From silks to science: The history of hematoxylin and eosin staining

Vidhi Naik
Aberdeen, Scotland

 

A slice of logwood, notably depicting its deeply colored heartwood, atop different fabrics stained by logwood dye. Image obtained and published with permission from Botanical Colors.

Introduction

Hematoxylin and eosin, dyes used to stain tissue samples, collectively known as H&E, form the benchmark for histological stains. These dyes possess a profound and alluring history, which includes stories of the fabric industry, pirates, fine art, and eventually histology.

 

The development of hematoxylin

The origins of hematoxylin can be traced back to the logwood tree, also known as Haematoxylum campechianum. Also known as the “blood wood tree,” its heartwood—the central, dead wood that provides structural support—is a crimson color.1 The species name, campechianum, refers to the tree’s origin, a city named Campeche in Mexico. The tree is native to Mexico, Belize, and Guatemala.2 The logwood tree possesses a high concentration of hematoxylin in its heartwood and roots and was used to produce a dye that was extracted from the tree by water boiling. This technique was created by the indigenous peoples and was recorded by Spanish conquistadors in the early 1500s.3 The dye was then used to stain silks and other textiles a rich purple color.

In the time of the conquistadors, purple dyes were coveted as they were rare and expensive. The discovery of a plant that produced a purple dye was highly significant. After colonizing Campeche, the conquistadors returned to Spain with large quantities of logwood that were then exported across Europe. Logwood dye was banned in Elizabethan England, possibly because the dye was purported to be substandard, or as a means of establishing favorites amongst suppliers, allowing exclusive distribution rights.4 In the late 1600s, the ban was lifted, so the Anglo-Spanish quest for domination over the logwood supply commenced. English pirates invaded Campeche, and competition arose for control over logwood production and exportation. Spanish ships were frequently looted by English pirates. The English had an advantage, as the colony of Jamaica was a stronghold for reconnaissance and regrouping. Eventually, the age of buccaneers faded, and the pirates who returned to land to exploit the lucrative logwood industry became “baymen,” though determining precisely when this change happened is difficult.5 The logwood industry contributed tremendously to the history of Belize.6 In the eighteenth century, the work of baymen proved to be the buttress for the success of the logwood industry of Belize, a former British colony. This engendered so much socioeconomic growth for the country that the baymen are honored on Belize’s flag.7

In his 1855 book, John Thomas Quekett, an English microscopist, first mentioned the use of logwood as a histological stain for preparing wood samples. He noted that if slices of wood were too transparent to be seen under a microscope, “they may be dyed with a tincture of iodine, or in a decoction of fustic or logwood.”8 This discovery, albeit just a brief mentioning of logwood in the context of microscopy, was the harbinger for the use of hematoxylin in histology. The first use of hematoxylin as a stain for human tissue is accredited to Heinrich Wilhelm Gottfried von Waldeyer-Hartz, a German anatomist.9 In a journal article in 1863, Waldeyer described his use of “Campeche wood” in the staining of neuronal axons.10 Although Waldeyer preferred the use of alkanet, he stated that hematoxylin distinguished cellular nuclei.11

More favorable results may have evaded Waldeyer because he did not use a mordant. Mordants help a dye “fix” to a fabric or tissue, intensifying and maintaining its colorfastness. The first use of hematoxylin with a mordant was credited to Friedrich Böhmer, a German general practitioner. In 1865, Böhmer detailed his approach of combining hematoxylin with alum, a sulfur-based mordant, commenting that tissue samples stained with this mixture could be well preserved.12 This discovery laid the groundwork for many widely used formulations of hematoxylin today. Hematoxylin’s journey of innovation around world has maintained its roots, as the dye is still produced from trees grown in Campeche and Belize.

 

Field with Irises near Arles by Vincent Van Gogh, May 1888. Oil on canvas, 54 cm x 65 cm. Van Gogh Museum, Amsterdam (Vincent van Gogh Foundation). No known restrictions on publication.

