HEMATOXYLIN & EOSIN

Their importance and Role in Histological Studies

by
Azis Kemal Fauzie
HEMATOXYLIN
..... from dye to stain
 HEMATOXYLIN
..... a tree
• Hematoxylin is extracted from the logwood tree Haematoxylon
campechianum L. which can grow up to 50 feet tall.
• It comes from a family of the legumes—which is the third largest in the plant
kingdom. Related to it are the peanut, soybean, and clover, which harbor
nitrogen-fixing bacteria; other dye-yielding plants, including indigo; the
balsam used in present-day shampoos; and the licorice plant.
Regnum: Plantae
Cladus: Angiosperms
Cladus: eudicots
Cladus: core eudicots
Cladus: rosids
Cladus: eurosids I
Ordo: Fabales
Familia: Fabaceae
Subfamilia: Caesalpinioideae
Tribus: Caesalpiniaceae
Genus: Haematoxylum
Species: Haematoxylum campechianum
 HEMATOXYLIN
..... its name
• “Haematoxylon campechianum” is a native tree of Mexico and Central
America.
• “Haematoxylon” is derived from Greek: ”haimatodec“ (bloodlike) and
“xylon“ (wood), and "campechianum" because both the tree and the
Nahuatl Indians who discovered it are native to the part of Mexico
bordered by the Bay of Campeche.
• In Spanish, the tree is known as "pinta de tinto" (tree of color).
• The Germans dubbed it "der Bliltezweig" for its deep blood-red color.
 HEMATOXYLIN
..... its history as dye

• It was started by the expedition of Spanish explorers to Yucatan, Mexico in 1502.

• When Hernando Cortez came to Mexico in 1519, he was struck by the lustrous violet
and black colors used by an Indian tribe, the Nahuatl or Aztec. He found that the
dye they used came from a prickly hedge, which grew into a medium-sized tree.
• The Spaniards introduced this new dye to Europe, where it became the sole
distributors of hematoxylin for nearly 60 years.
 HEMATOXYLIN
..... its history as dye

• After the sinking of Spanish Armada in 1588, English merchants were able to ship the logwood.
• At1590s, the use of logwood was banned because the color was impermanent. In 1620, Cornelius
Drebbel in Holland developed a process called metal mordanting which involved pretreating the
fabric with a metal salt. The English dyers learned it and in 1661 the law was lifted.
• By 1662, the English began cutting logwood in areas of Campeche Bay not inhabited by the
Spanish, particularly in the Laguna de Terminos, making England as growing competitor to Spain.
 HEMATOXYLIN
..... its history as dye

