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Chromic Acid

Fixing Agent

Chromic Acid


Chemical Formula:

Dry: CrO3
Solution: H2Cr2O7

State: Dry: Dull brown-orange plates.
Solution: Orange solution
Concentration: 1 - 3%
Fixation Time: Several hours
Aftertreatment: Wash well
Acid Dyes: Improves
Basic Dyes: Reduced
Additive: Yes
Coagulant: Yes
Hardens: No

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.


Chromium trioxide, when dissolved in water, makes chromic acid. It is a strong oxidizing agent and the dry material must be stored remotely from reducing agents and easily oxidizable materials, particularly those that are flammable. The solution is quite acidic, a 1% solution having a pH of about 1.2.

How it Fixes


Fixation is likely by oxidation, with attachment of chromium or one of its compounds to the tissue. Proteins are coagulated, and so is nucleoprotein. DNA is precipitated and hydrolyzed, so that a Feulgen reaction may be positive without further treatment with hydrochloric acid.


Carbohydrates are oxidized. Glycogen may give a positive reaction with Schiff’s reagent without further oxidation. It should be remembered that chromic acid is used as the oxidant in Bauer’s method for glycogen and Grocott’s method for fungi, both of which depend on the production of aldehydes from complex carbohydrates by oxidation. It may cause some carbohydrates to become metachromatic when they are not usually so.


Lipids may be oxidized and may become insoluble in lipid solvents. In other words, they may be fixed, and chromium trioxide is one of a very few chemicals that can do this. Due to it being a strong oxidizing agent, this is easily overdone, however, and it is more usual to use potassium dichromate for the purpose.


Tissues are well preserved morphologically. Hardening is not excessive with minimal shrinkage.


Chromic acid is a slowly penetrating fixing agent. Once it reaches the tissue, fixation then requires some time. Thin pieces are recommended so as to reduce penetration time.

Simple Solution

Chromic acid is not usually used alone. Since it is a strong oxidizing agent it would appear to make sense that it not be combined with reducing agents, but this is sometimes done, especially with formaldehyde and ethanol. The chemical composition of the solution in such cases may be difficult to discern, and the chemical reactions taking place are very difficult to explain.


Wash the tissues very well, preferably overnight, with running tap water. Transferring to ethanol without this being done is likely to cause precipitation of chromium compounds, which appear as dark deposits in the sections. It should be noted that extensive washing does not remove all chromium as some appears to be bound to the tissues.


  1. Baker, John R., (1958)
    Principles of biological microtechnique
    Methuen, London, UK.
  2. Disbrey, B. and Rack, J.H., (1970)
    Histological Laboratory Methods
    E. & S. Livingstone, London, UK.