BIO111: Diffusion and Osmosis

Learning Objectives
 Explore the relationship between surface area and volume of cells
 Explore the factors that affect the movement of molecules through semi-permeable
membranes
 Relate information gathered to membrane dynamics in cells

Introduction
Membrane Transport: Diffusion and Osmosis
The cell membrane is a fluid, dynamic structure made of phospholipids and protein. It is
extremely important to the cell: the membrane maintains the internal environment of the cell
by acting as a selectively permeable barrier for the cell that regulates movement of substances
into and out of the cell.

Several methods of transport that allow substances to move between the internal portion of
the cell and the external environment. The simplest is diffusion, which relies on the random
movement of molecules resulting in net movement from areas of high concentration to areas of
low concentration. Molecules will move from areas where there are many collisions with other
particles (high concentration) to areas where there are fewer collisions with other particles (low
concentration). These collisions will continue until there is an even distribution of the molecules
throughout the space available. Molecules that diffuse across a lipid bilayer have a small
molecular size and usually be nonpolar (i.e. have no net charge). Small polar molecules can
diffuse along their concentration gradient through protein transport channels in the
membrane.

A specialized case of diffusion is the movement of water through a membrane. This movement
of water is called osmosis. Water will diffuse through a membrane from areas with a lot of free
water to areas with fewer free water molecules (water moves to dilute). If you were to
separate a distilled water solution from a 20% glucose solution by a semi-permeable
membrane, the water would move from the distilled to the glucose solution (from areas of high
water concentration to areas of low concentration) to dilute the glucose solution to the same
concentration as on the other side of the membrane. There is a pressure exerted by the water
on the membrane as the water moves into the glucose solution. The pressure will eventually be
great enough to force some of the water back towards the distilled water’s side of the
membrane. This pressure is known as osmotic pressure.

Surface Area to Volume Ratio

One of the interesting developments observed from an evolutionary standpoint is that the size
of individual cells does not really increase dramatically as an organism gains in developmental
complexity. You just do not see 200-pound cells making up a 200-pound person! Instead, you
see that the size of the cell remains very small. Cells must exchange materials with their
surroundings across the cell membrane. Therefore, to achieve the best exchange efficiency, the
surface area of a cell is large in comparison to its volume.

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Pre-Lab Activity - The size and shape of a cell; relationship between surface area and volume

Background reading: Campbell, “A Tour of the Cell”, section on Prokaryotic cells and eukaryotic
cells differ in size and complexity.

1. Observe the drawings of several cells below. How does the shape of the cells change as cells
get larger? How does this affect the ratio of surface area to volume?

Figure 1: Various cells. From top and then left to right: nerve cell, muscle cell, plant cell,
bacterium, kidney epithelial cell, cheek cell, fungal filament.

2. Now let us explore the relationship between surface area and volume of cells. For simplicity
of comparison, let us assume the "cells" that we are going to compare are cubes. Each cube
has sides of equal length. Mathematically, the volume of a cube is the length multiplied by
width multiplied by height. Therefore, if all sides are of equal length (x), then volume = x3.
The surface area of a cube is 6x2, or six times the surface area of each face of the cube.
Therefore, to compare the surface area (SA) to the volume (V) of a cube, we can compare
their ratio, or SA/V ratio.
a. Fill in the following table using the formula for SA/V = 6x2/ x3.

x= x= x= x= x= x= x= x= x= x=
1 2 cm 3 cm 4 cm 5 cm 6 cm 7 cm 8 cm 9 cm 10
cm cm
SA (6x2)

V (x3)

SA/V
(6x2/ x3)
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b. Graph x vs. SA/V ratio below, where x is the independent variable.

c. Observations about the relationship of the length of the side on our "cells" and the SA/V
ratio:
i. What happens to the SA/V ratio as x gets larger?

ii. What happens to the SA/V ratio as x gets smaller?

iii. Which surface area to volume ratio is most efficient exchange of materials between
a cell and its environment? Explain your answer in terms of cell size versus SA/V
ratio.

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Cell Membrane Function: Lab Activity
In this lab, you will investigate factors affecting the movement of molecules through semi-
permeable membranes. You will be able to relate this information to cell membrane dynamics.
Part 1: Selective Permeability of Membranes – a dialysis bag model
Another function of the membrane is to be selective about what can pass through it. In this
portion of the laboratory, you will use dialysis tubing, which is a semi-permeable membrane.
There will be a solution of starch and dextrose (glucose) inside the dialysis tubing. There will be
a solution of water and iodine outside the dialysis tubing (in the glass beaker). Lugol’s iodine
(I2KI) turns blue-black in the presence of starch.

