10% Formal Alcohol
(4% Formaldehyde Alcohol)

10% Formal alcohol
Strong formalin 100 mL
Ethanol 95% as required
Tap water to make 1 Litre
Calcium acetate 0.5 g (optional)

Formal alcohol varies considerably in its alcohol content, so the fixation characteristics vary considerably as well. With lower concentrations of ethanol, in the 20% range, there is very little effect from it other than to depress the freezing point. This makes the fixative valuable in parts of the world where specimens have to be transported in unheated vehicles during winter.

An ethanol content of approximately 20% – 30% will depress the freezing point sufficiently so that the solution does not freeze solid until the temperature drops below -20°C to -30°C, although it may turn to a slush. As long as the tissue does not freeze solid the formation of ice crystal holes is avoided. These can be seen in the final sections when they have formed, appearing as thin irregular rectangular spaces, mostly in cellular tissues. If formal alcohol is used for this purpose, it is recommended that the tissues be fixed for 24 hours minimum before being transported, partly as a means of protecting the tissue and partly because cold temperatures slow down fixation.

If higher concentrations of ethanol in the 70% to 95% range are used there may be noticeable fixation effects. In the case where the formal alcohol is composed of strong formalin and 95% ethanol only, there will be distinct characteristics of ethanol fixation. Generally, this means tissues that are harder, more brittle and perhaps more shrunken that those fixed in formalin alone. As the concentration of ethanol is decreased these effects will lessen until there is little discenible difference from plain formalin fixation at the 20% range.

Variants of formal alcohol with the lower ethanol concentrations have some use for transporting specimens, but otherwise the fixative is not that popular. Erythrocytes will not usually be damaged. The purpose of the calcium acetate is to make the solution slightly alkaline which should inhibit the formation of formalin pigment if it is added.

This fixative should be applied overnight, and for high ethanol concentration variants this will be sufficient. Lower ethanol concentration variants may require longer.

There is no special aftertreatment. Tissues should be transferred to 70% ethanol or 95% ethanol depending on the ethanol content of the fixative. It is pointless to transfer tissues to ethanol of a lower concentration than the fixative itself contains.

Secondary fixation
Other fixatives may be applied after formalin fixation, and some of their characteristics will be obtained. It must be realised that secondary fixation in any fixative will not give the same results as would have been obtained if the secondary fixative had been applied to fresh tissue.


Culling, C.F.A.,
Handbook of Histopathological and Histochemical Techniques, Ed. 3,
Butterworths, London, UK.

Bancroft, J.D. and Stevens, A.,
Theory and Practice of Histological Techniques, Ed. 2,
Churchill Livingstone, Edinburgh,, UK.



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