1

HEMATOXYLIN & EOSIN STAINING (H & E) 3. Without an oxidizing agent, the rate of conversion of
STAINING hematoxylin to hematein is determined:
 The process that renders the different tissue components 3.1 pH of the solvent
more visible through variation in color, thereby promoting 3.2 type of solvent
easier optical differentiation & identification of the cell pH & Type of Solvent
& tissue components. - rate of oxidation of hematoxylin to hematein in aqueous
I. INTRODUCTION neutral solution as compared to other solvents.
1. Aqueous Solvent:
1.1 Neutral pH- hematein forms in a few hours
1.2 Acid pH- hematein forms more slowly
1.3 Alkaline pH- hematein forms more rapidly
2. Alcoholic Solvent:
 Hematein forms more slowly
3. Alcoholic Solvent:
 Hematein forms even more slowly with
glycerin
USE OF MORDANT
CLASSIFICATION OF MORDANT:
Nuclear Stain 1. Mordant w/ Iron
HEMATOXYLIN: 2. Mordant containing other Metallic ions such as:
 Most widely used nuclear stain 2.1 Aluminum
 Haematoxylin campechianum logwood 2.2 Potassium
 Requires oxidation to Hematein to become a weak 2.3 Ammonium
anionic dye or ripening 3. Mordant w/ Sulfate ions
 ALUM is a mordant that contains aluminum salts
THREE METHODS OF RIPENING: Use of Bluing Solutions: The alkaline pH of the bluing solution
1. Air oxidation causes the mordant dye-lake to reform in the tissue and become
1.1 Ehrlich’s Hematoxylin more permanent
1.2 Delafield’s Hematoxylin 1. Ammonia (Ammonia H2O) used in routine H & E
2. Addition of Oxidizing agents Staining method
2.1 Sodium iodate 2. Warm H2O (40-50 OC) or distilled H2O
2.2 Mercuric Oxide 3. Lithium carbonate
2.3 Potassium Permanganate 4. Bicarbonate
2.4 Hydrogen peroxide 5. Potassium or sodium acetate
2.5 Calcium Hypochlorite

