MICROTOMY

BY

DR. A. B. AJILEYE.
 • MICROTOMY
• Microtomy is a process of cutting thin sections from tissues with a
precision machine called microtome. Thin sections are necessary
because before sections can be examined under the microscope, light
has to pass through them and only thin sections allow light to pass
through them. The mechanism of cutting sections is designed in such
a way that after each cut, tissue or knife automatically moves forward
at a predetermined distance. This enables sections to be cut at every
activation of the cutting mechanism.
 Many types of microtome are available, the type of microtome used
for cutting sections depends on four major factors, and these are:

 The substance to be demonstrated in the tissue

 The type of tissue
 The embedding medium
 The urgency

• Microtomes are classified into various types, but all of them should
fulfil the following requirements:-
 Rigid support for the knife and the tissue block.
 Means of moving either the tissue block across the fixed knife-edge
or the knife edge across the block
 Means of accurately advancing the tissues to cut each section at the
predetermined thickness
 Types of Microtome
• There are several kinds of microtomes:
 Cambridge Rocking microtome – for cutting serial sections of large
blocks of paraffin embedded tissues.
 Rotary microtome - for cutting paraffin embedded sections.
 Sliding microtome - for cutting celloidin embedded sections.
 Base Sledge Microtome
 Freezing microtome -for cutting unembedded frozen sections.
 Cryostat or cold microtome – for cutting frozen sections
 Ultrathin microtome - for cutting sections for Electron Microscopy.
 1. Rocking (Cambridge) Microtome

• The simplest among the different types of microtomes.

• It’s a small light weight machine, which is spring-operated with a few
moveable parts. It can cut sections from wax, celloiding and many
other embedding media except from synthetic resins, which are
extremely hard. Sections produced from the Cambridge rocker are
often slightly curved. This is because movement of tissue against
knife is not vertical but through an arc. However, the account of
cutting curved sections is noticed only when the sections are thick.
• The Cambridge rocker has two (2) major disadvantages.
• Large pieces of tissue cannot be cut on it due to its light weight.
• It often moves when sections are been cut, although this problem
can be overcome if it is placed on a non-slip mat, damp cloth or it is
fitted unto a material which has been clamped onto a work bench.
Rocking Microtome
 2. Rotary (Minot) Microtome
• The instrument is most ideal for routine and research work. The
microtome is smaller in size than the sledge microtome and has a smaller
knife area which limits the size of tissue blocks to about 3×4cm. the Rotary
microtome has a wheel at the side. As the wheel is rotated, it actuates the
feed mechanism on the micrometer screw causing the tissue to move
vertically up and down, and a forward movement according to a set
distance, which is equivalent to the desired thickness of section. Flat
sections are cut as strokes of tissue come in contacts with knife, which is
fixed with cutting edge uppermost. Many rotary microtomes are provided
with knife guards which protect the finger from being cut during
sectioning.
• The advantages of rotary microtome are:-
• Large blocks of tissue can be cut from it
• It is heavy; therefore it is stable and does not vibrate during section
cutting.
• Serial and ribbon sections can be produced with ease from paraffin wax
Rotary Microtome
 3. Sliding Microtome
• The block remains stationary while the knife is moved backward and
forward during the process of sectioning.
• It was developed mainly for cutting celloidin embedded tissue blocks
and is inherently more dangerous because of the movable knife,
which makes it difficult to attach knife guards.
 4. BASE SLEDGE MICROTOME
• The knife is stationary and horizontally placed. The feed mechanism
is actuated by pushing the handle to and fro on runners. As the tissue
block comes across the knife, section is cut and the tissue block
passes beneath the knife. Large wedge-shaped knives are usually
used with the base sledge microtome. Frozen sections can be cut
when thermo-electric cooling devices are incorporated onto the
cutting stage of the microtome.
ADVANTAGES
• It is heavy therefore it does not vibrate during section cutting
• Very large pieces of tissue can be cut with it.
• The knife holder is adjustable so that knife can be obliquely
positioned for the cutting of celloidin sections and paraffin wax
sections
• Frozen sections can be cut with it.
 5. FREEZING MICROTOME
• The freezing microtome generally consists of a central advancing
screw on which the block holder is situated. A cylinder of CO2, the
cooling agent, is connected by means of a reinforced flexible lead, is
hollow and perforations around the perimeter. These perforations are
an essential part of the cooling allowing the gas to flow and freely
escape, thus providing uniform freezing of the tissue. To facilitate
section cutting, the knife edge is kept cool by means of another
cooling device. The recent models of freezing microtomes are
connected to a thermo-electric cooling units called thermal modules
instead of CO2. The module works on the principles of the peltier
effect. With thermal module, there is a continuous thawing and
freezing of tissue. The freezing microtome has its greatest application
when
• Urgent diagnosis is required
• Fat is to be demonstrated histologically
• In the absence of a cryostat
• When enzymes and neurological structures are to be demonstrated
It should be noted that with a freezing microtome, serial sections
cannot be cut, structural details are somehow distorted, staining of
frozen section of unfixed tissue is not very satisfactory and freezing
artefacts such as ice crystals in the tissue, these microtomes cut
alternate thick and thin sections.
Freezing Microtome
 6. CYOSTAT or COLD MICROTOME
• Before the advent of cryostat, the preparation of frozen section was
confined to freezing microtomes which were operated at room
temperature with CO2 as the tissue refrigerant. This method has
now been replaced by the cryostat. The cryostat is a precision
machine which is housed in a deep freeze cabinet maintained at a
temperature of about -150C to -300C. The major advantage of the
cryostat is that it maintains the tissue block, knife and the section at
the same temperature. This eliminates difficulties such as cutting of
unfixed tissue with knife freezing attachment and obtaining thin
sections. There is no more thawing and freezing of the tissue and
knife. The cryostat enjoys the same advantages as the freezing
microtome.
Cryostat
 7. ULTRA MICROTOME:-

