Evolution of Hematoxylin and eosin stain complete guide

1. Hematoxylin & Eosin stai
PRESENTED by
Dr.GPR.Abhinav
MEDICURE DIAGNOSTICS
&RESEARCH CENTER. HYDERABAD

2. HAEMATOXYLINAND EOSIN
The hematoxylin and eosin stain is the most widely used
histological stain because……
 Its comparative simplicity
 Ability to demonstrate clearly an enormous number of
different tissue structures.
 The hematoxylin stains cell nuclei blue / black
 Eosin stains cell cytoplasm and most connective tissue
fibres
MEDICURE

3. Why to stain
 The purpose of staining is that of outlining the tissue and
cellular components
 To identify tissue
 To establish the presence or absence of disease processes.

4. Most commonly used stains
 Histopathology – Routine Hematoxylin(H) &
Eosin(E),
 In microbiology – Gram’s Method and Ziehl-
Neelson’s method,
 In hematology- Romanowsky stain ,
 In cytopathology -Papanicoloau stain.

5. Some important terminology
 Basophilic- The entities stainable with basic dye &
are substances which are usually acidic in nature.
 Acidophilic- The entities stainable with acidic dye
& are substances which are usually basic in
nature.
 Sudanophilic - The entities stainable with oil
soluble dyes e.g. sudan III, IV

6.  Argyrophilic- The entities stainable with silver
nitrate solution.
E.g. AgNOR
 Argentaffin- The entities staininable with silver
nitrate solutions without chemical reduction
procedures
 Metachromatic -The entities will stain in a color
or hue different from that of staining solution itself.

7. Definition
 Stains:
Stains are chemical substances used to achieve visible color
contrast in the microscopic picture of a prepared tissue.
 Staining:
Staining may be loosely defined as treating tissue or cells with a
reagent or series of reagents so that it acquires a color; usually,
no particles of dyes are seen and the stained element is
transparent.

8. DYES
These are essentially aromatic
benzene ring compounds or
derivatives that possess the twin
properties of color and ability to bind to
tissue.

9. Classification
 According to the origin of a dye.
1)Natural
e.g. hematoxylin, Carmine, and Saffron
2)Synthetic
e.g. Benzene, toluene, and naphthalene
or phenols

10. Acidic dyes
Acid dyes usually stain basic components such
as cytoplasm, acidophil granules etc.
e.g. Eosin, Acid fuchsin
Basic dyes
Usually stain acidic components such as nucleus,
basophil granules etc.
e.g. Hematoxylin, Basic Fuchsin, Methylene blue.

11. Neutral dyes
These consist of mixtures of basic and
acidic dyes. Both cations and anion contain
chromophoric groups and both have colored
radicles.
e.g. Romanowsky dyes formed by the interaction
of polychrome methylene blue and eosin.

12. Types of Staining reactions
 Absorption or direct staining – tissue penetrated
by dye solution
 Indirect staining– using intermediary 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 compound.

13. Staining methods
 Vital staining
 Routine staining
 Special staining
Other classification
Regressive staining
Progressive staining

14. Vital staining
 Applied to living tissue
 Accomplished by injecting the staining solution into
some part of animal body
 By mixing the stain with living cells.
 Primarily used for research purpose

15. Routine Staining
 One that stains the different tissues with little
differentiation except between nucleus & cytoplasm.
 General relationship among cells, tissues & organs
are demonstrated.
 Eg. Hematoxylin & eosin stain

16. Special staining
 Special or selective staining demonstrate special
feature of tissue such as
 particular cell products,
 Microscopic intracellular & intercellular structure.
e.g. PAS stain for mucopolysaccharide

17. Differentiation
 Removal or washing out of excess stain until the color
is retained only by tissue component that are to be
studied.
 Generally done with Acid alcohol, Ethyl alcohol.
 Exposure to air may oxidized & improve the process.

18. Regressive staining
 In a regressive stain, the tissue is first overstained &
then partially decolorized.
 The process of partial declourization is
(differentiation).
 Differentiation is controlled visually by examination
with microscope.

19. Progressive staining
 Once the dye taken up by the tissue it is not removed
 Differentiation in progressive staining relies solely on
selective affinity of dyes for different tissue element
 The tissue is left in dye solution only until it retains
the desired amount of coloration.

