MLS 410 LAB–HISTOPATHOLOGIC AND CYTOLOGIC

TECHNIQUES
PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES
2ND SEMESTER | FINALS | PROF. JOHN MARKE BERNARDO

● Once sections are cut, they are floated on a warm

NOTE FROM TRANSERS water bath that helps remove wrinkles.
The transes for this lesson will be seperated into lab ● Then they are picked up on a glass microscopic slide.
manual and book. Book-based information will be coming WHAT IS THE CORRECT KNIFE ANGE?
from Chapter 12 and Chapter 13. Thank you and happy ● As a general principle, excessive bending of the
studying! section at the cut line is NEVER advantageous in
histology or material sectioning.
LAB MANUAL OUTLINE ○ Errors of too shallow and too steep an angle
I. Microtomy are damaging to the specimen.
II. What is the correct knife angle? ● This leads to inevitably to the conclusion that the
III. Errors
correct knife angle should always be set in the
IV. Procedure Preparation
following manner:
○ Position the lower bevel face parallel to the
MICROTOMY
specimen block and the plane of motion
● Also known as sectioning or cutting
○ Then raise the angle slightly above the bevel
● A process where tissues are cut into uniformly thin
angle to avoid having the lower bevel face
slices to facilitate microscopic studies.
slide over the specimen block and possible
○ Most paraffin embedded = 4-8 microns
produce friction damage.
● Basic instrument for this procedure is the
● The correct knife angle positioning should be
MICROTOME.
consistent regardless of the type of microtome, or
● The microtome mechanism slowly moves a block into
any specimen property.
the path of an extremely sharp steel knife.
● The clearance angle prevents contact between the
● Microtome knives may be generally grouped into four
knife facet and the face of the block.
types:
● The facet or bevel angle is the angle between the
○ Standard thick metal
two facets that form the cutting edge.
■ Allows sharpening to your liking
● For routine use knives and disposable blades are
■ Considered the most expensive
made with a facet angle of approximately 35°.
○ Thin disposable blades
○ But this angle can vary with the blade type
■ Does the same job as STMK and
and from manufacturer to manufacturer
probably better for a much lesser
● Therefore, for each blade type the clearance angle
cost.
must be optimally set.
○ Glass knives
● For rotary microtome knives and blade holders a
■ A glass knife can section down to
clearance angle between 0° and 15° is
about 1 micron.
recommended.
○ Diamond knives
■ Tissue sections that are embedded
in plastic materials such as
methacrylate, araldite, or epon
are sectioned with a glass or a
diamond knife.
■ For electron microscope, think
sections are preferred and they are
sectioned to about ¼ of a micron
utilizing a diamond knife.
● Histotechnologists are the artists of the laboratory.
● It is important to have a properly fixed and embedded Note: a) Correct angle knife, b) too shallow. c) too steep
block or much artifact can be introduced in the
sectioning. ● The consequences of incorrect knife angle will vary
○ Common artifacts included tearing, ripping, because of differences in specimen types, fixation and
holes, folding and etc. processing qualities and knife flexibility.

