The document discusses the hematoxylin and eosin stain, which is the most widely used histological stain. It stains cell nuclei blue or black using hematoxylin, and stains cell cytoplasm and connective tissue fibers pink using eosin. The purpose of staining is to identify tissue structures and the presence or absence of disease. Common stains discussed include hematoxylin and eosin, Gram's method, Ziehl-Neelson's method, and Papanicolaou stain. The document also provides details on the chemistry and procedures for hematoxylin and eosin staining.
H and E staining is most important part of the histopathological diagnosis, this presentation is to highlight some important basic concept of the Staining.
H and E staining is most important part of the histopathological diagnosis, this presentation is to highlight some important basic concept of the Staining.
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2. HAEMATOXYLINAND EOSIN
The hematoxylin and eosin stain is the most widely used
histological stain because……
Its comparative simplicity
Ability to demonstrate clearly an enormous number of
different tissue structures.
The hematoxylin stains cell nuclei blue / black
Eosin stains cell cytoplasm and most connective tissue
fibres
MEDICURE
3. Why to stain
The purpose of staining is that of outlining the tissue and
cellular components
To identify tissue
To establish the presence or absence of disease processes.
4. Most commonly used stains
Histopathology – Routine Hematoxylin(H) &
Eosin(E),
In microbiology – Gram’s Method and Ziehl-
Neelson’s method,
In hematology- Romanowsky stain ,
In cytopathology -Papanicoloau stain.
5. Some important terminology
Basophilic- The entities stainable with basic dye &
are substances which are usually acidic in nature.
Acidophilic- The entities stainable with acidic dye
& are substances which are usually basic in
nature.
Sudanophilic - The entities stainable with oil
soluble dyes e.g. sudan III, IV
6. Argyrophilic- The entities stainable with silver
nitrate solution.
E.g. AgNOR
Argentaffin- The entities staininable with silver
nitrate solutions without chemical reduction
procedures
Metachromatic -The entities will stain in a color
or hue different from that of staining solution itself.
7. Definition
Stains:
Stains are chemical substances used to achieve visible color
contrast in the microscopic picture of a prepared tissue.
Staining:
Staining may be loosely defined as treating tissue or cells with a
reagent or series of reagents so that it acquires a color; usually,
no particles of dyes are seen and the stained element is
transparent.
8. DYES
These are essentially aromatic
benzene ring compounds or
derivatives that possess the twin
properties of color and ability to bind to
tissue.
9. Classification
According to the origin of a dye.
1)Natural
e.g. hematoxylin, Carmine, and Saffron
2)Synthetic
e.g. Benzene, toluene, and naphthalene
or phenols
10. Acidic dyes
Acid dyes usually stain basic components such
as cytoplasm, acidophil granules etc.
e.g. Eosin, Acid fuchsin
Basic dyes
Usually stain acidic components such as nucleus,
basophil granules etc.
e.g. Hematoxylin, Basic Fuchsin, Methylene blue.
11. Neutral dyes
These consist of mixtures of basic and
acidic dyes. Both cations and anion contain
chromophoric groups and both have colored
radicles.
e.g. Romanowsky dyes formed by the interaction
of polychrome methylene blue and eosin.
12. Types of Staining reactions
Absorption or direct staining – tissue penetrated
by dye solution
Indirect staining– using intermediary treatment
with mordant.
Physical staining – simple solubility of dye in
element of tissue.
Chemical staining– formation of new substance.
E.g. PAS
Adsorption phenomenon– accumulation on the
surface of compound.
13. Staining methods
Vital staining
Routine staining
Special staining
Other classification
Regressive staining
Progressive staining
14. Vital staining
Applied to living tissue
Accomplished by injecting the staining solution into
some part of animal body
By mixing the stain with living cells.
Primarily used for research purpose
15. Routine Staining
One that stains the different tissues with little
differentiation except between nucleus & cytoplasm.
General relationship among cells, tissues & organs
are demonstrated.
