Summary
The present paper gives a review of the actual state of standardization of biological dyes and stains. In a first part general information is given on practical problems encountered by the routine user of dyes with special emphasis on dye contamination. Some theoretical aspects of standardization are discussed. The second part of the paper gives more detailed information on commercial batches of hematoxylin-eosin-, Giemsa- and Papanicolaou-stains and on their standardization. Special problems arising with the application of image analysis techniques are briefly mentioned. User-oriented specifications for the standardization of dyes, stains and staining procedures are given. Fluorescent dyes and dyes used in chromogenic reagents such as the Feulgen-Schiff reaction are not included in this review.
Similar content being viewed by others
References
Allison RT (1982) The Biological Stain Commission. A model for the UK? Gazette IMLS 1:3–4
Allison RT (1984) The need to standardise biological dyes. Gazette IMLS 28:487–488
Allison RT (1987) Personal communication
Bahr GF, Bartels PH, Bibbo M, de Nicolas M, Wied GL (1973) Evaluation of the Papanicolaou stain for computer assisted cellular pattern recognition. Acta Cytol 17:106–122
Baker JR, (1966) Cytological technique: the principles underlying routine methods. Methuen, London
Balaton A (1987) Applications des micro-ondes dans les techniques histologiques. Ann Pathol 7:242–245
Bartels PH, Bahr GF, Bibbo M, Richards DL, Sonek MG, Wied GL (1974) Analysis of variance of the Papanicolaou staining reaction. Acta Cytol 18:522–531
Bennion PJ, Horobin RW, Murgatroyd LB (1975) The use of a basic dye (Azur A or Toluidine Blue) plus a cationic surfactant for selective staining of RNA: a technical and mechanistic study. Stain Technol 50:307–313
Bentley SA, Marshall PN, Trobaugh FE (1982) Standardization of the Romanowsky staining procedure. An overview. Anal Quant Cytol 2:15–18
Böck P (1988) Romeis mikroskopische Technik. Urban & Schwarzenberg, München Wien Baltimore
Böhmer F (1865) Zur pathologischen Anatomie der Meningitis cerebromedullaris epidemica. Aerztl Intelligenzbl 12:539–550
Burger G, Jütting U, Rodenacker K (1981) Changes in benign populations in cases of cervical cancer and its precursors. Analyt Quant Cytol 3:261–271
Burger G, Jütting U, Soost HJ (1983) Notwendigkeit und Aufbau von zytologischen Datenbanken. Microsc Acta 6 [Suppl]:103–120
Clark G (1973) Staining procedures used by the Biological Stain Commission, 3rd edn. Williams & Wilkins, Baltimore London
Clark G (1974) Comparison of various oxidants for alum hematoxylin. Stain Technol 49:225–227
Clark G, Kasten FH (1983) History of staining, 3rd edn. Williams and Wilkins, Baltimore London
Clemens HJ, Toepfer K (1968) Physical-chemical properties of commercial thiazine dyes. 2. Quantitative and qualitative analysis of the dyes, and the reprecipitation method for obtaining higher degrees of purity. Acta Histochem 31:126–134
Colour Index (1971) Published by the Society of Dyers and Colourists and the American Association of Textile Chemists and Colorists. Lund Humphries, Bradford London
Conn HJ (1980) The history of the Stain Commission: I. Preliminaries. Stain Technol 55:327–352
Conn HJ (1981) The history of the Stain Commission: III. Birth of the Stain Commission. Stain Technol 56:1–17
Curtis DJ, Horobin RW (1975) Staining banded human chromosomes with Romanowsky dyes: some practical consequences of the nature of the stain. Humangenetik 26:99–105
Dean WW, Stastny M, Lubrano GJ (1977) The degradation of Romanowsky-type blood stains in methanol. Stain Technol 52:35–46
