What you need to know
- 2.3Core Practical 3: temperature and alcohol effects on membrane permeability.
Practical study guide for a supervised school laboratory. The teacher must set reagent strengths, operating ranges and local risk controls before use. This is original preparation and evaluation material, not a replacement for the school’s approved laboratory protocol.
By the end, you should be able to…
- Plan separate temperature and alcohol investigations.
- Control tissue preparation and exposure conditions.
- Interpret absorbance or transmission correctly and evaluate a leakage proxy.
Build your understanding
Why beetroot can reveal membrane changes
Beetroot pigment is held inside cell vacuoles. To reach the surrounding liquid from an intact cell, it must cross the tonoplastThe membrane around the vacuole in a plant cell. and the cell surface membraneThe partially permeable membrane around a cell that controls which substances enter and leave.. Disruption of membrane structure can increase leakage; the cell wallA rigid layer outside the cell membrane. In plants it is made mainly of cellulose; it supports the cell and resists bursting. is not the main partially permeableLets some molecules pass through but not others. barrier.
Heating can change lipid organisation and disrupt membrane proteins; alcohol can disrupt membrane lipids and protein interactions. Measuring released pigment provides an indirect indicator of membrane permeability, not a direct count of pores or proof of one molecular mechanism.
Prepare comparable tissue
Use one beetroot or distribute pieces from several beetroots evenly between treatments. A cork borer and ruler help produce cylinders with matched diameter and length. Cut safely on a tile, then rinse until loose pigment from cut cells is removed and blot consistently.
Use the same number and size of pieces, treatment volume, pHA measure of how acidic or alkaline a solution is, based on its H⁺ ion concentration. Below 7 is acidic, above 7 is alkaline., exposure time and mixing procedure. Independent tissue pieces provide repeats. Cutting some pieces much smaller changes surface areaThe total area of a surface. A larger surface area gives a faster rate of diffusion. and cell damage, so equal mass alone may not make them fully comparable.
Change temperature alone
Place equal volumes of water in labelled tubes in a teacher-approved range of water baths. Allow the liquids to reach their temperatures, measure actual temperatures with a thermometer, then add matched beetroot pieces. Keep exposure time equal before removing the tissue.
Include a comparison temperature, use at least three independent pieces per treatment where feasible and repeat the procedure consistently. Keep ethanol concentration at zero throughout this investigation. Choose a range that reveals a trend without exceeding local safe operating limits.
Change alcohol concentration alone
Prepare a measured range of aqueous ethanol concentrations including a 0% water control. Add matched tissue pieces to equal liquid volumes, keeping temperature, pH and exposure time fixed. Remove the tissue at the same elapsed time before measuring the liquid.
Keep ethanol away from flames and other ignition sources, close containers and do not heat alcohol solutions for this comparison. The temperature investigation and alcohol investigation answer separate questions; changing both at once would confound their effects.
Measure, process and evaluate
Use a suitable blue/green filter or wavelength and a colorimeter with a blank containing the same solventA liquid in which other substances (solutes) dissolve. Water is a good solvent for ions and polar molecules. mixture but no pigment. For each ethanol concentration, use its matching solvent blank. Set zero absorbance, equivalent to 100% transmission, according to the instrument instructions. Keep cuvettes clean, similarly oriented and free of bubbles.
More pigment generally gives higher absorbance and lower percentage transmission. Record which scale you used. Plot mean response against actual temperature or alcohol concentration, show repeat variability and investigate anomalies. Dilute readings beyond the usable instrument range consistently and record the dilution.
Exposure-time pigment release is a proxy affected by initial pigment content, cutting damage and solvent effects as well as permeability. Repeats, matched tissue and solvent blanks reduce these limitations but do not make the model identical to every animal or plant membrane.
Risk controls
Wear eye protection. Handle blades and borers away from fingers on a stable cutting surface; obtain prepared pieces if needed. Use holders for hot tubes and guard against hot water and steam. Keep electrical equipment dry and follow local disposal instructions for ethanol mixtures.
Graph skills: SLAPUK and choosing a scalePlotting mean response against temperature: choose a scale that is not the raw data.
The beetroot membrane lab
Change temperature or ethanol concentration, measure pigment leakage with a colorimeter and interpret the trend. All readings are fictional.
Simulation only. Cut on a tile with care, keep ethanol away from flames and never heat alcohol. A teacher sets safe temperatures, ethanol strengths and colorimeter settings.
Method 7 steps
- Cut beetroot into cylinders of the same diameter and length with a cork borer and knife, on a tile.
- Rinse the pieces in distilled water to remove pigment from cut cells, then blot them dry.
- Put each piece into the same volume of water at a set temperature (water baths), or into an ethanol concentration at room temperature.
- Leave each piece for the same time (for example 20 minutes), then remove it.
- Zero the colorimeter with a blank of the same solvent, using a blue-green filter.
- Measure the absorbance (or % transmission) of each liquid.
- Repeat with fresh pieces at each temperature or concentration, then plot mean absorbance against the independent variable.
Aligned with the order of the specification practical. Simulation only: a teacher sets the real volumes, concentrations and risk controls; this is not a laboratory protocol.
