Investigating the Effect of Temperature on Rate of Respiration in Yeast

Investigating the Effect of Temperature on Rate of Respiration in Yeast
Plan Yeasts are unicellular organisms, which belong to the fungi group. {Autotrophs are organisms that can produce their own stored energy resources, they can capture sunlight and use it in the process of photosynthesis. Animals, fungi and most other organisms cannot produce their own food, they can only consume their energy sources, these organisms are given the name heterotrophs [taken from resource 3]}. Yeast can be used for baking, because the carbon dioxide released causes the food to expand or �rise�. Yeasts also has other uses such as making alcohol. In the mitochondria of the yeast various biological pathways take place, including the Links reaction, Krebs cycle and the electron transport chain. The structure of the mitochondria is fairly simple, there are basically 4 main parts to it. There is the envelope, which consists of the outer and inner membrane. {The outer membrane is slightly permeable to small molecules, but the inner membrane is less permeable. The inner membrane contains proteins, which are necessary to carry out the electron transport chain and oxidative phosphorylation (the inner membrane is also the site for the electron transport chain and oxidative phosphorylation).The inner membrane also has small spheres attached to it, which are atp synthase enzymes. There are the cristae, which is parts of the inner membrane that have been folded inwards. In active cells, the cristae are densely packed. The matrix is the site of the links reaction and the Krebs cycle, and also contains the relevant enzymes for these pathways. The matrix also contains ribosomes. The intermembrane space works in conjunction with the matrix to produce atp, as the energy required to produce the atp comes from the hydrogen ion gradient between the intermembrane space and the matrix [taken from biology 2 book]}. {Under aerobic conditions, yeast mitochondria are involved in atp synthesis coupled to oxidative phosphorylation. Under anaerobic conditions, mitochondria seem to be dispensable, at least for respiratory function. Mitochondria perform other functions in yeast cell physiology, implicating that mitochondria are relevant to intact cell metabolism even under anaerobic conditions, such as synthesis and desaturation of fatty acids and lipids, or stress responses and adaptations to stresses. [http://biochemie.web.med.uni-muenchen.de/Yeast_Biology/02_Cellfunction.htm]} [image] {The �energy currency� for cells is known as atp (adenosine triphosphate). atp contains adenine and ribose which make up adenosine. And the adenosine is combined with three phosphate groups. When a phosphate group is removed energy is released and adp (adenosine diphosphate) is formed. Removing of a second phosphate releases the same amount of energy, and amp (adenosine monophosphate) is formed. Removal of the final phosphate group releases about half the energy, and leaves only adenosine. These reactions are all reversible, but require the same energy to convert them back to the original state. [Biology 2 book]} [image] In respiration organic molecules are broken down in different steps which release chemical potential energy, which is used to synthesise atp. Glucose is the main energy source, and can be broken down in four different stages: Glycolysis, the link reaction, the Krebs cycle, and oxidative phosphorylation. Yeast is an organism that actively respires aerobically. But it can respire anaerobically, when deprived of oxygen. But anaerobic respiration is not as efficient as aerobic respiration, because oxidative phosphorylation requires a constant supply of oxygen for it to occur; also the link reaction and the Krebs cycle need oxygen. Therefore the link reaction, Krebs cycle and oxidative phosphorylation only work in aerobic conditions, whereas glycolysis can work in anaerobic conditions also. Glycolysis means �sugar-splitting�. In glycolysis, there is a net gain of 2 atp molecules. You must start with a hexose sugar, such as glucose. The glucose molecule will undergo phosphorylation. Phosphorylation involves atp molecules being broken down into adp and their phosphates are attached to the glucose molecule. The first atp causes the hexose sugar to be turned into hexose phosphate, and the second atp molecule makes it hexose bisphosphate. Phosphorylation raises the energy of the glucose, and reduces the activation energy barrier for the pathway. After phosphorylation, the hexose bisphosphate is split into 2 other identical molecules of triose phosphate. Hydrogen is removed from the triose phosphate using dehydrogenase enzymes, and the hydrogen is taken by an nad molecule. nad is a hydrogen acceptor or a coenzyme molecule, and is reduced when it takes hydrogen. Also, the phosphate groups on the triose phosphate molecules are taken by adp molecules to produce atp. During these final stages of glycolysis 4 molecules of atp are produced. Since only 2 molecules of atp are used initially, and 