Ethanol

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

Wikipedia has an article about ethanol and another about denatured alcohol.

PropertyData
ChemicalEthanol
SynonymsAlcohol
Methylated spirits
Denatured alcohol
FormulaC2H5OH
StateColourless fluid
ConcentrationAbsolute
Fixation timeSeveral hours
AdditiveNo
CoagulantYes
HardensYes
Acid dyesNeutral
Basic dyesNeutral
AftertreatmentNone

Ethanol, in the form of brandy, is likely one of the oldest fixatives. In the earliest days of microscopy it was common to preserve materials in brandy for later investigation.

Ethanol used to be graded according to its "degrees proof". The concept of "proof" spirits is an old one and comes from the practice of burning rum soaked gunpowder to prove the rum was strong enough. A concentration of 57.15% ethanol is needed for it to burn and in Britain this was designated as 100° proof spirits. In the United States 100 proof spirits was specified as 50% ethanol. As a consequence the proof designations for 95% and absolute ethanols differed depending on which system was used. At present, throughout the world, alcohol concentration is expressed as a percentage. A proof designation, if needed, is now twice the alcohol concentration (the US system).

By volume96% ethanol100% ethanol
Britain168° proof175° proof
USA192 proof200 proof

How it fixes
Ethanol is a non-additive precipitant fixative. It fixes proteins by dehydration and precipitation, the degree to which this is done being dependant on the amount of water present and the solubility of the materials in the mixture. Fixatives containing ethanol are usually, but not exclusively, water free or contain only minor amounts of water so that precipitation is a major effect.

Carbohydrates
These are not specifically fixed, although carbohydrates attached to proteins will be precipitated along with the protein. Glycogen is precipitated and trapped within a surrounding mass of precipitated protein, so it is well preserved during processing, it being unusual to return to water after fixation in an alcoholic fixative. High concentration ethanol fixatives are often recommended for preserving glycogen.

Lipids
Lipids are not preserved and some may be dissolved. Many lipids, however, do not dissolve in ethanol and may remain unchanged within the tissue.

Morphology
Nuclear preservation is satisfactory, but cytoplasmic preservation is only fair as some constituents are destroyed. There may be considerable shrinkage overall.

Denatured alcohol
Denatured alcohol, or methylated spirits, can replace ethanol in fixatives, although since the denaturing agent can vary trials should be conducted before doing so. The author used denatured alcohol for most purposes in a histology laboratory in the UK for several years with no discernable difference from results obtained using absolute ethanol in other laboratories.

Time
Several hours for a 3 mm thick piece of tissue should be satisfactory. Thinner tissues such as fine needle biopsies will be fixed within an hour or two, bearing in mind that fixation continues during dehydration.

Simple solution
Ethanol is rarely used alone as a fixative because of its shrinkage and hardening effects. An exception is when inadequate formalin fixation is used, and the tissue is transferred to ethanol for dehydration. Since the tissue is not properly fixed, or may be unfixed, the ethanol fixes as it dehydrates with all the hardening and shrinkage obtained when it is used alone. This is often called parched earth artifact.

It is compatible with formaldehyde, acetic acid, mercuric chloride, picric acid, methanol, acetone and other agents. Strong oxidising agents such as chromium trioxide should be used with caution as they may oxidise ethanol to acetaldehyde and acetic acid.

Aftertreatment
No particular aftertreatment is needed. It is unusual to transfer to water after treating with ethanol, and tissues are more appropriately transferred to a clearing agent directly or to a higher concentration of ethanol if the fixative used was a mixture.

 

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

 


 

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