UNDERSTAND MICROTOMY AND THE MAINTENANCE OF

MICROTOMY TOOLS

The word “microtomy” is originated from the Greek language. Mikros means
small and temnein means to cut. So the word “microtomy” means to cut the
tissue in thin sections. For successful microscopic examination, it is necessary
to have thin sections of the tissue by microtomy.

microtome: - is a device used to cut extremely thin slices of material, known as

sections.

Microtome is a mechanical instrument used to cut biological specimens into

very thin segments for microscopic examination.

Chevalier (1839) first introduced the term “microtome” in the scientific

terminology. Rudolf Thoma, a Heidelberg pathologist in concert with Rudolf
Jung, a precision engineer, designed the first microtome in series production
called “Thoma microtome” enabling the beginning of a new era in histology.
Modern microtomes are precision instruments designed to cut uniformly thin
sections of a variety of materials for detailed microscopic examination. With
practice, sections which are quite thin and translucent could be produced. For
light microscopy, where magnifications can reach up to 1,800x, the thickness of
a section can vary between 1 and 10 microns (thin sections). For electron
microscopy, where magnifications of about 10,000,000x is possible, the
thickness of a section is usually of the order of 10 nanomicrons (ultra-thin
sections).

PARTS OF A MICROTOME

Microtomes consist of three main parts:

 Base (microtome body)

 Knife attachment and knife
  Material or tissue holder

With most microtomes a section is cut by advancing the material holder towards
the knife whilst the knife is held rigidly in place. The cutting action which can
be either in a vertical or horizontal plane is coupled with the advance
mechanism, so that the material holder is moved after each cut. The distance
moved is pre-selected using a scale setting on the microtome body and usually
extends between 0.5 and 50 microns on microtomes cutting thin sections and
from less than 60 nm to over 500 nm on machines cutting ultra-thin sections (6).

CLASSIFICATION OF MICROTOMES

There are different types of microtomes available, depending on the

manufacturer’s specifications and based on the application required for
sectioning of the samples.

Microtomes can be classified as:

 Manual microtomes
 Semi- automatic microtomes
 Automatic microtomes

Manual microtomes:

o Rocking microtome
o Rotary microtome
o Sledge microtome
o Freezing microtome
o Vibrating microtome
o Ultra microtome
o Cryostat
o Sliding microtome
o Saw microtome
 o Hand microtome

Automatic microtomes:

o Laser microtome
o Computer microtome
o Ultra-thin computer microtome

ROCKING MICROTOME

The rocking microtom is also known as Cambridge rocking microtome. This

instrument is one of the oldest in design, relatively cheap, and is exclusively
designed for sectioning paraffin blocks (Figure 1). The name of the microtome
derives from the rocking action of the crossarm and is simple to use. This
microtome comprises of three moving parts, which is extremely reliable and
requires minimum maintenance. In this type of microtome, the knife is static,
and the block of tissue moves in a rocking motion (arc-like movement of the
block).

However, the disadvantages are its tendency to move during cutting because of
the lightness of the frame and very thin sections are difficult to obtain. The
rocking microtome has largely been replaced by the more precise rotary
microtome although it is reappearing in portable cryostats.

Advantages:

1. Thin section

2. Easy to operate

3. Low-cost instrument and reliable

Disadvantages:

1. Tissue is curved and the microtome does not provide flat section.
2. As the microtome is of light weight so vibration may occur.

ROTARY MICROTOME

This is the most commonly used microtome in routine laboratory. This

microtome derives its name from the rotary action of the hand-wheel which
actuates the cutting movements. Machines of this sort are general purpose
microtomes for cutting semi-thin to thin sections for light microscopy (Figure
2). The operation is based upon the rotary action of a hand wheel activating the
advancement of a block towards a rigidly held knife. The block moves up and
down in a vertical plane in relation to the knife and therefore cuts flat sections.
It has the advantage of being heavier and therefore more stable than the
rocking type, and is ideal for cutting serial sections. Larger blocks of tissue may
be cut on this machine, and the cutting angle of the knife (tilt of knife) is
adjustable. Since a heavier and larger knife is used with this type of microtome,
there is less likelihood of vibration when cutting. Section thickness settings
range from 0.5μm to 60μm on most machines. Sections of paraffin wax
embedded tissues are normally cut within the range 3 to 5μm, while resin
sections are attained between 0.5 to 1μm.