4 are made in the end, there is a net production of 2 atp molecules. Glycolysis [image] {Pyruvate enters the link reaction next. But this pathway occurs aerobically. The pyruvate passes from the cytoplasm of the cell into the matrix of a mitochondrion by active transport. In the matrix, the pyruvate is decarboxylated using decarboxylase enzymes and it is dehydrogenated also, which produces more reduced nad. After that it is combined with coenzyme A to produce acetyl co enzyme A. [Biology 2 book]} [image] [image] The Krebs cycle is also known as the citric acid cycle, and it also occurs in the matrix of a mitochondrion. Oxygen is not required for this pathway, but it is part of aerobic respiration, so it must be carried out in aerobic conditions. There are a few main products of the Krebs cycle. Acetyl coenzyme A, from the link reaction, combines with oxaloacetate to form citrate; the citrate is decarboxylated and dehydrogenated, carbon dioxide is a waste gas and is diffuses out of the cell and is excreted, there is production of 2 carbon dioxide molecules per cycle. But the hydrogens are accepted by nad and fad, one fad is reduced per cycle and three nad molecules are reduced. Oxaloacetate is then formed and the cycle starts again. Reduction of nad and fad is a key factor for the final pathway, Oxidative phosphorylation. The electron transport chain is a system of electron carriers held within the inner membrane of the mitochondrion. {Here, the fad and nad molecules lose their hydrogens and give them to the hydrogen carriers, the hydrogens are split into its constituent H+ ions and electron, the hydrogen ion remains in the matrix. The electron is transferred to a series of electron carriers, the electron is soon transferred to an oxygen molecule (which is present in the mitochondrial matrix), and hydrogen ions combine with the oxygen molecule to reduce the oxygen to water. The transfer of these electrons creates energy which is used to convert adp into atp. When an electron passes from a higher energy carrier, to a lower energy carrier, energy is released. On average, 2½ atp molecules are made for each nad molecule entering the chain, and 1½ molecules of atp for every molecule of fad used in the chain. [Biology 2 book]} [image] Enzymes are also present in respiration. They are in the form of dehydrogenase enzymes, decarboxylase enzymes and atp synthetase enzymes. atp synthetase enzymes are used in the synthesis of atp from adp. atp synthetase is a protein that spans the phospholipids bi-layer. atp synthetase has 3 binding sites. The 3 binding sites allow the atp to pass through 3 different stages, binding adp and a phosphate group, forming a tightly bound atp molecule, and releasing atp. As you can see enzymes have very important roles in respiration. Enzymes are basically biological catalysts, which have a main role of breaking down substances into smaller molecules. Enzymes also lower the activation energy of reactions. Most enzymes have an optimum temperature of 37º, but optimum temperatures can vary from enzyme to enzyme. Enzymes and their substrates form enzyme-substrate complexes when joined together. [image] There are a few factors which affect the rate of reaction with enzymes. I will discuss these, because we now understand that the rate of respiration must also be dependant on the rate that the enzymes work. First we must consider the collision theory. The collision theory states that when two molecules react, they must have sufficient energy on impact to overcome the activation energy barrier and react. This energy should be enough to break the old bonds and form new bonds for the new molecule. Temperature Enzymes work up to their optimum temperature proportionally to the rate of reaction. Increase in temperature increases the kinetic energy of the particles which causes them to move faster and faster. If they begin to go too fast, atoms begin to vibrate too vigorously and the chemical begins to break down. When this happens the shape of the enzyme is altered and it is said to be denatured, and an enzyme cannot repair itself after it is denatured. When the temperature goes very low, the kinetic of the particles is very low too, and the particles mostly do not have enough kinetic energy to overcome the activation energy barrier, so the rate of reaction should be very low at this temperature. [image] PH level Enzymes all have optimum pH levels at which they work at. PH is basically a measure of hydrogen ions in a solution. If the pH level gets too high, the protein structure of the enzyme will be denatured. Smaller pH level creates changes in the R groups, which also can change the shape of the enzyme. The changes cannot be reversed. Enzyme Concentration If the enzyme concentration is high the rate of reaction will be a straight line graph, but at a point the graph will slowly level off. This plateau will be due to the fact that the amount of substrates is limited. Substrate concentration If the substrate concentration is high the graph for rate of reaction will be fast. But as the substrate