Difference Between Hematoxylin and Eosin

The key difference between hematoxylin and eosin is that hematoxylin is a basic dye, whereas eosin is an acidic dye.

Hematoxylin and eosin are important in histology for staining purposes. These two dyes are used as counterstains in the H and E staining process.

CONTENTS

1. Overview and Key Difference
2. What is Hematoxylin
3. What is Eosin
4. Side by Side Comparison – Hematoxylin vs Eosin in Tabular Form
5. Summary

What is Hematoxylin?

Hematoxylin is an organic compound having the chemical formula C₁₆H₁₄O₆. This compound is extracted from the heartwood of the logwood tree. This is a natural dye that has been long used as a histologic stain, ink and as a dye in the textile and leather industry. As a dye, this material has several names such as Palo De Campeche, longwood extract, bluewood and blackwood.

Generally, in histological applications, hematoxylin stain is paired with eosin through the H and E staining process, and it is a common combination in this field of histology. Moreover, hematoxylin is a component of Papanicolaou stain, which has many uses in studying cytology specimens.

The active colourant of hematoxylin is haematin, the oxidized form that can form strongly colourful complexes containing metal ions such as Fe(III) and Al(IIII) salts. When hematoxylin is pure, it appears as a colourless crystalline solid substance having typically light to dark brown appearance in commercial grade due to the presence of impurities.

Considering the extraction and the purification of hematoxylin dye, it is accomplished by boiling the wood chips in the French process or American process (which includes steam and pressure). This dye is sold as a liquid concentrate, a dried material, or as a crystalline substance once extracted. However, there are some modern methods of production, which includes the use of water, ether, or alcohol as the solvent.

What is Eosin?

Eosin is a group of fluorescent acidic compounds having bonds with basic or eosinophilic compounds to form salts. The salts are formed with compounds such as proteins which contain amino acid residue including arginine, and lysine. Eosin can stain protein residues dark red or pink colour as a result of the action of bromine on fluorescein. Additionally, we can use this dye to stain proteins in the cytoplasm and to stain collagen and muscle fibres for studying under the microscope. In this context, we can name microstructures that readily stain with eosin as eosinophilic structures.

When considering different types of eosin, there are two closely related compounds of eosin as eosin Y and eosin B. Among them, Eosin Y has a slightly yellow cast, while Eosin B has a faint bluish cast. We can use these dyes interchangeably.

There are many applications of eosin in histology as a counterstain to haematoxylin in H and E staining. It is the most common technique in histology. When a cytoplasm is stained using H and E staining, we can observe its colour change as stained in pink-orange and nuclei stained in a dark colour in blue or purple colour.

What is the Difference Between Hematoxylin and Eosin?

Hematoxylin and eosin are important dye compounds in histology. These are counterstain material for the H and E staining process. Hematoxylin is an organic compound having the chemical formula C₁₆H₁₄O₆ while eosin is a group of fluorescent acidic compounds having bonds with basic or eosinophilic compounds to form salts. The key difference between hematoxylin and eosin is that hematoxylin is a basic dye, whereas eosin is an acidic dye.

Below is a summary of the difference between hematoxylin and eosin in tabular form.

Summary – Hematoxylin vs Eosin

Hematoxylin and eosin are important dye compounds in staining microstructures such as proteins in the cytoplasm. The key difference between hematoxylin and eosin is that hematoxylin is a basic dye, whereas eosin is an acidic dye.

Reference:

1. Helmenstine, Anne Marie. “What Histology Is and How It’s Used.” ThoughtCo, Aug. 27, 2020, Available here.

Image Courtesy:

1. “Hämatoxylin” By NEUROtiker (talk) – Own work (Public Domain) via Commons Wikimedia
2. “EosinY” By WilliamsChemistry – Own work (CC BY-SA 4.0) via Commons Wikimedia