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Microtomy
1.
2. Microtomy :Microtomy :
Is the means by which tissue can be sectionedIs the means by which tissue can be sectioned
and attached to a surface for further microscopicand attached to a surface for further microscopic
examination.examination.
Microtome:Microtome:
Basic instrument used in microtomy.Basic instrument used in microtomy.
Mechanical device for cutting thin uniform slicesMechanical device for cutting thin uniform slices
of tissue – sections.of tissue – sections.
3. Types of microtomesTypes of microtomes
There are 5 basic types of microtomes namedThere are 5 basic types of microtomes named
according to the mechanism-according to the mechanism-
Rocking microtomeRocking microtome
Rotary microtomeRotary microtome
Base sledge microtomeBase sledge microtome
Sliding microtomeSliding microtome
Freezing mictotome.Freezing mictotome.
5. Name derived from the rocking action of theName derived from the rocking action of the
cross arm.cross arm.
Oldest in degisn, cheap , simple to use.Oldest in degisn, cheap , simple to use.
Extremely reliable.Extremely reliable.
Very minimum maintenance.Very minimum maintenance.
6. Mechanism of action:Mechanism of action:
Knife is fixed, the block of the tissue movesKnife is fixed, the block of the tissue moves
through an arc to strike the knife.through an arc to strike the knife.
Between strokes the block is moved towards theBetween strokes the block is moved towards the
knife for the required thickness of sections byknife for the required thickness of sections by
means of a ratchet operated micrometer thread.means of a ratchet operated micrometer thread.
Steady backward and forward movement of theSteady backward and forward movement of the
handle gives ribbons of good sections.handle gives ribbons of good sections.
7. Disadvantage:Disadvantage:
Size of the block that can be cut is limited.Size of the block that can be cut is limited.
Sections are cut in a curved plane:Sections are cut in a curved plane:
( Microtomes designed to cut perfectly( Microtomes designed to cut perfectly
flat sections; the block moving through an arc atflat sections; the block moving through an arc at
right angles to the knife edge are available.)right angles to the knife edge are available.)
Light instrument : advisable to fit it into a trayLight instrument : advisable to fit it into a tray
which is screwed to the bench , or to place it on awhich is screwed to the bench , or to place it on a
damp cloth to avoid movement during cutting.damp cloth to avoid movement during cutting.
8. Rotary microtomeRotary microtome
First machine designed by Professor Minot,henceFirst machine designed by Professor Minot,hence
often referred to as the “Minot Rotary”.often referred to as the “Minot Rotary”.
9. Mechanism:Mechanism:
The hand wheel rotates through 360 degreeThe hand wheel rotates through 360 degree
moving the specimen vertically past the cuttingmoving the specimen vertically past the cutting
surface and returning it to the starting position.surface and returning it to the starting position.
Block holder is mounted on a steel carriage whichBlock holder is mounted on a steel carriage which
moves up and down in grooves and is advancedmoves up and down in grooves and is advanced
by a micrometer screw- cutting perfectly flatby a micrometer screw- cutting perfectly flat
sections.sections.
10.
ManualManual (completely manipulated by the(completely manipulated by the
operator).operator).
Semi-automated (one motor to advance eitherSemi-automated (one motor to advance either
the fine or coarse hand –wheel)the fine or coarse hand –wheel)
Fully automated ( two motors that drive both theFully automated ( two motors that drive both the
fine and the coarse advace hand-wheel)fine and the coarse advace hand-wheel)
Mechanism of block advancement: retracting orMechanism of block advancement: retracting or
non retracting.non retracting.
Retracting action moves the tissue block awayRetracting action moves the tissue block away
from the knife on upstroke, producing a flat facefrom the knife on upstroke, producing a flat face
to the tissue block.to the tissue block.
11. Advantages:Advantages:
Ability to cut thin 2-3 mm sections.Ability to cut thin 2-3 mm sections.
Easy adaptation to all types of tissues ( hard,Easy adaptation to all types of tissues ( hard,
fragile, or fatty) sectioning.fragile, or fatty) sectioning.
Ideal for cutting serial sections: large number ofIdeal for cutting serial sections: large number of
sections from each block.sections from each block.
Cutting large blocksCutting large blocks
Cutting angle of knife is adjustable.Cutting angle of knife is adjustable.
Large and heavier knife used-less vibration whenLarge and heavier knife used-less vibration when
cutting hard tissue.cutting hard tissue.
Heavier and more stable .Heavier and more stable .
13. Originally designed for cutting sections of veryOriginally designed for cutting sections of very
large blocks of tissue (eg. whole brains)large blocks of tissue (eg. whole brains)
Used primarily forUsed primarily for
Large blocks,hard tissues,whole mounts.Large blocks,hard tissues,whole mounts.
Especially useful in neuropathology andEspecially useful in neuropathology and
ophthalmic pathology.ophthalmic pathology.
