Microtomy Flashcards
how does a rotary microtome work
moving the block over a stationary blade and advancing the block each cycle
why is tissue cut 4-5um
single layer of cells
what tissues are cut 2-3um
lymph and kidney
what tissues are cut 6-8um
breast and brain
what are disposable blades not sharp enough to cut. what is used instead?
resin embedded blocks use glass or diamond to cut <1um
What is facing
trimming away excess wax to expose the full face of tissue
how long does the block have to cool for
at least 15 minutes
what is levelling
section is made, then trim 60-100 um and another section is made
what is serial sections
block is faced and every ribbon is put on slide without skipping sections
what are deepers or recuts from a pathologist
levels are made
what steps for microtomy
- locate safety mechanism
- adjust microtome setting
- ensure all levers are tight
- patient soaking the block
- short ribbons are fine
- keep it clean
when should the handwheel be locked
inserting/removing a block and not actively cutting
what are the microns for facing
20-30
what steps are done prior to fine cutting
fixaation
processing
microtome
temperature of block is softened
what steps happen during fine cutting
cutting speed = 1 rotation per second
ribbons
water bath = 10 degrees below MP of wax
section retrieval
Causes of curved sections
block edge not parallel
dull blade
ununiform tissue density
solution to curved sections
face until parallel
new blade
causes of chatter
loose lever vibration
high clearance angle
hard tissue
OVERDEHYDRATED tissue
dull blade
solutions to chatter
tighten levels
lower clearance angle
surface decal/ rehydrate tissue
new blade
causes of holes in section
facing aggressively
hard particles in the tissue
solution of holes in section
lower micrometer setting
soak block in water
section splitting perpendicular to blade
nick in blade
calcification
hard particles
solution to splitting perpendicular to blade
new blade
surface decal
reembed
fail to form ribbons causes
too hard wax
wrong clearance angle
solution to failing form of ribbons
warm the block with thumb
increase speed
adjust clearance angle
sections attach to block on return stroke causes
low clearance angle
debris
microtome retraction now working
STATIC ELECTRICITY
solution to section attaching to block
increase clearance angle
HUMIDIFY the room
causes of incomplete sections
not fully faced
incomplete paraffin infiltration
solution to incomplete sections
reface block
reembed or reprocess
causes of compression
dull blade
soft or warm paraffin
solution to compression
new blade
cool block
reembed in harder wax
causes of expanding section in waterbath
incomplete tissue
high water bath temp
soluition to expansion in waterbath
reprocess
decrease water bath temp
causes of section rolling into cylinder instead of flat
dull blade
too thick sections
solution to rolling sections
new blade
thinner sections
what temperature is the water bath
41-45 degrees
or
10 degrees lower than MP of wax
too cold waterbath causes what to sections
wrinkles not come out
warm waterbath will cause what to section
melt and disintegrate section
how long and temperature of the oven
30 min - 1 hr at <60C
usually 37C
what is a disadvantage of using microwaves to dry slides
cooking the water and fat of the tissue
purpose of the oven
remove any water from section
NOT MELT WAX
what happens if water is on the slides when staining
deparrifinization is incomplete
when are adhesive or charged slides used
when slides undergo harsh solutions or pretreatments during staining
eg silver stains or IHC (moisture and enzymes)
what are common adhesives
gelatin
albumin
elmers glue
poly L lysine
disadvantage of adhesives
produce background saining
T/F charge slides are positively charged
T
what method to make charge slides
3-aminopropyltriethoxysilane (APTES)
how does APTES work
covalent bind to glass making an amino group coating
causes of parched earth
improper processing
tissue chilled too rapidly (cryosprays)
floating on waterbath too long
