MLS 410 LAB–HISTOPATHOLOGIC AND CYTOLOGIC

TECHNIQUES
HEMATOXYLIN & EOSIN STAINING
2ND SEMESTER | FINALS | PROF. JOHN MARKE BERNARDO

OUTLINE ● H&E is a useful all-purpose stain that has stood the test
I. LEARNING CONTENT of time and is now the most frequently used tissue stain
II. THE SCIENCE BEHIND H&E worldwide
III. THE BASIC STEPS IN STAINING AND THE BASIC STEPS IN STAINING AND MOUNTING
MOUNTING PARAFFIN SECTIONS PARAFFIN SECTIONS:
A. DEPARAFFINIZATION
B. HYDRATION 1.
DEPARAFFINIZATION
C. REMOVAL OF MERCURY PIGMENTS DEPARAFF INIZATION ● The removal of wax with xylene is essential to
D. STAINING ·removal of way (paraffin) ensure complete removal. At least 2 to 3 changes
E. DEHYDRATION AND CLEARING xylene in
in 2.3 charges
xylene are needed to ensure complete
of

Patches:presence Wax. removal, and sections should appear clear and

of

NOTE FROM TRANSERS mustbe immersedi n Xuleuc

transparent. Patches indicate the presence of
for longer periods.
This section includes the information sourced from the wax and sections should be kept longer in the
Learning Activity No. 10: Hematoxylin and Eosin xylene.
Staining Method.
LEARNING CONTENT HYDRATION 2.
● The most important details in this text are that a tissue HYDRATION: ● Xylene is not miscible with aqueous
specimen must be stained for microscopic evaluation, removal
solutions, so it is removed with graded alcohol
Xyleue using of

as unstained tissue lacks contrast and all of the fixed Decreasing Grades of from higher to lower concentrations starting
materials have a similar refractive index and color. The
↑
Alcohol followed by immersion
from 90%, 70% alcohol and finally distilled water.
in distilled water.

H14E:
staining process uses various dyes to stain particular cell absolute
Sections should now appear opaque, and any
Clear Areas:return to
· mostpreferred

Differentiate
components within tissues, so that it is easier to alcohol b rehydrate.clear areas should be returned to absolute
·
Cell Parts
From one another.
distinguish different cell parts from each other. alcohol and rehydrated.
History:
·
Bohmer (1865) -Hematoxylin
● H&E is the preferred staining method for routine
·
Escher (1875) -
Eosin
used in
microanatomical examination, producing colours for 3. REMOVAL OF MERCURY PIGMENTS (WHENEVER
Wissowzky (1876).
·

combination
different tissue structures to provide a detailed view of the REMOVAL OF NECESSARY)
tissue. MERCURIC PIGMENTS
● Mercury pigments, such as Zenker, are
procedure:
● H&E stain is a combination of two dyes, haematoxylin precipitated on sections and must be removed
Mercuric pigments
and eosin, introduced by Böhmer and Fischer in 1865 Iodine
↓
before staining is done. Removal involves
and 1875 respectively. Wissowzky described their use in iodine compound
treatment with iodine solutions, which changes
oThiosulfate
combination as a tissue staining method in 1876. ↓)

ictrathionate
mercury to an iodine compound. This is then
wash with
↓ converted to tetrathionate by thiosulphate, which
THE SCIENCE BEHIND H&E running water.
is readily soluble in water. Slides are placed in
●
Staining does not produce colour randomly; instead, running water to wash out extraneous chemicals.
SCIENCE:
the dyes exploit differences in the chemistry of the tissue STAINING: 4. STAINING
HBE does s tain
not

to differentially colour various components.

·

randomly. occurs in hydrated stage. ● Staining reagents are applied during the
Bonding
● Ionic bonding is the most important type of bonding hydrated stage, exposing tissues to
. Ionic
i mportant
most
bondi n

chemical in histologic staining techniques, as it involves hematoxylin, eosin Y and strong chemicals. Care
· electrostatic attraction
between
charges.
electrostatic attraction between opposite charges, one of
opposite
must be taken to avoid detachment of tissues.
Hematoxylin: which is fixed in the tissue and the other in the dye.
"dye" catonic
● Hematoxylin is not a dye, so it must be combined with a 5.
DEHYDRATION AND CLEARING
·

