What is the primary purpose of using an isotonic solution during cell fractionation?

An isotonic solution has the same water potential as the organelles, preventing the net movement of water by osmosis. This prevents the organelles from bursting or shrinking, which would alter their structure and function.

Why must the tissue be kept in a cold solution during the homogenisation process?

Cold temperatures reduce the kinetic energy and activity of enzymes released from the cells. This prevents these enzymes (such as proteases) from breaking down or damaging the organelles that are being isolated.

What is the difference between the 'homogenate' and the 'filtrate'?

The homogenate is the raw mixture of broken cells produced by the blender. The filtrate is the liquid obtained after passing the homogenate through a gauze to remove large debris and unbroken cells.

In what order are the nucleus, mitochondria, and ribosomes isolated during ultracentrifugation?

They are isolated from heaviest to lightest: first the Nucleus (lowest speed), then the Mitochondria (medium speed), and finally the Ribosomes (highest speed).

What is the 'supernatant' in the context of centrifugation?

The supernatant is the liquid layer that remains above the solid pellet after a mixture has been centrifuged. It contains the lighter components that did not sediment at that specific speed.

What error occurs if the centrifuge speed is not increased between cycles?

If the speed is not increased, no new organelles will sediment into a pellet. The centrifugal force must be higher in each subsequent step to pull down the lighter, less dense organelles remaining in the supernatant.

Why is a buffer solution added to the tissue before processing?

A buffer maintains a constant pH, preventing fluctuations that could denature proteins or enzymes within the organelles. This ensures that the biological activity of the isolated components remains intact.

How does the pellet differ from the supernatant?

The pellet is the solid sediment of denser organelles at the bottom of the tube, while the supernatant is the liquid above it containing lighter, unsedimented structures.

What is a common mistake when describing the isolation of chloroplasts?

A common mistake is forgetting that chloroplasts are only found in plant cells. In animal cell fractionation, the step for chloroplast isolation is skipped, moving directly from nuclei to mitochondria.

When should you use a higher speed vs. a lower speed in a centrifuge?

Use a lower speed to isolate large, heavy organelles like the nucleus. Use a higher speed to isolate smaller, lighter organelles like ribosomes that require more force to sediment.

Cell Fractionation & Ultracentrifugation | AQA A-Level Biology

1. Definition & Core Concepts

Cell Fractionation is the process of separating different parts of a cell, specifically organelles, to allow for detailed study of their individual functions and structures. This is essential because many organelles are too small or complex to be analyzed effectively while still contained within the intact cell.
The process relies on the physical properties of organelles, primarily their mass and density, which determine how they behave when subjected to external forces. By isolating these components, scientists can perform biochemical assays or observe them using high-resolution electron microscopy.
The procedure is typically divided into three distinct phases: homogenisation (breaking the cells), filtration (removing debris), and ultracentrifugation (separating the organelles by speed).

2. Critical Preparation Conditions

Before fractionation begins, the tissue must be placed in a specific solution that is cold, isotonic, and buffered to preserve the integrity of the organelles. Each of these three conditions serves a vital protective role during the harsh mechanical process of cell disruption.
Cold temperatures are maintained to reduce the kinetic energy of enzymes, specifically lysosomal enzymes that might be released during homogenisation. This prevents these enzymes from digesting and damaging the organelles intended for study.
An isotonic solution ensures that the water potential of the fluid matches that of the organelles. This prevents osmotic lysis (bursting) or crenation (shriveling) of membrane-bound structures like mitochondria or chloroplasts due to the net movement of water.
A buffered solution is used to maintain a constant pH throughout the process. Fluctuations in pH can alter the tertiary structure of proteins and enzymes within the organelles, leading to denaturation and loss of biological activity.

3. Homogenisation & Filtration

Homogenisation is the mechanical breaking of cells using a device called a homogeniser or blender. This process disrupts the plasma membrane and cell walls, releasing the internal contents into the surrounding solution to create a mixture known as the homogenate.
Following homogenisation, the mixture is passed through a gauze or fine mesh in a step called filtration. This removes large, unfragmented debris such as intact cells, large pieces of cell wall, or connective tissue that would interfere with the subsequent separation steps.
The resulting liquid that passes through the filter is called the filtrate. This filtrate contains a suspension of all the individual organelles (nuclei, mitochondria, ribosomes, etc.) ready for the final separation phase.

Diagram of a centrifuge tube showing the separation of the liquid supernatant from the solid pellet at the bottom.

4. Ultracentrifugation Methodology

Ultracentrifugation involves spinning the filtrate at very high speeds in a centrifuge. The centrifugal force generated causes the components of the mixture to move toward the bottom of the tube at different rates based on their mass.
The heaviest and densest organelles are forced to the bottom first, forming a solid mass called the pellet. The remaining liquid above the pellet, which still contains lighter organelles, is known as the supernatant.
To isolate lighter organelles, the supernatant is carefully removed and transferred to a new tube to be spun again at a higher speed for a longer duration. This process of differential centrifugation is repeated until the desired organelle is isolated.

5. Order of Organelle Isolation

Organelles are separated in a specific sequence from heaviest to lightest. The nucleus is the largest and densest organelle, so it is always the first to form a pellet at the lowest centrifugal speeds.
In plant cells, chloroplasts are the next to be isolated, followed by mitochondria in both plant and animal cells. These require medium speeds to be pulled out of the suspension.
The lightest components, such as the endoplasmic reticulum, lysosomes, and finally ribosomes, require the highest speeds and longest spinning times to form a pellet. Ribosomes are the smallest and least dense, making them the final fraction in the sequence.

6. Key Distinctions

It is vital to distinguish between the homogenate (the raw mixture of broken cells) and the filtrate (the mixture after debris has been removed). Only the filtrate is used for ultracentrifugation to ensure purity.

Feature	Pellet	Supernatant
State	Solid/Sediment at bottom	Liquid above the sediment
Content	Heaviest organelles from that spin	Lighter organelles still in suspension
Next Step	Collected for study	Re-spun at higher speed

Another critical distinction is the relationship between speed and mass: lower speeds isolate high-mass structures (nuclei), while higher speeds are required to overcome the buoyancy of low-mass structures (ribosomes).

7. Exam Strategy & Tips

The 'Why' behind the 'What': Exams frequently ask for the justification of the cold, isotonic, and buffered conditions. Always link 'cold' to enzyme activity, 'isotonic' to osmosis/organelle damage, and 'buffered' to protein denaturation.
Sequence Memorization: Use a mnemonic to remember the order of isolation (Nuclei → Chloroplasts → Mitochondria → Lysosomes → Ribosomes). A common error is swapping mitochondria and lysosomes.
Terminology Precision: Ensure you use the terms 'pellet' and 'supernatant' correctly. If a question asks how to get mitochondria after the nuclei have been removed, the answer must involve spinning the supernatant at a higher speed.
Sanity Check: If a question describes spinning at a very high speed first, recognize this as a mistake in the procedure, as it would clump all organelles together into a single, inseparable pellet.

Cell Fractionation & Ultracentrifugation

Summary

1. Definition & Core Concepts

2. Critical Preparation Conditions

3. Homogenisation & Filtration

4. Ultracentrifugation Methodology

5. Order of Organelle Isolation

6. Key Distinctions

7. Exam Strategy & Tips

Cell Fractionation & Ultracentrifugation

Summary

1. Definition & Core Concepts

2. Critical Preparation Conditions

3. Homogenisation & Filtration

4. Ultracentrifugation Methodology

5. Order of Organelle Isolation

6. Key Distinctions

7. Exam Strategy & Tips