FAULTS IN SECTIONING

• FAULTS OBSERVED DURING SECTION CUTTING

  • Sections expand or disintegrate on water bath
  • Sections roll into a coil instead of remaining flat on knife edge
  • Grooved, Scored, smeared and deformed sections
  • Sections fail to form ribbons
  • Sections roll up on cutting so that they adhere and get broken against the knife edge
  • Ribbon is curved, crooked or uneven instead of straight
  • Sections are compressed wrinkled or jammed
  • Sections are squashed (width of each section is less than that of block)
  • A hole is formed in the section
  • Sections of unequal thickness are produced
  • Sections adhere to the knife or other parts of the microtome
  • Ribbon is split or lengthwise vertical scratches are seen on sections
  • Sections are lifted from the knife on the upstrokes
  • Resistance is felt on the lower part of the section during cutting
  • Horizontal or parallel lines or furrows across the section (chatters) are seen, forming thin and thick zones
  • Section cut is sometimes thin sometimes thick
  • Knife makes a hard metallic scrapping or ringing sound on backstroke when section is cut
  • Frozen tissue crumbles and comes off the block holder when cut
  • Frozen tissue chips into fragments when cut
  • Pleated folded and crooked ribbons
• FAULTS OCCURRING DURING TISSUE PROCESSING

  • Brittle or hard tissues
  • Clearing agent turns milky as soon as tissue is placed in it
  • On trimming, tissue smells of clearing agent
  • Tissue is opaque, section cutting is difficult due to presence of alcohol
  • Tissue shrinks away from wax when trimmed
  • Tissue is soft when block is trimmed
  • Airholes are found on tissue during trimming
  • On trimming wax appears crystalline
  • Paraffin block, after cooling is moist and crumbles
  • Hard, dense tissues
  • On embedding, tissue is soft and mushy in the center
• Sections expand or disintegrate on water bath

  Reason: Poor impregnation of tissue

  Remedy: Re process tissue
• Sections roll into a coil instead of remaining flat on knife edge


  Reason: Blade dull

  Rake angle too small

  Section too thick

  Remedy: Use a new blade

  Reduce blade tilt if clearance angle is excessive

  Reduce section thickness
• Grooved, Scored, smeared and deformed sections

  Reason: Dull knife/blade edge

  Too warm room

  Remedy: Re-sharpen the knife/blade or move the existing blade to an unused surface or replace it with a new one

  Cool/Change the temperature of the room or cool the block in ice water
• Sections fail to form ribbons


  Reason: Surfaces and edges of the block are not parallel

  Horizontal surface of the block is not parallel to the knife

  Paraffin wax is too hard

  Knife is tilted too much

  Sections are too thick

  Knife is dull

  Remedy: Re-trim the block

  Re-adjust and re-orient the block

  Coat horizontal edges of the block with wax of lower melting point

  Reduce the tilt

  Re-adjust thickness of the section

  Hone and strop
• Sections roll up on cutting so that they adhere and get broken against the knife edge

  Reason: Knife is blunt

  Tilt of knife is too great

  Knife edge is dirty

  Remedy: Sharpen the knife

  Reduce the tilt

  Clean the knife edge
• Ribbon is curved, crooked or uneven instead of straight


  Reason: Blunt of dull spot on the knife, producing an irregular knife edge

  Edges of the block are not parallel but round or wedge shape

  Knife is not parallel to the block

  Paraffin is impure

  Remedy: Adjust the knife so that knife edge will present a uniformly sharp edge to the block or sharpen

  Re-trim the block

  Re-adjust knife and block

  Repeat impregnation using pure wax
• Sections are compressed wrinkled or jammed


  Reason: Knife is blunt or dull

  Paraffin block is warm and soft

  Knife is coated with paraffin

  Sections are too thin

  Microtome set screw is loose

  Tilt of knife is too vertical

  Too rapid cutting

  Too warm room

  Unparallel block edges

  Remedy: Re-sharpen the knife

  Cool the block on ice water until firm

  Clean the knife edge

  Re-adjust thickness of section

  Tighten the screw

  Reduce the tilt

  Decrease cutting speed for every thin section

  Cool/change the room temperature or cool the block in ice water

  Trim the block edges until they are parallel
• Sections are squashed (width of each section is less than that of block)


  Reason: Bevel of knife is lost due to incorrect sharpening

  Remedy: Re-sharpen using a knife back or automatic knife sharpener
• A hole is formed in the section

  Reason: Bubble or dirt formed in the embedding medium

  Hard spot in tissue due to calcium

  Excessive dehydration or improperly processing tissue

  Remedy: Re-embed in freshly filtered wax if necessary

  Once embedded in paraffin wax, decalcification is impractical, use a base sledge microtome with a wedge knife

  Expose the issue then soak the block in ice water or with a piece of cotton before sectioning
• Sections of unequal thickness are produced

  Reason: Tilt of knife is too great or bevel is not cleared hence object is compressed against the knife edge

  Clamp set screw on knife or blockholder is loose

  Blocks are too large

  Blocks are too hard

  Remedy: Reduce the tilt

  Tighten the screw

  Cut blocks into smaller fragments

  Soften the blocks in detergent or phenol
• Sections adhere to the knife or other parts of the microtome