·needs form
to

complexiti mordant to stain tissues. The mordant is typically a ● Dehydration is done using graded alcohols from
"due"to make ita
DEAYDRATION

negatively
·
metal cation, such as aluminum. Hematoxylin in complex
stains ·removal 70% to absolute alcohol. It can remove some
of water using

·charqcal basophilic increasing grades a lcohol

with aluminum salts is cationic and acts as a basic dye. stains, so time must be modified to minimize
of

organelles (70% absolute) -

·
nuclei, DNA
EOSIN
· anionic
It reacts positively with negatively charged, basophilic cell
(-) dye:
Notelime be
must fading. Alcohol is miscible in xylene, so it is
facilitated to
stains
preventr emoval of

positively-charged
stains
components, such as nucleic acids in the nucleus, used for clearing sections. Any sections from
·

aciduphilic organelles.
CLEARING:
resulting in blue staining. which water has not been completely removed
·
amino groups in

proteins in cytoplasm
of
removal water re
·

● Eosin is an anionic acidic dye that can be used to stain Note:

if water completely not
should be returned to absolute alcohol and the
removed,

pink when exposed to positively charged amino groups in returntoabsoluteanear process repeated. Mounting is done after 2nd
MOUNTING:
proteins in the cytoplasm, resulting in pink staining. ·

