Picric Acid

Please read this explanation about safe working with picric acid and cleanup of spills.

Wikipedia has an article about picric acid.

PropertyData
ChemicalPicric acid
Synonyms2,4,6-trinitrophenol
FormulaC6H3N3O7
StateYellow crystals, wet
ConcentrationOften saturated
Fixation timeSeveral hours
AdditiveYes
CoagulantYes
HardensNo
BrittleNo
Acid dyesImproves
Basic dyesReduced
Aftertreatment70% ethanol

Picric acid is used as both a dye and as a fixing agent, and is a valued reagent in both cases.

When used as a fixing agent it leaves tissues in a condition that makes them particularly receptive to staining with acid dyes, and methods such as Masson's trichrome stain quite brilliantly as a result. This is so distinctive that many reference texts include in their instructions for this and other trichrome staining methods, a recommendation that sections from formalin fixed tissues be refixed in a picric acid fixative in order to improve the intensity of staining. The solution used is often Bouin's fluid, although a simple saturated aqueous solution of picric acid is as effective and does not have the complication of giving off formalin fumes.

This improvement in staining does not extend to basic dyes, unfortunately, and results from using them may be disappointing. It should be noted that since picric acid is a fairly strong acid, it can hydrolyse DNA if tissues are left too long, and nuclear staining may be reduced. In some cases, a Feulgen stain for DNA may be positive without further hydrolysis in hydrochloric acid. For that reason it should be avoided if nuclei are of particular interest as that method is unreliable on picric acid fixed material.

It leaves tissues in a reasonably soft state and sectioning is not usually impaired.

How it fixes
Picric acid is both an additive and precipitant fixing agent. It is believed that it forms picrates with basic amino acids and in the process causes the proteins to precipitate. It is suggested that this is the reason that acid dye staining is so effective following picric acid fixation: that basic proteins, to which acid dyes would attach, are well preserved, but acid proteins are not adequately fixed and may be removed. Nuclear protein is also precipitated, but the DNA itself remains water soluble. This means that nuclear structure may be shown with acid dyes rather than basic dyes, and that DNA methods are unreliable.

There is no direct reaction with carbohydrates, although the protein component of carbohydrate-protein complexes may be fixed. Glycogen is unaffected directly but is either physically trapped within precipitated protein or, if it is bound to protein, is preserved along with the protein as it is fixed. This preservation of glycogen is striking enough that picric acid fixation is recommended when glycogen is of special interest, particularly if it is used in conjunction with high concentration of ethanol.

Lipids are not affected, but the protein component of lipid-protein complexes may be fixed.

Morphology
Tissues tend to shrink. It is often stated that the glomeruli in mammalian kidney are so severly affected that picric acid fixation should never be employed for them. This may be somewhat overstated, and comparisons should be made if this is a concern. Formalin and other fixatives also causes shrinkage of glomeruli, yet are considered to be quite adequate for use. Compare the three photographs of a glomerulus below.

glomerulus fixed with Bouin's fluid
Bouin's fluid
glomerulus fixed with formal sublimate
Formal sublimate
glomerulus fixed with Zenker's fluid
Zenker's fluid

Time
Picric acid penetrates slowly but still a 3mm to 5 mm thick piece of tissue will be fixed after about 8 hours, and certainly thoroughly fixed overnight. Tissues should not be left longer than necessary in the fixative due to the likelihood of acid hydrolysis of DNA.

Simple solution
Except when sometimes used to secondary fix sections before trichrome staining, picric acid is never used alone. It is commonly mixed with ethanol, formalin and acetic acid and is considered compatible with most other fixing agents.

Aftertreatment
Conflicting advice is given for the aftertreatment of picric acid fixed tissues. This has arisen due to the assumption, often repeated, that picric acid forms protein picrates during fixation and that these protein picrates are water soluble. The advice given as a consequence is that picric acid fixed tissues should never be washed in water after fixation as it will cause some of the material to be washed out. This is quite overstated, and washing picric acid fixed tissues in running tap water appears to have little effect on the final morphology. I encourage all technologists to test this out by fixing a 3 mm thick piece of mammalian kidney in Bouin's fluid overnight, then washing in running tap water for three days before processing and staining. Compare to a similarly treated piece of tissue fixed with 10% NBF.

Following picric acid fixation the tissue will be bright yellow from residual picric acid and many technologists want to remove this as they consider it to be a nuisance during processing, discolouring the dehydrating ethanols and clearing fluids. As it dissolves more efficiently in ethanol than water, it is more efficient to remove this discolouration with ethanols, so placing the tissues directly into 70% ethanol removes the yellow colour more effectively than water washing.

Rather than wash extensively, some technologists briefly wash off the excess picric acid, then place the tissue in a dilute carbonate solution to more quickly eliminate the yellow discolouration by converting it to sodium or lithium picrate which quickly dissolves.

Other technologists do not consider the yellow colour to be a problem and simply soak the tissue in 70% ethanol until it is time for routine processing to begin, then process normally. The tissue remains yellow and appears yellow in the block, thereby indicating the fixative used. If it interferes with staining, any residual yellow colour may be removed by treating the sections with carbonate for a few minutes prior to staining. In most cases it causes no problems, being replaced by the first acid dye applied.

 

Reference
Baker, John R., (1958)
Principles of biological microtechnique
Methuen, London, UK.


 

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