How can the permeability of a cell membrane be investigated in the lab?

A typical experiment to investigate permeability of cell membranes involves the use of beetroot. A factor affecting the integrity of the cell membrane needs to be changed, such as different temperatures or different alcohol concentrations. When beetroot cells are exposed to conditions damaging the cell membrane, a pigment (betalain) leaks out through the cell membranes. The more damage occurs to the cell membrane, the more pigment leaks out. This can be measured as absorbance using a colorimeter. A colorimeter gives the absorbance value which is the amount of light that is absorbed by each sample. Higher absorbance values indicate more pigment that has leaked out (greater permeability) and therefore more damage to the cell membrane.

Cell membrane consists of a phospholipid bilayer which makes the membrane partially permeable. Permeability can be altered by various different variables (e.g. temperature, solvent concentration like ethanol). Cell membrane permeability can be measured by utilising beetroot cells, containing betalain (a purple pigment). At higher permeability of the cell membrane, more pigment leaks out of cells. The permeability can therefore be measured by the amount of pigment leaked from beetroot cells into an aqueous solution using a colorimeter. 

Common variables to investigate are the effect of solvents or temperature because both of these factors can change the fluidity of the membrane. An increase in membrane fluidity will cause the pigment to leak out of the cell, and the amount of pigment can be measured simply by using a colorimeter.

The permeability of a cell membrane refers to how easily molecules can flow from inside to outside of a cell, or vice versa. The more permeable a membrane, the more easily cross membrane transport occurs.

There are several ways to investigate the permeability of membranes in a laboratory, however for the biology A level you will need to discuss the classic "Beetroot experiment".

In its most simple form, this experiment involves place slices of raw beetroot into a liquid such as water or ethanol, and then recording how readily the red/purple pigment inside the beetroot cells, moves out of the cells and into the surround liquid. The darker the liquid becomes, the greater the degree of pigment transfer between the cells and the liquid, and so the greater the permeability of the membrane.

In an assessed practical, you will be expected to conduct a semi-quantitative experiment to assess how a named variable impacts the permeability of the beetroot cell membrane. Here a variable can be taken to mean any thing that you can measure during an experiment that can be changed by the person conducting an experiment and can include things like, temperature of incubation, the length of incubation, concentration of ethanol used to suspend the beetroot, etc.

Let us assume that we are interested in how the concentration of ethanol impacts the permeability of beetroot cell membranes, we would design an experiment as follows.

1. First, we must create a set of standards by which we can score colour change / cell membrane permeability. To do this, we will use 100% pure beetroot extract, and create a dilution series in some test tubes. We will begin with 5ml of 100% pure beetroot extract, serving as our highest standard, or standard 10. Next, we will use 5ml of pure water to service as our lowest standard, or standard 0. Now, we must create a range of standards between these two. We will use 20% beetroot extract mixed with 80% pure water to be standard 2, 40% beetroot extract with 60% water to be standard 4, and so on until we have standards 0, 2, 4, 6, 8, and 10.
2. Next, we must create a similar dilution series for our ethanol solutions. We will use 100% ethanol as our strongest solution, 100% pure water as control solution with no ethanol, and a range of combinations of ethanol and water of known concentrations between these two. All solutions should be of equal final volume.
3. We will now use a water bath to heat our solutions to a uniform temperature, removing the impact of temperature as a variable.
4. We will now add identical pieces of beetroot, most likely discs, into our solution ethanol tubes, the tubes will be left in the water bath for a specified period of time and shaken to precise, predetermined intervals.
5. Once our incubation time is over, we will remove the solution from the pieces of beetroot into a clean test tube for each solution tested. We can then score each solution for the presence of beetroot pigments against our predetermined standards that we created earlier. If our solutions fall between two different standards, such as 2 and 4, we can give the solution and intermediate score of 3.
6. We can then plot our data to determine how the concentration of ethanol in a solution impacts the degree of colour transfer and so the level of cell membrane permeability.

