CHAPTER

12
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 is known as Microtomy. The basic
instrument used is a microtome that is 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. The
microtome consists of three essential parts, namely: 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.

Whatever the type of microtome is used, the principle remains essentially
the same, that is, 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.

There are five (5) kinds of microtomes:
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
This was invented by Paldwell Trefall in 1881, the 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.

Fig. 12-1. Rocking Microtome

When the lever is pulled forward, the pawl is brought in contact with the
ratchet wheel to which the millhead micrometer screw is attached. The ratchet
wheel is turned, rotating the micrometer screw. The lower arm is elevated, which
in turn raises the upper arm at its fulcrum, thereby carrying the chuck or block
holder forward, towards the knife. As the pressure on the operating handle or
lever is released, the tension on the spring causes the upper arm to return to its
normal position; in an arc of a circle. A section is thereby cut as the tissue passes
to the knife edge in a slightly curved plane, in 10-12 u thickness.
The Cambridge rocking microtome, available in two sizes, has been used to
cut small and large blocks of paraffin tissues. It is theoretically not
recommended for serial sections since tissues are cut in slightly curved planes. It
is 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.

B. Rotary (Minot) Microtome
This was invented by Minot in 1885-86 to cut paraffin embedded tissues,
and is 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.
 Fig. 12-2. Rotary Microtome

In the rotary microtome, 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 and the principle of section cutting is
shown in the image above. Although the flywheel in many microtomes can be
operated manually, they are generally automated or semi-automated. Typically,
sections are cut between 3 and 5 µm using paraffin wax for diagnostic histology
although thinner sections can be attained if samples are embedded in synthetic
resin.
It is different from the rocking microtome in that the knife and the block
holder are brought together by upward and vertical motions, cutting sections in a
perfectly flat plane, thereby allowing excellent serial sections to be cut. It is
heavier and more stable than the rocking microtome, is more complex in design
and construction, and is therefore more expensive. It may be used for cutting
large blocks of tissues although results are better when the sliding microtome is
used. The knife is placed in a blade-up position and is therefore relatively
dangerous. Both manual and electrically driven models are now available for
cutting ultrathin sections and for cryostat use.
A heavier knife is used, so there is less vibration. The cutting angle (tilt) of knife
is adjustable, so it can cut harder tissue. It can cut celloidin-embedded sections
by using a special holder to set the knife obliquely.

3. Sliding microtome
This was developed by Adams in 1789. There are two types of this
microtome. One model, the 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. The chuck or block holder is set on a heavy
metal base which can be moved backwards and forwards under the knife.
The Base-Sledge microtome is favored in laboratories where very hard
tissue or large blocks are usually sectioned. Such a machine is 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. It was originally designed for cutting sections of
very large blocks (whole brain). Sections are cut in a perfectly flat plane, thereby
making excellent serial tissue sections. It is comparatively heavier and more
stable than the ordinary sliding microtome. The angle of the knife is adjustable.
The knife used is long (24 cm), hence it requires less honing. The knife holding
clamps are adjustable and allow the tilt and the angle (slant) of the knife to be
easily set. Modern models of heavy duty base sledge microtomes are electrically
driven and are ideal for resin-embedded decalcified bone.
The second type, the 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.
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.

Fig. 12-3. Sliding Microtome

In both of these machines, the knife can be set obliquely for celloidin
sections or straight for large refractory paraffin blocks, cutting both large and
small tissues with ease; it is especially recommended for cutting extremely hard
and rough tissue blocks. It is the most dangerous type of microtome due to the
movable exposed knife. A slow but very steady motion is therefore required to
manipulate the instrument.

D. Freezing Microtome

Fig. 12-4. Freezing Microtome

This was invented by Queckett in 1848. 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. A simple lever operated valve
allows the release of rapid, intermittent bursts of carbon dioxide which will
freeze the block holder and the tissue evenly. A second cooling device for
lowering the temperature of the knife is also incorporated in most machines to
facilitate sectioning. The knife holder is attached to a lever which is in turn
connected to the pawl. When the operating handle is moved back, the knife is
moved back to its original position, away from the block. The lever, is in turn,
moved causing the pawl to get in contact with the ratchet feed wheel and thereby
turn the micrometer screw. The block holder is then raised towards the knife at a
predetermined thickness. By pulling the operating handle forward, a section is
cut as the knife edge slices thru the raised tissue block. The microtome is firmly
clamped on to the edge of the bench for use, or mounted on especially
constructed shelf, with C02 cylinder below.
It is 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. Although other microtomes can be modified for
cutting frozen section, this type will give the best results and is used almost
universally. The freezing microtome is 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.

