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Justine Gayon (jgayon102@gmail.com)

 MICROTOMY
The process by which processed tissue, most commonly
a paraffin embedded tissue, is trimmed and cut into
uniformly thin slices or "sections" to facilitate studies
under the microscope.
Microtome
- capable of cutting a section at a predetermined
thickness by sliding the block into a cutting tool,
usually a steel knife, glass or diamond blade,
which is fixed and attached to the machine.
Three (3) essential parts:
1. Block Holder - where the tissue is held in position.
2. Knife Carrier and Knife - for actual cutting of
tissue sections.
3. Pawl, Ratchet Feed Wheel and Adjustment
Screws - to line up the tissue block in proper
position with the knife, adjusting the proper
thickness of the tissue for successive sections.
PRINCIPLE:
A spring-balanced teeth or pawl is brought into contact
with, and turns a ratchet feed wheel connected to a
micrometer screw, which is in turn rotated, moving the
tissue block at a predetermined distance towards the
knife for cutting sections at uniform thickness
1. Rocking microtome – for cutting serial sections of
large blocks of paraffin embedded tissues.
2. Rotary microtome - for cutting paraffin embedded
sections.
3. Sliding microtome - for cutting celloidin embedded
sections.
4. Freezing microtome -for cutting unembedded
frozen sections.
5. Cryostat or cold microtome – for cutting frozen
sections
6. Ultrathin microtome - for cutting sections for
Electron Microscopy.
1. Rocking (Cambridge) Microtome
- invented by Paldwell Trefall in 1881
- simplest among the different types of microtomes.
- This consists of a heavy base and two arms
- The lower arm resting on pivots and a supporting
column, and attached to the micrometer screw, at the
base of which is found the ratchet wheel with feed
mechanism.
- The upper arm, carrying the block holder on one
end by means of a screw, is connected to a lever by a
piece of nylon thread.
- A section is thereby cut as the tissue passes to the
knife edge in a slightly curved plane, in 10-12 um
thickness
- available in two sizes
- has been used to cut small and large blocks of
paraffin tissues.
- not recommended for serial sections since tissues are
cut in slightly curved planes.
- not currently favored by most laboratories because
of the restrictions in size of tissue block that can be
cut, and the difficulty of reorienting the block.
2. Rotary (Minot) Microtome
- invented by Minot in 1885-86 to cut paraffin
embedded tissues
- Currently the most common type used for both
routine and research laboratories, especially for
sectioning paraffin-embedded tissues.
- Electrically driven rotary microtomes are also now
available and can be ideally used to produce ribbons
for serial sections.
- the device operates with a staged rotary action such
that the actual cutting is part of the rotary motion. In
a rotary microtome,
- the knife is fixed in a horizontal position
3. Sliding microtome
- developed by Adams in 1789.
There are two types:
 Base-Sledge Microtome,
- consists of two movable pillars holding the
adjustable knife clamps, allowing the knife to be
set at an angle for cutting celloidin sections.
- favored in laboratories where very hard tissue or
large blocks are usually sectioned
- suited for sectioning specimens embedded in all
forms of media, especially for cutting sections
from tough tissue blocks which may offer great
resistance to the knife.
- Larger sections are more easily cut with the
knife, set at an angle due to less resistance
offered by the block.
- was originally designed for cutting sections of
very large blocks (whole brain)
 Standard Sliding microtome
- is different from the base sledge microtome
because with this instrument, the block remains
stationary while the knife is moved backward
and forward during the process of sectioning.
- 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. Freezing Microtome
- invented by Queckett in 1848.
Justine Gayon (jgayon102@gmail.com)
- The stage for block holder is hollow and perforated
around its perimeter, attached to a reinforced flexible
lead pipe thru which carbon dioxide passes from a
cylinder
- used to cut undehydrated thin to semi-thin sections
of fresh, frozen tissues, especially in instances when
rapid diagnosis is required, when histological
