Microscopy Practical and Size Estimation

What this investigation is for

A light microscope allows cells to be observed directly, but an observation is scientifically useful only if it is recorded clearly. In this required practical, you use a light microscope to observe a selection of plant and animal cells, then make labelled drawings that include a magnification scale.

A typical comparison uses onion epidermal cells as the plant cells and cheek cells as the animal cells. Onion epidermal cells usually form a regular, brick-like arrangement and have visible cell walls. Cheek cells tend to have less regular outlines and no cell wall. With suitable staining, a nucleus, cytoplasm and cell boundary may be distinguishable. Only record structures that are genuinely visible: cells from the underground bulb of an onion are not expected to contain visible chloroplasts.

Preparing a temporary plant-cell slide

Use forceps to remove a very thin layer of onion epidermis and lay it flat on a clean glass slide. A thin specimen lets light pass through and prevents several layers of cells from obscuring one another. Add a drop of iodine solution to increase contrast.

Lower a coverslip gently at an angle, using a mounted needle or forceps to control it. Lowering one edge first helps push air out rather than trapping bubbles. The coverslip keeps the specimen flat and retains the liquid beneath it. Blot away excess liquid carefully if necessary.

Preparing a temporary animal-cell slide

Use a clean cotton bud to collect cells gently from the inside of the cheek, then smear them onto a clean slide. Add a drop of methylene blue stain and lower a coverslip carefully. The stain makes otherwise colourless structures, particularly nuclei, easier to distinguish.

Glass slides and stains must be handled carefully, and the laboratory instructions for the particular stains must be followed. A cheek-cell sample and the cotton bud used to collect it must not be shared.

Finding and focusing the cells

Place the slide securely on the microscope stage and select the lowest-power objective lens first. Looking from the side, position the objective close to the slide without allowing it to touch. Then look through the eyepiece and use the coarse-focus control to find the image. Adjust the light and use the fine-focus control to sharpen it.

Low power gives a wide field of view, so cells are easier to locate and the general arrangement of a tissue can be seen. Centre the area of interest before changing to a higher-power objective. Higher power gives a smaller field of view but can reveal more detail in one part of the specimen. Refocus carefully, normally using the fine-focus control.

The microscope's total magnification is found by multiplying the eyepiece magnification by the objective-lens magnification. For example, a $\times 10$ eyepiece used with a $\times 40$ objective gives a total magnification of $\times 400$. This describes how enlarged the image appears; it is not a measurement of the cell's real size.

Recording a biological drawing

Choose one or a small group of clearly focused cells and make the drawing large enough for their proportions to be shown. Use clear, unbroken pencil lines and avoid artistic shading. Draw what is visible rather than adding features from memory. Put labels outside the drawing and connect them to the relevant structures with straight, uncrossed lines.

Give the specimen a title and include a magnification scale. This might be the total magnification used, such as $\times 400$. If the image has been calibrated, a scale bar can instead show a stated real length, such as $50\,\mu\text{m}$. A scale bar must be a sensible size relative to the drawing and must accurately represent its stated distance.

A low-power drawing may show the arrangement of regions or groups of cells. A high-power drawing shows more detail in a smaller part of the slide. These have different purposes, so the power should be chosen to match what is being recorded.

Estimating relative size

An estimate is appropriate when an exact measurement is unnecessary or impossible—for example, when a structure has an irregular edge, the boundary is blurred, part of it is hidden, or the task asks only which structure is larger. Estimation is also helpful for checking whether a calculated answer is sensible.

If two structures are in the same image, their dimensions can be compared directly because they share the same scale. You might judge that a nucleus is roughly one-third of the cell's width, or that one chloroplast is about twice as long as another. If the structures appear in different images, compare their scale bars or stated magnifications first; their printed sizes alone are not reliable.

The field of view can provide a reference. If a cell spans about one-quarter of a field whose real diameter is known, its diameter is approximately one-quarter of that value. Several cells can also be counted across the field and the field diameter divided by that approximate number. These methods provide estimates because cells may overlap, vary in size or fail to fit exactly across the field.

Estimating areas and ratios

Area comparisons concern how much surface in the image each structure occupies, not merely its longest dimension. For a roughly rectangular structure, multiplying estimated length by estimated width gives an approximate area. For an irregular structure, imagine a grid over the image: count complete squares and combine partly covered squares to estimate the total.

Areas can then be expressed as a ratio. If the cytoplasm occupies approximately three times the area of the nucleus in a section, the estimated cytoplasm-to-nucleus area ratio is about $3:1$. Such a ratio is useful for comparing cells even when their overall sizes differ. It remains an estimate because the micrograph is a two-dimensional section of a three-dimensional cell and the boundaries may not be exact.

Improving confidence in an estimate

Base the judgement on several suitable cells rather than one unusual cell. Keep the magnification and method consistent, repeat measurements where possible and calculate a representative value such as a mean. State sensible limits: an answer such as “about one-quarter of the cell area” better reflects uncertain boundaries than an unjustifiably precise percentage.

Required practical

• Observe a selection of plant and animal cells using a light microscope.
• Prepare thin specimens on slides, add a suitable stain and lower a coverslip carefully.
• Iodine solution can increase contrast in plant-cell preparations; methylene blue can stain cheek cells.
• Begin with the lowest-power objective, focus, centre the cells and then increase the magnification if needed.