Biological Principles Name: _________________________________
Lab: Diffusion and Osmosis
To observe the motion of molecules
To investigate the relative rates of diffusion of molecules of different sizes through a selectively
To determine the rates of osmosis through a selectively permeable membrane when the relative
concentration of water on the two sides of the membrane is known
Diffusion requires no work by a cell. Diffusion results from the random motion (kinetic energy) of
atoms and molecules. All particles move in a random fashion and the hotter the material, the more the
motion. If one particular molecule is more highly concentrated in one area than in a neighboring area,
over time, some of these molecules will migrate from the area of higher concentration to the area of lesser
concentration. This movement is diffusion.
MODULE 1: Effect of molecular weight on rates of diffusion
Many factors change the rate of diffusion, but the direction is always from an area higher
concentration to an area of lesser concentration. Another factor is mass of the particle. The more massive
a particle is the greater its inertia and the slower it will diffuse. In this Module, you will compare the rates
of diffusion of two molecules with different masses. The atomic mass number (the sum of the number of
protons and neutrons) will be used for comparison. Potassium permanganate has a molecular weight of
158 g and the molecular weight of malachite green is 929 g.
Petri dish with agar crystals of malachite green
crystals of potassium permanganate metric ruler
1. Formulate and state your hypothesis in Question 1 of the lab report.
Before lab, one crystal each of potassium permanganate and malachite green were placed on the surface
of the agar in a Petri dish. The agar consists of water with 1% of thickening material extracted from sea
weed. The crystals of potassium permanganate and malachite green used were of similar size.
2. Measure the radius of the zone of diffusion to the nearest mm. Start at the center of the crystal
and measure to the edge of the colored zone. Record your data in the table in Question 2 of the
3. Answer Question 3.
MODULE 2: Diffusion through a selectively permeable membrane
Diffusion occurs through materials such as water and air but it may also occur through structures
such as the cell membranes. The size of a particle may slow or prevent its diffusion through a particular
membrane. If a membrane allows some items to diffuse through but slows or stops others it is described
as a selectively permeable membrane.
In this module, you will compare the diffusion rates through a membrane of four molecules with
two pieces of hydrated dialysis flask of distilled water with 10-ml pipette
membrane, 15 cm long sodium hydroxide solution (1 M)
four unlabeled dialysis clips dropper bottle of Lugol’s iodine
two 250-ml beakers dropper bottle of phenolphthalein
two glass stirring rods two paper towels
starch suspension with 10-ml pipette
1. Fill each of the beakers to the 200-ml mark with cool tap water.
2. Add 10 drops of iodine solution to one beaker and stir the contents of the beaker with a glass
stirring rod to obtain a uniform yellow color. The color will allow you to identify this beaker as
3. Add 10 drops of the sodium hydroxide (NaOH) solution to the other beaker and stir with a clean
glass rod. Caution: Concentrated NaOH is very caustic.
4. Open the hydrated membrane by rubbing it between your thumb and forefinger. When opened,
thoroughly wet the inside of the tube by allowing running tap water to pass through it.
5. Flatten the tube and fasten a dialysis clip about 1 cm from one end of each of the tube to form
6. Label one piece of paper toweling starch and the other phenolphthalein. Place one sack on each
and use the paper to transport the sack and to label it.
7. Pipet 10 ml of starch solution into one sack. With your fingers, gently squeeze the air out of the
sack. Fasten a dialysis clip about 1 cm from the top end of the sack. The “cell” thar you have
created should be rather slack to prevent pressure from being a variable in your experiment.
8. Rinse the outside of the entire sack under running tap water to remove any starch from the
outside of the sack and replace it on the labeled paper toweling.
9. Pipet 10 ml of water into the other sack and add 3 drops of phenolphthalein solution, squeeze out
the air, fasten the second clip, rinse and place it on the labeled paper toweling as in steps 7 and 8
10. Submerge the sack containing starch into the beaker containing iodine.
11. Submerge the sack containing phenolphthalein into the beaker containing NaOH.
12. Let the sacks incubate in their respective beakers for 5-10 min.
13. Answer Questions 4-6 in the lab report.
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MODULE 3: Osmosis through a selectively permeable membrane
Diffusion occurs with any particle as long as there is a concentration gradient and kinetic energy.
