Osmosis Practical Report PDF

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OSMOSIS

Case Study You are a veterinarian working in Indooroopilly and return from lunch to find a nurse treating a six year old kelpie, Baxter. Baxter is very lethargic, has an increased heart rate, and when you pinch his skin the fold remains visible. The owner tells you that they knew something was wrong when, upon returning to the outdoor car park from a three hour shopping trip, they saw Baxter passed out on the back seat. The temperature in the car would have been very high and Baxter will have lost a lot of water through evaporation as he panted to stay cool. This loss of water would have reduced his blood volume, thereby increasing the concentration of the ECF.

Hypothesis This is the hypothesis that you developed before conducting your experiment. If red blood cells are placed into a hypotonic solution, the absorbance of the extracellular fluid will increase.

Materials and Methods First a stock solution of 1M NaCl was made and then used to make 6 working solutions of 0mM, 50mM, 120mM, 150mM, 180mM and 450mM. Each NaCl working solution was then mixed with sheep’s blood at a ratio of 1:19. Each sample was then placed into the centrifuge at 10,000 RPM for 3 minutes. After this, the extracellular fluid was extracted and placed into cuvettes and the absorbance was measured for each sample at a 540nm wavelength. Following this, results were gathered from two other groups in order to compare data. The negative control used in the experiment was the 150mM solution as this is isotonic to the red blood cells and it is known that it will not cause any changes in the cells. The positive control used in the experiment was the 0mM solution, in that it is hypotonic, and it is known that all cells will lyse in this solution which will increase absorbance as the extracellular fluid will be at its darkest. Preparation of Stock NaCl 1M Stock solution = 5.84 g of NaCl dissolved in 100 ml of H2O

Working Solutions – Dilution Calculations

Table 1: Calculations for NaCl working solutions C1 V1 Concentration of Stock Added Volume of Stock (M)

(ml)

C2 Desired Dilution Concentration (mM)

1

0

0

4

1

0.2

50

4

1

0.48

120

4

1

0.6

150

4

1

0.72

180

4

1

1.8

450

4

1

V2 Required Total Final Volume (ml)

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Prediction Complete Table 2, including the osmolarity of your chosen dilution concentrations, and the effect these solutions will have on the osmotic movement of water and red blood cell integrity. Table 2: Predicted osmotic effects of NaCl solutions on sheep red blood cells Predicted osmolarity NaCl concentration (mM) Predicted effect on red blood cells (mOsmol/L) 0

0

All cells will lyse

50

100

Most cells will lyse

120

240

Some cells will lyse

150

300

No change in cells

180

360

Some cells will shrivel

450

900

Most cells will shrivel

Results Individual Results

Record your own group’s results in Table 3, including the effect on cell integrity as suggested by these data. Provide a professional title beside “Table 3:” Table 3: NaCl concentration (mM) 0

Value of dependant variable (with units) 3

50

2.766

Most cells lysed

120

2.878

Some cells lysed

150

0.172

Cells remain normal

180

0.169

Some cells shrivelled

450

0.165

Most cells shrivelled

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Effect on red blood cells All cells lysed

OSMOSIS

Comparative Analysis Plot the results you collected from the three groups in Figure 1 below (right-click on the chart; Edit Data). Remember to provide axes labels and a figure legend. N.B. If the data table doesn’t appear, save the file, close it, then open the file and try again.

Absorbance of Extracellular Fluid

3.5 3 2.5 2 1.5 1 0.5 0 0

50

100

150

200

250

300

350

400

450

500

Concentration (mM) Figure 1: Effect of hypotonic, isotonic and hypertonic NaCl solutions on sheep’s red blood cells. Data represents the absorbance of the extracellular fluid, as found by three different experiment groups.

Description of Findings Write a paragraph of text in the box below, describing the important relationships demonstrated by all of the results you collected and presented, and summarising the major findings: The experiment found that there was an increase in absorbance of the extracellular fluid in the solutions hypotonic to the red blood cell, as well as a decrease in absorbance as the solutions became isotonic, with little change as the solutions became more hypertonic. The negative control solution with an osmolarity of 0mOsmol/L had the highest absorbance, reading at a 3 in the cuvette. The next two readings came in at 2.76 and 2.878 respectively for the other hypotonic solutions of 100mOsmol/L and 240mOsmol/L. The positive control isotonic solution at 300mOsmol/L had a reading of 0.172 indicating low absorbance and this decreased slightly in the last two solutions of 360mOsmol/L and 900mOsmol/L with readings of 0.169 and 0.165 respectively. As well as these results, data was gathered from two additional groups for the purpose of comparison. These additional results followed the same trend of absorbance increasing with solutions hypotonic to the cells however group three’s results differed in that the readings were consistently lower than the results in the original experiment in all of the working solutions, indicating less of an increase in absorbance in the first two solutions and less of a decrease in the final two solutions.

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Discussion Remember to treat these questions like short answer questions in the final exam: be specific, clear and concise. 1. Briefly describe (in complete sentences) whether the results of your experiment confirm or disconfirm your original hypothesis? The results of the experiment confirmed the original hypothesis that if a red blood cell is placed into a solution hypotonic to the cell then the absorbance of the extracellular fluid would increase.

