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Lab

Paraffin Microtomy Trouble-Shooting

QuestionAnswer
Irregular or skipped sections may be caused by: Too little/much blade tilt, poorly adjusted chuck, cutting too fast or at inconsistent speeds, old or out of adjustment microtome, loose screws/parts
Crooked ribbons may be caused by: Blocks not trimmed parallel, irregular knife edge, uneven block temp
Sections sticking to knife may be caused by: Dirty knife - clean w/ xylene, knife angle too small, dull knife
Smeared/deformed sections may be caused by: Dull knife
Washboarding/undulations in section may be caused by: Block not clamped tightly, too much knife tilt, loose screws/parts, old microtome, under/overfilled block not sitting properly, often happens w/ hard tissues (e.g. uterus)
Ribbon may not form because: Cold room - warm knife slightly, too much knife angle, too thick sections, dull knife
Sections compressed, wrinkled, or jammed together may be caused by: Dull knife, paraffin build-up on blade, too little knife tilt, cutting too fast, warm room, warm block
Knife rings on upstroke & sections are scratched Knife angle too great > decr. angle Material too hard > soak in water to soften
Sections may be lifted from knife on upstroke because: Too little knife angle, dull knife, warm room - cool block, soft paraffin
Knife may not hold edge because: Knife angle is too severe
Scratches or splits in ribbon may be caused by: Defect in block (e.g. calcium deposit), defect in knife
Fragmented or torn sections may be caused by: Poor processing or fixation, too hot water bath, treat ribbon more gently
Sections flying due to static may be helped by: Breathing on section/blade, humidifer in room, dryer sheet behind blade
Chatter may be caused by: Dry, over-processed tissue, too much knife tilt, cutting too fast, block holder shaft over-extended - vibration
Holes in section may be caused by: Facing block too roughly, tears tissue, can try to face further & smooth
Parched earth artifact caused by: Too hot bath, using cryo spray to cool blocks, facing too roughly, dry sections
Wrinkles in section may be caused by: Not laying ribbon on bath carefully, bath not warm enough
Prevent skin cells in sections by: Wearing gloves, not sticking fingers in bath
Prevent cross-contamination by: Cleaning tools between blocks, cleaning bath between blocks
To help keep sections on slide may use: Slides coated w/ poly L lysine, aminoalkylsilane, or albumin, (may get background staining artifact), charged/plus slides
Created by: CCF