Neutral Stains 241 



stain is slowly dissolved again; but as a rule neutral stains are less 

 soluble in excess of basic dye than in excess of acid dye. 



As simple aqueous solutions of these compound dyes are im- 

 possible 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 stain in 

 solution; sometimes (as in the original Romano vsky stain) the 

 compound dye is used immediately after mixing, before the re- 

 action is complete or precipitation 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 may 

 be diluted with water. This latter method is particularly effica- 

 cious, possibly because a temporary state of supersaturation 

 occurs on addition of the water, and there are other known in- 

 stances where such a state enhances staining power. It should 

 be remarked that when water is mixed with such an alcoholic 

 solution, dissociation must take place and an unstable equilibrium 

 must exist until the compounds insoluble in the mixture of water 

 and methanol have been precipitated; under such circumstances 

 the staining reactions may be rather complicated. 



It is assumed that these compound dyes act on the protoplasm 

 somewhat as follows: certain parts of the cell have an affinity for 

 the neutral stain and take it up as such; others, having an affinity 

 for the basic dye, break up the neutral stain so as to obtain the 

 basic portion of it, or if dissociation has taken place, take up the 

 basic ion directly; while other parts of the cell with an affinity for 

 acid dyes similarly combine with the acid portion of the stain. 

 These three types of cell structures are known as neutrophile, 

 basophile and oxyphile elements, respectively. The differentia- 

 tion thus produced gives the neutral dyes their great value. 



Ehrlich's "Tri-acid Stain." 



The first neutral stain proposed for microscopic work was the 

 "tri-acid stain" of Ehrlich (1910, II, 313). In forming this com- 

 pound dye, acid fuchsin and orange G are mixed in solution and to 

 the mixture is then added such a quantity of methyl green that 

 there is still an excess of the acid dye. This excess of the acid dye 

 allows the neutral stain to stay in solution. The dye thus formed 

 is a useful blood stain, and brings out finely the different structures 

 in the leucocytes. 



The explanation of the name of this stain (which was based on a 

 mistaken chemical theory) is explained on a preceding page. 



Slight modifications of the stain have been used for tissues. The 

 best known is that of Biondi and Heidenhain. (See Krause, 1926-7, 

 p. 457, or Lee, 1937, p. 166). 



