Microtome & Knifes
Lecturer Mohamed Serar
Msc, Bsc, Histopathologist
Sudan
What are Microtomes?
• Microtomes are machines that will advance an
object for a predetermined distance then slide the
object to the cutting tool, usually a steel knife or
blade, and then force the object through the knife
thus producing a section.
• The thickness of which is the predetermined
distance previously set.
•All microtomes consist of three main parts:
- Base (microtome body).
- Knife attachment and knife
- Material or tissue holder.
Types of Microtome and their Uses
•Microtome styles of manufacture vary
depending upon the application
required.
Cambridge Rocking Microtome
•Produced in large numbers in the early 20th century, the
rocking microtome are small, lightweight instruments
which are very suitable for class work, and the teaching of
microtomy.
•Designed only for cutting paraffin sections the tissue
moves towards the knife.
• (the slightly biconcave heiffor knife is used) which is held
rigid causing the sections to be cut in a curved plane.
Rocking Microtome
• Very thin sections are difficult to obtain and one
major disadvantage is a limit to the size of block
which can be cut.
• Because of the lightness of the frame the
microtome has a tendency to move during
cutting.
Rocking Microtome
• The rocking microtome has largely been replaced
by the more precise rotary microtome although it
is re-appearing in portable cryostats.
• Now mainly used for botanical applications.
• Sections of 5 µm in thickness are possible.
Base Sledge Microtome
• These are designed for cutting large blocks of
paraffin and resin embedded material including
whole organs for neuropathology, for light
microscopy.
• This base is moved and runners against the knife
to produce sections.
• The feed mechanism being either automatic or
activated manually.
Base Sledge Microtome
•The microtome, which is very heavy for stability
and not usually subject to vibration, can also be
used to cut materials from various industrial
applications (wood, plastics).
•They are not suitable for cutting very hard resins.
Sliding Microtome
• Is used mainly for the cutting cellulose nitrate
embedded tissue with the obliquely/set knife
being drawn across the surface of the object.
• It is essentially more dangerous because it is
more difficult to attack knife guard.
Rotary Microtome
•Machines of this sort are general purpose
microtomes for cutting semi-thin to thin sections for
light microscopy.
•The microtome operation is based upon the rotary
action of a hand wheel activating the advancement of
a block towards a rigidly held knife.
•The block moves up and down in a vertical plane in
relation to the knife and therefore cuts flat sections.
Rotary Microtome
• Available machines range from light weight, rotary
microtomes suitable for cutting paraffin wax embedded
material in a continuous ribbon to heavy duty.
• Motor driven instruments used with a slow, continuous
speed and retracting advance movement to section hard
material embedded in synthetic resin.
• The rotary microtome can also be found in most
cryostats for cutting frozen sections.
Rotary Microtome
• Section thickness settings range from 0.5µm to
60µm on most machines.
• Sections of paraffin wax embedded tissues are
normally cut within the range 3 to 5µm whilst resin
sections are between 0.5 to 1µm.
• Rotary microtomes are especially suited to
cutting sections using disposable steel knives.
Rotary Microtome
Freezing Microtome
• This form of microtome is used for cutting thin to
semi-thin sections of fresh, frozen tissue and semi-
thin sections from industrial products such as some
paper, leather, soft plastics, pastes and food products.
• 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.
Freezing Microtome
•Some cooling systems also allow the knife to be cooled
at the same time.
• 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.
• The block moves by a pre-set amount, in microns, at
the end of each cut.
• Consistent, high quality, thin sections are very difficult
to obtain with this type of microtome.
Freezing Microtome
• A major disadvantage of CO2-freezing microtome is
the constantly changing temperature of the tissue
caused by repeated freezing.
• This freezing and thawing results in:
- In activation of some enzymes.
- An inability to prepare multiple sections of uniform
thickness.
Freezing Microtome
Ultra Microtome
• The ultra microtome is used to prepare ultra thin
sections(0.005- 0.1 micrometer) for light and electron
microscopy.
• Very small samples of tissue or industrial product are
usually embedded in hard resin before cutting.
• It has been reported that sections can be cut as thin as
10 nanometers.
Ultra Microtome
• The cutting stroke is motor driven to provide a regular,
smooth motion for sections of even thickness and
constant reproducibility.
