4-microtomy

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Microtomy :

• Is the means by which tissue can be sectioned and attached to


a surface for further microscopic examination.

Microtome:
• Basic instrument used in microtomy.
• Mechanical device for cutting thin uniform slices of tissue –
sections.
Basic Parts of a Microtome
Types of microtomes
• There are several types of microtomes named according
to the mechanism.
• Each designed for a specific purpose.

• Rocking microtome
• Base sledge microtome
• Sliding microtome
• Freezing microtome
• Ultramicrotome
• Rotary microtome
Rocking microtome:
• Name derived from the rocking action of the cross arm.
• The retracting action moves the tissue block away from
the knife on the upstroke
• Oldest in design, cheap , simple to use.
• Extremely reliable.
• Very minimum maintenance.
Mechanism of action:
• Knife is fixed, the block of the tissue moves through an
arc to strike the knife.
• Between strokes the block is moved towards the knife for
the required thickness of sections by means of a ratchet
operated micrometer thread.
• Steady backward and forward movement of the handle
gives ribbons of good sections.
Disadvantage:
• Size of the block that can be cut is limited.
• Sections are cut in a curved plane: (
Microtomes designed to cut perfectly flat sections; the block
moving through an arc at right angles to the knife edge are
available.)
• Light instrument : advisable to fit it into a tray which is
screwed to the bench , or to place it on a damp cloth to avoid
movement during cutting.
Base Sledge Microtome
• Originally designed for cutting sections of very large
blocks of tissue (eg. whole brains )
• Used primarily for large blocks, hard tissues, whole
mounts (eg. Prostate gland).
• Especially useful in neuropathology and ophthalmic
pathology.
Mechanism of action:

• The block holder is mounted on a steel carriage which


slides backwards and forwards on guides against a fixed
horizontal knife.
Advantages:
• Heavy , very stable, not subject to vibration.
• Knife large(24 cm in length) and usually wedge shaped –less
vibration .
• Adjustable knife holding clamps allow tilt and angle of the
knife to the block to be easily set– used for cutting celloidin
sections by setting the knife obliquely
- paraffin wax embedded sections are more easily cut .
Disadvantages
• Slower in use than rocker or rotary microtome- true only
when change from one instrument to another is made .
• With practice, sections from routine paraffin blocks can
be cut as quickly as on any other type of microtome.
Sliding microtome

• Designed for cutting celloidin-embedded tissue blocks.


• The knife or blade is stationary, specimen slides under it
during sectioning.
• Also used for paraffin –wax embedded sections.
Freezing microtome

• Gives best results for


cutting frozen sections.
• Machine is clamped to
the edge of a bench and
connected to a cylinder
of CO2 by means of a
specially strengthened
flexible metal tube.
Freezing microtome

• Knife freezing attachment is supplied with most


machines.
• Separately controlled flow of CO2 on the edge of the
knife - to delay the thawing of sections on the knife and
make it possible to transfer them directly from knife to
slides.
• Sections thickness gauge is graduated in units of 5
micrometer instead of 1micrometer.
Vibrating microtome

• Designed to cut tissue which has not been fixed,


processed or frozen.
• Used in enzyme histochemistry ,ultrastructural
histochemistry.
• During sectioning, the tissue is immersed in either water
,saline or fixative.
• It is cut by a vibrating razor blade , at thickness generally
greater than used for paraffin wax.
• Tissues are cut at a very slow speed to avoid
disintegration.
Rotary microtome

• First machine designed by Professor Minot, hence often


referred to as the “Minot Rotary”.
Mechanism:
• The hand wheel rotates through 360 degree moving the
specimen vertically past the cutting surface and
returning it to the starting position.
• Block holder is mounted on a steel carriage which moves
up and down in grooves and is advanced by a
micrometer screw- cutting perfectly flat sections.
• Manual (completely manipulated by the operator).
• Semi-automated (one motor to advance either the fine
or coarse hand –wheel)
• Fully automated ( two motors that drive both the fine
and the coarse advace hand-wheel)
• Mechanism of block advancement: retracting or non
retracting.
• Retracting action moves the tissue block away from the
knife on upstroke, producing a flat face to the tissue
block.
Advantages:
• Ability to cut thin 2-3 mm sections.
• Easy adaptation to all types of tissues ( hard, fragile, or
fatty) sectioning.
• Ideal for cutting serial sections: large number of sections
from each block.
• Cutting large blocks
• Cutting angle of knife is adjustable.
• Large and heavier knife used-less vibration when cutting
hard tissue.
• Heavier and more stable .
Microtome knives