• After 1715, logwood was successfully cultivated in Jamaica and Belize. A report
in 1852 shows that logwood was being imported mostly from Mexico but some
substantial amounts also from Haiti, the West Indies, Honduras, and the US.
• British and Spanish remained the major distributors of logwood until the 1870s.
 HEMATOXYLIN
..... its history as biological stain
• Robert Hooke in his 1665 book, Micrographia, was the first to observe that
logwood dyes stained hair and wool fibers in microscopy.
• Thomas Andrew Knight, an English botanist, published drawings in 1803 of
microscopic sections of potato plants stained with logwood.
• In 1810, Chevrevil isolated hematoxylin from logwood as yellow to orange
crystals.
• Quekett had briefly mentioned staining animal tissues with logwood dye in
his 1848 book published in London.
• In 1863, a German anatomist, Heinrich Waldeyer published article in which
he became the first man to use hematoxylin to stain human tissue.
• Frederick Böhmer in an article in 1865 introduced both mordant and
metachrome staining methods using metal salts (potassium alum).
• A formula containing logwood extract with alum and copper sulphate was
suggested by Cook (1879). The combination of hematoxylin and iron was
introduced originally by Benda (1886) before known by Heidenhain’s (1892).
• Since then numerous formulations have appeared like Harris’, Gill’s, Mayer’s
(1891), Weigert’s (1884), Delafield’s (1885) and Ehrlich’s (1926).
 HEMATOXYLIN
..... its chemical structure
• CAS Registry Number: 517-28-2
• IUPAC Name: 7,11b-Dihydroindeno[2,1-c]chromene-3,4,6a,9,10(6H)-pentol
• Other Names: Hematoxiline; (+)-Hematoxylin; Haematoxylin; Hematoxylin;
Hematoxyline; Hydroxybrasilin; Hydroxybrazilin; C.I. 75290; C.I. Natural Black
1; NSC 270085
• Physical Form: White to yellowish crystals, turns red on exposure to light
• Solubility: Soluble in water, ethanol, ethylene glycol, methyl cellosolve
• pH Range: 0.0–1.0; 5.0–6.0
• Chemical/Dye Class: Flavone
• Molecular Formula: C16H14O6
• Molecular Weight: 302.28
• Melting Point: 140C; 200C (decompose)
• Boiling Point: 58050C,
 HEMATOXYLIN
..... its application
• Industrial Applications: Textile dyeing; paper dyeing; plasma display panel
• Staining Applications: Aluminum; antigen; blood smears; cells; collagen;
epithelial cells; eye lens; fish; genes; gluten structure; horny cells; lipid; liver
tissues; malignant melanoma; myocardial biopsies; neurons; nucleic acids;
nucleus; oil droplets; proteins; starch granules; tissues; hairs; keratin fibers
• Biological Applications: Detecting breast cancer, collagen, genes,
microorganism; treating age-related macular degeneration, burns, cancer,
diabetes, obesity, gastroesophageal reflux disease, peripheral neural and
vascular ailments, prostate cancers, skin disorders, viral diseases
• Laboratory Applications: pH indicator
red (pH 0.0) to yellow (pH 1.0)
pale yellow (pH 5.0) to violet (pH 6.0)
 How to extract HEMATOXYLIN?
• It is extracted from the bark of a tree, ”hematoxylom campechianum”.
The hematoxylin which we buy is extracted from this bloodwood tree.
• To obtain the bark of freshly logged tree is chipped off, then boil the chips
in water. An orange red solution is obtained, which turns yellow, then
on cooling. The water is evaporated leaving crude hematoxylin.

• Hematoxylin by itself cannot stain. It must first be oxidized to hematein, to

retain its staining ability longer. The process is referred to as ripening.
• Ripening can proceed spontaneously and slowly by exposure to
atmospheric oxygen and natural light for 3-4 months, or rapidly by added
chemical oxidants such as mercuric oxide (Harris) or sodium iodate (Gill).
 HEMATOXYLIN Lake
• The combination of hematoxylin plus mordant is called a hematoxylin lake,
and lakes with different metals have different colors.
• The aluminum lake formed with ammonium alum (aluminum ammonium
sulfate) is particularly useful for staining nuclei. It is purple in acid solution,
but blue in alkaline solution.
• Either aluminum potassium sulphate (potassium alum) or aluminum sodium
sulphate (sodium alum) may also be used to create aluminum lakes.
• Hematoxylin recipes using any of these mordants are called alum
hematoxylin or hemalum.
METAL COLOUR
Aluminum Purple to blue
Potassium Purple
Iron Blue-black
Chromium Blue-black
Copper Blue-green to purple
Nickel Violet shades
Tin Red
Lead Dark brown
Osmium Green brown
 Commercial HEMATOXYLIN
Hematoxylin Chemical Formulations
Mayer Hematoxylin, Potassium aluminium sulfate (potassium alum), Sodium iodate,
Chloralhydrate, Citric acid, HCl (25%), Sodium hydrogen carbonate, Ammonia
aqueous, Distilled water
Harris Hematoxylin, Potassium aluminium sulfate (potassium alum), Ethanol, Mercury (II)
oxide (mercuric oxide), Glacial acetic acid, HCl (25%), Sodium hydrogen carbonate,
Ammonia aqueous, Distilled water
Gill Hematoxylin, Aluminium sulfate (alum), Ethylene glycol, Sodium iodate, Glacial
acetic acid, HCl (25%), Sodium hydrogen carbonate, Ammonia aqueous, Distilled
water
Ehrlich Hematoxylin, Potassium aluminium sulfate (potassium alum), Potassium iodate,
Isopropanol, Glycerol, Glacial acetic acid, HCl (25%), Sodium hydrogen carbonate,
Ammonia aqueous, Distilled water
Delafield Hematoxylin, Ammonium aluminium sulfate (ammonium alum), Sodium iodate,
Glycerol, Sodium hydrogen carbonate, Ethanol, Distilled water
Weigert Hematoxylin, Iron (III) chloride (ferric chloride), Ethanol, HCl (25%), Sodium hydrogen
carbonate, Distilled water
Heidenhain Hematoxylin, Ammonium ferric sulfate (iron alum), Ethanol, Sodium iodate, Glacial
acetic acid, Sulfuric acid, Sodium hydrogen carbonate, Distilled water
Hansen Hematoxylin, Ammonium ferric sulfate (iron alum), Ethanol, HCl (25%), Sodium
hydrogen carbonate, Distilled water
 How to prepare HEMATOXYLIN?
Preparation of Harris’s Preparation of Mayer’s
hematoxylin hematoxylin
Ingredients : Ingredients :