De Campos-Vidal B, Schlüter G, Moore GW (1973) Cell nucleus pattern recognition: influence of staining. Acta Cytol 17:510–521
DIN, Deutsches Institut für Normung (1987) DIN-Taschenbuch Medizinische Mikrobiologie. Beuth, Berlin Köln
Friedrich K, Seiffert W, Zimmermann HW (1990) Romanowsky dyes and Romanowsky-Giemsa effect. 5. Structural investigations of the purple DNA-AB-EY dye complexes of Romanowsky-Giemsa staining. Histochemistry 93:247–256
Freudenberg N (1980) Zytopathologie. Farbatlas und Kurzlehrbuch. Schattauer, Stuttgart New York
Galbraith W, Marshall PN, Lee ES, Bacus JW (1979) Studies on Papanicolaou staining. I. Visible-light spectra of stained cervical cells. Analyt Quant Cytol 1:160–168
Galbraith W, Marshall PN, Bacus JW (1980) Microspectrophotometric studies of Romanowsky stained blood cells. J Microsc 119:313–330
Galbraith W, Marshall PN (1984) Studies on Papanicolaou staining. III. Quantitative investigations of orangeophilia and cyanophilia. Stain Technol 59:133–142
Gill GW, Frost JK, Miller KA (1974) A new formula for a half oxidized hematoxylin solution that neither overstains nor requires differentiation. Acta Cytol 18:300–311
Gilliland JW, Dean WW, Stastny M, Lubrano G (1979) Stabilized Romanowsky blood stain. Stain Technol 54:141–150
Green MR, Patewska JV (1979) The cationic carbocyanine dyes Stains-all, DBTC, and Ethyl-Stains-all, DBTC-3,3′,9′-triethyl. J Histochem Cytochem 27:797–799
Green IE, Weintraub HA, Donnelly BS, Mordecai BG (1979) Sample preparation variation and its effect on automated blood cell differential analysis. Analyt Quant Cytol 1:187–201
Gunzer U, Harms H, Haucke M, Gerlach B, Thieme S, Aus HM, Meulen V ter (1984) Computeranalysen in der Hämatologie: Ein Beitrag zur Früherkennung von bösartigen hämatologischen Systemerkrankungen. In: Deutsche Forschungs- und Versuchsanstalt für Luft- und Raumfahrttechnik (Hrsg) Automation der zytologischen Diagnostik. Verlag TÜV Rheinland, Köln, pp 115–136
Harms H (1965) Handbuch der Farbstoffe für die Mikroskopie. Staufen, Kamp-Lintfort
Hiddemann W, Schumann J, Andreeff M, Barlogie B, Herman CJ, Leif RC, Mayall BH, Murphy RF, Sandberg AA (1984) Convention on nomenclature for DNA cytometry. Cytometry 5:445–446
Hollander DH (1975) De gustibus non disputandum: an examination of Papanicolaou staining. Acta Cytol 19:404–406
Holmquist J, Imasato Y, Bengtsson E, Olsen B, Stenkvist B (1976) A microspectrophotometric study of Papanicolaou-stained cervical cells as an aid in computerized image processing. J Histochem Cytochem 24:1218–1224
Hopwood D (1985) Cell and tissue fixation, 1972–1982. Histochem J 17:389–442
Hopwood D, Coghill G, Ramsay J, Milne G, Kerr M (1984) Microwave fixation: its potential for routine techniques, histochemistry, immunocytochemistry and electron microscopy. Histochem J 16:1171–1191
Horobin RW (1969) The impurities of biological dyes: their detection, removal, occurrence and histological significance — a review. Histochemical J 1:231–265
Horobin RW (1973) Effects on staining of the presence of various kinds of impurities. Acta Histochem 13 [Suppl]:269–280
Horobin RW (1980) The use of pure dyes for biological staining. Koch-Light, Colnbrook
Horobin RW, Boon ME (1988) Understanding microwave-stimulated Romanowsky-Giemsa staining of plastic embedded bone marrow. Histochem J 20:329–334
Horobin RW, Curtis D, Pindar L (1989) Understanding Romanowsky-Giemsa staining. II. The staining mechanism of suspension fixed cells, including influences of specimen morphology on the Romanowsky-Giemsa effect. Histochemistry 91:77–80
Horobin RW, Goldstein D (1972) Impurities and staining characteristics of Alcian Blue samples. Histochem J 4:391–399
