Microtomy Flashcards
Rotary microtome
- can cut 2-3 um thin sections
- good for a wide variety of tissue types (hard, fatty, fragile etc)
- can ribbon and do serial sections
- can be fully automated
- improve section quality
- increase productivity
- less chance of musculoskeletal disorder dev or techs — elbow, shoulder, wrist
Sliding microtome
- block is stationary and knife moves
- good for:
- large blocks
- hard sections
- can do whole mounts (placing a whole organism or specimen on a slide for exam) (neuropathology, ophthalmic pathology) (RESEARCH)
- cannot cut as thin as rotary microtome
- cannot ribbon
- can do serial sections
Knives
- EM used glass and Diamond because they cut VERY thin on plastics
- plastic techniques for bigger sections eg bone use Ralph knives (type of glass knife)
- has tool edge
Disposable blades
- need sharp, defect free edge; dull/nicked knives cause artifacts
- big or low profile - type chosen depends on microtome blade holder being used
- advs. :
- reliable sharpness
- ease of use
- high and low profile available
- don’t have to sharpen
CUTTING ANGLES (slide 7)
- Bevel
- very tip of knife
- 27- 32 degrees - Wedge
- main knife angle
- 15 degrees -
Clearance
- angle between bevel and block face
- 2–5 degrees - Rake
- inverse of clearance angle
- 90degrees minus clearance minus bevel angles
Clearance angles issues
A. Tilt too slight
- missed or skipped sections
- alternating thick and thin (vibrating against block )
- wrinkled tissue
- tissue lifted on upstroke
B. Tilt too large (knife tip almost perpendicular to block); less support for knife
- chatter
-microchatter
- washboarding
- may not be able to get a ribbon
Floatation bath
- should be able to control temp (kept at 10 degrees below melting point of paraffin)
- must keep bottom free of bubbles (clean with tissue wipes, coin, slide)
- for compression issues can add surfactant
- alcohol
- detergent
- most places can use tap water but some places have too much rust, sulfur or endemic microorganisms in the water; use distilled water
- must be kept clean
- can grow mold and bacteria
- clean with soap and water after each use
Drying oven
- goal is to remove water between section and slide
- If not done tissue will not stay adhered to slide; floats off during sectioning
- not to melt the paraffin
- if using horizontal staining need to let slides drain vertically before start heating
- temp should be around or a little above melting point of parafffin
- if too hot can cause distortion (>70deg)
- dark pyknotic nuclei ( misrepresentation of state of tissue since this usually marks apoptosis)
- nuclear bubbling from steam made by water pushing the nuclear proteins around
- cells completely devoid of nuclear detail
- usually 60 deg for 20-30 min
- can do 37 deg for 1 to 2 hrs could let it sit overnight for best results
- can do room temp overnight (aka air drying)
- can be done on newer platform automated H&E stainers
Slide Types
- variety of sizes
- routine : 76* 25mm, 1-1.2 mm thick
Section adhesives - used when tissue may detach from slide
- exposure to strong alkaline solution during staining
- frozen sections
- special procedures eg IHC, IF or special stains
- sections that will be exposed to hot or cold temps (oven)
- CNS tissue
- tissue with increased blood and mucus
- hard and crunchy tissue (esp decalcification tissue)
Adhesives
- gelatin (Knox or capsule), albumin (egg white) and starch : prone to microbial growth and increased background staining
- poly L-lysine: bought as a solution, diluted out 1:10 with water; slides are dipped in solution to coat the allowed to dry; only good for few days after being made
- 2-amiopropylthiethoxysilane (APES or Silane coated) : slides dipped in 2 % solution of APES in acetone, drained dipped in acetone, drained again, rinsed in distilled water and dried upright; good for cytology specimens that are bloody or have increased amount of protein
- pre-manufactured-charged slides
- commonly used, have a permanent positive charge
- utilizes a polymer that contains amino groups to bind and stick to silicone in glass
- esp good for IHC antigen retrieval methods
- will go bad, need to rotate stock and check expiration dates
Sectioning artifacts/issues
most common issues:
- dull knife
- something loose
- wrong tilt
Ribbon not forming
- Wax too hard:
- hard wax (high melting point paraffin; get lower melting point paraffin)
- breath on block to warm up
- don’t place on ice
- cut at room temperature - Wrong clearance angle
- knife clearance angle
- too small or to large >15<
- readjust
Compression
- Dull blade: move blade to a new area/ change blade
- Soft wax: use harder paraffin/ice block
