34 H. HAY6 BULLARD 



upon the length of time left in this second solution. The red 

 staining takes place rapidly. The blue staining is sometimes well 

 marked after fifteen minutes but may not appear for several hours 

 or may not occur at all. The color is also influenced by the reac- 

 tion of the mounting medium. In any alkaline medium the pale 

 blue droplets tend to become red, while in an acid medium the 

 red droplets tend to become blue. The entire mass of droplets 

 may stain uniformly either red, blue, or in various shades of purple. 

 On the other hand, a droplet colored red may include within its 

 mass one or several clearly marked smaller droplets which stain 

 an intense blue. Again, the periphery of a droplet maj^ show 

 blue staining while the center is red. After preparations have 

 been mounted for a variable number of days, droplets which at 

 first were red may assume an intense blue color. In other prepa- 

 rations the red color is retained for months. I have observed a 

 certain amount of blue staining of the fat droplets in every species 

 of animal from which material was taken, including man. The 

 blue staining compound is found in muscle fibers taken from well 

 nourished animals as well as in fibers from animals poorly nour- 

 ished. 



It is difficult to interpret the color reactions of Nile blue upon 

 the fatty substances of muscle fibers. The blue color of fat drop- 

 lets may possibly indicate that they are, in part, either fatty acid 

 or a lipoid substance, while the red color indicates the presence 

 of neutral fats. However, the blue color of the fat droplets may, 

 as we have seen, indicate the presence of a neutral fat which is 

 easily hydrolyzed, the fatty acid then forming a color soap. When 

 the blue staining takes place only after sections have been mounted 

 several days, it is, in all probability, dependent upon the gradual 

 hydrolysis of neutral fat. With fresh tissues treated with the 

 dye for a few minutes only, the blue staining, which is sometimes 

 immediately apparent, may be due to similar changes in the 

 neutral fat. It is possible that blue staining of fat droplets in 

 fresh unfixed tissue or in tissue obtained some time after the death 

 of the animal, may be due, in part, to the solution in the fat 

 droplets of the blue staining substance of the true interstitial 

 granules. It was pointed out by Smith ('07) that fat droplets in 