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TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES
● The correct knife angle is a property of the type of
knife and the final bevel, and may be different on
knives from different manufacturer’s and may change
after re-sharpening.
● To reduce the likelihood of changes in knife angle with
sharpening, the knife should always be
professionally sharpened.
ERRORS
If the ribbon is crooked rather than straight:
● The upper and lower edges of the block face are not
parallel to each other or to the knife edge.
● The knife edge may be uneven. Try another section
of the knife edge.
If a ribbon will not form and each section comes off
separately.
● The blade edge is dull. Sharpen it.
● The knife has the wrong tilt. Adjust it.
● The upper and lower edges of the paraffin block face
are crumbled or rounded. Retrim with a sharp razor
blade.
If the sections are compressed or folded:
● The knife is dull. Sharpen it.
● The knife angle is set too close to the vertical.
Increase the angle.
● The knife is clogged up with paraffin. Clean the edge
with chloroform.
● The sections are too thin. Increase the thickness
setting.
● The paraffin is too soft because the room is warm.
Pack the blade with ice cubes. Sometimes holding an
ice cube against the face of the block helps.
● The paraffin is still contaminated with xylene.
Re-embed.
If the specimen crumbles or falls out of the paraffin
section :
● The tissue is inadequately dehydrated or cleared.
Dissolve off the paraffin, rehydrate, and clear.
● The tissue is too hard and compact (e.g., liver). Soak
it in water or 70 % ethanol to soften it.
● The tissue was in the paraffin oven too long at too
high a heat. Throw it away.
● The tissue is too hard for a paraffin matrix (e.g., bone,
cuticle, heavy plant fibers, or xylem). Try celloidin
embedding.
If the ribbon continuously splits or scratches
vertically:
● Crystals or dirt particles are caught on the knife edge
or on the paraffin face. Wipe the knife edge
carefully and clean it with chloroform.
● The knife edge is nicked. Use another section of the
knife or sharpen it..
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If the ribbon wraps itself around your finger, the knife
blade or anything else:
● The problem is static electricity, which is often present
in very dry room.
● Let boiling water stand nearby, section in the morning
hours or make short ribbons.
PROCEDURES
Preparation of the Block of Tissue for Sectioning
1. Remove excess wax with a scalpel of sharp knife as
demonstrated so that the tissue, surrounded by wax
stands out from the block.
2. Trim the upper and lower edges of the block face so
that they are parallel.
3. Trim the lateral surfaces so that the resultant face
resembles a trapezoid. The tissue should be
surrounded by about a 2-3 mm frame of wax.
Preparation of the Microtome
1. The microtome should be clean when you start.
Brush away all discarded paraffin ribbons.
2. Test the action of the hand wheel. If it is stiff, oil it and
clean out the interior of the mechanism.
3. If the block holder is advanced toward its maximum,
retract it fully and reset the mechanism.
4. Lock the hand wheel or leave in the position at which
the block holder is in its highest position.
Mounting the Block
1. Clamp the specimen into the chuck of the block
holder.
2. Adjust the square face of the paraffin block vertically
and horizontally.
3. Make sure the block's upper and lower edges are
parallel with each other and parallel with the base of
the microtome.
4. The care with which the block is initially adjusted is
important to the success of sectioning.
Blade Preparation
1. Make sure the microtome is clean. Wipe the blade
carefully with chloroform or xylene to clean the edge,
if used.
2. Place the microtome blade in the blade holder (or
razor blade holder with its clamped razor blade).
3. Carefully check the angle of the blade, the firmness of
its seating in the holder, and its clearance of the
paraffin block and block holder. The angle of the
center line of the blade with the face of the block
should be about 20 degrees. DO NOT CHANGE THE
ANGLE
4. If necessary, readjust the paraffin block.
5. Some very careful trimming can be done with a razor
blade.
6. Watch your fingers- the blade is sharp!
7. The upper and lower edges of the block must be
parallel to each other and to the edge of the knife.
TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES

Sectioning blade and put it its box. Many laboratory accidents

1. Unlock the hand wheel and lower the paraffin block result from carelessness with the microtome blade.
until its face is level with the edge of the microtome ● Clean up the wax around the microtome.
blade. ● Put away all instruments.
2. Unlock the blade and bring it up the but not in contact
with the face of the block.
3. Relock the microtome blade firmly in position.
4. Set the thickness scale as desired. Students should
start at 10 microns.
5. Relax and turn the hand wheel with a steady rhythm.
Allow the first inch or two of paraffin ribbon to move
down the blade until the ribbon begins to bow up near
the edge of the blade.
6. Slip your brush or a dissection needle under the
ribbon at this point and lift the ribbon, still attached to
the edge, clear of the knife surface. Hold the ribbon
up but do not put any pull on its attachment with the
blade, thereby separating the ribbon from the cutting
edge or making a weak joint with the next section to
be cut. This will take a little practice.
7. When the ribbon is about 8 inches long, detach it from
the edge of the blade with an upward stroke of your
paint brush. Even the bristles of the paint brush will
nick the edge of the blade, so always brush away
from the cutting of the blade.
8. Lay the ribbon, dull surface up, in your ribbon box and
cut another strip.

