Description
This comprehensive academic note on The Haematoxylins & Eosin (H&E) stain provides foundational knowledge for tertiary students in Histotechnology, Pathology, and Laboratory Medicine. Derived from the authoritative text "Bancroft's Theory & Practice of Histological Techniques" by Dr. S.K. Suvarna et al., the document details the components and methodology of the most widely used histological stain. It explains that haematoxylin, extracted from logwood, must be oxidized to hematein and combined with a mordant, such as aluminum, iron, or tungsten salts, to effectively stain cell nuclei. The note classifies haematoxylin solutions by their mordant, covering alum haematoxylins like Harris's and Mayer's for routine nuclear staining, iron haematoxylins like Weigert's for detailed structures, and specialized types like PTAH for neuroglia and muscle striations. It describes Eosin Y as the standard cytoplasmic counterstain, producing shades of pink and red. The step-by-step staining protocol is outlined, from deparaffinization and rehydration through nuclear staining, differentiation, bluing, counterstaining, dehydration, clearing, and mounting. A troubleshooting section addresses common issues like over-differentiated nuclei or poor cytoplasmic staining. This material is essential for diploma and degree students to understand the principles, execution, and quality control of the H&E stain, which is critical for accurate tissue diagnosis in histopathology laboratories. Download this guide to master the standard staining technique that forms the basis of microscopic evaluation.
What's inside
The Haematoxylins & Eosin Objectives At the end of the lecture, you must be able to: 1. Explain the function of eosin in H &E staining 2. Describe the main properties of haematoxylin and staining using haematoxylin solutions 3. Describe haematoxylin and eosin staining method Introduction • H&E stain is the most widely used histological stain. • Simplicity and ability to demonstrate clearly an enormous number of different tissue structures. • The haematoxylin component stains the cell nuclei blue-black • The eosin stains cell cytoplasm and most connective tissue fibers in varying shades and intensities of pink, orange, and red. Routine Staining (Hematoxylin & Eosin) Nuclear detail - Hematoxylin Contrasting counterstain - Eosin Eosin Stain • The eosins are xanthene dyes and have the following types: eosin Y (eosin yellowish, eosin water-soluble), ethyl eosin (eosin S, eosin alcohol- soluble), eosin B (eosin bluish, erythrosin B). • Eosin Y is the most widely used, and despite its synonym it is also satisfactorily soluble in alcohol. • Usually used as a 0.5 or 1.0% solution in distilled water(addition of crystal thymol). Eosin Stain • Most suitable stain to combine with an alum haematoxylin. • With proper differentiation, it can distinguish between the cytoplasm of different types of cell, and the different types of connective tissue fibers and matrices, by staining them differing shades of red and pink. Haematoxylin • Extracted from the heartwood (‘logwood’) of the tree Haematoxylon campechianum (A) Logwood, (B) cut end of same logwood and hematoxylin powder. Haematoxylon campechianum Tree. Insert: leaves on branch. Hematoxylin.., • Extracted from logwood with hot water, and then precipitated out from the aqueous solution using urea. • Haematoxylin itself is not a stain. • It has to be oxidized, a process referred to as ripening. • The major oxidization product is hematein, a natural dye that is responsible for the color properties. • Hematein can be produced from haematoxylin in two ways. Natural oxidation by exposure to light and air • Is a slow process (sometimes 3-4 months) • Resultant solution seems to retain its staining ability for a long time. • Examples: Ehrlich’s and Delafield’s haematoxylin solutions. Chemical oxidation • Chemical oxidizing agents used are: i. sodium iodate (e.g. Mayer’s haematoxylin) or ii. mercuric oxide (e.g. Harris’s haematoxylin) • Converts the haematoxylin to hematein almost instantaneously, so these haematoxylin solutions are ready for use immediately after preparation. Chemical oxidation.., • Hematein is anionic, having a poor affinity for tissue, and is inadequate as a nuclear stain without the presence of a mordant. • The most useful mordants for haematoxylin are salts of aluminum, iron, and tungsten. Classification • Haematoxylin solutions can be classified according to which mordant is used: • Alum haematoxylins • Iron haematoxylins • Tungsten haematoxylins • Molybdenum haematoxylins • Lead haematoxylins
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