Microtomy

Microtomy is the technique of cutting tissues in very small sections. ... A tissue (or small part of it)
is embedded in paraffin wax and then cut using a microtome (a machine) to get thin sections. These
sections are stained with appropriate dyes and observed under the microscope.

PRINCIPLE:

Microtome is a sectioning instrument that allows the cutting of extremely thin slices of a material
known as section . Microtome are used in microscopy , allowing for the preparation of sample for
observation under transmitted light or electrons radiation .

Microtomy and Paraffin Section Preparation:

Creating great paraffin sections using a rotary microtome takes a great deal of skill and
experience. "Microtomy and Paraffin Section Preparation" is a great training aid for new
microtomists and an excellent refresher for experienced operators. All of the essential aspects of
cutting paraffin sections are covered, including:

Safety
Microtome setup
Microtome blades
Trimming, facing and roughing blocks
Techniques for consistent paraffin sections
Microtome maintenance
Common microtomy faults
 MICROTOME

Microtome is an instrument with the help of sections of tissues are cut and the process.

The thickness of sections produced during microtomy may be between fractions of 50-100 nm, in
ultramicrotomy, to several 100 microns.

The common range is between 5-10micron but both the maximum and minimum thickness is limited
by the consistency of relation of the thickness of sections to the nature of tissues.

These sections are stained using suitable staining techniques followed by observing them under
the microscope.
TYPES OF MICROTOMES –

1.) Rotary microtome

The Rotary microtome is called this because of a rotary action of the handwheel responsible for
the cutting moment. The block holder is mounted on a steel carriage, which makes up and down in
groves this type of instrument is the most ideal for routine and research work it is excellent for
cutting serial sections.

Parts of the rotary microtomes

Block holder
Knife clamp screw
Knife clamps
Block adjustment
Thickness gauge
The angle of tilt adjustment
Operating handle
Here the feed mechanism is activated by turning a wheel on one side of the machine. The knife is
fixed with its edge fixed upwards and the object is moved against the knife rising and falling vertically.

One rotation of the operating wheel produces a complete cycle downwards cutting stroke and an
upward return stroke and activation of the advanced mechanism. It is often modified to cut ultrathin
sections between 50Å – 200Å.

The wheel may be electrically operated or manually. In the former case the hands may be made free
for tissue maintenance, makes it available for incorporation in automated cryostats.

Advantages of the Rotary microtome

•Heavy and stable.

•Ideal for serial sections in large numbers.
•Paraffin-embedded tissues are cut by a rotary microtome.
•The knife holder is movable.
•The sections are cut are flat.
•It is useful for routine and research papers.
2.) Sliding or Base Sledge Microtome

This is a large heavy instrument with a fixed knife beneath which the object moves mounted on a heavy
sliding base containing the feed mechanism used primarily for cutting the sections of cellulose nitrate
embedded tissues with an obliquely set knife.

Parts of Base-sledge microtome

•Angular tilt adjustment

•Knife clamps
•Block holder
•Coarse feed adjustment
•Operating handle
•Thickness gauge
•Adjustment locking nut
•Block adjustment screw
•Split nut clasp
The blocks holder is mounted on a steel carriage which slides backward and forwards on groups
against a fixed horizontal knife this microtome is heavy and very stable. The block is raised towards
the knife at a predetermined thickness. This type of microtome is designed for cutting sections of
very large blocks of tissues for example whole brain, this microtome has become popular for routine
use.

Advantages of Base-sledge microtome

•It is useful for cutting extremely hard blocks and large sections.
•The microtome is heavy and stable.
•The knife used is sledge shaped which requires less honing.
Sliding/sledge Microtome
3.) Cambridge rocking microtome

The instrument is so named because the arm has to move in a rocking motion while cutting the
sections. The instrument was invented by Sir Horace Darwin in 1881 was developed by
Cambridge company hence it is called the Cambridge rocking microtome. It is a simple machine
in which the knife is held by means of microtome thread. The rocking microtome was designed
primarily for cutting paraffin wax sections but in an emergency use frozen section by inserting a
wooden block in which the tissue is frozen.

Parts of the rocking microtomes

•Knife holder
•Block holder or chuck
•Upper arm
•Screw
•Lever
•Pawl
•Ratchet wheel
•Mil head microtome screw
•Sleeve
•Lower Arm
•Scale
It cuts the sections between 1 to 20 microns. The knife is fixed with the edge, while the object is
moved against this knife circularly, producing a sharply curved surface to the block with each
stroke the tissue holder automatically moves vertically towards the life. Cutting stroke is Spring
operated and is easy to handle. The microtome must be placed on a solid non-slippery surface
to allow a better hold

Advantages of Cambridge rocking microtomes

•The cost of a knife and microtome is low.

