3. 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
4. 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.
5. 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.
6. 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.
7. 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.
8. 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).
9. 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: 140C; 200C (decompose)
• Boiling Point: 58050C,
11. 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).
12. 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
14. How to prepare HEMATOXYLIN?
Preparation of Harris’s
hematoxylin
Ingredients :
Hematoxylin 5 gm
Distilled water 1000 ml
Ammonium alum 100 gm
Mercuric oxide 2.5 gm
Absolute alcohol 50 ml
Glacial acetic acid 40 ml
Preparation of Mayer’s
hematoxylin
Ingredients :
Hematoxylin 1.0 gm
Distilled water 1000 ml
Ammonium alum 50 gm
Sodium iodate 0.2 gm
Citric acid (reduces pH) 1.0 gm
Chloral hydrate (preservative) 50 gm
Method - Dissolve hematoxylin in absolute
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.
Method - Hematoxylin is dissolved in distilled
water using gentle heat. Then alum is added and
dissolved. Then sodium iodate, citric acid and
chloral hydrate are added respectively.
15. 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-ent
and not prone to errors
nuclear detail stands out brighter
and crisper
Disadvantage nuclei may not stain brighter and
crisper
over differentiation will cause pale
nuclei , or under different-iation will
obscure fine detail
Examples Mayer, Gill No.1, Gill No.2 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.
16. 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.
19. 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.
20. 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: 295C
21. 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: 295.5C
23. 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)
(CI 26905)
0.4 gm
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.
25. 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.
26.
27. Procedure for Progressive Staining
using Mayer’s, Gill’s 1 or 2
8
Hema-
toxylin
6-15
min
9
Amonia
Water
1-5 min
1
Flame
slide on
burner
2
Xylene
3-5 min
3
100%
Alcohol
1-3 min
4
90%
Alcohol
1-3 min
5
80%
Alcohol
1-3 min
6
70%
Alcohol
1-3 min
7
Distilled
Water
1-5 min
Deparaffinization Hydration
10
Distilled
Water
2 min
11
95%
Alcohol
1 min
12
Eosin
2-10
min
14
95%
Alcohol
1-2 min
15
100%
Alcohol
1-2 min
16
Xylene
2 min
13
Tap
Water
1-5 min
Dehydration
Bluing Clearing
28. Procedure for Regressive Staining
using Harris’, Delafield’s, Ehrlich’s
1
Flame
slide on
burner
2
Xylene
3-5 min
3
100%
Alcohol
1-3 min
4
90%
Alcohol
1-3 min
5
80%
Alcohol
1-3 min
6
70%
Alcohol
1-3 min
7
Distilled
Water
1-5 min
8
Hema-
toxylin
3-5 min
10
Acid
Alcohol
1-5 min
Deparaffinization Hydration
11
Amonia
Water
1-5 min
12
Distilled
Water
2 min
13
95%
Alcohol
1 min
14
Eosin
2-10
min
16
95%
Alcohol
1-2 min
17
100%
Alcohol
1-2 min
18
Xylene
2 min
15
Tap
Water
1-5 min
Dehydration
Differentiation Bluing Clearing
9
Tap
Water
1-5 min
29. 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
34. 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
35. 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.
