H & e staining part 2

Hematoxylin & Eosin Staining
Presentedby
Dr.ShrikantSonune
Guided by
DrAshokPatil,
DrShilpa Kandalgaonkar,
DrMayurChaudhary,
DrSuyogTupsakhare,
DrMaheshGabhane.

Hematoxylinand eosintechnique
 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

 Removal of paraffin wax (dewaxing)
 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

Hematoxylin and eosin technique…..
 Hydration – 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

 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

 Blueing
 Slides after draining off hematoxylin is transferred to water
for 10min. Sections when removed from hematoxylin or
acid alcohol are pink in color
 Washing turns them blue
 Counterstain
 Transfer the slides to 1% aqueous eosin for 2min. Wash in
running water
 Dehydration
 Slides are taken through 3 stages of acid alcohol

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

 Results
 Cell nuclei – blue
 Muscle fibres – red
 Collagen fibres – pink
 RBC – bright red

Hematoxylin:
 Most commonly used stain.
 It is derived from core of the logwood tree, Haematoxylon
compechianum,
 It is not a true dye until partially oxidized (ripened)
----by exposure to air
----by chemical means with an oxidizing agent such as sodium
iodate or mercuric oxide.

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 --cell nuclei blue / black,
 Eosin stains cell cytoplasm and most connective tissue fibres

Thehematoxylinmaybeusedinthedemonstrationof:
 Intracellular substances
 Chromosomes
 Keratohyaline granules
 Extracellular substances – Elastin
 Minerals – calcium, copper etc.
 Central nervous system – myelin, neuroglia
fibers.

Hematoxylin
 Dark red color
 Extracted from the heartwood of the tree Hematoxylin
camechianum
 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.

 Hematoxylin itself is not a stain.
On oxidation it produces haematin - a poor dye but
Metallic mordant, forms the most powerful stain.
 When aluminum salts– will stain blue
 When ferric salt– will stain blue-black.

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

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.

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.

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.

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.

Classification according to which mordant is
used.
 Alum haematoxylins
 Ehrlich’s
 Mayer’s
 Harris
 Cole’s
 Delafield
 Carazzi’s

Classification according to which mordant is
used.
 Iron haematoxylins
 Weigert
 Heidenhain’s
 Loyez
 Verhoeff
 Tungesten
 Molybdenum
 Lead
 Hematoxylin without mordant

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 nuclei 1st become a red colored,
 Differentiation is carried out
 Then, Converted to familiar blue-black when the
section is washed in weak alkali.
 The alum hematoxylin can be used regressively or
progressively.

The tomes for hematoxylin staining and for satisfactory
differentiationwill varyaccording to:
 The type and age of alum hematoxylin used.
 The type of tissue.
 The personal preference of the pathologist.
 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.

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.

Preparation of solution:
 Hematoxylin - 1 g
 Absolute alcohol - 10 ml
 Ammonium or potassium alum - 20 g
 Distilled water - 200 ml
 Mercuric oxide - 0.5 g

 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.

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.

 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.

Mayer’s hematoxylin: (1903)
 Preparation of solution:
 Potassium or aluminium alum - 50 g
 Sodium iodate - 0.2 g
 Citric acid - 1 g
 Chloral hydrate - 50 g

 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.

Ehrlich’s hematoxylin: (1886)
 This is a naturally ripened alum hematoxylin, most
commonly used in both normal and morbid histology.
 Absolute alcohol - 100 ml
 Glycerol - 100 ml
 Glacial acetic acid - 10 ml
 Potassium alum - 10 – 14 g
( in excess )

 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.

 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.

Delafield’s hematoxylin: (1885)
 This is a naturally ripened alum hematoxylin, which has similar
longevity to Ehrlich’s hematoxylin.
 Solution A:
Hematoxylin - 4 g
Absolute alcohol - 25 ml
 Solution B:
Alumunium alum - 60 g
Distilled water - 400 ml
 Solution C:
Glycerol - 100 ml
Absolute alcohol - 100 ml

 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.

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.
 Hematoxylin - 1.5 g
 1%iodine in absolute ethanol - 50 ml
 Saturated aqueous potassium alum - 700 ml

 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,

Carazzi’s hematoxylin: (1911)
 This is an alum hematoxylin which is chemically
ripened using potassium iodate.
 Hematoxylin -5 gm
 Glycerol -100 ml
 Potassium alum - 25 gm
 Potassium iodate - 0.1 g

 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.

 Carazzi’s hematoxylin may be used as a
progressive nuclear counter stain using a short
staining time followed by blueing in tap water.
 It is particularly suitable since its pale and precise
nuclear stain, does not stain any of the cytoplasmic
components.
 It is particularly used for the frozen section (due to
rapid staining)

Gill’s hematoxylin: (1974)
 Preparation of solution:-
 Ethylene glycol - 250 ml
 Sodium iodate - 0.2 g
 Aluminium sulphate - 17.6 g
 Glacial acetic acid - 20 ml

 The reagents are added in the order given and the
mixture stirred for 1 hour at room temperature.
 The solution is ready for immediate use.
 Double or triple concentrations of ethylene glycol
may be used as preferred
 Gill’s 1 (normal),
 Gill’s 2 (double conc of ethylene glycol),
 Gill’s 3 (triple conc of ethylene glycol).

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.

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.