14. Mechanism of action:Mechanism of action:
TThe block holder is mounted on a steel carriagehe block holder is mounted on a steel carriage
which slides backwards and forwards on guideswhich slides backwards and forwards on guides
against a fixed horizontal knife.against a fixed horizontal knife.
15. Advantages:Advantages:
Heavy , very stable, not subject to vibration.Heavy , very stable, not subject to vibration.
Knife large(24 cm in length) and usually wedgeKnife large(24 cm in length) and usually wedge
shaped –less vibration .shaped –less vibration .
Adjustable knife holding clamps allow tilt andAdjustable knife holding clamps allow tilt and
angle of the knife to the block to be easily setangle of the knife to the block to be easily set
– used for cutting celloidin sections by– used for cutting celloidin sections by
setting the knife obliquelysetting the knife obliquely
- paraffin wax embedded- paraffin wax embedded
sections are more easily cut .sections are more easily cut .
16. DisadvantagesDisadvantages
Slower in use than rocker or rotary microtome-Slower in use than rocker or rotary microtome-
true only when change from one instrument totrue only when change from one instrument to
another is made .another is made .
With practice, sections from routine paraffinWith practice, sections from routine paraffin
blocks can be cut as quickly as on any other typeblocks can be cut as quickly as on any other type
of microtome.of microtome.
17. Sliding microtomeSliding microtome
Designed for cutting celloidin-embedded tissueDesigned for cutting celloidin-embedded tissue
blocks.blocks.
The knife or blade is stationary, specimen slidesThe knife or blade is stationary, specimen slides
under it during sectioning.under it during sectioning.
Also used for paraffin –wax embedded sections.Also used for paraffin –wax embedded sections.
18. Freezing microtomeFreezing microtome
Gives best results forGives best results for
cutting frozencutting frozen
sections.sections.
Machine is clamped toMachine is clamped to
the edge of a benchthe edge of a bench
and connected to aand connected to a
cylinder of CO2 bycylinder of CO2 by
means of a speciallymeans of a specially
strengthened flexiblestrengthened flexible
metal tube.metal tube.
19. Freezing microtomeFreezing microtome
Knife freezing attachment is supplied with mostKnife freezing attachment is supplied with most
machines.machines.
Separately controlled flow of CO2 on the edge ofSeparately controlled flow of CO2 on the edge of
the knife - to delay the thawing of sections onthe knife - to delay the thawing of sections on
the knife and make it possible to transfer themthe knife and make it possible to transfer them
directly from knife to slides.directly from knife to slides.
Sections thickness gauge is graduated in units ofSections thickness gauge is graduated in units of
5 micrometer instead of 1micrometer.5 micrometer instead of 1micrometer.
20. Vibrating microtomeVibrating microtome
Designed to cut tissue which has not been fixed,Designed to cut tissue which has not been fixed,
processed or frozen.processed or frozen.
Used in enzyme histochemistry ,ultrastructuralUsed in enzyme histochemistry ,ultrastructural
histochemistry.histochemistry.
During sectioning, the tissue is immersed inDuring sectioning, the tissue is immersed in
either water ,saline or fixative.either water ,saline or fixative.
It is cut by a vibrating razor blade , at thicknessIt is cut by a vibrating razor blade , at thickness
generally greater than used for paraffin wax.generally greater than used for paraffin wax.
Tissues are cut at a very slow speed to avoidTissues are cut at a very slow speed to avoid
disintegration.disintegration.
21. Microtome knivesMicrotome knives
Developed to fit specific types of microtomes andDeveloped to fit specific types of microtomes and
cope with different degrees of hardness of tissuescope with different degrees of hardness of tissues
and embedding media.and embedding media.
Paraffin-wax embedded tissues knives are madeParaffin-wax embedded tissues knives are made
of steel.of steel.
Resin-embedded tissue is normally cut usingResin-embedded tissue is normally cut using
glass knives.glass knives.
22. Knives are classified according to their shapeKnives are classified according to their shape
when viewed in profile as:when viewed in profile as:
Wedge.Wedge.
Planoconcave.Planoconcave.
Biconcave.Biconcave.
Tool edge or D profile.Tool edge or D profile.
23. Wedge :Wedge :
Originally designed for cutting frozen sectionsOriginally designed for cutting frozen sections
Gives great rigidity to the knifeGives great rigidity to the knife
Used for cutting all types of section on anyUsed for cutting all types of section on any
microtome.microtome.
24. Plano-concave:Plano-concave:
Used primarily for cutting nitrocellulose –Used primarily for cutting nitrocellulose –
embedded tissues.embedded tissues.
Available with varying degrees of concavity.Available with varying degrees of concavity.
25. Biconcave :Biconcave :
Classical knife shapeClassical knife shape introduced by Heiffor.introduced by Heiffor.
Used with the rocking microtome.Used with the rocking microtome.
Relatively easy to sharpen.Relatively easy to sharpen.
Less rigid , prone to more vibrations.Less rigid , prone to more vibrations.
With gradual adoption of more substantialWith gradual adoption of more substantial
microtomes , this knife design has lost popularity.microtomes , this knife design has lost popularity.
