Processing, Embedding, and Microtomy
From water to paraffin
Processing removes water and replaces it with paraffin: graded ethanol dehydration, xylene clearing (miscible with both alcohol and paraffin), then paraffin infiltration, often under vacuum and heat. Fatty tissue needs longer processing; dense fibrotic tissue also resists infiltration. Incomplete dehydration causes poor clearing and blocks with holes or cracking; over-dehydration makes tissue brittle. Dehydration usually begins at 70% ethanol rather than absolute alcohol to avoid harsh shrinkage.
Embedding and sectioning
Embedding orientation is a major source of diagnostic error: orient mucosal surfaces, epidermis, and the lesional edge so they are cut in the desired plane; avoid bubbles and multiple pieces at different depths. Routine paraffin sections are 3–5 µm, most often 4 µm. Maintain a blade clearance angle of about 5–10°, a parallel block face, and a water bath at 40–45 °C. Watch for folds, chatter, compression, knife lines, and thickness variation. Frozen sections are cut in a cryostat at about −20 to −25 °C using OCT, useful for rapid diagnosis and lipid staining.
Traps and a worked example
Questions often test the fact that thicker sections stain more intensely simply because more nuclei lie in the light path. A water bath that is too hot can expand or distort tissue; a dull blade causes chatter and vertical knife marks.
Worked example: A 4-µm section and a 10-µm section of the same block are stained identically. The 10-µm section appears darker and more cellular, but this reflects section thickness, not superior staining. If nuclei are pale across all sections, look at hematoxylin quality and differentiation rather than cutting thinner.