Microtomy - Lecture notes 11 PDF

Preview

Summary

microtomy...

Description

MICROTOMY  Process of trimming and cutting processed tissue (mostly, paraffin embedded tissue) into uniformly thin slices or sections to facilitate studies under the microscope.  skilled process that requires precision and hand - eye coordination combine with a delicate touch by the experience histotechnologist/cian Microtome  The basic instrument 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.  Principle o 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 uniform thickness. PARTS: 1. 2. 3.

4.

5.

Block holder / chuck  where the tissue is held in position Knife carrier and knife  for actual cutting of tissue sections Pawl, Ratchet feedweel and adjustment screw  Properly line up tissue block with the knife  Adjust proper thickness for successive sections Rotary wheel  Needs to be manipulated, moved mechanically to start the cutting process Microtome gauge  Determines the thickness of sections

TYPES: 1.

Rocking / Cambridge microtome  Paldwell Trefall, 1881  For cutting sections of small or large blocks of paraffin embedded tissue  available in two sizes  Simplest, with heavy base and two arms o Lower arm  resting on pivots and a supporting column, attached to a micrometer screw o Upper arm  connected to a lever by a piece of nylon thread  In an arc of a circle, a section is cut to 10-12μ thickness in a slightly curve plane  In practice, it is used to prepare serial sections of large paraffin blocks DISADVANTAGES: 1. Theoretically, not recommended for serial sections since tissues are cut into slightly curve planes 2. Not favored by most laboratories due to the restrictions in size of block that can be cut 3. Difficulty of reorienting the block

2.

Rotary / Minot microtome  Minot, 1885-1886  For cutting paraffin embedded tissues, 4-6μ  Most common type for both routine and research laboratories  Operated by the rotation of the flywheel  Block holder and knife: Brought together by upward and vertical motions  Manual and electrical models: for ultrathin sections and cryostat use



Electrically driven microtome is also now available and can be ideally used to produce ribbons for serial section.

ADVANTAGES: 1. Cut sections in a perfectly flat plane 2. Excellent serial sections to be cut 3. Heavier and more stable than rocking microtome 4. For cutting of large block of tissues DISADVANTAGE:

1.

Knife in blade-up position thus relatively dangerous

3.

Sliding microtome  Adams, 1789  Thickness  7-9μ  Most dangerous type and requires slow but very steady motion  for cutting celloidin embedded sections ADVANTAGES (If both used): 1. Ease cutting of large and small tissues 2. For extremely hard and tough tissue blocks 3. Obliquely knife = for celloidin sections 4. Straight knife = for large refractory paraffin blocks

4. 5.

Base sledge o With two movable pillars holding the adjustable knife clamps 6. o Allows knife to be set to an angle for cutting celloidin sections 7. o Block holder: Movable 8. o Knife: Stationary 9. Base sledge 10. o With two movable pillars holding the adjustable knife clamps 11. o Allows knife to be set to an angle for cutting celloidin sections 12. o Block holder: Movable 13. o Knife: Stationary 14. Base sledge 15. o With two movable pillars holding the adjustable knife clamps 16. o Allows knife to be set to an angle for cutting celloidin sections 17. o Block holder: Movable 18. o Knife: Stationary 1. Base sledge o With two movable pillars holding the adjustable knife clamps o Allows knife to be set to an angle for cutting celloidin sections o favored in laboratories where very hard tissue or large blocks are usually sectioned. o Block holder: Movable o Knife: Stationary ADVANTAGES: 1. For sectioning of very hard tissues or large blocks, offering great resistance to knife 2. For sectioning specimens embedded in all forms of media and tough tissue blocks 3. Easy cutting of large sections 4. Section are in a perfectly flat plane= excellent tissue sections 5. More stable than the ordinary sliding microtome 6. Electrical modern model; for resin embedded decalcified bone 2.

Standard sliding o For celloidin embedded tissue blocks \ requires slow and steady motion o Block holder: Stationary o Knife: Moved backward and forward DISADVANTAGES: 1. More dangerous because of the movable knife 2. Difficult to

attach knife guards Freezing microtome  Queckett, 1848  Thickness  10-15μ  for cutting unembedded frozen sections.  Hollow and perforated stage for block holder  Allows passage of CARBON DIOXIDE in a flexible lead pipe from a cylinder  Second cooling device is incorporated to lower the temp. of the knife to facilitate sectioning ADVANTAGES: 1. For cutting undehydrated tissues in a frozen state use for rapid diagnosis 2. For histological fat demonstration 3. For neurological studies 4. For damaged or destroyed sensitive tissue constituents due to heat 5. Ultrathin microtome  For cutting tissue sections at 0.5μ for ELECTRON MICROSCOPY  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  Sections are stained with an aqueous solution of an appropriate heavy metal salt and examined with a transmission electron microscope (TEM).  Knife: Selected fragments of broken plate glass  Specimen:  small, fixed in Osmium Tetroxide and embedded in Plastic

1.

4.





Plane-Concave knife (25mm length)  One side is flat and the other is concave  Less concave sides: for cutting celloidin-embedded tissue blocks (sliding microtome)  More concave sides: for cutting paraffin sections (basesledge, rotary, rocking microtome)



Biconcave knife (120mm length)  Both sides are concave  For cutting paraffin embedded sections (rotary microtome) Plane-Wedge knife (100mm length)  Both sides are straight/flat  For frozen sections or extremely hard and tough spx embedded in a paraffin blocks (sledge type or sliding microtome) Disposable blades  Razor-sharp blade: flawless cut section of the diagnostic tissue  2-4μ tissue section is needed  Magnetic knives : for cryostat Glass knives  For trimming and semi-thin sectioning of tissue blocks for Electron Microscopy  Prepared commercially, 40 x 2.5 cm \ washed with detergent, rinsed with distilled water and alcohol \ dried with lint-free paper  Scored with a tungsten carbide wheel, cracked to form 25 x 25 mm squares  Broken into two triangular-shaped knives with even pressure Diamond knives  For cutting any type of resin block for Electron Microscopy

6.

Cryostat / cold microtome  for cutting frozen sections  Refrigerated apparatus, used in fresh tissue microtomy  Freeze tissue to correct degree of hardness for easier and faster sectioning  With rotary microtome inside, kept inside a cold chamber (5 to -30°C or -20°C average)  Freeze tissues within 2-3 minutes  Section size =  4μ ADVANTAGES: 1. Prepare thin sections of fresh frozen tissues for fluorescent antibody staining or histochemical enzyme studies 2. Commonly used for INTRAOPERATIVE DIAGNOSIS, for rapid preparation of urgent tissue biopsies 3. Close to the operating room, inside the frozen section room 4. Sections are directly transferred to the slide or coverglass,at low temp.





 Microtome Knives:  Good cutting edge – should be made of good quality steel \ must be able to cut good sections from a paraffin wax block about 2-3μ thick without any serration  Too soft edges – becomes dull easily  Too hard edges – produces nicks or jagged edges, irregularities, tears or striations  Safety razor blades  ADVANTAGES:  1. For partially calcified materials, paraffin and frozen sections  2. Readily replaced when dull  3. Produce similarly good sections as those cut with microtome knives ADVANTAGES:

For partially calcified materials, paraffin and frozen sections 2. Readily replaced when dull 3. Produce similarly good sections as those cut with microtome knives DISADVANTAGE: 1. Unsatisfactory for sections...