Comparative Cell Membranes and TransportHands-On Labs, Inc. Version 42-0033-00-03
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:
You will investigate transport across cell membranes and the processes of diffusion and osmosis, a specific type of diffusion. You will explore the concept of osmosis by collecting data on the mass of potato samples placed in sucrose solutions with a range of concentrations. Diffusion of molecules through a semipermeable membrane will be simulated with dialysis tubing filled with starch and glucose solutions.
Experiment Comparative Cell Membranes and Transport
MaterialsStudent Supplied Materials
Quantity Item Description1 Bottle of distilled water1 Coffee cup or mug1 Cutting board1 Dish soap1 Pair of scissors1 Plastic wrap1 Raw white potato1 Resealable plastic bag1 Roll of paper towels1 Sharp knife1 Sheet of paper1 Source of hot water1 Source of tap water1 Stopwatch, timer, or watch with a second hand1 White granulated sugar (C H O )12 22 11
HOL Supplied Materials
Quantity Item Description1 Dialysis tubing, 6 inches1 Digital scale, precision1 Funnel, 70 mm1 Glass beaker, 100 mL1 Glass stirring rod1 Graduated cylinder, 25 mL1 IKI Indicator, 7 mL, 2.1%1 Metal forceps or tweezers2 Pair of gloves1 Pair of safety goggles1 Permanent marker3 Plastic cup, 9 oz2 Rubber band1 Ruler2 Short stem pipet1 Small graduated pipet, 5 mL
Experiment Comparative Cell Membranes and Transport
Quantity Item Description1 Test tube cleaning brush1 Test tube rack, 6 x 13 mm9 Test tube, 13 x 100 mm1 Thermometer1 Experiment Bag: Comparative Cell Membranes and Transport
3 - Pipets, empty, short-stem1 – Benedict’s Reagent, 15 mL in dropper bottle1 - Glucose solution, 20%, 5 mL1 - Starch solution, 1% stabilized, 7 mL in dropper bottle
Note: To fully and accurately complete all lab exercises, you will need access to:
1. A computer to upload digital camera images.
2. Basic photo editing software such as Microsoft Word® or PowerPoint®, to add labels, leader lines, or text to digital photos.
3. Subject-specific textbook or appropriate reference resources from lecture content or other suggested resources.
Note: The packaging and/or materials in this LabPaq kit may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List included in your LabPaq kit.
Experiment Comparative Cell Membranes and Transport
BackgroundPlasma Membrane Structure and Function
The movement of materials in and out of the cells of living organisms is vitally important. Chemical substances move into cells to promote and sustain cellular functions, and products and wastes produced within the cells must exit. In order to enter or exit the cell, substances must pass the plasma membrane which acts as a selective barrier and regulates the movement of substances between the intracellular and extracellular environments. Plasma membranes are present in the cells of all living beings including bacteria, fungi, plants, and animals. Figure 1 shows the plasma membrane of a typical animal and plant cell. The plasma membrane is fundamental in maintaining homeostasis (constant internal conditions) because it acts as a selective doorway.
The plasma membrane is composed of a bilayer (two layers) of phospholipids, as shown in Figure 2. Each phospholipid molecule has a polar, hydrophilic (“water-loving”) head and a nonpolar, hydrophobic (“water-fearing”) tail. The two layers are arranged like a sandwich: hydrophilic heads are exposed to the extracellular fluid; intracellular fluid and the hydrophobic tails are sequestered on the inside of the plasma membrane. Due to the polarity of the phospholipids, the plasma membrane is selectively permeable (sometimes called differentially permeable); only molecules with certain characteristics are able to pass through the membrane while others are blocked.
Experiment Comparative Cell Membranes and Transport
Figure 2. Plasma membrane structure. Note that animal membranes have a greater concentration of cholesterol than other organisms.
Protein molecules embedded in the phospholipid bilayer serve a variety of functions. Transporter proteins power active movement of molecules into and out of the cell. Receptor proteins bind chemicals in the extracellular fluid to detect chemical levels outside the cell. Enzymes, a specific type of protein, catalyze various reactions. Although the illustration in Figure 2 depicts a static and rigid plasma membrane, the components of the plasma membrane are in reality quite mobile and fluid, comparable to a liquid.
Transport Across Membranes
Molecules and ions pass through the cell membrane in different ways; the particular method depends on the size, shape, and chemical structure of the substances entering and exiting the cell, as well as the concentration gradient. The concentration gradient is the difference between the concentrations of particles outside the cell versus inside the cell.
