TISSUE

PROCESSING
 Biopsy…
▪ Word biopsy originates from the Greek terms bios (life) and opsis
(vision):
vision of life.
▪ Biopsy is the removal of tissue from a living organism for the purpose of
microscopic examination in order to establish a diagnosis.
▪ Types of biopsy
4 major types of biopsy routinely used in and
around the oral cavity:
I. Cytology
II. Aspiration biopsy
III. Incisional biopsy
IV. Excisional biopsy
• Cytology: It is the study of cells which exfoliate or abrade from
body surface.

• Aspiration Biopsy: Is the use of a needle and syringe to remove

sample of cells or contents of a lesion.To determine the presence
of fluid within a lesion.

• Incisional biopsy: This is a type of biopsy that examines only a

representative part of the lesion. Suspected lesions larger than
1cm in diameter

• Excisional biopsy: This procedure removes the lesion entirely at

the time that the diagnostic procedure is being performed.
INTRODUCTION
• Microscopic analysis of cells and tissues requires the preparation of very
thin, high quality sections (slices) mounted on glass slides and
appropriately stained to demonstrate normal and abnormal structures.

• Most fresh tissue is very delicate and easily distorted and damaged, and
it is thus impossible to prepare thin sections from it unless it is
chemically preserved or “fixed” and supported in some way whilst it is
being cut.
Broadly 2 methods that can be employed to provide this support:

Freeze the tissue and keep it frozen while we cut our sections.
These sections are called “frozen sections”.

Infiltrating tissue specimen with a liquid agent- subsequently converted

into a solid -appropriate physical properties, - allow thin sections to be
cut from it.
Paraffin wax- so-called “paraffin sections”.
• “Tissue processing” describes the steps required to take an
animal or human tissue from fixation to the state where it is
completely infiltrated with a suitable histological wax and can be
embedded ready for section cutting on the microtome.

• Tissue processing can be performed manually (hand processing), but

where multiple specimens must be dealt with, it is more convenient
and much more efficient to use an automated tissue processing
machine (a “tissue processor”).

• There are two main types of processors: tissue-transfer (or “dip and
dunk”) machines- specimens are transferred from container to
container to be processed; fluid-transfer (or “enclosed”) types -
specimens are held in a single process chamber or retort and fluids are
pumped in and out as required.
• Treatment of tissue specimens to allow paraffin wax
embedding is called Tissue Processing

• Aim of tissue processing: to embed the tissue in a

solid medium, firm enough to support the tissue and
give it sufficient rigidity to enable thin sections to be
cut, which can be viewed under microscope
Steps in soft tissue processing
• Collection of specimen
• Fixation
• Dehydration
• Clearing
• Impregnation
• Embedding
• Sectioning
• Staining
• Mounting
Collection of specimen/ Obtaining the specimen

Specimens taken must be removed carefully without crushing

 Fixation

• Physical or chemical process of treating a dead tissue

for the preservation of cells and tissue constituents in a
condition identical to that existed during life to facilitate the
preparation of thin, stained sections for histochemical
examination.
• Aim: to maintain the +ssue in as much a life like state as possible
by preven+ng autolysis and putrefac+on
• Principle: to coagulate +ssue proteins(cross linking of proteins)
thus reducing altera+on of +ssue.
• With minimum loss of architecture.
• 10% formalin
• Achieved by exposing the +ssue to chemical compounds, called
fixa+ves.
 Aim/Objectives of fixation
• To preserve the living structure as closely as possible.
• To prevent autolysis of tissue
• To inhibit bacterial or fungal growth
• To make the tissue resistant to damage during subsequent
stages of processing
• To prevent alteration of tissue volume and distort any part of
the tissue structure.

• Mechanism of action of fixatives:: Most fixatives act by denaturing or

precipitating proteins which then form a sponge or meshwork, tending to
hold the other constituents.

