Lab 3: Diffusion, Osmosis and Cell Permeability

Week of September 16, 2013

Name: __________________________________

Section: _________________

Pre-Lab Exercises
(To be completed and turned in to your TA at the beginning of Lab)
1. Read through the lab and the corresponding chapter in the textbook.
What is meant by the following terms?
Concentration gradient:

Diffusion:

Osmosis:

Active transport:

2. Here are some diagrams of the model cells you will make during Exercise 3 of this
weeks lab. Look at the procedure for Exercise 3 and label cells B, C, and D. Next,
label the solution in beakers B, C, and D according to how you will fill them in the lab.

More on the next page.

Pre-lab continued
3. Using the same type of drawing given for you in pre-lab question 2 from the
previous page, draw the set-up for Exercise 4 of this weeks lab.

Lab 3: Diffusion, Osmosis and Cell Permeability

Week of September 16, 2013

Background: Solutions and Diffusion

In every living cell, the internal structures of the cell are bathed in water in which many
small molecules (and some not-so-small) are dissolved, and the cell is surrounded by
water, again with various particles dissolved in it. Molecules such as nutrients or drugs
that enter the cell move through the cell membrane from the water outside to the water
inside. Things such as cellular wastes and hormones that leave the cell move out
through the membrane to the water outside.
Solutions are uniform in composition and have their components so thoroughly mixed
and mutually electrically attracted (remember the picture from your text of the NaCl
solution?) that they do not separate by any simple physical means (settling, filtering,
etc.) However, on the scale of a cell, a solution is not smooth and homogeneous, but is
more like a collection of marbles of different sizes and shapes, all bobbing about and
bumping into each other. Water-molecule "marbles" are quite different from sugarmolecule "marbles", and both are very different from protein-molecule "marbles". All of
the marbles are in motion, the smaller ones moving fastest and the larger ones moving
more slowly. Each moves until it bounces off something else (or is pushed by something
bouncing against it), and then moves in a new direction until it bounces off something
again, and so on. Of course, many of the marbles are a little sticky because of charge
attractions and hydrogen bonds, but they still move quite a bit relative to each other.
In diffusion, each kind of particle tends to spread from where particles of that
kind are more concentrated to where particles of that kind are less concentrated
(i.e. from high concentration to low concentration).
When something is added to a solution, particles of that substance diffuse outward from
the place they were put in, while whatever was already there (including the solvent)
diffuses into that area. Note that it does not make sense to talk about the solution
diffusing, because different parts of the solution do different things.
Thinking about the particles (molecules and ions) in a solution also makes it easier to
see that we can talk about the concentration of the solvent, just as we talk about the
concentration of solutes. There are particles of solvent and particles of solute in
each unit of volume.

Before starting Exercise 1, set up Exercises 3 and 4. While you are

waiting to make your observations for Exercises 3 and 4, you will
have time to complete Exercises 1 and 2.
Exercise 1: Brownian motion
Any substance or solution, whether it is the air around you, the fluid in your cells, or a
drop of water on a microscope slide, possesses a certain amount of heat energy, which
is actually kinetic energy. When you measure the temperature of the air, or of water in
a beaker, or of a metal rod, you are really measuring the energy of the random motion
of its particles. You can observe such molecular movements with a microscope by
viewing small particles, such as those of the powdered carmine dye that we will be
using in this exercise. The randomly moving water molecules, which are too small for
us to see with regular light microscopes, crash into the particles of carmine powder from
all sides causing them to bounce around. This random movement of molecules, often
called Brownian motion, shows us the underlying cause of diffusionthe random
movement of molecules, or the kinetic energy of the system.
Procedure for Observing Brownian motion:
1. Obtain a clean microscope slide, a cover slip, and a dropper bottle of carmine
mixture (the powder has already been mixed with water for you).
2. Place a drop of the carmine mixture on the clean microscope slide and gently cover
the drop with a cover slip.
3. As with all slides, view it first under low power and then under high power. You will
need to use your high power 40X objective lens (400X total magnification) to
observe Brownian motion.
4. Answer the following questions:
a. Is the movement random, or do the particles move only in one direction?

b. Do smaller particles of carmine move more rapidly than larger particles?

c. How can such movement lead to diffusion? (Remember the marbles!)

