Comprehensive Analysis of Haematoxylin & Eosin Staining in Biology

This assignment provides an overview of Haematoxylin & Eosin (H&E) staining, a widely used technique in biology for visualizing cells. It discusses the principle of H&E staining, which involves the use of eosin, an acidic dye, and hematoxylin, a basic dye. The document explains how these dyes interact with cell components, such as DNA and proteins, to produce contrasting colors. It covers the H&E staining protocol, including dewaxing, dehydration, differentiation, and bluing. The assignment also highlights the different types of H&E staining, such as progressive, regressive, and modified progressive methods, and mentions the importance of mordants and eosin mixtures. The document references key scientific articles to support the information provided. This assignment is a valuable resource for students studying histology and cell biology, offering a clear understanding of H&E staining techniques.

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Student name
Student No
Unit
Title: Haematoxylin & Eosin Staining

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Most of the body cells are colourless and therefore need staining for better visibility.
The technique used for staining could either be specific, where some cell components are stained
with bright colour and a counterstain on the rest of the cells or non-specific, where the cells are
stained with a uniform colour. There are different types of staining. Basophilic and acidophilic
staining and the H & E staining are the most common. In basophilic and acidophilic staining,
acidic dyes react with basic cell components (proteins and other cytoplasm components). Basic
dyes react components of the cell such as nucleic acids (and therefore said to be basophilic).
Haematoxylin and Eosin are the commonly used techniques for cell staining. It contains
eosin and haematoxylin as the dyes. Eosin is the acid dye, with a positive charge (acidic ion).
The general formula for acid dyes is Na+ dye-. It stains acidophilic structures pink or red, also
said to be eosinophilic. Haematoxylin on the other hand is a basic.. Haematoxylin is a simple
dye-stain extracted from Hematoxylon campechianum tree. Oxidation of haematoxylin leads to
formation of hematein and is combined with Al+ ions and staina acidic cell component to
purplish blue. Haematoxylin has to be used with aluminium salt (a mordant) so as to give it the
characteristic features of a basic dye, since it is not a basic dye. The aluminium salts combines
with the tissue while the haematoxylin with the salt, resulting to a tissue mordant haematoxylin
linkage (University of Leeds, 2018). The DNA in the nucleus, RNA in the rough endoplasmic
reticulum and ribosomes are acidic and therefore bind with haematoxylin to form a purple stain.
The level or deepness of the color is associated with the time the sample spends in hematoxylin
and also the amount of DNA. Most cytoplasmic proteins (intracellular membranes, extracellular
fibres cytoplasmic filaments) and are basic and bind with eosins forming a pink stains.
The Haematoxylin & Eosin staining follows the following protocol dewaxing,
dehydration, hematoxylin, differentiation, bluing, eosin, dehydration, cleaning and cover-

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slipping. Li et al. (2018) claim that H & E stains are extensively used in histology labs.
Hematoxylins are grouped according to the mordant used before staining. The mordant
influences boding to the ionic tissue component as well as final colour of the stained cells. Eosin
Y is mostly used and ca be used in alcohol and water. Addition of small amount of acetic acid to
eosin gives a sharper stain. Most labs prefer ordering their stains for repeatable and consistency
in quality. Eosin mixtures like EA50 and EA65 are used for cytology. Addition of these dyes
leads to color variation of the cytoplasm from pale blue to pink. Differentiation of the stains
involve a selective removal of stain from the tissue to match the viewer’s taste. Bluing involves
using some reagents to change the haematoxylin from red to blue (Sampias and Rolls, 2019). To
attain a good stain, the type of H & E stain used plays a big role. These types are of three types,
which include progressive, regressive and modified progressive. Progressive staining involves
adding hematoxylin to the tissue without a differentiator to do away with excess dye hence
background stain occur with treated slides. A differentiator is used in both regressive and
modified progressive staining.

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References
Li, Y., Li, N., Yu, X., Huang, K., Zheng, T., Cheng, X., Zeng, S., & Liu, X. (2018). Hematoxylin
and eosin staining of intact tissues via delipidation and ultrasound. Scientific reports, 8(1),
12259. https://doi.org/10.1038/s41598-018-30755-5
Sampias, C. and Rolls, G. (2019). H&E Staining Overview: A Guide to Best Practices. Retrieved
from: https://www.leicabiosystems.com/knowledge-pathway/he-staining-overview-a-guide-to-
best-practices/
University of Leeds. (2018). What is H&E? Retrieved from: https://histology.leeds.ac.uk/what-
is-histology/H_and_E.php