Microtomy

Microtomy :Microtomy : Is the means by which tissue can be sectioned Is the means by which tissue can be sectioned

and attached to a surface for further microscopic and 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 slices Mechanical device for cutting thin uniform slices

of tissue – sections.of tissue – sections.

Types of microtomesTypes of microtomes

There are 5 basic types of microtomes named There are 5 basic types of microtomes named according to the mechanism-according to the mechanism-

Rocking microtome Rocking microtome Rotary microtome Rotary microtome Base sledge microtome Base sledge microtome Sliding microtome Sliding microtome Freezing mictotome.Freezing mictotome.

Rocking microtome:Rocking microtome:

Name derived from the rocking action of the cross Name derived from the rocking action of the cross

arm.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.

Mechanism of action:Mechanism of action: Knife is fixed, the block of the tissue moves Knife 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 the Between strokes the block is moved towards the

knife for the required thickness of sections by knife 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 the Steady backward and forward movement of the handle gives ribbons of good sections.handle gives ribbons of good sections.

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 flat ( Microtomes designed to cut perfectly flat sections; the block moving through an arc at right sections; the block moving through an arc at right angles to the knife edge are available.)angles to the knife edge are available.)

Light instrument : advisable to fit it into a tray Light instrument : advisable to fit it into a tray which is screwed to the bench , or to place it on a which 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.

Rotary microtomeRotary microtome

First machine designed by Professor Minot,hence First machine designed by Professor Minot,hence often referred to as the “Minot Rotary”.often referred to as the “Minot Rotary”.

Mechanism: Mechanism: The hand wheel rotates through 360 degree The hand wheel rotates through 360 degree

moving the specimen vertically past the cutting moving 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 which Block holder is mounted on a steel carriage which moves up and down in grooves and is advanced moves up and down in grooves and is advanced by a micrometer screw- cutting perfectly flat by a micrometer screw- cutting perfectly flat sections.sections.

ManualManual (completely manipulated by the operator).(completely manipulated by the operator). Semi-automated (one motor to advance either the Semi-automated (one motor to advance either the

fine or coarse hand –wheel) fine or coarse hand –wheel) Fully automated ( two motors that drive both the Fully 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 or Mechanism of block advancement: retracting or

non retracting.non retracting. Retracting action moves the tissue block away Retracting action moves the tissue block away

from the knife on upstroke, producing a flat face from the knife on upstroke, producing a flat face to the tissue block.to the tissue block.

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 of Ideal 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 when Large and heavier knife used-less vibration when

cutting hard tissue.cutting hard tissue. Heavier and more stable .Heavier and more stable .

Sledge microtomeSledge microtome

Originally designed for cutting sections of very Originally 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 and Especially useful in neuropathology and

ophthalmic pathology.ophthalmic pathology.

Mechanism of action:Mechanism of action:

TThe block holder is mounted on a steel carriage he block holder is mounted on a steel carriage which slides backwards and forwards on guides which slides backwards and forwards on guides against a fixed horizontal knife.against a fixed horizontal knife.

Advantages:Advantages: Heavy , very stable, not subject to vibration.Heavy , very stable, not subject to vibration. Knife large(24 cm in length) and usually wedge Knife large(24 cm in length) and usually wedge

shaped –less vibration .shaped –less vibration . Adjustable knife holding clamps allow tilt and Adjustable knife holding clamps allow tilt and

angle of the knife to the block to be easily set angle 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 obliquely setting the knife obliquely - paraffin wax embedded sections - paraffin wax embedded sections are more easily cut .are more easily cut .

DisadvantagesDisadvantages Slower in use than rocker or rotary microtome- Slower in use than rocker or rotary microtome-

true only when change from one instrument to true only when change from one instrument to another is made .another is made .

With practice, sections from routine paraffin With practice, sections from routine paraffin blocks can be cut as quickly as on any other type blocks can be cut as quickly as on any other type of microtome.of microtome.

Sliding microtomeSliding microtome

Designed for cutting celloidin-embedded tissue Designed for cutting celloidin-embedded tissue blocks.blocks.

