MUHAMMED E MEENU ELDHOSE MSc Pathology JIPMER

1. Muhammed.EMuhammed.E
Meenu EldhoseMeenu Eldhose
MSc MLT PATHOLOGYMSc MLT PATHOLOGY
JIPMERJIPMER
H & E staining &H & E staining &
Trouble shootingTrouble shooting

2. INTRODUCTIONINTRODUCTION
• If something have color that helps to know things,
differentiate & express , particular entity
• Dyes are the agents which are get absorb into on which
it applied and resist to soap washing and sunlight
• Colors are agent which only adsorb on surface and
dose not resist to sunlight and soap
• “Biological dyes” - highlight and differentiate
tissue components and allow them to be seen under
the microscope

3. • The tissue section is colorless because the fixed protein
has the same refractive index as that of glass
• The sections,, are colorless and different components
can’t be appreciated
The Need to Stain
• Outlines tissues and cellular components.
• Identification of tissues.
• Establishes the presence or absence of
disease processes.

4. • Staining them by different coloured dyes, having
affinities of specific components of tissues, makes
identification and study of their morphology possible.
• stains give a contrast to the microscopic slide.

6. • Stain is the reagent used to generate the color
• Staining : process of coloring cells, cellular constituent
& tissue fibers to facilitate optical differentiation by
microscopic examination.
• Is the union between a colored dye & a tissue substrate
which resists simple washing

8. NATURAL DYESNATURAL DYES
• Dyes derived from plants, invertebrates or minerals.
• Majority natural dyes are derived from vegetables
1.Cochineal
2.Orcien
3.Saffron
4.Haematoxylin

9. 1. Cochineal1. Cochineal
• Source from dried female insect : Coccus cacti
• Cochineal dye was used by the Aztec and Maya peoples
of Central and North America
• Synonyms : cochineal extract", "carmine", "crimson
lake", "natural red 4", "C.I. 75470", "E120“
• Biological Morphology :Three pairs of legs and seven
pairs of antenna

10. Female
Male

11. • During egg development stage insects are collected.
• They are killed by water boiling or stove burning it gives
purplish black colors.
• When they are killed by charcoal fumes or burning with
sulphar it develops purplish grey color.
• Chemical constituent : 10% anthorquinone dye known
as carminic acid which brilliant purple.

12. • Carmine was introduced in 1849 by Goppert
and Cohn.

13. 2.2. OrceinOrcein
• Derived from lichens
• Demonstrate elastic fibers and, following permanganate
oxidation (Shikata's modification), hepatitis B surface
antigen, copper associated protein, and sulfated mucins

14. 3.3. SaffronSaffron
• From the dried stigmata of Crocus sativus
flower("saffron crocus“)
• In 1714 Leeuwenhoek employed saffron on sections of
muscle fibres
• Coloring substance is termed as “ crocin “

15. Synthetic DyesSynthetic Dyes
• Alizarin in 19th century -1st synthetic dye
• 1.Acidic dyes
• Dyes that have affinity towards basic part of tissue
components
• Acids have a negative charge in an electric field, they
migrate towards anode (anions)
• Tissue having a positive charge will attract with dyes with
a negative charges
• Such tissues are acidophilic
• Eg: Orange G, Eosine, Nigrosine

16. 2.Basic dyes
• Dyes have affinity towards acid part of tissues
• Bases have a positive charge ,migrate towards Cathode
(cations )
• Tissue carrying negative charge will attract dyes of
positive charge
• These tissue are basophilic
• Eg: Methylene blue, Crystal violet,
Basic fuchsin,Malachite green,
Hematoxylin

17. • 3.Neutral Dyes
• Compound stains
• Produced by neutralizing suitable acid dyes with suitable
Basic dyes
• When aqueous solutions of two dyes brought together ,an
interchange of ions takes place
• Precipitate is formed which is a two color- dye
• It is insoluble in pure water but dissolve in water
containing excess of acid/basic dye from which they are
formed
• Eg: Romanowsky stains

