HISTOPATHOLOGY - 10 - Haematoxylin & Eosen Technique

Cellular Pathology
Lecture #10
Histochemistry:
Haematoxylin & Eosin Technique
Procedural document:
Rare disease nomenclature in English
Ephraim Imhotep Zulu, BSc BMS, MSc Path

University of Zambia
School of Health Sciences
Dept. of www.orpha.net www.orphadata.org
Biomedical Sciences
Lecture Outline
• Hematoxylin stain
• Eosin stain
• Principle of Hematoxylin and Eosin staining
• Alum Hematoxylins
• Iron Hematoxylins
• Phosphotungstic Acid Haematoxylin
• Molybdenum Hematoxylins
• Lead Hematoxylins
• Hematoxylin Without A Mordant
• Staining protocol of H&E
Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 2

Learning Objectives:
At the end of this lecture, the student is expected to:
• Know the Principles, applications and limitations of H&E technique
• Appreciate the Classification of Hematoxylins
• Know the staining procedure of H&E technique
• Differentiate/Compare and contrast:
• Harris vs. Mayer's Hematoxylin

Synopsis
• The hematoxylin and eosin stain (H&E) is the most widely used stain in histology
and histopathology laboratories.
• The H&E stain provides a comprehensive picture of the microanatomy of organs and
tissues.
• Its popularity is based on its comparative simplicity and ability to demonstrate clearly
an enormous number of different tissue structures.
• The hematoxylin component stains the cell nuclei blue-black, showing good
intranuclear detail, The eosin stains cell cytoplasm and most connective tissue fibers
in varying shades and intensities of pink, orange, and red.
• When it is properly performed it has the ability to demonstrate a wide range of

normal and abnormal cell and tissue. There are a number of different hematoxylin and
eosin formulations in popular use, each with various advantages and disadvantages.

Eosin Stain
• Eosin is the most suitable stain to combine with an alum hematoxylin to
demonstrate the general histological architecture of a tissue.
• With proper differentiation, it can distinguish between the cytoplasm of

different types of cell, and the different types of connective tissue fibers and
matrices, by staining them differing shades of red and pink.
• The eosins are xanthene dyes and have the following types: eosin Y (eosin
yellowish, eosin water-soluble), ethyl eosin (eosin S, eosin alcohol-soluble),
eosin B (eosin bluish, erythrosin B).
• Of these, eosin Y is the most widely used, and despite its synonym it is also
satisfactorily soluble in alcohol.
• As a cytoplasmic stain, it is usually used as a 0.5 or 1.0% solution in distilled

water, with a crystal of thymol added to inhibit the growth of fungi.

Hematoxylin
• Extracted from the heartwood (‘logwood’) of the tree Hematoxylon
campechianum
Haematoxylon campechianum Tree logs. hematoxylin powder.

Hematoxylin.,

Hematoxylin..,
• Extracted from logwood with hot water, and then
precipitated out from the aqueous solution using urea.
• Hematoxylin itself is not a stain.
• The major oxidization product is hematein, a natural dye

that is responsible for the color properties.
• Hematein can be produced from hematoxylin in two

ways.

Natural oxidation by exposure to light and air
• AKA ripening, is a slow process

• Sometimes taking as long as 3–4 months
• Resultant solution seems to retain its

staining ability for a long time.
• Ehrlich’s and Delafield’s hematoxylin
solutions are examples of naturally
ripened hematoxylins.
Chemical oxidation
• Uses sodium iodate (e.g. Mayer’s hematoxylin) or mercuric oxide (e.g. Harris’s hematoxylin) as
chemical oxidizing agents
• The chemical oxidizing agents converts the hematoxylin to hematein almost instantaneously, so
these hematoxylin solutions are ready for use immediately after preparation.
• In general, they have a shorter useful life than the naturally oxidized hematoxylins, probably
because the continuing oxidation process in air and light eventually destroys much of the
hematein, converting it to a colorless compound. Hematein is anionic, having a poor affinity for
tissue, and is inadequate as a nuclear stain without the presence of a mordant.
• The mordant/metal cation confers a net positive charge to the dye-mordant complex and enables
it to bind to anionic tissue sites, such as nuclear chromatin.
• The type of mordant used influences strongly the type of tissue components stained and their final
color.
• The most useful mordants for hematoxylin are salts of aluminum, iron, and tungsten.

