, Volume 85, Issue 5, pp 353–364 | Cite as

Application of current chemical concepts to metal-hematein and-brazilein stains

  • H. Puchtler
  • S. N. Meloan
  • F. S. Waldrop


Current chemical concepts were applied to Weigert's, M. Heidenhain's and Verhoeff's iron hemateins, Mayer's acid hemalum stain and the corresponding brazilein compounds. Fe+++ bonds tightly to oxygen in preference to nitrogen and is unlikely to react with lysyl and arginyl groups of proteins. Binding of unoxidized hematoxylin by various substrates has long been known to professional dyers and was ascribed to hydrogen bonding. Chemical data on the uptake of phenols support this theory. Molecular models indicate a nonplanar configuration of hematoxylin and brazilin. The traditional quinonoid formula of hematein and brazilein was revised. During chelate formation each of the two
groups of the dye shares an electron pair with the metal and contributes a negative charge to the chelate. Consequently, the blue or black 2:1 (dye:metal) complexes are anionic. Olation of such chelates affects the staining properties of iron hematein solutions. The color changes upon oxidation of hematoxylin, reaction of hematein with metals, and during exposure of chelates to acids can be explained by molecular orbital theory.

Without differentiation or acid in dye chelate solutions, staining patterns are a function of the metal. Reactions of acidified solutions are determined by the affinities of the dye ligands. Brazilein is much more acid-sensitive than hematein. This difference can be ascribed to the lack of a second free phenolic −OH group in brazilein, i.e. one hydrogen bond is insufficient to anchor the dye to tissues. Since hematein and brazilein are identical in all other respects, their differences in affinity cannot be explained by van der Waals, electrostatic, hydrophobic or other forces.