Horobin RW, Murgatroyd LB (1968) The composition and properties of Gallocyanin-chrome alum stains. Histochem J 1:36–54
Horobin RW, Murgatroyd LB (1969) The identification and purification of Pyronin and Rhodamine dyes. Stain Technol 44:297–302
Horobin RW, Walter K (1987) Understanding Romanowsky staining. I: The Romanowsky-Giemsa effect in blood smears. Histochemistry 86:331–336
ICSH (1984) International Committee for Standardization in Haematology: ICSH reference method for staining of blood and bone marrow films by azure B and eosin Y (Romanowsky stain). Br J Haematol 57:707–710
Imes NK, Sanders DC, Crane CR, Clark G (1969) Assaying actual hematein content of commercial hematoxylins and hemateins. Stain Technol 44:167–172
Jakobsen P, Andersen AP, Lyon H (1984) Preparation and characterization of methyl green tetrafluoroborate. Histochemistry 81:177–179
Kok LP, Boon ME, Ouwerkerk-Noordam E, Gerrits PO (1986) The application of a microwave technique for the preparation of cell blocks from sputum. J Microsc 144:193–199
Liao JC, Ponzo JL, Patel C (1981) Improved stability of methanolic Wright's stain solutions with additive reagents. Stain Technol 56:251–263
Liao JC, Ponzo JL, Patel C (1982) Improved stability of methanolic Wright's stain solution with dual additives. Stain Technol 57:23–30
Lillie RD (1944) The deterioration of Romanowsky stain solutions in various organic solvents. Public Health Report 178:1–14
Lillie RD (1974) The hematoxylin shortage and the availability of synthetic substitutes. Am J Med Technol 40:455–461
Lillie RD (1977) Conn's biological stains, 9th edn. Williams & Wilkins, Baltimore
Lillie RD, Pizzolato P, Donaldson PT (1975) Hematoxylin substitutes: a survey of mordant dyes tested and consideration of the relation of their structure to performance as nuclear stains. Stain Technol 51:25–41
Löhr W, Grubhofer N, Sohmer I, Wittekind D (1975) The azure dyes: their purification and physicochemical properties. II. Purification of azure B. Stain Technol 50:149–156
Lubrano GJ, Dean WW, Heinsohn HG, Stastny M (1977) The analysis of some commercial dyes and Romanowsky-stains by high-performance liquid chromatography. Stain Technol 52:13–23
Lyon H, Andersen AP, Andersen I, Clausen PP, Herold B (1982) Purity of commercial non-certified European samples of Pyronin Y. Histochem J 14:621–630
Marshall PN (1976) The composition of stains produced by the oxidation of methylene blue. Histochem J 8:431–442
Marshall PN (1978) Romanowsky-type stains in haematology. Histochem J 10:1–29
Marshall PN (1983) Papanicolaou staining — a review. Microscopica Acta 87:233–243
Marshall PN, Bentley SA, Lewis SM (1975a) An evaluation of some commercial Romanowsky stains. J Clin Pathol 28:680–685
Marshall PN, Bentley SA, Lewis SM (1975b) A standardized Romanowsky stain prepared from purified dyes. J Clin Pathol 28:920–923
Marshall PN, Bentley SA, Lewis SM (1975c) A procedure for assaying commercial samples of eosin. Stain Technol 50:107–113
Marshall PN, Bentley SA, Lewis SM (1978a) Staining properties and stability of a standardized Romanowsky stain. J Clin Pathol 31:280–282
Marshall PN, Bentley SA, Lewis SM (1978b) Standardization of Romanowsky stains: the relationship between stain composition and performance. Scand J Haematol 20:206–212
Marshall PN, Galbraith W, Bacus JW (1979) Studies on Papanicolaou staining. II. Quantitation of dye components bound to cervical cells. Analyt Quant Cytol 1:169–178
Marshall PN, Galbraith W, Navarro EF, Bacus IW (1981) Micro-spectrophotometric studies of Romanowsky stained blood cells. II. Comparison of the performance of two standardized stains. J Microsc 124:197–210
