Staining ( Chapter 6 )


The sections, as they are prepared, are colourless and different components cannot be appreciated. Staining them by different coloured dyes, having affinities of specific components of tissues, makes identification and study of their morphology possible. Certain terminologies used in the following account are given below.


Substances stained with basic dyes


Substances stained by acid dyes

Vital staining

Staining of structures in living cells, either in the body (in vivo) or in a laboratory preparation (in vitro). e.g. Janus green is taken up by living cells and stains the mitochondria.

Metachromatic staining

There are certain basic dyes belonging to aniline group that will
differentiate particular tissue components by staining them a different color to that of original dye. The phenomenon is known as metachromasia. The tissue element reacting in this manner are said to be exhibiting metachromasia. The generally accepted explanation of this phenomenon is that change in color is due to polymerization.

Sulfated substances are highly metachromatic e.g. Mast cell granules. These contain Heparin which is highly sulfated.
Some of the common metachromatic dyes are :
Methylene blue Methyl violent
Thionin Crystal violent
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. Tissue components often demonstrated by metachromatic stains :
Anyloid material, Mast cell granules
Mucin Cartilage

Direct staining

Application of simple dye to stain the tissue in varying shades of colours.

Indirect staining

It means use of mordant of facilitate a particular staining method or the use of accentuator to improve either the selectivity or the intensity of stain.

Progressive staining

Stain applied to the tissue in strict sequence and for specific times. The stain is not washed out r decolorised because there is no overstaining of tissue constituents. Staining is controlled by frequent observation under microscope.

Regressive staining

Tissue is first overstained and then the excess stain is removed from all but the structures to be demonstrated. This process is called differentiation and should always be controlled under microscope.


Partial or complete removal of stain from tissue sections. When the colour is removed selectively (usually with microscopic control) it is called differentiation. In case decolourization is to restain the selection with some other stain, acid alcohol treatment is the method of choice.


In regressive staining differentiation is the removal of washing out of the excess stain until the colour is retained only in the tissue components to be studies.


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