, Volume 86, Issue 3, pp 331–336 | Cite as

Understanding Romanowsky staining

I: The Romanowsky-Giemsa effect in blood smears
  • R. W. Horobin
  • K. J. Walter


Normal blood smears were stained by the standardised azure B-eosin Y Romanowsky procedure recently introduced by the ICSH, and the classical picture resulted. The effects of varying the times and temperature of staining, the composition of the solvent (buffer concentration, methanol content, & pH), the concentration of the dyes, and the mode of fixation were studied. The results are best understood in terms of the following staining mechanism. Initial colouration involves simple acid and basic dyeing. Eosin yields red erythrocytes and eosinophil granules. Azure B very rapidly gives rise to blue stained chromatin, neutrophil specific granules, platelets and ribosome-rich cytoplasms; also to violet basophil granules. Subsequently the azure B in certain structures combines with eosin to give purple azure B-eosin complexes, leaving other structures with their initial colours. The selectivity of complex formation is controlled by rate of entry of eosin into azure B stained structures. Only faster staining structures (i.e. chromatin, neutrophil specific granules, and platelets) permit formation of the purple complex in the standard method. This staining mechanism illuminates scientific problems (e.g. the nature of ‘toxic’ granules) and assists technical trouble-shooting (e.g. why nuclei sometimes stain blue, not purple).


Eosin Normal Blood Blood Smear Buffer Concentration Scientific Problem 
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  1. Baker JR (1958) Principles of biological microtechnique. Methuen, LondonGoogle Scholar
  2. Bentley SA, Marshall PN, Wade MJ, Galbraith W (1979) Azure B-eosin staining of blood cells. The effects of variation in stain formulation and staining technique on stain performance. Anal Quant Cytol 1:179–186Google Scholar
  3. Bessis M (1973) Living blood cells and their ultrastructure. (Translated by RI Weed) Springer, Berlin Heidelberg New YorkGoogle Scholar
  4. Boon ME, Druijver JS (1986) Routine cytological staining techniques, theoretical background and practice. Macmillan, LondonGoogle Scholar
  5. Clark G, Kasten FH (1983) History of staining, 3rd edn. Williams and Wilkins, BaltimoreGoogle Scholar
  6. Comings DE (1975) Mechanisms of chromosome banding. IV. Optical properties of the Giemsa dyes. Chromosoma 50:89–110Google Scholar
  7. Elliott K, Knight J (eds) (1975) Biochemistry and pharmacology of platelets. Ciba Foundation Symposium, New Series No. 35. Elsevier, AmsterdamGoogle Scholar
  8. Fedorko ME, Morse SI (1965) Isolation, characterisation, and distribution of acid mucopolysaccharides in rabbit leucocytes. J Exp Med 121:39–48Google Scholar
  9. Goldstein DJ (1963) An approach to the thermodynamics of histological dyeing, illustrated by experiments with azure A. Qu J Microsc Sci 104:413–439Google Scholar
  10. Goldstein DJ (1965) Relation of effective thickness and refractive index to permeability of tissue components in fixed sections. J R Microsc Soc 84:43–54Google Scholar
  11. Hardin JH, Spicer SS (1971) Ultrastructural localisation of dialysed iron-reactive mucosubstances in rabbit heterophils basophils, and eosinophils. J Cell Biol 48:368–386Google Scholar
  12. Horn RG, Spicer SS (1964) Sulfated mucopolysaccharides and basic protein in certain granules of rabbit leukocytes. Lab Invest 13:1–15Google Scholar
  13. Horobin RW (1974) A preliminary quantitative study of the ‘rate of staining’ model for rationalizing various mixed acid dye stains (‘trichromes’). Proc R Microsc Soc 9:110Google Scholar
  14. Horobin RW (1982) Histochemistry: an explanatory outline of histochemistry and biophysical staining. Gustav Fischer, Stuttgart; Butterworths, LondonGoogle Scholar
  15. International Committee for Standardisation in haematology (1984) ICSH Reference Method for staining of blood and marrow films by azure B and eosin Y (Romanowsky-Giemsa stain) Br J Haematol 57:707–710Google Scholar
  16. James J (1986) Paper delivered at The First Course in Standardisation and Quantitation of Diagnostic Staining in Cytology, Breda, The NetherlandsGoogle Scholar
  17. Lillie RD (1943) Some experiments on the Romanowsky staining of blood films. J Lab Clin Med 28:1872–1875Google Scholar
  18. Lillie RD (1944) Factors influencing the Romanowsky staining of blood films and the role of methylene violet. J Lab Clin Med 29:1181–1197Google Scholar
  19. McCall CE, Katayama I, Cotran RS, Finland M (1969) Lysosomal and ultrastructural changes in human “toxic” neutrophils during bacterial infections. J Exp Med 129:267–282Google Scholar
  20. Marshall PN (1978) Romanowsky-type stains in haematology. Histochem J 10:1–29Google Scholar
  21. Marshall PN (1980) Romanowsky and reticulocyte stains. In: Schmidt RM (ed) CRC Handbook Series in Clinical Laboratory Science. Section I: Hematology, vol 2: CRC Press, Cleveland, pp 63–77Google Scholar
  22. Perrin DD, Dempsey B (1974) Buffers for pH and metal ion control. Champan and Hall, LondonGoogle Scholar
  23. Proctor GB, Horobin RW (1986) A widely applicable analytical system for biological stains: reverse phase thin layer chromatography. Stain Technol 60:1–6Google Scholar
  24. Spitznagel JK, Dalldorf FG, Leffell MS, Folds JD, Welch IRH, Cooney MH, Martin LE (1974) Character of azurophil and specific granules purified from human polymorphonuclear leukocytes. Lab Invest 30:774–785Google Scholar
  25. Sumner AT (1980) Dye-binding mechanisms in G-banding of chromosoms. J Microsc 119:397–406Google Scholar
  26. Sumner AT, Evans HJ, Buckland RA (1973) Mechanisms involved in the banding of chromosomes with Quinacrine and Giemsa. II. The interaction of the dyes with the chromosomal components. Exp Cell Res 81:223–236Google Scholar
  27. Wittekind DH (1979) On the nature of Romanowsky dyes and the Romanowsky-Giemsa effect. Clin Lab Haematol 1:247–262Google Scholar
  28. Wittekind DH (1983) On the nature of Romanowsky-Giemsa staining and its significance for cytochemistry and histochemistry: an overall view. Histchem J 15:1029–1047Google Scholar
  29. Wittekind DH, Kretschmer V (1980) Zur Histochemie der Leukozytengranula—Untersuchungen mit reinen Farbstoffen, insbesondere an den neutrophien Granula. Acta Histochem (Suppl) 21:39–45Google Scholar
  30. Zanker V (1981) Grundlagen der Farbstoff-Substrat-Beziehungen in der Histochemie. Acta Histochem (Suppl) 24:151–168Google Scholar

Copyright information

© Springer-Verlag 1987

Authors and Affiliations

  • R. W. Horobin
    • 1
  • K. J. Walter
    • 1
  1. 1.Department of Anatomy and Cell Biology, The UniversitySheffieldEngland

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