Microspectrophotometric Studies of Romanowsky Stained Blood Cells. III. The Action of Methylene Blue and Azure B

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Abstract
The performances of 2 standardized Romanowsky stains (azure B/eosin and azure B/methylene blue/eosin) were compared with each other and with a methylene blue/eosin stain. Visible-light absorbance spectra of various hematological substrates have been measured. These have been analyzed in terms of the quantities of bound azure B, methylene blue and eosin dimers and monomers, and in terms of the CIE [Commission Internationale de l''Eelairage] color coordinates. The addition of methylene blue to azure B/eosin produces little change in performance, at least using these 2 analytical methods. Methylene blue/eosin does not produce the purplish colorations typical of the Romanowsky effect. This is due not to differences between the spectra of methylene blue and azure B, but to the fact that methylene blue does not facilitate the binding of eosin to cellular substrates to the same extent as azure B.