BIO 101 Lab 6

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Apr 3, 2024

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'(%:‘\ i X4 LABORATORY ’ STUDENT OUTCOME OBJECTIVES At the completion of this lab, the student will be able to: 1. Discuss the general makeup of the plasma membrane. 2. Explain selectively permeable (semipermeable) as it pertains to the plasma membrane. 3. Define and explain passive transport processes including diffusion and osmosis. 4. Explain factors that affect the movement of molecules across the plasma membrane. 5 List several means by which the rate of diffusion can be increased. Background Information The primary function of the plasma membrane of a cell is to control the movement of substances into and out of the cell (Figure 6.7). This is essential for maintaining the homeostatic condition of the cell and to allow the cell to function properly. The phospholipid bilayer and specialized proteins of the plasma membrane maintain the selectively permeable (semipermeable) condition of the plasma membrane allowing certain molecules (including O,, CO,, plus water) and ions to pass through, while restricting other molecules. Diffusion is the movement of a substance from an area of higher concentration to an area of lower concentration. The substance is moving down a concentration gradient, a difference in concentration in two associated areas. Small nonpolar molecules 5 v 2 Small and hydrophobic such as oxygen gas and carbon dioxide gas can diffuse through Giilscilas the phospholipid bilayer of the plasma membrane. Diffusion is also gteriod hormones assisted by the random vibration of the fluid particles in a solution. 02, €02, N2 Water Water molecules are more restricted in their diffusion through cell 88 membranes because of their polar condition. However, the small size of water and the motion of the phospholipid molecules in the phospholipid bilayer allow water molecules to slip through the membrane. Osmosis is the diffusion of solvent molecules such as water through a semipermeable membrane. Water and other small 8 —H M —~0 kD0 6 or nonpolar molecules that diffuse down a concentration gradient through plasma membranes are moving by passive transport. This movement of molecules across a membrane does not require cellular energy. When the concentration of particles is the same on both sides 88 g 8 of a membrane, equilibrium has been reached. Equilibrium is an 8 a equal concentration of a substance on either side of a membrane. Simple diffusion Figure 6.1. Passive Transport. yratory | Cellular Transport Mechanisms © M. Sugermeyer 91

m TERM LIST « Selectively permeable - Concentration gradient - Passive transport - Diffusion « Osmosis «+ Equilibrium EXERCISE 1: Diffusion Objectives After completing this exercise, the student will be able to: 1. Describe the physical conditions necessary for diffusion to occur. 2. Describe several means by which the rate of diffusion can be increased or decreased. 3. Discuss diffusion through a selectively permeable membrane. Introduction to Exercise 1 You have noticed diffusion happening many times when you smelled delicious food odors or various types of fragrances. No doubt you have seen coffee, tea, or other beverages being made. In Lab 2, you observed salt diffusing into water producing a solution. The rate of diffusion increases if the concentration gradient is greater on one side of the membrane than the other. Increasing temperature, stirring, or shaking speeds up the motion of molecules (kinetic energy) and therefore the rate of diffusion. i O SR R S R i AR S R S AR S L D e =5 LR e A e '4{5 Figure 6.2. Diffusion through a Liquid. 92 Cellular Transport Mechanisms | Laboratory 6 © M. Sugermeyer

Begin your study of diffusion using potassium permanganate (KMnQ,). Potassium permanganate is a salt that divides into K* and MnO, ions in solution. It can be used as an oxidizer and as a disinfectant. E“ TERM LIST - Potassium permanganate (KMnQO,) MATERIALS AND EQUIPMENT + 250-mL Beaker « Electronic balance « 100-mL Graduated cylinder « KMnO, PROCEDURE 1. Place a piece of paper on the electronic balance and press tare. Weigh 2.0 grams of potassium permanganate crystals. Measure 100 mL of water using the graduated cylinder. Pour the water into a 250-mL beaker. Add the KMnO, to the water. o ok owoN Note the pattern of diffusion of KMnO, from the area of high concentration to the bottom of the beaker. TOPIC REVIEW 1. What color are the dry crystals of potassium permanganate? 2. What color is the potassium permanganate in solution? 3. How could the rate of diffusion of KMnO, be increased? Cellular Transport Mechanisms 93

