MICROTOMY AND SECTION CUTTING

• Tissue processing


  1. Tissue is trimmed

  2. Tissue is cut into uniformly thin slices or "sections" to facilitate studies under the microscope
• Paraffin embedded tissue


  Most commonly used tissue for processing
• Microtome


  Basic instrument used for cutting tissue sections
• Microtome


  • Capable of cutting a section at a predetermined thickness
  • Sections produced are around 4-6 micro in thickness

  
• Microtome operation


  A spring-balanced 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 the knife for cutting sections at a uniform thickness

  
• Essential parts of a microtome

  • Block Holder
  • Knife Carrier and Knife
  • Pawl, Ratchet Feed Wheel and Adjustment Screws
• Types of microtomes


  • Rocking microtome
  • Rotary microtome
  • Sliding microtome
  • Freezing microtome
  • Ultrathin microtome
• Rocking microtome


  Also called "Cambridge microtome", invented by Paldwell Trefall in 1881, simplest microtome, tissue sections produced are 10 to 12 micro in thickness, for cutting serial sections of large blocks of paraffin embedded tissues

  
• Rocking microtome


  • Consists of a heavy base and two (2) arms: lower arm and upper arm
• Rocking microtome operation


  1. When the lever is pulled forward, the pawl is brought in contact with the ratchet wheel

  2. The ratchet wheel is turned, rotating the micrometer screw

  3. The lower arm is elevated, which in turn raises the upper arm at its fulcrum

  4. As the pressure on the operating handle or lever is released, the tension on the spring causes the upper arm to return to its normal position
• Rotary microtome


  Also called "Minot microtome", invented by Minot in 1885-1886, for cutting paraffin-embedded tissues, most common type used for both routine and research laboratories

  
• Rotary microtome operation


  1. Operated by rotation of the fly wheel

  2. The thickness of the section is automatically regulated by the ratchet feed wheel

  3. The pawl is used to come in contact with the ratchet feed wheel
• Rocking microtome


  Operated by pulling the lever forward
• Rotary microtome


  Operated by the fly wheel by upward and vertical motions
• Rocking microtome


  Sections are cut in an arc of a circle
• Rotary microtome


  Sections are cut in a perfectly flat plane
• Rocking microtome


  Less heavy and less stable
• Rotary microtome


  Heavier and more stable
• Rocking microtome


  Less complex in design and construction
• Rotary microtome


  More complex in design and construction
• Rocking microtome


  Less expensive
• Rotary microtome


  More expensive
• Sliding microtome


  Developed by Adams in 1789, for cutting celloidin embedded sections, knife can be set obliquely or straight, especially recommended for cutting extremely hard and rough tissue blocks, most dangerous type of microtome, slow but very steady motion is required to manipulate the instrument

  
• Types of sliding microtomes

  • Base – Sledge Microtome
  • Standard Sliding Microtome
• Base-Sledge microtome


  • Favored in laboratories where very hard tissue or large blocks are usually sectioned, suited for sectioning specimens embedded in all forms of media, larger sections are more easily cut with the knife set at an angle due to less resistance offered by the block, sections are cut in a perfectly flat plane, comparatively more stable than the ordinary sliding microtome, modern models are electrically driven

  
• Base-Sledge microtome


  • Consists of two (2) movable pillars holding the adjustable knife clamps, chuck or block holder is set on a heavy metal base
• Standard sliding microtome


  • Unlike the Base-Sledge microtome, the block remains stationary while the knife is moved backward and forward during sectioning, developed mainly for cutting celloidin-embedded tissue blocks, inherently more dangerous than the Base-Sledge microtome

  
• Freezing microtome


  Invented by Queckett in 1848, for cutting unembedded frozen sections, a second cooling device for lowering the temperature of the knife is also incorporated in most machines, the microtome is firmly clamped on to the edge of the bench for use, or mounted on especially constructed shelf with carbon dioxide (CO2) cylinder below
• Situations where freezing microtomes are used

  • Cutting undehydrated tissues in a frozen state
  • Rapid diagnosis is required
  • Histological demonstration of fat is needed
  • Certain neurological structures are to be studied
  • Sensitive tissue constituents to be studied are damaged or destroyed by heat
• Cryostat


  Refrigerated apparatus used in fresh tissue microtomy, consists usually of a rotary microtome, capable of freezing fresh tissues within 2-3 minutes, cuts sections of 4 micro in thickness with ease, most commonly used for rapid preparation of urgent tissue biopsies for intraoperative diagnosis, provides a means of preparing thin sections of fresh frozen tissues, sections are usually transferred directly from the microtome knife to a slide or cover glass

  
• Ultrathin microtome


  For cutting sections for Electron Microscopy, produces tissue sections at 0.5 micro in thickness, diamond knife is used for ultrathin sections
• Microtome maintenance


  1. All the accumulated paraffin and small pieces of tissues must be brushed away with a soft brush after sectioning

  2. Parts should be wiped with xylol after carefully drying the machine and knife holder

  3. Movable portions should be oiled thoroughly to prevent rusting

  4. The microtome must always be covered when not in use
• Types of microtome knives

  • Plane-Wedge Knife
  • Plane-Concave Knife
  • Biconcave Knife
• Microtome knife


  • Angle formed between the cutting edges is normally about 27° to 32°, maintained for each knife by means of a slide-on back or knife back, 15° angle is theoretically the perfect and optimum cutting angle, 5-10° clearance angle is made to prevent uneven, alternate thin, and thick sections
• Microtome knife


  • Made of good quality steel, avoid too soft cutting edges or too hard cutting edges, able to cut good sections from a paraffin wax block, about 2-3 micro thick
• Disposable blades


  May be used for partially calcified materials, paraffin and frozen sections, readily replaced when dull, produce similarly good tissue sections as those cut with microtome knives, unsatisfactory for sections less than 10 micro
• Knife sharpening - Hard sharpening

  1. Coarse honing

  2. Honing Proper
• Honing


  Makes use of a hone (natural sharpening stone or hard grinding surface like Carborundum), purpose is to remove nicks and irregularities on the knife edges, for manual sharpening when cutting edge has been rendered blunt or nicked
• Types of hones


  • Belgium Yellow
  • Arkansas
• Honing procedure


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

  2. Cover the hone with a thin film of mineral and clove oil, xylene, liquid paraffin, or soapy water for lubrication

  3. Fit the knife to its corresponding knife back

  4. Draw the knife obliquely or diagonally: HEEL TO TOE direction

  5. Knife is turned over and process is repeated on the other side