Xylene.
done after and
or 3rd xylene.
or 3rd

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TOPIC: HEMATOXYLIN & EOSIN STAINING
OUTLINE
I. INTRODUCTION
F. General Information
G. Routine H&E Staining
IV. HEMATOXYLIN STAINS
A. ALUM HEMATOXYLIN
1. EHRLICH’S H.
2. HARRIS’ H.
3. COLE’S H.
B. IRON HEMATOXYLIN
C. COPPER HEMATOXYLIN
V. EOSIN STAIN
NOTE FROM TRANSERS
This section includes the information sourced from the
book, Histopathologic Techniques by Bruce-Gregorios
(2017). All of the Information presented in this document
can be found in the following chapters:
1. Chapter 16 - Principles of Staining
2. Chapter 17 - Stains & Staining Solutions
INTRODUCTION
● Hematoxylin and Eosin (H&E) staining is the
cornerstone of tissue-based diagnosis. The process
stains thin tissue sections so that pathologists can
visualize tissue morphology.
● The process uses a hematoxylin dye to stain cell nuclei
(and other parts) blue and an eosin dye to stain other
structures pink or red. Hematoxylin binds strongly to acids
and consequently binds to nuclear DNA and stains nuclei
blue. Properly applied, this technique provides
exceptional detail of tissue structure and the makeup of
the cells. This detail is required for tissue-based
diagnosis, particularly in the detection and classification
of infection, cancer or metabolic disease.
● Routine H&E staining plays a significant role in
tissue-based diagnosis by coloring otherwise
transparent tissue sections, and allowing cell structures
including the cytoplasm, nucleus, and organelles and
extra-cellular components to be clearly visible under the
microscope. In a histology laboratory, all specimens are
initially stained with H&E and additional stains are only
ordered if additional information is needed to provide a
more detailed analysis.
● Staining with H&E is very reliable although it does
show some variation depending on the exact
formulation of the stain, and the stain density is
considerably affected by the thickness of the
sections – thicker sections take up more stain. I
● It is also generally done before any additional staining
techniques, because histology with H&E can confirm the
basic tissue type and help to localize the lesion. Since
most cell structures are transparent, very little detail of the
structure can be seen, unless the cells are stained. The
same is true of components of the extracellular matrix.
Because different parts of the cell are biochemically
different, they take up specific stains to varying degrees.
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GENERAL INFORMATION
● Hematoxylin is a natural dye derived by extraction
from the core or the heartwood of a Mexican tree
known as "Hematoxylin Campechianum”.
● It is by far the most valuable staining reagent used by
the cytologist due to its powerful nuclear and
chromatin staining capacity, and its striking
polychrome properties which may be produced with
proper differentiation.
○ Note: Hematoxylin itself is not a true basic
dye.
● HEMATIN
○ The active coloring agent of Hematoxylin,
○ formed by the oxidation of hematoxylin, a
process known as "ripening."
■ This is usually accomplished by
exposing the substance to air and
sunlight, thereby oxidizing hematoxylin
(natural ripening). Such a process is
slow and takes as long as 3-4 months,
but it can be accelerated by adding
strong oxidizing agents such as:
● hydrogen peroxide
● mercuric oxide,
● Potassium permanganate
● sodium perborate
● sodium iodate
■ These agents convert hematoxylin to
hematin almost instantaneously by
chemical oxidation (artificial ripening),
so that the staining solution is ready for
use immediately after preparation.
○ It is essential that the oxidant be used in the
correct amount, since excessive oxidation
(over-ripening) leads to production of other
useless compounds.
○ Using the least amount of oxidant will result
in satisfactory staining and longer life of the
stain.
○ Ripened hematoxylin is seldom used alone
due to its inherent low affinity for the tissue
itself. It is most frequently used in combination
with alum, iron, chromium and copper salts,
which act as mordants catalyzing or forming
links between the hematin stain and the tissue.
TOPIC: HEMATOXYLIN & EOSIN STAINING
ROUTINE H&E STAINING IN PARAFFIN EMBEDDED
TISSUES (REGRESSIVE STAINING)
● FIXATION:
○ Most fixatives can be used except osmic acid
solutions which inhibit hematoxylin.
● PROCEDURE
1. Clear paraffin embedded sections in the first
xylene bath for 3 minutes.
2. Transfer to a second xylene bath for 2 to 3
minutes.
3. Immerse in the first bath of absolute ethyl alcohol
for 2 minutes.
4. Transfer to a bath of 95% ethyl alcohol for 1 or 2
minutes.
5. Rinse in running water for 1 minute.
6. Stain with Harris alum hematoxylin for 5 minutes
(Ehrlich's hematoxylin requires 15-30 minutes).
7. Wash in running tap water to remove excess stain.
8. Differentiate in 1% acid-alcohol (1 mL
concentrated HCl to 99 ml. of 80% ethyl alcohol)
for 10-30 sec. monitoring the changes in color
microscopically until only the nuclei are stained.
9. Rinse in tap water.
10. Blue in ammonia water (average of 5 minutes) or
1% aqueous lithium carbonate until the sections
appear blue (about 30 seconds).
11. Wash in running water for 5 minutes.
12. Counterstain with 5% aqueous eosin for 5
minutes. If alcoholic eosin is used, the time can be
reduced to 30 seconds or 1 minute.
13. If aqueous eosin is used, wash and differentiate in
tap water under microscope control until the
nuclei appear sharp blue to blue black and the
rest of the tissue appears in shades of pink.
a. If alcoholic solution is used, differentiate
with 70% alcohol.
14. Dehydrate, clear and mount
● NOTE
○ For tissues fixed with mercuric chloride:
■ The staining time in hematoxylin should
be increased slightly while the duration
of eosin staining should be reduced.
■ The mercury should be removed using a
0.5% solution of iodine in 80 to 95%
alcohol and rinsed in water.
■ The iodine is then removed by placing the
slide in 3% sodium thiosulfate solution
for 1 to 5 minutes and washing it well in
running water for 3 to 5 minutes.
■ Alternatively, mercury deposits may be
removed after sections are hydrated, by
immersing the sections in Gram's or
Lugol's iodine for 5 minutes, followed
by sodium thiosulfate and subsequently
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washing the section in water prior to
staining.
■ Staining may be prolonged for chromium
and osmium fixed tissues (e.g.
Flemming's fluid), for tissues subjected to
long acid decalcification, and after
prolonged storage in acid formalin or 70%
alcohol.
HEMATOXYLIN
● Hematoxylin is the staining solution most commonly
used for routine histologic studies.
● Stains Acid (or Basophilic) Structures Purplish-Blue
○ Note: Hematoxylin is not strictly a basic dye, but it is
used with a 'mordant' that makes this stain act as a
basic dye.
● Considered as a basic dye called HEMATIN:
○ Obtained from Log-Wood Trees
○ Formula: Cl - dye
● ALUM & IRON:
○ Mordants
○ Forms Lakes or Colored Complexes
(Dye-Mordant-Tissue Complexes)
■ Note: color of which will depend on the salt
used.
● Aluminum Salts = Blue
● Ferric Salt Lakes = Blue-Black
ALUM HEMATOXYLIN
● Recommended for Progressive Staining
○ Also used for Regressive Staining
● Counterstained with:
○ Eosin
○ Congo Red
○ Safranin
● Aluminum salts give a blue lake, and increase the
selectivity for nuclei, especially if acid is added or is
used as a differentiating agent.
● The two main alum hematoxylin solutions employed
are:
○ Ehrlich's hematoxylin
■ Ripened with Sodium Iodate
○ Harris hematoxylin
■ Ripened with Mercuric Chloride.
● Alum or potassium aluminum sulfate, when used as
the mordant, usually dissociates in an alkaline
solution, combining with -OH of water to form
insoluble aluminum hydroxide.
○ In the presence of excess acid, aluminum
hydroxide cannot be formed, with ultimate failure
of aluminum hematoxylin dye-lake to form, due
to lack of -OH ions. Hence, acid solutions of
alum hematoxylin become red.
● BLUEING PROCEDURE
TOPIC: HEMATOXYLIN & EOSIN STAINING

