Making temporary slides for the light microscope

You met the light microscope at IGCSE.

At AS Level you are expected to prepare a specimen yourself, quickly and well, because Paper 3 (Advanced Practical Skills) can ask you to make a slide, find particular cells on it and then draw or measure them.

A good slide is the start of every other microscope skill in this unit.

A light microscope makes an image from light that passes through the specimen.

Three things follow from that.

• The specimen must be thin, ideally one layer of cells. Light then passes through every cell, and cells do not lie on top of each other and hide each other's outlines.

• Most cell contents are almost colourless. A stain binds to some structures more than others, so those structures absorb more light and stand out from the background. Stain increases contrast.

• The specimen must stay flat and wet. A drop of liquid stops the cells drying and shrinking, and a coverslip holds the specimen flat in one plane of focus. The coverslip also keeps liquid off the objective lens.

A slide made this way is called a temporary preparation (or temporary mount), because the specimen is alive or freshly killed, sits in liquid and dries out within an hour or so.

Permanent slides, such as the prepared slides of stems or blood smears you will draw later in the course, are made by a longer process of fixing, cutting very thin sections, staining and sealing.

Onion epidermis is the standard plant specimen because it peels off as a sheet one cell thick.

Figure 1.1 shows the key step.

1. Put one or two drops of iodine in potassium iodide solution in the middle of a clean slide.

2. Cut a small piece of one fleshy leaf (scale leaf) from an onion bulb. With forceps, peel the thin, transparent layer of epidermis from its inner (concave) surface.

3. Lay a piece about 5 mm square flat in the drop. Use a mounted needle to unfold it, so there are no folds or overlaps.

4. Hold a coverslip at about 45° with one edge touching the slide beside the drop. Support the raised edge with a mounted needle and lower it slowly, so the liquid spreads out ahead of it and pushes the air out.

5. Draw off any extra liquid at the edge of the coverslip with a piece of filter paper or paper towel.

6. Leave the stain a minute or two to soak in, then view with the low-power objective first.

Table 1.1 Two stains for temporary slides

Both stains are on the list of materials Cambridge expects schools to have for Paper 3.

If you see a different stain in a question, use the information given: you are not expected to know every stain.

An animal specimen is made the same way.

Cells scraped gently from the inside of the cheek with a clean cotton bud are smeared in a drop of methylene blue on the slide, then covered as in step 4.

Many schools no longer use human cheek cells, so you may use another animal tissue instead.

The cotton bud goes straight into disinfectant after use.

The microscopes in Paper 3 have a ×10 eyepiece lens and two objective lenses, ×10 (low power) and ×40 (high power).

The total magnification is the eyepiece magnification multiplied by the objective magnification, so ×100 on low power and ×400 on high power.

1. Start with the low-power (×10) objective in place and the slide clipped on the stage, with the specimen over the hole.

2. Looking from the side, turn the coarse focus knob to bring the objective close to the coverslip without touching it.

3. Now look through the eyepiece and turn the coarse focus to move the objective away from the slide until the image is sharp. Then sharpen it with the fine focus.

4. Adjust the light (the diaphragm or the lamp) until the cells are clear and not washed out. Too much light hides pale structures.

5. Move the part you want into the centre of the field of view, then turn the high-power (×40) objective into place and use only the fine focus.

Low power first gives a wide field of view to find the right area; high power then shows detail in a small part of it.

The coarse focus is never used at high power, because the ×40 objective sits very close to the slide and can crack the coverslip.

Table 1.2 Faults on a temporary slide

Paper 3 can ask you to assess the risk of a procedure as low, medium or high.

A hazard is something that could cause harm; the risk is how likely and how serious that harm is in your procedure.

Both stains mark skin and clothing and can irritate the eyes.

Cambridge's materials list codes methylene blue as a health hazard and a moderate hazard, and codes both stains as hazardous to the aquatic environment, so neither goes down the sink in quantity.

With small volumes, eye protection and care, the risk is low.

Scalpels and broken coverslips carry a risk of cuts: cut downwards onto a tile, away from your fingers.

A light microscope does not see a cell the way your eye sees a chair across the room.

It sees differences: the image is built from places where more light is blocked and places where less gets through.

Most of a living cell is water, and the rest (proteins, membranes, the nucleus) absorbs and bends light almost exactly as water does, so an unstained cell is nearly invisible, like a clear glass marble dropped into a glass of water.

A stain works by giving some structures a property the background lacks.

Iodine is a neat example: in solution it forms short chains of iodine atoms that slot inside the coiled chains of starch (you will meet the coil in Topic 2), and starch packed with iodine absorbs most visible light, so the grains turn blue-black.

Researchers who need to watch living cells without stain use special optics, such as phase contrast, that turn tiny differences in how much light is slowed into differences in brightness.

That is not examined, but it is how most videos of living cells are filmed.

There is a second reason for working thin, which becomes obvious at high power.

A lens is only sharp at one distance, and with the ×40 objective the slice of specimen that is in focus at any moment is only about a micrometre deep.

Everything above and below that slice is still in the path of the light, but blurred, and the blur is laid over the sharp part.

Even one layer of cells is several micrometres deep, but wherever you place the sharp slice in a single layer it passes through just one set of cells, with nothing else stacked above or below.

A fold or a clump stacks other cells over the sharp slice, and their blur is laid over it, so you see grey mush instead of outlines.

Turning the fine focus moves the sharp slice up and down through the cells, which is why experienced microscopists keep a hand on the fine focus as they look: they are exploring the cell in depth, one thin layer at a time.

Starting on low power is not just tidiness.

Magnification spreads a smaller patch of specimen over the same circle of view, so the more you magnify, the less of the slide you see at once: doubling the magnification halves the width of the field of view and leaves you only a quarter of the area.

Searching for one good cell at high power is like looking for a friend in a crowded stadium through a drinking straw.

Low power gives you the wide view to find the right place; high power then spends the whole field on a small part of it.

The drop of liquid under the coverslip does more than stop the cells drying.

Light bends each time it crosses a boundary between glass, air and specimen, and every air gap scatters some of it.

A film of water fills those gaps, so the light passes through more evenly and the image is clearer.

It is also why an air bubble looks so dramatic: its curved edge bends light sharply away from the objective, giving the thick black ring described in Table 1.2.

1. Why is a coverslip lowered onto a specimen at an angle? [Recall] [1]
A. to avoid trapping air bubbles under the coverslip
B. to spread the stain evenly over the specimen
C. to squash the specimen so that it is one cell thick
D. to stop the objective lens touching the specimen

2. A student stains onion epidermis with iodine in potassium iodide solution. Which row is correct? [Recall] [1]

3. Explain why the specimen on a temporary slide should be only one layer of cells thick. [Recall] [2]

4. Outline how to change from viewing a specimen with the ×10 objective to viewing it with the ×40 objective. [Recall] [3]

5. A student looks at onion bulb epidermis cells and sees no chloroplasts. Suggest why. [Apply] [1]

6. Neutral red is a stain that collects in the large vacuoles of living plant cells; dead cells do not take it up. A student wants to see the vacuoles in onion epidermis. Suggest one advantage of neutral red over iodine solution for this, and one reason the slide must be viewed soon after it is made. [Apply] [2]

7. A microscope has a ×10 eyepiece. With the ×10 objective, the field of view is 1.8 mm across. The diameter of the field of view is inversely proportional to the total magnification.

(a) Calculate the total magnification with the ×40 objective. [Analyse] [1]

(b) Calculate the diameter of the field of view with the ×40 objective, in µm. [Analyse] [2]