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To study the process of diffusion through a membrane, you will use dialysis tubing. This is the same material that is used to treat kidney dialysis patients because it allows their blood to flow from their bodies into a machine that can cleanse their blood. Dialysis tubing serves well to simulate cell membranes because it too is selectively permeable. When dry, dialysis tubing resembles a rectangle of plastic. After being immersed in water, the ends of the tubing can be opened by rubbing the tubing between your fingers allowing you to form it into a bag. m TERM LIST - Dialysis tubing MATERIALS AND EQUIPMENT + 250-mL beaker - lodine - String « Starch solution (stock) - Dialysis tubing « Scissors HYPOTHESIS PROCEDURE 1. Filla 250-mL beaker to the 200 mL line with water. 2. Place one piece of dialysis tubing into the water for 2 minutes, allowing it to soften. 3. Remove the dialysis tubing from the water and tie a piece of string securely around one end of the dialysis tubing. 4. Open the dialysis tubing by rubbing the other end gently between your fingers. 5. Fill the dialysis tubing about half full with starch solution from the stock bottle. 6. Tie the other end of the tubing securely to create a bag. 7. Rinse the bag to remove any starch from the outside of the bag. 8. Place the bag into the beaker of water. 9. Remove the dropper from the iodine bottle and observe the color of the solution in the dropper. 10. Add enough iodine to the beaker of water to make the solution outside the bag a light amber. 11. Observe any changes at 5 minutes and 10 minutes after adding the iodine to the water. 12. Record your observations in the chart. (Next page) 13. Remove the bag and place it on a paper towel. 14. Observe any changes since first putting the bag into the beaker. 94 Cellular Transport Mechanisms | Laboratory 6

Table 6.1. Diffusion of lodine into a Starch Bag. TIME | OBSERVATION START 5 Minutes 10 Minutes TOPIC REVIEW 1. Was there any change in the color of the iodine outside the bag? 2. Did the starch solution leave the bag? What evidence did you use to draw your conclusion? 3. The color change in the starch solution showed that diffused into the bag. aboratory o | Cellular Transport Mechanisms 95

EXERCISE 2: Osmosis Objectives After completing this exercise, the student will be able to: 1. Compare and contrast osmosis to simple diffusion. 2. Determine the effect of a solute’s (sucrose) concentration gradient on osmosis. 3. Use the following terms to describe various solutions and their relationship to each other: solvent, solute, hypertonic, hypotonic, and isotonic solutions. 4. Determine the effect of hypertonic and hypotonic solutions on blood cells and on Elodea leaf cells. Introduction to Exercise 2 Osmosis (diffusion of water through a semipermeable membrane) occurs wherever there is a difference in the concentration of solutes on two sides of a selectively permeable membrane. Tonicity is the difference in concentration of solutes on either side of a semipermeable membrane. A solute concentration outside a cell that is LOWER than the solute concentration inside the cell is hypotonic to the cell, so water moves into the cell. A solute concentration outside the cell that is HIGHER than the solute concentration inside the cell is hypertonic to the cell, so water moves out of the cell. I the solute concentration is equal outside the cell and inside the cell, the condition is isotonic. Water molecules will move in both directions in equal amounts in an isotonic condition. Refer to Figure 6.3. L e e e e e | R e, R e o ST ey RE e e e EESRY S R Solution Solution e Solution 90% H,0 80% H,0 95% H,0 Cell 10% 20% coll 5% 90%H,0 Solutes Solutes 90% H,0 Solutes 10% 10% gelies Movement Movement bollites Movement of H,0 of H,0 _—~0f H,0 o > W £ A ey W o A Isotonic Hypertonic Hypotonic = Figure 6.3. Tonicity. m TERM LIST - Tonicity « Hypotonic 96 Cellular Transport Mechanisms « Hypertonic - Isotonic Laboratery 6

