Toluidine blue staining for mast cells

1. Toluidine Blue Staining
Histopathology
Presenters
Mr. Naeem Bukhari & Muhammad Waseem
Undergraduate Students, Dep't Of Medical Lab Technology;
University of Haripur, KPK, Pakistan.

2. Toluidine Blue Staining
• Properties:
• Toluene blue is a metachromatic stain, Which stain tissue Mast cell
granules.
• Monomers of metachromatic stain polymerize to form dimer and
stain polymer changing colour form blue to purple.
• Metachromasia: is the property of stain/dye, which stains certain
component of the tissue (Mast cell granules) in the form of colour
shade; totally different from original solution colour. It depends upon:
1. Nature of the dye
2. Nature of the tissue component to be stained.

3. 1. Nature of the dye
Meta-chromatic stains like, Toluidine blue, safranin, thionines, crystal
violet are basic in nature with large number of cationic radicals.
2. Nature of the Tissue Component
• Due to acidic nature, tissue components are called as acid
polysaccharides as larger number of an-ionic radicals; e.g. Sulphates,
phosphates and Carboxylic group are present.
- There is dual kind of bonding found in meta-chromatic stains. One
between tissue components. Secondly, stain polymerize itself and
changes its colour from blue to purple. Further polymerization leads
towards red colour.
Fig; A) POLYMERIZATION

4. Principle
The dual bonding is based upon acid base reactions, in which basic stain bonds with acidic
tissue components and dye itself polymerizes to give secondary bonding. Colour of the dye
changes from blue to purple/red.
• Composition
- 1 % toluidine blue in 50 % isopropanol. Standard solution of toluidine blue stain.
- Procedure
• Dewax and hydrate the paraffin sections.
• Stain with Toluidine blue for 30 min at 37 C.
• Differentiate the sections in absolute isopropanol / till clear background.
• Dehydrate and mount the stain for microscopy.
Results
Mast cell granules+ cartilage matrix+ muco-Polysaccarides-------purple-red colour.

5. Mast cells
• Tissue mast cells resemble basophils, but they are connective tissue
cells.
• Unlike basophils, they have a long life span of between 9 and 18
months.
• The mast cell, like the basophil, plays a role in hypersensitivity
reactions by binding IgE.

6. Nissle Substance/ Granules
• In the nerve cells there is abundant basophilic material in the form of
clumps and aggregate known as ribonucleoprotien. Which displays
granular ERC. The floating basophilic material in cytoplasm is called
as nissele substance/granules.
• Demonstration:
- Nissele granules are demonstrated by basic dyes like toluidine blue,
while best results are obtained with cresyle fast violet.

7. Its your time to Ask for Question
????????????????????? Either Blue/ Red……? ?????????????????

8. Sure, Now I have right to ask you
some questions
1. Define Metachromatic staining?

9. 2. Nature of toluidine dye?
3. Mast Cell?

10. 4. Why Staining blue is called blue, but it
stains tissue into purple-red colour

11. Thank you for listening …...
May Allah Bless You>>>>>>>>>>>>>>