A microtome cuts extremely thin slices, or “sections,” from a sample. These sections, often biological tissues, are mounted on microscope slides for detailed examination. Microtomes are used in medical diagnostics (histology, pathology) and scientific research. Producing uniform sections is important for accurate analysis. This guide outlines microtome use.
Types and Components
Microtome types vary by material and application. Rotary microtomes, widely used for paraffin-embedded tissues, move the sample block past a fixed blade, producing 0.5 to 60 micrometers (µm) thick sections. Sliding microtomes, with a blade sliding across a stationary sample, are used for larger or harder materials like wood, yielding 1 to 60 µm sections. Cryostats are freezer-housed rotary microtomes for frozen tissues without wax embedding, producing 4 to 15 µm sections. Vibrating microtomes use a vibrating blade to cut fresh or fixed tissues without embedding, producing thicker slices (over 30 µm for fresh, over 10 µm for fixed).
Most microtomes share core components for precise sectioning. A specimen holder grips the sample block for stability. The blade holder positions the blade at the correct angle, adjustable for optimal cutting.
An advance mechanism controls specimen movement toward the blade, determining section thickness. Coarse and fine adjustment knobs control position and sectioning parameters. Blades vary by material: steel for routine histology, glass for light and electron microscopy, and diamond for hard materials like bone or teeth.
Preparing for Use
Proper preparation is important before sectioning. Specimens are often embedded in a supportive medium. For routine histology, tissues are dehydrated and infiltrated with paraffin wax, which hardens them for thin slicing. For cryosectioning, fresh or fixed tissues are embedded in an aqueous medium (e.g., OCT compound) and rapidly frozen. Cooling paraffin blocks on ice beforehand improves sectioning quality by making tissue and wax more consistent.
Once prepared, mount the specimen block securely onto the microtome’s specimen holder. Firmly clamp the block to prevent movement during sectioning, which causes inconsistencies. Insert a sharp blade into the blade holder. Position and clamp the blade tightly, ensuring security without over-tightening, which can deform it.
Set initial machine parameters, including desired section thickness. For trimming, a thicker setting (10-30 µm) quickly exposes the tissue. For fine sectioning, thicknesses typically range from 2-10 µm, with 3-5 µm common for routine histological examinations. Maintain a clean working area and check all instrument connections.
Sectioning Techniques
Sectioning begins with coarse trimming to expose the tissue within the embedded block. Set the microtome to a thicker cut (20-30 micrometers) and advance the block until the tissue surface is revealed. This trimming ensures subsequent fine sections contain the desired tissue. After trimming, adjust section thickness for fine sectioning, typically 3-7 micrometers for most applications.
During fine sectioning, turn the handwheel smoothly and consistently (ideally one revolution per second) to produce uniform sections. Sections often form a continuous ribbon, especially with paraffin-embedded tissues. Collect ribbons carefully using a fine brush or forceps, avoiding direct blade contact. For cryosections, collect individual sections directly onto slides. Float collected sections on a warm water bath to flatten them before picking them up.
Challenges can impact section quality. Compression (sections shorter than the block) results from a dull blade or soft wax. Chatter (lines or thickness variations) indicates loose components or vibrations. Tears or nicks usually point to blade imperfections or debris. Troubleshooting involves checking clamp tightness, replacing the blade, or adjusting block temperature by cooling with ice if wax is too soft.
Care and Safety
After sectioning, proper microtome care and safety protocols are important. Clean the microtome thoroughly to remove paraffin wax shavings and tissue debris, which can corrode or interfere with its function. A dry brush removes most residue; some areas benefit from cleaning with alcohol or specialized paraffin cleaners. Avoid liquids inside the microtome’s internal mechanisms.
Handle and dispose of used blades with caution due to sharpness. Never touch blades directly; use forceps or a magnetic tool to remove them. Place used blades immediately into an approved sharps container for safe disposal.
Follow safety measures when operating a microtome. Always wear personal protective equipment (PPE), including a lab coat, safety glasses, and disposable gloves. Cut-resistant gloves are advisable when handling or changing blades.
Lock the microtome’s handwheel when not sectioning, and keep the blade guard in place when exposed or unattended. Maintain a clear distance between hands and the blade, use tools like brushes or forceps to manipulate sections, and ensure the work area is free of clutter. Regular maintenance and professional servicing ensure safe and effective microtome operation.