E. The Cryostat or Cold Microtome

Fig. 12-5. Cryostat

The Cryostat is 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. All the controls in the
microtome are operated from outside the refrigerated cabinet.
The 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. Sections are usually transferred directly from the microtome
knife to a slide or cover glass, all of which are maintained at a low temperature.

E. Ultrathin microtome

Fig. 12-6. Ultrathin Microtome

An 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. These semi-thin sections are generally 0.5 to 1 µm thick and are
mounted on a glass slide and stained to locate areas of interest under a light
microscope prior to thin sectioning for the TEM. Thin sectioning for the TEM is
often done with a gem quality diamond knife.

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.
Even cellulose fibers can cause damage to the blade.
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
fragments 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
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.

Plane-wedge and plane-concave knives are usually provided with backs, to
maintain the correct bevel angle throughout honing. Detachable handles may be
attached to the knife during sharpening.
There is a cutting facet (bevel) found on the tapered edge of all knives, the
sides of which are more acutely inclined towards each other than the side proper,
forming the actual cutting edge of all knives. The angle formed between the
cutting edges is known as the "Bevel Angle", 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. Each knife should have its
own corresponding back which should not be interchanged with another, to keep
the bevel angle.
A good cutting edge should be made of good quality steel. Too soft cutting
edges are likely to become dull easily, while too hard edges are likely to produce
nicks or jagged edges and irregularities on the knife edge, thereby producing
tears or striation on the tissue sections during cutting. A good cutting edge must
be able to cut good sections from a paraffin wax block about 2-3 microns thick,
without any serration noted on examination.
Safety razor blades may be used for partially calcified materials, paraffin
and frozen sections. They are readily replaced when dull, and produce similarly
good tissue sections as those cut with microtome knives. They are, however,
unsatisfactory for sections less than 10 µ.
Theoretically, the perfect and optimum cutting angle is obtained when the sides
of the wedge knife are inclined at an angle of about 15°, causing maximum
penetration of the tissues and minimizing distortion. To prevent uneven sections,
or alternate thin and thick sections, the knife should be inclined with a 5-10°
clearance angle from the cutting plane so that the cutting facet will not compress
the block during the process of cutting. The cutting edge must be thinner than
the section being cut. A good cutting edge must be sharp enough to cut good
sections from a paraffin wax block at 4 µ thick without causing serrations.
The knife or blade should be removed from the microtome when the
instrument is left unattended or when cleaning the instrument. This is best done
by unclamping the blade, then using the blade ejector on the left side of the
guard to start moving the blade laterally out of the clamp. It can then be grasped
with forceps (not fingers) and safely removed. Used blades should be disposed
of appropriately in a “sharps” container or into the “used blades” slot in the base
of the blade dispenser. Never place a knife or blade on the bench or in a box with
the cutting edge facing up.

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)
Honing 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.

 .
Fig. 12-7. Honing technique

The knife complete with handle and backing sheath is laid on the Hone with
the cutting edge facing away from the operator and the heel roughly at the center
of the nearest end of hone. The knife is held with thumb on the back and
forefinger on the front surface. The knife is pushed forward diagonally from heel
to toe to the other end of the hone, turned over on its back and moved across the
hone until the heel is in the center with the cutting edge leading and then brought
back diagonally. It is then turned across the hone to its original position Such
sequence forms a double stroke, with a knife held obliquely, taking the same
precaution to hone the entire length of the knife. Honing is then continued until
all the teeth in the knife edge have been eradicated. 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.
A flat circular glass plate with finely powdered aluminum oxide made into paste
with water (used as an abrasive) may be used for grinding and removing nicks.
Diamantine may also be used for final polishing. The plate glass is usually 1/4 to
3/ 8 inch thick, about 14 inches long and 1-2 inches wider than the length of the
knife blade to be sharpened. Due to the plate's relatively greater width, the knife
blade does not have to be held obliquely, but is pushed and pulled forward and
backward at right angles to the transverse diameter of the plate.
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. It is then washed, preferably with water, to remove all metal
particles that may have been collected during the process. The washing
fluid used must flow rapidly enough so that the metal chips are removed
between strokes and a clean hone is presented every time.
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. Badly nicked
knives require greater and longer honing than less irregular knives.
The hone should be cleaned before, during, and after use. A black film
that develops in the hone usually is imparted by the knife that is being
sharpened, and should be brushed out with a good nailbrush in running
water, which may either be plain or soapy, until the hone is thoroughly
cleaned. After its use, the hone must be washed with warm soapy water,
dried, and kept in a box to protect it from dust while it is not in use.
After honing, wipe off the oil or soap from the knife with xylene. Then
strop it thoroughly.



