make temporary preparations of cellular material suitable for viewing with a light microscope

Making Temporary (Wet) Preparations for Light‑Microscopy

Learning outcome

Students will be able to make a temporary (wet) preparation of cellular material, observe it with a light microscope and then:

• measure structures,
• calculate magnifications and actual sizes,
• draw accurate, labelled sketches, and
• compare light‑microscopy with electron‑microscopy.

Why use temporary (wet) preparations?

• Preserve the natural colour, shape and movement of living cells.
• Allow observation of growth, division and responses to stimuli.
• Require only simple, inexpensive equipment and can be prepared in minutes.

Key equipment & safety

Safety reminders

• Handle sharp tools away from your body; use a blade guard where possible.
• Never look directly at the microscope light source.
• Wear gloves when handling stains; dispose of stained slides according to school waste‑policy.
• Use oil‑immersion lenses only with immersion oil and clean the oil off afterwards.

Design‑your‑own wet‑mount task (teacher prompt)

Choose one specimen from the list below, justify your choice in 2‑3 sentences (e.g., “onion epidermis shows a clear cell wall and large vacuole, ideal for measuring cell size”), then carry out the full wet‑mount procedure.

• Onion (Allium cepa) epidermis – thin, transparent cells with visible nuclei.
• Human cheek cells – easy to obtain, show nucleus and cytoplasm.
• Pond water – contains a variety of unicellular algae, protozoa and bacteria.
• Elodea leaf peel – displays chloroplasts moving in live cells.
• Yeast suspension – convenient for observing budding and cell division.

General procedure for a wet‑mount preparation

1. Clean the slide and cover slip. Rinse with distilled water, dry with lint‑free tissue, and inspect for scratches.
2. Place the specimen. Using a needle or tweezers, transfer a tiny fragment (≈1 mm²) to the centre of the slide. Aim for a single, thin layer.
3. Add liquid. Place a 1 mm‑diameter drop of distilled water, saline, or the chosen stain onto the specimen.
4. Apply the cover slip. Hold the cover slip at a 45° angle, let one edge touch the liquid first, then lower it gently to push out air bubbles.
5. Remove excess liquid (optional). Blot the slide edges with tissue; seal with a thin line of petroleum jelly if the slide will be observed for more than a few minutes.
6. Observe. Start with the lowest‑power objective (4×), centre the field, then switch to higher magnifications. Use the fine focus knob for clarity.

Staining techniques for temporary preparations

Observing & focusing tips

• Always start with the 4× objective to locate the specimen before moving to higher powers.
• When switching objectives, use the coarse focus only on low power; fine focus is sufficient for higher magnifications.
• Adjust the diaphragm to improve contrast – a slightly closed diaphragm increases resolution for transparent specimens.
• If the image is dim, increase the illumination intensity rather than opening the diaphragm too far.

Magnification, resolution & comparison with electron microscopy

Key definitions

• Magnification (M) = ocular power × objective power.
• Resolution = smallest distance between two points that can be distinguished as separate.
  Light microscope (theoretical limit) ≈ 0.2 µm; electron microscope ≈ 0.001 µm.

Light microscope vs. electron microscope (quick comparison)

Measuring with an eyepiece graticule

1. Calibrate the graticule. Place the stage micrometer on the stage, focus with the 40× objective, and count how many graticule divisions correspond to a known distance (e.g., 10 µm). Record the conversion factor (e.g., 1 division = 0.5 µm).
2. Measure the structure. Switch to the objective you will use for observation, align the structure with the graticule, count the divisions and multiply by the conversion factor.
3. Record the result. Write the measurement in micrometres (µm); convert to nanometres (nm) if required (1 µm = 1000 nm).

Calculating actual size from a photomicrograph (worked example)

1. Microscope settings: 10× ocular, 40× objective → total magnification M = 400×.
2. On the printed image a cell measures 2 cm.
3. Convert the image length to the same unit as magnification: 2 cm = 20 mm.
4. Actual size = (image size) ÷ M = 20 mm ÷ 400 = 0.05 mm.
5. Convert to micrometres: 0.05 mm × 1000 µm mm⁻¹ = 50 µm.
6. Result: the cell’s true diameter is 50 µm.

Sketching what you see

Accurate drawings are a core skill for the Cambridge syllabus.

• Use a ruler or graph paper to keep proportions.
• Label at least three identifiable structures (e.g., cell wall, nucleus, chloroplast, vacuole, starch grain).
• Indicate the magnification used and, if measured, write the size next to the structure.
• Sketch in stages: outline → internal details → annotation.

Sketching checklist

Metric units – quick reference

Typical size ranges of biological structures

Common pitfalls and how to avoid them

• Air bubbles – lower the cover slip at a 45° angle; never drop it straight onto the liquid.
• Specimen movement – use a thin layer of specimen and a small cover slip; seal edges with petroleum jelly if observation will be prolonged.
• Over‑staining – rinse briefly after staining; excess dye obscures fine detail.
• Incorrect magnification calculation – always multiply ocular and objective powers; double‑check the ocular rating.
• Mis‑reading the graticule – calibrate with a stage micrometer each time you change the objective.

Summary of the wet‑mount procedure