4.11 Practicals: Diffusion & Osmosis

Objective

• To observe the selective diffusion of small molecules (e.g., glucose) across a partially permeable membrane (Visking tubing) and to understand how molecular size influences diffusion.

Materials Needed

• Visking (dialysis) tubing (~15 cm)
• Starch solution
• Glucose solution
• Distilled water
• Test tubes or boiling tubes
• Benedict’s solution (for glucose testing)
• Iodine solution (for starch testing)
• Optional: Colorimeter or color standards (for semi-quantitative analysis)

Method Overview

1. Prepare the Visking Tubing:
   • Fill a section of Visking tubing with a mixture of starch and glucose solutions.
   • Tie off both ends tightly to prevent any leakage.
2. Set Up the Experiment:
   • Suspend the filled Visking tubing in a test tube or boiling tube containing distilled water.
   • Allow the setup to sit undisturbed for a set period (e.g., 30 minutes) to enable diffusion.
3. Testing for Diffusion:
   • At regular intervals (e.g., every 10 minutes), test the water outside the tubing for the presence of glucose and starch:
     • Glucose Test: Add Benedict’s solution to a sample of the water outside the tubing and heat it in a water bath. A color change from blue to green, yellow, or red indicates the presence of glucose.
     • Starch Test: Add iodine solution to a sample of the water. If starch is present, the solution will turn blue-black; if no color change occurs, it indicates starch has not diffused out.

Expected Results

• Glucose should diffuse out of the Visking tubing into the surrounding water because it is a small molecule capable of passing through the semi-permeable membrane.
• Starch should remain inside the tubing because its larger molecular size prevents it from diffusing through the membrane pores.

Extension: Quantitative Analysis of Glucose Diffusion

1. Setting up a Time Series for Glucose Testing:
   • Collect samples from the water surrounding the Visking tubing at multiple time intervals (e.g., every 5 or 10 minutes).
   • Perform a semi-quantitative Benedict’s test on each sample and observe the color intensity as an estimate of glucose concentration.
2. Using Color Standards or Colorimeter:
   • Compare each sample’s color intensity with a set of glucose color standards or use a colorimeter for a more precise measurement of glucose concentration.
   • Plot a graph of glucose concentration over time to visualize the rate of diffusion, demonstrating how the concentration gradient between the inside and outside of the tubing affects the diffusion rate.

Enzyme Activity Variations (Optional)

• Sucrose and Sucrase: Place sucrose and sucrase inside the Visking tubing. Test for glucose in the surrounding water to observe how enzymatic breakdown (sucrase converting sucrose into glucose) affects diffusion.
• Starch and Amylase: Place starch and amylase in the tubing. Test the water outside for maltose using Benedict’s solution to observe how enzyme activity (starch breakdown) influences diffusion of smaller breakdown products.

Exam Advise:

• Interpreting Results: Explain that glucose diffuses due to its small molecular size, while starch does not, due to its larger molecular structure. Highlight how molecular size and membrane selectivity are key factors in selective permeability.
• Use Precise Terminology: Clearly differentiate between measuring the rate of diffusion (based on glucose concentration over time) and observing the extent of color change as an indicator of glucose presence.

Objective

To explore the effect of environmental factors (temperature and chemical exposure) on cell membrane permeability using beetroot tissue, which releases red pigment (betalain) when its membranes are compromised.

Materials Needed

• Fresh beetroot
• Distilled water
• Ethanol solutions at varying concentrations (e.g., 0%, 20%, 50%, and 70%)
• Heat source (e.g., adjustable water bath)
• Test tubes
• Colorimeter or color standards (optional, for quantitative analysis)

Method Overview

1. Prepare the Beetroot:
   • Cut beetroot into equal-sized pieces (e.g., cubes or discs) for consistency.
   • Rinse thoroughly under running water to remove any excess surface pigment released from cutting.
2. Set Up Treatment Conditions:
   • Temperature Test: Place beetroot pieces in separate test tubes containing distilled water at different temperatures (e.g., 0°C, 20°C, 40°C, 60°C, and 80°C).
   • Chemical Test: Place beetroot pieces in test tubes containing different ethanol concentrations (e.g., 0%, 20%, 50%, and 70%).
3. Observe and Measure Pigment Diffusion:
   • Allow the beetroot samples to sit in their respective conditions for a set time (e.g., 10–15 minutes).
   • Qualitative Observation: Arrange the test tubes by color intensity to visually assess membrane permeability.
   • Quantitative Measurement (optional): Use a colorimeter to measure absorbance or create a color scale from 0 (clear water) to 10 (darkest solution) for semi-quantitative assessment.

