Histology and Histological Techniques

The branch of biology that deals with the study of the structure and composition of tissues are termed histology. Histology is also known as microscopic anatomy or microanatomy as it involves studying tissues under an electron microscope or a light microscope. Special staining techniques are used to colourise a thin section of tissue. After that, it is examined under a microscope. It is important to stain the tissues before examining them under a microscope as it helps distinguish between various structures of the tissue. The histological sections can be prepared through various techniques. There are three types of histological techniques that can be used to prepare tissue sections if light microscopes are being used:

• Paraffin technique
• Frozen section technique
• Semithin section technique

The paraffin technique is the most commonly used histological technique.

Paraffin Technique

In this histological technique, tissues are fixed and embedded into wax. This helps maintain the firmness of the tissues, which makes slicing the tissue easier. The following steps are involved in the Paraffin technique:

• Fixation

In this step, chemical fixation is used to fix square blocks of tissues or whole organs. Chemicals are added to the tissues, which bind and cross-link some proteins. Some other proteins are denatured due to dehydration by the chemicals. The fixation process inactivates enzymes that prevent degradation and hardens the fixed tissues. 

• Dehydration and Embedding

After the first step, dehydration and embedding of the tissue are done. All water is removed from the tissues through the process of dehydration, which allows embedding of the tissue. Embedding is done in ‘xylene’, which removes paraffin from the tissue. Xylene is used because paraffin does not dissolve in water. The tissue solidifies after it is embedded in paraffin.

• Sectioning

After dehydration and embedding of the tissue section, it solidifies. It is then trimmed and cut into a thin section using a microtome. A microtome is a precision cutting instrument that cuts tissue into sections as thin as 100 μm. After the tissue is cut, it is stained.

Several methods can be used to stain the tissue. The stained section of the tissue is put on a slide and studied under a microscope, and a report is finally made.

Frozen Section Technique

In this histological technique, the tissues are rapidly cooled with liquid nitrogen and then cut in a cryostat. A cryostat is a refrigerated cabinet that is used to freeze the tissue samples and then cut them into microscopic sections 5 – 10 µm thick. The microscopic sections of the tissue are then stained and observed under a microscope. 

A doctor typically requests a frozen section during surgery as it is a faster technique. The results obtained from a frozen section help the doctor make decisions during surgery.

A typical use of the frozen section technique is examining the tissue surrounding a tumour. The tumour in such cases is extremely microscopic that cannot be seen with the naked eye. Frozen sections are also used to confirm that a diseased section of tissue has been removed. 

Semithin Section Technique

In this histological technique, the embedding of the tissues is done in acrylic resin or epoxy. This creates microscopic sections that are thinner than 2 µm. This technique is mostly used when more detailed tissues are needed, as minute details are sometimes missed in thicker sections.

Histology stains

When tissue sections are observed under a microscope, differentiating between cells and tissues becomes difficult as they are almost colourless. A differential colouration is created when the tissue section is stained with colour. Thus, precise observation and analysis of the section can be obtained.

In histology, a huge range of stains, from labelled antibodies to dyes and metals, are used. When added to the tissues, some stains result in a different colour than their original colour. This phenomenon is termed metachromasia. Some common types of stains used in histology are as follows:

• H&E (Haematoxylin and eosin) stain

This stain is used on tissue sections taken from the skin and is majorly useful in classifying and diagnosing cancer. The haemotoxylin stain adds blue colour to certain parts of the cell, the nucleus, for instance. On the other hand, the eosin stain adds a red or pink colour to other parts of the cell, the cytoplasm, for instance.

• Mucin stain

This stain is used mostly in mucopolysaccharides for their detection and dyeing. Some examples of the mucin stain are Mucicarmin, Period acid-Schiff (PAS) and Alcian blue.

• Melanin stain

This stain is used for dyeing melanin and is used to diagnose melanoma. The Fontana-Masson is a common example of a melanin stain.

• Trichrome stain

This stain is used for dyeing lipids. It uses a combination of three different dyes, as suggested by its name (tri). Gomori trichrome, Mallory trichrome and Sudan stains are some common examples of this stain. 

Conclusion 

Histology is the branch of biology that deals with studying tissues and their structure. In this article discussing the histological techniques notes, we understood the three different histological techniques: the Paraffin technique, the Frozen section technique, and the Semithin section technique.

In the paraffin technique, chemical fixation of the tissue is done, followed by dehydration and embedding. The tissue is then cut using a microtome, stained and finally mounted on a slide to be observed under a telescope. In the frozen section technique, the tissue is frozen and cut into microscopic sections in a cryostat. In the semithin section technique, the tissue is embedded in acrylic resin or epoxy, creating microscopic sections of the tissue to obtain a detailed view. In histology, the tissue is stained to distinguish between different tissue types and cells. H&E stain, mucin stain, Melanin stain and trichrome stain are some common types of stains.