Leung & Gibbon's
Alcian Yellow-Toluidine Blue for Helicobacter


Solution A
Periodic acid 1 g
Distilled water 100 mL
Solution B
Sodium metabisulphite 5 g
Hydrochloric acid, 1N 1 mL
Distilled water 100 mL
Solution C
Alcian yellow 1 g
Ethanol, 50% 100 mL
Acetic acid, glacial 3 mL
Filter before use
Solution D
Toluidine blue 1% aqueous 0.5 mL
Sodium hydroxide, 3% aqu. 2 drops
Distilled water 50 mL

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives have not been evaluated.


  1. Dewax and hydrate sections.
  2. Oxidize in solution A for 10 minutes.
  3. Wash well with water.
  4. Treat with solution B for 5 minutes.
  5. Wash in water for 2 minutes.
  6. Stain in solution C for 5 minutes.
  7. Wash well with water.
  8. Stain in freshly prepared solution D for 3 minutes.
  9. Wash well with water.
  10. Blot dry.
  11. Dehydrate, clear and mount.

Expected results

Toluidine blue - alcian yellow



  1. This is most useful as a screening method.
  2. Alcian yellow is a monoazo dye that stains similarly to alcian blue. After oxidation gastric mucin is stained yellow and cannot later be replaced by toluidine blue. The final result is blue bacteria on a yellow background.


Leung, K., & Gibbon, K.J., (1996)
A rapid staining method for Helicobacter pylori in gastric biopsies
Journal of Histotechnology, v. 19, pp. 131




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