Histology Techniques - Fixation

This process has two phases:
1) the coagulation or precipitation of the various components of the tissues and cells and
2) their preservation in a state as nearly as possible like the living condition by forming stable chemical compounds.
The first phase carries with it an intrinsic source of difficulty and error. The precipitation may be uneven and cause deposits to form where no structure existed in the living cell. These are called "fixation artifacts". The second phase also carries a source of difficulty because the compounds formed by some fixatives will not take up some stains.

It has not been possible to find an ideal fixative that
1) penetrates quickly,
2) renders all parts of all cells permanent and
3) allows the use of all kinds of stains.
The reason for this is not difficult to understand. The cell is a highly complex mixture of proteins, carbohydrates and fats. The ideal fixative would not only have to form stable compounds with all of these, but also render them insoluble both in fat solvents and in water. Some fixatives not only fail to preserve certain parts of the cell but actually dissolve or destroy them. For example, acetic acid destroys mitochondria. Moreover, some fixatives change the shape and relationship of parts of a tissue by shrinkage.


This is a good general fixative. Its effect is to cross-link membrane proteins by forming covalent bonds. It is made by dilution of commercial formaldehyde (which is a 40% solution of formaldehyde gas in water) in an aqueous phosphate buffer. The usual strength is 10% (or 4% of the gas). It penetrates rapidly, causes little distortion, does not destroy any of the cellular constituents and can be followed by almost all staining methods. It hardens the tissues very slowly, however, and does not protect them from the shrinking agents employed in embedding and sectioning. For this reason it is often combined with other fixing agents.


Osmium tetroxide (OsO4) preserves the cell in a form closer to the living than any other fixative. Its great disadvantage is that it penetrates poorly and cannot be followed by many stains. It is also used as a stain because it blackens fat and various lipid-containing materials such as the myelin sheaths of nerve fibers, and makes them insoluble both in water and in fat solvents. Osmium tetroxide solution, in various buffers, is a standard fixative for electron microscopy.