The development of eosin

The story of eosin began with Heinrich Caro, a German chemist. Caro worked at BASF, a German gasworks, when he undertook a collaborative project with Adolf von Baeyer.13 In 1874, eosin was created.14 Eosin, or tetrabromofluorescein, was made by the bromination in ethanol of fluorescein, a coal tar derivative, created by Baeyer.15 The name eosin is derived from the name for the Greek goddess of the dawn, Eos, which was also a nickname of a girl whom Caro admired in his youth.16 Although Heinrich Caro is credited with the discovery of eosin, Baeyer noted the involvement of his student and fellow Nobel Prize winner, Emil Fischer.17 In an 1875 report, Baeyer commented that Fischer “incidentally, included eosin in his investigations a year ago – albeit without publishing anything about it.”18

Popular uses of eosin were in silk dyeing, lipstick production, and ink and lake production.19 Eosin-based dyes are still widely used today in cosmetics. Eosin was sold as a lake, a pigment for use in paints, under the name of “geranium lake” and was used by famed expressionist painter Vincent van Gogh.20 Van Gogh admired the vibrancy of geranium lake and used it in his painting titled Field with Irises near Arles.21 Unfortunately, the lake lost its vibrancy when exposed to sunlight, resulting in fading and color warping.22

The use of eosin as a histological stain was first described by Ernst23 Fischer in 1876. Fischer commented on eosin’s ease of use and ability to distinguish tissue types with clarity when used to stain muscle and axonal tissues.24 Fischer detailed that “eosin can be used in both aqueous and alcoholic solutions to produce a stain for microscopic preparations.”25 Finally, Fischer gave his recommendation of the dye, stating that it could be used to “achieve a permanent, clear and beautiful staining.”26 Thus, it was with Fischer’s discovery that a new horizon in histology dawned upon eosin.

 

H&E staining in histology

The combination of hematoxylin and eosin for use in histological staining was first discussed by the chemist Wissozky in 1877.27 In a paper about histological investigations on hemoglobin in embryonic tissues, Wissozky noted that he combined eosin and hematoxylin in a solution to prepare slides.28 He credited Ernst Fischer for the inspiration to use eosin in his investigation.29 The use of both hematoxylin and eosin allowed Wissozky to view the protoplasm of cells, which were stained a pink-orange color, and the nuclei of cells, which were stained a deep purple.30 Wissozky, with an air of unbeknown prescience, stated: “this fact promises widespread application in the future.”31 Henceforth, the use of H&E staining found its inauguration in the realm of histology. Its legacy continues in many forms, from the work of today’s histopathologists to the preparation of slides to be photographed, printed, and haunt the pages of medical students’ textbooks.

 