6. Scott’s tap H2O substitute: (potassium carbonate,
magnesium sulphate, and water)
Classification of Hematoxylin Solution
 Based on whether the mordant is mixed or not mixed with
hematoxylin stain
1. “DIRECT” HEMATOXYLIN STAIN:
 Mordant plus hematoxylin are mixed together
1.1 Alum mordant hematoxylin:
1.1.1 Harris Hematoxylin Compositions:
1) Hematoxylin- stain
2) Ethyl Alcohol,95% - stain solvent
3) Ammonium/Potassium Alum - mordant
4) Distilled H2O- mordant solvent
5) Mercuric Oxide - oxidizing solvent
6) Glacial Acetic Acid – Accentuator
Harris’s hematoxylin (1900)
 This alum hematoxylin was traditionally chemically
ripened with mercuric oxide (sodium or potassium iodate
is frequently used as a substitute for oxidation)
 It gives particular clear nuclear staining
 It is used as a regressive stain in routine histology
practice
 It is used as a progressive stain in diagnostic exfoliative
cytology
 When using It is a progressive stain, an acetic acid-
alcohol rinse provides more controllable method in
removing excess stain from tissue components and the
glass slide.
1.1.2 Ehrlich’s Hematoxylin
1) Hematoxylin – 2g
2) 95% Alcohol – 100 ml
3) Distilled water – 100 ml
2
4) Ammonium aluminum sulfate or Potassium
aluminum sulfate – 3g (acts as mordant)
5) Glycerin / Glycerol – 100 ml
Ehrlich’s Hematoxylin (1886)
 Naturally ripened strong alum hematoxylin
 Stains nuclei intensely and crisply – stained sections fade
much more slowly
 Stains mucin in salivary glands, cartilage and cement lines
of the bones
 Suitable for tissue subjected to acid decalcification
 Suitable for tissues that have been stored in formalin for a
long period ` which have gradually become acidic over the
storage period
 Suitable for Bouin’s fixed tissue
 Not ideal for frozen sections
1.1.3 Delafield’s Hematoxylin
1) Solution A: Saturated ammonium sulfate
 Ammonium aluminum crystals
 Sulfate (mordant) – 180 g
 Distilled water – 1000 ml
2) Solution B: Hematoxylin – 4g
 95% Alcohol
3) Solution C: Glycerol (Stabilize oxidation) –
100 ml
 95% Ethyl alcohol
Delafield’s Hematoxylin (1885)
 The hematoxylin is dissolved in 25 ml of alcohol, and then
added to the alum solution
 This mixture is allowed to stand in light and air for 5 days
and then filtered
 Glycerin and further 100 ml of 95% alcohol are added to
the mixture
 Allow the stain to stand exposed to light and air for about
3-4 months or until sufficiently dark in color
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 Filter before use  Solutions A & B are not mixed. If they are, stain
deteriorates. During staining procedure, Solution A, the
1.2Iron - Mordant Hematoxylin: mordant precedes Solution B, the stain.
1.2.1 Weigert’s Iron Hematoxylin Heidenhain’s hematoxylin (1896)
Solution A: MORDANT (Iron Solution)  This iron hematoxylin uses ferric ammonium sulfate as
• Anhydrous Ferric chloride, 30% - 4 ml oxidant/mordant
• Concentrated Hydrochloric Acid – 1 ml  It used as the differentiating fluid
• Distilled H2O – 95 ml  It is a cytological stain
Solution B: STAIN  It is used regressively
• Hematoxylin – 180 g  After staining, all components are dark gray-black.
• Absolute Ethyl Alcohol – 100 ml  The hematoxylin staining is removed progressively from
 Mix equal Volume of Solutions A and B before use for best different tissue structures at different rates using iron alum
results. solution.
 It may be used to demonstrate
a. Chromatin
Weigert’s Iron Hematoxylin b. Chromosomes
 An iron hematoxylin used as a nuclear stain techniques c. Nuclei
where acidic staining solutions are applied to the sections d. Centrosomes
subsequently. e. Mitochondria
 e.g Van Gieson stain- picric acid is a constituent which f. Muscles striations
have marked decolorizing action on nuclei stained with g. Myelin
alum hematoxylin Other Types of Hematoxylin:
 It is a useful stain, with eosin, for CNS tissues. 1. Mayer Hematoxylin:
1) Hematoxylin – 1g
2. “MORDANT “HEMATOXYLIN –IRON MORDANTS: 2) Distilled water – 1000 ml
2.1 Heidenhain’s Iron Hematoxylin 3) Sodium Iodide (oxidizer) – 0.2 g
Solution A: MORDANT (differentiator) 4) Ammonium aluminum sulfate or Potassium
• Ferric Ammonium sulfate (Iron Alum) – 5g aluminum sulfate (mordant) – 50 g
• Distilled H2O – 100 ml 5) Citric acid (pH adjustment & preservative) – 1g
Solution B: STAIN 6) Chloral hydrate (preservative) – 50g
• Hematoxylin – 0.5 g
• Ethyl Alcohol, 95% - 10 ml Mayer Hematoxylin
• Distilled H2O – 90 ml  Widely used hematoxylin stain
 Chemically ripened with sodium iodate
 More vigorous in action than Ehrlich’s hematoxylin
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 Used as both progressive and regressive stain  A plasma stain, staining cytoplasm of the cells and other
 Used as nuclear counterstain in the demonstration of tissue components red or pink
glycogen (PAS, mucicarmine) in various enzyme  Used for differentially staining connective tissue &
histological techniques. cytoplasm
 Stain applied for short period 5-10 mins until nuclei are  Routinely used in histopathology as a counterstain after
stained and then blued without any differentiation which hematoxylin and before methylene blue.
might destroy decolor the stained cytoplasmic components.  It stains rapidly & brilliantly depending on:
1. fixative used
2. Gill Hematoxylin:  Mercuric salts of Zenker’s fixative attach to the (-)
• Hematoxylin anhydrous – 2g charged groups of the tissue components.
• Distilled water – 730 ml o This increases attraction of the negatively
• Ethylene glycol (solvent, preservative) – 250ml charged eosin stain to the positively charged
• Aluminum sulfate (mordant) – 17.6 g groups of the tissue components.
• Sodium iodate (oxidizer) – 0.2 g  Formalin fixed tissue: place tissue sections in a
• Glacial acetic acid – 20 ml saturated mercuric chloride 2-3 minutes prior to
counterstain to eosin
Gill Hematoxylin (1974)
 Available in 3 concentrations Forms of Eosin
Gill’s I (normal) 1. Eosin Y (Yellowish)
Gill’s II (double)  1% aqueous solution – 200 ml
Gill’s III (triple) -> most concentrated  95% Ethyl alcohol – 600 ml
 More frequently used rather than Mayer’s hematoxylin for  Glacial acetic acid – 4 ml
routine H and E staining Eosin Y
 More stable than Harris’s hematoxylin, as auto-oxidation is  Most commonly used
inhibited to the extend  Readily soluble in water
Other Nuclear Stains  Satisfactory soluble in alcohol
1. Methylene Blue Preparation:
2. Toluidine Blue  Eosin Y, water soluble – 5 gm
3. Crystal Violet  Distilled water – 1000ml
 Crystals of thymol added to inhibit fungal growth.
I. INTRODUCTION: Cytoplasmic Stains  Addition of little acetic acid (0.5-1000ml stain)
1. EOSIN: sharpens the staining
 Mainly a synthetic stain
 used as a counterstain to the nuclear stain 2. Eosin B, Erythrosin B ( Bluish)
 Gives a deeper blue color