demonstration of fat is needed, when certain
neurological structures are to be studied, and when
sensitive tissue constituents to be studied are
damaged or destroyed by heat.
- this type will give the best results and is used almost
universally.
- equipped with a stage upon which tissue can be
quickly frozen using either liquid carbon dioxide,
from a cylinder, or a low temperature recirculating
coolant.
- 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,
same as sliding microtome
5. The Cryostat or Cold Microtome
- a refrigerated apparatus used for freezing the
tissue into the block holder to the correct degree of
hardness that allows for easier and faster sectioning.
- It consists of a microtome, usually a rotary
microtome, kept inside a cold chamber which has
been maintained at a temperature between -5° to -
30°C (average is -20°C) by an adjustable
thermostat, capable of freezing fresh tissues within
2-3 minutes, and cutting sections of 4 μ with ease.
- cryostat provides a means of preparing thin
sections of fresh frozen tissues especially for
fluorescent antibody staining techniques or
histochemical enzyme studies.
- It is most commonly used for rapid
preparation of urgent tissue biopsies for
intraoperative diagnosis.
- It is often housed in the frozen section room
close to the operating room to allow direct
consultation between surgeon and
pathologist.
6. Ultrathin microtome
- equipped with a glass or gem grade diamond
knife
- is used to cut very thin sections (typically 60
to 100 nanometer) of tissue embedded in
epoxy resin.
- Sections are stained with an aqueous solution
of an appropriate heavy metal salt and
examined with a transmission electron
microscope (TEM).
- The ultrathin microtome is also used with its
glass knife or an industrial grade diamond knife
to cut semi-thin sections prior to thin
sectioning.
- semi-thin sections are generally 0.5 to 1 μm
thick
CARE OF THE MICROTOME
 Brush away all accumulated paraffin and small
pieces of tissues which may interfere in cutting
tissue blocks.
 Wipe with xylol after drying the machine and knife
holder
 Oil movable portions to avoid rusting.
 Remove knife or blade before cleaning,.
SAFETY MEASURES
 Staff should avoid distractions to prevent injury
from extremely sharp blades.
 Use forceps or brush instead of fingers to pick up
sections or wax fragments from blade or block face.
 Use hand wheel lock when changing blocks.
Remove blade always when not in use
MICROTOME KNIVES
Trimming and section-cutting are done with a
microtome knife, which is available in three basic types
or shapes:
1. Plane-Concave Knife (usually 25 mm. in length) –
- One side of the knife is flat while the other is
concave.
- Less concave sides are recommended for
cutting celloidin-embedded tissue blocks on a
sliding microtome.
- More concave sides are used to cut paraffin
sections on base-sledge, rotary or rocking
microtome.
2. Biconcave Knife (usually 120 mm. in length) –
- with both sides concave
- recommended for cutting paraffin - embedded
sections on a rotary microtome.
3. Plane-Wedge Knife (usually 100 mm. in length)
- have both sides straight
- recommended for frozen sections or for cutting
extremely hard and tough specimens
embedded in paraffin blocks, using a base
sledge type or sliding microtome.
Justine Gayon (jgayon102@gmail.com)
 Plane-wedge and plane-concave knives are usually
provided with backs, to maintain the correct bevel
angle throughout honing
Bevel Angle
- The angle formed between the cutting edges
- normally about 27° to 32°
- Such angle is maintained for each knife by
means of a slide-on back, a spring-loaded semi-
circular metal sheet slipped on to the knife with
one or more plane surfaces (plane-wedge or
plane-concave) to hold the cutting edge at a
constant, correct angle during the process of
honing and stropping.
HONING AND STROPPING
Badly nicked knives with blunted ends have to undergo
sharpening in order to ensure optimum sectioning of
tissue blocks and prevent gross irregularities on the
tissue sections. Jagged edges, if not corrected, will
produce tears or striations in tissue sections.
Sharpening of the knife involves two stages:
 HONING (HARD SHARPENING)
involves
- the removal of gross nicks on the knife edge
(Coarse Honing) to remove blemishes, and
- grinding the cutting edge of the knife on a stone
