Microscopy

1 Plan and risk assessment

A light microscope magnifies twice: the objective (×4, ×10 or ×40) and the eyepiece (×10), so the total is ×40, ×100 or ×400. The eyepiece scale has no units of its own, so it is calibrated against a stage micrometer for each objective. Light passes up through the specimen, so it must be thin; a stain makes the colourless parts show.

magnification = size of image ÷ size of real object

Prediction

2 Apparatus

• ✓Light microscope (eyepiece ×10; objectives ×4, ×10 and ×40) with a lamp Magnifies the cells ×40, ×100 or ×400.
• ✓Eyepiece graticule (a 100-division scale in the eyepiece) (resolution 1 division) A scale seen on top of the cells, to measure them.
• ✓Stage micrometer slide (1 mm in 100 divisions) (resolution 0.01 mm) A known length to calibrate the graticule.
• ✓Microscope slides and coverslips Hold the specimen flat under the microscope.
• ✓Onion, white tile and scalpel The plant tissue, and a safe surface to cut on.
• ✓Forceps and a mounted needle Peel and flatten the tissue; lower the coverslip.
• ✓Dropping pipette and water One drop of water to mount the tissue in.
• ✓Iodine solution in a dropper bottle Stains the nucleus and cytoplasm yellow-brown.
• ✓Filter paper Soaks up extra liquid from round the coverslip.
• ✓Sterile cotton buds, methylene blue and a beaker of disinfectant Cheek cells: collect, stain, then dispose of the buds safely.
• ✓Prepared slide of cheek cells Animal cells, if your school does not take cheek cell samples.
• ✓Safety goggles Protect your eyes from iodine solution.
• ✓Sharp pencil, ruler and plain paper For the labelled drawing.

3 Method: your choices

• Which version are you doing?: AQA required practical 1: onion and cheek cellsMake your own onion slide, measure five cells and compare plant and animal cells.
• Put on safety gogglesSafety: DoneIodine solution irritates the eyes, and a dropper can flick a drop up.
• Take a thin layer of onion: Peel the thin epidermis with forcepsA layer one cell thick: light passes straight through and every cell stands out on its own.
• Make sure the layer is flat: DoneA fold puts two layers on top of each other and stretches the cells along it.
• Stain the onion: Two drops of iodine solutionNuclei and cytoplasm stained yellow-brown: clear cell outlines to measure, and nuclei that show at ×400.
• Lower the coverslip: At an angle, lowered with a mounted needleFew or no air bubbles: most slides have none.
• Soak up the extra liquid: DoneExtra liquid lets the coverslip float and slide about, and iodine on top of the coverslip gets onto the stage and the objective lens.
• Choose your animal cells: Your own cheek cells, stained with methylene blueFresh cells you prepared yourself. Use only your own cells and a new cotton bud, and put the used bud and slide in disinfectant.
• Focus at high power: Fine focus onlyThe image sharpens slowly and safely, and the cells snap into focus.
• Calibrate the eyepiece graticule: Calibrate at ×10, the objective you measure withEach division is worth what you worked out: lengths in µm are right to within about 1 %.
• Measure along the cell: Turn the scale along each cellYou measure each cell from one end wall to the other.

• 0.0 Struck through: the anomaly, retaken; the new reading is beside it.

Anomaly: Length of cell 4, retaken. Likely cause: it was really two cells: the wall between them had torn (or the cell was stretched at a fold).

5 Graph

This practical has no graph: the results are in the table above.

6 Analysis

1. One eyepiece division

   length of one division = length on the stage micrometer ÷ number of eyepiece divisions

   With the ×10 objective, 0.50 mm (500 µm) on the stage micrometer lined up with 49 eyepiece divisions. Work out the length of one eyepiece division.

   10.2 µm Right first time

2. Lengths in µm

   Cell 1 245 · Cell 2 286 · Cell 3 245 · Cell 4 214 · Cell 5 194 Right first time

3. Mean length

   mean = sum of the lengths ÷ number of lengths

   Calculate the mean length of your onion cells in µm.

