7 Microtomy

• Microtome is used for trimming and actual section-cutting
• Three essential parts of microtome:

  • Block holder – where the tissue is held in position
  • Knife carrier & Knife – for actual cutting of tissue sections
  • Pawl, Ratchet Feed Wheel, & Adjustment Screws – to line up the tissue block in proper position with the knife, adjusting the proper thickness of the tissue for successive sections
• Principle of a microtome:

  • Spring-balance teeth or pawl is brought into contact with, and turns a ratchet feed wheel connected to a micrometer screw, which is in turn rotated, moving the tissue block at a predetermined distance towards for cutting sections at uniform thickness
• Rocking microtome is the simplest.

  • For serial sections of large paraffin-embedded sections
  • 10-12 micra thickness
  • Consists of heavy base and 2 arms:
  • Lower arm – supporting column and attached to micrometer screw.
  • Upper arm – carrying block holder, connected to lever by piece of nylon thread.
• Rotary microtome is the most common.

  • For paraffin-embedded sections
  • Parts:
  • Rotational flywheel → reciprocal motion of the knife over the block.
  • Ratchet feed wheel → thickness of the section being regulated
  • Pawl → rotates the micrometer screw making the block towards the knife
• Sliding microtome is the most dangerous.

  • For celloidin-embedded sections
• Sliding microtome type:

  Base-sledge microtome for cutting celloidin sections.

  • Chuck or block holder moved backwards and forwards under the knife.
• Sliding microtome type:

  Standard sliding microtome, the blocks remains stationary, the knife is move backward and forward → dangerous type
• Freezing microtome is used for frozen sections.

  • For unembedded frozen section
  • 4 micra thickness
  • Used to cut undehydrated tissue in a frozen state
  • Used for rapid diagnosis, histological demonstration of fat, neurological studied and sensitive tissue
• Freezing microtome has flexible lead pipe which carbon dioxide passes from a cylinder, this will freeze the block holder and the tissue evenly.
• Cryostat or Cold microtome is used for rapid frozen section.

  • Refrigerated apparatus used in fresh tissue examination.
  • Consists of rotatory microtome inside in a cold chamber.
  • Used for fluorescent antibody staining technique and histochemical enzyme studies.
  • Rapid preparation of urgent tissue biopsies for interoperative diagnosis.
• Cryostat or Cold microtome

  • Maintained temp = -5 to -30 °C
  • Average = -20 °C
  • Capable of freezing fresh tissue → 2-3 minutes
  • 4 micra thickness
• Ultrathin microtome used for electron microscopy → 0.5 micra.

  • Should be fixed in Osmium Tetroxide and embedded within a plastic.
  • Uses Glass knives and Diamond knives
• Ultrathin microtome knives:

  • Glass Knives → semi-thin
  • Section thickness: 0.5-1 micron
  • Diamond knives → ultra-thin
  • Section thickness: 500-1200 angstrom (50-120 nm)
• Plane Concave knife used for base sledge, rotaryand rocking microtome.

  • 25 mm
  • Less concave side → for Celloidin embedded tissue blocks
  • More concave side → for Paraffin embedded tissue blocks
• Biconcave knife for rotary microtome.

  • 120 mm
  • Most common microtome knife.
  • Both sides are concave for cutting paraffin embedded sections.
• Plane Wedge knife used for base sledge or sliding microtome.

  • 100 mm
  • Both sides are straight.
  • For frozen sections or very hard and toughparaffin embedded tissues.
• Bevel angle is the angle formed between the cutting edge, normally 27-32 °
• Wedge angle is the angle formed by the sides of the wedge knives, normally 14-15 °
• Clearance angle is used to prevent uneven sections or alternate thin and thick sections, the knife should be inclined with 5-10 ° clearance angle.

  • 2-3 microns thick
• Honing or sharpening, from heel to toe.
• Coarse Honing is the removal of gross nicks
• Honing Proper is when even edge is acquired
• Belgium Yellow is a hone with manual sharpening; gives best result
• Arkansas is a hone with more polishing effect
• Fine Carborundum is a hone that is much coarser; for badly nicked
• Stropping or polishing, from toe to heel.

  • Burrs formed during honing is removed
  • Cutting edge of knife is polished/sharpened
  • 40-120 double stokes
• Stropping

  • Treated with vegetable oil → Castor Oil
  • Should never be treated with Mineral oil
  • Should not be used 24-48 hours after treatment
  • Wax should not come in contact with the strop
• In trimming, excess wax is cut off from the block to expose the tissue surface in preparation for actual cutting.

  • Sides, top and bottom of tissue block are trimmed
  • Old knives or blades may be used for this procedure
  • Blocks are place on ice cold water or refrigerated → for ease to cut
  • 15-25 micra but it depends on the type of tissue
• Sectioning is the process whereby tissues are cut into uniformly thin slices or “sections” with the aid of a machine.

  • To facilitate the studies under the microscope
• Paraffin Sections may be cut by rocking or rotary microtome
• Celloidin Sections is usually cut by Sliding microtome
• Frozen Sections cut from tissues that have been fixed and frozen with carbon dioxide or fresh or fixed tissue frozen with cryostat
• Paraffin sections

  • Block is allowed to harden for cutting proper by facing then down in ice cold water or refrigerator for 5-10 minutes.
  • Cooling both tissue and wax will give them a similar consistency and make sectioning easier.
  • This block is then placed in the microtome for final trimming and cutting
  • Knife usually tilted at 0-15 ° on microtome
  • Biconcave knives require smaller clearance angle than wedge-shaped knives
• Removing paraffin ribbon from the knife:

  • Incomplete sections are discarded
  • Complete ribbons are pick up at once
  • use camel hairbrush, a pair of forceps or the fingers
  • Tissues tend to crumble
  • blood clots, bone marrow, spleen
  • Maintain cold temp of the block → forming a ribbon
• Floating out / Fishing out on a water bath is set at 45-50 °C or 5-10 °C below the melting point of the wax
• 50% alcohol flooded on slide may be used to promote flattening of section to slide in fishing out
• After fishing out, sections are place in metal rack with 25-slide division.

  • Place to paraffin oven with 2-5 °C above melting of the wax for 5 minutes → to dissolve excess wax
• Celloidin sections are cut between 10-15 micra thickness; same thickness in trimming as Paraffin section.

  • Does not require hardening by chilling before cutting
  • Used in sliding microtome
  • Section usually cut by the wet method → moist with 70% alcohol to avoid dehydration and shrinkage
• Characteristics of a ribbon should be:

  • Thin
  • Transparent
  • Without irregularities
  • Not wrinkled
  • Uniform in thickness
  • Can easily be separated from each other
  • Continuous