024 – Observation of animal cells

The compound light microscope is the instrument that made cell biology possible, and the cells lining the inside of the human cheek are the classic first specimen. Buccal epithelium is a stratified squamous tissue: its outermost cells are continuously shed, so a sample can be collected without cutting, staining, or sectioning anything. Cytologists still use exactly this material for oral cancer screening and for buccal swabs in genetic testing, which makes it a real diagnostic specimen rather than a teaching convenience.

Living animal cells are almost invisible in a light microscope for a simple physical reason: they are mostly water, and their refractive index is barely different from the water they sit in, so they absorb and bend very little of the transmitted light. What a microscopist sees under those conditions is a faint outline and little else. A stain solves the problem by binding selectively to some structures and not others, converting a difference in chemistry into a difference in colour that the eye can read. Lugol’s solution — iodine dissolved in potassium iodide — is the standard choice for this preparation: it tints the cytoplasm a pale yellow-brown and picks out the nucleus as a distinctly darker body, and it does so instantly and without heating.

In this laboratory you will prepare the same specimen twice and compare the two. The first slide is a plain wet mount in water; the second receives a drop of Lugol’s solution before the coverslip goes on. You will switch on the transmitted illuminator, start at the lowest magnification and work up through 40×, 100× and 400×, focusing with the coarse knob only at low power and the fine knob thereafter, and save an image at each step. The comparison between the two slides is the point of the exercise: it shows what a stain does, and why almost no microscopy of animal tissue is done without one.

Educational Goals

Familiarization with the laboratory environment

• Locate the microscope and its power switch, the three objectives, the coarse and fine focus knobs, the stage, the slides, the coverslips, the droppers and the absorbent paper before beginning.

Operation of a compound light microscope

• Set up transmitted (bright-field) illumination from below and explain why it suits a thin transparent specimen and would be useless for an opaque one.
• Change objectives in ascending order — 4×, then 10×, then 40× — and calculate the total magnification each gives with a 10× eyepiece.
• Use the coarse focus only at the lowest magnification and the fine focus at higher ones, and explain what would happen to the slide and the objective if the coarse knob were used at 400×.

Preparation of a wet mount

• Deposit a single drop of cell suspension on a clean slide, lower a coverslip onto it without trapping air bubbles, and blot the excess liquid so the coverslip sits flat against the specimen.
• Explain why the specimen must be thin and flooded with liquid for light to pass through it.

Use of a biological stain

• Add Lugol’s solution to a duplicate slide and state which structures it makes visible and which it does not.
• Compare the stained and unstained preparations directly, and account for the difference in terms of contrast rather than magnification.

Observation and recording

• Save a microscope image at each magnification for both slides, and annotate the cell membrane, the cytoplasm and the nucleus on the 400× stained view.
• Estimate the size of a cell and of its nucleus from the known field of view rather than guessing.

Protocol

Preparation

1. Turn on the microscope by pressing the switch on the front of the device. You will turn on the illuminator, so that the transmitted light passes through the sample from below, which is ideal for transparent specimens.
2. Place two clean slides on your work area.

Preparation of the first slide

1. Use the dropper to gently place 1 drop of buccal epithelium on the first slide.
2. Cover the slide with a coverslip.
3. Carefully blot the excess water with absorbent paper.

Preparation of the second slide

1. Use the dropper to gently place 1 drop of buccal epithelium on the second slide.
2. Place a drop of Lugol on the second slide.
3. Cover the slide with coverslip.
4. Carefully blot the excess water with absorbent paper.

Observation of the first slide

1. Place the slide on the microscope stage.
2. Click the « Microscope » button on the tablet to view the microscope image.
3. You can save an image of the observed microscope view by clicking the « Save image « button located in the lower left area of the « Microscope « section.

Microscope adjustment

1. Adjust the magnification by touching the microscope objectives. Begin observation at a magnification of 40x (red objective – labeled Plan 4/0.10).
2. Refine the focus with the coarse adjustment knobs located on the left and right of the microscope. Use the coarse knobs only at this magnification.
3. Gradually increase the magnification from 40x to 100x (yellow objective – labeled Plan 10/0.25), then to 400x (blue objective – labeled Plan 40/0.65), refocusing each time with the fine adjustment knob only. At these magnifications the coarse knob can drive the objective into the slide.

Note: A magnification of 1000x (white objective – labeled Plan 100/1.25) requires the use of oil between the objective and the coverslip. We therefore do not use this objective in this laboratory.

Observation of the second slide

1. Replace the first slide with the second slide containing the cells and Lugol.
2. Observe at a magnification of 400x (blue objective – labeled Plan 40/0.65) to identify a cell nucleus, which should appear colored yellow by the Lugol.
3. Recording observations : Document or record the important observations.

Turning off the microscope

1. Turn off the microscope by pressing the switch on the front of the device.

Anticipated Outcomes

A set of reference images of the expected microscope views is available in this document (PDF).