Permeability:
To "permeate" something is to move through it. If particles (whether they are solute
particles or solvent particles) are able to move through a barrier such as a membrane,
the membrane is said to be permeable to those particlesable to be permeated by
them.
A barrier that does not allow anything to pass through it is impermeable. No diffusion
occurs through an impermeable barrier.
A membrane (or other barrier) that permits some things to permeate it but does not
permit others is semi-permeable ("half-permeable") or selectively permeable or
differentially permeable (different authors use different terms). Solvent molecules
especially waterare often much smaller than solute molecules. Thus they may be able
to pass through openings in a membrane that are too small for most solute molecules.
This is what causes osmosis, when water diffuses from higher to lower concentration
through a membrane but solutes dissolved in it are prevented from diffusing because
they cannot pass through the membrane.
Osmosis is a type of diffusion that describes the diffusion of water molecules
across a semi-permeable membrane from areas of high concentration to low
concentration.
Cell membranes are much more permeable to water than to most other substances. It is
very important for the stability of a cell that the water concentration be the same outside
the cell as it is inside, because the volume of the cell changes when there is net
movement of water between inside and outside.

Tonicity:
If a cell shrinks when it is put into a solution, the solution is said to be hypertonic as
compared to the cytosol (the inside environment of the cell). More water molecules are
leaving the cell than are entering it. This is generally because the solute concentration
outside the cell is higher than the solute concentration inside the cell, making the water
concentration outside the cell lower than the water concentration inside the cell. Since
water permeates cell membranes more easily than most solutes, diffusion of water has
a more rapid effect on the cell than does diffusion of the solutes. The solution (outside
of the cell) is hypertonic compared to the cytosol, and the cytosol is hypotonic compared
to the solution.
If a cell swells when it is put into a solution, the solution is said to be hypotonic to the
cell. More water molecules are entering the cell than are leaving it, and the cell swells.
Again, this generally reflects a difference in solute concentration. The solution is
hypotonic to the cytosol, and the cytosol is hypertonic to the solution.

If a cell does not change size when it's put into a solution, it must mean that water
molecules are entering the cell at the same rate as they are leavingwhich must mean
the concentration of water is the same inside the cell as outside the cell. The solution is
said to be isotonic to the cell"same strength".
In every case, the cytosol also has a relationship to the solution outside the cell. The
terms hypertonic, etc., always describe one solution in relation to another solution
two solutions on opposite sides of a membrane (whether it is an artificial membrane or a
cell membrane). The net movement of water is always from the hypotonic side to the
hypertonic side of the membrane that separates the solutions.
The following drawings represent a cell being placed into three different solutions. In
the original solution, the solution is isotonic to the cell, and the cell is isotonic to the
solution. There is no net movement of water, but water is flowing equally into and out of
the cell. The other two images represent the same cell being placed into new solutions.
Note the effect of the solution on the size of each cell. Below each image, fill in the
blanks using the terms hypertonic and hypotonic.

Original solution
Cell

Solution is _________________ to the cell

Solution is _________________ to the cell

Cell is _________________ to the solution

Cell is _________________ to the solution

Cells that do not live in an isotonic environment have ways of coping with the loss or
gain of water. Plant cells, Protistan algae cells and fungus cells have a stiff wall (cell
wall) that prevents excessive expansion. They also have a large vacuole whose
contents are usually hypertonic to the cytosol. If the environment outside the cell is
hypotonic to the cytosol, water entering the cell by osmosis moves into the vacuole.
Similarly, if the cell is in a hypertonic environment, as the cytosol loses water to the
environment its relationship to the vacuole changes and water may move osmotically
from the vacuole to the cytosol. Thus the vacuole may dramatically change size,

depending on the environment the cell is in. Since the vacuole accounts for a large
percentage of the volume of the cell, the volume of the cell may also change.
Some Protistans (the ones called Protozoanspre-animals) have a contractile
vacuole that pumps out excess water. But bacteria and animal cells are generally not
very tolerant of environments that are not isotonic. Bacteria have no good protection
against the effects of a hypertonic environment, which is why jam or jelly is not likely to
support bacterial growth although they can become fuzzy with fungi, whose cells are
protected by their large vacuoles.
Most of the cells of an animal are on the inside of the body, bathed in an isotonic tissue
fluid that is kept at the right water concentration by the constant work of organs such as
the kidney. Water loss (or water entry) through the body surface is minimized by an
impermeable epidermis.

