M. A. Lane 413 



black specks in the islet cells of the guinea pig; a fact indicating tho 

 presence in the islet cells of a substance which unites with silver in a 

 reducible form,, which does not occur so far as Mankowski's experiments 

 show in the pancreas cell itself. These indications, however, were not 

 pursued farther by any of the above-mentioned observers. Negative 

 results as to granules were found by Hanseman, Stangl, and Ssobelew, 

 although Mankowski, however, confirmed Laguesse's observations of gran- 

 ules in the islets (of the guinea pig's pancreas) after fixation in Flem- 

 ming's fluid. Eeferences to the publications of these observers will be 

 found in Sauerbeck's review above mentioned. 



TECHNIQUE. 



Out of a rather wide range of fixing and staining fluids, three fixations 

 and one stain were found to be the most valuable in the present investi- 

 gation. The fixations used were (1) alcohol-chrome-sublimate, a fluid 

 consisting of equal parts of a solution of potassium bichromate (3.5 per 

 cent in water) and a saturated alcoholic (95 per cent) solution of mercuric 

 chloride; (2) 70 per cent alcohol; and (3) Mueller's fluid with 5 per cent 

 mercuric chloride added a fluid called here aqueous-chrome-sublimate. 

 Very small pieces of the pancreas (preferably from the splenic end) are 

 taken from the living animal and quickly transferred to a generous quan- 

 tity of fluid. For small pieces two hours (with one change) is sufficient 

 in the alcohol-chrome-sublimate fluid. Tissues are left in the 70 per cent 

 alcohol twenty-four hours. In the aqueous-chrome-sublimate from three 

 to four hours is sufficient. It is of the utmost importance in all this 

 technique that acetic acid be carefully avoided, as I have found that even 

 a few drops of this acid, after repeated trials with numerous fluids con- 

 taining acetic acid, were enough to vitiate the entire work. The tissues, 

 after fixation, were hardened in the customary graded alcohols, cleared in 

 bergamot oil, and imbedded in paraffin. Sections were cut three to five 

 micra thick and were fixed to slides by the water method. Out of a score 

 of stains I found the most effective to be Bensley's neutral gentian, I. To 

 a saturated aqueous solution of gentian violet is added a saturated equeous 

 solution of orange G. The acid dye precipitates the basic one. This is 

 filtered and thoroughly washed and dried. The precipitate is dissolved 

 in 25 or 30 cc. of absolute alcohol. For staining purposes enough of this 

 stock solution is added to 20 per cent alcohol to color the alcohol solution 

 a deep violet. 



The sections were stained 25 hours in this stain, blotted quickly and 

 thoroughly with thick blotting paper or several sheets of filter paper in 



