Since Blum’s proposal of formalin as a useful general biological fixative in 1893 it has become an unsurpassed standard. For 119 years zoologists, botanists, histologists, and pathologists have used formalin to preserve their materials for a detailed anatomical, histological or cytological study. To pathologists it represents until now the only admissible standard, in spite of the denounced toxicity of the product. Some time ago diluted solutions of formalin were proposed to anesthetize Monogenea, and in a recent article I proposed more diluted concentrations as relaxants for many micro invertebrate groups.

 In spite of this being stated in the article, I received some inquiries about how I expressed the concentrations, whether as formalin or as formaldehyde. The answer must be unambiguous and I think that it could be interesting to share it with other readers of Micscape. I want to start with a summary of what is intended by the two words.

Formaldehyde (French, formaldéhyde; Spanish, formaldehido; Italian, formaldeide, German, formaldehyd; Portuguese, formaldeído) is methanal, the aldehyde produced by partial oxidation of methanol(methyl alcohol). It is a gas with a molecular formula of  HCHO, that with further oxidation produces formic acid, a very common acid produced as a defensive weapon by many different kinds of invertebrates. As a gas it is used in many chemical industries, but it is very difficult to apply to biological tasks, and it is utilized by dissolving it in water. The most used dilution is formalin.

Formalin (a.k.a. Formaline?) (French, formol, formaline; Spanish, formol, formalina; Italian, formalina; German, formalin; Portuguese, formalina) is an aqueous solution of formaldehyde. The typical concentration is 37 - 40 %. That is, it is expected that a commercial formalin has 370 - 400 g of formaldehyde in each 1000 g of commercial solution. The intended dilution is w/w, not vol/vol. As formaldehyde is self-reactive, and continues to oxidize in aqueous solution producing formic acid, and in older solutions may even form a precipitate of paraformaldehyde (a solid polymerized formaldehyde), it is common to add to formalin 10% or so of methanol as a stabilizer. Formalin solutions thus really contain formaldehyde, paraformaldehyde, formic acid, and methanol.

 So formalin has a target composition but only a detailed analysis could tell us which is the real concentration of any component in a given batch, of a given age. Moreover, the variations are in a certain limited range, and biologists for more than 100 years were confident that by using some further dilutions of formalin, they can achieve an efficient fixation of the gross morphological, histological, and even cytological structure of the materials.

 The most common formulation is a 10% v/v formalin dilution in water. (10 ml of formalin, 90 ml of distilled water). As the formalin is almost certainly acidic, and this could impair its performance, pathologists use as their standard fixative NBF, Neutral Buffered Formalin, by adding to the 10% solution some buffers (monobasic sodium phosphate, and dibasic sodium phosphate for a pH of 7.0 - 7.2, or sodium borate (borax) for a pH of about 7.7) that balance the changes that could be occurring and stabilizes the behavior of the fixative.

 An infinite number of formulae use formalin as one of its components. Because of the very nature of the dilution, it is no more than an academic exercise, and never more than an gross approximation, to calculate the real concentration of the active ingredient formaldehyde.

 Although one site on the Web states that it is permissible to use the words formaldehyde and formalin, interchangeably, and many microscopists do it sometimes, you can see that this is not true. Both words have their own and very different meaning.

 There is even a web site on reptiles, which teaches that you must have a 10% concentration of formaldehyde in your fixative, so for your 10% fixative you must use 1 part formalin (of 40% concentration I suppose) and 3 parts water. This is against all the rules set up by old good zoologists, and is born surely by the partial knowledge of the subject (formalin is a 40% sol. of formaldehyde) and the false concept that the active ingredient (formaldehyde) is that which is involved in the formula. Certainly any one that applies this recipe will finish with a poor coiled snake that could only be straightened by breaking it into many pieces…and would expend 3 times more than necessary buying his formalin.

 When you read your books on microscopical techniques be aware that all of them speaking on formalin or on formaldehyde solution, intend to do the same thing: a dilution v/v of formalin in the dilutor, whichever it will be. And, also, that the best relaxant I found for some selected micro invertebrates was a dilution 1:8000 v/v of formalin (1 ml of formalin in 7999 ml of water).

Comments to the author, Walter Dioni, are welcomed.