Name _____________________________ Period _________

AP Biology Date ______________________

1

LAB _____A. DIFFUSON AND OSMOSIS: DIFFUSION

INTRODUCTION The life of a cell is dependent on efficiently moving material into and out of the cell across the cell membrane. Raw materials — such as oxygen and sugars — needed for the production of energy and other cellular products must enter the cell. Whereas waste products — such as carbon dioxide and ammonia — which are generated during energy production and cellular digestion must be removed from the cell. Most of these materials move passively — costing the cell no energy — through the process of diffusion.

Diffusion is the movement of molecules from an area of higher concentration of those molecules to an area of lower concentration. A good metaphor for this molecular motion is what happens if you were to open a bottle of hydrogen sulfide gas (H2S smells like rotten eggs) in one corner of a room. It would not be long before someone in the opposite corner of the room would smell the odor of rotten eggs. The molecules appear to be moving from an area of higher concentration of H2S gas (the bottle) to an area of lower concentration of H2S gas (the opposite corner of the room). Eventually a dynamic equilibrium is reached; the concentration of H2S gas will be approximately equal throughout the room and no net movement of H2S will occur from one area to the other.

Since all life takes place in water — either external waters or internal waters — we must also address the special case of the movement of water across cellular membranes. The diffusion of water through a selectively permeable membrane is referred to as osmosis. Water moves from a region of higher concentration of water to a region of lower water concentration. This is often also stated as movement from a region of higher water potential to a region of lower water potential. Distilled water (pure water) has the highest concentration of water or the highest water potential.

However, diffusion and osmosis do not entirely explain the movement of ions and molecules into and out of the cell. Some molecules are valuable enough to the cell to expend energy transporting them across the cell membrane. This active transport uses energy from ATP to move substances through the cell membrane. Active transport usually moves substances against a concentration gradient, from regions of low concentration of that substance into regions of higher concentration.

DIFFUSION In this experiment you will measure the diffusion of small molecules through dialysis tubing, an example of a selectively permeable membrane. Small dissolved molecules (solutes) and water molecules can move freely through a selectively permeable membrane, but larger molecules will pass through more slowly, or perhaps not at all. The size of the minute pores in the dialysis tubing determines which substances can pass through the membrane. We will explore the process of diffusion through a semi-permeable membrane in this activity.

Name _____________________________ AP Biology

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PROCEDURE, DAY 1 1. Using a pipette, place a 2mL sample of 15% glucose/1% starch solution into a clean test

tube. Test the solution for the presence of glucose using Benedict’s solution (remember to heat it). Record the results in Table 1 (Experimental Observations).

2. Using a pipette, place another 2mL sample of the 15% glucose/1% starch solution into a clean test tube. Test the solution for the presence of starch using Lugol’s iodine (IKI). Record the results in Table 1.

3. Obtain a 30cm piece of 2.5cm dialysis tubing that has been soaking in water. Tie off one end (like a balloon) of the tubing to form a bag. To open the other end, rub the end between your fingers until the edges separate.

4. Place 15mL of the 15% glucose/1% starch solution in the bag. Tie off the other end of the bag, leaving sufficient space for expansion of the contents in the bag. Record this initial color of the solution in Table 1

5. Place 200mL of water in a 250mL beaker. Place a 2mL sample of the water into a clean test tube and test for the presence of starch. Record the results in Table 1.

6. Place a second 2mL sample of the water into a clean test tube and test for the presence of glucose (remember to heat it). Record the results in Table 1.

7. Add Lugol’s iodine to the water in the beaker. Add only enough iodine to turn the water a medium amber color. Record the color of the solution in Table 1.

8. Immerse the dialysis bag in the beaker. Allow the experiment to stand overnight.

9. Table 2 is to be used to make predictions of what you expect to occur overnight in the beaker. Fill in the Initial Observations columns (Column A) from your observations today. Then complete your predictions for each section in Column B.

10. Complete the “Initial State” diagram in Figure 1.

PROCEDURE, DAY 2 1. The next day, record the final color of the solution in the bag and the final color of the

solution in the beaker in Table 1 (Experimental Observations).