3. Ethyl Eosin – (Eosin S)
 Eosin alcohol soluble
Eosin
 Xanthine dyes which stains connective
tissues and cytoplasm in varying intensity
and shades (red to pink)
 Available in the following types:
 Eosin Y (Eosin Yellowish, Eosin water
soluble) -most widely available
 Ethyl Eosin (Eosin S, eosin alcohol
soluble)
 Eosin B (Eosin in bluish, Erythrosine
B)
 Ethyl eosin and eosin B are now rarely
used, although occasional old methods
specify their use- e.g the Harris stain for
Negri bodies.
4. Eosin-Phloxine B Counterstain:
 1% Aqueous solution Eosin Y – 100 ml
 1% Aqueous solution Phloxine B – 10 ml
 95% Ethyl alcohol - 780 ml
 Glacial acetic acid – 5 ml
 Provides more vivid pink shade in the
cytoplasm
 Must be in acidic medium to develop the
appropriate charge on proteins
Types of Eosin
1. Alcoholic Eosin consists of :
• Eosin Y or Eosin B
• 95% Ethyl Alcohol
• Glacial Acetic Acid
5
2. Aqueous Eosin
3. Eosin – Orange G
EOSIN Y:
 most common counterstain to alum hematoxylin in H & E
method.
 stains satisfactorily from both aqueous and ethanolic
solution.
 one of the dyes in Papanicolaou’s EA solution for staining
exfoliative cytology.
 Used as a counterstain in the Gram-Weigert’s method for
gram (+) bacteria & fibrin
 Sometimes called Eosin Y , spirit soluble because the free
coloured acid radical of this dye is Eosinol Y , can be made
from Eosin Y, the sodium salt, by treatment with HCl
 Soluble in ethanol, slightly soluble in xylene but not in H2O
 Sometimes useful for staining difficult tissues
EOSIN B:
 sometimes used instead of Eosin Y in staining techniques
 The free colored radical of this dye, Eosinol B, can be made
from Eosin B, the sodium salt, by treatment with HCl
 Sometimes called Eosin B, spirit soluble
Other Counterstains
1. Acid Fuchsin– Magenta color
2. Aniline Blue– Blue Color
3. Congo red – Red
4. Orange G – Orange
5. Phloxine B - Red Purple