26. Tool edge(D-profile):Tool edge(D-profile):
Called ‘chisel edge’, similar to a woodworker’sCalled ‘chisel edge’, similar to a woodworker’s
chisel.chisel.
Used primarily to section exceptionally hardUsed primarily to section exceptionally hard
tissue.tissue.
Decalcified dense cortical bone.Decalcified dense cortical bone.
Undecalcified bone.Undecalcified bone.
Stouter than conventional knives to give addedStouter than conventional knives to give added
rigidity.rigidity.
Edge may be coated with tungsten-carbide forEdge may be coated with tungsten-carbide for
increased life.increased life.
27. Disposable bladesDisposable blades
Used for routine microtomy and cryotomy.Used for routine microtomy and cryotomy.
Provide a sharp cutting edge, produce flawless 2-Provide a sharp cutting edge, produce flawless 2-
4 mm sections.4 mm sections.
Disposable blade holders incorporated into theDisposable blade holders incorporated into the
microtome or an adapter.microtome or an adapter.
28. Disposable bladesDisposable blades
Blade is coated with PTFEBlade is coated with PTFE
(polytetrafluoroethylene) allowing ribbons to be(polytetrafluoroethylene) allowing ribbons to be
sectioned with ease.sectioned with ease.
Over-tightening the disposable blade in theOver-tightening the disposable blade in the
clamping device may cause cutting artifact suchclamping device may cause cutting artifact such
as thick and thin sections.as thick and thin sections.
29. Glass and diamond knivesGlass and diamond knives
Used in electron microscopy and withUsed in electron microscopy and with
plastic resin-embedded blocks.plastic resin-embedded blocks.
30. Knife anglesKnife angles
Clearance angle:Clearance angle: angle formed by a line drawnangle formed by a line drawn
along the block surface and the lower bevel of thealong the block surface and the lower bevel of the
knife.knife.
Rake angle:Rake angle: angle between the upper bevel of theangle between the upper bevel of the
knife and a line at 90 degrees to the blockknife and a line at 90 degrees to the block
surface.surface.
Angles asociated with the knife edge.Angles asociated with the knife edge.
A:rake angle; b:bevel ; c:clearance angle.A:rake angle; b:bevel ; c:clearance angle.
31. Sharpness orSharpness or acuityacuity:reflection of light by the knife:reflection of light by the knife
edge when viewed under the microscope.edge when viewed under the microscope.
Figures of 0.3and 0.1 micrometer – necessary forFigures of 0.3and 0.1 micrometer – necessary for
maximal acuity or sharpness.maximal acuity or sharpness.
Bevel angle:Bevel angle: angle of facets which meet to formangle of facets which meet to form
the edge.the edge.
Vary between 15 and 35 degrees.Vary between 15 and 35 degrees.
Longer facets and smaller bevel would give rise toLonger facets and smaller bevel would give rise to
a keener edge.a keener edge.
32. Microtome knife sharpeningMicrotome knife sharpening
Manual procedure or automatic procedure.Manual procedure or automatic procedure.
1) Abrasive grinding of the facets [HONING]1) Abrasive grinding of the facets [HONING]
2)Polishing [STROPPING]2)Polishing [STROPPING]
33. Abrasive grinding of the facets [HONING]Abrasive grinding of the facets [HONING]
Naturally occuring slabs of stone with varyingNaturally occuring slabs of stone with varying
abrasive properties:abrasive properties:
Stones :Stones : belgian black vein and arkansas,belgian black vein and arkansas,
Aloxite and carborundum-composites.Aloxite and carborundum-composites.
Lubricated with soapy water or light oil duringLubricated with soapy water or light oil during
use.use.
34. Abrasive grinding of the facetsAbrasive grinding of the facets
[HONING][HONING]
Glass plates:Glass plates:
hand sharpeninghand sharpening
Readily available ,cheapReadily available ,cheap
Surface roughened to enable particles of abrasiveSurface roughened to enable particles of abrasive
to adhere to the glass .to adhere to the glass .
Easily cleaned after use.Easily cleaned after use.
Copper and bronze plates:Copper and bronze plates: automatic knifeautomatic knife
sharpening machines.sharpening machines.
Expensive , superior propertiesExpensive , superior properties
36. Manual methodManual method
Knife with a ‘back’ effectively raises the nonKnife with a ‘back’ effectively raises the non
cutting edge up off the hone.cutting edge up off the hone.
Back of the knife is ground simultaneously withBack of the knife is ground simultaneously with
the edge , hence reserved for use only with thatthe edge , hence reserved for use only with that
particular knife.particular knife.
37. Manual methodManual method
Hone is placed on the bench on a non-skidHone is placed on the bench on a non-skid
surface (damp cloth) to prevent moving duringsurface (damp cloth) to prevent moving during
honing.honing.
Small quantity of light oil or soapy water appliedSmall quantity of light oil or soapy water applied
to the hone and smeared over the surface.to the hone and smeared over the surface.
