Histopathology FixationHistopathology Fixation

Histopathology Fixation

Histopathology Fixation, Fixation process is very important to preserve the tissue from changes in the histopathology section. Fixation refers to the process by which the tissue is protected from the effects of bacteria and the process of destruction. As soon as the tissue is placed in the fixative. The process of changing the structure of the tissue stops and the tissue becomes preserved. Another reason for placing the tissue in a fixative is to make the tissue a little harder. When the received tissue is cut into different parts. And then it is put in fixative. The amount of fixative should be about 10-20 times the size of the tissue. Filter If the tissue sample is very small, it should be wrapped in a special material and placed in a fixative.

Classification of Fixative:

Fixatives are divided into two groups:

  • Simple/Single Fixative
  • Compound Fixative

Simple / Single Fixative:

A fixative which consists of one chemical component is called simple/single fixative. The following Fixative are used in Histopathology.

Histopathology Fixation

Name of Simple Fixative:

  1. Formal dehyde (Formalin 10%)
  2. Mercuric Chloride
  3. Osmium Tetra Oxide
  4. Acetic Acid
  5. Ethanol
  6. Citric Acid
  7. Potassium Dichromate

Ideal Fixative:

There are many fixatives used in histopathology. A good and ideal fixative should have the following properties,

  • A good fixative quickly embeds into the tissue and preserves all the cells of the tissue evenly.
  • Ideal Fixative is easy to prepare.
  • The ideal fixative is inexpensive.
  • The ideal fixative creates an adequate degree of stiffness in the tissue. Which makes sectioning easier.
  • Ideal Fixative prevents the effects of bacteria on the tissue. Prevents tissue changes.
  • Ideal Fixative does not cause contraction or swelling of the tissue.
  • Ideal Fixative is non-toxic and does not cause inflammation or irritation to the human body.
  • Ideal Fixative preserves the tissue in its own color. No one adds color on their own.
  • Ideal Fixative can preserve tissue for long periods of time.

The following side effects have been reported due to Fixative.

  • Formaldehyde leaves a brown pigment on the tissue.
  • Chloride causes black deposits to accumulate on the tissue.
  • Some fixatives cause tissue shrinkage.
  • If the penetration of the fixative into the tissue is low, many useful substances are lost from the tissue and the inner cells of the tissue are not protected.
  • The size and thickness of the tissue is important for the fixative.
  • If there is too much mucus or fat on the tissue, the penetration of the fixative is reduced.
  • Temperature also affects the performance of the fixative.


Formaldehyde is usually used as a fixative. Formaldehyde is a gas. Which is dissolved in water to make a solution. 40% of formaldehyde available in the market is found in this solution. A 10% solution is prepared by treating this 40% solution as 100%. It is used to store samples in the laboratory.

Advantage / Merits:

  • Formaldehyde is a cheap chemical. It is easy to prepare.
  • It preserves the tissue in its natural color.
  • Does not produce any kind of scar in the tissue.
  • Preserves Lipid and Protein in the tissue.
  • Staining tissue preserved in formalin gives better results.

Disadvantage / Demerits:

  • Formalin Fixative is toxic. Long-term use causes inflammation of the human body known as dermatitis.
  • Formalin fumes cause sinusitis, an inflammation of the nose.
  • The use of formalin causes asthma in allergic patients.
  • If formalin is allowed to sit for a long time, it forms a white precipitate. If these precipitates do not affect the ability of the solution to protect the tissue. These sediments are separated by a filtration process.
  • Formalin available in the market contains 11 to 16% Methanol to inhibit the formation of young sediments.
  • Formalin Fixative penetrates the tissue with medium speed. And it takes 12 to 24 hours for a tissue to be completely preserved.
Histopathology Fixation
Histopathology Fixation


Sometimes it is useful to re-immerse the fixed tissue in fixative for four hours. This process is called Secondary Fixation.

Name of Compound Fixative:

A compound fixative is a fixative that contains two or more simple fixatives Prepared by mixing fixative. Or there are fixatives that are made by mixing two or more chemicals.

Formal Sublimate:

  • Formalin 40% 100 ml Reagent
  • Mercuric Chloride

Formal Calcium:

  • Formalin 40% 100 ml Reagent
  • Cacl2 10% 100 ml Reagent
  • Distill Water 900 ml

Zenker Fixative:

  • Mercuric Chloride 5.0 gram
  • Potassium Dichromate 2.5 gram
  • Sodium Sulphate 1.0 gram
  • Distill Water 100 ml

Bouin Fluid:

  • Saturated Picric Acid 75 ml
  • Formalin 40% 25 ml
  • Glacial Acetic Acid 0.5 ml

Gross Examination:

In this step, the sample received in the laboratory is visually inspected. Its size is noted for its color and texture. The amount of mucus, blood, etc. present on the sample is checked. If the sample size is large, two or three specific parts are selected and cut. And if the sample size is small, it is passed through the complete next steps.

De Calcification:

This is a process in which samples containing calcium have accumulated. Calcium is extracted from it. And the ingredients used for this purpose are called Decalcifying Agents. Bones and teeth present in human bone contain a high amount of inorganic salts like Calcium, Phosphate, Calcium Carbonate Fluoride, and Magnesium, etc. and due to these salts, the parts become hard. Sometimes calcium is also deposited in other tissues such as thyroid or large blood vessels. And hardens them. When such samples are received in the laboratory, they must be softened for microscopic examination. So that Sectioning can be easily obtained from them. This process is done after the fixation step and before tissue processing. The following chemicals are used as decalcifying agents:

  1. Formic acid
  2. Nitric acid
  3. Hydrochloric acid
  4. Trichloroacetic acid

De calcification process is checked by following methods:

  • By the Rays.
  • By entering ‘Pin’ in the sample.
  • By bending the bones or specimen.
  • By weighing the sample before and after decalcification.

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