MICRTOTOMY

• Microtomy


  The process by which processed tissue, most commonly a paraffin embedded tissue, is trimmed and cut into uniformly thin slices or "sections" to facilitate studies under the microscope
• Microtome


  • Capable of cutting a section at a predetermined thickness by sliding the block into a cutting tool, usually a steel knife, glass or diamond blade, which is fixed and attached to the machine
  • Consists of three essential parts: Block Holder, Knife Carrier and Knife, Pawl, Ratchet Feed Wheel and Adjustment Screws
• Types of microtomes


  • Rocking microtome
  • Rotary microtome
  • Sliding microtome
  • Freezing microtome
  • Cryostat or cold microtome
  • Ultrathin microtome
• Rocking (Cambridge) Microtome


  1. Lever pulled forward brings pawl into contact with ratchet wheel, turning micrometer screw

  2. Lower arm elevated raises upper arm, carrying chuck/block holder forward towards knife

  3. Pressure released, upper arm returns to normal position, cutting section in slightly curved plane
• Rocking Microtome


  • Simplest among different types of microtomes
  • Used to cut small and large blocks of paraffin tissues
  • Not recommended for serial sections as tissues are cut in slightly curved planes
  • Restrictions in size of tissue block that can be cut, and difficulty of reorienting the block
• Rotary (Minot) Microtome


  1. Knife fixed in horizontal position, cutting part of rotary motion

  2. Sections cut between 3-5 μm using paraffin wax for diagnostic histology, thinner sections possible with synthetic resin

  3. Cuts sections in perfectly flat plane, allowing excellent serial sections
• Rotary Microtome


  • Most common type used for both routine and research laboratories, especially for sectioning paraffin-embedded tissues
  • Heavier and more stable than rocking microtome, more complex in design and construction, therefore more expensive
  • Can be used for cutting large blocks of tissues, better results with sliding microtome
  • Knife placed in blade-up position, relatively dangerous
  • Heavier knife used, less vibration, cutting angle (tilt) of knife adjustable
• Sliding Microtome


  1. Base-Sledge Microtome: Knife clamps allow knife to be set at angle for cutting celloidin sections, block holder moves backwards and forwards under knife

  2. Standard Sliding Microtome: Block remains stationary, knife moved backwards and forwards during sectioning
• Sliding Microtome


  • Favored for sectioning very hard tissue or large blocks, suited for all embedding media
  • Larger sections more easily cut with knife set at angle due to less resistance
  • Sections cut in perfectly flat plane, making excellent serial tissue sections
  • Comparatively heavier and more stable than ordinary sliding microtome
  • Knife angle adjustable, long knife (24 cm) requires less honing
  • Most dangerous type of microtome due to movable exposed knife
• Freezing Microtome


  1. Block holder attached to flexible lead pipe, CO2 passed through to freeze block

  2. Knife holder attached to lever, moved back to original position, pawl contacts ratchet feed wheel to raise block towards knife

  3. Pulling operating handle forward cuts section as knife edge slices through raised tissue block
• Freezing Microtome


  • Used to cut undehydrated thin to semi-thin sections of fresh, frozen tissues
  • Gives best results for cutting frozen sections, used almost universally
• Cryostat


  Refrigerated apparatus used for freezing tissue into block holder to correct hardness for easier and faster sectioning
• Cryostat


  • Microtome, usually rotary, kept inside cold chamber maintained at -5° to -30°C
  • Allows rapid preparation of urgent tissue biopsies for intraoperative diagnosis
• Ultrathin Microtome


  Used to cut very thin sections (60-100 nm) of tissue embedded in epoxy resin, for examination with transmission electron microscope
• After sectioning, accumulated paraffin and tissue pieces must be brushed away, parts wiped with xylol, movable portions oiled to prevent rusting
• Microtome must be covered when not in use to prevent dust and dirt accumulation
• Microtome should be placed on stable bench, away from air drafts, to prevent section handling difficulties
• Knife or blade must be removed before cleaning, avoid dragging anything along cutting edge
• Microtome should be inspected annually by qualified technician
• Staff must not be distracted when using microtome due to injury risk from sharp blades, non-slip flooring is preferable
• Use forceps or brush instead of fingers to pick up sections or wax fragments, remove knife/blade when microtome left unattended or being cleaned
• Types of microtome knives

  • Plane-Concave Knife
  • Biconcave Knife
  • Plane-Wedge Knife
• Bevel Angle


  Angle formed between cutting edges of knife, normally about 27° to 32°, maintained by slide-on back
• Good cutting edge must be made of quality steel, not too soft or too hard, able to cut 2-3 μm paraffin sections without serration
• Safety razor blades may be used for partially calcified materials, paraffin and frozen sections, but unsatisfactory for sections less than 10 μ
• Optimum cutting angle is 15°, to allow maximum penetration and minimize distortion, knife should have 5-10° clearance angle from cutting plane
• Cutting edges


  • Too soft edges are likely to become dull easily
  • Too hard edges are likely to produce nicks or jagged edges and irregularities on the knife edge, thereby producing tears or striation on the tissue sections during cutting
• Good cutting edge


  Able to cut good sections from a paraffin wax block about 2-3 microns thick, without any serration noted on examination
• Cutting tools


  • Safety razor blades
  • Microtome knives
• Optimal cutting angle


  Sides of the wedge knife are inclined at an angle of about 15°, causing maximum penetration of the tissues and minimizing distortion
• Clearance angle


  1. 10° from the cutting plane so that the cutting facet will not compress the block during the process of cutting
• The cutting edge must be thinner than the section being cut
• A good cutting edge must be sharp enough to cut good sections from a paraffin wax block at 4 μ thick without causing serrations
• Removing knife/blade from microtome

  1. Unclamping the blade

  2. Using the blade ejector on the left side of the guard to start moving the blade laterally out of the clamp

  3. Grasping with forceps (not fingers) and safely removing

  4. Disposing of used blades appropriately in a "sharps" container or into the "used blades" slot in the base of the blade dispenser
• Never place a knife or blade on the bench or in a box with the cutting edge facing up
• Honing (Hard Sharpening)


  1. Coarse Honing to remove gross nicks on the knife edge

  2. Honing Proper to grind the cutting edge of the knife on a stone to acquire an even edge
• Types of hones


  • Belgium Yellow
  • Arkansas
  • Fine carborundum
• Honing procedure


  1. Wiping the hone surface clean with a soft cloth moistened with xylene

  2. Covering the hone with a thin film of Mineral and Clove Oil, Xylene, Liquid Paraffin or Soapy Water for lubrication

  3. Placing the knife on one end of the hone, cutting edge first, and drawing it obliquely or diagonally towards the operator until the "toe" is reached

  4. Turning the knife over and drawing it forward, edge first, with a heel to toe direction

  5. Honing until all the teeth in the knife edge have been eradicated
• Alternative honing method


  1. Using a flat circular glass plate with finely powdered aluminum oxide made into paste with water as an abrasive

  2. Pushing and pulling the knife forward and backward at right angles to the transverse diameter of the plate
• Mechanical honing


  1. Using a vibrating frosted glass plate or a wheel driven by an electrical motor

  2. Pressing the knife against the flat side of the rotating glass wheel with gentle pressure for approximately 30 double strokes on each side