1.09 Practicals: Organelles, Cells and Viruses

How Does a Centrifuge Work?

Role of Density

• Density is the mass per unit volume of a substance.
• In a mixture, components with different densities will separate when spun in a centrifuge.
  • Heavier (denser) particles move outward to form a pellet at the bottom of the tube.
  • Lighter (less dense) substances remain in the supernatant (the liquid above the pellet).

Centrifugal Force

• The spinning creates centrifugal force, which enhances the natural settling due to gravity.
• Higher speed and longer time increase the separation based on density differences.

What Does a Centrifuge Do?

• Separates Mixtures: Divides liquids and solids by spinning them rapidly.
• Isolates Components: Obtains pure substances like cells, proteins, or DNA from complex mixtures.
• Concentrates Particles: Gathers small, dense particles into a pellet for easier analysis.

How to Use a Centrifuge

1. Prepare Samples:
   • Place your sample in centrifuge tubes.
   • Ensure all tubes have the same volume and are balanced in weight.
2. Load the Centrifuge:
   • Place tubes opposite each other in the rotor to maintain balance.
3. Set Parameters:
   • Choose the appropriate speed (RPM) and time based on your protocol.
4. Start the Centrifuge:
   • Close the lid securely and start the machine.
   • Wait until the centrifuge stops completely before opening.
5. Retrieve Samples:
   • Carefully remove the tubes to avoid mixing the separated layers.

What Can You Use a Centrifuge For?

1. Blood Separation

• Purpose: To separate blood into its different components: plasma, red blood cells (RBCs), white blood cells (WBCs), and platelets.
• How It Helps:
  • Plasma (the liquid part) is less dense and remains in the supernatant.
  • Red Blood Cells are denser and form a pellet at the bottom.
  • Layering: After centrifugation, distinct layers can be easily extracted for medical tests or transfusions.

2. Cellular Fractionation

• Purpose: To separate different parts of cells, such as nuclei and mitochondria, for study.
• How It Helps:
  • Nuclei are larger and denser, forming a pellet first.
  • Mitochondria and other organelles pellet at higher speeds.
  • Sequential Centrifugation: By increasing speed and time, specific organelles can be isolated from the cell mixture.

3. DNA/RNA Extraction

• Purpose: To purify genetic material from cells for genetic testing and research.
• How It Helps:
  • Cell Debris and Proteins are denser and form a pellet.
  • DNA/RNA remains in the supernatant or can be pelleted separately under specific conditions.
  • Purification: This separation allows for the collection of clean genetic material for further experiments.

4. Protein Purification

• Purpose: To isolate specific proteins for biochemical experiments.
• How It Helps:
  • Cellular Components are separated based on density.
  • Proteins can be concentrated in the pellet or remain in the supernatant, depending on their properties.
  • Isolation: This allows researchers to obtain pure proteins for studying their functions and interactions.

5. Virus Isolation

• Purpose: To concentrate viruses from samples for research and vaccine development.
• How It Helps:
  • Viruses are small but can be pelleted using ultracentrifugation.
  • Concentration: This increases the number of viral particles in a sample, making them easier to study or use in vaccine production.

Key Points to Remember

• Balance is Crucial: Always balance the tubes to prevent damage and ensure safety.
• Choose the Right Settings: Use the correct speed and time for your specific experiment.
• Handle with Care: After centrifugation, handle samples gently to keep the separated layers intact.
• Understand Density: Knowing the densities of your sample components helps predict and achieve effective separation.

Safety Tips

• Check for Damage: Inspect tubes and the centrifuge for any signs of wear before use.
• Wear Protective Gear: Use gloves and safety glasses to protect against spills.
• Follow Instructions: Always adhere to the manufacturer’s guidelines and lab protocols.

