Fixatives
Today, the most common primary fixatives for electron microscopy (EM) are the aldehydes. Originally, osmium tetroxide was used as the primary fixative, but following Sabatini, Bensch and Barnett in 1963, aldehydes became the preferred fixative with osmium applied as a secondary fixation for the purposes of good electron contrast and lipid preservation. Fixatives form cross-links between molecules, stabilising them, and are used in combination with buffering agents that should be as close as possible to solute concentrations and osmolarity of the tissue or cells being fixed.
Glutaraldehyde
- Glutaraldehyde is probably the most commonly used EM fixative and is normally supplied at concentrations of 25 or 50% and used at concentrations of 0.5-2%.
- The molecular weight of glutaraldehyde is 100.12 g/mol and at a 1% concentration will have a molarity of 0.1 mols/litre.
Formaldehyde
- Formaldehyde is a common fixative for light microscopy and is often used on its own. However, for EM it often doesn't provide enough stability for all of the sample preparation steps and ultrastructure can be poor. For EM it is routinely used in combination with glutaraldehyde*.
- The molecular weight of formaldehyde is 30.03 g/mol
*interesting note
The combination of formaldehyde and glutaraldehyde was presented by a scientist called Karnovsky at a conference and never published as a paper. However, the combination proved so popular that this combination has been termed "Karnovsky's fixative", often used in a modified form with lower aldehyde concentrations than originally proposed. A link to the story as told by the author is found here: Karnovsky M J. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. CellBiol. 27:137-8A, 1965.
Links to chemical MSDS
The chemicals used in electron microscopy (EM) are optimsed for fixing and preparing biological tissue. For this reason, you really do not want your own biological tissue exposed to them as it will be fixed. The use of personal protective equipment (PPE), i.e. lab coat, chemical resistant gloves and fume hood are recommended for all EM chemical use.
Below are links to Material Safety Data Sheets (MSDS) from Sigma-Adrich for the most commonly used EM chemicals. You should always read the MSDS and the standard operating procedures in your own labs before using any of these or any other chemicals.
Glutaraldehyde
Formaldehyde
Osmium Tetroxide
Uranyl acetate
Below are links to Material Safety Data Sheets (MSDS) from Sigma-Adrich for the most commonly used EM chemicals. You should always read the MSDS and the standard operating procedures in your own labs before using any of these or any other chemicals.
Glutaraldehyde
Formaldehyde
Osmium Tetroxide
Uranyl acetate