Hydrogen Phenol Hydrogen Bond Hematoxylin Negative Charge 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Abrahart EN (1977) Dyes and their intermediates. Edward Arnold, LondonGoogle Scholar
  2. Arshid FM, Desai JN, Duff DJ, Giles CH, Jain SK, Macneal IR (1954a) A study of certain natural dyes. I The adsorption of brazilwood and logwood colouring matters by fibres. J Soc Dyers Colour 70:392–401Google Scholar
  3. Arshid FM, Connelly RF, Desai JN, Fulton RG, Giles CH, Kefalas JC (1954b) A study of certain natural dyes. II The structure of the metallic lakes of the brazilwood and logwood colouring matter. J Soc Dyers Colour 70:402–412Google Scholar
  4. Baeyer A (1905) Dibenzalaceton and Triphenylmethan. Ber Dtsch Chem Ges 38:569–590Google Scholar
  5. Bailar JC, Busch DH (1956) A general survey of the coordination compounds. In: Bailar JC, Busch DH (eds) The chemistry of coordination compounds. Reinhold, New York, pp 1–99Google Scholar
  6. Baker JR (1962) Experiments on the action of mordants. 2 Aluminum-haematein. Qu J Microsc Sci 103:493–517Google Scholar
  7. Bancroft JD, Stevens A (1982) theory and practice of histological techniques. Churchill Livingstone, Edinburgh London Melbourne New YorkGoogle Scholar
  8. Basolo F, Johnson R (1964) Coordination chemistry. WA Benjamin, New York AmsterdamGoogle Scholar
  9. Bielig HJ (1956) Farbstoffe, natürliche. In: Foerst W (ed) Ullmann's Encyklopädie der technischen Chemie, 3 Aufl, Bd 7. Urban & Schwarzenberg, München Berlin, pp 84–144Google Scholar
  10. Brown GI (1958) An introduction to electronic theories of organic chemistry. Longmans, Green & Co, London New York TorontoGoogle Scholar
  11. Candlin JP, Taylor KA, Thompson DT (1968) Reactions of transition-metal complexes. Elsevier, Amsterdam London New YorkGoogle Scholar
  12. Cotton FA, Wilkinson G (1972) Advanced inorganic chemistry, 3rd edn. Interscience, New YorkGoogle Scholar
  13. Culling CFA (1963) Handbook of histopathological techniques, 2nd edn. Butterworths, WashingtonGoogle Scholar
  14. Dähne S (1970) Die historische Entwicklung der Farbstofftheorien I. Z Chemie 10:133–140Google Scholar
  15. Drew RL (1937) The application of logwood to the rayons. Am Dyestuff Rep 26:623–625Google Scholar
  16. Dunkel H (1961) Färben von Polyamidfasern mit Blauholzschwarz. Textil-Praxis 16:1129–1131Google Scholar
  17. Eichhorn GL (1973) Complexes of polynucleotides and nucleic acids. In: Eichhorn GL (ed) Inorganic biochemistry, vol 2. Elsevier. Amsterdam London New York, pp 1210–1243Google Scholar
  18. Elias JM (1974) Chemistry of H & E staining. Am J Med Technol 40:513–514Google Scholar
  19. Endres H (1961) Die Gerbwirkung niedermolekularer Polyhydroxyphenole. Leder 12:294–297Google Scholar
  20. Feeman JF (1978) Leather dyes. In: Venkataramen K (ed) The chemistry of synthetic dyes, vol VIII. Academic Press, New York San Francisco London, pp 37–80Google Scholar
  21. Fischer-Bobsien CH (1955) Metallisierbare and metallisierte Farbstoffe bereichern fortlaufend die moderne Färbereitechnik (III). Z Ges Textil Ind 57:156–158, 209–211Google Scholar
  22. Förster T (1939) Farbe and Konstitution organischer Verbindungen vom Standpunkt der modernen physikalischen Chemie. Z Elektrochemie 45:548–573Google Scholar
  23. Frey H (1868) Diek Hämatoxylinfärbung. Arch Mikrosk Anat 4:345–346Google Scholar
  24. Gábor M, Dux E (1952) Die experimentelle Beeinflussung der Kapillarresistenz mit Hämatoxylin und verwandten Derivaten. Acta Physiol Acad Sci Hung 3:409–413Google Scholar
  25. Gábor M, Horváth B (1953) Untersuchungen über die Wirkung von “P-Vitamin-Artigen” pflanzlichen Farbstoffen auf das isolierte und in situ arbeitende Froschherz. Arch Int Pharmacodyn 95:68–77Google Scholar
  26. Gábor M, Szórády I (1952) Die Beeinflussung der experimentellen Senfölentzündungen mit Hämatoxylin. Acta Physiol Acad Sci Hung 3:405–407Google Scholar
  27. Gábor M, Horváth B, Kiss L, Dirner Z (1952) Über den Adrenalineffekt prolongierende Wirkung der Glieder der Hämatoxylingruppe an überlebenden Organen und in vivo. Acta Physiol Acad Sci Hung 3:585–590Google Scholar
  28. Gierke H (1884) Färberei zu mikroskopischen Zwecken. Z Wiss Mikrosk 1:62–100, 372–408, 497–557Google Scholar
  29. Giles CH (1959) Studies in hydrogen bond formation: Part VIII Hydrogen bonding in adsorption processes. In: Hydrogen bonding: Symposium Liubljana, pp 449–460Google Scholar