Question 1

Define density and explain its role in the separation of mixtures using a centrifuge. (5 marks)

Mark Scheme:

1. Definition of Density:
   • Density is the mass per unit volume of a substance. (1 mark)
2. Role in Separation:
   • In a mixture, components with different densities will separate when spun in a centrifuge. (1 mark)
3. Heavier Particles:
   • Heavier (denser) particles move outward to form a pellet at the bottom of the tube. (1 mark)
4. Lighter Substances:
   • Lighter (less dense) substances remain in the supernatant (the liquid above the pellet). (1 mark)
5. Centrifuge Function:
   • The centrifuge exploits density differences to effectively separate components based on their mass-to-volume ratios. (1 mark)

Question 2

Explain how centrifugal force enhances the natural settling of particles in a centrifuge. (5 marks)

Mark Scheme:

1. Centrifugal Force Creation:
   • The spinning motion of the centrifuge generates centrifugal force, pushing particles outward. (1 mark)
2. Enhancement of Settling:
   • Centrifugal force increases the rate at which heavier particles settle compared to natural gravity-driven settling. (1 mark)
3. Separation Efficiency:
   • Higher speed and longer centrifugation time enhance the separation based on density differences. (1 mark)
4. Pellet Formation:
   • Heavier particles are rapidly moved to the bottom, forming a pellet, while lighter substances remain in the supernatant. (1 mark)
5. Overcoming Weak Forces:
   • Centrifugal force overcomes weaker intermolecular forces, ensuring a clear separation of components. (1 mark)

Question 3

List and describe three common uses of a centrifuge in biological laboratories. (6 marks)

Mark Scheme:

1. Blood Separation:
   • Purpose: To separate blood into its different components: plasma, red blood cells (RBCs), white blood cells (WBCs), and platelets.
   • Function: Plasma remains in the supernatant, while RBCs form a pellet. (2 marks)
2. DNA/RNA Extraction:
   • Purpose: To purify genetic material from cells for genetic testing and research.
   • Function: Cell debris and proteins form a pellet, allowing DNA/RNA to remain in the supernatant or be pelleted separately. (2 marks)
3. Protein Purification:
   • Purpose: To isolate specific proteins for biochemical experiments.
   • Function: Proteins are either concentrated in the pellet or remain in the supernatant, depending on their properties. (2 marks)

Question 4

Describe the steps involved in using a centrifuge to separate a mixture. (6 marks)

Mark Scheme:

1. Prepare Samples:
   • Place the sample in centrifuge tubes and ensure all tubes have the same volume and are balanced in weight. (1 mark)
2. Load the Centrifuge:
   • Arrange the tubes opposite each other in the rotor to maintain balance. (1 mark)
3. Set Parameters:
   • Select the appropriate speed (RPM) and time based on the protocol or experiment requirements. (1 mark)
4. Start the Centrifuge:
   • Close the lid securely and start the machine, allowing it to spin for the set duration. (1 mark)
5. Wait for Completion:
   • Allow the centrifuge to complete the spin and stop completely before opening the lid to ensure safety. (1 mark)
6. Retrieve Samples:
   • Carefully remove the tubes, avoiding mixing the separated layers of pellet and supernatant. (1 mark)

Question 5

Explain how a centrifuge can be used to concentrate particles in a sample. (5 marks)

Mark Scheme:

1. Spinning the Sample:
   • The centrifuge spins the sample rapidly, creating centrifugal force. (1 mark)
2. Movement of Particles:
   • Dense particles are forced outward and settle at the bottom of the tube as a pellet. (1 mark)
3. Separation of Supernatant:
   • The supernatant (liquid) remains above the pellet, separating from the concentrated particles. (1 mark)
4. Concentration Effect:
   • By removing the supernatant, the pellet containing the concentrated particles can be isolated for further analysis or use. (1 mark)
5. Enhanced Analysis:
   • Concentrated particles are easier to analyze or manipulate in subsequent experiments due to their increased density and volume. (1 mark)

Question 6

Compare the processes of centrifugation in blood separation and DNA extraction. (6 marks)

