AQA A-level Biology: Microscopy, Magnification and Cell Fractionation (7402) – Revision Notes

Short notes for the final weeks. For full explanations, start with the study guide. These notes follow section 3.2.1.3, Methods of studying cells, in the AQA AS and A-level Biology specification (A-level 7402), for first teaching 2015, exams June 2016 onwards. Nothing here is marked A-level only. Test yourself with the practice questions, find the AQA A-level Biology hub, tick off topics on the checklist, and use the free diagnostics to find weak spots. The cell structure notes cover the organelles themselves.

Key definitions

Term	Meaning
Magnification	The factor by which the image is enlarged compared with the specimen
Resolution	The smallest separation at which two neighbouring points still look like two
Artefact	A feature in an image that is not part of the living specimen, made by preparation or observation
Homogenate	The mixture of cell parts made when tissue is broken up
Pellet	The solid that settles at the bottom after centrifuging
Supernatant	The liquid left above the pellet
Graticule	A scale in the eyepiece with arbitrary units
Stage micrometer	A slide ruled with a scale of exactly known size, used as the reference when calibrating

Microscope comparison

	Optical	TEM	SEM
Beam	Light	Electrons pass through	Electrons scan the surface
Resolution	Lowest	Highest	Between the two
Specimen	Thin; can be alive	Extremely thin; dead; in a vacuum	Any thickness; dead; in a vacuum
Image	Colour, 2D	Black and white, 2D	Black and white, 3D surface
Shows	Cells, nuclei, large organelles	Ultrastructure (membranes, ribosomes)	Surface shape
Main limitation	Resolution limited by light’s wavelength	Dead specimen, thin sections, artefacts	Surface only; lower resolution than TEM

Why electron microscopes resolve more: electrons have a much shorter wavelength than light, and shorter wavelengths separate closer points.

Typical figures, for comparison only: optical about 200 nm, SEM about 3 to 10 nm, TEM about 0.5 nm.

Must-know distinctions

• Magnification is not resolution. Enlarging a blurred image gives a bigger blur. Detail comes from resolution.
• TEM is not SEM. TEM goes through a slice and shows inside; SEM bounces off a surface and shows 3D outside.
• Pellet is not supernatant. Pellet is solid and is the heavier part at that speed.
• Isotonic is not buffered. Isotonic protects against osmosis; buffered protects against pH change.
• Artefact is not organelle. An artefact is made by the method, an organelle is part of the cell.

Units

From	To	Do this
mm	µm	multiply by 1000
µm	nm	multiply by 1000
µm	mm	divide by 1000
nm	µm	divide by 1000

Write very small sizes in standard form when asked: 3.4 µm = 3.4 × 10⁻⁶ m.

Method box: magnification

1. Measure the image (or scale bar) with a ruler, in mm.
2. Convert the image and the real size into the same unit.
3. Divide: M = I ÷ A.
4. Rearrange for image size or real size if needed. Check the answer is sensible.
5. Give no unit for magnification. Give a unit for sizes.

Worked reminder: a cell is 8 µm long and appears 24 mm long. Image = 24 000 µm. Magnification = 24 000 ÷ 8 = ×3000.

Scale-bar reminder: bar of 20 mm is labelled 5 µm, and a cell is 70 mm across. Real width = 70 ÷ 20 × 5 = 17.5 µm. Magnification = 20 000 ÷ 5 = ×4000.

Method box: calibrating a graticule

1. Line up the graticule and stage micrometer.
2. Find where two lines coincide, then count the divisions on both.
3. Real length of one eyepiece division = (stage divisions × length of one stage division) ÷ eyepiece divisions.
4. Measure the specimen in eyepiece divisions and multiply.
5. Repeat for each objective lens.

Worked reminder: 25 eyepiece divisions match 10 stage divisions of 10 µm. One eyepiece division = 100 ÷ 25 = 4 µm.

Method box: cell fractionation

1. Homogenise the tissue in a blender, in a solution that is cold, isotonic and buffered.
2. Filter to remove cell debris and unbroken cells.
3. Centrifuge at low speed. The heaviest organelles form the pellet.
4. Pour off the supernatant and spin it faster. Repeat at increasing speeds.

Solution property	Reason
Cold	Slows enzymes, so lysosomal enzymes do not digest organelles
Isotonic	No net movement of water, so organelles do not burst or shrink
Buffered	Keeps pH constant, so enzymes do not denature

Order of sedimentation: nuclei, chloroplasts (plants only), mitochondria, lysosomes, endoplasmic reticulum, ribosomes. The big and dense come out first.