(Honing Proper) to acquire an even edge.
- The degree of sharpness is proportional to the
fineness of the abrasive used in sharpening.
- This procedure makes use of a hone, a natural
sharpening stone or hard grinding surface
(carborundum), which serves to remove nicks
and irregularities on the knife edges.
Several types of hones may be used:
1. Belgium Yellow - for manual sharpening when
cutting edge has been rendered blunt or nicked.
This type usually gives the best result.
2. Arkansas - gives more polishing effect than the
Belgium Yellow.
3. Fine carborundum - is much coarser than the
first two types and is used only for badly nicked
knives followed by either one of the first two
knife sharpeners.
 The surface of the hone is wiped clean with a soft
cloth moistened with xylene in order to remove the
scattered small particles of stones and metal.
 It is then covered with a thin film of Mineral and
Clove Oil, Xylene, Liquid Paraffin or Soapy Water
for lubrication.
 The knife is fitted to its corresponding back, placed
on one end of the hone, and with the cutting knife
edge first, the "heel" (handle end) is drawn obliquely
or diagonally towards the operator on the stone until
the "toe" (head portion) is reached.
 The knife is then turned over, and the other surface
is again drawn forward, EDGE FIRST, with a HEEL
TO TOE direction.
 Hone is placed on non-skid surface. A damp cloth
may be used-to prevent movement of the hone. Light
lubricating oil or soapy water is used for lubrication.
 In the case of the Minot or plane-wedge knife, the
knife is turned over so as to sharpen the other
surface every I0-20 strokes. For plane-concave
knives, only the concave surface should be rubbed
on the Hone.
 Mechanical honing with machines may make use of
a vibrating frosted glass plate or a wheel driven by
an electrical motor.
 The knife is pressed against the flat side of a rotating
glass wheel which is being driven by a mechanical
device.
 Approximately 30 double strokes are given each side
of the knife to which very gentle pressure is applied.
The use of knife sharpening machines, although
quite expensive, is usually time-saving and
produce well sharpened knives with uniform
bevels.
PRECAUTIONS DURING HONING:
 The hone should be long enough (about 8" x 3") to
allow the whole length of the knife edge to be
sharpened in a single stroke and wide enough to
sufficiently support and prevent the rocking of the
knife.
 The hone should be lubricated with warm soapy
water or fine oil before using.
 The pressure on the knife should be gentle and
steady to keep it from rocking. The number of
strokes usually amounts to 20-30 times in each
direction, depending upon the condition of the knife
 The hone should be cleaned before, during, and
after use.
 After honing, wipe off the oil or soap from the
knife with xylene. Then strop it thoroughly.
 STROPPING
- process whereby the "burr" formed during
honing is removed and the cutting edge of the
knife is polished.
Justine Gayon (jgayon102@gmail.com)
 - The purpose of stropping is to polish and
sharpen the cutting edge, while that of honing is
to remove the irregularities from the knife.
- If the knife has become dull and blunt, but is
free from nicks or teeth, it is usually only
necessary to strop it.
- For delicate work, the knife is stropped before
every object is sectioned.
 A paddle strop made up of the best quality horse
leather, firmly attached to a solid back, in order to
prevent sagging is preferred.
 The procedure is the reverse of honing.
 The knife is first fitted with its appropriate knife
back, then laid obliquely on the strop and with the
cutting edge behind, (EDGE LAST) is pushed
backward and drawn forward in a TOE TO HEEL
direction.
 Around 40 - 120 double strokes are usually
required.
 In the case of plane-wedge or Minot knives, the
knife is turned around at the end of each stroke so as
to sharpen each surface alternately. For plane
concave knives, only the concave surface should be
stropped
PRECAUTIONS FOR STROPPING:
 The knife should always be wiped clean with a soft
cloth before and after a series of stropping strokes
and before changing from a coarse to a fine strop to
remove particles which may have been taken off the
knife. After stropping is satisfactorily completed, the
knife edge is then oiled or greased to prevent it from