•Celloidin embedded tissues can be sectioned easily.
Cambridge rocking microtome
4.) Freezing microtomes

This type has been designed for the production or preparation of frozen sections of fluid and
non-fluid tissues usually without preliminary embedding. The object stage is connected to the
cylinder of compressed carbon dioxide for the rapid cooling of the tissues and provisions are
also made for the cooling of the knife.

Part of freezing type microtome

•Knife clamps
•Operating handle
•Thickness gauge
•Stage
•Stage valve
•Coarse adjustment
The movement of the knife takes place horizontally across the surface of the tissues. Ribbon
sections cannot be prepared using this microtome. All freezing microtomes have the feature of
employing a non-movable tissue block and cooling system.

Advantages of Freezing microtome

•It is used for sections required for Rapid diagnosis

•It cuts non-dehydrated fresh tissue in a frozen state.
•The method is useful for Rapid histopathological diagnosis during operation
•This type of microtome is also used when lipids, enzymes, and neurological structures are to
be demonstrated.

Nowadays, the most commonly used type of microtome is a Rotary microtome which is easy to
operate and ideal for routine use for diagnosis and research purposes.
Freezing microtomes
WORKING PRINCIPLE OF ROTARY MICROTOME –

⇒ It is used for slicing paraffin tissue sections of uniform thickness.

⇒ This method is designed to cut 1-60 micron thick sections.
⇒ A knob on the device (typically at the backside) is used to modify the thickness of the sections.
⇒ A knife is constant inside the knife holder and clamped tightly.
⇒ The tissue block is drawn throughout the knife-edge and it is mechanically advanced. The top and
bottom of the block have to be parallel and horizontal and as a minimum 1mm of paraffin has to be
present in all aspects beyond the tissue.
⇒ The trimming of the edges of the block is usually completed with a single-sided razor blade and
the block face is trimmed with the microtome knife.
⇒ The technician decides the type of section to be made in line with the nature of tissue and
instructions received from the pathologist. At some stage in section slicing, as the wheel of the
microtome turns, sections are cut and slide on the knife. A ribbon of sections is produced.
⇒ The ribbon of sections is transferred to warm water inside the tissue floatation bath to put off any
wrinkles present in the section.
⇒ The best quality section that is free from any scratches and cracks can be decided on from the
tissue ribbons. The tissue ribbons are then taken on smooth glass slides with a respective
identification number.
⇒ The slides are pulled from the water and the preferred sections are positioned flat on the surface
of glass slides. The slides with the sections are positioned on a rack in a hot air oven to dry.
 WORKING PRINCIPLE OF MICROTOMY
(Steps to Better Microtomy + Flotation + Section Drying)

1. High quality, sharp blades are always used for cutting.

2. Knife tilt angle is always optimized for each microtome and blade type
3. Blocks are carefully trimmed to expose tissue. The last few sections are always cut at
what will be the final thickness to polish the block face.
4. Avoid Freezing Damage
Blocks are chilled on a cold wet surface and are always cold when cut (the surface of melting ice is
excellent).
5. Use Cold Blocks (Blocks are always cold when cut).
6. Cut Sections Slowly
The final sections from each block are cut gently with a uniform, slow rotation.
7. Use Clean Water
The water in the flotation bath is replaced regularly.
8. Ensure Slides are Clean
The cleanliness of slides is always checked before they are used. Handling of slides is kept to a minimum
to avoid contamination with squamous cells prior to flotation.
9. Avoid Cross-contamination
The water surface is always skimmed between specimens to avoid contamination of one
section with cells from another.
10. Avoid Contamination with Squames
Care is taken not to brush hair or hands while floating-out sections (squames can contaminate sections).
11. Don’t Float from Multiple Blocks
Sections from more than one block (case) are never simultaneously floated on the water bath.
12. Check Water Temperature
Flotation bath temperature is carefully checked. A temperature 6–10°C below the melting point of the
wax is optimal. Sections should readily flatten but the wax should not melt.
13. Avoid Wrinkles in Sections
Sections flatten readily on the flotation bath.
14. Avoid Over-expanding Sections
Sections are left on the flotation bath for just long enough to flatten then promptly picked up on a slide.
15. Don’t Damage Floating Sections
Extreme care is taken to avoid damaging floating sections when mechanically removing wrinkles with a
brush or forceps.
16. Carefully Choose Sections
The first one or two sections in a ribbon are never picked up on slides.
17. Prevent Bubbles Under Sections
Care is taken to avoid the formation of air bubbles in the flotation bath. Any visible bubbles are dislodged
before the sections are laid on the water.
18. Prevent Section Lifting
The use of “sticky” (charged) slides or section adhesives such as AAS is considered and used appropriately.
19. Drain Before Drying
Sections are drained briefly before being placed in the slide dryer or onto a hotplate.
20. Monitor Drying Temperature
The temperature of the slide dryer is carefully monitored.
21. Dry for Appropriate Time
The minimum and maximum slide drying time is monitored.
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