Microtomy

Question 1

What is the purpose of microtomy (a microtome)?

Answer 1

FOR A PATHOLOGIST TO MAKE A DIAGNOSIS………

TISSUE MUST BE CUT THINLY AND PLACED ON A GLASS SLIDE

Question 2

What are the main items to be done for cleaning a microtome?

Answer 2

CLEANING
Dispose blade in sharps
Brush large bits and ribbons into the garbage
Use pressure to remove wax. Can use a xylene substitute
Note: The textbook says to clean the microtome thoroughly at the end of each day or shift.

Question 3

How should maintenance be done on a microtome?

Answer 3

MAINTENANCE
Must have regular maintenance by company.
Note: Textbook says apply microtome oil or grease to all sliding parts as indicated by the manufacturer and according to their schedule on a routine basis.

Question 4

What are the type of microtome most commonly found in hospitals?

Answer 4

Hospital sliding microtome, where the block stays stationary and the blade can be moved. Once knife is back in original position the block advances.

Question 5

What type of microtome is used for plastics or electron microscopy.

Answer 5

Ultramicrotome.

Question 6

What is the main factor determining good quality slides?

Answer 6

A SHARP, WELL ALIGNED KNIFE

Question 7

What kind of knife is used to cut paraffin blocks? Thickness of sections?

Answer 7

Disposable stainless steel blades for paraffin blocks – cut 3 to 5 µm

Question 8

What kind of knives are used for light microscopy? Thickness of sections?

Answer 8

Glass knives- used to cut thin (0.5 to 1.0 µm) epoxy resin (plastic) sections for light microscopy

Question 9

What are diamond knives typically used for? Thickness of sections achieved?

Answer 9

Diamond knives- used to cut ultra-thin sections (70-90 nm) for electron microscopy.

Question 10

What is the clearance angle and what typically should it be set at?

Answer 10

The clearance angle is angle between the vertical (block face) and the back of the blade knife edge.
Clearance set between 3 - 8 degrees (5 degrees at RRC) – may have to alter for different tissues

Question 11

How are the slides pre-prepared after the blocks are embedded? What are the typical next steps in the technologist’s job?

Answer 11

1. Once blocks are embedded, labels are printed out to match the cassettes. Slides are pre-labeled and laid out in a slide tray.
2. The cassettes that go with the tray of slides are put together.
3. A technologist will grab a set and go to their microtome.
4. They will rough in all the blocks then start sectioning.

Note: In my lab experience she did not want us to get too far ahead on slide labelling.

Question 12

What is roughing in?

Answer 12

ROUGHING IN”
Remove wax and expose full face of tissue
The flatter the tissue is embedded the easier this is to do.

Question 13

What is best practice for ‘roughing in’?

Answer 13

Best to rough in several blocks at a time then switch to a new part of a blade (or new blade to do the fine cutting

Question 14

What are the best practices and/or steps for ‘fine cutting’?

Answer 14

FINE CUTTING

1. Best to work with blocks that have been on ice/water block
2. Cut around one revolution per second – different tissues may prefer faster or slower
3. Like it to be one cell thick (3 to 5 µm) – keeps all nuclei in one plane of focus
4. Get ribbon of sections
5. Lay out on flotation bath

Question 15

What defects are you trying avoid when you lay the ribbon section on the flotation bath?

Answer 15

Try to avoid wrinkles/folds, large bubbles, tears, splits in tissue

Question 16

What is one of the most important safety rules to remember when working on the microtome?

Answer 16

Locking lever
Microtome safety is paramount. 
This is a simple slide lock. 
prevents the drive wheel from moving.
Must be in the locked position (12 o’clock) when not turning the drive wheel, start to lock at the 10 o’clock position and as the drive wheel moves to the 12 o’clock position the lock will click into place.

Question 17

What is the purpose of the flotation bath?

Answer 17

PURPOSE – float out ribbons to remove compressions. If you act fast you may be able to stretch out wrinkles/folds.

Question 18

What are issues that can be encountered in the flotation bath as you work on the ribbon?

Answer 18

1. As you float and try to stretch out out wrinkles/folds - if you pull too hard it can tear the tissue.
2. May be able to remove tiny bubbles by gently poking but do not poke a hole in the tissue.
3. You don’t want to leave tissues in bath too long. Separation of tissue will start to occur.

Question 19

What kind of water is used in the flotation bath? How often is it replenished?