concentration gets more and more the graph will again reach a plateau, where the enzymes will be constantly working to react the substrate molecules. When the reaction reaches this point of maximum output, the point is known as the Vmax. Therefore substrate concentration highly affects the rate of reaction. [ here write about methylene blue ] Prediction I predict that as I increase the temperature of the yeast suspension, the rate of reaction will increase. The rate of reaction will increase constantly until the optimum temperature is reached. As the temperature gets hotter still, the enzymes will start to denature and the graph will experience a decline. Graph for effect of temperature on enzymes. [image] My prediction is brought on by the fact that respiration involves many enzyme-controlled pathways. The kinetic energy of the particles increase as the temperature increases, which means that the temperature should constantly increase the rate of respiration. But if the temperature get�s too high, the enzymes may become denatured. If the enzymes are denatured, then the respiration should slowly come to a halt. This theory can be proven using an experiment, because enzymes are involved in some of the biological pathways for respiration. Some of the main enzymes are dehydrogenase enzymes and decarboxylase enzymes, the dehydrogenase enzymes take hydrogen and pass them onto nad molecules, or simply let them stay in the matrix, and wait to be bonded with an oxygen molecule. Another important enzyme is atp synthetase, which synthesises atp from adp. Method I can choose from a number of methods, all of which are reasonably accurate in their own ways. I could use a Gas burette, which could collect bubbles of carbon dioxide that are produced during respiration. I could use a manometer, which pushes a liquid up as more carbon dioxide is produced. I could use a hydrogen-carbonate indicator, which turns from red to yellow as carbon dioxide is added to it. I also have the choice to use redox indicators. I could use ttc which slowly turns to pink, and continues changing to darker shades of pink without stopping. I also have the choice to use methylene blue (redox indicator), this changes form its original blue colour, to the colour of the yeast as the yeast respires. I had the choice of using these different techniques. But my background research indicates that methlyene blue indicator is the best test; which I will justify later. First I will gather all of the apparatus required and set it in a safe position and one that will allow easy reading for the results. I will examine all of the equipment to ensure everything is in complete working order and there are no faults. I will place my test tubes in a test tube rack, so that they are safe. I will put my thermometer in the beaker for ease of use, and to make sure it doesn�t break by falling down. I will gather some yeast suspension and put it into a 100cm³, and fill it roughly two thirds of the volume of the beaker. I will take 5cm³ using a 10cm³ syringe of the yeast suspension and place it in a test tube, I will do this very carefully so I can gain precise results. I will set a thermostatically controlled water bath to the required temperature and place my beaker (which also contains water) in it, and wait for the water to equilibrate with the water in the water bath. In the case that the thermostatically controlled water bath cannot be set to the required temperature, I will have to use a kettle to heat water, or ice to make the water colder. I will mix hot and cold water until I get the required temperature, and I will keep my thermometer in it during the reaction so that I can see if the temperature goes any lower or too high, so I can adjust it accordingly. I will put the yeast suspension which is in the test tube, into the beaker that is at the set temperature. I will allow it to sit in the beaker for 1 minute, to allow it to equilibrate with the water in the beaker. This is done so that the yeast suspension is at the right temperature before the methylene blue is added. I will then use the pipette containing the methylene blue and add three drops of it into the yeast suspension which is at the set temperature. I will then immediately place a bung over the test tube and invert it twice. After inverting it I will remove the bung and place the test tube carefully back into the beaker. I will then start the analogue stop-watch. I will wait for the methylene blue indicator to disappear and for the original colour of the yeast to return, and then I will stop the analogue stop clock. I will record the results in an appropriate results table. I will then wash the test tube that I used and allow it to dry while I carry out the experiment at the other temperatures. I will carry the experiment out at 5 different temperatures: 35ºC, 40ºC, 45ºC, 50ºC and 55ºC.I will repeat the experiment 3 times for each temperature used; this is simply to improve the reliability of my results. Because I am using such temperatures I will not need to use ice. Justification of Method I have explained what each method involves above, I will now justify why I used the chosen method and not the others. The gas burette would provide accurate amounts of carbon dioxide and the amount will be reliable. But the measurements will be hard to record accurately because the meniscus takes up a large space and curves as well. The Manometer should be able to provide accurate results but it would be risky. This is because the manometer tube is very thin, and the carbon dioxide produced may cause the liquid to seep out of the manometer and ruin the apparatus. This is also a health and safety hazard. So the Manometer will not be used. The hydrogen-carbonate indicator was a good experiment. It is reliable, accurate and simple. The only problem I found was that the indicator takes a very long time to change colour. The indicator is sensitive to pH changes and indicates when decarboxylase enzymes release carbon dioxide. If the carbon dioxide production is not sufficient, the indicator may even turn purple, which is a totally unwanted result. So the hydrogen-carbonate indicator is not very reliable either. ttc could have been used also. ttc is a redox indicator which goes into the yeast by diffusion. ttc accepts the hydrogen that is taken off by the dehydrogenase enzymes, which are usually given to the nad. However, ttc is sensitive to sunlight. Due to this sensitivity ttc may begin to turn pink when stored in any conditions which contain sunlight, and alterations of the amount of sunlight could provide very unreliable and anomalous results. Also the ttc changes to a darker and darker shade of pink. We would have to stop the stop clock at a certain colour of ttc. Trying to remember and stop the stop clock at a certain point is probably impossible by the naked eye. Even a colorimeter cannot be used. So ttc is not a very good indicator to use. I believe that methylene blue is the best indicator to use. It is the most reliable and does not require copious amounts of anything, and it doesn�t take a long amount of time. A shorter period of time gives a higher reliability. Methylene blue is also a hydrogen acceptor, as ttc. But methylene blue reaches a definite point where it has all gone, unlike the ttc which continues reacting for a long time. I will use 3 drops of methylene blue for my experiment because it is not too much so that the experiment takes an extremely long time to be carried out. But it is not too small so that at higher temperatures I do not get an unreliable amount of time. Variables The variables have been discussed earlier, but I will link them to the chosen experiment to make it clearer on what I plan to do. PH level will have to be controlled because it effects the structure of enzymes. The pH level should remain constant throughout the experiment without my need to interfere. The yeast concentration (linked to enzyme concentration) must to be kept constant. If different yeast concentrations are used then the results will be extremely unreliable. As I have explained earlier, the enzyme concentration greatly affects the rate of reaction to a certain extent. So if the concentration of the yeast varies, it will affect the rate of respiration in the yeast too. I will therefore have to use the exact same yeast suspension for my entire experiment, or my results will be unreliable. I will also use the same amount of yeast for each temperature in every repetition. The glucose concentration (linked to substrate concentration) must also be kept constant. If the concentration does not stay the same it will take different amounts of time for the methylene blue to decolourise. Earlier I explained how substrate concentration affects rates of reaction. So if there is more glucose present, the reaction will go very fast, because rate of respiration will increase. If this varies then the results will be entirely unreliable. I will therefore use a constant concentration of glucose in my yeast suspension I keep the amount of times I invert the test tube constant. I will keep this constant, because the more I invert it, the faster the kinetic energy of the particles, which could cause a faster rate of respiration. I will use 2 inversions for each temperature. I will keep the amount of methylene blue constant. I will do this because if I put more methylene blue in, then it will take very long to decolourise, which will greatly affect my results. So I will use 3 drops of methylene blue, which is just right to get reliable results. The only thing I will be varying is the temperature of the yeast. I will use 5 different temperatures to find the rate of respiration of the yeast suspension at high and low temperatures. I will use a kettle and a thermostatically controlled water bath to adjust the temperatures, and I will use tap water to cool any water baths down, if needed. I will also use a thermometer to help maintain the temperatures required. Apparatus:
Methylene Blue
Yeast suspension
Water
Thermostatically Water Bath
Kettle
5 test tubes
5 bungs
Test tube rack
0-10 cm³ syringe
100 cm³ beaker and 200cm ³ beaker.
