SEMINAR

ON

HAEMATOXYLIN AND EOSIN STAINING

SUBMITTED BY,

Dr.Lakshmi S Anand

II MDS
INTRODUCTION

Haematoxylin and eosin is probably the most widely used histological stain. Its popularity

is based on its comparative simplicity and wide range of applications. This is due to the fact that

haematoxylin and eosin show most histological structures, and are particularly suitable for the

demonstration of nuclei which are the most important structures in almost every section. Even

when not sufficient by themselves, they usually provide information to indicate which other

staining methods are to be used.

Essentially, the hematoxylin component stains the cell nuclei blue/black with good intra-

nuclear detail, while the eosin stains cell cytoplasm and most connective tissue elements in

varying shades and intensities of pink. Both of these however, have many more uses than in H&E

combinations.

HAEMATOXYLIN

Haematoxylin is extracted from the heart-wood (logwood) of the tree (Haematoxylin

campechianum). Pure haematoxylin is colourless but it can be readily be oxidized to the reddish

dye haematein which is the active dyestuff in all the so called haematoxylin solutions. This

change occurs as soon as the logwood is exposed to air so that pure colourless haematoxylin is

never used. Haematein itself is not entirely stable, being rendered colourless by further oxidation.

Haematein, which is itself a poor dye, in the presence of a metallic mordant (usually aluminium,

iron or tungsten) forms a most powerful stain. Oxidation of haematoxylin gives rise to a

paraquinoid structure which is responsible for the staining properties of haematein.

Oxidation

1. Natural oxidation (natural ripening): involves exposure of solutions of heamatoxylin to

sunlight and air for about 6-8 weeks. Alternatively, oxygen or air may be bubbled through the

solution for 3-4 weeks. Eg: Ehrlich’s and Delafield’s hematoxylin.

 Advantage: Once oxidation has reached an acceptable level, the staining solution may be used

and though the stock will continue to oxidize, it is unlikely to proceed too far. Also, the

solution has longer shelf-life and fading of color with time is less.

Disadvantage: The process is slow. Planning and organization is required to ensure that a

usable solution is always available.

2. Chemical oxidation (artificial ripening): is achieved by the addition of oxidizing agents such as

mercuric oxide, sodium iodate and potassium permanganate. Sodium iodate is preferred as it

does not require boiling and hence may contribute to an increased shelf-life.

Advantage: It can be used soon as it is made up.

Disadvantage: Has shorter shelf-life.

Glycerol has been incorporated into many formulas, referred to as a stabilizer, as it

prevents over-oxidation and reduces evaporation.

Blueing:

When sections are first stained with an aluminium haematoxylin, nuclei are a dark red

colour. In order to change it to blue and to stabilize the dye, the sections must be treated with a

weak alkali. Tap water, if it is alkaline, may be used for this purpose. This process is referred to

as Blueing.

Staining

For almost all nuclear staining schedules, haematoxylin is used as a regressive stain. There

are two exceptions:

1. It is occasionally expedient to use haematoxylin progressively for urgent methods.

2. Haematoxylin is sometimes used as a counterstain and may then follow a stain that would be

removed if the haematoxylin is differentiated.

 Whenever possible, progressive staining is to be avoided because it either leaves the nuclei

too pale or colors the cytoplasm with the haematoxylin.

Types of haematoxylin

According to the mordant used, it is classified into:

1. Alum haematoxylin

2. Iron haematoxylin

3. Tungsten haematoxylin

4. Molybdenum haematoxylin

5. Lead haematoxylin

6. Haematoxylin without mordant

Many haematoxylin mixtures, which are known by the name of the worker who first used

them, are used.

I. Alum haematoxylins

This group comprises most of the hematoxylins used routinely in H&E stains. The mordant

is usually in the form of potash alum (aluminium potassium sulphate) or ammonium alum

(aluminium ammonium sulphate). All alum hematoxylins stain the nuclei a red color, which is

converted to blue by the process of blueing.

