5. Investigate the effect of temperature on beetroot membrane permeability. Flashcards
Background information one BEETROOT.
Beetroot cells contain the red pigment Betalain. The pigment is stored in the vacuoles of the cells. Betalain molecules are too large to pass through the phospholipid bilayer of the tonoplast.
Method:
- Prepare eight water baths pre-set to a range of temps between 0 degrees C and 70 degrees C.
- Use a syringe to add 10 cm cubed of distilled water to eight test tubes. Label each test tube with temp.
- Place each tube in the water bath set to the corresponding value for 5 mins.
- check temp of each bath using thermometer. record actual temperature and use this in table and graph.
- Cut eight beetroot cylinders using a cork borer. Using a knife, ruler and white tile, trim them all to the same length (1cm). Wash cylinders thoroughly with water until water runs clear and pat dry gently with paper towel.
- Add one beetroot cylinder to each of the eight tubes. Leave in water bath for 15 mins.
- Shake tubes once. Using forceps, remove cylinders from each tube. Discard cylinders, keeping supernatant liquid.
- Set colorimeter to blue/ Green filter and percentage transmission. Calibrate colorimeter using a blank cuvette with distilled water in.
- Transfer liquid from each test tube in turn into a colorimeter cuvette, place into colorimeter and read percentage transmission, recording results in table.
- Plot a graph of transmission against temperature.
Risks:
Scalding from hot water in water bath:
- Take care when removing lids to allow steam to escape, away from face or body.
- Take care with sharp items.
Analysis of results:
- record results in a table showing repeats and calculate a means for each temp.
- Highlight the maximum and minimum values at each temp and decide if any values are anomalies. repeat tests that have produced anomalous results and calculate a new mean.
- plot a line graph of mean percentage transmission against temperature. Add ERROR bars of the maximum and minimum to show the range of transmission values at each temperature.
Describe the results.
The percentage transmission decreases as the temperature rises. Initially, there is little increase but at around 40-60°C, the percentage transmission decreases sharply. At higher temperatures, the rate of decrease usually levels.
Explain the results:
what happens to the beetroot membrane
At lower temperatures, the tonoplast and plasmalemma are intact and betalain molecules are too large to pass through the membranes easily, meaning light transmission remains high.
The higher the temperature, the greater the kinetic energy and the faster the movement and diffusion of pigment molecules. Greater kinetic energy also causes the phospholipids of the membrane to become more fluid and bonds between the fatty acid tails can begin to separate.
This means the membrane is more permeable, allowing betalain pigment to leave the cells and diffuse back into the surrounding solution. This decreases the percentage transmission through liquid.
The point of sudden increase in percentage transmission occurs when proteins in the membrane begin to lose their tertiary structure. At higher temps, the protein molecules in the membrane become completely denatured, and the membrane develops gaps through which pigment can flood out.
Eventually, the change in transmission levels out as the concentration of pigment is the same inside and outside the cells.
Why are cylinders placed in water baths for five minutes before the cylinders are added?
The temperature must be equilibrated to ensure the tubes contain water at the correct temperature before starting the experiment.
Allows confidence that the correct temperature is being assessed.
Why are beetroot cylinders washed with distilled water and dried before?
To remove excess surface pigment from the cut cells at the edge. This could distort transmission readings, giving inaccurate results.