Experiment Comparative Cell Membranes and Transport
When molecules or ions move from an area of greater concentration to one of lesser concentration, the movement is called diffusion; this is also referred to as “moving down the concentration gradient.”If the molecules are small enough, they move freely through the plasma membrane. The process is a type of passive transport because it does not require any energy. Passive transport proceeds until molecules are distributed equally on either side of the cell membrane, resulting in a state of balance called equilibrium. When active transport occurs, molecules and ions are able to travel from an area of lesser concentration to an area of greater concentration (against the concentration gradient). Active transport is a process which requires energy in the form of ATP.
One type of diffusion is osmosis; the diffusion of water across a selectively permeable membrane. Water attempts to be equal in concentration on the two sides of a membrane. Thus, water moves down its concentration gradient from an area with a lesser concentration of particles to an area of greater concentration of particles. See Figure 3. Particles that are dissolved in solution are called solutes. It is often easier to think of the process of osmosis as one of dilution because the water molecules seem to move across the selectively permeable membrane in an attempt to dilute the more concentrated particles. Osmosis does not require energy in the form of ATP.
Figure 3. The process of osmosis, whereby water molecules move from an area of lesser solute concentration to an area of greater solute concentration.
Osmosis in a cell occurs when the concentration of solute inside a cell is greater than or less than its external environment. For example, when fingers are placed in water for an extended period, the oils on the finger tips (called sebum) wash off. Since the concentration of solute is greater in the skin cells of the fingers than in the surrounding water, water moves into the cells. Essentially, the cells become waterlogged. The skin swells and appears wrinkly because it is connected to deeper dermal layers in some areas. Water may also move out of a cell when the cell is placed in an environment that has greater solute concentration.
Experiment Comparative Cell Membranes and Transport
Hypertonic, Hypotonic, and Isotonic
Osmosis in cells results in three primary cell states, all related to solute concentration inside versus outside the cell: hypertonic, isotonic, or hypotonic. In a hypertonic solution (hyper- meaning “over” and tonic meaning “tone”), the concentration of solutes outside the cell is greater than inside the cell and water diffuses out of the cell. In a hypotonic solution (hypo- meaning “under”), the concentration of solutes inside the cell is greater than outside the cell and water diffuses into the cell. In an isotonic solution (iso- meaning “same”), the concentration of solutes inside the cell equals the concentration of solutes outside the cell. In an isotonic solution, water may move across the membrane, but concentration inside and outside the cell remain constant. Figure 4 illustrates each of these osmotic states.
Figure 4. Osmosis in cells: hypertonic, hypotonic, and isotonic solutions.
Experiment Comparative Cell Membranes and Transport
Exercise 1: Plant Cells and Osmosis In this exercise, you will use potatoes to explore the properties of a living membrane system. Potato strips will be placed in sucrose solutions of varying concentrations. Initial mass and mass after soaking in the solutions for 24 hours will be compared. You will determine whether water enters or leaves the potato cells, and you will classify each solution as hypotonic, hypertonic, or isotonic.
Procedure
Note: This procedure requires 2 days to perform. The procedures for the first day will require about 1 hour followed by a 24 hour incubation period. The procedures for the second day will require about 20 minutes.
Part 1: Day 1
1. Use the permanent marker to label 6 test tubes a – f. In future steps, the test tubes will contain the following solutions: a) distilled water, b) 0.2 M sucrose, c) 0.4 M sucrose, d) 0.6 M sucrose, e) 0.8 M sucrose, f) 1.0 M sucrose.
2. Place the 100 mL beaker on the digital scale and tare (zero) the scale. Ensure that the scale is set to grams.
Note: To tare the scale, press the power button once. The scale should read 0.00 g with the beaker on the weighing platform. Thus, you will be able to determine the weight of a chemical substance without the added weight of the beaker.
3. Measure 17.10 g table sugar (sucrose, C12H22O11) in the beaker.
4. Use a 25-mL graduated cylinder to measure a total of 50 mL of distilled water. Make small additions of water into the beaker. Stir the solution with the glass stir rod until the sugar dissolves and all 50 mL of water has been added. The beaker now contains 50 mL of a 1.0 M sucrose solution.
5. Use the permanent marker to label a short stem pipet “DW,” representing distilled water. This pipet will be used throughout the experiment, and should only be used for distilled water and no other chemicals.