Mounting Sections on Slides

1. Use clean glass slides. Slides should be rinsed in
95% alcohol. Wipe them dry with a clean soft lintless
cloth.
2. Use a diamond-tipped pencil or lead pencil to write
the identity of the tissue and your initials on one end
of the slide.
3. Spread a thin film of adhesive (egg albumin) on the
slide surface.
4. Float some tissue ribbons. in a waterbath. DO NOT
float out ribbons from different blocks at the same
time.
5. Select a tissue section by fishing it out of the
waterbath using the adhesive-coated slide. Paraffin
expands about 20% so do not use too many sections.
Remember that they must be covered by a 22 by 22
mm coverslip later after staining (although large sizes
are available).
6. Heat the slide gently on the slide warmer (40 to 45ºC)
until the sections spread and flatten out. Do not melt
the wax.
7. Drain off the excess water with a paper towel or
tissue; do not drain off any sections with it.

When Finished with the Microtome:

● Place your ribbon box in a cool place.
● When you are through sectioning, or when you leave
the microtome for any reason. Remove the microtome

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TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES

BOOK OUTLINE CHAPTER 12 - MICROTOMY 4. Freezing Microtome - Cutting unembedded frozen

I. Microtomy sections
II. Kinds of Microtomes 5. Cryostat or Cold Microtome - cutting frozen
A. Rocking Microtome sections
B. Rotary Microtome 6. Ultrathin Microtome - Cutting sections for Electron
C. Sliding Microtome Microscopy
D. Freezing Microtome ROCKING (CAMBRIDGE) MICROTOME
E. Cryostat or Cold Microtome
F. Ultrathin Microtome
III. Care of the Microtome and Safety Measures
IV. Microtome Knives
A. Plane-Concave Knife
B. Biconcave Knife
C. Plane-Wedge Knife
D. Other details about Microtome
Knives
V. Honing and Stropping ● Invented by Paldwel Trefall in 1881
A. Honing ● Simplest among all the types of microtomes
B. Stropping
● Consists of a heavy base and two arms
C. Knives
○ Lower Arm - Resting on pivots and a
VI. Other Equipment
supporting column, and attached to the
micrometer screw, at the base of which is
MICROTOMY
found the rached wheel with feed
mechanism
● A process which processed tissue (paraffin embedded
○ Upper Arm - Carries the block holder on one
tissue) is trimmed and cut into uniformly thin slices or
end by means of a screw, and is connected
“sections”
to a lever by a piece of nylon thread
● The basic instrument is called a MICROTOME and is
● Mechanism
capable of cutting predetermined thickness of tissue
1. When the lever is pulled forward, the pawl is
sections.
brought in contact with the ratchet wheel to
● It has THREE (3) essential parts
which the millhead micrometer screw is
○ Block Holder - Tissue is held in position
attached.
○ Knife Carrier and Knife - Actual cutting of
2. The ratchet wheel is turned, rotating the
tissue sections
micrometer screw. The lower arm is
○ Pawl, Rachet Feed Wheel and Adjustment
elevated, which in turn raises the upper arm
Screws - To line up the tissues block in
at its fulcrum, thereby carrying the chuck or
proper position with the knife, adjusting the
block holder forward, towards the knife.
proper thickness of the tissue for successive
3. As the pressure on the operating handle or
sections.
lever is released, the tension on the spring
● The principle of microtomy will be the same across all
causes the upper arm to return to its normal
the different types of microtome.
position; in an arc of a circle.
○ A spring-balanced teeth or pawl is brought
4. A section is thereby cut as the tissue passes
into contact with, and turns a rachet feed
to the knife edge in a slightly curved plane, in
wheel connected to a micrometer screw,
10-12 µm thickness.
which is in turn rotated, moving the tissue
● The Cambridge rocking microtome, available in two
block at a predetermined distance towards
sizes, has been used to cut small and large blocks
the knife for cutting sections at a uniform
of paraffin tissues.
thickness.
● It is theoretically not recommended for serial
sections since tissues are cut in slightly curved
KINDS OF MICROTOMES
planes.
1. Rocking Microtome - Cutting serial sections of large
● It is not currently favored by most laboratories due to
blocks of paraffin embedded tissues
reasons such as:
2. Rotary Microtome - Cutting paraffin embedded
○ Restrictions in size of tissue block that can
sections
be cut
3. Sliding Microtome - Cutting celloidin embedded
○ Difficulty of reorienting the block.
sections
ROTARY (MINOT) MICROTOME

