Undulations seen in a tissue section.
As mentioned in the introduction, the tissue to be sectioned must be properly fixed and processed in order to achieve an optimal microtomy product. But successful microtomy also depends on factors associated with instrumentation as well as the working environment. We have all heard the saying that "the microtome is rarely the cause of poor sections unless it is old or damaged." Let us consider the following factors that affect microtomy:
- Stable work surface to prevent microtome vibration: The bench must be able to withstand the weight of the microtome and flotation bath, as well as the movement of the microtome during sectioning. Benches must be secured to the floor and/or wall. Microtomes should never be used on carts, card tables, or tables on wheels. Microtome vibration will introduce artifacts into the sections, causing undulations, chatter, and thick/thin sections.
- Properly maintained microtome: Although the microtome may rarely be the cause of poor sections, it should be the priority when it comes to maintenance and service. A broken microtome may be the cause of many sectioning problems and should be one of the first considerations during troubleshooting when problems arise.
- All microtome parts are clamped down and finger tight to prevent vibration: Vibration is the most common cause of undulations (washboarding) in tissue sections, shown in the image. This includes the blade clamp, knife holder base, and knife tilt. Also, the block holder adjustments should be tightened properly to prevent the block from moving during sectioning. Tighten all microtome parts if washboarding is grossly seen on tissue sections.
- Proper knife tilt (clearance angle 3-8°) dependent on blade or knife used: Once the optimal knife tilt is obtained, this adjustment should rarely if ever be changed.
- Nick-free blade or knife: A new blade should be used after blocks have been faced. Also, tissue that is calcified or has a lot of hair will reduce the life of a blade tremendously. A blade should be changed as soon as streaks or tears are noticed in tissue sections on the flotation bath.
- Consistent cutting speed (one revolution per second): Faster cutting may introduce artifacts such as undulations (washboarding) or thick and thin sections, while slower cutting may allow the tissue block to expand and create thick and thin sections. The same cutting speed should be used for all sections in one ribbon or tissue sections will vary in thickness and sections may not form a complete ribbon.
- Clean water bath at proper temperature (5-10° C below melting point of paraffin): Use distilled water free of microbes and check the water temperature at least once per day.
- Properly fixed, processed, and embedded blocks: If any of these pre-microtomy steps are suboptimal, the final product may be compromised and the diagnosis may not be attained. In under-fixed and under-processed tissue, it may be difficult to get a complete section. Tissue that is not embedded completely flat or at the proper orientation will not allow the microtomist to cut a complete and representative section for diagnosis.
- Chilled and hydrated blocks: Most tissue samples are over-dehydrated and can benefit from ice-water. Trays of ice covered with distilled water cool the paraffin and tissue to make microtomy easier, especially in warm climates. It is not recommended that blocks be placed in the freezer since the paraffin and tissue can crack. Freezing blocks will introduce artifacts to tissue sections. Blocks that have calcium deposits should have the exposed block surface decalcified prior to sectioning.
- Work environment: A room that is not too cold, hot, humid, or drafty is best. Although this ideal environment is difficult to find in most laboratories, measures should be taken to prevent temperature extremes, as well as drafts. Chasing thin ribbons around the bench is not as enjoyable as it looks! Even one's breath can ruin a great ribbon before it hits the water bath.
- Coordination of microtomist: This skill takes patience and practice.