MICROTOMY

o Process by which previously processed tissue, is trimmed and cut into uniformly thin slides or sections
o One of the most important in tissue processing
o Most important thing: Tissue Block (Paraffin blocks commonly)  Sections (Thin & Uniform)
MICROTOME
o Capable of producing thin sections from blocks.
o Cutting sections at a predetermined thickness by sliding the block into a cutting tool, usually a steel knife or blade which is fixed and attached to the machine.
3 Basic Parts of a Microtome:
1) Block Holder – where the tissue is held in position.
2) Knife and Knife Carrier/Holder – for actual cutting of tissue sections.
3) Pawl Ratchet Feed Wheel and Adjustment Screw – to line up the tissue block in proper position with the knife, adjusting the proper thickness of the tissue for successive
sections; parallel alignment of block holder and knife.
PRINCIPLE of MICROTOME
o A spring-balanced teeth or pawl is brought in contact with, and turns a ratchet feed wheel connected to a micrometer screw, which is in turn rotated, moving the tissue block at a
predetermined distance towards the knife for cutting sections at uniform thickness.

TYPES OF MICROTOME
Microtome Description
1) Rocking o Can only be adjusted to a
(Cambridge) certain extent
Microtome o Simplest type
o Consists a heavy base and 2
arms (upper and lower arm)
o 2 sizes: to cut small and
large blocks
2) Rotary (Minot) o Heavier & more stable
Microtome o Most commonly used for
both routine & research
laboratories.
o Up & down vertical
movement = perfectly flat
plane
o More complex in design
Use(s)/Function Inventor Tissue Size Advantage(s) Disadvantage(s)
o For cutting sections o Paldwell Trefall o 10-12 µ o Size restriction (difficulty of
of small & large o 1881 reorienting the block).
paraffin-embedded o Cannot be used for serial
blocks. sections since tissues are cut
in slightly curved planes.
o For cutting paraffin- o Minot o 4-6 µ o Produces serial o More expensive
embedded sections o 1885-86 sections o Relatively dangerous since
o Maybe used for the blade is oriented in an
cutting large blocks. *Both manual & upward position.
electrically driven
models: for cutting
ultrathin sections &
for cryostat use
 Microtome Description Use(s)/Function Inventor Tissue Size Advantage(s) Disadvantage(s)
3) Sliding o Recommended for o Adams o 4-9 µ o Most dangerous type
Microtome cutting extremely hard o 1789
& rough tissue blocks.
2 Types:

a) Standard o Block remains o Mainly for cutting o More dangerous because of

Sliding stationary while the celloidin-embedded the movable knife (difficult
knife is moved tissues. to attach knife guards).
backward & forward o Can be used in small &
during process of large paraffin blocks
sectioning. & celloidin sections.

*Modern models of heavy duty

b) Base- o Block is movable o Favored for hard & o Produces serial base-sledge microtome are
Sledge o Sections are cut in a tough tissue blocks in sections electrically driven and are ideal
perfectly flat plane. all forms of media o Comparatively for resin-embedded decalcified
more stable bone.
4) Ultrathin o Uses broken plate o For electron o 0.5µ
Microtome glass knives/diamond microscopy.
edge knife
o Special process
required in fixation –
use Osmium
Tetroxide
o Embedding Medium
 Plastic
5) Freezing o Stage is hollow & o To cut undehydrated o Queckett o 10-15µ (right
Microtome perforated in order to tissues in a frozen state. o 1848 thickness for
freeze the block/tissue. o For fats & tissue neurological
o Freezing agent: CO2 constituents that may be tissues)
 Release into the damaged
stage & will o For rapid diagnosis in
evenly freeze the processing of
block holder & neurological structures
tissue. & heat sensitive tissue
constituents
Cryostat or Cold Microtome 3) Biconcave Knife: usually 120 mm in length
o For cutting paraffin-embedded sections
o Refrigerated apparatus used in fresh tissue microtomy, for freezing the tissue into
 Rotary Microtome
the block holder to the correct degree of hardness to facilitate easier and faster
B. Sections to be focused under Electron Microscope (very thin sections)
sectioning.
1) Diamond-edge Knife
o Consists of a microtome, usually rotary microtome, kept inside a cold chamber
o Resin blocks for Ultrathin Microscopy & for EM
which has been maintained at a temperature between −𝟓℃ 𝐭𝐨 − 𝟑𝟎℃ (average is
2) Glass Knives
−𝟐𝟎℃) by an adjustable thermostat.
o Generally used for trimming semi-thin sections
o Capable of freezing tissues within 2-3 minutes.
C. Disposable Blades
o Can be used if thin sections are required (4-6µ)
 Very thin cutting edge, 2-4µ
o Provides a means of preparing thin sections of fresh frozen tissues for:
 Cheaper, can be disposed of easily
 Fluorescent staining antibody techniques
 Magnetic Knife: Type of disposable blades; Used for Cryostat
 Histochemical enzyme studies D. Safety Razor Blades
o Most commonly used for rapid preparation of urgent tissue biopsies for  Much thicker sections
intraoperative diagnosis.  For partially calcified materials & paraffin & frozen sections
o Housed in the frozen section room close to the operating room to allow the direct
consultation between surgeon and pathologist. Microtome Knives

