Haematoxylin Staining

Haematoxylin Stain is a natural dye extracted by boiling the wood of log tree Haematoxylon campechianum and purified through re crystallization. It is one of the best known Nuclear stains. It can also be used to detect metals in tissues, to stain myelin sheath, mitotic stages, muscle fibers etc.The word Haematoxylin is derived from two Greek words Haimato meaning Blood and Xylon meaning wood.

Haematoxylin powder is poorly soluble in water but readily soluble is ethyl alcohol. The active dye used for staining is the oxidized form of haematoxylin called Haematein.The Ripening process for six weeks to several months makes the stain very good for staining purpose. Oxidization or ripening can be achieved very fast by adding oxidizing agents such as sodium iodate or mercuric oxide. But by adding these substances, over oxidation may result which can be prevented by adding glycerin.

Role of Mordants

Haematoxylin stain alone is very poor in action but its action can be increased by adding metallic salts called mordants. The mordant couples the stain to the tissues. The combination of haematoxylin and mordant is called Haematoxylin Lake. To form the haematoxylin lake, various metals like Ammonium alum (Ammonium ammonium sulphate), Aluminium potassium sulphate (potassium alum), or Aluminium sodium sulphate (sodium alum) etc are used. The Haematoxylin Lake formed of these alums is generally called as Alum Haematoxylin or Haemalum. The colour of the stain depends on the alum used. Various colours like Purple (Aluminium), Blue black (Iron), Blue black (Chromium), Blue green (copper), Red (Tin), Green brown (Osmium) are produced depending on the alum used as mordant.

When using as a nuclear stain, addition of Acetic acid will enhance the colour. The acetic acid reacts with the chromatin and gives a crispy appearance. Two methods are employed to use Haematoxylin stain. In Progressive staining, the tissue is kept for long time in the stain with periodic observation to assess the colour. In Regressive staining, the tissue is over stained first then destained until the tissue reaches the end point colour. Regressive staining is more acceptable because, the staining time is not important and also remove the stain adhered in the tissue adhesive during destaining and gives a clear transparent background. A well stained slide will show the nuclei as Blue after completing the staining procedure.

The stained slides should be differentiated using alcohol to remove excess stain. Due to prolonged differentiation, sometimes, the tissue loose stain. This can be rectified by placing the slide in the stain for few seconds. Differentiation can be stopped by washing the slide in distilled water. If properly stained, only the Nuclei will take the stain leaving a clear or pale background. Since the stain contains acid, the nuclei will stain purple first. Nuclear colour can be changed by dipping the slide in ammonia water or dilute sodium carbonate. Nuclear colour can also be enhanced by washing the slide in tap water for few minutes. Washing in tap water also removes excess alum, so that the slide remains as such without fading for long period.

Many factors cause weakening of the quality of stain. As the stain is kept for long periods, oxidation occurs, causing precipitation which looses the strength of the stain. So the stain must be filtered before using. Oxidation process also changes the colour of the stain.

Red Counter staining

Counter staining will give contrast to the nuclei against a light back ground. Usually Eosin Y is used as the counter stain. Eosin Y is soluble in 95% alcohol, so that if the slide is kept in 95% alcohol for long time it will remove all the eosin from the tissue. To avoid this, the slide should be run quickly through the eosin stain to the clearing solution. Eosin stain is yellow- orange in colour and by adding little acetic acid will make the stain more red in appearance to give good contrast with the blue nuclei.

Staining procedure

1.      Deparaffinize the slides and hydrate with water, through graded alcohol( 95%,80%,70%,50%,30% alcohol).

2.      If the tissue is fixed in Zenker’s fluid, remove the mercuric chloride using Iodine and clear it with sodium thiosulphate.

3.      Mordant in 3% Iron alum for 30-45 seconds

4.      Wash in distilled water

5.      Over stain the tissue in Haematoxylin for 30-45 minutes

6.      Wash well in distilled water

7.      Differentiate in 3% Alum

8.      Wash in running tap water until the nuclei changes to blue

9.      Dehydrate up to 70% alcohol

10.  Counter stain with Eosin Y (in 70% alcohol) for 15 seconds to 2 minutes depending on the age of the eosin watching the end point colour.

11.  Dehydrate in 95% alcohol. Keep the slide for two minutes and change the alcohol two times.

12.  Clear the slide in Xylol for two minutes. Two changes are required

13.  Mount the slide in DPX

14.  Nuclei will appear as Blue with some metachromasia

15.  Cytoplasm shows various shades of Pink.

Preparation of Stain – Heidenhain’s Iron – Haematoxylin

Solution I – Mordant

Iron Alum – 2.5g

Distilled water – 100ml

Solution II – Stain

Haematoxylin – 0.5g

Ethyl alcohol( 25%) – 10ml

Distilled water – 90ml

Dissolve Haematoxylin in Alcohol and add distilled water. Plug the bottle with cotton and allow the solution to ripe for 4-5 weeks.

 Counter Stain

Eosin Y in 70% alcohol

Simple PBS method

Haematoxylin- Eosin Staining – PBS method

Materials required

Paraffin sections, Ethyl alcohol, Phosphate Buffer Solution (PBS), Haematoxylin stain, Eosin stain, Xylol, DPX, Cover slips, Nail polish, Distilled water, Microscope.

  1. Deparaffinise the sections by incubating at 65 degree for 30 minutes in an oven.
  2. Place the slide in Xylene for 30 minutes. Repeat the process with new xylene.
  3. Place the slide in Absolute alcohol for 10 minutes. Repeat the process with new alcohol.
  4. Place the slide in 90% alcohol for 5 minutes.
  5. Place the slide in 70% alcohol for 1 minute.
  6. Place the slide in 50% alcohol for 1 minute.
  7. Place the slide in 30% alcohol for 1 minute.
  8. Rinse well in PBS for 5 minutes.
  9. Wipe out excess liquid present around the tissue using filter paper.
  10. Add a few drops of Haematoxylin stain over the tissue using a dropper and incubate for 5 minutes at room temperature.
  11. Wash the slide in running tap water from reverse slide.
  12. Rinse in PBS for 1 minute.
  13. Wipe out excess liquid present around the tissue using filter paper.
  14. Add a few drops of Eosin Y stain over the tissue using a dropper and stain for 30 seconds.
  15. Wash in running tap water from reverse slide.
  16. Rinse in PBS for 1 minute.
  17. Dehydrate in Absolute alcohol for 2 minutes– Two change.
  18. Clear in Xylene for 10 minutes – 2 changes.
  19. Mount in DPX and seal the rim of cover slip with Nail polish.
  20. Observe under high power objective of  microscope

Preparation of Phosphate Buffer

KCL 2 gms

KH2PO4 – 2.49gms

NaCl – 80 gms

Na2HPO4 – 11.45 gms

Dissolve all in 800 ml distilled water and make up to 1000 ml. Store at room temperature.



One response to “Haematoxylin Staining

  1. Thanks a lot for your notes…really appreciate it..
    it help me to completed my lab report…:)