Microtomy

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Microtomy

  1. 1. 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.
  2. 2. 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.
  3. 3. Rocking microtome:Rocking microtome:
  4. 4.  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.
  5. 5. 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.
  6. 6. 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.
  7. 7. 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”.
  8. 8. 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.
  9. 9.  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.
  10. 10. 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 .
  11. 11. Sledge microtomeSledge microtome
  12. 12.  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.
  13. 13. 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.
  14. 14. 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 .
  15. 15. 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.
  16. 16. 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.
  17. 17. 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.
  18. 18. 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.
  19. 19. 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.
  20. 20. 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.
  21. 21. 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.
  22. 22. 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.
  23. 23. 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.
  24. 24. 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.
  25. 25. 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.
  26. 26. 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.
  27. 27. 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.
  28. 28. 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.
  29. 29. 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.
  30. 30.  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.
  31. 31. 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]
  32. 32. 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.
  33. 33. 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
  34. 34. AbrasivesAbrasives  Aluminium oxide(alumina)Aluminium oxide(alumina)  Iron oxide(Jeweller’s rouge)Iron oxide(Jeweller’s rouge)  Silicon carbideSilicon carbide  DiamondDiamond
  35. 35. 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.
  36. 36. 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.
  37. 37. 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.
  38. 38. 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
  39. 39. 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.
  40. 40. Automatic knife sharpnersAutomatic knife sharpners
  41. 41. 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.
  42. 42. 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.
  43. 43. 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.
  44. 44. 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).
  45. 45. 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.
  46. 46. 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.
  47. 47. 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.
  48. 48. 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.
  49. 49. 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,
  50. 50. 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.
  51. 51.  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.
  52. 52.  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.
  53. 53.  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.
  54. 54. Flotation (water bath)Flotation (water bath)
  55. 55. 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.
  56. 56. 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.
  57. 57. 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.
  58. 58. Drying oven or hot plateDrying oven or hot plate
  59. 59. 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.
  60. 60. 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.
  61. 61. 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.
  62. 62. 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.
  63. 63.  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.
  64. 64. 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.
  65. 65. 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.
  66. 66. 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.
  67. 67. 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.
  68. 68. 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.
  69. 69. 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.
  70. 70. 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
  71. 71.  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.
  72. 72. 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.
  73. 73. 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
  74. 74. 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
  75. 75. 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.
  76. 76. 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.
  77. 77. 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.
  78. 78. 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.
  79. 79. 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
  80. 80. ReferencesReferences  Cellular pathology technique – C.F.A.Culling.Cellular pathology technique – C.F.A.Culling.  Histological techniques- J.D.Bancroft.Histological techniques- J.D.Bancroft.

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