The time for staining with alum hematoxylins will vary according to the type and age of

the hematoxylin used, the type of tissue and the preference of the pathologist. The commonly used

alum hematoxylins in routine lab work are Ehrlich’s, Harris’s, Mayer’s, Gill’s, Cole’s and

Delafield’s. Carrazzi’s hematoxylin is occasionally used, particularly for urgent frozen sections.

1. Mayer’s Haematoxylin (Mayer 1903)

Formula

Haematoxylin 1 g.
 Distilled water 1000 ml.

Ammonium alum 50 g.

Sodium iodate 0.2 g.

Citric acid 1 g.

Procedure

Dissolve the haematoxylin in distilled water using gentle heat. Add the alum, shaking to

dissolve, followed by the sodium iodate (oxidizing agent). Continue heating whilst adding the

citric acid and chloral hydrate. The stain is ready for use when cooled.

Chloral hydrate – acts as a preservative while citric acid is used to acidify the stain which

is believed to sharpen nuclear staining.

Uses

It can be used as a regressive stain like all alum H. however, it is most useful as a

progressive stain, particularly in situations where a nuclear counter-stain is needed to emphasize a

cytoplasmic component which has been demonstrated using any special stain, and where the acid-

alcohol differentiation might destroy or de-color the special stain. Thus it is used as a counterstain

in procedures such as PAS, mucicarmine and many enzyme histochemical procedures. The

staining time in these cases is usually 5-10 min.

2. Harris’s Haematoxylin (Harris 1900)

Formula:

Haematoxylin 2.5 g.

Absolute alcohol 50 ml

Ammonium alum 50 g

Distilled water 500 ml

Mercuric oxide 1.5 g.

 Glacial acetic acid 20 ml

Procedure

Dissolve the haematoxylin in the alcohol and the alum in hot water. Mix the two solutions

together and heat to boiling. Add the mercuric oxide (Oxidizer) and cool rapidly by plunging the

flask into cold water. The solution is ready for use. Glacial acetic acid, added after cooling gives

more precise nuclear staining.

Mercuric oxide is highly toxic, environmentally unfriendly, and can have detrimental,

corrosive long-term effects on automated staining machines. Therefore, the HgO of original

Harris’s H has currently been replaced by Sodium or Potassium Iodate (0.5gm).

Use

It is a popular method for routine use. The heamatoxylin does not need time to ripen.

Results are consistent and staining schedule is fairly rapid. It gives particularly clear nuclear

staining and therefore has been used as a regressive stain in routine histology with a staining time

of 10-15min. When used thus, it is usually differentiated in Acid-alcohol (1% HCl in 70%

Alcohol). However, if used progressively, it is better to use a milder differentiating agent like

acetic acid/alcohol for better control.

Common Errors

i) Tiny dark grey or black granules may be seen due to precipitation of haematoxylin. They can

be avoided by filtering the stain once a week.

ii) Dull red or brown staining of nuclei is due to either over-ripe haematoxylin or insufficient

blueing after differentiation.

iii) Nuclei appear as blue rings with colourless centres when the haematoxylin staining time has

been short.

iv) Uniformly weak staining of the nuclear membrane and chromatin is usually due to over-

differentiation.
v) Sections in which the nuclei are blue and congested without proper chromatin detail have not

had sufficient differentiation. In very bad cases, cytoplasm is an unpleasant slaty-blue colour.

3. Ehrlich’s Haematoxylin (Ehrlich 1886)

Formula

Haematoxylin 6 g.

Absolute alcohol 300 ml.

Distilled water 300 ml.

Glycerol 300 ml.

Glacial acetic acid 30 ml.

Potassium alum–to saturation 10-14 g.

