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Potassium Dichromate

Fixing Agent

Potassium Dichromate

Synonyms:

Chemical Formula:

K2Cr2O7

State: Orange crystals & solution
Concentration: 1 - 3%
Fixation Time: Hours to weeks
Aftertreatment: Wash well
Acid Dyes: Improves
Basic Dyes: Reduced
Additive: Yes
Coagulant: No
Hardens: Moderately

Before You Begin

Please consult the following guide to safe working with this chemical fixing agent, including how to safely clean up spills.

Safety Note

We use two chromium compounds for fixation. They are chromium trioxide, which produces chromic acid when dissolved in water, and potassium dichromate. Potassium dichromate is reasonably safe to handle and easy to work with and presents few problems. It should be handled with the same care as any other chemical in a laboratory.

Chromium trioxide is another matter, as when it is dissolved in water, it forms a strong acid (chromic acid). This acid used to be used for cleaning glassware in the past, although the cleaning solution was made by adding sulphuric acid to potassium dichromate solutions. It was considered to be the ultimate cleaner, removing just about all known materials. If glassware could not be cleaned with chromic acid it was usually discarded as being beyond recovery.

Just a few granules of the dry chemical will discolor the skin, even when washed off immediately. This emphasizes how much caution needs to be taken to ensure that skin is not contaminated with dry chemical or solutions of this acid. It is a strong oxidizing agent and must be handled and stored with some care. The crystals give off corrosive fumes, and this has to be taken into account when storing the chemical. It should not be stored close to reducing agents, or with chemicals that are easily oxidized into other compounds.

If the dry chemical is spilled, the first step is to put on protective gloves and an apron for safety. The excess dry chemical may then be scooped into a plastic or glass container. Finally, the area should be washed down with a cloth or towels and copious amounts of water. Before doing so, ensure that all objects are removed so that no dry chemical is trapped under them. All contaminated material should be disposed of according to local regulations.

Description

Potassium dichromate fixes differently than does chromic acid, although both contain chromium. This is due to the differing pH at which they are used. A simple 1% potassium dichromate solution has a pH of just over 4. Chromic acid is much lower than that.

The pH difference results in different fixation characteristics. It should be noted that if potassium dichromate is used in solutions with a pH lower than 3.4, it will have the same characteristics as chromic acid, since the ions present will be the same.

To be explicit, if used at a pH below 3.4, potassium dichromate fixes the same as chromic acid. If used at a pH above 3.8, it fixes as described below.

How it Fixes

Proteins

Proteins are converted into a gel rather than precipitated. DNA is dissolved, but the histones of the nucleus are preserved and nuclear structure may be demonstrated with appropriate techniques.

Carohydrates

There is little to no effect on carbohydrates.

Lipids

Potassium dichromate is a very valuable fixing agent where lipids are concerned. This is best observed with the post chroming procedure, which makes many unsaturated lipids insoluble. During this procedure the chromium attaches to the lipid and may be used as a mordant for some dyes, including hematoxylin. It has to be stressed that this is a slow process, and post chroming may need to be applied for weeks to be fully effective. For faster demonstration of lipids in paraffin sections, post osmication should be used.

Morphology

Morphological preservation is reasonable when combined with other fixing agents, although it is rarely used alone as the primary fixative. Fixatives containing potassium dichromate may also contain an indifferent salt. This is the term given to a compound included in a fixative which appears to have no function. The presence of an indifferent salt in a simple aqueous solution of potassium dichromate improves the quality of the morphological preservation.

Time

It fixes relatively slowly, but mixtures containing it are usually effective overnight. Post chroming may take up to six weeks or so in some cases, although seven to ten days is more common.

Simple Solution

It is not usually used alone as a primary fixative, but applied as a 1 – 3% aqueous solution for post chroming.

Compatibility

Potassium dichromate is compatible with mercuric chloride and osmium tetroxide. It is incompatible with ethanol as it causes chromic oxide to be precipitated into the tissue. That is the reason for extended washing after fixation.

Formaldehyde is a reducing agent, while potassium dichromate is an oxidizing agent. The reaction between them is relatively slow and mixtures containing both are not unusual, i.e. Helly’s fluid. These solutions deteriorate so they should be prepared immediately before use and fixation in them should not be extended.

Too high an acid content in a mixture containing potassium dichromate may reduce the pH to below 3.4 and change the characteristics of the preservation. Acetic acid, picric acid and chromium trioxide can all reduce the pH to below 3.4 if used in other than very small amounts.

Aftertreatment

Wash the tissues very well with running tap water, preferably overnight. Transferring to ethanol without this being done is likely to cause precipitation of chromium compounds, which appear as dark deposits in the sections.

References

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