2. Why do we preserve tissue samples ???
• For gross display in the Museum .
• For microscopic evaluation .
3. For Microscopic Study
The Major tissue component ,
• Is initially fixed using a suitable fixative .
• Undergoes adequate change in solutions , at adequate time - PROCESSING .
• Embedded in paraffin wax as BLOCKS .
• From the blocks , thin tissue sections are cut using specialized equipment
called MICROTOME .
• Stained & studied under a microscope
4. MICROTOMY
• The art of section cutting.
• This is a process of cutting thin sections of tissue embedded in
the wax block using a microtome .
MICROTOME
• Basic instrument / Mechanical device for cutting thin uniform
slices of tissue .
5. HISTORY
• In the beginning of microscopy , sections were made with Razor blades.
• This method failed due to the impossibility of light transmission through
thicker tissue .
• Only with proper clean reproducible cuts of 100 μm , light can be
transmitted , allowing precise observation of tissues to be carried out .
6. With this idea , one of the first
devices for such cuts were
designed by George Adams in the
year 1770 & was further developed
by William Cunnings .
Named as CUTTING ENGINE .
Hand operated machine
A cylinder to hold sample & a hand
crank to cut .
7. • Then the word Microtome was derived from Greek mikros, meaning
"small", & temnein, meaning "to cut" .
• It is a tool used to cut extremely thin slices of material, known as Sections.
• Important in science to cut sections for microscopical observation.
• Microtomes use steel , glass , or diamond blades depending upon the
specimen type & desired thickness of the sections to be cut .
• Sections can be made as thin as , breadth of a human hair , between
50 – 100 μm .
8. • After several modifications , today we use microtomes of
KNIFE BLOCK DESIGN.
• It has a
Changeable knife , &
Specimen holder.
• Automatic machines are also available , where the sample moves
over the knife with a precision control of tissue thickness.
12. TYPES OF MICROTOMES
Based upon the mechanism ,
• Rotary
• Rocking
• Base sledge
• Freezing
• Vibrating
• Saw
• Sliding
• Cryostat
• Ultra-Microtome
13. ROTARY MICROTOME
So called because of the Rotary action of hand wheel used for cutting
movement.
The block holder is mounted on a steel carriage, which moves in grooves ,
up & down.
Most ideal for routine & research work .
Excellent for cutting serial sections.
14. PARTS OF THE ROTARY MICROTOMES
• Block holder
• Knife clamp screw
• Knife clamps
• Block adjustment
• Thickness gauge
• Angle of tilt adjustment
• Operating handle
15. • The feed mechanism is activated by turning a wheel on one side of
the machine.
• The wheel operation may be electrical / manual .
• The knife is fixed with its edge fixed upwards , while the object
moves against the knife .
• 1 rotation of the operating wheel produces , 1 complete cycle with a
downward cutting stroke & an upward return.
• It is often modified to cut ultrathin sections between 50Å – 200Å.
16. Advantages of the Rotary microtome
• Heavy & stable.
• Used for large number of serial sections .
• Ideal for Paraffin-embedded tissues .
• Movable knife holder .
• The sections cut are flat.
• It is useful for routine & research papers .
• Currently most common type of microtome in practice.
17. Base Sledge Microtome
• Large & heavy instrument .
• Very stable & popular for routine use .
• The feed mechanism has a fixed knife , beneath which , the object
mounted on a heavy sliding base moves .
• Mainly used for cutting sections of cellulose nitrate embedded tissues
with an obliquely set knife.
20. • The block holder is mounted on a steel carriage which slides backward
& forwards in groups against fixed horizontal knife .
• The block is raised towards the knife at a predetermined thickness.
Advantages of Base-sledge microtome
• Useful to cut extremely hard blocks & large sections ( eg : brain ).
• The microtome is heavy & stable.
• The knife used is sledge shaped which requires less honing.
22. • A simple machine invented by Sir Horace Darwin in 1881 .
• Developed by Cambridge company .
• The instrument has an arm that moves in a rocking motion while cutting the
sections.
• Hence named as Cambridge rocking microtome.
• The knife is held by means of microtome thread.
• The rocking microtome was designed primarily for cutting paraffin wax sections .
• When needed , it can also be used to cut frozen section tissues .
