Formaldehyde Terminology

In concert with the very common use of formaldehyde in histology over many decades some terminology has developed along with it. Unfortunately, this terminology was not rigidly defined in the early years of its use, and today we have a situation where the same terms may be used to mean slightly different things. This is especially the case where different areas of histological applications are involved: diagnostic versus research, university anatomists versus drug testing, and so on. Despite this, there has been a consensus develop as to how the different terms should be applied, although it is still not rigidly enforced by scientific journal editors.

Formaldehyde

Formaldehyde is a gas. It has the formula HCHO or CH₂O and other variations of that. The word formaldehyde is always reserved for the gas itself. It is never applied to solutions of the gas. For that reason, the only time formaldehyde and fixation should be used together is when formaldehyde vapor fixation takes place, i.e. when smears are placed in a closed contained with a source of formaldehyde fumes and the gas itself fixes the cells.

Formalin

Solutions of formaldehyde gas dissolved in water are called formalin. This name is used regardless of how much of the gas is dissolved, and it contains no information nor inference about the concentration.

When the expression formalin or formalin fixed is encountered, it should not be interpreted to mean 10% formalin, 10% formal saline, 10% neutral buffered formalin nor any other formalin variant. It just means that fixation was done with a solution which included formalin. The solution may, or may not, have included some other agents capable of fixation in their own right. The context may give more information, but an assumption should not be made. Authors should be clear as to what is meant and give the full descriptive text.

To specify the concentration of the formaldehyde gas in the water a convention is applied based on the saturated solution being called 100% formalin rather than giving the concentration of formaldehyde directly. Formaldehyde dissolves into water to 37% w/v. This solution is variously called 100% formalin, strong formalin, concentrated formalin, or saturated formalin. The first expression, 100% formalin, is clearest, but the others will be understood and are frequently used.

When diluting formalin solutions, the final concentration is expressed based on the 100% formalin reference. In other words 10% formalin is a 1:10 dilution of 100% formalin, i.e. it is 10 mL of 100% formalin plus 90 mL water, or equivalent volumes. The terminology 10% formalin is most commonly used. However, the expression 4% formaldehyde is sometimes encountered. This expression is based on the actual formaldehyde content of the solution. It is less common than 10 formalin, but the terms are equivalent and refer to the same thing.

There was considerable confusion between the terms formaldehyde and formalin in the past. Before a consensus arose as to how the terminology should be used, 10% formalin, 10% formaldehyde, 4% formalin and 4% formaldehyde could all refer to the same thing – a 1:10 dilution of the concentrated formaldehyde gas solution in water. The terms formalin and formaldehyde were often used interchangeably and this caused considerable confusion when it was not made clear what was meant.

Paraformaldehyde

Formaldehyde can polymerize. The polymeric form is called paraformaldehyde (para-formaldehyde, p-formaldehyde) and is usually obtained as a fine, white powder. When heated it depolymerizes back to formaldehyde. If 4 grams of paraformaldehyde are added to 100 mL of water and heated it depolymerizes and the formaldehyde immediately dissolves making a formalin solution. This is often referred to as 4% paraformaldehyde or 4% PFA. However, once it has been heated long enough to depolymerize all the paraformaldehyde back to formaldehyde, the resulting solution is simply 10% formalin (or 4% aqueous formaldehyde) and no paraformaldehyde remains.

The difference between such a solution and a 1:10 dilution of 100% formalin is in the presence of a small amount of methanol. This alcohol is added to 100% formalin solutions at about 10% concentration as it can inhibit polymerization during storage, so all formalin solutions made by diluting 100% formalin will contain some methanol. If the methanol is likely to interfere with a histological reaction or process, 10% formalin may be made by adding 4 grams paraformaldehyde to some water containing a base, and heating to above 60°C until it depolymerizes. This solution of 10% formalin from paraformaldehyde should be identified as methanol free 10% formalin or methanol free 4% formaldehyde. The expressions 4% PFA and 4% paraformaldehyde are misleading.

Formol or Formal

The term formalin has often been shortened to formol in compound expressions and names, as in formol-saline or formol-alcohol. This arose many years ago and has become entrenched in the literature and common practice. Unfortunately the term may give a false impression. The ending -ol signifies a hydroxyl as in phenol or ethanol, and that does not apply to either formalin or formaldehyde. Formaldehyde is an aldehyde, so the short form formal would be more appropriate and is often used instead. Unfortunately, formal is used in chemistry to refer to dimethoxymethane (methylal). Nevertheless, formal is the expression used on this site, but both formol and formal are likely to be encountered in the literature.