Theory of Staining 49 



staining. It is difficult to explain wholly on the basis of various 

 isoelectric points the fact that certain basic dyes have stronger 

 affinities for certain parts of the nuclei than for others, and that of 

 the various cytoplasmic structures outside the nucleus some are 

 more readily stained by certain acid dyes and some by others. 

 In the Flemming triple stain for example, which employs the 

 acid dye orange G and the two basic dyes safranin and gentian 

 violet, with intervening alcoholic differentiation, it is possible to 

 stain the chromatin with gentian violet and the rest of the nucleus 

 with safranin. Before such a phenomenon as this can be explained 

 on a purely chemical basis, it is obvious that we must have a much 

 more detailed knowledge of histo- and cytochemistry than is yet 

 available. 



Discussion of the Chemical Theory. The weaknesses of the 

 chemical theory show up particularly in reactions such as this 

 triple stain. Besides the difficulty of accounting for the stronger 

 affinity of certain portions of the nucleus for certain basic dyes and 

 other portions of the nucleus for other basic dyes, there is the 

 difficulty of explaining the action of solvents. The differential 

 staining secured in the Flemming stain, and in fact in the ma- 

 jority of other similar procedures, is brought about not by the 

 staining itself but by the action of solvents which extract some 

 of the dyes more readily from certain portions of the cell than 

 from others. In case a chemical union has taken place between 

 the tissue and the dye, the alcohol or other solvent used must 

 have the effect of breaking down the chemical compound formed 

 between the tissue proteins and the dye molecule, or else it must 

 actually dissolve out the compound in question. It is, however, 

 difficult on the one hand to conceive of alcohol breaking down 

 such a compound; while, on the other hand, if the compound 

 formed is dissolved out of the tissue, it is hard to understand how 

 restaining of decolorized structures is possible. 



A further difficulty of the chemical theory arises from the fact 

 that it assumes ionization of the compounds stained, while the 

 tissue is not in solution. Thus, the hypothesis above given as- 

 sumes a definite isoelectric point for the protein of the tissue, and a 

 physical chemist defines this point as the reaction where the min- 

 imum dissociation occurs. Now a protein occurring in a solid form 

 as in sections of fixed tissue can hardly be expected to dissociate, 

 and accordingly, it is a question whether it can properly be said to 

 have an isoelectric point. Under the chemical hypothesis, in short, 

 it is very difficult to conceive how an insoluble solid can behave 

 as an electrolyte and take part in the reactions assumed for it. 



One can, however, assume that differential staining indicates 

 differences in chemical nature of the different parts of the cell with- 

 out adopting the generally accepted chemical theory. Even if 

 the dye is actually taken up by a process of adsorption, this pro- 