20. Armamentarium of staining

21. Armamentarium in staining
 Specially designated bench
 Staining bench Should be facing window
 Slide washing tray made of stainless steel
 Bunsen burner – to heat up the stain
 Thermostatically controlled hot plate to melt the wax
 Microscope to control staining reaction

22. Armamentariuminstaining
 Slides are stained in following ways
1. Using staining dishes
 Small grooved couplin jars with glass lids
 Large staining troughs
2. Using staining racks
 Two pieces of stout glass rods 2-4 cm apart
3. Using staining machine
 Same as processing machine but carry slide racks

23. Requirementsforstaining
 All glassware should be thoroughly cleaned
 Correct solvent should be used
 Silver and osmic acid solutions should be kept in dark
bottles
 Solutions like dilute ammonia should be freshly prepared
 Constituents of stain dissolved should follow the formula
 Alcoholic solutions of the stain should be kept in glass
stoppered bottles
 All dyes should be filtered before use

24. PROCEDURE
FOR
STAINING

25. Hematoxylin and eosin technique
 Principle
 H and E are principle stain for demonstration of nucleus and cytoplasm.
 Alum acts as a mordant and the hematoxylin containing alum stains the
nucleus light blue which turns red in the presence of acid.
 The cell differentiation is achieved by treating the tissue with acid solution.
The counterstaining is performed using eosin which imparts pink color to
cytoplasm

26. Hematoxylin and eosin technique
 Removal of paraffin wax (Deparaffinization)
 Removed with xylene (impermeable to stains)
 2-3min of xylene immersion sufficient for sections of 10 µ
thickness
 First facilitated by warming the slides at 60 degrees oven to melt
the wax
 Removal of xylene
 Xylene is not miscible with water or low grade alcohols, hence
dipped in two changes of absolute alcohol

27.  Hydration(High to low)
 After removal from xylene sections are transferred to
absolute alcohol for 1-2min until it becomes opaque
 Sections rinsed in second bath of alcohol, drained and taken
to water
 Any pigments or deposits should be removed at this stage

28. Hematoxylinand eosin technique
 Staining
 Slides immersed in hematoxylin (Mayer s, Harris, Gills)
 If regressive stain is used longer time is used to overstained
the structures
 Differentiation
 Sections are dipped in Acid alcohol, agitated and washed in
tap water
 Observed under microscope
 If underdifferentiated- returned to acid alcohol
 If overdifferentiaited – retured to hematoxylin and
differentiated again

29. Hematoxylinandeosintechnique
 Blueing
 Slides after draining off hematoxylin is transferred to
ammonia water for 2 min. Sections when removed from
hematoxylin or acid alcohol are pink in color
 Washing turns them blue
 Counterstain(Eosin)
 Transfer the slides to 1% aqueous eosin for 2min. Wash in
running water
 Dehydration( low to high)
 Slides are taken through 3 stages of Alcohol

30. Hematoxylinandeosintechnique
11. Clearing
 Sections transferred to xylene and left until clear
 Tested for clarity by being held against a dark background
12. Mounting
 Surplous xylene wiped off from slide surface
 This step completed quickly to avoid section drying
 Whole operation takes 5-10 seconds

31. Hematoxylinandeosintechnique
 Results
 Cell nuclei – Blue
 Muscle fibres – Red
 Collagen fibres – Pink
 RBC – Bright red

32. THEORY OF H & E
STAINING

33.  The word hematoxlin is drived from old Greek word
Haimato(blood) and Xylon(wood), reffering to its dark red
color in natural state and to its origin(wood).
 A natural dye extracted from the
log wood of tree Haematoxylon
Campechianum .
 Basic in nature and stains acidic component of the
tissue, nucleus, mitochondria etc.

34.  Theory of H & E staining
The introduction of
hematoxylin is attributed to
Waldeyer in 1862 that used
it as a watery extract but
without very much success

35. Historical aspect of hematoxylin
 Two years later Bohmer combined haematoxylin
with alum as a mordant and obtained more specific
staining.

36. Historical aspectof hematoxylin
 In 1891 Heidenhain introduced his classical Iron
alum-haematoxylin method which today is still the
standard technique of the cytologist.

