Microtome troubleshooting and some embedding

Crooked Ribbon Causes

-edges of block not parallel to blade

-block is unevenly chilled

-imperfections in blade

Crooked Ribbon Resolutions

-make block parallel to blade

-ensure even chilling

-change blade

Uneven Sections Causes

-block holder not aligned to blade
-loose block angle lever
-improper embedding

Uneven Sections Resolutions

-adjust block holder
-use microtome calibration tools
-tighten levers
-re-embed

Holes in Section Causes

-aggressive sectioning/facing
-tissue is under-hydrated
-tissue is under decalcified
-unexpected calcifications, staples, or sutures

Holes in Section Resolution

-slower speed
-face blocks when warm
-properly hydrate
-recognize natural holes in section

Ribbon will not Form/Lifting Sections Causes

-dull blade/paraffin buildup behind blade
-paraffin too hard/sticky
-room /block too warm
-thick sections
-incorrect clearance angle
-loose parts
-static

Ribbon will not Form/Lifting Sections Resolutions

-replace blade/remove buildup
-use different wax
-adjust room temp
-chill block
-decrease microns
-change clearance angle
-tighten parts
-score block
-scroll method

Water Trapped Under Section Cause

scooping section when picking up

Water Trapped Under Section Resolution

use pulling method (angle slide at 45 degrees to pick up tissue than rotate it to 90 degrees to remove from bath)

Washboarding/Undulation/Venetian Blind Causes

-worn/loose parts
-blade/block not clamped correctly
-aggressive sectioning
-block holder overextended
-clearance angle too high
-underdecalcified tissue
-dense/fibrous tissue

Washboarding/Undulation/Venetian Blind Resolutions

-tighten parts/clamp sufficiently
-retract holder
-decrease clearance angle
-surface decal
-soften tissue (fabric softener/dish soap)

Chatter Causes

-underhydrated tissue
-clearance angle too high
-aggressive sectioning
-dull blade

Chatter Resolutions

-longer hydration
-decrease dehydration time on processor
-if tissue is overprocessed: float on bath, and add alcohol to block
-decrease clearance angle
-slower speed

Thick and Thin Causes

-block not secure
-blade not tight
-clearance angle too low
-loose/worn parts
-aggressive sectioning
-overextended chuck

Thick and Thin Resolutions

-increase clearance angle
-tighten parts
-slow speed
-retract chuck

Compression Causes

-dirty/dull blade
-clearance angle too low
-sectioning too rapidly
-room/block too warm
-underhydrated tissue

Compression Resolutions

-change blade
-increase angle
-slower speed
-chill block/hydrate

Knife Lines Causes

-blade defect
-underdecalcified tissue
-staples, sutures, or foreign particles present

Knife Lines Resolutions

-replace blade
-reposition block in chuck holder
-surface decal
-melt block to remove debris

Fragmented, Separated, or Torn Sections Causes

-incomplete fixation, processing
-over manipulation on bath
-bath too warm
-extreme overhydration

Fragmented, Separated, or Torn Sections Resolutions

-reprocess
-super chill block
-add freeze spray, alcohol, or detergent to bath
-decrease bath temp

Blade Gouges out of Block Causes

-improper embedding (air bubbles)
-knife/parts not clamped
-block not secure

Blade Gouges out of Block Resolutions

-re-embed
-tighten parts
-re-secure block

Cracking/Parched Earth Causes

-under-hydrated tissue (blood)
-bath too warm
-overuse of freeze spray
-over manipulation on bath

Cracking/Parched Earth Resolutions

-soak tissue
-ensure proper hydration
-decrease bath temp

Folds/Wrinkles Causes

-bath too cold
-not allowing section to flatten on bath
-section not adhered to slide
-slide not properly dried

Wavy Section Causes

-poor adhesion to slide

-incomplete drying before staining

Wavy Sections Resolutions

-use charges slides
-completely dry before staining

Floaters/Contamination/Debris Causes

-dirty bath
-squames
-debris in sections
-ppt from staining solutions

Floaters/ Contamination/ Debris Resolutions

-use a kimwipe to clean bath
-use lotion
-filter staining solutions

Nuclear Bubbling/Distortion/Cell Shrinkage Causes

-excessive heat

-under fixed tissue

Nuclear Bubbling/Distortion/Cell Shrinkage Resolutions

-monitor oven temp

-ensure proper fixation

Cracks in Paraffin Block Causes

-overuse of freeze spray
-block chilled too fast
-block left on embedding center cold plate too long
-dirty molds

Cracks in Paraffin Block Resolutions

-avoid freeze spray

-re-embed if necessary

Tissue has a White Appearance Causes

-excessive hydration

-underprocessed tissue

Tissue has a White Appearance Resolutions

-allow tissue to dry
-limit soaking time
-re-process

Soft Mushy Tissue Causes

-tissue grossed too thick
-inadequate processing
-saturated/contaminated processing reagents
-inadequate fixation

Soft Mushy Tissue Resolution

-re-process/re-embed

Soft Mushy Tissue Prevention

-gross thinner sections

-change processor reagents

Paraffin Cracking Causes

-cold plate too cold (wax hardens too quickly)
-molds are dirty (may cause wax to stick)
-block removed from mold too soon

Paraffin Cracking Prevention

-decrease cold plate temp or place paper towel on surface if temp cannot be changed
-clean molds
-do not remove blocks from mold until they are sufficiently chilled