   237 µm Right first time

4. Is it sensible?

   Onion epidermis cells are usually 100–400 µm long. Your mean is 237 µm. Is that sensible?

   Yes: it is in the usual range for onion epidermis cells Right first time

5. Estimate from the field of view

   length of one cell ≈ diameter of the field of view ÷ number of cells across it

   At ×100 the field of view is 1.8 mm (1800 µm) across. You counted 7 cells end to end across it. Estimate the length of one cell in µm.

   257 µm Right first time

6. Draw and label

   Labelled drawing; cell 1 drawn 82 mm long Right first time

7. Magnification of your drawing

   magnification = size of image ÷ size of real object

   In your drawing, cell 1 is 82 mm long. Its real length is 245 µm. Calculate the magnification of your drawing.

   335 Right first time

8. Plant and animal cells

   Which structures did the onion cells have that the cheek cells did not?

   A cell wall and a large vacuole Right first time

7 Conclusion and evaluation

Conclusion

Sources of error

Improvement

Repeatable or reproducible

9 Exam questions on your data

On your own readings, self-marked against the mark scheme, with error carried forward where it applies. Total 32/32.

1. 01.1[3 marks]

   A student made a slide of onion cells. Three of the steps were:

   1. Peel a thin layer of epidermis from the onion.

   2. Add two drops of iodine solution.

   3. Lower the coverslip at an angle.

   Give one reason for each step.

   Your answer

   1. So that light can pass through and you can see individual cells. 2. The iodine stains the nucleus and cytoplasm so they can be seen. 3. To stop air bubbles getting trapped under the coverslip.

   3/3 self-marked

   • Thin layer: so light can pass through / so individual cells can be seen (allow one cell thick)
   • Iodine: to stain the nucleus / cell wall / parts of the cell so they can be seen (ignore "to stain the cell")
   • Coverslip at an angle: to prevent (or reduce) air bubbles

   Self-marked: 3 of 3 marks. This is your own judgement against the mark scheme.

   Model answer: Thin layer: so light can pass through and individual cells can be seen. Iodine: stains the nucleus and cytoplasm so they can be seen. Coverslip at an angle: so no air bubbles are trapped.

2. 01.2[2 marks]

   The student used a scalpel to cut the onion.

   Give one risk of using the scalpel and one way to reduce this risk.

   Your answer

   You could cut your fingers with the blade. Cut the onion on a white tile, cutting away from your body.

   2/2 self-marked

   • Risk: cutting yourself (fingers, hand or skin) or someone else
   • Control: cut on a white tile (board), away from the body, keeping fingers behind the blade (ignore "be careful"; do not accept goggles)

   Self-marked: 2 of 2 marks. This is your own judgement against the mark scheme.

   Model answer: You could cut your fingers. Cut on a white tile, away from your body, with your fingers behind the blade.

3. 02.1[2 marks]

   When you calibrated the eyepiece graticule with the ×10 objective, 0.50 mm on the stage micrometer lined up with 49 eyepiece divisions.

   Calculate the length of one eyepiece division in micrometres (µm).

   Your answer

   10.2

   Working: 0.50 mm = 500 µm 500 ÷ 49 = 10.2 µm

   2/2

   • 0.50 mm ÷ 49 (or 500 µm ÷ 49): 1 mm = 1000 µm
   • = 10.2 µm

   Full marks. Clear and precise.

   Model answer: 0.50 mm = 500 µm 500 ÷ 49 = 10.2 µm

4. 02.2[1 mark]

   Table 1 shows the first lengths you recorded. One division was 10.2 µm.

   Which cell’s result is anomalous? Select the row.

