Cell Fractionation and Ultracentrifugation
How organelles are separated from tissue: homogenisation, filtration, then ultracentrifugation in isotonic, cold, buffered solution, with pellets forming in order of density. Includes the 5-mark exam question, a worked model answer and the examiner trap on which organelles pellet first.
Chapters (8)
Exam question [5 marks]
Describe how organelles can be separated from a sample of liver tissue by cell fractionation. Explain why the solution used must be isotonic, cold and buffered.
Try it on paper first, then check the mark scheme below. The question is original and was written for this lesson.
Mark scheme
| Mark | Mark point | Also accept | Do not accept |
|---|---|---|---|
| 1 | Homogenise (blend / break up) the tissue to break open cells and release organelles; filter to remove debris / unbroken cells (either homogenising or filtering earns the mark) | "grind", "blend"; "remove large pieces" | "cut the cells" with no release of organelles |
| 2 | Isotonic solution, so no net osmosis / water potential same, so organelles do not burst or shrink | "same water potential", "prevents osmotic damage" | "isotonic" alone with no reason |
| 3 | Cold, to reduce enzyme activity so organelles are not digested / damaged | "slows enzymes", "prevents autolysis" | "cold to stop enzymes working" with no link to damage; "kills enzymes" |
| 4 | Buffered, to keep pH constant so enzymes / proteins are not denatured | "maintains pH" | "buffer stops acid" with no consequence |
| 5 | Centrifuge at low speed, heaviest organelles (nuclei) form a pellet; remove supernatant and spin at higher speed, repeating to separate the next organelles (low-speed pellet of heaviest organelles OR repeat at higher speed earns the mark; both not required) | "denser organelles first"; "mitochondria next" | "lightest organelles first"; "one spin separates all organelles" |
The question asks to explain isotonic, cold and buffered, so marks 2, 3 and 4 each need their reason. Order by density, heaviest first, speed increasing each spin. Further credit-worthy detail that does not add marks beyond 5: the later pellets are mitochondria, lysosomes, then endoplasmic reticulum and ribosomes.
Model answer (5/5)
The tissue is homogenised in a blender to break open the cells and release the organelles, then filtered to remove debris and unbroken cells. The solution is isotonic, so there is no net osmosis and the organelles do not burst or shrink. It is cold, which reduces enzyme activity so the organelles are not digested. It is buffered, which keeps the pH constant so enzymes are not denatured. The filtrate is spun in a centrifuge at low speed so the heaviest organelles, the nuclei, form a pellet. The supernatant is removed and spun at a higher speed, and this is repeated, so the next organelles, such as mitochondria, pellet in turn.
Examiner trap
- Forgetting the solution must be isotonic, cold and buffered and why
- Saying the heaviest organelles are in the supernatant first
- Not giving the order of organelle separation
Key terms
- homogenisation
- filtration
- ultracentrifugation
- isotonic
- cold
- buffered
- supernatant
- pellet
- artefact
- density
Lesson notes
A written version of the video, chapter by chapter, for revision. Times link to the video.
0:21Homogenisation and filtration
First, the aim. We want to pull a cell apart and sort its organelles, without damaging them. Step one is homogenisation. The tissue is blended, which breaks open the cells and releases the organelles. Step two is filtration. The mixture is filtered to remove large debris and any unbroken cells. What passes through is called the homogenate, and it contains all the organelles, mixed together.
0:49Isotonic, cold, buffered
But the solution you blend in matters hugely. It must be isotonic, cold and buffered. Isotonic means the same water potential as the cells. So there is no net movement of water by osmosis, and the organelles do not burst or shrivel. Cold, because breaking open cells releases enzymes, and they could digest the organelles. Low temperature reduces enzyme activity.
Buffered, because a buffer keeps the pH constant, so enzymes and other proteins are not denatured.
Isotonic, cold, buffered. Each one needs its reason.
1:28Ultracentrifugation
Now ultracentrifugation. The homogenate goes into a tube and is spun in a centrifuge. The spinning force pushes the heaviest, most dense organelles to the bottom. They form a pellet. The liquid left above is the supernatant. The pellet is one fraction. The supernatant still holds all the lighter organelles.
1:50Spinning faster: the order
So we repeat the spin. Pour the supernatant into a new tube and spin it faster. At the lowest speed, the heaviest organelles pellet first. These are the nuclei. Pour off the supernatant, spin faster, and the next pellet is mitochondria. Faster again, lysosomes. And at the highest speeds, the lightest, endoplasmic reticulum and ribosomes.
The order is by density, heaviest first, and each time the speed goes up.
2:20Artefacts versus organelles
One more idea from the specification. For a long time, scientists argued. Were the small structures seen under the microscope real organelles, or artefacts? An artefact is something produced by the preparation, such as damage from staining or fixing, and not part of the living cell. Fractionation helped, because organelles that could be isolated and shown to do a job were real.
3:24Examiner trap and recap
The trap. The heaviest organelles do not stay in the supernatant. They pellet first. Recap. Break it, filter it, keep it isotonic, cold and buffered, then spin faster and faster, densest first.
Next, a different question. How does a cell divide? The cell cycle and mitosis.
Watch first
This lesson builds on:
- Optical, TEM and SEM Microscopes Compared · AQA 7402 · 3.2.1.3
Keep going
More from 3.2 Cells
- Nucleus, Mitochondria and Chloroplasts · AQA 7402 · 3.2.1.1
- Rough ER, Golgi and Lysosomes Explained · AQA 7402 · 3.2.1.1
- Cell Wall, Vacuole and Specialised Cells · AQA 7402 · 3.2.1.1
- Prokaryotic vs Eukaryotic Cells · AQA 7402 · 3.2.1.2
Independent revision resource, not affiliated with or endorsed by AQA. The exam question is original and was written for this lesson; it is not an AQA question. Spotted an error? Email info@medservatory.com with the lesson code AB-029.