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LAB 3: DIFFUSION, OSMOSIS, AND CELL PERMEABILITY
From a Nonrandom to a Random Distribution of Molecules
– without the use of energy!
Learning objectives (What we expect you to get out of this lab):
Concepts & techniques
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Vocabulary:
Understand diffusion and osmosis
Understand how diffusion and osmosis are caused by concentration gradients
Understand the importance of diffusion and osmosis to biological organisms.
Setup laboratory apparatus
Measure movement of solutions and molecules to observe osmosis and diffusion
Calculating rate from of processes
Developing graphs to show data patterns
Drawing conclusions from data
concentration gradient
diffusion
osmosis
cell membrane
cytoplasm
cell wall
Introduction:
On a rainy Sunday afternoon, the smell of baking cookies slowly spreads through the house. The scent
molecules given off by the cookies are initially strongest in the kitchen, but over the course of the
afternoon, the entire house begins to take on the delicious aroma of vanilla, butter and sugar. The
random movement of scent molecules through out the house occurs as the molecules gradually spread
down the concentration gradient. When the cookies begin baking, the molecules are concentrated in the
oven, but over time, they move to areas lacking scent molecules- from areas of higher concentration to
lower concentration until the distribution of molecules throughout the house is fairly equivalent (this is
why the scent of cookies is strongest in the kitchen when they first come out of the oven, and becomes
weaker both over time and also the farther you get from the initial area of concentration).
1 minute
30 minutes
60 minutes
Diffusion, the movement of a substance from a region of high to a region of low concentration, is the
process by which nutrients and wastes move toward and away from cells. If molecules could not pass
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through cell membranes, life would end because there would be no continuous uptake of energy rich
food molecules. Life will also cease if toxic materials, such as the wastes produced by each cell,
accumulate to lethal levels in the immediate vicinity of the cell. In this lab, you will observe several
demonstrations of DIFFUSION, each of which emphasizes that this is a spontaneous process driven by
random molecular motion.
Osmosis is a special type of diffusion that refers to the net movement of water through a selectively
permeable membrane. A selectively permeable membrane only allows certain molecules to cross it. In
the context of the living cell, the selectively permeable membrane is the outer boundary of the cell.
Osmosis is used to maintain appropriate concentrations of molecules in the cell, which is one feature of
the organization of matter that is required to maintain life. Analyzing osmosis lets you explore the idea
that a disorganizing process, the diffusion of water, can be coupled to an organized structure, like a
semipermeable membrane, to do work that increases or maintains the organization of part of a system. If
you have ever attempted to control slugs in your garden by sprinkling salt on them, you have used
osmosis. By the end of this activity, you’ll be able to explain what is happening in the case of the salted
slug!
Three components must be present for osmotic
pressure to exist: a selectively permeable membrane
and two solutions that differ in their concentrations
of a substance that cannot cross the membrane. In
this investigation, and in most biological situations,
the substance that cannot cross the membrane is
dissolved in water. In this diagram, the solution on
the left of the selectively permeable membrane has a
higher concentration of molecules, indicated by dots,
than the solution on the right side of the membrane.
The molecules cannot cross the membrane and
are dissolved in water, which can cross the
membrane. There is a concentration gradient of molecules; that is, a given volume of solution on the
left has a larger number of molecules than the same volume of the solution on the right.
You will begin this lab by investigating two physical processes that control the movement of nutrient
and waste molecules relative to cells. By the end of the investigation, you should be able to define
diffusion and osmosis; you will need to understand these concepts to discuss how they apply to
observations of you will make on living cells during next week’s lab.
☞ NOTE: Both lab procedures today will take at least one hour once you get the
experiments set up. You should get the osmometer set up and running and then set up the
acid diffusion experiment. Do not wait to finish collecting osmometer data before you set
up the diffusion experiment. You can collect data simultaneously on both experiments.
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Part 1: Measuring Osmosis
1. An osmometer is a device for measuring osmotic pressure. To conceptualize the impact of osmotic
pressure, consider the following questions about a bag of solution of sucrose (C12H22O11) dissolved
in water (H2O) enclosed by a semipermeable membrane that is immersed in a beaker of solution that
is less concentrated.
1A. Can the sucrose molecules spontaneously diffuse
down this gradient? Why or why not?
1B. There is also a concentration gradient of water. Is the
water concentration higher inside or outside of the bag?
1C. Will water diffuse down its concentration gradient?
Why or why not?
1D. Will the net movement of water be into or out of the bag?
1F. What will happen to the volume of solution inside the bag?
2. Use your answers in Procedure 1 above to make a hypothesis about what will happen to the level of
fluid in a tube that can carry excess liquid out of bag. How do you hypothesize that the concentration
of the solution inside the bag will influence the volume change in the bag and subsequent fluid
movement in the tube?
Hypothesis:
3. Construct your osmometer (see figure below)
• Get a dialysis bag (made of a semipermeable material) from your instructor and securely tie off one
end of a soaked tube.
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Fill the bag with 10 mL of a solution containing sucrose, which even when dissolved cannot cross
the membrane. There are three different sucrose solutions, 10%, 20%, or 40%. Be sure to record
which solution your table is using.
Tie off the open end of the bag to the end of the surgical tubing using the string. The liquid from the
bag must completely cover the end of tube, with no air gap. It is necessary to be certain that there are
no leaks in the membrane and that the outside has been washed free of any spilled solution.