Hematoxylin 5 gm Hematoxylin 1.0 gm

Distilled water 1000 ml Distilled water 1000 ml

Ammonium alum 100 gm Ammonium alum 50 gm

Mercuric oxide 2.5 gm Sodium iodate 0.2 gm

Absolute alcohol 50 ml Citric acid (reduces pH) 1.0 gm

Glacial acetic acid 40 ml Chloral hydrate (preservative) 50 gm

Method - Dissolve hematoxylin in absolute Method - Hematoxylin is dissolved in distilled

alcohol and ammonium alum in hot water. Mix
the two solutions and heat to boiling. Remove
from flame, and add mercuric oxide and cool
rapidly. Glacial acetic acid if added gives
brisk nuclear staining, but it should be added
in working solution.
water using gentle heat. Then alum is added
and dissolved. Then sodium iodate, citric
acid and chloral hydrate are added
respectively.
 Progressive and Regressive Staining
Aspect Progressive Regressive

Hemalum concentration Less (1 to 4 gm/L) More (5 gm/L or more)

Rate of uptake Slow (15 minutes-2 hours) Rapid (1.5-6 minutes)

Easily controlled? Yes No

Overstaining? No Yes

Differentiation required? No Yes

Advantage nuclear staining is more consist- nuclear detail stands out

ent and not prone to errors
Disadvantage nuclei may not stain brighter
and crisper
Examples Mayer, Gill No.1, Gill No.2
brighter and crisper
over differentiation will cause
pale nuclei , or under different-
iation will obscure fine detail
Harris, Ehrlich, Delafield

Progressive stains are generally less concentrated and work slowly to

avoid overshooting the endpoint.
Regressive stains are more concentrated and many can achieve
overstaining in less time, while differentiation by acid alcohol is required
to decolorize the cytoplasm and to remove excess dye from chromatin.
 BLUING

• Bluing is necessary to convert nuclear coloration from reddish purple to a

crisp blue color that will give a much better contrast with the usual red
counterstains.
• Bluing can be done in one of the following ways:
1. The slides may be dipped for a few seconds into a weakly alkaline
solution such as ammonia water or sodium acetate or dilute sodium
carbonate or saturated lithium carbonate solution. Using ammonia
water, the pH may be too high, so section loss may occur .
2. They may be washed for 2-5 minutes in tap water. Tap water tends to
be slightly acid, with a pH in the range of 5.4 to 9.8 and is more alkaline
than the pH of alum hematoxylins (2.6 - 2.9). Tap water can wash out
any excess alum, give a crisper nuclear stain and prevent fading
during storage, but the pH may be unpredictable.
3. They may be rinsed in Scott's tap water substitute. It was made by
dissolving 2 g of sodium bicarbonate and 20 g of magnesium sulphate
in 1 litre of deionised water and store in room temperature. Using
Scott’s tap water are more effective at maintaining the optimal pH,
produces crisp blue/purple chromatin stain, gentle for tissues and
excellent for use with frozen sections.
BLUING
 EOSIN
..... a counterstain
 EOSIN
..... its history as synthetic dye
• Heinrich Caro was a Polish chemist working for the chemical company
which later became BASF. Working with a sample of fluorescein, Caro
synthesised a yellow-red dye which he named ‘Eosin’ (after the nickname
of a girl he admired!).
• Eosin, the potassium salt of tetrabromofluorescein, was also synthesized by
Baeyer and his coworkers in 1871. The name itself is derived from a Greek
word “eos” meaning “dawn or morning red”.
• Emil Fischer was another German chemist working in the field and in 1875
he published a paper on Eosin Y which is the commonly used eosin dye in
histology. The ‘Y’ stands for ‘yellowish’.
• Skip forward a year, Wissowzky published his work on a combined staining
using both haematoxylin and eosin.
• In 1876 Dreschfeld and Fischer described the usefulness of eosin as a tissue
stain.
• A few months later, Busch reported on the double staining of the
ossification border with eosin and hematoxylin.
• Over a century later, these still remain the most commonly used materials
for tissue staining.
 EOSIN B
..... its chemical structure

