SECTIONING/MICROTOMY

• 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 is known as Microtomy.
  • The basic instrument used is a microtome that is 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.
• Block Holder

  • Where the tissue is held in position
• Knife Carrier and Knife

  • For actual cutting of tissue sections.
• Pawl, Ratchet Fee 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.
• Rocking microtome- For cutting serial sections of large blocks of paraffin embedded tissues.
• Rotary microtome- For cutting paraffin embedded sections
• Sliding microtome- For cutting celloidin embedded sections
• Freezing microtome For cutting unembedded frozen sections
• Cryostat or cold microtome For cutting frozen sections
• Ultrathin microtome For cutting sections for Electron Microscopy
• Rocking (Cambridge) Microtome

  • invented by Paldwell Trefall in 1881, the simplest among the different types of microtomes
  • This consists of a heavy base and two arms
  • A section is thereby cut as the tissue passes to the knife edge in a slightly curved plane
  • 10-12 u thickness
  • available in two sizes, has been used to cut small and large blocks of paraffin tissues.
  • not recommended for serial sections
  • It is not currently favored by most laboratories because of the restrictions in size of tissue block that can be cut, and the difficulty of reorienting the block.
• Rotary (Minot) Microtome

  • invented by Minot in 1885-86 to cut paraffin embedded tissues, and is currently the most common type used for both routine and research laboratories, especially for sectioning paraffin-embedded tissues.
  • ideally used to produce ribbons for serial sections.
  • the device operates with a staged rotary action
  • knife is fixed in a horizontal position
  • generally automated or semi-automated
• Sliding microtome

  • This was developed by Adams in 1789. There are two types of this microtome, 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 in laboratories where very hard tissue or large blocks
  • originally designed for cutting sections of very large blocks (whole brain).
  • comparatively heavier and more stable than the ordinary sliding microtome.
  • 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.
• Sliding microtome

  • knife can be set obliquely for celloidin sections or straight for large refractory paraffin blocks, cutting both large and small tissues with ease; it is especially recommended for cutting extremely hard and rough tissue blocks. It is the most dangerous type of microtome due to the movable exposed knife. A slow but very steady motion is therefore required to manipulate the instrument.
• Rotary (Minot) Microtome

  • 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.
  • different from the rocking microtome in that the knife and the block holder are brought together by upward and vertical motions
  • heavier and more stable than the rocking microtome
  • A heavier knife is used, so there is less vibration
• Freezing Microtome

  • This was invented by Queckett in 1848.
  • The stage for block holder is hollow and perforated around its perimeter, attached to a reinforced flexible lead pipe thru which carbon dioxide passes from a cylinder
  • intermittent bursts of carbon dioxide which will freeze the block holder and the tissue evenly.
• Freezing Microtome

  • used to cut undehydrated thin to semi-thin sections of fresh, frozen tissues, especially in instances 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.
• Freezing Microtome

  • this type will give the best results and is used almost universally.
  • The cutting action of the freezing microtome differs from those described previously as in this case the knife is moved whilst the tissue block remains static, same as sliding microtome.
• The 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
  • maintained at a temperature between -5° to -30°C (average is -20°C)
  • capable of freezing fresh tissues within 2-3 minutes, and cutting sections of 4 µ with ease.
  • All the controls in the microtome are operated from outside the refrigerated cabinet.
• The Cryostat or Cold Microtome

  • most commonly used for rapid preparation of urgent tissue biopsies for intraoperative diagnosis.
  • direct consultation between surgeon and pathologist
  • Sections are usually transferred directly from the microtome knife to a slide or cover glass, all of which are maintained at a low temperature.
• Ultrathin Microtome

  • equipped with a glass or gem grade diamond knife is used to cut very thin sections (typically 60 to 100 nanometer) of tissue embedded in epoxy resin
  • examined with a transmission electron microscope (TEM)
  • These semi-thin sections are generally 0.5 to 1 µm thick
  • Thin sectioning for the TEM is often done with a gem quality diamond knife.
• Plane-Concave Knife (usually 25 mm. in length)

  • One side of the knife is flat while the other is concave. Less concave sides are recommended for cutting celloidin-embedded tissue blocks on a sliding microtome. More concave sides are used to cut paraffin sections on base-sledge, rotary or rocking microtome.
• Biconcave Knife (usually 120 mm. in length)