• Developed to fit specific types of microtomes and cope


with different degrees of hardness of tissues and
embedding media.
• Paraffin-wax embedded tissues knives are made of steel.
• Resin-embedded tissue is normally cut using glass knives.
Knives are classified according to their shape when
viewed in profile as:
• Wedge.
• Planoconcave.
• Biconcave.
• Tool edge or D profile.
Disposable blades

• Blade is coated with PTFE (polytetrafluoroethylene)


allowing ribbons to be sectioned with ease.
• Over-tightening the disposable blade in the clamping
device may cause cutting artifact such as thick and thin
sections.
Glass and diamond knives

• Used in electron microscopy.


• Tissue sections that are embedded in plastic
materials such as methacrylate, araldite, or
epon.
Knife angles

• Clearance angle: angle formed by a line drawn along the


block surface and the lower bevel of the knife.
• Rake angle: angle between the upper bevel of the knife
and a line at 90 degrees to the block surface.

Angles asociated with the knife edge.


A:rake angle; b:bevel ; c:clearance angle.
• Sharpness or acuity: reflection of light by the knife edge
when viewed under the microscope.
• Figures of 0.3and 0.1 micrometer – necessary for
maximal acuity or sharpness.
• Bevel angle: angle of knife’s facets which meet to form
the edge.
• Vary between 15 and 35 degrees.
• Longer facets and smaller bevel would give rise to a
keener edge.
Microtome knife sharpening

• Manual procedure or automatic procedure.

• 1) Abrasive grinding of the facets [HONING]


• 2)Polishing [STROPPING]
Microtomy- paraffin wax

• Factors involved in producing good paraffin-wax sections


:
Temperature:
• Tissues are more easily sectioned at a lower temperature
than that of the atmosphere.
• Lowering temperature brings tissues of differing
composition to a more uniform consistency,degree of
hardness-ensures a uniform cutting process.
• Blocks are cooled by keeping , face down on ice-tray (2-
3min).
Knife angle

• Greater the rake angle(flatter the knife)more likely is a


smooth plastic flow type cutting action.
• Higher rake angles are more suitable to softer tissues
• Lower rake angles for harder tissues.
Speed of cutting

• Soft tissues are cut more easily at a slow speed.


• Hard tissues are cut easily at a little fast rate.
• If sections are cut at too fast speed, compression will
become more marked.
• If cut too slowly, difficult to maintain the rhythmic action
required.
Slant

• Commonly used to refer to the relationship of the knife


edge to the block when cutting nitrocellulose-embedded
tissue on a sliding microtome.
• Advantages: larger area of the edge is employed.
• Resistance to cutting force is applied more gently.
Paraffin Section Cutting
• Equipment required:
• Microtome.
• Flotation(water bath)
• Slide drying oven or hot plate
• Fine pointed or curved forceps.
• Sable or camel haired brush.
• Scalpel.
• Slide rack.
• Clean slides.
• Teasing needle.
• Ice tray.
• Chemical resistant pencil or pen.
Cutting technique

