32 H. HAYS BULLAUD 



4. Osmium tetroxide. It is well known that osmic acid is reduced 

 onl}^ by the unsaturated fata, the reduction depending upon the 

 oxidation of the fat. Therefore it has been considered that fat 

 droplets that are blackened by osmic acid consist, wholly or in 

 part, of unsaturated fats. However, unsaturated fats present in 

 small amounts as mixtures with saturated fats may fail to blacken 

 with osmic. The fat droplets in muscle fibers of many animals do 

 not reduce osmic acid. "While in certain individuals the fat 

 droplets blaken with osmic acid, in other individuals of the same 

 species the reduction does not occur. This shows that the unsat- 

 urated fat in the droplets is variable in amount. 



5. Results with the Nile blue method. Smith ('07) explained 

 the staining of fats with the basic anilin dyes as depending upon 

 the formation of color-soaps by the action of fatty acids and color 

 bases. Neutral fat, as such, can not be stained by basic anilin 

 dyes, but after hydrolysis the free fatty acid combines with the 

 color base. This observer also found that neutral fat in the tis- 

 sues is in a very unstable condition, being hydrolyzed by dilute 

 acids and even by the carbon dioxide of the air. Thus neutral fat 

 droplets in stained sections, after having been hydrolyzed into 

 fatty acid and .glycerine by exposure to the air were observed to 

 take the color of the basic stain. Smith found that Nile blue 

 sulphate initially gives a red color to both neutral fat and fatty 

 acid. Neutral fat retains the red staining quality but subse- 

 quently fatty acids form color-soaps with the Nile blue base, 

 the deep blue of the soap obscuring the comparatively faint red 

 color of the fatty acid. Aschoff ('09) found that the phosphatid 

 lipoids and cerebroside are also colored blue by Nile blue sulphate. 

 McCrae and Klotz ('10), who used Nile blue on sections of fatty 

 liver, state that they experienced some difficulty in interpreting 

 their results. According to Klotz ('09) ''The blue coloration 

 obtained in staining sections with Nile blue is not to be depended 

 upon as indicating in every instance the presence of fatty acids, 

 as the shade of the color is influenced by external circumstances. " 

 He, however, does not specifically state by what circumstances 

 the color is influenced. 



Nile blue has been used as a means of investigating the chemical 

 nature of fat droplets in tissue sections, but in so far as I know 