23. PARTS OF THE ROCKING MICROTOME
• Knife holder
• Block holder / chuck
• Upper & lower arms
• Screw
• Lever
• Pawl
• Ratchet wheel
• Mill head microtome screw
• Sleeve
• Scale
24.
25. • It cuts the sections between 1 - 20 microns.
• The knife is fixed with the edge.
• The object moves against the knife circularly , producing a sharply curved
surface to the block .
• With each stroke the tissue holder automatically moves vertically towards the
knife.
• Cutting stroke is Spring operated & is easy to handle .
• This microtome must be placed on a solid non-slippery surface to allow a
better hold .
26. Advantages of Cambridge rocking microtomes
• The cost of knife & microtome is low.
• Celloidin embedded tissues can be sectioned easily.
27. VIBRATING MICROTOMES
• Cuts using a vibrating blade .
• The resultant cut is made with less pressure.
• Used for difficult samples .
• Thickness around 30 -500nm for fresh tissues & 10 – 500 nm for
fixed tissues .
28.
29. SAW MICROTOME
• Used for hard tissues ( teeth & bones ).
• Has a recessed rotating saw which slices
through the sample .
• Minimal cutting thickness is 50 μm .
30. ULTRA MICROTOMES
• Designed to cut very thin sections .
• Parts :
Low power Binocular microscope .
object stage
• Has both manual / cutting actions.
31. How does it differ from conventional
microtomes ?
• Fine and precise feedback mechanism .
• Mechanical / thermally controlled
• Thickness range 5 – 100 nm
• Special knives - plate glass / plastic
• Can cut even small blocks .
• Can be used even in hard embedding media like araldite
32. Freezing Microtomes
• Designed for the preparing frozen sections of fluid & non-fluid tissue .
• Preliminary embedding is not needed .
• A cylinder of compressed carbon-dioxide for rapid cooling of the tissues is
connected with the object stage .
• Provisions to cool the knife is also present .
• The knife moves horizontally across the tissue surface .
• Characterstic features - Non-movable tissue block & cooling system.
33. Parts of freezing type microtome
• Knife clamps
• Operating handle
• Thickness gauge
• Stage
• Stage valve
• Coarse adjustment
34. Advantages of Freezing Microtome
• Used to cut sections for Rapid diagnosis .
• It cuts non-dehydrated fresh tissue in a frozen state.
• Used for Rapid histopathological diagnosis during surgery .
• This type of microtome is also used when lipids, enzymes, &
neurological structures are to be demonstrated.
35. DRAWBACKS OF FREEZING MICROTOME
• Inactivation of some enzymes
• Ribbon sections cannot be prepared using this microtome
• An inability to prepare multiple sections of uniform thickness .
Ultimately led to the invention of CRYOSTAT.
36. CRYOSTAT
• Pearse 1960 ; Hollands 1962 .
• Also called as Cold microtome / Deci-slicer in refrigerator .
• Device by which temperature can be maintained at low level .
• As described by Linderstorm – Lang and Mogeson ,
The cryostat consists of a microtome contained within a
refrigerated cabinet designed to operate at - 5 to - 30 ° C .
37. PROCEDURE
• Block tissue - up to 3 cm thick
Fresh tissue .
• Block is mounted in a small quantity of 1% glucose / water on the platform
of the chuck standing vertically on a bench .
• Immediately frozen - liquid carbon dioxide / solid carbon dioxide (dry ice).
• The chuck with frozen tissue is transferred to microtome maintained at -15 to
-20° C in the refrigerated chamber .
• The knife is moved & sections are cut at 4-5 μm with anti-roll guide plate
in position .
59. In earlier models of
freezing microtomes ,
temperature controlled
manually
In Cryostat ,
temperature control is
automatic .
60. I DON’T NEED TO CHECK
THE TEMPERATURE OR
TISSUE THICKNESS..
That’s a relief !!
61. DISADVANTAGES
• Complete defrosting of machine is required from time to time.
• Slow fixation reaction .
• Cannot be used for fixed tissues.
• Not useful if the tissue requies lower temperature than the
setting temperature of the machine .
• Morphological details less accurate.
• Pigment formation is too prominent.
• Dermatitis of hand & irritation of nostrils to the microtomist.
62. In simple words ,
Microtome cuts a tissue
of only one millimeter
into million pieces …