   Table 1

   Cell	Length of cell in eyepiece divisions	Length of cell in µm
   1	24	245
   2	28	286
   3	24	245
   4	56	571
   5	19	194

   Your answer

   cell 4

   1/1

   • Cell 4: about twice as long as the others

   Full marks. Your answer meets all the points for this question. Look for the length that does not fit: about twice the others.

   Model answer: Cell 4: it is about twice as long as the others (probably two cells whose dividing wall tore).

5. 02.3[2 marks]

   Use Table 1.

   Calculate the mean length of the onion cells in µm. Do not include the anomalous result.

   Give your answer to the nearest µm.

   Your answer

   243

   Working: (245 + 286 + 245 + 194) ÷ 4 = 970 ÷ 4 = 243 µm

   2/2

   • Leaves out cell 4: (245 + 286 + 245 + 194) ÷ 4
   • = 243 µm

   Full marks. Your answer meets all the points for this question.

   Model answer: (245 + 286 + 245 + 194) ÷ 4 = 970 ÷ 4 = 243 µm

6. 02.4[1 mark]

   Another student calibrated the eyepiece graticule with the ×40 objective. They then measured the cells with the ×10 objective.

   What effect does this have on the lengths they calculate?

   Tick (✓) one box.

   • ✓ Every length is about four times too small. (your choice)
   • Every length is about four times too big.
   • The lengths are more spread out, but their mean is right.
   • No effect: the graticule is in the eyepiece, so it does not change.

   Your answer

   Every length is about four times too small.

   1/1

   • Every length is about four times too small.

   Full marks. That is exactly what the mark scheme is looking for.

   Model answer: Every length is about four times too small: a division at ×10 is worth four times as much as at ×40.

7. 03.1[3 marks]

   In your drawing, cell 1 is 82 mm long. You measured the real length of cell 1 as 245 µm.

   Calculate the magnification of the drawing.

   Your answer

   335

   Working: 82 mm = 82000 µm magnification = 82000 ÷ 245 = 335

   3/3

   • Same units: 82 mm = 82000 µm (or 245 µm = 0.245 mm)
   • 82000 ÷ 245
   • = 335 (no unit)

   Full marks. Well done.

   Model answer: 82 mm = 82000 µm magnification = 82000 ÷ 245 = 335

8. 03.2[2 marks]

   The magnification of your drawing is ×335. You decide to add a scale bar that stands for a real length of 100 µm.

   Calculate how long the scale bar should be drawn on the page, in millimetres (mm).

   Your answer

   33.5

   Working: size of image = magnification × size of real object = 335 × 100 µm = 33500 µm = 33.5 mm

   2/2

   • size of image = magnification × size of real object: 335 × 100 µm = 33500 µm (or 335 × 0.1 mm)
   • = 33.5 mm (µm ÷ 1000)

   Full marks. Well done.

   Model answer: size of image = magnification × size of real object = 335 × 100 µm = 33500 µm = 33.5 mm Draw a line 33.5 mm long beside the drawing and write “100 µm” on it.

9. 03.3[3 marks]

   A photograph of a cheek cell taken through a light microscope is 4 cm across. The magnification is ×400.

   Calculate the real width of the cell in micrometres (µm).

   Your answer

   100

   Working: real size = size of image ÷ magnification = 4 ÷ 400 = 0.01 cm = 0.1 mm = 100 µm

   3/3

   • Real size = image ÷ magnification: 4 ÷ 400 = 0.01 cm
   • Converts to µm: 0.01 cm = 0.1 mm = 100 µm (or 4 cm = 40 mm = 40000 µm)
   • = 100 µm

   Full marks. Well done.

   Model answer: real size = size of image ÷ magnification = 4 ÷ 400 = 0.01 cm = 0.1 mm = 100 µm

10. 03.4[1 mark]

    A typical onion epidermis cell is 250 µm long.

    Which of these is the length of the cell in metres, written in standard form?

    Tick (✓) one box.

