AP Biology: Unit 2: Biochemistry & Cells: AP Lab #3: Diffusion & Osmosis

Objectives
Use dialysis tubing to model diffusion across the cell membrane
Investigate the influence of solute concentration on osmosis
Investigate the concept of water potential in relation to water movement into or out of plant cells
Background to Activities A and B
The cell membrane is a cell's interface with its surroundings. In one sense, this membrane must function as a barrier: it must
keep together in one bundle the enzymes, DNA, and metabolic pathways that make life possible. The cell membrane must
also function as a gateway: waste products must be discharged through it and essential materials (oxygen, water, etc.) must
enter through it. A membrane that allows some molecules to pass through while blocking the passage of others is said to
be semipermeable. Molecules pass through the cell membrane either through processes that require the cell to expend
energy (active transport), or through processes driven by the kinetic (thermal) energy of molecules (passive transport).
In these lab activities, you will investigate the passage of materials through a semipermeable membrane by passive transport.
The membrane you will use, dialysis tubing, is semipermeable because it has submicroscopic holes through it. Molecules
are in constant random motion. By chance, a molecule's motion may move it toward the membrane (Figure 1). If it collides
with the membrane wall, it rebounds. If its motion takes it toward a pore, it may either pass through the pore, or it may
rebound, depending upon the size of the molecule relative to the diameter of the pore. Molecules that are small enough
to pass through the pores can pass through in either direction. Notice that on one side of the membrane solute molecules
have displaced some of the water molecules. Thus, there is a higher concentration of water molecules on the opposite side
of the membrane. More water molecules are available to collide with the membrane on the side having the higher
concentration of water. Thus, although water molecules will move in both directions across the membrane, more will
move from the side having the higher concentration to the side having the lower concentration. The movement of
molecules from areas of higher concentration to areas of lower concentration is called diffusion.
The diffusion of water molecules across a semipermeable membrane is termed osmosis. A process that depends upon
random motion might seem inefficient, but so many water molecules are involved and they move so fast, that it is
estimated that a red blood cell floating in blood plasma gains an amount of water equal to 125 times its own volume every
second. It also loses the same amount of water each second, all by osmosis. This occurs because the concentration of
solutes in the blood plasma is the same as the concentration of solutes in red blood cells. Solutions that have the
same solute concentration are isotonic. If we took a sample of whole blood and added salt to the plasma,
increasing its solute concentration, the plasma becomes hypertonic to the solution in the red blood cells, and the
cells lose water and shrink. If we add water to the blood plasma, decreasing its solute concentration, the plasma
becomes hypotonic to the solution in the red blood cells. The cells gain water, swell, and may even burst.

Activity A: Diffusion
Materials
Dialysis tubing, plastic cup, glucose/starch solution, distilled water, iodine-potassium iodide (IKI) solution,
dropping pipet, glucose test strips, funnel.
Caution: KI solution can irritate the skin, mouth, and eyes, and can stain skin or clothing.
Introduction
In Activity A, you will explore the diffusion of different molecules through dialysis tubing, a semipermeable
membrane. You will use glucose test strips to check for the presence of glucose and IKI solution to test for the presence of
starch. As you probably know, IKI reacts with starch to give a dark blue, almost black color. When IKI reacts with
starch, it becomes part of the starch molecule and is removed from solution,

Procedure
1. Pour 160-170 ml, of distilled water into a plastic cup. Add approximately 4 mL of IKI solution to the water and mix
well. Record the initial solution color in Table 1 in your notebook (see example below). Dip a glucose test strip into the
solution and record the initial glucose test results in Table 1 in your notebook. Use the + symbol to indicate a positive
test result for glucose and the symbol to indicate a negative result. Discard the used glucose test strip.
2. Dip a fresh glucose test strip into the glucose/starch solution. Record the initial results in Table 1 in your notebook.
Discard the used glucose test strip.
3. Obtain a piece of dialysis tubing that has been soaked in water. The tubing should be soft and pliable. Roll the
tubing between your thumb and index finger to open it. Close one end of the tube by knotting it or tying it off with
string. This will form a bag.
4. Using a small funnel, pour 15 mL of glucose/starch solution in the dialysis bag. Smooth out the top of the bag,
running it between your thumb and index finger to expel the air. Tie off the open end of the bag. Leave enough
room in the bag to allow for expansion. Record the initial color of the glucose/starch solution in Table 1 in your
notebook.
5. Immerse the dialysis bag in the solution in the cup. Make sure that the portion of the bag that contains the
glucose/starch solution is completely covered by the solution in the cup at all times.
6. Wait 30 minutes. While waiting, complete the following exercise.
7. Copy figure 2 into your lab notebook. Indicate on your Figure 2 the initial locations (inside or outside of the bag) of all
the kinds of molecules that are available for diffusion through the dialysis membrane.
8. For each of the molecules you list on your Figure 2, predict in the space below their direction of net (overall) diffusion: into
the bag, out of the bag, both into and out of the bag equally, or none (will not diffuse across the dialysis membrane). Give a
reason for each prediction.