18. • Sections stained with acidic or basic dyes are mounted in
non aqueous media to prevent loss of dye

19. Theory of staining
• 1.Physical theory
• Solubility: stain is more soluble in fat than in the solvent
• Adsorption :large body attracts minute particles from the
surrounding medium

20. • Chemical theory
• Basic structure of all the stains is Benzene
• Benzene has a ring structure, it give flexibility
• Chromophore :Chromophore :Chemical group introduced into benzene
ring by substituting Hydrogen
• Benzene + chromophoreBenzene + chromophore ChromogenChromogen

21. • Along with chromogen it is necessary to introduce
Ionizing groups-Auxochromes
• After the addition of Auxochromes ,colored compound
stains the tissue and be resistant to simple washing
• STAIN = AuxochromeAuxochrome+ChromogenChromogen

22. Principle of staining
1.Electrostatic Bonding1.Electrostatic Bonding
•Affinity between Opp.ionic groups of dye and tissue
2.Hydrogen Bonding2.Hydrogen Bonding
•Form of dye tissue attraction
•Arises when hydrogen atom lies between two
electronegative atoms
•Weak bonds, occur readily in water, Occur between dye
and water

23. • 3.Van der waal forces3.Van der waal forces
• Occur between reagent and tissue substrates
• Involves various intermolecular attractions
Dipole-dipole
 Dipole induced dipole
 Dispersion forces
• These forces are polar attractions
• Effective over a short distance
• Eg : Staining of elastic fibres by orcein

24. • 4.Covalent Bonding4.Covalent Bonding
• Can occur between the stain and the tissue
• Covalent bonding involves sharing of electrons
• Sharing of 2,3 or more electrons between 2 atoms –
Multiple covalent bonds
• This bonding is of significance in mordant dying process

25. MordantsMordants
• “To bite”
• A polyvalent metal ion which forms coordination
complexes with certain dyes
• A substance which acts as an intermediary between dye
and tissue, thus increasing the affinity between them
• Strictly applicable to salts and hydroxides of divalent
and trivalent metals
• A mordant is a metal with a valency of at least two
• Commonly used in histotechnology are aluminum and
ferric iron (Valencies 3 )

26. PRINCIPLE OF MORDANT
• Hematin is anionic.
• Tissue is also anionic.
• Therefore hematin has poor affinity for tissue
• Making hematin adequate as a nuclear stain with the
presence of a 3rd element (mordant)
• Mordant forms a link between the “tissue and the stain”

27. • The chelate formed from a mordant dye and a metal is
called a lake

28. Mordant-Dye application
• Mordant is applied first, followed by the dye.
e.g Heidenhain’s iron hematoxylin
• Mordant and dye are mixed together and then applied.
Commonly done in histotechnology
e.g Alum hematoxylin solutions
• Dye applied first, followed by the mordant.
Hardly done in histotechnology

29. AccentuatorsAccentuators
• Substances which increase the staining intensity ,
crispness and selectivity of dye.
• Eg : KOH in Lofflers methylene blue
phenol in carbol fuschin & carbol thionin
Aniline dye in gentian violet
• They do not form lakes with dyes
• They act by changing pH

31. AcceleratorsAccelerators
• Accentuators used in metallic impregnation technique for
the nervous system.
• e.g chloral hydrate, Barbital
Trapping agentsTrapping agents
• Agents which inhibit removal of dyes from tissue
• e.g Iodine in Gram stain
NaCl in Gram Weigert method

33. RipeningRipening
• Oxidation of haematoxylin
• This process converts the haematoxylin to haematin
1.Natural
• Carried out by exposure to light and air
• Slow process – takes about 3-4 months
• Resultant solutions seem to retain its staining ability for a
long time
• e.g Ehrlich’s & Delafield’s haematoxylin solutions

34. 2.Chemical oxidation
• Oxidation occurs instantaneously
• These solutions have a shorter shelf life
• Oxidation using sodium iodate – most commonly used
e.g. Mayer’s hematoxylin
• Oxidation by mercuric oxide e.g. Harris’s hematoxylin
• Ferric chloride and potassium permanganate can also used

35. Staining ReactionsStaining Reactions
• Absorption or direct staining – tissue penetrated by
dye solution.
• Indirect staining – using intermediate treatment with
mordant
• Physical staining – simple solubility of dye in element
of tissue.
• Chemical staining – formation of new substance e.g.
PAS
• Adsorption phenomenon – accumulation on the surface
of the compound.