  30. Giles CH (1975) Dye-fibre bonds and their investigation. In: Bird CL, Boston WS (eds) The theory of coloration of textiles. Dyers Company Publication Trust, Bradford, pp 41–110Google Scholar
  31. Grassmann W, Endres H, Oppelt M, El Sissi H (1959) Affinität von Gerbstoffen und gerbstoffähnlichen Substanzen zur CONH-Gruppierung in Polyamiden. Leder 10:149–156Google Scholar
  32. Gray HB (1964) Molecular orbital theory for transition metal complexes. J Chem Educ 41:2–12Google Scholar
  33. Griffiths J (1976) Colour and constitution of organic molecules. Academic Press, London New York San FranciscoGoogle Scholar
  34. Griffiths J (1981) Recent developments in the colour and constitution of organic dyes. Rev Progr Coloration 11:37–57Google Scholar
  35. Gurd FRN, Wilcox PE (1947) Complex formation between metallic cations and proteins, peptides and amino acids. Adv Protein Chem 11:311–427Google Scholar
  36. Gustavson KH (1956) The chemistry of tanning processes. Academic Press, New YorkGoogle Scholar
  37. Harms H (1957) Handbuch der Farbstoffe für die Mikroskopie, Teil II, 2. Lieferung. Staufen Verlag, Kamp-LintfortGoogle Scholar
  38. Harris CM, Livingstone SE (1964) Bidentate chelates. In: Dwyer FP, Mellor DP (eds) Chelating agents and metal chelates. Academic Press, New YorkGoogle Scholar
  39. Heidenhain M (1890) Beiträge zur Kenntnis der Topographie und Histologie der Kloake und ihrer drüsigen Adnexe bei den einheimischen Tritonen. Arch Mikrosk Anat 35:173–274Google Scholar
  40. Heidenhain R (1885) Eine neue Verwendung des Hämatoxylin. Arch Mikrosk Anat 24:468–470Google Scholar
  41. Herrmann K (1970) Nucleinsäuren. In: Sund H (ed) Grosse Moleküle. Suhrkamp, Frankfurt am Main, pp 11–34Google Scholar
  42. Hikino H, Taguchi T, Fujimura H, Hiramatsu Y (1977) Antiinflammatory principles of Caesalpina Sappan wood and of Haematoxylon campechianum wood. Planta Med 31:214–220Google Scholar
  43. Hrapchak BB (1976) Selective staining with hematoxylin, applications and theory: a review. Am J Med Technol 42:371–379Google Scholar
  44. Holzner G (1884) Zur Geschichte der Tinktionen. Z Wiss Mikrosk 1:254–256Google Scholar
  45. Hübner J (1913) A contribution to the history of dyeing. J Soc Dyers Colour 29:344–357Google Scholar
  46. Hückel W (1941) Organische Chemie, 2. Aufl. Akademische Verlagsgesellschaft, LeipzigGoogle Scholar
  47. Hummel JJ, Hasluck PN (1906) Colouring matters for dyeing textiles. Cassel and Co. LondonGoogle Scholar
  48. Hummel JJ, Knecht E (1888) Die Färberei und Bleicherei der Gespinstfasern. Springer, BerlinGoogle Scholar
  49. Iyer SRS (1974) Physical chemistry of dyeing: kinetics, equilibrium, dye-fiber affinity and mechanism. In: Venkataraman K (ed) The chemistry of synthetic dyes, vol VII. Academic Press, New YorkGoogle Scholar
  50. Johnson RD, Callis CF (1956) Water softening through complex formation. In: Bailar JC, Busch DH (eds) The chemistry of coordination compounds. Reinhold, New York, pp 768–783Google Scholar
  51. Kauffmann H (1907) Die Auxochrome. In: Ahrens FB (ed) Sammlung chemischer und chemisch-technischer Vorträge, Bd 12. Ferdinand Enke. Stuttgart, pp 1–111Google Scholar
  52. Keller RN, Parry RW (1956) Modern developments: the electron pair bond and structure of coordination compounds. In: Bailar JC, Busch DH (eds) The chemistry of coordination compounds. Reinhold, New York, pp 157–219Google Scholar
  53. Knecht E, Rawson C, Loewenthal R (1933) A manual of dyeing, 9th edn, vol I. Charles Griffin, LondonGoogle Scholar
  54. Krähenbühl E (1949) Kupferhaltige Färbungen und Farbstoffe. Textil-Rundschau 4:157–168Google Scholar
  55. Kratzert W, Peichert R (1981) Farbstoffe. Quelle & Meyer, HeidelbergGoogle Scholar
  56. Larsen EM (1965) Transitional elements. WA Benjamin, New York AmsterdamGoogle Scholar
  57. Legget WF (1944) Ancient and mediveal dyes. Chemical Publishing Co, BrooklynGoogle Scholar
  58. Lestourgeon WM, Forer A, Yang Y-Z, Bertram JS, Rusch HP (1975) Contractile proteins: major components of nuclear and chromosome non-histone proteins. Biochim Biophys Acta 379:529–552Google Scholar
  59. Lillie RD (1965) Histopathologic technic and practical histochemistry, 3rd edn. McGraw-Hill, New YorkGoogle Scholar