Figure 2
9. After 30 minutes, remove the bag from the cup, blot it on paper towel, and cut a slit in the bag large enough to insert

a glucose test strip. Fill in the final columns of Table 1 in your notebook.
Table 1
Solution Color
Location
Dialysis Bag
Cup

Solution

Initial

Final

Glucose Test Results

Initial

Final

Glucose/
Starch
IKI

Analysis of Results, Activity A: Diffusion

a.

Compare your results with your predictions. Do you find any conflicts that would cause you to revise your
predictions? If so, explain.

b.

Does this activity account for the diffusion of all the molecules that you listed on Figure 2 and in your
predictions? If not, what data could have been collected to show the net direction of diffusion of this molecule or
molecules?

c. What does your data tell you about the sizes of the molecules relative to the pore size of the dialysis tubing?
Activity B: Osmosis
Materials
Dialysis tubing, plastic cups, distilled water, funnel, sucrose solutions, paper towels, balance, calculator (optional).
Introduction
In Activity B, you will investigate the influence (if any) of solute concentration on the net movement of water molecules
through a semipermeable membrane. The solute you will use is sucrose (cane or table sugar) in the following molar
concentrations:
0.0 M (distilled water)
0.2 M
0.4 M
0.6 M
0.8 M
1.0 M
Procedure
Complete the following steps for each sucrose solution that you are assigned to test.
1. Pour 160-170 mL of distilled water into a plastic cup. Label the cup with the concentration of sucrose that you
will test.
2. Obtain a piece of dialysis tubing that has been soaked in water. The tubing should be soft and pliable. Roll the
tubing between your thumb and index finger to open it. Close one end of the tube by knotting it or tying it off with
string. This will form a bag.
3. Using a small funnel, pour 25 mL of sucrose solution into the dialysis bag. Smooth out the top of the bag, running it
between your thumb and index finger to expel the air. Tie off the open end of the bag. Leave enough room in the
bag to allow for expansion.
4. Dry the bag on paper towels and then determine its mass. Record this as the initial mass in Table 2 in your notebook
(example below).
5. Immerse the dialysis bag in the water in the cup. Make sure that the portion of the bag that contains the sucrose
solution is completely covered by the water in the cup at all times. Wait 30 minutes before continuing to the next
step.
6. After 30 minutes, remove the bag from the cup and dry it on paper towels. Mass the bag and record the final mass in

Table 2 in your notebook. Finally, determine the change in the mass of the bag and record this data in Table 2 in
your notebook.
Table 2
Contents in
Dialysis Bag
0.0 M sucrose
(distilled water)
0.2 M sucrose
0.4 M sucrose
0.6 M sucrose
0.8 M sucrose
'1.0 M sucrose

Initial
Mass

Final
Mass

Change
in Mass

Percent
Change in Mass

Analysis of Results, Activity B: Osmosis

I. Calculate the percent change in mass. Use the following formula. Record the results in Table 2 in your notebook.
% Change in Mass = Change in Mass Initial Mass x 100%
2. Obtain and record the class averages for change in mass and percent change in mass.
Questions:
1.
2.
3.
4.

What does the change in mass indicate?

What variable is being tested in this experiment?
List at least three variables (other than the one listed in your answer to #5) that could influence the outcome of this
experiment. Briefly describe the method of control used for each of these variables.
Graph the percent change in mass for your group data and for the class averages. Title the graph and supply the
following information:
a. The independent variable is
b. The dependent: variable is
c. Plot the independent variable on the x-axis, and the dependent variable on the y-axis.

5.

Explain any differences between the graph for your group and the graph of class averages.

6.

On the basis of your data and graph, has this experiment adequately tested the variable you listed under #3?

7.

On the basis of your results, write a statement that expresses the relationship of solute concentration and direction
of net movement of water molecules in osmosis.

8.

In which, if any, of the experimental setups were the solutions in the bag and outside of the bag isotonic to each
other?

9.

If the experimental setup specified that only distilled water be used to fill the dialysis bag and that the sucrose
solutions be used to fill the cup, how would that change your results?

10. When you drink a glass of water, most of it is absorbed by osmosis through cells lining your small intestine. Drinking
seawater can actually dehydrate the body. How?