36. Progressive stainProgressive stain
• Tissue is left in the stain just enough to reach the
endpoint
• Frequent monitoring of stain quality may be needed to
determine when staining is complete
• The staining intensity is controlled by the time it is
immersed in the solution
• Eg : progressive staining using Gill’s Hematoxylin

37. Regressive StainingRegressive Staining
• Involves over-staining where the dye completely
saturates all tissue elements
• The tissue is then selectively de-stained using a process
called differentiation
• Eg :Harris Hematoxylin is popularly used
regressively in many histology labs for routine H & E
staining
• Sharper degree of staining is obtained

38. DiffrentiationDiffrentiation
• Selective De-staining
• Removal or washing out of excess stain until the color is
retained only by the tissue component that is to be
studied
• Differentiation is achieved by using a dilute acid, most
typically 1% acid alcohol
• Differentiation is halted by washing in water when the
desired endpoint is reached
• Exposure to air may oxidize and improve the process.

39. BlueingBlueing
• Blueing is the process of shifting the color
from reddish to purple blue by the application of
a weak alkaline solution
• Blueing is utilized in both progressive and
regressive techniques
• Eg :Tap water , Scott’s tap water(Magnesium
sulfate ,Sodium bicarbonate )Saturated Lithium
carbonate, Ammonia in distilled water

41. MetachromasiaMetachromasia
• Dye has the ability to change its color without changing
chemical structure
• Eg : Thiazine group like thionin,toludine blue,Azure A,B
• When negatively charged groups on the tissue react with
cationic dyes
• Enhanced when intermolecular distance reduced

43. • Polychromasia:Polychromasia: Exhibit two or more shades of color
reactions Eg : Leishman,Giemsa
• Orthochromasia :Orthochromasia : Color of the dye doesn’t change

44. History of hematoxylin
• It was independently introduced in 1865 and 1875, by
Böhmer and Fischer respectively.
• Primary diagnostic technique in the histo-pathology
laboratory.
• Waldeyer established the use of
hematoxylin in histology in 1862.

45. Cont…
• Bohmer combines hematoxylin with
alum as a mordant & obtained more
specific staining in 1864.
• Heindenhan introduced classical Iron -
Alum hematoxylin method
• Ehrlich overcame the instability of alum
hematoxylin by adding glacial acetic acid.

46. HEMATOXYLIN
• The word hematoxylin is derived from old Greek word
Haimato (blood) & Xylon (wood) , referring to its dark red
colour in natural state.
• A natural dye extracted from the log wood of the tree
Haematoxylon campechianum, mainly seen in Campeche
state of Mexico & also available in West Indies

47. Extraction
• Small pieces of the log wood is boiled in water
• First orange- red solution is formed.
• On cooling it turn into black solution.
• Then precipitated with urea or ether.
• Purified and sold commercially.

48. • Hematoxylin itself is not a stain
• On oxidation it produces hematein (poor dye).
• Commonly used dye in histopathology, cytology,
immuno histochemistry.
• Best nuclear stain.
• Basic in nature, stains acidic component of the
tissue, nucleus, mitochondria etc.

