Summary
The chances of Romanowsky-Giemsa (RG) staining becorning a reliable and useful histochemical procedure are reviewed, based on the now proven fact that RG staining requires two dyes only, namely, cationic Azure B and anionic Eosin Y. These two dyes differe from otherwise similar dye combinations in that they give, on distinct biological substrates, one additional colour, purple, which cannot be obtained by the use of either dye alone. The purple colour characterizes the Romanowsky-Giemsa effect (RGE), which is the essential feature of RG staining.
Consideration is given to the physico-chemical and morphological implications of RGE. Of primary importance is the nature of the biological substrates where RGE occurs, and also of those where it has never been observed. The way substrates react to RG stains largely depends on the kind of pretreatment they have received; for instance, alcoholic fixation preserves RGE but formaldehyde may inhibit it. Physico-chemical factors are considered which, by altering either the biological substrates or the composition of the staining solutions, may modify the RG staining pattern.
This review also serves as an introduction for a series of experimental papers that will follow and which are intended to consolidate the basis of RG staining, a method which holds much promise as a useful histochemical tool.
Similar content being viewed by others
References
Baker, J. R. (1958)Principles of Biological Microtechnique. London: Methuen.
Bentley, S. A., Marshall, P. N. &Trobaugh, F. E. (1980) Standardization of Romanowsky procedure: an overview.Analyt. Quant. Cytol. 2, 15–8.
Bretz, V. &Baggiolini, M. (1974) Biochemical and morphological characterization of azurophil and specific granules of human neutrophil leukocytes.J. Cell Biol. 63, 251–69.
Comings, D. E. (1975) Mechanisms of chromosome banding. IV. Optical properties of Giemsa dyes.Chromosoma 50, 98–100.
Conn, H. J. &Lillie, R. D. (1977)Biological Stains. A Handbook on the Nature and the Uses of the Dyes Employed in the Biological Laboratory. Baltimore: Williams & Wilkins.
Cramer, H. D., Rogers, E. R., Parker, J. W. &Lukes, R. J. (1973) The Giemsa stain for tissue sections: an improved method.J. Clin. Path. 60, 148–56.
Curtis, D. J. &Horobin, R. W. (1975) Staining banded human chromosomes with Romanowsky dyes: some practical consequences of the nature of the stain.Humangenetik 26, 99–104.
Duijndam, W. A. L. &Van Duijn, P. (1975) The influence of chromatin compactness on the stoichiometry of the Feulgen-Schiff procedure studied in model films. I. Theoretical kinetics and experiments with films containing isolated deoxyribonucleic acid.J. Histochem. Cytochem. 23, 882–90.
Eickhorn, R. (1982)Über Färbungseffekte kationischer Oxazinfarbstoffe in der Cytologie und über Zusammenwirken mit anionischen Farbstoffen am Substrat. Inaugural dissertation, University of Freiburg i. Br.
Feder, N. &Wolf, M. K. (1965) Studies on nucleic acid metachromasy. II. Metachromatic and orthochromatic staining by toluidine blue of nucleic acids in tissue sections.J. Cell Biol. 27, 327–36.
Fedorko, M. &Morse, S. E. (1965) Isolation, characterization and distribution of acid mucopolysaccharides in rabbit leukocytes.J. exp. Med. 121, 39–48.
Filkuka, J. &Kleinwächter, V. (1981)Basic Staining of Cell Nuclei. Brno: Purkyne University, Medical Faculty.
Flax, M. H. &Himes, M. H. (1952) Microspectrophotometric analysis of metachromatic staining of nucleic acids.Physik. Zool. XXV, 297–311.
Fraenkel-Conrat, H. (1954) Reaction of nucleic acid with formaldehyde.Biochim. Biophys. Acta 15, 307–9.
Galbraith, W., Marshall, P. N. &Bacus, I. W. (1980) Microspectrophotometric studies of Romanowsky stained blood cells.J. Microsc. 119, 313–30.
Giemsa, G. (1902a) Färbemethoden für Malariaparasiten.Zbl. Bakteriol. 31, 429–30.
Giemsa, G. (1902b) Färbemethoden für Malariaparasiten.Zbl. Bakteriol. 32, 307–13.
Giemsa, G. (1904) Eine Vereinfachung und Vervollkommnung meiner Methylenazurmethylenblau-Eosinfärbung zur Erzielung der Romanowsky-Nochtschen Chromatinfärbung.Zbl. Bakteriol. 37, 308–11.
Giemsa, G. (1910) Über die Färbung von Schnitten mittels Azur-Eosin.Dtsch. Med. Wschr. 12, 552–3.
Giemsa, G. (1934) Geschichte, Theorie und Weiterentwicklung der Romanowsky-Färbung.Med. Welt 8, 1432–4.
Gilliland, J. W., Rogers, C. H. &Sage, B. H. (1974) Blood stain for automatic differential analysis of leukocytes.Clin. Lab. Products 3, 2–8.
