BIO 1140 Lab 2 - lab 1 PDF

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SimBio Virtual Labs®: OsmoBeaker®

Osmosis Mixing Blood and Water: Osmosis and Cells A patient is brought into the emergency room unconscious, with blood gushing from a wound. Among the first things you must do is get fluid into her circulatory system. If too much blood drains out, her veins could collapse. So, you stick a needle into a large vein in her arm and hook up an intravenous (IV) system for delivering fluids into veins. You're probably familiar with IVs from movies and TV shows: they are the bags hanging from a metal pole with a tube running into the patient. But what is in that bag? Is it just water, or does the water contain something? If so, does it matter what else is in the water? Later in the day, another patient comes into your clinic. He’s here to check his intestine for polyps, a possible sign of colon cancer. But the intestines are usually a bit obstructed with the remains of the last few meals. Therefore, you gave the patient pills to take with lots of water the day before his exam. The pills are made of small molecules that are indigestible and therefore will pass right through the digestive system. This causes water to leak out into the intestine and wash it out. It’s not very pleasant for the patient, but necessary to do the exam. But why should some indigestible material cause water to exit his body? These are two of many of possible examples that involve osmosis, a process by which water moves across membranes in response to differences in the concentration of other molecules dissolved in the water. In this lab, you'll take advantage of a fictional invention from a fictional bio-engineering company called Fictional Science, Incorporated. Their “Cell-O-Scope” simulator allows you to create tiny “SimCells” made out of a material that is similar to the membranes that surround animal cells. The Cell-O-Scope lets you see molecules moving around inside of the SimCells. By conducting experiments on SimCells and making observations of how the SimCells respond to different extracellular fluids, you will learn how osmosis works, and why it is an important property to consider when designing IV fluids.

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Concentration and Diffusion: A Brief Review Before you begin this lab, it is important that the meaning of concentration and the process of diffusion make sense to you.

Concentration Concentration is the amount of a substance in a particular volume or space. Concentration is a relative measure that can be expressed as a quantity of the substance (the solute) per unit of volume of the solvent (usually water in biology). The quantity of solutes is generally expressed as grams, or as moles. One mole (symbol mol) corresponds to 6.02x1023 molecules of the solute. Depending on the atom composition of the solute, one mole will have a different weight (or mass), which is called molecular weight (for example, the molecular weight of Glucose is 180.16 g/mol and that of Sodium Chloride NaCl is 58.44 g/mol). The concentration standard unit is mole per litre or mol/L (or mol.L-1), symbolized by M. Other units are used for the concentration such as percentage (%), which is often used in medical context. For instance, percentage (%) of weight per volume (w/v) refers to a quantity of solute in grams per 100 ml of solvent (5% NaCl = 5g of NaCl in 100ml of water). If the solute is a liquid, the % can be volume/volume (v/v). In this case the solute is not weighed, but a certain volume of the solute is measured and then water is added to complete to 100ml. Therefore, a 20% salt solution contains twice as many salt molecules in any given volume (e.g., a liter) than a 10% salt solution. In this series of exercises, concentration is calculated as relative concentration, which is the number of molecules of a solute divided by the total number of molecules in each cell compartment (intracellular or extracellular) and expressed as a percentage.

Diffusion Diffusion is the movement of molecules from areas of higher concentration to lower concentration. This lab explores osmosis, a special case of diffusion. Figure 1 illustrates the process of diffusion. In each case, molecules move from areas of higher to lower concentration. (Note that in fig.1.3A, the initial number of molecules is the same on both sides of the membrane, but the concentration of molecules is different.)

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Figure 1. Diffusion of Molecules Across a Permeable Membrane. A membrane that does not restrict the movement of molecules divides each box into two compartments. Comparing A to B, you can see that in each of the three examples, the number of molecules decreased on the side of the box that started out more concentrated, and increased on the side of the membrane that started out less concentrated.

A common misconception about diffusion is that molecules are somehow directed or “want” to move to where there is more space. This is not the case! Groups of molecules tend to spread out over time, but individual molecules are in constant random motion and are equally likely to move in any direction. You will be able to see this for yourself as you conduct experiments in this lab.

Exercise 1: Pressure and Equilibrium Create a SimUText account and install the SimUText program following instructions on Brightspace. Open the program by double-clicking the SimUTEXT application icon on your desktop (Windows) or in the Applications folder (Mac OSX), or by selecting it from the Start Menu > All Programs (Windows). When the SimUTEXT sign in window opens, enter your information, then click on Osmosis Simulation Questionnaire, then Osmosis.