○ During staining, alum hematoxylin stained

● Glycerin
sections are usually passed on to an alkaline ● Distilled water
solution (e.g. 1% hydroxide) in order to ● Glacial acetic acid
neutralize the acid and free the OH group, to
form an insoluble blue aluminum MAIN STAINING REGRESSIVE STAINING
hematin-tissue-lake. METHOD
○ For blueing of alum-hematoxylin -stained
sections, warm (40° to 50°C) tap water is DIFFERENTIATING 70% ALCOHOL (ACID-ALCOHOL)
AGENT
commonly used, since it is generally
sufficiently alkaline.
ARTIFICIAL SODIUM IODATE
○ When tap water is not sufficiently alkaline, or RIPENER
is even acid, and is unsatisfactory for
blueing hematoxylin, Use: STAINING TIME: 15-40 MINUTES
■ lithium carbonate (1% w/v in water)
■ Bicarbonate (0.2 to 0.5% w/v in tap MAIN USES ● Tissues subjected to
water) Acid Decalcification
■ Potassium or Sodium acetate ● Tissues that have
become acidic during
○ Alternatively, Scott's Tap Water Substitute
prolonged storage in
(T.W.S.), which consists of 33.5 gm. NaHCO4 hematoxylin
and 20 grams MgS04 , in 1000 cc of water, ● Mucopolysaccharide
with thymol (to inhibit the formation of molds), is Substances (e.g.
used to accelerate blueing of thin paraffin Cartilage & Cement Lines
sections. of Bones)
○ Stains it Blue
○ Blueing with ammonia, lithium carbonate or
Scott's Tap Water Substitute has more rapid
action (about 15, 30 and 60 seconds NOT USED FOR: FROZEN SECTIONS
respectively), compared to the 5 to 15
minutes required for warm tap water to PROCEDURE
"blue" hematoxylin. 1. Dissolve hematoxylin in absolute ethyl alcohol
○ Ammonia water: with gentle heat.
■ used to blue stains, 2. Dissolve the potassium alum in distilled water
and glycerin with gentle heating and shake
■ Prepared by mixing 2 cc. of strong
a. Glycerin is added to slow the oxidation
ammonium hydroxide with 98 cc of tap process and prolong the shelf life of
water. hematoxylin
● Note that Ammonia (0.5 to 1% in 3. Mix and add glacial acetic acid.
80% alcohol) may be "hard" on 4. Expose to air and sunlight for several weeks or
delicate tissues and may loosen months in a flask lightly plugged with cotton,
and cause sections to fall off the shaking daily.
5. Transfer in a well-stoppered bottle and store in
slides during staining.
a warm place.
○ Lithium carbonate has a tendency to form a. Alum hematoxylin takes about 2
crystalline deposits unless the slides are months to ripen naturally, but it's
agitated in it and washed well afterwards. staining property will last for months
■ The use of very cold water slows down or years.
the process while warming accelerates NOTE:
it. ● Hematoxylin may be partially oxidized iodate to
hasten ripening by addition of 0.3 gm Sodium,
■ In fact, the use of very cold water
but this will also inevitably shorten the shelf life
(below 10°C) for blueing sections may of the stain.
even produce pink artifact ● As hematoxylin solution becomes oxidized, the
discolorations on the tissue. color of the solution will change from purplish
to deep red, while the pungent odor of acetic
acid will be replaced by a pleasant aroma.
b. Glycerin acts as a stabilizer, retards
EHRLICH’S HEMATOXYLIN evaporation of the solution, and
appears to slow down ripening, so that it
FORMULA ● Hematoxylin may be added 4-6 weeks after the initial
● Absolute Ethyl Alcohol preparation.
● Aluminum potassium Sulfate

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TOPIC: HEMATOXYLIN & EOSIN STAINING

HARRIS’ HEMATOXYLIN ■ Should be filtered off before use.