 Progressively or regressively method.
e.g. Mayer’s hematoxylin used progressively 5-10
min, used regressively 10-20 min.
 Pretreatment of tissues or sections
e.g. length of time in fixative or acid decalcifying
solution or whether paraffin or frozen section.
 Post treatment of sections
e.g. subsequent acid stains such as Van Gieson.
 Personal preference.

Disadvantages of alum hematoxylin
 Sensitivity to any subsequently applied acidic
staining solutions
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.

 Nuclear staining can be achieved by using an iron –
mordanted hematoxylin e.g.Weigert’s hematoxylin,
 Alternative is the combination of celestine blue
staining solution with an alum hematoxylin.
 Celestine blue is resistant to the effects of acid and
ferric salt.
 Strengthens the bond between the nucleus and alum
hematoxylin.

Celestine blue- alum hematoxylin:
 Preparation of solution:
 Celestine blue solution
 Celestine blue B -2.5 g
 Ferric ammonium sulphate -25 g
 Glycerol -70 ml

 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.

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.

Iron haematoxylin
 Iron salts such as ferric chloride or ferric ammonium
sulphate are used both as oxidizing agent and as mordant.
 Demonstrating a much wider range of tissue structures than
the alum haematoxylins,
The most common iron haematoxylins are
 Heidenhain’s hematoxylin
 Weigert’s hematoxylin
 Verhoeff’s hematoxylin
 Loyez hematoxylin

 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. Weigert’s hematoxylin
 To use them consecutively (e.g. Heidenhain’s and
Loyez haematoxylins).
 It may be used as
 Differentiating fluid after hematoxylin staining,
 Mordanting fluid before it.

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.
 Weigert hematoxylin which is mordanted to iron salt
(ferric chloride) has sufficient avidity to withstand this
treatment.

Preparation:
The iron and hematoxylin solutions are prepared separately and
are mixed immediately before use.
Solution A (stain) Solution B
(mordant)
Hematoxylin – 1 g 30% aqueous ferric chloride
Absolute alcohol – 100 ml ( anhyd. ) -
4 ml
Conc. HCl - 1 ml
Dist. Water - 95ml
The color of the mixture should be a violet black.
If muddy – brown, it must be discarded.

Heidenhain’s hematoxylin (1896):
 Ferric ammonium sulphate as oxidant/ mordant.
 The same solution is also used as a differentiating
fluid.
 The iron solution is used first
 The section is treated with hematoxylin solution
until it is over stained,
 Then it is then differentiated with iron solution under
microscopic control.

 Heidenhain’s hematoxylin is a cytological stain.
 It is used regressively
 Requires careful differentiation, for which reason it is
only completely successful on thin sections.
 It may be used to demonstrate
 chromatin,
 chromosomes,
 nuclei,
centerosomes,
mitochondria,
muscle striations
myelin.

Heidenhain’s hematoxylin (1896):
Preparation:
Hematoxylin solution (stain)
Hematoxylin - 0.5g
Absolute alcohol- 10ml
Distilled water- 90ml
Iron solution (mordent and differentiator)
Ferric ammonium sulphate- 5g
Distilled water - 100ml

 Dissolved hematoxylin in alcohol
 add water.
 Allow to ripen for a few weeks and store in a tightly
stoppered bottle.

Loyez hematoxylin (1910):
 This is an iron hematoxylin in which ferric ammonium
sulphate is used as the mordant.
 The mordant and hematoxylin solution are used
consecutively,
 Differentiation is by Weigert’s differentiator (borax and
potassium ferricyanide).
 It is used to demonstrate myelin and can be applied to
paraffin, frozen or nitrocellulose sections.

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.

 Introduced by Mallory in 1897 for demonstration of
neuroglia.
 It is now widely used for other purposes (fibrin,
muscle striation).
 It is unique among hematoxylin stains in the
number of structures that may be demonstrated,
together with the two color staining (shades of blue
and red) from the single solution.
Tungsten hematoxylin

 Mallory first advocated a 1: 10 ratio of hematoxylin
to mordant, later a 1: 20 ratio should be used with
pure mordant due to earlier mordant was impure.
 Later Turner et al proposed that this proportion is
too high and does not give the most satisfactory
staining.

 Natural ripening of phosphotungstic acid
hematoxylin is slow and several chemical oxidizing
agents have been used to hasten the conversion of
hematoxylin to haematein. As an alternative to
chemical oxidation,
 Haematein may be used initially, instead of
hematoxylin and provides a stain that is usable 24
hours after preparation.

Molybdenum hematoxylin:
 Molybdenumic acid as the mordant are rare,
 Technique which gained any acceptance was the
Thomas (1941) technique which was mentioned by
MacManus and Mowry (1964).
 Demonstration of collagen and coarse reticulin,
 More valuable and accepted for these connective
tissue fibre exist

Lead hematoxylin:
 Hematoxylin solutions which incorporate Lead
salts have recently been used in the demonstration
of the granules in the endocrine cells of the
alimentary tract and other regions

Hematoxylin without a mordant
 Freshly prepared hematoxylin solutions, used
without a mordant,
 Used to demonstrate various minerals in tissue
sections – Mallory described a method for lead, iron
and copper.
 The basis of the Mallory methods is the ability of
unripened hematoxylin to form blue black lakes with
these metals.

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.

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

Eosin
 Eosin, a red dye,
 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)

 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.

 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.

 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.

H & e staining part 2

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