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.
4. It may also be used for cutting celloidin embedded sections with the help
of special holder to set the knife.
5. Good-quality 2–3-μm-thin section is possible.
6. Good tissue ribbon production.
7. Easy-to-cut various types of tissue: firm, fragile, small biopsy, etc.

Disadvantages:
1. Expensive.

2. Unsuitable to cut large block.

3. Knife faces up and so may be dangerous to the technical staff.

SLEDGE MICROTOME

Sledge microtome This microtome was originally designed for cutting sections
of very large blocks of tissue (e.g. whole brain). The sledge microtome has
become a popular machine for routine use since the World War II. The block-
holder is mounted on a steel carriage which slides backwards and forwards on
guides against a fixed horizontal knife. This microtome is heavy and
consequently very stable and not subjected to vibration. A large knife is used
(24 cm in length) and the knife is usually wedge-shaped which reduces the
possibility of vibration and requires less honing. The knife-holding clamps are
adjustable and allow the tilt and the angle (slant) of the knife to the block to be
adjusted with ease. Its action is much slower when compared to rocking or
rotary microtome which is its major disadvantage.

Advantages:

1. Hard tissue can be cut.

2. Large tissue sample can be cut.

3. The best microtome for ophthalmology and large neuropathology section.

Disadvantages:

1. Difficult to get thin section.

2. Large slides are required.

SLIDING MICROTOME
In a sliding microtome, the knife is moved horizontally against a fixed block
which progresses against it in an inclined plane (Figure 4). The sliding
microtome can be used for paraffin-wax embedded sections although it was
designed for cutting celloidin-embedded sections.

Advantages:

1. Large sections can be cut.

2. Mainly used for celloidin-embedded tissue.

3. Simpler design and easy maintenance.

4. Brain sections can be cut better by this type of microtome.

Disadvantages:

1. The knife may glide in case of hard tissue and may jump.

2. Long knives are difficult to sharpen.

FREEZING MICROTOME

Although other microtomes can be modified for cutting frozen sections, this
type of microtome was known to be efficient, producing the best results and was
used almost universally. The machine is clamped to the edge of a bench and is
connected to a cylinder of CO 2 by means of a specially strengthened flexible
metal tube. The cutting action of the freezing microtome differs from those
described previously as in this case the knife is moved whilst the tissue block
remains static. The block moves by a pre-set amount, in microns, at the end of
each cut. However, consistent, high quality, thin sections are very difficult to
obtain with this type of microtome. This device enables tissue to be frozen
without the necessity of solid carbon dioxide or liquid nitrogen. This method
has acquired popularity before the cryostat became widely used for frozen
sections. In this instrument, two dissimilar metals are placed in apposition with
one another and when a direct electric current passes through them, heat is
generated on one surface and lost from the other. This phenomenon is known as
‘Peltier’ effect.

Vibrating Microtome

he 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 µm for live tissue and
10500 µm for fixed tissue.

Originally thought to replace the hand microtome, the vibrating microtome was
conceived as a microtome which could produce high quality sections of fresh,
unfixed material from animal or botanical sources. This instrument has been
designed to cut tissue which has not been fixed, processed or frozen and has the
greatest application in enzyme histochemistry and ultrastructural
histochemistry. The name of the instrument was derived from the high speed
vibration produced by a safety razor blade which provided the cutting power.
The amplitude of vibration is adjusted by altering electrical voltage applied to
the ‘knife’. To prevent tearing, soft material is cut whilst immersed in a fluid
which also aids in dissipating heat produced at the vibrating edge of the razor
during cutting.

ULTRA MICROTOME

The ultra-microtome is used to prepare ultra-thin sections for light and electron
microscopy. Very small samples of tissue or industrial product are usually
embedded in hard resin before cutting. It has been reported that sections can be
cut as thin as 10 nanometers. Two forms of advance mechanism have been
developed in this style of microtome. The thermal mechanism relies upon heat
induced expansion in a bifurcated metal strip. Whereas in the mechanical form a
microprocessor coupled to a precise stepping motor controls the advance
mechanism. The cutting stroke is motor driven to provide a regular, smooth
motion for sections of even thickness and constant reproducibility. Knives are
usually made from glass, diamond or sapphire. The block is brought to the knife
edge under microscopical control and as each section is cut it is floated on to a
water bath adjacent to the knife edge.