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TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES
● Invented by Minot in 1885-1886
● Currently the most common type of microtome 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.
● 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
● Generally, rotary microtomes are either automated or
semi-automated, although the flywheel in many
microtomes can be operated manually,
● Typically, sections are cut between 3 and 5 μm using
paraffin wax for diagnostic histology
○ Thinner sections can be attained if samples
are embedded in synthetic resin.
● Mechanism
○ 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.
● Characteristics of Minot Microtome
○ Heavier and more stable than the rocking
microtome
○ More complex in design and construction
○ More expensive
○ May be used to cut large blocks of tissues
■ Better results are seen if sliding
microtome is used instead
○ 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.
○ Heavier knife is used = less vibrations
○ The cutting angle (tilt) is adjustable to cut
hardere tissues
○ It can cut celloidin-embedded sections by
using a special holder to set the knife
obliquely.
SLIDING MICROTOME
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● Developed by Adams in 1789
● Two models - Base-Sledge Microtome and
Standard Sliding Microtome
A. 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.
○ 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, and 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.
B. Standard 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.
C. Both Base-Sledge and Standard
○ The knife can be set obliquely for celloidin
sections or straight for large refractory
TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES
paraffin blocks, cutting both large and small
tissues with ease
○ Recommended for cutting extremely hard
and rough tissue blocks.
○ It is the most dangerous type of microtome
due to the movable exposed knife.
i. A slow but very steady motion is
therefore required to manipulate the
instrument.
FREEZING MICROTOME
● Invented by Queckett in 1848.
● Features
○ 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.
● Mechanism
○ 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, espeically in
instances such as:
○ Rapid diagnosis is required
○ Histological demonstration of fat is needed,
○ Certain neurological structures are to be
studied
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○ 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 STANDARD sliding microtome.
CRYOSTAT OR COLD MICROTOME
● 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 4μm 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.
ULTRATHIN MICROTOME
● An ultrathin microtome equipped with a glass or gem
grade diamond knife is used to cut very thin sections
TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES

(typically 60 to 100 nanometers) of tissue when the instrument is left unattended or when
embedded in epoxy resin. cleaning the instrument.
● Sections are stained with an aqueous solution of an MICROTOME KNIVES
appropriate heavy metal salt and examined with a ● Trimming and section-cutting are done with a
transmission electron microscope (TEM). microtome knife, which is available in three basic
● Also used with its glass knife or an industrial grade types or shapes:
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 AND SAFETY
MEASURES
● After sectioning, all the accumulated paraffin and MICROTOME KNIVES (ACCORDING TO SHAPES)
small pieces of tissues must be brushed away with a
soft brush and not allowed to stay in the microtome, PLANE-CONCAVE ● 25 mm in length
since this may later interfere with the cutting of tissue KNIFE ● One side of the knife is flat
blocks. while the other is concave.
● After carefully drying the machine and knife holder, ○ Less Concave (flat) -
the parts should be wiped with xylol. Prolonged and Celloidin-Embedded
tissues on a Sliding
continuous application of the painted parts with xylene Microtome
should, however, be avoided since this reagent is ○ More concave -
capable of removing the paint. Paraffin sections on
● Movable portions should be oiled thoroughly to base-sledge, rotary or
prevent rusting rocking microtome.
● The microtome must always be covered when not in
use, to prevent accumulation of dust and other dirt BICONCAVE KNIFE ● 120 mm in length
which may later on interfere with the normal ● Both sides concave
sectioning of tissues. ● For cutting paraffin -
● The microtome should be placed on a stable bench, embedded sections on a
away from air drafts, doorways and passing staff. Any rotary microtome.
air movement from air conditioners or other causes
PLANE-WEDGE KNIFE ● 100 mm in length
can make section handling very difficult.
● Both sides straight
● Always remove the knife or blade before cleaning.
● For frozen sections or for
The knife holder can easily be removed to facilitate
cutting extremely hard
access for cleaning. No fluid must enter the inside of
and tough specimens
the instrument during cleaning.
embedded in paraffin
● When cleaning the blade avoid dragging anything
blocks, using a base
along the cutting edge. Even cellulose fibers can
sledge type or sliding
cause damage to the blade.
microtome.
● Have the instrument inspected at least once a year by
a qualified service technician. ● Plane-wedge and plane-concave knives are usually
Safety Measures provided with backs, to maintain the correct bevel
● It is very important that staff are not distracted when angle throughout honing. Detachable handles may be
using the microtome because of the risks of injury attached to the knife during sharpening.
from extremely sharp blades. It is preferable to have OTHER DETAILS ABOUT MICROTOME KNIVES
non-slip flooring in the vicinity of microtomes because, ● BEVEL ANGLE - angle formed between cutting
inevitably, wax fragments will find their way onto the edges
floor where they can produce a slippery surface. ○ About 27° to 32°
● Use forceps or brush instead of fingers to pick up ○ Cutting facet (bevel) found on the tapered
sections or wax fragments from blade or block face. edge of all knives, the sides of which are
● Use hand wheel lock when changing blocks. The more acutely inclined towards each other
knife or blade should be removed from the microtome