Mark Scheme:

1. Purpose of Centrifugation:
   • Blood Separation: To separate blood into plasma, RBCs, WBCs, and platelets.
   • DNA Extraction: To purify DNA from cells by removing cell debris and proteins. (1 mark)
2. Sample Preparation:
   • Blood Separation: Blood is placed in centrifuge tubes and spun to separate components based on density.
   • DNA Extraction: Cell lysate containing DNA, proteins, and other cellular components is centrifuged. (1 mark)
3. Centrifugation Speed and Time:
   • Blood Separation: Typically uses a moderate speed and time to form distinct layers.
   • DNA Extraction: May require different speed and time settings to pellet specific components like cell debris. (1 mark)
4. Outcome of Centrifugation:
   • Blood Separation: Plasma remains as the supernatant, while RBCs form a pellet.
   • DNA Extraction: Cell debris and proteins form a pellet, allowing DNA to remain in the supernatant or be pelleted under specific conditions. (1 mark)
5. Post-Centrifugation Handling:
   • Blood Separation: Layers are carefully extracted for medical tests or transfusions.
   • DNA Extraction: DNA is either in the supernatant or pelleted for further purification. (1 mark)
6. Final Utilization:
   • Blood Separation: Extracted plasma and cells are used for diagnostics or transfusions.
   • DNA Extraction: Purified DNA is used for genetic testing and research. (1 mark)

Question 7

Explain the importance of balancing centrifuge tubes before spinning and the potential consequences of imbalance. (5 marks)

Mark Scheme:

1. Ensuring Stability:
   • Balancing centrifuge tubes ensures that the rotor remains stable during spinning, preventing vibrations. (1 mark)
2. Preventing Damage:
   • An imbalance can cause mechanical stress on the centrifuge, leading to damage of the rotor or machine components. (1 mark)
3. Safety Risks:
   • Imbalanced tubes can result in excessive vibrations or spinning out of control, posing safety hazards to the user. (1 mark)
4. Effective Separation:
   • Properly balanced tubes ensure that centrifugal force is evenly applied, allowing for effective and consistent separation of sample components. (1 mark)
5. Longevity of Equipment:
   • Maintaining balance reduces wear and tear, extending the lifespan of the centrifuge and its parts. (1 mark)

Question 8

Describe how a centrifuge is used in cellular fractionation and the benefits of this process. (6 marks)

Mark Scheme:

1. Purpose of Cellular Fractionation:
   • To separate different parts of a cell, such as nuclei, mitochondria, and other organelles, for detailed study. (1 mark)
2. Initial Centrifugation:
   • Cells are lysed to release organelles and then spun at low speeds to pellet large components like nuclei. (1 mark)
3. Subsequent Centrifugation:
   • The supernatant is centrifuged at higher speeds to pellet smaller organelles like mitochondria and lysosomes. (1 mark)
4. Sequential Separation:
   • By increasing speed and time in sequential steps, specific organelles can be isolated from the cell mixture. (1 mark)
5. Benefits of the Process:
   • Allows researchers to study individual organelles in detail, understand their functions, and perform biochemical analyses. (1 mark)
6. Enhanced Purity:
   • Achieves high purity of isolated organelles, minimizing contamination from other cellular components. (1 mark)

Question 9

Explain the role of centrifugal force in concentrating viral particles for vaccine development. (5 marks)

Mark Scheme:

1. Centrifugation Process:
   • Viral samples are spun at ultrahigh speeds in a centrifuge to concentrate viral particles. (1 mark)
2. Separation Based on Density:
   • Viral particles are small but dense enough to be pelleted using high centrifugal force. (1 mark)
3. Concentration Effect:
   • Pelleting increases the number of viral particles in a smaller volume, making them easier to study or use in vaccine production. (1 mark)
4. Enhanced Yield:
   • Concentrated viruses ensure a higher yield for effective vaccine formulation, improving immunogenicity. (1 mark)
5. Facilitation of Downstream Processes:
   • Concentration allows for efficient extraction, purification, and formulation of viral components necessary for vaccine development. (1 mark)

