Über Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt

Summary

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed.

The nucleus of RG stained LM cells, pH≃7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates.

In the spectrum of RG stained nuclei, pH≃7, three absorption bands are distinguishable: They are A1 (15400 cm⁻¹, 649 nm), A2 (16800 cm⁻¹, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm⁻¹, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of assoziation p>2) and eosin Y. The complex is primarily held together by electrostatic interaction: Binding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both moleculare species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm⁻¹ is due to the cosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei.

Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y. Its absorption spectrum is identical with the spectrum of Romanowsky stained nuclei. This experiment demonstrates that the whole Romanowsky-Giemsa staining pattern of the nucleus is primarily produced by DNA, azure B and eosin Y.

The spectrum of RG stained cytoplasm, pH≃7, consists of three absorption bands A1′ (15300 cm⁻¹, 654 nm), A2′ (16900 cm⁻¹, 592 nm), A3′ (17600 cm⁻¹, 568 nm). They are attributed to monomers, dimers and polymers of azure B bound to RNA. In general A2′ and A3′ overlap strongly and generate a broad absorption band A′ (∼17000 cm⁻¹, 588 nm). Some experimental results can be interpreted in terms of a RNA-azure B-eosin Y complex in the cytoplasm. But its concentration must be very small.

Variations in the biological materials and the experimental staining conditions may alter the position and the intensity of the absorption bands but does not change the underlying molecular concept of the Romanowsky-Giemsa effect.