BIOLOGY HOMEWORK

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Cell Membranes and Transport Hands-on labs, inc. Version 42-0033-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will learn how various cell membrane proteins function and will study both passive and active transport. Students will study how temperature affects the rate of diffusion across a differentially permeable membrane and how osmosis is affected by various concentrations of sucrose. They will calculate osmolality and study the effect of various solvents on the structural integrity of membranes by measuring the amount of betacyanin release from beets.

ExpErimEnt

ObjEctivEs ● To understand diffusion and be able to give examples of this process

● To learn about osmosis and its importance in the cell

● To familiarize yourself with isotonic, hypertonic, and hypotonic concentrations. and be able to give examples of each

● To predict the effects of isotonic, hypertonic and hypotonic NaCl solutions on cells

● To learn about the impact of solvents on membrane systems

Time Allocation: 5 Hours. Exercise 2 requires a 24-hour incubation period.

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Experiment Cell MeMbranes and TransporT

matErials

MATERiAlS FRoM:

lABEl oR BoX/BAg: QTy iTEM DESCRiPTioN:

Student Provides 1 Beet, raw 1 Coffee cup 1 Board, cutting 1 Dish 1 Water, distilled, 1 L 1 Egg white 1 Isopropyl alcohol, 70% ~20 mL 1 Knife, kitchen 1 Olive oil, few drops (~ 2 mL) 3 Paper towels 1 Plastic wrap, piece 1 Potato, raw 1 Refrigerator 1 Pan, small, cooking 1 Stove top or other heat source 1 Sugar, white granulated

From LabPaq 1 Beaker, 50 mL 1 Corn Starch, 1 gram 1 Beakers, glass 250 mL 1 Digital scale-500g 3 Pipets, short, thin stem

Dissection-kit with 7-tools - including the following: Bent Probe, Dropping Pipet, Probe, Ruler in pocket, Scalpel with 2 Blades - Note blades are in the pocket, Scissors, Tweezers

1 Graduated cylinder, 10 mL, plastic 1 IKI Indicator, 2.1% - 7 mL 1 Marking pencil, wax 2 Cup, Styrofoam, 8 oz 4 Cup, graduated, 1 oz 1 Test Tube, 6 at 13 x 100 mm

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Experiment Cell MeMbranes and TransporT

1 Test-tube- cleaning- brush 1 Test-tube-rack, 6x13-mm 1 Thermometer in cardboard tube 1 Stir Rod 3 Bag- 2x3-Zip Lock - Stir

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment Cell MeMbranes and TransporT

DiscussiOn anD rEviEw The cellular plasma membrane delineates cell boundaries, maintaining a homeostatic environment within the cell through the regulation of entering and exiting molecules. This membrane is composed of phospholipids arranged in a bilayer. These two layers are arranged like a sandwich with distinct polar and nonpolar regions. Phospholipids are molecules composed of a phosphate group attached to a glycerol subunit. Each phospholipid molecule has a polar, hydrophilic (“water- loving”) head and a nonpolar, hydrophobic (“water-fearing”) tail.

The hydrophilic heads line the external region of the cell adjacent to the extracellular fluid. They also line the internal surface of the cell adjacent to the intracellular fluid. The fatty acid tail subunits are hydrophobic. These are sandwiched in the middle of the cell lining so they are protected from contact with water, and also prevent polar molecules from penetrating the membrane. See Figure 1.

Figure 1: Cell membrane phospholipid bilayer

Embedded in this lipid bilayer are protein molecules which serve a variety of functions. Transporter proteins power active movement of molecules into and out of the cell. Receptor proteins bind to chemicals in the extracellular fluid to detect chemical levels outside the cell. Enzymes in the cell membrane catalyze various reactions.

Distinct molecules and ions pass through the cell membrane in different ways. The particular method depends on the size and shape of entering and exiting materials, as well as on their concentration gradient. The concentration gradient is the difference between the amount, or concentration, of a substance outside the cell versus the concentration inside the cell.

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Experiment Cell MeMbranes and TransporT

Transport

If molecules or ions are moving from an area of greater concentration to one of lesser concentration, the movement is called diffusion. If the molecules are small enough, they can pass through the membrane freely, a process called passive transport. Because the substance is following the Second Law of Thermodynamics (entropy) in this case, no energy is required for passive transport. Passive transport continually occurs, and eventually molecules are distributed equally on either side of the cell membrane, a state called equilibrium.

