1.

MICROTOMY, TRIMMING AND SECTIONING

TRIMMING MANUAL AUTOMATED
• Optional step (same with decalcification)
• Water/Floatation bath – to flatten the sections and prepare them for

mounting onto the slides

• Cutting off the excess wax from the tissue block to expose the

tissue surface in preparation for actual cutting

SECTIONING
• Process whereby tissues are cut into uniformly thin slices or
“sections” with the aid of a microtome to facilitate studies under the
microscope
Pawl and Rachet Feed Wheel
MICROTOMY jk • Found inside the microtome
• 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

3 ESSENTIAL PARTS OF A MICROTOME

Block Holder where the tissue is held in position
Knife Carrier and Knife actual cutting of tissue sections
to line up the tissue block in proper position
Pawl, Ratchet Feed
with the knife, adjusting the proper
Wheel and Adjustment
thickness of the tissue for successive
Screws • Lock/open
sections • Used to cover the knife
the blade

• Micron adjustment
• Reflects the size of the tissue
• Set at 10 um in thickness

KINDS OF MICROTOME
Rotary paraffin-embedded sections
Rocking cutting serial sections of large blocks of
paraffin embedded tissues
Sliding cutting celloidin embedded sections
Freezing cutting unembedded frozen sections
Cryostat or Cold cutting frozen sections Tissue block holder Coarse Hand Wheel Advancement Hand Wheel
Ultrathin cutting sections for Electron Microscopy • With screws • Moves the tissue block • Turning the wheel vertical
• To forward or away from or upward motion will give
1. ROCKING (CAMBRIDGE) MICROTOME adjust/tighten/loosen the knife sections
• used for cutting serial sections of small and the holder • Minor/small movement Safety Lock
large blocks of paraffin embedded tissues • Lock the tissue block
• invented by Paldwell Trefall in 1881

• simplest among the different types of 3. SLIDING MICROTOME

microtome • for cutting celloidin-embedded sections
• 10-20 u thickness • Invented by Adams in 1789

• not recommended for serial sections since tissues are cut in • especially recommended for cutting
slightly curved planes. extremely hard and rough tissue blocks.
• restrictions in size of tissue block that can be cut • (!) MOST DANGEROUS type of microtome

• difficulty of reorienting the block due to the movable exposed knife

• This consists of a heavy base and two arms the lower arm resting on

pivots and a supporting column, and attached to the micrometer a. BASE-SLEDGE MICROTOME
screw, at the base of which is found the ratchet wheel with feed • Very hard tissue or large blocks

mechanism. The upper arm, carrying the block holder on one end by • suited for sectioning specimens embedded in all forms of media,

means of a screw, is connected to a lever by a piece of nylon thread. especially for cutting sections from tough tissue blocks which may
offer great resistance to the knife.
2. ROTARY MICROTOME • originally designed for cutting sections of very large blocks (whole

• invented by Minot in 1885-1886 brain sections)

• for cutting paraffin embedded tissue sections

• most common type used for both routine and research laboratories,

especially for sectioning paraffin-embedded tissues. b. STANDARD SLIDING MICROTOME

• sections are cut between 3 and 5 μm using paraffin wax for • different from the base sledge microtome

diagnostic histology • the block remains stationary while the knife is moved backward

• 2 types: semi-automated and automated and forward during the process of sectioning
• different from the rocking microtome in that the knife and the block • developed mainly for cutting celloidin embedded tissue blocks

holder are brought together by upward and vertical motions, cutting • more dangerous because of the movable knife

sections in a perfectly flat plane

• heavier and more stable, more complex design, more expensive
 4. FREEZING MICROTOME 3. PLANE-WEDGE KNIFE
• Used for cutting unembedded tissues • usually 100 mm
• Invented by Queckett in 1848 • both sides are straight

• used to cut undehydrated thin to semi-thin • recommended for frozen sections or for cutting extremely hard and

sections of fresh, frozen tissues especially in tough specimens embedded in paraffin blocks, using a base sledge
instances when: type or sliding microtome.
o rapid diagnosis is required
o histological demonstration of fat is needed
PARTS
o certain neurological structures are to be
Frozen with CO2 a. Cutting facet (Bevel)
studied • cutting edge found on the tapered edge of all knives
o sensitive tissue constituents to be studied are damaged or
• the sides of which are more acutely inclined towards each other than
destroyed by heat the side proper
• A good cutting edge
5. COLD MICROTOME (CRYOSTAT) o should be made of good quality steel
• a refrigerated apparatus used for freezing the o must be able to cut good sections from a paraffin wax block about
tissue into the block holder 2-3 microns thick, without any serration noted on examination.
• consists of a microtome (rotary microtome)

