MicrotomyAll

Cards (73)

  • 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
    The basic instrument used for microtomy, 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
  • Essential parts of a microtome
    • Block Holder - where the tissue is held in position
    • Knife Carrier and Knife - for actual cutting of tissue sections
    • Pawl, Ratchet Feed Wheel and Adjustment Screws - to line up the tissue block in proper position with the knife, adjusting the proper thickness of the tissue for successive sections
  • Types of microtomes
    • Rocking microtome
    • Rotary microtome
    • Sliding microtome
    • Freezing microtome
    • Cryostat or cold microtome
    • Ultrathin microtome
  • Rocking (Cambridge) Microtome
    • Simplest among the different types of microtomes
    • Consists of a heavy base and two arms, the lower arm resting on pivots and a supporting column, and attached to the micrometer screw, at the base of which is found the ratchet wheel with feed mechanism
    • The upper arm, carrying the block holder on one end by means of a screw, is connected to a lever by a piece of nylon thread
    • When the lever is pulled forward, the pawl is brought in contact with the ratchet wheel to which the millhead micrometer screw is attached
    • The ratchet wheel is turned, rotating the micrometer screw, elevating the lower arm which in turn raises the upper arm at its fulcrum, thereby carrying the chuck or block holder forward, towards the knife
    • 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; in an arc of a circle, cutting a section as the tissue passes to the knife edge in a slightly curved plane, in 10-12 u thickness
  • Rotary (Minot) Microtome
    • Operates with a staged rotary action such that the actual cutting is part of the rotary motion
    • The knife is fixed in a horizontal position
    • Sections are cut between 3 and 5 µm using paraffin wax for diagnostic histology although thinner sections can be attained if samples are embedded in synthetic resin
    • Allows excellent serial sections to be cut as the knife and the block holder are brought together by upward and vertical motions, cutting sections in a perfectly flat plane
    • Heavier and more stable than the rocking microtome, more complex in design and construction, and therefore more expensive
    • May be used for cutting large blocks of tissues although results are better when the sliding microtome is used
    • The knife is placed in a blade-up position and is therefore relatively dangerous
    • Both manual and electrically driven models are now available for cutting ultrathin sections and for cryostat use
  • Sliding Microtome

    • Two types: Base-Sledge Microtome and Standard Sliding Microtome
    • Base-Sledge Microtome: Consists of two movable pillars holding the adjustable knife clamps, allowing the knife to be set at an angle for cutting celloidin sections. The chuck or block holder is set on a heavy metal base which can be moved backwards and forwards under the knife. Favored for sectioning very hard tissue or large blocks, suited for cutting sections from tough tissue blocks with the knife set at an angle due to less resistance offered by the block. Sections are cut in a perfectly flat plane, making excellent serial tissue sections. Comparatively heavier and more stable than the ordinary sliding microtome. The angle of the knife is adjustable. The knife used is long (24 cm), hence it requires less honing.
    • Standard Sliding Microtome: The block remains stationary while the knife is moved backward and forward during the process of sectioning. Developed mainly for cutting celloidin embedded tissue blocks and is inherently more dangerous because of the movable knife, which makes it difficult to attach knife guards.
  • Freezing Microtome
    • The stage for block holder is hollow and perforated around its perimeter, attached to a reinforced flexible lead pipe through which carbon dioxide passes from a cylinder. A simple lever operated valve allows the release of rapid, intermittent bursts of carbon dioxide which will freeze the block holder and the tissue evenly. A second cooling device for lowering the temperature of the knife is also incorporated to facilitate sectioning. Used to cut undehydrated thin to semi-thin sections of fresh, frozen tissues, especially when rapid diagnosis is required, when histological demonstration of fat is needed, when certain neurological structures are to be studied, and when sensitive tissue constituents to be studied are damaged or destroyed by heat.
  • Cryostat or Cold Microtome
    • A refrigerated apparatus used for freezing the tissue into the block holder to the correct degree of hardness that allows for easier and faster sectioning. Consists of a microtome, usually a rotary microtome, kept inside a cold chamber which has been maintained at a temperature between -5° to -30°C (average is -20°C) by an adjustable thermostat, capable of freezing fresh tissues within 2-3 minutes, and cutting sections of 4 µ with ease. Provides a means of preparing thin sections of fresh frozen tissues especially for fluorescent antibody staining techniques or histochemical enzyme studies. Most commonly used for rapid preparation of urgent tissue biopsies for intraoperative diagnosis.
  • Ultrathin Microtome