Procedure

The haematoxylin should be fully dissolved in the alcohol before the other ingredients are

added. Finally, the potassium alum is added until there is a deposit of alum crystals on the bottom

of the stock container. The incorporation of glycerol gives more even and precise staining and

also stabilizes the stain against over-oxidation and reduces evaporation. The solution is allowed to

ripen in clear glass bottles over 6-8 weeks. The ripening time can be shortened somewhat by

placing the bottles un-stoppered in a warm sunny place. Artificial ripening by the addition of 0.9 g

sodium iodate to the solution allows it to be used immediately.

Uses

It is a naturally ripened hematoxylin and so stained sections fade much slowly than the

chemically ripened alum hematoxylins. It is a good, strong hematoxylin and stains nuclei intensely

and crisply. It also stains mucin in salivary glands and goblet cells and the ground substance of

cartilage. Because of its high hematoxylin content, it is particularly useful for staining tissues that

have been subjected to acid-decalcification or been in formalin fixatives for very long time.

Advantages:
i) Fine nuclear chromatin is shown more precisely.

ii) The stained sections are even less liable to fading.

iii) It stains some sections that others will not, like sections from tissue which has been stored too

long in fixative or subjected to fierce acid decalcification.

Disadvantages:

i) The long period needed for ripening. The hastening procedures are not recommended as they

spoil the keeping properties of the stain.

ii) The length of time required for staining (45 min). This is due to the inhibitory effect of the

glycerol.

iii) Ehrlich’s haematoxylin is unsuitable for frozen sections because of its high alcohol content.

iv) The high concentration of haematoxylin makes it expensive and hence, is not economical

Common Errors

In addition to the errors found in Harris’s haematoxylin, an additional cause of dull red or

brown nuclear staining is the use of unripened stain.

4. Delafield’s Haematoxylin (Delafield 1885)

This solution is similar to Ehrlich’s in that it also ripens naturally and thus will last

indefinitely.

5. Carazzi’s Haematoxylin (Carrazzi 1911)

It is an alum haematoxylin chemically ripened using potassium iodate. Similar to Mayer’s,

it is usually used as a progressive nuclear counterstain in special stains. It is a pale precise nuclear

stain that does not stain any of the cytoplasmic components. It is not intended for use with eosin as

a histological stain although smears can be stained satisfactorily.

Uses

May be used either as a progressive stain or as a regressive stain. It is used a progressive

stain in methods like Congo red, PAS and histochemical methods for enzymes.
 It is more often used as a regressive stain for fat-stained frozen sections, for smears, or in

conjunction with such stains as alcian blue and mucicarmine.

A particular use of Carrazzi’s H is in rapid staining of frozen sections in case of an urgent

surgical biopsy. In this scenario, it is usually used at double or triple strength solution and

provides good clear nuclear staining with a very short staining time (1min).

Gill’s Haematoxylin (Gill et al. 1974)

It is fast in action, stable for atleast 12 months produces little or no surface precipitate, and

its preparation does not involve boiling the solution. The major disadvantage with Gill’s H is

excessive background staining where it staing the gelatin adhesive and even the glass itself. It is

thought that the mordant used (Aluminium sulfate) is responsible for this effect.

6. Cole’s haematoxylin (Cole 1943)

This solution has good keeping qualities, but will require filtering before use.

Staining Times with Alum Hematoxylins:

The staining time with all alum H vary depending on a number of factors.

1. Type of Haematoxylin: eg. Ehrlich’s H – 45min, Harris’s H – 10-15min.

2. Age of Stain: Staining time will need to be increased for older solutions.

3. Degree or Usage: heavy usage leads to quicker loss of staining power and the staining time

will have to be increased.

4. Whether used progressively or regressively: eg. Mayer’s H used progressively – 5-10min;

used regressively – 10-20min.

5. Pre-treatment of tissues: long time in fixative, acid decalcified tissues etc need longer

staining time. Frozen sections need less staining time.

6. Post-treatment of sections: eg. Subsequent acid stains such as Van Gieson remove

hematoxylin. Increase staining time with alum H or use other H.