• kept in a cold chamber maintained at a Too soft cutting edges Dull easily
temperature between -5 to -30 degrees Celsius Too hard Produce nicks or jagged edges
(average of: -20 degrees Celsius
• Cutting sections of 4 u
a.1. Bevel Angle
• angle formed between the cutting edges

• 27° to 32°
6. ULTRATHIN MICROTOME
• maintained for each knife by means of a slide-on back, a spring-
• Used for cutting sections for electron microscopy
loaded semi-circular metal sheet
• also used with its glass knife or an industrial grade diamond knife to
slipped on to the knife with one or
cut semi-thin sections prior to thin sectioning
more plane surfaces (plane-wedge or
• 0.5 to 1 um sections
plane-concave) to hold the cutting
• Sections are stained with an aqueous solution of an appropriate
edge at a constant, correct angle
heavy metal salt and examined with a transmission electron
during the process of honing and
microscope (TEM)
stropping
CARE OF THE MICROTOME
• After sectioning, all the accumulated paraffin and small pieces of
b.1. Clearance Angle
tissues must be brushed away with a soft brush and not allowed to
• angle between knife and surface of
stay in the microtome
tissue block set at 5-10° to prevent
• After carefully drying the machine and knife holder, the parts should
uneven sections
be wiped with xylol. Prolonged and continuous application of the
• cutting facet will not compress the
painted parts with xylene should, however, be avoided since this
block during the process of cutting.
reagent can remove the paint.
• Movable portions should be oiled thoroughly to prevent rusting.

• The microtome must always be covered when not in use. Theoretically, the perfect and optimum cutting angle is obtained when
o prevent accumulation of dust and other dirt which may later on the sides of the wedge knife are inclined at an angle of about 15°
interfere with the normal sectioning of tissues causing maximum penetration of the tissues and minimizing distortion
• Always remove the knife or blade before cleaning.

• Have the instrument inspected at least once a year by a qualified

service technician. HONING AND STROPPING HONING (HARD SHARPENING)

• The purpose of stropping is to polish and sharpen the cutting edge,
MICROTOME KNIVES while that of honing is to remove the irregularities from the knife.
Trimming and section-cutting are • Badly nicked knives with blunted ends have to undergo sharpening

done with a microtome knife, in order to ensure optimum sectioning of tissue blocks and prevent
which is available in three basic gross irregularities on the tissue sections.
types or shapes: • Jagged edges, if not corrected, will produce tears or striations in

1. Plane-Concave Knife tissue sections.

2. Biconcave Knife
3. Plane-Wedge Knife HONING (HARD SHARPENING)
• removal of gross nicks on the knife edge to remove blemishes, and
1. PLANE-CONCAVE KNIFE grinding the cutting edge of the knife on a stone to acquire an even
• 25 mm edge
• one side flat, one side concave • the degree of sharpness is proportional to the fineness of the

less cutting celloidin-embedded tissue blocks on a abrasive used in sharpening

• uses a hone - serves to remove nicks and irregularities on the knife
concave side sliding microtome
edges
more cut paraffin sections on base-sledge, rotary or • Types:
concave side rocking microtome. a. Belgium yellow
b. Arkansas
2. BICONCAVE KNIFE c. Fine Carborundum
• usually, 120 mm
• both sides are concave
a. BELGIUM YELLOW
• recommended for cutting paraffin-embedded sections on a rotary
• for manual sharpening when cutting edge has been rendered blunt
microtome or nicked. This type usually gives the best result.

b. ARKANSAS
• gives more polishing effect than the Belgium Yellow
 c. FINE CARBORUNDUM OTHER EQUIPMENT REQUIRED IN SECTIONING
• much coarser than the first two types and is used only for badly Waterbath
nicked knives followed by either one of the first two knife • thermostatically controlled type is preferred

sharpeners • temperature of the water should be between 5

and 10°C below the melting point of the paraffin

• The surface of the hone is wiped clean with a soft cloth moistened wax
with xylene to remove the scattered small particles of stones and
metal Drying oven or hot plate
• It is then covered with lubricants: • temperature setting at the melting point of the wax to

o mineral oil allow sections to dry

o clove oil

o xylene

o liquid paraffin Forceps and squirrel hair brush

o soapy water • needed for handling sections during

cutting, and for removing folds and

creases on the sections during
HONING (HEEL TO TOE) "floating out" in water bath
• fine-pointed or curved type

Clean Slides
• For routine work, 76 x 25 mm. slides that are 1.0 -1.2 mm thick are

usually preferred
• Frost-ended slides are generally used

Slide Rack
• made on the assumption that regular slides

have been used.

