Instrumentation: Microtomy Flashcards
Microtomy
Cutting thin sections of tissue for microscopic examination
Micron
0.001mm (one thousandth of a millimeter)
1uM
Bevel Angle
Angle of the cutting edge of the microtome blade
Clearance Angle
Angle formed by the intersection of the block face and the cutting facet of the knife
Optimum Angle is 3-8degrees
Function of the flotation bath
Used for floating out paraffin ribbons, helps to remove wrinkles
Proper care for microtome
Routine maintenance, clean daily, oil according to manufacturer instructions
Keep covered to protect from dust when not in use
Proper care for flotation bath, and temperature
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
Rotary Microtome
Commonly used in paraffin sectioning, also commonly used inside a cryostat
Sliding Microtome
Used for sectioning Celloidin (brain) and large paraffin blocks, not used in routine histology
Ultra Microtome
Used in electron microscopy, cuts sections 50-90nM
Clinical Freezing Microtome/Cryostat
Obtains free floating sections (no ribbons) required for some special stains
Safety precautions when disposing of blades
All blades are discarded in red puncture proof sharps containers
Crooked Ribbons
Block and blade are not parallel, also uneven chilling of the block
Too Thick Sections
Incorrect clearance angle, too high or low
Too Thin Sections
Incorrect clearance angle, too high or low
Compressed Sections
Dull blade paraffin sticking behind the blade too little clearance angle too rapid cutting too warm room
Venetian Blind Effect/Chatter
Over-dehydration of the tissue
Dull blade
too high clearance angle
too fast cutting
Knife Marks
Dull or damaged blade, move to a new section or new blade
Failure to form a Ribbon
Dull blade
Paraffin too sticky or too hard
Decrease clearance angle
Room temperature
Holes in the Ribbons
Facing too aggressively, air bubbles during embedding or processing
Block is Sectioning Unevenly
Nonparallel orientation of chuck/block relative to blade
Scratches in the ribbon
Nicks in the blade, move to a new spot or new blade
Or calcium deposits, use surface decal
Skipped Sections
Check tightness of screws and clamps?
Washboarding
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
Section Fly Away
Static, wipe down microtome with dryer sheet, increase humidity in room, or breeze caused by air vents or people walking by/breathing
Thick/Thin Sections
Too much or too little blade tilt; adjust clearance angle
Section lifts from blade
dull blade
too little blade tilt
too soft paraffin
Case Study: Possible Causes?
Quality Control Slide Acceptable
Inconsistent staining on your slides, different colors
Thick/thin sectioning
Cut too thick
Check microtome to make sure thickness is consistent and at correct setting
Case Study: How to fix?
Breast core biopsy is shredding, contains micro-calcifications
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
Can’t get a ribbon while sectioning, what are 5 potential causes?
blade angle block isn't cold enough dull blade paraffin is too hard/high melting point sections are too thick
Crooked ribbons, why?
blade is undamaged
block has been evenly chilled
non-parallel orientation of chuck relative to blade
make sure you faced and sectioned the block in the same orientation
Static issues, 3 ways to fix
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
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?
Warm the block 30 seconds, cold water, then cut again
May need to repeat this process several times
Warm the block with your thumb
You are cutting fatty tissue and it explodes in the water bath. Why and how to fix?
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
You keep getting knife marks, even with fresh blades
Why and how to fix?
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
