Microtomy Flashcards
What is the purpose of microtomy (a microtome)?
FOR A PATHOLOGIST TO MAKE A DIAGNOSIS………
TISSUE MUST BE CUT THINLY AND PLACED ON A GLASS SLIDE
What are the main items to be done for cleaning a microtome?
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.
How should maintenance be done on a microtome?
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.
What are the type of microtome most commonly found in hospitals?
Hospital sliding microtome, where the block stays stationary and the blade can be moved. Once knife is back in original position the block advances.
What type of microtome is used for plastics or electron microscopy.
Ultramicrotome.
What is the main factor determining good quality slides?
A SHARP, WELL ALIGNED KNIFE
What kind of knife is used to cut paraffin blocks? Thickness of sections?
Disposable stainless steel blades for paraffin blocks – cut 3 to 5 µm
What kind of knives are used for survey sections viewed by light microscopy in prep for electron microscopy? Thickness of sections?
Glass knives- used to cut thin (0.5 to 1.0 µm) epoxy resin (plastic) sections for light microscopy
What are diamond knives typically used for? Thickness of sections achieved?
Diamond knives- used to cut ultra-thin sections (70-90 nm) for electron microscopy.
What is the clearance angle and what typically should it be set at?
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
How are the slides pre-prepared after the blocks are embedded? What are the typical next steps in the technologist’s job?
- Once blocks are embedded, labels are printed out to match the cassettes. Slides are pre-labeled and laid out in a slide tray.
- The cassettes that go with the tray of slides are put together.
- A technologist will grab a set and go to their microtome.
- 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.
What is roughing in?
ROUGHING IN”
Remove wax and expose full face of tissue
The flatter the tissue is embedded the easier this is to do.
What is best practice for ‘roughing in’?
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
What are the best practices and/or steps for ‘fine cutting’?
FINE CUTTING
- Best to work with blocks that have been on ice/water block
- Cut around one revolution per second – different tissues may prefer faster or slower
- Like it to be one cell thick (3 to 5 µm) – keeps all nuclei in one plane of focus
- Get ribbon of sections
- Lay out on flotation bath
What defects are you trying avoid when you lay the ribbon section on the flotation bath?
Try to avoid wrinkles/folds, large bubbles, tears, splits in tissue
What is one of the most important safety rules to remember when working on the microtome?
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.
What is the purpose of the flotation bath?
PURPOSE – float out ribbons to remove compressions. If you act fast you may be able to stretch out wrinkles/folds.
What are issues that can be encountered in the flotation bath as you work on the ribbon?
- As you float and try to stretch out out wrinkles/folds - if you pull too hard it can tear the tissue.
- May be able to remove tiny bubbles by gently poking but do not poke a hole in the tissue.
- You don’t want to leave tissues in bath too long. Separation of tissue will start to occur.
What kind of water is used in the flotation bath? How often is it replenished?
WATER –filled fresh every morning, distilled water is used or Millipore-filtered deionized water. Tap water is not used.
What should the water temperature be maintained at in the flotation bath?
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).
How do you move the ribbons from the microtome to the flotation bath?
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.
What do some labs add to the water of the flotation bath and why?
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.
What does the colour coding of slides mean?
Colour coded– varies with facility, can mean surgical, autopsy, stat, etc.
Where are slides labeled?
Frosted area – area that is labeled
Place tissue on the labeled side of the slide