Eg. Hematoxylin & eosin stain
16. Special staining
Special or selective staining demonstrate special
feature of tissue such as
particular cell products,
Microscopic intracellular & intercellular structure.
e.g. PAS stain for mucopolysaccharide
17. Differentiation
Removal or washing out of excess stain until the color
is retained only by tissue component that are to be
studied.
Generally done with Acid alcohol, Ethyl alcohol.
Exposure to air may oxidized & improve the process.
18. Regressive staining
In a regressive stain, the tissue is first overstained &
then partially decolorized.
The process of partial declourization is
(differentiation).
Differentiation is controlled visually by examination
with microscope.
19. Progressive staining
Once the dye taken up by the tissue it is not removed
Differentiation in progressive staining relies solely on
selective affinity of dyes for different tissue element
The tissue is left in dye solution only until it retains
the desired amount of coloration.
21. Armamentarium in staining
Specially designated bench
Staining bench Should be facing window
Slide washing tray made of stainless steel
Bunsen burner – to heat up the stain
Thermostatically controlled hot plate to melt the wax
Microscope to control staining reaction
22. Armamentariuminstaining
Slides are stained in following ways
1. Using staining dishes
Small grooved couplin jars with glass lids
Large staining troughs
2. Using staining racks
Two pieces of stout glass rods 2-4 cm apart
3. Using staining machine
Same as processing machine but carry slide racks
23. Requirementsforstaining
All glassware should be thoroughly cleaned
Correct solvent should be used
Silver and osmic acid solutions should be kept in dark
bottles
Solutions like dilute ammonia should be freshly prepared
Constituents of stain dissolved should follow the formula
Alcoholic solutions of the stain should be kept in glass
stoppered bottles
All dyes should be filtered before use
25. Hematoxylin and eosin technique
Principle
H and E are principle stain for demonstration of nucleus and cytoplasm.
Alum acts as a mordant and the hematoxylin containing alum stains the
nucleus light blue which turns red in the presence of acid.
The cell differentiation is achieved by treating the tissue with acid solution.
The counterstaining is performed using eosin which imparts pink color to
cytoplasm
26. Hematoxylin and eosin technique
Removal of paraffin wax (Deparaffinization)
Removed with xylene (impermeable to stains)
2-3min of xylene immersion sufficient for sections of 10 µ
thickness
First facilitated by warming the slides at 60 degrees oven to melt
the wax
Removal of xylene
Xylene is not miscible with water or low grade alcohols, hence
dipped in two changes of absolute alcohol
27. Hydration(High to low)
After removal from xylene sections are transferred to
absolute alcohol for 1-2min until it becomes opaque
Sections rinsed in second bath of alcohol, drained and taken
to water
Any pigments or deposits should be removed at this stage
28. Hematoxylinand eosin technique
Staining
Slides immersed in hematoxylin (Mayer s, Harris, Gills)
If regressive stain is used longer time is used to overstained
the structures
Differentiation
Sections are dipped in Acid alcohol, agitated and washed in
tap water
Observed under microscope
If underdifferentiated- returned to acid alcohol
If overdifferentiaited – retured to hematoxylin and
differentiated again
29. Hematoxylinandeosintechnique
Blueing
Slides after draining off hematoxylin is transferred to
ammonia water for 2 min. Sections when removed from
hematoxylin or acid alcohol are pink in color
Washing turns them blue
Counterstain(Eosin)
Transfer the slides to 1% aqueous eosin for 2min. Wash in
running water
Dehydration( low to high)
Slides are taken through 3 stages of Alcohol
30. Hematoxylinandeosintechnique
11. Clearing
Sections transferred to xylene and left until clear
Tested for clarity by being held against a dark background
12. Mounting
Surplous xylene wiped off from slide surface
This step completed quickly to avoid section drying
Whole operation takes 5-10 seconds
33. The word hematoxlin is drived from old Greek word
Haimato(blood) and Xylon(wood), reffering to its dark red
color in natural state and to its origin(wood).
A natural dye extracted from the
log wood of tree Haematoxylon
Campechianum .
Basic in nature and stains acidic component of the
tissue, nucleus, mitochondria etc.
