Core Practical 3: Investigating Membrane Structure & Permeability

what factors affect permeability of cell membranes

• The permeability of cell membranes is affected by several factors such as

  • Temperature

  • pH

apparatus

• Scalpel

• Cork borer 

• Cutting board

• Ruler

• Digital balance

• Test tubes

• Measuring cylinder

• Water baths

• Digital stopwatch

• Pipettes

• Colorimeter and cuvettes

method tot est for effect of permeability on different temperatures

1. Using a cork borer and scalpel, cut five equal-sized sections of beetroot

   • The pieces must have the same dimensions so that they all have equal surface areas and volumes, as these factors could affect the rate at which the pigment leaks out

   • A cork borer enables cores of equal diameter to be cut, which can then be cut to the same length

2. Rinse the beetroot pieces

   • To remove any pigment released during cutting

3. Add the beetroot pieces to five different test tubes, each containing the same volume of water, e.g. 5 cm³

4. Put each test tube in a water bath at a different temperature, e.g. 10 ℃, 20 ℃, 30 ℃, 40 ℃, and 50 ℃, for the same length of time

   • The time should be long enough to allow the pigment to diffuse into the water, e.g. around 30 minutes

   • check temp of ater bath using thermometer

5. Remove the beetroot pieces, leaving just the coloured liquid in the five test tubes

6. Use pipettes to transfer samples of the coloured liquid to colorimeter cuvettes

   • Cuvettes are small cuboid containers that hold the liquid to be measured in a colorimeter

   • A different pipette should be used for each sample to avoid the transfer of pigment molecules between samples

   • skae tube once. Discard cylinders but keep the supernatant liquid

7. Use a colorimeter to measure how much light is absorbed as it passes through each of the five samples of coloured liquid

   • The higher the absorbance, the more pigment must have been released due to a greater membrane permeability

   • set it to a blue/green filter and prcenatge transmission. Zero colorimeter usign a blank cuvette wiht distilled water

analysis of effect of temperature

• highlight the maximum and minimum values at each temeprature and decide if any values are anomalies. If possible repeat the tests that produced these results and calculate a new means

• plot a graph of mean percentage transmission against temeprature.

Describe the effect of temperature at 0°C to 40°C on membrane permeability

• As temperature increases, the phospholipids within the cell membrane move more because they have more kinetic energy; increased movement means the phospholipids are not as tightly packed together, increasing the permeability of the membrane

Describe the effect of temperature above 40°C on membrane permeability

1. Proteins in the membrane deform/denature at high temperatures, meaning they cannot control what goes in and out of the cell. This results in increased membrane permeability.
2. Phospholipids start to melt.
3. Expanding water inside the cell puts increasing pressure on the membrane

testing permeability of membranes using alcohol

1) Take five test tubes and add 10cm3 of ethanol to each one. Use a different concentration of ethanol in each tube (distilled water can be used for 0% concentration)

2) Use a cork borers to cut five beetroot cylinders. Usse a knife, ruler and white tile to trim them all to teh same lenght. Wash the cylinders thoroughly with water until the water runs clear, then gently pat dry with a paper towel

3) Add one beetroot cylinders to each of the five tubes and leave for 15 minutes

4) Shake the tubes once. Then, working quickly and carefully, use forceps to remove the cylinder from each tube. Discard the cylinders but keep the supernatant liquid (the clear liquid above the soid). It may be easier to decant this liquid into clean test tubes

5) Set the colorimeter to a blue/green filter and percentage transmission. Zero the colorimeter using a blank cuvette filled with distilled water

6) Transfer liquid from each test tube in turn into a colorimeter cuvette, place in the colorimeter and take the percentage transmission reading. Record your results in a suitable table

results of alcohol on permeability

• As alcohol concentration increases the permeability of the membrane increases

• This is because alcohol dissolves lipids in the cell surface membrane so the membrane loses its structure and the beetroot pigment can leak out

analysis of effect of alcohol

• highlight the maximum and minimum values at eah concentration and decide if any values are anomalies (results that show a substantial deviation from teh general pattern of results.) If possible, repeat the tests that produced these results and calculate a new mean

• Plot a graph of mean percentage transmission against concentration. Add error bars to show the rance of transmission values at each concentration of alcohol

limitations of this experiment

• Cuvettes may differ in thickness; a thicker cuvette will absorb slightly more light than a thinner cuvette

  • Solution: use the same cuvette for every reading, or repeat the investigation many times and find a mean

  • Note that scratched cuvettes can have the same impact on absorbance as thicker cuvettes

• The beetroot pieces may not be identical in size and shape, meaning some test tubes could contain slightly more beetroot tissue than others

  • Solution: cut the discs as accurately as possible using a scalpel and ruler, and repeat each investigation several times to find a mean

• Some parts of beetroot tissue have more pigment in their cells than others

  • Solution: conduct several repeats, using different parts of the beetroot and find a mean

Size and surface area of beetroot pieces

Type of variable: Controlled variable

Why controlled: The surface area affects how much pigment can diffuse out. If the pieces are different sizes, the amount of leakage won’t be due to just alcohol or temperature.

How controlled: A cork borer was used to cut uniform cylinders of beetroot, and a ruler or scalpel was used to ensure all pieces were the same length (e.g., 1 cm each).

volume of solution used

Type of variable: Controlled variable

Why controlled: Different volumes can affect the concentration gradient and how much pigment is diluted.

