Biology RP4 - Investigation into the effect of a named
variable on the permeability of cellsurface membranes.
Method:
1.​ Preparation
●​ Cut Beetroot: Use a scalpel to cut the beetroot into 6-10 identical cubes
(approximately 1 cm³ each).
●​ Rinse: Rinse the beetroot cubes to remove any surface pigment.
2.​ Setting up
●​ Prepare Solutions: Prepare a range of ethanol solutions with varying
concentrations (e.g., 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100% ethanol in distilled water).
●​ Label Boiling Tubes: Label boiling tubes for each ethanol concentration.
3.​ Experiment
●​ Place Beetroot in Solutions: Place one beetroot cube into each boiling tube
containing the different ethanol concentrations.
●​ Incubate: Leave the tubes in a water bath at a constant temperature (e.g.,
30°C) for 20 minutes to allow the pigment to leak out of the beetroot cells.
4.​ Filtration
●​ Filter Solutions: After 20 minutes, use filter paper to filter each solution into a
separate cuvette, removing the beetroot pieces.
5.​ Measurement
●​ Set Up Colorimeter: Set the colorimeter to a blue filter and zero it using a
cuvette with distilled water.
●​ Measure Absorbance: Measure the absorbance of each filtered solution using
the colorimeter. Record the absorbance values.
6.​ Analysis
●​ Plot Graph: Plot a graph of absorbance against ethanol concentration.
●​ Interpret Results: Higher absorbance indicates higher permeability of the cell
membrane, meaning more pigment has leaked out.
Background
a) Structure of Plasma Membranes
The plasma membrane aka cell membrane, is a crucial structure in all living cells. It is
primarily composed of a phospholipid bilayer, where each phospholipid molecule has
a hydrophilic (water-loving) head and hydrophobic (water-fearing) tails. This bilayer
forms a semi-permeable barrier that regulates the entry and exit of substances [1].
Membrane proteins are embedded within the bilayer and perform various functions
such as transport, acting as receptors, and providing structural support. Cholesterol
molecules are also interspersed within the bilayer, contributing to membrane stability
and fluidity. Carbohydrates attached to proteins (glycoproteins) or lipids (glycolipids)
play roles in cell recognition and signaling [2].
b) Structure of a Typical Beetroot Cell
Beetroot cells are plant cells that have a rigid cell wall, a large central vacuole, and
are rich in a red-purple pigment called betacyanin, which is contained within the
vacuole. The cell membrane surrounds the cytoplasm and regulates the movement of
substances in and out of the cell.
Beetroot is often used in experiments investigating cell membrane permeability
because betacyanin is a visible pigment that leaks out when the cell membrane is
damaged. This makes it easy to observe changes in membrane integrity [3].
c) Factors Affecting Membrane Integrity
The permeability of cell membranes can be affected by several factors, including pH,
temperature, alcohol concentration, and detergent concentration. Here, we will focus
on the effects of alcohol concentration.
1.​ pH: Extreme pH levels can disrupt the interactions within membrane proteins
and lipids, leading to increased membrane permeability. Acidic or basic
conditions can denature proteins, altering their structure and function [4].
2.​ Temperature: High temperatures increase the kinetic energy of molecules,
making the membrane more fluid and permeable. Conversely, low
temperatures can make the membrane too rigid, which can also affect its
function [5].
3.​ Alcohol Concentration: Alcohols, such as ethanol, interact with the lipid
bilayer, disrupting the interactions that hold the membrane together. This
disruption increases membrane permeability by making the membrane more
fluid [6].
4.​ Detergent Concentration: Detergents can insert themselves into lipid bilayers
and solubilize membrane proteins and lipids, disrupting the membrane
structure and increasing permeability [7].
Variables
a. Independent and Dependent Variables:
●​ Independent Variable:
○​ Alcohol Concentration: variable you are investigating. You will use
different concentrations of ethanol (e.g., 0%, 10%, 20%, 30%, etc.) to
observe its effect on cell membrane permeability.
●​ Dependent Variable:
○​ Membrane Permeability: variable you are measuring. You will measure
the extent of betacyanin leakage from beetroot cells, which indicates
changes in membrane permeability. This can be quantified by
measuring the absorbance of the solution using a colorimeter.
b. Control Variables and How to Control Them:
1.​ Beetroot Sample Size and Shape:
○​ Control: Use beetroot cubes of the same size and shape (e.g., 1 cm³) to
ensure consistency in surface area and volume, which could affect the
rate of betacyanin leakage.
2.​ Temperature:
○​ Control: Maintain a constant temperature throughout the experiment
(e.g., using a water bath set at 30°C). Temperature fluctuations can
affect membrane fluidity and permeability.
3.​ Incubation Time:
○​ Control: Keep the incubation time constant for all samples (e.g., 20
minutes). This ensures that all beetroot samples are exposed to the
ethanol solutions for the same duration.
4.​ pH of the Solutions:
○​ Control: Ensure that the pH of the ethanol solutions is consistent. Use
buffered solutions if necessary to maintain a stable pH, as changes in
pH can affect membrane integrity.