OUTLINE FOR GENERAL STAINING PROCEDURE
BASIC PROCEDURES FOR STAINING PARAFFIN INFILTRATED
& EMBEDDED TISSUES:
II. H&E PROCEDURE:
1. Deparaffinization (Decerate)
 Removal of paraffin so the stain can permeate the
tissue section w/ highly organic solvents
 2 changes of xylene, 2 mins each
2. Section to Alcohol
 Absolute Alcohol removes xylene from the tissue
sections (xylene is not miscible with lower
concentrations of Alcohol & H2O)
6
 One or two changes of absolute alcohol for about 1
minute is adequate
 Gradual “run” through a series of decreasing
concentration of alcohols (95, 80, 70% for delicate
tissues, even 50 & 30% to H2O
Section to Alcohol (Rehydration)
 PURPOSE:
 Prevents rapid movement of fluids into & out of
the tissue sections & consequent loss of the
sections from the glass slides
 DURATION: A minute or 10 dips
 95% Ethyl alcohol is used for hydration &
dehydration but isopropyl alcohol or tertiary butyl
alcohol may be employed
3. Washing
 This is the step where the tissue sections are
brought to the solvent of the stain, usually H2O or
the concentration of alcohol in which the stain is
dissolved
 If H2O is the solvent, the washing is accomplished
in slowly dripping tap H2O for about 3 minutes
4. Staining and Counterstaining
 Follow the order of applying the stain and
counterstain and the use of mordants, differentiating
or decolorizing agents to obtain properly stained
tissue sections.
 Mordant acts to hold stain properly to the tissue to
prevent the stain from being lost from the tissue
sections when they are placed in H2O or low
concentrations of Alcohol, as in dehydration
5. Dehydration (run slides up)
 Accomplished w/ a series of gradually increasing
concentrations of alcohol and absolute alcohol

 70, 95% 2 or 3 changes of 100% alcohol before
passing to xylene
 REASON: Dehydrating in ascending grades of alcohol as
stained sections are mounted in amounting medium
soluble in an organic solvent, such as xylene
6. Clearing
 Assures the removal of the alcohol, thereby making
tissue transparent
 XYLENE: the most commonly used clearing agent
and it serves as the solvent for the mounting medium
 2 or 3 changes of xylene for 1 minute (10 dips) which
are placed in different staining dishes to prevent carry
– over.
7. Mounting
NOTE in STAINING:
 Time in each alcohol is very short (10 dips) as
many stains are soluble in low alcohol
concentration & therefore can be extracted from
tissue sections during the process
 Other remedy: the use of an alcoholic counterstain and
differentiating fluids
II. H&E PROCEDURE: 2 TYPES
1. H&E Progressive Staining
2. H&E Regressive Staining
1. ROUTINE H&E STAINING: Regressive step
Procedure:
1. Deparaffinize sections on slides: 60℃ oven for 15
minutes
2. Xylene: 3 changes, 15-20 dips each
3. Absolute Ethanol: 2 changes, 15-20 dips each
4. 95% Ethanol: 2 changes, 15-20 dips
7
5. Tap water: rinse several times until oily appearance in
slides disappear
6. Harris Hematoxylin: 15 minutes
7. Tap water: wash several times until sections on slides
visible
8. Acid-alcohol (1% HCl in 70% alcohol): 1 quick dip
9. Immediately wash in tap water: 5 dips
10. Neutralize in Ammonia-water: 10-15 dips or until dark
blue color develops
11. Tap water: 15 dips
12. 95% Ethanol: 15 dips
13. Eosin: 5 minutes
14. 95% Ethanol: 2 changes, 15-20 dips each
15. Absolute Alcohol: 3 changes, 15-20 dips each
16. Xylene: 2 changes, 15-20 dips each
ROUTINE H&E STAINING: Automated
Features:
 Linear strainers that transfer 1 or more slides from
1 container to the next
 Leaves the slide in each container for the same
amount of time
 Time that slides are in any given solution can be
changed by altering the number of containers of that
solution
 Containers must be kept filled at all times
 Filter hematoxylin daily & rotate containers
every 2-3 days depending on the volume of the slides
stained
 Change all solutions except Eosin & Hematoxylin
daily
 Eosin should be changed weekly
 8