37.  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.
Historical aspect of hematoxylin

38. Hematoxylin
 Dark red color
 The hematoxylin is extracted from log wood with hot water
and then precipitated out from the aqueous solution using
urea.
 It is sold commercially as a crude mixture of hematoxylin
and other, unidentified substance.
 It comes as a brownish tan powder which is poorly soluble
in water and somewhat more soluble in ethyl alcohol.

39.  Hematoxylin itself is not a stain.
On oxidation it produces HEMATIN - a poor dye but
metallic mordant, forms the most powerful stain.
 When aluminum salts– will stain blue
 When ferric salt– will stain blue-black.

40. Ripening
 This process of oxidation is often referred to as
ripening or maturing.
 This can be carried out in two ways
1. Natural oxidation
2. Chemical oxidation

41. Natural oxidation:
 Carried out by exposure to light and air.
 Slow process
 Resultant solutions seem to retain its staining ability for a
long time.
Advantage
 Ones oxidation has reached an acceptable level, the staining
solution may be used, and it last for longer,
Disadvantage
 In the planning and organization required ensuring that usable
solution is always available. For example: Ehrlich’s and
Delafield’s hematoxylin.

42. Chemical oxidation:
 It is achieved by the addition of the oxidizing agents
such as mercuric oxide, sodium iodate and
potassium permanganate.
 The use of chemical oxidizing agents converts the
hematoxylin to haematin almost instantaneously,
so these hematoxylin solutions are ready for use
after preparation.

43. Properties of chemically oxidized
hematoxylin
 Have a shorter useful life than the naturally oxidized
haematoxylins .
 However, the possibility of over – oxidation has been
clearly established that the production of oxyhaematein
inhibits successful staining.
 To prevent these, Glycerol has been incorporated in
many formulas. Glycerol acts as stabilizer by
preventing over oxidation and reducing evaporation.

44. Properties
 Haematin is anionic, having poor affinity for tissue.
 It is an inadequate stain without the presence of
mordant. (most useful mordant are salts of
aluminium,)
 Hematoxylin solution using Lead as a mordant are
occasionally used for demonstration of argyrophil
cells.

45. Mordant
 Mordant can be defined as polyvalent metal ion
which forms coordination complexes with
certain dyes.
 A substance which acts as an intermediary between
dye and tissue

46. TYPES OF HAEMATOXYLIN
1. Alum haematoxylins
2. Iron haematoxylins
3. Tungsten haematoxylins
4. Molybdenum haematoxylin
5. Lead haematoxylins
6. Haematoxylin without mordants

47. TYPES OF ALUM HEMATOXYLIN
Ehrilch’s haematoxylin.
Mayer’s haematoxylin.
Harris’s haematoxylin.
Gill’s haematoxylin.
Cole’s haematoxylin.
Delafield’s haematoxylin.
Carazzi’s haematoxylin.

48. Alum hematoxylin:
 Routinely used in the hematoxylin and eosin stain,
and produce good nuclear staining.
 The mordant is aluminium, in the form of ‘potash
alum’- aluminium ammonium sulphate.
 The alum hematoxylin can be used regressively or
progressively.

49.  The most commonly used hematoxylin are
Ehrlich’s, Mayer’s, Harris’s, Cole’s and Delafield’s
haematoxylins.
 Carazzi’s hematoxylin is occasionally used,
particularly for urgent frozen sections.

50. Harris’s hematoxylin: (1900)
 This is an alum hematoxylin which is traditionally chemically
ripened with Mercuric oxide (sodium or potassium iodate
may be used as substitutes for oxidation.).
 It is a powerful and selective nuclear stain giving clear
nuclear staining.
 It is used widely as a nuclear stain in exfoliative cytology.
 In routine histology practice it is used regressively, but in
exfoliative cytology it may be used as a progressive stain.

51. preparation
 Haematoxylin 2.5gm
 Absolute alcohol l25ml
 Potassium alum 50gm
 Distilled water 500ml
 Mercuric oxide 1.25gm
 Glacial acetic acid 20ml
 Acetic acid is optional but its inclusion gives more
precise and selective staining of nuclei

52. Harris’s hematoxylin: (1900)
 Dissolve hematoxylin in alcohol
 Add to it alum, previously dissolved in hot water.
 The mixture is rapidly brought to boil
 Mercuric oxide is then slowly and carefully added, when the
solution turns dark purple.
 The stain is rapidly cooled under tap water.
 Filter before use.

