M. A. Lane Al3 
black specks in the islet cells of the guinea pig; a fact indicating the 
presence in the islet cells of a substance which unites with silver in a 
reducible form, which does not oceur—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 fiuid. References 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) anda saturated alcohohe (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 fiuid 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 fiuids 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 
