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
- Make a smear of the broth
- Allow to air-dry
- Fix with absolute methanol for 1-2 minutes
- Stain with Toluidine blue for 30-60 seconds
- Wash off with water
- 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).