● Since most of the alcohol is evaporated in the
FORMULA ● Hematoxylin process of boiling, 10 ml. of ethyl alcohol may be
● Absolute Ethyl Alcohol added to the final solution, to help prevent the
● Ammonium/Potassium Alum growth of molds.
● Mercuric Oxide
● Distilled water
● Glacial acetic acid MAYER’S HEMATOXYLIN
MAIN STAINING REGRESSIVE STAINING FORMULA ● Hematoxylin
METHOD ● Sodium Iodate
● Potassium Alum
DIFFERENTIATING 70% ALCOHOL (ACID-ALCOHOL) ● Citric Acid
AGENT ● Chloral Hydrate
● Distilled water
ARTIFICIAL MERCURIC CHLORIDE
RIPENER PROCEDURE
1. Allow hematoxylin, alum and sodium iodate to
STAINING TIME: 5-20 MINUTES dissolve in water overnight.
● Depends on the batch and 2. Add chloral hydrate and citric acid. Boil for 5
age of stain, the nature of minutes and cool.
tissue, and the degree of 3. The addition of sodium iodate immediately
staining required. ripens the hematoxylin.
● Best results are obtained
when the solution is NOTES:
made every 2 or 3 ● Citric acid is usually added after potassium
months. alum has been dissolved (by shaking the
solution); however, the addition of 20 mL
MAIN USES ● Routine Nuclear Staining glacial acetic acid seems to give better nuclear
● Exfoliative Cytology staining and a more stable solution.
● Staining of Sex ● Chloral hydrate is added to the final solution as
chromosomes. a preservative.

DISADVANTAGE can be stored only for 3 to 6

PROCEDURE months at the most.
1. Dissolve hematoxylin in absolute ethyl alcohol
with gentle heating.
2. Dissolve ammonium or potassium alum in
distilled water on a large boiling flask or beaker. COLE’S HEMATOXYLIN
3. Add hematoxylin solution and boil.
4. Add mercuric oxide and plunge immediately
FORMULA ● Hematoxylin
into cold water for rapid cooling.
● 1% Iodine in 95% Alcohol
a. A large beaker should be used, because
● Sat. Aqueous Ammonium
the violent liberation of oxygen will
Alum
cause the solution to explode from a
● Distilled water
narrow-mouthed flask.
b. The solution should assume a dark
MAIN USE Routine Purposes
purple color when ripened by
mercuric oxide.
5. The addition of 4% glacial acetic acid will give a
more precise nuclear staining. ARTIFICIAL ALCOHOLIC-IODINE SOLUTION
6. The solution is then filtered and transferred into a RIPENER
well-stoppered bottle.
STAINING TIME 10 Minutes
NOTE:
● Harris hematoxylin may either be used PROCEDURE:
immediately or stored for future use, since it 1. Dissolve hematoxylin in warm distilled water
remains stable for a long time (about 6 and mix with iodine.
months). 2. Add alum solution and boil.
○ But formation of precipitate in the 3. Cool and filter before use.
stored staining solution indicates
deterioration in nuclear staining
properties. IRON HEMATOXYLIN
● Used only for Differential or Regressive staining,

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TOPIC: HEMATOXYLIN & EOSIN STAINING