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Osmosis is the movement of water through a selectively permeable membrane until equilibrium is established on both sides of the membrane. In solutions where there is a higher solute concentration, for example sucrose, on one side of the membrane than the other, the amount of water on that side will be less. The water will move by osmosis from the area of higher concentration of water to the area of lower concentration of water until the amount of water on either side of the membrane is equal. This principle can be tested using dialysis tubing (a semipermeable membrane), water, and a sucrose solution. Before you begin, write a hypothesis about the changes that will occur in each system (hypertonic, hypotonic, and isotonic) during the experiment on osmosis. MATERIALS AND EQUIPMENT - Dialysis tubing + Tap water « Graph (three 10 cm strips) + 30% sucrose solution « Paper towels - String « Electronic balance m TERM LIST « Osmosis PROCEDURE 1. Label and fill three 250-mL beakers according to the directions in Table 6.2. 2. Fill a fourth beaker with warm tap water. 3. Soak three strips of dialysis tubing in the warm water for several minutes to soften. 4 Remove the three strips of dialysis tubing and tie one end of each securely with string to form three bags. “n Fill each of the three bags one-half full with the solutions as indicated in Table 6.2. 6. Press excess air out of each bag and tie the other end of each bag securely with string to prevent leakage. Leave some slack area before the knot. Cut off excess string. 7. Rinse each bag with tap water and then place the bags on paper towels labeled 1, 2, and 3. 8. Carefully blot the bags dry with paper towels. 9. Weigh each bag on the electronic balance. Record the weights of the bags to the nearest tenth of a gram in Table 6.3. 10. Return the bags to the proper beakers. Be careful not to mix up the bags. 11. Every five minutes, repeat the weighing of the bags and record your data in Table 6.3. 12. Record the condition of tonicity (hypertonic, hypotonic, and isotonic) in the beaker for each system in Table 6.2. 13. Data Analysis—Create a line graph based on the data in Table 6.3. | Cellular Transport Mechanisms 97

Table 6.2. Contents of Beakers and Bags. System 1 150 mL Water Water System 2 150 mL Water 30% Sucrose Solution System 3 150 mL 30% Sucrose Solution | Water Table 6.3. Weight of Each Bag at 5 Minute Intervals. System # | Initial weight | +5 Min. | +10Min. | +20Min. +25 Min. System 1 System 2 System 3 DATA ANALYSIS Prepare a line graph the data from Table 6.3. Be sure to mark the x-axis and y-axis appropriately. The graph will reveal which system gained or lost weight over time. Plot the points using the KEY then connect the points with a line. Based on your data, you should be able to analyze and clearly explain what happens in each system using the appropriate terms (hypertonic, hypotonic, and isotonic.) Key: Isotonic—Circle O, Hypotonic—Triangle 2\, Hypertonic—Triangle Y/ Yaxis 18g. 16 g. 14 g. 12g. 10g. 8g. 649. 4q. 2g. X axis 98 Cellular Transport Mechanisms | Laboratory 6

TOPIC REVIEW 1. Did your experiment show that the bag in System 1 had a significant change in weight? 2. What type of condition is represented by System 1? 3. Which bag had the greatest change in weight? 4. Which system had a hypertonic condition in the beaker? Blood cells must be in an isotonic condition relative to blood plasma in order to maintain their biconcave disc shape. Blood cells placed in a hypotonic condition such as distilled water will expand as water moves into the cells. Eventually, the excess water moving into the cell will cause hemolysis or rupturing of the blood cells. Other animal cells will also lyse (rupture) when placed in a hypotonic condition. If there is a high concentration of a solute, such as salt, in the plasma of blood, water will be drawn out of blood cells causing the cells to crenate (shrink). mTERM LIS « Hemolysis - Lyse « Crenate Laboratory 6 | Cellular Transport Mechanisms 99

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PROCEDURE 1. Study the diagrams of dialysis bags in different conditions in Figure 5.4. 2. Record the condition (hypotonic, hypertonic, and isotonic) under each beaker in the diagram labeled a, b, and c. 3. Label the conditions of the blood cells in diagrams d and e. =T " D sucrose I © M. Sugermeyer Normal blood cells ¢ & Figure 6.4. Tonicity and the Osmotic Effects on Red Blood Cells. TOPIC REVIEW 1. What is the sucrose in beakers a and b in the illustration, solute or solvent? 2. What solute(s) might be in blood plasma that could cause blood cells to shrink such as condition d? 3. Which organ in the body is responsible for removing excess water from the blood? 100 cCellular Transport Mechanisms |