7. Personal preference.
The Celestine blue-Alum H Procedure:

The major disadvantage of Alum H’s is their sensitivity to any subsequently applied acidic

staining solutions. The most common examples are the Van Gieson and other trichome stains. The

application of the Picric acid-Acid fuschin mixture in Van Gieson stain removes most of the

hematoxylin so that the nuclei are barely discernible. The problem can be overcome by the use of

either more stronger H’s like Iron H or by combining Celestine blue staining with the Alum H. Of

these Celestine Blue/Alum H procedure is the more recent, more suitable and currently more

popular method.

Celestine Blue Preparation

Celestine Blue B 2.5gm

Ferric Ammonium sulfate 25gm

Glycerin 70ml

Distilled water 500ml

The ferric sulfate is dissolved in cold distilled water with stirring, Celestine blue is added to this

solution and the mixture boiled for a few minutes. After cooling, the stain is filtered and glycerin

is added. Usable for 5 months. Filter before use.

Staining Method

1. Dewax, and hydrate sections

2. Stain in Celestine blue solution for 5min

3. Rinse in distilled water

4. Stain in an Alum H (eg. Mayer’s, Harris’s)

5. Wash in water until blue

6. Proceed with the required staining technique.

Celestine blue is resistant to the effects of acid and the ferric salt in the Celestine blue solution

strengthens the bond between the nucleus and alum H. This provides a strong nuclear stain which

is reasonably resistant to acid used subsequently.

EOSIN

Eosin is the most suitable stain to combine with an Alum H to demonstrate the general

histological architecture of a tissue. Its particular value is its ability, with proper differentiation, to

distinguish between the cytoplasm of different types of cells, and between the different types of

connective tissue fibers and matrices, by staining them differing shades of pink and red.

Eosin, a xanthene dye, is tetrabromofluorescein which may be contaminated with mono-

and dibromo derivatives, affecting the shade of the dye. Of the various eosins available

commercially, water soluble Eosin Y is the most widely used one. It is usually used as a 0.5% or

1% solution in water (which can be diluted from a 5% stock solution as it is less susceptible to the

growth of moulds). The addition of a crystal of thymol or a few drops of formalin also inhibits

mould growth. Calcium chloride and acetic acid have been added to simple eosin solutions to

improve their performance

Differentiation of Eosin occurs in the subsequent tap water wash, and a little further

differentiation occurs during the dehydration through alcohols. The intensity of eosin staining and

the degree of differentiation is a matter of individual preference. Under certain circumstances,

eosin staining can be very intense and adequate differentiation may be difficult; this usually occurs

in tissues fixed using a mercuric fixative.

Uses

Eosin is used as a counter stain for haematoxylin and gives all structures except the nuclei

a uniform pink colour. It can also be used as a specific stain for red blood corpuscles and

eosinophils. This is by differentiating eosin from all other structures except the above.

Standard procedure for staining with haematoxylin and eosin

i) Dewax sections, hydrate through graded alcohols to water.

ii) Stain in Alum Haematoxylin of choice for required time.

iii) Wash in water

iv) If regressive stain is used, differentiate in acid alcohol.

v) Wash in water.

vi) Blue in running water or Scott’s tap- water substitute.

vii) Rinse in water.

viii) Stain in 1 % aqueous eosin – 3-10 minutes.

ix) Wash in running water – 1-5 minutes

x) Dehydrate in three changes absolute alcohol.

xi) Clear in two changes xylene

xii) Mount in suitable synthetic resin.

Results

Nuclei, RNA rich cytoplasm, calcium – Blue

Cytoplasm - Varying shades of pink

Muscle, fibrin, Keratin, Collagen - Deep Pink/Bright red

RBC’s – Orange/ red.

IRON HAEMATOXYLINS

In these haematoxylin solutions, iron salts are used both as the oxidizing agent and the

mordant. Ferric Chloride and Ferric Ammonium Sulfate are the commonly used Iron salts and the

most common Iron H’s are:

1. Weigert’s H

2. Heidenhain’s H

3. Loyez H for Myelin

4. Verhoeff’s H for Elastic fibers

 Over-oxidation of the H is a problem with iron H’s so it is usual to prepare separate

mordant/oxidant and haematoxylin solutions and mix them immediately before use eg.

