Week 5 - Microtomy

Question 1

What is microtomy

Answer 1

• 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

Question 2

what are some safety things to remember when using a microtome

Answer 2

• 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

Question 3

What are the components of a microtome

Answer 3

-firmly supported blade
-device to hold tissue
-mechanism to move blade across tissue or tissue across blade

Question 4

what do you need to get good quality sections

Answer 4

-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

Question 5

What is a rotary microtome

Answer 5

-has a rotary wheel with stationary blade holder and moving specimen holder- check rest on S 14

Question 6

What is a specimen block/clamp holder

Answer 6

-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

Question 7

what are the specimen orientation screws

Answer 7

-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

Question 8

What is the section thickness gauge and knob

Answer 8

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)

Question 9

what is the knife holder and base

Answer 9

-can be removed for cleaning
-has slides in 2 runners/tracks
-locked in place should only unlock when moving blade closer to block

Question 10

What is meant by the clearance angle

Answer 10

-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

Question 11

What is the drive wheel (fine trimming)

Answer 11

-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

Question 12

what does the drive wheel and coarse trimming wheel look like

Answer 12

-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

Question 13

what is the advanced mechanism

Answer 13

-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

Question 14

what is the drive wheel and what is the rate of cutting

Answer 14

-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

Question 15

How is the microtome cleaned

Answer 15

-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

Question 16

for proper set up what must be done

Answer 16

-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

Question 17

how to mount sections on a slide

Answer 17

-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

Question 18

how make better microtomy

Answer 18

-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

Question 19

why are cold blocks used in microtomy

Answer 19

warm blocks can cause compression
-sit on ice for 10-15 minutes

Question 20

why should you cut sections slowly

Answer 20

-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

Question 21

why should you use clean water in waterbath and also only float one sample at a time

use clean slides

Answer 21

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

Question 22

how could cross contamination occur during microtomy

Answer 22

-could occur in flotation bath
-sweep waterbath surface with tissue in between samples

Question 23

how can contamination with squames be avoided

Answer 23

• can occur on floatation bath
  -dont touch the water bath so cells dont shed off

Question 24

why should you always check the water temperature

Answer 24

-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

Question 25

how do we avoid wrinkles in sections

Answer 25

-only flotation will not fix the wrinkles in sections instead use a better cutting technique and warm water

Question 26

how to avoid over expanding sections

Answer 26

-dont float for too long on a waterbath that is too hot
-lymphoid and hematopoietic tissues to prone to over expansion damafe

Question 27

how can floating sections be damaged

Answer 27

-mechanical damage by gouging when trying to remove a fold in the section
-only touch wax not tissue

Question 28

how to choose sections to put on a slide

Answer 28

-on a ribbon the first sections can be wide or least compressed but as the block warms the sections can get narrow or more compressed. And even though the microtome is on 3 um those wide sections the first sections could be thicker due to thermal expansion causing darker staine
-better to use middle section

Question 29

how do we prevent bubble under sections

Answer 29

• making sure there are no bubbles under the section during flotation because it can prevent flattening and adhesion

Question 30

how to prevent tissue lifting

Answer 30

ensure you dont scoop up when putting the section on a slide

Question 31

why should you drain before drying

Answer 31

-you can have a raised out of focus area. This can be prevented by vertically picking up a section on the flotation bath and drying vertically for a bit before putting into the slide dryer.
-60 degree drying oven , slides may have to stay in for a hour there might wax droplets on the slide

Question 32

why should drying temperature be monitored

Answer 32

-can cause nuclear meltdown on the perimeter of samples if there is too much heat on the slide dryer
-affects primarily epithelial tissues
-The nuclei will show uneven staining appearing pink or blue
-can also cause cracking with prolonged heat exposure - parched earth mostly in highly cellular tissues like thymus spleen

these defects can also be caused by over tissue processing but you wouldnt know until you looked at stained slide

Question 33

how to troubleshoot under processed tissues

Answer 33

*Remove wax with xylene (2 changes)
* Remove xylene with 100% alcohol (2-4 changes)
* Remove 100% alcohol with xylene (2 changes)
* Infiltrate with wax (2-4 changes)

Question 34

how to troubleshoot over processed tissues

Answer 34

• Coarse trim the blocks
• Place block face on ice water or cold weak detergent solution or gauze wet with fabric
  softener

Question 35

how to troubleshoot under decalcified tissues

Answer 35

• Surface decal tissues for 10 min, rinse block face with cold water, chill on ice then cut.

Question 36

what are some Embedding artifacts will greatly affect microtomy:
* Floating artifact
* Stratification
* Wrong size mold .
* Air bubble
* Poor orientation

Answer 36

• Floating artifact will result in loss of tissue.
• Stratification could result in loss of block face.
• Wrong size mold will result in compression.
• Air bubble in the block will not cut.
• Poor orientation will result in tissue compression, scores or drag.