Toludine Blue

 Classification: connective tissue stain

Purpose: stains mast cells

Staining principle: Hydrogen bonding

Mechanism of staining: Metachromasia

Positive controls: small intestine, skin, any tissue containing mast cells

Well Stained Slide

Chromotropes stain purple

All other tissue stains various shades of blue or colorless if rinsed in 1% acetic acid.

 


REAGENT

REAGENT

PURPOSE

MECHANISM OF STAINING

SOURCE OF ERROR

Toluidine blue

 

Basic primary dye demonstrates carboxylated and sulphated acid mucopolysaccharides

(progressive)

Attaches to chromotropes by salt linkages. Metachromia occurs.

Omitted: Mast cells not demonstrated.

Too short: Will not affect the staining.

Too long: Will not affect the staining.

1% Acetic acid  (optional)

 

Differentiator

 

Omitted: Difficulty seeing mast cells.

Too short: No effect on the staining.

Too long: Other tissue components may be pale.

 

Special Considerations

Some labs use 1% Acetic acid to rinse the slide after Toluidine blue to make the background clear.

In order for merachormasia to occur the tissue’s reactive sites must be negatively charged, evenly spaced, close together, dye must be polymerized and water must be present.


Over powering. Mast cells are not demonstrated.

Treatment: dip in acetic acid, longer time in alcohol.

 


Well Stained Slide





Mast cells are demonstrated as purple.

Slide has been treated with acetic acid. Cell cytoplasm is clear and nuclei are blue.


References

Officer B. HIML251 Lecture notes: Elastic Stains: Toluidine blue staining method, March 19, 2009