The knife or blade is stationary, specimen slides The 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.

Freezing microtomeFreezing microtome

Gives best results for Gives best results for cutting frozen sections.cutting frozen sections.

Machine is clamped to Machine is clamped to the edge of a bench the edge of a bench and connected to a and connected to a cylinder of CO2 by cylinder of CO2 by means of a specially means of a specially strengthened flexible strengthened flexible metal tube.metal tube.

Freezing microtomeFreezing microtome

Knife freezing attachment is supplied with most Knife freezing attachment is supplied with most machines. machines.

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

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 ,ultrastructural Used in enzyme histochemistry ,ultrastructural histochemistry.histochemistry.

During sectioning, the tissue is immersed in During 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 thickness It 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 avoid Tissues are cut at a very slow speed to avoid disintegration.disintegration.

Microtome knivesMicrotome knives

Developed to fit specific types of microtomes and Developed to fit specific types of microtomes and cope with different degrees of hardness of tissues cope with different degrees of hardness of tissues and embedding media.and embedding media.

Paraffin-wax embedded tissues knives are made Paraffin-wax embedded tissues knives are made of steel.of steel.

Resin-embedded tissue is normally cut using Resin-embedded tissue is normally cut using glass knives.glass knives.

Knives are classified according to their shape Knives 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.

Wedge :Wedge :

Originally designed for cutting frozen sections Originally designed for cutting frozen sections Gives great rigidity to the knife Gives great rigidity to the knife Used for cutting all types of section on any Used for cutting all types of section on any

microtome.microtome.

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.

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 substantial With gradual adoption of more substantial

microtomes , this knife design has lost popularity.microtomes , this knife design has lost popularity.

Tool edge(D-profile):Tool edge(D-profile): Called ‘chisel edge’, similar to a woodworker’s Called ‘chisel edge’, similar to a woodworker’s

chisel.chisel. Used primarily to section exceptionally hard Used 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 added Stouter than conventional knives to give added

rigidity.rigidity. Edge may be coated with tungsten-carbide for Edge may be coated with tungsten-carbide for

increased life.increased life.

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 the Disposable blade holders incorporated into the

microtome or an adapter.microtome or an adapter.

Disposable bladesDisposable blades

Blade is coated with PTFE Blade 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 the Over-tightening the disposable blade in the clamping device may cause cutting artifact such clamping device may cause cutting artifact such as thick and thin sections.as thick and thin sections.

Glass and diamond knivesGlass and diamond knives

Used in electron microscopy and with Used in electron microscopy and with plastic resin-embedded blocks.plastic resin-embedded blocks.

Knife anglesKnife angles

Clearance angle:Clearance angle: angle formed by a line drawn along angle formed by a line drawn along the block surface and the lower bevel of the knife.the block surface and the lower bevel of the knife.

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

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 for Figures 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 form angle 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 Longer facets and smaller bevel would give rise

to a keener edge.to a keener edge.

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]

Abrasive grinding of the facets [HONING]Abrasive grinding of the facets [HONING]

Naturally occuring slabs of stone with varying Naturally 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 during Lubricated with soapy water or light oil during use.use.

Abrasive grinding of the facets Abrasive grinding of the facets [HONING] [HONING]

Glass plates:Glass plates: hand sharpening hand sharpening Readily available ,cheap Readily available ,cheap Surface roughened to enable particles of abrasive Surface 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 knife automatic knife

sharpening machines.sharpening machines. Expensive , superior propertiesExpensive , superior properties

AbrasivesAbrasives

Aluminium oxide(alumina)Aluminium oxide(alumina) Iron oxide(Jeweller’s rouge)Iron oxide(Jeweller’s rouge) Silicon carbideSilicon carbide DiamondDiamond

Manual methodManual method

Knife with a ‘back’ effectively raises the non Knife 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 with Back of the knife is ground simultaneously with the edge , hence reserved for use only with that the edge , hence reserved for use only with that particular knife. particular knife.