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The Cell-O-Scope Simulator (from Fictional Science, Inc.) has two monitors that let you see what happens when artificial cells (“SimCells”) are suspended in different extracellular fluids. The Cell View Monitor lets you watch the entire SimCell. The Membrane View Monitor zooms in on the SimCell membrane (the red stripe down the middle of the Membrane View Monitor), letting you watch molecules move around inside the SimCell (on the left side of the membrane) and in the extracellular fluid (on the right side of the membrane), as well as the position of the membrane. The membrane position is also measured under the monitor view as a number between 0 (leftmost position) and 40 (rightmost position). The first simulations start with the membrane at position 20. NOTE: In the real world, you could never actually see the molecules in a cell. If you could see the molecules, there would be a lot more of them! The Settings Panel beneath the monitors identifies the types of molecules present, and lets you see the approximate number of each type of molecule in the cell and in the extracellular fluid. The Control Panel at the bottom of the screen runs the Cell-O-Scope Simulator. Try clicking the GO button to run the simulator. The molecules should start moving. Notice that the movement of any individual molecule is random. Then click the STOP button. Make sure that the simulator is STOPPED, then push the cell membrane by clicking on the membrane and dragging it to the left. Watch the SimCell in the Cell View Monitor change shape as the membrane is compressed. Next, drag the membrane to the right to stretch the membrane. The relative areas you see in the Membrane View are proportional to the relative volumes of the SimCell and extracellular fluid. You can assume that when the membrane position is in the middle (at 20 on the Membrane Position scale), that the SimCell occupies half of the volume of the Cell-O-Scope. 4.1. What happens to the membrane, when you try pushing it all the way to the right (click on the red vertical line representing the membrane in the Membrane View Monitor and move it to the right)? (If you’re not sure what happened, click the RESET button in the Control Panel and try again while moving the membrane line more slowly.) The membrane disappears and there is a red flash that represents it when it is moved all the way to the right 4.2. To what position do you have to pull the membrane to burst the cell? The membrane had to pulled to about 28 so the cell would burst

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4.3. What happens to the SimCell (visible in the Cell View Monitor) when you try pushing the membrane all the way to the right? When pushing the membrane all the way to the right, the SimCell bursts Click the RESET button in the Control Panel. Then click the GO button. The molecules on either side of the membrane will begin to move around in random directions. à Every time a molecule happens to collide with the flexible membrane, the impact causes the membrane to move slightly. The total force of the impacts of all the molecules on the membrane is called pressure. 5.1. What is your prediction if you increase the number of water molecules in the extracellular fluid? will the membrane move to the right or the left in the Membrane View? Explain your reasoning in terms of pressure. If the number of water molecules increases in the extracellular fluid I think the membrane will move towards the left in the membrane view. This is because increasing the number of water molecules outside will mean more molecules hitting the membrane. Which will mean the membrane will move towards the left because of all the pressure put on it

In the Settings panel, increase the Initial Count of water molecules in the extracellular fluid from 250 to 500. Click the SET button and the simulation will reset with two times as many water molecules to the right of the membrane. Next, click the STEP button (to the right of the GO button) to run the simulation 100 time steps. [NOTE: you can see how many time steps have passed in the Time Elapsed box next to the Control Panel. 7.1. What was the position of the membrane at the beginning of the experiment? The position of the membrane was at 20 7.2. What is the position of the membrane after running 100 steps? The position of the membrane was at 18.3 after running 100 steps

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7.3. Was your prediction in 5.1 correct? What happened? Yes, my prediction was correct, the membrane was pushed to the left

Adding water molecules to the extracellular fluid results in more molecules colliding with the outside of the membrane. The additional pressure against the membrane should have caused it to move left. As the membrane moves to the left and the cell shrinks, there will be less and less space for the molecules inside the cell to move around, and this will cause them to bump into the membrane more frequently. Meanwhile, the molecules on the extracellular fluid side of the membrane will have more and more space and bump against the membrane less. Thus, if you run the simulation longer, the pressure on either side of the membrane should eventually equalize. Think about the above description. Then run the simulator for 500 additional time steps by clicking the STEP button five more times. For each 100-time steps, pay attention to whether the membrane consistently moves in one direction. 9.1. How can you tell when the pressures have equalized? When the membrane is no longer moving in any direction 9.2. What is the approximate “equilibrium” position of the membrane? The approximate equilibrium position seems to be between 13-13.5 9.3. If you continue to run the simulator after the system has reached equilibrium, why does the membrane continue to jiggle back and forth? Because the water molecules on either side of the membrane are still hitting the membrane

à NOTE: A system is at a stable equilibrium when it consistently returns to its original

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state after being pushed slightly in any direction. If the simulator is running, click the STOP button, but do NOT reset it. You can test whether the new membrane position is at a stable equilibrium by pushing it a little in one direction and seeing if it returns to that position. Without resetting the simulator, push the membrane by dragging it a little bit to the left or right, being careful not to push it past its breaking point. Then RUN the simulation for at least 500 time steps.

à NOTE: If you push the membrane past its breaking point and the membrane disappears, you’ll need to reset the simulator and let it run until the equilibrium position is reached before trying again.

10.1. Was the membrane at a stable equilibrium before you pushed it? How could you tell? Yes because it was not moving from its position no matter how many steps it went. Even after having moved the membrane to either the right or left the membrane would bounce back to its original position meaning it is at a stable equilibrium

Next, observe how temperature influences how molecules move by moving the temperature slider at the bottom of the screen to the right (high temperature) and to the left (low temperature) and running the simulator. Once you have a feel for how temperature influences the movement of molecules, click the RESET button. 11.1. Based on how the molecules moved at different temperatures, which of the below choices (a, b, or both) do you think is correct? [NOTE: You’ll experiment with this in the next step; for now simply make a prediction.] a. at higher temperatures, the equilibrium position of the membrane should shift to the right. b.

at higher temperatures, the system should equilibrate faster.

