Simple Metachromatic Stain

A metachromatic dye, 0.2% aqueous
Acetic acid, 1% aqueous

Suitable blue dyes are:
azure A
azure B
azure C
toluidine blue
polychromed methylene blue

Less common dyes are:
Neutral red,
Safranin O,
Bismarck brown Y

Tissue sample
Paraffin sections of neutral buffered formalin fixed tissue are suitable. Mercuric chloride fixatives are reputed to emphasise metachromasia. Other fixatives may be satisfactory.


  1. Bring sections to water via xylene and ethanol.
  2. Place in to staining solution for 5 minutes.
  3. Rinse well with tap water.
  4. If staining is too dark, differentiate with acetic acid until metachromasia is evident.
  5. Rinse well with tap water.
  6. a) Coverslip with water and examine wet, or
    b) Blot dry and immerse in xylene. Repeat until cleared. Coverslip using a resinous medium

Expected results

Dye colour Blue Red Brown
Nuclei Blue Red Brown
Acid mucopolysaccharides Red/purple Yellow Yellow
Background Blue Red Brown


  1. The actual dye concentration is not too important. It should be strong enough to stain within 5 minutes. Concentrations between 0.1% and 1% are usually suitable.
  2. The staining time can be varied. The staining solution should be applied for long enough to give dark staining. Usually this is about 2-5 minutes.
  3. Methylene blue may be polychromed by making a 1% w/v solution and leaving it for several months in an airy, bright location with a loose stopper of cotton wool. When it gives good metachromatic staining, stopper tightly and place in a cupboard.
  4. The acetic acid concentration may be varied. Usually a concentration of 0.1% to 1% is suitable. The higher the concentration, the faster dye is removed.
  5. Sections may require little or no differentiation. Always check the staining before applying acetic acid. Stop when a distinct contrast in colour is seen.
  6. To mount in a resinous medium, blot the wet section dry and immerse in xylene. Repeat until the section is cleared and becomes transparent. Mount using a resinous medium.
  7. Ethanol should be avoided as it may destroy any metachromasia.
  8. Metachromatically stained materials include intestinal and other mucins, cartilage, connective tissue ground substance and mast cell granules.


No specific reference.




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