Manual methodManual method

Hone is placed on the bench on a non-skid Hone is placed on the bench on a non-skid surface (damp cloth) to prevent moving during surface (damp cloth) to prevent moving during honing.honing.

Small quantity of light oil or soapy water applied Small 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 on Knife with handle and backing sheath is laid on

the hone with cutting edge facing away from the the hone with cutting edge facing away from the operator , heel roughly in the centre of the operator , heel roughly in the centre of the nearest end of the hone.nearest end of the hone.

Manual methodManual method

Handle of the knife is held betweenHandle of the knife is held between the thumb the thumb and the forefinger .and the forefinger .

Thumb and forefinger of other hand rest on the Thumb and forefinger of other hand rest on the other end of the knife to ensure even pressure other 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 to Knife is pushed forward diagonally from heel to toe ,turned over on its back and moved across toe ,turned over on its back and moved across the hone until the heel is in the centre with the the hone until the heel is in the centre with the cutting edge leading and then brought back cutting edge leading and then brought back diagonally.diagonally.

It is then turned over on its back and moved It is then turned over on its back and moved across the hone to its original position completing across the hone to its original position completing figure of eight movement.figure of eight movement.

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 from Types 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 is Back 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 small Strops should be kept soft by applying a small

quantity of vegetable oil into the back of the quantity of vegetable oil into the back of the leather leather

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 wooden Single leather strop stretched over a wooden

frame to give a standard tension or a block of frame to give a standard tension or a block of wood about 12x2x2 inches in size having a wood about 12x2x2 inches in size having a handle at one end with four grades of leather or handle 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 the The sides of these strops are numbered and the knife is stropped on No1, then No2 and so on knife is stropped on No1, then No2 and so on finishing on the finest leather.finishing on the finest leather.

Automatic knife sharpnersAutomatic knife sharpners

Automatic knife sharpnersAutomatic knife sharpners

Two basic designs available.Two basic designs available. 1) knife is held vertically with revolving sharpening 1) knife is held vertically with revolving sharpening

wheels grinding the cutting edge.wheels grinding the cutting edge. 2) knife is held horizontally against the surface of 2) knife is held horizontally against the surface of

a slowly rotating flat plate.a slowly rotating flat plate.

Automatic knife sharpnersAutomatic knife sharpners

Plates – glass , copper or bronze charged with an Plates – glass , copper or bronze charged with an abrasive.abrasive.

Glass plates need to be roughened before use to Glass plates need to be roughened before use to allow the abrasive particles to be held more allow the abrasive particles to be held more easily in place.easily in place.

Copper and bronze plates used in conjunction Copper and bronze plates used in conjunction with diamond paste, 6micrometer particle size with diamond paste, 6micrometer particle size being most appropriate for rough sharpening, and being most appropriate for rough sharpening, and 1 micrometer for fine polishing.1 micrometer for fine polishing.

StroppingStropping Technique:Technique:

Knife is laid on the near end of the strop with the Knife is laid on the near end of the strop with the cutting edge towards the operator (opposite cutting edge towards the operator (opposite direction to that used in honing.)direction to that used in honing.)

Knife held with forefinger and thumb to facilitate Knife 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 in Action is exact opposite to that used in honing,using full length of the strop and stropping honing,using full length of the strop and stropping evenly the whole of the blade.evenly the whole of the blade.

Microtomy- paraffin waxMicrotomy- paraffin wax

Factors involved in producing good paraffin-wax Factors involved in producing good paraffin-wax sections :sections :

Temperature:Temperature: Tissues are more easily sectioned at a lower Tissues are more easily sectioned at a lower

temperature than that of the atmosphere.temperature than that of the atmosphere. Lowering temperature brings tissues of differing Lowering temperature brings tissues of differing

composition to a more uniform composition to a more uniform consistency,degree of hardness-ensures a consistency,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).

Knife angleKnife angle

Greater the rake angle(flatter the knife)more Greater 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 softer Higher rake angles are more suitable to softer tissues tissues

Lower rake angles for harder tissues.Lower rake angles for harder tissues.

