Techniques in Microtomy

Sahaya Sukeetha D
Bon Secours College for
women
Thanjavur

MICROTOME

A microtome (from the Greek mikros, meaning small, and

temnein, meaning to cut) is a tool used to cut extremely thin
slices of material, known as sections.
Various types of microtomes are available.
Rotary Mictrotome
Sledge Microtome
Ultramicrotome
Vibrating microtome
Laser microtome
Saw microtome
Most commonly used microtome for routine histopathology
is rotary microtome.

Rotary Mictrotome
In a rotary microtome, the knife is typically fixed in a
horizontal position.
A rotary action of the hand wheel perform the cutting
movement.
Hard tissues can be cut without vibration.
The block holder or block is mounted on the steel carriage
that moves up and down and is advanced by a micrometer
screw.
Auto-cut microtome has built in motor drive with foot and hand
control.
With suitable accessories the machine can cut thin sections of
paraffin wax blocks and 0.5 to 2.0 micrometer thin resin
sections.
Advantages:
1. The machine is heavy, so it is stable and does not vibrate
during cutting.
2. Serial sections can be obtained.
3. Cutting angle and knife angle can be adjusted.

Sledge Microtome
Sledge Microtome is a device where the sample is placed into a
fixed holder (shuttle).
The sledge placed upon a linear bearing, a design that allows for
the microtome to readily cut many coarse sections.
Typical cut thickness achievable on a sledge microtome is between
is 10 and 60 micron.
Applications
Design of microtome are of the preparation of large samples, such
as those embedded in paraffin for biological preparations.

Cryomicrotome
For the cutting of frozen samples, many rotary microtomes can be
adapted to
cut in a liquid nitrogen chamber, in a so-called cryomicrotome.
The reduced temperature allows for the hardness of the sample to
be increased, such as by undergoing a glass transition, which allows
for the preparation of semi thin samples.
However the sample temperature and the knife temperature must
be controlled in order to optimize the resultant sample thickness.

Ultramicrotomes
An ultramicrotomes is a main tool of ultramicrotomy.
It can allow for the preparation of extremely thin
sections, with the device functioning in the same manner
as a rotational microtome, but with very tight tolerances
on the mechanical construction.
As a result of the careful mechanical construction, the
linear thermal expansion of the mounting is used to
provide very fine control of the thickness.
These extremely thin cuts are important for use with
transmission electron microscope (TEM).
The typical thickness of these cuts is between 40 and
100 nm for transmission electron microscopy.

Diamond knives (preferably) and glass knives are used with

ultramicrotomes.
To collect the sections they are floated on top of a liquid as they
are cut and are carefully picked up onto grids suitable for TEM
specimen viewing.
The thickness of the section can be estimated by the thin-film
interference colors of reflected light that are seen as a result of
the extremely low sample thickness.

Vibrating microtome
The vibrating microtome operates by cutting using a vibrating
blade, allowing the resultant cut to be made with less pressure
than would be required for a stationary blade.
The vibrating microtome is usually used for difficult biological
samples.
The cut thickness is usually around 30-500 m for live tissue and
10- 500 m for fixed tissue.
Saw microtome
The saw microtome is especially for hard materials such as teeth
or bones.
The microtome of this type has a recessed rotating saw, which
slices through the sample.
The minimal cut thickness is approximately 30 m, and can be

Laser
microtome

The device operates using a cutting action of an infra-red laser.

As the laser emits a radiation in the near infra-red, in this
wavelength regime the laser can interact with biological
materials.
The combination of high power with a high faster rate allows the
scanner to cut large areas of sample in a short time.
In the laser microtome the laser-microdissection of internal
areas in tissues, cellular structures, and other types of small
features is also possible.

MICROTOME KNIFE
Important instrument used to cut uniform thin serial sections of the
tissue.
Various types of knives are used with different microtomes.
The size varies from 100 mm to 350 mm in length.
Microtome knives are made of good quality of high carbon or steel
which is
tempered at the tip.
Hardness of knife is essential to obtain good tissue sections.
Sharpening of microtome knife - To achieve good sections
knife should be
very sharp. The knife is put in the knife back to sharpen. Knife can be
sharpened
manually or by the use of automatic machine.
Stropping - The purpose of stropping is to remove the burr
formed during

Preparation of Biological Samples

Various techniques have been developed to prepare tissues for study so
that they closely resemble their natural, living state.
The steps involved are
Fixation,
Dehydration
Clearing
Embedding - suitable medium,
Sectioning - thin slices to permit viewing by transillumination,
Mounting - sections onto a surface for ease of handling, and
Staining - so that the various tissue and cell components
may be differentiated.