Expected Results

• Higher Temperatures or Alcohol Concentrations: Likely to increase membrane permeability, resulting in more red pigment diffusing out of beetroot cells.
• Lower Temperatures or Alcohol Concentrations: Expected to maintain membrane integrity, resulting in minimal or no color change in the surrounding solution.

Explanation of Results

• Diffusion: The red pigment, betalain, moves from an area of high concentration within the beetroot vacuole to the surrounding solution due to diffusion.
• Membrane Permeability:
  • Normally, the cell membrane restricts the movement of betalain.
  • Heat and Chemicals (e.g., ethanol) disrupt membrane structure, increasing permeability and allowing pigment to leak out.
  • Heat denatures membrane proteins, and ethanol dissolves membrane lipids, both compromising the cell’s selective barrier.

Extensions and Experimental Design Ideas

1. Time Variation: Test the effect of different exposure times (e.g., 5, 10, 15 minutes) at the same temperature or ethanol concentration.
2. Different Alcohol Types: Compare the effect of other alcohols, such as methanol or isopropanol, on membrane permeability.
3. Gradual Temperature Changes: Observe pigment diffusion over time with gradually increasing temperatures to simulate environmental changes.

Conclusion

• This experiment visually demonstrates how environmental stressors, such as temperature and chemicals, affect cell membrane integrity and permeability.
• By using the visible diffusion of beetroot pigment, students can directly observe the impact of stress on cellular membranes, making this a powerful tool for understanding membrane dynamics.

Objective

• To investigate how environmental factors, such as temperature and alcohol concentration, affect cell membrane permeability by observing the release of red pigment (betacyanin) from beetroot cells.

Method

1. Preparation:
   • Use a cork borer to cut beetroot pieces into uniform sizes for consistent results.
   • Rinse the beetroot pieces thoroughly to remove any surface pigment released during cutting.
2. Treatment:
   • Place beetroot pieces in test tubes filled with distilled water.
   • Apply two experimental conditions:
     • Temperature Variation: Place tubes in water baths at different temperatures (e.g., 0°C, 20°C, 40°C, 60°C, and 80°C).
     • Alcohol Concentration Variation: Place tubes in solutions with varying alcohol concentrations (e.g., 0%, 20%, 50%, and 70% ethanol).
3. Observation:
   • Allow beetroot samples to sit in their respective conditions for a set time (e.g., 10–15 minutes).

Data Collection

• Qualitative Data: Observe the color intensity of the solution surrounding each beetroot sample, which indicates the extent of pigment release.
  • Arrange test tubes in order of color intensity to assess relative membrane permeability.
• Quantitative Data (optional):
  • Use a colorimeter to measure the absorbance of red pigment in each solution, providing an objective measure of concentration.
  • Alternatively, compare the color of each sample to a set of color standards to assign a relative intensity level (e.g., 0 to 10).

Explanation

• Membrane Permeability:
  • Under normal conditions, the cell membrane is partially permeable, containing the betacyanin pigment within the large central vacuole.
  • High Temperatures: Cause denaturation of membrane proteins, disrupting the membrane structure and increasing permeability.
  • Alcohol: Disrupts the lipid bilayer by dissolving membrane lipids, which also increases permeability.
  • When the membrane is compromised by these factors, it allows the pigment to diffuse out of the cell.
• Diffusion Process:
  • The red pigment moves from an area of high concentration (inside the vacuole) to an area of low concentration (the surrounding solution), following the concentration gradient.

Conclusion

• This experiment demonstrates how cell membrane permeability changes in response to external stressors, such as heat and alcohol, using the visible release of red pigment as an indicator of membrane damage.
• It highlights the impact of environmental factors on cellular integrity and the process of diffusion across damaged membranes.

Investigating Diffusion Using Agar: Effect of Surface Area-to-Volume Ratio

Objective:
To investigate how changing the surface area-to-volume ratio affects the rate of diffusion in different-sized agar cubes.