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TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES
than the side proper, forming the actual
cutting edge of all knives.
○ 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 - dull easily
○ Too hard cutting edges - likely to produce
nicks or jagged edges and irregularities on
the knife edge, producing tears/striations on
the tissue sections
○ 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.
○ 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 μm.
● CUTTING ANGLE
○ 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.
● CLEARANCE ANGLE
○ 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 μm 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
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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 (e.g.
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 -
a. Much coarser than the first two
types
b. 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.
● Honing Technique
○ 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.
TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES
○ 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.
● Step-by-Step Process in Honing
1. 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.
2. 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.
3. 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.
4. Honing is then continued until all the teeth in the knife
edge have been eradicated.
a. In the case of the Minot or plane-wedge
knife, the knife is turned over so as to
sharpen the other surface every 10-20
strokes.
b. For plane-concave knives, only the
concave surface should be rubbed on the
Hone.
5. 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.
a. Diamantine may also be used for final
polishing.
6. 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.
7. 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
Transes Warriors: Lim, A., Nadong │ 2K │Page 9 of 18
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
STROPPING
● 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.
● PADDLE STROP
○ Preferred option: Made from the best quality
horse leather firmly attached to a solid back,
in order to prevent sagging
● 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.
TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES
○ 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 planeconcave knives, only the concave
surface should be stropped.
● PRECAUTIONS DURING HONING
○ 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.
Transes Warriors: Lim, A., Nadong │ 2K │Page 10 of 18
○ 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.
BLADES/KNIVES
● 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 μm 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
● Waterbath - The thermostatically controlled type is
preferable, but if this is unavailable, water from a hot
TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES

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/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, 37 degrees Celcius
for 24 hours or longer is recommended.
● Forceps & Squirrel Head 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.
● Slide Rack - 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.

