Introduction to microtomy

Question 1

What is microtomy?

Answer 1

The process by which thin sections of tissue measuring only a few micrometers in thickness are excised and mounted onto microscope slides.

Question 2

What is the first thing when starting microtomy straight from embedded?

Answer 2

Clean the block - remove excess wax from the edges of the cassette using a blunt scraper.

Question 3

Why should excess wax on the outside of the cassette be removed for microtomy?

Answer 3

Excess wax can cause the block to wobble during microtomy which results in uneven sections coming off the blade.

Question 4

What is the next step after excess wax is removed from the block?

Answer 4

Mount the block by pulling the lever forward and check the block is attached without movement.

Question 5

Should a block be mounted if a blade is in position?

Answer 5

No (though safety measures are put in place to allow this for efficiency).

Question 6

What should be done after the blade has been clamped in place?

Answer 6

Check safety measures - the blade-guard and handwheel lock should always be engaged when not cutting.

Question 7

What should always be engaged on the microtome if not actively cutting tissue?

Answer 7

The handwheel lock.

Question 8

What is the purpose of the handwheel lock?

Answer 8

Prevents accidental dragging down of the block holder (AKA chuck) and hence dragging of fingers onto the blade.

Question 9

What is the name and function of the wheel on the left side of the microtome?

Answer 9

Coarse feed wheel - at the first notch it moves the chuck forward 10 μm, and at the second notch it moves it forward 10X (30 μm).

Question 10

You have mounted a block onto the microtome and are ready to begin. The block is ~1cm away from the blade - what should be done next?

Answer 10

Use the coarse feed wheel to move the block forward until it is just touching the blade.

Question 11

What is the name and function of the dial on the right side of the front face of the microtome?

Answer 11

Section thickness indicator - it is used to change the thickness of the tissue sections that will be cut.

Question 12

What is the most common section thickness (for the purposes of this unit)?

Answer 12

3 μm

Question 13

Why are most tissues (for the purposes of this unit) cut into 3 μm sections?

Answer 13

3 μm sections usually gives one layer of cells per section.

Question 14

What is ‘facing the section’?

Answer 14

Facing the section is the trimming/removal of wax from the surface of the block until the entire face of the embedded tissue is exposed/completely showing through.

Question 15

You have begun facing the section and want to check that the entire face of tissue is exposed. What are your next three actions?

Answer 15

Engage the hand wheel lock, engage the blade guard, and remove the block from the block holder to examine.

Question 16

You have safely removed a block to check if you have completely faced the section. A tiny corner of the section is shiny and still covered by a thin layer of wax, but the majority of the tissue face is exposed. What should you do?

Answer 16

Return the block to the block holder and continue facing the section until you are ready to check the block again.

Question 17

You have finished facing the section and have removed the block to confirm this. What is your next action?

Answer 17

Place the block back on the cooling plate and clean the wax from the microtome while the block solidifies.

Question 18

Do microtomes need to be cleaned when switching between blocks?

Answer 18

Yes

Question 19

Why should microtomes be cleaned when switching between blocks?

Answer 19

Cleaning between different blocks prevents cross-contamination of samples in both diagnostic and research settings.

Question 20

How should wax be cleaned off a microtome?

Answer 20

Use a brush to flick the wax up and away from the blade and into the well.

Question 21

You have faced the section and allowed it to cool before remounting and cutting a few sections. After a few slices you see that the sections are crumpling and appear folded. What does this mean and what should your next actions be?

Answer 21

This indicates that the block was not cooled enough before returning to the microtome; the wax is too soft. Engage the handwheel lock and blade guard, remove the block from the holder and return it to the cooling plate for another 5 - 10 minutes.

Question 22

You have faced the section and allowed it to cool before remounting and cutting a few sections. After a few slices you see that the sections are pulling to one side. What does this mean?

Answer 22

This suggests that the blade may be getting blunt or that there may be some material stuck to the blade.

Question 23

How should you transfer section ribbons to the water bath?

Answer 23

Cradle the ribbon between forceps and a thin brush.

Question 24

What motion should be avoided when mounting sections in a water bath on a glass slide?

Answer 24

Scooping actions should be avoided - use an upwards motion instead.

Question 25

How should you separate a section ribbon in the water bath?

Answer 25

There are three methods - use the point of a non-stick (yellow) slide to separate the ribbon, use the long edge at the notch and lift, or use forceps to separate.

Question 26

Describe the process of mounting a section in a water bath on a slide.

Answer 26

Touch the bottom of the water bath with the slide, tilt the slide forward 45', move it to the edge of the target section, and lift up (maintaining the angle).

Question 27

What should be done with unwanted sections in the water bath after desired sections have been mounted on slides?

Answer 27

Remove unwanted sections by dragging a tissue across the surface of the bath, repeating if necessary.

Question 28

What should be done immediately after mounting sections on slides?

Answer 28

Label the slides.

Question 29

Slides should be ____ immediately after mounting.

Answer 29

Labelled

Question 30

What information must be included when labelling a slide?

Answer 30

Name (surname, initial) Date of staining Tissue (if known) Stain type (if known)

Question 31

Should an old/blunt blade be kept?

Answer 31

Yes, old blades can be kept for use as trimming blades.

Question 32

When replacing a microtome blade, should you push the blade all the way into place?

Answer 32

No - slide the blade most of the way into place then use a pencil or brush to push it the rest of the way.

Question 33

What is the purpose of the small handle to the right of the blade on a microtome?

Answer 33

Clamps the blade in place. The handle is pulled towards the user to release the blade and pushed away to secure the blade.

Question 34

How long should a section ribbon be?

Answer 34

4 - 5 sections is adequate - don't get too greedy!

Question 35

What should you be aware of when transferring a section ribbon from the microtome to the water bath?

Answer 35

Air currents in the room. Remember to cradle the ribbon gently between forceps and brush, and be sure to close the door!

Question 36

What is the difference between yellow and white slides?

Answer 36

Yellow slides are non-stick slides. White slides are charged slides used when long or harsh treatments will be applied to the section.

Question 37

Are gloves and safety glasses required for microtomy?

Answer 37

No - gloves and glasses are only required when solvents are being used.

Question 38

What are the six rules of safety for microtomy?

Answer 38

- Lock at 12 o'clock - Knife guard up - Use forceps - Take care placing/removing knives - Clean up *after* knife removal - Don't walk around with sharps