Sometimes, if a molecule or ion is large, a channel or carrier is necessary for diffusion to occur. This is called facilitated diffusion, and it only occurs when a channel or carrier is positioned to allow the diffusion. Sometimes, a molecule or ion travels from an area of low concentration to an area of high concentration (against its concentration gradient). This process requires energy, and is called active transport.

osmosis

A subset of diffusion is osmosis, the diffusion of water across a selectively permeable membrane. Selectively permeable membranes only allow the passage of certain molecules. It is often easier to visualize this process as one of dilution. If an area has more solutes such as sodium chloride (NaCl; table salt), water will move into the area to dilute the solutes. It is important to note that concentrations refer to the amount of solute dissolved in the solvent. For instance, a 5% NaCl solution contains 5 g NaCl in 100 mL of H2O. (Although the solution contains 95% water, it is not stated as such.)

The following terms are used to describe osmotic concentrations that exist in cells. See Figure 2.

Figure 2: Osmotic concentrations of a solution

● isotonic: In this condition, there is no net movement of solutes into or out of a cell. For example, IV fluids contain the same concentration of solutes as blood plasma, so that they can coexist in the bloodstream without interfering with normal body functions.

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Experiment Cell MeMbranes and TransporT

● Hypertonic: A hypertonic solution is one that has a higher concentration of solutes than another solution. Cells placed in a hypertonic solution have a lower concentration of solutes inside the cell than exists outside the cell. To equalize internal and external concentration, water will leave the cell, resulting in a shrinking of the cell. For example, red blood cells have an internal concentration of 0.9% NaCl. If they are placed in a solution containing more than 0.9% NaCl, water will rush out of the cell, shriveling the cell.

● Hypotonic: A hypotonic solution is one that has a lower concentration of solutes than another solution. When cells are placed in a hypotonic solution, the interior of the cell has a higher concentration of solutes than exists outside the cell. To equalize internal and external solute concentrations, water will pass into the cell, expanding its size and often rupturing the cell membrane as a result. For example, if red blood cells are placed in a solution containing less than 0.9% NaCl, water will move into the cells, and they will eventually burst.

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Experiment Cell MeMbranes and TransporT

Exercise 1: Diffusion Molecules are in constant motion, and this movement causes them to collide with each other. The size of the molecules and the temperature of the solution play a role in diffusion. The larger the molecules, the more they bump into each other, and the slower they diffuse. Temperature increases the speed of the molecules, increasing the rate of diffusion.

prOcEDurE In this exercise, you will use temperature changes to study rates of diffusion.

1. Before beginning, set up a data table similar to the Data Table 1 in the Lab Report Assistant section to record your observations for this procedure.

2. Use the marking pencil to label the three small zip-lock bags as Cold, Ambient, and Hot.

3. Set up a water bath in a small pan with 6 cm of water using a stove or other heat source, boil the water and then reduce the heat to maintain the water at a simmer. Only a few small bubbles may rise slowly upward through the water from the bottom of the pan.

4. Use the thermometer to record the temperature inside your refrigerator (“Cold) and the work room (“Ambient”). Record these data into your data table.

5. Measure and record the temperature of this water bath one minute after it stabilizes at a simmer.

6. Use the graduated cylinder to pour 100 mL of ambient temperature distilled water into two Styrofoam cups and one 250-mL glass beaker. The cups and beaker should contain 100 mL of distilled water each.

7. Add 20 drops of IKI indicator to each of the three containers. Mix the solutions by gently swirling the cups.

8. Place the glass beaker in the simmering water bath. Place one Styrofoam cup in the refrigerator. Place the other Styrofoam cup on an open surface in the room. Leave the cups in their three different environments for 5 minutes.

9. Use a 10 mL graduated cylinder to measure out 45.5 mL of distilled water into the 50 mL beaker.

10. Prepare a 1% corn starch solution. Measure out 0.5 g of corn starch onto paper placed on the digital scale. Add the corn starch to the 50 mL beaker of water. Stir the corn starch until it dissolves.

11. Measure 10 mL of the 1% corn starch solution into the graduated cylinder.

12. Pour this solution into one of the three zip-lock bags. Securely seal the bag to prevent leakage of the solution. Repeat this procedure for the other two zip-lock bags. Record the color of each bag in Data Table 1.

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Experiment Cell MeMbranes and TransporT

13. Place the “Cold” bag into the cup in the refrigerator. Place the “Hot” bag into the water bath beaker. Place the “Ambient” bag into the ambient temperature workspace cup.

14. Record the start time. Immediately record the color of the solution in the bag. Record the changes of solution color for each bag every 5 minutes for 30 minutes. Then take a final reading at 60 minutes. See Figure 3.

