Hematoxylins and

Eosin
Hematoxylin and Eosin Stain
 Most widely used histological stain
 Hematoxylin- blue-black nuclei
 Eosin- pink, orange, red cytoplasm and
C.T. fibers

E.M.MANGADA, RMT 2
 Extracted from H. campechianum
 Hematein- responsible for the color
properties
 Anionic and inadquate as a nuclear stain

 Chemical oxidation- using sodium iodate,

mercuric oxide

 Natural oxidation- exposure to light and air

◦ Takes as long as 3-4 months
◦ Ex. Erlich’s and Delafield’s

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MORDANTS FOR HEMATEIN
 Aluminum, irom, tungsten, lead
 are cations that binds the dye to tissue


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Gill’s Hematoxylin
 Double or triple hematoxylin
concentrations
 More frequently used for routine H and E
than Mayer’s
 More stable than Haris’s
 Disadvantage:
◦ staining of adhesive and tha glass
◦ Stain mucus darkly

E.M.MANGADA, RMT 5
ALUM HEMATOXYLINS

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ALUM HEMTOXYLINS
 Mordans is usually ‘potash alum’
 Nuclei is red, then, turns blue-black using
the procedure of ‘bluing’
 Can be used regressively and
progressivley
 Erlich’s, Mayer’s, Harris’s, Gill’s, Cole’s and
Delafield’s, Carazzi’s

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Ehrlichs’s hematoxylin
 Takes 2 months to ripen
 An excellent nuclear stain
 Also stains mucins and recommended for
staining of bone and cartilage
 Can be chemically oxidized using sodium
iodate
 Stains nuclei intensely and crisply
 Stained sections fade much slowly than
other hemtaoxylins

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Ehrlichs’s hematoxylin
Preparation of Solution
Hematoxylin 2g
Absolute Aclohol 100 ml
Glycerin 100 ml
Distilled Water 100 ml
Glacial acetic acid 10 ml
Potassium alum 15 g approx.

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Ehrlichs’s hematoxylin
 Suitable for tissues that have been
exposed to acids
 NOT ideal for frozen sections

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Delafield’s hematoxylin
 Naturally ripened with similar longevity
with Ehrlich’s

Preparation of Solution
Hematoxylin 4g
95% alcohol 125 ml
Sat. aq. Ammonium alum (15g/100ml) 400 ml
Glycerin 100 ml

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Mayer’s hematoxylin
 Chemically ripened with sodium iodate
 Useful as a progressive stain
 Used as nuclear counterstain in demonstration
of glycogen
 Applied for 5-10 minutes
 Used in Celestine blue hemalum method of
Nuclear staining

E.M.MANGADA, RMT 12
Mayer’s hematoxylin in Celestine blue
hemalum method
 Deparaffinize sections, rehydrate
 Stain in celestine’s blue for 5 minutes
 Rinse in distilled water
 Stain in Mayer’s hematoxylin for 5 minutes
 Wash in water until blue
 Proceed with required staining technique

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Mayer’s hematoxylin
Preparation of Solution
Hematoxylin 1g
Distilled water 1000 ml
Potassium or ammonium alum 50 g
Sodium iodate 0.2 g
Citric aci 1g
Chloral hydrate SLR 50 g
Chloral hydrate Ar 30 g

E.M.MANGADA, RMT 14
Harris’s hematoxylin
 Tradiotionally ripened using mercuric oxide
 Useful as general purpose hematoxylin-
diagnostic stain for exfoliative cytology
 Most often used for progressive staining with
acetic acid-alcohol rinse

E.M.MANGADA, RMT 15
Harris’s hematoxylin
Preparation of Solutions
Hematoxylin 2.5 g
Absolute alcohol 25 ml
Potassium alum 50 g
Distilled water 500 ml
Mercuric oxide 1.25 g or
Sodium iodate 0.5 g
Glacial acetic acid 20 ml

E.M.MANGADA, RMT 16
Cole’s hematoxylin
 Artificially ripened with an alcoholic iodine
solution
Preparation of Solutions
Hematoxylin 1.5 g
Sat. aq. Potassium alum 700 ml
1% iodine in 95% alcohol 50 ml
Distilled water 250 ml