6. Create the following solutions for test tubes a – f:
a. Distilled water solution: Use a 25-mL graduated cylinder to measure 5 mL of distilled water. Transfer the distilled water to test tube “a.” Set the test tube aside in the test tube rack. Shake any residual water droplets from the graduated cylinder.
b. 0.2 M sucrose solution: Use the graduated cylinder to measure 1 mL of the 1.0 M sucrose solution held in the 100-mL beaker. Use the pipet labeled “DW” to add 4 mL of distilled water to the graduated cylinder. Transfer the solution to test tube “b.” Rinse the graduated cylinder with distilled water and shake to dry.
Experiment Comparative Cell Membranes and Transport
c. 0.4 M sucrose solution: Use the graduated cylinder to measure 2 mL of 1.0 M sucrose solution held in the 100-mL beaker. Use the pipet labeled “DW” to add 3 mL of distilled water to the graduated cylinder. Transfer the solution to test tube “c.” Rinse the graduated cylinder with distilled water and shake to dry.
d. 0.6 M sucrose solution: Use the graduated cylinder to measure 3 mL of 1.0 M sucrose solution. Use the pipet labeled “DW” to add 2 mL of distilled water to the graduated cylinder. Transfer the solution to test tube “d.” Rinse the graduated cylinder with distilled water and shake to dry.
e. 0.8 M sucrose solution: Use the graduated cylinder to measure 4 mL of the 1.0 M sucrose solution. Use the pipet labeled “DW” to add 1 mL of distilled water to the graduated cylinder. Transfer the solution to test tube “e.” Rinse the graduated cylinder with distilled water and shake to dry.
f. 1.0 M sucrose solution: Use the graduated cylinder to measure 5 mL of the 1.0 M sucrose solution. Transfer the solution to test tube “f.”
Note: Each test tube will contain an equivalent volume of 5 mL total.
7. Locate a cutting board or plate, a sharp kitchen knife, a ruler, a potato, and a small piece of plastic wrap. Slice 12 equally sized strips of potato. Use the ruler to ensure each strip measures 5 × 5 × 20 mm. The strips should not include any of the outer brown skin of the potato. Place each potato strip in the piece of plastic wrap immediately after cutting to prevent dehydration.
8. Cover the digital scale with another piece of plastic wrap, as shown in Figure 6. The plastic wrap will protect the scale from juice as you find the mass of the potato strips.
Figure 6. Plastic wrap over digital scale.
9. While the plastic wrap is over the weighing platform, tare the scale. Place 2 potato strips on the weighing platform. Record the mass in grams in Data Table 1 of your Lab Report Assistant under “Initial Mass” for test tube “a.”
Experiment Comparative Cell Membranes and Transport
10. Place the 2 weighed potato strips into test tube “a.”
11. Repeat the process for test tubes b – f, recording the mass of a pair of potato strips and placing the pair into its respective test tube. See Figure 7.
Figure 7. Test tubes in rack.
12. Cover the test tubes with a piece of plastic wrap to prevent evaporation of the solutions.
13. Place the test tube rack in an area where it will not be disturbed and allow the setup to stand overnight at room temperature.
14. In the final row of the Data Table 1, record a hypothesis as to whether the potato strips will gain weight or lose weight in each of the sucrose solutions.
15. Pour the remaining sucrose solution down the drain and wash the 100 mL beaker with dish soap and water. Wash and dry the graduated cylinder.
Part 2: Day 2
16. After the test tube setup has incubated for 24 hours, find the final mass of each pair of potato strips. Work sequentially with 1 test tube at a time and do the following:
a. Pour the entire contents of test tube “a” into a clean 100 mL beaker.
b. Plastic a piece of plastic wrap over the scale, and tare the scale.
c. Use the tweezers to remove the 2 potato strips from the beaker and then place the strips on a paper towel in order for excess liquid to drain off or be absorbed. Do not rub or blot the strips.
Experiment Comparative Cell Membranes and Transport
d. Place the 2 potato strips on the weighing platform of the scale. Weigh and record the total mass of the 2 strips for each test tube in Data Table 1 on the appropriate row.
e. Discard the solution in the beaker. Rinse the beaker with distilled water.
f. Repeat the preceding steps (a–d) for each test tube.
17. Calculate and record the mass difference of the potatoes in Data Table 1. The formula for this is:
Mass Difference = Final Mass – Initial Mass
Note: Potato strips that gained weigh will have a positive mass difference and potatoes that lost weight will have a negative mass difference.
18. Calculate the percentage change in mass of the potatoes and record in Data Table 1. The formula for this is:
−= ×
(Final Mass Initial Mass)Percent Change in Mass 100Initial Mass
19. Create a bar graph to show the percent change in potato mass for each solution. Plot molarity of sucrose (M) on the independent axis (x-axis) and the change in potato mass (%) on the dependent axis (y-axis). (The molarity of sucrose in distilled water is 0.) Insert your graph into Data Table 2 of your Lab Report Assistant.