• Good fixative is one of the most important factors in the production of

satisfactory results in histopathology.
• The fixation can be carried out at room temperature

• The speed of fixation of most fixative is almost 1 mm/hour

• Amount of fixative fluid should be approximately 10-20 times the volume

of the specimen

• Standard ratio of fixative to tissue volume is 10:1

Factor affecting fixation:

1. Size and thickness of piece of tissue.

2. Tissue covered by large amount of mucous fix slowly.
3. Tissues covered by blood or organ containing very large
amount of blood fix slowly
4. Fatty and lipomatous tissue fix slowly.
5. Fixation is accelerated by agitation.
6. Fixation is accelerated by maintaining temperature around 60oc.
Factors affect fixation:
• PH
• Temperature
• Penetration of fixative
• Time

• According to previous factors we can determine the concentration of

fixative and fixation time.
Properties of an Ideal Fixative:

1. Prevents autolysis and bacterial decomposition.

2. Preserves tissue in their natural state and fix all components.
3. Preserves tissue volume.
4. Avoid excessive hardness of tissue.
5. Allows enhanced staining of tissue.
6. Should be non-toxic and non-allergic for user.
7. Should not be very expensive.

Classification of Fixatives
•Aldehydes:- Eg: Formaldehyde, Gluteraldehyde
•Oxidising Agents:- Eg: Osmium tetroxide, Potassium permanganate
•Protein denaturing agent or Coagulant:- Eg: Acetic acid, Methyl alcohol, Ethyl
alcohol
•Other cross linking agents:- Eg:Carbodimides
•Physical:- Eg: Heat, Microwave oven
•Miscellaneous:- Eg: Mercuric chloride, Picric acid
Most commonly used fixative is- Formalin (10%)
 Dehydration
• Done to remove the water content from tissues to allow the
penetration of paraffin wax (Paraffin and water do not mix)
• Done by passing through ascending grades of alcohol (to
prevent sudden shrinkage of tissue)
• E.g.- 50%, …..70%, 90% & 100%
• Volume: 50-100 times that of tissue
• Solutions used
• Methyl & Ethyl alcohol
• Isopropyl alcohol
• Acetone
 Clearing
• Paraffin and alcohol are not miscible. So impregnation of tissue
by paraffin is not possible unless alcohol is replaced by a fluid
that is miscible with both alcohol & paraffin. This process is called
clearing

• Xylene is one of the solutions that is miscible with both paraffin

and alcohol

• Term “clearing” comes from the fact that the clearing

agents often have the same refractive index as proteins.
As a result, when the tissue is completely infiltrated with
the clearing agent, it becomes translucent/clear.

• Presence of opaque areas indicates incomplete

dehydration.
Ideal requirements of a clearing solution
• Speedy removal of alcohol
• Minimum tissue damage & toxicity
• Cost factor

Reagents used
• Xylene (most commonly used)
• Chloroform
• Toluene
• Benzene
• Methyl salicylate (oil of wintergreen).
• Food oil derivatives
• Cedar wood oil
Impregnation with wax
• Saturation of tissue cavities and cells by a supporting substance
which is generally, but not always, the medium in which they
are finally embedded

• Replaces xylene with paraffin by immersion in molten wax (600

C)

• Factors affecting impregnation

• Size & type of tissue
• Clearing agent employed
• Vacuum embedding
 EMBEDDING/BLOCKING
• Process by which tissues are surrounded by a medium such as agar, gelatin, or wax which
when solidifies will provide sufficient external support during sectioning.

• Impregnated tissue transferred from wax bath to a mould filled with molten wax to get a
block of wax with the tissue specimen at the center with the cutting surface facing the
base of the block
• Done using
• Leuckhart’s L shaped pieces
• Ice trays
• Paper boats
• Embedding cassette
• Make sure that there are no air bubbles trapped b/w the tissue and the molten wax.
• Wax-filled mold containing the tissue is then allowed to cool.
• Wax blocks are labeled to make easier identification.
• Wax hardened block removed from the mould and trimmed.
• Wax block to be fixed on to a wooden/metal block to prevent wax block from crumbling
during sectioning.
 SECTIONING

