Quarterly Journal of Microscopical Science, Vol s3-103, 519-530, Copyright © 1962 by Company of Biologists

An Investigation of the Anomalous Staining of Chromatin by the Acid Dyes, Methyl Blue and Aniline Blue

¹ Cytological Laboratory, Department of Zoology, University Museum, Oxford, and the Colour Chemistry Research Laboratory, Department of Chemical Technology, The Royal College of Science and Technology, Glasgow, C. 1

Methyl blue and aniline blue, though acid dyes, stain the chromatin of the spermatogenetic cells of the mouse (especially of the primary spermatocytes) strongly. Extraction of the basiphil nucleic acid constituents from the chromatin causes loss of this property, while destruction of acidophilia in the protein constituents does not. It has been concluded that the dyes interact with the nucleic acids. Further, they appear to react with both DNA and RNA in the chromatin, although they show no affinity for the cytoplasm of the exocrine cells in sections of pancreas, which is rich in RNA.

The mechanism of the reaction has not been fully elucidated, although apparently the dyes do not behave as basic dyes towards the nucleic acids, and the interaction is non-ionic. Methyl blue and aniline blue stain strongly other ‘acidic’ substrates, such as cellulose and nitrocellulose, and attempts have been made to relate the staining of nucleic acids to the staining of these substrates, particularly cellulose; for the staining properties of this substrate have been intensively investigated elsewhere. No satisfactory correlation, however, has been obtained, for nitrocellulose has been found to be less strongly stained at pH 3.0 than at pH 7.1, while the reverse is true for cellulose. Further, only one of 3 direct cotton dyes used appears to have any affinity for the chromatin of the spermatogenetic cells. Direct cotton dyes have large flat molecules with a high degree of conjugation. It is suggested that these characteristics are essential for interaction with nucleic acids, and also that the molecule must be reasonably compact.

Finally, it has been shown that methyl blue, aniline blue, and 3 direct cotton dyes of the azo type have no ability to stain the glycogen in liver cells, yet glycogen is very closely related to cellulose.