- Hematoxylin and eosin (H&E) staining is the most widely used staining technique in histopathology. Hematoxylin stains cell nuclei blue-black, while eosin stains cytoplasm and other tissues shades of pink-red. - Hematoxylin is extracted from the logwood tree and must be oxidized to hematin to function as a stain. It is used with a mordant like aluminum or iron salts to bind to cell nuclei. Alum hematoxylins are the most common and produce red nuclei that are "blued" after staining. - Different hematoxylin solutions exist based on the mordant used, including alum, iron,

1. MR:MAHMOUD IBRAHIM
HEMATOXYLIN AND
EOSIN (H&E)

2. HISTORICAL ASPECT OF
HEMATOXYLIN
•The introduction of hematoxylin is
attributed to Waldeyer in 1862 that
used it as a watery extract but without
very much success.

3. •Two years later Bohmer combined
haematoxylin with alum as a mordant
and obtained more specific staining.

4. •Ehrlich (1886) who overcame the
instability of hematoxylin and alum by
the additions of glacial acetic acid and at
the same time produced his formula for
haematoxylin as it is used today.

5. HAEMATOXYLINS AND
EOSIN (H&E)
•H&E stain is the most popular stain
(routine) in histopathology field.
•Compare the simplicity and demonstrate
clearly different types of tissue
structures.

6. • Hematoxylin stains the nucleus blue-black
with clear chromatin particles.
• Eosin stains cytoplasm and most connective
tissue fibers and muscles in different shades
of colours varying from pink to red.

7. •Hematoxylin is a natural dye extracted
from the log wood (heart wood) of
Haematoxylon campechianum tree.
•Hematoxylin extracted from log wood by
hot water and precipitated by urea.

8. • Hematoxylin original country is southern
Mexico and cultivated for commercial
purposes in Jamaica and Indies.
• Hematoxylin its self is not a stain, unless it
oxidizes to haematein

9. •Hematoxylin converted to haematein
through oxidation

13. OXIDATION OF
HEMATOXYLIN
•Hematoxylin Oxidized to haematin by
two ways:
Naturally :
•This is a slow process, sometimes taking
as long as 3–4 months, but the resultant
solution seems to retain its staining
ability for a long time.

14. •Ehrlich’s and Delafield’s hematoxylin
solutions are examples of naturally
ripened hematoxylins.

15. Chemically :
•(chemical oxidizing agents) using
sodium iodate , potassium iodate,
mercuric oxide or alcoholic iodine.

16. •Chemical oxidation takes short time but
the hematoxylin useful life is short when
compared to naturally oxidized
hematoxylin.

17. •Haematein is an anionic dye having a
poor affinity for tissue ( nucleus), unless
mordant is used.

18. •Mordant with hematoxylins is alkaline
substance (metal), added to haematein to
link between tissue( nuclei) and the dye (
haematein).

19. •Most mordants are incorporated into the
hematoxylin staining solutions, although
certain hematoxylin stains required the
tissue section to be pre-treated with the
mordant before staining; such as
Heidenhain’s iron hematoxylin.

20. TYPES OF HEMATOXYLINS
Hematoxylin solutions can be classified
according to which mordant is used into:
•1. Alum hematoxylins
•2. Iron hematoxylins

21. •3. Tungsten hematoxylins
•4. Molybdenum hematoxylins
•5. Lead hematoxylins
•6. Hematoxylins without mordant

22. 1- ALUM HEMATOXYLINS
•Mordant is aluminum either in the form
of aluminum potassium sulfate or
aluminum ammonium sulfate.
•Oxidation either naturally or chemically.
•Stain the cell nucleus red.

23. •Converted into familiar blue color by
process of bluing.
•Bluing is the conversion of the red color
into blue as a result of changing the pH
from acid to alkaline, done by:

24. •R.T.W, 0.05% Ammonia in water,
Alkaline solution such as Lithium
Carbonate, Scott's tap water

25. •The alum hematoxylins can be used
regressively, meaning that the section is
over-stained and then differentiated in
acid alcohol, followed by ‘blueing’,

26. •Or progressively, i.e. stained for a
predetermined time to stain the nuclei
adequately but leave the background
tissue relatively unstained.

27. •The most common alum hematoxylins
are:
•Ehrlich’s H, Delafield H, Mayer’s H,
Harris's H , Cole’s H, Carazzi’s H, and
Gill’s H .

28. 1- EHRLICH'S HEMATOXYLIN
•Naturally ripened alum hematoxylin.
•Composes of : hematoxylin powder (dye)
, absolute ethanol (solvent),)

29. •glycerin (stabilizer: prolonged half life
and slow oxidation rate), D.W (solvent),
potassium alum (mordant), and G.A.A
(accentuator)

30. •Uses: good nuclear stain, mucins,
cartilage and bone.
•Advantages: useful for staining sections
from tissues that have been exposed to
acid.