Abrasive is applied to the glass or metal plate.Abrasive is applied to the glass or metal plate.
Knife with handle and backing sheath is laid onKnife with handle and backing sheath is laid on
the hone with cutting edge facing away from thethe hone with cutting edge facing away from the
operator , heel roughly in the centre of theoperator , heel roughly in the centre of the
nearest end of the hone.nearest end of the hone.
38. Manual methodManual method
Handle of the knife is held betweenHandle of the knife is held between the thumbthe thumb
and the forefinger .and the forefinger .
Thumb and forefinger of other hand rest on theThumb and forefinger of other hand rest on the
other end of the knife to ensure even pressureother end of the knife to ensure even pressure
along the whole edge of the knife.along the whole edge of the knife.
Knife is pushed forward diagonally from heel toKnife is pushed forward diagonally from heel to
toe ,turned over on its back and moved acrosstoe ,turned over on its back and moved across
the hone until the heel is in the centre with thethe hone until the heel is in the centre with the
cutting edge leading and then brought backcutting edge leading and then brought back
diagonally.diagonally.
It is then turned over on its back and movedIt is then turned over on its back and moved
across the hone to its original position completingacross the hone to its original position completing
figure of eight movement.figure of eight movement.
39. StroppingStropping
Process of polishing an already fairly sharp edge.Process of polishing an already fairly sharp edge.
Types of strop: best strops made from hide fromTypes of strop: best strops made from hide from
the rump of the horse marked ‘shell horse’.the rump of the horse marked ‘shell horse’.
2 types: flexible(hanging) and rigid.2 types: flexible(hanging) and rigid.
Flexible type:Flexible type:
Back of the strop is made of canvas and isBack of the strop is made of canvas and is
intended to support the leather during stropping.intended to support the leather during stropping.
Strops should be kept soft by applying a smallStrops should be kept soft by applying a small
quantity of vegetable oil into the back of thequantity of vegetable oil into the back of the
leatherleather
40. StroppingStropping
Strops should be kept free from grit and dust.Strops should be kept free from grit and dust.
Rigid type:Rigid type:
Single leather strop stretched over a woodenSingle leather strop stretched over a wooden
frame to give a standard tension or a block offrame to give a standard tension or a block of
wood about 12x2x2 inches in size having awood about 12x2x2 inches in size having a
handle at one end with four grades of leather orhandle at one end with four grades of leather or
even a soft stone cemented on each side.even a soft stone cemented on each side.
The sides of these strops are numbered and theThe sides of these strops are numbered and the
knife is stropped on No1, then No2 and so onknife is stropped on No1, then No2 and so on
finishing on the finest leather.finishing on the finest leather.
42. Automatic knife sharpnersAutomatic knife sharpners
Two basic designs available.Two basic designs available.
1) knife is held vertically with revolving1) knife is held vertically with revolving
sharpening wheels grinding the cutting edge.sharpening wheels grinding the cutting edge.
2) knife is held horizontally against the surface of2) knife is held horizontally against the surface of
a slowly rotating flat plate.a slowly rotating flat plate.
43. Automatic knife sharpnersAutomatic knife sharpners
Plates – glass , copper or bronze charged with anPlates – glass , copper or bronze charged with an
abrasive.abrasive.
Glass plates need to be roughened before use toGlass plates need to be roughened before use to
allow the abrasive particles to be held more easilyallow the abrasive particles to be held more easily
in place.in place.
Copper and bronze plates used in conjunctionCopper and bronze plates used in conjunction
with diamond paste, 6micrometer particle sizewith diamond paste, 6micrometer particle size
being most appropriate for rough sharpening, andbeing most appropriate for rough sharpening, and
1 micrometer for fine polishing.1 micrometer for fine polishing.
44. StroppingStropping Technique:Technique:
Knife is laid on the near end of the strop with theKnife is laid on the near end of the strop with the
cutting edge towards the operator (oppositecutting edge towards the operator (opposite
direction to that used in honing.)direction to that used in honing.)
Knife held with forefinger and thumb to facilitateKnife held with forefinger and thumb to facilitate
easy rotation at end of each stroke.easy rotation at end of each stroke.
Action is exact opposite to that used inAction is exact opposite to that used in
honing,using full length of the strop andhoning,using full length of the strop and
stropping evenly the whole of the blade.stropping evenly the whole of the blade.
45. Microtomy- paraffin waxMicrotomy- paraffin wax
Factors involved in producing good paraffin-waxFactors involved in producing good paraffin-wax
sections :sections :
Temperature:Temperature:
Tissues are more easily sectioned at a lowerTissues are more easily sectioned at a lower
temperature than that of the atmosphere.temperature than that of the atmosphere.
Lowering temperature brings tissues of differingLowering temperature brings tissues of differing
composition to a more uniformcomposition to a more uniform
consistency,degree of hardness-ensures aconsistency,degree of hardness-ensures a
uniform cutting process.uniform cutting process.
Blocks areBlocks are cooled by keeping , face down on ice-cooled by keeping , face down on ice-
tray (2-3min).tray (2-3min).
46. Knife angleKnife angle
Greater the rake angle(flatter the knife)moreGreater the rake angle(flatter the knife)more
likely is a smooth plastic flow type cutting action.likely is a smooth plastic flow type cutting action.
Higher rake angles are more suitable to softerHigher rake angles are more suitable to softer
tissuestissues
Lower rake angles for harder tissues.Lower rake angles for harder tissues.
47. Speed of cuttingSpeed of cutting
Soft tissues are cut more easily at a slow speed.Soft tissues are cut more easily at a slow speed.
Hard tissues are cut easily at a little fast rate.Hard tissues are cut easily at a little fast rate.
If sections are cut at too fast speed, compressionIf sections are cut at too fast speed, compression
will become more marked.will become more marked.
If cut too slowly, difficult to maintain theIf cut too slowly, difficult to maintain the
rhythmic action required.rhythmic action required.
48. SlantSlant
Commonly used to refer to the relationship of theCommonly used to refer to the relationship of the
knife edge to the block when cuttingknife edge to the block when cutting
nitrocellulose-embedded tissue on a slidingnitrocellulose-embedded tissue on a sliding
microtome.microtome.
Advantages: larger area of the edge is employed.Advantages: larger area of the edge is employed.
Resistance to cutting force is applied more gently.Resistance to cutting force is applied more gently.
49. Paraffin section cuttingParaffin section cutting
Equipment required:Equipment required:
Microtome.Microtome.
Flotation(water bath)Flotation(water bath)
Slide drying oven or hot plateSlide drying oven or hot plate
Fine pointed or curved forceps.Fine pointed or curved forceps.
Sable or camel haired brush.Sable or camel haired brush.
Scalpel.Scalpel.
Slide rack.Slide rack.
Clean slides.Clean slides.
Teasing needle.Teasing needle.
Ice tray.Ice tray.
Chemical resistant pencil or pen.Chemical resistant pencil or pen.
50. Cutting techniqueCutting technique
Insert apprropriate knife in the knife-holder of theInsert apprropriate knife in the knife-holder of the
microtome and screw it tightly in position.microtome and screw it tightly in position.
Correctly set the adjustable knife angles.Correctly set the adjustable knife angles.
Fix the block in the block holder of the microtomeFix the block in the block holder of the microtome
Move the block holder forward or upward until theMove the block holder forward or upward until the
paraffin wax is almost touching the knife edge.paraffin wax is almost touching the knife edge.
Ensure that the whole surface of the block willEnsure that the whole surface of the block will
move parallel to the edge of the knife,move parallel to the edge of the knife,
51. Cutting techniqueCutting technique
Trim the excess wax from the block surface andTrim the excess wax from the block surface and
expose the tissue,advance the block by settingexpose the tissue,advance the block by setting
the thickness to about 15 micrometer.the thickness to about 15 micrometer.
Care should be taken not to trim too coarsely asCare should be taken not to trim too coarsely as
A)Small biopsies may be lost.A)Small biopsies may be lost.
B) tissue in the block may be torn giving rise toB) tissue in the block may be torn giving rise to
considerable artefact.considerable artefact.
C) unsuspected small foci of calcification mayC) unsuspected small foci of calcification may
cause tears in the tissue and nicks in the knife.cause tears in the tissue and nicks in the knife.
52. Once the surface of tissue has been revealedOnce the surface of tissue has been revealed
proceed to trim the next block.proceed to trim the next block.
Replace the trimming edge by a sharp one andReplace the trimming edge by a sharp one and
check it is tightly secured.check it is tightly secured.
Reset the thickness gauge to 4-5 micrometer.Reset the thickness gauge to 4-5 micrometer.
Insert the block to be cut and tighten securely.Insert the block to be cut and tighten securely.
Bring the block face up until it nearly touches theBring the block face up until it nearly touches the
knife edge.knife edge.
53. Paraffin-wax embedded tissue , sections areParaffin-wax embedded tissue , sections are
normally cut at a thickness of 4-5 micrometer.normally cut at a thickness of 4-5 micrometer.
Thicker sections(10-20 micrometer) :demonstrateThicker sections(10-20 micrometer) :demonstrate
certain features of the central nervous system.certain features of the central nervous system.
Thin sections(1-2 micrometer): for examiningThin sections(1-2 micrometer): for examining
highly cellular tissue such as lymph nodes.highly cellular tissue such as lymph nodes.
The amount of advance is operator determinedThe amount of advance is operator determined
most commonly in graduated 1 micrometermost commonly in graduated 1 micrometer
stages.stages.
54. Paraffin wax embedded tissue: the properties ofParaffin wax embedded tissue: the properties of
the wax causes each section to adhere by itsthe wax causes each section to adhere by its
edge to the previous forming a ribbon of sectionsedge to the previous forming a ribbon of sections
Ribbons should be held gently with a fineRibbons should be held gently with a fine
moistened brush or with a pair of fine forceps.moistened brush or with a pair of fine forceps.