• Insert apprropriate knife in the knife-holder of the


microtome and screw it tightly in position.
• Correctly set the adjustable knife angles.
• Fix the block in the block holder of the microtome
• Move the block holder forward or upward until the
paraffin wax is almost touching the knife edge.
• Ensure that the whole surface of the block will move
parallel to the edge of the knife,
Cutting technique
• Trim the excess wax from the block surface
and expose the tissue, advance the block by
setting the thickness to about 15 micrometer.
• Care should be taken not to trim too coarsely
as
A)Small biopsies may be lost.
B) tissue in the block may be torn giving rise to
considerable artefact.
C) unsuspected small foci of calcification may
cause tears in the tissue and nicks in the
knife.
• Once the surface of tissue has been
revealed proceed to trim the next block.
• Replace the trimming edge by a sharp one
and check it is tightly secured.
• Reset the thickness gauge to 4-5
micrometer.
• Insert the block to be cut and tighten
securely.
• Bring the block face up until it nearly
touches the knife edge.
• Paraffin-wax embedded tissue sections are
normally cut at a thickness of 3-5 micrometer.
• Thicker sections(10-20 micrometer)
:demonstrate certain features of the central
nervous system.
• Thin sections(1-2 micrometer): for examining
highly cellular tissue such as lymph nodes.
• Paraffin wax embedded tissue: the properties
of the wax causes each section to adhere by its
edge to the previous forming a ribbon of
sections Ribbons should be held gently with a
fine moistened brush or with a pair of fine
forceps.
• Holding the ribbons with the finger is to be
discouraged : section and water bath may
become contaminated with the operator’s
exfoliated squames.
• Before being attached to the slides the creases
must be removed and the sections flattened.
• This is achieved by floating them on warm
water.
Flotation (water bath)
Flotation(water bath)
• Thermostatically controlled water baths for
floating out tissue ribbons after sectioning.
• To remove the creases and flatten the sections.
• Temperature of water in the bath should be 10
degree celsius below the melting point of paraffin
employed.
• Distilled water may be used to prevent water
bubbles from being trapped under the sections.
• Alcohol or a small drop of detergent may be
added to the water to reduce the surface tension-
to flatten out the sections with ease.
Flotation(water bath)
• Sections which are curled will flatten on
warm water, creases removed.
• To remove air bubble, thick sections of
wax which curl into a roll during trimming
are used.
• Hold one roll in the end of a pair of forceps
and bring the end of the wax roll up under
the section to touch the air bubble.
• The bubble will adhere to the wax roll and
come away with it when removed.
Mounting the section on a slide:

• A clean slide is half submerged in water and brought into


contact with the edge of the section.
• Section approached from the side, straight approach will push
the section away.
• Section oriented on wet slide using the edge of the forceps or
dissecting needle.
• Section should be centrally positioned on the slide.
• Slide should be identified by inscribing the appropriate no.on
the slide with a diamond pencil.
Drying oven or hot plate
Drying oven or hot plate

• Drying oven :
• Mounted section placed in an oven at 50 degree celsius
for 1 hour to dry.
• Hot plates:
• Slide complete with section may be transferred directly
to the surface of the hot plate maintained at a
temperature of 55-60 degree celsius and left for 15 min.
• Section left face up until water evaporates then turned
over to prevent dust settling.
• Small creases disappear as the section warms up.
Brush and forceps
• Forceps, brushes or teasing needles for removal of folds,
creases and bubbles that may form during the floating
out of the section on water bath.
• Manipulating the section as it passes acrosss the edge of
the blade.
Slides
• For normal routine work, 76x25 mm slides universally
used.
• Thickness :1-1.2mm,do not break as easily.
• Larger slides for tissues such as eyes or brain.
• Chemical resistant pens and pencils routinely used to
label the slide.
Section adhesives

Sections may detach from the slides:


• Exposure to strong alkali solutions during staining.
• Cryostat sections for immunofluorescense,
immunohistochemistry ,or intraoperative diagnosis.
• Central nervous system tissues.
• Decalcified tissues.
• Tissues containing blood and mucus.
• Sections submitted to extreme temperatures.
• For sections from ester or polyester –wax embedded
tissue , adhesives are mandatory.
Albumen
• Equal parts of glycerin,distilled water and egg white are
mixed filtered through coarse filter paper.
• A crystal of thymol is added to inhibit the growth of
moulds, solution kept in refrigerator.
• Small quantity of the solution is smeared over the
surface of the slide immediately before mounting
sections from the water bath.
Gelatin :
• May be used as a 0.5% solution in distilled water.
• Liable to be contaminated with moulds ,needs to be
melted with gentle heat before use.
Araldite:
• Clean slides are coated with 1 in 10 dilution of the resin
in acetone immediately before use.
• As section dries ,resin polymerizes forming a rigid bond
between tissue and slide.
Starch:
• Successful adhesive .
• Lost popularity due to staining reactivity with many dyes.
Poly-L-lysine :
• As 0.1% solution, diluted further for use 1 in 10 with
distilled water.
• Effectiveness diminishes in few days.
3-aminopropyltriethoxysilane(APES):
• Slides dipped in 2% solution of APES in
acetone,drained,dipped in acetone, drained again.
• The process is complete when the slides are dipped in
distilled water.
• Useful for cytology,for specimens that may be bloody or
contain proteinaceous material.
Charged or plus slides: slides manufactured with a
permanent positive charge.
• Coating slide with basic polymer in which a chemical
reaction occurs leaving the amino groups linked by
covalent bonds to silicon atoms of the glass.
• Superior in their resistance to cell and tissue loss during
staining.
Tissues cut reasonably well if
• Properly fixed
• Decalcified if necessary.
• Completely dehydrated.
• Cleared and impregnated with paraffin wax.
• Sharp knife is rigidly held in a properly adjusted
microtome.
Cutting dificult tissue