Method:

1. Preparation:
   • Cut agar into cubes of various sizes (e.g., 0.5 cm³, 1 cm³, and 2 cm³).
   • Purple Indicator Agar: Prepare the agar using a dilute solution of sodium hydroxide with Universal Indicator to color it purple.
2. Diffusion Setup:
   • Place each agar cube in a boiling tube containing dilute hydrochloric acid.
   • The acid diffuses into the agar, causing a color change as it neutralizes the sodium hydroxide.
3. Measurements:
   • Measure the time it takes for the acid to completely change the agar’s color.
   • Alternatively, measure the distance the acid travels into the agar (shown by the color change) in a set time period (e.g., 5 minutes).
4. Calculating Rate of Diffusion:
   • Convert times to rates by using the formula Rate=1time taken\text{Rate} = \frac{1}{\text{time taken}}Rate=time taken1​.
   • Plot a graph showing how the rate of diffusion varies with the surface area-to-volume ratio.

Explanation:

• Surface Area-to-Volume Ratio: Smaller cubes (higher surface area-to-volume ratio) show faster diffusion rates, as a larger surface area relative to volume allows more efficient molecular exchange.

Examiner Tips

Surface Area-to-Volume Ratio: As an object (e.g., an agar cube or cell) increases in size, its volume grows faster than its surface area. A larger volume relative to surface area reduces diffusion efficiency. Therefore, a higher surface area-to-volume ratio results in a faster diffusion rate.

Objective

To study how the surface area to volume (SA) ratio affects the rate of diffusion using agar cubes as models for cells of different sizes.

Method Summary

1. Preparation:
   • Cut agar into cubes of varying dimensions (e.g., 0.5 cm, 1 cm, and 2 cm).
   • Indicator Setup: Use agar made with sodium hydroxide and Universal Indicator, making it purple. The color changes as hydrochloric acid (HCl) diffuses into the cubes.
2. Diffusion Setup:
   • Place each agar cube in a boiling tube filled with dilute hydrochloric acid.
   • As HCl diffuses into the cube, it neutralizes the sodium hydroxide, causing the agar to go colorless.
3. Measurements:
   • Record the time taken for each cube to turn completely colorless. This indicates that diffusion has reached the center of the cube.
   • Calculate rate of diffusion as Rate=1time taken\text{Rate} = \frac{1}{\text{time taken}}Rate=time taken1​.

Data Analysis

1. Calculate SARatios:
   • For each cube, calculate the surface area and volume to determine the SAratio.
   • Example from the data:
     • Cube A (smallest): SA= 12:1, time = 176 seconds.
     • Cube B (medium): SA= 6:1, time = 259 seconds.
     • Cube C (largest): SA= 3:1, time = 384 seconds.
2. Calculate Diffusion Rates:
   • Rate = 1time\frac{1}{\text{time}}time1​ in seconds−1^{-1}−1.
   • Example Rates:
     • Cube A: 0.0057 s−1^{-1}−1
     • Cube B: 0.0039 s−1^{-1}−1
     • Cube C: 0.0026 s−1^{-1}−1
3. Graphing:
   • Plot a graph of rate of diffusion against SAratio. The graph shows a decreasing rate of diffusion as the SAratio decreases.

Conclusion and Explanation

• Relationship Between SAand Diffusion:
  • Higher SAratios (smaller cubes) allow faster diffusion rates, indicating that materials enter and exit more quickly.
  • Lower SAratios (larger cubes) result in slower diffusion rates, as less surface area is available per unit of volume for exchange.
• Biological Implication:
  • Cells rely on a high SAratio for efficient material exchange. Larger cells or organisms need adaptations, like circulatory systems or folded surfaces, to maintain efficient exchange.

Examiner Tip

Understanding this relationship helps explain why cells are small and why larger organisms have specialized exchange structures.

Be sure to mention that higher SAratios enhance diffusion rates due to more surface area relative to volume, allowing quicker access for molecules.



Objective

• To explore how the size (surface area-to-volume ratio) of an object affects the rate of diffusion using agar blocks containing an alkaline solution and pH indicator.

