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Preparation of tissues
for study
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HISTOLOGY :
Is the branch of science which deals with microscopic
study of normal tissue .
HISTOPATHOLOGY :
Is the branch of science which deals with microscopic
study of tissue affected by disease.
Tissue can be obtained from:
Biopsies
Autopsies
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Microtechnique
Microtechnique : is tissue preparation for
microscopic examination.
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Microtechnique
Most tissues are thick , so they must be sliced to obtain thin , translucent sections.
It usually involves hardening of the tissue followed by sectioning (cutting).
There are different methods used, however the basic principles are similar.
- Paraffin technique
- Freezing technique
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Histological Techniques:
1. Paraffin technique:
- Tissues are hardened by replacing water with paraffin.
- The tissue is then cut in the microtome at thicknesses
varying from 2 to 25 µm thick.
- The tissue can be mounted on a microscope slide,
stained and examined using a light microscope.
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2. Freezing technique:
Water-rich tissues are hardened by freezing and cut frozen sections. Sections are stained and examined with a light microscope.
This technique is much faster than paraffin technique (5 -20 minutes vs. 16 hours).
Are used in operations to achieve a quick diagnosis.
Cryosections can also be used in immunohistochemistry as freezing tissue does not alter or mask its chemical composition as much as preserving it with a fixative.
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Histological Technique
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Protocols followed in Histotechniques:
1. Receipt & Identification.
2. Labeling of the specimen with numbering.
3. Fixation.
4. Dehydration.
5. Clearing.
6. Impregnation (infiltration).
7. Embedding.
8. Section / cutting.
9. Staining.
10. Mounting.
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Specimen Dissection - the stage
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Dissecting and Specimen Sampling
Taking a representative sample of the tissue
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Mickey Mouse Rt nasal polyp
Dr. Makewell 26/11/13 18/11/28 DOB:
Specimen
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Fixation
Is the process by which the constituents of cells and
tissue are fixed in a physical and chemical state so
that they will withstand subsequent treatment with
various reagents with minimum loss of architecture .
This is achieved by exposing the tissue to chemical
compounds, call fixatives.
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Properties of an Ideal Fixative Prevents autolysis and bacterial decomposition.
Preserves tissue in their natural state and fix all
components.
Make the cellular components insoluble to
reagent used in tissue processing.
Preserves tissue volume.
Avoid excessive hardness of tissue.
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Important Factors in fixation:
• Fresh tissue.
• Proper penetration of tissue by fixatives.
• Correct choice of fixatives.
This should be approximately 10-20 times
the volume of the specimen.
Fixative should surround the specimen on
all sides.
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Tissue fixatives
There are many tissue fixatives i.e
Buffered formalin (light microscope preparation)
Buffered gluteraldehyde ( electron microscope preparation).
Osmium tetraoxide (electron microscope preparation,
preserve and stain).
Zenker’s formal saline
Bowen’s fluid
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No fixative will penetrate a piece of tissue
thicker than 1 cm.
For dealing with specimen thicker than this, cut slices not thicker than 5 mm are recommended.
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Cassette
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Specimen bits are placed in porous cassettes
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Cassettes are collected in fixatives 10% formalin
1mm/hour fixation ~ 6 hour
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It can be subdivided into:
a) Dehydration
b) Clearing
c) Impregnation (infiltration)
Tissue Processing
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Dehydration ( removal of water)
It is the process in which the water content in the tissue is
completely removed by passing the tissue through increasing
concentrations of dehydrating agents.
Tissues are dehydrated by using increasing strength of alcohol; e.g.
80%, 95% and 100%.
Replace water by diffusion.
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During dehydration water in tissue has been
replaced by alcohol.
The next step alcohol should be replaced by
paraffin wax.
As paraffin wax is not alcohol soluble, we
replace alcohol with a substance in which wax is
soluble. This step is called clearing.
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Clearing
Replacing the dehydrating fluid with a fluid that is
totally miscible with both the dehydrating fluid and
the embedding medium.
Some clearing agents:
- Xylene.
- Toluene.
- Chloroform.
- Benzene.
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Impregnation (infiltration):
In this the tissue is kept in a wax bath containing
molten paraffin wax.
The wax is infiltrated in the tissue which
increases the optical differentiation & hardens
the tissue & helps in easy sectioning of the
tissue.
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Types of tissue processing
There are two types :
1. Manual Tissue Processing
2. Mechanical Tissue Processing
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Manual Tissue Processing
In this process the tissue is changed from one
container of reagent to another by hand.
Note:
The processing, whether manually or mechanically, involves the same steps and reagents in same sequence.
