How Romanowsky stains work and why they remain valuable — including a proposed universal Romanowsky staining mechanism and a rational troubleshooting scheme

Abstract

An introduction to the nomenclature and concept of “Romanowsky stains” is followed by a brief account of the dyes involved and especially the crucial role of azure B and of the impurity of most commercial dye lots. Technical features of standardized and traditional Romanowsky stains are outlined, e.g., number and ratio of the acidic and basic dyes used, solvent effects, staining times, and fixation effects. The peculiar advantages of Romanowsky staining are noted, namely, the polychromasia achieved in a technically simple manner with the potential for stain intensification of “the color purple.” Accounts are provided of a variety of physicochemically relevant topics, namely, acidic and basic dyeing, peculiarities of acidic and basic dye mixtures, consequences of differential staining rates of different cell and tissue components and of different dyes, the chemical significance of “the color purple,” the substrate selectivity for purple color formation and its intensification in situ due to a template effect, effects of resin embedding and prior fixation. Based on these physicochemical phenomena, mechanisms for the various Romanowsky staining applications are outlined including for blood, marrow and cytological smears; G-bands of chromosomes; microorganisms and other single-cell entities; and paraffin and resin tissue sections. The common factors involved in these specific mechanisms are pulled together to generate a “universal” generic mechanism for these stains. Certain generic problems of Romanowsky stains are discussed including the instability of solutions of acidic dye–basic dye mixtures, the inherent heterogeneity of polychrome methylene blue, and the resulting problems of standardization. Finally, a rational trouble-shooting scheme is appended.

Key words::

• AT-rich genome
• bacteria
• blood film
• carmine
• G-banded chromosome
• Giemsa
• Leishman
• malaria parasite
• marrow smear
• microorganism
• paraffin section
• parasite
• resin section
• trouble-shooting
• Wright

Acknowledgments and dedications

I wish to acknowledge Prof. I. McGrath, Division of Integrated Biology, FBLS, University of Glasgow, for providing facilities, and P.N. Marshall, A.T. Sumner and Richard Hartley (Department of Chemistry, University of Glasgow) for helpful discussion and comment. I dedicate this paper to R.D. Lillie and D.W. Wittekind, two colorful giants of histotechnology. I had the benefit and, sometimes, pleasure of meeting both men. The former told me long ago “Young men like theories, but I think you should go back to the lab and do more experiments, Horobin,” while the latter said to me many times “But Dick…” Perhaps I should have listened to them more attentively.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.