Immerse the filled bag into a beaker of distilled water. Make sure the bag is completely immersed in
the beaker of water- you can adjust and secure the apparatus using the clamps at the top of the stand.
Record the time and the height of the fluid in the glass tube once you have completed setting up the
osmometer.
At five-minute intervals over the next 60 minutes, measure and record in the data table the height of
the fluid column in the osmometer.
Record your group’s data in the table and on the class data sheet at the front of the room. Once all of
the class data has been recorded, make sure you copy it down so that you have data for all three
glucose concentrations.
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Data Table: Osmosis in the Presence of Three Different Concentrations of Sucrose:
10% Sucrose
Time
(minutes)
Table 1
Table 2
20 % Sucrose
Table 3
Table 4
40 % Sucrose
Table 5
Table 6
5
10
15
20
25
30
35
40
45
50
55
60
4. As your team records the data for your osmometer, you can begin to construct a graph that shows the
change as time goes by in the height of the fluid column in the osmometer. Plot the data for your
osmometer first; you can add in the other solutions when the other groups make their data available.
Be sure to use a different symbol (for example, ✕,,or Δ) for each solution. Do not forget to label
your graph and both axes!
4A. What should be plotted on the X-axis of your graph?
On the Y-axis?
4B. What are the units of the item plotted on the X-axis?
On the Y-axis?
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5. Do the data points on the graph you plotted for your group’s osmometer data seem to fit a straight
line or a curved one?
5A. Is this what you would predict from thinking about what is happening in the experiment? Why?
(SUBTLE HINT: As time passes, what happens to the size of the concentration gradient that drives
osmosis?)
6. Calculate the rate of osmosis for your osmometer. (Show your work.)
7. How did you select the part of the graph to use in answering the last question?
8. Tabulate the rates of osmosis calculated by all other team in your lab section, including the team that
used the same concentration that you did.
9. Is there a consistent relationship between sucrose concentration and rates of osmosis?
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Part 2: Measuring Diffusion
1. Obtain three petri dishes which have been filled with agar dyed with bromothymol blue.
Bromothymol blue is an indicator that will turn yellow-green in the presence of an acid.
2. Bring the petri dishes back to your lab bench. Use the cork borer to make a shallow depression in
the center of each block. Be sure that your depression does not extend all the way to the glass bottom
of the dish.
3. Use what you have learned about diffusion to make a hypothesis about what will happen when the
acids are added to the depressions in the agar in the petri dishes.
Hypothesis:
4. You will be using acids of three different molecular weights. How do you hypothesize that the
molecular weight will influence (or not) diffusion of the acid through the agar? The molecular
weight of each acid is:
Hydrochloric acid = 37
Acetic acid = 60
K Acid Phthalate = 204
Hypothesis:
5. Put on your goggles and carefully use the droppers to fill the depression in the first agar block with
hydrochloric acid, and fill the depressions in the second and third agar blocks with acetic acid and
Potassium acid phthalate, respectively. Record the time at which each depression is filled. Once you
are done with the droppers, you may take your goggles off.
6. At five minute intervals over the next 60 minutes, measure and record in the data table in Part 1 of
Results, the diameter of the yellow circle that forms in each block as the acid diffuses out of the
depression.
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Data Table: Diffusion of Acid in an Agar Block:
Time
(min)
5
Acetic
Acid
Hydrochloric
Acid
K Acid Phthalate
Aid
10
15
20
25
30
35
40
45
50
55
60
7. Plot the data for all three acids on a single time series graph, using a different symbol (circles, “x”s,
triangles) for each acid. Be sure to label your graph and both axes!
7A. What should be plotted on the X-axis of your graph?
On the Y-axis?
7B. What are the units of the item plotted on the X-axis?
On the Y-axis?
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8. Review your graph. Do the data points on your graph seem to fit a straight line or a curved one?
Is this what you would predict from thinking about what is happening in the experiment?
Why?
(SUBTLE HINT: As time passes, what happens to the size of the concentration gradient that drives
diffusion? NOT-SO-SUBTLE HINT: Will the diameter of the yellow circle continue to increase if
one of the acids diffuses to the edges of the agar block?)
9. Remember that the molecular weight of each acid is:
Hydrochloric acid = 37
Acetic acid = 60
K Acid Phthalate = 204
Is there a consistent relationship between the size of the acid molecules and their diffusion rates?
How did you select the part of each acid’s graph to use in answering the last question?
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Questions
Name____________________
1. Define “osmosis”
2. Define “diffusion”
3. Describe the primary differences between osmosis and diffusion.
4. In the lab, you observed acids with different molecular sizes diffusing through three agar plates, all of which
had the same density of agar. Suppose you took three agar plates with different densities: one low density, one
medium density, and one high density. You prepared a hole in each plate and dropped in one drop of hydrochloric
acid in each plate. What would be the results? Explain the basis for your answer:
5. Imagine that you wanted to examine diffusion of a gas within a closed room. You use three gasses, each with a
different molecular weight. Gas X has a molecular weight of 41, Gas Q has a molecular weight of 320, and
Gas K has a molecular weight of 12. Which of the three gasses would you expect to diffuse most rapidly
through the room? Why?
6. Use what you learned about osmosis to explain:
a. a slug shrivels when you put salt on it (think about what is happening to semipermeable cells of the slug)?
b. carrot sticks get crisp when you put them in a bowl of water in the refrigerator
c. Why it is a bad idea to drink saltwater when you are already dehydrated