How controlled: The same volume (e.g., 10 cm³) was measured for each test tube using a measuring cylinder or pipette.

source of beetroot

Type of variable: Controlled variable

Why controlled: Different beetroots can contain different amounts of pigment, which would affect results.

How controlled: All samples were taken from the same beetroot and the outer slices were discarded to avoid damaged cells.

Time beetroot is left in the solution

Type of variable: Controlled variable

Why controlled: Longer exposure allows more pigment to leak, regardless of alcohol or temperature.

How controlled: A stopwatch was used to make sure each sample stayed in the solution for the same amount of time (e.g., 10 minutes).

Washing the beetroot before the experiment

Type of variable: Controlled variable

Why controlled: Cutting the beetroot can release pigment from damaged outer cells, which would affect the starting absorbance.

How controlled: All pieces were rinsed thoroughly with distilled water before being placed in test tubes.

Volume of beetroot sample

Type of variable: Controlled variable

Why controlled: Larger or heavier samples can release more pigment.

How controlled: The same size and mass of beetroot was used in each test.

Wavelength setting on the colorimeter

Type of variable: Controlled variable

Why controlled: Different wavelengths can give different absorbance readings.

How controlled: The same wavelength (typically around 520 nm for red pigment) was used for all readings.

pH of the solution

Type of variable: Controlled variable

Why controlled: Extreme pH levels can also damage membranes, which would interfere with the results.

How controlled: The solutions were kept at a neutral pH or buffers were used if needed

Light intensity during measurement (if using a colorimeter)

Type of variable: Controlled variable

Why controlled: External light can affect the accuracy of colorimeter readings.

How controlled: Readings were taken in a consistent lighting environment or within the enclosed colorimeter.

Suggest why the tubes were placed in the water baths for 5 mins before the cylinders were added.

The test tubes were placed in the water baths for 5 minutes before adding the beetroot cylinders to allow the solutions to reach the desired temperature.

This step is important because:

It ensures that the only variable affecting the membrane permeability is the set temperature — not the gradual heating of the solution.

If the beetroot were added before the solution reached the target temperature, the cells would be exposed to a range of temperatures, making the results inconsistent and less reliable.

Pre-equilibrating the solution makes sure that all samples start under the same conditions for a fair comparison.

So basically, it improves the accuracy and validity of the experiment by making sure temperature is the only factor causing changes in permeability at that stage.

Why were the beetroot cylinders washed with distilled water and dried before the experiment began?

The beetroot cylinders were washed with distilled water and dried before the experiment began to:

Remove any pigment released during cutting

Cutting the beetroot can damage some cells on the surface, causing betalain pigment to leak out immediately.

If this pigment isn’t washed off, it could affect the starting absorbance and give inaccurate results, since the leakage would not be due to temperature or alcohol.

Ensure a fair test:
All beetroot pieces need to start with clean, undamaged surfaces, so any pigment released during the experiment reflects the effect of the independent variable (e.g., temperature or alcohol concentration), not leftover pigment from cutting.

Prevent contamination between samples:
Washing and drying the beetroot helps avoid cross-contamination and ensures each test is independent and reliable.

In short, this step helps improve the accuracy, validity, and consistency of the experiment.

Explain how (a) high temperatures and (b) ethanol damages cell membranes. Make reference to the fluid mosaic model in your answer.

🥵 (a) High Temperatures

How it damages the membrane:

• High temperatures give the phospholipids more kinetic energy, causing the bilayer to become more fluid and unstable.

• As temperature continues to rise, membrane proteins (which help transport and support the structure) begin to denature.

• The disruption of the proteins and excessive fluidity lead to the breakdown of membrane integrity, making it more permeable.

• Eventually, gaps form in the bilayer, allowing substances (like beetroot pigment) to leak out.

Fluid mosaic reference:

• The “fluid” part of the model increases with heat, but too much disrupts the carefully arranged mosaic of lipids and proteins.

• Proteins lose their shape and function, disturbing the structure of the membrane.

🍷 (b) Ethanol

How it damages the membrane:

• Ethanol is a non-polar solvent that can dissolve lipids in the phospholipid bilayer.

• It disrupts the orderly arrangement of the phospholipids, making the bilayer more leaky or even breaking it down entirely.

• Ethanol also denatures membrane proteins, further weakening the structure and control of what enters or exits the cell.

• This causes substances inside the cell (like beetroot pigment) to leak out more easily.

Fluid mosaic reference:

• Ethanol affects the phospholipids (fluid layer) by breaking up their structure and removing their ability to form a selective barrier.

• The "mosaic" is disturbed because proteins lose their proper shape (denature) and may no longer function in transport or signaling.

The cellulose cell walls of plant cells are permeable whereas cell membranes of plant cells are partially permeable. Explain the meaning of the terms (a) permeable and (b) partially permeable

🌿 (a) Permeable

A permeable structure allows all substances to pass through it freely, regardless of their size or type.

There is no restriction — substances can move in or out without being selectively filtered.

🧬 (b) Partially Permeable

A partially permeable (or selectively permeable) membrane allows some substances to pass through, but blocks others, usually based on size, charge, or solubility.

It acts like a filter, letting only certain molecules in or out — often essential for maintaining homeostasis.