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To study organelles, scientists must isolate them from cells (cell fractionation) and visualise them using microscopy. This lesson covers the techniques of cell fractionation, the principles of light and electron microscopy, and the key calculations you need to master for AQA A-Level Biology (specification 3.2.1).
Cell fractionation is the process of separating the different organelles of a cell so that they can be studied individually. It involves two main stages: homogenisation and differential centrifugation.
The tissue is placed in a cold, isotonic, buffered solution and broken up using a blender or homogeniser (e.g., a Potter–Elvehjem homogeniser) to produce a homogenate — a suspension of disrupted cells and their contents.
The solution must be:
| Condition | Reason |
|---|---|
| Cold (ice-cold, around 4 °C) | Reduces the activity of enzymes (especially hydrolytic enzymes from lysosomes) that could digest the organelles |
| Isotonic (same water potential as the cell contents) | Prevents organelles from swelling and bursting (in a hypotonic solution) or shrinking (in a hypertonic solution) by osmosis |
| Buffered (maintained at a constant pH, typically around pH 7.4) | Prevents changes in pH that could denature enzymes or alter the structure of organelles |
The homogenate is then filtered through gauze or cheesecloth to remove large debris such as connective tissue fibres and intact cells.
The filtered homogenate is placed in a centrifuge tube and spun at progressively increasing speeds.
Key Definition: Differential centrifugation is a technique that separates organelles based on their size and density. The heaviest and densest organelles sediment first at low centrifugal forces; lighter organelles require higher forces.
Exam Tip: Remember the order of sedimentation: Nuclei → Mitochondria → Lysosomes/ER → Ribosomes. A useful mnemonic: Naughty Mice Love Running.
For greater resolution, a density gradient (e.g., of sucrose solution, with the densest solution at the bottom) can be used. The sample is layered on top and centrifuged at very high speeds. Organelles migrate through the gradient until they reach the point where their density equals that of the surrounding solution (the isopycnic point), forming distinct bands. This allows separation of organelles with similar sizes but different densities.
Two key concepts underpin all microscopy:
Key Definition: Resolution (resolving power) is the ability to distinguish between two closely spaced objects as separate entities. It depends on the wavelength of the radiation used — shorter wavelengths provide better resolution.
The formula triangle to remember is:
I = A × M
Where:
Rearranged:
Exam Tip: Always ensure that units are consistent before calculating. Convert all measurements to the same unit (µm is usually most convenient). Remember: 1 mm = 1000 µm; 1 µm = 1000 nm.
A mitochondrion has an actual length of 2 µm. On an electron micrograph it appears as 50 mm long.
Magnification = image size ÷ actual size = 50 mm ÷ 0.002 mm = ×25 000
(Note: 2 µm = 0.002 mm, so the calculation uses consistent units.)
Electron micrographs often include a scale bar rather than stating magnification directly. To use a scale bar:
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