Neutral Stains 245 



Mixed powdered dyes of approximately this formula are available 

 from some of the American manufacturers. 



MacNeal (1922) proposed a stain, intended to give similar re- 

 sults, although prepared on different principles. He showed that 

 by mixing Bernthsen's methylene violet and azure I with meth- 

 ylene blue and eosin Y in definite proportions, dissolving in 

 methyl alcohol, and employing like a Leishman or Wright stain, 

 one could obtain results almost identical with those secured by 

 these other blood stains without the uncertainties introduced by 

 their methods of manufacture. Soon after publishing this paper 

 he realized that methylene azure could be a mixture and certainly 

 was by the methods he had used in preparing it; (MacXeal, 1925). 

 He showed it to contain both azure A and azure B, whose chem- 

 istry is discussed in Chapter \ I of this book. He regarded the 

 former as the more important ingredient, and stated that the stain- 

 ing effects of Azure B are little different from those of a mixture 

 of azure A and methylene blue. This statement of his gave rise 

 to the impression that azure B was not a satisfactory stain; and 

 only recently has this impression come to be corrected in the minds 

 of users of these stains. MacNeal's tetrachrome stain is ordinarily 

 made up according to the following formula: 



Methylene blue chloride (dye content about 90%) 1-0 g. 



Azure A 0.6 g. 



Methylene violet, Bernthsen, free base 0.2 g. 



Eosin Y, (dye content 80-85%) 10 g. 



It will readily be seen that the dry tetrachrome stain is a mixture 

 rather than a chemical compound like the Leishman stain and its 

 modifications. The four dyes mixed together combine but very 

 slowly even after dissolving in the alcohol; and the first pro- 

 nounced chemical reaction occurs only after diluting with water in 

 actual application. There is evidence, however, that certain chemi- 

 cal reactions (and of an undesirable nature) take place in the 

 alcoholic solution, and thus interfere with the keeping qualities of 

 the solution. Manufacturers of tetrachrome stain realize this and 

 give directions for allowing the alcoholic solution to stand 48 

 hours at 50°C and then filtering; this practice seems to remove 

 whatever harmful compound may have formed, and the solution 

 thus obtained is reasonably permanent. Undoubtedly, more- 

 over, methyl alcohol alone is an undesirable solvent for this mixture 

 of dyes, just as it is in the case of Giemsa stain. There can be little 

 question but that the addition of 40% glycerol, by volume, as 

 recommended by Giemsa, would make the solvent for the tetra- 

 chrome stain more satisfactory and would improve the keeping 

 qualities of the solution. 



It can readily be inferred that the thiazin eosinates, made up by 

 any of the above mentioned procedures, must be very complex 

 mixtures. Theoretically each of the azures or other oxidation 