Materials Needed

• Agar prepared with dilute sodium hydroxide solution and Universal indicator (colored purple due to alkaline pH)
• Containers to set agar (e.g., ice cube trays or small dishes)
• Dilute hydrochloric acid solution (higher molarity than sodium hydroxide)
• Ruler
• Stopwatch

Method Overview

1. Prepare Agar Blocks:
   • Pour the prepared agar solution into an ice cube tray or shallow container and allow it to set.
   • Cut agar blocks of different sizes (e.g., 2 cm × 2 cm, 1 cm × 1 cm, and 0.5 cm × 0.5 cm).
   • Measure and record the dimensions of each block to calculate surface area (SA) and volume (V) and determine the surface area-to-volume ratio (SA) for each size.
2. Set Up the Experiment:
   • Place each agar block in a container and cover it with dilute hydrochloric acid.
   • The acid will begin to diffuse into the agar, neutralizing the sodium hydroxide and causing the indicator to change color as the pH shifts from alkaline to acidic.
3. Measure Diffusion Rate:
   • Option 1: Time how long it takes for the acid to completely change the color of each agar block.
   • Option 2: After a set time (e.g., 5 minutes), measure the distance the acid has diffused into each block (indicated by the color change).
   • Record all times or diffusion distances for each block size.
4. Calculate and Plot Results:
   • For each block, calculate the rate of diffusion based on time or distance measurements.
   • Calculate the surface area-to-volume ratio (SA) for each block size.
   • Plot the rate of diffusion against the SAratio to observe any relationship.

Expected Results

• Smaller Blocks (with a higher SA ratio): Expected to show a faster rate of diffusion, indicated by a quicker color change or greater distance traveled by the acid.
• Larger Blocks (with a lower SA ratio): Expected to show a slower diffusion rate.

Explanation of Results

• Surface Area-to-Volume Ratio:
  • Smaller objects with a higher SA ratio have more surface area relative to their volume, allowing more molecules to diffuse across the surface per unit volume.
  • This results in faster diffusion rates in smaller blocks compared to larger blocks.
• Implications for Cells:
  • Cells maintain small sizes to maximize diffusion efficiency, which is essential for nutrient uptake, gas exchange, and waste removal.
  • Larger organisms develop specialized structures (e.g., lungs, blood vessels) to overcome the limitations of low SA ratios.

Extension Ideas

Effect of Concentration Gradient: Change the hydrochloric acid concentration to test how the steepness of the concentration gradient affects diffusion rate.

Different pH Indicators: Experiment with different pH indicators in the agar to visually assess diffusion rates using varying color changes.

1. Surface Area (SA)

• Definition: The total area of an organism or cell that is exposed to its environment.
• Importance: The surface area allows for the exchange of materials (e.g., nutrients, gases) with the surroundings.

2. Volume (V)

• Definition: The total internal space within an organism or cell.
• Importance: Represents the amount of material (e.g., cytoplasm, organelles) inside a cell or organism that requires resources and produces waste.

Key Concept: Size and SA Ratio

• Size and Ratio Relationship:
  • As an organism’s or cell’s size increases, its volume grows faster than its surface area.
  • This causes a decrease in the SA ratio as size increases.
• Implications:
  • Smaller Organisms or Cells: Have a higher SAratio, which facilitates efficient material exchange across the cell membrane.
  • Larger Organisms or Cells: Often require specialized structures (e.g., lungs, blood vessels) to maintain efficient exchange of materials due to a lower SAratio.

Calculating SA Ratios for Different Shapes

1. Cube

1. Cuboid

1. Cylinder

Practical Application and Exam Advice:

• Relate SA ratios to real-life biological systems, explaining why certain adaptations are necessary for larger organisms.
• Implications of SA Ratio:
  • Larger Organisms: Often need adaptations (e.g., circulatory systems, specialized respiratory surfaces) to support effective exchange processes due to their lower SA ratios.
• Exam Focus:
  • Be able to calculate and interpret SA ratios for various shapes.
  • Understand that as cells grow, their volume increases faster than surface area, impacting material exchange rates.

Objective

• To estimate the water potential of potato cells by observing changes in mass after submerging potato cylinders in sucrose solutions of varying concentrations.