Holding the ribbons with the finger is to beHolding the ribbons with the finger is to be
discouraged : section and water bath maydiscouraged : section and water bath may
become contaminated with the operator’sbecome contaminated with the operator’s
exfoliated squames.exfoliated squames.
Before being attached to the slides the creasesBefore being attached to the slides the creases
must be removed and the sections flattened.must be removed and the sections flattened.
This is achieved by floating them on warm water.This is achieved by floating them on warm water.
56. Flotation(water bath)Flotation(water bath)
Thermostatically controlled water baths for floatingThermostatically controlled water baths for floating
out tissue ribbons after sectioning.out tissue ribbons after sectioning.
To remove the creases and flatten the sections.To remove the creases and flatten the sections.
Temperature of water in the bath should be 10Temperature of water in the bath should be 10
degree celcius below the melting point of paraffindegree celcius below the melting point of paraffin
employed.employed.
Distilled water may be used to prevent waterDistilled water may be used to prevent water
bubbles from being trapped under the sections.bubbles from being trapped under the sections.
Alcohol or a small drop of detergent may be addedAlcohol or a small drop of detergent may be added
to the water to reduce the surface tension-toto the water to reduce the surface tension-to
flatten out the sections with ease.flatten out the sections with ease.
57. Flotation(water bath)Flotation(water bath)
Sections which are curled will flatten on warmSections which are curled will flatten on warm
water, creases removed.water, creases removed.
To remove air bubble, thick sections of wax whichTo remove air bubble, thick sections of wax which
curl into a roll during trimming are used. Holdcurl into a roll during trimming are used. Hold
one roll in the end of a pair of forceps and bringone roll in the end of a pair of forceps and bring
the end of the wax roll up under the section tothe end of the wax roll up under the section to
touch the air bubble.The bubble will adhere to thetouch the air bubble.The bubble will adhere to the
wax roll and come away with it when removed.wax roll and come away with it when removed.
58. Mounting the section on a slide:Mounting the section on a slide:
A clean slide is half submerged in water andA clean slide is half submerged in water and
brought into contact with the edge of the section.brought into contact with the edge of the section.
Section approached from the side, straightSection approached from the side, straight
approach will push the section away.approach will push the section away.
Section oriented on wet slide using the edge ofSection oriented on wet slide using the edge of
the forceps or dissecting needle.the forceps or dissecting needle.
Section should be centrally positioned on theSection should be centrally positioned on the
slide.slide.
Slide should be identified by inscribing theSlide should be identified by inscribing the
appropriate no.on the slide with a diamondappropriate no.on the slide with a diamond
pencil.pencil.
60. Drying oven or hot plateDrying oven or hot plate
Drying oven :Drying oven :
Mounted section placed in an oven at 50degreeMounted section placed in an oven at 50degree
celcius for 1 hour to dry.celcius for 1 hour to dry.
Hot plates:Hot plates:
Slide complete with section may be transferredSlide complete with section may be transferred
directly to the surface of the hot plate maintaineddirectly to the surface of the hot plate maintained
at a temperature of 55-60 degree celcius.and leftat a temperature of 55-60 degree celcius.and left
for 15 min.for 15 min.
Section left face up until water evaporates thenSection left face up until water evaporates then
turned over to prevent dust settling.turned over to prevent dust settling.
Small creases disappear as the section warms up.Small creases disappear as the section warms up.
61. Brush and forcepsBrush and forceps
Forceps, brushes or teasing needles for removalForceps, brushes or teasing needles for removal
of folds, creases and bubbles that may formof folds, creases and bubbles that may form
during the floating out of the section on waterduring the floating out of the section on water
bath.bath.
Manipulating the section as it passes acrosss theManipulating the section as it passes acrosss the
edge of the blade.edge of the blade.
62. SlidesSlides
For normal routine work, 76x25 mm slidesFor normal routine work, 76x25 mm slides
universally used.universally used.
Thickness :1-1.2mm,do not break as easily.Thickness :1-1.2mm,do not break as easily.
Larger slides for tissues such as eyes or brain.Larger slides for tissues such as eyes or brain.
Chemical resistant pens and pencils routinelyChemical resistant pens and pencils routinely
used to label the slide.used to label the slide.
63. Section adhesivesSection adhesives
Sections may detach from the slides:Sections may detach from the slides:
Exposure to strong alkali solutions duringExposure to strong alkali solutions during
staining.staining.
Cryostat sections for immunofluorescense,Cryostat sections for immunofluorescense,
immunohistochemistry ,or intraoperativeimmunohistochemistry ,or intraoperative
diagnosis.diagnosis.
Central nervous system tissues.Central nervous system tissues.
Decalcified tissues.Decalcified tissues.
Tissues containing blood and mucus.Tissues containing blood and mucus.
Sections submitted to extreme temperatures.Sections submitted to extreme temperatures.
