Homogenisation: Cells are broken up using a homogeniser (essentially a high-speed blender). This physical disruption breaks the plasma membrane, releasing the organelles into the surrounding liquid to form a mixture called the homogenate.
Filtration: The homogenate is passed through a fine gauze or filter. This step is crucial for removing large, unwanted debris such as unbroken cells, connective tissue, or cell wall fragments.
The resulting liquid, which contains the suspended organelles, is known as the filtrate.
Ultracentrifugation involves spinning the filtrate in a centrifuge at very high speeds. The centrifugal force causes the densest components to move to the bottom of the tube.
After the first spin at a low speed, the heaviest organelles form a solid mass at the bottom called the pellet. The remaining liquid above it, containing lighter organelles, is the supernatant.
The supernatant is carefully poured off into a new tube and spun again at a higher speed to isolate the next densest organelle. This cycle repeats until all desired components are separated.
The order of sedimentation from heaviest (first pellet) to lightest (last pellet) is generally: Nuclei Chloroplasts (in plants) Mitochondria Lysosomes Endoplasmic Reticulum Ribosomes.
| Term | Definition | Role in Process |
|---|---|---|
| Homogenate | The raw mixture of broken cells. | The starting material before filtration. |
| Filtrate | The liquid passed through a gauze. | Contains organelles but lacks large debris. |
| Supernatant | The liquid above the pellet after spinning. | Contains the remaining lighter organelles. |
| Pellet | The solid sediment at the bottom. | Contains the isolated, heavier organelles. |
The 'CIB' Checklist: Always check if a question mentions the conditions. If asked why a solution is cold, the answer is always 'to reduce enzyme activity'. If asked why it is isotonic, the answer is 'to prevent osmotic damage'.
The Sedimentation Order: Memorize the sequence: Nuclei Chloroplasts Mitochondria Lysosomes Ribosomes. A common exam trick is to ask which organelle would be in the second pellet of a plant cell (Chloroplasts) versus an animal cell (Mitochondria).
Verification: If a question asks how to isolate a specific organelle, ensure you describe the removal of the previous pellets first. You cannot get a pure sample of mitochondria without first removing the nuclei at a lower speed.