  • with both sides concave, recommended for cutting paraffin - embedded sections on a rotary microtome.
• Plane-Wedge Knife (usually 100 mm. in length)

  have both sides straight, recommended for frozen sections or for cutting extremely hard and tough specimens embedded in paraffin blocks, using a base sledge type or sliding microtome.
• MICROTOME KNIVES

  • Plane-wedge and plane-concave knives are usually provided with backs, to maintain the correct bevel angle throughout honing
  • There is a cutting facet (bevel) found on the tapered edge of all knives,
  • The angle formed between the cutting edges is known as the "Bevel Angle", normally about 27° to 32°.
  • Too soft cutting edges are likely to become dull easily, while 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.
• MICROTOME KNIVES

  • good cutting edge must be about 2-3 microns thick, without any serration noted on examination.
  • unsatisfactory for sections less than 10 µ.
  • perfect and optimum cutting angle is obtained when the sides of the wedge knife are inclined at an angle of about 15°, causing maximum penetration of the tissues and minimizing distortion.
• MICROTOME KNIVES

  • To prevent uneven sections, or alternate thin and thick sections, the knife should be inclined with a 5-10° clearance angle.
  • 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.
• Honing (Hard Sharpening)

  • removal of gross nicks on the knife edge (Coarse Honing) to remove blemishes, and grinding the cutting edge of the knife on a stone (Honing Proper) to acquire an even edge.
  • The degree of sharpness is proportional to the fineness of the abrasive used in sharpening.
  • serves to remove nicks and irregularities on the knife edges.
• Belgium Yellow- for manual sharpening when cutting edge has been rendered blunt or nicked. This type usually gives the best result.
• Arkansas gives more polishing effect than the Belgium Yellow.
• Fine carborundum is much coarser than the first two types and is used only for badly nicked knives followed by either one of the first two knife sharpeners.
• Honing

  • The surface of the hone is wiped clean with a soft cloth moistened with xylene in order to remove the scattered small particles of stones and metal.
  • It is then covered with a thin film of Mineral and Clove Oil, Xylene, Liquid Paraffin or Soapy Water for lubrication
  • EDGE FIRST, with a HEEL TO TOE direction.
  • Hone is placed on non-skid surface
• Honing

  • In the case of the Minot or plane-wedge knife, the knife is turned over so as to sharpen the other surface every I0-20 strokes. For plane-concave knives, only the concave surface should be rubbed on the Hone.
• Honing

  • flat circular glass plate with finely powdered aluminum oxide made into paste with water (used as an abrasive) may be used for grinding and removing nicks. Diamantine may also be used for final polishing. The plate glass is usually 1/4 to 3/ 8 inch thick, about 14 inches long and 1-2 inches wider than the length of the knife blade to be sharpened
  • Mechanical honing with machines may make use of a vibrating frosted glass plate or a wheel driven by an electrical motor. Approximately 30 double strokes are given each side of the knife to which very gentle pressure is applied.
• TROPPING

  • process whereby the "burr" formed during honing is removed and the cutting edge of the knife is polished.
  • The purpose of stropping is to polish and sharpen the cutting edge, while that of honing is to remove the irregularities from the knife.
  • If the knife has become dull and blunt, but is free from nicks or teeth, it is usually only necessary to strop it.
• • Tropping
  • The procedure is the reverse of honing.
  • TOE TO HEEL direction.
  • Around 40- 120 double strokes are usually required.
  • In the case of plane-wedge or Minot knives, the knife is turned around at the end of each stroke so as to sharpen each surface alternately.
  • For plane concave knives, only the concave surface should be stropped.
• DISPOSABLE BLADES

  • They have a sharp cutting edge that can cut 2-4 µ thick sections with ease
  • manufacturers have also now incorporated a disposable blade holder in place of a knife holder. Magnetic knives are also now available that can attach to some blade holders and are particularly suitable for use in the cryostat.
• GLASS KNIVES

  • used for trimming and semi-thin sectioning of tissue blocks for electron microscopy. They are prepared from commercially available 40 x 2.5 cm. plate glass strips that have been washed with detergent, rinsed in distilled water and alcohol, and dried with lint-free paper. Cleaned strips are clamped into a knife maker, scored with a tungsten carbide wheel, cracked to form 25 x 25 mm square pieces, and further broken into two triangular shaped knives using even pressure.