• CUTTING THE TISSUE INTO THIN SLICES FROM THE TISSUE

BLOCK IS TERMED: SECTIONING
• Embedded tissues, to be cut into thin sections of 3- 5 µ with a
microtome.
• Cut sections are, floated on a warm water bath
• Helps to spread the specimen and remove wrinkles.
• Floated sections are picked up on an adhesive coated glass
slide.
• Glass slide kept on a slide warmer at 580 temp for 20 min to
ensure adhesion
• Egg albumin with additives - commonly used adhesive

• 2 types of microtome- manual & automatic

 Staining
• Biochemical technique of adding a class-specific
dye to a substrate (DNA, proteins, lipids,
carbohydrates) to qualify or quantify the presence
of a specific compound is known as staining.

Commonly used Staining Techniques

Hematoxylin and Eosin (H&E) staining

Gram staining
Papanicolaou staining
Periodic Acid Schiff (PAS) staining
Masson's Trichrome staining
 H & E stain/ Haematoxylin & Eosin stain
• Most popular staining method in histology.
• Most widely used stain in medical diagnosis.
• Involves application of basic dye haematoxylin, which colors
acid molecules with blue-purple hue, and alcohol-based
acidic eosin-Y, which colors basic molecules bright pink.
• Basophilic structures are usually the ones containing nucleic
acids, such as the ribosomes and the chromatin-rich cell
nucleus, and the cytoplasmic regions rich in RNA.

• Eosinophilic structures are generally composed of intracellular or

extracellular protein. Most of the cytoplasm is eosinophilic.

• Red blood cells are stained intensely red.

HEMATOXYLIN
• Hematoxylin is a natural dye which is extracted from the
heartwood of the tree Hematoxylon campechianum.

• Dye is usually used in conjunction with a mordant which

helps the stain to bind to the tissue

Oxidation Mordant
Hematoxylin Haematin STAIN

EOSIN
• Second component of H & E, the counterstain
• Eosin is a red dye resulting from the action of bromine on
fluorescein.
• Eosin Y-Commonly used form of eosin
• Used to stain cytoplasm, collagen and muscle fibers
• Both water and ethanol soluble.
Staining Procedure:
Mounting
What is mounting ?
The stained section on the slide must be covered with a thin
glass coverslip to protect the tissue from being scratched.
Why is mounting done?
To provide better optical quality for viewing under the
microscope,
To preserve the tissue section for years to come.
How is mounting done?
Mounting medium is used to adhere the coverslip to the slide
Distal Mounting Media/ Mountant
Two types of mounting media
• Water based mounting media
Eg: Gelatin media, Gum Arabic media

• Resinous mounting media

Eg: DPX & Canada balsam
 DPX (Dibutylphthalate Polystyrene Xylene)
• DPX is a synthetic non-aqueous mounting medium for
microscopy.
• A traditional resin-based slide mountant with xylene solvent.
• Medium viscosity; dry in ~ 15 minutes.

To mount a slide

A. Apply drops of mounting medium upon tissue

section.
B. Hold coverslip at 45o allowing the drop to spread
along the edge of the slip.
C. Let go of slip and allow the medium to spread slowly.

Allow it to dry and section viewed under microscope

 Special Stains:
1. PAS (Periodic Acid Schiff) stain: demonstrates glycogen and neutral mucous
substances, outlines basement membranes and reticulin and makes evident most
types of fungi and parasites.

2. Stains for micro-organism:

a. Gram-stain: allows the separation of bacteria those that retain the crystal-
violet-iodine complex (gram-positive) and those that are decolorized by alcohol
treatment and counterstained by eosin, safranin or fuchsin.
b. Ziehl_Neelsen stain: detect acid fast bacilli.
c. PAS stain: used for fungi, amoeba and Tricomonas.
d. Modified Giemsa (2% Giemsa in water): detects Helicobacter pylori.