Goldstein, D. J. (1968) On the affinity of dyes for histological substrates. InCell Structure and its Interpretation (edited byMcGree-Russel, S. M. andRoss, K. F. A.), pp. 67–77. London: Arnold.
Good, E., Winget, G. D., Winter, W., Conolly, T. N., Izawa, S. &Singh, R. M. M. (1966) Hydrogen buffers for biological research.Biochemistry 5, 467–77.
Grossman, L., Levine, S. L. &Allison, W. S. (1961) The reaction of formaldehyde with nucleotides and T2 Bacteriophage DNA.J. Molec. Biol. 3, 47–60.
Halaby, S. A. &Vance, M. E. (1979) Computer controlled spectral measurements of blood cells IEEE.Trans. Biomed. Eng. 26, 34–43.
Harms, H. (1965)Handbuch der Farbstoffe für die Mikroskopie. Kamp-Lintfort: Staufen-Verlag.
Horobin, R. W. (1980) Structure-staining relationships in histochemistry and biological staining. I. Theoretical background and a general account of correlation of histochemical staining with the chemical structure of the reagents used.J. Microsc. 119, 345–55.
Horobin, R. W. (1981) Structure-staining relationships in histochemistry and biological staining. Part 3. Some comments on the intentional and artifactual staining of lipids.Acta histochem. Suppl. Bd. XXIV, 237–46.
Horobin, R. W. &Flemming, L. (1980) Structure staining relationships in histochemistry and biological staining. II. Mechanistic and practical aspects of the staining.J. Microsc. 119, 357–72.
ICSH-Expert Panel for Standardization of Stains in Haematology, Bilthoven (1976): ICSH-Publications, 1983.
Jenner, L. (1899) A new preparation for rapidly fixing and staining blood.Lancet 1, 370.
Krause, R. (1926)Enzyklopädie der mikroskopischen Technik III. Berlin, Wien: Urban-Schwarzenberg.
Lapen, D. (1982) A standardized differential stain for hematology.Cytometry 2, 309–15.
Leeman, U., Weiss, S. &Schmutz, E. (1971) Centrifugation technique for cytochemical preparations.J. Histochem. Cytochem. 19, 758–60.
Lennert, K. (1952) Zur Praxis der pathologisch-anatomischen Knochenmarksuntersuchung.Frankf. Ztschr. Rath. 63, 267–99.
Lennert, K. (1978)Methodological Prerequisites for a Differential Diagnosis of Lymphomas. Berlin, Heidelberg, New York: Springer-Verlag.
Lillie, R. D. (1944a) Purification of the thiazines and spectra of purified dyes.Pub. Health Rep., Suppl.178, 17–21.
Lillie, R. D. (1944b) Factors influencing the Romanowsky staining of blood films and the role of methylene violet.J. Lab. clin. Med. 29, 1181–97.
Lison, L. (1935) Sur la détermination du pH intracellulaire par les colorants vitaux indicatuers: L'erreur métachromatique.Protoplasma 24, 453–65.
Loach, K. W. (1971) Thin layer chromatographic separation of methylene blue and related thiazine dyes.J. Chromatogr. 60, 119–26.
Macneal, W. J. (1925) Methylene violet and methylene azure A and B.J. Infect. Dis. 36, 538–46.
Malachowsky, E. (1891) Zur Morphologie des Plasmodium Malasiae.Cbl. Klin. Med. 12, 601–3.
Marshall, P. N. (1978) Romanowsky-type stains in haematology.Histochem. J. 10, 1–29.
Marshall, P. N. (1979) Romanowsky staining: state of the art and ideal techniques. InDifferential Leukocyte Counting, pp. 205–6. College of American Pathologists.
Marshall, P. N., Bentley, S. A. &Lewis, S. M. (1975) A standardized Romanowsky stain derived from purified dyes.J. clin. Path. 28, 920–3.
Marshall, P. N., Galbraith, W., Navarro, E. F. &Bacus, I. W. (1981) Microspectrophotometric studies of Romanowsky stained blood cells. II. Comparison of the performance of two standardized stains.J. Microsc. 124, 197–210.
Mcnamara, W. L. (1933) Giemsa stain for tissue, rapid method.J. Lab. clin. Med. 18, 752–3.
May, R. &Grünwald, L. (1902) Uber Blutfärbungen.Cbl. inn. Med. 11, 265–70.
Nerenberg, C. &Fischer, R. (1963) Purification of thionin, azur A, azur B and methylene blue.Stain Technol. 38, 75–84.
Pappenheim, A. (1912) Die kombinierte May-Giemsa-Essigsäure-Färbungsmethode als histologische Universalübersichtsfärbung.Anat. Anz. 42, 525–8.
Pauluhn, J., Grèzes, R. &Zimmermann, H. W. (1981) Modellversuche zur Bindung von Ethidium bromid an DNA als Grundlage für die stöchiometrische Färbung der Biopolymeren in Zellen.Acta histochem., Suppl.24, 169–80.