Diffusion of water through a selectively permeable membrane is such a common and important thing in
living organisms that it has been given a name, osmosis. A good definition of osmosis is diffusion of
water through a selectively permeable membrane. Please note that osmosis is specifically the diffusion of
water and that, as with all diffusion, the water is always moving from an area of its higher concentration to
an area of its lower concentration. If we are told that the ocean is about 3.5% salt it is important to realize
that it is about 96.5% water. In these parts of the lab you will observe osmosis and compare the rate of
diffusion of water through a membrane when there are various concentrations of water an each side of
the membrane. There are several ways to calculate a concentration gradient. In part 6 of the lab you will
determine the percentage of water on each side of the membrane and the difference in percentage of
water will represent the concentration gradient. Since the concentration gradient represents energy,
which does work but does not require the cell to use ATP, it is called a free-energy gradient. The free-
energy gradient is potential energy and the rate of diffusion is directly proportional to this gradient.
four pieces of hydrated dialysis 50% sucrose solution with 10-ml pipette
membrane, 15 cm long small bowl
four dialysis clips labeled A, B, C, and D large bowl
four unlabeled dialysis clips digital balance with weigh boat
1% sucrose solution with 10-ml pipette paper towel
25% sucrose solution with 10-ml pipette
1. Fill the small bowl to a depth of about 3 cm with 50% sucrose solution.
2. Fill the large bowl to a depth of about 3 cm with 1% sucrose solution
3. Set both bowls near the center of our work area.
4. Open a piece of hydrated dialysis membrane by rubbing it between your thumb and forefinger.
When opened, thoroughly wet the inside of the tube by allowing running tap water to pass
5. Flatten the tube and fasten the dialysis clip with “A” on it about 1 cm from one end of the tube to
form a sack.
6. Do the same with the other three pieces of membrane and dialysis clips B, C, and D.
7. Pipet 10 ml of 1% sucrose solution into sack A. With your fingers, gently squeeze the air out of
the sack. Fasten a dialysis clip about 1 cm from the other end of the sack. The “cell” that you
have created should be rather slack to prevent pressure from being a variable in your experiment.
8. Rinse the entire sack under running tap water to remove any sucrose from the outside of the sack
and place it on a piece of paper toweling.
9. Pipet 10 ml of 1% sucrose solution into sack B, 10 ml of 25% sucrose into sack C, and 10 ml of
50% sucrose into sack D following the same procedure as in steps 7 and 8 above.
10. Obtain a digital balance, turn it on, place the weigh boat on the scale and press the tare button.
Check to make sure that the scale is reporting the weight in grams. Place a few paper towels
near the scales.
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11. Simultaneously submerge sack A in the small bowl and sacks B, C, and D into the larger bowl
and cover with the solution in the bowl.
12. Immediately remove sack A from the small bowl. Gently blot it on to a folded paper towel and
then lie it in the weigh boat. Record the weight in the data table in Question 9 on the lab report.
This is your 0 time measurement for this experiment. Immediately return sack A to the small bowl
and cover with the solution. Retare the scales if it is no longer registering 0 g.
13. Weigh sack B following the same procedure, record the weight in the table, return the sack to its
solution. Do the same with sacks C and D.
14. After 15 minutes, repeat the weighings in the same order and using the same procedure. During
this time, answer Questions 7 and 8 on the lab report.
15. At 30, 45 and 60 min (from your start time), weigh all four sacks. Record your data in the table.
16. Prepare a graph of your collected data and answer the remaining questions.
Return the Petri dishes of agar and metric rulers to the instructor’s bench.
Discard all solutions down the drain
Take the bowls to the sink, remove the dialysis clips and rinse them well under running water.
Place them in the basket to dry.
Discard any used dialysis membrane in the trash. (Please check the sinks for any dialysis
membrane and remove it.)
Rinse the beakers and bowls with tap water and leave them by the sink to dry.
Return the pipettes and pipette pumps to the instructor’s bench.
Gently wipe down the balances with a damp paper towel.
Wipe down your lab bench with cleanser and paper towels.
Push in your chair when you leave.
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