Formalin Fixation: How It Works and Why It’s Used

Formalin fixation is a method used in biology and medicine to preserve tissues by stopping decay and maintaining cellular structure. This process allows for detailed examination and long-term storage of biological samples. By preventing decomposition, fixation ensures that tissue samples remain stable for diagnostic and research applications that require the analysis of cellular and tissue architecture.

What is Formalin?

Formalin is the most common fixative used in laboratories, consisting of an aqueous solution of formaldehyde gas. Commercial formalin contains 37-40% formaldehyde in water, with 10-15% methanol added as a stabilizer to prevent polymerization. This process involves formaldehyde molecules linking together and settling out of the solution.

The standard working solution is 10% neutral buffered formalin (NBF), a dilution of the commercial stock that results in a 3.7-4% formaldehyde solution. It is buffered to a neutral pH of 6.8 to 7.4 to prevent the formation of formic acid, an acidic byproduct that can damage tissue. The small size of the formaldehyde molecule allows it to penetrate tissue quickly, while its reactive nature stabilizes cellular components.

How Formalin Preserves Tissues

Formalin preserves tissue by forming chemical bonds, or cross-links, with its components, primarily proteins. When formalin penetrates a sample, formaldehyde molecules react with various parts inside and outside the cells. This reaction creates a network of stable chemical bridges that lock the cellular structures in place.

This cross-linking occurs most readily with proteins, as formaldehyde reacts with amino acids like lysine. These reactions link protein chains together, creating a stable mesh that gives the tissue mechanical strength. This process halts both autolysis (self-digestion by cellular enzymes) and putrefaction (decomposition by bacteria).

Formaldehyde also reacts with nucleic acids like DNA and RNA, as well as some fats, further stabilizing the entire cellular environment. The formation of these cross-links hardens the tissue, changing its consistency from soft to firm. While formaldehyde penetrates tissue quickly, the cross-linking process takes several hours, which is why specimens are often left in formalin to ensure complete fixation.

Common Uses of Formalin Fixation

One of the most frequent applications of formalin fixation is in diagnostic pathology. When a patient has a biopsy or tissue removed during surgery, the specimen is placed in formalin. This preserves the tissue architecture, allowing pathologists to slice, stain, and examine it under a microscope to diagnose diseases like cancer.

In scientific research, formalin is used to preserve tissues from experimental models. This allows researchers to investigate cellular structures to understand biological processes or disease mechanisms. Fixed tissues can be stored for long periods, creating archives for retrospective studies on disease progression or treatment effects.

Formalin is also used in anatomy and education to preserve whole organs or entire organisms for study in medical schools and for display in museums. The hardening effect of fixation maintains the three-dimensional structure of these specimens. This provides students and the public with a detailed view of biological structures.

Safety and Handling of Formalin

Working with formalin requires strict safety protocols, as formaldehyde is a hazardous chemical and a known human carcinogen. Exposure can pose health risks, as its vapors irritate the eyes, nose, and respiratory tract. Direct contact with the liquid can cause skin irritation or allergic reactions, so handling is restricted to controlled laboratory or medical environments.

Personnel must use personal protective equipment (PPE), including gloves, safety goggles or face shields, and lab coats to prevent skin and eye contact. To minimize inhalation, formalin is handled in well-ventilated areas or inside a chemical fume hood, which pulls vapors away from the user.

Regulatory bodies like the U.S. Occupational Safety and Health Administration (OSHA) have established permissible exposure limits (PELs) for formaldehyde in the air. Laboratories must also follow strict guidelines for the storage and disposal of formalin waste to protect both personnel and the environment.