Bio 1 Lab Report

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BIOL:1411 LAB REPORT 1 Bethel Tewabe Team Members: Gavin Smith, Colten Kuhn, Ashley Jokovich Lab section: 0A29 10/03/23

Experiment 1 Question & Hypothesis The overarching question for the first experiment conducted was: How much mitochondrial activity is present within a specific cell fraction? To do this, we performed cell fractionation of our chosen plant cell and centrifuged them to determine the amount of specific organelles present, but more specifically the mitochondrial activity present with the use of biochemical markers. We hypothesized that our final pellet would have the most mitochondrial activity, most prominent color change, because mitochondria is one of the smallest organelles and with each centrifuge the organelles being filtered out were getting smaller. Results Once the centrifuging was done and we obtained all of our cell fractions, we had a series of cuvettes that received a specific amount of: cell fraction, for measurement purposes, sodium azide, to prevent the transfer of electrons to the electron transport chain, DCIP, which is now acting as the electron acceptor and undergoes a color change when reduced, and succinate, which is the biochemical marker for mitochondrial presence. Once all of these amounts were added to the cuvettes, they were put into the spectrometer to obtain an absorbance reading. Figure 1 Depicted in the graph is the difference in absorbance values at each timed interval.

Figure 2 Depicted in this graph is the overall change in percent value of each cell fraction. Shown in both figure 1 and figure 2, there is no change whatsoever within the E1 cuvette and this is because E1 was the control. Noticeable from figure 1, our E2 cuvette had the largest absorbance reading in comparison to all of the other cuvettes. Only cuvettes E2 and E3 had a significant decrease as opposed to E1 which had none and E4 which had the smallest and, overall, insignificant decrease. E3 had the largest decrease in both graphs, but more prominently seen in figure 2. Discussion Our hypothesis, that P3 would have the highest mitochondrial activity, was supported by our data. This is due to the fact it had the highest decrease in absorbance indicating that the SDH accounted for the presence of many mitochondria in comparison to the other cuvettes. An additional experimental condition could be to repeat this experiment with animal cells and compare data or to implement changes in pH, temperature, substrate/enzyme concentration, competitive/noncompetitive inhibitors which is what we performed in our second experiment (Grewe, 2023).

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Experiment 2 Question & Hypothesis This experiment, designed to build off the initial experiment, is where we added the variability of temperature. The big question for this one was: how will different temperatures effect the mitochondrial activity within a specific cell fraction and which one will have the highest? To answer this question, cell fractionation was conducted and, based off of the data from experiment 1 determining which fraction had the highest mitochondrial activity, the P3 cell fraction was used. P3 was divided into different cuvettes and placed in varied temperature conditions, and a spectrometer was used to determine absorbance values. We hypothesized that the cuvette placed in the hot bath would have the most mitochondrial activity because a slight increase in temperature increases the rate of most reactions (Hillis, 2022). Results Once the P3 cell fraction was obtained, it was divided into three separate cuvettes to be placed in various temperatures. One was placed in an ice bath, at about 4 degrees Celsius, one was left at room temperature, about 22 degrees Celsius, and the final one was inserted into a hot bath, at 65 degrees Celsius. We used the spectrometer to take an initial reading before placing them in their temperature conditions. After seven minutes had passed, another reading was taken. The cuvettes were again placed back in their conditions and after another seven minutes had passed, 14 minutes total, a final reading was recorded. Figure 3 Depicted in the graph to the left are obtained absorbance values for specified temperatures conditions.

Figure 4 Depicted in the figure to the left are overall absorbance values changes in percentages. Shown in the above graphs, but more prominently in figure 4, the decrease in absorption was still substantial across all the cuvettes. The cuvette placed in the hot bath had the largest decrease with a 9% drop overall, but the biggest decrease is seen between the 0 minute and 7 minute mark in figure 3. The cuvette placed in the ice bath had the smallest decrease but not too far behind the others. Discussion Our hypothesis that the cuvette placed in the hot bath would have the largest absorbance proved to be correct as seen in the data. Although all the cuvettes had a considerable decrease, this was expected since all the cuvettes has cell fraction from P3 which had the most mitochondria in it, indicated by experiment 1. Cooler temperatures slows reactions which is why the ice bath had the lowest of the three (Hillis, 2022). Higher temperatures, to a certain extent, speed up the rate of reaction so this is why trend is as follows: ice bath, room temp, hot bath. Another experimental aspect one could try would be to introduce another variable into a similar experiment such as adding a specific amount of acid/base solute to see how that effects the mitochondrial activity within their specified temperature conditions.

Works Cited Grewe, Brenda, et al. “Wet Lab 2A.” Foundations of Biology 1411 Lab Manual , Macmillan Learning, 2023, pp. 51–62. Hillis, David M., et al. Principles of Life: Digital Update . Macmillan Learning, 2022.

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