34. Theory of H & E staining
The introduction of
hematoxylin is attributed to
Waldeyer in 1862 that used
it as a watery extract but
without very much success
35. Historical aspect of hematoxylin
Two years later Bohmer combined haematoxylin
with alum as a mordant and obtained more specific
staining.
36. Historical aspectof hematoxylin
In 1891 Heidenhain introduced his classical Iron
alum-haematoxylin method which today is still the
standard technique of the cytologist.
37. Ehrlich (1886) who overcame the instability of hematoxylin
and alum by the additions of glacial acetic acid and at the
same time produced his formula for haematoxylin as it is
used today.
Historical aspect of hematoxylin
38. Hematoxylin
Dark red color
The hematoxylin is extracted from log wood with hot water
and then precipitated out from the aqueous solution using
urea.
It is sold commercially as a crude mixture of hematoxylin
and other, unidentified substance.
It comes as a brownish tan powder which is poorly soluble
in water and somewhat more soluble in ethyl alcohol.
39. Hematoxylin itself is not a stain.
On oxidation it produces HEMATIN - a poor dye but
metallic mordant, forms the most powerful stain.
When aluminum salts– will stain blue
When ferric salt– will stain blue-black.
40. Ripening
This process of oxidation is often referred to as
ripening or maturing.
This can be carried out in two ways
1. Natural oxidation
2. Chemical oxidation
41. Natural oxidation:
Carried out by exposure to light and air.
Slow process
Resultant solutions seem to retain its staining ability for a
long time.
Advantage
Ones oxidation has reached an acceptable level, the staining
solution may be used, and it last for longer,
Disadvantage
In the planning and organization required ensuring that usable
solution is always available. For example: Ehrlich’s and
Delafield’s hematoxylin.
42. Chemical oxidation:
It is achieved by the addition of the oxidizing agents
such as mercuric oxide, sodium iodate and
potassium permanganate.
The use of chemical oxidizing agents converts the
hematoxylin to haematin almost instantaneously,
so these hematoxylin solutions are ready for use
after preparation.
43. Properties of chemically oxidized
hematoxylin
Have a shorter useful life than the naturally oxidized
haematoxylins .
However, the possibility of over – oxidation has been
clearly established that the production of oxyhaematein
inhibits successful staining.
To prevent these, Glycerol has been incorporated in
many formulas. Glycerol acts as stabilizer by
preventing over oxidation and reducing evaporation.
44. Properties
Haematin is anionic, having poor affinity for tissue.
It is an inadequate stain without the presence of
mordant. (most useful mordant are salts of
aluminium,)
Hematoxylin solution using Lead as a mordant are
occasionally used for demonstration of argyrophil
cells.
45. Mordant
Mordant can be defined as polyvalent metal ion
which forms coordination complexes with
certain dyes.
A substance which acts as an intermediary between
dye and tissue
46. TYPES OF HAEMATOXYLIN
1. Alum haematoxylins
2. Iron haematoxylins
3. Tungsten haematoxylins
4. Molybdenum haematoxylin
5. Lead haematoxylins
6. Haematoxylin without mordants
48. Alum hematoxylin:
Routinely used in the hematoxylin and eosin stain,
and produce good nuclear staining.
The mordant is aluminium, in the form of ‘potash
alum’- aluminium ammonium sulphate.
The alum hematoxylin can be used regressively or
progressively.
49. The most commonly used hematoxylin are
Ehrlich’s, Mayer’s, Harris’s, Cole’s and Delafield’s
haematoxylins.
Carazzi’s hematoxylin is occasionally used,
particularly for urgent frozen sections.
50. Harris’s hematoxylin: (1900)
This is an alum hematoxylin which is traditionally chemically
ripened with Mercuric oxide (sodium or potassium iodate
may be used as substitutes for oxidation.).
It is a powerful and selective nuclear stain giving clear
nuclear staining.
It is used widely as a nuclear stain in exfoliative cytology.
In routine histology practice it is used regressively, but in
exfoliative cytology it may be used as a progressive stain.