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Carazzi’s hematoxylin
 Chemically ripened with potassium iodate
 Suitable as progressive nuclear counterstain since it is a
pale and precise nuclear stain
 Used for frozen sections from urgent surgical biopsy
 Excellent and clear nuclear staining when used in double
and triple strength
Preparation of Solutions
Hematoxylin 5g
Glycerol 100 ml
Potassium alum 25 g
Distilled water 400 ml
Potassiujm iodate 0.1 g
E.M.MANGADA, RMT 18
Carazzi’s hematoxylin and Eosin for
Frozen Sections
 Freeze suitable tissue block onto a chuck
 Cut cryostat section at 3-6 mm thickness
 Fix section in 10% NBF at RT for 20 seconds
 Rinse in tap water
 Stain in double strength Carazzi’s hematoxylin
for 1 minute
 Wash well in tap water for 10-20 s
 Stain in 1% aq. Eosin for 10 s
 Rinse in tap water
 Dehydrate, clear and mount
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Staining Times with Alum
Hematoxylins
 Factors affecting staining time with Alum
hematoxylins:
◦ Type of hematoxylin used
◦ Age of stain
◦ Intensity of use of stain
◦ Whether stain is used progressively or regressively
◦ Pre-treatment of tissues or sections
◦ Post-treatment of sections
◦ Personal preference

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Staining Times with Alum
Hematoxylins
Cole’s 20-45 min
Delafield’s 15-20 min
Ehrlich’s (progressive) 20-45 min
Mayer’s (progressive) 10-20 min
Mayer’s (regressive) 5-10 min
Haris’s (progressive in cytology) 4-30 s
Haris’s (regressive) 5-15 min
Carazzi’s (progressive) 1-2 min
Carazzi’s (regressive) 45 s
Carazzi’s (frozen section) 1 min
Gill’s I (regressive’s) 5-15 min

E.M.MANGADA, RMT 21
Disadvantages of Alum
Hematoxylins
 Nuclear stains are sensitive to any subsequently
applied acidic stains
 Satisfactory nuclear staining can be done in
combination with celestine blue staining
solution

Celestine Blue solution

Celestine blue B 2.5 g
Ferric Ammonium Sulfate 25 g
Glycerin 70 ml
Distilled Water 500 ml

E.M.MANGADA, RMT 22
◦ Most suitable stain to combine with an alum
hematoxylin
◦ Has the ability of differentiating cytoplasm of different
types of cells, CT fibers and matrices
◦ Differentiation occurs in subsequent tap water wash
and during dehydration with alcohol
◦ Xanthine dyes:
 Eosin Y
 Ethyl eosin
 Eosin B

E.M.MANGADA, RMT 23
Eosin Y
 Most widely used
 Water and alcohol soluble
 Usually used as 0.5%-1.0% solution in
distilled water with crystal thymol

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Routine Staining Procedure using
Alum Hematoxylin
 Deparaffinize sections, rehydrate.
 Remove fixation pigments
 Stain in alum hematoxylin of choice for suitable time
 Wash with running water until sections ‘blue’ for 5
minutes or less
 Differentiate in 1% acid alcohol for 5-10 seconds
 Wash well in tap water until sections again blue 10-15 min
 Blue by dipping in alkaline solution
 Dip in tap water for 5 min
 Stain in 1% eosin Y for 10 min
 Wash in running water for 1-5 min
 Dehydrate through alcohols, clear and mount
E.M.MANGADA, RMT 25
Routine Staining Procedure
 Expected Results:
◦ Nuclei – blue/black
◦ Cytoplasm- varying shades of pink
◦ Muscle fibers- deep pink/red
◦ Red blood cells- orange/red
◦ Fibrin- deep pink

