Required and core practicals

6.1 · Free notes

Practical: preparing temporary slides, using a light microscope and calibrating an eyepiece graticule

Key points

• A temporary mount (wet mount) is a very thin piece of tissue, e.g. onion epidermis peeled with forceps, placed on a slide in a drop of water or stain so that light can pass through it.
• Lower the coverslip at an angle using a mounted needle to avoid trapping air bubbles. Blot away excess liquid with filter paper.
• Stains increase contrast because most cell structures are colourless. Iodine in potassium iodide stains starch blue-black and also shows nuclei. Methylene blue and eosin stain other structures.
• Focusing: start on the lowest-power objective (×4), use the coarse focus first, then the fine focus. On higher powers use only the fine focus, so the lens does not crack the slide.
• Total magnification = eyepiece magnification × objective magnification, e.g. ×10 × ×40 = ×400.
• Magnification = image size ÷ actual size (I = A × M). Convert both to the same unit first: 1 mm = 1000 µm and 1 µm = 1000 nm.
• Resolution is the minimum distance at which two points can be seen as separate. For a light microscope it is limited by the wavelength of light to about 0.2 µm (200 nm). Higher magnification alone does not improve it.
• An eyepiece graticule is a ruler in the eyepiece with arbitrary units. It must be calibrated against a stage micrometer, a slide with a known scale, usually 10 µm divisions.
• To calibrate, line up the zero marks, then find a second place where the scales match exactly. 1 graticule division = length on stage micrometer (µm) ÷ number of graticule divisions.
• Recalibrate for each objective lens. The graticule stays the same size but the image is magnified differently, so the value of one division changes. Measure by counting divisions × calibration value.
• Biological drawings need sharp continuous lines, no shading, a title, the magnification or scale, and labels using ruled lines that do not cross.

In more detail

• Worked calibration: 100 µm on the stage micrometer lines up with 25 eyepiece units at ×400, so 1 unit = 4 µm. A cell 18 units long is 72 µm.
• Stains: iodine for starch and plant cells; methylene blue for animal cells and nuclei; eosin for cytoplasm; toluidine blue for lignin and general tissue.
• Drawing rules: draw only what you see, use clear single lines, label lines with a ruler that do not cross, and no arrowheads.
• Why recalibrate: at higher power each eyepiece unit covers a smaller real distance, so a value calibrated at ×100 is wrong at ×400.