    • 2.5 × 10⁻³ m
    • ✓ 2.5 × 10⁻⁴ m (your choice)
    • 2.5 × 10⁻⁷ m
    • 25 × 10⁻⁵ m

    Your answer

    2.5 × 10⁻⁴ m

    1/1

    • 2.5 × 10⁻⁴ m

    Full marks. Well done.

    Model answer: 250 µm = 250 × 10⁻⁶ m = 2.5 × 10⁻⁴ m. (25 × 10⁻⁵ m has the right value but is not standard form; 2.5 × 10⁻³ m is the length in mm written as metres.)

11. 03.5[2 marks]

    Figure 1 shows another student’s drawing of onion cells seen with a light microscope.

    Give two ways the drawing could be improved.

    Your answer

    Do not shade the cells. Add the magnification.

    2/2 self-marked

    • Include the magnification (or a scale bar)
    • Use single, continuous (unbroken) lines
    • Do not shade (or colour) the cells
    • Only label structures that can be seen (no ribosomes or chloroplasts in onion cells)
    • Draw label lines with a ruler, straight, not arrows, not crossing, each touching the structure it names (the ribosome and nucleus lines end on the wrong part)
    • Draw it larger (use more of the space)
    • Add a title
    • Draw the cells the correct shape, with no gaps between them and walls drawn as double lines

    Self-marked: 2 of 2 marks. This is your own judgement against the mark scheme.

    Model answer: Any two from: add the magnification or a scale bar; use single continuous lines; do not shade; only label structures that can be seen (not ribosomes or chloroplasts); draw label lines with a ruler, each touching the structure it names; draw it larger; add a title.

12. 03.6[2 marks]

    Figure 2 shows a drawing of onion cells made at a magnification of ×100. Lay the on-screen ruler on the figure: it reads in millimetres at the size the drawing is printed.

    Use the ruler to measure the length of cell X on the drawing. Then calculate the actual length of cell X in micrometres (µm).

    The reading is right if it is within 1 mm of the true length. Put one end of the ruler on each end wall of the cell.

    Your answer

    25 mm; 250

    2/2

    • Ruler laid edge to edge: a reading of 25 mm, within 1 mm
    • Actual length of cell X in µm = 250 µm

    Both ends on the edges and the calculation carried through correctly.

    Model answer: length on the drawing = 25 mm actual length = size of image ÷ magnification = 25 ÷ 100 = 0.25 mm = 250 µm

13. 03.7[2 marks]

    Give two advantages of looking at the onion cells with an electron microscope instead of a light microscope.

    Your answer

    It has a much higher magnification. It also has a higher resolution, so you can see more detail.

    2/2 self-marked

    • Higher magnification (ignore "bigger" or "zoom")
    • Higher resolution or resolving power (allow see more detail / smaller structures such as ribosomes)

    Self-marked: 2 of 2 marks. This is your own judgement against the mark scheme.

    Model answer: It has a higher magnification and a higher resolution, so sub-cellular structures such as ribosomes can be seen in detail.

14. 04[6 marks]

    A student wants to find the length of an onion epidermis cell using a light microscope.

    Describe how the student should prepare a slide of onion cells and use the light microscope to find the length of one cell.

    Your answer

    1. Peel a thin layer of epidermis from the inside of an onion layer with forceps. 2. Put it flat on a drop of water on a microscope slide. 3. Add two drops of iodine solution to stain it. 4. Lower a coverslip at an angle with a mounted needle so no air bubbles are trapped. 5. Put the slide on the stage with the lowest power objective. Watching from the side, use the coarse focus to bring the objective close to the slide, then look through the eyepiece and use the coarse focus to move them apart until the cells are in focus. 6. Change to a higher power objective and focus with the fine focus. 7. Calibrate the eyepiece graticule with a stage micrometer at that objective. 8. Count the eyepiece divisions along the length of a cell and multiply by the length of one division. 9. Measure several cells and calculate a mean.