2. Observe the color changes as a test for starch and record the results for starch in Table 1.

3. Test the liquid in the beaker and in the bag for the presence of glucose. Record the results for glucose in Table 1.

4. Compare your predictions to your actual data. Any surprises? If so, discuss with the class.

5. Complete the “Final State” diagram in Figure 1.

6. Complete the Summary Questions for this section of the lab.

Name _____________________________ AP Biology

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TABLE 1. EXPERIMENTAL OBSERVATIONS 1

Color of Solution 2

Presence of Glucose 3

Presence of Starch Initial contents A

Initial B

Final A

Initial B

Final A

Initial B

Final

Bag 15% glucose 1% starch

Beaker H2O & IKI

TABLE 2. PREDICTIONS 4

Color of Solution 5

Presence of Glucose 6

Presence of Starch Initial

contents A Initial

from Table 1

B Predicted

Final

A Initial

from Table 1

B Predicted

Final

A Initial

from Table 1

B Predicted

Final

Bag 15% glucose 1% starch

Beaker H2O & IKI

FIGURE 1. Label the diagrams below to identify the contents of the bag and the beaker in the initial state and in the final state of the experiment. Also draw arrows to indicate the movement of molecules during the experiment.

INITIAL STATE FINAL STATE

Name _____________________________ AP Biology

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SUMMARY QUESTIONS

A. DIFFUSION 1. Which substance(s) are entering the dialysis bag and which are leaving the bag? What

experimental evidence supports your answer?

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2. Explain the results you obtained by discussing concentration differences and membrane pore size.

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3. Although we didn’t measure it, what other molecule can we assume also moved across the membrane?_________________________________

4. In assessing the movement of glucose, starch and iodine across the membrane in this experiment, you generated qualitative data. Quantitative data use numbers to measure observed changes. How could this experiment be modified so that quantitative data could be collected to show that water diffused into the dialysis bag.

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Name _____________________________ AP Biology

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5. Based on your observations, compare the size of each of the following molecules (glucose molecules, water molecules, IKI molecules, starch molecules) to the size of the pores in the dialysis membrane (greater than, less than):

The size of glucose molecules are _________________________ the membrane pore size.

The size of water molecules are____________________________ the membrane pore size.

The size of IKI molecules are _____________________________ the membrane pore size.

The size of starch molecules are ___________________________ the membrane pore size.

6. What results would you expect if the experiment was set up incorrectly: the water and IKI solution was placed inside the dialysis bag and the starch and glucose solution was placed in the beaker.

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7. In many animals, glucose, rather than starch, is transported by the blood through the body to all cells. In the digestive system, starches are digested by amylase to yield glucose. Based on the findings of this lab, explain why the digestion of starch to glucose is necessary.

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8. Summarize the process of diffusion.

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Name _____________________________ Period _________

AP Biology Date ______________________

6

LAB _____B. DIFFUSON AND OSMOSIS: OSMOSIS

OSMOSIS Using the principles you learned in the first exercise on diffusion, we will now investigate the movement of water in and out of a model. Water moves from areas of higher concentration of water to a region of lower water concentration. Water is at its highest concentration as distilled water. The concentration of water decreases as solutes are dissolved in the water.

If a model cell (like our dialysis tubing) is filled with a salt solution and placed in a beaker of distilled water, water will diffuse into the cell until a dynamic equilibrium is reached. At that point, no net movement of water will occur between the cell and the beaker. If a model cell is filled with a distilled water and placed in a beaker of salt solution, water will diffuse out of the cell until a dynamic equilibrium is reached. Once again, at that point, no net movement of water will occur between the cell and the beaker.

Let us now see these principles demonstrated using sucrose and salt solutions.

PROCEDURE: DAY 1 1. Take a 30cm piece of 2.5cm dialysis tubing that has been soaking in water. As you did

before, tie off one end of the tubing to form a bag, your “cell”.

2. Measure out 15mL of concentrated (1.0 molar) sucrose solution and pour it into the dialysis bag using a funnel. Tie off the other end of the bag, leaving space for expansion of the contents in the bag.

3. In case any solution spilled on the outside, rinse off the model “cell” you just made by holding it under running water.

4. Carefully blot the outside of the “cell” with a paper towel. Mass the sucrose solution “cell” (in grams) on a scale and record the Initial Mass in Table 3.

5. Place the “cell” in an empty 250mL beaker and label the beaker with tape. Now fill the beaker with distilled water so that the sucrose “cell” is completely submerged.

