Faults in Processing

During the processing and cutting of sections, several difficulties may be encountered, due to some faults which may have been made in the previous procedures. A good medical technologist should be alert in taking note of such faults, which, if not immediately corrected shall cause entire failure in the processing, poor sectioning, and ultimately improper evaluation of the tissue in question.

The following are the most common difficulties observed during tissue processing, together with their reasons and possible remedy for each.

FAULTS	REASON	REMEDY
Brittle or hard tissue	Prolonged fixation	Tissues may be softened by soaking in a small dish or bowl containing water with detergent, phenol or Molliflex.
	Prolonged dehydration
	Prolonged clearing
	Prolonged paraffin infiltration in overheated paraffin oven
	Drying out of tissue before actual fixation
	Failure to give special handling of tissues when treated with routine processing methods
Clearing agent turns milky as soon as tissue is placed in it	Water not completely removed due to incomplete dehydration	Repeat dehydration with absolute alcohol, then clear again.
On trimming, tissue smells of clearing agent	Clearing agent not completely removed due to insufficient impregnation	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	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	Insufficient dehydration, therefore incomplete clearing and impregnation	Repeat the whole procedure
Tissue is soft when block is trimmed	Incomplete fixation	Repeat fixation
Airholes found on tissue during trimming	Incomplete impregnation	Repeat impregnation
On trimming, wax appears crystalline	Contaminated wax	Re–embed in freshly filtered wax
Paraffin block after cooling is moist and crumbles	Insufficient paraffin impregnation	Repeat paraffin impregnation then re–embed

FAULTS OBSERVED DURING SECTION – CUTTING

Difficulties encountered during cutting of sections are mostly due to faults encountered during the processing of tissues or due to some faults in the technique or cutting itself, and therefore must be evaluated and corrected on a case to case bases, if good tissue sections are to be made.

FAULTS	REASON	REMEDY
Sections fail to form ribbons	Surfaces and edges of the block are not parallel	Re–trim the block
	Horizontal surface of the block is not parallel to the knife	Re–adjust and re–orient
	Paraffin wax is too hard	Coat horizontal edges of the block with wax of lower melting point
	Knife is tilted too much	Reduce the tilt
	Sections are too thick	Readjust the thickness of the sections
	Knife is dull	Hone and strop
Sections roll up on cutting so that they adhere and get broken against the knife edge	Knife is blunt	Sharpen the knife
	Tilt of knife is too great	Reduce the tilt
	Knife edge is dirty	Clean the knife edge
Ribbon is curved, crooked or uneven instead of straight	Blunt or dull spot on the knife producing an irregular knife edge	Adjust the knife so that knife edge will present a uniformly sharp edge to the block; or sharpen
	Edges of the block are not parallel but round or wedge shaped	Re–trim the block
	Knife is not parallel to the block	Readjust the knife and the block
	Paraffin is impure	Repeat impregnation using pure wax
Sections are compressed, wrinkled or jammed	Knife is blunt or dull	Re–sharpen the knife
	Paraffin block is warm and soft	Cool the block on ice water until firm
	Knife edge is coated with paraffin	Clean the knife edge
	Sections are too thin	Readjust thickness of section
	Microtome set screw is loose	Tighten the screw
	Tilt of knife is too vertical	Reduce the tilt
Sections are torn and crumble when cut	Incomplete dehydration, clearing, and/or infiltration tissue with wax	Remove paraffin with clearing of agent, pass thru decreasing grade of alcohol, then repeat dehydration, clearing and embedding
	Paraffin is warm and soft	Cool and harden paraffin in ice for ¼ to ½ hour
	Knife is blunt	Sharpen the knife
Sections are squashed (width of each section less than that of block)	Bevel of knife is lost due to incorrect sharpening	Re–sharpen, using a knife back or automatic knife sharpener
A hole is formed in the section	Bubble or dirt formed in the embedding medium	Re–embed in freshly filtered wax if necessary
	Hard spot in tissue possibly due to calcium	Once embedded in paraffin wax, decalcification is impractical; use base–sledge microtome with a wedge knife
Selections of unequal thickness are produced	Tilt of knife is too great or bevel is not cleared, hence object is compressed against the knife edge	Reduce the tilt
	Clamp set screw on knife or blockholder is loose	Tighten screw
	Blocks are too large	Cut blocks into smaller fragments
	Blocks are too hard	Soften blocks in detergent or phenol
Sections adhere to the knife or other parts of the machine	Static electricity due to low atmospheric humidity	Breath out or blow gently on the block and knife to break up static electricity or boil water in the room to increase the humidity
	Knife edge is dirty	Clean the knife edge
	Knife edge is dull	Sharpen the knife
	Knife edge is too great	Reduce the tilt
Ribbon is split or lengthwise vertical scratches are seen on sections	Nicks are damage on the knife edge	Sharpen the knife
	Dirty embedding medium	Re–embed in filtered wax
	Knife edge is dirty	Clean knife edge with xylene
	Tilt of knife is too great	Reduce tilt
Resistance is felt on the lower part of the section during cutting	Tilt of knife is insufficient; paraffin block is therefore compressed against the base of the knife towards the end of stroke	Increase the tilt
Horizontal or parallel lines or furrows across the section (chatters) are seen, forming thin and thick zones	Knife edge vibrates due to: (a)  hardness of the tissue (b)  tilt of the knife is too great	Treat with phenol during processing; or colloidionize Reduce the tilt
Section cut is sometimes thin, sometimes thick	Knife is blunt	Sharpen knife
	Knife is not clamped properly	Adjust the knife
	Tilt of knife is too great	Reduce the tilt
	Knife or blockholder is loose	Tighten adjusting and locking screw
	Knife tilt is too small that block is compressed by bevel and section is not cut	Increase the tilt
Knife makes a hard metallic scraping or ringing sound on backstroke, when section is cut	Tilt of knife is too slanted or too big	Readjust the angulation of the knife
	Tissue is too hard	Take fresh block treated with phenol during processing
	Knife blade is too thin	Charge the knife
Frozen tissue crumbles and comes off the blockholder when cut	Freezing not adequate	Refreeze the tissue block
Frozen tissue chips into fragments when cut	Tissue is frozen too hard	Warm the tissue with the fingers