20. Optional: Share data among the entire class or a group of classmates. Determine the average for the percent change in mass for each molarity of sucrose. (Consult your professor to determine whether this step is required of you.)
21. Wash all equipment with dish soap and dry for later use. Use the test tube cleaning brush to thoroughly clean the test tubes.
QuestionsA. Define osmosis. How did you observe osmosis in Exercise 1?
B. List molar concentrations (0.0 M, 0.2 M, etc.) at which water entered the potato strips. Why did water move into the potato strips? Were these solutions hypotonic, hypertonic, or isotonic?
C. List the molarities at which water exited the potato strips. Why did water move out of the potato strips? Were these solutions hypotonic, hypertonic, or isotonic?
D. Was the hypothesis you recorded in Data Table 1 supported or refuted by your experimental results? Discuss your results in relation to your hypothesis. (It is acceptable if your hypothesis was refuted, as long as you can explain WHY.)
E. If you were asked to place potato strips in a solution that would cause the potato to neither gain or lose mass, what molarity would you choose and why? Would this solution be hypotonic, hypertonic, or isotonic?
Experiment Comparative Cell Membranes and Transport
Exercise 2: Diffusion Across a MembraneIn this exercise, you will examine diffusion across a semipermeable membrane.
Caution: Chemicals used in this exercise may cause permanent staining to clothes and work surfaces. Wear safety gloves and goggles through the entirety of the exercise.
Procedure
1. Gather the short, clear plastic cups provided in your kit. Use the permanent marker to label the cups “1” and “2.”
Note: If the labels from the previous exercise did not wash off of the cups, cross-out the previous labels before writing the new labels.
2. Use the graduated cylinder to add exactly 150 mL of distilled water to cup 1.
3. Place the dialysis tubing in the distilled water in cup 1. Allow the tubing to soak for at least 5 minutes or until the tubing becomes soft and pliable.
Note: The dialysis tubing is provided in your kit. The tubing is clear, nearly invisible, and packaged within a sealable plastic bag. Take care when opening the sealable plastic bag so as to not damage the dialysis tubing. It will act as a membrane in this exercise.
4. Use the permanent marker to label a short stem pipet “DW”. Use this pipet to add 4 mL of distilled water to the graduated cylinder.
5. Add 2 mL of starch solution directly from the dropper bottle to the graduated cylinder. Keep the tip of the dropper bottle clear of the rim of the graduated cylinder to avoid contamination. The final volume for this step is 6 mL.
6. Add 2 mL of 20% glucose solution directly from the dropper bottle to the graduated cylinder. The final volume for this step is 8 mL.
7. Transfer the solution in the graduated cylinder to cup 2.
8. Use the glass stir rod to mix the solution thoroughly. Wash the stir rod with dish soap and tap water and dry the stir rod with paper towels.
9. Use scissors to snip a rubber band in 1 place, as shown in Figure 8. Repeat for a second rubber band and set the second rubber aside until future steps.
Experiment Comparative Cell Membranes and Transport
Figure 8. Snipped rubber band.
10. Remove the dialysis tubing from the water in cup 1.
11. Set cup 1 aside with the water still inside it for later use.
12. Fold the dialysis tubing about 1 ½ cm from the end. Tie the snipped rubber band around the folded end of the tubing, creating a seal.
13. Test the seal with a small amount of distilled water. Use the following procedures as a guide:
a. To open the unsealed end of the dialysis tubing, carefully rub the tubing between your fingers until the middle of the tubing opens, as shown in Figure 9.
b. Use the pipet labeled “DW” to add a small amount of distilled water to the dialysis tubing.
c. If the tube leaks, tighten the knot in the rubber band and repeat the test.
d. Discard the distilled water used to test the dialysis tubing.
Figure 9. Opening the dialysis tubing.
14. Place a funnel in the open end of the dialysis tubing.
15. While holding the dialysis tubing around the funnel, slowly pour the glucose/starch solution from cup 2 into the funnel.
Experiment Comparative Cell Membranes and Transport
16. Use your fingers to press any air from the top of the dialysis tubing. Fold the end of the tubing and tie the end closed with a rubber band. Ensure that there are no leaks from the dialysis tubing.
17. Rinse the outside of the dialysis tubing with distilled water in case any portion of the glucose/starch solution was spilled during the sealing.
18. Set the dialysis tube aside on a paper towel.
19. Observe the solution in the dialysis tubing and record your findings under “Initial observations” in Data Table 3 of your Lab Report Assistant.
