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microtomy
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- 2. 2 Microtomy. Techniqueby which tissues can be sliced into thin sections and then attached to a surface for microscopy Microtomes. These are instruments used to cut uniformly thin sections of the tissues for further study. Microtomes use sharp glass or steel blades/diamond knives to cut the tissues into thin sections of uniform thickness Introduction
- 3. Thickness ofthe sections is predetermined at regular distance on the microtome The sections are attached to a surface like glass slides for staining Tissues like muscles, bones, hair are cut of thickness from 50 nm and 100 µm Thickness of the sections may be fixed according to the need of the study and material involved The stained sections can be studied by light or electron microscopy Introduction
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- 5. Types of microtomy Main types include ◦ Rotary microtome ◦ Cryotome ◦ Ultra microtome ◦ Laser microtome Use depends on the material under study and the nature of the work Simple studies involve rotary microtome while detailed studies may involve ultra microtome, etc. Similarly cryomicrotome involves sectioning of the frozen tissues.
- 6. The mostcommon type of the microtome Rotary action involves the sectioning process at predetermined thickness on every rotation of the flywheel Blade is fixed at horizontal position The sample holder moves the sample ahead by the fixed distance for cutting Flywheel of the instrument may be automatic or manual Section thickness may vary from 0.5 µm to 60 µm Types of microtomy
- 7. Types of microtomy Used for making extremely thin sections (40 nm to 500 nm) Used mostly for biological samples but samples may also be processed The sections are studied in transmission or scanning electron microscopy The cut sections are floated on the top of a liquid These are then mounted on a copper, nickel, gold, or other metal grid
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- 9. It consistsof following main steps Fixation Processing Dehydration Clearing Embedding Section cutting Staining Deparaffinization Method (fixation and processing)
- 10. 10 It isthe most important step in histological studies The histological details will only be demonstrated if the tissue is promptly and adequately fixed It is the process of preserving the tissues in the natural condition Poorly fixed specimens are more difficult to section than the well fixed ones Method (fixation and processing)
- 11. Commonly usedfixatives are alcohol, formalin, glutaraldehyde, etc. Factor affecting fixation are temperature, change in pH, penetration of the fixative, volume, time, etc. The lowest concentration of the fixative is preferred than the higher one 10% formalin or 2.5% glutaraldehyde is used Method (fixation and processing)
- 12. 12 It isa process in which tissues are treated to make the thin sections A specimen is generally processed as follows Dehydration. It is the removal of water from the tissues by immersing serially in 50%, 70%, 85% and 100% alcohol for some time Clearing. It is removal of the dehydrant from the tissues by immersing in paraffin miscible medium like xylene Method (fixation and processing)
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- 14. 14 It isthe process of placing the processed samples into molds like paraffin blocks, etc. The blocks are then used on the microtome to make thin sections There are a number of techniques that can be used to make sections from difficult blocks. If the block is hard or friable, the exposed surface can be soaked in cold water or a softening agent Method (embedding)
- 15. 15 Embedding isan important step that always requires a care Any excess wax on the outside of a cassette should be removed to ensure the block is firmly held during the sectioning Specimen orientation is very important Paraffin embedded specimens may be stored indefinitely for future use Method (embedding)
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- 17. 17 It isprocess of cutting the embedded samples into thin sections It requires great care as tissues of the diagnostic importance can easily be lost or the block surface may be damaged. We should be sure that all of the clamping mechanisms are tightened securely Method (sectioning and staining)
- 18. 18 The thicknessof the sections is normally between 10 μm to 30 μm. The cuts sections are then floated on warm water (at 40 °C) bath that helps to remove wrinkles These are then placed on the glass microscopic slides or some other support for staining, etc. Method (sectioning and staining)
- 19. 19 The slidesmust always be grease and dust free and stored and handled correctly. It involves reversal of the process of embedding to remove paraffin and then staining by water soluble stains The slides are treated with xylene and alcohol to remove the paraffin Method (sectioning and staining)
- 20. 20 Some ofthe stains need mordants (Iodine acts as a mordant in Gram staining of bacteria) The stains used depend on their differential affinity for various cellular organelles Some of the special stains are used in special cases Slides must always be properly labeled Method (sectioning and staining)