not indicate that they are neutral in reaction any more than do the 

 corresponding terms acid and basic dyes. A dye chemist, in fact, 

 uses the term neutral dye in an entirely different sense; but it is 

 frequently employed by biologists, especially by Ehrlich, to refer 

 to the basic dye salts of dye acids. In this chapter these compound 

 dyes will be called neutral stains, as this latter term is not employed 

 by dyers or dye chemists in any sense, and is unlikely, therefore, to 

 be misunderstood. Erhlich also uses another term to apply to 

 some of these compounds, namely "tri-acid dyes." He uses this 

 term on the assumption that, while in the ordinary basic dyes only 

 one of the three affinities of the dye for acid is satisfied, it is pos- 

 sible to satisfy all three and in this wav to saturate the basic dve 



«. »-■ t, 



with acid. This assumption of his seems to be incorrect; but 

 Ehrlich's "tri-acid stain" (see below) is so well-known that an 

 explanation of the term is necessary. 



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

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

 the basic dyes the most suitable for this purpose are methylene 

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

 among the acid dyes eosin and the sulphonic acids (e.g., orange G 

 and acid fuchsin). 



Altho the neutral stains are insoluble in water, they are soluble 

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

 dye. Thus if a watery solution of acid fuchsin is neutralized by 

 adding drop by drop a watery 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 

 dye is precipitated and the solution becomes nearly colorless. Then 

 if more methyl green is added the neutral dye is slowly dissolved 

 again; but as a rule neutral dyes are less soluble in excess of base 

 than in excess of acid. 



As simple aqueous solutions of these dyes are impossible and as 

 alcoholic solutions of dyes do not stain well, various methods are 

 employed to secure their action on the tissues. In some instances 

 they are kept dissolved by the presence of an excess of acid or base 

 (particularly the former) ; in others a certain quantity of acetone or 

 methylal is used to hold the neutral dye in solution ; sometimes (as 

 in the original Romanovsky stain) the compound dye is used im- 

 mediately after mixing, before the reaction is complete or pre- 

 cipitation has taken place; or again (as in the Wright stain) methyl 

 or ethyl alcohol may be used as a solvent, and then after applying 

 the alcoholic solution to the slide it mav be diluted with water. 

 This latter method is particularly efficacious, because the disso- 

 ciation which takes place upon the addition of water causes the 

 production of various dye compounds which may stain intensively 

 and very selectively. 



87 