51. preparation
Haematoxylin 2.5gm
Absolute alcohol l25ml
Potassium alum 50gm
Distilled water 500ml
Mercuric oxide 1.25gm
Glacial acetic acid 20ml
Acetic acid is optional but its inclusion gives more
precise and selective staining of nuclei
52. Harris’s hematoxylin: (1900)
Dissolve hematoxylin in alcohol
Add to it alum, previously dissolved in hot water.
The mixture is rapidly brought to boil
Mercuric oxide is then slowly and carefully added, when the
solution turns dark purple.
The stain is rapidly cooled under tap water.
Filter before use.
53. Mayer’s hematoxylin: (1903)
A next widely used hematoxylin
Chemically ripened with sodium iodide.
It is more vigorous in action than Ehrlich’s
hematoxylin and gives little or no staining of muco-
polysaccharide material.
54. It is used as a nuclear counter stain in the
demonstration of glycogen (PAS, mucicarmine) in
various enzyme histological techniques.
The stain is applied for short period (progressive
stain usually 5-10 min.) until nuclei are stained, and
is then blued without any differentiation.
Differentiation might destroy or decolour the stained
cytoplasmic components. It can be used as a
regressive stain like any alum hematoxylin.
56. The hematoxylin, potassium alum, sodium iodate is
dissolved in distilled water by warming and stirring, or
by allowing to stand at room temperature overnight.
The chloral hydrate and citric acid are added, and the
mixture is boiled for 5 minutes, then cooled and
filtered.
Chloral hydrate acts as a preservative and citric acid
sharpens nuclear staining.
57. Ehrlich’s hematoxylin: (1886)
This is a naturally ripened alum hematoxylin, most
commonly used in both normal and morbid histology.
Preparation of solution:
Hematoxylin - 2 g
Absolute alcohol - 100 ml
Glycerol - 100 ml
Distilled water - 100 ml
Glacial acetic acid - 10 ml
Potassium alum - 10 – 14 g
( in excess )
58. Dissolve the hematoxylin in the alcohol.
The incorporation of glycerol.
Finally, add potassium alum (till saturation).
The stain may be ripened naturally by allowing to
stand in large flask, loosely stoppered with cotton
wool.
Filter before use.
59. It may be partially oxidized
Stain used immediately by the addition of 0.3 g
sodium iodate to the above.
It also stains mucin in salivary glands, some
muco-polysaccharide substances such as cartilage,
and ‘cement lines’ of bone etc. Ehrlich’s
hematoxylin is not ideal for frozen sections.
61. The hematoxylin is dissolved in 25 ml of alcohol
added to solution ‘B’ (alum solution).
Mixture is allowed to stand in light and air for 5 days
and is then filtered.
Added to solution ‘C’.
Allowed to stand exposed to light and air for about 3-4
months or until the stain is sufficiently dark in color,
Then filtered and stained.
62. Cole’s hematoxylin: (1943)
This is an alum hematoxylin,
Artificially ripened with an alcoholic iodine solution.
It has good keeping qualities and is suitable for use in
sequence with celestine blue, unlike Ehrlich’s
hematoxylin.
64. The hematoxylin is dissolved in warm distilled water
and mixed with iodine solution.
The alum solution is added, and the mixture
brought to boil, then cooled quickly and filtered.
The solution is ready for immediate use,but may
need on occasion filtering after storage,
66. Carazzis Haematoxylin
Preparation
Haematoxylin 0.5gm
Glycerol 100ml
Potassium alum 25gm
Distilled water 400ml
Potassium iodate 0.1gm
Usable for 6 months
Used as progressive nuclear counter stain
Largely confined to use with frozen sections
because it gives excellent and clear nuclear
staining with a very short staining time
67. Hematoxylin is dissolved in the glycerol
and the alum is dissolved in most of the water
overnight.
The alum solution is added slowly to solutition.
Potassium iodate is dissolved in the rest of the water
with gentle warming and is then added to the
haematoxylin-alum-glycerol mixture.
The final staining solution is mixed well and is then
ready for immediate use, it remains usable for about six
months.
