GRANULES AND FAT OF STRIATED MUSCLE 35 



tissues may be hydrolyzed by the action of the formahn fixatives. 

 It follows that in formalin fixed material the blue staining with 

 Nile blue is often an expression of neutral fat which has under- 

 gone hydrolysis. 



The fat droplets of the muscle fibers of many individuals of 

 a species show no blue staining after a prolonged exposure to 

 the stain. From this we may conclude that the droplets in these 

 individuals contain little or no free fatty acids, phosphatid lipoids, 

 or cerebroside. In no case, in so far as I have observed, does 

 the staining with Nile blue afford convincing proof that any sub- 

 stance other than neutral fat is normally present in the fat drop- 

 lets of muscle fibers during the life of the animal. Contrary'' 

 indiqations may be due to postmortem changes, 



6. Results with the methods of Benda, Fischler and Klotz for free 

 fatty acids and soaps. According to Benda, neutral copper acetate 

 forms, with free fatty acids, colored copper soaps which for the 

 most part are insoluble in fat solvents. The methods of Benda, 

 Fischler and Klotz depend upon this reaction. Fischler ('04) 

 found that the fatty acid copper compound forms a lake with 

 hematoxylin. He also stained soaps in the tissues by the same 

 methods, the soluble potassium and sodium soaps being first 

 converted into insoluble calcium soaps by the action of calcium 

 salicjdicum. Klotz ('06) suggested further modifications. Bell 

 ('11) used the above methods on preparations from a considerable 

 number of muscles, but in no case was able to get the color reac- 

 tion for free fatty acids or soaps. He concludes that the lipo- 

 somes (fat droplets) of muscle fibers do not contain either fatty 

 acids or soaps. I have used these methods only to a limited extent 

 and my results are in agreement with those of Bell. However 

 a positive result with the methods of Fischler and Klotz should not 

 be taken as certain proof of the existence of free fatty acid during 

 life. The fixative employed in both these methods contains 

 formalin and in the method of Klotz, acetic acid is added. It is 

 thus possible that the staining occasionally depends upon the 

 previous hydrolysis of neutral fat. 



7. Results with the Weigert method and related methods. We 

 have already discussed these methods and seen that they give 



