9-Respiration_Report_2021

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Biology

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Jan 9, 2024

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LIFE 212 Lab Report 9- “Respiration” - Rosenberg sections Name:_____________________________ Due:_______________________________ Section: ____________________________ Isolation of Mitochondria and Respiration 50 pts total plus Notebook Conclusion (4 pts) 1. Describe the pellet in tube 1 (after the 200 x g and 700 x g spins) and how its appearance is consistent with the theory for separation by differential centrifugation. Support your answer with specific cell components and their expected order in the tube, including detail of differential centrifugation principles. (2 pts) 2. List and describe the contents of the resuspended pellet in tube 3. Include specific cell components that are expected to be present. How did this pellet differ from the pellet in tube 2? Which component is present in both fractions? Explain. (4 pts) 3. Describe the contents of tube 4 supernatant, as seen under the microscope, with and without adding the Janus green stain. Note that, upon homogenization, mitochondria clump together into masses, now made blue by the stain. Which is the best objective lens to see stained individual mitochondria? Explain. (6 pts)

LIFE 212 Lab Report 9- “Respiration” - Rosenberg sections Name:_____________________________ Due:_______________________________ Section: ____________________________ 4. EXPERIMENTAL CONCLUSION ( 14 pts total )- After evaluating the color of the reaction tubes after the incubation period, did you successfully isolate functional mitochondria? Justify your conclusion. Conclusion (6 pts) - What is the expected result for tube 1? (3 pts) - What was the reason for the inclusion of reaction 2? (3 pts) - Suppose that tube 3 remained blue after 24 hours. Give plausible explanations for why this occurred. (2 pts) - 5. The homogenization buffer for this experiment contains the following ingredients: 70 mM Sucrose, 220 mM mannitol, 0.5 g/L Bovine serum albumin, 2.0 mM Hepes pH 7.4. Explain the purpose of each of the buffer ingredients. Feel free to consult online resources. ( 12 pts )

LIFE 212 Lab Report 9- “Respiration” - Rosenberg sections Name:_____________________________ Due:_______________________________ Section: ____________________________ 6. A. What conditions are critical to ensure the success of this qualitative assay of mitochondrial function? Provide 2 reasons. Explain. ( 2 pts ) B. Why do we consider the respiration assay to be qualitative rather than quantitative? ( 2 pts ) 7. Explain the purpose for the mineral oil in this experiment. ( 4 pts ) 8. Assume that the respiration assay was performed using fractions of organelles that were separated by isopycnic (equilibrium) centrifugation in sucrose gradients extending from 0.9 (top) to 1.9M sucrose (bottom). Components should float in the layer with similar density. Based on this information, where would the mitochondria be located? How would that compare to the location of chloroplasts? ( 4 pts )

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LIFE 212 Lab Report 9- “Respiration” - Rosenberg sections Name:_____________________________ Due:_______________________________ Section: ____________________________ Notebook Conclusion (4pts) The conclusion should be a concisely written summary of the experimental results using technical language. Describe each objective from your Introduction individually and frame in context with the experimental results. Vague and/or incomplete conclusions will not receive any credit.

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