6. Take a second 30cm piece of 2.5cm dialysis tubing (that has been soaking in water) and tie it off to make another “cell”. However, this time fill the bag with 15mL of distilled water.

7. Carefully blot the outside of the bag with a paper towel. Mass the water “cell” (in grams) on a scale and record the Initial Mass in Table 3.

8. Place the “cell” in an empty 250mL beaker and label the beaker with tape. Now fill the beaker with starch solution so that the “water cell” is completely submerged.

9. Set the beakers aside. Cover them and let them sit undisturbed overnight.

Name _____________________________ AP Biology

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PROCEDURE: DAY 2 1. Retrieve your Osmosis beakers with the “sucrose solution cell” and “water cell”.

2. Carefully remove each cell from its beaker, gently blot the outside of the “cell” with a paper towel. Mass each “cell” (in grams) on a scale and record the Final Mass in Table 3.

3. Calculate the Change in Mass for each “cell”. Record in Table 3.

Change in Mass = Final Mass – Initial Mass

a. What does it mean if the Change in Mass is a positive number?

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b. What does it mean if the Change in Mass is a negative number?

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4. Obtain data from the other lab groups in your class to complete Table 4. Calculate the class average.

5. Complete the diagrams in Figure 2. Be sure to indicate the movement of water in each case.

6. Complete the Summary Questions for this section of the lab.

TABLE 3: OSMOSIS INDIVIDUAL DATA

Set Up Initial Mass Final Mass Change in Mass

A Sucrose solution in cell; Water in beaker

B Water in cell; Sucrose solution in beaker

Name _____________________________ AP Biology

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OSMOSIS DATA TABLE

TABLE 4: OSMOSIS CLASS DATA

Change in Mass of Model Cells Set Up Group

1 Group

2 Group

3 Group

4 Group

5 Group

6 Total Class Average

Sucrose solution in cell; Water in beaker

Water in cell; Sucrose solution in beaker

FIGURE 2. MOVEMENT OF WATER ACROSS A MEMBRANE Label the diagrams below to identify the contents of the “cell” and the beaker. Clearly draw arrows to indicate the movement of water during the experiment.

SUCROSE SOLUTION IN CELL; WATER IN BEAKER

WATER IN CELL; SUCROSE SOLUTION IN BEAKER

Name _____________________________ AP Biology

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SUMMARY QUESTIONS

B. OSMOSIS 1. Describe what happened to the water cell in the 1.0 Molar sucrose solution.

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2. Explain why this result occurred.

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3. Describe what happened to the sucrose cell in the distilled water.

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4. Explain why this result occurred.

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5. Summarize the process of osmosis.

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Name _____________________________ Period _________

AP Biology Date ______________________

10

LAB _____C. DIFFUSON AND OSMOSIS: PLANT CELL PLASMOLYSIS

Cells lose or gain water due to the difference in solute concentrations between the cytoplasm (the intracellular fluid) and the solution surrounding the cell (the extracellular fluid). The movement of water in and out of a cell is governed by the laws of diffusion: water flows from a region of higher water concentration to a region of lower concentration.

When a cell is in a hypertonic solution, it will experience a net loss of water. A hypertonic solution contains a higher concentration of solutes than the cell and therefore a lower concentration of water. Consequently, water will flow out of the cell from the region of higher water concentration to the region of lower concentration.

When a cell is in a hypotonic solution, it will experience a net gain of water. A hypotonic solution contains a lower concentration of solutes than the cell and therefore a higher concentration of water. Consequently, water will flow into the cell from the region of higher water concentration to the region of lower concentration.

When a cell is in an isotonic solution, it will experience neither a net gain or loss of water. A isotonic solution contains an equal concentration of solutes as the cell and therefore an equal concentration of water. Consequently, water will flow equally into and out of the cell.

Plasmolysis is the shrinking of the cytoplasm of a plant cell in response to diffusion of water out of the cell and into a hypertonic solution surrounding the cell as shown below in Figure 3. During plasmolysis the cell membrane pulls away from the cell wall. In this lab exercise, you will examine this process by observing the effects of a highly concentrated salt solution on plant cells.

Figure 3.

HYPOTONIC SOLUTION ISOTONIC SOLUTION HYPERTONIC SOLUTION

Name _____________________________ AP Biology

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PROCEDURE 1. Prepare a wet mount of red onion epidermis. Locate a good section of leaf under low power

and then observe under high power. Sketch a single red onion cell in the space below and describe the appearance of the plant cell.

