Microtomy Procedure
- Fill water bath with distilled water and allow time for the appropriate temperature to be reached which is 5-10°C below the melting point of the wax used in the block (approximately 45-48ºC).
- The hand-wheel must always be locked in place except when using the microtome to make sections.
- Turn back the coarse feed wheel back almost as far as it will go as a starting point.
- Install a disposable knife in the microtome and immediately put up the knife guard.
- Set the correct clearance angle (knife tilt), normally 3 to 8º to prevent compression or alternate thick and thin sections. The clearance angle is that between the cutting facet of the knife and the block.
- Remove excess wax from all edges of the block.
- Check to make sure knife guard is up.
- Fit the block into the block holder making sure it is secure and completely flat in the holder.
- Release the clamping lever on the front side of the microtome base plate and slide knife holder to a position where the block just touches the knife edge.
- Re-tighten the clamping lever on the base plate and make sure the block and all levers are tight before beginning.
- Release hand-wheel lock and lower knife guard.
- Coarse cut the block at 10-50 µm using the lever for activating mechanical trimming until the “full face” has been trimmed for a single section. “Full face” means the entire piece of tissue is visible when mounted on a slide. Do NOT cut “through” any tissue in the block. Sometimes “levels” or “serial sections” will be required for a tissue (***see notes below).
- After full face has been achieved, release the coarse trimming lever and ‘polish’ the block with several rotations at 4-5 µm to remove any rough trim artefact. (this is especially important with dense tissue such as uterus)
- Lock hand-wheel and put up knife guard.
- Hydrate the block face by briefly (~ 30 seconds) laying the tissue surface on a clean Kimwipe that has been dipped in the water bath. (Do not overhydrate or the tissue will begin to swell out of the block
- Return the trimmed block to the cold surface.
- Continue to “coarse in” the remaining blocks while the “faced in” blocks are chilling.
- Label slide (frosted edge at the top) with a pencil or solvent resistant marker including the surgical/autopsy/PM number, date, your initials, the name of the stain (except H&E) and if there are levels or serial sections. Pre-label only slides for one block at a time (the block you are working on). Select charged slides for immunohistochemistry, silver methods and for sections containing high concentrations of blood (blood clot, spleen, bone marrow), nervous tissue (brain, spinal cord), skin and decalcified tissues as these are also prone to detaching from slides.
- If there is wax built up on the back of the blade this will interfere with obtaining a good section. Carefully remove by pushing upwards on the back of the blade holder to dislodge the wax (this can be done with Kimwipes but also with a camel hair brush). If the camel hair brush comes “down” across the surface of the blade it can cause “scores” on the knife blade resulting in poor sectioning.
- Make sure the knife guard is up.
- Move the knife to a new, unused area or install a fresh, sharp blade in the microtome.
- Set the control knob for advance feed to the appropriate tissue section thickness (usually 4 to 5µm.
- Before putting the chilled block in the block holder double check that the block and slide number match.
- Re-install the cold block in the block holder. Use the coarse wheel adjustment knob to retract the block holder so the block can clear the knife.
- Release hand-wheel lock and lower knife guard.
- Cut a series (or ribbon) of sections at the required thickness. The formation of the ribbon is due to heat generated during cutting which causes the edges of the sections to adhere. Gently breathing upon the sections as they are cut dissipates static electricity, flattens the section and facilitates movement of the ribbon down the knife. Obtaining a ribbon is dependent on a well-chilled block. Sections must be collected quickly (in the first or second attempt at ribboning) before the block must be returned to ice to chill again! Single sections can also be collected, when necessary.
- Separate the ribbon from the knife edge with a moist applicator stick (or pick) and gently lay it across the surface of a warm water bath. If using a pick be extremely careful not to let it touch the edge of the blade as this will cause nicks and affect future sectioning. Bubbles under the surface of the section cause non-adherence to the slide in that spot. Small air bubbles trapped beneath the wax can be removed by careful prodding using some wax between skinny forceps under the tissue in the waterbath. The water bath bottom should have bubbles removed/disturbed on a regular basis with the end of a camel hair brush and a Kimwipe.
- Make sure the brake is on and the knife guard is up.
- Separate and collect sections on a clean, labeled glass slide. If you are unable to produce a section as block needs more chilling, place the block on the cooling tray and place that labelled slide directly under the block, to keep them together.
- Tap the slide and lean it against the water bath to remove excess water.
- Make sure the brake is on and the knife guard is up.
- Remove the block from the microtome and once again check to make sure the block and slide numbers match!!
- Dry slides for at least 10 minutes in a hot air oven to ensure the section is firmly attached. Slides needed for immunohistochemical studies are to be dried in oven no hotter than 55-60°C. For delicate tissues more prolonged drying in a hot air oven at a lower temperature may prove beneficial. Overnight drying at 37°C may be necessary for maximum section adhesion. Slides left in the open for drying will accumulate dust. If not staining slides right away store them in slide holder in cupboard labeled with your name.
- Clean surface of the water bath with a Kimwipe between each slide to eliminate “carryover” between specimens!!
- Many tissues contain small amounts of calcium which can make sectioning difficult. Blocks can be surface decalcified using a small amount of decalcifying solution in a lid of a glass coplin jar in the fume hood. After trimming, place blocks face down in the decalcifying solution for 5-10 minutes. Rinse with distilled water, wipe dry and place on the cold tray before sectioning. (eg. placenta, bone marrow, breast). Do not decalcify tiny biopsies or nerve tissue.
- Tissues that are “bloody”(eg liver) may also be hard to section. You may have more success if after taking the block off of the cold plate, giving it a quick in-and-out dip in the water bath to warm the tissue slightly. Alternatively, you can hold your thumb over the tissue surface for a few seconds before putting the block in the holder. A slower cutting speed may also be helpful.
- When you are finished with the microtome, carefully take out knife and dispose of in the safety disposal slot on the bottom on the knife box. NEVER leave a microtome at the end of lab with a blade in!! Collect any loose wax/tissue with camel hair brush and dispose in bio waste. Wipe off any remaining wax buildup on microtome and especially knife holder. Disinfect area and put instruments and supplies away.
- Clean and dry waterbath and cover daily to prevent the growth of organisms and to prevent rust formation.
Labelling Slides
Slides must be labelled with the following information:
- Accession number
- Patient’s Last name, First name
- Block (A1, A2, B1, B2, etc) and slide number (-1, -2, -3 etc)
- Staining method that the slide is intended for (only if other than H&E). If no staining method is indicated, it is assumed to be H&E.
- Section thickness (only if other than routine 4-5 µm). If none is indicated, it is assumed to be routine thickness.
- Your initials
Example: A-1 block with one routine slide cut for H&E staining