TISSUE FIXATION
Fixation is a complex series of chemical events that differ for the
different groups of substance found in tissues.

Fixation refers to treatment of the tissue with chemical

agents that not only retard the alterations of tissue
subsequent to death (or after removal from the body) but
also maintain its normal architecture.

The aim of fixation

To prevent autolysis and bacterial attack.

To fix the tissues so they will not change their volume and shape
during
processing.
To prepare tissue and leave it in a condition which allow clear
staining of sections.
To leave tissue as close as their living state as possible, and no
small molecules should be lost.

Factors affect fixation

1. pH (Isoelectric point)
2. Total ionic strength of
reagents

Types of fixative
There are five major groups of
fixatives, classified according to
mechanism of action:

3. Osmolarity
4. Temperature
5. Length of fixation
6. Method of application of
fixative

1. Aldehydes
2. Mercurials
3. Alcohols
4. Oxidizing agents
5. Picrates

Aldehydes

These include formaldehyde (formalin) and glutaraldehyde.

Formaldehyde: Tissue is fixed by cross-linkages formed in
the proteins, particularly between lysine residues.
This cross-linkage does not harm the structure of proteins
greatly, so that antigenicity is not lost.
Therefore, formaldehyde is good for immunoperoxidase
techniques.
Formalin penetrates tissues well, but is relatively slow.
Glutaraldehyde : Causes deformation of alpha-helix
structure in proteins so is not good for immunoperoxidase
staining.
However, it fixes very quickly so is good for electron
microscopy.
It penetrates very poorly, but gives best overall
cytoplasmic and nuclear.

Mercurials
These fix tissues by an unknown mechanism.
They contain mercuric chloride and include such wellknown
fixatives as B-5 and Zenkers.
These fixatives penetrate relatively poorly and cause some tissue
hardness, but are fast and give excellent nuclear.
Their best application is a for fixation of hematopoietic and
reticuloendothelial tissues.

Alcohols
Including methyl alcohol (methanol) and ethyl alcohol (ethanol),
are protein denaturants and are not used routinely for tissues
because they cause too much brittleness and hardness.
However, they are very good for cytologic smears because they
act quickly and give good nuclear.

Oxidizing Agents
These include permanganate fixatives (potassium
permanganate), dichromate fixatives (potassium dichromate),
and osmium tetroxide.
They cross-link proteins, but cause extensive denaturation.

Picrates
These include fixatives with picric acid.
It has an unknown mechanism of action.
It does almost as well as mercurials with nuclear detail but
does not cause as much hardness.
Picric acid is an explosion hazard in dry form.
As a solution, it stains everything it touches yellow, including
skin.

Dehydration
Removal of fixative and water from the tissue and replacing
them with dehydrating fluid is called deydration.
There are a variety of compounds many of which are alcohols.
several are hydrophilic so attract water from tissue.

To minimize tissue distortion from diffusion currents, delicate

specimens are dehydrated in a graded ethanol series from water
through 10%-20%-50%-95%-100% ethanol.

In the paraffin wax method, following any necessary post fixation

treatment.
Dehydration from aqueous fixatives is usually initiated in 60%-70%
ethanol,
Progressing through 90%-95% ethanol, then two or three changes of
absolute ethanol before proceeding to the clearing stage.

Types of dehydrating agents:

Ethanol, Methanol, Acetone.

Duration of dehydration should be kept to the minimum

consistent with the tissues being processed.

Tissue blocks 1 mm thick should receive up to 30 minutes in

each alcohol, blocks 5 mm thick require up to 90 minutes or
longer in each change.

Tissues may be held and stored indefinitely in 70% ethanol

without harm

Clearing
Replacing the dehydrating fluid with a fluid that is totally miscible with both
the dehydrating fluid and the embedding medium is known as Clearing.
Choice of a clearing agent depends upon the following
- The type of tissues to be processed, and the type of processing to be
undertaken.
- The processor system to be used.
- Intended processing conditions such as temperature, vacuum and
pressure.
- Safety factors.
- Cost and convenience.
- Speedy removal of dehydrating agent .
- Ease of removal by molten paraffin wax .
- Minimal tissue damage .