31. •It is suitable for tissues that have been
subjected to acid decalcification or, more
valuably, tissues that have been stored
for along period in formalin fixatives.

32. •Disadvantages: Not suitable for frozen
section

33. 2- DELAFIELD'S HEMATOXYLIN
•Naturally ripened alum hematoxylin.
•Composes of : hematoxylin powder (dye)
,95% ethanol (solvent), glycerin
(stabilizer: prolonged half life and slow
oxidation rate), D.W (solvent), potassium
alum (mordant).
•Similar to Ehrlich,s.

34. 3- MAYER'S HEMATOXYLIN
•Chemically ripened alum hematoxylin.
•Composes of : hematoxylin powder (dye)
, D.W (solvent), potassium alum
(mordant), sodium iodate (oxidizing
agent), citric acid (accentuator), chloral
hydrate (preservative).

36. •Uses: nuclear stain, nuclear counter stain
in glycogen and Immunohistochemisty ,
Enzymehistochemistry.

37. 4- HARRIS'S HEMATOXYLIN
•Chemically ripened alum hematoxylin.
•Composes of : hematoxylin powder (dye)
, absolute ethanol (solvent), D.W
(solvent), ammonium alum (mordant),
mercuric oxide (oxidizing agent), G.A.A
(accentuator).

38. •Uses: nuclear stain, especially in
diagnosis of exfoliative cytology.

41. 5- COLE'S HEMATOXYLIN:
• Chemically ripened alum .
• Composes of : hematoxylin powder (dye) ,
saturated aqueous alcoholic iodine(oxidizing
agent), potassium alum (mordant), D.W
(solvent),
• Uses: nuclear stain.

42. 6- CARAZZI'S HEMATOXYLIN
• Chemically ripened alum.
• Composes of : hematoxylin powder (dye) ,
glycerin (stabilizer: prolonged half life and
slow oxidation rate), D.W (solvent),
potassium alum (mordant), potassium iodate
(oxidizing agent).

43. •Uses: nuclear stain.
•Advantages: suitable for frozen section.

44. 7- GILL'S HEMATOXYLIN:
•Chemically ripened alum hematoxylin.
•Compose of : hematoxylin powder (dye) ,
aluminum sulfate (mordant), D.W
(solvent), ethylene glycol (preservative),
sodium iodate (oxidizing agent), G.A.A
(accentuator).

45. •Uses: nuclear stain, mucin darkly.
•Disadvantages: stain gelatin, stain slide.

46. DISADVANTAGES OF
ALUM H.
•Sensitivity to any subsequently applied
acidic staining solutions .
•This problem overcame by use iron H , or
combination of alum H with Celestine
blue B

47. STAINING TIME WITH ALUM H.
Depend on:
•Type of H used.
•Age of stain.
•Intensity of use of stain.
•Whether the stain used progressively or
regressively

48. •Pretreatment of tissue or section.
• Post-treatment of section
• Personal preference

49. EOSIN
•Eosin is the most suitable stain to
combine with an alum hematoxylin to
demonstrate the general histological
architecture of a tissue.

50. •Its particular value is its ability, with
proper differentiation, to distinguish
between the cytoplasm of different types
of cell,

51. • and between the different types of
connective tissue fibers and matrices, by
staining them differing shades of red and
pink.

52. TYPES OF EOSIN
•Eosin B.
•Eosin Y.
•Ethyl eosin.
•The Eosin Y is the most common stain
used as counter stain with hematoxylin,
because it colors back ground by color
vary from pinkish to reddish

53. •1 g or 0.5 g Eosin in 100 ml D.W(1% or
0.5% Aqueous Eosin), 0.05 ml G.A.A and
small amount crystal
thymol(preservative).

54. •Differentiation of the eosin staining
occurs in the subsequent tap water wash,
and a little further differentiation occurs
during the dehydration through the
alcohols.

55. •The intensity of eosin staining, and the
degree of differentiation required, is
largely a matter of individual taste.

60. CELESTINE BLUE-ALUM
HEMATOXYLIN
•Is popular method used overcome
disadvantage of alum hematoxylin
Celestine blue is resistant to the effects of
acid, and the ferric salt in the prepared
Celestine blue solution strengthens

61. •The bond between the nucleus and the
alum hematoxylin to provide a strong
nuclear stain which is reasonably
resistant to acid.

62. IRON HEMATOXYLIN
•In these hematoxylin solutions, iron salts
are used both as the oxidizing agent and
as mordant. The most commonly used
iron salts are ferric chloride and ferric
ammonium sulfate, and the most
common iron hematoxylins are:

64. • Over-oxidation of the hematoxylin is a
problem with these stains, so it is usual to
prepare separate mordant/oxidant and
hematoxylin solutions and mix them
immediately before use e.g. in Weigert’s
hematoxylin)

65. •or to use them consecutively (e.g.
Heidenhain’s and Loyez hematoxylins).
Because of the strong oxidizing ability of
the solution containing iron salts,.