ylin is a good
i bluish black. (a) Natural: This is done by exposing the haema-
Hematoxylin & Eosin Principle
agenous mate- toxylin powder in sunlight and air. This is also
OH OH
O O
HO CH2 HO CH2
C OH C OH
the bark of CH CH2 CH CH2
that is mainly
xico. Presently Oxidation
HO OH HO
O
Haematoxylin Haematin
n, the freshly
pechianum is
oiled in water. Dye-mordant
formed which binds with tissue
O O
ion. The com- O O
H 2O AI OH2
cipitated with H2O AI OH2
O O
brownish tan H2O OH2
O PO O P O
r purified and Dye-mordant complex O OH
ound haema- Base
pability unless DNA strand
its staining
Fig. 8.1 Schematic diagram showing oxidation of hae-
matoxylin and its combination with aluminium

Hematoxylin & Eosin Principle

Classification
• Hematoxylin solutions can be arbitrarily classified
according to which mordant is used:
• Alum hematoxylins
• Iron hematoxylins
• Tungsten hematoxylins
• Molybdenum hematoxylins
• Lead hematoxylins
• Hematoxylin without mordant

Alum Hematoxylins
• Comprises most of those that are used routinely in the H&E stain,
and produce good nuclear staining.
• The mordant is aluminum, usually in the form of ‘potash alum’

(aluminum potassium sulfate) or ‘ammonium alum’ (aluminum
ammonium sulfate).
• All stain the nuclei a red color, which is converted to the familiar
blue-black when the section is washed in a weak alkali solution (eg.
Tap water).
• This procedure is known as ‘blueing’.
• Can be used regressively or progressively

Alum Hematoxylins.,
70 8 Haematoxylin and Eosin Stain of the Tissue Section
Table 8.1 Essential types of haematoxylin

Stain type Mordant used Application Time to stain Comments
Mayer’s Potassium or Popular nuclear 5–10 min in progressive
haematoxylin ammonium alum counterstain stain and 10–20 min in case
of regressive stain
Ehrlich’s Potassium alum Nuclear stain and also 20–30 min Natural ripening.
haematoxylin stains mucin, bone and Stain is more
cartilage long-lasting
Harris Ammonium or Good nuclear stain in 5–15 min in case of Ripening by
haematoxylin potassium alum exfoliative cytology regressive stain and 5–30 s mercuric oxide
in progressive stain
Gill’s Aluminium Good nuclear stain Regressive stain: 5–15 min The stain is stable
haematoxylin sulphate for 1 year
Cole’s Potassium alum Good nuclear stain Regressive stain: 30 min Stable for 3
haematoxylin months
known as ripening. The oxidation process is Table 8.1 highlights different types of haema-
slow and takes approximately 3 months. toxylin with their mordant and properties.
However, the useful staining life of the dye is
longer by natural ripening process.
Tuesday, April 19, 2022
(b) Chemical: This is done by treating the dye 8.3
Ephraim Zulu - HISTOPATHOLOGY
Bluing 15
Alum Hematoxylins.,

able for native is the combination of a celestine blue staining
his and Staining
solution withtimes with
an alum alum hematoxylins
hematoxylin. Celestine blue
Table 10.1 Staining times with alum hematoxylins

Cole’s 20–45 min
oxylins
ed var- Delafield’s 15–20 min
Ehrlich’s progressive 20–45 min
Mayer’s progressive 10–20 min
ng time
Mayer’s regressive 5–10 min
Harris’s progressive in cytology 4–30 s
d hema-
rapidly Harris’s regressive 5–15 min
y or, in Carazzi’s progressive 1–2 min
ine the Carazzi’s regressive 45 s
rvals.
Carazzi’s with frozen sections, see text 1 min
regres-
Gill’s I regressive 5–15 min