• This microtome is a highly specialized precision instrument, it is

designed to cut sections from hard synthetic resins, it cuts in the
order of 0.005µm to 0.1µm [50-1000A], which is suitable for electron
microscopy work. It can also cut sections which are 1-2µm thick for
examination with the light microscope. The knife is stationary with
cutting edge uppermost while the tissue moves up and down
vertically as sections are cut. A low power binocular microscope is
attached to the front of the microtome above the tissue block. It is
used for easy manipulation of tissue block and sections. The feed
mechanism can be actuated, by turning the wheel at the side of the
microtome. The knife is prepared from diamond or plate glass
suitable for cutting tissues embedded in hard synthetic resin media.
When sections are cut, they fall into a trough beneath the knife from
where they are picked up and prepared for electron microscopy.
Ultrathin Microtome
 CARE OF THE MICROTOME
• After sectioning, all the accumulated paraffin and small pieces of
tissues must be brushed away with a soft brush and not allowed to
stay in the microtome, since this may later interfere with the cutting
of tissue blocks.
• After carefully drying the machine and knife holder, the parts should
be wiped with xylol. Prolonged and continuous application of the
painted parts with xylene should however be avoided since this
reagent is capable of removing the paint.
• Movable portions should be oiled thoroughly to prevent rusting
• The microtome must always be covered when not in use, to prevent
accumulation of dust and other dirt which may later on interfere with
the normal sectioning of tissues.
• The microtome should be placed on a stable bench, away from air
drafts, doorways and passing staff. Any air movement from air
conditioners or other causes can make section handling very difficult.
• Always remove the knife or blade before cleaning. The knife holder
can easily be removed to facilitate access for cleaning. No fluid must
enter the inside of the instrument during cleaning.
• When cleaning the blade, avoid dragging anything along the cutting
edge.
• Have the instrument inspected at least once a year by a qualified
service technician.
 Safety Measures:
• It is very important that staff are not distracted when using the
microtome because of the risks of injury from extremely sharp
blades. It is preferable to have non-slip flooring in the vicinity of
microtomes because, inevitably, wax fragments will find their way
onto the floor where they can produce a slippery surface.
• Use forceps or brush instead of fingers to pick up sections or wax
fraagments from blade or block face.
• Use hand wheel lock when changing blocks. The knife or blade should
be removed from the microtome when the instrument is left
unattended or when cleaning the instrument.
 MICROTOME KNIVES

