Toluidine Blue staining in Microbiology: Procedure and uses

Bacteria are easier to detect in broth culture if the preparation is stained using toluidine blue 0.5% w/v, rather than stained by the Gram staining technique.

Procedure of Toluidine Blue Staining

  1. Make a smear of the broth
  2. Allow to air-dry
  3. Fix with absolute methanol for 1-2 minutes
  4. Stain with Toluidine blue for 30-60 seconds
  5. Wash off with water
  6. Allow the smear to air dry and examine microscopically.

Results of Toluidine Blue Staining

  • Corneybacterium diphtheria contains granules with polymerized inorganic polyphosphate, which stains red violet color.
  • Helicobacter stains dark blue against a variably blue background. The concentration of Toluidine Blue used is 1%.

Note: When bacteria are detected, examine also a gram stained smear to identify whether the organisms are Gram positive or Gram negative (unless the organism can be recognized by their morphology in the Toluidine blue preparation.


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