68. Gill’s hematoxylin: (1974)
Preparation of solution:-
Distilled water - 730 ml
Ethylene glycol - 250 ml
Hematoxylin - 2 g
Sodium iodate - 0.2 g
Aluminium sulphate - 17.6 g
Glacial acetic acid - 20 ml
69. Advantages
They are fast in action,
Stable for at least 12 months,
Produce little or no surface precipitate,
Their preparation does not involve boiling the
solution.
70. Staining Times with alum
haematoxylins:
It will vary according to following factors:
Type of hematoxylin used:- e.g. Ehrlich’s hematoxylin
20-45 min, Mayer’s hematoxylin 10-20 min.
Age of stain: as the stain ages the staining time will
need to be increased.
Intensity of use of stain: A heavily used hematoxylin will
loose its staining power rapidly and longer staining
times will be necessary.
71. Celestine blue- alum hematoxylin:
Preparation of solution:
Celestine blue solution
Celestine blue B -2.5 g
Ferric ammonium sulphate -25 g
Glycerol -70 ml
Distilled water - 500 ml
72. Ferric ammonium sulphate is dissolved in cold
distilled water with stirring.
The celestine blue B is added to this solution and
the mixture is boiled for few minutes.
Filtered
glycerin is added.
Filter before use.
73. Celestine blue B,
an oxazine dye,
Has little useful colouring property of its own.
It forms an additional strong mordant with certain
haematoxylins .
Celestine blue B is used as a preliminary to alum
hematoxylin staining.
74. TYPES OF IRON HEMATOXYLINS
Weigert’s hematoxylin
Heidenhain’s
hematoxylin
Verhoeff’s
hematoxylin
Loyez hematoxylin
75. Iron haematoxylin
Iron salts such as ferric chloride or ferric ammonium
sulphate are used both as oxidizing agent and as
mordant.
Over oxidation is a problem with these stains
76. Therefore mordant/oxidant & haematoxylin
solution are prepared separately and mixed
before use
Capable of demonstrating wider range of
tissue structures compared to alum
haematoxylin but techniques are more time
consuming and needs microscopic control
for accuracy
77. Weigert’s hematoxylin (1904):
An iron hematoxylin used as a nuclear stain in
techniques where acidic staining solutions are
applied to the sections subsequently (e.g. Van
Gieson stain).
In Van Gieson stain, picric acid is one of the
constituents which have marked decolorizing action
on nuclei stained with alum hematoxylin.
78. preparation
Hematoxylin solutions
Haematoxylin 1gm
Absolute alcohol 100ml
This is allowed to ripen naturally for four weeks before use
Iron solution
30%aqueous ferric chloride 4ml
Hydrochloric acid(conc.) 1ml
Distilled water 95ml
79. Ferric ammonium sulphate as oxidant/ mordant.
Heidenhain’s hematoxylin is a cytological stain.
It is used regressively
It may be used to demonstrate
chromatin,
chromosomes,
nuclei,
centerosomes,
mitochondria,
muscle striations
myelin.
Heidenhain’s hematoxylin (1896):
81. Loyez haematoxylin
Ammonium sulphate is used as mordant
Differentiation is byWeigert’s differentiator
(borax and potassium ferricyanide)
Used to demonstrate myelin
Can be applied to frozen, paraffin and nitro
cellulose sections
82. Verhoeff’s hematoxylin (1908):
Verhoeff’s hematoxylin is used to demonstrate
elastic fibers after all routine fixative.
Coarse fibres are intensely stained, but the staining
of fine fibers may be less than satisfactory.
The differentiation step is critical to the success of
this method.
