Microtomy and sectioning
PROFESSOR: SIR BILLY AFRICA Rmt2023
OBJECTIVES: is rotated, moving the tissue block at a known distance towards the
At the end of this module, the students should be able to: knife for cutting sections at uniform thickness
1. Demonstrate the proper way of using the rotary microtome.
2. Correctly identify the different parts of the rotary microtome 3 ESSENTIAL PARTS
along with its functions. a. Block holder – the tissue is held in position
3. Demonstrate the creation of a good and quality section. b. Knife carrier and knife – actual cutting of tissue sections
(We can use knife or blade. In a knife, it needs to be hone.
DISCUSSION: While in blade, it can be disposed after use)
AKA : sectioning or cutting tissue processing c. Pawl, ratchet feed wheel and adjustment screws – to line
⮚ Microtomy, otherwise known as Sectioning, is a process up the tissue block in proper position with the knife.
whereby the paraffin embedded tissue (before microtomy
step→ impregnation,embedding and trimming of section) is 5 KINDS OF MICROTOME
cut into thin and uniform sections(to easily observe the a. Rocking Microtome→ for large blocks of paraffin embedded
architecture or morphology of sections under microscope). tissue (it has a old/primitive structure. Very simple in structure)
The instrument used is called the microtome. Difficulties b. Rotary Microtome→ paraffin embedded tissue (as mentioned
encountered during cutting of sections are mostly due to in impregnation and embedding, the most common agent is
faults encountered during the processing of tissues or due to paraffin wax)
some faults in the technique or cutting itself. Many microtomy c. Sliding Microtome→ celloidin embedded tissue/tissue blocks
problems have several causes so it is best to address them with hard consistency
one at a time until the problem is resolved. Steps that causes d. Freezing Microtome→ cutting unembedded frozen sections
failure in microtomy: (used whenever results are needed immediately or urgent
a. Fixation → overfixation can cause hard and brittle biopsies)
tissue e. Ultrathin Microtome→ for EM (produces very thin slices of
b. Dehydration → hard and brittle tissue, difficulty in the section) Electron microscopy is the most powerful type of
cutting microscope and you need to have very thin slices for you to
c. Skipped decalcification → failure to remove easily study the specimen in a microscope.
calcium which makes the specimen hard to cut.
d. Improperly performed decalcification → Discussion:
incomplete decalcification MATERIALS/ APPARATUS
e. clearing → make the tissue hard and brittle if exact ⮚ Tissue Block
time is not followed ⮚ Rotary Microtome
f. Impregnation → uses hot temp to maintain the
paraffin wax in melted state. If the temperature is not PROCEDURES:
controlled specimens can result in hard consistency. 1. Put the embedded tissue on the specimen holder.Set up the
Hard and brittle specimens can damage microtome. micro knife on the knife holder. Block holder of the microtome
g. Due to some faults in the techniques is also known as the chuck
⮚ The microtome is the principal instrument used that is 3. Start with a large thickness 15-25 um (Mechanical trimming
capable of cutting a section at a predetermined thickness or trimming of excess wax from tissue block). Go to the section
by sliding the block into a cutting tool, usually a steel knife, you want to cut.
glass or diamond blade, which is fixed and attached to the 4. Place your hand on the hand wheel and start rotating the
machine. wheel until you can see the exposed tissue surface.(part of
trimming and the target is to expose the tissue from tissue
⮚ Microtomy/Sectioning is the process by which the processed block)
tissue, most commonly a paraffin embedded tissue is 5. Adjust the cutting thickness to 3-5 um (Sectioning/ribboning).
trimmed and cut into uniformly thin slices or "sections" to 6. Place your hand on the hand wheel and start rotating the
facilitate studies under the microscope. wheel. Each rotation of the wheel equals one section
produced.
7. Do step 6 continuously until there are series of sections
produced (ribbons) the target is to produce ribbons. Ribboning
is the term when the sections produced are stuck together in a
series. Hence, the ribbon-like appearance.
8. Using an applicator stick, forcep or a brush, gently
transfer the ribbons to the water bath to prepare for “fishing”
The purpose of putting it to waterbath is to remove foldings
wrinkles of the ribbon. Then after that using a slide, get the
tissue through the process called fishing out. Let it dry and
now ready for staining.
Image: Rotary Microtome → most commonly used used type of CARE OF THE MICROTOME:
microtome ⮚ After sectioning, all the accumulated paraffin and small
PRINCIPLE: a spring balanced pawl is brought into contact with a pieces of tissues must be brushed away with a soft brush and
ratchet feed wheel connected to a micrometer screw (which not allowed to stay in the microtome (it can adhere to the knife
determines the thickness of the specimen that will produce), which
OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I HISTOPATH 1
or blade which affects the next microtomy procedures), since
this may later interfere with the cutting of tissue blocks.
⮚ After carefully drying the machine and knife holder, the parts
should be wiped with xylol (to remove even the small chips
of the paraffin wax and sections into the microtome) .
Prolonged and continuous application of the painted parts
with xylene should, however, be avoided since this reagent is
capable of removing the paint.
⮚ Movable portions should be oiled thoroughly to prevent
rusting.
⮚ The microtome must always be covered when not in use, to
prevent accumulation of dust and other dirt which may later
on interfere with the normal sectioning of tissues.
⮚ The microtome should be placed on a stable bench, away
from air drafts, doorways and passing staff. Any air
movement from air conditioners or other causes can make
section handling very difficult.
⮚ Always remove the knife or blade before cleaning. The knife
holder can easily be removed to facilitate access for
cleaning. No fluid must enter the inside of the instrument
during cleaning.
⮚ When cleaning the blade avoid dragging anything along the
cutting edge. Even cellulose fibers can cause damage to the
blade.
⮚ Have the instrument inspected at least once a year by a
qualified service technician.
SAFETY MEASURES:
⮚ It is very important that staff are not distracted when using the
microtome because of the risks of injury from extremely sharp
blades. (one of the most encountered accident in histopath
lab is the cutting of one’s finger using microtome)
⮚ It is preferable to have non-slip flooring in the vicinity of
microtomes because, inevitably, wax fragments will find
their way onto the floor where they can produce a slippery
surface.
⮚ Use forceps or brush instead of fingers to pick up sections or
wax fragments from the blade or block face.
⮚ Use hand wheel lock when changing blocks. The knife or
blade should be removed from the microtome when the
instrument is left unattended or when cleaning the instrument.
OUR LADY OF FATIMA UNIVERSITY I PAMPANGA CAMPUS I COLLEGE OF MEDICAL LABORATORY SCIENCE I HISTOPATH 2