64. For sections from ester or polyester –waxFor sections from ester or polyester –wax
embedded tissue , adhesives are mandatory.embedded tissue , adhesives are mandatory.
AlbumenAlbumen
Equal parts of glycerin,distilled water and eggEqual parts of glycerin,distilled water and egg
white are mixed filtered through coarse filterwhite are mixed filtered through coarse filter
paper.paper.
A crystal of thymol is added to inhibit the growthA crystal of thymol is added to inhibit the growth
of moulds, solution kept in refrigerator.of moulds, solution kept in refrigerator.
Small quantity of the solution is smeared over theSmall quantity of the solution is smeared over the
surface of the slide immediately before mountingsurface of the slide immediately before mounting
sections from the water bath.sections from the water bath.
65. Gelatin :Gelatin :
May be used as a 0.5% solution in distilled water.May be used as a 0.5% solution in distilled water.
Liable to be contaminated with moulds ,needs toLiable to be contaminated with moulds ,needs to
be melted with gentle heat before use.be melted with gentle heat before use.
AralditeAraldite::
Clean slides are coated with 1 in 10 dilution of theClean slides are coated with 1 in 10 dilution of the
resin in acetone immediately before use.resin in acetone immediately before use.
As section dries ,resin polymerizes forming a rigidAs section dries ,resin polymerizes forming a rigid
bond between tissue and slide.bond between tissue and slide.
66. Starch:Starch:
Successful adhesive .Successful adhesive .
Lost popularity due to staining reactivity withLost popularity due to staining reactivity with
many dyes.many dyes.
Poly-L-lysine :Poly-L-lysine :
As 0.1% solution, diluted further for use 1 in 10As 0.1% solution, diluted further for use 1 in 10
with distilled water.with distilled water.
Effectiveness diminishes in few days.Effectiveness diminishes in few days.
67. 3-aminopropyltriethoxysilane(APES):3-aminopropyltriethoxysilane(APES):
Slides dipped in 2% solution of APES inSlides dipped in 2% solution of APES in
acetone,drained,dipped in acetone, drainedacetone,drained,dipped in acetone, drained
again.again.
The process is complete when the slides areThe process is complete when the slides are
dipped in distilled water.dipped in distilled water.
Useful for cytology,for specimens that may beUseful for cytology,for specimens that may be
bloody or contain proteinaceous material.bloody or contain proteinaceous material.
68. Charged or plus slides:Charged or plus slides: slides manufacturedslides manufactured
with a permanent positive charge.with a permanent positive charge.
Coating slide with basic polymer in which aCoating slide with basic polymer in which a
chemical reaction occurs leaving the aminochemical reaction occurs leaving the amino
groups linked by covalent bonds to silicon atomsgroups linked by covalent bonds to silicon atoms
of the glass.of the glass.
Superior in their resistance to cell and tissue lossSuperior in their resistance to cell and tissue loss
during staining.during staining.
69. Tissues cut reasonably well ifTissues cut reasonably well if
Properly fixedProperly fixed
Decalcified if necessary.Decalcified if necessary.
Completely dehydrated.Completely dehydrated.
Cleared and impregnated with paraffin wax.Cleared and impregnated with paraffin wax.
Sharp knife is rigidly held in a properly adjustedSharp knife is rigidly held in a properly adjusted
microtome.microtome.
70. Cutting dificult tissueCutting dificult tissue
Alternate sections being thick and thin.Alternate sections being thick and thin.
Only part of the tissue being cut.Only part of the tissue being cut.
Sections extremely compressed.Sections extremely compressed.
Divided into two groups:Divided into two groups:
1) tissue exceptionally hard and tough.1) tissue exceptionally hard and tough.
2) fragmentation of tissue occurs as it is cut.2) fragmentation of tissue occurs as it is cut.
71. Cutting dificult tissueCutting dificult tissue
Hard tissuesHard tissues
Decreasing the rake angle.Decreasing the rake angle.
Resharpening the knife.Resharpening the knife.
Softening agents:Softening agents:
solution of 4 % phenol,solution of 4 % phenol,
Mollifex(British drug houses Ltd)soak the blockMollifex(British drug houses Ltd)soak the block
for 30-60 minutesfor 30-60 minutes
72. Fragmentation of tissue:Fragmentation of tissue:
Blood clots and lymphoid tissues :Blood clots and lymphoid tissues :
increasing theincreasing the
rake angle. coating therake angle. coating the
block with celloidin by a camel hair brush inblock with celloidin by a camel hair brush in
between the sections.between the sections.
73. Serial sectionsSerial sections
Necessary to cut and preserve every section fromNecessary to cut and preserve every section from
a piece of tissue or from a specific area .a piece of tissue or from a specific area .
Required:Required:
To identify small ulcerTo identify small ulcer
Presence of malignant cells tracking along aPresence of malignant cells tracking along a
lymphatic or neural sheath.lymphatic or neural sheath.
Scarce organisms such as acid fast bacilli.Scarce organisms such as acid fast bacilli.
