BIOM444 Histopathology

Practical lecture: Slide preparation

Dr. Amal Al-Haidose

Slide Preparation Steps
1. Receipt & Identification
2. Labeling of the specimen with numbering
3. Fixation
4. Dehydration
5. Clearing
6. Impregnation
7. Embedding
8. Section cutting
9. Staining
10. Mounting
Specimen identification and labeling:
Tissue specimen received in
the Histopathology laboratory
have a request form that lists
the patient information and
history along with a
description of the site of
origin.

The specimen are accessioned

by giving them a number that
will identify each specimen for
each patient
Fixation
 It is a process in which a specimen is treated by exposing it
to a fixative for a particular period of time in order to
facilitate the succeeding steps.
 The purpose of fixation is to preserve tissues structure
 The fixative should be 15 – 20 times more in volume than
the specimen.

Pre-Fixation Procedures & precaution:

 Tissues should be washed in physiological saline
 Excessive blood & mucous should not be there
 Early dispatch of the specimens to histology laboratory
 Best thickness 3-5mm
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.
Characteristics and Aims of Fixation:
1. It should prevent autolysis & decay of the cell.
2. It should penetrate evenly and rapidly.
3. It should harden the tissues
4. Should not cause shrinkage or swelling of the cells
5. Must not react with the receptor sites & thus
must not interfere with the staining procedure.
6. It must be cheap and easily available.
 Factors involved in fixation
1. Buffers & hydrogen ion concentration:
 Best fixation occurs between pH 6-8
 Buffers used – phosphate, s-collidine, bicarbonate.

2. Temperature:
 Most tissues fixed– room temp
 Electron microscopy & histochemistry (0-4˚C)

3. Penetration of tissues:
 Blocks should be small or thin to ensure adequate
penetration.
Continue…

4. Volume changes:
 Due to inhibition of respiration, membrane
permeability changes, changes in ion transport
through membrane.

 Tissues fixed in formaldehyde & embedded in paraffin

shrink by 33%
5. Duration of fixation:
• Most tissues should remain in fixative for 24 hours.
• Formaldehyde – prolonged fixation – shrinkage & hardening
of tissue.
• Gluteraldehyde – prolonged fixation – advantageous.
• Long fixation in aldehydes - inhibits enzyme activity.
• Long fixation in oxidizing fixatives – degrade the tissue.

Small intestine well preserved Autolyzed Small intestine

 Classification of fixatives

1. Aldehydes: formaldehyde, gluteraldehyde, acrolein

2. Oxidizing agents: osmium tetroxide, potassium

permanganate, potassium dichromate

3. Protein denaturing agents: acetic acid, methyl

alcohol, ethyl alcohol

4. Other cross linking agents: Carbodiimides,

dimethlysuberimidate.

5. Unknown mechanism: Mercuric chloride, Picric acid

 Formalin
The most commonly used fixative is Formalin .

 It is prepared by mixing 40 % Formaldehyde gas in 100 w/v

of distilled water.

The resultant mixture is 100 % Formalin.

 Routinely, 10 % formalin is used which is prepared by

mixing 10 ml of 100 % formalin in 90 ml of distilled water.

 Mechanism of action:
It forms cross links between amino acids of proteins
thereby making them insoluble.
It fixes 4 mm thick tissue in 8 hours .
Advantages:

• Formalin is cheap
• Easy to prepare
• Relatively stable
• Frozen sections can be prepared with ease.
• Staining of fat and tissue enzymes.
• Penetrates tissues well.
• Beneficial hardening with little shrinkage
Disadvantage:
• Unpleasant vapour irritation
to eyes & respiratory
epithelium

• Formalin dermatitis
 GLUTERALDEHYDE

• Used for Electron Microscopy

with osmium tetroxide

Advantage:
• Most efficient cross linking
agent for collagen
• More rapid fixation than
formalin.

Disadvantage:
• Poorer penetration
• More costly
 ETHYL ALCOHOL

• Colour-less.
• Powerful dehydrating agent.
• Causes shrinkage & hardening
• Used for preservation of
glycogen.
• Used in histochemical method
for enzymes.
Cut up of Tissue.
After the specimen is properly fixed, the pathologist selects the
tissue from the specimen for diagnosis.
The cut up is done in a fume hood (2 types):
1. It is mainly used for cutting specimens and it has an outside
venting.
2. It is a Compact, portable hood, fixed on a bench and it has
no outside venting, but it has a special filter, it is used for
preparing solutions & reagents.
We need for cut up:
• Scalpel handle and blades
• Scissors
• Knife
• Forceps with and without tooth tips
• Gloves
• Tissue paper(Neutra pad)
• Dishes
• Embedding cassettes
• Apron
• After the section are selected we put in embedding
cassettes, with identifying number, we should ensure
that we use soft pencil which stands all fluids used in
processing tissue.
Tissue Processing
• Tissue processing refers to any treatment of tissue
necessary to impregnate them with a solid medium to
facilitate the production of sections for microscopy
• It requires 24hrs and done in many stages.
• It can be subdivided into
a) dehydration
b) clearing
c) impregnating
d) embedding.
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.
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.

• Note:
The processing, whether manually or mechanically,
involves the same steps and reagents in same sequence.
Processing
Formalin  Alcohol  Xylene  Wax

Wax

Xylene
Formalin

Alcohol
Sequence of tissue processing
Dehydration
• During dehydration water in tissue has been replaced by
alcohol.

• Tissues are dehydrated by using increasing strength of

alcohol; 50%, 70%, 90% and 100%.

• The duration for which tissues are kept in each strength of

alcohol depends upon the size of tissue, fixative used and
type of tissue.

• The volume of alcohol should be 50-100 times that of tissue.