Transes Warriors: Lim, A., Nadong │ 2K │Page 11 of 18

TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES
OUTLINE CHAPTER 13 - CUTTING SECTIONS
I. Sectioning AND TYPES OF TISSUE
SECTIONS
A. Paraffin Sections
1. Faults/Problems during
Sectioning
B. Celloidin Sections
SECTIONING
● 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:
○ PARAFFIN SECTIONS - For paraffin
embedded tissue blocks which may be cut
by rocking and rotary microtome.
○ CELLOIDIN SECTIONS - for celloidin
embedded tissues whichare usually cut by
means of the sliding microtome.
○ FROZEN SECTIONS - which may be cut
from tissues that have been fixed and frozen
with CO2 or for fresh or fixed tissues
frozenwith the cryostat.
PARAFFIN SECTION
● 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.
● Process of Sectioning under paraffin sections
○ 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 μm
■ Samples of small biopsy tissue may
be trimmed only to the depth of the
first representation of several levels
that will be collected.
● COARSE FACING/TRIMMING
○ Since tissue is completely surrounded by
paraffin, it is useful to uncover the surface of
the block to reveal the tissue.
Transes Warriors: Lim, A., Nadong │ 2K │Page 12 of 18
○ 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.
● USE OF THE HEATED SPATULA
○ 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
○ 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.
○ 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.
TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES
● 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.
● 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.
Transes Warriors: Lim, A., Nadong │ 2K │Page 13 of 18
● 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.
● FLOATATION
○ 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
TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES
flotation bath to prevent floaters or
cross-contamination.
○ The mounted section is then placed in a 70
degrees Celsius 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 37 degrees C for a
longer time (severalhours 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 completely 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
Transes Warriors: Lim, A., Nadong │ 2K │Page 14 of 18
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 OBSERVED DURING SECTIONING
These are the 23 common faults seen during sectioning:
1. Section failed to form ribbons
2. Sections roll up on cutting so that they adhere and get
broken against the knife edge
3. Ribbon is curved, crooked, or uneven instead of
straight
4. Sections are compressed, wrinkled or jammed
5. Sections are squashed (width of each section is less
than that of the block)
6. A hole is formed in the section
7. Sections of unequal thickness are produced
8. Sections adhere to the knife or other parts of the
microtome
9. Ribbon is split or lengthwise vertical scratches are
seen on sections
10. Sections are lifted from the knife on upstrokes
11. Resistance is felt on the lower part of the section
during cutting
12. Horizontal or parallel lines or furrows across the
section ("chatters") are seen
13. Section cut is sometimes thin, sometimes thick
14. Knife makes a hard metallic scraping or ringing sound
on backstroke, when section is cut
15. Frozen tissue crumbles and comes off the block
holder when cut
16. Frozen tissue chips into fragments when cut
17. Ribbons are crooked
18. Sections are too thick
19. On trimming, tissue smells of clearing agent
20. Tissue is opaque, section cutting is difficult due to
presence of alcohol
21. Tissue shrinks away from wax when trimmed
22. On trimming, wax appears crystalline
23. Paraffin block, after cooling, is moist and crumbles
Presented below are their reasons as to why it happens and
their corresponding remedies.
PROBLEM REASONS REMEDY
Surface and
Edges are not Re-trim the block
parallel
1.
SECTION Horizontal
FAILED TO surface Re-adjust and
FORM of the block is not re-
RIBBONS parallel to the orient the block
knife
TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES

Coat horizontal Microtome set Tighten the

edges of the screw is loose screw
Paraffin wax is too
block
hard
with wax of lower Tilt of knife is too
melting point Reduce the tilt
vertical

Knife is tilted too 5.

Reduce the tilt
much SECTIONS
ARE
Re-sharpen,
Readjust the SQUASHED Bevel of knife is
Sections are too using a knife
thickness of the (WIDTH OF lost due to
thick back or
sections EACH incorrect
automatic knife
SECTION IS sharpening
sharpener
Knife is dull Hone and strop LESS THAN
THAT OF THE
2. Sharpen the BLOCK)
Knife is blunt
SECTIONS knife
ROLL UP ON Bubble or dirt Re-embed in
CUTTING SO Tilt of knife is too formed in the freshly filtered
THAT THEY great Reduce the tilt embedding wax if
ADHERE AND medium necessary
GET BROKEN
AGAINST THE Clean the knife Tissue is not
KNIFE EDGE Knife edge is dirty processed
edge
properly and will Re-process
Adjust the knife not form a section tissue
so (especially if
that knife edge 6. center is raw)
Blunt or dull spot A HOLE IS
will
on the knife, FORMED IN Under-processed
present a
producing an THE SECTION portion of tissue Re-process
uniformly
irregular knife bursts on contact tissue
sharp edge to
3. edge with warm water
the
RIBBON IS block, or
CURVED, sharpen Once embedded
CROOKED, OR in paraffin wax,
UNEVEN decalcification is
Edges of the block Hard spot in tissue
INSTEAD OF impractical; use
are not parallel due to calcium
STRAIGHT a base-sledge
but Re-trim the block
round or wedge microtome with
shaped a wedge knife