• Alternate sections being thick and thin.


• Only part of the tissue being cut.
• Sections extremely compressed.
• Divided into two groups:
• 1) tissue exceptionally hard and tough.
• 2) fragmentation of tissue occurs as it is cut.
Cutting dificult tissue

Hard tissues
• Decreasing the rake angle.
• Resharpening the knife.
• Softening agents: solution
of 4 % phenol,
• Mollifex(British drug houses Ltd)soak the block for 30-60
minutes
• Fragmentation of tissue:
• Blood clots and lymphoid tissues :
increasing the rake angle. coating
the block with celloidin by a camel hair brush in between
the sections.
Serial sections

Necessary to cut and preserve every section from a piece


of tissue or from a specific area .
Required:
• To identify small ulcer
• Presence of malignant cells tracking along a lymphatic or
neural sheath.
• Scarce organisms such as acid fast bacilli.
• In embryology.
Problems and Remedies for paraffin section.
Problem: Ribbon/consecutive Remedy
sections curved.
1) Block edges not parallel
2) Dull blade edge. 1) Trim block until parallel.
3) Excessive paraffin. 2) Replace balde.
3) Trim away excess paraffin.
4) Tissue varying in 4) Re-orient block
consistency
Problem: Thick and thin Remedy
sections

1) Paraffin too soft for tissue


1) Remove excess paraffin

2) Insufficient clearance angle


2) Increase clearance angle.
3) Faulty microtome
3) Check for faults in
mechanisms
microtome.
4) Blade or block loose in
4) Tighten block and blade
holder.
Problem: Remedy
Sections will not form
ribbons

1) Paraffin too hard for 1) Re-embed in lower


sectioning. melting point paraffin.
2) Debris on knife edge. 2) Clean blade and back of
blade holder
3) Clearance angle incorrect. 3) Adjust to optimal angle.
Problem: Remedy
sections attach to block
on return stroke
1) Insufficient clearance 1) Increase clearance angle.
angle.
2) Debris on blade edge. 2) Clean blade edge.
3) Debris on block edge. 3) Trim edges of block
4) Static electricity on ribbon. 4) Humidify the air around
the microtome.
Problem: incomplete Remedy
section

1) Incomplete impregnation 1) Re-process tissue block.


of tissue with paraffin. 2) Re-embed tissue;make
2) Tissue incorrectly sure orientation is correct
embedded. and tissue is flat in mould.
3) Sections superficially cut. 3) Re-face block,cut deeper
into the tissue.
Problem: Remedy
chatter-thick and thin
zones parallel to blade
edge
1) Knife or block loose in 1) Clean blade edge to
holder remove excess paraffin.
2) Excessive knife tilt 2) Replace or use new area
of blade.
3) Paraffin too hard for
sectioning. 3) Tighten the blade levers.
4) Calcified areas in tissue. 4) Reduce angle.
5) Overhydration of tissue. 5) Rehydrate .
6) Dull blade. 6) Re-embed in fresh
paraffin.
Problem: Remedy
Splitting of sections at right
angles to knife edge
1) Nicks in blade. 1) Use different part of blade
or replace.
2) Hard particles in tissue. 2) Calcium deposit-surface
decal.

3) Hard particles in paraffin. 3) Mineral or other particle-


remove with fine sharp
pointed forceps

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