66. •it is often used as a subsequent
differentiating fluid after hematoxylin
staining, as well as for a mordanting
fluid before it

67. •The iron hematoxylins are capable of
demonstrating a much wider range of
tissue structures than the alum
hematoxylins, but the techniques are
more time-consuming.

68. •and usually incorporate a differentiation
stage which needs microscopic control
for accuracy.

69. WEIGERT,S HEMATOXYLIN
• This is an iron hematoxylin in which ferric
chloride is used as the mordant/oxidant. The
iron and the hematoxylin solutions are
prepared separately and are mixed
immediately before use. Used to stain nuclei

70. Preparation:
The iron and hematoxylin solutions are
prepared separately and are mixed
immediately before use.
Solution A:
1 g hematoxylin dissolve in 100 ml of absolute
alcohol

71. •Solution B (Mordant and oxidizing):
•30% ferric chloride 4ml
•Conc Hcl 1ml
•D.W 95 ml

72. The color of the mixture should be a violet
black. If muddy – brown, it must be
discarded.
Differentiator used 1% acid alcohol

73. HEIDENHAIN’S HEMATOXYLIN
•This iron hematoxylin uses ferric
ammonium sulfate as oxidant/mordant,
and the same solution is used as the
differentiating fluid.

74. The iron solution is used first
The section is treated with hematoxylin
solution until it is over stained,
Then it is then differentiated with iron
solution under microscopic control.

75. •Heidenhain’s hematoxylin can be used to
demonstrate many structures according
to the degree of differentiation.

76. • It may be used to demonstrate
chromatin,
chromosomes,
nuclei,
centrosomes,
mitochondria,
muscle striations
myelin

77. LOYEZ HEMATOXYLIN
• This iron hematoxylin uses ferric ammonium
sulfate as the mordant. The mordant and
hematoxylin solutions are used consecutively,
and differentiation is by Weigert’s
differentiator ((borax and potassium
ferricyanide)

78. •It is used to demonstrate myelin and can
be applied to paraffin, frozen, or
nitrocellulose sections.

79. VERHÖEFF’S HEMATOXYLIN
•This iron hematoxylin is used to
demonstrate elastic fibers. Ferric chloride
is included in the hematoxylin staining
solution,

80. •together with Lugol’s iodine, and 2%
aqueous ferric chloride is used as the
differentiator. Coarse elastic fibers stain
black.

81. TUNGSTEN HEMATOXYLINS
• Mallory phosphotungstic acid hematoxylin
(PTAH) is only one widely used tungsten
hematoxylin. combined hematoxylin with 1%
aqueous phosphotungstic acid, the latter
acting as the mordant.

82. •Its use is applicable to both CNS material
and general tissue structure, and to
tissues fixed in any of the standard
fixatives.

83. MOLYBDENUM HEMATOXYLINS
•Hematoxylin solutions that use molybdic
acid as the mordant are rare. Used to the
demonstration of collagen and coarse
reticulin.

84. LEAD HEMATOXYLINS
• Hematoxylin solutions that incorporate lead
salts have recently been used in the
demonstration of the granules in the
endocrine cells of the alimentary tract and
other regions.

85. • The most practical diagnostic application is in
the identification of endocrine cells in some
tumors, but it is also used in research
procedures such as in the localization of
gastrin-secreting cells in stomach.

86. HEMATOXYLIN WITHOUT A
MORDANT
•Freshly prepared hematoxylin solutions,
used without a mordant, have been used
to demonstrate various minerals in tissue
sections (Iron, Copper).

87. •The basis of the method is the ability of
hematoxylin to form blue black lakes
with these metals

88. TEST FOR STAINING POWER OF
HEMATOXYLIN
•Adding few drops of hematoxylin to
50ml of tap water will turn a bright,
clear purple or blue violet color.
•Exhausted solutions will not be clear &
bright & the color will be rusty or green

90. APPLICATION OF H&E
•1-Cell biology
•2-Primary diagnostic technique in the
histopathology laboratory.
•3-Primary technique for the evaluation
of morphology.

91. •4-Counter stain in
immunohistochemistry
•5-Counter stain in many special stains
•6-Demonstration Carbohydrate, lipid

92. •Connective tissue fiber and nervous
tissue
•7-Diagnostic procedure in cytology
•8-Inflammation

93. TROUBLESHOOTING OF H& E
STAIN
Problem Causes Solvents
White spots are seen
in the section after
deparaffinization
step. If they are not
recognized at this
point, spotty or
irregular staining
will be seen
microscopically on
the stained section.
A. The section was
not dried properly
before beginning
deparaffinization.
B. the slide did not
remain in xylene
long enough for
complete removal of
the paraffin.
A. The slides must
be treated with
absolute alcohol to
remove the water
and then retreated
with xylene to
remove the paraffin.
If incomplete drying
is severe, the
sections may loosen
from the slides.