  60. Lillie RD (1969) H.J. Conn's biological stains, 8th edn. Williams & Wilkins, BaltimoreGoogle Scholar
  61. Lillie RD, Pizzolato P (1972) Mechanisms of iron II and iron III sequence hematoxylin stains. J Histochem Cytochem 20:116–129Google Scholar
  62. Marsden RJB, Urquhart AR (1942) The swelling of cellulose acetate in aqueous phenol solutions. J Textile Inst 33:T105-T136Google Scholar
  63. Marshall PN, Horobin RW (1972) The oxidation products of haematoxylin and their role in biological staining. Histochem J 4:493–503Google Scholar
  64. Marshall WJ, Peters RH (1947) The heats of reaction and affinities of direct cotton dyes for cuprammonium rayon, viscose rayon and cotton. J Soc Dyers Colour 63:446–461Google Scholar
  65. Martell AE (1973) Artificial enzymes. In: Sigel H (ed) Metal ions in biological systems, vol 2. Marcel Dekker, New York, pp 207–268Google Scholar
  66. Martell AE, Calvin M (1962) Chemistry of the metal chelate compounds. Prentice Hall, Englewood CliffsGoogle Scholar
  67. Mason SF (1970) Color and the electronic states of organic molecules. In: Venkataraman K (ed) The chemistry of synthetic dyes, vol III. Academic Press, New York, pp 169–221Google Scholar
  68. Mayer F (1943) The chemistry of natural coloring matters. Reinhold, New YorkGoogle Scholar
  69. McManus JFA, Mowry RW (1960) Staining methods: histologic and histochemical. Paul B. Hoeber, New YorkGoogle Scholar
  70. Mellor DP (1964) Historical background and fundamental concepts. In: Dwyer FP, Mellor DP (eds) Chelating agents and metal chelates. Academic Press, New YorkGoogle Scholar
  71. Meloan SN, Puchtler H (1974) Iron alizarin blue S stain for nuclei. Stain Technol 49:301–304Google Scholar
  72. Moon CK, Ha BJ, Lee CC (1984) Effects of brazilin and haematoxylin on the lipidperoxidation in the rat liver mitochondria. Arch Pharm Res 7:63–64Google Scholar
  73. Morsingh F, Robinson R (1970) The synthesis of brazilin and hematoxylin. Tetrahedron 26:281–289Google Scholar
  74. Murmann RK (1964) Inorganic complex compounds. Reinhold, New YorkGoogle Scholar
  75. Muspratt S (1860) Chemistry, theoretical, practical and analytical, vol I. Mackenzie, Glasgow Edinburgh LondonGoogle Scholar
  76. Napier J (1875) A manual of dyeing and dyeing receipts, 3rd edn. Charles Griffin & Co, LondonGoogle Scholar
  77. Neifakh SA, Avranmov JA, Gaitskhoki VS, Kazakova TB, Monakhov NK, Repin VS, Turovski VV, Vassiletz IM (1965) Mechanism of the controlling function of mitochondria. Biochim Biophys Acta 100:329–343Google Scholar
  78. Nietzki R (1901) Chemie der organischen Farbstoffe, 4th edn. Springer, BerlinGoogle Scholar
  79. Ogiwara Y, Kubota H (1969) Combination of cellulosic materials and metallic ions. J Polymer Sci 7:2087–2095Google Scholar
  80. Ohnishi T, Kawamura H, Takeo K, Watanabe S (1964a) Propriété des protéines contractiles resemblantes à l'actine et à la myosine extraites des mitochondries du foie. J Biochem (Tokyo) 56:273–278Google Scholar
  81. Ohnishi T, Kawamura H, Tanaka Y (1964a) Die Aktin und Myosin ähnlichen Proteine im Kalbsthymuszellkern. J Biochem (Tokyo) 56:6–15Google Scholar
  82. Perkin AG, Everest AE (1918) The natural organic colouring matter. Longmans, Green & Co, LondonGoogle Scholar
  83. Ploss EE (1962) Ein Buch von alten Farben. Impuls-Verlag Heinz Moos, HeidelbergGoogle Scholar
  84. Pratt LS (1948) The chemistry and physics of organic pigments. John Wiley & Sons, New YorkGoogle Scholar
  85. Pratt R, Yuzuriha Y (1959) Antibacterial activity of the heartwood ofHaematoxylon braziletto. J Am Pharm Assoc 48:69–72Google Scholar
  86. Price B (1970) The chemistry of metal complex dyestuffs. In: Venkataraman K (ed) The chemistry of synthetic dyes, vol III. Academic Press, New York London, pp 303–383Google Scholar
  87. Puchtler H, Meloan SN (1985) On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions. Histochemistry 82:201–204Google Scholar
  88. Puchtler H, Sweat F (1964) On the mechanism of sequence ironhematein stains. Histochemie 4:197–208Google Scholar
  89. Puchtler H, Waldrop FS (1979) On the mechanism of Verhoeff's elastica stain: a convenient stain for myelin sheaths. Histochemistry 62:233–247Google Scholar
  90. Puchtler H, Waldrop FS, Meloan SN, Terry MS, Conner HM (1970) Methacarn (methanol-Carnoy) fixation: practical and theoretical considerations. Histochemie 21:97–116Google Scholar
