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Fixing Agent



Methylated spirits
Denatured alcohol

Chemical Formula:


State: Colorless fluid
Concentration: Absolute
Fixation Time: Several hours
Aftertreatment: None
Acid Dyes: Neutral
Basic Dyes: Neutral
Additive: No
Coagulant: Yes
Hardens: Yes

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

Fortunately, ethanol is not generally considered to be a dangerous material. It can cause harm, even death, but the circumstances needed for that are not usually met and harm from ethanol is not a major concern in histotechnology.

Much ethanol is manufactured by distillation from fermentation of sugar by yeast, although it can be manufactured from other sources, such as oil, in chemical processes. Ethanol made industrially from fermentation should not be considered comparable to that made in small batches for human consumption as whiskey, vodka and so on. The scale is much larger, and the likelihood of some non-potable contamination is of concern. These aspects are strictly controlled in authorized distilleries for the production of drinking ethanol and the products are potable. This may not be true in the case of ethanol produced industrially for industrial purposes.

It is not unknown for laboratory workers to dilute some ethanol for consumption on the basis that ethanol is just ethanol and when diluted with water it is the same as vodka. It is not the same, and some words of caution are needed.

The process is much the same for potable or industrial production except for the scale. A sugar source is fermented with yeast, and the low-concentration ethanol is then distilled. It may be distilled more than once to get the maximum concentration of ethanol possible. From these solutions of water and ethanol only, ethanol of about 95% may be obtained. To get higher concentrations of ethanol, other materials may be added, and this may be of major concern if the resulting ethanol is consumed. The other material used is usually benzene, and from a mixture of ethanol, water and benzene distillation can produce 100% ethanol, or as near to it as makes no difference. It does mean, however, that 100% ethanol contains trace amounts of benzene, a known carcinogen. This kind of ethanol should never be consumed.

Ethanol has been controlled by many governments throughout the world, some ban its sale and consumption completely while others tax it to varying degrees. So those having a legitimate need for high-concentration ethanol may use it without paying tax. Some governments make it available without tax if it is adulterated to make it undrinkable. A common means of adulteration is to add methanol, and this mixture has given rise to the name “methylated spirits”. Today, materials other than methanol may be used and it is more properly named “denatured alcohol”.

Denatured alcohol is usually relatively easy to obtain, and is suitable for dehydration during paraffin processing. Whether it is suitable for other purposes should be determined by trial in a specific case, but it often is. It is usually possible to obtain unadulterated ethanols but doing so comes with some inconvenience, as strict record-keeping is usually involved.

A second concern with ethanol is its flammability. This is particularly so as modern automatic tissue processors often have heated chambers to improve dehydration and clearing. Firefighting materials should always be readily available for use when needed, although large fires should be left for professional firefighters.

Ethanol spills may be cleaned up by soaking into a cloth or paper towels, then washing out the cloth and wiping down the area with a wet cloth. Other than sufficiently large quantities of water being used, no further treatment is necessary.

The disposal of used ethanol may be problematic, although many jurisdictions permit it to be discharged into their wastewater systems. The amount may be reduced by redistilling if your jurisdiction permits that. Recovered ethanol may be used during the dehydration step in processing. Keep in mind that both waste and recovered ethanols may be contaminated with materials from the tissues dehydrated.


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).

Ethanol by VolumeBritish TerminologyAmerican Terminology
96% Ethanol168° proof192 proof
100% Ethanol175° 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 dependent 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.

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.


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


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


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 oxidizing agents such as chromium trioxide should be used with caution as they may oxidize ethanol to acetaldehyde and acetic acid.


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.


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