Price, D. L. (1968) An-improved method for Giemsa staining of formalin-fixed tissue sections.Milit. Med. 133, 363–7.
Roe, M. A., Lillie, R. D. &Wilcok, A. (1940) American azures in the preparation of satisfactory Giemsa stains.Publ. Hlth. Rep. 55, 1272–8.
Romanowsky, D. L. (1891)On the question of parasitology and therapy of malaria. Dissertation No. 38 (in Russian), Imperial Medical Military Academy, St. Petersburg.
Romeis, B. (1948)Taschenbuch der mirkroskopischen Technik. München: Oldenburg.
Saal, J. R. (1964) Giemsa stain for the diagnosis of bovine babesiosis. I. Staining properties of commercial samples and their component dyes.J. Protozool. 11, 573–82.
Scheibe, F. &Zanker, V. (1958) Physikochemische Grundlagen der Metachromasie. Suppl. Bd. I,Acta Histochem. 6–37.
Schulte, E. (1982) Uber Färbungseffekte kationischer Phenazinfarbstoffe in der Cytologie und über ihr Zusammenwirken mit anionischen Farbstoffen am biologischen Substrat. Inaugural dissertation, University Freiburg i. Br.
Steel, F. (1980) Personal communication.
Sternberg, C. (1905) Eine Schnittfärbung nach der Romanowsky'schen Methode.Cbl. Allg. Path. Path. Anat. 16, 293–4.
Sumner, A. T. (1980) Dye binding mechanisms in G-banding of chromosomes.J. Microsc. 119, 397–406.
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.
Tas, I. (1977) Polyacylamide films as a tool for investigating qualitative and quantitative aspects of the staining of glycosaminoglycans with basic dyes.Histochem. J. 9, 267–76.
Taylor, K. B. (1961) The influence of molecular structure of thiazine and oxazine dyes on their metachromatic properties.Stain Technol. 36, 73–83.
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.
Von Moellendorff, W. (1920) Vitale Färbungen an tierischen Zellen.Erg. Physiol. 18, 141–306.
Von Prowazek, S. (1914) Zur Kenntnis der Giemsafärbung vom Standpunkt der Zytologie.Z. Mikros. 31, 1–16.
Wittekind, D. (1979) On the nature of Romanowsky dyes and the Romanowsky-Giemsa-effect.Clin. Lab. Haemat. 1, 247–62.
Wittekind, D. (1980) Farbungen in der Haematologie und ihre Beiträge zur Kenntnis der Morphologie des Blutes und der blutbildenden Organe.Verh. Anat: Ges. 74, 183–201.
Wittekind, D. &Kretschmer, V. (1978) Über Farbstoffwirkungen auf fixierte Zellen. Untersuchungen an Zellkulturen mit gereinigten Thiazinfarbstoffen und Eosin.Verh. Anat. Ges. 72, 383–4.
Wittekind, D. &Kretschmer, V. (1980) Zur Histochemie der Leukozytengranula, Untersuchungen mit reinen Farbstoffen, insbesondere an Leukozytengranula.Acta histochem., Suppl. Bd.21, 39–45.
Wittekind, D., Kretschmer, V. &Löhr, W. (1978) Kann Azur B-Eosin die May-Grünwald-Giemsa-Färbungersetzen?Blut 32, 71–8.
Wittekind, D., Kretschmer, V. &Sohmer, I. (1982) Azure B-eosin Y stain as the standard Romanowsky-Giemsa-stain.Br. J. Haemat. 51, 391–3.
Zanker, V. (1973) Definition der Farbstoffe.Acta histochem., Suppl.13, 233–49.
Zanker, V. (1981) Grundlagen der Farbstoff-Substrat-Beziehungen in der Histochemie.Acta histochem., Suppl.24, 151–68.
Zipfel, E., Grèzes, J. R., Seiffert, W. &Zimmermann, H. W. (1981) Über Romanowky-Farbstoffe und den Romanowsky-Giemsa-Effekt. 1. Mitteilung: Azur B, Reinheit und Gehalt von Farbstoffproben, Assoziation.Histochemistry 72, 279–90
Zipfel, E., Grèzes, J. R., Seiffert, W. &Zimmermann, H. W. (1982) Über Romanowsky-Farbstoffe un den Romanowsky-Giesma-Effekt. 2. Mitteilung: Eosin Y, Erythrosin B, Tetrachlorfluoreszein. Spektroskopische Charakterisierung der reiner Farbstoffe, Assoziation von Eosin.Histochemistry 75, 539–55.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Wittekind, D.H. On the nature of Romanowsky-Giemasa staining and its significance for cytochemistry and histochemistry: an overall view. Histochem J 15, 1029–1047 (1983). https://doi.org/10.1007/BF01002498
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF01002498