Exercise 2: Observing Plasmolysis in Plant cells

Elodea is a freshwater plant commonly found in ponds and fresh water aquaria. The
cells of Elodea, like all other cells, contain cytosol, which is essentially water with
dissolved solutes. Usually, the cytosol contains more dissolved solutes than the
surrounding water in which the plant is living. Thus, there is a concentration gradient
allowing free osmosis of water into the cells. However, the dimensions of the cell wall
restrict movement of water into the cells. Water enters the cell, and to a large extent is
stored in the central vacuole, filling the cell and exerting pressure against the cell wall.
This pressure is called turgor pressure, and it is partly responsible for giving the plant
structural support.
Procedure for observing Plasmolysis:
1. Make a wet mount of an Elodea leaf.
a. Place two drops of water on a clean microscope slide.
b. Gently pluck a leaf from near the tip of a branch of an Elodea plant.
c. Place the leaf bottom-side-up in the water on your microscope slide.
d. Carefully cover the leaf with a cover slip making sure to avoid air bubbles.
2. Examine the leaf under scanning power, then low power (10x objective), and then
increase the magnification to high power (40x objective).
3. Study a single cell under high power. Use the fine adjustment and continually focus
up and down to perceive depth. Observe the cell wall completely enclosing each cell.
The cell membrane adheres to the inside of the cell wall and is probably not visible.
Much of the cell consists of a large, central vacuole containing cell sap (mostly water).
The cytosol is present as a thin, nearly transparent fluid layer restricted to the edges of
the cell because of the space filled by the central vacuole. The chloroplasts are seen as
green, oval bodies in the cytoplasm. You may see them passively moving around the

cell as the cytoplasm flows about in what is known as cytoplasmic streaming or cyclosis.
Draw and label the Elodea leaf in the spaces provided.
The nucleus of the cell may be obscured by the chloroplasts in the cell. If you focus up
and down with the fine adjustment and scan the cytoplasm bordering the cell wall, you
may detect the nucleus as an enlargement in the cytoplasm. You may have to adjust
the amount of light using the iris diaphragm, to see the nucleus or other cellular
components.
Label:
Cell wall
Cell membrane
Chloroplasts
Central Vacuole

Elodea (water) 400X

4. Next, we will change the usual environment of Elodea by placing the leaf into a
solution of concentrated salt water (5% NaCl), that is, a solution that is hypertonic to the
cytosol of the cells in the leaf. What do you think will happen? ____________________
______________________________________________________________________
a. Remove the cover slip of the mount of your Elodea leaf and blot some of the
water surrounding the leaf.
b. Place a couple drops of 5% NaCl on the leaf and replace the cover slip.
c. Wait a minute or two.
d. Examine the Elodea leaf under high power and make another drawing of the
cells that you see. Indicate with arrows the direction you think water is
flowing (i.e. into or out of cells).

Label:
Cell wall
Cell membrane
Chloroplasts
Central Vacuole
Arrows indicating water movement
Elodea (5% NaCl) 400X

Plasmolysis is the shrinkage of the cytoplasm and subsequent pulling away of the cell
membrane from the cell wall. What caused the plasmolysis you observed in the Elodea
leaf?__________________________________________________________________
______________________________________________________________________

5. After observing Elodea in salt solution, remove the salt water by adding a drop or two
of distilled water to one side of the slide right next to the coverslip. Draw the distilled
water under the cover slip using the corner of a paper towel to absorb the salt water
from the other side of the cover slip.
Does plasmolysis appear to be reversible? ______________________________
Clean Up:

Microscope slides - wash and dry them and return them to your table.
Cover slips dispose in regular trash
Microscopes using lens cleaner and lens paper clean the Carmine off of the
objective lenses. Then, put the lowest power objective down and return scope to
the cabinet.

Exercise 3: Studying Osmosis Using Artificial Membranes

For the major investigation in this lab, we will use dialysis tubing as a model of the cell
membrane. It is chemically different than the cell membrane, and it does not have all of
the characteristics of the cell membrane as a permeability barrier. However, it is about
as permeable as the cell membrane to water and to small organic molecules. This
allows us to focus on these aspects of membrane behavior without having to deal with
all of the other things that happen at once in real cell membranes.
Procedure for using dialysis tubing cells to observe diffusion and osmosis:
Your instructor will demonstrate how to fill the tubing and fasten the ends.
1. Make 4 model cells using dialysis tubing by opening the water-soaked tubing and
fastening one end, as was shown by your instructor. At the sink, carefully fill each cell
approximately one third to one half full with the following solutions:
Cell A - 5% sucrose
Cell B - distilled water
Cell C - 10% sucrose
Cell D - 25% sucrose
Make sure to keep track of which cell is what; ask your instructor for suggestions
if needed.

2. Gently squeeze the open end of each cell to push out trapped air. Twist the open
end and then fasten it, as was shown by your instructor. The cell should be limp.
3. Squeeze your cells gently to check for leaks. Leaks are not acceptable. Rinse
your cells with tap water and blot each cell with a paper towel.
4. *Weigh your cells before putting them in the beakers*. Record the initial weights
of your model cells to the nearest 0.1 grams in Table 1.
5. Place cell A in a beaker with about 125 mL of 25% sucrose and cells B, C, D in
separate beakers with about 125 mL of distilled water.
Record time here: __________________
6. Hypothesize and predict what will happen to the weight of each cell. Fill in the
column labeled predicted weight change in Table 1.

