Some Clarity on Aldehydes Used as Biological Fixatives by Courtney Drayer

Aldehydes are carbonyl groups (C = O) that are bonded to either 2 hydrogen atoms (formaldehyde) or a hydrogen and an alkyl or phenol group (higher aldehydes). Aldehydes are known for their pungent smells but also include some culinary favorites such as vanilla, cinnamon, and spearmint oil. In the biological sciences, certain aldehyde solutions are used to fix tissue samples for analysis. Chemical fixatives preserve biological specimens by halting enzymatic degradation, preventing microbial colonization, and increasing cellular strength. Common aldehydes used in chemical fixation include formaldehyde and gluteraldehyde.

There are many misconceptions surrounding the definitions of these chemicals. Formaldehyde (HCOH) is a gas that hydrates when dissolved in water forming methylene hydrate (HOCH2OH). The maximum solubility of formaldehyde in water is 37% by weight and 40% by volume. Solutions that contain the maximum amount of formaldehyde dissolved in water are defined as 100% formalin. A 10% formalin solution is 10% of a 37-40% formaldehyde solution or 3.7-4% formaldehyde solution.

As formalin solutions age, the methylene hydrate can polymerize. Precipitation of these polymers can form a flammable white powder, paraformaldehyde. Likewise, the dissolution of 4g of paraformaldehyde into 100ml of water will form a 10% formalin solution. Many researchers add methanol (usually 10%) to their formalin solutions to stabilize the solution and prevent polymerization. Additionally, monobasic sodium phosphate monohydrate and dibasic sodium phosphate anhydrate can be added to formalin solutions to buffer the low pH caused mainly by the oxidation of formaldehyde to formic acid.

Gluteraldehyde (OHCCH2CH2CH2CHO) is also a fixative used for preservation in preparations for electron microscopy and other applications. Gluteraldehyde solutions fix samples more quickly than formalin solutions due to the dialdehyde structure of the molecule, but its larger molecular structure causes penetration of the fixative to slow.

While formalin solutions without significant alcohol, and thus higher flash points, are not regulated by the EPA, the characteristic that make formalin a useful fixative, namely the ability to kill the useful  bacteria used in sewage treatment plants make it unsuitable for drain disposal in sewer systems. Other reasons for the collection of formalin are the toxic and carcinogenic effects caused by the off-gassing of formaldehyde from the solution. This is the rationale for OSHA regulations that apply to regular use of formalin, including the requirement that all users be properly trained.  See  Glutaraldehyde is not regulated in this manner but the same operational mandates apply due to its acute health effects. Fortunately there are many aldehyde-free fixation alternatives available on the market today. EH&S can provide consultation in selecting appropriate substitutes for an array of toxic substances.  Additional information on fixative alternatives can be found by searching

Health and Safety Manual is Online by Kevin McGhee

The newest edition of the Columbia University Health and Safety Manual for laboratories has been posted on the EH&S website at  EH&S chose to publish this edition online rather than in print to save paper and to allow for the flexibility of referring users to abundant online resources.  The Manual serves as a central reference point for Columbia University laboratory policies and procedures, and should be primary safety resource for those working in University laboratories, whether new or experienced.


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