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, compression If sections are cut at too fast speed, compression

will become more marked.will become more marked. If cut too slowly, difficult to maintain the rhythmic If cut too slowly, difficult to maintain the rhythmic

action required.action required.

SlantSlant

Commonly used to refer to the relationship of the Commonly used to refer to the relationship of the knife edge to the block when cutting knife edge to the block when cutting nitrocellulose-embedded tissue on a sliding nitrocellulose-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 Resistance to cutting force is applied more

gently.gently.

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.

Cutting techniqueCutting technique

Insert apprropriate knife in the knife-holder of the Insert 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 the Move 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 will Ensure that the whole surface of the block will

move parallel to the edge of the knife, move parallel to the edge of the knife,

Cutting techniqueCutting technique

Trim the excess wax from the block surface and Trim the excess wax from the block surface and expose the tissue,advance the block by setting expose 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 to B) tissue in the block may be torn giving rise to

considerable artefact.considerable artefact. C) unsuspected small foci of calcification may C) 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.

Once the surface of tissue has been revealed Once 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 and Replace 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 the Bring the block face up until it nearly touches the

knife edge.knife edge.

Paraffin-wax embedded tissue , sections are Paraffin-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) :demonstrate Thicker 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 examining Thin 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 determined The amount of advance is operator determined most commonly in graduated 1 micrometer most commonly in graduated 1 micrometer stages.stages.

Paraffin wax embedded tissue: the properties of Paraffin wax embedded tissue: the properties of the wax causes each section to adhere by its the wax causes each section to adhere by its edge to the previous forming a ribbon of sections edge to the previous forming a ribbon of sections Ribbons should be held gently with a fine Ribbons 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 be Holding the ribbons with the finger is to be discouraged : section and water bath may discouraged : section and water bath may become contaminated with the operator’s become contaminated with the operator’s exfoliated squames.exfoliated squames.

Before being attached to the slides the creases Before 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.

Flotation (water bath)Flotation (water bath)

Flotation(water bath)Flotation(water bath)

Thermostatically controlled water baths for floating Thermostatically 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 10 Temperature of water in the bath should be 10

degree celcius below the melting point of paraffin degree celcius below the melting point of paraffin employed.employed.

Distilled water may be used to prevent water Distilled 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 added Alcohol or a small drop of detergent may be added to the water to reduce the surface tension-to flatten to the water to reduce the surface tension-to flatten out the sections with ease.out the sections with ease.

Flotation(water bath)Flotation(water bath)

Sections which are curled will flatten on warm Sections which are curled will flatten on warm water, creases removed.water, creases removed.

To remove air bubble, thick sections of wax which To remove air bubble, thick sections of wax which curl into a roll during trimming are used. Hold one curl into a roll during trimming are used. Hold one roll in the end of a pair of forceps and bring the roll in the end of a pair of forceps and bring the end of the wax roll up under the section to touch end of the wax roll up under the section to touch the air bubble.The bubble will adhere to the wax the air bubble.The bubble will adhere to the wax roll and come away with it when removed. roll and come away with it when removed.

Mounting the section on a slide:Mounting the section on a slide:

A clean slide is half submerged in water and A 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, straight Section 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 of Section oriented on wet slide using the edge of the forceps or dissecting needle.the forceps or dissecting needle.

Section should be centrally positioned on the Section should be centrally positioned on the slide.slide.

Slide should be identified by inscribing the Slide should be identified by inscribing the appropriate no.on the slide with a diamond appropriate no.on the slide with a diamond pencil.pencil.

Drying oven or hot plateDrying oven or hot plate

Drying oven or hot plateDrying oven or hot plate

Drying oven :Drying oven : Mounted section placed in an oven at 50degree Mounted 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 transferred Slide complete with section may be transferred

directly to the surface of the hot plate maintained directly to the surface of the hot plate maintained at a temperature of 55-60 degree celcius.and left at a temperature of 55-60 degree celcius.and left for 15 min.for 15 min.

Section left face up until water evaporates then Section 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.

