Types of fixation

Fixation of tissues can be achieved by chemical or physical means. Physical

methods include heating, micro-waving and cryo-preservation (freeze
drying). Heat fixation is rarely used on tissue specimens, its application being
confined to smears of micro organisms. However, microwave fixation, which
can be regarded as a form of heat fixation, is now widely practiced in routine
laboratories . Cryo-preservation, usually in the form of freeze drying has
some applications in histochemistry but is not usually applied to diagnostic
tissue specimens.2

Chemical fixation is usually achieved by immersing the specimen in the

fixative (immersion fixation) or, in the case of small animals or some whole
organs such as a lung, by perfusing the vascular system with fixative
(perfusion fixation). For some specialised histochemical procedures fixatives
have occasionally been applied in the vapour form. For example
paraformaldehyde and osmium tetroxide can be used to vapour-fix freeze-
dried tissues.

Fixative solutions may contain a single fixative agent dissolved in a solvent

such as water or alcohol or more commonly, a buffer solution to stabilize pH.
Some popular fixative solutions contain several different fixing agents in
combination, the rationale being that the defects in one agent can be
compensated for by the addition of another. For example acetic acid is
present in some formulations to counter the shrinkage caused by other
agents such as ethanol. 5

Theoretical basis of fixation

Fixation may be considered “a complex series of chemical events”.6 Although
we can now define some of these “events” our understanding of much of
what happens during fixation is still incomplete. Cells and extracellular
components contain peptides and proteins, lipids and phospholipids
(membranes), carbohydrates and carbohydrate complexes, various types of
RNA and DNA and so on. Just how these elements will react during fixation
will depend on the type of fixation, the fixing agent used and the fixation
conditions. Some tissue elements will chemically react with the fixative, be
stabilised by cross-linking and thus preserved, others may be unaffected by
the fixative but trapped within a cell or tissue by other fixed elements.