Figure 3:

NoTE: Some bag solutions may not change color or will change slowly. Make certain to record the color for each interval regardless of any lack of color change.

15. Thoroughly clean and dry all equipment for future reuse. Discard the plastic bags and their contents.

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Experiment Cell MeMbranes and TransporT

Exercise 2: osmosis Osmosis is the process of water movement across a selectively permeable membrane from an area of low to high solute concentration. The purpose of the water movement is to balance the concentration of the solute. The size of the solute particles does not influence osmosis, because the particles themselves do not move across the membrane. Equilibrium is reached once sufficient water has moved to equalize the solute concentration on both sides of the membrane.

In this exercise you will use potatoes to explore the properties of a living membrane system.

NoTE: This procedure requires two days to perform. This includes a 24-hour incubation period at the end of the Day 1 procedure.

prOcEDurE Day 1

1. Before beginning, set up a data table similar to the Data Table 2 in the Lab Report Assistant section to record your observations.

2. Label six small, 13 × 100 mm test tubes a through f:

Tube Contents Tube Contents

a Distilled Water d 0.6 M Sucrose b 1.0 M Sucrose e 0.4 M Sucrose c 0.8 M Sucrose f 0.2 M Sucrose

3. Use the digital scale to measure 17.1 g of sugar. Put the sugar into the 50-mL beaker. Use the graduated cylinder to a measure 50 mL of distilled water. Add the water to the beaker. Stir the solution with the stir rod until the sugar dissolves. The beaker now contains 50 mL of a 1.0 M solution.

4. Measure 5 mL of distilled water into the graduated cylinder. Pour this into test tube a. Set this tube aside in the test tube rack. Rinse and dry the graduated cylinder.

5. Measure 5 mL of the 1.0 M sucrose solution into test tube b. Set this tube aside in the test tube rack. Rinse and dry the graduated cylinder.

6. Label four small plastic cups: 0.8 M Sucrose; 0.6 M Sucrose; 0.4 M Sucrose; and 0.2 M Sucrose.

7. Prepare in the labeled plastic cups 0.8 M, 0.6 M, 0.4 M, and 0.2 M solutions from the above prepared 1.0M solution as follows:

a. To create the 0.8 M sucrose solution, use the graduated cylinder to measure 8 mL of the 1 M sucrose solution and pour it into its labeled plastic cup. Measure and add 2 mL distilled water for a solution of 10 mL of 0.8 M. Rinse and dry the graduated cylinder.

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Experiment Cell MeMbranes and TransporT

b. To create the 0.6 M sucrose solution, use the graduated cylinder to measure 6 mL of the 1 M sucrose solution and pour it into its labeled plastic cup. Measure and add 4 mL distilled water for a solution of 10 mL of 0.6 M. Rinse and dry the graduated cylinder.

c. To create the 0.4 M sucrose solution, use the graduated cylinder to measure 4 mL of 1 M sucrose solution and pour it into its labeled plastic cup. Measure and add 6 mL distilled water for a solution of 10 mL of 0.4 M. Rinse and dry the graduated cylinder.

d. To create the 0.2 M sucrose solution, use the graduated cylinder to measure 2 mL of 1 M sucrose solution and pour it into its labeled plastic cup. Measure and add 8 mL distilled water for a solution of 10 mL of 0.2 M. Rinse and dry the graduated cylinder.

8. Measure 5 mL of the 0.8 M sucrose solution from the cup and pour it into test tube c. Set this tube aside in the test tube rack. Rinse and dry the graduated cylinder.

9. Measure 5 mL of the 0.6 M sucrose solution from the cup and pour it into test tube d. Set this tube aside in the test tube rack. Rinse and dry the graduated cylinder.

10. Measure 5 mL of the 0.4 M sucrose solution from the cup and pour it into test tube e. Set this tube aside in the test tube rack. Rinse and dry the graduated cylinder.

11. Measure 5 mL of the 0.2 M sucrose solution from the cup and pour it into test tube f. Set this tube aside in the test tube rack. Rinse and dry the graduated cylinder.

12. Use a cutting board and sharp kitchen knife to safely slice a potato into 12 equally-sized strips. Use the ruler to ensure each strip measures 5 mm × 5 mm × 20 mm.

13. Remove any skin from the potato strips. Keep the potato strips in a covered dish until ready to use.

iMPoRTANT: Potato cells are naturally water-filled; thus leaving them uncovered may cause dehydration and affect test results.