Analogue stop clock
Justifying apparatus I have justified why I will use methylene blue. The yeast suspension is something that cannot be helped. I will use water to control the temperature because it is extremely easy to get a hold of and manipulate its temperature. Also I will use a thermostatically controlled water bath because it gives a very precise temperature measurement. It also allows me to sit back and watch while the reaction happens and the methylene blue decolourises. I chose a kettle because at some temperatures I will not be able to use the thermostatically controlled water bath due to certain restrictions applied by the supervisor, and I will only be provided with two water bath, each of which will stay at one temperature. The kettle gives very hot water, and it is fairly easy to manipulate the temperature of the water by simply adding tap water to it. I will use 5 test tubes because I will be doing 5 repetitions. Using a clean test tube for each temperature will stop any test tubes being contaminated by any extra yeast, methylene blue or any other unwanted substance. I will wash the test tubes after one repetition. I will use 5 bungs because if a bung is used for one test tube and used for another one immediately after, it may have some extra yeast or methylene blue on the bung, therefore I will use a different bung for each temperature and wash them for the next repetition. The test tube rack is for safety purposes, so that the test tubes are safely held and will not fall and break. I will use a 0-10cm³ syringe because it can give me an accurate measurement of my required 5cm³ of yeast suspension. A larger syringe may not give me a very reliable amount. Without a syringe at all it is even less reliable and should not even be considered. I will be using a 100cm³ beaker to put my yeast suspension in; I will fill it to about 70cm³. This will give me just enough yeast for all of the repetitions, and if I require more I can fill it back up. I will use a 200cm³ beaker for the water bath. A 200cm³ beaker is sufficient in the way that it doesn�t lose very much heat as much as if it were smaller. But it also isn�t too large so that when setting the temperature, the temperature is consistent throughout the water in the beaker. I will be using an analogue stop clock due to restrictions in the lab. But the analogue stop clock is fairly easy to use and gives accurate and reliable results. It measures to each second, which is all I require for my experiment. Risk assessment Methylene Blue � This may stain clothing if spilled. To prevent any spillages I will place the bottle cap on the bottle at all times when it is not in use. I will also keep it slightly distant from myself to prevent me from knocking it over. I will wear goggles to protect my eyes from any slashes that may result from moving the methylene blue around. I will wear a lab coat to prevent any spillages from staining my clothes. I will simply wipe any dropped methylene blue. Yeast � I will keep the yeast in a safe place, where it cannot get knocked over by accident. In the case of yeast falling in the table top or floor, I will immediately clear it up. Glass breakages � I will handle the glass equipment with extra care to prevent any breakages which can be very dangerous. I will place all test tubes in a test tube rack. I will place beakers in a safe area, where they will be same from accidental knocking. I will place the thermometer in the appropriate beaker so that the thermometer and beaker stay safe, and in the position required for the experiment. I will immediately clear up any breakages and inform the supervisor. I will also immediately inform the supervisor of any cuts that I may have got from breakages. Electrical apparatus � I will handle electrical instruments with care. I will ensure all electrical appliances, the kettle and thermostatically controlled water bath, are insulated properly. I will also use them with care, and replace the plug in and out of the mains socket with care also. High Temperatures � I will handle hot temperatures with care, so as I do not burn myself. In the case of burning something I will immediately inform the supervisor and clear up the spillage, or run my burnt hand through the tap to cool it down. Water spillage on the floor can also be a hazard as people may slip. I will try my best to stop water falling onto the floor, but in the case that water does spill, I will immediately wipe it up and keep that area dry. Conclusion My graph shows that as the temperature increases, the time taken for the yeast to return to original colour also decreases. This clearly shows that as the temperature increases the rate of respiration also increases. But after 40ºC the rate of respiration does not increases as much as before 40ºC. Also, after 45ºC the rate of respiration gets slower still. I can now conclude that as temperature increases, the kinetic energy of the particles increases, which in turn increases the rate of reaction until optimum temperature, is reached. Once the optimum temperature has been reached, the enzymes begin to denature and the rate of respiration slows down. This proves that my scientific theory and prediction were actually