SECTION ONE 



amphoteric properties, as does acid fuchsin and certain other acid 

 dyes. It can only combine with basic dyes or acid dyes which 

 possess basic groups attached directly to the aromatic nuclei. 

 The structure of eosin Y is such that union with a basic dye 

 or an amphoteric dye would not result in the decolorization of 

 the eosin. With a suitable basic dye it would form an eosinate, 

 an alcoholic solution of which, applied to microscopic tissue 

 preparations, would produce a polychrome picture. If the eosin 

 were to combine with an acid aminotriarylmethane dye containing 

 one or more basic groups, or certain other acid dyes of the 

 quinonoid group, it might be expected to decolorize or partially 

 decolorize such dyes by disrupting their resonance sequence of 

 double and single bonds. Water soluble nigrosin is a highly 

 sulphonated (acid) dye of the azine series, the exact structure of 

 which is not known. Azine dyes also belong to the quinonoid group. 

 The dye-ion of nigrosin possesses a basic nitrogen atom as 

 well as sulphonic acid groups. In view of statements made 

 above, one might reasonably expect the eosin to decolorize the 

 nigrosin either wholly or partially, or at least to modify its colour. 

 In fact in vitro tests which I have recently carried out show that 

 this actually happens. Eosin Y unites chemically with water- 

 soluble nigrosin to produce a water soluble compound dye 

 which is much deeper in colour than eosin itself but lighter in 

 colour than nigrosin. The compound is in fact bluish violet. It 

 might be asked how then does nigrosin " decoloriz;e " the eosin in 

 Button's (1928) technique? It should be remembered that the 

 concentration of the solid methylene blue eosinate of Wright's 

 stain solution is only 0-3%. This means that the actual eosin 

 content is only about i'5%. Only a fraction of the Wright's 

 stain, and consequently the eosin component, applied to the 

 specimen, is taken up by the tissues. The bulk of the stain is 

 washed away. The nigrosin is applied in a concentration which 

 is many times greater than that of the eosin of the Wright's 

 stain solution, and still greater than the concentration of the 

 eosin left in the cytoplasm. Therefore the number of molecules 

 of nigrosin present would be infinitely greater than the number 

 of molecules of eosin. If it takes one, two, three or even as many 

 as twenty molecules of nigrosin to unite with each of the eosin 

 molecules present in the tissues and these nigrosin molecules are 

 reduced in colour as a result of the union, then there is still an 



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