The actual process of microtomy is filled with so many little steps and skilled movements that it would take longer to describe than to physically do it from beginning to end. A seasoned histotechnologist can complete one slide in about 30 seconds. The reason for the quick turn-around is not rapid movements, but an organized approach that produces consistent sections with high quality. A sectioning novice may feel like a "bull in a china shop" when engaging in the craft of microtomy; their every movement, even their breathing, seems to create chaos. The ribbons explode everywhere and attach to their clothes and hair. Many first time students cut through an entire tissue block without successfully producing a slide. Everyone can recall their first few attempts at microtomy and it was not graceful or pretty. But once the skill is learned, it's like riding a bike...no one forgets! It is a skill for a lifetime.
The following steps are recommendations for the process of microtomy. Although these steps cover the basics, each individual is encouraged to explore various ways of doing each step. Sometimes the best teacher is trial and error.
- Expose or "face" the tissue blocks: Since tissue is completely surrounded by paraffin, it is useful to uncover the surface of the block to reveal the tissue. Coarse facing is done on the microtome at approximately 30 microns at a time until the entire tissue surface is exposed. Care should be taken to avoid removing too much tissue in this step. Tissue that was embedded improperly may not reveal the entire tissue surface and will have to be re-embedded. It is critical to have the entire flat tissue surface revealed so that the section is complete, representative, and diagnosible.
- Chill, hydrate, and decalcify blocks: Chilling paraffin blocks makes sectioning easier and faster. Histology laboratories are typically equipped with ice trays, ice cubes, crushed ice, or cold plates for this purpose. Soaking faced tissue blocks in a little bit of ice water also makes sectioning much easier, especially if tissue has been overly dehydrated during processing. Tissue types that benefit the most from hydration are: bloody samples, bone, uterus, brain, liver, heart tissue, and colon biopsies. Re-hydrating tissue prevents tissue chatter or splintering, something that often occurs with bloody tissue. At times, bone or microcalcifications in tissue can make microtomy difficult or impossible. Once the tissue block has been gently faced, it can be placed in a decalcifying solution for 30 minutes to an hour. Decalcified tissue should be rinsed well in tap water and then placed in ice water prior to sectioning.
- Cut one block at a time: Although it may seem obvious to cut one block at a time and pick up the sections from that block before moving on to the next block, it is worth reiterating. When histotechnologists feel the pressure to produce, some will find creative ways to get the job done! Cutting several blocks and laying down several different ribbons on one flotation bath, as show in the top image, increases the chances for major errors. These are errors that the pathologist would not recognize. Being organized during microtomy includes taking preventive measures to ensure the quality and integrity of your slides.
- Pick up sections on the correct slides: After the block is sectioned and the ribbon is on the water relaxing, the next step is to pick up the desired sections on the slides. If the ribbons seem to run away from the slide, use a tool to guide the section onto the slide, or even anchor one end of the ribbon to the water bath wall. Once the ribbon is stable, picking up sections is effortless. Next, the individual will glance at the floating ribbon and choose the section that is as close to flawless as possible. Routine tissue requires at least one good section on a slide. Biopsies are often small and several sections will fit on one slide. When picking up sections, always check the slide number and/or letter to make certain that the tissue label matches the slide label. This is a common source of errors that also compromises the quality and integrity of your slides.
- Allow slides to drip dry: Drying slides upright helps remove the water from underneath the paraffin sections and prepares them for deparaffinization. Slides that are not drained and dried properly may lose their tissue sections prior to staining. Special drying racks are available to assist in this process.