• Knives are usually made from glass, diamond or
sapphire.
• The block is brought to the knife edge under
microscopical control (low power binocular) and as each
section is cut it is floated on to a water bath adjacent to
the knife edge.
Ultra Microtome
Cryostat
• A cryostat is primarily used for cutting sections of
frozen tissue as well as pastes, powders and some food
substances.
• The cryostat commonly consists of a microtome
contained within a refrigerated chamber, the
temperature of which can be maintained at a preset
level. A recent innovation has the body of the microtome
positioned outside the refrigerated chamber.
Cryostat
• The cryostat usually contains a rotary
microtome although some portable units utilize a
rocking microtome.
• With the object, object holder and knife all at
the same temperature and all other conditions
for cutting the material optimal.
• Sections as thin as 1 micron are possible.
Cryostat
Vibrating Microtome
• Originally conceived as a microtome which
could produce high quality sections of fresh,
unfixed material from animal or botanical
sources and to replace the hand microtome.
• The name of the instrument derives from the
high speed vibration produced in a safety razor
blade to provide the cutting power.
Vibrating Microtome
• The amplitude of vibration is adjusted by altering
electrical voltage applied to the 'knife'. Different degrees
of vibration are required to produce sections from
varying densities of material.
• To prevent tearing, soft material is cut whilst immersed
in a fluid which also aids in dissipating heat produced at
the vibrating edge of the razor as it cuts.
Microtome knives
Steel Knifes:
• Steel microtome knives are manufactured from high
quality carbon which is heat treated to harden the edge.
• The steel should be free from impurities, contain anti-
corrosives and be rust-resistant.
• The best knives are those that are fully hardened.
• Those which are only surface hardened lose the cutting
edge very quickly once the hardened area is removed
through repeated re-sharpening.
Profile of Steel Knives
Microtome knives of hardened steel are made to
four different profiles for cutting various materials.
Profile A, B: Strongly Plano Concave/Biconcave
• One surface of the Plano concave knife is straight whilst
the other is hollow ground.
• The bi-concave knife has two hollow ground surfaces.
• Both knives are extremely sharp and are used for cutting
soft, celliodin embedded material and wax embedded
sections.
• These knives are not suitable for relatively hard
materials, which cause the edge to vibrate and produce
the phenomenon known as chattering. To obtain the best
result the knife should always be oblique to the object
when cutting sections.
Profile C: Wedge Shaped
-Wedge shaped is plane surface on both sides.
-The wedge shaped knife has more rigidity than profile A
or B knives and can therefore be used for cutting harder
materials.
- Because of the extra thick nature of the wedge at the tip
this type of knife cannot be ground as sharp as profile A or
B knives.
Profile C: Wedge Shaped
- Commonly used for cutting sections from paraffin wax
embedded material, frozen sections, cryostat sections and
for small, synthetic resin embedded material this knife can
also cut soft plastics, rubber, and wood.
Profile D: Plane Shaped
- This knife will cut hard and tough material as it has
greater stability than any of the other profile knives.
- It is commonly used for cutting synthetic resin blocks,
hard materials embedded in paraffin wax, large wax
blocks and various substances used in industry.
Sharpening of Steel Knives
• A sharp knife edge free from imperfections is essential
for the production of good sections.
• However, with the introduction of disposable knives the
practice of sharpening traditional microtome knives has
all but disappeared.
• Bluntness is caused through the edge becoming rounded
and takes the form of large (macroscopical) or
small(microscopical) teeth, or serrations, caused by
breaking or rearrangement of the metal edge.
Sharpener
Sharpening of Steel Knives
•This can be achieved manually or by using an automatic
knife sharpening machine.
•Automatic machines tend to remove more metal during
sharpening so that knives become worn quickly.
•Knife sharpening consists of two operations:
- Honing( Arkansas, carborundum, Belgian yellow stone).
- Stropping or polishing.
Sharpening of Steel Knives
• Manual methods on the other hand
remove far less metal but require more
skill, experience and time to produce a
satisfactory edge.
Automatic
• There are two types of automated knife sharpening
machine:
1. Rotating wheel type made by Temtool.
2. Glass plate type made by Shandon.
• Abrasive powders such as aluminium oxide(alumina).
