Microtome Cutting Tips: What Can I Do When Sections Crumble?
Microtome Cutting Tips
Welcome to our first installment of microtome cutting tips where we explore common sectioning issues and solutions to them. Below you will find generic microtome cutting recommendations along with specific solutions to solve crumbling sections.
Regardless of the type of material or blade being used, the most important factors influencing section quality are the condition of the edge of the blade, the clearance angle and expertise of the histotechnician.
The clearance angle in relation to the block face is typically 3-8 degrees but can vary with the model of the microtome. The blade clearance angle prevents contact between the blade facet and the face of the block. The facet angle is the angle between two facets that form the cutting edge. The facet angle is approximately 35 degrees but can vary with different OEM blades. Check the operating manual of your microtome for the suggested angle.
Successfully obtaining a section from a poorly processed tissue is possible if the knife or blade is sharp whereas taking a section from a properly processed tissue with a dull knife or blade is often very difficult.
Below we explore the causes and solutions to a common microtome cutting error – crumbling sections.
What Causes Sections to Crumble?
Sections can crumble due to the following:
- Insufficient dehydration of tissue by alcohols during processing.
- Insufficient clearing of tissue during processing.
- Incomplete infiltration of tissue with embedding medium during processing.
- Extended time of tissue cassette in paraffin either on the tissue processor, or embedding center/paraffin too hot.
- Type of clearing reagent used.
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Solutions to Crumbling Sections
- If the specimen is still intact, try reprocessing backward to alcohols. However, salvage is rarely possible.
- Return tissue to several changes of paraffin, may want to replace last paraffin with “new”.
- Check the processing schedule to make sure tissues are in paraffin the correct amount of time. This will differ for biopsies versus or larger specimens. Make sure first paraffin is not contaminated with previous solutions. May need to reprocess tissue back through clearing agent to alcohols. May need to change first reagent container of each solution on the tissue processor and bump up reagents accordingly /or add new reagents.
- Do not leave cassette basket with tissue cassettes in the embedding center for an extended period of time before starting the embedding process. Check temperature of paraffin in processor, embedding center, and paraffin dispenser to make sure it is 2-4 degrees above the melting point of the paraffin wax.
- Use appropriate clearing agent for tissue type.