Week 5 - Microtomy Flashcards
What is microtomy
- cutting tissues with precision into small slices with microtome :small” + “to cut”
-a microtome is a precise cutting tool that cuts slices from a block of embedded tissue
-embedded in paraffin for routine and plastic for electron microscopy
what are some safety things to remember when using a microtome
- use forceps and brush in order to pick up sections
-lock wheel at 12
-raise blade guard
-remove blade when not in use
-place all blades in sharps container
-dont catch falling blade
-dont have a microtome away from drafty areas because your ribbons can fly away
What are the components of a microtome
-firmly supported blade
-device to hold tissue
-mechanism to move blade across tissue or tissue across blade
what do you need to get good quality sections
-embedded blocks
-sharp blade with cold tissue block
-microtome (either rocking, rotatory microtome or sliding microtome( hold block stationary only blade moves used for large hard tissues))
-skilled tech
What is a rotary microtome
-has a rotary wheel with stationary blade holder and moving specimen holder- check rest on S 14
What is a specimen block/clamp holder
-holds paraffin block tightly
-why you paratrim so it can be tightly fit
-otherwise itll vibrate because its uneven
-can fit all 4 orientation but we face label left
what are the specimen orientation screws
-moves the specimen in relation to edge of microtome knife
-vertical (N/S) or Horizontal (E-W)
-dont move until the microtome is calibrated the next day
What is the section thickness gauge and knob
1-5 micromilimetres
-section cut from 4 micron
kidneys and cellular tissues are cut the thinnest at 3u
Brain is cut at 6u
DNA material at 10u
Congo red stain 8-10 u (amyloid sample)
what is the knife holder and base
-can be removed for cleaning
-has slides in 2 runners/tracks
-locked in place should only unlock when moving blade closer to block
What is meant by the clearance angle
-it is the angle between the block surface and blade bevel
-prevents the blade coming into contact with the block
-adjustments are from 0-10 but the usual is 3-8 degrees
-if angle too steep itll deform the specimen
What is the drive wheel (fine trimming)
-handle must be at 12 o clock
-ensure the levers are locked when floating out sections (towards you is locked)
-blade guard should be up
-crank clockwise at 4 microns
what does the drive wheel and coarse trimming wheel look like
-connected to brass cylinder where the specimen clamp is.
-if you see too much of the brass or a red line- retract
-consists of a feed mechanism
-used for trimming blocks
-ensure you load blocks in the same direct you cut
-moves clockwise toward you and vice versa
what is the advanced mechanism
-pawl and gear= ratchet
-continuous rotary motion in one direction clockwise
-gears that are attached to feed mechanism of cylinder and can be moved by coarse trimming wheel or drive wheel
-get stripped when it reaches its limit or when it is turned counterclockwise
what is the drive wheel and what is the rate of cutting
-part of advance mechanism
-if you turn 180 degrees the specimen is lowered
-if you turn 360 specimen in raised
-crank away from you and its one revolution per second while alternating with coarse trimmer at 1/8 turn
-if you crank to fast youll create heat which can warm up tissue block faster
-make sure you crank the drive wheel 5-10x and set the micro meter at 4um to remove the moth eaten effect
How is the microtome cleaned
-blade is removed and disposed in sharps
-wax is cleaned off with gauze
-water droplets are wiped off
-runners are oiled as required
-cover microtome - lock drive wheel, knife holder base, clearance and retract the holder clamp
-RELEASE pressure tension plate
make sure you complete the clean up checklist
for proper set up what must be done
-retract the feed mechanism cylinder
-calibrate the microtome so the block face is parallel to blade edge
-make sure block is push to the back of the specimen clamp to avoid two angles on block face or vibrations during cutting
-all blocks should be coarse trimmed and placed face down on ice for 15 mins. old wax gives better support
-fine trim with drive wheel cold block and label corresponding slides
-drive wheel must be at 12 o clock
-do not leave blade in the middle of the block
-when there is no blade remove wax
-if you put on a new blade, wipe upwards with gauze to remove oil
-make sure everything is tightened before cutting
-4-5 sections on a ribbon
how to mount sections on a slide
-ensure there are no bubbles in waterbath
-float ribbon on far end of waterbath in one smooth motion
-only pick artifact free sections dont worry about artifacts in the wax
-use pull technique -dont scoop sections on slide
-put in 60 degree oven for an hour , 2-4 degrees above paraffin melting point
-leave thermometer in the waterbath dont need to hold it
-label slides AFTER picking up the tissue section
how make better microtomy
-use high quality knives - shitty knives leave fine lines which can be seen during flotation
-have proper clearance angle otherside you can get short sections of ribbons
-trim blocks properly if not then you can get holes in the sample if youre too rough on the para trimmer
-avoid freezing damage if not there can be cracking. This will make sectioning and floatation hard because the wax is not bound to the tissue
-tissue will crack after frozen after coarse trimming
-use the whole blade dont start in the middle other wise youll have one sharp end and one dull end causing crooked ribbons
why are cold blocks used in microtomy
warm blocks can cause compression
-sit on ice for 10-15 minutes
why should you cut sections slowly
-you can get fine chatter if a cold block of brittle tissue is cut too fast. Try to warm the block a little first before cutting very slowly
-chatter can also happen if block or blade isnt secured
why should you use clean water in waterbath and also only float one sample at a time
use clean slides
extra organisms may turn up on the slide
-dont float too many samples at once to avoid inaccurate identification
-hold the frosted end adhesives are added to the waterbath - gelatin based - dont scoop slides and allow slides to dry
how could cross contamination occur during microtomy
-could occur in flotation bath
-sweep waterbath surface with tissue in between samples
how can contamination with squames be avoided
- can occur on floatation bath
-dont touch the water bath so cells dont shed off
why should you always check the water temperature
-if the bath is too warm the section can show cracks and separation of layers
-dont float ribbons while checking temps it can lead to artifact
-cold = holes (better for fatty tissues)
hot=spread apart
