BIOM1051 Osmosis prac 2 PDF

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OSMOSIS

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 the sheep's red blood cells were placed in a hypertonic solution, the PCV (%) will be less than that of an isotonic solution.

Materials and Methods Firstly, to make the NaCl stock solution, put 5.8g of NaCl into the funnel and fill it up with 100ml of deionised water. To make the dilutions, label all the test tubes then set the pipette volume to the desired amount and pipette the amount of stock needed by using a tip into the labelled test tube. By using a different tip, add the desired amount of deionised water. To combine the sheep blood with NaCl working solutions, pipette 1 part sheep’s blood to 3 parts dilution into the tube. Once blood and solution are combined, close the cap and gently roll the solutions to mix the solution. Following the mixing, difference in transparency can be observed which indicates the different levels of tonicity. Take out a test tube and take out the first solution to fill the haematocrit tubes. Tilt the test tube on an angle and insert and gently pulse until the solutions fills approximately ¾ of the tube. Place your finger on top to seal it, then plug it into the clay to seal the bottom of the tube. Ensure that each haematocrit tube is labelled and place them on the plate. Once all solutions are filled up, place them into the haematocrit centrifuge by orienting the clay on the outside and blue line on the inside. Ensure that the tubes are evenly spaced. Once done, place the glass lid on top, close the lid and spin at 10,000 RPM for 10 minutes and press start. Once solutions are out of the centrifuge, the PCV can be measured by using the critocap reader. In order to do this, place one of the samples on the scales and line the clay side with 0, and top of the solution with 100. Once its aligned, the top of the blood cells can be read, which generates the PCV of that sample. The positive control is 0. The negative control is the isotonic solutions.

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Preparation of Stock NaCl 1M Stock solution = 5.8 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) 1

(ml) 0

C2 Desired Dilution Concentration (M) 0

1

0.2

0.05

4

1

0.48

0.12

4

1

0.6

0.15 (neg control)

4

1

0.72

0.18

4

1

1.8

0.45

4

2

V2 Required Total Final Volume (ml) 4

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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 Predicted effect on red blood cells NaCl concentration (mM) (mOsmol/L) 0 0 Burst 100

200

Burst

150

300

Normal/no change

200

400

shrivel

250

500

shrivel

300

600

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: Effect of different levels of NaCl concentrations on the PCV (%) NaCl concentration Value of dependant variable Effect on red blood cells (with units) (mM) 0% 0 Burst 4% 50 Burst 120

8%

Burst

150

7%

Normal/no change

180

5.5%

Shrivel

450

5%

Shrivel

Comparative Analysis

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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.

[NaCl] vs PCV Group 1

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Group 2

Group 3

9 8 7 PCV (%)

6 5 4 3 2 1 0 0

1

2

3

4

5

6

7

8

9

10

NaCl concentration (mM)

Figure 1: Different levels of NaCl concentrations vs PCV

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 results above demonstrate that PCV depends on the osmolarity of the solution. Given that the sheep’s blood cells are isotonic at 300mOsmol/L, this indicates that 300mOsmol/L is in a hypertonic solution. Identifying in which tonicity they are in determines the effect on the cell (see table 3). If the cells are in hypotonic solution, this would cause the PCV to be low as the cell loses water following the burst, hence why cells that have a NaCl concentration below 150mM (see figure 1; (50,0), (50,1), (50,4), (120,8), (120,2.5), (120,2)) has lower PCV compared to other level of tonicity. As aforementioned, the cell is at isotonic at 300mOsmol/L, i.e. 150mM. Thus, this implies that PCV at 150mM will be at its highest as illustrated by figure 1 ((150,7), (150,9)). Hypertonic solutions contain NaCl concentration greater than 150mM ((180,4), (180,5.5), (180,7.5) (450,5), (450,4), (450,3.5)). It can be observed that PCV for hypertonic solutions are relatively higher than that of hypotonic but less than isotonic 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 above (refer to figure 1) indicate that the PCV in hypertonic solution is indeed less than isotonic, hence supporting the original hypothesis. This is because when a cell is placed in a hypertonic solution, water will exit out of the cell, resulting in loss of cell volume and shrivelled cells. Conversely, a cell in an isotonic solution will not lose or gain water, resulting in a stable cell volume and no effect on the cells.

2. What happens to the parameter you have measured when sheep red blood cells are placed in both hypotonic and hypertonic environments?

