The molecules of cholesterol embedded in the membrane reduce the fluidity of the cell membrane by preventing the movement of phospholipids because it makes the fatty layer more viscous and more repellent to water. Another way for the cell to control the fluidity of its membrane is to regulate the ratio of saturated to unsaturated hydrocarbon chains of the phospholipids. Saturated hydrocarbons are straight-chains so it fits together and can pack close together tightly and hold the phospholipids to each other. Some are unsaturated fatty acids, so it bent and fit together loosely. The more unsaturated they are, the more fluid the membrane becomes. The lower the temperature is, the less fluid it is as well.
We know that when we heat something, you give it energy. Molecules start to spin and vibrate faster. The water will expand too. This will have a disruptive effect on any membrane in its way. To make things worse, lipids become more fluid as temperature goes up so membranes become more fragile.
Proteins are remarkable “machines”. They are formed of coiled and folded strings of amino acids, held together by hydrogen bonds and disulphide bridges. If it is heated too much, it will untangle and break apart causing more vibrations. When this happens to the proteins spanning a lipid membrane, they will form holes that will destroy the delicate structure so any pigments in the innermost compartment will spill out and that is why colour leak out of cooked beetroot as proteins denaturing at high temperature.
Aim:
To use beetroot to examine the effect of temperature on cell membranes and relate the effects observed to membrane structure.
Hypothesis:
An increase in temperature will damage and denature the plasma membrane and cause cytoplasm of beetroot and other substances contained within the membrane to leak out.
Independent Variables:
Temperature- use water baths and measure the actual temperature to allow enough time for the reactions to reach indicated temperature, stated according to the time it responds to.
Volume of water/liquid used- amount needed to put in with beetroot in the labelled boiling tubes are evenly distributed just like the rest.
Beetroot- to maintain rate of reactions for each beetroot on each different temperatures, the size, and cross-sectional area should be in same sectional cut, to provide accurate results and less inaccuracies.
Reaction Vessels- Other reactants with the beetroot, e.g. Distilled water
Time- how long you are going to react the beetroot for, ask the questions: “Why that given time is chosen and relevant.
Range- what range of readings you are going to take, it should be sufficient for reliable support.
Dependent Variables:
Absorbency- What are you going to measure this with?, Why digital colorimeter is suitable and set up the filter that is most appropriate to the experiment?.
Beetroot's Reaction- because beetroot's/plant's temperature doesn't follow human respond to temperature.
Safety Precautions/Risk Assessment:
A) Take care when using a knife when cutting beetroot.
B) Be careful when placing boiling tubes at water bath as it might be too hot for your skin
C) Apply extra handling with apparatus/equipments involved in the experiment
D) Mop spills straight way to prevent further accident and not to spill beetroot juice on skin or clothing as it stains badly.
E) Any student who suffer from accidental cut or any other body accidental contact with hot water must seek medical attention immediately and report to teacher to take appropriate further precautions.
Methods:
1. Cut sections from single beetroot using a sized cork borer. Cut eight 1cm length slices from these sections. Be careful not to spill beetroot juice on skin/clothing as it stains badly.
2. Place the slides in a beaker of distilled water. Leave overnight to wash away excess dye.
3. Next day, place eight labelled boiling tubes each containing 5 cm3 distilled water into water baths at 0, 10,20,30,40,50,60,70/ºC. Leave for 5 minutes until water reaches required temperature. Place one of the beetroot section into each of the boiling tubes leave for 15-20 minutes in water baths.
4. Remove beetroot sections; shake the water/solution to disperse the dye.
5. Switch on the colorimeter and set it to read absorbency percentage.
6. Set the filter dial to the 490 Absorption- blue/green filters.
7. Using a pipette accurately. Measure 2 cm3 distilled water into a cuvette. Place the cuvette into the colorimeter making sure that the light is shining through smooth glides.
8. Adjust the colorimeter to read 0 absorbance for clear water. Do not alter the setting again during the experiment.
9. Place 2 cm3 of the dye solution into a colorimeter cuvette and take reading for absorbency. Repeat readings for all the temperature.
10. Present your results in appropriate way.
11. Identify any trends or patterns in your results.
12. Explain any trends or patterns supporting your statements with evidence from your data and scientific knowledge.
13. Describe how you could have improved this experiment to give more reliable results.
Results:
A table of results to show Average light percentage absorbency of the dye solution in responds to the temperature and time taken.
A GRAPH TO SHOW THE AVERAGE LIGHT ABSORBENCY IN RESPOND TO THE TEMPERATURE INDICATED.
Analysis:
From the graph, the overall trend seems to be that as temperature increases, the percentage absorbency of the light absorbed increases gradually although there were points in the graph that didn't lie on the line of best fit. This is suggesting that membrane permeability decreases with temperature and gradually pigment leaks out. The darker the colour means more pigments have leaked out and higher percentage of absorbency is. As expected, the hottest temperatures were the deepest colour. I understand that this is due to the damage and denaturing of the cell membranes at high temperature allowing the pigment to be released from the vacuole. Results were obtained using a colorimeter, a device used to measure the absorbency of a specific solution.
At the temperature 0ºC, or “freezing point”, the freezing of beetroot first burst the cell membranes and kills the cells thus allowing the pigment to be extracted much more quickly. That is why at the beginning of the results, light absorbency of temperature 0ºC is higher than those of 10-40ºC the so-called in between “room temperatures”.