Background to Activity C
As you have seen, in animal cells, movement of I-120 into and Out of a cell is influenced by the relative concentration of
solute on either side of the cell membrane. If water moves out of the cell, the cell will shrink or crenate. if water- moves
into the cell it will swell and may even burst, or cytolyze. In plants, the situation is complicated by the presence of a
rigid or semi-rigid cell wall. Consider a potato cell suspended in pure water. As water enters the cell, the cell
membrane is pressed against the cell wall; thus, a physical or hydrostatic pressure develops that opposes the entrance

of additional water. When the pressure inside the cell becomes great enough, no additional water can accumulate in the
cell, even though the cell still has a higher solute concentration than does pure water. Thus, movement of water through
plant tissue cannot be predicted simply by knowing the relative solute concentrations on either side of the plant cell
membrane. It is necessary to also account for any differences in pressure between the two sides. The concept of water
potential is used to combine the differences in solute concentration and pressure to predict the direction in which water
will diffuse through living plant tissues.
In a general sense, water potential is a quantification of the tendency of water to diffuse from one area to another under a
given set of perimeters. Traditionally, water potential is expressed in bars, a metric unit of pressure equal to 10 newtons
per cm2 or about 1 atmosphere. Water potential is abbreviated by the Greek letter psi () and has two major components:
solute potential (s), which is dependent: on solute concentration, and pressure potential (p), which results from the
exertion of pressureeither positive or negativeon a solution. Thus:

Water
Potential

=
=

p
+
s
Pressure + Solute
Potential
Potential

The water potential of pure water at 1 atmosphere is by definition zero. If we dissolve a substance in the water, the water
potential drops below zero. This may seem a bit counterintuitive, but remember that we are talking about the tendency of
water molecules to diffuse. This is driven by the kinetic energy of free water molecules. As solute concentration goes up,
water concentration goes down, there are fewer water molecules available to diffuse, so water potential must go down.
Notice that solute potential must: be either zero (for pure water), or negative, if the water contains a solute. (As
solute concentration increases, solute potential decreases.) Pressure potential can be positive, negative, or zero.
Although water diffuses in all directions, the net movement of water will always be from an area of higher water
potential to an area of lower water potential.
Returning to our example of at potato cell suspended in pure water: the system is open to the atmosphere, so p = 0. We
will assign a value of -9 for s. The initial conditions can then be described as follows:
Pure Water
= p + s
or = 0+0=0
Potato Cell
= p + s

or = 0 + (-9) = -9

Since -9 is less than 0, water will diffuse into the cell.

At equilibrium, the following condition exists;
Pure Water
= p + s
or = 0+0=0
Potato Cell
= p + s

or = 9 + (-9) = 0

Since 0 = 0, there will be no net movement of water into or out of the cell.
Suppose we add solute to the water in the cup until we produce the following initial conditions:
Water + Solute
= p + s
or = 0 + (-15) = -15
Potato Cell
= p + s

or = 0 + (-9) = -9

Since -15 is less than -9, water will diffuse out of the cell. If this continues, the cell will shrink and the cell
membrane may pull away from the cell wall, a condition known as plasmolysis. This often injures the cell and
may kill it.

If we know the molarity of a sucrose solution that will produce equilibrium between the solution and the contents of the
potato cell, we can determine the solute potential by using the following formula.
s = -iCRT
where
I = ionization constant (for sucrose, this value is 1)
C = molar concentration of sucrose per liter at equilibrium (must be determined experimentally)
R = pressure constant (0.0831 liter bar/mole K)
T = temperature of solution in kelvin (K =Celsius + 273)
Example: If C is experimentally determined to be 0.300 and T is 293 K, then
s = -(1) (0.300 mole/liter) (0.0831 liter bar/mole K) (293 K)
s = -7.304 bars

Activity C: Plant Cells and. Water Potential

Materials
Plastic cups, distilled water, sucrose solutions, potato cores (or cork borer, scalpel, and potato), plastic
wrap, paper towels, balance, thermometer (optional), calculator (optional).
Introduction
In this activity, you will investigate water potential by immersing potato cores in sucrose solutions and determining the
change in mass, if any, of the cores. You will graph your data and use the graph to determine a value for C. Using the
experimentally determined value for C, you will then calculate a value for s.
Your group will be assigned one or two sucrose solutions to test. For each solution you test, complete the following
procedure.
Procedure
1. Label a cup with the concentration of sucrose that you will test.
2. Use a cork borer to cut four cylinders of potato tissue from the potato. Do not: stab your hand with the cork borer.
Trim both ends of each cylinder to remove the skin. Cut each cylinder into sections that are approximately 3 cm in
length. Use caution when slicing the cylinders so you do not injure yourself or others.
3. If you cannot immediately use a balance to mass your cores, place them in a beaker or cup and cover- them with a lid or
with plastic wrap. Why do you think this is necessary?
4. Use a balance to determine the total mass of all the potato sections. Record this as the initial mass in Table 3 in your
notebook (see example below).
5. Place all of the potato sections in the labeled cup. Pour 100 mL of the assigned sucrose solution into the cup.
6. Cover the cup with plastic wrap. Why do you think this step is necessary?
7. If you have been assigned a second concentration of sucrose to test, repeat steps 1-6 using that solution.
8. Stop. Allow the potato sections to remain in the sucrose solution overnight. Then, proceed to Step 9. Continue with
Step 9 only after the potato sections have been in the sucrose solution overnight. Perform these steps for each
solution you are testing.
9.