Disadvantages of alum hematoxylins
• Their sensitivity to any subsequently applied acidic staining solutions. The most
common examples are in the van Gieson and other trichrome stains.
• The application of the picric acid acid fuchsin mixture in van Gieson’s stain removes
most of the hematoxylin so that the nuclei are barely discernible.
• In this case satisfactory nuclear staining can be achieved by using an iron-mordanted

hematoxylin such as Weigert’s hematoxylin, which is resistant to the effect of picric
acid.
• A suitable, and now more popular, alternative is the combination of a celestine blue
staining solution with an alum hematoxylin.
• Celestine blue is resistant to the effects of acid, and the ferric salt in the prepared
celestine blue solution strengthens the bond between the nucleus and the alum
hematoxylin to provide a strong nuclear stain which is reasonably resistant to acid.

–3 laboratory.
he Table 8.2 Differences between Mayer’s and Harris
ain haematoxylin
on, Mayer’s
nd Factors Harris haematoxylin haematoxylin
Solvent Absolute alcohol Distilled
water
Oxidant Mercuric oxide Sodium
iodate
Substance added Glacial acetic acid Chloral
to sharpen the hydrate
sa stain
te- Stain type Both progressive Progressive
is- and regressive stain stain

Change stains frequently.
Iron Hematoxylins
Iron hematoxylins
These hematoxylin solutions use iron salts, usually
ferric chloride and ferric ammonium sulfate, both as
the oxidizing agent and the mordant. The most com-
mon iron hematoxylins are:
• Weigert’s
• Heidenhain’s
• Loyez for myelin
• Verhöeff’s for elastin fibers.
Over-oxidation Ephraim
Tuesday, April 19, 2022
of the hematoxylin is a problem21
Zulu - HISTOPATHOLOGY
• Over-oxidation of the hematoxylin is a problem with these stains, so it is usual to
prepare separate mordant/oxidant and hematoxylin solutions and mix them
immediately before use (e.g. in Weigert’s hematoxylin) or to use them consecutively
(e.g. Heidenhain’s and Loyez hematoxylins).
• Because of the strong oxidizing ability of the solution containing iron salts, it is often
used as a subsequent differentiating fluid after hematoxylin staining, as well as for a
mordanting fluid before it.
• The iron hematoxylins are capable of demonstrating a much wider range of tissue
structures than the alum hematoxylins,
• but the techniques are more time-consuming and usually incorporate a differentiation
stage which needs microscopic control for accuracy.

Phosphotungstic Acid Haematoxylin (PTAH)
• Can be prepared using chemical oxidation or natural ripening of the
tungsten hematoxylin solution in light and air.
• A routine progressive stain for nervous tissue owing to its ability to
stain astrocytes, fibroglia, myoglia, muscle striations, collagen,
reticulin, fibrin, etc in shades of blue and red
• Tissue sections may be treated with Mallory bleach to suppress
staining of myelin.

Table 8.4 Applications of different haematoxylin stains • Low visc
for different purposes bubble fo
Substances Haematoxylin coverslip.
Routine stain in histology Harris colour and
sections and cytology smears haematoxylin
Carbohydrates Mayer’s
haematoxylin
Phospholipid Baker’s acid
haematein Box 8.1Cle
technique • Xylene
Fibrin, cross striations of skeletal Tungsten
muscle haematoxylin
Connective tissue fibres Verhoeff’s iron Basic prop
haematoxylin
Amoeba, microfilaria Iron haematoxylin • Colour
Nuclear chromatin Gill’s • Refract
haematoxylin the mo
Photomicrography Heidenhain’s iron
• It gives
haematoxylin
Counterstaining in Ehrlich’s
immunohistochemistry and haematoxylin Warnin
cytochemistry

Molybdenum Hematoxylins
• Uses molybdic acid as the mordant
• Eg the Thomas technique for the demonstration of

collagen and coarse reticulin
• Thomas method also stains argentaffin cell granules.
• Gives the following Results: Collagen and coarse reticulin

violet to black, Argentaffin cells black, Nuclei pale blue
and Paneth cells orange

Lead Hematoxylins
• Incorporate lead salts as mordants
• have recently been used in the demonstration of the

granules in the endocrine cells of the alimentary
tract and other regions.
• The most practical diagnostic application is in the

identification of endocrine cells in some tumors,
• it is also used in research procedures such as in the

localization of gastrin-secreting cells in stomach.