• Heel (handle) to toe (head) direction
• 20-30 double strokes
A. 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.
B. The knife is then turned over, and the other surface is again drawn
forward, EDGE FIRST, with a HEEL TO TOE direction.
• Larger size of slides are used for sections
of eyes or CNS tissues when these will not
fit on the regular
SECTIONING
• process whereby tissues are cut into uniformly thin slices or
"sections" with the aid of a microtome, to facilitate the studies under
the microscope
A. Paraffin Sections
• paraffin-embedded tissue blocks, cut

STROPPING (TOE TO HEEL) using rocking or rotary microtome

• process whereby the "burr" formed during honing is removed and the • 4-6 um

cutting edge of the knife is polished.

• TOE to HEEL B. Celloidin Sections
• for celloidin embedded tissues which are usually cut by means
• 40-120 double strokes
of the sliding microtome
• Require oiling before use: • 10-15 um

o Vegetable oil (castor oil)

o Mineral Oil – NEVER!!! C. Frozen Sections
-tend to blister and destroy the leather • which may be cut from tissues that have been fixed and frozen

with CO2 or for fresh or fixed tissues frozen with the cryostat
• 0.5 um
• Paddle strop made up of the best quality horse leather

• differ in terms of embedding and impregnating medium that were

used
BLADES
TRIMMING
DISPOSABLE BLADES • Once the tissues have been embedded and the wax has solidified,
• cheaper the wax block is removed from the mold, the identification number is
• ease in cutting noted and the excess wax is cut off from the block to expose the
• have a sharp cutting edge (2 to 4 u thick sections)
tissue surface in preparation for actual cutting.
• cutting excess wax from the block to expose the tissue surface and

prepare for actual cutting

• the sides, top and bottom of the tissue block are trimmed until
GLASS KNIVES perfectly level and all sides are parallel
• generally used for trimming and semi-thin sectioning of tissue • Only thin slices are taken out at a time to prevent the block from
blocks for electron microscopy cracking.
DIAMOND KNIVES COARSE TRIMMING
• used to cut any type of resin block for electron microscopy • done on microtome at approximately 30 microns at a time until the
• already mounted in a metal block designed to fit directly into the knife
entire tissue surface is exposed
holder of the ultrathin microtome
• brittle and expensive, but very durable
FINE TRIMMING
• done by either setting the thickness adjuster at 15 mm or by