  91. Puchtler H, Waldrop FS, Meloan SN (1980) On the mechanism of Mallory's phosphotungstic acid-hematoxylin stain. J Microsc 119:383–390Google Scholar
  92. Rattee ID, Breuer MM (1974) The physical chemistry of dye adsorption. Academic Press, London New YorkGoogle Scholar
  93. Reinicke R (1934) Bestimmt gerichtete, schwache Nebenvalenzkräfte als die wirklichen Farbträger (Chromophore). Z Physik Chem 26B:159–186Google Scholar
  94. Remy H (1957) Lehrbuch der Anorganischen Chemie, I Bd. Akademische Verlagsanstalt, LeipzigGoogle Scholar
  95. Rifkind JM (1973) Hemoglobin and myoglobin. In: Eichhorn GL (ed) Inorganic biochemistry, vol 2. Elsevier, Amsterdam London New York, pp 832–901Google Scholar
  96. Robinson R (1962) Synthesis in the brazilin group. In: Gore TS, Joshi BS, Sunthanker SV, Tilak BD (eds) Chemistry of natural and synthetic colouring matters and related fields. Academic Press, New York, pp 1–11Google Scholar
  97. Rollinson CL (1956) Olation and related chemical processes. In: Bailar JC, Busch DH (eds) The chemistry of coordination compounds. Reinhold, New York, pp 448–471Google Scholar
  98. Rosenthal SI, Puchtler H, Sweat F (1965) Paper chromatography of dyes; method to investigate vagaries of staining. Arch Pathol 80:190–196Google Scholar
  99. Rys P, Zollinger H (1972) Fundamentals of the chemistry and application of dyes. Wiley-Interscience, London New York Sydney TorontoGoogle Scholar
  100. Schweizer HR (1964) Künstliche organische Farbstoffe und ihre Zwischenprodukte, Springer, Berlin Göttingen HeidelbergGoogle Scholar
  101. Schweppe H (1976) Farbstoffe, natürliche. In: Ullmann's Encyklopädie der technischen Chemie, 4 Aufl, Bd 11. Verlag Chemie, New York Weinheim, pp 99–134Google Scholar
  102. Sheehan DC, Hrapchak BB (1973) Theory and practice of histotechnology, CV Mosby, St. LouisGoogle Scholar
  103. Smythe CV, Schmidt CLA (1930) Studies on the mode of combination of iron with certain proteins, amino acids and related compounds. J Biol Chem 88:241–269Google Scholar
  104. Spiro TG (ed) (1980) Nucleic acid-metal ion interactions. John Wiley, New York Chichester Brisbane TorontoGoogle Scholar
  105. Staab HA (1962) Einführung in die theoretische organische Chemie, 3 Aufl. Verlag Chemie, WeinheimGoogle Scholar
  106. Steiner RF (1965) The chemical foundations of molecular biology. Van Nostrand, Princeton Toronto New York LondonGoogle Scholar
  107. Stockert JC (1979) Observations on the chromatin staining by aluminum-hematoxylin. Z Naturforsch 34C:1285–1286Google Scholar
  108. Stocks HB (1926) The reaction between tannin and casein. J Soc Leather Trades Chem 10:409–410Google Scholar
  109. Taylor FS, Singer C (1956) Pre-scientific industrial chemistry. In: Singer C, Holmyard EJ, Hall AR, Williams TI (eds) A history of technology, vol II. Oxford University Press, New York, pp 347–382Google Scholar
  110. Tisdale HR (1941) Natural dystuffs for textile fibers. Can Textile J 57:44–47Google Scholar
  111. Trotman ER (1970) Dyeing and chemical technology of textile fibres, 4th edn. Griffin, LondonGoogle Scholar
  112. Tu AT, Heller MJ (1974) Structure and stability of metal-nucleoside-phosphate complexes. In: Sigel H (ed) Metal ions in biological systems, vol I. Marcel Dekker, New YorkGoogle Scholar
  113. Verhoeff FH (1908) Some new staining methods of wide applicability; including a rapid differential stain for elastic tissue. J Am Med Assoc 50:876–877Google Scholar
  114. Vial, Quiles JP, Ianakieva M, Chêne M (1969) Sur la rétention des ions ferriques et ferreux par la cellulose. Cellulose Chem Technol 3:21–31Google Scholar
  115. Vickerstaff T (1954) The physical chemistry of dyeing. Oliver & Boyd, LondonGoogle Scholar
  116. Weigert K (1904) Eine kleine Verbesserung der Hämatoxylin-van Gieson Methode. Z Wiss Mikrosk 21:1–5Google Scholar
  117. Windus W (1973) Tanning In: Hampel CA, Hawley CG (eds) The encyclopedia of chemistry, 3rd edn. Van Nostrand Reinhold, New York, pp 1078–1080Google Scholar
  118. Wizinger R (1933) Organische Farbstoffe. Ferdinand Dümmlers Verlag, Berlin BonnGoogle Scholar

Copyright information

© Springer-Verlag 1986

Authors and Affiliations

  • H. Puchtler
    • 1
  • S. N. Meloan
    • 1
  • F. S. Waldrop
    • 1
  1. 1.Department of PathologyMedical College of GeorgiaAugustaUSA

Personalised recommendations