammonium molybdate. Heat 

 in a flask with the required amount of distilled water till the 

 molybdate is entirely dissolved. Then add the hydrochloric 

 acid. A yellowish white precipitate is formed which dissolves 

 upon shaking. Place the fluid in the vessel in which it is to be 

 used and surround it with ice, since both at the time of its 

 application and during the first hours of its action it must be 

 kept at a temperature below 10° C. 



1. In the meantime the tissue to be used is stained either by 

 injection (preferably) or by immersion, and the stain developed 

 by exposure to the air as directed under the procedure chosen. 



2. As soon as examination under the microscope shows the 

 nerve elements satisfactorily stained, the tissue must be placed 

 immediately in 20-30 volumes of the cold Bethe's fixing fluid 

 and kept in the fluid for 12-24 hours. During the first 6-8 



■The Bardeen Freezing Microtome, manufactured by the Bausch & Lomb 

 Optical Co., is recommended. Directions as to its use may be obtained with the 

 apparatus. 



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