correct. I would have used percentage differences but they would have confused the results table and graph. This is because with higher numbers there will not be much of a percentage difference even if the actual difference is great, but if you were given lower numbers with an equal difference, the percentage difference would be still be higher. This will confuse my results and provide an unclear analysis of them; therefore I will not use percentage differences My scientific knowledge backs up my results entirely. The kinetic energy of the particles increased as the temperature was raised, which cause the rate of respiration to increase substantially. But as the temperature got higher than the optimum temperature for the enzymes, the rate of respiration decreased. This was because the kinetic of the particles was increased they started to vibrate vigorously and the enzymes were slowly denatured. So if the yeast was left in the temperature for longer more enzymes would have denatured, causing the rate of respiration to decrease even more. From 35˚C � 40˚C the rate of respiration is fairly fast, and is the steepest slope in the entire graph. This proves my theory that the optimum temperature for most enzymes is 37˚C. From 40˚C ? 45˚C the rate of respiration decreases, but only a slight decrease is noticeable. But in my theory it is written that at this point the enzymes should be denaturing. I still believe my theory to be true, because I believe that the enzymes were not given enough time to denature fully. From 45˚C and onwards there is quite a clear decrease in the intensity of the slope of the graph, this is because the temperature is high enough so that the kinetic energy of the particles is large enough for the enzymes to denature quite quickly. I was required to how the temperature affects the rate of respiration in yeast. I can conclude that as the temperature increases, the rate of respiration increases due to the faster motion of the particles. But after the optimum temperature of between 35ºC � 40ºC is reached, the enzymes will slowly begin to denature and the rate of respiration will decrease from there onwards. It will take longer and longer for the methylene blue indicator to disappear. Evaluation Overall my experiment was a success. My hypothesis was correct and the experiment was a predicted. It was a good way of finding the rate of respiration in yeast if all factors were taken into account. But even though my experiment was a success, I believe there was definitely room for improvement. My experiment was also carried out in two different days, with the same yeast suspension which were made again. I must explain the definition of �reliable results� and anomalous results before I go on to say more. Reliability means that the experiment will go as in the theory, and no variations; it must fit into the theory and provide me with accuracy. Reliability must also have a correct theoretical pattern. Anomalous result means that a result is totally wrong and doesn�t fit into any pattern whatsoever. Results do not necessarily need to fit in with a specific theoretical pattern, but the pattern in the results which is slightly different to theory, but still experimentally adequate. The pattern of the results may be theoretically wrong in some cases due to inadequate technique and other sources of error; this is unreliability. Anomalous results I experienced two anomalous results altogether, both were in my third repetition. Both anomolous were suspiciously the final repetitions of my experiment. The anomalous results could have been for a number of different reasons. The first anomaly, which occurred in the second to last temperature, had a higher rate of respiration than the other two repetitions; the rate of respiration for the anomaly was 90 seconds, whereas the average of the other two was 124.5 seconds. The last anomaly occurred on the final temperature, this rate of respiration was also far greater than the other two; the rate of respiration for the anomaly was 83 seconds, but the average rate of respiration was 106.5 seconds. Limitations/ Sources of Error I could have experienced these anomalies for a variety of reasons. The reasons are the limitations that I was stuck with, and other sources of error. The main source of error would be human error involved with reading the apparatus, and measuring. When drawing the required amount of yeast suspension, I decided to take 5cm³ because this was adequate. The syringe was accurate, but when I looked at it, I may have made small error in calculating the exact measurement of the syringe, the actually volume taken may have been slightly higher than 5cm³. Also, when I checked the thermometer, to check that the required temperature was what it read; my eye-level was sometimes not level with the meniscus, this could have given an anomalous result. These two sources of error could have easily been responsible for the anomalous results I gained. The temperature of the surroundings may not have been the same on the two days, or even on one single day they may have fluctuated. Other fluctuations of temperature of the water baths did occur. This was a major drawback. The thermostatically controlled