TROPPING
Stropping is the process whereby the "burr" formed during honing is
removed and the cutting edge of the knife is polished. 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.

 Fig. 12-8. Stropping Technique

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 Observed in 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. Then, the knife is kept covered in a
suspension box to prevent the settling of dust and grit on its surface,
causing damage to the knife edge. The knife should not be allowed to
rest on its sides since this may also damage the cutting edge.
Pressure during the first stropping strokes should be quite light, since the
natural compressibility of the leather is what actually does the work.
Only a gentle pressure should be applied while the knife is held steady on
the strop, since a slip may cut the strop and damage the cutting edge.
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. Hence, strops are usually mounted on a wooden canvass
and covered with a flat pad to prevent them from sagging.
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. The knife is then dried off, by wiping the knife on a soft paper or
cloth (NEVER wipe the paper or cloth on the knife). The procedure is
again repeated with fresh xylene and a fresh sheet of paper or both, until
all the wax has been removed.

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
Glass knives are 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. Cleaned
strips are clamped into a knife maker, scored with a tungsten carbide wheel,
cracked to form 25 x 25 mm square pieces, and further broken into two
triangular​ shaped knives using even pressure. Glass knives should be prepared
and stored in dust-free boxes with lids, just before use, to avoid contamination.


Diamond Knives
Diamond knives are 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. Diamond knives 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.

OTHER EQUIPMENT
In addition to the microtome and the microtome knife, the following items
are also required during the process of 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. Alcohol or small quantities of
detergent may be added for reducing surface tension and allowing the
section to flatten out with greater ease.
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 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.
Equipment such as a slide rack is made on the assumption that regular
slides have been used. Larger size of slides are used for sections of eyes
or CNS tissues when these will not fit on the regular
The quality of sections cut on a microtome suffer badly from several
(avoidable) causes. 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.

REFERENCES
Bancroft JD. (1975) Histochemical Technique, 2nd ed. Butterworths, London.
Bancroft JD, Cook HC. (1994) Manual o Histological Techniques and their Diagnostic Application.
Churchill Livingstone, Edinburgh.
Bancroft JD, Hand NM. (1987) Enzyme Histochemistry. Royal Microscopical Society Handbook 14.
Oxford Science, Oxford.
Brown RW, ed. (2009) Histologic Preparations: Common Problems and Their Solutions. Northfield, IL:
CAP Press; 2009.
Carson FL. (1997) Histotechnology. A Self-Instructional Text, 2nd ed. ASCP Press, Chicago.
Carson FL, Hladik C. (2010) Histotechnology, A Self-Instructional Text. 3rd ed. Chicago, IL: ACSP
Press.
Culling CFA, Allison RT, Barr WT. (1985) Cellular Pathology Technique. 4th ed. London:
Butterworths.
Leica Microsystems. (2008) Instruction Manual Leica RM2235 V1.3 Nussloch: Leica Microsystems,
2008.
Leica Microsystems website. Available at: http://www.leica-microsystems.com.
Lubatschowski H. (2007) Laser Microtomy, Wiley-VCH Verlag, Biophotonics, S. 49-51.
Mailhiot MA. (2005) Microtomy. It’s all about Technique! National Society for Histotechnology.
Merryman HT. (1960) Principles of freeze drying. Annals of the New York Academy of Sciences. 85,
630.
Peachey Lee D. (1958) "Thin Sections: A study of section thickness and physical distortion produced
during microtomy" J. Biophysic. & Biochem. Cytol. 4(3): 233–242
Pearse AGE. (1980) Histochemistry, Theoretical and Applied, 4th ed. Vol 1. Churchill Livingstone,
Edinburgh.
Porter KR, Blum J. (2005) Article first published online: The Anatomical Record. Vol 117, Issue 4
Rolls G. (2008) 101 Steps to Better Histology. Melbourne: Leica Microsystems, 2008.
Rolls GO, Farmer NJ, Hall JB. (2008) Artifacts in Theory and Practice of Histotechnology. 2nd ed.
Columbus, OH: Battelle Press.
Santoianni RA, Hammami A. (1990) Nuclear Bubbling an Overlooked Artifact. The Journal of
Histotechnology, 13; 135-136.
Sheehan DC, Hrapchak B. (1980) Theory and Practice of Histotechnology, 2nd ed. C.V. Mosby Co., St.
Louis, pp 79-82.