2. What happens to the parameter you have measured when sheep red blood cells are placed in both hypotonic and hypertonic environments? When sheep red blood cells are placed in a hypotonic environment the absorbance of the extracellular fluid increases, though when placed in a hypertonic environment the absorbance will decrease, though it was found in the experiment that it will not decrease significantly more than the fluid in an isotonic environment.

3. Explain the biological mechanisms which cause these effects. When sheep red blood cells are placed into a hypotonic environment the cells will take in water quicker than they are releasing it and when their membranes cannot withstand the pressure any longer, the cell will lyse (Urry et al., 2017). It will release the pigments inside into the extracellular fluid and therefore create a fluid that appears more opaque. On the other hand, when sheep red blood cells are placed into a hypertonic environment the cells will lose water and the membrane will shrink in response, decreasing the surface area (Urry et al., 2017) and therefore leave the extracellular fluid more clear. 4. Discuss the biological reasons that could explain the variations between data sets in your comparative analysis (Figure 1)? A biological reason for variations in the comparative data is the age of the blood samples. Over time sheep’s red blood cells mature and become more susceptible to hypotonic haemolysis (Sziegoleit, Fussle & Wellensiek, 1980), therefore it is possible that group three had a batch of younger sheep’s blood which did not lyse as easily as the other two groups cells. As well as this, sheep’s red blood cells are comparative to human red blood cells (Donald et al., 2012) and over a 100-day lifespan human red blood cells lose approximately 30% of their volume (Malka, Delgado, Manalis et al., 2014), a factor which could also have an effect on the results. Another biological reason for variations could be the sex of the sheep in which the blood sample came from. In some species of sheep there is a discernible difference in red blood cell circumference between male and female sheep (Adili, Melizi & Belabbas, 2016). 5. Some of the signs Baxter showed in the case study were increased heart rate and lethargy. How does severe dehydration result in these symptoms? Severe dehydration can result in increased heart rate due to the reduced blood volume (University of Queensland, 2020). This means that Baxter’s heart is working much harder than usual to pump enough blood around his body with the reduced volume therefore resulting in an increased heart rate. As well as this, due to the lack of hydration, blood is being pulled to the most important parts of the body in order to maintain vital functions. This would draw blood away from Baxter’s muscles and therefore cause lethargy.

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OSMOSIS

6. Would you administer water intravenously or isotonic saline intravenously to treat Baxter? Use your results and knowledge from lectures to support your answer. It is important to administer isotonic saline intravenously to treat Baxter as the solution will contain the same concentration of solutes that are already in Baxter’s blood. Water on the other hand is hypotonic and would dilute these concentrations which could result in further dehydration and even lead to death. Just like the sheep’s red blood cells, Baxter’s cells can react in the same way by either lysing in extracellular fluid that is hypotonic to the cell or by shrivelling up in extracellular fluid that is hypertonic to the cell. 7. Baxter’s owner does not have a strong science background. Clearly describe and explain (using complete sentences) the cause of Baxter’s problems and the process of osmosis to them, using language they can understand. By leaving Baxter in a hot car for three hours he has become very dehydrated. This is because he did not have access to water, and he was panting in order to release heat and attempt to cool down his body which results in evaporation of water. This lack of hydration is what has caused Baxter’s heart to race and his body to become lethargic. Through the process of osmosis, Baxter’s cells have lost water without receiving their usual replenishment and therefore the concentrations of salt and other solutes normally in his blood have become unbalanced. Osmosis is when water moves across a membrane in order to find equilibrium, in Baxter’s case water has moved out of his cells in order to dilute the extracellular fluid as he lost water. This is why it is necessary to replenish Baxter’s water stores with saline intravenously, to balance his solute concentration levels and rehydrate him.

References List any references you have used in your answers, in the panel below. Adili, N., Melizi, M. & Belabbas, H. (2016). Species determination using the red blood cells morphometry in domestic animals. Vet World, 9(9). 960-963. doi: 10.14202/vetworld.2016.960-963 Donald, M. M., Mathews, N. I., Zhu, S., Strauss, R. G., Schmidt, R. L., Zimmerman, M. B., … Widness, J. A. (2012). Comparison of red blood cell survival in sheep determined using cells labelled with either biotin at multiple densities or cyanate: validation of a model to study human physiology and disease. Transfusion, 52(5). 963-973. doi: https://doi.org/10.1111/j.1537-2995.2011.03512.x Malka, R., Delgado, F. F., Manalis, S. R. & Higgins, J. M. (2014). In Vivo volume and haemoglobin dynamics of human red blood cells. PLOS Computational Biology, 10(10). doi: https://doi.org/10.1371/journal.pcbi.1003839 Sziegoleit, A., Fussle, R. & Wellensiek, H., J. (1980). Influence of the age of sheep red blood cells on virusinduced and hypotonic hemolysis. Medical Microbiology and Immunology, 168. 211-216. doi: https://doi.org/10/1007/BF02122855 University of Queensland. (2020). Osmosis Case Study [document]. Learn U.Q. https://learn.uq.edu.au/ Urry, L.A., Meyers, N., Cain M.L, Wasserman, S. A., Minorsky, P. V. & Reece, J. B. (2017). Campbell Biology, 11th Ed, Pearson Education, Australia.

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