83. TUNGSTEN’S HAEMATOXYLINS
Widely usedTungsten’s haematoxylin is PTAH
(PHOSPHOTUNGSTICACID HEMATOXYLIN )
Used to demonstrate fibrin, muscle striations,
cilia and glial fibres
Myelin can also be demonstrated
Widely used as CNS stain
84. Preparation
PTAH solution using haematin
Haematin 0.5gm
Phosphotungstic acid 5gm
Distilled water 500ml
Stain is ready after 24hours
PTAH solution (KMnO4)
Haematoxylin 0.5gm
Phosphotungstic acid 10gm
Distilled water 500ml
Aqueous KMnO4 25ml
Peak staining activity after 7 days
85. RESULTS
Muscle striations, neuroglia fibres, fibrin, amoeba
– Dark blue
Nuclei, cilia, RBC – Blue
Myelin – Lighter blue
Collagen, osteoid, cartilage, elastic fibres – Deep
brownish red
Cytoplasm - Pale pinkish brown
86. Molybdenum haematoxylins
Haematoxylin solution using molybdic acid as
mordant. Its a rare stain
Used as demonstration of collagen, coarse
Reticulin and also stains Argentaffin cell
granules
87. Preparation
Haematoxylin solution
Haematoxylin 2.5gm
Dioxane 49ml
Hydrogen peroxide 1ml
Phosphomolybdic acid solution
Phosphomolybdic acid 16.5gm
Distilled water 44ml
Diethylene glycol 11ml
Resultant solution shouled be dark violet and
allowed to stand for 24hrs
88. Results
Collagen and coarse reticulin – violet/black
Argentaffin cells – black
Nuclei – pale blue
Paneth cells – orange
Tissue fixed in dichromate do not give good
results
89. Lead Haematoxylin
Used in demonstration of granules in endocrine
cells of alimentary tract and other regions
Most practical diagnostic application is in
identification of endocrine cells in tumors of
doubtful origin
Also used in localisation of gastrin
secreting cells in stomach
90. Haematoxylin without a
mordant
Freshly prepared haematoxylin is used to
demonstrate various minerals in tissue
sections
These methods are now replaced with more
specific techniques
91. Test for staining power of hematoxylin…
Adding few drops of hematoxylin to 50ml of tap
water will turn a bright, clear purple or blue violet
color.
Exhausted solutions will not be clear & bright & the
color will be rusty or green.
92. Hematoxylin Specific use
Harris;’s hematoxylin Exfoliative cytology
Mayers hematoxylin Nuclear counter stain in PAS
Ehrlichs hematoxylin Mucin & other mucopolysaccharide
Carazzis hematoxylin Progressive nuclear stain & frozen section
Celestine blue- alum hematoxylin Where subseqent acidic stains are to be
used e.g. van Gieson stain
Weigerts hematoxylin Where subseqent acidic stains are to be
used e.g. van Gieson stain
Heidenhains hematoxylin Differentiating cytological fluid stain
Loyes hematoxylin To demontrate myelin, frozen section
Tungsten hematoxylin Fibrin , & muscle straitions
Molybdenum hematoxylin Demonstration of collegen fibre
Lead hematoxylin Granules of endocrine cell
Hematoxylin without mordant Pigment lead , iron
93. Eosin
Stains connective tissue and cytoplasm in
varying intensity and shades (red to pink)
Eosin is derived from fluorescein and is
available in following types:
Eosin Y (eosin yellowish, eosin water soluble)
Ethyl eosin (eosin S, eosin alcohol soluble)
Eosin B (eosin bluish, erythrosine B)
94. Eosin Y is most commonly used and is readily
soluble in water, less so in a alcohol thus it is
sometimes sold as ‘water and alcohol soluble’
Preparation
Eosin Y, water soluble 5gm.
Distilled water 1000ml.
Crystals of THYMOL added to inhibit the growth of
fungi.
95. Alcohol soluble eosin is employed as a 0.5 % solution
in alcohol.
Eosin Y water &
Alcohol soluble 10gm
Distilled water 50ml
95%ethyl alcohol 940ml
In use, sections should be treated with 95% alcohol
before staining with alcoholic eosin, and the excess
stain washed out in the same solvent.
96. The addition of little ACETIC ACID (0.5 ml to 1000
ml stain) is said to sharpen the staining.
Ethyl eosin and eosin B are now rarely used,
although occasional old methods specify their use,
e.g. the Harris stain for Negri bodies.
97. Bibiliography
John D. Bancroft and Alan StevensTheory and
practice of histological techniques (3rd ed)p9
no 174.186 vol3Hematoxylins and eosin. John
D. Bancroft • Christopher Layton.
Lynch’s Medical laboratory technology (4th ed)
Internet sources