    6/6 self-marked

    • Peel a thin layer of onion epidermis (with forceps)
    • Place it (flat) on a drop of water on a slide
    • Add iodine solution (a stain)
    • Lower a coverslip (at an angle, with a mounted needle, to avoid air bubbles)
    • Start with the lowest power objective and focus with the coarse focus
    • Change to a higher power objective and focus with the fine focus
    • Calibrate the eyepiece graticule with a stage micrometer
    • Count the eyepiece divisions across the length of the cell and convert to µm (or use the field of view)
    • Measure several cells and calculate a mean

    Self-marked: Level 3, 6 of 6 marks. You ticked 9 of 9 indicative points. This is your own judgement against the mark scheme.

    Model answer: 1. Peel a thin layer of epidermis from the inside of an onion layer with forceps. 2. Put it flat on a drop of water on a microscope slide. 3. Add two drops of iodine solution to stain it. 4. Lower a coverslip at an angle with a mounted needle so no air bubbles are trapped. 5. Put the slide on the stage with the lowest power objective. Watching from the side, use the coarse focus to bring the objective close to the slide, then look through the eyepiece and use the coarse focus to move them apart until the cells are in focus. 6. Change to a higher power objective and focus with the fine focus. 7. Calibrate the eyepiece graticule with a stage micrometer at that objective. 8. Count the eyepiece divisions along the length of a cell and multiply by the length of one division. 9. Measure several cells and calculate a mean.

Unseen data: the same skills, new apparatus

1. 01[2 marks]

   A student calibrates an eyepiece graticule with the ×40 objective: 0.10 mm on the stage micrometer lines up with 42 eyepiece divisions. With the same objective, a guard cell from a leaf is 14 eyepiece divisions long.

   Calculate the length of the guard cell in µm.

   Your answer

   33.3

   Working: 100 / 42 = 2.38 µm 14 * 2.38

   2/2

   • One division = 100 ÷ 42 = 2.38 µm.
   • The guard cell’s length in µm.

   Full marks. That is exactly what the mark scheme is looking for.

   Model answer: 0.10 mm = 100 µm, so one division = 100 ÷ 42 = 2.38 µm. Length = 14 × 2.38 = 33.3 µm.

2. 02[2 marks]

   A photomicrograph shows a red blood cell. In the photograph the cell is 21 mm across. The magnification is ×3000.

   Calculate the real diameter of the red blood cell in µm.

   Your answer

   7

   Working: 21 mm = 21000 µm 21000 / 3000

   2/2

   • 21 mm = 21000 µm.
   • Real size = size of image ÷ magnification, in µm.

   Full marks. That is exactly what the mark scheme is looking for.

   Model answer: 21 mm = 21000 µm. Real size = 21000 ÷ 3000 = 7 µm.

3. 03[2 marks]

   A student makes a slide of a leaf from the pondweed Elodea to look at its cells with a light microscope.

   Which TWO steps help the cells show clearly? Tick (✓) two boxes.

   • ✓ Use a single, thin leaf (one or two cells thick), so light can pass through it. (your choice)
   • ✓ Lower the coverslip at an angle with a mounted needle, so that no air bubbles are trapped. (your choice)
   • Focus at ×400 with the coarse focus, so the image comes into focus quickly.
   • Fold the leaf over twice, so more cells are in view.
   • Start with the ×40 objective, so the cells look as big as possible straight away.

   Your answer

   Use a single, thin leaf (one or two cells thick), so light can pass through it.; Lower the coverslip at an angle with a mounted needle, so that no air bubbles are trapped.

   2/2

   • Use a single, thin leaf (one or two cells thick), so light can pass through it.
   • Lower the coverslip at an angle with a mounted needle, so that no air bubbles are trapped.

   Full marks. Your answer meets all the points for this question.

   Model answer: A thin layer lets light through, so single cells show; a coverslip lowered at an angle pushes the air out, so there are no bubbles. Start on the lowest power; use only the fine focus at high power, or the objective can crack the slide.