Hematoxylin & Eosin Stain BY ENGR. UMAIR RAZA

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Topic:
Hematoxylin & Eosin Stain
Introduction:
The H&E stain (Hematoxylin & Eosin stain) is the usually used as histological stain because
 Comparative Simplicity
 Capability to demonstrate clearly more number of assorted structure
 Hematoxylin stains cell nuclei blue black because it indicates good nucleus
 Eosin stains cell cytoplasm & most connection tissue fibers in varying shades & intensities of
orange, red & pink.
 Eosin is the most suitable stain.
History:
Hematoxylin is attributed to Waldeyer in 1862 that used it as a watery
Abstract but without very much success. Two years later Bohmer combined
Hematoxylin with potash alum as a harsh and obtained more specific staining
Ehrilch (1886) who conquer the unpredictability of Hematoxylin & potash alum by the additions of glacial
acetic acid & at the same time produced his formula for Hematoxylin as it is used today.
Staining:
A technique in which cells or thin sections are colored (a material or object) by applying a penetrative
dye or chemical.
Extraction of Hematoxylin stain:
 Tinny pieces of the log wood is boiled in water.
 First orange-red solution is formed.
 On cooling it turn into black solution.
 Then precipitated with urea or ether.
 Purified & sold commercially.
 Hematoxylin itself is not a stain.
 On oxidation it produces hematein (This process is called Ripening )
 Commonly used dye in histopathology, cytology.
 Best nuclear strain.
 Basic in nature, stains acidic constituent of the tissue, nucleus, mitochondria etc.
Oxidation of Hematoxylin stain:
 Oxidation of hematein is called Ripening.
 Hematine can be produced in 2 ways.
 Natural ripening (sunlight & air is used in
this process).
Hematoxylin is converted to
hematein through oxidation.

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 Chemical ripening (chemicals are used like sodium iodate, mercuric oxide are used
commonly).
Types of Hematoxylin stain :
Based upon mordant used
 Alum Hematoxylin
 Iron Hematoxylin
 Tungsten Hematoxylin
 Molybdenum Hematoxylin
 Lead Hematoxylin
Advantages & Disadvantagesof Ripening:
Structural formula of
Hematoxylin:
Eosin Stain:
 Most suitable stain to combine with potash alum Hematoxylin .
 Eosins are xanthine dyes( tetrabromofluorescein ).
Types of Eosinstain:
They can prepare commercially
 Eosin Y
Advantages Disadvantages
 Once oxidation has reached an
acceptable level, the staining solution
may be used & it lasts for longer.
 Requires a considerable period of time.
 Two groups of naturally ripened
(Sunlight & air is used in this process)
product may not produce the same
staining qualities.

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 Ethyl eosin
 Eosin B
EosinY:
 It is yellow in color.
 It is mostly used.
 It is alcohol soluble.
 It stains the cytoplasm-0.52-1% solution in distilled water with the crystal of thymol (It prevents the
growth of fungus).
 Addition of CH3COOH sharpens the staining.
 EosinY is an acidic dye which stains the erythrocytes and cilia.
Ethyl Eosin:
Eosin alcohol- soluble
EosinB:
Eosin bluish, erythrosine B
Substitute of Eosin:
 Phloxine
 Bierbrich scarlet(gives more intense red color to the tissues).
 Safranin
Structural formula of
Eosin:

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Principle of H&E stain:
 H&E is used for the better indication of nucleus & cytoplasm.
 Potash alum( KAl(SO₄ )₂ ·12H₂ O) used as the mordant & Hematoxylin satin the nucleus light blue
in the presence of alum which turns into red in the presence of acid.
 Cell differentiation is achieved by treating the tissue with acid solution& Eosin used as counterstain
which gives pink color to cytoplasm.
Method of staining:
1. De-paraffinization of sections (removal of paraffin wax using xylene for 10-20 minutes).
2. Then sections are rehydrated:
a. 100% alcohol for 1-2 minutes.
b. 95% alcohol for 1-2 minutes.
3. Rinse in tap water.
4. Rinse in the distilled water.
5. Stain with the Hematoxylin for 3-5 minutes.
6. Wash in the tap water.
7. Differentiate the sections with 1% hydrochloric acid in 70% alcohol(1-2 dip & check it under the
microscope). If it is essential return the slide to hydrochloric acid for more differentiation.
8. Watch the slide with tap water for 15 minutes.
9. Stain the slide with Eosin for 1-4 minutes.
10. Dehydration & differentiation :
a. 95% alcohol 5-6 dips.
b. 100% alcohol 5-6 dips.
c. Clear with xylene (2 times).
d. Use DPX ( Distyrene plasticizer xylene).

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