3 Histotechnology

• Microscope is used for the magnification of the tissues and cells
• Microtome is used for the cutting of the wax and tissues/cell blocks
• There are 3 essential parts of the microtome:

  • Block Holder (Chuck) – holds the tissue in position.
  • Knife & Knife Carrier – for actual cutting of tissue sections.
  • Pawl, Ratcher Feed Wheel & Adjustment Screw – lines up the tissue block in proper position with knife, adjusting thickness of tissues for successive sections.
• Microtome is one of the most important instrument in Pathology section
• The parts of the microtome should be wiped by using xylol, a clearing agent used for deparaffinization
• Rocking (Cambridge) Microtome is invented by Paldwell Tirefall in 1881; it is the “simplest” among types of microtome and is used for cutting serial sections of “large Blocks” of paraffin embedded tissues.
• Rotary (Minot) Microtome is invented by Minot 1885-1886; it is the “most Common” type used for both routine and research laboratories and for sectioning paraffin embedded
• Sliding Microtome developed by Adams in 1789 is the “most dangerous” type of microtome due to its movable exposed knife; it is used for cutting celloidinor embedded tissues.
• The 2 types of sliding microtome are:

  • Base Sledge Microtome
  • Standard Sliding Microtome
• Freezing Microtome is invented by Queckett in 1848; its is used to cut un-dehydrated tissues in frozen state (for unembedded frozen section) and for unprocessed specimen
• Cryostat/Cold Microtome is the most commonly used for “rapid preparation” and a usual type of microtome inside “Rotary Microtome”; it is a refrigerated apparatus used in “fresh tissue microtomy”.
• The cryostat/cold microtome has an adjustable thermostat at -5 to - 30°C (average of 20°)
• Freshly collected sample is the most used sample type in cryostat/cold microtome
• Cryostat/cold microtome is used for rapid diagnostic of cancer
• Ultra-thin Microtome is used to produce sections “electron microscopy" where tissues are embedded in “plastic resins”
• The Knives used in ultra-thin microtome are Glass (semi-thin) and Diamond Knives (ultra-thin)
• The section thickness used in ultra-thin microscope are:

  • Semi-thin → 0.5-1 micron
  • Ultra-thin → 500-1200 angstrom (50-120nm)
• Plane Concave conventional steel knives with 25mm length are used for base sledge, rotary and rocking microtome where:

  • Less Concave side → for Celloidin embedded tissue blocks
  • More Concave side → for Paraffin embedded tissue blocks
• Biconcave conventional steel knives with 120mm length are used for rotary microtome where both sides are concave for cutting paraffin embedded sections; it is the most common microtome knives
• Plane wedge conventional steel knives with 100mm length are used for base sledge or sliding microtome where both sides are straight; for frozen sections or very hard and tough paraffin embedded tissues.
• Honing or hard sharpening of the conventional steel knives is the removal of gross nicks.

  • It always uses mineral oils.
  • 20-30 double strokes in each direction; but depends on the condition of knife.
  • Heel to toe direction
• There are 3 Types of Hones used:

  • Belgium yellow – manual sharpening; it gives best results.
  • Arkansas – gives more polishing effect.
  • Fine Carborundum – much coarser than the first two; used only for badly nicked knives by either one of the first 2 knife sharpeners.
• Stropping or polishing of the conventional steel knives is the removal of burr (formed during honing).

  • It is done to polish and sharpen the cutting edge.
  • Toe to heel direction
  • 40-120 double stroke are usually required
• Disposable Blades are more commonly used microtome knives where Honing and stropping process is eliminated; it is cheaper to use than conventional steel knives and it is used in Rotary microtome
• Magnetic Knives can attach to some blade holders; it is particularly suitable for use in the cryostat
• Glass Knives are used for trimming and semi-thin “survey” sectioning of tissue blocks for electron microcopy; with a section thickness of 0.5-1 micron
• Diamond Knives is used only for cutting “ultra-thin” sections for Electron microscope and also used in any type of resin block for Electron Microscopy; it is brittle, expensive, but very durable; has a thickness of 500-1200 angstrom
• The bevel of the cutting angle of the microtome is 27-32°; the perfect and optimum Cutting Angle is 15°
• the Clearance angle used in microtome is 0-15°:

  • To prevent uneven sections or alternate thin and thick sections, the knife should be inclined with 5-10° clearance angle.
  • Once above 15°, the angle will not be even
• Automatic Tissue Processor only has 4 steps in histopath section:

  • Fixation
  • Dehydration (most critical stage)
  • Clearing
  • Infiltration
• The Advantage of automatic tissue processor is Constant agitation
• Wax impregnation is a vacuum embedding apparatus under negative atmospheric pressure inside an embedding oven.
• Vacuum embedding gives the fastest result.
• Orientation is the most important step in embedding
• Floatation Water Bath used to float out and flatten paraffin ribbons; it prevents folding/stretching out the paraffin
• Floatation Water Bath is set at a temperature of 45-50°C; approx. 6-10°C lower than melting point of the wax used.
• Oven (processes):

  • Infiltration process → regular at 55-60°C
  • Drying process → done at 56-60°C (2 hours)
  • Impregnation → if automatic processor cannot be used
• Hot Plate may be used instead of drying oven; it is not recommended since they can cause overheating and there is a risk of dust falling onto the section during drying period.
• Forceps (Fine Pointed/Curved) and Squirrel Hair Brush are used in handling sections during cutting, and for removing folds and creases on the sections during “floating out” in water bath.
• For clean slides:

  • For routine work → 76x25mm slides that are 1.0-1.2 thick are preferred.
  • Frosted end slides are preferred, where the ID number of the section can be inscribed with a pencil.
  • Slide refractive index → 1.518 (standard)