In embryology.In embryology.
74. Problems and solutions for paraffin section.Problems and solutions for paraffin section.
Problem:Problem:
Ribbon/consecutiveRibbon/consecutive
sections curved.sections curved.
1)1) Block edges notBlock edges not
parallelparallel
2)2) Dull blade edge.Dull blade edge.
3)3) Excessive paraffin.Excessive paraffin.
4)4) Tissue varying inTissue varying in
consistencyconsistency
SolutionsSolutions
1)1) Trim block untilTrim block until
parallel.parallel.
2)2) Replace balde.Replace balde.
3)3) Trim away excessTrim away excess
paraffin.paraffin.
4)4) Re-orient blockRe-orient block
75. Problem: Thick and thinProblem: Thick and thin
sectionssections
1)1) Paraffin too soft forParaffin too soft for
tissuetissue
2)2) Insufficient clearanceInsufficient clearance
angleangle
3)3) Faulty microtomeFaulty microtome
mechanismsmechanisms
4)4) Blade or block loose inBlade or block loose in
holder.holder.
SolutionsSolutions
1)1) Remove excessRemove excess
paraffinparaffin
2)2) Increase clearanceIncrease clearance
angle.angle.
3)3) Check for faults inCheck for faults in
microtome.microtome.
4)4) Tighten block andTighten block and
bladeblade
76. Problem:Problem:
Sections will not formSections will not form
ribbonsribbons
1)1) Paraffin too hard forParaffin too hard for
sectioning.sectioning.
2)2) Debris on knife edge.Debris on knife edge.
3)3) Clearance angleClearance angle
incorrect.incorrect.
SolutionsSolutions
1)1) Re-embed in lowerRe-embed in lower
melting pointmelting point
paraffin.paraffin.
2)2) Warm surface ofWarm surface of
blockblock
3)3) Clean blade and backClean blade and back
of blade holderof blade holder
4)4) Adjust to optimalAdjust to optimal
angle.angle.
77. Problem:Problem:
sections attach tosections attach to
block on returnblock on return
strokestroke
1)1) Insufficient clearanceInsufficient clearance
angle.angle.
2)2) Debris on bladeDebris on blade
edge.edge.
3)3) Debris on blockDebris on block
edge.edge.
4)4) Static electricity onStatic electricity on
ribbon.ribbon.
SolutionsSolutions
1)1) Increase clearanceIncrease clearance
angle.angle.
2)2) Clean blade edge.Clean blade edge.
3)3) Trim edges of blockTrim edges of block
4)4) Humidify the airHumidify the air
around thearound the
microtome.microtome.
5)5) Place static guard orPlace static guard or
dryer sheets neardryer sheets near
microtome.microtome.
78. Problem:Problem:
incomplete sectionincomplete section
1)1) IncompleteIncomplete
impregnation ofimpregnation of
tissue with paraffin.tissue with paraffin.
2)2) Tissue incorrectlyTissue incorrectly
embedded.embedded.
3)3) Sections superficiallySections superficially
cut.cut.
SolutionsSolutions
1)1) Re-process tissueRe-process tissue
block.block.
2)2) Re-embedRe-embed
tissue;make suretissue;make sure
orientation is correctorientation is correct
and tissue is flat inand tissue is flat in
mould.mould.
3)3) Re-face block,cutRe-face block,cut
deeper into thedeeper into the
tissue.tissue.
79. Problem:Problem:
chatter-thick andchatter-thick and
thin zones parallel tothin zones parallel to
blade edgeblade edge
1)1) Knife or block looseKnife or block loose
in holderin holder
2)2) Excessive knife tiltExcessive knife tilt
3)3) Paraffin too hard forParaffin too hard for
sectioning.sectioning.
4)4) Calcified areas inCalcified areas in
tissue.tissue.
5)5) Overhydration ofOverhydration of
tissue.tissue.
6)6) Dull blade.Dull blade.
SolutionsSolutions
1)1) Clean blade edge toClean blade edge to
remove excessremove excess
paraffin.paraffin.
2)2) Replace or use newReplace or use new
area of blade.area of blade.
3)3) Tighten the bladeTighten the blade
levers.levers.
4)4) Reduce angle.Reduce angle.
5)5) Rehydrate .Rehydrate .
6)6) Re-embed in freshRe-embed in fresh
paraffin.paraffin.
80. Problem:Problem:
Splitting of sections atSplitting of sections at
right angles to kniferight angles to knife
edgeedge
1)1) Nicks in blade.Nicks in blade.
2)2) Hard particles inHard particles in
tissue.tissue.
3)3) Hard particles inHard particles in
paraffin.paraffin.
SolutionsSolutions
1)1) Use different part ofUse different part of
blade or replace.blade or replace.
2)2) Calcium deposit-Calcium deposit-
surface decal.surface decal.
3)3) Mineral or otherMineral or other
particle-remove withparticle-remove with
fine sharp pointedfine sharp pointed
forcepsforceps
