10% Formalin & Formal Saline
(4% Formaldehyde & Formaldehyde Saline)

10% Formalin
Strong formalin 100 mL
Tap water 900 mL
10% Formal Saline
Strong formalin 100 mL
Tap water 900 mL
Sodium chloride 8.5 g

These are probably the simplest fixatives and have been in common use for many years, which should be thought of as a testament to their effectiveness. 10% formalin is not isotonic and there is a possiblilty that erythrocytes may be damaged by lysis. Formal saline is a little more effective than 10% formalin as it is isotonic and there is less likelihood that erythrocytes will be damaged. Both solutions are prone to the formation of formalin pigment, especially in bloody tissues, sometimes quite rapidly. This is due to formic acid being produced from the formaldehyde causing the pH of the solution to be lowered.

To neutralise the formic acid as it is produced, the fixative should be stored over a layer of marble chips or crushed oyster shells. Ensure that any gas produced has a means of escape. 100% formalin should never be stored in this way, as carbon dioxide will be produced from the reaction between formic acid and the carbonate, and if the contents are in a tightly sealed container the pressure could cause the container to break, sending concentrated formalin all over the floor.

Both are useful as fixatives for museum and photography specimens as they permit restoration of natural colour to the specimen.

Time
These fixatives should be applied overnight as a minimum, but fixation is not complete until applied for a few days, and a week or two is not too long. For thorough fixation the proteins in the tissue need to be crosslinked, but it is well known that simple formalin mixtures discolour tissues well before they crosslink the proteins. For that reason visual observation does not give any indication as to the degree of fixation, and discolouration should never be used as an indicator that it is complete.

It is essential that the time in fixative be noted, and sufficient time be allowed for the chemical reactions to occur. Time measured in a few hours is not adequate, and the lack of crosslinking in tissues treated for such a short time will not give adequate protection to the tissue from the fixation effects of dehydrating ethanols. Smaller pieces of tissue do not fix appreciably faster than larger pieces. Fine needle biopsies require the same length of time as 3 mm thick slices of solid organs.

On a practical basis in a diagnostic laboratory the slowness of simple formalin fixation is a distinct drawback. If the constraints of making a diagnosis are a major problem, then consideration should be given to increasing the temperature of the fixative solution. It must be emphasised that, although increasing the temperature of the fixative may increase the speed of fixation, it will cause a reduction in the quality of the final stained section and there may also be effects on some staining or immunohistochemical reactions. The temperature increase should be kept to a minimum consistent with accomplishing the goal of faster fixation, since heat itself is a means of fixation. If increased temperatures are used then formaldehyde fumes will be generated, more so as the temperature is increased, so fixation should be done in a fume chamber to protect personnel.

Complete organs are often placed in containers filled with a simple formalin variant. Although formalin penetrates fairly rapidly, it will not reach the centre of a large mass for some time and the delay may permit some autolysis in the middle of the tissue. Large organs should be described as soon as possible, then sliced into 1 cm or so slices to allow the fixative to gain access. Similarly, intestines should be opened and cleaned of any contents, then returned to a container large enough to permit the fixative to contact all the tissue surfaces, or pinned out on a board with rust free pins and placed into the fixative.

Tissues may be stored in this solution for long periods, but the fixative should be changed at least every six months.

Aftertreatment
There is no special aftertreatment.

Secondary fixation
Other fixatives may be applied after formalin fixation, and some of their characteristics will be obtained. It must be realised that secondary fixation in any fixative will not give the same results as would have been obtained if the secondary fixative had been applied to fresh tissue.

 

Reference
Drury, R.A.B. and Wallington, E.A.,
Carleton's Histological Technique, Ed. 5,
Oxford University Press, Oxford, UK.

Almost all texts for animal histology contain references to these fixatives.

 


 

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