Question 10

Discuss the safety precautions that must be taken when using a centrifuge in the laboratory. (5 marks)

Mark Scheme:

1. Check for Damage:
   • Inspect centrifuge tubes and the machine for any signs of wear or damage before use. (1 mark)
2. Wear Protective Gear:
   • Use gloves and safety glasses to protect against potential spills or breakage. (1 mark)
3. Balance the Tubes:
   • Ensure that all tubes are balanced in weight to prevent imbalance and damage. (1 mark)
4. Follow Manufacturer’s Guidelines:
   • Adhere to the centrifuge’s instructions and lab protocols for safe operation. (1 mark)
5. Secure the Lid:
   • Always close the centrifuge lid securely before starting to prevent spillage and ensure safe operation. (1 mark)

Question 11

A researcher wants to isolate mitochondria from plant cells. Describe the centrifugation steps they should follow and explain why each step is necessary. (6 marks)

Mark Scheme:

1. Cell Lysis:
   • Break open plant cells to release organelles, including mitochondria. (1 mark)
2. Low-Speed Centrifugation:
   • Spin the lysate at a low speed to pellet large components like nuclei. (1 mark)
3. Discard Pellet and Supernatant:
   • Remove the pellet containing nuclei and retain the supernatant containing smaller organelles. (1 mark)
4. Higher-Speed Centrifugation:
   • Spin the supernatant at a higher speed to pellet mitochondria and other medium-sized organelles. (1 mark)
5. Isolation of Mitochondria:
   • Carefully collect the mitochondria pellet, which is now separated from other organelles. (1 mark)
6. Purity Verification:
   • Optionally, perform a further centrifugation or use marker enzymes to confirm the purity of the isolated mitochondria. (1 mark)

Question 12

Explain why centrifuges are essential tools in molecular biology laboratories. Provide two specific examples of their applications. (6 marks)

Mark Scheme:

1. Essential for Separation:
   • Centrifuges are used to separate components of a mixture based on density differences, which is crucial for various molecular biology techniques. (1 mark)
2. Blood Separation Example:
   • Application: Separating plasma from red blood cells for medical diagnostics and transfusions.
   • Importance: Allows for the analysis of blood components and preparation for transfusions. (2 marks)
3. DNA/RNA Extraction Example:
   • Application: Purifying DNA or RNA by separating it from cell debris and proteins.
   • Importance: Provides clean genetic material for genetic testing and research. (2 marks)
4. Protein Purification Example:
   • Application: Isolating specific proteins from a complex mixture for biochemical studies.
   • Importance: Enables detailed study of protein functions and interactions. (1 mark)
5. Virus Isolation Example:
   • Application: Concentrating viral particles for vaccine development or research.
   • Importance: Facilitates the study and production of vaccines by increasing the viral load. (Optional for additional detail)

Question 13

A mixture contains two types of particles: Particle A with a density of 1.2 g/cm³ and Particle B with a density of 0.8 g/cm³. After centrifugation, where will each particle be located, and why? (5 marks)

Mark Scheme:

1. Particle A (Denser):
   • Location: Moves outward to form a pellet at the bottom of the centrifuge tube. (1 mark)
2. Particle B (Less Dense):
   • Location: Remains in the supernatant above the pellet. (1 mark)
3. Reason for Separation:
   • Density Differences: Centrifugal force causes heavier particles (A) to move outward and settle, while lighter particles (B) stay in the liquid. (1 mark)
4. Centrifugal Force Effect:
   • The centrifugal force enhances the settling of denser particles beyond what gravity alone would achieve. (1 mark)
5. Outcome:
   • The mixture is separated based on density, allowing for the isolation of Particle A from Particle B. (1 mark)

Answer:
Particle A (1.2 g/cm³) will move outward and form a pellet at the bottom of the tube due to its higher density, while Particle B (0.8 g/cm³) will remain in the supernatant above the pellet.