End notes

  1. Carlos Ortiz-Hidalgo and Sergio Pina-Oviedo, “Hematoxylin: Mesoamerica’s Gift to Histopathology. Palo de Campeche (Logwood Tree), Pirates’ Most Desired Treasure, and Irreplaceable Tissue Stain,” International Journal of Surgical Pathology, 27, no.1 (2019): 4-14. https://doi.org/10.1177/1066896918787652. Accessed November 15, 2022.
  2. J Rojas-Sandoval and P Acevedo-Rodríguez, “Haematoxylum campechianum (logwood)”, CABI Compendium (2013). https://doi.org/10.1079/cabicompendium.26332. Accessed November 13, 2022.
  3. Ortiz-Hidalgo and Pina Oviedo, “Hematoxylin: Mesoamerica’s Gift,” 5.
  4. Gilbert M. Joseph, “British Loggers and Spanish Governors: The Logwood Trade and Its Settlements in the Yucatan Peninsula: Part I,” Caribbean Studies, 14, no.2 (1974): 7-37. http://jstor.org/stable/25612609. Accessed November 17, 2022.
  5. Joseph, “The Logwood Trade and Its Settlements I,” 30.
  6. Robin Kunkel, “Logwood – A History of Palettes, Pirates, and Pathology,” Department of Pathology at University of Michigan (2018): paragraph 6. https://pathology.med.umich.edu/news/574. Accessed November 17, 2022.
  7. Wayne P. Armstrong, “Logwood and Brazilwood: Trees That Spawned 2 Nations,” Pacific Horticulture, 53, no.1 (1992): 38-43, paragraph 16). https://www2.palomar.edu/users/warmstrong/ecoph4.htm. Accessed November 17, 2022.
  8. John Thomas Quekett, A Practical Treatise on the Use of the Microscope: Including the Different Methods of Preparing and Examining Animal, Vegetable, and Mineral Structures, 3rd ed. (London: H. Baillière, 1855): 365. https://wellcomecollection.org/works/fes2zq8f. Accessed November 17, 2022.
  9. E. Butt and I. Ashraf, “H01: The haematoxylin and eosin stain: from piracy to pathology,” British Journal of Dermatology, 185, no.1 (2021): 161. https://doi.org/10.1111/bjd.20155. Accessed November 18, 2022.
  10. Heinrich Wilhelm Gottfried von Waldeyer-Hartz, “Untersuchungen über den Ursprung und den Verlauf des Axencylinders bei Wirbellosen und Wirbeltieren, sowie über dessen Endverhalten in der quergestreiften Muskelfaser” (Investigations into the origin and course of the axon cylinder in invertebrates and vertebrates, and into its final behavior in the striated muscle fiber), Zeitschrift für rationelle Medicin, 1863: 193-257. http://opacplus.bsb-muenchen.de/title/2999004/ft/bsb10086962?page=208. Accessed November 17, 2022]. All translations by author.
  11. Waldeyer-Hartz, “Untersuchungen über den Ursprung und den Verlauf des Axencylinders,” 200.
  12. Friedrich Böhmer, “Zur pathologischen Anatomie der Meningitis cerebro-medullaris epidemica” (On the pathological anatomy of meningitis cerebro-medullaris epidemica), Aerztliches Intelligenz-Blatt, 1865: 539-50. Google Play Books. https://play.google.com/books/reader?id=vFpEAAAAcAAJ&pg=GBS.PA548&hl=en_GB&q=b%C3%B6hmer. Accessed November 17, 2022. Translations by author.
