Glutaraldehyde
–
C5H8O2
Before You Begin
Please consult the following guide to safe working with this chemical fixing agent, including how to safely clean up spills.
Safety Note
Glutaraldehyde is not a particularly dangerous reagent to use, and simple precautions make it quite safe. These are basically the same precautions that would be used with any other laboratory reagent, but with extra attention paid to ensuring that breathing in the fumes and exposing mucous membranes to the fumes are avoided. That can be done by dispensing and using solutions containing glutaraldehyde under a fume hood. To avoid skin contamination, gloves should be worn when diluting the concentrate for use and when removing the tissue from the container.
Spills are likely to be minor, particularly if small bottles of the concentrate are purchased. This is advisable to ensure the reagent is fresh, since glutaraldehyde fixation for light microscopy is not often done in most laboratories. Small spills may be cleaned up with paper towels and lots of water. Ensure adequate ventilation while doing so.
In the event that there is a large spill with inadequate ventilation and any distress due to the fumes, err on the side of caution and safety and get someone experienced in the use of a mask and air tanks to clean up the spill.
How it Fixes
Proteins
Glutaraldehyde cross-links proteins in a manner analogous to formaldehyde, but targeting different components, such as amino groups. During this process, it stabilizes the protein mass and preserves the morphology. It fixes somewhat faster than formaldehyde, but its penetration rate is poorer. Since it is extensively used as a primary fixative for electron microscopy, it was thought it might function as effectively for light microscopy, but this did not prove to be the case, and it is not popular as a routine fixative.
For histological purposes, purified glutaraldehyde should be purchased. It is available as a 25% solution.
Carohydrates
Carbohydrates are not fixed, although any protein bound to them will be, i.e. mucins.
Lipids
Generally, triglycerides remain unfixed but the protein part of lipoprotein may be fixed, and these may resist extraction by solvents.
Morphology
Morphological preservation is fair to good. The results are similar to formaldehyde.
Time
For electron microscopy, tiny pieces are fixed quite rapidly, but the 3 mm pieces required for light microscopy require longer. Generally, several hours to overnight will be adequate.
Simple Solution
Glutaraldehyde is usually used alone as a 4% solution, freshly made, in a pH 7.4 buffer. Once made, it should be used within 8 hours.
Aftertreatment
There is no particular aftertreatment for the tissue block, and it may be placed into ethanol at similar concentrations to those used with formaldehyde fixed tissue.
A problem does arise when a staining method is required on sections that depend on the production of aldehydes, such as a PAS or a methenamine silver reduction for fungi etc. Following fixation, there are invariably some reactive free aldehyde groups present. These are sufficient in number to interfere with staining techniques which depend on aldehydes to recolor or reduce other solutions.
The resolution to this problem is to carry out an aldehyde block before staining any sections with one of these methods. The aniline-acetic block is suggested as a convenient method that does not take too long. Prior to the oxidation step in the method, block the pre-existing aldehyde by placing a dewaxed and hydrated section into a mixture of 9 parts glacial acetic acid and 1 part aniline for about 30 minutes at room temperature. Then wash well and continue with the oxidation step of the technique.
References
- Culling, C.F.A., (1999)
Handbook of histopathological and histochemical techniques, 3rd ed.
Butterworths, London. pp. 38 – 39 - Kiernan. J.A., (1999)
Histological and histochemical methods: Theory and practice, 3rd ed.
Butterworth Heinemann, Oxford, UK. pp. 23 – 24