53. Mayer’s hematoxylin: (1903)
 A next widely used hematoxylin
 Chemically ripened with sodium iodide.
 It is more vigorous in action than Ehrlich’s
hematoxylin and gives little or no staining of muco-
polysaccharide material.

54.  It is used as a nuclear counter stain in the
demonstration of glycogen (PAS, mucicarmine) in
various enzyme histological techniques.
 The stain is applied for short period (progressive
stain usually 5-10 min.) until nuclei are stained, and
is then blued without any differentiation.
 Differentiation might destroy or decolour the stained
cytoplasmic components. It can be used as a
regressive stain like any alum hematoxylin.

55. Preparation
 Haematoxylin 1gm
 Distilled water 1000ml
 Potassium/ammonium alum 50gm
 Citric acid 1gm
 Chloral hydrate 50gm
 Sodium iodate 0.2gm

56.  The hematoxylin, potassium alum, sodium iodate is
dissolved in distilled water by warming and stirring, or
by allowing to stand at room temperature overnight.
 The chloral hydrate and citric acid are added, and the
mixture is boiled for 5 minutes, then cooled and
filtered.
 Chloral hydrate acts as a preservative and citric acid
sharpens nuclear staining.

57. Ehrlich’s hematoxylin: (1886)
 This is a naturally ripened alum hematoxylin, most
commonly used in both normal and morbid histology.
Preparation of solution:
 Hematoxylin - 2 g
 Absolute alcohol - 100 ml
 Glycerol - 100 ml
 Distilled water - 100 ml
 Glacial acetic acid - 10 ml
 Potassium alum - 10 – 14 g
( in excess )

58.  Dissolve the hematoxylin in the alcohol.
 The incorporation of glycerol.
 Finally, add potassium alum (till saturation).
 The stain may be ripened naturally by allowing to
stand in large flask, loosely stoppered with cotton
wool.
 Filter before use.

59.  It may be partially oxidized
 Stain used immediately by the addition of 0.3 g
sodium iodate to the above.
 It also stains mucin in salivary glands, some
muco-polysaccharide substances such as cartilage,
and ‘cement lines’ of bone etc. Ehrlich’s
hematoxylin is not ideal for frozen sections.

60. Delafield’s haematoxylin
 Naturally ripened
 Similar longevity
 PREPARATION
1. Haematoxylin 4gm
2. 95% alcohol 125ml
3. Saturated aq. ammonium alum 400ml
4. Glycerin 100ml

61.  The hematoxylin is dissolved in 25 ml of alcohol
 added to solution ‘B’ (alum solution).
 Mixture is allowed to stand in light and air for 5 days
and is then filtered.
 Added to solution ‘C’.
 Allowed to stand exposed to light and air for about 3-4
months or until the stain is sufficiently dark in color,
 Then filtered and stained.

62. Cole’s hematoxylin: (1943)
This is an alum hematoxylin,
Artificially ripened with an alcoholic iodine solution.
It has good keeping qualities and is suitable for use in
sequence with celestine blue, unlike Ehrlich’s
hematoxylin.

63. Coles Haematoxylin
Preparation
 Haematoxylin 1.5gm
 Saturated Aq. Potassium alum 700ml
 1% iodine in 95% alcohol 50ml
 Distilled water 250ml

64.  The hematoxylin is dissolved in warm distilled water
and mixed with iodine solution.
 The alum solution is added, and the mixture
brought to boil, then cooled quickly and filtered.
 The solution is ready for immediate use,but may
need on occasion filtering after storage,

65. Carazzi’s hematoxylin: (1911)
 This is an alum hematoxylin which is chemically
ripened using potassium iodate.

66. Carazzis Haematoxylin
Preparation
 Haematoxylin 0.5gm
 Glycerol 100ml
 Potassium alum 25gm
 Distilled water 400ml
 Potassium iodate 0.1gm
 Usable for 6 months
 Used as progressive nuclear counter stain
 Largely confined to use with frozen sections
because it gives excellent and clear nuclear
staining with a very short staining time

67.  Hematoxylin is dissolved in the glycerol
 and the alum is dissolved in most of the water
overnight.
 The alum solution is added slowly to solutition.
Potassium iodate is dissolved in the rest of the water
with gentle warming and is then added to the
haematoxylin-alum-glycerol mixture.
 The final staining solution is mixed well and is then
ready for immediate use, it remains usable for about six
months.

