Faults Flashcards by PATRICIA ONG

Faults: Sections expand or disintegrate on water bath
Reason:

Poor impregnation of tissue
Water temperature too high in flotation bath

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Faults: Sections expand or disintegrate on water bath
Remedy:

Re-process tissue
Turn down the temperature of the flotation bath

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Faults: Sections roll into a coil instead of remaining flat on knife edge
Reason:

Blade dull
Rake angle too small
Section too thick

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Faults: Sections roll into a coil instead of remaining flat on knife edge
Remedy:

Use a new blade
Reduce blade tilt if clearance angle is excessive
Reduce section thickness

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Faults: Grooved, Scored, smeared and deformed sections
Reason:

Dull knife /blade edge
Too warm room

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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

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Faults: Sections fail to form ribbons
Reasons:

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

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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

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

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Fault: 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

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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

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Faults: 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

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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

Readjust knife and block

Repeat impregnation using pure wax

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Faults: Sections are compressed wrinkled or jammed
Reason:

knife is blunt or dull
Paraffin block is warm and soft
Knife edge 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

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Reason:
Knife is blunt or dull
Paraffin block is warm and soft
Knife edge 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 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

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Faults: Sections are squashed (width of each section is less than that of block)
Reason:

Bevel of knife is lost due to incorrect sharpening

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Reason: Bevel of knife is lost due to incorrect sharpening

Remedy:

Re-sharpen using a knife back or automatic knife sharpener

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Fault: 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 processed tissue

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Reason:

Bubble or dirt formed in the embedding medium
Hard spot in tissue due to calcium
Excessive dehydration or improperly processed 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 tissue, then soak the block in ice water or with a piece of cotton before sectioning

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Faults: Sections of unequal thickness are
produced

Reasons:

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

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Reasons:
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

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Faults: Sections adhere to the knife or other parts of the microtome

Reason:

Static electricity due to low atmospheric humidity
Knife edge is dirty
Knife edge is dull
Tilt of knife is too great

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Reason:

Static electricity due to low atmospheric humidity
Knife edge is 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 ü Reduce tilt

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Fault: 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

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Reason:
Nicks or damage on the knife edge
Dirty embedding
Knife edge is dirty
Tilt of knife is too great

Sharpen the knife
Re-embed in filtered wax
Clean knife edge with xylene
Reduce the tilt

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Fault: Sections are lifted from the knife on the upstrokes Reason:

Knife tilt is too great Knife is dull Paraffin is too soft or room temperature is warm

Reason: Knife tilt 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

Fault: 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

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

Fault: 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

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 it 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

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 adjusting and locking screw Increase the tilt

Faults: Knife makes a hard metallic scrapping or ringing sound on backstroke, when section is cut Reasons:

Tilt of knife is too slanted or too big Tissue is too hard Knife blade is too thin

Remedy for Tilt of knife is too slanted or too big Tissue is too hard Knife blade is too thin

Readjust the angulation of the knife Take fresh block treated with phenol during processing Change the knife

Faults: Frozen tissue crumbles and comes off the block holder when cut Reason:

Freezing is not adequate

Reason: Freezing is not adequate Remedy:

Refreeze the tissue block

Faults: Frozen tissue chips into fragments when cut Reason:

Tissue is frozen too hard

Tissue is frozen too hard Remedy:

Warm the tissue with the fingers

Faults: 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

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 the edge of blocks is parallel to the knife edge Try another part of the knife edge

Fault: Brittle or Hard tissues REASON:

Prolonged fixation Prolonged dehydration Prolonged clearing Prolonged paraffin infiltration Overheated paraffin oven Drying out of tissue before actual fixation

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 du to incomplete dehydration

Water not completely removed du 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

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

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

Insufficient dehydration therefore incomplete clearing and impregnation Remedy:

Repeat the whole procedure

Tissue is soft when block is trimmed Reason:

Incomplete fixation

Incomplete fixation Remedy:

Repeat whole procedure

Fault: Airholes are found on tissue during trimming Reason:

Incomplete impregnation

Incomplete impregnation Remedy

Repeat impregnation

On trimming wax appears crystalline Reason:

Contaminated wax Block not cooled rapidly enough

Reason: Contaminated wax Block not cooled rapidly enough

Re-embed in freshly filtered wax

Paraffin block, after cooling is moist and crumbles Reason:

Insufficient paraffin impregnation

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

Fatty tissues should be sampled and cut thinly for easy reagent action. Ethanol is a poor fat solvent. To ensure complete dehydration, 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 for: Tough keratin component of certain tissues

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: Inadequate processing due to very thick or fatty sections

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