94. B. The slides should be
returned to xylene for a
longer
time.
The nuclei are too pale (the
hematoxylin is too light).
A. The sections were not
stained long enough in
hematoxylin.
B. The hematoxylin was
over oxidized and should
not have been used.
C. The differentiation step
was too long.
D. Pale nuclei in bone
sections may be the result
of over decalcification
A. The section must be
restrained. When sections
have been placed in an
extremely acidic fixative
such as Zenker solution,
the ability to stain the
nucleus may be
impaired and the time in
the hematoxylin may have
to be increased, or a
method to increase tissue
basophilia may be needed.

95. B. Discard hematoxylin
and replace with fresh.
C. Run back and restrain
D. no solution
The nuclei are overstained
(the hematoxylin is too
dark), or
diffuse hematoxylin
staining of the cytoplasm
has occurred
A. The sections were
stained too long in
hematoxylin.
B. The sections are too
thick.
C. The differentiation step
was too short.
A. Decolorize the section
and restain, making
appropriate adjustments in
the staining time of
hematoxylin.
B. Recut the section.
C. . Decolorize the section
and restain, making
appropriate adjustments in
the differentiation times

96. Red or red-brown nuclei. A. The hematoxylin is
breaking down.
B. The sections were not
blued sufficiently
A. Check the oxidation
status of the hematoxylin.
B. Allow a longer time for
bluing of the sections; it is
impossible to over blue the
sections.
Pale staining with eosin. A. The pH of the eosin
solution may be above 5.0,
possibly caused by
carryover of the bluing
reagent.
B. The sections may be too
thin.
C. Slides may have been
left too long in the
dehydrating
solutions
A. Check the pH of the
eosin solution, and adjust it
to a pH of 4.6 to 5.0 with
acetic acid if necessary. Be
sure the bluing reagent is
completely removed before
transferring the slides to
the eosin.
B. Check the thickness of
the section.

97. C. Restain with eosin and
do not allow the stained
slides to stand in the lower
concentrations of alcohols
Cytoplasm is overstained,
and the differentiation is
poor.
A. The eosin solution may
be too concentrated,
especially if
phloxine is present.
B. The section may have
been stained for too long.
C. The sections may have
been passed through the
dehydrating alcohols too
rapidly for good
differentiation of the
eosin to occur.
A. Dilute the eosin
solution.
B. Decrease the staining
time.
C. Allow more time in each
of the dehydrating
solutions for
adequate differentiation of
the eosin.
(Also, check the section
thickness.)

98. Blue—black precipitate on
top of the sections.
The metallic sheen that
develops on most
hematoxylin
solutions has been picked
up on the slide
Filter the hematoxylin
solution daily before
staining
slides
Water bubbles are seen
microscopically in the
stained
sections.
The sections were not
completely dehydrated,
and
water is present in the
mounted section.
Remove the cover glass
and mounting medium
with xylene. Return the
slide to fresh absolute
alcohol (several changes).
After the sections are
dehydrated, clear with
fresh xylene and mount
with synthetic resin.
All dehydrating and
clearing solutions should
be changed before staining
any more sections

99. Difficulty bringing some
areas of the tissue in focus
with
light microscopy.
Mounting medium may be
present on top of the cover
glass.
Remove the cover glass
and remount with a clean
cover glass. Review the
method used for mounting
sections, and modify if
needed
The mounting medium has
retracted from the edge of
the
cover glass.
A. The cover glass is
warped.
B. The mounting medium
has been thinned too much
with xylene.
A. Remove the cover glass
and apply a new cover
glass.
B. Apply a new cover glass
with fresh mounting
medium. Keep the
mounting medium
container tightly
capped when not in use.
Use a small container for
the mounting medium and
discard when it becomes
too thick

100. The water and the slides turn
milky when the slides are
placed in the water following
the rehydrating alcohols.
Xylene has not been removed
completely by the
alcohols
Change the alcohols, back the
slides up to absolute
alcohol, and rehydrate the
sections
The slides are hazy or milky
in the last xylene rinse prior
to coverslipping.
Water has not been
completely removed from the
sections before being placed
in the xylene.
Change the alcohol solutions,
especially the anhydrous
or absolute reagents.
Redehydrate the sections and
clear in fresh xylene
The mounted stained sections
do not show the usual
transparency and crispness
when viewed by light
microscopy.
The mounting medium may
be too thick, causing the
cover glass to be held too far
above the tissue.
Remove the cover glass and
mounting medium with
xylene. Remount the section
with fresh mounting
medium.