49. oxidation
• Oxidation of hematoxylin is called RIPENING.
• Hematein can be produced in 2 ways
i. Natural ripening
ii. Chemical ripening

50. Cont…
Natural ripening
• By exposure to sunlight & air
• Slow process, takes approximately 3 months.
• Staining life of the dye is longer.
Chemical ripening
• Chemical agents converts hematoxylin to hematein
instantly.
• Sodium iodate , mercuric oxide commonly used, Ferric
chloride and potassium permanganate can also be used.
• Short useful lifespan

51. Natural oxidation Chemical oxidation
Ehrlich’s hematoxylin Mayer’s hematoxylin
Delafield’s hematoxylin Harri’s hematoxylin
Cole’s hematoxylin
Carazzi’s hematoxylin
Gill’s hematoxylin

52. MORDANT
• Hematein is a weak basic dye having a poor affinity for
tissue
• Mordant is a chemical substance that increases the
affinity of dye for tissue and its staining efficiency by
forming link between the stain and the tissue.
• It’s a metal with valence of atleast two.
• Various mordants used are aluminium ,iron, lead,
phosphotungstic acid etc.

53. Types of hematoxylin
According to the mordant used
 Alum hematoxylin
 Iron hematoxylin
 Tungsten hematoxylin
 Molybdenum hematoxylin
 Lead hematoxylin
 Hematoxylin with out mordant

54. ALUM HEMATOXYLIN
• Mordant used are aluminum salts, either aluminum
potassium sulphate (potash alum) or aluminum
ammonium sulphate (ammonium alum).
• Mainly used in routine H and E staining.
• Has different types but all of them stain the nuclei blue-
black.
• These are used when counter stain does not contain an
acid.
• Can be used progressively or regressively.

55. 1. Ehrlich’s hematoxylin
• Strong stain for nuclei, stains intensely & crisply
• Oxidized naturally
• Stained sections fade slowly.
• Not ideal for frozen sections
e

56. 2. Harri’s hematoxylin
• Widely used in exfoliative cytology as a nuclear
stain
• Chemical oxidation – mercuric oxide
• Life span is short : 2-3 months

57. 3. Mayer’s hematoxylin
• Used as anuclear counterstain where the cytoplasmic
material needed to demonstrate.
• Used as a progressive stain
• Chemical oxidation – sodium iodate
• Gives little or no staining of mucopolysaccharide
material.

58. 4. Delafield’s hematoxylin
• Naturally ripened
• Similar properies of Ehrlich’s hematoxylin

59. 5. Cole’s hematoxylin
• Chemically ripened with alcoholic iodine solution

60. 6. Carazzi’s hematoxylin
• Chemically ripened with potassium iodate
• Used as a progressive nuclear stain.
• Largely confined to use with frozen sections

62. Disadvantage of alum hematoxylin
• Alum hematoxylin nuclear stain is sensitive
to the subsequently applied acidic solutions.
• Common examples are Van Gieson & trichrome
stain.
• Satisfactory staining can be achieved by using Iron
mordanted hematoxylin, which resist effect of picric
acid.
• Combination of celestine blue with an alum

63. Celestine blue
• Oxazine Dye
• Has little useful coloring property of its own
• It forms an additional strong mordant with certain
hematoxylins.
• Used as a preliminary to alum hematoxylin staining.
• Resistant to the effects of acid.
• Ferric salt in the celestine blue solution strengthens the
bond between the nucleus and the alum hematoxylin to
provide a strong nuclear stain which is reasonably
resistant to acid.

64. Preparation
• Celestine blue B : 2.5g
• Ferric ammonium suphate : 25g
• Glycerine : 70ml
• Distilled water : 500ml
 Ferric ammonium sulphate is dissolved in cold distilled water with
stirring.
 The celestine blue B is added to this solution and the mixture is
boiled for few minutes.
 Filtered
 Glycerine is added, filter

65. Iron hematoxylin
• Iron salts used both as mordant & oxidizing agent
• Commonly used salts are ferric chloride & ferric
ammonium sulphate.
• Over oxidation is the problem : to avoid this prepare
mordant/ oxidant & hematoxylin seperately & mix them
immediately before use.

