Lab 1 Report

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Apr 3, 2024

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Lab Report 1 Arya Sajadian, Ian Johnson, Tom Ohl, Thomas Harold Section 0A09 20 Feb 2024
1 Experiment 1 (Parts I and II) Question and Hypothesis The question posed by this lab was to test for mitochondria in cell fractionates that were separated based on mass via centrifugal force. In order to test how much mitochondria was present, an artificial electron acceptor, DCIP, was used to show a visible change in any mitochondria undergoing the citric acid cycle. The DCIP loses color due to being reduced from SDH-FADH transferring electrons to it, therefore indicating the presence of mitochondria. The hypothesis postulated is that pellet 2 would have the most mitochondria, due to the fact that most of the organelles did congregate there, but did not contain any larger structures like nuclei. Results
2 The goal of the experiment was to see how to observe organelle functions such as the citric acid cycle, understand how the internal workings of the cell are able to be divided up, and be able to see how electron reduction and oxidation occur with SDH’s reduction into SDH-FADH and the subsequent oxidation, as the electrons get transferred and reduce DCIP. The general steps that were taken were to first prepare and divide a cell homogenate, then to prepare several cuvettes to monitor the absorbance. Four cuvettes were used to monitor DCIP absorbance reduction with no homogenate, pellet 2, pellet 3, and supernatant 3, and an additional 4 were used as blanks that contained no DCIP. The data from this experiment showed some initial change in the blank, but little throughout, which can be attributed to a slight error in being unfamiliar with the Spec-200, leading to that discrepancy. Pellent 2 had some notable descent in absorbance, totaling in a 0.08 difference. Pellet 3 had significant descent, totaling in at 0.17 absorbance difference. Supernatant 3 exhibited no change whatsoever throughout the experiment. On the whole, there is not much of a trend to follow due to the small sample, although one could argue a possible bell curve shape, with pellet 3 having significantly more than both other “sides”, being pellet 2 and supernatant 3.
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3 Discussion 1. The hypothesis was disproven, as the fractionate with the most disparity in absorbance was P3. The hypothesis was postulated as such due to an overestimation in mitochondria size and mass. 2. One possible additional variable that could be tested is attempting to split up cell homogenates even further to see how divided up the organelles can get, since the type of cell fractionate being used is the experimental variable. References Zawily El A, McAllister F. B, Grewe B. Spring 2024. Foundations of Biology 1411 Laboratory Manual. New York (NY), Macmillan Learning Curriculum Solutions. Experiment 1 (Part III) Question and Hypothesis Temperature was chosen as the experimental variable as enzyme reaction rate has a unique relation with temperature. Rather than being a simple linear relationship, enzymes have an ideal equilibrium temperature, and significant deviations in either direction impact it negatively, leading to a sort of bell curve for the ideal temperature for an enzyme. Of the 5 possible environments (0 C (ice), 2-4 C (fridge), 20-25 C (room temp), and 37 C and 65 C hot water baths, the hypothesis postulated was that 37 degrees would be the most ideal for enzyme activity, and therefore have the largest absorbance disparity. The reasoning for this is that internal body temperature is usually around 98.6 F, which is very close to 37 C and is a possible scenario in this experiment.
4 Results
5 The goal of this experiment was to see how the pellet with the most mitochondria was influenced by temperature and monitor the reaction via absorbance disparity in DCIP. The structure was fairly similar to that of the previous experiment, but only required one blank as what was being solely tested was the pellet 2 fractionate. 5 other identical cuvettes were also prepared, but instead of being stored on ice like in experiment 1, each cuvette was designated a certain environment to be placed in to see how it influenced the absorbance readings. 0 C exhibited little change, totaling at 0.03 absorbance difference. 2-4 C had some difference, totaling at 0.06 absorbance difference. 20-22 C exhibited notable difference at 0.08 total. 37 C had significant difference at 0.14 total. 65 C also had notable difference at 0.09 total. Something else significant about 65 C is, unlike the other trends at other temperatures, 65 does not linearly descend throughout. Rather, it has a faster rate of decay that then plateaus. Discussion 1. The hypothesis was not disproven, as the condition with the highest amount of absorbance disparity was the 37 C. Since inside the organism is where these reactions are meant to take place, it would follow that the closest temperature to that value would provide the best rate of reaction. 2. Another condition would be testing additional positive temperatures to better showcase how straying too much in either direction of temperature negatively impacts the reaction. Currently, it feels as though the values are skewed to the left, rather than an even normal distribution. It would
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6 also allow for a second possible experiment to see if higher temperatures explicitly mirror the unique behavior exhibited by 65 C in terms of its absorbance difference. References Zawily El A, McAllister F. B, Grewe B. Spring 2024. Foundations of Biology 1411 Laboratory Manual. New York (NY), Macmillan Learning Curriculum Solutions. Assistance from TA Hindle Cumulative References Zawily El A, McAllister F. B, Grewe B. Spring 2024. Foundations of Biology 1411 Laboratory Manual. New York (NY), Macmillan Learning Curriculum Solutions. Assistance from TA Hindle

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