Xanthene Dyes 185 



and Holmes (1926) ; by reference to which paper it will be seen that 

 in color the dyes above listed increase in depth in the following 

 order: eosin Y, ethyl eosin, eosin B, erythrosin B, phloxine, rose 

 bengal. In the case of each of these, the color is lighter or deeper 

 according to the number of halogen atoms present. Eosin B is 

 the strongest acid in the group, and eosin Y the next strongest; 

 erythrosin is weaker, phloxine weaker than eosin and possibly 

 weaker than erythrosin; while rose bengal is the weakest of all. 



To interpret this information into terms of actual use, it is neces- 

 sary to remember that there are two main types of histological pro- 

 cedures in which eosin is used: first as a general counterstain 

 (usually in alcoholic solution) following a basic dye; secondly, as a 

 cytoplasm stain (always in aqueous solution) preceding a basic dye. 

 In the first of these types of procedure it is important that the 

 dye be one with diffuse staining properties and with a color show- 

 ing good contrast to the basic dye employed (generally methylene 

 blue or hematoxylin). The more acid and lighter colored dyes in 

 the series (eosin Y, ethyl eosin, and eosin B) seem to possess these 

 properties to the greatest extent and accordingly to be best adapt- 

 ed to such procedures. The particular dye to choose depends 

 undoubtedly on the exact shade desired. 



The second type of procedure (i.e. preceding a basic dye) is rep- 

 resented by the Mallory phloxine-methylene-blue stain and the 

 Held erythrosin-methylene-blue technic. In such procedures as 

 these, both dyes are used in aqueous solution; and there is there- 

 fore much more opportunity for chemical interaction between the 

 acid dye already in the tissue and the basic dye subsequently 

 added than when the acid dye is used in alcoholic solution. (It is 

 well known that acid and basic dyes in alcoholic solution do not 

 form insoluble reaction products.) In procedures like the Mallory 

 technic, there seems to be a tendency (possibly on account of this 

 interaction) for the acid dye to stain the cytoplasm too weakly 

 when followed by a basic dye, in case the very acid eosin Y is used. 

 Thus Held employs erythrosin, and Mallory who formerlyjspeci- 

 fied eosin discovered later that his original eosin was not a true 

 eosin Y, phloxine giving better results in his technic than one of the 

 true eosins. Now phloxine and erythrosin are not only deeper in 

 color than eosin Y, but are also less strongly acidic; and it is pos- 

 sible that their chemical nature rather than their color may be the 

 decisive factor in determining their superiority for such pro- 

 cedures as this. Rose bengal is even deeper in color, and is still 

 less strongly acidic; in fact, it seems to be too purple to contrast 

 well with methylene blue and of such a weakly acid character that 

 it tends to remain in the nuclei when used by the technic in ques- 

 tion. It might prove valuable preceding some basic dye; but it 

 has been found to give poor results preceding methylene blue in 

 the Mallory technic. 