    • Equipped with a glass or gem grade diamond knife, used to cut very thin sections (typically 60 to 100 nanometer) of tissue embedded in epoxy resin. Sections are stained with an aqueous solution of an appropriate heavy metal salt and examined with a transmission electron microscope (TEM). Also used with its glass knife or an industrial grade diamond knife to cut semi-thin sections prior to thin sectioning for the TEM.
  • Care of the Microtome
    1. Brush away accumulated paraffin and small pieces of tissues after sectioning
    2. Wipe with xylol after drying the machine and knife holder, but avoid prolonged and continuous application of xylene
    3. Oil movable portions thoroughly to prevent rusting
    4. Cover the microtome when not in use to prevent accumulation of dust and dirt
    5. Place the microtome on a stable bench, away from air drafts, doorways and passing staff
    6. Remove the knife or blade before cleaning, avoid dragging anything along the cutting edge when cleaning the blade
    7. Have the instrument inspected at least once a year by a qualified service technician
  • Safety Measures
    • Staff should not be distracted when using the microtome due to the risks of injury from extremely sharp blades
    • Use forceps or brush instead of fingers to pick up sections or wax fragments from blade or block face
    • Use hand wheel lock when changing blocks, remove the knife or blade when the instrument is left unattended or when cleaning
  • Types of Microtome Knives
    • Plane-Concave Knife
    • Biconcave Knife
    • Plane-Wedge Knife
  • Bevel Angle
    The angle formed between the cutting edges of a microtome knife, normally about 27° to 32°, maintained by means of a slide-on back
  • Safety razor blades may be used for partially calcified materials, paraffin and frozen sections. They are readily replaced when dull, and produce good sections.
  • Bevel angle
    Angle maintained for each knife by means of a slide-on back, a spring-loaded semi-circular metal sheet
  • Good cutting edge
    • Made of good quality steel
    • Not too soft (dull easily)
    • Not too hard (produce nicks or jagged edges)
  • Safety razor blades

    • 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 µ
  • Optimum cutting angle
    Sides of the wedge knife 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
  • Cutting edge
    • Thinner than the section being cut
    • 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
  • 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
  • Hone
    • Natural sharpening stone or hard grinding surface (carborundum)
    • Removes nicks and irregularities on the knife edges
  • 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. Fitting the knife to its corresponding back
    4. Placing the knife on one end of the hone, with the cutting knife edge first, and drawing the "heel" (handle end) obliquely or diagonally towards the operator on the stone until the "toe" (head portion) is reached
    5. Turning the knife over and drawing the other surface forward, EDGE FIRST, with a HEEL TO TOE direction
  • Alternative honing method

    1. Using a flat circular glass plate with finely powdered aluminum oxide made into paste with water (used 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 a rotating glass wheel
  • Precautions during honing

    • Hone should be long and wide enough to allow the whole length of the knife edge to be sharpened in a single stroke and prevent rocking
    • Hone should be lubricated with warm soapy water or fine oil before use
    • Gentle and steady pressure on the knife to prevent rocking
    • 20-30 strokes in each direction, depending on the condition of the knife
    • Cleaning the hone before, during, and after use
  • Stropping
    Removing the "burr" formed during honing and polishing the cutting edge of the knife
  • Stropping procedure
    1. Fitting the knife with its appropriate knife back
    2. Laying the knife obliquely on the strop with the cutting edge behind
    3. Pushing the knife backward and drawing it forward in a TOE TO HEEL direction
    4. 40-120 double strokes usually required
    5. Turning the knife around at the end of each stroke for plane-wedge or Minot knives to sharpen each surface alternately
    6. Stropping only the concave surface for plane-concave knives
  • Precautions during stropping
    • Wiping the knife clean before and after stropping to remove particles
    • Oiling or greasing the knife edge after stropping to prevent rusting
    • Keeping the knife covered in a suspension box to prevent dust and grit settling
    • Applying only gentle pressure during stropping
    • Avoiding speed - allowing 1 full second per stroke
    • Treating the leather strop with vegetable oil, not mineral oil
    • Keeping the stropping surface firm and not loose
  • Disposable blades
    • Sharper cutting edge that can cut 2-4 µ thick sections with ease
    • Cheaper to use than conventional steel knives
  • Glass knives
    • Used for trimming and semi-thin sectioning of tissue blocks for electron microscopy
    • Prepared from commercially available plate glass strips
  • Diamond knives
    • Used to cut any type of resin block for electron microscopy
    • Brittle and expensive, but very durable
    • Cutting edge must be kept clean
  • Other equipment required
    • Waterbath
    • Drying oven or hot plate
    • Forceps and squirrel hair brush
    • Clean slides
  • Quality of sections cut on a microtome can suffer from fecal material, hair, sutures, thread or staples in the specimen
  • Slide rack
    Made on the assumption that regular slides have been used
  • Larger size of slides
    Used for sections of eyes or CNS tissues when these will not fit on the regular
  • Causes of poor quality sections cut on a microtome
    • Fecal material in intestine, especially in the colon where this material is very hard
    • Hair - it can be removed using a razor blade or clippers
    • Hair can also sometimes be inadvertently included with organs
  • Sutures, thread or staples should be removed from the specimen prior to cutting with the knife