Hematoxylin Without A Mordant
• Freshly prepared hematoxylin solutions, used without a
mordant, have been used to demonstrate various
minerals in tissue sections.
• The basis of the Mallory methods is the ability of

unripened hematoxylin to form blue-black lakes with
these metals.

Table 10.2 The uses of hematoxylin stains
Hematoxylin Applications Oxidant Mordant
Ehrlich Nuclear stain used with eosin and stains some mucins Natural Alum
Delafield Nuclear stain used with eosin Natural Alum
Mayer Nuclear stain used with eosin and nuclear counterstain Sodium iodide Alum
Harris Nuclear stain used with eosin Mercuric oxide Alum
Cole Nuclear stain used with eosin Iodine Alum
Carazzi Nuclear stain used with eosin in frozen sections Potassium iodate Alum
Gill Nuclear stain used with eosin Sodium iodate Alum
Weigert Nuclear stain used with acid dyes Natural Iron
Heidenhain Intranuclear detail, muscle striations Natural Iron
Verhöeff Elastic fibers Natural Iron
Loyez Myelin Natural Iron
Mallory PTAH Fibrin, muscle striations, glial fibers Natural Tungsten
Thomas Collagen, endocrine cell granules Hydrogen peroxide Molybdenum
Solcia Endocrine cell granules No oxidant Lead
Mallory Iron, copper, lead No oxidant No mordant
Weigert-Pal Myelin in block preparation No oxidant Chromium-copper

pH differences. Laboratory produced hematoxylin in tissues and/or paraffin wax blocks sent from
Staining protocol of H&E
8.6 Iron Haematoxylin 73
Fig. 8.2 Basic steps of 10 minutes in xylene, 3

haematoxylin and eosin Step 1 Deparaffinization changes
stain are highlighted
Step 2 Rehydration: Graded alcohol 1-2 minutes, 2 changes
Step 3 Nuclear stain: Haematoxylin 5 to 15 minutes
1% acid alcohol: 2 dips

Step 4 Differentiation and wash
Step 5 Bluing Running tap water
Eosin Y for 2 to 3
Step 6 Counterstain : Eosin minutes
Step 7 Graded alcohol 3-5

Dehydration minutes each
Step 8 Clearing and mounting Xylene 5 minutes
Fig. 8.3 Well-stained

tissue by haematoxylin
Step by Step Guide to Performing an H&E Stain
Step 1: Deparaffinization
• Following the preparation of a paraffin section, all the elements are infiltrated with
and surrounded by paraffin wax which is hydrophobic and impervious to aqueous
reagents.
• The first step in performing an H&E stain is to dissolve all the wax away with xylene.
Step 2: Hydrate the Section
• After thorough de-waxing, the slide is passed through several changes of alcohol to
remove the xylene, then thoroughly rinsed in water.
• The section is now hydrated so that aqueous reagents will readily penetrate the cells
and tissue elements.

Step 3: Apply the Hematoxylin Nuclear Stain
• The slide is now stained with a nuclear stain such as Harris hematoxylin, which
consists of a dye (oxidized hematoxylin or hematein) and a mordant or binding agent
(an aluminum salt) in the solution.
• Initially this stains the nuclei and some other elements a reddish-purple color.
Step 4: Complete the Nuclear Stain by “Blueing”
• After rinsing in tap water, the section is “blued” by treatment with a weakly alkaline
solution. This step converts the hematoxylin to a dark blue color.
• The section can now be rinsed and checked to see if the nuclei are properly stained,
showing adequate contrast and to assess the level of background stain.

Step 5: Remove Excess Background Stain (Differentiate)
• On most occasions when Harris hematoxylin is employed, a differentiation (destaining) step

is required to remove non-specific background staining and to improve contrast. A weak
acid alcohol is used. After this treatment, blueing and thorough rinsing is again required.
Step 6: Apply the Eosin Counterstain
• The section is now stained with a solution of eosin. This colors many nonnuclear elements
in different shades of pink.
Step 7: Rinse, Dehydrate, Clear and Mount (Apply Cover Glass)
• Following the eosin stain, the slide is passed through several changes of alcohol to remove
all traces of water, then rinsed in several baths of xylene which “clears” the tissue and
renders it completely transparent. A thin layer of polystyrene mountant is applied, followed
by a glass coverslip.