5.​ Volume of Ethanol Solution:
○​ Control: Use the same volume of ethanol solution for each beetroot
sample (e.g., 10 mL per boiling tube) to ensure that the concentration
effect is consistent across all samples.
6.​ Type of Beetroot:
○​ Control: Use the same beetroot for all samples to minimize biological
variability. Different beetroots may have varying levels of betacyanin
and membrane properties.
7.​ Measurement Technique:
○​ Control: Use the same colorimeter and set it to the same wavelength
(blue filter) for all measurements. Calibrate the colorimeter before use
and ensure it is clean to avoid measurement errors.
Hypothesis
I hypothesize that increasing the concentration of alcohol (ethanol) will increase the
permeability of beetroot cell membranes, leading to more betacyanin pigment
leakage, which results in higher absorbance readings in the colorimeter. This
expectation is based on the fact that higher ethanol concentrations disrupt the lipid
bilayer, making the membrane more fluid and permeable. Additionally, ethanol can
denature membrane proteins, further compromising membrane integrity.
Consequently, higher ethanol concentrations are expected to cause more pigment to
leak from the beetroot cells, as indicated by the higher absorbance readings.
Equipment list
Equipment
Justification
Scalpel
To precisely cut beetroot cubes of uniform
size, ensuring consistency.
Ruler
For accurately measuring the dimensions of
beetroot cubes (1 cm³ each).
Forceps
For safely handling beetroot pieces and
preventing contamination.
Cutting board
A clean, flat surface to ensure accurate and
safe cutting of beetroot.
250ml beaker
Adequate size to contain sufficient solution
for immersing beetroot cubes.
10 ml syringe
Ensures precise measurement of liquids to
maintain consistent concentrations.
Boiling tubes
Suitable size for immersing beetroot cubes
and allows for easy handling.
Boiling tube racks
To hold boiling tubes upright and securely
during the experiment. Keeps samples
organized and prevents spillage.
Distilled water
prepare ethanol solutions and rinse
beetroot pieces, ensuring no impurities that
could affect the experiment.
Ethanol
To investigate the effect of alcohol on
membrane permeability, providing different
concentrations to assess the impact on
beetroot cells.
Water bath
To maintain a constant temperature (e.g.,
30°C) during incubation, ensuring
consistent temperature conditions for all
samples.
Filter paper
To filter solutions and remove beetroot
pieces before measuring absorbance,
providing clear solutions for accurate
colorimeter readings.
Thermometer
To accurately monitor and maintain the
water bath temperature, ensuring precise
control of experimental conditions.
Timer
To control incubation times precisely,
ensuring each sample is exposed to ethanol
for the same duration.
Cuvettes
To hold filtered solutions during colorimeter
measurements, ensuring accurate and
consistent measurement of absorbance.
Colorimeter
To measure the absorbance of solutions,
providing precise and accurate data on
membrane permeability.
Blue filter
For use with the colorimeter to ensure
consistent wavelength, ensuring accurate
absorbance measurements.
Risk assessment
Hazard
Risk
Control measures
Scalpel use
Cuts or injury while cutting
beetroot
Use the scalpel carefully and
cut away from the body.
Wear gloves for protection
and use forceps to hold
beetroot while cutting.
Ethanol
Chemical burns or irritation
from ethanol
Wear safety goggles, gloves,
and a lab coat to protect
skin and eyes. Work in a
well-ventilated area to avoid
inhaling fumes.
Rinse with distilled water
Slips or spills due to water
on the bench
Clean up spills immediately.
Keep the workspace
organized and dry. Use
absorbent pads if necessary.
Boiling tubes and rack
Glass breakage leading to
cuts or injury
Handle boiling tubes
carefully. Use a boiling tube
rack to secure tubes.
Dispose of broken glass
properly.
Water bath
Burns or scalds from hot
water
Use tongs to handle boiling
tubes in the water bath.
Monitor the temperature
with a thermometer. Ensure
water bath temperature is
not excessively hot.
Using colorimeter
Electrical hazards or
equipment malfunction
Ensure the colorimeter and
cables are in good
condition. Follow the
manufacturer's instructions
for safe use.
Filter paper and cuvettes
Contamination or incorrect
readings
Use clean, dry filter paper
and cuvettes for each
sample. Handle cuvettes
with forceps to avoid
fingerprints.
Results table
Ethanol Concentration / %
References:
Absorbance / Arbitrary Units (AU)
[1] Fullick A (2015). Edexcel A Level Biology Student Book 1. Pearson Education. [2] Toole, G., & Toole, S. (2014). AQA Biology A Level
Student Book. Oxford University Press. [3] Jones, M., & Fellowes-Freeman, H. (2016). Cambridge International AS and A Level Biology
Coursebook. Cambridge University Press. [4] Fullick, A. (2015). Edexcel A Level Biology Student Book 2. Pearson Education. [5] Smith, R., &
Bailey, M. (2016). OCR A Level Biology A Student Book 1. Hodder Education. [6] Toole, G., & Toole, S. (2014). AQA Biology A Level Student
Book. Oxford University Press. [7] Jones, M., & Fellowes-Freeman, H. (2016). Cambridge International AS and A Level Biology Coursebook.
Cambridge University Press.