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

Solutions

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.

Method

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

• Helicobacter  –  blue
• Mucin  –  yellow
• Background  –  pale blue

 

Notes

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.

 

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