investigate simple diffusion and osmosis using plant tissue and non-living materials, including dialysis (Visking) tubing and agar

Movement into and out of Cells

Learning Objective

Investigate simple diffusion and osmosis using plant tissue and non‑living materials (dialysis/Visking tubing, agar) and relate the observations to the underlying theory, quantitative relationships and the wider range of membrane‑transport mechanisms required by the Cambridge International AS & A Level Biology (9700) syllabus.

Transport Mechanisms Required by the Syllabus

Theoretical Background

Simple Diffusion

Fick’s first law describes the flux (J) of a solute:

\( J = -D \dfrac{\Delta C}{\Delta x} \)

• D – diffusion coefficient (m² s⁻¹)
• \(\Delta C\) – concentration difference across the membrane
• \(\Delta x\) – membrane thickness (distance travelled)

Facilitated Diffusion

The flux is still proportional to the concentration gradient, but the proportionality constant is the permeability of the carrier protein (P), which is far larger than that of the bare lipid bilayer:

\( J = -P \, \Delta C \)

Typical carrier proteins:

• GLUT‑1 – transports glucose into cells.
• Aquaporin – provides a rapid pathway for water.
• Na⁺ channel – allows Na⁺ ions to move down their electrochemical gradient.

Osmosis & Water Potential

Water moves from higher to lower water potential:

\( \Psiw = \Psis + \Psi_p \)

• \(\Psi_s\) – solute (osmotic) potential (always ≤ 0).
• \(\Psi_p\) – pressure potential (≥ 0, e.g., turgor pressure).

For dilute sucrose solutions, \(\Psi_s \approx -i C R T\) (where i = 1, C = molarity, R = 0.0831 L bar mol⁻¹ K⁻¹, T = temperature in K).

Active Transport

Energy‑dependent transport can be expressed as:

\( \Delta G = RT \ln\!\left(\dfrac{C{\text{in}}}{C{\text{out}}}\right) + zF\Delta\Psi \)

When \(\Delta G > 0\) the cell must supply ATP (directly or via a secondary carrier) to move the solute.

Endocytosis & Exocytosis

Both processes involve vesicle formation or fusion and are driven by the cytoskeleton and ATP.

• Phagocytosis – uptake of large particles (e.g., bacteria) by specialised cells.
• Pinocytosis – non‑selective uptake of extracellular fluid.
• Receptor‑mediated endocytosis – selective uptake of specific ligands (e.g., LDL, hormones).
• Exocytosis – vesicle fuses with the plasma membrane to release contents (e.g., hormones, enzymes).

Factors Influencing Diffusion and Osmosis

Quick Maths – Surface‑Area‑to‑Volume Ratio (SA/V)

Experimental Design

Materials – Non‑living

• Dialysis (Visking) tubing – cut into 5 cm lengths
• Agar slabs (1 cm thick, 5 cm diameter) – optional diffusion medium
• Coloured dye (e.g., methylene blue) – for agar diffusion activity
• Sucrose solutions (0 %, 5 %, 10 %, 15 % w/v) – prepared with distilled water
• Distilled water
• 250 mL beakers (minimum 5)
• Analytical balance (±0.01 g)
• Timer/stopwatch
• Thermometer (±0.5 °C) and, if possible, a water‑bath to control temperature

Materials – Plant tissue

• Fresh potato (or carrot) strips – 1 cm × 1 cm × 2 cm
• Sucrose solutions as above
• Beakers, balance, timer, thermometer

Method 1 – Simple Diffusion (Dialysis Tubing)

1. Fill each piece of dialysis tubing with 5 mL of 10 % sucrose solution; seal both ends securely (use a knot or heat‑sealer).
2. Record the initial mass of each sealed tube (including its contents).
3. Place each tube in a separate beaker containing 100 mL of distilled water.
4. Maintain the beakers at a constant temperature (≈22 °C). Start the timer.
5. After 30 min, remove the tubes, gently blot the exterior dry, and record the final mass.
6. Calculate the change in mass (Δm). A gain indicates net water influx; a loss indicates net water efflux.

Method 2 – Osmosis (Potato Strips)

1. Weigh each potato strip and record its initial mass.
2. Place each strip in a beaker containing 100 mL of one of the sucrose solutions.
3. Keep the temperature constant (use a water‑bath if required).
4. After 30 min, remove the strip, blot gently, and weigh again.
5. Calculate percentage mass change:
   

   %Δm = ((mfinal – minitial) / m_initial) × 100

Method 3 – Optional Agar Diffusion Activity

1. Prepare agar slabs (1 cm thick, 5 cm diameter) and allow them to set.
2. Use a 5 mm cork borer to make a central well in each slab.
3. Add 0.5 mL of 0.1 % methylene‑blue solution to the well.
4. Leave the slab at room temperature and record the distance the colour front travels every 2 min for 20 min.
5. Plot distance (cm) against √time (min^½). The slope (k) is proportional to \(\sqrt{D}\), allowing a rough estimate of the diffusion coefficient for the dye.

Observations & Data Recording

Analysis

• Osmosis in potato strips – Plot %Δm against external sucrose concentration. The concentration at which %Δm = 0 is the isotonic point; at this concentration \(\Psiw\) inside = \(\Psiw\) outside.
• Diffusion in dialysis tubing – A gain in mass indicates net water influx (water moving from higher Ψ_w outside to lower Ψ_w inside). A loss indicates net water efflux.
• Agar diffusion (optional) – The linear relationship between distance and √time confirms Fickian diffusion and permits a rough calculation of D for methylene blue.
• Relate the observed trends to the equations presented above, discussing how each factor in the “Factors Influencing” table quantitatively affects the measured rates.
• Use the SA/V calculations to explain why thin potato strips (high SA/V) show larger % mass changes than thicker pieces.

Safety Considerations

• Handle scissors and scalpel blades with care when cutting dialysis tubing or agar.
• Wear a lab coat, safety goggles and disposable gloves when preparing concentrated sucrose solutions and coloured dyes.
• Dispose of sugar‑containing waste according to school laboratory guidelines; do not pour large volumes down the sink without checking local regulations.
• Use heat‑proof gloves when handling hot agar.

Conclusion Points

• Simple diffusion and osmosis are passive processes driven solely by concentration or water‑potential gradients.
• Facilitated diffusion provides a selective route for polar or charged molecules that cannot cross the lipid bilayer directly.
• Active transport, endocytosis and exocytosis require cellular energy and are essential for the uptake or export of large or charged substances.
• Experimental data with plant tissue (potato) and non‑living models (dialysis tubing, agar) illustrate how surface area, membrane thickness, temperature and gradient magnitude quantitatively affect rates of movement.
• The isotonic sucrose concentration for the potato tissue corresponds to the point where solute potential inside equals that outside, giving \(\Psi_w\) equilibrium.

Extension Activities

1. Replace agar with gelatin of varying concentrations to explore how gel density (analogous to membrane thickness) influences diffusion rates.
2. Determine the diffusion coefficient (D) of a coloured solute (e.g., methylene blue) in agar using the distance‑versus‑√time method described above.
3. Repeat the diffusion and osmosis experiments at three temperatures (5 °C, 22 °C, 35 °C) and analyse the effect of temperature on D and water flux.
4. Design a simple active‑transport experiment (e.g., uptake of glucose by yeast cells in the presence/absence of metabolic inhibitors) to contrast with the passive processes studied.