GRANULES AND FAT OF STRIATED MUSCLE 33 



the method has not been applied to muscle fibers. I have fol- 

 lowed directions given by Smith and Mair ('11) for the use of 

 Nile blue sulphate but the results have not been very satisfactory. 

 Apparently the fat droplets are decolorized by the 2 per cent acetic 

 acid used as a differentiating fluid. The method which I have 

 used with best success is given here under the heading ''Material 

 and methods." In brief it consists in setting free the color base 

 by the addition of a small amount of alkali to the medium, in 

 which the section is mounted, or by washing in slightly alkaline 

 water after differentiating in distilled water. Precipitates are 

 formed if sections are not carefully washed before being placed in 

 the alkaline solution. With a little care in making the prepara- 

 tions with Nile blue chlorhydrate, and apparently with the sul- 

 phate also, it is possible to stain all the fat droplets to be found 

 in muscle fibers. That is to say, Nile blue will stain droplets 

 that are not stained by simple alcoholic solutions of Sudan iii 

 and Scharlach II or by osmic acid. With Nile blue, as with other 

 stains, fresh tissue should be used if all the droplets are to be 

 stained, for they frequently disappear in fixed tissue. 



As has already been said, Nile blue sulphate stains not only the 

 fat droplets but, in formalin fixed material at least, it also stains 

 the true interstitial granules. This is best shown by staining 

 a section from the pectoral muscle of a pigeon or bat. The true 

 interstitial granules are especially abundant and stain a bright 

 blue, the shade depending upon the length of time in the stain and 

 upon the reaction of the mounting medium. The fat droplets 

 which may also be present in large numbers, usually stain a some- 

 what faint red but sometimes in various shades of purple or blue. 

 In formalin fixed specimens of material containing large true inter- 

 stitial granules, it is not difficult to distinguish the latter from, 

 fat droplets since the granules do not have the globular form of the 

 droplets and for the most part present color differences. 



The color assumed by the fat droplets when stained with Nile 

 blue is of some importance as it may help in identifying the fats 

 chemically. The color of the droplets depends, to a considerable 

 extent upon the time occupied in staining and upon the alkalinity 

 of the solution to which the stained section is exposed, as well as 



THE AMERICAN JOURNAL OF ANATOMY, VOL. 14, NO. 1 