- Dense tissue: ice block longer
Ribbon comes off in a “C”
- Top and/or bottom of block
- not parallel to knife, trim with safety razor - Dull blade over part of block
- compression of block in dull area
- non compression in sharp area - Tissue is more dense in one area
- less dense in other
- more compression on dense side
- ice the block
Sections attach to block face on return stroke
- Too small of a clearance angle; make larger
- Tissue embedded at the top of block
- Top of block “ragged”
- Block vertical, not clear knife
- Static electricity
Sections roll up on knife edge
- Dull knife (move or replace)
- Sections too thick
- make thinner section
- use lower melting point wax =softer - Too large clearance angle
- make smaller - Too sharp knife
- brains
- dull the knife (dull block or edge of pencil)
Chippers/divots/moth eaten
Rough trim too aggressively
- knife pulls chunks of wax from block
-rough trim at smaller intervals
- do not use fist 2-4 sections on ribbon
Thick and thin sections
See on on floatation bath where you have difference in size and shades of white in sections
1. Something loose
- tighten all levers and knobs
2. Too small clearance angle
- increase clearance angle
3. Wax too soft
- use higher melting point paraffin
- ice block
Thick and thin sections
- Tissue too dense for wax
- overprocessed (cut down on time or temperature )
- wax too soft (ice block; use higher melting point paraffin)
- tissue naturally dense (uterus/ muscular structure) - “Light-weight” microtome
- need a sturdier microtome or blade/knife - Hand rhythm
- need to be uniform
- not speed up, slow down - Faulty microtome
- least likely
- call repair person
Venetian blind/washboarding
- thick and thin zones within each section
- visible with eye after staining
1.something loose
-not as loose as thick and think
- tighten all levers and knobs
- Too much knife tilt
- reduce clearance angle - Tissue is too dense for wax
A. tissue is naturally dense: uterus, cervix, prostate
- ice block
- use higher melting point wax
B. Over processed
- reduce time or temperature of processing
C. Wax too soft
- ice block
- use higher meting point wax
Microchatter
- thick and thin microscopically
- most often seen in:
A. Epithelial layers of GI
B. Fluid cysts
C. Thyroid
Microchatter troubleshooting
- Something loose
- not as likely
- tighten everything - Tissue slightly hard
- same actions as overprocessed - slow down
- hand movement too fast; do even slow rotations - Tissue slightly over-processed
- removed too much bound H2O
- running biopsies on long cycle
- correct:
A. Soak block face in water for 5-60 secs
B. Reduce processing time
C: reduce temp on processor stations
Tear/split/knife line
- Nicks in knife edge
- be carful removing sections from knife edge
- previous/ current section/ bloc has something hard in it
- line goes through entire section - Something hard in paraffin
- particle of dust, dirt
- remove from block
- filter wax before use in embedding station - Something hard in tissue eg.:
- calcium = use surface decal (good probs in breast and kidney cause it guides the docs to where the bad tissue is)
- keratin = use surface NairTM
- suture = metal or cloth
- glass, sand, vegetative material
- tissue component = keratin layer, muscle
- cut thru last
- line starts on hard particle
- may eventually become entire line
- remove hard particle (probe or forceps)
Tear/ Crack
- Pulling on tissue hard
- laying on water bath
- removing from knife - Splitting along tissue lines (eg blood vessels, connective tissue splits from squamous tissue)
- flotation bath too hot;
- too long of time on water bath - Face plate holding blade
- may be uneven
- catches tissue sections
dragging = raking on micro sections
- commonly seen in connective tissue layers (lymphatic tissue)
- dull knife
- to correct, move, replace blade
Parched earth (seen in liver, placenta, blood vessels containing blood)
- Random cracks in different directions
- seen in:
- overprocessed tissues
- bloody tissue
- lymphoid tissue
- reduce time/temp of processors
- cryogenic spray
To correct: soak block
- water
- water + glycerin
- water + dish detergent
- water +wetting agent
Folds/ wrinkles
- Poor technique
- anchor at one end
- remove with forceps
- pick up other sections - Curved tissue
E.g. artery, appendix
Solution: ended straight, leave on flotation bath a longer time - Too hot flotation bath
- wrinkles/ folds melt in place
- not enough time to remove wrinkles
- solution = cool water temperature
Waves
- 3D old
- seen in curved tissue
- action: increase temp of flotation bath; increase time on flotation bath
=make sure to clean water bath as well because endemic bacteria can grow in it
