Instrumentation: Microtomy

Question 1

Microtomy

Answer 1

Cutting thin sections of tissue for microscopic examination

Question 2

Micron

Answer 2

0.001mm (one thousandth of a millimeter)

1uM

Question 3

Bevel Angle

Answer 3

Angle of the cutting edge of the microtome blade

Question 4

Clearance Angle

Answer 4

Angle formed by the intersection of the block face and the cutting facet of the knife
Optimum Angle is 3-8degrees

Question 5

Function of the flotation bath

Answer 5

Used for floating out paraffin ribbons, helps to remove wrinkles

Question 6

Proper care for microtome

Answer 6

Routine maintenance, clean daily, oil according to manufacturer instructions
Keep covered to protect from dust when not in use

Question 7

Proper care for flotation bath, and temperature

Answer 7

Keep temperature 5-10C lower than melting point of your paraffin
Must clean after each block by skimming the surface of the water with a kimwipe to remove debris and contaminants

Question 8

Rotary Microtome

Answer 8

Commonly used in paraffin sectioning, also commonly used inside a cryostat

Question 9

Sliding Microtome

Answer 9

Used for sectioning Celloidin (brain) and large paraffin blocks, not used in routine histology

Question 10

Ultra Microtome

Answer 10

Used in electron microscopy, cuts sections 50-90nM

Question 11

Clinical Freezing Microtome/Cryostat

Answer 11

Obtains free floating sections (no ribbons) required for some special stains

Question 12

Safety precautions when disposing of blades

Answer 12

All blades are discarded in red puncture proof sharps containers

Question 13

Crooked Ribbons

Answer 13

Block and blade are not parallel, also uneven chilling of the block

Question 14

Too Thick Sections

Answer 14

Incorrect clearance angle, too high or low

Question 15

Too Thin Sections

Answer 15

Incorrect clearance angle, too high or low

Question 16

Compressed Sections

Answer 16

Dull blade
paraffin sticking behind the blade
too little clearance angle
too rapid cutting
too warm room

Question 17

Venetian Blind Effect/Chatter

Answer 17

Over-dehydration of the tissue
Dull blade
too high clearance angle
too fast cutting

Question 18

Knife Marks

Answer 18

Dull or damaged blade, move to a new section or new blade

Question 19

Failure to form a Ribbon

Answer 19

Dull blade
Paraffin too sticky or too hard
Decrease clearance angle
Room temperature

Question 20

Holes in the Ribbons

Answer 20

Facing too aggressively, air bubbles during embedding or processing

Question 21

Block is Sectioning Unevenly

Answer 21

Nonparallel orientation of chuck/block relative to blade

Question 22

Scratches in the ribbon

Answer 22

Nicks in the blade, move to a new spot or new blade

Or calcium deposits, use surface decal

Question 23

Skipped Sections

Answer 23

Check tightness of screws and clamps?

Question 24

Washboarding

Answer 24

Typical in uterus and other dry/over-hardened tissues. Soak for a long time to try and rehydrate
Also check that all clamps and screws are properly tightened

Question 25

Section Fly Away

Answer 25

Static, wipe down microtome with dryer sheet, increase humidity in room, or breeze caused by air vents or people walking by/breathing

Question 26

Thick/Thin Sections

Answer 26

Too much or too little blade tilt; adjust clearance angle

Question 27

Section lifts from blade

Answer 27

dull blade
too little blade tilt
too soft paraffin

Question 28

Case Study: Possible Causes?
Quality Control Slide Acceptable
Inconsistent staining on your slides, different colors

Answer 28

Thick/thin sectioning
Cut too thick
Check microtome to make sure thickness is consistent and at correct setting

Question 29

Case Study: How to fix?

Breast core biopsy is shredding, contains micro-calcifications

Answer 29

Brief surface decal of approx 30 minutes, then re-attempt to section

If it is a research specimen check to see if they are specifically looking for calcium; if that is the case just soak the block longer and do your best to get a section

Question 30

Can’t get a ribbon while sectioning, what are 5 potential causes?

Answer 30

blade angle
block isn't cold enough
dull blade
paraffin is too hard/high melting point
sections are too thick

Question 31

Crooked ribbons, why?
blade is undamaged
block has been evenly chilled

Answer 31

non-parallel orientation of chuck relative to blade

make sure you faced and sectioned the block in the same orientation

Question 32

Static issues, 3 ways to fix

Answer 32

get a huididfier in the lab or your work station
wipe down your hands and microtome with a dryer sheet
lower the heat in the room
keep your forceps cold in your ice-bath
wet gauze and keep it behind the blade holder to increase humidity

Question 33

Cutting bloody tissue (spleen, lymph node, currettings, placenta) that is shredding
You soaked it and got a fresh blade, but its still shredding
What next?

Answer 33

Warm the block 30 seconds, cold water, then cut again
May need to repeat this process several times

Warm the block with your thumb

Question 34

You are cutting fatty tissue and it explodes in the water bath. Why and how to fix?

Answer 34

Water bath is too warm, turn it down/add a little ice to rapidly cool
Under-processed tissue, reprocess
Melt down, squeeze out excess xylene with a paper towel and re-embed

Question 35

You keep getting knife marks, even with fresh blades

Why and how to fix?

Answer 35

Bad lot of blades, manufacturing defect, get a new case of blades or try another brand
calcium deposits in the block keep damaging your new blades, do surface decal then re-cut
staples missed during grossing, will cause bad noise while sectioning
cartilage/bone present