  Reason: Static electricity due to low atmospheric humidity

  Knife edge is too dirty

  Knife edge is dull

  Tilt of knife is too great

  Remedy: Breathe out or blow gently on the block and knife to breakup static electricity, or boil water in the room to increase humidity

  Clean knife edge

  Sharpen the knife

  Reduce tilt
• Ribbon is split or lengthwise vertical scratches are seen on sections


  Reason: Nicks or damage on the knife edge

  Dirty embedding

  Knife edge is dirty

  Tilt of knife is too great

  Remedy: Sharpen the knife

  Re-embed in filtered wax

  Clean knife edge with xylene

  Reduce the tilt
• Sections are lifted from the knife on the upstrokes


  Reason: Knife is too great

  Knife is dull

  Paraffin is too soft or room temperature is warm

  Remedy: Reduce the tilt

  Sharpen the knife

  Cool paraffin wax in ice water
• Resistance is felt on the lower part of the section during cutting


  Reason: Tilt of knife is too small, paraffin block is therefore compressed against the base of the knife towards the end of stroke

  Remedy: Increase the tilt
• Horizontal or parallel lines or furrows across the section (chatters) are seen, forming thin and thick zones


  Reason: Knife edge vibrates due to hardness of tissue

  Tilt of knife is too great

  Overhydration or lack of moisture in tissue

  Use of dull knife/blade

  Cutting too quickly

  Remedy: Treat with phenol during processing or collodionize

  Reduce tilt

  Restore moisture of tissue by letting the block stand and soaking it briefly in ice water or by placing ti face down on an ice tray

  Resharpen the knife/blade

  Decrease cutting speed
• Section cut is sometimes thin sometimes thick


  Reason: Knife is blunt

  Knife is not clamped properly

  Tilt of knife is too great

  Knife or block holder is loose

  Knife tilt is too small that block is compressed by bevel and section is not cut

  Remedy: Sharpen the knife

  Adjust the knife

  Reduce tilt

  Tighten the adjusting and locking screw

  Increase tilt
• Knife makes a hard metallic scrapping or ringing sound on backstroke when section is cut


  Reason: Tilt knife is too slanted or too big

  Tissue is too hard

  Knife blade is too thin

  Remedy: Readjust the angulation of the knife

  Take fresh block treated with phenol during processing

  Change the knife
• Frozen tissue crumbles and comes off the block holder when cut

  Reason: Freezing is not adequate

  Remedy: Refreeze the tissue block
• Frozen tissue chips into fragments when cut

  Reason: Tissue is frozen too hard

  Remedy: Warm the tissue with the fingers
• Pleated folded and crooked ribbons

  Reason: Imperfections in the blade edge, i.e. the horizontal edges are not parallel or the lower block edge is not parallel to the knife edge when sectioning

  Remedy: Trim the block parallel to the other edge or re-embed them in molds so that edge of blocks is parallel to the knife edge

  Try another part of the knife edge
• Brittle or hard tissues

  Reason: Prolonged fixation

  Prolonged dehydration

  Prolonged clearing

  Prolonged paraffin infiltration

  Overheated paraffin oven

  Drying out of tissue before actual fixation

  Remedy: Tissue may be softened by soaking in a small dish or bowl, containing water with detergent phenol or molliflex
• Clearing agent turns milky as soon as tissue is placed in it


  Reason: Water not completely removed due to incomplete dehydration

  Remedy: Repeat dehydration with absolute alcohol then clear again
• On trimming, tissue smells of clearing agent

  Reason: Clearing agent not completely removed due to insufficient impregnation

  Remedy: Block is trimmed down nearest to the tissue. Remaining wax is melted on embedding oven and paraffin impregnation is repeated, changing the paraffin at least once before blocking
• Tissue is opaque, section cutting is difficult due to presence of alcohol


  Reason: Insufficient clearing

  Remedy: Repeat clearing if object has already been embedded, prolong clearing up to 12 hours, then re-embed
• Tissue shrinks away from wax when trimmed


  Reason: Insufficient dehydration therefore incomplete clearing and impregnation

  Remedy: Repeat the whole procedure
• Tissue is soft when block is trimmed

  Reason: Incomplete fixation

  Remedy: Repeat the whole procedure
• Airholes are found on tissue during trimming

  Reason: Incomplete impregnation

  Remedy: Repeat impregnation
• On trimming wax appears crystalline


  Reason: Contaminated wax

  Block not cooled rapidly enough

  Remedy: Re-embed in freshly filtered wax
• Paraffin block, after cooling is moist and crumbles


  Reason: Insufficient paraffin impregnation

  Remedy: Repeat paraffin impregnation then re-embed
• Inadequate processing of fatty tissues

  Reason: Routine schedule and reagents may not process fat adequately

  Remedy: Fatty tissues should be sampled and cut thinly for easy reagent actions. Ethanol is a poor fat solvent. To ensure complete dehydrations, a far better lipid solvent (acetone or isopropanol) should be inserted before using the final absolute ethanol
• Hard, dense tissues


  Reason: Tough keratin component of certain tissues

  Remedy: Treat fixed tissues using 4% aqueous phenol for 24-72 hrs to soften
• On embedding, tissue is soft and mushy in the center

  Reason: Inadequate processing due to very thick or fatty sections

  Remedy: At gross, cut thin sections especially fatty tissues, ensure complete fixation