CHAPTER 13
CUTTING SECTIONS

Sectioning is a process whereby tissues are cut into uniformly thin slices or
"sections" with the aid of a microtome, to facilitate the studies under the
microscope. Three general types of tissue sections may be made: 1. PARAFFIN
SECTIONS - for paraffin embedded tissue blocks which may be cut by rocking
and rotary microtome.
2. CELLOIDIN SECTIONS - for celloidin embedded tissues which
are usually cut by means of the sliding microtome.
3. FROZEN SECTIONS - which may be cut from tissues that have
been fixed and frozen with CO2 or for fresh or fixed tissues frozen
with the cryostat.

PARAFFIN SECTIONS
Once the tissues have been embedded and the wax has solidified, the
wax block is removed from the mold, the identification number is noted and
the excess wax is cut off from the block to expose the tissue surface in
preparation for actual cutting. This procedure is known as TRIMMING. Only
thin slices are taken out at a time to prevent the block from cracking.
The sides, top and bottom of the tissue block are trimmed until perfectly
level and all sides are parallel, almost to the edge of the tissue. An old knife or
blade may be used for this procedure, but it must still be relatively sharp to avoid
damage to the tissue. When using the coarse feed, avoid cutting unintentional
thick sections as this will damage the knife and possibly the block face.
Depending upon the size and orientation of the tissue sample, shave
conservatively into the block surface taking appropriate cuts that may measure
between 4-60 micrometers. Samples of small biopsy tissue may be trimmed only
to the depth of the first representation of several levels that will be collected.
Since tissue is completely surrounded by paraffin, it is useful to uncover the
surface of the block to reveal the tissue. Coarse facing is done on the microtome
at approximately 30 microns at a time until the entire tissue surface is exposed.
Care should be taken to avoid removing too much tissue in this step. Tissue that
was embedded improperly may not reveal the entire tissue surface and will have
to be re-embedded. Since tissue is completely surrounded by paraffin, it is useful
to uncover the surface of the block to reveal the tissue. Coarse facing is done on
the microtome at approximately 30 microns at a time until the entire tissue
surface is exposed. Care should be taken to avoid removing too much tissue in
this step. Tissue that was embedded improperly may not reveal the entire tissue
surface and will have to be re-embedded.
After coarse trimming, a heated spatula is held between the tissue block
and the block holder until the wax begins to melt. The spatula is then withdrawn
and the block is gently pressed into position. The block is allowed to harden for
cutting proper by facing them down in ice cold water or refrigerator for 5-10
minutes. Placing blocks in a freezer can cause surface cracking, where the friable
tissue separates from the surrounding wax cohesive sections become difficult to
obtain. Cooling both the tissue and the wax will give them a similar consistency,
and make sectioning easier. Re-chilling of the block may be required if the block
face becomes warm or if deeper levels are required. The block is then placed in
the microtome for fine trimming and cutting.
Fine trimming may be done by either setting the thickness adjuster at 15
mm or by advancing the block using the coarse feed mechanism. The knife is
usually tilted at 0-1 5° angulation on a microtome to allow a clearance angle
between the cutting facet and the tissue block. Biconcave knives require smaller
clearance angles than wedge-shaped knives.