E.M.MANGADA, RMT 26
Papanicolaou stain for cytological
preparations
Papanicolaou Formula
Harris’s Hematoxylin
Orange G 6
10% aq. Orange G 50 ml
Alcohol 950 ml
Phosphotungstic acid 0-15 g
EA 50
0.04 M light green Sf 10 ml
0.3 M eosin Y 20 ml
Phosphotungstic acid 2g
Alcohol 750 ml
Methanol 250 ml
Glacial acetic acid 20 ml
E.M.MANGADA, RMT 27
Papanicolaou Staining Method
 Remove PEG fixative in 50% alcohol, 2 min
 Hydrate in 95% alcohol, 2 min, and 70% alcohol, 2 min
 Rinse in water, 1 min
 Stain in Haris’s hematoxylin, 5 min
 Rinse in tap water, 2 min
 Differentiate in 0.5% aq. HCl, 10 s
 Rinse in tap water, 2 min
 ‘Blue’ in Scotts tap water substitute, 2 min
 Rinse in water, 2 min
 Dehydrate in 70%, 95%, 95% alcohol for 2 min in each solution
 Stain in OG 6
 Rinse in two changes of 95% alcohol for 2 min each
 Stain in EA 50, 3 min
 Rinse in 95% alcohol E.M.MANGADA, RMT 28
Papanicolaou Staining Results
◦ Nuclei- blue/black
◦ Cytoplasm (non-keratinizing squamous
cells)- blue/green
◦ Keratinizing cells- pink/orange

E.M.MANGADA, RMT 29
IRON HEMATOXYLINS

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IRON HEMATOXYLINS
 Wiegert’s hematoxylin
 Heidenhain’s hematoxylin
 Loyez hematoxylin for myelin
 Verhoeff’s hematoxylin for elastin fibers

 Problem with these stains is overoxidation

 Demonstrates wider range of tissue structures
than alum hematoxylins, but, time consuming

E.M.MANGADA, RMT 31
Weigert’s Hematoxylin
 Uses ferric chloride as a mordant/oxidant
 Iron and hematoxylin solutions are prepared
seperately and are mixed immediately before
use
 Working solution remains active for 1-2 days
 Mixture is violet-black
 Use as a nuclear stain where acidic stains are to
be applied to tissue subsequently
 Useful stain for CNS tissues
 Staining tine: 15-30 minutes

E.M.MANGADA, RMT 32
Heidenhain’s Hematoxylin
 Mordant/oxidant/differentiating fluid: ferric
ammonium sulfate
 Use to demonstrate structures according to
degree of differentiation
 Results: all components are black or dark
gray black
 Mitochondria, muscle and nuclear chroatin and
myelin can all be demonstrated

E.M.MANGADA, RMT 33
Heidenhain’s Hematoxylin
 Staining time for tissue fixed in Susa’s, Bouin’s, Carnoy’s:
1 hour
 Staining time for tissue fixed in Helly’s or Zenker: 3
hour
 Staining time for tissue fixed in Osmiun tetroxide and
Flemming’s fluid: 24 hour
 Cytoplasmic counterstain: aq. Eosin or Orange G
 Sections should be wash well after differentiation stage
to resist fading of sections

E.M.MANGADA, RMT 34
Loyez Hematoxylin
 Mordant: ferric ammonium sulfate
 Differentiator: Weigert’s differentiator
(borax and potassium ferricyanide)
 Used to demonstrate myelin, and can be applied
to paraffin, frozen, or nitrocellulose sections
 Two methods:
◦ Heidenhain myelin stain
◦ Weil technique

E.M.MANGADA, RMT 35
Verhoeff’s hematoxylin
 Use to demonstrate elastic fibers
 Contains ferric chloride, Lugol’s iodine, 2% aq.
Ferric chloride (differentiator)
 Intense black staining produced is the most
ideal for photomicrography

E.M.MANGADA, RMT 36
TUNGSTEN
HEMATOXYLINS

E.M.MANGADA, RMT 37
TUNGSTEN HEMATOXYLINS
 Most widely used tungsten hematoxylin:
Mallory PTAH

E.M.MANGADA, RMT 38
Mallory PTAH
 Mallory combine hematoxylin with 1% aq.
Phosphotungstic acid (mordant)
 Oxidant: potassium permanganate (usable
for 24 hours only)
 Ripening: usable for years
 Applicable for CNS tissues and general tissue
structure

E.M.MANGADA, RMT 39
MOLYBDENUM
HEMATOXYLINS

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MOLYBDENUM HEMATOXYLINS
 Rare
 Most accepted technique: Thomas technique
 Recommended for demonstration of collagen
and coarse reticulin, and argentaffin cell granules
 Differentiator: picro-acetic alcohol

E.M.MANGADA, RMT 41
LEAD HEMATOXYLINS

E.M.MANGADA, RMT 42
Lead Hematoxylins
 Used in demonstration of granules and
tumors in the endocrine cells of doubtful
origin
 Also used in the localization of gastrin
secreting cells in stomach

E.M.MANGADA, RMT 43