Skip to main content
SpringerLink
Log in

On the nature of Romanowsky-Giemsa staining and the Romanowsky-Giemsa effect. I. Model experiments on the specificity of Azures B-Eosin Y stain as compared with other thiazine dye-Eosin Y combinations

  • Papers
  • Published:
The Histochemical Journal Aims and scope Submit manuscript

Summary

After incorporation into a polyacrylamide matrix, the biopolymers DNA, RNA, heparin, hyaluronic acid, collagen and the synthetic polymers poly(U) and poly(A, U) were stained with the pure thiazine dyes, Methylene Blue, the Azures and Thionin alone and combined with Eosin Y. Satisfactory spectrophotometric agreement was obtained between the staining reactions of the biopolymers in the artificial matrix and those in their natural surroundings. This was especially true with respect to the specificity of the Azure B-Eosin Y dye-pair, which is based on the generation, on suitable substrates, of a purple colour, the Romanowsky-Giemsa effect (RGE), with an absorbance maximum near 550 nm. In the model experiments, DNA, heparin, hyaluronic acid and collagen were found to be RGE-positive and poly(U), poly(A, U) and RNA RGE-negative.

A theory of RGE is proposed which complies with the new and earlier observations: after saturation of available anionic binding sites and aggregate formation by Azure B, electron donor acceptor complexes are formed between Eosin Y and Azure B via hydrogen-bridge formation of the aminosubstituent proton of Azure B and between Eosin Y and the biopolymer surface. Charge-transfer complex formation may also account for the qualitative identity of Azure B-Eosin Y and Azure A-Eosin Y spectra of substrates, which are coloured purple. Quantitatively, Azure A-Eosin Y is less efficient in giving RGE. The generation of RGE is time-dependent. Equilibrium staining is attained after about 120 h. The implications of the results for the biological application of Romanowsky-Giemsa staining are discussed briefly.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bergeron, J. A. &Singer, M. (1958) Metachromasy: an experimental and theoretical evaluation.J. biophys. biochem. Cytol. 4, 433–56.

    Google Scholar 

  • Comings, D. E. (1975) Mechanisms of chromosome banding. IV. Optical properties of Giemsa dyes.Chromosoma 50, 98–100.

    Google Scholar 

  • Darzynkiewicz, Z., Evenson, D., Kapuszincki, J. &Melamed, M. (1983) Denaturation of RNA and DNAin situ induced by Acridine Orange.Expl Cell Res. 148, 31–46.

    Google Scholar 

  • De Leenheer, A. P., Nelis, H., Van Liederke, A. & Vandamme, V. (1983) Purity and stability of Romanowsky dyes (Azure B). Liquid chromatographic analysis of Romanowsky stains. Report to the European Community Bureau of Reference (in press).

  • Duijndam, W. A. L., Hermans, J. &Van Duijn, P. (1973) Application of the method of kinetic analysis of staining and destaining processes to the complex formed between hydrolyzed desoxyribonucleoprotein and Schiff's reagent in model films.J. Histochem. Cytochem. 21, 729–36.

    Google Scholar 

  • Flax, M. H. &Himes, M. H. (1952) Microspectrophotometric analysis of metachromatic staining of nucleic aicds.Physiol. Zool. XXV, 297–311.

    Google Scholar 

  • Galbraith, W., Marshall, P. N. &Bacus,J. W. (1980) Microspectrophotometric studies of Romanowsky stained blood cells I. subtraction analysis of a standardized procedure.J. Microsc. 119, 313–30.

    Google Scholar 

  • Goldstein, D. (1969) The fluorescence of elastic fibres stained with Eosin and excited by visible light.Histochem. J. 1, 187–98.

    Google Scholar 

  • Horobin, R. W. (1982)Histochemistry. Stuttgasrt, New York: G. Fischer; London, Boston, Sydney, Wellington, Durban, Toronto: Butterworths.

    Google Scholar 

  • Horobin, R. W. &Bennion, P. W. (1973) The interrelation of size and substantivity of dyes; the role of vander Waals' attractions and hydrophobic bonding in biological staining.Histochemie 33, 191–204.

    Google Scholar 

  • International Committee For Standardization In Haematology (1984) ICSH Reference Method for staining of blood and bone marrow films by azure B and eosin Y (Romanowsky-Giemsa-stain).Br. J. Haemat. (in press).

  • Laboratory Manual (1983)Polyacrylamide Gel Electrophoresis. Laboratory techniques Uppsala Pharmacia Fine Chemicals AB.

  • Ladik, J. J. (1983) Charge-Transfer-Reaktionen in Biomolekülen. InBiophysik. 2nd edn. (edited byHoppe, W., Lohmann, W., Marki, H. andZiegler, H.), pp. 239–42. Berlin, Heidelberg, New York: Springer.

    Google Scholar 

  • Larsson, R. &Norden, B. (1970) On the dimerization of the acridine orange cation. A potentiometric and a spectrophotometric proof that the dimerization does not involve counterions.Acta chem. scand. 24, 2583–92.

    Google Scholar 

  • Lerman, L. S. (1961) Structural considerations in the interaction of DNA and the acridines.J. molec. Biol. 3, 18–30.

    Google Scholar 

  • Löber, G. (1969) Zur Komplexbildung von Farbstoffen mit Nucleinsäuren.Z. Chem. 9, 252–65.

