Hypothesis: As temperature increases, the rate of aerobic respiration should increase proportionally till optimum temperature is reached at which point enzyme activity (i.e. frequency of sucsessfull collisions with the maximum E-S complexes formed,) is at its maximum From there i believe as temperature moves beyond that optimum temperature, due to the ionic and hydrogen bonds between peptide chains in the tertiary structure of the enzyme starting to break, the active site of various respiratory enzymes (NAD, FAD) will begin to change shape to be no longer complementary to the H+ substrate, leading to less FAD and NAD reduced meaning less H+ ions entering the ETC and thus a slower rate of decolourisation of the methylene blue dye. Apparatus: - Test tubes - Yeast solution - Water bath - Thermometer - Timer - Glucose solution - Methylene Blue dye. - pH buffer - tongs Method 1. Set up a warm water bath at 30°C. 2. Add equal volumes of Yeast and glucose solution (6 cm3 each) in to three test tubes, swirl gently to ensure both solutions are mixed together evenly. 3. Then, to ensure only the independent variable (temperature) has an effect on the dependent variable (rate of respiration), add 2 cm cubed of pH buffer to each test tube. 4. Then place the test tubes into the water bath with a thermometer and leave the solution to equilibrate for 10 minutes to ensure a constant temperature and by extension, a constant rate of respiration. 5. Once a constant temperature has been established, add 2cm cubed of Methylene Blue to each test tube. 6. Start the timer and time how long it takes for the dye substrate to turn blue to colourless Methylene Blue is a redox dye and acts as an alternate electron acceptor of the electrons transferred during ATP synthase in the electron transport chain. The reduction of Methylene blue converts the blue colour to colourless denoting respiration has taken place. 7. Record the time taken for each test tube in a suitable table 8. Then, take a mean time for all three test tubes. 9. Calculate the mean rate of respiration by calculating 1/mean time (for methylene blue to decolourise) for the specific temperature 10. Then repeat the experiment (steps 1-9) at temperatures 30°C,40°C,50°C,60°C,70°C 11. Find the mean of the results for each temperature and use to calculate the average rate of respiration. Risk Assessment Hazard Risk Methylene Blue Flammable liquid and vapour Harmful if inhaled Hot liquids Scalding Broken glass Cuts from sharp object Safety Precaution Keep away from heat, hot surfaces, sparks, open flames and other ignition sources. Wear protective clothing, gloves, eye and face protection Handle with care; use tongs to remove boiling tubes from water bath; wear eye protection Take care when handling glass objects; keep away from edge of desk In emergency Risk level Immediate first aid is imperative. IF ingested, Do not induce vomiting unless under the direction of medical personnel. If in doubt, get medical attention promptly. Run burn under cold water; seek medical assistance 3/5 Elevate cuts; apply pressure; do not remove glass from wound; seek medical assistance 2/5 Yeast: not considered hazardous, no additional precautions required. 2/5 Table Temperature (°C) Time for Methylene Blue to decolourise /s Average rate (1/mean time) 2dp. 30 40 50 60 70 Calculate average rate of respiration overall: 𝐀𝐯𝐞𝐫𝐚𝐠𝐞 𝐫𝐚𝐭𝐞 𝐨𝐟 𝟑𝟎°𝐂, 𝟒𝟎°𝐂, 𝟓𝟎°𝐂, 𝟔𝟎°𝐂, 𝟕𝟎°𝐂 𝐢𝐧 𝐬 5 Reference: Pmt.physicsandmathstutor.com. 2017. AQA Biology A-Level Required Practical 9 Investigation into the effect of a named variable on the rate of respiration of cultures of single-celled organisms.. [online] Available at: <https://pmt.physicsandmathstutor.com/download/Biology/A-level/Notes/AQA/PracticalSkills/RP%2009%20-%20Respiration%20in%20Single%20Celled%20Organisms.pdf> [Accessed 16 January 2022][18:24] Brecklandscientific.co.uk. 2018. SAFETY DATA SHEET According to 1907/2006/EC, Article 31 Yeast - Dried. [online] Available at: <https://www.brecklandscientific.co.uk/v/vspfiles/MSDS/S0001649.pdf> [Accessed 16 January 2022][18:26] Pro-lab.com. 2016. Methylene Blue. [online] Available at: <https://www.pro-lab.com/wpcontent/uploads/2016/11/Methylene-Blue-SDS.pdf> [Accessed 16 January 2022][18:27]