SAW MICROTOME

Saw microtomes cut sections from very hard material such as undecalcified
bone, glass or ceramics. The samples, commonly embedded in resins, are
moved extremely slowly against a diamond coated saw rotating at
approximately 600 rpm. It is possible to produce sections of 20 μm or greater,
provided the saw blade is in perfect condition. The saw microtome is not
capable for producing very thin sections.

HAND MICROTOME

The hand microtome is limited to sectioning intrinsically rigid botanical

material and it is difficult to obtain thin sections from animal tissues.

LASER MICROTOME

Laser microtome is used for precise, non-contact sectioning and was designed to
slice samples with high precision. It’s equipped with state of-the-art
femtosecond laser technology. It enables non-contact cutting inside biological
tissues and various materials without causing thermal damage. Depending on
the material being processed, slice thicknesses of about 5 to 100 μm are
feasible. Non- contact processing, sub micrometer precision, cutting of the
tissue in its native state, no thermal damage, fewer artifacts and less time
consumption in tissue preparation are the added advantages of this laser
microtome.
 MICROTOME KNIFE

It is the important instrument used to cut uniform thin serial sections of the
tissue. The microtome knife is important to cut uniform and thin section of
tissue. 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.

Microtome knives can be classified according to the material used for making
the knife or based on the shape of the knife edge.

Based on shape of the knife edge (Profile):

– Profile A: Strongly plano-concave/biconcave

– Profile B: Plano-concave

– Profile C: Wedge Shaped

– Profile D: Plane Shaped

Based on material of the knife:

– Steel knives

– Non-corrosive knives for cryostat

– Disposable blades

– Tungsten carbide knives

– Glass knives

– Diamond knives

– Sapphire knives

BASED ON SHAPE OF THE KNIFE EDGE (PROFILE)

1. Plano concave (Profile A): One side of the knife is plain and the other side
is concave. Originally these knives were used for cutting celloidin-embedded
tissue. This is a very sharp knife and is used for cutting soft tissues. However,
presently these knives are less frequently used.

2. Biconcave (Profile B): The knife is concave on both sides. The knife was
used for rocking microtome. The concavity of the knife is often difficult to
identify. This is a less rigid type of knife and often vibrates during cutting.

3. Wedge (Profile C): This knife is plain on both sides. This is the most widely
used knife for routine microtomy and it is compatible with the different type of
microtome. This type of knife is also easy to sharpen.

4. Tool edge (Profile D): The knife resembles chisel used in wood working.
Both sides of the knife are plain; however, the cutting edge is steep. The tool
edge knife is mainly used to cut the hard tissue such as decalcified bone. The
knife is difficult to sharpen and is not recommended presently.

BASED ON MATERIAL OF THE KNIFE

Steel Knives

Steel microtome knives are manufactured from high quality carbon or tool
grade steel which is heat treated to harden the edge. The steel should be rust
resistant, free from impurities and contain anti-corrosives. The best knives are
those that are fully hardened. Those which are only surface hardened lose the
cutting edge very quickly once the hardened area is removed through repeated
re-sharpening.

Diamond Knives

Diamond knives are manufactured from gem quality diamonds without flaws.
Although the diamond knives are very expensive, the knives are extremely
durable, due to its hardness. These knives are used primarily for cutting very
thin sections

Glass Knives

The cutting edge of glass knives (‘Ralph knives’ with edges of 25 or 38 mm)
used for conventional sectioning is parallel to one surface of the glass while
knives used for ultramicrotomy is positioned against / across the thickness of
the glass. Different profiles of ‘Ralph knife’ for cutting sections from different
embedding media can be produced very quickly. Glass knife holders are
available so that ‘Ralph knives’ can be used with a rotary microtome. Glass
knives are hard but brittle and therefore require care when handling. These
knives deteriorate with storage due to changes in the ‘flow’ or ‘strain’ of the
glass after fracture and from oxidation impurities remaining in the hardened
glass after manufacture. Knives should thus be prepared immediately before
use.

Tungsten Carbide

Knives manufactured from high quality tungsten carbide (Figure 15) are
practically nonmagnetic and 100 times harder than hardened tool steel (25). The
knives have excellent resistance to wear but are brittle because of their extreme
hardness and should be handled carefully. But, it has been reported that up to
30,000 serial sections of undecalcified bone embedded in methacrylate can be
obtained per sharpening