Artefacts and organelles

Scientists took a considerable time to decide which structures seen in early microscope images were real organelles and which were artefacts. Evidence that something is real:

• it appears after different preparation methods;
• it is seen with different microscopes;
• other research groups repeat the observation;
• it is linked to a function, for example by testing a fractionated sample.

Common artefacts: folds, scratches and air bubbles on a slide, shrinkage from dehydration, stain deposits, and damage from the electron beam.

Choosing a microscope

You need to see	Best choice	Reason
Moving, living cells in pond water	Optical	Only the optical microscope can examine living material
The 3D surface of a pollen grain or leaf	SEM	Electrons scattered from the surface build a 3D image
Cristae inside a mitochondrion, or ribosomes	TEM	Highest resolution, and it shows internal structure
Starch grains in a potato cell	Optical, with iodine in potassium iodide solution	The grains are large enough to see and stain blue-black

Order of magnitude

Compare two sizes by dividing the larger by the smaller. A ratio near 10 is one order of magnitude, near 100 is two, near 1000 is three. Example: a nucleus of 6 µm in a cell of 60 µm differs by one order of magnitude.

Reading pellet data

If a question gives a table of what each pellet contains or can do, match the property to the organelle:

• DNA present, no chlorophyll or respiration: nuclei.
• Chlorophyll and photosynthesis: chloroplasts.
• Uses oxygen in respiration: mitochondria.
• Contains hydrolytic enzymes: lysosomes.

Then say why: the first pellet formed at the lowest speed, so it holds the largest, densest organelle.

Practical skills linked to this section

• Use low power first, then high power, focusing with the fine focus.
• Ease the coverslip down from one edge with a mounted needle so air bubbles are not trapped.
• Iodine in potassium iodide solution turns starch grains blue-black.
• A scientific drawing uses sharp, single lines, no shading, no colouring, and label lines that do not cross.

Quick self-test

1. An image on a print is 36 mm long. The real object is 12 µm. Calculate the magnification.
2. A cell of length 7.5 µm is viewed at ×400. How long is the image, in mm?
3. A micrograph has magnification ×1500. A structure measures 45 mm on it. Find its real size in µm.
4. 25 eyepiece divisions match 10 stage divisions, each 10 µm. How long is one eyepiece division?
5. Convert 0.0034 mm to µm and to metres in standard form.
6. State two reasons why a TEM cannot be used to study living cells.
7. Why is the homogenising solution kept cold?
8. After centrifuging, which organelle is found in the first pellet from liver tissue?
9. Explain why an electron microscope has a higher resolution than an optical microscope.
10. Suggest why a structure seen after one fixing method but not after another might be an artefact.

Answers

1. 36 mm = 36 000 µm; 36 000 ÷ 12 = ×3000.
2. 7.5 × 400 = 3000 µm = 3 mm.
3. 45 mm = 45 000 µm; 45 000 ÷ 1500 = 30 µm.
4. 10 × 10 = 100 µm; 100 ÷ 25 = 4 µm.
5. 0.0034 × 1000 = 3.4 µm; 3.4 × 10⁻⁶ m.
6. The specimen must be in a vacuum, and it must be in extremely thin sections after fixing, so it is dead.
7. To reduce enzyme activity, so enzymes from lysosomes do not break down the organelles.
8. Nuclei.
9. Electrons have a much shorter wavelength than light, so points that are closer together can be seen as separate.
10. If the structure depends on the preparation method, it is more likely to have been produced by that method than to be part of the living cell.

Where marks are usually lost

• Answers that say “higher magnification” when the question is about seeing more detail, instead of “higher resolution”.
• Dividing without converting units, so the answer is out by a factor of 1000.
• Quoting a calibration from one objective lens for another.
• Writing “to stop osmosis” for the isotonic solution, when the idea is to avoid a net movement of water into or out of the organelles.
• Writing “to stop enzymes working” for the buffer, when it keeps the pH constant so enzymes are not denatured.
• Mixing up TEM and SEM on 2D versus 3D.
• Giving a magnification with a unit, or a size without one.
• Treating every unfamiliar structure in an image as an organelle, rather than considering preparation artefacts.

Official syllabus

AQA AS and A-level Biology specification (7401 and 7402), for first teaching 2015, exams June 2016 onwards, Version 1.5, published by AQA. Section 3.2.1.3 Methods of studying cells.