# Practical: Investigating Water Potential ## Introduction - It's possible to investigate the effects of immersing plant tissue in **solutions of different water potentials** - Results can then be used to **estimate the water potential** of the plant tissue itself - The most common practical involves cutting cylinders of potato and placing them into solutions with a range of different water potentials - Usually **sucrose solutions of increasing concentration** are used - At least **5 different concentrations** are typically required ## Practical 1: Potato Cylinder Investigation ### Apparatus - Scalpel - Cork borer (optional) - Cutting board - Ruler - Digital balance - Test tubes - Measuring cylinder - Water baths - Digital stopwatch ### Method - Using a scalpel, cut **five equal-sized cubes** of potato - The pieces must have the **same dimensions** so they all have equal surface areas and volumes - These factors could affect the rate at which pigment leaks out - A cork borer can also be used, as long as the cores are cut to the same length - You should also use a **digital balance** to check that all pieces have the same mass - **Rinse** the potato pieces - To remove any pigment released during cutting - Add the potato pieces to five different test tubes, each containing the **same volume of water** (e.g. $5 \text{ cm}^3$) - Put each test tube in a **water bath** at a different temperature (e.g. 10°C, 20°C, 30°C, 40°C, 50°C) for the **same length of time** - The time should be long enough to allow water movement by osmosis (e.g. around 30 minutes) - The water bath is typically set at around 30°C - Remove the potato pieces and **dry** them to remove excess liquid - The **final length and mass** of each potato cylinder is then measured and recorded

Always blot potato cylinders dry before measuring to remove excess surface water that would affect mass readings

### Calculating Percentage Change in Mass - The **percentage change in mass** for each potato cylinder is calculated - To find the percentage change in mass: $$\text{Percentage change in mass} = \frac{\text{final mass} - \text{initial mass}}{\text{initial mass}} \times 100$$

A **positive** percentage change indicates water gain by osmosis; a **negative** percentage change indicates water loss

### Results and Interpretation **Positive percentage change:** - The potato has **gained water by osmosis** - Net movement of water from the solution into the potato - The solution had a **higher water potential** than the potato - The gain of water makes potato cells **turgid** as water exerts turgor pressure (hydrostatic pressure) on cell walls - The potatoes will feel **hard** **Negative percentage change:** - The solution had a **lower water potential** than the potato - The potato cylinder in the **strongest sucrose concentration** will have decreased in mass the most - This is because there is the **greatest concentration gradient** between potato cells (higher water potential) and sucrose solution (lower water potential) - More water molecules move out of potato cells by osmosis, making them **flaccid** and decreasing mass - The potato cylinders will feel **floppy** - If examined under a microscope, cells might be **plasmolysed** (cell membrane pulled away from cell wall) **No change in mass:** - There was **no overall net movement** of water into or out of potato cells - The solution had the **same water potential** as the solution in the cytoplasm of potato cells - No concentration gradient, therefore no net movement of water

Temperature affects the rate of osmosis but not the final equilibrium position - ensure consistent temperature throughout

### Analysis Using Graphs - The concentration of sucrose inside the potato cylinders can be found by plotting a graph - Plot **percentage change in mass** against **concentration of sucrose solution** - Draw a **line of best fit** - The point where the line crosses the **x-axis** (where percentage change = 0) indicates the concentration of sucrose inside the potato cylinders - At this point, the external solution and internal cytoplasm have equal water potential | Sucrose concentration (mol dm⁻³) | % change in mass | Interpretation | |---|---|---| | 0.00 (distilled water) | +9.4 | Water gained - hypotonic solution | | 0.25 | +1.9 | Water gained - slightly hypotonic | | 0.50 | -1.0 | Water lost - slightly hypertonic | | 0.75 | -3.8 | Water lost - hypertonic solution | | 1.00 | -5.1 | Water lost - very hypertonic | The graph below shows typical results for this investigation. Notice how the line crosses the x-axis at approximately 0.35 mol dm⁻³ - this represents the sucrose concentration (and therefore water potential) inside the potato cells:

## Practical 2: Onion Cell Investigation ### Introduction to Plasmolysis - Evidence of osmosis in plant cells can be shown when cells undergo **plasmolysis** - If a plant cell is placed in a solution with **lower water potential** than the cell (e.g. concentrated sucrose solution): - Water leaves the cell through its partially permeable cell surface membrane by osmosis - As water leaves the **vacuole**, the volume of the cell **decreases** - The **protoplast** (living part inside cell wall) gradually shrinks and no longer exerts pressure on cell wall - The protoplast begins to **pull away from the cell wall** - This process is **plasmolysis** - the plant cell is **plasmolysed** ### Apparatus - Epidermal strips (e.g. red onion, rhubarb petioles, red cabbage) - Microscope slides and coverslips - Forceps/tweezers - Scalpel - Range of sucrose or sodium chloride solutions (gradually decreasing water potential) - Light microscope - Paper towels

Plants with coloured sap like red onion make observations much easier than colourless tissues

### Method - Epidermal strips are placed in a **range of molarities** of sucrose solution or sodium chloride solutions - Solutions should have **gradually decreasing water potential** - The strips are then viewed under a **light microscope** - The **total number** or **percentage** of onion cells that have undergone plasmolysis can be counted - **Plasmolysis may take several minutes** to occur ### Results - The unknown monosaccharides can be identified by **comparing** and **matching** them with chromatograms of known standard solutions - Count cells in each category: - Normal turgid cells - Incipient plasmolysis (just starting) - Plasmolysed cells - Calculate percentage plasmolysed for each concentration - The concentration at which 50% of cells are plasmolysed approximates the water potential of the tissue ## Key Considerations ### Safety and Technique - Cut potato cylinders **underwater** initially to prevent air entering xylem (if investigating transport) - Always use the **same volume** of solution in each test tube - Ensure potato pieces are **thoroughly dried** before final measurements - Use a **digital balance** accurate to at least 0.01g - Keep temperature **constant** using water baths - Allow sufficient time for equilibrium (typically **30 minutes**) ### Variables to Control - **Same plant tissue** source (same potato, same onion) - **Same volume** of solution - **Same surface area/mass** of tissue - **Same temperature** - **Same time** period - Use **fresh solutions** each time ### Common Errors - Not drying potato pieces properly → inaccurate mass readings - Using different sizes of potato → unequal surface area affects rate - Not allowing enough time → equilibrium not reached - Air bubbles in microscope slides → obscures view of plasmolysis - Using old/oxidised plant tissue → cell damage affects results ## Limitations ### Practical Limitations | Limitation | Impact | Solution | |---|---|---| | Potato pieces not identical | Different surface area:volume ratios affect rate | Use cork borer for uniform cylinders; measure mass of each piece | | Solution concentration changes | Osmosis alters external water potential slightly | Use large volume of solution relative to tissue | | Temperature fluctuations | Affects rate of osmosis | Use water baths with thermostatic control | | Cell damage during cutting | Releases cell contents, affects osmosis | Use sharp implements; cut quickly; rinse immediately | | Time for equilibrium varies | May not reach true endpoint | Pilot study to determine appropriate time; use consistent timing | ### Biological Variability - Different regions of the same potato may have **different sugar concentrations** - Use tissue from the same region of one potato - Calculate **mean values** from multiple repeats - Cell size varies within tissue - Count sufficient cells to get representative sample - Use at least 50-100 cells for plasmolysis counts - Age and storage conditions affect water potential - Use fresh tissue where possible - Keep all tissue under same storage conditions

Never assume all cells in a tissue have identical water potential - biological variation always exists

## Extensions and Applications ### Determining Water Potential Values - To convert sucrose concentration to actual water potential values: - Use published data tables showing relationship between sucrose molarity and water potential - At 20°C, pure water has water potential of 0 kPa - Adding solute decreases water potential (becomes more negative) - Example: 0.5 mol dm⁻³ sucrose ≈ -1300 kPa ### Real-World Applications - Understanding crop irrigation needs - Predicting plant survival in different soil water conditions - Food preservation using high solute concentrations (e.g. jam, salted fish) - Explaining why plants wilt in salty soils - Medical applications (e.g. IV fluid composition) ### Comparing Different Plant Tissues - Different plant tissues have different water potentials: - **Root cells**: relatively high water potential (take up water from soil) - **Leaf mesophyll cells**: lower water potential (lose water by transpiration) - **Storage tissues** (potato tubers): intermediate water potential - This can be investigated by comparing percentage change graphs for different tissues