● Differentiating Agent:
MAIN USE ● ROUTINE
○ Acid-Alcohol ○ STANDARD IRON
● The dye lake obtained when ferric salts are used HEMATOXYLIN STAIN
as mordants is an intense blue-black one. They
can be applied to tissues fixed in virtually all ● MUSCLE FIBERS &
fixatives, producing permanent stains, provided CONNECTIVE TISSUES
all iron mordants have been wiped out.
● recommended when the
● Tissues that have been stored in alcohol for years and
preceding stains contain
which would ordinarily fail to stain, will normally take
acid (e.g. Van Gieson
iron hematoxylin. Tissue structures are stained
stain containing picric
blackish or grayish, according to the extent of
acid) which decolorizes
differentiation, producing minimal eyestrain; hence,
nuclei stained with alum
making it useful for photomicrography.
hematoxylin.
● Solutions prepared with correct or optimal
●
amounts of iron salts (0.5 g. metallic iron for each
1 gram of hematoxylin) are used for dense,
regressive staining (e.g. myelin methods). The MORDANT FERRIC AMMONIUM CHLORIDE
stain becomes more selective for nuclei if acid or
an excess of ferric salt is added. PROCEDURE:
● Ferric salts ripen hematoxylin rapidly and are active 1. Hematoxylin is dissolved in alcohol with gentle
oxidizing agents; hence, they do not keep well as a heating, while ferric chloride, hydrochloric acid
prepared mixture. In mixtures of hematoxylin and and water are mixed in a different container.
ferric salts, the insoluble lake gradually precipitates 2. Both solutions are stable and may be stored
out, so that premixed stains are not very stable. separately for 6 weeks before use.
3. Ferric chloride is usually added to the staining
solution just before use, by mixing equal parts of
REGAULD’S METHOD FOR MITOCHONDRIA
the two solutions to produce a deep black mixture.
PREFERRED POTASSIUM DICHROMATE & NOTE:
FIXATIVE FORMALIN ● The working solution will remain active for 1-2
days.
MORDANT DICHROMATE ● COLOR CHANGE:
○ deep blue black-violet, -> violet. purple,
MAIN USES ● MITOCHONDRIA in Light brown yellowish brown within 2 to 3
Microscopy weeks,
■ Discard if Solution becomes
brown
DISADVANTAGES Results are not uniform: some
cells will be over-stained and
some under-stained.
● Therefore a number of HEIDENHAIN’S HEMATOXYLIN
microscopic fields should
FORMULA MORDANT DIFFERENTIATOR:
be examined.
● Ferric ammonium sulfate
● Distilled water
IC TI
HEMATOXYLIN STAIN:
WEIGERT’S HEMATOXYLIN SOLUTION ● Hematoxylin
● 95% ethyl alcohol
FORMULA SOLUTION A: ● Distilled Water
● Hematoxylin
● Absolute Ethyl Alcohol STAINING METHOD REGRESSIVE STAINING OF THIN
SOLUTION A: SECTIONS
● 30% anhydrous ferric
chloride MAIN USE ● STUDY OF MITOSIS
● Concentrated hydrochloric ● Demonstration of both
acid Nuclear and Cytoplasmic
● Distilled Water Inclusions such as:
○ CHROMATIN
■ Stains

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TOPIC: HEMATOXYLIN & EOSIN STAINING

Blue-Black ○ Immediate Ripening

○ CHROMOSOMES can be Obtained by
○ NUCLEOLI Adding 50 ml of 0.25%
○ CENTROSOMES
aqueous potassium
○ MITOCHONDRIA.
● VOLUNTARY MUSCLE permanganate
STRIATIONS and MYELIN
are also well stained STAINING TIME 12-24 Hours

MORDANT FERRIC AMMONIUM SULFATE
(IRON ALUM)
STAINING:
1. Hematoxylin is dissolved in ethyl alcohol and
added with water, allowed to ripen for 4-5
weeks, and stored in tightly stoppered bottles.
a. The mordant differentiator is used
separately during the process of staining,
instead of being added to the solution.
2. After staining, all components are black or dark
grey - black.
3. The hematoxylin staining is moved progressively
from different tissue structures at different rates
using the iron alum solution.
4. Differentiation can be more easily controlled if
the differentiating iron alum solution is diluted
with an equal volume of distilled water or an
alcoholic picric acid solution.
PHOSPHOTUNGSTIC ACID HEMATOXYLIN (PTAH)
FORMULA ● Hematoxylin
● Phosphotungstic acid
● Distilled Water
STAINING METHOD PROGRESSIVE STAINING
● microscopic examination of
the materials every hour is
recommended
STAINING:
1. Dissolve the solids in separate portions of distilled
water.
2. Add together and stand in the light to ripen for
several weeks.
a. Immediate Ripening can be Obtained
by Adding 50 ml of 0.25% aqueous
potassium permanganate after the two
solutions are mixed, so that stain can be
used the next day, although peak
staining activity is not reached until
after 7 days.
3. The color of the solution ranges from
reddish-brown to purple, although this is not a
reliable guide for the study of stained tissues.
NOTES:
● When hematin is used instead of hematoxylin to
prepare a staining solution, the oxidation process is
not necessary and the staining solution can be
used immediately, but its staining activity is
comparatively short-lived.
● 95% Alcohol usually removes the red
component of the stain, so that dehydration and
rinsing of sections should be brief.
● Phosphotungstic acid hematoxylin stain usually
demonstrates structures in paraffin as well as
celloidin and frozen sections.

