Microtomy Flashcards
1
Q
Rotary microtome
A
- 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
2
Q
Sliding microtome
A
- 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
3
Q
Knives
A
- 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
4
Q
Disposable blades
A
- 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
5
Q
CUTTING ANGLES (slide 7)
A
- 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
6
Q
Clearance angles issues
A
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
7
Q
Floatation bath
A
- 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
8
Q
Drying oven
A
- 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
9
Q
Slide Types
A
- 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)
10
Q
Adhesives
A
- 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
11
Q
Sectioning artifacts/issues
A
most common issues:
- dull knife
- something loose
- wrong tilt
12
Q
Ribbon not forming
A
- 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
13
Q
Compression
A
- Dull blade: move blade to a new area/ change blade
- Soft wax: use harder paraffin/ice block
- Dense tissue: ice block longer
14
Q
Ribbon comes off in a “C”
A
- 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
15
Q
Sections attach to block face on return stroke
A
- 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