 The section is place in xylene for 30 minutes to

remove the remaining balsam & then brought down
to H2O
 Place in a 0.5 Potassium permanganate sol’n for 5-
10 mins
 Rinse in tap H2O
 Subsequently immerse in 5% oxalic acid for 5 mins.
or until the section is decolorized
RESULTS:  Wash again in running tap H2O for another 5 mins
Nuclei : Blue  Re-stain with the appropriate staining technique
Cytoplasm : pink TROUBLE SHOOTING IN H&E:
PROBLEM CAUSE PREVENTION
White spots in Water left in tissue Dry section properly
tissue due to or incomplete before paraffinization.
incomplete drying Slides can be treated
deparaffinizatio with absolute alcohol
Precautions in Staining: n then re-treated with
 Stains on the skin should be avoided: xylene
 Not just a sign of poor technique but are health Nuclear stain is Weak acid alcohol Check correct % of acid
hazards per se, being slowly absorbed by the skin too blue alcohol used
eventually producing side effects Nuclear stains is Not leaving the Leave the slides for an
 Failure of sections to remain on the slide during too pale slides in adequate length of time
staining due to: hematoxylin long
 dirty/oily slide enough
 Faulty solution, hematoxylin may not have been There are red to Breaking down of Ensure that the
properly and sufficiently ripened red-brown hematoxylin & sections are blued
 Paraffin, fixative or decalcifying solution that has not nuclei improperly done properly.
been thoroughly washed out and removed bluing step Check oxidation of
Re-Staining of Old Sections: hematoxylin
 Old, bleach or faded sections may be restrained : There are dark Overstaining or Avoid concentrated
 The slide is usually immersed in xylene for 24 cytoplasmic poor eosin solution. If
hours, or gently heated until the mounting begins staining differentiation necessary, dilute the
to bubble eosin solution.
 The coverslip may then be removed by lifting it with Blue-black Metallic sheen Filter the hematoxylin
a dissecting needle precipitate atop developing on daily before using.
 9

sections on slide most hematoxylins

picked up on the
slides
Poor contrast Poor staining Check pH of the
between nucleus either the nuclear staining & adjust if
& cytoplasm or cytoplasmic necessary.
stain Determine whether the
nuclear stain or the
cytoplasmic stain is
inadequate & adjust
staining time
Tissue Falling off Slides not dry Check the temperature
the slides enough before of the oven. Characteristics of A Good Mounting Medium:
staining 1. The refractive index of the mountant should be as near as
Faded slides Stain on archival Demount the coverslip, possible to that of the glass which is 1.518.
slides fading over hydrate section and re- 2. It should be freely miscible with xylene and toluene.
time stain if necessary. 3. It should not quickly dry.
4. It should not crack or produce artefactual granularity on
the slide upon drying.
IV: MOUNTING
5. It should not dissolve out of fade tissue sections.
 Is the placing of the coverslip over the stained tissue
6. It should not cause shrinkage and distortion of tissues.
sections using a syrupy fluid applied between the section
7. It should not bleach out any stain or affect staining.
and the coverslip, setting the section firmly, preventing the
8. It should not change in color or pH.
movement of the coverslip
9. It should set hard, thereby producing permanent mounting
PURPOSE:
of sections.
1. To make a permanent preparation
2. To provide a clear visualization of tissue components to be
studied using a medium which has a refractive index close
METHODS OF COVERSLIPPING: (Hand Coverslipping)
to that of glass slide and near the average refractive of the
1. Forceps method
tissue (Rf =1.518) during microscopic study. Rf of glass
2. Inverted slide method
slide and tissues (Rf=1.53 -1.54).
3. Inverted coverslip method
3. To protect the specimen from physical injury, bleaching
and deterioration due to oxidation.
4. To facilitate easy handling and storage
 10