Fig. 13-1. Knife Clearance and Bevel Angles

The block that is clamped on the chuck must be retracted enough to ensure
that the knife does not touch the chuck or block on initial down stroke. The
surface block is then trimmed away until the entire tissue surface has been
partly exposed. The block is advanced into the knife and cutting is continued
until complete sections come out of the block and a regular cutting rhythm is
maintained. The cutting rate depends upon the type of the tissue, the size of the
block, and the model or type of the microtome that is used. Sections usually
form ribbons due to slight heat generated between the block and the knife edge
during the process of cutting.
Sections are cut between 4-6µ in thickness for routine histologic procedures,
after the block has been fixed and secured to the block holder. The micrometer
gauge is set to the required thickness and the knife is positioned in such a way
that the center of the blade is in line with the block and the knife has been
securely clamped in place .
The actual thickness of the first couple of sections in a ribbon may be
thicker than indicated because of thermal expansion when cutting a cold paraffin
block. Using the microtome handle, try to cut in a slow and consistent manner -
don’t start and stop while the blade is cutting a block as this may produce
horizontal lines across the block and the sections (and very slight changes in
thickness). Sectioning is generally improved when the specimen and the wax are
well matched in hardness. It is for this reason that most paraffin blocks must be
cold when sections are cut. The actual method used to chill the block is
important.
Cold wax provides better support for the harder elements in a specimen
allowing thinner sections to be obtained. Place the blocks on a cold plate or a
cold wet surface for a few minutes (such as the surface of melting ice). Water
penetrates a small distance into the block face, swelling tissues and making them
more amenable to cutting. This is particularly important to over-dehydrated, dry
or crumbly tissues.
Incomplete sections are discarded. Complete ribbons are picked up at once
with a camel hair brush, or a pair of forceps. Tissues which tend to crumble (e.g.
blood clots, bone marrow) or do not form a smooth flat surface can be sectioned
with ease, by exhaling gently into the block surface while the section is being cut
slowly, to reduce the effects of static electricity. Successive sections will usually
stick edge-to-edge due to local pressure with each cutting stroke, thereby
forming a ribbon.
Generally a slow, uniform cutting stroke produces the best results and the
least compression. Do not stop and restart during a cutting stroke as this will
produce bands of different thickness across the section.

Fig. 13-2. Cutting Paraffin-Embedded Sections

The practice of gently breathing on the face of a chilled block immediately
before cutting each section, is common in some laboratories. The application of
warm, moist breath tends to make sections more cohesive, but it also causes
thermal expansion thus making the section thicker. Debris adhering to upper or
lower edges of the block, or the back of the blade, can make it difficult to obtain
cohesive ribbons and cause the ribbon to lift off the blade on the upstroke. If
debris is present clear it away, re-chill the block and start again.
Sections are removed in ribbons of ten to allow easy location of serial
sections. The sections are then floated out on a water bath set at 45-50°C,
approximately 6-10°C lower than the melting point of the wax used for
embedding the tissue. This is to flatten the sections and prepare them for
mounting onto the slides. Folds and creases may be removed by stretching the
sections gently with a pair of dissecting needles or forceps. Bubbles may be
teased out from beneath the sections by means of the same needle. Sections
should not be left on the water bath for a long time (30 seconds will be enough)
to avoid undue expansion and distortion of tissue.
A section is selected for staining and picked up onto a clean slide in a
vertical position. The slide is immersed in the water bath in a near vertical
position as close as possible to the section. When the slide touches the section, it
is lifted vertically out of water and drained. Sections may also be flattened out
by placing them on a slide which has been flooded with 20% alcohol, producing
convection currents which will serve to remove the creases in the tissue within a
few seconds. Sections are very easily damaged when dislodging wrinkles or
bubbles with brush or forceps. Examine each section as it floats on the water
surface as imperfections can be readily seen. Leave the section on the water
surface just long enough for it to flatten. Overexpansion can spoil the
morphology in susceptible sections.