14. Determine the mass of two potato strips by weighing them with the digital scale. Record the mass in Data Table 2 for row a. Put these two potato strips into test tube a. Repeat this process for test tubes b-f, putting two potato strips (weight recorded first) into each test tube of solution. Cover the test tubes with plastic wrap to prevent evaporation.

15. Place the rack of test tubes in an area where they will not be disturbed and let them stand overnight at room temperature. See Figure 4.

Experiment Cell MeMbranes and TransporT

Figure 4: Test tubes with potato strips

Day 2

16. The next day (24 hours later), work sequentially with one test tube at a time. Pour the potato strip and solution into a clean cup. Use forceps from the dissection kit to remove the two potato strips from the cup, and then blot the strips gently on a paper towel. Discard the solution and clean the cup.

17. Weigh and record the total mass of the two strips for each test tube into Data Table 2 on the appropriate row. Repeat this process for each test tube.

18. Calculate and record the percentage change in the potato strips’ mass for each solution. The formula for this is:

19. Wash, rinse, and dry all materials for later use. Use the test tube cleaning brush to thoroughly clean the test tubes.

Experiment Cell MeMbranes and TransporT

Exercise 3: The Effect of Solvents on a Membrane System Living membrane systems are susceptible to stress under changing conditions of temperature, pH, and chemicals. When a membrane is compromised, the ability to regulate the entry and exit of substances into the cell is affected.

In this exercise, you will investigate the effect of an organic solvent on membrane systems. Beet cells contain a red pigment called betacyanin. This pigment is stored in the central vacuole of the cell. The outer membrane of a vacuole has a similar structure to that of the cell plasma membrane. If this structure is damaged, the betacyanin pigment will leak out.

prOcEDurE 1. Before beginning, set up a data table similar to the Data Table 3 in the Lab Report Assistant

section to record your observations.

2. Use a cutting board and sharp knife to safely slice a beet into four circular discs that are 0.5 cm wide. Use a metric ruler to cut 4 equal-sized beet strips from these discs. Each should measure approximately 5 mm × 5 mm × 20 mm.

3. Place the beet strips into a coffee cup and gently rinse them with slowly running tap water for 5 minutes. Drain and discard the colored rinse water, and set the beet strips aside.

4. Label six test tubes a through f.

5. These procedures require making isopropyl alcohol solutions using a volume-to-volume percent solution. Use a 10-mL graduated cylinder to measure 5 mL of 70% isopropyl alcohol (rubbing alcohol) usually found in a grocery store or pharmacy. Add the 5 mL of 70% isopropyl alcohol to test tube a. Place test tube a into the test tube holder.

6. Add 5 mL of 70% isopropyl alcohol to a 10-mL graduated cylinder. Then add 5 mL of distilled water to dilute the solution down to a 35% isopropyl alcohol solution. Gently swirl the graduated cylinder to mix the solution.

7. Pour 5 mL of the 35% isopropyl alcohol into test tube b. Place the test tube into the test tube holder.

8. Add 5 mL of distilled water to the remaining 5 mL of the 35% isopropyl alcohol in the 10-mL graduated cylinder. This creates a 17.5% isopropyl alcohol solution.

9. Pour 5 mL of the 17.5% isopropyl alcohol solution into test tube c and place the test tube into the test tube holder. In case it is needed later, save the remaining 17.5% isopropyl alcohol in a clean, labeled plastic cup. Thoroughly rinse and dry the graduated cylinder.

10. Use the graduated cylinder to add 5 mL of distilled water into test tube d.

11. Place a beet strip into each of the four test tubes a through d. Allow the samples to remain at ambient room temperature for 30 minutes. Occasionally stir the test tubes by gently swirling their contents. See Figure 5.

Experiment Cell MeMbranes and TransporT

Figure 5: Test tubes with beet strips

12. After 30 minutes, observe the color of each solution, and the height of the color in the test tube. Observe and quantify the relative color of the solutions based on a 0 to 10 scale, with 0 being colorless and 10 being the darkest. Record these observations in Data Table 3.

13. Gently invert the test tube and empty the solution into a cup. Remove the beet from the cup with the tweezers. Record observations of the condition of the beet in Data Table 3.

14. Use a clean pipet to add 20 drops of 70% isopropyl alcohol into test tube e and 20 drops into test tube f. Place tubes e and f into the test tube holder.

15. Use a clean pipet to add 10 drops of raw egg white to test tube e. Swirl the test tube and observe the results.

16. Use a clean pipet to add 5 drops of olive oil to test tube f. Swirl the test tube and observe the results.

Experiment Cell MeMbranes and TransporT