68. Gill’s hematoxylin: (1974)
 Preparation of solution:-
 Distilled water - 730 ml
 Ethylene glycol - 250 ml
 Hematoxylin - 2 g
 Sodium iodate - 0.2 g
 Aluminium sulphate - 17.6 g
 Glacial acetic acid - 20 ml

69. Advantages
 They are fast in action,
 Stable for at least 12 months,
 Produce little or no surface precipitate,
 Their preparation does not involve boiling the
solution.

70. Staining Times with alum
haematoxylins:
It will vary according to following factors:
 Type of hematoxylin used:- e.g. Ehrlich’s hematoxylin
20-45 min, Mayer’s hematoxylin 10-20 min.
 Age of stain: as the stain ages the staining time will
need to be increased.
 Intensity of use of stain: A heavily used hematoxylin will
loose its staining power rapidly and longer staining
times will be necessary.

71. Celestine blue- alum hematoxylin:
 Preparation of solution:
 Celestine blue solution
 Celestine blue B -2.5 g
 Ferric ammonium sulphate -25 g
 Glycerol -70 ml
 Distilled water - 500 ml

72.  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
 glycerin is added.
 Filter before use.

73. Celestine blue B,
 an oxazine dye,
 Has little useful colouring property of its own.
 It forms an additional strong mordant with certain
haematoxylins .
 Celestine blue B is used as a preliminary to alum
hematoxylin staining.

74. TYPES OF IRON HEMATOXYLINS
Weigert’s hematoxylin
Heidenhain’s
hematoxylin
Verhoeff’s
hematoxylin
Loyez hematoxylin

75. Iron haematoxylin
 Iron salts such as ferric chloride or ferric ammonium
sulphate are used both as oxidizing agent and as
mordant.
 Over oxidation is a problem with these stains

76.  Therefore mordant/oxidant & haematoxylin
solution are prepared separately and mixed
before use
 Capable of demonstrating wider range of
tissue structures compared to alum
haematoxylin but techniques are more time
consuming and needs microscopic control
for accuracy

77. Weigert’s hematoxylin (1904):
 An iron hematoxylin used as a nuclear stain in
techniques where acidic staining solutions are
applied to the sections subsequently (e.g. Van
Gieson stain).
 In Van Gieson stain, picric acid is one of the
constituents which have marked decolorizing action
on nuclei stained with alum hematoxylin.

78. preparation
 Hematoxylin solutions
Haematoxylin 1gm
Absolute alcohol 100ml
 This is allowed to ripen naturally for four weeks before use
 Iron solution
30%aqueous ferric chloride 4ml
Hydrochloric acid(conc.) 1ml
Distilled water 95ml

79.  Ferric ammonium sulphate as oxidant/ mordant.
 Heidenhain’s hematoxylin is a cytological stain.
 It is used regressively
 It may be used to demonstrate
 chromatin,
 chromosomes,
 nuclei,
centerosomes,
mitochondria,
muscle striations
myelin.
Heidenhain’s hematoxylin (1896):

80. Preparation
 Haematoxylin solution
Haematoxylin 0.5gm
Absolute alcohol 10ml
Distilled water 90ml
 Iron solution(5% iron alum)
Ferric ammnonium sulphate 5gm
Distilled water 100ml

81. Loyez haematoxylin
 Ammonium sulphate is used as mordant
 Differentiation is byWeigert’s differentiator
(borax and potassium ferricyanide)
 Used to demonstrate myelin
 Can be applied to frozen, paraffin and nitro
cellulose sections

82. Verhoeff’s hematoxylin (1908):
 Verhoeff’s hematoxylin is used to demonstrate
elastic fibers after all routine fixative.
 Coarse fibres are intensely stained, but the staining
of fine fibers may be less than satisfactory.
 The differentiation step is critical to the success of
this method.