Answer 19

WATER –filled fresh every morning, distilled water is used or Millipore-filtered deionized water. Tap water is not used.

Question 20

What should the water temperature be maintained at in the flotation bath?

Answer 20

TEMPERATURE – usually maintained 5°C to 10°C below the melting point of the wax. With some waxes it must be a bit lower than that. If the wax starts to swell and separate (parched earth) it is probably too hot. (May also be an issue with getting too hard and brittle in processor or embedding station).

Question 21

How do you move the ribbons from the microtome to the flotation bath?

Answer 21

Use a wet applicator stick to move the ribbon from the blade of the microtome to the water bath. You can stretch the ribbon slightly to help remove wrinkles. You must clean the bath between specimens to avoid carryover of samples.

Question 22

What do some labs add to the water of the flotation bath and why?

Answer 22

Some labs use a chemical in the water to help get wrinkle free sections by reducing the surface tension. There are commercial agents available. Some add 2 mL of 95% alcohol to the waterbath.

Question 23

What does the colour coding of slides mean?

Answer 23

Colour coded– varies with facility, can mean surgical, autopsy, stat, etc.

Question 24

Where are slides labeled?

Answer 24

Frosted area – area that is labeled

Place tissue on the labeled side of the slide

Question 25

How are slides labeled?

Answer 25

Labelling
– done before tissue is put on the slide,
- slides are filed with the frosted end up.
- Must be able to read the label.
- Label must match the information on the block.
We label the slides with the Histo pencil. Other pencils or markers will wash off in the staining process.

Question 26

How should slides be handled?

Answer 26

Pre-cleaned, should use some sort of adhesive
Do not handle with bare fingers – will contaminate with squamous cells
Never use a dirty/contaminated slide.

Question 27

What are the purpose of slide adhesives?

Answer 27

Allow tissue to stay on slide during staining process

Question 28

What are the different types of slide adhesives?

Answer 28

TYPES
Egg Albumin – coated on slide
Glue Derivative - coated on slide
Gelatin (added to the waterbath or on slides)
Poly-L-Lysine, SILANE (AAS)- used for special stain methods and IHC, Frozen sections of fixed tissue, in situ hybridization
POSITIVELY CHARGED SLIDES (attracts electrostatically)- excellent for silver stains, IHC, and enzyme histochemical techniques.

Question 29

What are the disadvantages of slide adhesives?

Answer 29

1. Excessive adhesive causes background staining as most are protein in nature.
2. It may make the slide look messy and it may interfere with examining the tissue.
3. Some of these can become contaminated with microorganisms.

Question 30

Why are slides heated after they are made?

Answer 30

Slides are heated after microtomy to remove any traces of water. The wax will start to melt but the purpose is not to remove the wax. If this is not done the section may float off in staining solutions. Water also contaminates xylene in the first steps to perform staining techniques.

Question 31

What is the process of heating the slides?

Answer 31

This can be achieved by a slide warmer or in a dryer or oven. The best temperature is 60°C. They are left for 15 to 60 minutes (the shorter time is for forced air dryers). Higher temperatures will affect antigens and cannot be used for IHC.
Can be left overnight at 37°C as a more gentle way of removing the water.

Question 32

What is the most common reason for faulty ribbons?

Answer 32

MICROTOMY – most faults are caused by a dull knife or a warm block. When you are starting out make sure all levers are tight before and you back up the block holder between blocks.

Question 33

What are “routine” sectioning rules for larger specimens?

Answer 33

ROUTINE – large specimens – one section per slide. Place in the middle of the slide. Follow facility rules - may want small ribbon for smaller tissues.

Question 34

What are recuts / deepers?

Answer 34

RECUTS / DEEPERS – may be requested following viewing by pathologist. There may be something of interest that may lay deeper in the tissue or not all of the specimen is present on the slide.

Question 35

What are level / steps?

Answer 35

LEVELS / STEPS – several sections put on the slide. Rough in, take a section. Rough in further and take another section. Rough in further and take another section. Large specimens will be put on separate slides, small specimens all on one.

Question 36

What are serial sections?

Answer 36

SERIAL SECTIONS – all of the specimen is put on slides, serially. Minimal roughing in (no tissue goes in garbage)

Question 37

How are tissue blocks stored?

Answer 37

Tissue cassettes are stored in boxes in the hospital on shelves and then when that is full, items are moved off-site for storage.
Note: In another class we had discussed that storage was done for a really long time - nearly forever but likely just a lifetime.  She didn't have exact years or anything.