• CAS Registry Number: 548-24-3

• IUPAC Name: 4',5'-dibromo-3',6'-dihydroxy-2',7'-dinitro-1-spiro
[isobenzofuran-3,9'-xanthene]one, sodium salt (1 : 2)
• Other Names: Eosin B, Eosin Bluish, Dibromodinitrofluorescein sodium,
Saffrosine, Eosin Scarlet, Acid Red 91, Imperial Red, C.I. 45400
• Physical Form: Red-brown to green crystals or powder
• Solubility: Freely soluble in water; soluble in ethanol
• Chemical/Dye Class: Xanthene
• Molecular Formula: C20H6Br2N2Na2O9
• Molecular Weight: 624.06
• Melting Point: 295C
 EOSIN Y
..... its chemical structure
• CAS Registry Number: 17372-87-1
• IUPAC Name: 2-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen-9-yl)
benzoate, sodium salt (1 : 2)
• Other Names: Eosin Y, Eosin Yellowish, Acid Red 87, Bromoeosine,
Bromofluoresceic acid, D&C Red No. 22 , Japan Red 103 , C.I. 45380
• Solubility: Freely soluble in water; slightly soluble in ethanol, methanol;
insoluble in ether
• pH Range: Non-fluorescence (0.0) to green fluorescence (3.0)
• Physical Form: Red-brown crystals or powder
• Chemical/Dye Class: Xanthene
• Molecular Formula: C20H6Br4Na2O5
• Molecular Weight: 691.85
• Melting Point: 295.5C

EOSIN B
• Staining Applications: Brain; cells;
microorganisms; nucleic acids;
peptides; proteins; enzyme
substrates; hairs.
• Biological Applications: Antimalarial
agent; protein assay; detecting
enzyme activity; treating cancer,
malaria, diabetes, a variety of
conditions affecting skin, mouth,
digestive tract, urinary tract,
reproductive tract, respiratory tract,
circulatory system, head, neck,
endocrine system, lymphoreticular
system; dental materials.
• Industrial Applications: Color filters;
liquid crystal displays; inks; NLO
materials; photographic materials;
laundry detergent; textiles.
EOSIN
..... its application
EOSIN Y
• Staining Applications; Blood; blood
smears; bone marrow; cells; nucleus;
cytoplasm; membrane; candies;
drinks; keloid; orthodontic adhesives;
proteins; tissues; thrombocytes; eye
lens; eye shadow; lips; skin; hairs;
keratin fibers.
• Biological Applications: Treating
age-related macular degeneration,
burns, cancer, diabetes, obesity,
dental bone defects, gastro-
esophageal reflux disease, prostate
cancer, viral diseases; stents; wound-
healing materials.
• Industrial Applications: Solar cell;
semiconductor devices; color filters;
light-emitting devices; photovoltaic
devices; electrochromic devices; thin
films; sol–gel materials; inks; colored
bubbles.
 How to prepare EOSIN?

Preparation of Eosin Y
Ingredients :

Eosin Y (CI 45380) 5 gm

Phloxin B (CI 45410) 2.1 gm

Biebrich scarlet (water soluble) 0.4 gm

(CI 26905)
95% ethanol 200 mL

Distilled water 800 mL

Method - Mix the above reagents together, and stir well.

Phloxin B is sometimes added to eosin formulations to increase the range of
red colors. However, phloxin B is exceedingly “bright” and can be visually
overpowering if too much is used. Therefore, one needs to be cautious
when using phloxin B.
HEMATOXYLIN & EOSIN
..... balance of coloration
 The H&E Stain
..... its principle
• Hematoxylin and Eosin (H & E) staining is used for demonstration of
nucleus and cytoplasmic inclusions in clinical specimens. Eosin is
formulated to produce optimal contrast with hematoxylin.
• Alum acts as mordant and hematoxylin containing alum stains nucleus
light blue. This turns red in presence of acid, as differentiation is achieved
by treating the tissue with acid solution. Bluing step converts the initial
soluble red color within the nucleus to an insoluble blue color. The
counterstaining is done by using eosin which imparts pink color to the
cytoplasm.
• Ideally, hematoxylin should color chromatin blue. Depending on the
mordant, mucin may also be colored blue. The depth of color should be
deep enough to make small particles visible and shallow enough to not
obscure fine details. Cytoplasm should be colored scarcely at all.
• Eosin should color nucleoli red, and stain collagen, muscle and cytoplasm
varying shades of orange/pink. When present, erythrocytes and cilia
should be colored varying shades of red/pink.
Procedure for Progressive Staining
using Mayer’s, Gill’s 1 or 2