This is a semi-quantitative experiment, meaning that as the researcher, you will be able to make observations about how much the colour changes, but we lack a definitive scale by which to judge the true amount of permeation across the cell membrane. As we are manually classifying colours, it is subjective and can be open to a degree of interpretation. The use of a colourimeter to determine the colour of the solutions removes this subjectivity and is one way in which this experiment could be strengthened. Another way this experiment could be improved is to use replicates of each tested solution concentration, preferably at least 3, to increase our confidence in our collected data.

Permeability of a cell membrane = how easily molecules can flow from inside to outside of a cell, or from outside to inside.

More permeable a membrane = the easier cross membrane transport occurs. For Biology A level you will need to talk about the Beetroot experiment

BEETROOT EXPERIMENT

• place slices of raw beetroot into a liquid such as water or ethanol
• Record how readily the red/purple pigment inside the beetroot cells, moves out of the cells and into the surround liquid.
• The darker the liquid becomes, the greater the degree of pigment transfer between the cells and the liquid, and so the greater the permeability of the membrane.

A simple test is done by using beetroots and a condition damaging the cell membrane (e.g. alcohol or changing temperature).

When the beetroot membrane is exposed to conditions that damage it, the pigment leaks out. The more pigment that leaks out (indicative of more damage) can be measured by absorbance into a colorimeter. This produces an absorbance value which tells us the amount of light absorbed by each sample.

Higher absorbance values tells us that there has been more damage done to the membrane as reflected by more pigment leaking out.

By doing an experiment using beetroot.

Take any plant sample for example onion. Peel the inner layer and place in different solutions of isotonic, hypertonic and hypotonic.

Results will show the permeability of cell membrane.

Using Beetroot and a colorimeter

Cell-surface membranes are made up of a ​phospholipid bilayer​​ which makes them selectively permeable​​. This permeability can be changed by different variables, such as ​temperature​​ and ​concentration of solvents,​​ like ethanol. 

The permeability of a membrane can be measured by using beetroot cells, which contain a purple ​pigment ​​called​ betalain. ​​

When the cell-surface membrane has a higher permeability, more pigment leaks out of cells. The permeability can therefore be measured by the ​amount of pigment leaked​​ from beetroot cells into an aqueous solution using a ​colorimeter​​. 

Results will typically be presented as a line graph

A simple way of investigating the permeability of a cell membrane in a lab is by using beetroots.

Beetroot cells contain betalain - a purple pigment. If the cell membranes have a high permeability, betalain will leak out of the cell, causing the external solution to become coloured. The amount of pigment which leaks out can be determined using a colorimeter.

For this experiment, the variables that can be investigated are the concentration of ethanol solution and the temperature.

The method used is:

1. Cut 7 cubes of the same size from the same beetroot.
2. Blot the cubes gently on a piece of paper towel to remove any pigment released during Step 1.
3. Create a dilution series of ethanol ranging from 0% - 100% in 20% increments.
4. Place one cube in each solution.
5. Let stand undisturbed for 30 minutes and then take the beetroot out of the solutions.
6. Zero the colorimeter with distilled water.
7. Measure the absorbance of each solution, noting down the results in a table.
8. Plot a graph of absorbance against ethanol concentration.

Using a coloured plant tissue, often beetroot, place cut, peeled and washed pieces into water at different temperatures or a range of ethanol concentrations, depending on what you are investigating. Leave for a few minutes and the colour inside the cells will leach out depending on the permeability of the cell membrane. Remove the pieces of tissue and assess the concentration of pigment in the liquid, either by sight, comparison to standards or preferably using a colorimeter. The greater the concentration the more permeable the cell membrane was.