66. Stain Mordant oxidation Application/
results
Staining time
Weigert’s
hematoxylin
Ferric chloride Natural Nuclear stain
with acid dye,
Stains nucleus
brown to black
15-30 min
Heidenhains’s
hematoxylin
Ferric
ammonium
sulphate
Natural mitochondria,
chromatin &
muscle fiber
striation stains
black or dark
grey- black
30-45 min at
60º C
12- 24 hrs at
RT
Verhoeff’s
hematoxylin
Ferric chloride Natural Stains elastic
fibers as black
25- 60 min
Loyez
hematoxylin
Ferric
ammonium
sulphate
Natural Myelin

67. Tungsten hematoxylin
• Mallory phosphotungstic acid hematoxylin (PTAH) is an
example
• Phosphotungstic acid is used as mordant.
• Possible to prepare a staining solution using hematein ;
instead of hematoxylin. Oxidation not required, can be
used immediately , but short lived.

68. • Can be naturally oxidized, takes months to ripen: usable
for many years
• Can be oxidized chemically using potassium
permanganate.
• Applicable to nervous tissue & also used to stain
muscle striations and fibers

69. Molybdenum hematoxylin
• Molybdic acid as the mordant.
• For the demonstration of collagen, coarse reticulin&
granules in endocrine cells.
• Hydrogen per oxide is used for oxidation.
Lead hematoxylin
• Lead salts act as the mordant.
• Identification of endocrine cells in some in tumors.

70. Alternatives to hematoxylin
• Celestine blue
• Gallo cyanine
• Solochrome cyanine

71. Eosin
• Most suitable stain to combine with alum hematoxylin.
• Eosins are xanthine dyes (tetrabromofluorescein)
• TYPES OF EOSIN - commercially available
 Eosin Y
 Ethyl eosin
 Eosin B

72. Cont…
o Eosin Y
• Eosin yellowish
• Most widely used
• It is water & alcohol soluble.
• Used as a cytoplasmic stain - 0.5-1% solution in distilled
water with a Crystal of thymol - prevent fungal growth.
• Addition of Acetic acid sharpens the staining
o Ethyl eosin (eosin alcohol-soluble)
o Eosin B (eosin bluish, erythrosine B)

73. Cont…
DIFFERENTIATION OF EOSIN
• Occurs in the subsequent tap water wash
• Further differentiation occurs during the dehydration
through the alcohols
ALTERNATIVES FOR EOSIN
 Phloxine
 Bierbrich scarlet
 saffranine

74. Basic steps of staining

75. RESULT

76. Troubleshooting in H&E staining
Problems Possible causes Remedies
Pale stained nuclei 1. Too much differentiation
2. Too less time in
haematoxylin
3. Due to excessive
decalcification
4. Haematoxylin is over
oxidized
1. Stain in haematoxylin
again
2. Keep in haematoxylin for
longer duration
3. Not possible to correct
4. Change the haematoxylin
solution
Darkly stained nuclei 1. Too short differentiation
2. Too much time in
haematoxylin
3. Thick section
1. Decolorize and do
optimum differentiation
2. Decolorize and give
appropriate time in
haematoxylin
3. Recut thin section
Nuclei looks reddish brown 1. Insufficient bluing
2. Haematoxylin is
degenerating
1. Restain by giving more
time in bluing step
2. Check the oxidation status
of haematoxylin

77. Problems Possible causes Remedies
Pale coloured cytoplasm
by eosin
1. Too thin section
2. The eosin solution has
pH more than 5
3. Too much dehydration
of
the section in alcohol
1. Recut the section
properly
2. May be due to dilution
of
eosin by the carryover
bluing solution. Check
pH of eosin solution
3. Do not keep the slide in
alcohol for a long time
Bluish-black precipitate May be due to precipitation
of haematoxylin
Filter the haematoxylin
staining solution
Staining is irregular and
spotty
Improper deparaffinization Keep the slide in xylene for
longer time to remove
the paraffin
Dark-blue stain at
the edge of the
tissue sections
Due to heating artefact for
using electrocautery
No solution
Water bubbles in the
section
Incomplete dehydration Remove the mounting
medium and coverslip.
Keep the section in
absolute alcohol for

78. Pale stained nuclei Too dark nuclear stain

79. Incomplete deparaffinization Cytoplasmic staining is dark

80. Weak staining of eosin Uneven staining

81. THANK YOUTHANK YOU