Marshall PN, Galbraith W (1984) On the nature of the purple coloration of leucocyte nuclei stained with Azure B-Eosin Y. Histochem J 16:793–797
Marshall PN, Horobin RW (1972) The oxidation products of hematoxylin and their role in biological staining. Hitochem J 4:493–503
Marshall PN, Horobin RW (1973a) The mechanism of action of “mordant” dyes — a study using preformed metal complexes. Histochemie 35:361–371
Marshall PN, Horobin RW (1973b) Measurements of the affinities of basic and “mordant” dyes for various tissue substrates. Histochemie 36:303–312
Marshall PN, Horobin RW (1973c) The influence of inorganic salts when staining with preformed metal complex dyes. Histochemie 37:299–311
Marshall PN, Horobin RW (1974) A simple assay procedure for mixtures of hematoxylin and hematein. Stain Technol 49:137–142
Marshall PN, Lewis SM (1974a) A rapid thin-layer chromatographic system for Romanowsky blood stains. Stain Technol 49:235–240
Marshall PN, Lewis SM (1974b) Batch variations in commercial dyes employed for Romanowsky-type staining: a thin layer chromatographic study. Stain Technol 49:351–358
Marshall PN, Lewis SM (1975a) A technique for distinguishing between Methylene Violet and Methylene Violet Bernthsen. Stain Technol 50:51–53
Marshall PN, Lewis SM (1975b) Metal contaminants in commercial thiazine dyes. Stain Technol 50:143–147
Meloan SN, Puchtler H (1974) Ion alizarin blue S stain for nuclei. Stain Technol 49:301–304
Meloan SN, Puchtler H (1978) Demonstration of amyloid with Mesitol WLS-Congo Red: application of a textile auxiliary to histochemistry. Histochemistry 58:163–166
Mowry RW, Kasten F (1975) The importance of dye purification and standardization in biomedicine. Stain Technol 50:65–81
Mowry RW, Longley JB, Emmel VM (1980) Only aldehyde fuchsin made from pararosaniline stains pancreatic B cell granules and elastic fibers in unoxidized microsections: problems caused by mislabelling of certain basic fuchsins. Stain Technol 55:99–103
Müller-Walz R, Zimmermann HW (1987) Über Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt. 4. Mittei-lung: Bindung von Azur B und DNA. Histochemistry 87:157–172
Nelis HJCF, de Leenheer A (1986) Quality control of azure B preparations by liquid chromatography and standardization with azure B tetrafluoroborate. Clinica Chimica Acta 156:247–258
Palmer RW, Lillie RD (1965) Spectroscopic and staining studies of the ripening and overripening of aluminum hematoxylins. Histochemie 5:44–54
Papanicolaou GN (1942) A new procedure for staining vaginal smears. Science 95:438–439
Pfuller U, Franz H, Preiss A (1977) Sudan Black B: chemical structure and histochemistry of the main blue components. Histochemistry 54:327–333
Preston K (1980) Automation of the analysis of cell image. Analyt Quant Cytol 2:1–14
Proctor GB, Horobin RW (1987) Basic fuchsin homologues. Are pure dyes really necessary? Med Lab Sci 44:398–400
Puchtler H, Meloan SN, Waldrop FS (1986) Application of current chemical concepts to metal-hematein and — brazilein stains. Histochemistry 85:353–364
Reinhardt ER (1983) Methodische und technologische Anforde-rungen an Zellbildanalysatoren. Microsc Acta 6 [Suppl]: 69–77
Rüter A, Wittekind D, Aus HM (1983) Kolorimetrische und zytophotometrische Untersuchungen einer modifizierten Papanicolaou-Färbemethode in Zervikalzellen. Microscopica Acta 87:245–269
Schulte E (1986a) Hematoxylin and the Feulgen reagent. In: Boon ME, Kok LP (eds) Standardization and quantitation of diagnostic staining in cytology. Coulomb Press, Leiden, pp 9–15
Schulte E (1986b) Air-drying as a preparatory factor in cytology: investigation of its influence on dye uptake and dye binding. Diagn Cytopathol 2:160–167
