Staining and Mounting.pptx53263611888787

HAEMATOXYLIN AND
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

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

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.

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.

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

Classificatio
n
 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

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.

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.

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.

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

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

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

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

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.

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.

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.

 Specially designated bench
 Staining bench Should be facing window
 Slide washing tray made of stainless steel
 Bunsen burner – to heat up the stain
 Microscope to control staining reaction
Requirements

Requirements for
staining
 All glassware should be thoroughly cleaned
 Correct solvent should be used
 Silver and osmic acid solutions should be kept in
dark bottles
 Solutions like acidalcohol 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

 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.

 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

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

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

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

Hematoxyli
n
 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.

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

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

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.

Eosi
n
 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

 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.

 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

 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.

Deparaffinize the section
• Place the slides in xylene solution for 3 to 4 times (for 2-5
minutes each)
Hydration
Hydrate the section by passing it through decreasing
concentration alcohol bath and water
• bath in 100 % alcohol for 2-3 minutes
Staining Procedure Staining

Staining
• Stain the section with hematoxylin solution for 3 to 5
minutes wash in running tap water.
• quickly dip the slide in 0.5% HCL/ acid alcohol.
• Quickly rinse the slide in tap water for 10-15 minutes for
bluing coloration to the nucleus.
• Agitate the slide in eosin solution for 2 to 5 minutes, drain
the staining solution.
Dehydration
• bath in 70 % alcohol for 30 to 60 second
Staining

Clearing
• Place the slide twice in xylene for 3-5 minutes.
Mounting
• Drain the excess xylene and mount DPX
(Dibutylphthalate Polystyrene Xylene) or canada balsam
with a cover slip.
Staining

AUTOSTAINER
HIGH THROUGHPUT STAINER COMPACT STAINER

Types
Dips slides into
the stains
Applies stain to
the slide
Linear design
• 1 slide at a time
• Slides are clipped to slide
holders, which are
attached to carrier
mechanism.
Batch design
• Linear / Carousal
• Multiple slides
• Slide racks are moved
through baths of staining
solution

Types
Dips slides into
the stains
Applies stain to
the slide
Capillary Gap
Stainer
• Force or draw the
stain between the
specimen slide and
another surface.
Centrifugal
Stainer
• Spray the stain as
the slide rotate past
the spray nozzle in
a spinning
chamber.
Flat Stainer
• Drops the stain
onto the slide,
while the slide lies
flat within the
stainer.
PAP, AFB, Haematology IHC

COVER SLIPPING
• Suctioning mechanism for picking up a
cover glass from a stack of cover
glasses.
• Exert a force onto the cover glass to
insure that it is released from the
selecting device and placed onto the
slide.
• After placement of the cover glass onto
the slide, capillary action pushes air
bubbles out from underneath the cover
glass.

LABELING
• Imprints - resistant to chemical exposure and physical wear.
• Easier to locate.
• Alphanumeric characters, barcodes or logos.

Microscopy
• Nuclei, fibrin, fibroglia and microglia : Blue
• Collagen : Yellow or Brownish
Red
• Coarse elastic fibrils : Purplish tint

Fibroblasts with dark nuclei [A] are
seen here along with thick collagen
fibers [B], thin elastic fibers [C] and
very fine reticular fibers [D].
Microscopic view of a histological
specimen of human lung tissue stained
with hematoxylin and eosin.
Microscopy

Microscopy
• Analysis, the cellular morphology and
tissue distribution to deduce whether
tissue regions has abnormalities.
• Standard for clinical diagnosis of
cancers, as well as for identification of
prognostic and therapeutic targets.
Images are taken at low magnification and include many objects of
interest, such as cells and prominent cellular structures (e.g., nuclei).
These are widely distributed in the images and surrounded by different
neighboring tissues (for example, in the cervix, epithelium, and
stroma).

Pitfalls
1. Knife marks (scratches perpendicular to knife edge)
2. Compression (waves parallel to knife edge)
Poor sectioning

Pitfalls
1. Folds & tears
2. Excess albumin (stain)
Mounting sections

Staining
1. Inadequate rehydration (uneven staining)
2. Too dark or Too light (timing off)
3. Inadequate agitation
Pitfalls

Coverslipping
1. Bubbles
2. Excess Per mount
3. Two cover slips
Pitfalls

Staining and Mounting.pptx53263611888787

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Staining and Mounting.pptx53263611888787