Toluidine Blue Staining in Microbiology: Procedure, Uses

Toluidine blue is a basic dye with its use in histology and cytology for staining acidic structures in cells. It is commonly employed to stain tissues for light microscopy. This dye is particularly useful for highlighting metachromatic elements, substances that exhibit different colors when viewed under a microscope.

Toluidine blue has a positive charge and readily binds to negatively charged molecules in the cell, such as nucleic acids and acidic proteins. This staining helps visualize various cellular components, including nuclei, mast cell granules, and cartilage matrix.
There are different forms of toluidine blue, such as toluidine blue O and toluidine blue O 0.1%, each with specific applications. Toluidine blue is widely useful in medical research and diagnostics to study tissues and cells under a microscope, providing valuable information about their structure and composition. Zolpidem

Bacteria are also 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).


  1. Winn, W. C., & Koneman, E. W. (2006). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Color Atlas & Textbook of Diagnostic Microbiology). Lippincott Williams & Wilkins
  2. Sridharan, G., & Shankar, A. A. (2012). Toluidine blue: A review of its chemistry and clinical utility. Journal of oral and maxillofacial pathology : JOMFP, 16(2), 251–255.
  3. Jordan, E. O., Caldwell, M. E., & Reiter, D. (1934). Bacterial Motility. Journal of bacteriology, 27(2), 165–174.

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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