A-Level Biology: Cell Fractionation and Ultracentrifugation

What is cell fractionation?

A process where cells are broken up and organelles are separated based on size/mass using gravity.

What are the three main stages of cell fractionation?

Homogenisation, filtration, and ultracentrifugation.

Why is tissue homogenised before cell fractionation?

To break open cell membranes and release organelles.

What is the purpose of using a cold solution in cell fractionation?

To reduce enzyme activity that may break down organelles.

Why must the solution used in cell fractionation be isotonic?

To prevent organelles from bursting or shrinking due to osmotic gain/loss of water.

What does a buffered solution do in cell fractionation?

Maintains a constant pH to protect proteins and enzymes.

What is the first step in the centrifugation process?

Centrifuging at a low speed to allow larger fragments to form a pellet.

What is the supernatant in the context of centrifugation?

The liquid remaining above the pellet after centrifugation.

What happens to the supernatant during subsequent centrifugation?

It is re-spun at a higher speed to collect smaller organelles forming new pellets.

What organelles are typically found in the first pellet after low-speed centrifugation?

The heaviest organelles, such as the cell membrane, nucleus, and vacuoles.

What is the significance of using higher speeds in centrifugation?

To isolate smaller organelles by increasing the force applied.

What is the role of a blender in the cell fractionation process?

To homogenise the tissue by breaking down cell membranes.

Why is filtration necessary after homogenisation?

To remove large debris that would interfere with the separation of organelles.

What does the term 'homogenate' refer to?

The mixture obtained after homogenisation that contains broken cells and released organelles.

What is the purpose of using an ice-cold buffer in cell fractionation?

To prevent autolysis and preserve organelle integrity.

How does an isotonic solution affect organelles during cell fractionation?

It prevents osmotic pressure changes that could damage the organelles.

What is the final goal of cell fractionation?

To isolate specific organelles for study.

What is the process of differential centrifugation?

A method to separate organelles based on their size and density using varying centrifuge speeds.

What happens to organelles at higher centrifuge speeds?

Smaller organelles are collected in a pellet as the force increases.

What is the importance of maintaining a constant pH during cell fractionation?

To ensure that proteins and enzymes remain functional and are not denatured.

What type of organelles might be isolated after centrifugation at high speeds?

Smaller organelles such as chloroplasts and endoplasmic reticulum.

What does the term 'pellet' refer to in centrifugation?

The solid mass of organelles that collects at the bottom of the centrifuge tube.

How does temperature affect enzyme activity during cell fractionation?

Lower temperatures reduce enzyme activity, preventing degradation of organelles.

What is the significance of using buffered solutions in biological experiments?

They help maintain a stable pH, which is crucial for enzyme and protein function.

What is the role of the supernatant in the isolation of organelles?

It contains smaller organelles that remain suspended after the larger ones have settled.

What is the expected outcome of the first centrifugation step?

The heaviest organelles will form a pellet at the bottom of the tube.

Why is a cold solution used in cell fractionation?

To reduce enzyme activity that may break down cells.

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

To prevent organelles from shrinking or swelling due to osmosis.

What is the role of a buffer solution in cell fractionation?

To maintain a stable pH, as changes in pH can alter the structure of organelles.

What does homogenisation involve?

Breaking up the tissue to release the organelles.

What is the purpose of filtration in cell fractionation?

To remove large debris from the homogenate.

How does centrifugation work in cell fractionation?

It uses gravity to separate substances by their weight, with larger fragments collecting at the bottom of the tube.

What happens during the first centrifuge spin?

Nuclei are separated and form a pellet at the bottom of the tube.

At what speed are mitochondria separated during centrifugation?

3,500 revolutions per minute (revs min-1).

What is the lightest organelle that can be separated during centrifugation?

Ribosomes.

Why must the homogenate be filtered?

To remove any large cells or large pieces of cell membrane.

What is the heaviest organelle that can be separated during centrifugation?

Nucleus.

What is the purpose of increasing the speed of centrifugation?

To separate smaller organelles from larger ones by increasing the G force.

What is the supernatant in centrifugation?

The liquid remaining above the pellet after centrifugation.

What is the significance of using an ice-cold, isotonic, buffered solution in cell fractionation?

It slows enzyme activity, prevents damage to organelles, and controls pH.

What is the process of homogenisation?

The breaking of a cell in order to separate different organelles in a homogeniser.

What is the role of the cell wall in bacteria?

It acts as a physical barrier to some substances, protecting against mechanical damage and osmotic lysis.

What is the function of the bacterial capsule?

It protects against other cells and allows bacteria to stick together for protection.

What is the role of the cell-surface membrane in bacteria?

It is differentially permeable and controls the entry and exit of chemicals.

What does circular DNA do in bacteria?

It contains the genetic information necessary for the replication of bacteria.

What is the purpose of centrifugation in cell fractionation?

To separate organelles based on their size and density.

What is the term for the blended material after homogenisation?

Homogenate.

What happens to organelles during centrifugation?

They fall to the bottom of the tube in order of their weight, forming a pellet.

What is the importance of studying cell components through fractionation?

It allows for the examination of the structure and function of individual organelles.