Weigert’s H or to use them consecutively eg. Heidenhain’s and Loyez H. because of the strong

oxidizing ability of iron salts solutions, it is often used as a subsequent differentiating fluid

after H staining, as well as for a mordanting fluid before it.

The iron H’s are capable of demonstrating a much wider range of tissue structures than

alum H. the techniques are however, time consuming and usually incorporate a differentiation

stage which needs microscopic control for accuracy.

1. Weigert’s haematoxylin: (Weigert 1904)

This is an iron H using ferric chloride as the mordant/oxidant. The iron and haematoxylin

solutions are prepared separately and are mixed immediately before use.

Formula:

Solution A

Haematoxylin 1g.

Absolute alcohol 100 ml.

Use gentle heat to dissolve

Solution B

30% aqueous ferric chloride 4 ml.

Conc. Hcl 1 ml.

Distilled water 100 ml.

Mix equal quantities of A and B immediately before use. The mixture should be a violet

black color and must be discarded it it is brown.

Use

i) Sometimes used with eosin especially for sections of brain

ii) Chief use is in methods where it is used as a nuclear counterstain followed by a ‘searching’

counterstain such as picric acid (in Van Gieson’s stain). Picric acid can effectively remove

alum haematoxylin from cell nuclei but Weigert’s haematoxylin which is mordanted to an iron

salt, has sufficient affinity to withstand this treatment.

2. Heidenhain’s Haematoxylin: (Heidenhain 1896)

The most intense of haematoxylins, Heidenhain’s is used as a regressive stain.

Counterstains are unnecessary and the results lend themselves admirably to photomicrography.

Nuclear detail and muscle striations are beautifully demonstrated and the stain is of sufficient

intensity to be successful on very thin sections. The iron alum solution is used as both mordant

and differentiator. Heidenhain’s haematoxylin is resistant to fading provided that iron alum has

been thoroughly removed by washing.

This haematoxylin produces a dark grey or black colour. What structures are stained will

depend on the degree of differentiation. Mitochondria are decolourized very quickly and cross-

striations of muscles rather more slowly while nuclei are relatively resistant to differentiation.

Keratin and RBC’s remain heavily stained and the cytoplasm of other cells remains dull grey.

It can be used for staining of sections that reject other haematoxylin mixtures like tissue

fixed in osmium tetroxide mixtures, or tissue that spent too long in decalcifying fluid, or was

stored for a very long time in fixative or in alcohol. It may be used with orange G for

demonstration of mitoses and other fine nuclear detail.

Limitations:

1. The tissue section needs to be treated with the mordant solution first followed by the

haematoxylin. The length of time in each is usually very long (1hour-24hours).

2. Hematoxylin staining is followed by differentiation in the iron solution. The degree of

differentiation needs to be microscopically controlled until desired structure is viewed

satisfactorily.

3. If the differentiation proceeds too far then staining has to be started again.

4. If the iron solution is not removed completely after differentiation by washing, the slides will

fade out very quickly.

3. Other Iron Haematoxylins

1. Loyez haematoxylin – used to demonstrate myelin. Iron alum is the mordant used.

2. Verhoeff’s haematoxylin – used to demonstrate elastic fibers. Ferric chloride with Lugol’s

Iodine is the mordant.

TUNGSTEN HAEMATOXYLIN

The most widely used tungsten haematoxylin, Mallory’s phosphotungstic acid

haematoxylin (PTAH) is a connective tissue stain particularly useful for demonstration of muscle

striations and fibrin though originally devised as a technique for CNS. An unusual feature is the

concomitant staining of various structures in two colours - shades of red and blue.

MOLYBDENUM HAEMATOXYLIN

This method is used for the demonstration of collagen and coarse reticulin, although more

valuable and widely accepted techniques for these connective tissue fibers exist.