    Google Scholar 

  • Löhr, W., Grubhofer, N., Sohmer, I. &Wittekind, D. (1975) The azure dyes: their purification and physicochemical properties. II. Purification of azure B.Stain Technol. 50, 149–56.

    Google Scholar 

  • Marshall, P. N., Galbraith, W., Navarro, E. F. &Bacus, J. W. (1981) Microspectrophotometric studies of Romanowsky stained blood cells II. Comparison of the performance of two standardized stains.J. Microsc. 124, 197–210.

    Google Scholar 

  • Mulliken, R. S. (1952a) Molecular compounds and their spectra.J. Am. chem. Soc. 74, 811–24.

    Google Scholar 

  • Mulliken, R. S. (1952b) Molecular compounds and their spectra. III. The interaction of electron donors and acceptors.J. phys. Chem. 56, 801–22.

    Google Scholar 

  • Newman, R. E. (1949) The amino acid composition of gelatins, collagens and elastins from different sources.Arch. Biochem. 23/24, 289–98.

    Google Scholar 

  • Rattee, I. D. &Breuer, M. M. (1974)The Physical Chemistry of Dye Adsorption. London, New York: Academic Press.

    Google Scholar 

  • Rigler, R. (1966) Microfluorometric characterization of intracellular nucleic acids and nucleoproteins by acridine orange.Acta phys. scand. 67, Suppl. 267.

    Google Scholar 

  • Steiner, R. F. &Beers, R. F. Jr. (1961)Polynucleotides. Amsterdam.: Elsevier Publishing Company.

    Google Scholar 

  • Stone, A. L. &Bradley, D. F. (1961) Aggregation of acridine orange bound to polyanions: the stacking tendency of desoxyribonucleic acids.J. Am. Chem. Soc. 83, 3627–34.

    Google Scholar 

  • Stork, W. H. J., De Hasseth, P. L., Schippers, W. B., Körmeling, C. M. &Mandel, M. (1973) Interaction between crystal violet and poly(methacrylic acid) in aqueous solutions. I. Results from spectroscopic measurements and dialysis.J. phys. Chem. 77, 1772–7.

    Google Scholar 

  • Sumner, A. T. &Evans, M. J. (1973) Mechanisms involved in the banding of chromosomes with quinacrine and Giemsa. II. The interaction of dyes with the chromosome components.Expl Cell Res.,81, 223–36.

    Google Scholar 

  • Tas, J. &Westermeng, G. (1981) Fundamental aspects of the interaction of propidium diiodide with nucleic acids studied in a model system of polyacrylamide films.J. Histochem. Cytochem. 29, 929–36.

    Google Scholar 

  • Van Duijn, P. &Van Der Ploeg, M. (1970) Potentialities of cellulose and polyacrylamide films as vehicles in quantitative cytochemical investigations on model substances. InIntroduction to Quantitative Cytochemistry (edited byWied, G. L. andBahr, G. F.), pp. 232–63. New York, London: Academic Press.

    Google Scholar 

  • Wagner, D. (1969) Zusammenhänge zwischen der Löslichkeit und der Assoziation von Farbstoffen in binären, wässrigen Systemen (Hydrotropie).Textil-Praxis 5, 310–14,6, 383–8.

    Google Scholar 

  • Wittekind, D. (1983) On the nature of Romanowsky-Giemsa staining and its significance for cytochemistry and histochemistry: an overall view.Histochem. 15, 1029–47.

    Google Scholar 

  • Wittekind. D. & Kretschmer. V. (1983) Unpublished results.

  • Wittekind, D., Kretschmer, V. & Schmidt, G. (1983) Unpublished results.

  • Wittekind, D., Schmidt, G. & Kretschmer, V. (1982) Unpublished results.

  • Zanker, V. (1981) Grundlagen der Farbstoff-Substrat-Beziehungen in der Histochemie.Acta histochem. Suppl.24, 151–68.

    Google Scholar 

  • Zimmermann, H. W. (1983) Physikalisch-chemische Grundlagen der Färbung für manuelle und apparative Zytodiagnostik.Microsc. Acta Suppl. 6, 45–58.

    Google Scholar 

  • Zipfel, E., Grèzes, J. R., Seiffert, W. &Zimmermann, H. W. (1981) Über Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt. 1 Mitteilung: Azur B, Reinheit und Gehalt von Farbstoffproben, Assoziation.Histochemistry 72, 279–90.

    Google Scholar 

  • Zipfel, E., Grèzes, J. R., Seiffert, W. &Zimmermann, H. W. (1982) Über Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt. 2. Mitteilung: Eosin Y, Erythrosin B, Tetrachlorfluoreszein. Spektroskopische Charakterisierung der reinen Farbstoffe, Assoziation von Eosin, Y.Histochemistry 75, 539–55.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institut für Anatomie II der Universität, Albertstrasse 17, 7800, Freiburg i. Br., West Germany

    D. H. Wittekind & T. Gehring

Authors
  1. D. H. Wittekind
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. T. Gehring
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wittekind, D.H., Gehring, T. On the nature of Romanowsky-Giemsa staining and the Romanowsky-Giemsa effect. I. Model experiments on the specificity of Azures B-Eosin Y stain as compared with other thiazine dye-Eosin Y combinations. Histochem J 17, 263–289 (1985). https://doi.org/10.1007/BF01004591

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01004591

Keywords

Search

Navigation