3. Congo-red: used for identification of amyloid.

4. Sudan-Black: used for fat staining.
5. Masson’s Trichrome: used for differentiation of connective tissue elements.
6. Papanicolaou’s stain: used to stain cells in cervical and sputum smear for
cytology.
 GROUND SECTIONS

• Ground section preparation consists of cutting a thin slice of

bone/teeth with a suitable saw followed by manual grinding on
a flat abrasive surface like arakansa stone.

Equipments required
• Laboratory lathe
• Carborundum disc
• Arakansa stone
• Stream of water
• Natural teeth
• Xylene
• Camel’s hair brush
• Mounting medium
• Microscopic slides & cover glasses
 Ground sections - Indications
For study purposes
✓ For viewing abnormal
For viewing normal tooth structures
Structures that can be viewed includes tooth structure
➢ Dead tracts
• Rods, with their directions
• Incremental lines of Retzius ➢ Cracks
• Enamel lamellae ➢ Caries
• Enamel tufts
• Dentinoenamel junction
• Enamel spindles
• Dentinal tubules
• Cementocytes
• Primary & secondary cementum
• Pulp chambers, canals
 Ground Sections -Procedure
• Tooth cut longitudinally in mesiodistal plane:

• Coarse-abrasive lathe wheel is attached to lathe, water is directed onto the wheel

• Tooth is held securely in fingers; buccal surface is applied firmly to that flat surface of
rapidly rotating wheel.

• Tooth is ground down to desired section.

• Coarse wheel is then exchanged for fine-abrasive lathe wheel; cut surface of tooth is
ground again until desired section is reached

• At this point, piece of adhesive tape is wrapped around the wooden block- sticky side of
tape is directed outwards

• Ground surface of tooth is wiped dry & is pressed onto the adhesive tape
• Block held securely in fingers; lingual surface of the tooth is also reduced as above to
desired section
• Finish sectioning using manual grinding on a flat abrasive surface like arakansa stone,
using pumice and water.
• It is then mounted on a microscope slide
 MOUNTING:
• A drop of mounting medium is placed on the slide, section is
lifted with a camel’s hair brush & placed on the drop, another
drop of mounting medium is put on the top of the section, & a
cover glass is affixed for microscopic study.

Ground sections - Precautions

• Teeth used for ground sections should not be allowed to dry out after
extraction.

• Extracted tooth should be preserved in 10% formalin or normal saline until

use.
 DECALCIFICATION
PREPARATION OF DECALCIFIED SECTIONS -
Technique
I. Selection of specimen

II.Fixation

III.Decalcification

IV.Neutralisation

V. Washing
 PREPARATION OF DECALCIFIED SECTIONS
• Presence of calcium salts in the tissue prevents the preparation of
good sections by routine method.

• Incomplete removal of these salts results in torn & ragged sections

& also cause damage to the cutting edge of the microtome knife

• Teeth/bone/pathologically calcified soft tissue require the removal

of calcium salts before sectioning.

AGENTS FOR DECALCIFICATION

I. ACIDS
Strong acids (Nitric acid, Hydrochloric acid)
Weak acids (Formic acid, Acetic acid, Picric acid)

II. ION EXCHANGE RESINS WITH ACID DECALCIFYING FLUIDS

III. ELECTROPHORETIC DECALCIFICATION

IV. CHELATING AGENT

EDTA
 TESTS FOR COMPLETE DECALCIFACATION
I. Piercing hard tissue with a needle – when needle enters the bone &
tooth easily, tissue is ready for further treatment
II. Precipitation test – whether there is calcium present in the nitric acid in
which specimen is immersed for decalcification
Precipitate forms if appreciable amount of calcium is present; then
acid covering the specimen should be changed, & a couple of days
later the test should be repeated
If no precipitate is detected, it may be assumed that the specimen is
completely decalcified

• End point of decalcification is sometimes difficult to determine, but it is

important
• Specimens left in the acid too short time are not completely decalcified &
cannot be cut successfully
• Specimens left in the acid too long time do not stain well
 Previous year questions
• Ground section of teeth
• H & E staining procedure
• Microtome
• Steps in histologic processing
• Fixatives/ Fixation of tissue
• Formalin

• Clearing