MAIN USE ● NUCLEI, FIBRIN, MUSCLE COPPER HEMATOXYLIN

STRIATIONS, MYOFIBRILS: ● are utilized for the STUDY OF
○ Stained Blue SPERMATOGENESIS

● COLLAGEN, BONE,
CARTILAGE:
○ Stained
orange-red or
brownish red to
deep brick-red

MORDANT 1% AQUEOUS
PHOSPHOTUNGSTIC ACID

RIPENER NATURAL METHODS (LIGHT &

AIR)
● Takes Months of Ripen

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TOPIC: HEMATOXYLIN & EOSIN STAINING

EOSIN
STAINING PROCEDURE:
● Eosin is one of the most valuable stains used for
1. Dissolve Eosin Y in water by gentle heating.
differentially staining connective tissues and cytoplasm. 2. Cool and add alcohol.
● It is a red general cytoplasmic stain that combines 3. For use, one part of the stock solution is usually
with hemoglobin to give an orange color. diluted with three parts of 80% alcohol.
● It may be used after any fixative and is routinely used in 4. Addition of 0.5 ml. glacial acetic acid for every
histopathology as a counterstain to hematoxylin, 100 ml. of stain will usually give a deeper red
imparting a pink or red color to cytoplasmic material, stain to the tissue.
cell membranes, and some extracellular structures.
NOTE:
● It is commonly used as a background stain because it 5. Differentiation of the eosin staining occurs in
gives a pleasing and colorful contrast to nuclear stains, the subsequent tap water wash, and a little
particularly in chromate and picric acid fixed tissues, and further differentiation occurs through the alcohols.
in acid decalcified materials which are strongly stained 6. Combining eosin Y and phloxine B produces a
with eosin. cytoplasmic stain that demonstrates various
tissue components more dramatically.
● Yellowish (Eosin Y)
○ is the most commonly used Eosin Dye.
○ It is readily soluble in water, less in alcohol,
EOSIN-PHLOXINE B SOLUTION
available in both aqueous and alcoholic
FORMULA ● 1% phloxine
solutions, showing a green yellow fluorescence ● 1% eosin Y
especially in alcoholic medium. ● Glacial Acetic Acid
○ The aqueous stain is generally used as a I % ● 95% Alcohol
solution for 15 seconds to 3 minutes, depending
on the tissue, type of fixative and intensity of color
desired. ROMANOWSKY STAINS
○ Slightly longer staining time is required after formalin
than after Zenker’s solution. FORMULA ● 1% phloxine
● 1% eosin Y
● EOSIN B (EOSIN BLUISH OR IMPERIAL RED) ● Glacial Acetic Acid
● 95% Alcohol
○ Has a very faint bluish cast.
○ The two dyes are interchangeable, and the use of
MAIN USES ● Examine BLOOD or BONE
one or the other is more a matter of preference and MARROW SAMPLES
tradition. Eosin S and Eosin B are now rarely ● Detect BLOOD-BORNE
used. PARASITES (e.g.
MALARIA)
○ Preferred over H&E
5% AQUEOUS EOSIN Y for inspection of
blood cells because
FORMULA ● Eosin Y
different types of
● Distilled Water
○ Dissolve in water by leukocytes (white
gentle heating. Cool blood cells) can be
and filter. readily distinguished.
○ Thymol crystals may
be added to prevent ● Based on a Combination of Eosinate
formation of molds. (chemically reduced eosin) and Methylene Blue
(sometimes with its oxidation products azure A
EOSIN STOCK ALCOHOL
SOLUTION and azure B).
● Eosin Y ● ROMANOWSKY VARIANTS:
● Distilled Water ○ Wright’s Stain
● 95% Alcohol ○ Jenner’s Stain
○ Leishman Stain
○ Giemsa Stain

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