Schulte E, Wittekind D (1989a) Standardization of the Feulgen-Schiff technique: staining characteristics of pure fuchsin dyes. Histochemistry 91:321–331
Schulte E, Wittekind D (1989b) Standardized Thionine-Eosin stain in bronchial cytology: a substitute for Hematoxylin-Eosin Y. Anal Quant Cytol Histol 11:131–139
Schulte E, Wittekind D (1989c) The influence of Romanowsky-type stains on nuclear and cytoplasmic features of cytological specimens. Anal Cell Pathol 1:83–86
Schulte E, Wittekind D (1989d) Standardized Thionin-Eosin Y: a quick stain for cytology. Stain Technol 64:255–256
Schulte E, Wittekind D (1989e) A quick and standardized Giemsa stain for wet-fixed cytological material. Stain Technol 64:253–254
Schwarz G (1983) Ein Konzept für die routinemäßige Herstellung von automatengerechten gynäkologischen Präparaten. Microsc Acta 6 [Suppl]:29–36
Schwarzmann P, Bloss WH (1984) System FAZYTAN. In: Deutsche Forschungs-und Versuchsanstalt für Luft- und Raumfahrttechnik (Hrsg) Automation der zytologischen Diagnostik. Verlag TÜV Rheinland, Köln, pp 35–54
Scott BE, French RW (1924) Standardization of biological stains. Milit Surgeon 5:229–243
Scott JE (1967) On the mechanism of the Methyl Green-Pyronin stain for nucleic acids. Histochemie 9:30–47
Scott JE (1972a) Lies, damned lies — and biological stains. Histochem J 4:387–389
Scott JE (1972b) The histochemistry of Alcian Blue, note on the presence and removal of boric acid as a major diluent in Alcian Blue 8GX. Histochemie 29:129–133
Scott JE, Willet IH (1966) Binding of cationic dyes to nucleic acids and other biological polyanions. Nature 209:985
Soost HJ, Falter EW, Otto KH (1979) Comparison of two Papanicolaou staining procedures for automated prescreening. Anal Quant Cytol Histol 1:37–42
Sprenger E (1984) Automation in der Zytodiagnostik. In: Deutsche Forschungs-und Versuchsanstalt für Luft- und Raumfahrttechnik (Hrsg) Automation in der zytologischen Diagnostik. Verlag TÜV Rheinland, Köln, pp 7–16
Stoward PJ (1967) Studies in fluorescence histochemistry. 1. The demonstration of sulphomucin. J R Microsc Soc 87:215–222
Toepfer K (1972) Analyse der May-Grünwald-Giemsafärbung als Beitrag zur Notwendigkeit des Arbeitens mit reinen, definierten Farbstoffen. Verh Dtsch Ges Inn Med 78:272–276
Turner JN, Collins DN (1987) Standard specimens for stain calibration and their application to the Papanicolaou stain. Analyt Quant Cytol Histol 6:521–530
Turner JN, Weir B, Collins D (1990) Standard specimens for stain calibration: application to Romanowsky-Giemsa staining. Stain Technol 65:55–67
Wied G, Bartels P (1988) Image analysis and quantitative cyto- and histopathology. Techn Rep Int Acad Cytol 2:1–27
Wittekind D (1979) On the nature of Romanowsky dyes and the Romanowsky-Giemsa effect. Clin Lab Haematol 1:247–262
Wittekind D (1981) Biologische Farbstoffe: Definition, Zugehörigkeit zu Chemie und Morphologie; Farbstoffe als Werkzeuge der Histochemie. Acta Histochem 24 [Suppl]:133–181
Wittekind D (1983) On the nature of Romanowsky-Giemsa staining and its significance for cytochemistry and histochemistry: an overall view. Histochem J 15:1029–1047
Wittekind D (1984a) Probleme der Standardisierung von Ferbstoffen und zytologischen Färbungen. In: Deutsche Forschungsund Versuchsanstalt für Luft- und Raumfahrttechnik (Hrsg) Automation der Zytologischen Diagnostik. Verlag TÜV Rheinland, Köln, pp 81–92
Wittekind D (1984b) Zukünftige Entwicklungsmöglichkeiten und zukünftige Fähigkeiten der Zytoautomaten. In: Deutsche Forschungs- und Versuchsanstalt für Luft-und Raumfahrttechnik (Hrsg) Automation der Zytologischen Diagnostik. Verlag TÜV Rheinland, Köln, pp 151–166
