HAEMATOXYLIN AND EOSIN

                        HAEMATOXYLIN AND EOSIN

Haematoxylin is extracted from the logwood of the tree Heamatoxylon çampechianum, Oxidation of this extract produces, a colored substance hamatin, which is itself a poor dye, but which in the presence of a metallic mordant forms a powerful stain. It is a nuclear stain. The routine haematoxylin used is Ehrlich's Haematoxylin


Ehrlich's Haematoxylin

                                                Haematoxylin              -           6gm
                                                Absolute alcohol            -           300ml
                                                Glycerol                       -           300ml 
                                                 Glacial acetic acid      -           30ml
                                                Potassium alum             -        10-14gm (modant).

Dissolve haematoxylin in alcohol, alum in distilled water. Mix it together. Add glycerol and acetic acid. Shake well and keep it under sunlight for natural ripening. It takes around 3 months to get ripened.

It is a regressive stain and requires differentiation with 1% acid alcohol. Before ripening, the solution will be light purple and it changes to thick dark purple colour after ripening.

EOSIN
                        Eosin               -           1 gm
                        Distilled water    -           100ml
Few crystals of thymol is added as preservative. The color of the stain will be light orange.

Acid Alcohol 1%

            Hydrochloric acid                  -           1ml
            70% alcohol                           -           99ml

Procedure

1)   Deparaffinise the section in xylene for 5-10mnts
2)      Bring the section to water through descending series of alcohol starting from 100% to 50%.
3)      Keep it in Haematoxylin for 15-20min.
4)      Wash it in water and differentiate in 1% acid alcohol.
5)      Keep it in running tap water for bluing for 1Omnts. Bluing is the step by which the acid is neutralized by the alkali in running tap water. Before bluing the section will be purple in colour. It turns blue in color after keeping it in running tap water for 5-10mts.
6)       Counter stain with eosin for 1-2mnts.
7)      Wash it in water. dehydrate, clear and mount it with DPX.


Result

Nuclei, RNA rich cytoplasm, calcium                         -            Blue
 Muscle, fibrin, keratin                                     -           Bright red
 Collagen                                                         -           Pink
 Red blood cell                                                -          Orange / Red.


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