SPECIMEN COLLECTION &

TISSUE PROCESSING FOR

LIGHT MICROSCOPY

DR ROZINA KHAN
M.B.B.S, MPHIL SCHOLAR HEMATOLOGY, BIH
LECTURER, DEPARTMENT OF PATHOLOGY, BMC
LEARNING OBJECTIVES
 At the end of the practical the students will be
able to know:

1. How to collect & examine Specimens properly.

2. Define Tissue Processing and list its various

steps.

3. Identify the use of different Reagents & Stains.

4. What is H & E Staining System & why Staining

is done.

5. Identify the Morphological Features of Types of

Cell Injury.
SPECIMEN COLLECTION
 The first step before tissue processing is proper specimen collection which includes the
following:

1. The Preservative: The specimen should be received in a preservative commonly Formalin.

2. Request Form & Its Contents: Tissue Specimens received in the Pathology Lab have a Request
Form that must include :
I. Identification of the patient including Name, Age, Gender etc.
II. History of the patient to help correlate the findings.
III. Date, Time, Type, Site as well as Clinical details of the Specimen.

3. Numbering of Specimens: The specimens are given a Identification Number once received in
the lab that will help to differenciate the specimens of one patient from another.
SPECIMEN EXAMINATION
 After Proper Specimen Collection and Identification, the specimen is examined grossly as well as
microscopically.

1. Gross Examination of specimen by naked eye will provide information about the General
Appearance, Colour, Size, Shape, Consistency and also about any Abnormality visible by the
naked eye like ulceration or nodularity.

2. Microscopic Examination of specimen will provide information about the tissue at cellular level.
• For Microscopic Examination Tissue Processing is done.
• First a piece of the tissue is cut and is placed in a Cassette. Tiny biopsies can be wrapped in a filter
paper and then put in the cassettes.
• Then the tissue specimen is passed through various steps.
TISSUE PROCESSING STEPS
 1. FIXATION:
• The tissue specimen in the cassette is
exposed to a fixative solution to preserve
the tissue from Internal (autolysis) and
external (microorganisms) damage and
retain it in as life like state as possible.
• The best fixative which is commonly used
is 10% Buffered Formalin.
• Other fixatives include Zenkers Solution,
Bouins Solution etc
TISSUE PROCESSING STEPS
 2. DEHYDRATION:
• To remove the water content of the tissue, it
is passed through Dehydrating Agents like
Ethanol (Ethyl Alcohol) such that the
dehydrating agent takes the place of water.
• The tissue is exposed to increasing
concentrations of Alcohol instead of 100%
Alcohol, to prevent it from shrinking
suddenly and getting damaged.
• Dehydration is done so that the Paraffin Wax
used later on can easily infilterate the tissue.
• Other dehydrating reagents include
Methanol, Acetone etc
TISSUE PROCESSING STEPS
 3. CLEARING
(DEALCOHOLIZATION):
• Clearing is done to remove the alcohol in
the tissue by replacing it with Clearing
Agents, most commonly Xylene which is
mixable in the Paraffin Wax.
• Other clearing reagents include Chloroform,
Benzene, Petrol etc
TISSUE PROCESSING STEPS

 4. PARAFFIN WAX INFILTERATION & EMBEDDING:

• This step is done to harden the tissue for easy cutting.
• The tissue is kept in a mould containing molten Paraffin Wax which helps it
in infilterating the tissue.
• It is then allowed to cool and solidify.
• After solidification the mould is removed and a block of hard Paraffin Wax
along with the embedded tissue is obtained.
Moulds

Tissue embedded
in Paraffin Wax Block
supported by cassette
 TISSUE PROCESSING STEPS
 5. SECTION CUTTING:
• By using a Microtome (Micro: Small / Tome: Cutting) the embedded tissue is sliced
into very thin sections to obtain Wax and Tissue Ribbons.

Block Holder Drive Wheel

Blade Wax & Tissue Ribbon

Microtome Machine
TISSUE PROCESSING STEPS
 6. WATER BATH:
• After section cutting, the Ribbon
of Wax containing the thin
sections of the tissue are picked
up from the Microtome and
floated on the warm Water Bath
which helps in separating the
Paraffin Wax from the tissue.
TISSUE PROCESSING STEPS
 7. MOUNTING:
• The sections of the tissue are then
picked up on clean glass slides from
the water bath. This is called
Mounting.
TISSUE PROCESSING STEPS
 8. STAINING:
• Most of the tissues/cells are
colourless and therefore have to be
stained to make the structure of the
cells visible.
• After staining the tissue section is
covered by a coverslip and the slide
is ready to be examined under the
microscope.
STAINING SYSTEMS
 The most commonly used staining system is called H&E Staining System.
 This set contains the two dyes Haemotoxylin and Eosin.

 1. Eosin is an Acidic Dye and stains Pink.

• It reacts with Basic Components in cells such as Proteins in the cytoplasm.
• In other words cytoplasmic proteins are Acidophilic (i.e Acid liking) and thus they bind to acidic
dyes.

 2. Haemotoxylin is a Basic Dye and stains Purple.

• It reacts with Acidic Components in cells like Nucleic Acids such as DNA in the Nucleus and RNA
in the cytoplasm.
• In other words Nucleic Acids are Basophilic (i.e Base liking) and thus they bind to Basic Dyes.


 CYTOPLASM STAINED PINK
BY EOSIN DYE

NUCLEUS STAINED PURPLE

BY HAEMOTOXYLIN DYE
CELL INJURY
CELL INJURY

 Any Internal or External Stimuli that cause a variety of stress causing

changes in the structure or function of the cell result in Cell Injury.

 The cellular response to the injury depends upon the nature or type of the
injury, its duration and severity.

 All the cells of the body have an internal system that helps them to adapt to
the changes in homeostasis.
CAUSES OF CELL INJURY

1. Genetic Derangements.
2. Ischemia / Hypoxia (Oxygen Deprivation).
3. Infections.
4. Immune Reactions.
5. Nutritional Imbalances.
6. Physical Agents.
7. Chemical Agents.
REVERSIBLE CELL INJURY
1. Definition: Type of Cell Injury which occurs
when the Structural and Functional changes are
reversible if the damaging stimulus is removed.
2. Membrane: Damage of membrane cytoskeleton
resulting in Blebbing.
3. Cytoplasm: Eosinophilic and vacuolated
appearance.
4. Organelles: Generalized Swelling of the cell
and its organelles due to increased permeability. 4. Organelles: Rupture, Breakdown and loss of organelles due
Detachment of Ribosomes from ER resulting in
halting of protein synthesis.
5. Nucleus: Clumping of Chromatin material due
to decreased PH as a result of Anaerobic
Respiration induced lactic acid.
IRREVERSIBLE CELL INJURY
1. Definition: Type of Cell Injury which occurs when there is
persistent stimulus resulting in irreparable cell damage to a
point of no return (lethal hit) such that the cell eventually
progresses to Cell Death.
2. Membrane: Disruption of membrane due to digestion of
cytoskeleton.
3. Cytoplasm: Formation of Myelin Figures which are Whorls
of damaged cell and organelle membranes.
to digestion by Lysosomal Enzymes.
5. Nucleus: Pyknosis, Karyorrhexis, Karyolysis.
i. Pyknosis: Nuclear Shrinkage due to Chromatin Clumping.
ii. Karyorrhexis: Nuclear Fragmentation due to nuclear
membrane damage.
iii. Karyolysis: Nuclear Dissolution/Fading by enzymes.
MYELIN FIGURES
PRACTICAL TASK

 Q. Tabulate the Elaborate Differences between Reversible and

Irreversible Cell Injury.
Remember,
You’re not studying to pass an exam,
You’re studying to save lives…

THANK YOU :)