The PCV in hypotonic solution will be relatively lower than that of hypertonic (see figure 1). This can be explained due to the cells in hypotonic solutions bursting as a consequence of the cell gaining water. The cell bursts when it has reached its maximum capacity, thus producing a lower PCV. Alternatively, in hypertonic solutions, the cells will shrivel as a consequence of water exiting out of the cell. Although water is exiting out of the cell, it does not lose as much water compared to when a cell is lysed. Hence, cells in hypertonic solutions will have a greater PCV than that of hypotonic.

3. Explain the biological mechanisms which cause these effects.

Water moves from high concentration to low concentration through a semi-permeable membrane, which results in the water exiting the cells or entering the cells. A cell that is in an isotonic environment will not have net movement of water across the membrane as the concentration of solutes and water molecules are at equilibrium on either side of the membrane. A cell in a hypotonic environment will gain water, eventually leading to a burst when there’s too much water in the cell, decreasing the extracellular fluid. A cell in a hypertonic environment will lose water, eventually making the cell shrivel as water exits the cell into the extracellular fluid.

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4. Discuss the biological reasons that could explain the variations between data sets in your comparative analysis (Figure 1)?

The biological reasons may include the surface area of cells, temperature and illness of the sheep. Cells are not the exact same size; thus, some cells could absorb or lose water at a quicker rate, affecting the PCV. Moreover, the temperature of the experiment was not controlled which signifies that when the temperature was higher at a certain time than the other, the faster the process occurs, which also affects the PCV. Lastly, it is unknown whether the sheep has any other conflicting issues that affect the red blood cells, such as sickle cell anaemia.

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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? When severe dehydration occurs, there is an inverse correlation between heart rate and blood pressure. That is, when heart rate increases, blood pressure deceases. This happens due to decreased blood volume, making the heart pump faster to compensate for the lost water, thereby increasing heart rate. When there is a sudden drop in blood pressure, this causes poor circulation and limited blood flow to the brain, causing lethargy. Coupled with that, the body is fighting to ensure that all nutrients and fluid are restored back into the cells, resulting in weak muscles and cramps. Since the muscles lack these nutrients and fluid, this may also lead to lethargy.

6. Would you administer water intravenously or isotonic saline intravenously to treat Baxter? Use your results and knowledge from lectures to support your answer. I would administer isotonic saline intravenously as saline contains the same concentration of NaCl as that of the blood plasma, thereby not affecting the size of cells and increasing the extracellular fluid. Although Baxter’s body comprises majorly of water, I would not administer water intravenously as this may dilute the essential electrolytes in his blood. This would also create an osmotic effect in blood cells, whereas saline comprises of the same electrolytes as plasma, sodium and chloride ions, which results in less of an osmotic effect.

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. Leaving Baxter in the car that is very hot has caused him to lose water through sweating, also known as evaporation in this case. When he loses water in excess, he becomes dehydrated. Since he has lost a lot of water, his heart needs to pump blood quickly to restore the water in his body, causing his heart rate to increase. Simultaneously, his blood pressure drops, making him fatigue and lethargy. As the fold is visible after pinching him, this indicates he is experiencing hypertonic dehydration. This happens when he loses more water than sodium. When your cells are in a hypertonic environment, water will exit the cells, resulting in decreased cell volume. When the cell’s volume decreases, the cell shrinks. This is why you are able to see the fold on his skin as his cells have shrunk. All this happens through the process of osmosis. Osmosis is the movement of water from high concentration to low concentration via a semipermeable membrane. This happens so water is balanced out on either side of the membrane.

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References List any references you have used in your answers, in the panel below.

Drip Drop, 2020. How To Stop Feeling Tired: How Dehydration Can Cause Fatigue. [Online] Available at: https://dripdrop.com/blogs/news/how-to-stop-feeling-tired [Accessed 17 March 2021]. Physiopedia, 2021. Dehydration. [Online] Available at: https://www.physio-pedia.com/Dehydration [Accessed 16 March 2021]. Registered Nurse RN, 2021. Isotonic, Hypotonic & Hypertonic IV Fluid Solution NCLEX Review Notes. [Online] Available at: https://www.registerednursern.com/isotonic-hypotonic-hypertonic-iv-fluid-solutionoverview-for-nursing-students-with-quiz/ [Accessed 17 March 2020]. Vital Force, 2020. Why is saline used instead of water for IV fluids?. [Online] Available at: https://vitalforceal.com/iv-vitamins/why-is-saline-used-instead-of-water-for-iv-fluids/ [Accessed 16 March 2021].

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© 2017 The School of Biomedical Science

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