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Mechanical Tissue Processing
In this the tissue is moved from one jar to another by mechanical device.
Timings are controlled by a timer which can be adjusted in respect to hours and minutes.
Temperature is maintained around 60 ºC ( infiltration).
Automatic tissue processor:
Overnight
12 Baths.
16 hours .
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Tissue basket
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Tissues processor
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Mechanical Tissue Processing
Fixation 1. 10 % Formalin saline (I) for 1.5 hours 2. 10 % Formalin saline (II) for 1.5 hours
Dehydration 1. 80 % alcohol for 1 hour 2. 95 % alcohol (I) for 1 hour 3. 95 % alcohol (II) for 1 hour 4. Absolute alcohol (100%) (I) for 1 hour 5. Absolute alcohol (100%) (II) for 1 hour 6. Absolute alcohol (100%) (III) for 1 hour
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Clearing: 1. Xylene (I) for 1.5 hours 2. Xylene (II) for 1.5 hours
Infiltration: 1. Paraffin wax (I) for 1.5 hours
2. Paraffin wax (II) for 1.5 hours
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Is the process by which iimpregnated tissues are surrounded
by a medium such as agar, gelatin, or wax which when
solidified will provide sufficient external support during
sectioning.
Embedding
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Embedding:
It is done by transferring the tissue which has been
impregnated with wax to a mould filled with
molten wax & is allowed to cool & solidify.
After solidification, a wax block is obtained which is then sectioned to obtain ribbons.
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Embedding tools
Moulds Paraffin wax
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Paraffin wax:
Is a polycrystalline mixture of solid hydrocarbons. Paraffin
wax is traditionally marketed by its melting points which
range from 39°C to 68°C.
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Embedding Centre
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Embedding Centre
Molten wax (60°C) (reservoir)
Cold plates (-5°C) Wax dispenser
Wax flow adjuster
Heated chambers Used lids
Hot surface Cassette
Mould
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General Embedding
Procedure
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Open the tissue cassette.
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Fill the mould with paraffin wax.
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Using warm forceps select the tissue, taking care that
it does not cool in the air.
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Orienting the tissue in the mould.
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Correct orientation of tissue in a mould is the most important step in embedding.
1. cross section
2. longitudinal section
Incorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy.
Orientation Of Tissue In The Block
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Chill the mould on the cold plate and firming the
tissue into the wax with warmed forceps.
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Cool the block on the cold plate.
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Blocks on the cold plate.
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Remove the block from the mould.
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Blocks of embedded tissue are usually trimmed
to remove the excess wax on the surface.
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Sectioning
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Sectioning (Section Cutting) :
It is the procedure in which the blocks which have been
prepared are cut or sectioned and thin strips of uniform
thickness are prepared.
The instrument by which this is done is called as a
Microtome.
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TYPES OF MICROTOMES:
• Sliding microtome.
• Rotary microtome.
• Rocking microtome.
• Freezing (cryostat) microtome.
• Base sledge microtome.
• Ultramicrotome.
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Main parts of all microtomes :
1. Base ( microtome body).
2. Knife and its holder.
- Steel Knives / Disposable Blades.
- Non-corrosive Knife
- Glass Knives.
- Diamond Knives.
3. Block holder.
4. Micron adjustment (section thickness).
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Steel Knives (for rotary microtome)
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Disposable Blades (for rotary microtome)
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Glass Knives (for ultramicrotome)
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Rotary Microtome
Drive wheel
Knife holder
Block holder
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Paraffin Block
Micron adjustment
(section thickness)
1-60µ
Steel knife
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Rotary Microtome
The knife is stationary (fixed) and the block holder is moved up and down in a vertical plane by the rotary action of the hand wheel.
It is the most commonly used.
Suitable for paraffin embedded sections.
It is suitable for cutting of small tissues .
Ideal for cutting serial sections.
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Ultra Microtome
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Ultra Microtome
Used for very thin section (up to 1 µm).
The typical thickness of tissue cut is between
40 -100 nm for TEM.
Examine by Transmission electron microscpy
(TEM).
Knife: Diamond or Glass.
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Ultra Microtome
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Freezing (Cryostat) microtome
Frozen tissue embedded in a freezing
medium is cut on a microtome in a cooled
machine called a cryostat.
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Freezing (Cryostat) microtome
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Freezing (Cryostat) microtome
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Sectioning procedure
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Ribbon of sections
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Tissue floatation bath
It is a thermostatically
controlled water bath.
It is maintained at a
temperature maintained
5 – 6 degree below the
melting point of paraffin
wax.