Fig. 13-3. Floating Paraffin Embedded Sections

Flotation should expand the section to its original dimensions and ensure
that it is completely flat. The temperature will need to be 5 - 9 ˚C below the
melting point of the wax. Make sure the water is clean and free of bubbles. To
promote efficient drainage and to prevent the section from slipping down the
slide, remove slides vertically from the water. After floating and mounting the
paraffin sections from each block, use lint-free Kleenex or Kim-wipe to
thoroughly wipe clean the surface of the water and the edges of the flotation bath
to prevent floaters or cross-contamination.
The mounted section is then placed in a 70oC paraffin oven for 20 minutes
or until water droplets are no longer visible on the slides. Besides the paraffin
oven which is maintained at a temperature of 2-5°C above the melting point of
the paraffin used, small thermostatically controlled incubators may be used,
regulated at 37°C, and at 45-55°C, for enzyme digestion, chemical extraction,
metallic impregnation and enzyme localization techniques. Hot plates are not
recommended because they can cause overheating and there is a risk of dust
falling onto the section during the drying period. Excessive heat can cause
droplets of water underneath a section to boil and this will cause damage. Proper
drying ensures that sections are completely dehydrated, free of heat damage, flat
and unlikely to lift during staining. Drain excess water from beneath the section
before drying. Dry sections for between 5 and 30 minutes. Some delicate
specimens will produce best results when dried at 37oC for a longer time (several
hours to overnight).
Metal racks with 25-slide divisions are used to store the mounted sections
during the drying process which usually takes about 5 minutes in the heated
oven. Once dry, the whole rack of slides can be taken for manual batch staining
or placed on an automated staining machine. Staining of serial sections should
never be attempted unless they are completel y dried. Overheating should be
avoided because it will distort the tissue and melt some of the structures like
collagen.
Slides must always be grease- and dust-free and stored and handled
correctly. If staining is to include antigen retrieval (IHC), enzyme pretreatment
(ISH), or prolonged incubation steps, charged slides or an adhesive must be
used. Some special stains, particularly those that employ alkaline reagents, can
also cause sections to lift. Extended storage (usually more than 3 days) of
unstained formalin-fixed paraffin embedded slides should be avoided as this
may result in the loss of antigens. While not established, vacuum sealing and
refrigeration may help preserve some unstable antigens. For nucleic acid
extraction sections, allow the individual sections to roll up naturally and place
them directly into sterile microfuge tubes ready for nucleic acid extraction. The
extraction buffer can be added directly to the microfuge tube in order to
preserve the molecular integrity of the sample. When cutting sections for DNA
or RNA extraction, all instruments and equipment must be pre-cleaned and
wiped down with RNAse-away before and between each specimen. Gloves
must be worn. Molecular grade water must be used for floating sections for
RNA extraction.

Faults/Problems Observed during Section-Cutting
Difficulties encountered during cutting of sections are mostly due to faults
encountered during the processing of tissues or due to some faults in the
technique or cutting itself. Therefore, it must be evaluated and corrected on a
case-to​case basis, if good tissue sections are to be made. Below is a summary of
the most common problems that can be identified at the microtome. Many
microtomy problems have several causes so it is best to address them one at a
time until the problem is resolved.

