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UND 362 Microtomy

what are 3 additives to paraffin and what do they add? beeswax, rubber, plastic
what does beeswax do to paraffin reduces crystal size, increased stickiness and adhesion
what does rubber do to paraffin reduces brittleness, increased stickiness and ehances ribbon formation
what does plastic do for paraffin increased hardness and support
define plastic point lowest temp where perm. deformation can occur w/out fracturing
section quality is determined by what? sharp knife, proper clearance angle (3-8○), correct cutting speed (1 rev/sec)
what happens if cutting is done too fast/ too slow fast = increased section thickness slow = poor ribboning
if blade tilt is too slight it will cause what (4) missed sections, skipped sections, thick and thin sections, wrinkles and jammed sections
if blade tilt is too great what can occur chatter, washboarding, microvibrations, undulations
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
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
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*
what are the adhesives used on slides albumin, glue, gelatin, agar, poly L lysine, chrome alum, + charged slides
what can be caused if albumin is used as a flotation adhesive increase hematoxylin on slides
what are + charged slides good for and a drawback good for IHC, but are costly
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
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)
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)
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)
what can cause holes in sections and correction block faced too aggressively, excessive dehydration, brittle tissue FIX - face gentler, soak blocks
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
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)
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
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)
what causes compressed, wrinkled, jammed sections dull knife, or knife gummed w/paraffin fix- change knife clean off excess paraffin
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)
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)
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
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
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○
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)
what are 3 disadv to cryostat histology is less than optimal, some tissue should never be frozen, freeze artifact common
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)
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
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
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)
Explain the research technique with FS on formalin fixed tissue use cryoprotectant (20-30% sucrose\must sink) in PBS (phosphate) buffer
Created by: mustangvxd



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