Formal Sublimate

Please read this article about the disposal of used mercuric chloride first.
Then read these comments about safe working with mercury compounds and cleanup of spills.

Formal Sublimate
Mercuric chloride, sat aqueous 900 mL
Strong formalin 100 mL

Due to the very toxic nature of the mercuric chloride and the fact that the solution is completely colourless, a red dye is often added to draw attention to its not being plain water. A drop or two of 1% aqueous acid fuchsin is satisfactory. This also has the advantage of colouring small tissue fragments.

For a long time this was the fixative of choice for improved nuclear and cytoplasmic staining. Nuclear structure is well defined, and deep staining is obtained with alum hematoxylin solutions and most basic dyes. The staining with acid dyes is enhanced. Trichrome stains on tissues fixed with this mixture do not require the sections to be further fixed in Bouin's fluid nor treated with other solutions for staining enhancement. Although the staining results obtained are not as intense as those following Bouin's solution, the demarcation of elements is sharp and the colours distinct.

Formal sublimate and Lillie's B5 have similar formulas, but B5 includes sodium acetate to make the solution slightly alkaline. Unfortunately, the sodium acetate makes B5 unstable and it deteriorates over a period of about 24 hours. In practice there is little, if any, discernible difference between tissues fixed in B5 and those fixed in formal sublimate. In addition, formal sublimate is stable almost indefinitely, and a solution in excess of 10 years old will fix as well as a freshly made one. Since formal sublimate and B5 can usually be substituted for each other, the stability of formal sublimate makes it a better choice for intermittent or occasional use.

A second and major advantage to formal sublimate is that it is quite practical and very simple to reuse. It is a very strong protein precipitant, and filtering through a Whatman No 1 paper or equivalent to remove any tissue fragments is all that is required. Solution may be reused numerous times in this fashion with no discernable difference in fixation. The author has used a stock of four litres of formal sublimate for the fixation of lymphomas over a period of several years in this fashion, with no noticeable degrading of fixation or staining. Since the solution is reused it allows for mercury fixation with considerably fewer problems dealing with disposal of an environmentally damaging chemical. It is much easier to collect and safely dispose of the small amounts of wash fluids, filter papers etc, that this form of recycling generates in comparison to disposal of the full volume of fixative.

Time
Very tiny pieces can be fixed in half an hour, although generally a minimum of about two hours is suggested. Some of the enhanced staining effects will be obtained after that time. Full fixation effects, including nuclear definition, are not obtained unless overnight fixation (16 hours) is used.

This solution is sometimes considered to be an intolerant fixative, that is, extended fixation increases brittleness and hardening with consequent difficulties in sectioning and adherance to the slide. These are somewhat overstated. Fixation of seven to ten days has been recommended by Lendrum and his associated for renal tissues when small fibrin deposits are to be demonstrated with his less common methods. Leaving tissues a few days for routine paraffin processing will not usually be harmful.

Aftertreatment
The high mercuric chloride content causes mercury pigment to form. This is conveniently removed with the iodine-thiosulphate sequence.

Secondary fixation
This is one of the most valuable fixatives for secondary fixation following primary fixation in 10% formalin variants. Overnight treatment of trimmed and fixed tissues will give most of the effects noted and enhance staining considerably.

Warning
This fixative will corrode metal. Metal cassettes, metal jar lids, metal forceps, metal rulers, metal countertops and all other metal objects must be avoided. This includes stainless steel, which may resist corrosion better than other metals but will still be affected. Dry paper towels are an effective barrier. Do not forget to dispose of them safely.

 

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

 


 

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