Note: Be as precise as possible when making observations. Good observations include terms like “opaque,” “clear,” and “colorless.”
20. Use the graduated pipet to slowly add 20 drops of IKI solution to the distilled water in cup 1.
21. Use the glass stir rod to mix the solution.
22. Record the color of the contents in cup 1 in Data Table 3 under “Initial observations.”
Note: IKI indicator is used to test for the presence of starch. When IKI indicator comes in contact with starch, it turns a dark blue/black color. You will investigate color changes to determine the location of starch (inside the dialysis tube, in the cup-1 solution, or both).
23. Place the dialysis tubing containing the glucose/starch solution in cup 1, which contains the IKI solution.
24. Allow the dialysis tubing to sit in the cup for 1 hour. Wash cup 2 with dish soap and water and dry with paper towels.
25. When 1 hour has passed, record the color of the solution in the cup and the color of the solution in the dialysis tubing in Data Table 3 under “Final observations.”
26. Remove the dialysis tubing from the solution in cup 1 and hold it over cup 2. Use scissors to snip the dialysis tubing and transfer the entire contents of the dialysis tubing to cup 2.
27. Discard the empty dialysis tubing in a trash bin. Set the solutions in cup 1 and cup 2 aside for future steps.
28. Gather 3 test tubes. Use the permanent marker to label the test tubes “1”, “2”, and “3”. With a ruler, place a mark 2 cm and 3 cm from the bottom of each test tube.
29. Prepare each of the test tubes as follows:
a. To test tube 1, use a clean, short stem pipet to add the solution in cup 1 to the 2-cm mark of the test tube. Discard the pipet in a trash bin.
b. To test tube 2, use a clean, short stem pipet to add the solution from cup 2 (the dialysis tubing contents) to the 2-cm mark of the test tube. Discard the pipet in a trash bin.
Experiment Comparative Cell Membranes and Transport
c. To test tube 3, use the short stem pipet labeled “DW” to add distilled water to the 2-cm mark of the test tube.
d. Add Benedict’s reagent to 3-cm mark of each of the three test tubes. Swirl the test tube contents to mix.
e. Set the 3 test tubes aside. An empty cup, beaker, or 24-well plate work well as a test tube holder.
30. Record observations for the solution in each test tube under “Initial observations” in Data Table 4 of your Lab Report Assistant.
31. Create a hot water bath by filling a mug ½ full with water that is near boiling. Use a thermometer to ensure that the temperature is at least 90°C.
Note: Water may be heated in a saucepan over a hot plate or stove or water may be heated in the microwave. Handle all hot liquids with care.
32. Place test tubes 1–3 in the hot water bath.
33. Allow the solutions in the test tubes to incubate for 10 minutes.
Note: Benedict’s reagent is used to test for the presence of simple sugars, such as glucose. When Benedict’s reagent comes into contact with sugar, it changes color. You will investigate color changes to determine the location of sugar (inside the dialysis tube, in the cup-1 solution, or both). Distilled water is used as a control.
34. Record observations for the solution in each test tube under “Final Observations” in Data Table 4.
35. Cleanup: Place the dropper bottles of glucose, starch, and IKI back in your kit for possible future use. Rinse the graduated pipet and place in a sealable plastic bag labeled IKI. Carefully pour all mixed solutions down the drain with copious amounts of tap water. Wash all glassware and equipment with dish soap and tap water and dry. Place the pipet and all equipment back in the kit for future use.
QuestionsA. Define diffusion.
B. IKI indicator tests for the presence of which substance? Benedict’s reagent tests for the presence of which substance?
C. In the first part of Exercise 2, a glucose/starch solution was placed in dialysis tubing. Then, the tubing was placed in a cup containing IKI indicator. Did starch stay within the tubing or move out of the tubing? Did IKI indicator stay in the solution in the cup or move into the tubing? Support your answer with the observations you recorded in Data Table 3.
Experiment Comparative Cell Membranes and Transport
D. In the second part of Exercise 2, the IKI solution in the cup and the contents of the dialysis tubing were heated with Benedict’s reagent. Did sugar stay within the dialysis tubing or move out of the tubing? Support your answer with the observations you recorded in Data Table 4.
E. What was the purpose of testing distilled water with Benedict’s reagent?
F. How is a cell membrane similar to the dialysis tubing used in this experiment?
G. Is the movement of substances in this exercise active or passive?
H. From your data, might it be inferred that a) starch and glucose molecules are the same size, b) starch molecules are larger than glucose molecules, or c) starch molecules are smaller than glucose molecules. Use your data to explain your answer.