With your lab partners, discuss the differences in molecular size of iodine, water, glucose and
starch. How might these differences affect their ability to cross the membrane?

Materials
Note: Beakers look more like a glass but with a spout and
are used for many different tasks. Graduated cylinders are
tubes with a yellow plastic base and are used only for
measuring.

IF other groups have done lab before you, most of the

glassware you need will be on the drying cart. Please Beaker Graduated Cylinder
check there first.
 250 ml glass beaker  10% glucose solution  Distilled water
 Dialysis bag  1% starch solution  Benedict’s reagent
 Dental floss  Balance
 Plastic pipette  I2KI (Lugol’s Iodine)

Procedure
1. Place 50 ml of distilled water in a 250 ml glass beaker.
2. Soak a pre-cut 6-inch strip of dialysis tubing in water for 5 minutes to make it pliable, then
fold one end of the bag over and then tie the folded portion shut with dental floss.
3. Open the other end of the tubing to make a “bag”.
4. Wear goggles for the remainder of this activity. Use a plastic pipette to fill the bag with
equal parts (approximately 2 mL each) of the prepared 10 % glucose solution and 1% starch
solution. The pipettes may be disposed in the trash.
5. Fold over the other end of the bag and tie off with dental floss.
6. Rinse any excess starch or glucose solution off the outside of the bag, as these will
interfere with your test results.
7. Weigh the dialysis bag (in grams) and record the weight: _______________g
8. Place the dialysis bag inside a 250 ml glass beaker.
9. Check that the distilled water in the glass beaker just barely covers the bag (add/remove
water if needed) and drops of I2KI into the water until the water is dark yellow to the eye (at
least 12 drops).
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 a. I2KI is a qualitative indicator for starch. I2KI remains brown if no starch is present. If
starch is present it turning dark blue-black.
b. Is this a quantitative or qualitative test for polysaccharides?

10. Describe the solutions inside and outside of the tubing using the terms hypotonic, isotonic,
and hypertonic.

11. Make your predictions about the movement of each molecule across the membrane:
a. What will happen to the starch?

b. What will happen to the glucose?

c. What will happen to the water?

d. What will happen to the I2KI?

12. Observe the setup for 1 hour while you work on the other parts of this lab.
13. At the end of 1 hour, observe visible changes to the bag or the water in the beaker. Record
your observations here:

14. Weigh the dialysis bag, record the new weight, and compare it to the initial weight.______g
15. Perform a Benedict’s reagent for simple sugars with the water in the beaker. Benedict’s
reagent contains copper sulfate, which is reduced by many simple sugars. The reduced
copper sulfate forms a yellow, orange, green, red to brownish precipitate of copper oxide
on heating. The darker the precipitate, the more sugar there is in the sample.

Is this a quantitative or qualitative test for simple sugars?

a. Measure 3 ml of Benedict’s reagent in a small graduated cylinder and place it in a test

tube.
b. Measure 3 mL of beaker solution with the graduated cylinder and add it to the test tube.
c. Place the test tube in a 250 ml glass beaker, half-filled with tap water, and heat on a hot
plate to near boiling for approximately 3 minutes. Do not heat the graduated cylinder.
i. Make sure that no cords are touching the hot surface of the hot plate. Use test tube
holders or heat-resistant gloves to handle the hot materials.
d. Does the Benedict reagent indicate positive or negative for glucose?__________

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16. Dispose of waste in the appropriate containers provided in the hood (iodine waste and
Benedict’s waste go in different containers). Rinse the beakers and your graduated cylinder
and place them on towels on the cart to dry. Rinse the test tube and invert it in the rack in
the hood. Place the hot plate on the side counter to cool when you are done with it. Return
any other materials to their original location.

17. What conclusions can you draw from this experiment? Consider which environment has the
greater solute concentration – inside or outside of the dialysis tubing.

18. How do your results compare with your predictions?

19. What resulted in the bag’s change weight?

20. How does the size of a molecule affect its ability to move across a membrane?

21. Dispose of the dialysis tubing in the waste container for eggs, agar, and dialysis tubes.
Dispose of the iodine/water and Benedict’s test liquids in the appropriate (separate!) waste
containers. Rinse your test tubes and glassware and invert in the test tube rack in the hood
(test tubes) or on the cart (glassware).

