Fixation: History and Speed

By: Neil Macintyre

The art and science of preserving a once living object as close as possible to its natural state dates back to the earliest times, with Hippocrates discussing the merits of mercury and alcohol as far back as 400 B.C. Alcohol, (referred to at the time as “spirits of wine”) was probably the most common, being used as a preservative by early explorers when bringing back specimens on long sea voyages.

However, it wasn’t really until the advances in microscope optics that the study of tissues and the use of fixatives really took off with detailed systematic studies of fixatives beginning in the late 19th century. By the early 20th century, there were so many fixative formulae that it was becoming difficult to keep up and a halt was called to the introduction of new formulae, many of which were simply minor adjustments to earlier ones.

The ideal aims of fixation however, are never fulfilled. Fixation remains a compromise between the desire to preserve the cellular content in as lifelike a representation of the living state as possible and the constraints of the technique itself.

What do we mean by fixation and what is the difference between fixation and preservation?

A fixative actually "fixes" a specimen by preventing protein breakdown and stabilizing the proteins within the tissues in a manner which retains a semblance of their life like state, thus allowing detailed morphological studies to be carried out. In contrast to a fixative, a preservative is a solution in which the tissue can be stored and maintained without further degradation, for long periods of time. The most common preservative used being 70% ethanol.

The History of Formalin

In 1892 a German firm approached an industrial chemist named Blum for formalin to be tested as a possible antiseptic agent. He noticed that while handling the formalin, the skin on his fingertips became hardened, a very similar effect to the one alcohol had on tissue during fixation. Acting on this, Blum proceeded to test formalin as a potential histological fixative with excellent results.

Many workers are still confused over the meaning of the terms formaldehyde and formalin. What do they really mean when they say a 4% solution or a 10% solution?

Formaldehyde is a gas which when bubbled through water is soluble up to approximately 40%.

Most commercial concentrated solutions are sold at a concentration of between 37% - 39% but for all practical purposes are considered as 40% solutions. The standard routine fixative mixture is referred to as 10% formalin which contains approximately 4% formaldehdye. Due to the formation of larger insoluble polymers in the solution it is impossible to gauge the exact amount of formaldehyde present at any one time.

How Fast are Fixatives?

The original experiments of Medawar in 1941 used plasma clots and proved that fixatives obey the laws of diffusion. That is: the depth penetrated was proportional to the square root of time. Following his experiments he set out a coefficient of diffusibility for each fixative. This is known as the Medawar constant or K value. The higher the K value the faster the rate of penetration.

Baker in 1958 carried out a similar experiment using gelatin/albumin gel to more closely mimic solid tissue and as you would expect he determined slightly lower K values for the various fixatives.

This has important implications for fixing tissues. The time that an individual fixative takes to get to the centre of any given sample will vary according to the thickness. This is of particular importance when dealing with large tissue samples as the centre may be fixed in a manner quite differently to that of the extremities.

Fixation Rate of Formalin

Formalin has a high K rate, which means it is one of the fastest penetrating fixatives, so why is formalin considered a slow fixative compared to the others when it clearly has one of the fastest penetration rates?

Methylene glycol is a small molecule, making it easy for it quickly penetrate the tissue, the fixing process however, takes much longer.

When formaldehyde is dissolved in water, methylene glycol is formed and a small amount of dissociated formaldehyde. An equilibrium exists between methylene glycol and formaldehyde. This lies firmly in favour of the methylene glycol. As the small amount of formaldehyde is used up another small amount of formaldehyde dissociates from the methylene glycol. This is repeated throughout the tissue as the methylene glycol penetrates into the tissue.

Carbon labelled formaldehyde studies have indicated that complete equilibrium takes 24 hrs at room temperature or 18 hrs at 37 degrees C. This process can be sped up by the use of microwave and ultrasound technologies.

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