• Microtome knives are used for cutting sections when they are
properly attached to the microtome. They are heavy knives made
with rust proof carbon-steel materials. There are also Tungsten-
carbide tipped knives, some others are made from pyramidal plate
glass or diamond for use in ultra-microtomy. Microtome blades are
also available for cutting sections, they are fragile, thin, Teflon-
coated metals with very sharp cutting edge. The blade is attached
to a holder when it is to be used, the blades are also disposable.
The knives can be sharpened and re-used. The quality of cut
sections depends to a large extent on the sharpness of the knife
used, the correct orientation of knife on the microtome, the type
of tissue, type of embedding media used and the type of
microtome used.
 TYPES OF KNIFE
• BICONCAVE PROFILE:- The shape makes this knife confined to the Cambridge
rocker microtome. A Biconcave knife has slightly curved surfaces on both sides
of knife. The degree of concavity varies. It may be high or low. It is used mainly
for wax embedded tissues.

• PLANOCONCAVE:-is used with the base sledge microtome to cut all except the
toughest tissue embedded in paraffin wax. It is also suitable for low viscosity
nitrocellulose(LVN) embedded materials. When in use, the concave surface
should face upwards. It is almost completely vibration free and it is easy and
quick to sharpen.

• WEDGE SHAPED:- both surfaces of a wedge shaped knife are flat. It is used for
cutting sections from wax, cellulose nitrate hard tissues, frozen etc. it takes
longer time to sharpen than the planoconcave knife.

• TOOL EDGE:- the cutting facet of a tool edge is shaped like a chisel. It is used
for cutting very hard materials such as undecalcified bone.
• A: Biconcave
• B: Planoconcave
• C: Wedge- Shaped
• D: Tool-edge
 KNIFE SHARPENING PROCEDURE
• HONING:- is a knife sharpening procedure. It is done to remove nicks
from cutting facet and to sharpen a blunt knife. Knife back is worn on
knife and placed on a hone. The purpose of a knife sharpening back is
to raise the back of the knife so that the angle of the cutting facet
(bevel) is maintained. Each microtome knife has its specific knife
sharpening back. It must not be changed from one knife to another;
otherwise the bevel angle will be distorted. During honing, the knife
is moved from side to side and forward at the same time on the hone
with the cutting edge leading. At the end of the movement, knife is
moved backward the same way with the cutting edge leading on the
hone. The process is repeated until knife is sharp. Aluminum oxide or
soapy water is put on the hone to serve as a lubricant. Examples of
materials used as hone are Arkansas stone, plate glass, Belgian yellow
stone,carborundum and Aloxide.
 STROPPING
• Is a knife polishing procedure of a honed knife. Stropping is
performed in a manner similar to honing except that the knife is
reversed and lightly stroked back and forth over a leather surface.
The knife is moved from side to side and forward at the same time
but with the back of the knife leading. This is unlike in honing where
the cutting edge of the knife leads. The strop is made of leather,
which is supported beneath with a wooden block. To ease stropping,
Jeweler’s rough may be put on to the strop.
 • TYPES OF SECTION
• Ribbon sectioning:- ribbon sectioning is a process of cutting sections such
that cut sections adhere to each other at the edges as they cut. This results
in a chain like stretch of consecutive sections. They are useful for class
work especially when it is required to demonstrate the same part of a
tissue to a large number of students at the same time. Ribbon sections are
particularly obtained from tissue embedded in paraffin wax.
• SERIAL SECTIONING:-is cutting of sections from the same block and
arranging them in consecutive succession as they are cut. Cut sections are
numbered serially and are usually of the same thickness.
• STEP SECTIONING:- in step sectioning, at least three sections are cut from
a block, but tissue block is trimmed very briefly after each cut. Thus there is
an alternate trimming and cutting of sections. The sections are numbered
serially e.g 1st Step, 2nd step, 3rd step etc
• DEEPER LEVEL SECTIONING:- deeper level sections are required when it is
suspected that a tissue contains certain pathological condition in the
deeper part of the tissue. Therefore, tissue block is trimmed until sufficient
part of the tissue is exposed before sections are cut. Deeper levels are also
required when block is not adequately trimmed before sections are cut for