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2. Add 2 or 3 drops of 15% NaCl to one edge of the cover slip. Draw the salt solution across the slide by touching a piece of paper towel to the fluid under the opposite edge of the cover slip. Observe the plant cells in the microscope while you draw the salt water across the slide. Sketch a single red onion cell in the space below and describe what has happened to the plant cell.

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3. Remove the cover slip and flood the red onion tissue with fresh water. Observe under high power. Describe and explain what has happened.

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4. Complete the Summary Questions for this section of the lab.

Name _____________________________ AP Biology

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SUMMARY QUESTIONS

C. PLANT CELL PLASMOLYSIS 1. What is plasmolysis?

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2. Explain the changes observed in the red onion cells using the terms hypertonic and hypotonic.

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3. In the winter, icy roads are often salted to remove the ice and make them less slippery. Grasses and other herbaceous plants often die near the side of these roads. What causes this to happen?

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4. When a person is given fluid intravenously (an I.V.) in the hospital, the fluid is typically a saline solution isotonic to human body tissues. Explain why this is necessary.

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Name _____________________________ AP Biology

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5. What if the unthinkable happened at the hospital! A patient was given an I.V. bag with distilled water in it rather than saline solution. Describe what would happen to their red blood cells and explain why this would occur.

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6. Many freshwater one-celled organisms, like Paramecium, have contractile vacuoles. These structures collect and pump out excess water that accumulates in the cell. Explain why these organisms needs such a structure.

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7. Explain why contractile vacuoles would be of little value to one-celled organisms living in salt water.

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8. Popcorn sold at movie theaters is very salty, causing people to become thirsty and to buy soft drinks. Explain why salty popcorn causes this thirst.

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Name _____________________________ AP Biology

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9. Explain why soft-bodied invertebrates, like slugs, die when you pour salt on them.

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10. Label the following diagrams: Are the red blood cells in a hypertonic, isotonic, or hypotonic solution?

11. In the space below, draw a molecular diagram of the cell membrane. Illustrate the following

processes and the cellular structures and materials that are involved:

a. diffusion of water molecules across the cell membrane

b. active transport of a sodium ion across the cell membrane

c. diffusion of non-polar molecules (i.e., lipid hormone) across the cell membrane

Name _____________________________ Period _________

AP Biology Date ______________________

15

LAB _____D. DIFFUSON AND OSMOSIS: OSMOSIS CHALLENGES

You will now be given the opportunity to apply the principles you learned in the previous exercises.

CHALLENGE 1 In the front of the room are 6 flasks of different sucrose solutions. The flasks contain solutions that are either 0.0M, 0.2M, 0.4M, 0.6M, 0.8M, or 1.0M sucrose. Unfortunately my colleague, Professor I. B. Clueless, forgot to label the flasks! Devise a method for quantitatively determining which flask holds which solution.

1. Explain how you solved Challenge 1.

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2. Describe how you could improve on your initial solution.

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DATA TABLE 5

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Name _____________________________ AP Biology

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CHALLENGE 2 Let’s now consider the molarity of an actual cell. In front of the room is a bag of potatoes. My colleague, S. W. Gamgee, challenges you to quantitatively determine the molarity of potato cells (then he invites you to Second Breakfast).

3. Explain how you solved Challenge 2.

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4. Describe how you could improve on your initial solution.

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DATA TABLE 6

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Name _____________________________ AP Biology

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Title _________________________________________________________________________

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1. From the graph, determine the sucrose molar concentration equivalent to the molarity of the potato cores. __________________________

Explain how you determined this answer.

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Name _____________________________ AP Biology

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SUMMARY QUESTIONS

1. Zucchini cores placed in sucrose solutions at 27°C resulted in the following percent changes after 24 hours:

Sucrose Molarity % Change in Mass 0.0M 20%

0.2M 10%

0.4M -3%

0.6M -17%

0.8M -25%

1.0M -30%

Graph the results.

Title _________________________________________________________________________

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Name _____________________________ AP Biology

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2. From the graph, determine the sucrose molar concentration equivalent to the molarity of the zucchini cells. _____________________________________

3. Why did you calculate percent (%) change in mass of the potato cores rather than use the change in mass directly?

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