ALKALOIDAL REAGENTS, ETC. 147 



Evidence of the existence of protein dye compounds even 

 when these are freely soluble is afforded by the influence of added 

 protein upon the distribution of the dye between water and some 

 other appropriate solvent (100). Thus if erythrosin in acidulated 

 water is shaken up with ethyl acetate it passes completely into 

 the ethyl acetate, forming a yellow solution and leaving the 

 water colorless; if, however, neutral sodium caseinate be added 

 to the water, the watery layer remains pink upon shaking it up 

 with erythrosin. Similarly, when gentian violet in alkaline 

 watery solution is shaken up with ethyl acetate a large proportion 

 of the gentian violet passes into the ethyl acetate, forming a 

 red solution but leaving the water blue; if, however, casein or 

 gelatin be added to the water very little of the gentian violet 

 passes into the ethyl acetate and that which does so only stains 

 it faintly violet. Similarly neutral red can be shown to combine 

 with casein, and bismarck brown with casein and gelatin. I 

 have observed that casein forms insoluble compounds with acid 

 fuchsin and orange G, gelatin with crystal violet and acid fuchsin, 

 and protamin with orange G and carminic acid. The compound 

 of protamin with carminic acid is very interesting because it is 

 of a very different color from the free acid or its compounds 

 with inorganic bases; as is well known, free carminic acid, in 

 solution, is golden in color, while its salts with inorganic bases 

 have the familiar carmine tint; the compound with protamin 

 is, however, deep violet. I have observed in several cases, also, 

 that the colors of solutions of dyes to which the proteins have 

 been added are not identical with the colors of pure watery 

 solutions. 



Many authors have doubted the chemical nature of the process 

 of staining of tissues, preferring to regard it as a physical phe- 

 nomenon of "solid solution" or "adsorption." Having regard, 

 however, to the unquestionable chemical combinations between 

 dyes and proteins which occur, as we have seen, in vitro, and 

 recollecting that, as Mathews has shown, even proteins coagu- 

 lated by fixatives show similar phenomena, there can remain 

 very little doubt that similar combinations must frequently occur 

 between the dyes used by cytologists and the protein constituents 

 of the tissues.* 



* For a discussion of this interesting question Cf. Gustav Mann (75), also 

 Pelet-Jolivet, L., " Die Theorie des Farbeprozesses," Dresden (1910). 