Fig. 13-4. Section detached from the slide due to faulty processing
PROBLEM REASON REMEDY
Surfaces and edges
of the block are not Re-trim the block
parallel
Horizontal surface
Re-adjust and re-
of the block is not
orient the block
parallel to the knife
Coat horizontal
Sections fail to form Paraffin wax is too edges of the block
ribbons hard with wax of lower
melting point
Knife is tilted too
Reduce the tilt
much
Readjust the
Sections are too
thickness of the
thick
sections
Knife is dull Hone and strop
Sections roll up on Knife is blunt Sharpen the knife
cutting so that they Tilt of knife is too
Reduce the tilt
adhere and get broken great
against the knife edge Knife edge is dirty Clean the knife edge
Adjust the knife so
Blunt or dull spot
that knife edge will
on the knife,
present a uniformly
producing an
sharp edge to the
irregular knife edge
Ribbon is curved, block, or sharpen
crooked or uneven Edges of the block
instead of straight are not parallel but
Re-trim the block
round or wedge
shaped
Knife is not parallel Readjust the knife
to the block and block
Knife is blunt or
Re-sharpen the knife
dull
Paraffin block is Cool the block on
warm and soft ice water until firm
Knife edge is coated
Sections are Clean the knife edge
with paraffin
compressed, wrinkled
or jammed Readjust thickness
Sections are too thin
of the section
Microtome set
Tighten the screw
screw is loose
Tilt of knife is too
Reduce the tilt
vertical
Sections are squashed Re-sharpen, using a
Bevel of knife is
(width of each section knife back or
lost due to incorrect
is less than that of the automatic knife
sharpening
block) sharpener
Bubble or dirt Re-embed in freshly
formed in the filtered wax if
embedding medium necessary
Tissue is not
processed properly
and will not form a Re-process tissue
section (especially if
center is raw)
A hole is formed in Under-processed
the section portion of tissue
Re-process tissue
bursts on contact
with warm water
Once embedded in
paraffin wax,
decalcification is
Hard spot in tissue
impractical; use a
due to calcium
base-sledge
microtome with a
wedge knife
Tilt of knife is too
great or bevel is not
cleared, hence
Reduce the tilt
object is
compressed against
Sections of unequal the knife edge
thickness are Clamp set screw on
produced knife or block Tighten the screw
holder is loose
 Cut blocks into
Blocks are too large
smaller fragments
Soften the blocks in
Blocks are too hard
detergent or phenol
Breathe out or blow
gently on the bock
Static electricity due and knife to break up
to low atmospheric static electricity, or
Sections adhere to the humidity boil water in the
knife or other parts of room to increase
the microtome humidity
Knife edge is dirty Clean the knife edge
Knife edge is dull Sharpen the knife
Knife tilt is too
Reduce the tilt
great
Nicks or damage on
Sharpen the knife
the knife edge
Ribbon is split or Re-embed in freshly
Dirty embedding
lengthwise vertical filtered wax
scratches are seen on Clean knife edge
sections Knife edge is dirty
with xylene
Tilt of knife is too
Reduce the tilt
great
Knife tilt is too
Reduce the tilt
great
Sections are lifted
Knife is dull Sharpen the knife
from the knife on
upstrokes Paraffin is too soft
Cool paraffin wax in
or room temperature
ice water
is warm
Tilt of knife is too
small, paraffin
Resistance is felt on block is therefore
the lower part of the compressed against Increase the tilt
section during cutting the base of the knife
towards the end of
stroke
Knife edge vibrates Treat with phenol
Horizontal or parallel due to hardness of during processing or
lines or furrows across tissue collodionize
the section
("chatters") are seen Tilt of knife is too
Reduce the tilt
great
Knife is blunt Sharpen the knife
Adjust the knife so
that knife edge will
Knife is not
present a uniformly
clamped properly
sharp edge to the
Section cut is block, or sharpen
sometimes thin, Knife or block Tighten adjusting
sometimes thick holder is loose and locking screws
 Knife tilt is too
small that block is
compressed by Increase the tilt
bevel and section is
not cut
Tilt of knife is too
Readjust the tilt
Knife makes a hard slanted or too big
metallic scraping or Take fresh block
ringing sound on Tissue is too hard treated with phenol
backstroke, when during processing
section is cut Knife blade is too
Change the knife
thin
Frozen tissue
crumbles and comes Freezing is not Refreeze the tissue
off the block holder adequate block
when cut
Frozen tissue chips
Tissue is frozen too Warm the tissue with
into fragments when
much the fingers
cut
Top and bottom
edges of block are
Adjust the block
not parallel to edge
holder to make the
Ribbons are crooked of blade/sides of
block edges parallel
block are not
to the knife
perpendicular to the
blade
Wrong micrometer Microtome needs
Sections are too thick
setting recalibration
Block is trimmed
down nearest to the
tissue. Remaining
wax is melted on
Clearing agent not
On trimming, tissue embedding oven and
completely removed
smells of clearing paraffin
due to insufficient
agent impregnation is
impregnation
repeated, changing
the paraffin at least
once before
embedding
Repeat clearing; if
Tissue is opaque, object has already
section cutting is been embedded,
Insufficient clearing
difficult due to prolong clearing up
presence of alcohol to 12 hours, then re-
embed
Insufficient
Tissue shrinks away dehydration,
Repeat the whole
from wax when therefore
procedure
trimmed incomplete clearing
and impregnation
 On trimming, wax Contaminated wax Re-embed in freshly
appears crystalline filtered wax
Block not cooled Re-embed in freshly
rapidly enough filtered wax
Paraffin block, after Repeat paraffin
cooling, is moist and Insufficient paraffin impregnation, then
crumbles re-embed

