His To Path Tech

Preparation of
tissues for study

 HISTOLOGY :
It is the branch of science which deals with the microscopic
study of normal tissue
 HISTOPATHOLOGY :
It is the branch of science which deals with the microscopic
study of tissue affected by disease
Tissue for study can be obtained from:
 Biopsies
 Autopsies
Microtechnique
• Microtechnique: is tissue preparation for
microscopic examination
• There are different methods used, however

the basic principles are similar
• It usually involves hardening of the tissue
followed by sectioning (cutting)
- Paraffin technique
- Freezing technique
Histological Techniques:
1. Paraffin
Tissues are hardened by replacing water with
paraffin
2. Freezing technique:
Water-rich tissues are hardened by freezing and cut
frozen
Freezing technique is much faster than traditional
histology (20 minutes vs. 16 hours) and 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
Protocols followed in Histotechniques
1. Identification & Labeling of the specimen

2. Fixation
3. Dehydration
4. Clearing
5. Impregnation (infiltration)
6. Embedding
7. Section cutting
8. Staining
9. Mounting
Labeling
Cassette
Fixation
• This 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: fixatives
• Fixatives prevent autolysis and bacterial
decomposition and preserves tissue in their natural
state and fix all components
Tissue fixatives
• Buffered formalin (light microscope preparation)

• Buffered gluteraldehyde (electron microscope preparation)
• Osmium tetraoxide (electron microscope preparation,
preserve and stain)
• No fixative will penetrate a piece of tissue
thicker than 1 cm.
Specimen is placed in porous cassettes
Cassettes are collected in fixatives 10% formalin
1mm/hour fixation
Processing
Tissue Processing
 In order to cut thin sections of the tissues, it
should have suitable hardness and consistency
when presented to the knife edge.
 These properties can be imparted by infiltrating and

surrounding the tissue with paraffin wax, various
types of resins or by freezing.
 This process is called tissue processing.

Tissue Processing
It can be subdivided into:
a) Dehydration
b) Clearing
c) Impregnation (infiltration)
Types of tissue processing
• There are two types :
1. Manual Tissue Processing

2. Mechanical Tissue Processing
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
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
• Automatic tissue processor:

 Overnight
 12 Baths
 16 hours
Tissues processor
Tissue basket
Dehydration (removal of water)
It is the process in which the water content in the
tissue to be processed is completely removed by
passing the tissue through increasing concentrations of
dehydrating agents
• Tissues are dehydrated by using increasing strength of

alcohol; e.g.
70%, 90% and 100%
• Water is replaced by diffusion

• 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.
Clearing
• Replacing the dehydrating fluid with a fluid that is
totally miscible with both the dehydrating fluid
(alcohol) and the embedding medium (wax)
• Some clearing agents:

- Xylene
- Toluene
- Chloroform
- Benzene
Impregnation (infiltration):
 The tissue is kept in a wax bath containing

molten paraffin wax
The duration of the procedure can be noted
down as:
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
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
Embedding
Embedding: is the process by which impregnated
tissues are surrounded by a medium such as agar,
gelatin, or wax which when solidified will provide
sufficient external support during sectioning
Embedding:
 It is done by transferring the tissue 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
Embedding tools
Moulds Paraffin wax

Embedding Centre
Embedding Centre
Molten wax (60°C)
(reservoir)
Used lids Heated chambers
Wax flow adjuster Mould
Hot surface Cassette
Cold plates (-5°C)

Wax dispenser
General Embedding
Procedure
Fill the mould with paraffin wax
Using warm forceps select the tissue, take care
that it does not cool in the air
Orienting the tissue in the mould
Orientation Of Tissue In The Block
 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
Cool the block on the cold plate
Blocks on the cold plate
Remove the block from the mould
Blocks of embedded tissue are usually trimmed
to remove the excess wax on the surface
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
TYPES OF MICROTOMES:
• Rotary microtome
• Freezing (cryostat) microtome
• Ultramicrotome
Rotary Microtome
Micron adjustment
(section thickness)
1-30µm
Paraffin Block
Steel knife
Freezing (Cryostat) microtome
Ultra Microtome
Used for very thin section

 The typical thickness of
tissue cut is between 20 -
100 nm for TEM
 Knife: Diamond or Glass
Ultra Microtome
Diamond Knife
Ultra Microtome
Glass Knife
Sectioning: Ribbon of sections
Tissue floatation bath
 It is a thermostatically
controlled water bath
• It is maintained at a
temperature 5 – 6
degrees below the
melting point of paraffin
wax
Flattened paraffin sections
Taking the floating sections onto slide
Adhesives used for fixing the sections on the slides
Albumin solution ( Mayor’s egg albumin)
Taking the section
onto slide
Flat, no air bubbles,

no stretch or breaks
Staining
• Staining is a process by which we give colour to a
section. Staining of the section is done to bring out
the particular details in the tissue under study
• There are hundreds of stains available
• The most commonly used stain in routine practice is

Hematoxylin & Eosin stain
Staining
Classification of Stains:
• Acid stains (ex. Eosin)

• Basic stains (ex. Hematoxylin)
Acid Dyes
In an acidic 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

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

• Result :
The nucleus
stains Blue
The cytoplasm
stains pink
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.
Manual Staining
Automatic stainer
Mounting
• Stained section on microscope slide is
mounted using mounting medium
dissolved in xylene
• Examples of Mountants : DPX ( Distrene

Dibutyl phthalate Xylene )
 A coverslip is placed on top, to protect the sample

Light Microscopic Examination
Concerning Electron Microscopy
 Electron beam instead of light
 Gluteraldehyde 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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Preparation of

tissues for study

Tissue for study can be obtained from:

• There are different methods used, however

Cryosections can also be used in

1. Identification & Labeling of the specimen

• This is achieved by exposing the tissue to chemical

• Buffered formalin (light microscope preparation)

 These properties can be imparted by infiltrating and

 This process is called tissue processing.

It can be subdivided into:

• There are two types :

1. Manual Tissue Processing

• Timings are controlled by a timer which can be adjusted

• Temperature is maintained around 60 ºC

• Automatic tissue processor:

• Tissues are dehydrated by using increasing strength of

70%, 90% and 100%

• Water is replaced by diffusion

• The next step alcohol should be replaced by

• As paraffin wax is not alcohol soluble, we

• Some clearing agents:

 The tissue is kept in a wax bath containing

1. Paraffin wax (I) for 1.5 hours

 After solidification, a wax block is obtained which is

Moulds Paraffin wax

Wax flow adjuster Mould

Hot surface Cassette

Cold plates (-5°C)

 Incorrect placement of tissues

 It is the procedure in which the blocks

Used for very thin section

Flat, no air bubbles,

• There are hundreds of stains available

• The most commonly used stain in routine practice is

• Acid stains (ex. Eosin)

• The basic component is colored and the acid

• Acid dyes stain basic components e.g. eosin stains

• The color imparted is shade of red

• Basic dyes stain acidic components e.g.

• The color imparted is shade of blue

• There are two types of staining:

• Slides stained either manually or by

• Examples of Mountants : DPX ( Distrene

 A coverslip is placed on top, to protect the sample

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