Brush and forcepsBrush and forceps Forceps, brushes or teasing needles for removal Forceps, brushes or teasing needles for removal

of folds, creases and bubbles that may form of folds, creases and bubbles that may form during the floating out of the section on water during the floating out of the section on water bath.bath.

Manipulating the section as it passes acrosss the Manipulating the section as it passes acrosss the edge of the blade.edge of the blade.

SlidesSlides For normal routine work, 76x25 mm slides For 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 routinely Chemical resistant pens and pencils routinely

used to label the slide.used to label the slide.

Section adhesivesSection adhesives

Sections may detach from the slides:Sections may detach from the slides: Exposure to strong alkali solutions during Exposure to strong alkali solutions during

staining.staining. Cryostat sections for immunofluorescense, Cryostat sections for immunofluorescense,

immunohistochemistry ,or intraoperative immunohistochemistry ,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.

For sections from ester or polyester –wax For sections from ester or polyester –wax embedded tissue , adhesives are mandatory.embedded tissue , adhesives are mandatory.

AlbumenAlbumen Equal parts of glycerin,distilled water and egg Equal parts of glycerin,distilled water and egg

white are mixed filtered through coarse filter white are mixed filtered through coarse filter paper.paper.

A crystal of thymol is added to inhibit the growth A 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 the Small quantity of the solution is smeared over the surface of the slide immediately before mounting surface of the slide immediately before mounting sections from the water bath.sections from the water bath.

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 to Liable 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 the Clean 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 rigid As section dries ,resin polymerizes forming a rigid

bond between tissue and slide.bond between tissue and slide.

Starch:Starch: Successful adhesive .Successful adhesive . Lost popularity due to staining reactivity with Lost 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 10 As 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.

3-aminopropyltriethoxysilane(APES):3-aminopropyltriethoxysilane(APES): Slides dipped in 2% solution of APES in Slides dipped in 2% solution of APES in

acetone,drained,dipped in acetone, drained acetone,drained,dipped in acetone, drained again.again.

The process is complete when the slides are The process is complete when the slides are dipped in distilled water.dipped in distilled water.

Useful for cytology,for specimens that may be Useful for cytology,for specimens that may be bloody or contain proteinaceous material.bloody or contain proteinaceous material.

Charged or plus slides:Charged or plus slides: slides manufactured slides manufactured with a permanent positive charge.with a permanent positive charge.

Coating slide with basic polymer in which a Coating slide with basic polymer in which a chemical reaction occurs leaving the amino chemical reaction occurs leaving the amino groups linked by covalent bonds to silicon atoms groups linked by covalent bonds to silicon atoms of the glass.of the glass.

Superior in their resistance to cell and tissue loss Superior in their resistance to cell and tissue loss during staining.during staining.

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 adjusted Sharp knife is rigidly held in a properly adjusted

microtome.microtome.

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.

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 block for Mollifex(British drug houses Ltd)soak the block for

30-60 minutes 30-60 minutes

Fragmentation of tissue:Fragmentation of tissue: Blood clots and lymphoid tissues :Blood clots and lymphoid tissues :

increasing the rake increasing the rake angle. coating the block angle. coating the block with celloidin by a camel hair brush in between with celloidin by a camel hair brush in between the sections.the sections.

Serial sectionsSerial sections

Necessary to cut and preserve every section from Necessary 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 a Presence 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.

Problems and solutions for paraffin section.Problems and solutions for paraffin section. Problem: Problem:

Ribbon/consecutive Ribbon/consecutive sections curved.sections curved.

1)1) Block edges not Block edges not parallelparallel

2)2) Dull blade edge.Dull blade edge.3)3) Excessive paraffin.Excessive paraffin.

4)4) Tissue varying in Tissue varying in consistencyconsistency

Solutions Solutions

1)1) Trim block until Trim block until parallel.parallel.

2)2) Replace balde.Replace balde.3)3) Trim away excess Trim away excess

paraffin.paraffin.4)4) Re-orient blockRe-orient block

Problem: Thick and thin Problem: Thick and thin sectionssections

1)1) Paraffin too soft for Paraffin too soft for tissuetissue

2)2) Insufficient clearance Insufficient clearance angleangle

3)3) Faulty microtome Faulty microtome mechanismsmechanisms

4)4) Blade or block loose in Blade or block loose in holder.holder.