Knife is not Readjust the Tilt of knife is too

parallel knife great or bevel is
to the block and block not cleared,
hence object is Reduce the tilt
Knife is blunt or Re-sharpen the compressed
dull knife against the knife
edge
Cool the block 7.
Paraffin block is on ice water until SECTIONS OF Clamp set screw
Tighten the
warm and soft firm UNEQUAL on knife or block
4. screw
THICKNESS holder is loose
SECTIONS ARE
ARE PRODUCED Cut blocks into
Knife edge is Blocks are too
COMPRESSED, Clean the knife smaller
coated with large
WRINKLED edge fragments
paraffin
OR JAMMED
Readjust Soften the
Sections are too Blocks are too blocks in
thickness
thin hard detergent or
of the section
phenol

Transes Warriors: Lim, A., Nadong │ 2K │Page 15 of 18

TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES

Breathe out or ARE SEEN

blow
gently on the Sharpen the
Static electricity bock and knife Knife is blunt
knife
due to low to break up
atmospheric static electricity, Adjust the knife
8. humidity or boil water in so that knife
SECTIONS the room to edge will present
increase Knife is not
ADHERE TO a uniformly
humidity 13. clamped properly
THE KNIFE OR sharp edge to
OTHER PARTS SECTION CUT
the block, or
OF THE Clean the knife IS
Knife edge is dirty sharpen
MICROTOME edge SOMETIMES
THIN,
Tighten
SOMETIMES Knife or block
Sharpen the adjusting and
Knife edge is dull THICK holder is loose
knife locking screws

Knife tilt is too Knife tilt is too

Reduce the tilt
great small that block is
compressed by Increase the tilt
Nicks or damage Sharpen the bevel and section
on the knife edge knife is not cut
9.
Re-embed in 14. Tilt of knife is too
RIBBON IS Readjust the tilt
freshly KNIFE MAKES slanted or too big
SPLIT OR Dirty embedding
filtered wax A HARD
LENGTHWISE
METALLIC Take fresh block
VERTICAL
SCRAPING OR treated with
SCRATCHES Tissue is too hard
Clean knife edge RINGING phenol during
ARE SEEN ON Knife edge is dirty
with xylene SOUND ON processing
SECTIONS
BACKSTROKE,
Tilt of knife is too WHEN ● Change
Reduce the tilt SECTION IS Knife blade is too
great the
CUT thin
knife
Knife tilt is too
Reduce the tilt
great 15.
10. FROZEN
SECTIONS Sharpen the TISSUE
Knife is dull CRUMBLES
ARE LIFTED knife Freezing is not Refreeze the
FROM THE AND COMES
adequate tissue block
KNIFE ON Paraffin is too soft OFF THE
UPSTROKES Cool paraffin BLOCK
or room
wax in HOLDER
temperature
ice water WHEN CUT
is warm

11. Tilt of knife is too 16.

RESISTANCE small, paraffin FROZEN
Tissue is frozen
IS FELT ON block is therefore TISSUE CHIPS Warm the tissue
too much
THE LOWER compressed INTO with the fingers
Increase the tilt FRAGMENTS
PART OF THE against the base
SECTION of the knife WHEN CUT
DURING towards the end of
CUTTING stroke Top and bottom
edges of block are
Adjust the
12. Treat with not parallel to
Knife edge 17. block holder to
HORIZONTAL phenol during edge of blade OR
vibrates due to RIBBONS ARE make the block
OR PARALLEL processing or sides of block are
hardness of tissue CROOKED edges parallel to
LINES OR collodionize not
the knife
FURROWS perpendicular to
ACROSS the blade
Tilt of knife is too
THE SECTION Reduce the tilt
great 18. Wrong micrometer Microtome
("CHATTERS")

