Histology Sample Fixation: Principles and Methods

Posted on Jun 20, 2025 in Medicine & Health

Fixation is the first step of any histological and cytological laboratory technique. It is the process by which the cells in the tissue are fixed in a chemical and physical state, and all biochemical and proteolytic activities within the cells are prevented, allowing the cells or tissues to resist morphological change, distortion, or decomposition after subsequent treatment with various reagents.

Aims of Histological Fixation

  • To preserve the tissue as close to its living state as possible.
  • To prevent any change in the shape and size of the tissue during processing.
  • To prevent autolysis and bacterial growth in the tissue.
  • To make the tissue firm to hard.
  • To enable clear staining.

Ideal Fixative Properties

  • Prevention of autolysis in cells or tissue.
  • Maintaining cell volume and shape.
  • Cost-effective and non-toxic.
  • Rapid action.

Tissue Changes During Fixation

Volume Changes

  • Osmium tetroxide causes cell swelling.
  • Formaldehyde may cause volume shrinkage by up to 33%.
  • Similarly, glutaraldehyde also causes significant tissue shrinkage.
  • When glutaraldehyde followed by osmium tetroxide is used for fixation in epoxy resin, a 70% increase in cell size is noted.

Hardening of Tissue

Some hardening occurs due to fixation.

Interference with Staining

Fixation may hinder enzyme staining. Formaldehyde inactivates 80% of the ribonuclease enzyme. It has been noted that osmium tetroxide inhibits hematoxylin and eosin staining.

Changes in Optical Density

Fixation may alter the optical density of nuclei, causing them to appear condensed and hyperchromatic.

Types of Histology Fixation

Fixatives can be classified based on:

  • Nature of Fixation
  • Chemical Properties
  • Components Present
  • Action on Tissue Protein

Classification of Fixatives

  • A. By Nature of Fixation
    • Immersion Fixation
    • Coating Fixation
    • Vapor Fixation
    • Perfusion Fixation
    • Freeze-Drying
    • Microwave Fixation
  • B. By Chemical Properties
    • Aldehydes: Formaldehyde, Glutaraldehyde
    • Oxidizing Agents: Osmium Tetroxide
    • Protein Denaturing Agents: Ethyl Alcohol, Methyl Alcohol
  • C. By Components Present
    • Simple (Single Chemical)
      • Formaldehyde
      • Ethyl Alcohol
      • Glutaraldehyde
    • Compound (Multiple Chemicals)
      • Bouin’s Fluid
      • Carnoy’s Solution
  • D. By Action on Protein
    • Coagulative: Ethyl Alcohol, Picric Acid
    • Non-Coagulative: Formaldehyde, Osmium Tetroxide, Glutaraldehyde

Detailed Methods of Fixation

Immersion Fixation

  • This is the most common method of fixation in laboratories.
  • The entire specimen is immersed in the liquid fixative.

Coating Fixation

  • This is commonly used for cytology samples.
  • Spray fixatives are used for easy transportation of slides.
  • The main advantages of spray fixatives include:
    • Fixation of cells.
    • Imparting a protective covering over the smear.
    • No need to carry liquid fixative in a bottle or jar.

Vapor Fixation

  • Chemical vapors are used to fix either a smear or a tissue section.
  • Commonly used chemicals for vapor fixation include formaldehyde, osmium tetroxide, glutaraldehyde, and ethyl alcohol.
  • The vapor converts soluble material into insoluble material, which is then retained when the smear comes into contact with a liquid solution.

Perfusion Fixation

  • In this technique, the fixative solution is infused into the arterial system of an animal, fixing the entire animal.
  • Organs such as the brain or spinal cord can also be fixed by perfusion.

Freeze-Drying

The freeze-drying technique is primarily applicable for studying soluble materials and tiny molecules.

Advantages:

  • Excellent for enzyme studies.
  • No change in proteins.
  • Preservation of glycogen.

Microwave Fixation

  • Electromagnetic waves with frequencies between 300 MHz and 300 GHz.
  • Mechanism: Microwaves create an electromagnetic field, causing dipolar molecules to rapidly oscillate and generate heat through kinetic motion.

Advantages:

  • Uniform heat production.
  • No tissue volume change.
  • Good for electron microscopy after osmium tetroxide fixation.
  • Preservation of tissue antigens.

Disadvantages:

  • Tissue in formalin for microwave fixation may produce toxic gas.
  • Heat injury may occur from microwaves.

Applications:

  • In routine surgical pathology laboratories.
  • Electron microscopy.
  • Urgent processing of special biopsies.

Essential Precautions for Histology Fixation

  • Tissue should be free from excessive blood before immersion in a fixative.
  • Tissue should be thinly cut to 3-5 mm thickness. The volume of fixative fluid should be at least 20 times greater than the tissue volume.
  • Never heat the fixative solution to accelerate fixation, as this may cause tissue shrinkage.