A. HAND COVERSLIPPING SLIDES:


1. The slide is taken from the last xylene station & excess
xylene is wipe off the back of the slide (opposite the
specimen) and the edges of the tissue section.
2. The slide is placed horizontally on a tabletop, with the
tissue section facing upwards.
3. Drop one or two drops of mounting medium onto the
coverslip or beside the tissue section on the glass slide
 If too much mounting medium is used: it will ooze
out at the sides of the cover slip, should be carefully
wiped away with the fingernail covered by a fine
cloth dipped in xylene
 If too little or too dilute mounting medium: it will
draw away from the edges of the cover slip and the
section must be remounted
4. Lower a slide carefully onto the long edge of the coverslip
and allow it to cover the slide, spreading the mounting
medium evenly and without air bubbles. If air bubbles
form under the leading edge of the cover slip, move the
cover slip up slightly and remove the air bubbles.
5. Carefully wipe the back of the slide with a gauze pad or lint-
free tissue
6. The slides are kept flat overnight (ideally)
 Occasionally, one may note Cloudy areas in the mounted
sections , these are due to moisture  dehydration has not
been adequate
B. AUTOMATED COVERSLIPPING
IV: MOUNTING: Procedure
RE-COVER SLIPPING HAND COVERSLIPPED SLIDE:
1. If a slide needs to be re-covered, place the slide into the
Xylene Coplin jar and secure the lid.
2. Slide may need from a couple of minutes to several days
depending on how old the slide is.
11
3. Gently remove the cover slip once it has been loosened by
the Xylene.
4. If necessary re-cover slip by hand
Mounting of Broken Slides:
 The coverslip can be removed by soaking in xylene
 Place the broken slide in the incubator (37OC) until all the
mountant has been removed.
 Using a sharp scalpel blade, the hardened film is cut around
the section, and the slide is placed in cold H2O until the film
and section float off.
 The film containing the section is mounted on a clean slide,
placed in the 37OC incubator until dry
TYPES OF MOUNTING MEDIA:
1. Water – insoluble Mounting Media or resinous mounting
media (permanent mounting media)
1.1 Natural resinous Mounting Media
 Canada Balsam (Rf index 1.524)
• Medium of choice until 1939
 Gum Damar
1.2 Synthetic Resinous Mounting Media
 Permount or Clarite – (RF index 1.544)
• Most widely used in N. America
 Histoclad
 Kleermount
 Eukitt*
2. . Water –Soluble or aqueous Mounting Media
(Temporary mounts)
 Kaiser’s Glycerol Jelly (Rf index 1.47)
 Von Apathy’s Gum Syrup(Rf index 1.52)
 Farrant’s Medium (Gum Arabic)- (Rf index 1.43)
 Liquid Paraffin – best for Romanowsky stains
V: LABELING:
 12

 Each slide label must bear unique identification and

accession codes
 Includes Patient name
 Accession number usually include the year the specimen
collected with varying prefixes for different types of
specimen
 Labels may be written, etched or embosed on each slide
 Permanent pens which are chemical-resistant shoukd be
used
 Using of bar codes are recommended to minimize clerical
errors

REFERENCES:
 Bancroft, John D., Cook, Harry C.(2010).Theory and
Practice of Histological Techniques.(6th ed).
 Gregorios, Jocelyn H. (2005). Histopathologic
Techniques (2nd ed.).
 Raphael, Stanley S. et al. Lynch Medical Laboratory
Technology.( latest edition).
 Lo, Raymundo W. et al. (2015).Basic Histopathologic
Techniques
 Lamberg, Stanley G. Laboratory Manual of Histology and
Cytology