Histology: Emphasis on Histotechniques

Introduction to the meaning of Histology

Histology refers to the study of microscopic structure in biological material and its ways where individual components are both structurally and functionally related. It is vital in the study of biological and medical science as it is central in biochemistry, disease process, and molecular biology and physiology. It is usually performed through the examination of cells and tissues using a light or electron microscope. The sample is sectioned, stained, and mounted on a slide. Tissue culture may be used as an artificial environment that maintains the isolated cells. Trained professionals will them perform histopathological examination to provide a diagnosis based on their observations of the microscopic specimen. These trained personnel are known as histotechnicians, histotechnologists, histology technologists, scientists, histology technicians, laboratory technicians, or biomedical science support workers.


Marcello Malpighi, an Italian, invented one of the first microscopes in the 17th century. He used it to study the organs of animals such as bats and frogs. While he was analyzing the structure of the lung, he notices the membranous alveoli and the connections between veins and arteries and named them as capillaries. Through his discovery, it was established how oxygen enters the blood stream to supply the rest of the body. By the 19th century, histology became a specialized field of study. In 1801, Bichat, a French anatomist introduced the concept of tissue and by 1819, the term histology appeared in a book by Karl Meyer. Histologists Camillo Golgi and Santiago Ramon y Cajal won the Nobel Prize in 1906 for the interpretation of the neural structure of the brain.

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Histotechnique is a term that describes the processes involved in producing a microscopic slide from specimens examined in the pathology laboratory. Some of the histotechniques are the following:

a) Gross examination
The tissues that are obtained are first examined by a pathologist. Gross examination includes the description of the specimen and placing it into a cassette which are then fixed. If a malignancy is suspected, the specimen is covered with ink to mark the margins of the specimen. Different colors are used to identify different areas if necessary.

b) Fixation
The process of fixation aims to preserve the tissues permanently with as little protein denaturation as possible. Fixation is performed as soon as possible after the tissue is removed from the sample to prevent autolysis. Formaldehyde is the best fixative but there are may different fixatives available depending on the main use needed. the 5 major groups of fixatives are categorized according to the mechanism of action: aldehydes, mercurial, alcohols, oxidizing agents, and picrates. The factors that may affect fixation include buffering, penetration, volume, temperature, concentration, and time interval.

c) Tissue processing
Tissues taken from the specimen of study must be processed by the histology laboratory to produce slides that can be viewed under the microscope. The individual that is responsible for tissue processing and producing microscopic slides are known as histotechnologists. Specimens received usually lists the patient information and site of origin. A number is given to each specimen to help in the identification process of the sample. The process of getting the fixed specimen into paraffin blocks is called tissue processing. The main steps involved are dehydration and clearing.

The water from the specimen is first removed by dehydration using a series of alcohols. A mixture of alcohol and formalin can also be used. Although other dehydrants can be used, there are major disadvantages when compared to alcohol and formalin. The clearing process involves the removal of the dehydrant using a substance that mixes well with paraffin, the embedding medium. The commonest agent used is xylene. Toluene also works well but costs much more than xylene. Finally, the tissue is embedded in the agent, usually paraffin. The embedding process is important as the tissue embedded has to be aligned properly in the paraffin block.

Paraffin alternatives such as plastics allow thinner sections being cut. Some plastic examples are methyl methacrylate, glycol methacrylate, araldite, and epon. The disadvantage of using plastics is the requirement of special regents for the dehydration and clearing process that can be expensive. When a large volume of tissues is processed, the above methods can be automated.

d) Sectioning
To section the blocks, a special microtome is used. Once cut, the specimen is floated on a warm water bath and picked up on a glass microscopic slide. Slides are placed in a warm oven for 15 minutes to ensure the section adhere to the slide.

e) Frozen sections
This is performed using an instrument called a cryostat. The tissue is frozen in a cold liquid or environment, cut, and picked up on the glass slide.

f) Staining
Slides are first deparaffinized before staining starts. The routine stain used is hematoxylin and eosin.

g) Coverslipping
After staining, the slide is covered using a thin piece of plastic or glass to protect the specimen, provide better quality for viewing, and preservation of the tissue.

h) Decalcification
There are tissues that contain calcium deposits which can cause issues during the sectioning process. To remove it, mineral acids, organic acids, and electrolysis can be used.


Each step should be performed carefully to avoid issues in the histologic sections. Safety in the lab is of the utmost importance and must follow the protocols set by the Occupational Safety and Health Administration. Accidents are documented and investigated to help prevent future episodes.


Histotechniques. Accessed 3/2/2018. https://library.med.utah.edu/WebPath/HISTHTML/HISTOTCH/HISTOTCH.html

Lowe JS, Anderson PG. Histology. Stevens & Lowe’s Human Histology (4th Edition). 2015. https://www.sciencedirect.com/topics/neuroscience/histology

Histology. Wikipedia. Accessed 3/2/2018. https://en.wikipedia.org/wiki/Histology