Tonicity works the same for all living cells because water can cross the lipid bilayer of the plasma membrane. Plant cells have a defense against cells breaking when too much water tries to enter the cell. The cell wall of plant cells restrains the plasma membrane from being stretched to the bursting point. Turgor pressure is the hydrostatic pressure within the cell that holds the plasma membrane firmly against the cellulose cell wall keeping the plant erect. When the plant is standing erect, it is in turgor. When plant cells lose water, the plasma membrane shrinks away from the cell wall in the process of plasmolysis. If the water loss continues, the plant will enter terminal wilt. The water plant Elodea is a good specimen to study osmosis in plant cells because the leaves are only two cells thick and the condition inside the cells can be clearly seen with a microscope. Your instructor will set up fresh demonstrations of Elodea in conditions of turgor and plasmolysis. m TERM LIST « Turgor pressure « Turgor « Plasmolysis PROCEDURE 1. Observe the demonstrations of turgor and plasmolysis. 2. Make drawings of both of these conditions with the power of magnification. Tugor: X Plasmolysis: X TOPIC REVIEW 1. What conditions might cause plants in the wild to lose water? 2. Which condition do we associate with wilting of plant leaves? 3. What prevents plant cells from bursting when filled with water? shoratory 6| Cellular Transport Mechanisms 101

Name: Lab: OSMOSIS HOMEWORK—PART 1 1. Identify the condition of the solution outside the cell in each of the following. 0] H,0 Figure 6.5. Tonicity for Homework. 2. Identify the condition of the cell and the tonicity of the solution that caused the condition in each of the following. —.\ \ €a. NORMAL Condition of cell Tonicity of solution Condition of cell Tonicity of solution NORMAL Conditionofecell ___ Condition of cell Tonicity of solution Tonicity of solution Figure 6.6. Effect of Tonicity on Cells. Laboratory 6 | Cellular Transport Mechanisms 103

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OSMOSIS HOMEWORK—PART 2 1. 11. 12. 13. Take 2 small containers (bowls or cups). In each, put about 6 to 8 oz. of tap water. Mark one container and add 1T. of salt. Add the same amount of green leaves (lettuce, celery, or even grass) to each container. Allow to sit for at least 8 hours (overnight will do). Hypothesis of the condition of the leaves after the test: ANALYSIS: Test the condition of the leaves by picking them up. Describe the condition of the leaves in the unmarked container. This shows that the water has moved the leaves. The leaves were in a solution. The cells of the leaves are in a condition of Describe the leaves in the tap water plus salt in the marked container. This shows that water has moved the leaves. The leaves were in a solution. The cells of the leaves are in a condition of 104 Cellular Transport Mechanisms | Laboratery 6

e Name: Date: LABORATORY 6 Give an example of diffusion of a gas. Give an example of diffusion in a liquid. How does diffusion differ from osmosis? What part of a cell does the dialysis tubing simulate? Osmosis is the diffusion of across a Write in the meaning of: a. Hypotonic: b. Isotonic: c. Hypertonic: What happens to blood cells when they lose water? What plant was used to study the movement of water into and out of plant cells? Labotatocy 6| Cellular Transport Mechanisms 105

Name: Date: 1. What types of molecules or particles are unable to diffuse through the lipid bilayer of the 2. plasma membrane? 3. Molecules diffuse from areas of concentration to associated areas of concentration. 4. What happens to potassium permanganate when it is added in water? 5. Dialysis tubingis a membrane. 6. What does the term dialysis indicate? 7. Which is the largest, water, iodine, or starch? How did you come to this conclusion? 8. What would happen to blood cells if the amount of salt increased to a dangerous level? taburatory 6 | Cellular Transport Mechanisms 107

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10. 11. Name two ways that you could speed up and increase the changes in Exercise 2 Activity A that () uses sucrose solution. , , o How would excess salt or alcohol in your diet affect the cells of your body? Which condition (hypotonic, hypertonic) will ensure that the central vacuole of a plant cell is kept full of water? Cellular Transport Mechanisms | Laboratory 6

BIO 101 Lab 6

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