LEAD HAEMATOXYLINS

This is used in the demonstration of granules in the endocrine cells of the alimentary tract

and other regions.

HAEMATOXYLIN without MORDANT

1. Mallory and Parker: used freshly prepared haematoxylin solution for demonstrating

minerals like Copper, Iron and Lead in tissue sections. Based on the ability of unripened

haematoxylin to form blue/black lakes with these metals.

 2. Weigert-Pal Technique: used for myelin. Does not use a mordant with the haematoxylin,

but the tissue block for this is mordanted in a chromate solution before embedding and

sectioning. So not a true un-mordanted haematoxylin in strict sense.

TROUBLE SHOOTING IN HEMATOXYLIN AND EOSIN STAINING

Problems

1. Pale-stained nuclei

Causes :
1. Too much differentiation
2. Too less time in haematoxylin
3. Due to excessive decalcification
4. Haematoxylin is over oxidized
Remedies :
1. Stain in haematoxylin again
2. Keep in haematoxylin for longer duration
3. Not possible to correct
4. Change the haematoxylin solution
2. Darkly stained nuclei
Causes:
1. Too short differentiation
2. Too much time in haematoxylin
3. Thick section
Remedies:
1. Decolorize and do optimum differentiation
2. Decolorize and give appropriate time in haematoxylin
3. Recut thin section

3. Nuclei looks reddish brown

Causes:
1. Insufficient bluing
2. Haematoxylin is degenerating
Remedies :
1. Restain by giving more time in bluing step
2. Check the oxidation status of haematoxylin
4. Pale-coloured cytoplasm by eosin
Causes :
1. Too thin section
2. The eosin solution has pH more than 5
3. Too much dehydration of the section in alcohol
Remedies :
1. Recut the section properly
2. This may be due to dilution of eosin by the carryover bluing solution. Check pH of eosin
solution, and if necessary, adjust pH by adding acetic acid
3. Do not keep the slide in alcohol for a long time

5. Cytoplasmic staining is very dark

Causes :
1. Long duration in eosin solution
2. Overconcentrated eosin solution
3. Very quick dehydration in alcohol
Remedies :
1. Keep the section in eosin for shorter duration
2. Make optimally diluted eosin solution
3. Increase time duration in dehydration
6. Bluish-black precipitate
Causes :
1.It may be due to precipitation of haematoxylin
Remedies :
1.Filter the haematoxylin staining solution
7. Staining is irregular and spotty
Causes :
1.Improper deparaffinization
Remedies :
1.Keep the slide in xylene for longer time to remove the paraffin
8. Water bubbles in the sections
Causes :
1.Incomplete dehydration
Remedies :
1.Remove the mounting medium and coverslip. Keep the section in absolute alcohol for
dehydration. Do several changes and then remount

9. Milky section after the xylene rinse before putting the coverslip
Causes :
1. Incomplete dehydration
Remedies :
1. Change the alcohol solution. Please dehydrate the section properly before putting in xylene

CONCLUSION

Haematoxylin is probably the single most important dye employed in histological staining.

This dye acts as a morphological reference in many specialized histochemical methods. The value

of haematoxylin is not limited to nuclear staining as it can be used to demonstrate intracellular

substances (eg. chromosomes, keratohyaline), extracellular substances (eg. elastin), ground

substance (e.g. cement lines in bone), minerals (e.g. calcium, copper, etc) and neural elements

(e.g. myelin, neuroglia fibres etc). In exfoliative cytology, haematoxylin stained nuclei are the

most important diagnostic features of Papanicolaou stained smears. Understanding the various

types of haematoxylin and their specific uses will help in more judicious and informative use of

these in diagnostic pathology

REFERENCES

1. CFA Culling, RT Allison, WT Barr: Cellular pathology technique, 4th edition.

2. John D Bancroft: Theory and practice of histological techniques, 5th edition.

3. Basic and advanced laboratory techniques in histopathology and cytology- P.Dey