SolutionsSolutions

1)1) Remove excess Remove excess paraffinparaffin

2)2) Increase clearance Increase clearance angle. angle.

3)3) Check for faults in Check for faults in microtome.microtome.

4)4) Tighten block and Tighten block and bladeblade

Problem:Problem: Sections will not form Sections will not form

ribbonsribbons

1)1) Paraffin too hard for Paraffin too hard for sectioning.sectioning.

2)2) Debris on knife edge.Debris on knife edge.3)3) Clearance angle Clearance angle

incorrect.incorrect.

SolutionsSolutions

1)1) Re-embed in lower Re-embed in lower melting point melting point paraffin.paraffin.

2)2) Warm surface of Warm surface of blockblock

3)3) Clean blade and back Clean blade and back of blade holderof blade holder

4)4) Adjust to optimal Adjust to optimal angle.angle.

Problem: Problem: sections attach to sections attach to block on return block on return strokestroke

1)1) Insufficient clearance Insufficient clearance angle.angle.

2)2) Debris on blade Debris on blade edge.edge.

3)3) Debris on block Debris on block edge.edge.

4)4) Static electricity on Static electricity on ribbon.ribbon.

SolutionsSolutions

1)1) Increase clearance Increase clearance angle.angle.

2)2) Clean blade edge.Clean blade edge.3)3) Trim edges of blockTrim edges of block4)4) Humidify the air Humidify the air

around the around the microtome.microtome.

5)5) Place static guard or Place static guard or dryer sheets near dryer sheets near microtome.microtome.

Problem: Problem: incomplete sectionincomplete section

1)1) Incomplete Incomplete impregnation of impregnation of tissue with paraffin.tissue with paraffin.

2)2) Tissue incorrectly Tissue incorrectly embedded.embedded.

3)3) Sections superficially Sections superficially cut.cut.

SolutionsSolutions

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

2)2) Re-embed Re-embed tissue;make sure tissue;make sure orientation is correct orientation is correct and tissue is flat in and tissue is flat in mould.mould.

3)3) Re-face block,cut Re-face block,cut deeper into the deeper into the tissue.tissue.

Problem: Problem: chatter-thick and thin chatter-thick and thin zones parallel to zones parallel to blade edgeblade edge

1)1) Knife or block loose Knife or block loose in holderin holder

2)2) Excessive knife tiltExcessive knife tilt3)3) Paraffin too hard for Paraffin too hard for

sectioning.sectioning.4)4) Calcified areas in Calcified areas in

tissue.tissue.5)5) Overhydration of Overhydration of

tissue.tissue.6)6) Dull blade.Dull blade.

SolutionsSolutions

1)1) Clean blade edge to Clean blade edge to remove excess remove excess paraffin.paraffin.

2)2) Replace or use new Replace or use new area of blade.area of blade.

3)3) Tighten the blade Tighten the blade levers.levers.

4)4) Reduce angle.Reduce angle.5)5) Rehydrate .Rehydrate .6)6) Re-embed in fresh Re-embed in fresh

paraffin.paraffin.

Problem:Problem: Splitting of sections at Splitting of sections at

right angles to knife right angles to knife edgeedge

1)1) Nicks in blade.Nicks in blade.2)2) Hard particles in Hard particles in

tissue.tissue.3)3) Hard particles in Hard particles in

paraffin.paraffin.

SolutionsSolutions

1)1) Use different part of Use different part of blade or replace.blade or replace.

2)2) Calcium deposit-Calcium deposit-surface decal.surface decal.

3)3) Mineral or other Mineral or other particle-remove with particle-remove with fine sharp pointed fine sharp pointed forcepsforceps

References References Cellular pathology technique – C.F.A.Culling.Cellular pathology technique – C.F.A.Culling. Histological techniques- J.D.Bancroft.Histological techniques- J.D.Bancroft.