• The knife blade is automatically turned at preset
intervals so that each side is evenly sharpened.
Automatic
• Lubricants are essential for successful knife
sharpening.
• These agents cool the knife edge (heat generated by
the abrasive procedure can destroy the 'temper' of
the steel) and continually remove the fine metal
particles, produced by the abrasive process, away
from the knife edge.
Stropping
• Stropping polishes the knife edge and removes fine metal
burrs retained along the edge after honing.
• Leather, coated with a fine rouge powder or a lapping
stick coated with a diamond paste containing industrial
diamonds of less than 0.5 µm are effective for this
purpose.
• It is important that very little pressure is used when
stropping.
Disposable Blades
• Disposable microtome blades are essentially refined,
thickened razor blades.
• When held in a specially adapted knife holder the blades
consistently produce high quality sections and have
replaced conventional microtome knives in many
instances.
• All disposable blades are manufactured from high
quality stainless steel although there are different grades
according to the thickness of the blade.
Disposible Blades
Glass Knives
• Those used for ultramicrotomy.
• Glass knife holders are available so that 'Ralph knives'
can be used with a rotary microtome.
• Glass knives are hard but brittle and care is required
with their handling.
• These knives deteriorate with storage due to changes in
the 'flow' or 'strain' of the glass after fracture and from
oxidation impurities remaining in the hardened glass after
manufacture.
• Knives should thus be prepared immediately before use.
Section Cutting
There are many factors which affect the production of
good sections. Some of these are:
• Fixation and embedding: Animal and human tissues are
too soft when fresh to be cut thinly.
• Some form of pre-treatment is required to harden the
tissue to facilitate cutting thin sections. This consists of
either freezing or embedding tissues in a medium which
offers support for cutting
Section Cutting
• The hardness of the embedding compound: This
reflects the thickness at which sections can be cut.
• It is difficult to cut very thin sections from soft
embedding compounds.
Section Cutting
• The following is a guide to the thickness at which
sections can be obtained from different embedding
media ranging from soft (gelatin) to hard (resin).
- Gelatin - 50 to 200 µm.
- Paraffin wax - 1 to 15 µm.
- Paraffin wax/resin mixtures - 0.5 to 2 µm.
- Resin - 0.05 to 1 µm.
- Ice - 5 to 20 µm (frozen section).
Principles of Section Cutting
The basis of all good sectioning depends on:
- Properly prepared tissue.
- A suitable microtome in a good condition.
- The skill of microtomist.
- Sharp knife.
Relation of Knife to Object
• The basic principle of advancement of the specimen
against the knife edge at a predetermined thickness is
common to all microtomes with any embedding media.
Provision is made on most microtomes for variation of
the position of the knife in relation to object. Most
important are the angles of tilt and slant.
Tilt of the knife
• Is defined as the angle between the surface of the block
and a line bisecting the edge of the knife.
• Cutting angle: If the angle of bevel (cutting angle) is too
great it can cause compression in the cut section.
• If the angle is too fine the edge of the knife can vibrate
causing chatter in the section.
• Tilt angle may vary between 10-40 degrees.
Slant of the Knife
• May be defined as the angle its edge makes with the
line of section or direction of travel of the object.
• Usually two edges of the block must be parallel to each
other as well as to the knife edge.
Clearance Angle
• The clearance angle, is usually between 3_10 degrees.
• The correct clearance angle is also important to avoid
compression of the object against the back of the knife
after passing the edge.
Cutting Angle
Setting the Microtome
•All clamps and screws must be firmly tightened.
•All angles must be set.
•The block is fixed on block holder.
•Trimming of tissue block.
Cutting Sections
Once Sections are Cut, They are Floated on a Warm
Water bath
Section Adhesives
• Adhesive media either:
- Protein adhesives such as albumin, gelatin, starch, now
are not recommended as section adhesives.
- Poly-L-lysine.
- 3-AminoPropyltriEthoxySilane.
Tray of
sections
In drying with
oven
Methods of Drying
1. Drying for 16-24hours in an incubator at 37C°.
2. In an oven or on a hot plate at 45-50C° for two
or three hours.
3. In an oven or on a hot plate at temperature
high enough to melt the wax 55-60C° for 30-60
minutes.