Record the temperature as given by your teacher.

10. Remove the potato sections from the sucrose solution. Blot them on paper towels to remove excess solution. If you
cannot immediately use a balance to mass your cores, cover them as before to prevent evaporation.
11. Use a balance to determine the final mass. Determine the change in mass of the potato sections. Record this data in
Table 3 in your notebook.

Table 3: Water Potential

Contents
in Cup

T emp.

Initial
Mass

Final
Mass

Change in % Change
Mass
in Mass

0.0 M sucrose
(distilled water)

% Change in
Mass
(Class Average)

0.2 M sucrose
0.4 M sucrose
0.6 M sucrose
0.8 M sucrose
1.0 M sucrose

Analysis of Results, Activity C: Plant Cells and Water Potential

1. Calculate the percent change in mass of the potato sections and record this data in Table 3 in your notebook. Obtain
and record the class averages and record those as well.
2. Graph the class averages for percent change in mass. Plot the independent variable on the x-axis, and the
dependent variable on the y-axis.
3. On your completed graph, find the point where the line of your data crosses the 0 line (x-axis) of the grid. This is the
equilibrium point; at this point there is no net gain or loss of water from the potato cells. Read the corresponding
value of sucrose molarity for this point. This is the molar concentration of sucrose that produces equilibrium.
Below, record this concentration of sucrose as your experimentally determined value for C. Convert ambient
temperature from C to K.
4. Using the formula s = iCRT, calculate the solute potential at equilibrium. Show your calculations in your
notebook.
5. Using the formula = p + s, give the following:
a. The water potential of the solution at equilibrium.
a. The water potential of the potato cells at equilibrium
Questions:
1.

A chef chops vegetables into a bowl of water. Would you expect the vegetable slices to gain or lose water? Explain
your answer in terms of water potential.

2.

Imagine that you are an agri-science consultant: to a large corporate farm that raises 7,000 acres of wheat on desert
land adjoining the Mediterranean Sea. Just before the wheat matures, all the wells used for irrigation water run dry.
The farm manager wants to irrigate the fields with water drawn from the Mediterranean. From previous tests, you
know that the average solute potential of root tissue taken from the wheat fields is 11.1.3 bars. You test the seawater
and determine its solute potential to be 24.26 bars. What will you advise the farm manager and why?
A marine clam is mistakenly added to a freshwater aquarium. What will happen to the clam and why?

3.
4.

A 1 cm diameter, 6 cm long core is removed from a carrot (see Figure 3). The resulting hole is filled with corn
syrup (highly concentrated sugar), and a glass tube is inserted into the hole to make a watertight seal. The carrot is

suspended in a cup of pure water. Beginning with the carrot cells adjoining the hole, and going out to the carrot
epidermal cells exposed to the water, describe what will happen to the carrot cells. Also, what will happen to the level
of liquid in the glass tube and why?

Optional: Activity D: Observing Plasmolysis in Onion Skin Cells

Materials
onion section
scalpel
forceps
dropping pipets
absorbent paper
15% NaCI solution (dissolve 15 g NaC1 in 85 mL of water)

coverslips
distilled water
microscopes
microscope slides

Procedure
1. An onion is made up of several layers of thick scale leaves. You will need one portion of scale from a cut 'section of an
onion. Working on the concave surface of the scale, cut out a section about 1 cm'. Use forceps to remove the
epidermal layer (onion skin) from the concave surface of the section you have cut. Place the onion epidermis on a
microscope slide and smooth it to remove as many wrinkles as possible. Add one or two drops of distilled water and
a coverslip, and observe under a microscope.
2. You have probably observed onion cells before, but reacquaint: yourself with their structure and appearance. Note
especially the cell walls and the location of the nuclei.
3. Remove the slide from the microscope. Add a drop or two of 15% NaC1 solution to one edge of the coverslip. Use a piece
of absorbent paper to absorb water from the opposite edge of the coverslip. This should "pull" the salt solution under
the coverslip and bring it in contact with the onion cells.
4. Observe under the microscope for several minutes for signs of change in the onion cells. If after several minutes you
have seen no change, remove the coverslip, blot away excess water, and add one or two drops of 15% NaCl solution
directly to the square of onion epidermis. Replace the coverslip and observe again.
Once you have observed plasmolysis, remove the coverslip and blot: away the excess water. Flood the epidermis with distilled
water and blot again. Add another drop of distilled water and replace the coverslip. Observe under the microscope. Do you
see any change that would indicate that water is entering the cells? What is causing the change?