water bath did a good job in maintaining the temperature, but the temperature did rise and fall about 1˚C or so once in while. But when I manually measured the temperature, there were much more major fluctuations in temperature. When adding water from a kettle, the temperature rose alarmingly fast, so I had to add tap water to cool it down, this sometimes cooled it too much. Other than this, when I did find the wanted temperature, the temperature would fall again and I would have to add water from the kettle to warm it up, which would lead to the same routine of it getting to hot. This could have given me unreliable results. When adjusting the temperature I also found myself looking away from the methylene blue and yeast suspension, in some cases, when I looked back at the suspension, it had already returned to its original colour. This would have given me some anomolous results, especially for the lower temperatures, which ran for a longer period of time, and so required more work to be done to maintain the temperature. Variations in the total time that the methylene blue and the yeast were left to react, and the time taken for me to adjust the apparatus and start and stop the stop clock. Also another source of error would be the analogue stop-clock I was using, at some points it got slightly tricky to use and, sometimes, seconds could have been counted for the time it took me to adjust it. These variations in time were mostly due to human error. I started the timer after I added the yeast, placed the bung and inverted the test tube twice and then replaced the bung, I could have missed crucial seconds in my results due to this flaw. This could have given unreliable results, but if done the same way every time, the results would not contain anomalies. I mean �the same way� as saying that starting the timer after the bung is removed, and not before. This could have been the reason for my anomalous results because the particles may have gained extra kinetic energy and respired faster. The yeast suspension may have not been evenly distributed in the test tube. I did invert the test tube to ensure that, initially the yeast suspension didn�t stay settled at the bottom of the test tube and the methylene blue didn�t remain above. This allowed the two to mix properly and be evenly distributed throughout. But eventually I believe that the yeast may have begun to settle down again. If the yeast did eventually settle that would mean that it would take even longer for the yeast to return to its original colour. This would provide unreliable results. But I do not believe this gave me my anomalous results, as my anomalous results were faster than they should have been. If the yeast did settle then the rate of respiration would have been less, resulting in a slower time. A liable reason for my results being anomalous would be the way I inverted the test tube. As I may have put more effort into inverting one test tube than another, and one mixture may have gained more kinetic energy, so the rate of respiration would increase. This could have caused my anomalous results to come about. Adding methylene blue had to be done carefully so I didn�t accidentally add more than 3 drops, this was done accurately by me, so it could not have been a source of error. But the volume of the drops probably always varied from drop to drop. If three small drops were put into a yeast suspension which had more yeast suspension in it by human error, the methylene blue indicator would disappear much faster. This source of error could have been another reason for my anomalous results. Over the two days there was a variety of methylene blue bottles available to me. If I used two different bottles over the two days, the concentrations could be varied, due to contamination or dilution. This would have seriously affected my results, and could have led to my anomalous results. Other small things could have affected the results and could have caused anomalies. The syringe may not be entirely clean for every repetition, because it is used over about 15 times. I could not clean it with water, or else water may contaminate it, causing the yeast suspension to become slightly dilute on some occasions. There was a small amount of methylene blue remaining on top of the solution. This methylene blue was reacting with the oxygen in the air. The temperature in the water bath may not have been entirely uniformly distributed, due to little mixing. Accuracy of results Although I obtained 2 anomalous results, I believe that overall my experiment was decent and a success. The results came out as predicted, and the graph came out as suspected. The reliability of my results can be challenged, because there were many limitations that could have caused my reliability to be bad. But the only unreliable thing my results could have given was an overall higher rate of respiration, or overall lower rate of respiration. This would have not really affected my reason for doing this experiment, which was to see how temperature affects the rate of respiration in a yeast organism. Talk about measuring apparatus.

Investigating the Effect of Temperature on Rate of Respiration in Yeast 7.9 of 10 on the basis of 1833 Review.