Transes Warriors: Lim, A., Nadong │ 2K │Page 16 of 18

TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES

○ Structural relationships of the various types

SECTIONS setting needs
ARE TOO recalibration of tissue components can be seen clearly.
THICK ● Disadvantages of Celloidin embedding over paraffin
embedding
Block is ○ Longer time to cut
trimmed down ○ Thickness of the sections
nearest to the ○ Necessity for staining to be done on free
tissue. floating section
Remaining wax
19. ○ Inconvenience of having to store the blocks
Clearing agent is melted on
ON TRIMMING, in sealed jars with tight lids
not completely embedding
TISSUE ■ Prevent complete evaporation of
removed due to oven and
SMELLS OF
insufficient paraffin 70% ethanol
CLEARING
impregnation impregnation is ○ Restrictions on the type of staining methods
AGENT
repeated, that may be used.
changing the ● Celloidin may be purchased either as a solution or as
paraffin at least
once before a solid, damped with a liquid (usually ethanol) to
embedding reduce flammability.
○ The stock purchased as a solution may be
20. Repeat in an undesirable solvent and it is often the
TISSUE IS clearing; if practice to evaporate the solvent to
OPAQUE, object has obtain dry celloidin, which is then weighed
SECTION already been and re-dissolved in the appropriate
Insufficient
CUTTING IS embedded,
clearing solvent.
DIFFICULT prolong
DUE TO clearing up to ○ Celloidin is used in form of solution, usually
PRESENCE OF 12 hours, then in a 1:1 mixture of ethanol-ether at
ALCOHOL re-embed concentrations of 2%, 4% and 8%.
● Process of making a celloidin solution
21. Insufficient ○ The fastest way to dissolve celloidin is to
TISSUE dehydration, soak it first in half the final volume of
Repeat the
SHRINKS therefore
whole procedure anhydrous ethanol to soften it (50 mL for
AWAY FROM incomplete
WAX WHEN clearing and each 8 grams celloidin) with intermittent
TRIMMED impregnation mixing in a tightly stoppered container.
○ The next day, an equal volume of diethyl
22. Contaminated wax ether is added and intermittently mixed until
Re-embed in
ON TRIMMING, an evenly consistent solution is obtained.
freshly filtered
WAX APPEARS Block not cooled ○ The 2% and 4% solutions may then be made
wax
CRYSTALLINE rapidly enough by simple dilution of the 8% solution with an
equal parts mixture of ethanol and diethyl
23.
PARAFFIN ether.
Repeat paraffin ● The evaporation to dryness is done slowly at room
BLOCK,
impregnation, temperature, without additional heat and in an
AFTER Insufficient paraffin
then
COOLING, IS explosion safe environment as a fire safety
re-embed
MOIST AND precaution.
CRUMBLES ● The solution should be transparent, without
undissolved material, and should be stored in a
CELLOIDIN EMBEDDING completely closed container which is ether resistant.
● A slow process, usually taking weeks, and does not ● For delicate tissues, gradual dehydration with several
produce sections as thin as those produced by changes of alcohol is strongly recommended to avoid
paraffin embedding. distortion from removing the water too fast.
● Advantages of Celloidin embedding over paraffin ● No clearing agent is used with celloidin and
embedding: following dehydration with absolute ethanol, the tissue
○ Completely avoids the use of heat at any may be placed in ethanol-ether.
stage. ○ Ether is a lipid solvent and will remove much
■ As a consequence, heat of the fat from the tissue.
produced-artifacts are avoided. ○ Denser tissues take a longer to infiltrate it
○ Shrinkage is absolutely minimal must be left to infiltrate.

Transes Warriors: Lim, A., Nadong │ 2K │Page 17 of 18

TOPIC: PROPER TECHNIQUE OF SECTIONING TISSUE SAMPLES
● Casting and Hardening of Celloidin
○ 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.
● Celloidin Hardening & Shrinkage
○ 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.
● Celloidin Block Trimming
○ Block is then trimmed, leaving about 3-5 mm
of celloidin all around the tissue, then a
Transes Warriors: Lim, A., Nadong │ 2K │Page 18 of 18
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.
● Celloidin Sectioning
○ Tissues embedded in celloidin are usually
sectioned with a sliding microtome
■ 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.