  13. Anthony S. Travis, “Ambitious and Glory Hunting . . . Impractical and Fantastic”: Heinrich Caro at BASF,” Technology and Culture, 39, no.1 (1998): 105-15. https://doi.org/10.2307/3107005. Accessed November 15, 2022.
  14. Heinrich Caro, “Ueber die Entwickelung der Theerfarben-Industrie” (On the development of the tar-dye industry), Berichte der deutschen chemischen Gesellschaft, 25, no.3 (1892): 955-1105. https://doi.org/10.1002/cber.18920250399. Accessed November 13, 2022. All translations by author.
  15. CJ Cooksey, “Quirks of dye nomenclature. 10. Eosin Y and its close relatives,” Biotechnic & Histochemistry, 93, no.3 (2018): 211-19. https://doi.org/10.1080/10520295.2017.1413207. Accessed November 14, 2022.
  16. Carsten Reinhardt and Anthony S. Travis, Heinrich Caro and the Creation of Modern Chemical Industry, 1st ed. (Dordrecht: Springer, 2000): 181. https://doi.org/10.1007/978-94-015-9353-3. Accessed November 13, 2022.
  17. Caro, “Ueber die Entwickelung der Theerfarben-Industrie,” 1051.
  18. Adolf von Baeyer, “Zur Geschichte des Eosins” (On the history of eosin), Berichte der deutschen chemischen Gesellschaft, 8, no.1 (1875): 146-48. https://doi.org/10.1002/cber.18750080149. Accessed November 15, 2022. All translations by author.
  19. Isabella Blank-Elsbree, on behalf of BASF. 2022. Email correspondence to author. Referenced with permission.
  20. Cooksey, “Quirks of dye nomenclature,” 214.
  21. Silvia Centeno et al., “Van Gogh’s Irises and Roses: the contribution of chemical analyses and imaging to the assessment of color changes in the red lake pigments,” Heritage Science, 5, no.18 (2017): 1-11. https://doi.org/10.1186/s40494-017-0131-8. Accessed November 17, 2022.
  22. Centeno, “Van Gogh’s Irises and Roses,” 2.
  23. Not to be confused with Emil Fischer.
  24. Ernst Fischer, “Eosin als Tinctionsmittel für mikroskopische Präparate” (Eosin as a tincture agent for microscopy), Archiv für mikroskopische Anatomie, 12, (1876): 349-52. https://doi.org/10.1007/BF02933896. [accessed 17th November]. All translations by author.
  25. Fischer, “Eosin als Tinctionsmittel,” 349.
  26. Fischer, “Eosin als Tinctionsmittel,” 349.
  27. HC Cook, “Origins of… Tinctorial Methods in Histology,” Journal of Clinical Pathology, 50 (1997): 716-20. http://dx.doi.org/10.1136/jcp.50.9.716. Accessed November 14, 2022.
  28. N Wissozky, “Ueber das Eosin als Reagens auf Hämoglobin und die Bildung von Blutgefässen und Blutkörperchen bei Säugethier- und Hühnerembryonen” (On eosin as a reagent for haemoglobin and the formation of blood vessels and blood cells in mammalian and chick embryos, Archiv für mikroskopische Anatomie, 13 (1877): 479-96. https://doi.org/10.1007/BF02933947. Accessed November 15, 2022. All translations by author.
  29. Wissozky, “Ueber das Eosin,” 479.
  30. Wissozky, “Ueber das Eosin,” 482.
  31. Wissozky, “Ueber das Eosin,” 482.