advancing the block using the coarse feed mechanism

 PARAFFIN SECTIONS FAULTS OBSERVED DURING SECTION-CUTTING
• The cutting rate depends upon the type of tissue, the size of the PROBLEM REASON REMEDY
block, and the model or type of the microtome that is used. Surfaces and edges of the
Re-trim the block
block are not parallel
• Sections usually form ribbons due to slight heat generated between
Horizontal surface of the
the block and the knife edge during the process of cutting. Re-adjust and
block is not parallel to the
reorient the block
• 4-6 μ in thickness for routine histologic procedures knife
Sections fail to Coat horizontal edges of the
form ribbons Paraffin wax is too hard block with wax of lower
SECTIONING melting point
• cut in a slow and consistent manner Knife is tilted too much Reduce the tilt
• do not stop or start while the blade is cutting a block as this may Readjust the thickness of
Sections are too thick
the sections
produce horizontal lines across the block and the sections (and
Knife is dull Hone and strop
very slight changes in thickness). Sections roll up a. Knife is blunt Sharpen the knife
• Sectioning is generally improved when the specimen and the wax on cutting so that Tilt of knife is too great Reduce the tilt
are well matched in hardness. they adhere and
get broken
• most paraffin blocks must be cold when sections are cut Knife edge is dirty Clean the knife edge
against the knife
• COLD WAX – provides better support for harder elements in a edge
specimen allowing thinner sections to be obtained Blunt or dull spot on the
Adjust the knife so that knife
edge will present a
knife, producing an irregular
uniformly sharp edge to the
• Immediately, after cutting of sections, the ribbon is floated out in a Ribbon is curved knife edge
block, or sharpen
water bath set at 45-50oC (approximately 6-10oC lower than the crooked or
Edges of the block are not
uneven instead of
melting point of the wax) straight
parallel but round or wedge Re-trim the block
• The purpose of floating out is to flatten the sections and prepare
shaped
Knife is not
them for mounting. parallel to the block
Readjust the knife and block
• Folds and creases may be removed by gently stretching the sections Knife is blunt or dull Re-sharpen the knife
with dissecting needles (or forceps) Paraffin block is warm and Cool the block on ice water
• The ribbon is then placed in a slide with adhesive and is then placed soft until firm
Sections are Knife edge is coated with
in a 70oC paraffin oven for 20 minutes to dry. compressed, paraffin
Clean the knife edge
wrinkled or Readjust thickness of the
Sections are too thin
• Note: Hot plates are not recommended because they can cause jammed section
overheating and there is a risk of dust falling onto the section during Microtome set screw is
Tighten the screw
loose
the drying period. Tilt of knife is too vertical Reduce the tilt
• In between processing, remove tissue blocks before placing tissue
Sections are
sections squashed (width
Bevel of knife is lost due to
Re-sharpen, using a knife
• Process one tissue specimen at a time (prevent mixing specimens of each section is back or automatic knife
incorrect sharpening
less than that of sharpener
up/contamination) > misdiagnosis. the block)
Bubble or dirt formed in the
Re-embed in freshly filtered
Cleaning: embedding medium
wax if necessary
• After floating and mounting the paraffin sections from each block,
Tissue is not processed
use lint-free Kleenex or Kim-wipe to thoroughly wipe clean the properly and will not form a
surface of the water and the edges of the flotation bath to prevent section (especially if center
floaters or cross-contamination. A hole is formed
is raw) Re-process tissue
Under-processed portion of
in the section
tissue bursts on contact
Fishing out/Floating Out with warm water
• Done in a vertical position Once embedded in paraffin
• Promote drainage of water, prevent the tissue section from slipping wax, decalcification is
Hard spot in tissue due to
impractical; use a base-
down the slide calcium
sledge microtome with a
wedge knife
Note: Tilt of knife is too great or
• Tissue sections should not be left for a prolonged period of time – bevel is not cleared, hence
Reduce the tilt
object is compressed
cause undue expansion and distortion of tissue (30 seconds will be against the knife edge
Sections of
enough). unequal Clamp set screw on knife
Tighten the screw
• Metal racks with 25-slide divisions are used to store the mounted thickness are or block holder is loose
sections during the drying process produced Cut blocks into
Blocks are too large
smaller fragments
• Staining: manual, batch, automated staining
Soften the blocks in
Blocks are too hard
detergent or phenol
Breathe out or blow gently
on the bock and knife to
Static electricity due to low
Sections adhere break up static electricity, or
atmospheric humidity
to the knife or boil water in the room to
other parts of the increase humidity
microtome Knife edge is dirty Clean the knife edge
Knife edge is dull Sharpen the knife
Knife tilt is too great Reduce the tilt
Nicks or damage
Sharpen the knife
Ribbon is split or on the knife edge
lengthwise Re-embed in
Dirty embedding
vertical scratches freshly filtered wax
are seen on Clean knife edge with
Knife edge is dirty
sections xylene
Tilt of knife is too great Reduce the tilt
Sections are Knife tilt is too great Reduce the tilt
lifted from the Knife is dull Sharpen the knife
knife on Paraffin is too soft or room Cool paraffin wax in ice
upstrokes temperature is warm water
Tilt of knife is too small,
Resistance is felt
paraffin block is therefore
on the lower part of
compressed against the Increase the tilt
the section during
base of the knife towards
cutting
the end of stroke
 Horizontal or Knife edge vibrates due to Treat with phenol during
parallel lines or hardness of tissue processing or collodionize
furrows across
the section
Tilt of knife is too great Reduce the tilt
("chatters") are
seen
Knife is blunt Sharpen the knife
Adjust the knife so
Knife is not clamped that knife edge will present
properly a uniformly sharp edge to
Section cut is
the block, or sharpen
sometimes thin,
Knife or block holder is Tighten adjusting
sometimes thick
loose and locking screws
Knife tilt is too small that
block is compressed by Increase the tilt
bevel and section is not cut
Knife makes a Tilt of knife is
Readjust the tilt
hard metallic too slanted or too big
scraping or Take fresh block treated
ringing sound on Tissue is too hard with phenol during
backstroke, when processing
section is cut Knife blade is too thin Change the knife
Sections are too Microtome needs
Wrong micrometer setting
thick recalibration
Tissue is opaque, Repeat clearing; if object
section cutting is has already been
difficult due to Insufficient clearing embedded, prolong clearing
presence of up to 12 hours, then
alcohol reembed
Tissue shrinks Insufficient dehydration,
away from wax therefore incomplete Repeat the whole procedure
when trimmed clearing and impregnation
Paraffin block,
Repeat paraffin
after cooling, is
Insufficient paraffin impregnation, then re-
moist and
embed
crumbles

PROBLEMS DURING TRIMMING

PROBLEM REASON REMEDY
Block is trimmed down
nearest to the tissue.
Remaining wax is melted on
On trimming, Clearing agent not
embedding oven and
tissue smells of completely removed due to
paraffin impregnation is
clearing agen insufficient impregnation
repeated, changing the
paraffin at least once before
embedding
On trimming, wax Contaminated wax
Re-embed in freshly filtered
appears Block not cooled rapidly
wax
crystalline enough

PROBLEMS ON FROZEN SECTIONS

PROBLEM REASON REMEDY
Frozen tissue
crumbles and
comes off the
Freezing is not adequate Refreeze the tissue block
block holder
when cut when
cut
Frozen tissue
Warm the tissue with the
chips into Tissue is frozen too much
fingers
fragments