rusting.
 Pressure during the first stropping strokes should be
quite light, since the natural compressibility of the
leather is what actually does the work.
 Speed in stropping should be avoided. One full
second should be allowed for each stroke to avoid
injury to the strop and the knife.
 Leather strops are usually dry and require oiling
before they are used.
 Strops are usually treated with vegetable oil (e.g.
castor oil) applied into the back of the strop, NOT
the surface. The strop should not be used for at least
24-48 hours after treatment. Too much oil will make
the
 stropping surface slippery and will render the
procedure unsatisfactory.
 To remove excessive oil from the strop, its surface is
scraped with a blunt instrument, e.g. the back of the
knife.
 Mineral oil is not recommended and should NEVER
come in contact with a strop since it will tend to
blister and destroy the leather. One drop of mineral
oil will spoil the polish of that area, and produce a
permanent blemish on the strop.
 Stropping surfaces should be firm and not loose, to
prevent the turning of the knife's edge
 Wax must not be allowed to come in contact with
the strop. With an applicator, the used knife blade
should be washed and flushed with xylene
Disposable Blades
Sharpening (honing) and polishing (stropping) are no
longer common practice in most modern laboratories
because of the availability of disposable knives that are
cheaper to use than conventional steel knives. They have
a sharp cutting edge that can cut 2-4 μ thick sections
with ease. Some microtome manufacturers have also
now incorporated a disposable blade holder in place of a
knife holder. Magnetic knives are also now available
that can attach to some blade holders and are particularly
suitable for use in the cryostat.
Glass Knives
- generally used for trimming and semi-thin
sectioning of tissue blocks for electron
microscopy.
- They are prepared from commercially available
40 x 2.5 cm. plate glass strips that have been
washed with detergent, rinsed in distilled water
and alcohol, and dried with lint-free paper.
Diamond Knives
- used to cut any type of resin block for electron
microscopy.
- When supplied by manufacturers, they are
already mounted in a metal block designed to fit
directly into the knife holder of the ultrathin
microtome.
- are brittle and expensive, but very durable, and
the cutting edge must be kept clean to make it
cut longer and to avoid damage during
sectioning.
Waterbath - The thermostatically controlled type is
preferable, but if this is unavailable, water from a hot
water tap can be used although this can give rise to air
bubbles which may be trapped under cut sections. The
temperature of the water should be between 5 and 10°C
below the melting point of the paraffin wax.
Drying oven or hot plate - Small drying ovens are now
available, incorporating a fan, especially designed for
drying tissue section on slides. With a temperature
setting at the melting point of the wax no obvious
damage is done to the sections and drying is complete in
30 minutes. A hot plate may also be used instead of a
drying oven. For more delicate tissues such as brain, a
lower drying temperature is used to avoid splitting
Justine Gayon (jgayon102@gmail.com)
and cracking of the section due to excessive heat. In
such cases, 37oC for 24 hours or longer is
recommended.

Forceps (fine pointed or curved) and squirrel hair brush

- These tools are needed for handling sections during
cutting, and for removing folds and creases on the
sections during "floating out" in water bath.

Clean Slides - For routine work, 76 x 25 mm. slides that

are 1.0 -1.2 mm thick are usually preferred because they
do not break easily. Frost-ended slides are generally
used, where the identification number of the section can
be inscribed with a pencil. Automatic slide labeling
machines are also now available.

Things to avoid include:

 Fecal Material in intestine, especially in the colon
where this material is very hard
 Hair is particularly bad - it can be removed using a
razor blade or clippers. Hair can also sometimes be
inadvertently included with organs. Please be careful
during dissections
 Sutures, Thread Or Staples should be removed
from the specimen prior to cutting with the knife.

Justine Gayon (jgayon102@gmail.com)