Properties and Principles of Hematoxylin & Eosin staining technique

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Hematoxylin and eosin stain mltgeeks

In histopathology,the sections (microtomy) as they are prepared are colourless and different components cannot be appreciated. Staining of tissues(histochemical staining) with different coloured dyeshaving affinities for specific components of the  tissue, makes identification and study of the tissue morphology possible by histotechnologist. There are somany types of stain such as giemsa stain,pas stain,wright stain,immunofluorescence stain used in immunohistochemistry and Hematoxylin and Eosin (h and e stain) is one of  the most frequently used stain in histopathology.Hematoxylin solution or hematoxilin is neither a dye nor it has coloring properties. For nuclear staining with hematoxylin stain, it is necessary to oxidize the hematoxylin to a weak anionic purple dye called hematin

Hematin has no affinity for the nucleic acids of nuclei. Hence the addition of a  metallic salt or mordant to hematoxylin gives a positive charge to the dye by virtue of the metal action. Thus the cationic dye (metal complex) will then bind to the anionic nuclear chromatin.

Ammonium or potassium alum ferric salt, chrome alum and phosphotungstic acid are various mordants used. The tissue component which is most frequently demonstrated is the nuclear chromatin using alum mordant in the hematoxylin and eosin staining.

A combination of hematoxylin and mordant is known as  hematoxylin lake. The aluminium lake formed with ammonium alum is particularly useful for staining nuclei. Hematoxylin recipes using these mordants are called alum hematoxylin.

Oxidation of Hematoxylin tohematin
Oxidation of Hematoxylin tohematin

How to extract Hematoxylin

  • Hematoxylin is extracted from the bark of a tree known as  haematoxylum campechianum.
  • The hematoxylin which we buy is extracted from this bloodwood tree.
  • The bark of freshly logged tree is chipped off, then boil the chips in water which produces an orange red solution which turns yellow, then black on cooling.
  • The water is evaporated leaving crude hematoxylin. Further purification is done.
  • The solutions of the dye should be oxidized to retain its staining ability longer.
  • The dye may also be oxidized by exposing it to the natural light for about 3-4 months.
  • As for chemical oxidation,it is achieved by using either sodium iodate or mercuric oxide.
  • The chemical oxidation converts the dye almost instantaneously but the product does not have shelf life.
  • Sodium iodate is most commonly used oxidizing agent (0.2 gm oxidizes 1.0 gm hematoxylin).
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Extraction of Hemaxylin
Extraction of Hemaxylin


  • It  has no staining property
  • The hematin with a mordant such as potassium  or ammonium alum forms lake which functions as cationic dye and is able to stains anionic tissue components.
  • Also in an  aqueous solution, hematin can be an acidic or an alkaline dye depending on pH. 4 of the solution
  • Hematin has the affinity for several tissues with an appropriate mordant

Progressive and Regressive staining

Progressive staining  

It is when the  tissue is left in the stain just long enough to reach the proper end point. The slides have to be examined at different interval to find out when the staining is optimum.

Regressive staining

With this method, the tissue is overstained then destained (differentiated) until the correct endpoint is reached.An example of a regressive stain is Harris hematoxylin; here,the overstaining is removed using  acidified alcohol.The removal of this excess dye is called differentiation.

Because of it’s acid pH,Hematoxylin alum gives a reddish hue to the tissues. To convert this colour to the final blue, an alkaline pH is required and the  process is known as “blueing”. Which is done either by tap water or by ammonium hydroxide.

How to prepare Harris’s hematoxylin

The following ingredients are required to prepare Harris’s hematoxylin

Hematoxylin5 gram
Absolute alcohol50 ml
Ammonium alum100 gram
Distilled water1000 ml
Mercuric oxide 2.5 gram
Glacial acetic acid40 ml

preparation of harris’s hematoxylin

  1. Start by dissolving  the hematoxylin in absolute alcohol and ammonium alum in hot water.
  2. Then mix the two solutions and heat in order to boil.
  3. Remove from flame then  add mercuric oxide and cool rapidly.
  4. Glacial acetic acid if added gives brisk nuclear staining, but life of the solution is reduced. Hence if acetic acid is to be added, it should be added in working solution
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How to prepare  Mayer’s hematoxylin

The following ingredients are required to prepare Mayer’s hematoxylin

Hematoxylin1.0 gram
Distilled water1000 ml
Ammonium alum 50 gram
Sodium iodate0.2 gram
Citric acid (reduces pH)1.0 gram
Chloral hydrate (preservative)50 gram

Preparation of Mayer’s hematoxylin

  • Hematoxylin is dissolved in distilled water using gentle heat.
  • Then alum is added and dissolved.
  • Then sodium iodate, citric acid and chloral hydrate are added respectively.

Eosin stain or Eosin solution

Eosin stain the cytoplasm to rose colored and is used as a counterstain. The intensity of the eosin is of individual or laboratory choice.

The most widely used eosin is the eosin y stain or eosin yellow where as the “ Y “ stand for  yellowish. It is available either in alcohol soluble form or water soluble form.In most laboratories,water soluble form of eosin Y in an alcohol-water solution is used.

Eosin Y water soluble form reagents

Eosin Y (water soluble)1.0 gram
Distilled water80 ml
95% alcohol320 ml
Glacial acetic acid0.4 ml

Preparation of Eosin Y water soluble form

  1. Start by dissolving eosin in water then add this to 95% alcohol making a one in 5 dilution(one part eosin solution with 4 parts alcohol).
  2. To the final mixture add a few drops or 0.4 ml of acetic acid .This help to increase  the staining intensity of eosin.
  3. When ready to use, the stain should be cloudy; if the stain is  clear, add a few drops of the acetic acid.
  4. The solution should be standardized by staining with a control slides.

How to stain with Hematoxylin and Eosin stain (h&e staining protocol)

  1. Start by deparaffinize sections in xylene for 10-20 minutes.
  2. Filter Hematoxylin.
  3. Rehydrate sections in 100% alcohol for 1-2 minutes then 95% alcohol for 1-2 minutes
  4. Rinse in tap water
  5. Then rinse in distilled water 5
  6. Now stain with Hematoxylin for a period of 3-5 minutes
  7. Then wash in tap water
  8. Differentiate section with 1% HCl in 70% alcohol 1-2 dips and check under microscope. If necessary, return slides to HCl for further differentiation.
  9. Wash the slides in running tap water for 15 minutes
  10. Now stain the slides in Eosin for 1-4 minutes
  11. Dehydration and Differentiation in 95% alcohol 5-6 dips of the slide then 100% alcohol 5-6 dips
  12. Clear slides in xylene two times
  13. Mount  the slides with Permount or DPX
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  • At no stage of staining the section should be dry
  • H&E is a regressive stain in which a tissue is over-stained and then excess dye is removed to obtain desired intensity of stain
  • Remember to filter Hematoxylin each time before staining
  • Change most of alcohol and xylene each time before staining

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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