83. TUNGSTEN’S HAEMATOXYLINS
Widely usedTungsten’s haematoxylin is PTAH
(PHOSPHOTUNGSTICACID HEMATOXYLIN )
Used to demonstrate fibrin, muscle striations,
cilia and glial fibres
Myelin can also be demonstrated
Widely used as CNS stain

84. Preparation
 PTAH solution using haematin
Haematin 0.5gm
Phosphotungstic acid 5gm
Distilled water 500ml
 Stain is ready after 24hours
 PTAH solution (KMnO4)
Haematoxylin 0.5gm
Phosphotungstic acid 10gm
Distilled water 500ml
Aqueous KMnO4 25ml
 Peak staining activity after 7 days

85. RESULTS
 Muscle striations, neuroglia fibres, fibrin, amoeba
– Dark blue
 Nuclei, cilia, RBC – Blue
 Myelin – Lighter blue
 Collagen, osteoid, cartilage, elastic fibres – Deep
brownish red
 Cytoplasm - Pale pinkish brown

86. Molybdenum haematoxylins
 Haematoxylin solution using molybdic acid as
mordant. Its a rare stain
 Used as demonstration of collagen, coarse
Reticulin and also stains Argentaffin cell
granules

87. Preparation
 Haematoxylin solution
Haematoxylin 2.5gm
Dioxane 49ml
Hydrogen peroxide 1ml
 Phosphomolybdic acid solution
Phosphomolybdic acid 16.5gm
Distilled water 44ml
Diethylene glycol 11ml
Resultant solution shouled be dark violet and
allowed to stand for 24hrs

88. Results
 Collagen and coarse reticulin – violet/black
 Argentaffin cells – black
 Nuclei – pale blue
 Paneth cells – orange
Tissue fixed in dichromate do not give good
results

89. Lead Haematoxylin
 Used in demonstration of granules in endocrine
cells of alimentary tract and other regions
 Most practical diagnostic application is in
identification of endocrine cells in tumors of
doubtful origin
 Also used in localisation of gastrin
secreting cells in stomach

90. Haematoxylin without a
mordant
 Freshly prepared haematoxylin is used to
demonstrate various minerals in tissue
sections
 These methods are now replaced with more
specific techniques

91. 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.

92. Hematoxylin Specific use
Harris;’s hematoxylin Exfoliative cytology
Mayers hematoxylin Nuclear counter stain in PAS
Ehrlichs hematoxylin Mucin & other mucopolysaccharide
Carazzis hematoxylin Progressive nuclear stain & frozen section
Celestine blue- alum hematoxylin Where subseqent acidic stains are to be
used e.g. van Gieson stain
Weigerts hematoxylin Where subseqent acidic stains are to be
used e.g. van Gieson stain
Heidenhains hematoxylin Differentiating cytological fluid stain
Loyes hematoxylin To demontrate myelin, frozen section
Tungsten hematoxylin Fibrin , & muscle straitions
Molybdenum hematoxylin Demonstration of collegen fibre
Lead hematoxylin Granules of endocrine cell
Hematoxylin without mordant Pigment lead , iron

93. Eosin
 Stains connective tissue and cytoplasm in
varying intensity and shades (red to pink)
 Eosin is derived from fluorescein and is
available in following types:
 Eosin Y (eosin yellowish, eosin water soluble)
 Ethyl eosin (eosin S, eosin alcohol soluble)
 Eosin B (eosin bluish, erythrosine B)

94.  Eosin Y is most commonly used and is readily
soluble in water, less so in a alcohol thus it is
sometimes sold as ‘water and alcohol soluble’
 Preparation
Eosin Y, water soluble 5gm.
Distilled water 1000ml.
Crystals of THYMOL added to inhibit the growth of
fungi.

95.  Alcohol soluble eosin is employed as a 0.5 % solution
in alcohol.
 Eosin Y water &
Alcohol soluble 10gm
Distilled water 50ml
95%ethyl alcohol 940ml
 In use, sections should be treated with 95% alcohol
before staining with alcoholic eosin, and the excess
stain washed out in the same solvent.

96.  The addition of little ACETIC ACID (0.5 ml to 1000
ml stain) is said to sharpen the staining.
 Ethyl eosin and eosin B are now rarely used,
although occasional old methods specify their use,
e.g. the Harris stain for Negri bodies.

97. Bibiliography
 John D. Bancroft and Alan StevensTheory and
practice of histological techniques (3rd ed)p9
no 174.186 vol3Hematoxylins and eosin. John
D. Bancroft • Christopher Layton.
 Lynch’s Medical laboratory technology (4th ed)
 Internet sources

98. Thank you……