Hematoxylin is a natural dye extracted from the heartwood of the bloodwood tree. It is converted to hematein during preparation of staining solutions, usually using an oxidizing agent. Hematoxylin and eosin staining is commonly used in histology, staining nuclei blue with hematoxylin and cytoplasm red with eosin. The dyes require a mordant like aluminum or iron to bind to tissues. Sections are deparaffinized, stained, dehydrated and mounted for examination under a microscope using an aqueous or resinous mounting medium.

1. Hematoxylin & Eosin
staining
DR. SARA A.G. SEIF EL DIN

2. Hematoxylin
Hematoxylin is a natural dye, which is extracted from the heartwood
of the tree Haematoxylum campechianum, although
histotechnologists are probably more familiar with the name as
Hematoxylon campechianum. The genus name Hematoxylum is
derived from two Greek words: haimatos which means blood, and
xylon which means wood.

3. Leaves and flowers of H. campechianum

4. The hematoxylin which we buy is extracted from
the heartwood of this bloodwood tree. There
may be some differences in method, but one is
to chip the heartwood of freshly logged trees,
then boil the chips in water. An orange-red
solution is obtained, which turns yellow, then
black on cooling. The water is evaporated leaving
crude hamatoxylin. Further purification is
undoubtedly done

6. Although it is common practice to use hematoxylin, it is not
itself the dye. During the preparation of staining solutions
hematoxylin is converted into hematein. This is usually
accomplished with:
1- chemical oxidizing agents, Sodium iodate is the most
commonly used oxidizing agent for this purpose (0.2g will
oxidize 1g hematoxylin). Others are mercuric oxide (now
strongly deprecated because it is poisonous), potassium
permanganate and iodine.
2- Natural oxidizing atmospheric oxygen over time.
Hematein may also be referred to as hematoxein, although it
is not usually seen in histotechnology references. Hematein
is not incorporated directly into most staining solutions
because it continues to oxidize in solution and forms non-
staining products.

8. Mordants
Alum
Iron
Iodine
Lead
Tungsten
Molybdenum
Without mordant

9. The dye is usually used in conjunction with a mordant, the two
commonest being
aluminum (as ammonium or potassium alum),
or iron (ferric chloride or iron alum).
Other mordants are used much less frequently but include
chrome alum
and phosphotungstic acid. can demonstrate fibrin, muscle
striations and some neuroglia fibres.
The tissue component most frequently demonstrated is nuclear
chromatin using an aluminum mordant in the Hematoxylin and
Eosin general oversight staining method. Using ferric salts as the
mordant, it is also used for acid resistant nuclear staining, the
demonstration of muscle striations and numerous other
elements.

10. Hematoxylin and eosin
Hematoxylin and eosin staining is one of the most
common method used in histology and
histopathology. Basophilic nuclei, bacteria,
calcium, and so on are stained "blue" with
hematoxylin. Eosinophilic cytoplasm, connective,
and all other tissues are counterstained "red"
with eosin. Hematoxylin and eosin staining is
often used to detect inflammation or to
determine the integrity of a tissue.

12. Deparaffinize and hydrate to water
Mayer's hematoxylin for 15 minutes
Wash in running tap water for 10minutes
Counterstain with eosin from 15 seconds to 2 minutes depending on
the age of the eosin, and the depth of the counterstain desired. For
even staining results dip slides several times before allowing them to
set in the eosin for the desired time
Dehydrate in 95% and absolute alcohols, two changes of 2 minutes
each or until excess eosin is removed. Check under microscope
Clear in xylene, two changes of 2 minutes each
Mount in DPX

13. Mounting Media
Histological sections which need to be examined for any length of
time or to be stored, must be mounted under a cover-slip.
There are two types of mounting media :
1. Aqueous media - Used for material which is unstained, stained
for fat, or metachromatically stained.
2. Resinous media - For routine staining.

14. Aqueous Mounting Media
There are used for mounting sections from distilled water when the stains
would be decolorised or removed by alcohol and xylene, as would be the case
with most of fat stains (Sudan methods). Gome stains, e.g. methyl violet, tend
to diffuse into medium after mounting. This can be avoided by using
Highman's medium. Aqueous mountains require addition of bacteriostatic
agents such as phenol, crystal of thymol or sodium merthiolate to prevent the
growth of fungi.

15. 1. Apathy's medium (R.I- 1.52)
A very useful medium for mounting sections for
fluorescent microscopy.
2. Farrant's medium (R.I. 1.43) Recommended for fat
stains.
3. Glycerine jelly (R.I. 1.47) An excellent routine
mountant for fat stains.
4. Highman's medium (R.I. 1.52) Recommended with
the metachroamtic dyes especially methyl
violet.

16. Resinous mounting media
Natural or synthetic resins dissolved in benzene, toluene or xylene.
These are purchase readymade. In case they become too viscous they
may have to be diluted with xylene. Following are some of these media.

17. 1. Canada balsam - Natural resin (R.I. - 1.52) It is used
as 60% resin by weight in xylene. H.&E stained slides
are fairly well preserved but basic aniline dyes tend to
fade and prussian blue is slowly bleached. Slides take
few months to dry.
2. D.P.X. (R.I. 1.52) Polystyrene resin dissolved in
xylene as a 20% solution. It is most commonly used. 3.
There are many other synthetic resins sold under
various trade names e.g. Coverbond (R.I. 1.53), H.S.R.
(Harlew synthetic Resin), Histoclad (R.I. - 1.54),
Permount (r.I. 1.54), Pro-Texx (R.I. 1.495).

18. Criteria of acceptable mounting media
1. Refractive index should be as close as possible to
that of glass i.e. 1.5.
2. It should not cause stain to diffuse or fade.
3. It should not be crack or appear granular on setting.
4. It should be dry to a nonsticky consistency and
harden relatively quickly.
5. It should not shrink back from edge of cover-glass.
6. It should be free flowing and free bubbles.

19. To mount a slide, (A) Apply a single drop of mounting medium upon
tissue section. (B) Hold coverslip at 45o allowing the drop to spread along
the edge of the slip. (C) Let go of slip and allow medium to spread slowly.

20. Stepping forward and Looking
forward towards a brighter
future
Stepping forward Looking forward
Painted by: Eiman Yahia Painted by: Reem Yahia

21. Thanks