UND 362 Microtomy
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| what are 3 additives to paraffin and what do they add? | beeswax, rubber, plastic
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| what does beeswax do to paraffin | reduces crystal size, increased stickiness and adhesion
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| what does rubber do to paraffin | reduces brittleness, increased stickiness and ehances ribbon formation
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| what does plastic do for paraffin | increased hardness and support
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| define plastic point | lowest temp where perm. deformation can occur w/out fracturing
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| section quality is determined by what? | sharp knife, proper clearance angle (3-8○), correct cutting speed (1 rev/sec)
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| what happens if cutting is done too fast/ too slow | fast = increased section thickness
slow = poor ribboning
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| if blade tilt is too slight it will cause what (4) | missed sections, skipped sections, thick and thin sections, wrinkles and jammed sections
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| if blade tilt is too great what can occur | chatter, washboarding, microvibrations, undulations
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| when checking section thickness (4-6 microns) with a microscope what should be seen | all nuclei should stay in one plane when focusing up and down
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| what are the exceptions to sectioning | amyloid cut at 8-12 microns, brain at 6-8/10-15/or 20-30 for FS, biopsies (liver, lymph node) cut at 2-4 micron
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| what are 7 things to consider when facing in a block (rough cutting) | inspect face for issues, orient block in holder, orient to reduce resistance, secure and tighten all screws/clamps, advance on tissue, look for artifacts in section, *best to keep block holder close to microtome as possible*
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| what are the adhesives used on slides | albumin, glue, gelatin, agar, poly L lysine, chrome alum, + charged slides
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| what can be caused if albumin is used as a flotation adhesive | increase hematoxylin on slides
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| what are + charged slides good for and a drawback | good for IHC, but are costly
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| describe the artifacts that can occur if the temp is too high or too low | increased temp = edema/parched earth (ie dry mud cracking), decreased temp = sections won't decompress and will appear wavy
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| if a section is too thick what is the cause/correction and how is it shown | cause - excessive speed, therefore slow down
cells look layered (some in and others out of focus)
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| if a ribbon will not for what is cause/correction | cause = dull knife, excessive clearance angle
fix by change knife and reduce clearance angle. can also be caused by hard paraffin (melt pt too high or room temp too low)
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| crooked ribbons cause and correction | block is not parallel in holder, check for paraffin on edges of block, replace and reface
other causes (irregular chilling, dull knife)
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| what can cause holes in sections and correction | block faced too aggressively, excessive dehydration, brittle tissue FIX - face gentler, soak blocks
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| what are some additional causes for holes in sections and fix (besides excessive facing, excess dehydration, brittle tissue) | air bubbles during embedding, hollow tissue not infiltrated FIX - improve embedding technique and improve infiltration
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| what is the cause when sections lift from knife edge when block is raised and FIX | dull knife, too little knife tilt, warm room or paraffin too soft (get sharp knife, increase clearance angle, cool room, cool block or embed in higher melt pt paraffin)
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| what can cause washboarding or undulation and fix | cause - loose knife or hard tissue (uterus)
fix - tighten clamps, soak in water
undulation appears as whitish parallel lines on slide
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| what caused micro vibrations/chatter and what can fix. also give appearance | over dehydration, dull knife, excessive knife tilt FIX - soak in h2o, change knife, lower clearance angle (appears as mini parallel lines close together)
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| what causes compressed, wrinkled, jammed sections | dull knife, or knife gummed w/paraffin
fix- change knife clean off excess paraffin
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| artifact can appear similar to parched earth | an air bubble trapped under section (it will have the same cracking but will be darker surrounding it because it will have been stained on both sides)
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| what can cause lengthwise scratches or splits in a ribbon and give FIX | defect in knife or hard region in tissue
fix - move knife if the tear remains then it is in the tissue) (knife lines appear as straight lines fully through slide section)
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| give some info on the cryostat for FS | knife typically wedge shape, tilt usually 30○
sections picked up on glass slides, fixed in alcohol, acetone, carnoys, methanol OR air dried and post fixed
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| what are some facts pertaining to getting good sections with a cryostat | training, sharp knife, -20○C, good brush and practice. some cryostats have anti roll device
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| how does the temp vary per tissue with a cryostat | colder for fatty tissue, warmer for brain -12c, liver -14c, spleen -16c, lymph nodes -14c, fixed tissue -12○ - -17○
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| what are the ADV to a cryostat | immediate freezing and sectioning, immediate info for dr's, (sections fixed in alcohol and H/E performed, some IHC requires FS)
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| what are 3 disadv to cryostat | histology is less than optimal, some tissue should never be frozen, freeze artifact common
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| what is the freeze artifact for cryostats | caused by slow freezing of tissue. ice crystal size is proportional to speed of freezing (slower = larger holes) (appears as microscopic holes in tissue)
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| how can freeze artifacts in cryostats be avoided | using passive heat sink, cryo spray, snap freezing (isopentane bath in liquid nitrogen (ie muscle bx), use PPE, -15- produces least artifacts, preferred for muscle enzyme and lymph node surface markers
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| give safety and cleanup of cryostat | *blood borne pathogen potential*. keep cryo clean/disinfected, pick up tissue with alcohol gauze (put in bio bag), lube per manual. When defrosting cryo must be dry or it will freeze up and become inoperable
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| what are some things to avoid with a cryostat | avoid whipping handwheel quickly, avoid sprays that may blow back tissue (tissue may have HepB and HepC)
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| Explain the research technique with FS on formalin fixed tissue | use cryoprotectant (20-30% sucrose\must sink) in PBS (phosphate) buffer
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