There are certain basic dyes belonging to aniline group that will differentiate particular tissue components by giving them a different color to that of original dye. The phenomenon is known as metachromasia.

 METACHROMASIA:-Metachromasia takes place when certain negatively charged groups on the tissue react with cationic dyes. On polymerization the original colour of the dye changes to another colour (eg mast cell stain pink with toluidine blue).

Thionin and toluidine blue dyes are commonly used for quick staining of frozen selection using their metachromatic property to stain nucleus and cytoplasm
differently.Metachromasia is enhanced when intermolecular distances are reduced.

Factors which enhance metachromasia are:-

1. Increasing concentration of dye.
2. Decreasing temperature.
3. pH
4. Water a polar solvent, contributes to the efficiency of van der Waal’s forces by which the molecules are held together.

In tissues, where there is a high concentration of anions e.g. in sulphated mucopolysaccharides, the cationic dye molecules may be held in such close proximity to one another that van der Waal’s forces can exert their influence and cause the dye to polymerize. Consequently the colour changes from blue to red.

Tissue components often demonstrated by metachromatic stains:-

Amyloid material, Mast cell granules
Mucin Cartilage
Amyloid Stain -Various stains are used to demonstrate amyloid

CRYSTAL VIOLET STAIN FOR AMYLOID:-

Aim: To demonstrate amyloid in tissue sections.

Principle: Amyloid (a glycoprotein) exhibits metachromasia in tissue sections
when stained with crystal violet and other cationic dyes.

Control: positive control.

Reagents:-

Crystal violet solution

Stock solution

Crystal violet                                          14 gm
95% alcohol                                           100 ml
Working solution
Stock solution                                      10 ml                                                                                                                                                    Distilled water                                      300 ml                                                                                                                                                Concentrated hydrochloric acid        1 ml

Procedure:-

*Deparaffinize and bring the sections to water.
*Put working crystal violet solution for 1 to 2 minutes and check under microscope.
*Rinse in tap water.
*Mount in water or in water soluble media.
*Put on the coverslip seal the edges with nail polish (Do not let it dry.)

Result:-
Amyloid                                               purple violet
Other tissues                                      blue

CONGO-RED STAIN FOR AMYLOID:-

Aim:- To demontrate amyloid in tissues.

Principle:- Diazo dye attaches itself to amyloid fibrils. The union is affected by
H bonds between the OH groups of amyloid and amino side groups of the dye.
Congo red dye forms non-polar hydrogen bonds with amyloid. The green
birefringence of congo red stained amyloid by polarized light is considered
diagnostic of amyloid.

Control:- Known positive tissue.

Reagents

Congo red solution

Congo red                            1.0gm
Distilled water                     100ml

Saturated solution of Lithium Carbonate

Lithium carbonate 1.3gm
Distilled water 100ml

Procedure:-

*Bring section to water.

*Pour congo red solution for 20 minutes.

*Pour off the solution and cover the slide with lithium carbonate for 1.5
 minutes to differentiate.
*Wash with water.
*Counter-stain with hematoxyline for 5 minutes.
*Differentiate with 1% acid alcohol.
*Wash in running tap water.
*Dehydrate, clear in xylene and mount in DPX.

Result:-

Amyloid                                              bright red which gives apple green birefringence in polarized light.
Nuclei                                                 blue
Other structures                               unstained to yellow

Notes:-

1. Sections must be cut at 8 to 10 microns for birefringence
2. Solution must be filtered through glass wool, not paper filters for birefringence
    to occur
3. Tissue fixed in solutions other than formalin may display false positive
    birefringence.