B5
Buffered 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.

Stock solution A   Stock solution B
Mercuric chloride, sat aqueous 6 g Strong formalin (40% aqueous) 10 mL
Distilled water 100 mL
Sodium acetate 1.25 g
Add the strong formalin to the mercuric chloride solution just before it is used.

B5 and formal sublimate are very similar fixatives and B5 is sometimes referred to as buffered formal sublimate, since their formulae and fixation effect are so close. There are, however, two significant differences between them. The first is that the B5 working solution is made from two stock solutions just before being used and has a working life of about 16-24 hours. One of the stock solutions contains sodium acetate to ensure that the fixative has an alkaline pH, but this also ensures that the solution deteriorates.

The second difference comes about as a consequence of the first, and is that the fixative may not be recycled as formal sublimate may. The solution must be discarded after use. This should be done in accord with local regulations and consideration for the environment. 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 this stability makes it a better choice for intermittent or occasional use.

As with formal sublimate, B5 gives improved nuclear and cytoplasmic staining when compared to formalin mixtures. 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 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.

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.

Due to the deterioration of the solution after 24 hours, the question of fixative tolerance is not an issue. Tissues should be removed before that time as the solution is not effective.

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

Secondary fixation
Although this fixative may be used as a secondary fixative following primary fixation in 10% formalin variants, it is not usually the fixative chosen. If it is used, 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
Bancroft, J. D. and Stevens, A.,
Theory and Practice of Histological Techniques, Ed. 2,
Churchill Livingstone, London, UK.

 


 

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