What the cells look like, and why

Buccal epithelial cells are squamous — flattened and plate-like — and having been shed from the surface of the tissue they lie loose and unsupported on the slide. An animal cell has no cell wall, only a flexible plasma membrane, so nothing holds it in a fixed geometry: the cells spread out, take on irregular rounded-polygonal outlines, and often overlap or fold at the edges. Each is a large cell by animal standards, typically 50–70 µm across but only a few micrometres thick, with a single round nucleus of roughly 8–10 µm sitting near the centre. The contrast with the plant cells of laboratory 025 is worth drawing explicitly: those are rectangular, packed edge to edge in a rigid grid, because a cellulose wall dictates their shape.

Magnification, resolution and field of view

Total magnification is the product of the objective and the eyepiece. With the standard 10× eyepiece, M = M_obj × M_eye gives 4 × 10 = 40×, 10 × 10 = 100× and 40 × 10 = 400×. Magnification, however, only enlarges; what sets the finest detail that can be separated at all is the numerical aperture printed after the objective’s power. By the Abbe criterion, d = λ / (2 × NA); taking green light at λ = 550 nm, the 0.10 objective resolves d = 550 nm ÷ (2 × 0.10) = 2.8 µm, the 0.25 objective 1.1 µm, and the 0.65 objective 550 nm ÷ (2 × 0.65) = 0.42 µm. A nucleus 8 µm wide is therefore in principle resolvable even at 40× — it is contrast, not resolution, that keeps it invisible on the unstained slide.

The field of view shrinks in proportion as the objective power rises: for an eyepiece of field number 18 mm, the visible diameter is 18 ÷ 4 = 4.5 mm at the red objective, 1.8 mm at the yellow one and 18 ÷ 40 = 0.45 mm — that is, 450 µm — at the blue one. This gives a direct way to size a cell without a graticule: a 400× field about 450 µm wide spanned by roughly seven cells edge to edge puts each cell near 60 µm. It also explains why the specimen must be centred at low power first, because at 400× only about one four-hundredth of the low-power area is still in view.

Depth of field falls even faster than field width, from tens of micrometres at 40× to well under a micrometre at 400×. That is why the protocol restricts the coarse knob to the lowest magnification: at 400× the blue objective sits a fraction of a millimetre above the coverslip, and a coarse movement drives glass into glass, cracking the slide and damaging the front lens.

Why the stain works

Lugol’s solution is molecular iodine held in solution by potassium iodide as the triiodide ion: I₂ + I⁻ → I₃⁻. Iodine is strongly absorbing in the visible and binds readily to proteins and to nucleic acids, so it accumulates wherever the cell is densest in macromolecules. The nucleus, packed with DNA and histone, takes up far more iodine per unit volume than the watery cytoplasm around it and so appears markedly darker. The mechanism is chemical affinity, not magnification, which is the single most transferable idea in the laboratory: to see a structure you must first find something that binds to it and not to its surroundings.

Summary of Assignment by Grade Range

Grade 9–10

• Focus — operating the microscope safely and describing what is seen, with the vocabulary of the instrument and of the cell: objective, eyepiece, stage, coarse and fine focus, wet mount, coverslip, stain, membrane, cytoplasm, nucleus.
• Activities — prepare both slides, observe each at 40×, 100× and 400×, and save the six images. Draw the 400× stained view and label the membrane, the cytoplasm and the nucleus. Answer the guided questions: what can you see on the stained slide that you cannot see on the unstained one, and what does the stain therefore add? Why are the objectives changed from lowest to highest and never the other way round?

Grade 11

• Focus — quantifying the observation and connecting cell structure to cell function.
• Activities — calculate the total magnification of each objective with a 10× eyepiece; use the field of view (18 mm ÷ objective power) to estimate the diameter of a cell and of its nucleus in micrometres, and compare the results with the accepted 50–70 µm and 8–10 µm; explain the irregular shape of these cells by the absence of a cell wall, and contrast it with the plant cells of laboratory 025; and identify the structures that must be present in the cell but cannot be seen, saying why in each case.

Grade 12 / College Level

• Focus — the optics that set the limits of the instrument, and the chemistry that sets the limits of the stain.
• Activities — compute the Abbe resolution limit d = λ / (2 × NA) for all three objectives and state which cellular structures each could in principle resolve; distinguish resolution from contrast, and use that distinction to explain why an 8 µm nucleus stays invisible on the water mount even though every objective can resolve it; explain the chemistry of Lugol’s solution (triiodide formation, and why iodine concentrates in the nucleus) and propose a stain that would instead mark a different organelle, justifying the choice; estimate the depth of field at each magnification and relate it to the coarse-focus restriction; and design a control that would distinguish a genuine nucleus from a trapped air bubble or a debris particle.

Laboratory essentials

Instruments

• Compound light microscope with transmitted illuminator and three objectives (Plan 4/0.10, Plan 10/0.25, Plan 40/0.65) with a 10× eyepiece — 40×, 100× and 400× total
• 2 microscope slides
• 2 coverslips
• Droppers
• Tweezers (for handling coverslips)
• Beaker (50 mL)
• Absorbent paper

Products

• Human buccal epithelium cells in suspension (1 drop per slide)
• Lugol’s solution 2% (1 drop, second slide only)