 

Bibliography

  • Armstrong, Wayne P. 1992. “Logwood and Brazilwood: Trees That Spawned 2 Nations.” Pacific Horticulture 53 (1): 38–43. https://www2.palomar.edu/users/warmstrong/ecoph4.htm.
  • Baeyer, Adolf. 1875. “Zur Geschichte Des Eosins.” Berichte Der Deutschen Chemischen Gesellschaft 8 (1): 146–48. https://doi.org/10.1002/cber.18750080149.
  • Blank-Elsbree, Isabella. Letter to Vidhi Naik. 2022. “History at BASF.” Email, 2022.
  • Böhmer, Friedrich. 1865. “Zur Pathologischen Anatomie Der Meningitis Cerebro-Medullaris Epidemica.” Aerztliches Intelligenz-Blatt 1865: 529–50. Google Play Books. https://play.google.com/books/reader?id=vFpEAAAAcAAJ&pg=GBS.PA548&hl=en_GB&q=b%C3%B6hmer.
  • Butt, E., and I. Ashraf. 2021. “H01: The Haematoxylin and Eosin Stain: From Piracy to Pathology.” British Journal of Dermatology 185 (S1): 161–61. https://doi.org/10.1111/bjd.20155.
  • Caro, Heinrich. 1892. “Ueber Die Entwickelung Der Theerfarben‐Industrie.” Berichte Der Deutschen Chemischen Gesellschaft 25 (3): 955–1105. https://doi.org/10.1002/cber.18920250399.
  • Centeno, Silvia A., Charlotte Hale, Federico Carò, Anna Cesaratto, Nobuko Shibayama, John Delaney, Kathryn Dooley, Geert van der Snickt, Koen Janssens, and Susan Alyson Stein. 2017. “Van Gogh’s Irises and Roses: The Contribution of Chemical Analyses and Imaging to the Assessment of Color Changes in the Red Lake Pigments.” Heritage Science 5 (1). https://doi.org/10.1186/s40494-017-0131-8.
  • Cook, HC. 1997. “Origins of … Tinctorial Methods in Histology.” Journal of Clinical Pathology 50 (9): 716–20. https://doi.org/10.1136/jcp.50.9.716.
  • Cooksey, CJ. 2018. “Quirks of Dye Nomenclature. 10. Eosin Y and Its Close Relatives.” Biotechnic & Histochemistry 93 (3): 211–19. https://doi.org/10.1080/10520295.2017.1413207.
  • Fischer, Ernst. 1876. “Eosin Als Tinctionsmittel Für Mikroskopische Präparate.” Archiv Für Mikroskopische Anatomie 12 (1): 349–52. https://doi.org/10.1007/bf02933896.
  • Joseph, Gilbert M. 1974. “British Loggers and Spanish Governors: The Logwood Trade and Its Settlements in the Yucatan Peninsula: Part I.” Caribbean Studies 14 (2): 7–37. http://jstor.org/stable/25612609.
  • Kunkel, Robin. 2018. “Logwood – a History of Palettes, Pirates, and Pathology.” University of Michigan Medicine Department of Pathology. March 29, 2018. https://pathology.med.umich.edu/news/574.
  • Ortiz-Hidalgo, Carlos, and Sergio Pina-Oviedo. 2018. “Hematoxylin: Mesoamerica’s Gift to Histopathology. Palo de Campeche (Logwood Tree), Pirates’ Most Desired Treasure, and Irreplaceable Tissue Stain.” International Journal of Surgical Pathology 27 (1): 4–14. https://doi.org/10.1177/1066896918787652.
  • Quekett, John Thomas. 1855. A Practical Treatise on the Use of the Microscope: Including the Different Methods of Preparing and Examining Animal, Vegetable, and Mineral Structure. 3rd ed. London: H. Baillière. https://wellcomecollection.org/works/fes2zq8f.
  • Reinhardt, Carsten, and Anthony S. Travis. 2000. Heinrich Caro and the Creation of Modern Chemical Industry. 1st ed. Dordrecht: Springer Netherlands. https://doi.org/10.1007/978-94-015-9353-3.
  • Rojas-Sandoval, J, and P Acevedo-Rodríguez. 2013. “Haematoxylum Campechianum (Logwood).” CABI Compendium, March. https://doi.org/10.1079/cabicompendium.26332.
  • Travis, Anthony S. 1998. “‘Ambitious and Glory Hunting . . . Impractical and Fantastic’: Heinrich Caro at BASF.” Technology and Culture 39 (1): 105. https://doi.org/10.2307/3107005.
  • Waldeyer-Hartz, Heinrich Wilhelm Gottfried von. 1863. “Untersuchungen Über Den Ursprung Und Den Verlauf Des Axencylinders Bei Wirbellosen Und Wirbeltieren, Sowie Über Dessen Endverhalten in Der Quergestreiften Muskelfaser.” Zeitschrift Für Rationelle Medicin 1863: 193–257. http://opacplus.bsb-muenchen.de/title/2999004/ft/bsb10086962?page=208.
  • Wissozky, N. 1877. “Ueber Das Eosin Als Reagens Auf Hämoglobin Und Die Bildung von Blutgefässen Und Blutkörperchen Bei Säugethier- Und Hühnerembryonen.” Archiv Für Mikroskopische Anatomie 13 (1): 479–96. https://doi.org/10.1007/bf02933947.

 


 

VIDHI NAIK was raised in the tranquil southwest of Scotland. Having had a passion for science, which was nurtured by her parents from childhood, she decided to continue to study science in high school, furthering this passion by choosing to study medicine at university. She is currently an MBChB student at the University of Aberdeen, in the northeast of Scotland. Her interests lie in pathology, women’s health, and ophthalmology. Alongside her educational onus, she is a member of the FGM Education Project, creating the content for the project’s social media pages. She hopes to one day become a pathologist.

 

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