Beet Lab. Aim: To determine the effects of temperature on the permeability of the beetroot cell membrane.

It has to do with the fluidity of the membrane. If the temperature is warmer, the membrane will be more fluid and so more permeable (also a reason why bacteria can be killed by high heat, the membrane gets TOO fluid and ruptures). The lower the temperature, the less fluid the membrane becomes and so the less permeable the membrane is. Other things, such as saturated or unsaturated fatty acids in the lipid membrane part also affect fluidity. Beetroot tissue will be used as the model to investigate the types of environmental stresses that affect membrane integrity. {4}

Reference:

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Literature Graphs:

Hypothesis:

• If the temperature is increased, then the membrane will become more permeable and the absorbance will be greater because the extra energy of higher temperatures disrupts the structure of proteins in the membrane; the absorbance reading is directly proportional to the greater concentration of leaked pigments in the solution.
• Our hypothesis of the study was that as the amount of damage increased, the more betacyanin would be released.  The betacyanin would absorb light of a specific wavelength and we would be able to measure absorbance maximally.
• If the temperature is increased, then the membrane will become more permeable and the absorbance will be greater, because the extra energy of higher temperatures disrupts the structure of proteins in the membrane.
• The absorbance reading is directly proportional to the greater concentration of leaked pigments in the solution.
• The extreme temperatures that the beet cell membranes were put through should have caused denaturation of the proteins that make up the membrane and allow the purple-red pigment bound within the plasma membrane to leak out of the cell. 

• Temp. ranging 20° C to 30° C- The rate of diffusion would speed up due to the low heat, thus increasing the cell permeability.
• Temp. ranging 30° C to 40° C -  Vital proteins such as betacyanin and phospholipids will start to break down, hence making the membrane more permeable than before.
• Temp. ranging 40° C to 50° C - Integrity of the cell membrane becomes compromised, as betacyanin and other proteins become liquids, increasing the cell permeability.
• Temp. ranging 50° C to 60° C - Betacyanin and other proteins start leaking out, causing gaps in the membrane. Cell permeability continues increasing.

Variables:

Apparatus:

• Spectrophotometer
• 5 test tubes
• 5 water baths (20oC, 30oC, 40oC, 50oC, 60oC)
• Hot water
• Ice
• Labels
• Stopwatch
• Glass stirring rod

• Mounting needle/forceps to handle beetroot cores
• Syringe

• Thermometers for each water bath
• 5 cuvettes

Procedure:

1. Obtain the beetroot cores from the lab technician.
2. We put each of the beetroot cores in each of test tubes.
3. Using a syringe, we fill up 10 ml of water and pour it into the test tubes.
4. Meanwhile prepare the water baths (20oC, 30oC, 40oC, 50oC, 60oC).
5. When the water baths are ready, place each test tube in each of the water baths. Have your stopwatches ready.
6. Keep the test tubes for exactly 10 minutes in the water baths.  
7. After 10 minutes, take out the test tubes, discard the beetroot cores and pour out its contents, the pigmented water, into the cuvette.  
8. Ask the lab technician to help you calibrate and warm up the spectrophotometer to 480.5nm
9. Using one cuvette for each trial, measure out the absorbance and transmittance %.
10. Repeat steps 1-9 for all trials.
11. Record your observations in a table.

Precautions:

• Make sure you don’t spill out anything.
• Beware of the hot water baths; use a test tube holder to take out the test tube from the hot water baths.
• Be cautious when using the knife to cut out the beetroot cores.
• Report any spills so they may be cleaned up.
• After the experiment, all the apparatus should be cleaned and wiped properly. Equipments must be stored back to their respective places.
• Before starting the experiment, we should just wipe the apparatus to ensure zero error.
• Do not overtime the experiment.
• When the beetroot was cut some of the membrane was broken causing the pigment to leak out this must be thoroughly cleaned and washed off to secure the accuracy of results.

Data Collection:

Absorbance Table

Transmission % Table

Data Processing:

Absorbance Table

Transmission % Table

Uncertainty calculations of the apparatus

% error is calculated by (uncertainty/value) x 100

We see that as the temperatures increase, the percentage of random errors for uncertainties decreases.

• The total percentage error is calculated by taking the formula (lit value – gotten value/lit value) x 100
• Systematic error is found by taking the percentage error – random error

Transmittance %

Absorbance

Data Presentation:

Conclusion: Higher temperatures also lead to increases in absorbance at which the cell loses a large amount of its betacyanin. The hypothesis that increases in temperature lead to increases in membrane permeability has been shown to be valid. As the size of beetroot gets smaller, surface area is more, therefore permeability is more. Denser the colour, more pigment is present. If absorbance is higher, transmittance ray is lower and vice versa. The effect of temperature on membrane permeability is generally that increased temperatures cause more leakage through the membrane, with the most notable exception being the temperatures close to the freezing point of water. For most of the temperature range, higher temperatures appeared to effect greater permeability, hence higher betacyanin concentration in the solution, and thus greater absorbance.

The membrane becomes more permeable at higher temperatures. This was explained by the fact that proteins denature with high temperatures and the phospholipids structure changes and becomes less stable. When the temperature rises, the molecules have increased kinetic energy which means that the phospholipids and other substances move around more, making the membrane more 'leaky' which means that substances that wouldn't usually do so can enter and leave the cell. As long as the temperature does not go beyond what the membrane is supposed to withstand, the permeability of the plasma membrane should not be affected. Water expands, putting pressure on the membranes from within. The lipid part of the membrane liquefies, making it more prone to leakage. The proteins that span the membrane fall apart, creating holes in the fabric. All this combined will allow compounds to exit the cell.

Higher temperature makes all molecules shake and vibrate more. The faster movement disrupts any ordered structure there might have been, eventually destroying the structure altogether. The absorbance increases with a raise in temperature while the transmittance is quite the opposite it decreases with an increase in temperature.

 Evaluation: