BIOL2070 Lab 3 Cell membranes PDF

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Lab 3 in Cell Biology...

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Jordan Gilder 0665817 BIOL-2070H-A W12 February 11th 2020 Lab Partner (Part 2): Isabelle Nebesky

Understanding the Nature of Cell Membranes: Osmosis in Bovine Red Blood Cells and Pigment Concentration of Beet Roots

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Abstract This laboratory’s purpose was to observe cow’s red blood cells undergoing osmosis in different saline solutions and to observe the active diffusion of pigment that is found in beetroots after different concentrations of alcohol were added. Red blood cells mixed with different saline solutions were examined to determine the sodium concentration of each solution, all the while the other half of the team prepared solutions with pieces of beetroot submerged in differentiating Ethanol (EtOH) concentrations to release the beetroot’s pigmentation. A spectrophotometer was then used to identify the absorbency level of the pigment that leaked into the solution. The results from this lab showed that on average, as the alcohol concentration decreased as did the level of pigment measured in the spectrophotometer and the saline solutions observed did affect the osmotic pressure of the bovine red blood cells. To conclude, it was found that the Ethanol does break down the beet root’s cell walls and the concentrations of nutrients found outside of the cell do affect the osmotic pressure of the cell. Introduction A cell’s plasma membrane is composed of a phospholipid bilayer that has proteins throughout it (Alberts et al. 2014). The plasma membrane which is made of proteins and lipids provides channels for the cell that transports nutrients and waste into and out of the cell, helping to prevent specific molecules from reaching the inside (Alberts et al. 2014). There is an outer membrane and membranes surrounding each organelle within eukaryotic cells, similar to ones used in the lab. Active and passive transport is used to exchange nutrients, proteins and waste inside and outside of the cell as well as a method called diffusion (Alberts et al. 2014)

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Ethanol (EtOH) is a clear, colourless liquid that is quickly absorbed from the gastrointestinal tract and becomes distributed throughout the body (PubChem, 204). This compound is used often as a solvent or preservative for preparations in the pharmaceutical field as well as being the main ingredient found in alcoholic drinks (PubChem, 2004). Ethanol is considered to be a short-chain alcohol which represents a group that can exert influence on the properties of a cell membrane (Gurtovenko and Anwar, 2009). When ethanol is introduced, there are changes in the lipid metabolism and protein interactions and repeated exposure to this alcohol selectively alters lipid composition towards the resistance of ethanol upon being exposed (Gurtovenko and Anwar, 2009). Ethanol has amphiphilic characteristics due to it being a shortchain alcohol, however, its hydrophobicity is limited, therefore when this compound divides into a lipid membrane the ethanol molecules are mainly found within the lipid interface which causes a formation of hydrogen bonds with the hydrophilic lipid head groups (Gurtovenko and Anwar, 2009). This formation is accompanied by an increase in the fluidity of the membrane, changing the membrane structure. Due to ethanol’s ability to affect the fluidity of membranes, it was hypothesized that the concentration of alcohol will affect the permeability of the cell membrane and predicted that a stronger alcohol concentration will produce a higher pigment measurement due to an increase in ethanol molecules in the solution acting on the membranes. The rate of pigment diffusion will increase as the concentration of alcohol increases due to how ethanol affects the cell membrane of the beetroots. Methods This lab had two parts where two out of the four members of the group separately completed each part in unison (see Appendix A). One part of the lab required circular-shaped 3

beetroots that were provided in the lab to be cut into twelve pieces, all approximately the same size. After being given “alcohol” as our group's treatment, a total of twelve 10 ml test tubes were used to prepare four trials with three replicates of each, using 2% Ethanol to prepare the concentrations. Using the C1V1 = C2V2 equation, we calculated the concentration amounts for each of the three treatments. The control of this experiment was prepared with four treatments made from 10 ml of H2O, being composed of 100% tap water. The four trials of the first sample contained 10 ml of 2% Ethanol and were deemed as the “high concentration” sample due to no water being added. The medium concentration sample had four trials composed of 5 ml of 2% Ethanol, diluting the alcohol to 1% EtOH, and 5 ml of H2O. Lastly, the low concentration sample had four trials prepared with 2.5 ml of 2% Ethanol which diluted the alcohol to 0.5% EtOH and 7.5 ml of H2O. The pieces of beetroot from the start of the lab were then placed individually into each of the test tubed with the added solutions. After a duration of 30 minutes, the beetroot pieces were removed with forceps and the solutions were transferred into a cuvette to be analyzed by a spectrophotometer. After first measuring the absorbency of the control, each solution has its amount of absorbance measured and noted down. The raw data was transferred into an Excel sheet and the means and standard deviation was calculated (see Table 1). A line graph was created with a line of best fit to see the relationship between the concentration amount in each sample number and the amount of pigmentation recorded. Results When plotting the raw data into a graph, the strongest concentration average (sample 1) was plotted first with the decreasing concentrations following behind. As shown in Figure 1, the line of best fit starts to increase and then decreases gradually, representing a decline in the level of measured pigment as the concentration of alcohol decreased. The control of this experiment 4

was measured to have a mean AU of 0.3, and a standard deviation of 0.1. The first trial, the strongest concentration measurement that had an ethanol concentration of 2% was measured to have an average AU of 0.85 and a standard deviation of 0.5. The second trial that was considered to have a medium concentration of ethanol of 1%, had an average measured pigment of 1.0 AU with the largest calculated standard deviation of 0.36. The third and final trial was considered as the lowest amount of concentration (other than the control) and had an ethanol concentration of 0.5%, and an average measured pigment amount of 0.3 AU and the lowest calculated standard deviation of 0.1 (see Table 2). It was observed during the laboratory experiment that the test tube that contained the 100% EtOH solution turned the most intense shade of pink compared to the other solutions. There was an obvious difference in the colour of the 1% EtOH solution as it was almost a pale pink compared to the first sample. The lowest concentrated sample was a very light shade of pink, indistinguishable from the control that only contained water and it was only when placed into the spectrophotometer we were able to see there was a difference in their average measured pigment of beetroot.

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Discussion The results from this experiment highlight how higher concentrations of ethanol affect the rate of diffusion in the cells found in beetroots. This experiment supports the hypothesis that the concentration of alcohol will affect the permeability of the cell membrane, as well as the prediction that a stronger alcohol concentration will produce a higher pigment measurement from the spectrophotometer. As the concentration of the alcohol solution increased as did the impact EtOH had on the cell membranes due to the increased number of molecules weakening the membrane and allowing for a greater rate of diffusion. The lower concentration of the alcohol solution still affected the cell membrane of the beetroots however, it was not as intense as the diffusion rate was much slower due to a lower amount of EtOH molecules acting on the cell membrane. Using more uniform and smaller shaped beet root pieces would aid in the diffusion rate of the pigmentation, increasing the intensity of the solution’s pink colouration in all trials that were observed as well as a probable increase in the amount of pigment measured. Ethanol directly leads to an increase in cell membrane fluidity and changes in the membrane’s composition of proteins. These changes cause conformational changes to the cell itself, influencing the function and resistance to certain outside nutrients and protein (Tóth et al. 2013). The alterations in the lipid environment caused by ethanol influences the function of some membrane proteins such as receptors, ion channels and enzymes, which in turn alters the biological processes of the cell including the enzymatic reactions that occur, membrane transport and signalling pathways (Tóth et al. 2013). This allows for fluids to diffuse more easily into and out of the cell, and in this experiment, it is the pigmentation that is diffusing out of the cell. Throughout this experiment, there were many instances where human error could have skewed the results. One of these instances where a source of error was present was when 6

preparing the solutions for each of the four trials, the amount of solution varied across the twelve test tubes with ± 1 ml of the solution in each trial. This was due to using a 50 ml beaker when measuring out the 10 ml needed for the solution rather than using a pipette that would have given us more accuracy and minimized human error. There is a spike of pigmentation measured in the second trial (measured at 1.0 AU) which seems to be an outlier due to this sample having an increase in concentration when the pigmentation should have been reducing, not increasing. This could have been caused by uneven pieces of beetroot slices that caused a higher rate of diffusion in the second trial if the piece was larger than the others, allowing for more pigmentation. This experiment proved that ethanol will affect the permeability of the cell membrane by having the diffusion rate increase as the concentration of EtOH increased. The cell membrane’s permeability was disrupted the most during the second-highest concentration of ethanol, which as already stated was due to human error as the highest concentration of ethanol should have represented the highest rate of diffusion. However, the results do show that as the concentration of ethanol decreases as does the diffusion rate of the cell membrane, representing that ethanol does affect lipid bilayer of cell membranes.

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Appendix A

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References 2004-09-16. Ethanol. https://pubchem.ncbi.nlm.nih.gov/compound/Ethanol. Accessed on 202002-09. Alberts, B., Bray, D. Hopkin, K., Johnson, A., Lewis, J., Raff, M., and Walter, P. 2014. Essential Cell Biology, 4th ed. Garland Science, Taylor and Francis Group, New York, New York. p. 359-362, 372-373. Gurtovenko, A., and Anwar, J. 2009. Interaction of Ethanol with Biological Membranes: The Formation of Non-bilayer Structures within the Membrane Interior and their Significance. Journal of Physical Chemistry. 113(7):1983-1992. Tóth, E. M., Vígh, L., and Sántha, M. 2013. Alcohol stress, membranes and chaperones. Cell Stress Society International. 19(3):299-309

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