Histopathologic Techniques Introduction (LAB)

MICROTOMY
2nd SEM, 2022
MICROTOMY ● We are not simply cutting the specimen, very
● Microtomy, otherwise known as Sectioning, is important that the specimens have the same
a process whereby the paraffin embedded thickness
tissue is cut into thin and uniform sections. ● Rotary microtome - most commonly used
The instrument used is called the microtome. microtome
Difficulties encountered during cutting of
sections are mostly due to faults encountered PRINCIPLE
during the processing of tissues or due to ● A spring balanced pawl is brought into contact
some faults in the technique or cutting itself. with a ratchet feed wheel connected to a
Many microtomy problems have several micrometer screw, which is rotated, moving
causes so it is best to address them one at a the tissue block at a known distance towards
time until the problem is resolved. the knife for cutting sections at uniform
thickness
● The microtome is the principal instrument used
that is capable of cutting a section at a 3 ESSENTIAL PARTS
predetermined thickness by sliding the block
● Block holder – the tissue is held in position
into a cutting tool, usually a steel knife, glass
or diamond blade, which is fixed and attached ● Knife carrier and knife – actual cutting of tissue
to the machine. sections
● Microtomy/Sectioning is the process by which ● Pawl, ratchet feed wheel and adjustment
the processed tissue, most commonly a screws – to line up the tissue block in the
paraffin embedded tissue is trimmed and cut proper position with the knife.
into uniformly thin slices or "sections" to
facilitate studies under the microscope. ADDED NOTES:

ADDED NOTES: ● We can either use a knife or blade in

microtome
● MICROTOMY - also called sectioning or ○ Knife - the problem in the knife is you
cutting in tissue processing need to hone it when it becomes bland
● Before microtomy, we performed the (mapurol)
impregnation, embedding, and trimming ○ While the blade can be disposed after
● Uses a special machine (MICROTOME) to use
produce a thin and uniform section. This is
done to easily study architecture or 5 KINDS OF MICROTOME
morphology of the specimens under the ● Rocking Microtome- for large blocks of paraffin
microscope. embedded tissue
● Steps that can affect the microtomy or the
● Rotary Microtome- paraffin embedded tissue
sectioning in tissue processing
○ Fixation - over can lead to hard and ● Sliding Microtome- celloidin
brittle tissue
○ Dehydration - can also result to hard ● Freezing Microtome- cutting unembedded
and brittle tissue making it hard to be frozen sections
cut or section ● Ultrathin Microtome- for EM (electron
○ If you skip decalcification or you microscopy)
perform the decalcification but it is
improper, meaning there are still ADDED NOTES:
calcium present in the specimen
○ Clearing - can also make the tissue ● Rocking microtome - the one which holds for
hard and brittle especially if the time primitive structure. Has a very simple structure
and steps was not properly followed ● Sliding microtome - for celloidin or for tissue
○ Impregnation - not controlled block with have a hard consistency
temperature ● Freezing microtome - used when the results
● Hard and brittle specimen can damage the are needed immediately or for urgent biopsy
microtome ● Ultrathin microtome - can produce very thin
● microtomy is a skill slice of the section
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ARAULLO, ASGARE, BAIS, BALATBAT, BANAWA, BRIONES, DE CASTRO, DE LEON, DELOS TRINOS, DURAN, GALANG, MENDOZA, MUZADA, OLBES, ORDONA, OSDON, PUNZALAN, RASING, SALVO, RODRIQUEZ, TOLENTINO, VENTURA
 Histopathologic Techniques Introduction (LAB)
MICROTOMY
2nd SEM, 2022
● Electron microscope - most powerful type of ● Movable portions should be oiled thoroughly to
microscope therefore you need very thin slice prevent rusting.
of section for you to easily study it
● The microtome must always be covered when
DISCUSSION not in use, to prevent accumulation of dust and
● Materials/ Apparatus other dirt which may later on interfere with the
normal sectioning of tissues.
○ Tissue Block
● The microtome should be placed on a stable
○ Rotary Microtome
bench, away from air drafts, doorways and
PROCEDURES passing staff. Any air movement from air
1.Put the embedded tissue on the specimen holder. conditioners or other causes can make section
handling very difficult.
2. Set up the micro knife on the knife holder.
● Always remove the knife or blade before
3. Start with a large thickness 15-25 um (Mechanical cleaning. The knife holder can easily be
trimming). Go to the section you want to cut. removed to facilitate access for cleaning. No
fluid must enter the inside of the instrument
4. Place your hand on the hand wheel and start
during cleaning.
rotating the wheel until you can see the exposed tissue
surface. ● When cleaning the blade avoid dragging
anything along the cutting edge. Even
5. Adjust the cutting thickness to 3-5 um (used for the
cellulose fibers can cause damage to the
actual Sectioning).
blade.
6. Place your hand on the hand wheel and start
● Have the instrument inspected at least once a
rotating the wheel. Each rotation of the wheel equals
year by a qualified service technician.
one section produced.
ADDED NOTES:
7. Do step 6 continuously until there are a series of
sections produced (ribbons). Ribboning is the term ● xylol - to remove even the small chips of the
when the sections produced are stuck together in a paraffin wax and section in the microtome
series. Hence, the ribbon-like appearance.
SAFETY MEASURES
8. Using an applicator stick, forcep or a brush, gently ● It is very important that staff are not distracted
transfer the ribbons to the water bath to prepare for when using the microtome because of the
“fishing” risks of injury from extremely sharp blades.
ADDED NOTES: ● It is preferable to have non-slip flooring in the
vicinity of microtomes because, inevitably, wax
● Mechanical trimming - removing of excess wax
fragments will find their way onto the floor
using the microtome
where they can produce a slippery surface.
● the purpose of putting the ribbons in water
bath is to remove the wrinkles of the ribbons ● Use forceps or brush instead of fingers to pick
up sections or wax fragments from the blade
CARE OF MICROTOME
or block face.
● After sectioning, all the accumulated paraffin
and small pieces of tissues must be brushed ● Use hand wheel lock when changing blocks.
away with a soft brush and not allowed to stay The knife or blade should be removed from the
in the microtome, since this may later interfere microtome when the instrument is left
with the cutting of tissue blocks. unattended or when cleaning the instrument.
● After carefully drying the machine and knife ADDED NOTES:
holder, the parts should be wiped with xylol.
Prolonged and continuous application of the ● one the common accident that happened in
painted parts with xylene should, however, be histopath laboratory is cutting one's finger with
avoided since this reagent is capable of a microtome
removing the paint.

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ARAULLO, ASGARE, BAIS, BALATBAT, BANAWA, BRIONES, DE CASTRO, DE LEON, DELOS TRINOS, DURAN, GALANG, MENDOZA, MUZADA, OLBES, ORDONA, OSDON, PUNZALAN, RASING, SALVO, RODRIQUEZ, TOLENTINO, VENTURA