Wittekind D (1985) Standardization of dyes and stains for automated cell pattern recognition. Anal Quant Cytol Histol 7:6–30
Wittekind D, Gehring T (1985) On the nature of Romanowsky-Giemsa staining and the Romanowsky-Giemsa effect: I. Model experiments on the specificity of azue B-eosin Y stain as compared with other thiazine dye-eosin Y combinations. Histochem J 17:263–289
Wittekind D, Hilgarth M, Kretschmer V (1979) Die einfache und reproduzierbare Papanicolaou-Färbung. Geburtsh Frauenheilkd 39:969–972
Wittekind D, Hilgarth M, Kretschmer V, Seiffert W, Zipfel E (1982a) The new and reproducible Papanicolaou stain: morphologic and spectrophotometric observations on the influence of stain composition on staining results. Anal Quant Cytol Histol 4:286–294
Wittekind D, Kretschmer V, Löhr W (1976) Kann Azur-B-Eosin die May-Grünwald-Giemsa-Färbung ersetzen? Blut 32:71–78
Wittekind D, Kretschmer V, Sohmer I (1982b) Azure B-Eosin Y stain as the standard Romanowsky-Giemsa stain. Br J Haematol 51:391–393
Wittekind D, Kretschmer V (1987) On the nature of Romanowsky-Giemsa staining and the Romanowsky-Giemsa effect. II. A revised Romanowsky-Giemsa staining procedure. Histochem J 19:399–401
Wittekind D, Reinhardt ER, Kretschmer V, Zipfel E (1983) Influence of staining on fast automated cell segmentation, feature extraction and cell image analysis. Anal Quant Cytol Histol 5:55–60
Wittekind D, Schulte E (1987) Die Bedeutung der Standardisierung der Zell- und Gewebspräparation für bildanalytische Operationen. In: Eins S (Hrsg) Quantitative und strukturelle Bildanalyse in der Medizin. GIT-Verlag, Darmstadt, pp 5–12
Wittekind D, Schulte E, Kretschmer V (1989) The influence of Romanowsky-Giemsa type of stains on the nuclear texture of desoxyribonuclease-treated cytological preparations. Acta Histochem 37 [Suppl]:131–137
Wolman M (1955) Problems of fixation in cytology, histology and cytochemistry. Int Rev Cytol 4:79–102
Yasumatsu H (1977) Stain using celestine blue B as substitute nuclear stain in routine cytologic examinations. Acta Cytologica 21:173–174
Zanker V (1981) Grundlagen der Farbstoff-Substrat-Beziehungen in der Histochemie. Acta Histochem 24 [Suppl]:151–168
Zimmermann HW (1984) Physikalisch-chemische Grundlagen der Färbung für manuelle und apparative Zytodiagnostik. Microsc Acta Suppl 6:45–58
Zimmermann HW (1990) Personal communication
Zipfel E, Grèzes JR, Seiffert W, Zimmermann H (1981) Uber Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt: 1. Mitteilung: Azur B, Reinheit und Gehalt von Farbstoffen. Histochemistry 72:279–290
Zipfel E, Grèzes JR, Seiffert W, Zimmermann HW (1982) Uber Romanowsky-Giemsa-Farbstoffe und den Romanowsky-Giemsa-Effekt. 2. Mitteilung: Eosin Y, Erythrosin B, Tetrachlor-fluorescein. Spektroskopische Charakterisierung der reinen Farbstoffe, Assoziation von Eosin Y. Histochemistry 75:539–555
Zipfel E, Grèzes JR, Naujok A, Seiffert W, Wittekind DH, Zimmermann H (1984) Uber Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt: 3. Mitteilung: Mikrospektralphoto-metrische Untersuchungen der Romanowsky-Giemsa-Färbung. Spektroskopischer Nachweis eines DNA-Azur-B-Eosin-Y-Komplexes, der den Romanowsky-Giemsa-Effekt verursacht. Histochemistry 81:337–351
Author information
Authors and Affiliations
Additional information
This paper is dedicated to my academic teacher, Prof. Dr. D.H. Wittekind, on the occasion of his 70th birthday
Rights and permissions
About this article
Cite this article
Schulte, E.K.W. Standardization of biological dyes and stains: pitfalls and possibilities. Histochemistry 95, 319–328 (1991). https://doi.org/10.1007/BF00266958
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00266958