Method Overview

1. Preparation of Potato Cylinders:
   • Use a cork borer to cut multiple potato cylinders of the same diameter.
   • Trim each cylinder to the same length using a scalpel and ruler for consistency.
2. Measuring Initial Mass:
   • Measure and record the initial mass of each potato cylinder using a balance accurate to at least 0.01 g.
   • Optionally, record the initial length to observe any additional physical changes.
3. Osmosis Experiment Setup:
   • Prepare a series of sucrose solutions with different concentrations (e.g., 0.0 M, 0.25 M, 0.5 M, 0.75 M, 1.0 M).
   • Place each cylinder in a different sucrose solution, ensuring they are fully submerged.
   • Leave the potato cylinders in the solutions for a set period (e.g., 30 minutes to an hour) at a constant temperature to ensure consistent results.
4. Measuring Final Mass:
   • After the designated time, remove each potato cylinder, blot dry to remove excess solution, and measure the final mass.
   • Calculate the change in mass for each cylinder and determine the percentage change in mass to standardize the results.

Analysis

• Observing Water Movement:
  • At higher sucrose concentrations, potato cells lose water by osmosis, becoming flaccid and possibly plasmolysed if enough water leaves the cells.
  • At lower sucrose concentrations, the cells may gain water, becoming turgid.

Graphing and Interpreting Results

1. Plotting Data:
   • Create a graph of percentage change in mass (y-axis) against sucrose concentration (x-axis).
2. Identifying Water Potential:
   • The point where the line of best fit crosses the x-axis (where there is no change in mass) indicates the sucrose concentration at which the water potential inside the potato cells equals the water potential of the solution.
   • This concentration represents the water potential of the potato cells, as there is no net movement of water at this point.

Understanding Results

• Positive Percentage Change in Mass:
  • Indicates water gain due to osmosis, meaning the surrounding solution has a higher water potential than the potato cells.
• Negative Percentage Change in Mass:
  • Indicates water loss, meaning the surrounding solution has a lower water potential than the potato cells.

Summary

By determining the sucrose concentration where there is no net change in mass, we identify the water potential of the potato cells, enhancing our understanding of osmosis and water potential in plant cells.

This experiment demonstrates how to estimate the water potential of plant tissue by using osmosis and observing changes in mass.

Objective

• To observe the process of osmosis and plasmolysis in plant cells when placed in solutions with varying water potentials.

Method

1. Preparation of Plant Tissue:
   • Use epidermal strips from plants with naturally colored sap (e.g., red onion, rhubarb, or red cabbage) to make the process of plasmolysis easier to observe under a microscope.
2. Treatment:
   • Place the prepared strips in solutions of different sucrose molarities or sodium chloride concentrations to create varying water potentials.
3. Observation (Under Microscope):
   • Plasmolysis:
     • In a solution with a lower water potential than the plant cell (e.g., a concentrated sucrose or NaCl solution), water exits the cell by osmosis.
     • Process:
       • The vacuole shrinks as water leaves the cell, causing the protoplast (the living contents of the cell) to decrease in volume.
       • The protoplast gradually pulls away from the cell wall, a characteristic process called plasmolysis.
   • Observation Over Time:
     • Plasmolysis typically takes a few minutes to complete. Observe the tissue under the microscope at intervals to capture the stages of plasmolysis as it occurs.
4. Data Collection:
   • Compare the appearance of plasmolysed cells (where the protoplast has pulled away from the cell wall) to non-plasmolysed cells.
   • Note the degree of plasmolysis in different solution concentrations to see how varying water potentials affect the cells.

Explanation

• Osmosis: Water moves from an area of higher water potential (inside the cell) to a lower water potential (outside, in the concentrated solution).
• Plasmolysis: As water exits the cell, the vacuole shrinks, and the protoplast contracts, eventually detaching from the cell wall, resulting in plasmolysis. This is visible as the protoplast pulls away from the cell wall, creating gaps between them.

Understanding Results

• High Water Potential Solution (e.g., Distilled Water): Cells absorb water, become turgid, and do not undergo plasmolysis.
• Low Water Potential Solution (e.g., Concentrated Sucrose or Salt Solution): Cells lose water, resulting in plasmolysis as the protoplast pulls away from the cell wall.

Summary

By observing cells under different osmotic conditions, we can understand how water potential affects cell structure and water movement, illustrating the principles of osmosis and the importance of maintaining water balance in plant cells.

This practical activity visually demonstrates the process of osmosis and plasmolysis in plant cells.