From 10-40ºC, the points on the graph do not exactly are on the line-of-best-fit. This is because obviously, in between the readings is the room temperature, i.e. 20-30ºC. This is the point were the beetroot starts to receive chemical messages saying that heat is being applied and would cause a response to its function and structure. However it is clear that even though these anomalies occurred, it is obvious that light absorbency gradually increases.
Between the points of 50-60ºC, proteins are denaturing more and more as temperature increases, increasing the light percentage absorbency as more pigments are leaking out due to the pigment's innermost compartment of the proteins spanning a lipid form holes that will destroy the delicate structure. At 70ºC most of the proteins have already denatured and this explains the steep slope on the graph as the most colours has already leaked out and therefore resulting in the highest light percentage absorbency. The fact that because it is known that 70% of membrane is protein, the pigment is heat labile and this is why it is fragile to heat. Two main major reasons of the effect of temperature that had had caused this kind of trend is that at higher temperature, i.e 50-70ºC particles vibrate faster due to higher kinetic energy, so diffusion is also affected. In this case, the pigment has higher chance of being diffused out. Also, the cell membrane has small proteins embedded. Some of which occupy a whole small part. They are usually responsible for allowing substance to pass through. At high temperatures, the proteins denature and thus allowing more substances to pass through. That is to say that membrane is usually in a typical cell, and when heated become more dehydrated. So i would suspect that this was what had happened to the beetroot cells as temperature rise because if it becoming more dehydrated, then it starts to release the liquid(red pigment) from the membrane.(like in human, we lose heat in the form of sweat).
So, as membranes are composed mostly of lipids, when membrane is heated, the lipids will become more destabilized, they will transform into a more liquid like state. This allows the pigments within the cell to leak out through the membrane. This explains why points on the graph, specifically 40-70ºC are darker in colour and have higher light percentage absorbency.
Evaluation:
Throughout the experiment, the evidence produces from the method was good and we could see this from the line of best-fit which can help us to discuss how and why does the temperature affects on cell membranes and that as temperature increases, protein/ plasma membrane will denature and will be damaged and this causes the red pigments to leak, just like what I’ve mentioned on my hypothesis. The fact that lipids will become more fluid as temperature goes up.
Obviously, the anomalies in the graph certainly is present due to different reasons, primarily, as living organisms, beetroot’s respond to temperature do not follow human’s respond to temperature because beetroot has it own ability to react in certain stimuli, e.g heat.
The increase in temperature on the beetroot will also affect the diffusion of the colour dye in the beetroot which is why looking back at my graph, at high points are becoming steeper because absorbency increases with temperature. I believe that with the increase of temperature applied onto the plasma membrane, the structure of the membrane will become damaged and membrane will eventually denature and because membrane’s composition has been occupied by mostly proteins, say 50-60%, the damage to the proteins will be greater and therefore, as the disruption to the proteins gets higher, the more the red pigment will leak out and release darker solution at higher temperature.
As usual, there will be always being sources of different errors that caused the anomalies in the graph results. First problem, was keeping the water bath temperature at exact real temperature. This means during one-minute time, temperature was getting either lower or higher and sometimes needed some extra heating which could result with higher temperature than needed which we definitely experienced during our practical especially in the last two temperature, 60-70ºC because what the thermostat was reading was too low on beforehand. The best solution for this is to use the bath with thermostat readily installed and as a result there could be a possibility of improving the accuracy of experiment results and temperature would also possibly stay the same as we needed it to be and eliminate further systematic/random errors.
Also the temperature of the thermostatic controlled water baths aren’t always constant and might have been slightly under or over the desired temperature. This is because the heater on water baths only starts working once temperature has dropped below what is required, and once the heater has started working, once again it causes the temperature to rise above what it is required, then the bath cools below the optimum temperature.
Another problem that could be a source of error was to take beetroot out of the boiling tube after 30 minutes period of time. After heating the beetroot, it would become softer and there were cases when during taking it out, it has slightly broken so there was more pigment than expected. One good solution that we could have done was to pour the solution into another clean test tube and taking the beetroot out later. Technical nature will always play as one source of error. When we were using the colorimeter, it was acting peculiarly strange because during our testing, numbers on the screen were constantly changing even when we placed the control 0,(distilled water) in the cuvette to the reset the machine, there was some cases when it was reading 0.03 instead of just 0. In some cases, the changing would stop but that could be a not so reliable sign that it is the right colour density. To prevent this from happening, one good respond is to use more sensitive equipment or to get colorimeter checked because it could be broken and will result to equipment failure and false reading.
Human error can be the biggest source of error as well. Because on some occasions, we didn’t shake the boiling tubes with the equal efforts before we took the beetroot out and so could have been one of the reasons why the solution is darker or lighter than it should be. On inaccuracy that can be account to human error is the size of the beetroot. This could be explained by free hand sectioning because this would affect the surface area to volume ratio which in turn affects the diffusion. If the size was slightly smaller, it would have reduced the surface area therefore decreasing the amount of pigment leaked from the sample and makes the solution normal in respond to temperature. Another reason for the anomalies appearing could be due to the preparation sample. Many of the cells could have been damaged causing the pigment to leak during the experiment. A way of overcoming the inaccuracy of the measuring equipment used is to have rulers which are graded more accurately which may help to prevent inaccuracies occurring in the future.
Possible additional work that can be done to rectify errors and would make the experiment much improved than it was before and could make findings more accurate are the following: using digital thermometers which can feed information on temperature continuously to a computer system would be ideal also, the experiment should be repeated more than two or three times and by doing so, it would be easier for me to work out clear average from wide numbers of range values in between results.