Clearing
• 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 call clearing.
Continue….
• Clearing of tissue is achieved by any of the following
reagents:
• Xylene
• Chloroform
• Benzene
• Carbon tetrachloride
• Toluene
Note:
• Xylene is commonly used. Small piece of tissue are cleaned
in 0.5 – 1 hour; whereas larger (5cm or more thick) are
cleaned in 2-4 hours.
STEPS OF PROCESSING
Fixation Dehydration

Clearing
Impregnation
• Placement of tissues with medium that fill natural
cavities, spaces, replaces clearing agent
• Done in an oven heated to 52-54 ºC
• supports tissue
• firmness to tissue
Impregnation with Wax
• This is allowed to occur at melting point temperature of
paraffin wax, which is 54-60 ºC. Volume of wax should be
about 25-30 times the volume of tissues.

• The duration of impregnation depends on size and types

of tissues and the clearing agents employed.

• Longer periods are required for larger pieces and also for
harder tissue like bones and skin as compared to liver
kidney, spleen, lung etc.
Continue….
• Total duration of 4 hours is sufficient for routine
impregnation.

• Types of Wax employed for Impregnation:

1. Paraffin wax
2. Water soluble wax
3. Other material, like colloidin, gelatin, paraplast etc.

• Paraffin wax is used routinely. It has hard consistency, so

section of 3-4 micron thickness can be cut.
Embedding:
It is done by transferring the tissue which has been cleared
of the alcohol to a mould filled with molten wax & is allowed
to cool & solidify.
Dispense wax Align tissue

Cassette on
Cool in place
ID bead Top-up wax

Leave to set

Cool plate
Sectioning
• block is mounted in microtome which cuts very thin
slices from the tissue block (3-10 μm; micron = 1 X 10-6m
= 1/1000 mm). These slices are used in preparation of
microscope slides, after staining and mounting.
Microtomy
• For light microscopy, a glass knife mounted in a
microtome is used to cut 4-6 um-thick tissue sections
which are mounted on a glass microscope slide.

• For transmission electron microscopy, a diamond knife

mounted in an ultramicrotome is used to cut 50-nm-thick
tissue sections which are mounted on a 3-mm-diameter
copper grid. Then the mounted sections are treated with
the appropriate stain.

• Frozen tissue embedded in a freezing medium is cut on a

microtome in a cooled machine called a cryostat.
Cryostat Microtome
Manual Rotary Microtome
1 2

3
Ready to stain
Staining
• Staining of the section is done to bring out the
particular details in the tissue under study .

• Classification of Stains:
• Acid stains
• Basic stains
• Neutral stains
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.

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.

GENERAL CATEGORIES OF STAINS
1. Acid-Base Combinations =
Most sections are stained with both acidic and basic
dyes to enhance contrast by providing different colors.
The most common combination is:
Hematoxylin and Eosin (H & E)
Hematoxylin = basic dye, stains nuclear structures blue
Eosin = acidic dye, stains cytoplasmic and intercellular
structures pink
2. Trichrome Methods =
provides 3 colors, allows differentiation between
cytoplasmic and intercellular components.
Special stains
Stain specific components of tissue or structures:
Iron hematoxylin = useful in distinguishing finer cytologic
details (e.g., subcellular organelles)
• Mallory-Azan = trichrome method; stains collagen fibers and
mucus blue, stains nuclei and cytoplasmic components red
• Mason = trichrome method; collagen fibers stain green,
cytoplasmic components stain purplish-red
• Periodic-Acid Schiff = selectively stains carbohydrate-containing
molecules/substances red (e.g., glycogen, muco- and glycoproteins,
glycosaminoglycans)
• Silver Impregnation = selectively outlines reticular fibers
• Orcein, Resorcin-Fuchsin = selectively stains elasticfibers
• Sudan Black B = specifically stains fat
The general rule for staining paraffin
sections are:
• Deparaffinization
• Hydration
• Staining
• Dehydration
• Clearing
• Mounting
Procedure:
• Sections to water
• a. Paraffinize xylene
• b. Hydration alcohol
• 2 Stain in Harris’s Haematoxylin-5-10 mins.
• 3. Wash well in tap water.
• 4. Differentiate in 1% Acid alcohol. (5-10 sec.)
• 5. Wash in water.
• 6. Blue in 5% borax.
• 7. Wash in water.
• 8. Stain in eosin for 20-30 seconds.
• 9. Quick wash in water
• 10. Dehydrate clear mount.
Mounting
• Excess stain washed away with water (or alcohol for some
dyes, depending on dye solvent).

• Tissue slice dehydrated by passing through increasing

strengths of alcohol to absolute alcohol

• Alcohol removed by a clearing agent so that unstained

spaces appear transparent.

• Mounting medium is then added to tissue slice, covered

with a coverslip and allowed to dry, and "Ready to View"
Manual Staining
• In a small laboratory when a few slides are
stained daily, this is the method of choice.

• Although it is time consuming it is economical.

• Different reagent containers are placed in a

special sequence and the slides are removed
from one container to another manually.
Automatic staining
• In this procedure an automatic stainer is required.
• It has a timer, which controls the time.
• It has a mechanical device which shifts the slides from one
container to next after the specified time.

• Advantages of automated stainer are:

• It reduces the man power
• It controls the timing of staining accurately
• Large number of slides can be stained simultaneously
• Less reagents are used

• Note: Slides stained either manually or by automatic stainer,

pass through same sequences.
Automatic stainer
Automatic stainer
 Hematoxylin and Eosin (H & E).

• Result :

The nucleus stains Blue

The cytoplasm stains pink.

Useful videos

• https://www.youtube.com/watch?v=nUjK4n3_1C8

• https://www.youtube.com/watch?v=7-LIbAWPc-g&t=157s

• https://www.youtube.com/watch?v=4DJm4NLECQs