CELLOIDIN EMBEDDING
Celloidin embedding is a slow process, usually taking weeks, and does not
produce sections as thin as those produced by paraffin embedding. The
advantage of celloidin embedding is that it completely avoids the use of heat at
any stage. As a consequence, heat produced artifacts are avoided. In particular,
shrinkage is absolutely minimal, if there is any, and structural relationships of
the various types of tissue components can be seen clearly.
The disadvantages are the longer time to cut, the thickness of the sections,
the necessity for staining to be done on free floating section, the inconvenience
of having to store the blocks in sealed jars with tight lids to prevent complete
evaporation of 70% ethanol, the resulting restrictions on the type of staining
methods that may be used.
Celloidin may be purchased either as a solution or as a solid, damped with a
liquid (usually ethanol) to reduce flammability. The stock purchased as a
solution may be in an undesirable solvent and it is often the practice to evaporate
the solvent to obtain dry celloidin, which is then weighed and re-dissolved in the
appropriate solvent. Celloidin is used in form of solution, usually in a 1:1
mixture of ethanol-ether at concentrations of 2%, 4% and 8%. The fastest way to
dissolve celloidin is to soak it first in half the final volume of anhydrous ethanol
to soften it (50 mL for each 8 grams celloidin) with intermittent mixing in a
tightly stoppered container. The next day, an equal volume of diethyl ether is
added and intermittently mixed until an evenly consistent solution is obtained.
The 2% and 4% solutions may then be made by simple dilution of the 8%
solution with an equal parts mixture of ethanol and diethyl ether.
The evaporation to dryness is done slowly at room temperature, without
additional heat and in an explosion safe environment as a fire safety precaution.
The solution should be transparent, without undissolved material, and should be
stored in a completely closed container which is ether resistant. For delicate
tissues, gradual dehydration with several changes of alcohol is strongly
recommended to avoid distortion from removing the water too fast. No clearing
agent is used with celloidin and following dehydration with absolute ethanol, the
tissue may be placed in ethanol-ether. Ether is a lipid solvent and will remove
much of the fat from the tissue. Denser tissues take a longer to infiltrate it must
be left to infiltrate.
When infiltration is complete, the block has to be cast and hardened. Paper
boats have the advantage in that they may be cut off if the paper does not peel
away easily. Some thick celloidin is poured into the bottom of a boat, then the
tissue is oriented, and more celloidin poured in to cover the tissue. After filling
the boat with thick celloidin, it is placed under a bell jar with a base that ensures
air is excluded. Each day the top of the jar is lifted a little for a few minutes so
that evaporated ethanol-ether can escape. More solvent will evaporate from the
block each day, evaporating the solvent off slowly and ensuring that the celloidin
thickens and hardens evenly throughout the tissue. Evaporating it too fast will
result in the outside of the block becoming hard while the inside is still soft.
The slow method of hardening the block allows the increasing concentration
of celloidin to get into the block and give additional support to the tissue. The
embedding process takes time and is complete when the block is sufficiently
hard, often judged by pressing it with a finger nail without leaving an
impression. Hardening of the celloidin block may be hastened by placing a small
open container of chloroform under the bell jar. The chloroform will saturate the
atmosphere and harden the celloidin without further evaporation.
As the block hardens the celloidin will shrink. If at any time the celloidin
shrinks enough to expose the tissue, more celloidin should be poured in to cover
it. At the end of the hardening process there should be sufficient celloidin to
allow for trimming the back of the block flat so that it may act as a base for
glueing the block to a wooden holder for sectioning. Once hardened, the block is
removed from the paper boat, preferably by peeling, but it can be cut away with
a sharp blade if necessary.
The block is then trimmed, leaving about 3 -5 mm of celloidin all around the
tissue, then a distinctive cut is made on one corner for orientation. The back is
trimmed flat and the block is placed into 70% ethanol until ready to be
sectioned. The blocks are trimmed in the same manner as in paraffin blocks, but
they do not require hardening by chilling before cutting.
Tissues embedded in celloidin are usually sectioned with a sliding
microtome, where the block is mounted to a holding platform facing upwards
and some thick celloidin is placed onto the holder, positioning the block so that it
will meet the knife as wanted, and the assembly is left until the attachment is
firm. The knife is held at a significant slant so that most of the blade edge is used
during the cutting stroke, and is quite long, often in excess of 25 cm. The face of
the block is lubricated with 70% ethanol and the knife drawn across the top of
the block at a strong slant, shaving off a section, which is immediately removed
and placed in 70% ethanol. The surface of the block is then re-lubricated for the
next cutting stroke.
To avoid dehydration and shrinkage, section cutting is usually done wet,
which means that the block is lubricated with a fluid, usually 60-70% ethanol,
and is not allowed to dry out. This makes section cutting somewhat messy and
quite a bit slower than the dry sectioning used with paraffin. Celloidin sections
do not come off in ribbons and tend to roll up during cutting, and moistening
the block and section with alcohol by means of a camel hair brush will serve to
flatten the sections on the knife.
After cutting the sections, they are immediately collected into 70% alcohol
instead of being mounted on to glass slides. They are then stored in the same
solution in jars with tightly fitting lids, and finally mounted on to slides after
they have been stained. They are usually stained free floating and put on slides
at the same time as the coverslip is applied. This makes it difficult to prepare
serial sections as each section must be stored in individual, appropriately
numbered containers. Small batches of 5 or more sections may be prepared,
stored in the same alcohol that was used for lubrication, and never be allowed
to become dry.

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