First! we need to understand, 'What is permeability?'. A substance is permeable if it can allow molecules to pass thorough the membrane. This property is called 'Permeability'. And it mainly depends on the substance's electric charge and to some extent on the molecular mass also. So, Now Lets come to Cell membrane permeability, Cell membrane is selective in nature and only allows some molecules to pass through it. The best way to study the 'Permeability of a cell membrane in the lab is 'Beetroot test'. Keeping in mind, factors like optimum temperature, pressure, light exposure is necessary. "Why only Beetroot?', this question may come to our mind, So the answer to this question is, Beetroot is very rich in pigments which helps to understand the mechanism of cell membrane permeability, and thus Beetroot is used as a specimen for the lab investigation. Increase in temperature during the experiment increases the fluidity of the membrane which increases the mobility of the pigment and at a very high temperature, the pigment oozes out of the cell membrane. The quantity of the pigment can be measured with the help of a colorimeter.

By injecting a coloured dye into the cell of interest and measuring change in colour over time

Hi, quick practical method by OCR

The effect of temperature on membrane permeability                                                                                             STUDENT

Introduction

Beetroot cells contain the purple pigment, betalain, as shown in the diagram below:

This experiment investigates the effect of temperature on membrane structure by considering the leakage of betalain from the beetroot cells over a range of temperatures. The quantitative assessment of this is carried out using a colorimeter.

Aim

·        To investigate the effect of temperature on membrane structure.

·        To understand the use of a colorimeter.

Intended class time

·        1 hour

Chemicals

Not applicable for this activity.

Equipment (per class)

·        Thermostatically controlled water baths at 30°C, 40°C, 50°C, 60°C and 70°C containing test tube racks

Equipment (per 4-6 students/groups)

·        Colorimeter and green filter

Equipment (per student/group)

·        Thermometer

·        1 beetroot cylinders at least 30 mm long

·        Knife / Scalpel

·        Ruler

·        White tile

·        Paper towel

·        6 test tubes

·        6 cuvettes

·        Distilled water

·        10 cm³ syringe

·        Marker pen / Chinagraph pencil / OHP pen / Stickers

·        Forceps

·        Timer

·        Test tube rack

Health and Safety

Take care with the knife when trimming the cylinders of beetroot to the correct length. The water in the water baths over 40°C will be hotter than hand-hot so be aware of this.

Procedure

1.      Take the six test tubes and label each with one of the temperatures from 20°C to 60°C.

2.      Add 10 cm³ distilled water to each tube using the syringe.

3.      Place the correctly labelled test tube in its corresponding water bath for 5 minutes to allow the water to equilibrate to the correct temperature. Record the actual temperature with the thermometer. Leave the tubes in the water baths.

4.      Cut the beetroot cylinder to give six 5 mm cylinders using the knife and ruler on the white tile.

5.      Rinse the cylinders under a running tap and pat dry using paper towel.

6.      Add one 5 mm beetroot cylinder to a tube in each temperature and leave for 15 minutes.

7.      Remove the tubes from the water bath, carefully swirl once and use the forceps to remove the cylinders. Throw the cylinders into a waste receptacle.

8.      One tube at a time, carefully pour the remaining liquid into a cuvette.

9.      Use the colorimeter to measure the absorption for each temperature and record this information in a suitably designed table. The absorption is measured in arbitrary units (AU).

10.  Plot a graph of temperature against absorption and draw an appropriate line.

Extension questions

1.      Describe the relationship between temperature and the rate of leakage of the pigment from the beetroot cells.

2.      Explain your results and the shape of your graph using theory about membrane structure.

3.      What effect might a solvent such as ethanol have on membrane permeability? Explain your answer using ideas about membrane structure.

4.      State four limitations of this procedure.

What to record

As evidence for the Practical Endorsement, you should have your table and graph of the data as described above. All work should be clearly dated.

In addition you should have considered the above questions as the answers to these questions will aid you in preparation for your written examinations.

Kind Regards

Carrie Garbowska

Permeability of Cell membrane can be measured by using beetroot cells that contain betalain which is a purple pigment. If permeability of the cell membrane is higher, more pigment'll leak out of cells. This can therefore be measured by using a colorimeter.