Part 2. Molecular Movement (Diffusion)

Materials
 Two 250 ml glass beakers  Boiling water: water from tap heated
 Cold tap water from jug in refrigerator with a hot plate
 Food coloring
Procedure
1. Wear goggles for the remainder of this activity. Obtain two 250 mL glass beakers.
2. Fill one beaker with ~150 ml of tap water and use the hot plate to bring the water to boiling.
Use the heat-resistant gloves to protect your hands as you remove this beaker from the hot
plate.
3. Fill the second beaker with ~150 ml cold tap water (from the fridge).
4. Allow the contents of the glass beakers to come to a rest on the table.
5. Carefully drop a few drops of your “favorite” FD&C food coloring in the center of each
beaker and observe.
6. Record and explain your observations.

7. Dispose of this waste in the sink. Rinse your glass beaker and return it to the drying cart.

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Part 3: Investigating diffusion using an agar cube as a cell model.
You will take agar that contains a pH indicator called phenolphthalein and cut it into two cubes.
The phenolphthalein is colorless until it is in contact with solutions of pH 10 or greater, which
cause it to turn a bright pink. We will use a very basic solution of sodium hydroxide, NaOH, as a
solution with a pH > 10. Think of the cubes as cells and the NaOH as "nutrients" coming into the
cell. You will observe how the size and surface area of the cell influence the degree to which
“nutrients” move across the cell's surface into the cell.
Materials
 Knife  Two 100 ml glass beakers
 Ruler  0.1 M NaOH
 Agar with phenolphthalein  Paper towel

Procedure
1. Wear goggles for the remainder of this activity. Using a knife, cut two cubes of the prepared
agar, one large cube (where x = about 3 cm) and one smaller cube (x = about 1 cm). Record
the exact measurement of x (the length of a side of the cube) for both the large and the
small cube. Small _______cm Large ______cm
2. Place the cubes into a small (100 mL) glass beaker and CAREFULLY pour sodium hydroxide
(NaOH) solution over the cubes. Use enough NaOH to cover only the top surface of the
cubes. The NaOH will diffuse into the agar and you will see the bright pink color develop
where the phenolphthalein and NaOH come in contact. This is an effective model for how
far the "nutrients" have moved into the "cells".
3. Allow the cubes to soak for one (1) minute. Carefully pour the NaOH solution off the cubes
into the disposal jug in the hood.
4. Rinse the cubes with distilled water, pour off the water, and put the cubes on a dry paper
towel.
5. Cut the cubes in half with a knife or spatula.
6. Sketch what you see.

7. Write a sentence that describes the difference between the appearance of the large cube
and the small cube.

8. Given that the rate of absorption is the same in both cubes, what is the effect of surface
area to volume ratio?

9. Observe your results. Which "cell" size is the most efficient at moving nutrient molecules in
and waste molecules out?

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10. Where do the cubes fall on the graph you made earlier (in the pre-lab) of “x vs SA/V”? Does
your answer to the previous question (number 9) correspond with your answer for
questions 2c in the pre-lab? Explain.

11. Dispose of the agar cubes in the disposal container with the eggs and the NaOH waste in
the NaOH jug in the hood. Rinse the glass beakers and place them on the drying cart.

Part 4: Osmosis in Hypotonic and Hypertonic Solutions Using a Chicken Egg as a Cell Model
You will investigate the movement of water across the cell membrane of a shell-less chicken
egg. Chicken eggs can be used as a model of a single cell because of their permeable outer
membrane. After removing the shell by soaking the eggs in vinegar (the acid dissolves the
calcium carbonate), the chicken “cell” can be used to investigate the movement of molecules
across cell membranes.
Materials
 6 plastic beakers  Petri dish/weigh boat  Glucose solutions
 Tape and sharpie  Spoon (10%,20%,30%,40%,
 6 shell-less eggs  Balance and unknown)
 Paper towel  Distilled water

Procedure
1. Obtain six 250 mL plastic beakers and use tape and sharpie to label them #1 – 6.
2. Before weighing an egg, weigh the empty weigh boat (or petri dish) and tare (zero) the
balance!
a. Record the mass of the weight boat/petri dish ______________ g.
3. Remove a shell-less egg from the container provided and gently dry the egg on paper
towels.
4. Working quickly, so as not to allow the egg to dry out, weigh the egg by placing it in the
weigh boat/petri dish to get an initial weight for (weight at 0 min.) and record it in Table 1.
5. Immediately place the egg in plastic beaker #1 and fill the plastic beaker with distilled water
until the solution covers the egg.
6. Repeat steps 2-5 until you have 6 eggs in beakers with different soaking solutions: distilled
water (#1), 10% sucrose (#2), 20% sucrose (#3), 30% sucrose (#4), 40% sucrose (#5), and the
unknown solution (#6).
7. After 15 minutes remove the egg from the first plastic beaker, gently dry it with paper
towels, and weigh it. Enter the egg’s weight in Table 1. Repeat for each egg.
8. Repeat step 7 every 15 minutes for 75 minutes.
9. When finished, rinse the plastic beakers and place them on towels on the cart to dry;
dispose of eggs and sucrose waste in the containers provided. Use a damp cloth to carefully
wipe down your balance and countertop – coil the cord for the balance and return the
balance and cord to the bottom of the cart.