Question 14

Why is it important to handle centrifuged samples carefully after spinning? (5 marks)

Mark Scheme:

1. Prevent Mixing:
   • Care must be taken to avoid disturbing the separated layers, ensuring that the pellet and supernatant remain distinct. (1 mark)
2. Maintain Separation Integrity:
   • Gentle handling prevents resuspension of the pellet, which could compromise the purity of the separated components. (1 mark)
3. Safety Considerations:
   • Spun samples may contain pellets that could cause spills or contamination if not handled carefully. (1 mark)
4. Accurate Analysis:
   • Maintaining clear separation ensures that subsequent analyses or extractions are accurate and reproducible. (1 mark)
5. Sample Integrity:
   • Proper handling preserves the integrity of the isolated components, preventing degradation or loss of the desired substances. (1 mark)

Question 15

Explain how the principle of the pressure flow mechanism facilitates the transport of sugars in the phloem. (6 marks)

Mark Scheme:

1. Sugar Loading at Source:
   • Sugars (e.g., sucrose) are actively loaded into the phloem sieve tubes at the source (e.g., leaves). (1 mark)
2. Osmotic Gradient Formation:
   • High sugar concentration in the phloem creates an osmotic gradient, causing water to enter the phloem from the xylem via osmosis. (1 mark)
3. Turgor Pressure Increase:
   • The influx of water increases the turgor pressure within the phloem, generating a pressure gradient from source to sink. (1 mark)
4. Sap Movement:
   • The pressure gradient drives the flow of phloem sap, carrying sugars from the source to the sink tissues (e.g., roots, fruits). (1 mark)
5. Sugar Unloading at Sink:
   • At the sink, sugars are unloaded from the phloem, reducing the osmotic pressure and allowing water to exit the phloem back into the xylem. (1 mark)
6. Continuous Flow Maintenance:
   • The recycling of water and ongoing sugar loading at the source sustain the pressure flow, ensuring continuous nutrient transport. (1 mark)

Answer:
The pressure flow mechanism involves the active loading of sugars into the phloem at the source, creating an osmotic gradient that draws in water, increasing turgor pressure. This pressure drives the phloem sap towards the sink, where sugars are unloaded, reducing pressure and allowing water to exit, maintaining a continuous flow.

Question 16

What adaptations in phloem tissue enhance the efficiency of sugar transport? Provide two examples. (5 marks)

Mark Scheme:

1. Sieve Plates:
   • Perforated sieve plates allow for the continuous flow of sugars between sieve tube elements, facilitating efficient transport. (2 marks)
2. Companion Cells:
   • Companion cells provide metabolic support and energy (ATP) necessary for the active loading and unloading of sugars into the phloem. (2 marks)
3. Lack of Organelles in Sieve Tubes:
   • Sieve tube elements lack nuclei and most organelles, reducing flow resistance and maximizing space for nutrient transport. (1 mark)

Answer:
Phloem tissue is adapted for efficient sugar transport through sieve plates, which allow continuous flow between sieve tube elements, and companion cells, which provide the necessary metabolic support and energy for active sugar loading and unloading.

Question 17

A sample contains proteins in the supernatant after centrifugation. Explain how this outcome relates to the density of proteins compared to other components in the mixture. (5 marks)

Mark Scheme:

1. Density Relationship:
   • Proteins in the supernatant indicate that they are less dense compared to the pelleted components. (1 mark)
2. Centrifugal Force Effect:
   • During centrifugation, denser particles are forced to the bottom to form a pellet, while less dense substances remain in the supernatant. (1 mark)
3. Protein Density:
   • Proteins have a lower density than other components that have pelleted, such as cell debris or heavy organelles. (1 mark)
4. Separation Efficiency:
   • The centrifugation process has effectively separated components based on their density differences. (1 mark)
5. Implications for Analysis:
   • Proteins remaining in the supernatant can be collected for further analysis or purification without contamination from denser pelleted materials. (1 mark)

Answer:
Since proteins remain in the supernatant after centrifugation, it indicates that they are less dense than the other components that have pelleted, demonstrating effective density-based separation where denser particles settle while lighter proteins stay suspended in the liquid.