Some clearing agents

Zylene
Toluene
Chloroform
Benzene
Petrol.

Embedding
Is the process by which tissues are surrounded by a medium such as
agar, gelatin, or wax which when solidified will provide sufficient
external support during sectioning.
Paraffin wax
properties

Paraffin wax is a polycrystalline mixture of solid hydrocarbons

produced during the refining of coal and mineral oils.
It is about two thirds the density and slightly more elastic than
dried protein.
Paraffin wax is traditionally marketed by its melting points which
range from 39C to 68C.
The properties of paraffin wax are improved for histological
purposes by the inclusion of substances added alone or in
combination to the wax
- improve ribboning.
- increase hardness.
- decrease melting point
- improve adhesion between specimen and wax

Precaution while embedding in wax

The wax is clear of clearing agent.

No dust particles must be present.
Immediately after tissue embedding, the wax must be
rapidly cooled to reduce the wax crystal size.

Embedding and Sectioning

Paraffin Sectioning for Light Microscopy

 There are four main mould systems and

associated embedding protocols presently
in use :
1- Traditional methods using paper boats
2- Leuckart or Dimmock embedding irons or
metal containers
3- The Peel-a-way system using disposable
plastic moulds and
4- Systems using embedding rings or
cassette-bases which become an integral
part of the block and serve as the block
holder in the microtome.

Tissue processing
Embedding moulds:
(A) paper boat
(B) metal boat mould
(C) Dimmock embedding
mould
D) Peel-a-way disposable
mould
(E) base mould used with
embedding ring ( F) or
cassette bases (G)

General Embedding Procedure

1- Open the tissue cassette, check against worksheet entry to ensure

the correct number of tissue pieces are present.
2- Select the mould, there should be sufficient room for the tissue with
allowance for at least a 2 mm surrounding margin of wax.
3- Fill the mould with paraffin wax.
4 Using warm forceps select the tissue, taking care that it does not
cool in the air; at the same time.
5- Chill the mould on the cold plate, orienting the tissue and firming it
into the wax with warmed forceps. This ensures that the correct
orientation is maintained and the tissue surface to be sectioned is
kept flat.
6- Insert the identifying label or place the labeled embedding ring or
cassette base onto the mould.
7- Cool the block on the cold plate, or carefully submerge it under
water when a thin skin has formed over the wax surface.
8- Remove the block from the mould.
9- Cross check block, label and worksheet.

ORIENTATION OF TISSUE IN THE BLOCK

Correct orientation of tissue in a mould is the most important step in
embedding.
Incorrect placement of tissues may result in diagnostically important
tissue elements being missed or damaged during microtomy.
Elongate tissues are placed diagonally across the block
Tubular and walled specimens such as vas deferens, cysts and
gastrointestinal tissues are embedded so as to provide transverse
sections showing all tissue layers
Tissues with an epithelial surface such as skin, are embedded to
provide sections in a plane at right angles to the surface (hairy or
keratinised epithelia are oriented to face the knife diagonally)
Multiple tissue pieces are aligned across the long axis of the mould
and not placed at random

STAINING

Hematoxylin and Eosin (H

H & E is a charge-based,
& E)
general purpose stain.

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.

Fixation
Any well fixed tissue.

Staining Procedure
1- Deparaffinize and hydrate to water
2- If sections are Zenker-fixed, remove the mercuric chloride
crystals with iodine and clear with sodium thiosulphate
(hypo)
3- Mayer's hematoxylin for 15 minutes
4- Wash in running tap water for 20 minutes
5- Counterstain with eosin from 15 seconds to 2 minutes
depending on the age of the eosin, and the depth of the
counterstain desired. For even staining results dip slides
several times before allowing them to set in the eosin for the
desired time
6- Dehydrate in 95% and absolute alcohols, two changes of 2
minutes each or until excess eosin is removed. Check under
microscope
7- Clear in xylene, two changes of 2 minutes each
8- Mount in Permount or Histoclad
Results
Nuclei - blue - with some metachromasia
Cytoplasm - various shades of pink-identifying different tissue
components

Staining machine