hp lec

• Tissue block


  Expected output after embedding
• Purpose of trimming


  To even out the edges of our tissue block
• Trimming process


  1. Trimming of tissue

  2. Cooling the block with ice

  3. Cutting the tissue and making ribbons of tissue

  4. Floating the tissue in a water bath

  5. Picking up the tissue on the slide and drying
• Trimming


  Removal of excess wax using a knife or cutter after the wax block is removed from the tissue cassette or paper boat
• Ideal shape of trimmed tissue block

  • Truncated pyramid
• Coarse trimming


  Thicker chunk will be trimmed
• Fine trimming


  Small bits or just the edges of your tissue block will be trimmed
• Microtomy


  A process whereby tissues are cut into uniformly thin slices or "sections" with the aid of a machine (Microtome), to facilitate the studies under the microscope
• Tissue ribbons


  An indication that your sectioning is correct and you already cut thin slices of tissues which are ready to be transferred to the flotation bath
• Microtome


  Principle: Spring-balanced or pawl is brought into contact with, and turns the ratchet feed wheel connected to a micrometer screw, which is, in turn, rotated, moving the tissue black 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
• Rocking Microtome (Cambridge)


  Simplest, 10-12 μm thickness, tissues are cut in slightly curved planes thus not recommended for serial sections
• Rotary Microtome (Minot)


  Most common, used for routine and research laboratories, 3-5 μm thickness, excellent for serial sections
• Sliding Microtome


  Developed by Adams (1789), types: Base-Sledge Microtome and Standard Sliding Microtome, recommended for cutting "extremely" hard and rough tissue blocks, cannot make tissue ribbons, only tissue sections
• Freezing Microtome


  Invented by Quickett (1848), stage for block holder is hollow and perforated, attached to a flexible lead pipe containing carbon dioxide, used to cut undehydrated tissues in a frozen state
• Cryostat or Cold Microtome


  More common than freezing microtome, used for rapid preparation of urgent tissue biopsies for intraoperative diagnosis (STAT), consists of a rotary microtome kept inside a cold chamber at -5 to -30°C, average -20°C, cutting section 4 μm
• Ultrathin Microtome


  Cutting section 0.5 μm, media: plastic, for electron microscopy, tissues fixed with osmic acid, uses special diamond knife
• Care of the microtome

  1. Brush away accumulated paraffin and small pieces of tissues with a soft brush after sectioning

  2. Oil movable parts to prevent rusting

  3. Cover microtome to prevent accumulation of dust
• Types of tissue sections

  • Paraffin (4-6 μm)
  • Celloidin (10-15 μm)
  • Frozen Sections
• Microtome knives


  • Plane-Concave
  • Biconcave
  • Plane-Wedge
• Characteristics of microtome knives

  • Plane-Concave: One side is flat, other side is concave

  Biconcave: Both sides concave

  Plane-Wedge: Both sides straight
• Other knives and blades

  • Disposable Blades
  • Glass Knive/ Ralph Knives
  • Diamond Knives
• Microtome knives

  • Plane-concave
  • Biconcave
  • Plane-wedge
• Plane-concave knives


  • One side is flat, other side is concave
• Biconcave knives


  • Both sides concave
• Plane-wedge knives


  • Both sides straight
• Knife characteristics

  • Length
  • Embedding medium
  • Microtome
• Other knives and blades

  • Disposable blades
  • Glass knives/Ralph knives
  • Diamond knives
  • Safety razor blades
• Bevel angle


  • 27° to 32°, angle of the knife itself and between the cutting edges
• Cutting angle


  • 14°, angle between the upper facet of the knife and the surface of the tissue block
• Clearing angle


  • Most important, angle between the lower facet of the knife and the surface of the tissue block, should be inclined at 5-10° from the cutting plane
• Sharpening knives

  1. Honing

  2. Stropping
• Hones

  • Belgium yellow
  • Arkansas
  • Fine carborundum
  • Plate glass
• Lubricants

  • Soapy water
  • Mineral oil
  • Clove oil
  • Xylene
  • Liquid paraffin
• Knife sharpeners

  • Flat glass plate with finely powdered aluminum oxide
  • Diamantine
• Stropping


  In honing, the edge is first, while in stropping, the edge is last
• Paddle strop


  • Made of horse leather, attached to a solid back, usually dry thus requires oiling with vegetable oil
• Stropping procedure


  Toe to heel movement, edge last
• Adhesives


  • Mayer's egg albumin
  • Dried albumin
  • Starch paste
  • Plasma
  • 1% gelatin
  • 1% methyl cellulose
  • Poly-L lysine
  • Sodium silicate
  • Resins
• Mayer's egg albumin


  Most common adhesive, never used with Pap staining method, produces background staining