Routine Staining (Hematoxylin & Eosin) Nuclear detail/definition - Hematoxylin
Contrasting counterstain - Eosin
References & Credits
• Anderson J: An Introduction To Routine And Special Staining
• Avwioro Godwin (2011) histochemical uses of haematoxylin - a review, JPCS
vol (1)
• Brown Skip, The Science and Application of Hematoxylin and Eosin
Staining, Robert H. Lurie Comprehensive Cancer Center Northwestern
University
• Carson FL. Histotechnology: a self-instructional text. 2nd ed. Chicago:
American Society of Clinical Pathologists Press; 1996
• Pranab Dey (2018), Basic and Advanced Laboratory Techniques in

Histopathology and Cytology. © Springer Nature Singapore Pte Ltd
• Sampias C and Rolls G, (2022), H&E Staining Overview: A Guide to Best

Practices. Leica Biosystems Nussloch GmbH 2022
• Suvarna, S.K, Christopher L, Bancroft J.D, Bancroft`s Theory & Practice of

Histological Techniques, 7th edition, Churchill Livingstone, NY, U.S.A.

Ephraim Imhotep Zulu
End of Lecture
Cellular Pathology

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Cellular Pathology

Ephraim Imhotep Zulu, BSc BMS, MSc Path

• Principle of Hematoxylin and Eosin staining

• Phosphotungstic Acid Haematoxylin

• Hematoxylin Without A Mordant

• Staining protocol of H&E

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 2

• Know the Principles, applications and limitations of H&E technique

• Appreciate the Classification of Hematoxylins

• Know the staining procedure of H&E technique

• Differentiate/Compare and contrast:

• Harris vs. Mayer's Hematoxylin

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 3

• When it is properly performed it has the ability to demonstrate a wide range of

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 4

• With proper differentiation, it can distinguish between the cytoplasm of

• As a cytoplasmic stain, it is usually used as a 0.5 or 1.0% solution in distilled

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 5

Haematoxylon campechianum Tree logs. hematoxylin powder.

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 6

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 7

• Hematoxylin itself is not a stain.

• The major oxidization product is hematein, a natural dye

• Hematein can be produced from hematoxylin in two

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 8

• AKA ripening, is a slow process

• Resultant solution seems to retain its

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 10

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 11

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 12

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 13

• The mordant is aluminum, usually in the form of ‘potash alum’

• This procedure is known as ‘blueing’.

• Can be used regressively or progressively

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 14

Table 8.1 Essential types of haematoxylin

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 16

Table 10.1 Staining times with alum hematoxylins

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 17

• In this case satisfactory nuclear staining can be achieved by using an iron-mordanted

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 18

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 20

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 22

• Can be prepared using chemical oxidation or natural ripening of the

tungsten hematoxylin solution in light and air.

• A routine progressive stain for nervous tissue owing to its ability to

stain astrocytes, fibroglia, myoglia, muscle striations, collagen,

reticulin, fibrin, etc in shades of blue and red

• Tissue sections may be treated with Mallory bleach to suppress

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 23

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 24

• Eg the Thomas technique for the demonstration of

• Thomas method also stains argentaffin cell granules.

• Gives the following Results: Collagen and coarse reticulin

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 25

• have recently been used in the demonstration of the

• The most practical diagnostic application is in the

• it is also used in research procedures such as in the

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 26

• The basis of the Mallory methods is the ability of

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 27

Tuesday, April 19, 2022 Ephraim Zulu - HISTOPATHOLOGY 28

Fig. 8.2 Basic steps of 10 minutes in xylene, 3

Step 3 Nuclear stain: Haematoxylin 5 to 15 minutes

HISTOPATHOLOGY - 10 - Haematoxylin & Eosen Technique

HISTOPATHOLOGY - 10 - Haematoxylin & Eosen Technique