Toluidine Blue Staining in Microbiology: Procedure, 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 diphtheriae contains granules with polymerized inorganic polyphosphate, which stains red violet color.
  • Helicobacter pylori 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).

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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