CHAPTER XXIX. 359 



are always present together, as, for instance, in the cells of adipose 

 tissue, or in cells showing fatty degeneration. Mixtures of the true 

 fats and lipoids which constitute, for instance, many of the fat 

 globules of the adrenal cortex also take these stains. It is to be 

 noted, however, that Scharlach R and Sudan III. are applied in 

 alcoholic solution, and in the process of staining with these dyes some 

 of the fatty substances tend to be dissolved out. The true fats are 

 apparently not so readily dissolved as some substances, which show 

 a double refraction, presumably cholesterin fatty acid mixtures. 



The method of Herxheimer, in which Scharlach R is used in 

 strongly alkaline solution, has been recommended on the ground 

 that it is a more ' ' energetic ' ' fat stain for fatty substances in the 

 sense that it stains many cell inclusions which are not stained by 

 Scharlach or Sudan alone. The chemistry of the reaction has not 

 been worked out. It depends probably on the saponifying action 

 of the alkali, which liberates some fatty acids and then produces 

 mixtures of fatty acids and lipoids, which are more readily stained 

 by Scharlach. The range of staining by this method is, therefore, 

 probably as wide as that of osmic acid alone, and will be found to 

 comprise most fatty substances, but for the purpose of differentiating 

 between the different groups of fatty substances has lost the advantage 

 of restricted staining, which the ordinary staining with Sudan III. and 

 Scharlach R alone possesses. 



(2) OSMIC ACID METHODS.- -The true fats and the lipoids are all 

 blackened by osmic acid. This blackening indicates a reduction of 

 osmic acid to a lower oxide. It is due to the fact that all these 

 fatty substances have a double linkage (marked ii in table on p. 358) 

 in their molecule and are, therefore, more or less easily oxidised. 

 But the various groups of substances differ in the readiness with which 

 they are oxidised, and consequently in the rapidity with ivhich they are 

 blackened by osmic acid and the depth of the blackening produced. The 

 true fats are blackened most rapidly and most deeply, the phospha- 

 tides, lecithin and kephalin, come next in order, while cholesterin, the 

 cerebrosides and phospho-cerebrosides are least susceptible to the 

 oxidising action of osmic acid. In fact, these substances when 

 solid are not blackened at all, but are so when dissolved in an appro- 

 priate solvent, such as chloroform. In the myelin sheath of peri- 

 pheral nerves they are present in the form of a colloidal solution, 

 since it can be shown that they contribute to the blackening of the 

 sheath by osmic acid. 



This difference in the reducing power of the various fatty sub- 

 stances can be accentuated by using osmic acid together with bichro- 