Deparaffinization Hydration

1 2 3 4 5 6 7 8

Flame Xylene 100% 90% 80% 70% Distilled Hema-

slide on Alcohol Alcohol Alcohol Alcohol Water toxylin
burner

3-5 min 1-3 min 1-3 min 1-3 min 1-3 min 1-5 min 6-15
min

9 10 11 12 13 14 15 16

Amonia Distilled 95% Eosin Tap 95% 100% Xylene

Water Water Alcohol Water Alcohol Alcohol

1-5 min 2 min 1 min 2-10 1-5 min 1-2 min 1-2 min 2 min
min

Bluing Dehydration Clearing

 Procedure for Regressive Staining
using Harris’, Delafield’s, Ehrlich’s

Deparaffinization Hydration

1 2 3 4 5 6 7 8 9

Flame Xylene 100% 90% 80% 70% Distilled Hema- Tap

slide on Alcohol Alcohol Alcohol Alcohol Water toxylin Water
burner

3-5 min 1-3 min 1-3 min 1-3 min 1-3 min 1-5 min 3-5 min 1-5 min

10 11 12 13 14 15 16 17 18

Acid Amonia Distilled 95% Eosin Tap 95% 100% Xylene

Alcohol Water Water Alcohol Water Alcohol Alcohol

1-5 min 1-5 min 2 min 1 min 2-10 1-5 min 1-2 min 1-2 min 2 min
min

Differentiation Bluing Dehydration Clearing

 The H&E Stain
overstaining and understaining
• Overstained alum-
hematoxylin
- Lack of nuclear detail
- Poor nuclear/cytoplasmic
contrast
- Cytoplasmic detail is masked
• Light or washed out alum
hematoxylin
- Nuclei are not crisp
- Poor nuclear/cytoplasmic
contrast
• Overstained eosin Y
- Nuclei may lack crispness-
purple
- Poor nuclear/cytoplasmic
contrast
• Light or washed out eosin Y
- Cytoplasmic detail is lacking
- Acidophilic nuclei may not be
visible
- Poor nuclear/cytoplasmic
contrast
 Slide Match Project
4X 10X
 Slide Match Project
10X 40X
 Slide Match Project

unstable pH

stable pH
 Troubleshooting The H&E Stain
• If over differentiated, bring sections back through the alcohols, stain again
in eosin and differentiate rapidly through the dehydrating alcohols.
• If under differentiated, leave longer in the 95% alcohol before proceeding
through the dehydrating alcohols.

very weak eosin staining good color balance of H&E

 REFERENCES
• Brown S, The Science and Application of Hematoxylin and Eosin Staining.
• Conn HJ, Biological Stains – A Handbook on The Nature and Uses of The
Dyes Employed in The Biological Laboratory, Williams & Wilkins Co.,1953.
• Emge DJ, H&E Staining Troubleshooting.
• Ellis R, Hematoxylin and Eosin (H&E) Staining Protocol.
• Gill GW, H&E Staining: Oversight and Insights.
• Giri D, Hematoxylin and Eosin staining : principle, procedure and
interpretation, 2015.
• Gurecki JJ, The History of Hematoxylin, Laboratory Medicine 15(6), 1984.
• Hammeke E, Logwood Dye on Paper.
• King DF & King LAC, A Brief Historical Note on Staining by Hematoxylin and
Eosin, Am. J. Dermatopathology 8(2), 1986.
• Kuhlmann WD, Haematoxylin staining methods, 2006.
• Millikin PD, Hematoxylin Staining – Some Technical Notes, 2005.
• Sabnis RW, Handbook of Biological Dyes and Stains – Synthesis and
Industrial Applications, John Wiley & Sons Inc., 2010.
• Wilson M, H and E Part One: History, Background and Solutions, 2014.