Table 1: Weight changes in model cells over time

Predicted
Final
Initial
Weight change (Final Weight Initial Weight)
Cell weight change Weight Weight
Indicate + or (g)
(+, 0, -)
(g)
(g)
A
B
C
D

7. After 60 minutes, remove the cells from their beakers, gently blot excess water,
and weigh them to the nearest 0.1g. Record the final weights of each cell in Table 1.
Determine the change in weight of each cell and indicate weight gain with a (=) and
weight loss with a (-).
Conclusions:
We are examining the effects of concentration on weight gain/loss to better understand
osmosis.
1. What is the dependent variable in this experiment? _________________
2. What is the independent variable in this experiment? _______________
3. In this experiment, we expected the weight of the cells to change.
Is this change due to the movement of sugars or the movement of water?
Explain your answer. ___________________________________________
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4. Explain the weight changes in the cells based on the principle of osmosis. (If the
cells didnt change, try to explain that!) Be sure to refer to a water gradient and to the
net movement of water.

Cell A

Cell B

Cell C

Cell D

Exercise 4: Differential Permeability

This exercise makes use of the reaction of iodine with starch, which you observed in the
biological molecules lab. Recall that when starch and iodine are mixed, the iodine
molecules become stuck in the spirals of the starch molecule, turning the solution
purplish if the concentration of starch is low and intensely blue-black if the concentration
is high.
In lab, a model cell will be made by filling a bag made from dialysis tubing with a
starch solution (starch dissolved in water). The bag will then be put into a beaker
containing a water/iodine solution and observed over a span of 30 minutes.
Procedure for observing differential permeability:
1. Make a model cell using dialysis tubing by opening the water soaked tubing and
fastening one end, as was shown by your instructor. At the sink, carefully fill the
cell with approximately 20 mL of starch solution.

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2. Gently squeeze the open end of the cell to push out the trapped air. Twist the
open end and fasten it, as was shown by your instructor. The cell should be limp.
3. Squeeze the cell to check for leaks.
4. Place the cell in a beaker with 125 mL of water.
5. Add enough drops of Iodine to make the water dark (yet light enough to see your
cell).
6. Observe your cell over a span of 30 minutes. Record your observations in Table 2.
Table 2: Observations of Differential Permeability
Time

Observations

15 minutes
30 minutes
7. Answer the following questions:
a. What molecules will be inside the cell?

b. What molecules will be in the beaker outside the cell?

c. Which of the molecules named above is the largest?

d. Which of the molecules named above is the smallest?

e. How will you know if iodine is able to diffuse across the membrane?

f. How will you know if starch is able to diffuse across the membrane?

Conclusions:
1. Did Iodine diffuse across the membrane? How do you know?

2. Did starch diffuse across the membrane? How do you know?

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Clean Up:
Black clips for dialysis tubing Save and return them to the proper container
Dialysis tubing-pour the contents down the sink and throw away the tubing
Beakers-wash and return to table

Discussion Questions
1. Sharks can smell blood in water from quite a long distance from the source. How
might a shark locate its prey once it has detected blood in the water? (Hint: remember
what you know about diffusion.)

2. A man walks into the classroom wearing far too much cologne. Soon even the people
on the opposite side of the room can detect the aroma of his odor-enhancing product.
Will those who are farther away from him catch a whiff of his eau de toilette sooner in a
warm or cold room? Why?

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3. Some students have jokingly referred to learning by osmosis, which can mean anything
from picking up bits of knowledge from hanging around people who know a lot of things
to sleeping with a textbook under your pillow in hopes that information will diffuse into
your brain while you slumber. Lets pretend that information could diffuse from your
textbook across a pillow membrane. What is the maximum amount of the information
contained in the textbook that a student could learn by osmosis in this manner?

4. If you go to a pet store to buy tropical fish for the aquarium you might have in your dorm
room, you will find that they have salt-water fish and fresh water fish for you to choose
from. Think abut their different environments. The interior of the cells of the fresh-water
fish has more solute in them than the fresh-water surrounding them. The interior of the
cells in the salt-water fish has less solute in them than the salt-water surrounding them.
Explain what is happening with regard to osmosis in both situations. How might the fish
deal with their osmotic situations? (Hint: think about concentration gradients, passive
transport and active transport.

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