Osmium Tetroxide

Please read this explanation about safe working with osmium tetroxide and cleanup of spills.

Wikipedia has an article about osmium tetroxide.

ChemicalOsmium tetroxide
SynonymsOsmic acid
StatePale yellow crystals
ConcentrationUp to 1%
Fixation timeA few hours
Acid dyesPoor
Basic dyesGood
AftertreatmentWash well

Osmium tetroxide has an important, if limited, part in fixation for light microscopy as it is the only fixing agent known which completely and easily fixes fats. Although dichromate can do something similar with regards to lipid containing membranes and lipoproteins, the treatment necessary to accomplish it takes several days, whereas osmium tetroxide can accomplish the same thing much more effectively within a few hours.

Unfortunately osmium tetroxide has two very serious drawbacks. It is quite toxic and it is very expensive. For those reasons it is usual to use it only in circumstances in which other fixing agents are not suitable. Usually this is the fixation of fat for paraffin sectioning, as it is the only fixing agent that allows this to be done, although it also has value for fixation of lipid membranes and structures such as mitochondria.

Note that osmium tetroxide must always be used in a fume hood and care must be taken to ensure that the fumes are not inhaled. It causes a very distinctive, piercing and unpleasant headache. It can also dissolve in body fluids such as are found in the eyes and exposed mucous membranes of the nose and mouth. Such dissolved osmium tetroxide can then fix the underlying tissues and may blacken it in the process.

It is usually purchased sealed in small glass vials, not just because of its expense but also because it emits toxic fumes. It is customary to make a solution at an appropriate concentration, then to add the required volume of that to any other reagents to be combined with it in the fixative. Unused solution may be stored in a capped container, but is likely to deteriorate quite soon. Osmium tetroxide solutions are susceptible to contaminants and easily reduced to its other oxides. It should be stored in the dark as it can be reduced by light. Any solution which developes black deposits should be discarded and a fresh solution made. For that reason, it is advised to purchase vials containing an amount of chemical close to that required for a single use, if possible.

Osmium tetroxide penetrates slowly, and the outer fixed rim resists further penetration. In other words, as the outer rim is fixed the penetration rate declines. For that reason small, thin pieces are required.

How it fixes
According to Baker it likely links proteins through tryptophane and histidine side chains, and possibly blocks amino groups since acid dye staining is reduced. There does not appear to be any effect on DNA. Carbohydrates are largely unaffected.

In light microscopy osmium tetroxide is used largely for its ability to fix fats, including triglycerides. This is often accomplished by a form of secondary fixation known as post-osmication. During treatment and processing triglycerides are blackened, and the blackened area is seen in paraffin sections after treatment with strong fat solvents, which would normally remove them.

Osmium tetroxide reacts with the double bonds of unsaturated lipids, rendering them resistant to extraction by many solvents. Since lipids are often present as mixtures which include unsaturated compounds, it is to be expected that globules of triglycerides be blackened. In addition, saturated lipids can dissolve osmium tetroxide which can then be reduced to a black oxide by ethanol during processing. This material remains when the fat itself is dissolved away. A black globule in a paraffin section can be assumed to be a fat, but cannot be identified as saturated or unsaturated since both will be black.

This subject is not as clear cut as the paragraph above implies, so those interested in this subject should read Baker's and Kiernan's comments and refer to the bibliographies they give.

This is extremely good, particularly at the cytological level. Most structures are preserved extremely well. It is particularly recommended if mitochondria are the target contituent. Although it preserves cell morphology very well, subsequent paraffin processing may undo much of that.

It is fairly rapid, a few hours perhaps on suitably thin pieces. The progressive resistance to penetration means that thick pieces may not be fixed for some time.

Simple solution
It is not usually used alone, and is compatible with most other fixing agents with two notable exceptions. Formaldehyde slowly reduces it, and ethanol rapidly reduces it. Osmium tetroxide should never be used in mixtures with ethanol.

Tissues must be washed very well with running tap water (for hours) to ensure removal of all unbound osmium tetroxide. Subsequent treatment with ethanol will reduce any free osmium tetroxide to black deposits and these interfere with interpretation.


Baker, John R., (1958)
Principles of biological microtechnique
Methuen, London, UK.

Kiernan, J.A., (1999)
Histological and histochemical methods, theory and practice. Ed. 3,
Butterworth, Heinemann, Oxford, UK.



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