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10. Calculate relative weight changes: To normalize your data to the eggs’ initial weight (weight
at 0 minutes), subtract weight at time 0 (initial weight) from the recorded weight for each
time interval. For example:
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑐ℎ𝑎𝑛𝑔𝑒 𝑎𝑡 15 𝑚𝑖𝑛
= 𝑅𝑒𝑐𝑜𝑟𝑑𝑒𝑑 𝑤𝑒𝑖𝑔ℎ𝑡 𝑎𝑡 15 𝑚𝑖𝑛 − 𝑟𝑒𝑐𝑜𝑟𝑑𝑒𝑑 𝑤𝑒𝑖𝑔ℎ𝑡 𝑎𝑡 0 𝑚𝑖𝑛
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑐ℎ𝑎𝑛𝑔𝑒 𝑎𝑡 30 𝑚𝑖𝑛
= 𝑅𝑒𝑐𝑜𝑟𝑑𝑒𝑑 𝑤𝑒𝑖𝑔ℎ𝑡 𝑎𝑡 30 𝑚𝑖𝑛 − 𝑟𝑒𝑐𝑜𝑟𝑑𝑒𝑑 𝑤𝑒𝑖𝑔ℎ𝑡 𝑎𝑡 0 𝑚𝑖𝑛
Calculate the relative weight change of each egg at time 15, 30, 45, 60, and 75 minutes and
record the data in Table 2.
11. Plot a graph of relative weight change vs. time (Table 2), where time is on the X-axis. Your
graph will have six lines. What conclusions can you infer from this graph?
a. Use the terms hypotonic, isotonic, and hypertonic to describe the environments
inside and outside the eggs.

b. Which sucrose solutions are hypotonic, hypertonic or is most isotonic to the egg due
to the relative weight change.

c. Describe the direction of water movement across the egg (cell).

12. Plot a graph of total weight change (last column of data in Table 2) of the eggs (except for
the egg in the unknown solution) against the concentration of the solution.
b. Use this graph to determine which solute concentration(s) are isotonic, hypotonic
and hypertonic to the contents of the chicken eggs. Explain your logic.
c. Use this graph as a standard curve to determine the concentration of the unknown
solution. Unknown concentration: _______________g
d. Using this graph, a type of standard curve, visually read the sucrose concentration
that is isotonic to the chicken egg.

e. Calculate the sucrose concentration for the Unknown solution.

f. Make predictions using the standard curve. (e.g., If an egg is placed into a 25%
sucrose solution, visually read and calculate the weight of the egg?

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Table 1 – Weight of Eggs (grams)
Solution in Weight at Weight at Weight at Weight at Weight at Weight
plastic time 0 15 min. 30 min. 45 min. 60 min. at 75
beaker min. min.

Water
10%
sucrose
20%
sucrose
30%
sucrose
40%
sucrose

Unknown

Table 2: Relative Weight Change of Eggs (grams)

Solution in Relative Relative Relative Relative Relative Relative
plastic weight at weight at weight at weight at weight at weight at
beaker 0 min. 15 min. 30 min. 45 min. 60 min. 75 min.

0
Water
10%
0
sucrose
20%
0
sucrose
30%
0
sucrose
40%
0
sucrose

0
Unknown

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Clean Up
1. Drain as much liquid waste as possible into the designated waste container(s). Note: there
are different containers for different wastes!
2. Discard eggs, dialysis tubing, and agar in the appropriate container in the hood/on the dish
cart.
3. Return reagents and equipment to their proper tray on the cart.
4. Allow hot plates to cool and then return them to their proper storage place.
5. Place boiling chips on a piece of paper towel to dry and then return them to their container
in the lab box on the cart.
6. Remove all tape. Rinse test tubes and invert in the test tube rack in the hood. Rinse and
place any other items to be washed on the dish cart.
7. Place goggles into the sanitizing cabinet.
8. Carefully wipe any sucrose solution off the balances, coil the cord, and return them to the
bottom of the cart.
9. Wipe the benchtop with a damp towel. Leave the classroom prepared for the next class to
do lab.

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