11.2. Explain your choice(s). b. At higher temperatures, the system should equilibrate faster, because in the simulator it seemed that the cell reached equilibrium at fewer than the 5 steps it took at an average temperature.

You now will experiment with temperature to determine whether your answer to Question 11.1 was correct. ©2020, SimBio. All Rights Reserved.

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12.1. Describe experiment(s) to test and measure the effect of temperature. Does temperature influence the equilibrium position of the membrane? Does temperature influence how long it takes for the system to equilibrate? Hint: You will have to set some parameters in the setting panel and run the. Then, you will have to change the value of one parameter and measure the results you’re getting. For each experimental condition (=value of the changing parameter), you will have to repeat the experiment several times to get a significant number of results (at least 3). My experiment will consist of setting the temperature to the highest extremity, in the middle and the lowest extremity. Then at each temperature, see what happens after 500 time steps. The initial amount of water molecules in the extracellular fluid will be 500 and 250 in the intracellular fluid

Hot: At high temperatures the equilibrium was reached very fast. Instead of taking 500 steps it took 200. The temperature does not seem to affect the position of the equilibrium.

Medium: At a medium temperature the equilibrium took about 500 steps to get there, so it took an average amount of time. The membranes position was not affected by this temperature as well

Cool: At the lowest temperature extremity the molecules were moving very slowly so it took a much longer time for the membrane to equilibrate. After 500-time steps it still had not reached equilibrium.

12.2. What is your conclusion based on the results of the experiments described above? Based on the experiments done in the previous part, the membrane reaches equilibrium much faster when it is at a higher temperature than a cooler one. This is because at higher temperatures the molecules have more energy and so they are moving more and moving faster.

Click the TEST YOUR UNDERSTANDING button in the bottom right corner of the screen. You can try as many answers as you like to make sure that you understand why only one is correct.

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Exercise 2: Osmotic Pressure So far, your membrane has been impermeable (nothing can pass through it), and the SimCell and extracellular fluid have only contained water. Real cells contain water and lots of other types of molecules dissolved in the water. Water is referred to as the solvent in cells, because most of the molecules in real cells are water molecules. The molecules dissolved in the water are referred to as solutes. Real cells also have protein channels (such as aquaporins) that allow water to pass through their membranes. Osmosis is the diffusion of water across a semi-permeable membrane— a membrane that is permeable to some molecules (such as water) but not others (such as dextrose). If water molecules diffuse across a membrane into a cell, the pressure of the added molecules will cause the membrane to stretch and the cell to grow.

Select OSMOTIC PRESSURE 1 from the SELECT AN EXERCISE button

in the upper left-hand

corner of the screen. The new SimCell contains 200 water molecules and 50 dextrose molecules. The extracellular fluid contains 250 water molecules, so the total number of molecules is the same on either side of the membrane (250). Click the GO button to confirm that the pressure on either side of the membrane is the same. 15.1. How can you tell that there is no major difference in pressure on either side of the membrane? Since the position of the membrane stays the same once the “go” button is pressed, it indicates that the pressure on either side are the same

Next you will add channels to the membrane that will allow water molecules to pass through. Click the RESET button and then write down a prediction for what you think will happen to the position of the membrane in the Membrane View Monitor when you make the membrane permeable to water.

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16.1. Will the membrane move to the right, left, or stay in the same position? Explain your reasoning. I believe the membrane will move to the right because unlike the right side, which only has water molecules, the left side has water and dextrose and since dextrose cannot go through the membrane it would mean that it is able to push the wall the other way.

Add water channels by checking the box next to the water molecule icon in the Membrane Permeability column on the right. Then click the GO button to test your prediction. Run the Simulator for at least 1000 time steps. 17.1. Were you correct? What happened? I was correct that the membrane would move towards the right as oppose to staying the middle, however not only did it move to the right but after 1000 steps the membrane broke

Since the total number of molecules starts out the same on both sides of the membrane (250), the same number of molecules hit the membrane from each side as before, but this time, some water molecules pass through. If there are 250 water molecules in the extracellular fluid and only 200 inside of the cell, by random chance, more water molecules will move from the extracellular fluid to the cell than vice-versa. This movement results in a greater total number of molecules inside the cell, and thus greater pressure.

F Osmotic pressure is the pressure generated by osmosis. Technically, it is the minimum pressure that must be applied to a solution to prevent water from flowing across a semi-permeable membrane into the solution. If osmotic pressure in the SimCell increases, the membrane will stretch, and the cell will expand. If the cell membrane is stretched too far, it will break, as you saw if you ran the simulator long enough in Step 4.

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Next, reverse the conditions by loading OSMOTIC PRESSURE 2 in the SELECT AN EXERCISE menu. This time, the simulator will start out with the 50 dextrose molecules in the extracellular fluid rather than in the SimCell. Again, make the membrane permeable to water. 18.1.

Do you predict that the SimCell will e...