156 BULLETIN OF THE BUREAU OF FISHERIES. 



During the progress of this work a preliminary notice and final paper have been 

 published by Greene and Skaer*^ reinvestigating the fat absorption from the stomach 

 in mammals; also a paper by Weiss'" briefly presenting the fact of fat absorption by 

 the gastric mucosa in the snake and in mammals. 



EXPERIMENTS DEMONSTRATING THE ABSORPTION OF FATS. 



METHOD. 



.The method of determining the character and degree of fat absorption from different 

 portions of the alimentary tract of the king salmon has been that of microscopic exam- 

 ination. Tissues were examined fresh and after formalin fixation followed by the 

 newer fat stains, Sudan III, scariet red, etc. The chief reliance for staining the fat in 

 the cells has been on the alkaline scarlet red. These methods of observation have been 

 confirmed by more careful tissue fixation in Flemming's osmic acid mixture and by the 

 corrosive bichromate method of Bensley. Flemming's solution not only fixes the tissues 

 but gives the characteristic osmic acid staining of the fats. The Bensley fixation, when 

 followed up by paraffin sections and differential staining, gives a negative picture, since 

 the fats are dissolved out by the clearing fluids, leaving only fat vacuoles. 



The detail of procedure for staining with scariet red is as follows: The perfectly 

 fresh material, living tissue if possible, was dropped into a 10 per cent formalin for two 

 hours or more. Precautions were taken to insure penetration and proper fixation. The 

 material fixed in formalin was then frozen in a freezing microtome and cut as thin as 

 possible. The frozen sections were cut directly into 70 per cent alcohol, and stained in 

 alcoholic solutions of scariet red. The stain was made by heating an excess of scarlet 

 red in 70 per cent alcohol containing 2 per cent sodium hydroxide to a temperature 

 of about 80° C. This procedure, which is recommended by Bell,'' gives a stain which 

 on cooling leaves a saturated solution of greater staining powers than the ordinary alco- 

 holic scarlet red. The stain was always filtered into shallow dishes just before using. 

 Shallow oval bottom salt cellars were used, and these immediately covered to prevent 

 evaporation. The sections were lifted from the 70 per cent alcohol, the excess of fluid 

 quickly removed, and then they were dropped into the stain. Staining is compara- 

 tively rapid and requires only from 5 to 1 5 minutes for a successful impregnation. 



Sections were taken from the stain, the excess of adherent stain being removed by a 

 momentary immersion in 70 per cent alcohol, and then were immediately plunged into a 

 large dish of water. When the particular tissues were delicate, an intermediate grade of 

 35 per cent alcohol was used. In this case the sections must be in contact with the 

 alcohol only long enough to remove the adherent stain, otherwise the stain in the tissue 

 itself will be drawn. As a matter of routine practice it was found desirable to add to 

 the wash water bath a couple of drops of hydrochloric acid. The faint acidity was found 

 favorable to the more rapid removal of the traces of alkali. This step contributes 

 decidedly to the keeping powers and clearness of the sections after they are mounted 



o Greene, Charles W., and Skaer. William F.: Absorption of fat by the mammalian stomach, Proceedings American Physi- 

 ological Society, American Journal of Physiology, vol. 29, no. 4, 1912. p. x.\xvii. Evidences of fat absorption by the mucosa of 

 the mammalian stomach. American Journal of Physiology, vol. 3;. 1913, p. 358. 



s Weiss. Otto: Die Resorption dcs Fettes im >Iagcn. Pfluger's Archiv fiir die gesamte Physiologic, bd. 144. 1912, p. 540-543. 



'Bell. E. T.: The staining o( fats in epithelium and muscle fibers. Anatomical Record, vol. 4, 1910. p. 199-213. 



