240 Biological Stains 



Considering that salts of color acids are called acid dyes, and 

 salts of color bases are known as basic dyes, a theoretically satis- 

 factory name for the compound dyes in which a color acid is com- 

 bined with a color base would be "neutral dyes". The term is not 

 ordinarily used this way, however; in fact, the dye chemist uses 

 the same term in an entirely different sense. The chemist has not, 

 however, pre-empted the term "neutral stain"; and considering 

 that this class of compound dyes is employed for biological stain- 

 ing rather than general dyeing purposes, the name is quite ap- 

 propriate. The Stain Commission, therefore, has adopted the 

 use of the term neutral stain for this purpose and defines it as a 

 compound of an acid dye and a basic dye in which both the anion 

 and the kation contain chromophore groups. 



Ehrlich (1898) introduced a different term, "tri-acid dyes", 

 which he intended, apparently, to apply to the whole class of 

 neutral stains. He seems to have introduced this term on the 

 basis of two assumptions; first, that most of the basic dyes are 

 triamino compounds (and therefore potentially tribasic); second, 

 that in the ordinary mineral acid salts of these dyes only one of the 

 affinities for acid is satisfied, but when compounded with an acid 

 dye the other two affinities also become satisfied. The latter 

 assumption, at least, seems to be entirely incorrect. As just ex- 

 plained, the reaction between an acid dye and a basic dye seems to 

 be purely one of metathesis, and there is no reason whatsoever 

 for assuming that the dye anion attaches itself to any other place 

 in the basic dye molecule than that previously occupied by the dis- 

 placed mineral anion. Ehrlich's term, however, has persisted for 

 the particular "tri-acid stain" which he specially recommended 

 (see below); and since this particular neutral stain is no more a 

 tri-acid product than any other, some explanation of the term 

 seems necessary. 



It is possible to obtain an endless variety of such dyes; but in 

 practice only a certain number of them have proved useful. Among 

 the basic dyes the most suitable for this purpose are the thiazins 

 and the rosanilins (which act as strong ammonium bases) ; among 

 the acid dyes, the eosins and the sulfonic acids (e.g., orange G and 

 acid fuchsin). 



Although the neutral stains are insoluble in water, they are sol- 

 uble to a greater or less extent in excess of either the acid or the 

 basic dye. Thus if an aqueous solution of acid fuchsin is neu- 

 tralized by adding drop by drop an aqueous solution of methyl 

 green, there is at first no precipitation, because the methyl green 

 salt of acid fuchsin is kept in solution by the excess of acid fuchsin. 

 After the proper amount of methyl green has been added, however, 

 and the mixture has stood long enough for the reaction to take 

 place, the neutral stain is precipitated and the solution becomes 

 nearly colorless. Then if more methyl green is added the neutral 