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Flattens paraffin sections
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Ribbon sections are taken on hot water bath.
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Taking the floating sections onto slide.
Adhesives used for fixing the sections on the slides
Albumin solution ( Mayor’s egg albumin)
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Taking the section
onto slide.
Flat, no air bubbles,
no stretch or
breaks.
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Staining
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Staining
Staining is a process by which we give color to a section
which bring out the particular details in the tissue.
The most commonly used stain in routine practice is Haematoxylin & eosin stain.
Staining steps:
a. rehydration
b. stain
c. dehydration
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Classification of Stains:
Acid stains
Basic stains
Neutral stains
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Acid Dyes
In an acid dye:
The basic component is colored and the acid component is colorless.
Acid dyes stain basic components e.g. eosin stains cytoplasm.
The color imparted is shade of red.
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Basic Dyes
In a basic dye
The acid component is colored and the basic
component is colorless.
Basic dyes stain acidic components e.g.
Hematoxylin stains nucleus.
The color imparted is shade of blue.
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Hematoxylin and Eosin (H & E)
Hematoxylin: stains acidic molecules shades of blue.
Eosin: stains basic materials shades of red, pink
and orange.
H & E stains are universally used for routine histological
examination of tissue sections.
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Result :
The nucleus
stains Blue .
The cytoplasm
stains pink.
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Neutral Dyes
When an acid dye is combined with a basic dye a neutral dye is formed.
As it contains both colored radicals, it gives different colors to cytoplasm and nucleus simultaneously.
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Special stains When a specific components of tissue e.g. fibrous tissue,
elastic tissue, nuclear material is to be stained, certain
special stains are used which specifically stain that
component tissue.
Examples :
1. PAS.
2. Maisson’s trichrome.
3. Silver.
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Normal glomerulus of kidney is stained with H&E
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Normal glomerulus of kidney is stained with PAS
detects glycogen, glycoproteins, glycolipids and mucins in tissues
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Normal glomerulus of kidney is stained with
Masson_trichrome
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Normal glomerulus of kidney is stained with
methenamine silver. Silver stains reticular fibres (type III collagen).
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Normal glomerulus of kidney is stained with
methenamine silver
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Staining Tools
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Slide rack
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Slide rack
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Solutions
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Procedure of staining:
There are two types of staining:
Manual Staining
Automatic staining
Slides stained either manually or by
automatic stainer, pass through same
sequences of reagents.
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Manual Staining Different reagent containers are placed in a
special sequence and the slides are removed
from one container to another manually.
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Manual Staining
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Manual Staining
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Automatic stainer
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Automatic stainer
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Procedure : 1. Deparaffinization with xylene 2. Hydration (hydrate to water 100%, 95%, 70%
ethanol ) 3. Wash under water 4. Stain with Haematoxylin for 10-15 min 5. Wash with water 6. Differentiate with 1 % acid alcohol (lithium
carbonate) 7. Wash with water 8. Stain with 1% Eosin for 4-5 min 9. Dehydration ( 70%, 95% and absolute 100%
alcohols) 10. Clearing with xylene 11. Dry 12. Mount
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Deparaffinization
(xylene)
Hydration
(alcohol)
Haematoxylin
Eosin
Dehydration
(alcohol) Clearing
(xylene)
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Mountining
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Mountining:
Stained section on microscope slide then is mounted using mounting medium dissolved in xylene.
Examples of Mountants : DPX ( Distrene Dibutyl phthalate Xylene ).
Canada Balsam
Colophonium resin
Terpene resin
A coverslip is placed on top, to protect the sample.
Evaporation of xylene around the edges of the coverslip, dries
the mounting medium and bonds the coverlips firmly to the
slide.
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Imagination
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Light Microscopic Examination
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Microscopic Examination
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Concerning Light Microscopy
Use beam of transmission light.
Magnified images are typically from 10-
1000x.
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Concerning Electron Microscopy
Electron beam instead of light.
Magnified images are typically from 1000X to
50,000X.
Gluteraldehyde & Osmium tetraoxide fixative.
Embedding is in hard epoxy plastic.
Ultramicrotome.
Diamond or Glass knives.
Thin section (0.02 -0.1 µm).
Specimen is mounted on a metal grid.
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For transmission electron microscopy, a diamond knife
( or glass) mounted in an ultramicrotome is used to cut 0.02- 0.1 µ
thick tissue sections which are mounted on a 3-mm-diameter
copper grid. Then the mounted sections are treated with the
appropriate stain.
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Transmission Electron microscope (TEM)
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