Question 18

Why is it important to use the correct speed and time settings when centrifuging samples? (5 marks)

Mark Scheme:

1. Effective Separation:
   • Correct speed and time ensure that components with different densities are properly separated. (1 mark)
2. Avoiding Damage:
   • Using inappropriate settings can damage delicate components or cause breakage of centrifuge tubes. (1 mark)
3. Optimal Pellet Formation:
   • Proper settings facilitate the formation of a clear and compact pellet, enhancing purity of separated components. (1 mark)
4. Preventing Incomplete Separation:
   • Incorrect speed or time may result in incomplete separation, leaving some denser particles in the supernatant or causing light particles to pellet. (1 mark)
5. Reproducibility:
   • Consistent settings are crucial for reproducible results, allowing experiments to be replicated accurately. (1 mark)

Answer:
Using the correct speed and time settings ensures effective separation of mixture components based on density, prevents damage to samples and equipment, promotes optimal pellet formation, avoids incomplete separation, and ensures reproducible results essential for reliable experimental outcomes.

Question 19

What is the purpose of a pellet in centrifugation, and how is it formed? (5 marks)

Mark Scheme:

1. Purpose of Pellet:
   • The pellet contains the denser particles separated from the mixture, allowing for the isolation of specific components. (1 mark)
2. Formation Process:
   • During centrifugation, centrifugal force drives heavier particles outward, causing them to settle at the bottom of the centrifuge tube. (1 mark)
3. Pellet Characteristics:
   • The pellet is typically a compact mass of solid particles that have settled due to their higher density. (1 mark)
4. Supernatant Relationship:
   • The pellet forms beneath the supernatant, which contains the less dense or soluble components of the mixture. (1 mark)
5. Post-Centrifugation Handling:
   • After spinning, the pellet can be removed or processed further for specific analyses or applications. (1 mark)

Answer:
A pellet is formed at the bottom of the centrifuge tube during centrifugation as denser particles are driven outward by centrifugal force and settle. The pellet serves to isolate these heavier components from the supernatant, allowing for their collection and further analysis.

Question 20

Explain how a centrifuge can be used to isolate DNA from a cell lysate. (6 marks)

Mark Scheme:

1. Cell Lysis:
   • Break open cells to release DNA, proteins, and other cellular components. (1 mark)
2. First Centrifugation (Low Speed):
   • Spin the lysate at a low speed to pellet large debris like cell membranes and nuclei. (1 mark)
3. Discard Pellet and Collect Supernatant:
   • Remove the pellet containing debris, retaining the supernatant that contains DNA, proteins, and smaller organelles. (1 mark)
4. Second Centrifugation (Higher Speed):
   • Spin the supernatant at a higher speed to pellet proteins and other macromolecules, leaving DNA in the supernatant. (1 mark)
5. DNA Precipitation (Optional):
   • Add alcohol (e.g., ethanol) to precipitate DNA, then centrifuge to pellet the DNA. (1 mark)
6. Isolation of DNA:
   • Carefully collect the DNA pellet, wash if necessary, and resuspend in an appropriate buffer for further use. (1 mark)

Answer:
To isolate DNA, cells are first lysed to release DNA and other components. The lysate is then centrifuged at low speed to remove large debris. The supernatant is centrifuged at a higher speed to pellet proteins, leaving DNA in the supernatant. Optionally, alcohol is added to precipitate the DNA, which is then pelleted and isolated for further experiments.