Fixation

FIXATION

  • Fixation is the method of preserving the cell and tissue constituents as in life like condition as possible. Adequate and complete fixation are the foundation of good histological preparation. It is essential that tissues are fixed as soon as possible after death or removal from the body.
  • The amount of fixative (that is an agent used for fixation) should be 15 to 20 times the bulk of tissue to be fixed. Tissues or sectioning should be sufficiently thin to be adequately fixed throughout in a reasonable time. The best thickness for routine use is 3 to 5 m.m.

Aims and effects of fixation

  • Fixation is required to prevent putrefaction and autolysis to preserve and harden, to solidify colloid materials and to aid visual differentiation of structures. Certain fixatives have effects on subsequent staining and this should be remembered.
  • Fixation also renders cells insensitive to hypotonic and hypertonic solutions. Fixation alters the refractive indices of various components of cell and tissue into varying degrees so that details are easily seen. Fixative must be used in solutions either with normal saline or distilled water.

Common fixatives employed

  • Formalin is the most common fixative used in 5 to 10% strength in normal saline.
  • Mercuric chloride in combination with other fixatives.
  • Osmium tetroxide (Incorrectly named Osmic acid) to fix fat and lipids used as a 2 to 5% aqueous solutions).
  • Chromic acid: used as a 2% solution preserves carbohydrates.
  • Potassium dichromate used a 3% aqueous solution. This is another common fixative used in combination with others.
  • Picric acid used as a saturated aqueous solution in combination with others.
  • Acetic acid-used in varying strengths in combination with other.
  • Trichlor-acetic acid used with others in varying strengths.

Type of Fixatives

  • Generally the fixatives are named after their inventors
    1. Formal Saline 5-10%
    2. Heidenhain’s Susa
    3. Bouin’s fluid
    4. Zenker’s fluid and Zenker formaldehyde
    5. Flemming’s fluid
    6. Carnoy’s fluid
    7. Helly’s fluid
  • Of these Numbers 1,2,3 and 4 are micro anatomical fixatives, that is they accurately preserve the relations of tissue layers and large aggregates of cells to one another and are useful in routine work. Numbers 5, 6 and 7 are cytoplasmic and nuclear fixatives.
  • In general, fixatives are either;
    • Precipitant fixatives (that is they precipitate or coagulate colloidal constituents (e.g. mercuric chlorides, alcohol, picric, chromic acid) or
    • Non-precipitant fixatives (that is they fix proteins not by chemical combination but by denaturing them which involves a change in the shape of protein molecule and results in making it insoluble (eg.) Formalin, Osmium tetroxide and Potassium dichromate).
Last modified: Tuesday, 24 August 2010, 6:12 AM