Variables
- Independent
- Temperature of the water, or concentration of ethanol.
- Dependent
- Absorbance (or % transmission) of the liquid: the amount of pigment that leaked out.
- Controlled
- Size and surface area of the beetroot pieces (same borer and length)
- Volume of liquid and exposure time
- Rinsing and blotting before exposure
- Colorimeter filter and blank matched to the solvent
Safety
- Cut away from yourself on a tile, and take care with the cork borer and knife.
- Ethanol is highly flammable: keep it away from flames and never heat it.
- Hot water baths scald: use tongs. Beetroot juice stains, so wipe up spills.
Follow your school’s risk assessment; these notes are not a substitute for it.
Interpreting
- Higher absorbance means more pigment leaked, which is indirect evidence that the membranes are more permeable.
- Leakage usually rises slowly at first, then steeply once membrane proteins denature or the bilayer is disrupted.
- Use the means of repeats and look at the spread. Overlapping ranges mean a difference may not be real.
- Absorbance and transmission are inversely related; state which scale you used.
Guided method · step 1 of 8
- In 1 · Plan one investigation, just below these steps, set Water bath temperature (20–80 °C); start at 20 °C.
- Leave Exposure time at 20 min and Tissue preparation on Rinsed and blotted.
- In the Toolbox under the simulator, drag a Beetroot piece into the tube, or press it.
Red betalain pigment is stored in the vacuole. To reach the liquid it must cross the tonoplast and the cell-surface membrane, so the colour of the liquid shows how damaged the membranes are. Rinsing removes pigment from cells cut open during preparation.
1 · Plan one investigation
Change one independent variable. Pigment must cross the tonoplast and cell-surface membrane to leak out, so more colour suggests more disrupted membranes.
Toolbox
Exposure clock
One simulated minute passes every quarter second (slower in slow mode). The piece is removed automatically at the end.
Choose one investigation, set its level, then drag a beetroot piece into the tube.
2 · Measure and record
More pigment gives higher absorbance and lower transmission: %T = 100 × 10−A. Record which scale you use.
3 · Results, graph and interpretation
Record readings to fill the table. Aim for at least three independent pieces at each level.
Evaluate the claim
Independent pieces show biological variation. Unrinsed pieces add pigment from cut cells, a mismatched blank shifts every reading and a different exposure time breaks the comparison.
Make your reasoning visible
Keep optical direction correct
More leakage → more pigment → higher absorbance, but lower transmission. Do not swap the two scales.
Wash before the timed treatment
Rinsing removes pigment from cells damaged by cutting, reducing an uncontrolled starting contribution.
Use a matching blank
A solventA liquid in which other substances (solutes) dissolve. Water is a good solvent for ions and polar molecules. blank corrects background from water or ethanol mixtures; it is different from an untreated tissue control.
Avoid this misconception
The claim: Pigment leaves beetroot by osmosis.
The correction: Osmosis describes water movement. Pigment leakage depends on membrane permeability and movement of pigment down its gradient.
Check your understanding
Try each question before opening the suggested answer.
Practice question
Retrieval and explanation · suggested answer
Explain why unwashed beetroot pieces can give misleading results.
Show suggested answer
Pigment from cut cells can colour the liquid before treatment has affected intact membranes, adding uncontrolled background.
Practice question
Retrieval and explanation · suggested answer
A treatment gives higher absorbance than the water control. Predict transmission and suggest the membrane interpretation.
Show suggested answer
Transmission is lower. More pigment has entered the liquid, consistent with increased permeability under controlled conditions.
Practice question
Retrieval and explanation · suggested answer
Name two variables to hold constant when changing ethanol concentration and explain one.
Show suggested answer
Temperature and exposure time, for example. Longer exposure could release more pigment regardless of ethanol concentration, so use the same timed interval.
Take the idea further
Apply the same specification knowledge to an unfamiliar problem. Any extra assumptions are supplied in the question.
Stretch question
The most concentrated alcohol treatment has lower absorbance than a middle concentration. Give two checks before concluding that its membranes were less permeable.
Show a hint
Consider optical interference and the tissue itself.
Explore the reasoning
Check matched solvent blanks and whether the pigment response changes in that solvent; check tissue size, pigment content, precipitation and instrument range. A lower reading is not automatically less membrane damage.
Recall this lesson
Lesson recall questions
Question 1 of 5: List four variables that must be controlled when comparing alcohol concentrations as the independent variable.
Membrane permeability practical
Practice question
Retrieval and explanation · suggested answer
List four variables that must be controlled when comparing alcohol concentrations as the independent variable.
Lesson: CP3: Membrane Properties and Permeability
Try this from memory first.
Beyond the classroomA visible proxy for an invisible barrierResearch · cell models · Enrichment, not required recall
Practical Biology uses beetroot pigment to investigate temperature effects on membranes. An accessible model makes a hidden cellular property measurable through a visible outcome.
Back to the spec: Specification 2.3: evaluate how well an observed signal represents the property of interest.
Practical Biology: temperature and membranesQuestion
Why might beetroot results not transfer quantitatively to human airway membranes?
Explore a possible response
The cells differ in membrane composition, structure and experimental conditions. The model supports general principles, not an identical numerical response.