SOME OBSERVATIONS ON THE THEORY 

 OF STAINING 



When two dyes, one basic and the other acid, are brought 

 together in aqueous sohitions under suitable conditions, a pre- 

 cipitated compound dye is formed. If this precipitate is collected 

 and thoroughly washed with water to remove any excess of one or 

 the other dye and the colourless, water-soluble, by-product which 

 is an inorganic salt, it will then be found impossible to separate 

 the two component dyes by means of ordinary solvents such as 

 water, alcohol, cellosolve, xylol, chloroform, etc. The reason for 

 this is, of course, that the negatively charged (acid) dye-ion is 

 united with the positively charged (basic) dye-ion to form a 

 chemical compound. When such a dye is dissolved in alcohol 

 and applied to tissues the union of the acid and basic dye-ions 

 no longer holds in face of competition from various tissue elements 

 of the opposite charge. There is no doubt about the chemical 

 nature of the union between the two dyes in vitro, but there is 

 a doubt concerning the nature of the union of the liberated dye- 

 ions with the tissue elements. We know that the components of 

 a chemical compound cannot be separated from each other by 

 the action of ordinary solvents. 



Then it follows that any dye that can be extracted from stained 

 tissues, with ordinary solvents such as water or alcohol without 

 prolonged soaking, could not have been in chemical combination 

 with the tissue elements that were coloured with it. On the other 

 hand, if the dye cannot be extracted at all, or if it cannot be 

 completely removed from the tissues, this does not prove that 

 the dye remaining in the tissues has entered into chemical union 

 with the latter. Many basic dyes, used without mordants, can be 

 completely extracted from tissues by ordinary solvents, as can a 

 number of carboxylated (acid) dyes which are devoid of sulphonic 

 groups. Eosin is an example of the latter class of dyes. Many 

 but not all, sulphonated acid dyes hold on to the tissue elements 

 stained by them; that is, after the normal histological process of 

 washing away the excess dye, dehydration, etc. When any dye, 