KINDS OF MICROTOME KNIVES 1) Clearance Angle

o Between the edge of the knife & the tissue block
A. Sections to be focused under Compound Microscope o 5-15º (Optimum - 15º: Less distortion & maximum penetration of
1) Plane-Concave Knife: usually 25 mm in length tissues)
o One side of knife is flat while the other is concave. o 5-10º should be maintained in order to maintain even sections.
o Less concave sides are recommended for cutting CELLOIDIN- 2) Wedge Angle
EMBEDDED TISSUE BLOCKS (plane part) o Angle of cutting
 Use Sliding Microtome o 15º
o More concave sides are used to cut PARAFFIN-EMBEDDED 3) Bevel Angle
SECTIONS o Angle of cutting facet
 Base-Sledge Microtome o 27-32º (tapered end of knife)
 Rotary/Rocking Microtome 4) Rake Angle
2) Plane-Wedge Knife: usually 100 mm in length o 90º - (Bevel Angle + Clearance Angle)
o Have both sides straight
o For frozen sections Microtome Knives Sharpening
o For celloidin-embedded sections & extremely hard tissues  Cutting Edge – must be of good quality steel
 Base-Sledge Type or Sliding Microtome o Too soft: Doesn’t maintain the edge, easily becomes dull
o Too hard: likely to nick against hard objects
TESTS: HONING STROPPING
o Smooth stones o Horse leather
 Should cut a paraffin wax block at 2-4µm thickness w/o serrations when Material
o Machine hone
examined under the microscope (100x) o Soapy water, Oil (Mineral Oil, Castor o Vegetable Oil/Oiling
 Von Mol’s Criterion Oil, Clove Oil) or Xylene (Clove Oil, Castor
o Cutting edge is able to reflect light **Mineral oil is not recommended; Oil)
o Will split hair strand across to it with their own resistance. Lubricant should not be in contact with leather **Apply oil on the back
because it causes permanent blemishes of Strop  Removes
2 Stages of Sharpening
on the Strop. excess oil  Allow to
1) Honing stand for 24-48 hours
o Can be bypassed if no nicks in knife Direction o Heel-to-Toe, Edge First o Toe-to-Heel, Edge last
o Hard sharpening Motion o Zigzag o Zigzag
o To remove nicks & irregularities o 20-30 strokes if knife is not specified o 40-120 double strokes
o Machine Hone:
2 Stages under Honing  30 double stroke for each side
 Consistent pressure is applied
a) Coarse Honing: Removal of blemishes/gross irregularities No of
 Preserves uniform bevel
b) Honing Proper: Entails the grinding of cutting edge, using finer Strokes
o Minot/Plane-Wedge Knife:
hones in order to preserve/maintain the thickness of cutting edge.  10-20 strokes per sides
 For Plane-Concave knives, hone
HONES: Natural sharpening stones
the concave part only.
a) Carborundum: Most coarse
b) Arkansas: More polishing effect
HONES: Oilstones
c) Belgium Yellow: Usually for manual sharpening, best hone
d) Belgian Black Vein  The finer the grain, the harder the hone
2) Stropping  Plate Glass Honing
o Can be performed before sectioning of delicate specimens  Diamantine: used for final polishing
o To remove burrs during honing  Glass Knives
o Final polishing  Abrasives: Aluminum Oxide in Water
o To sharpen cutting edge  Paste
 Factory Grinding
 After repeated sharpening
 When there is widened Bevel Angle (>35º)
 Automatic Hones
PRECAUTIONS IN MICROTOMY
o Cutting edge must be sharp & smooth
o Cutting edge must be THINNER than the section to be cut
o Knife and its corresponding knife back should not be interchanged
o Honing
o Stropping
CARE OF THE MICROTOME
o The microtome knife is an expensive piece of equipment and great care must be
taken of it. The following points must be observed:
 Store the knife in its box when not in use.
 Never lay a knife flat on the bench.
 The edge of the knife should be “touched up” daily before use.
 Never lend or borrow a knife.
GENERAL STEPS IN FIXING SECTIONS ONTO SLIDE
1) Floating
2) Adhesion
3) Fishing Out
4) Orientation
5) Deparaffinization
 Alcohol lamp
 Paraffin oven
6) Drying sections
 Wax oven (56ºC-60ºC for 2 hours)
 Incubators (overnight)
 Hot plate (45ºC-55ºC for 30-45 minutes)
 Alcohol lamp/Bunsen flame
7) Post-mordanting
 Secondary fixation (post-chroming)
 Used primarily as mordant & secondary as fixative
 5-10 minutes in ether
 Aqueous solution of HgCl2
 Aqueous Picric Acid