DIFFUSION & OSMOSIS
Diffusion, or the movement of atoms, ions, or molecules from
areas of high concentration to areas of low concentration, is due to the
constant random motion of the individual particles. Diffusion is a crucial
process in living things. As examples: (1) oxygen diffuses from the
alveoli or air sacs in the lungs to the bloodstream, and carbon dioxide
diffuses from the blood into the alveoli, (2) wastes diffuse from cells to
the blood for eventual elimination by the excretory organs, and (3) ATP,
the energy currency within cells, is produced in the mitochondria and
diffuses away to provide the energy to drive biochemical processes
elsewhere in the cell. In this lab you will observe evidence of the
random motion of molecules and study factors which influence the rate
of diffusion.
Osmosis is a special case of diffusion; it is defined as the
movement of water from an area of high concentration to an area of low
concentration through a semi-permeable membrane. A semi-permeable
membrane allows only some molecules (including water) to pass. For
example, cell membranes allow the passage of water, oxygen and other
gasses, but do not permit the free passage of ions and large molecules
such as proteins. Since 70% of the body is water, understanding
osmosis, or the movement of water into and out of cells, is important.
Understanding osmosis (and diffusion) is also of practical importance in
many clinical situations- dialysis, the blood-cleansing procedure used to
treat patients with kidney disease, is an example.
Evidence of random motion
Brownian movement is the motion of small particles as a result of
collisions with other particles in motion. In the case you will examine,
small India ink (carbon) particles in water are caused to move by
collisions with the water molecules. The motion of the ink particles is
random, i.e., haphazard and non-directional, because the motion of the
water molecules is random.
Place a small drop of India ink on a microscope slide, apply a
coverglass and examine the preparation at high magnification. (Use the
microscopy procedures you learned last week!). The tiny carbon
particles may be flowing across the slide due to the pressure of the
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coverglass on the fluid - that's not Brownian movement. Let the
preparation stabilize, then look for a jiggling or tumbling motion by the
particles. That's Brownian movement, evidence that the water
molecules are in constant motion.
What do you think provides the energy for the movement of the
water molecules?
Effect of temperature on diffusion rate
In order to examine the effect of temperature (energy) on diffusion
rate, you will dissolve a small amount of potassium permanganate, a
purple chemical, in cold water and in hot water, and compare the rate at
which the color spreads through the water. Before you begin, heed
these warnings: (1) do not touch the potassium permanganate - it can
burn your skin, and (2) set up your experiment out of harm's way - you
want the color to spread by diffusion, not by someone shaking the
dishes.
Obtain two small specimen dishes from the supply table. Put ice
water in one and hot tap water in the other. Place them in an
undisturbed place, then, with forceps, drop a crystal of potassium
permanganate into the center of each dish. Over the next few minutes,
observe the spread of the purple color.
In which bowl does diffusion occur at a faster rate? Why?
Effect of molecular size on diffusion rate
The size and shape of a molecule might determine how fast it
diffuses. It's not unreasonable to predict that a small, compact molecule
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might move faster than a large, bulky molecule. To examine the effect
of size, we will look at the diffusion rates of similarly shaped, but
different sized molecules, specifically, a series of four dye molecules, all
dissolved in water, and diffusing through water.
Obtain an agar plate from the supply table. (The agar plate is 1%
agar and 99% water. The agar has no effect on the experiment
other than to prevent the water from splashing about, so think of
the plate as a dish of water in a very undisturbed location.) Cut 4
wells in the plate as demonstrated by your instructor, then place a few
drops of dye in each well as follows:
Well #1:
Well #2:
Well #3:
Well #4:
Orange G, Molecular weight (MW) = 452
Amido Black, MW = 616
Congo Red, MW = 697
Brilliant Blue G, MW = 854
Allow the plate to stand undisturbed for about an hour, then
observe how far the different dyes have diffused outward from the wells.
What is the relationship between molecular weight (or size) and the
rate of diffusion?
A demonstration of osmosis
Your lab instructor will demonstrate osmosis by using an artificial
semi-permeable membrane rather than a true biological membrane.
This artificial membrane allows any small molecule or ion to pass
through its minute pores, but does not allow the passage of large
molecules.
How is this semi-permeable membrane different from a true
biological membrane?
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The membrane will be formed into a (nearly) closed bag
containing molasses, a concentrated solution of both large (complex
carbohydrates) and small molecules (sugar) in water. One opening, to
a glass tube, will remain, as shown below. The bag will be lowered into
a beaker of water.
What do you think will happen as a result of diffusion and
osmosis? Hint: Think about the concentration of water inside and
outside the bag. What will happen to the large and small
molecules inside the bag?
Osmosis in plant tissue
Another simple demonstration of osmosis involves potato cells.
Each potato cell is bounded by a semi-permeable plasma membrane
and a semi-permeable cell wall, so osmosis should occur in potato
tissue. To show that water can either enter or exit cells, we will place
slices of potato into: (1) water, and (2) concentrated saline (or 10% salt
water), and observe the effect of each treatment on the size of the slice.
If water enters the cells, the slice should swell; if water exits, the slice
should shrink.
Use a corkborer (as demonstrated by your instructor) to obtain a
long cylinder of potato. Cut it into two equal lengths, and measure them
as accurately as possible (in millimeters). Immerse one in a dish
containing water, and the other in a dish containing 10% NaCl. Leave
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them for about 30-60 minutes, then remove them from the dishes and
measure their lengths. Record your results below.
Length of the slices at start: ________ mm
Length of slice in water (at finish): ________ mm
Length of slice in saline (at finish): ________ mm
Are these results consistent with your expectations?
Now feel the consistency (hardness/softness) of the potato slices.
Are the results consistent with the idea that water enters the potato
in one solution and exits in the other? Which method (length or
consistency) seems to be the best way to get at the truth here?
Effect of osmosis on red blood cells
Red blood cells (RBCs) are remarkable in that they have no nuclei
nor other cellular organelles with the exception of the plasma membrane. In fact, red blood cells are essentially membranous sacs of
hemoglobin, the oxygen-transporting protein, and a variety of enzymes,
ions, etc. dissolved in water. Their three-dimensional appearance can
be seen on page 113 in your text; they are biconcave, i.e., "dished in"
on both the top and bottom. RBCs in the blood are in an isotonic
environment, where the concentrations of water (the solvent) and
dissolved materials (solutes) inside and outside the RBCs are equal.
Thus, there is no net movement of water into or out of the cells, and
they retain their characteristic shape.
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RBCs placed in a solution of lower solute concentration (i.e.,
higher water concentration) are said to be in a hypotonic solution (The
cells are hypertonic to the fluid.). An example is distilled water, where
the solute concentration is zero.
How will water move with respect to the plasma membrane when
RBCs are placed in a hypotonic solution? What will happen to the
shape of the cells?
RBCs placed in a solution of higher solute concentration (i.e.,
lower water concentration) are said to be in a hypertonic solution (The
cells are hypotonic to the fluid.). Concentrated salt water (saline) is an
example of a hypertonic solution.
How will water move when RBCs are placed in a hypertonic
solution? What will happen to the shape of the cells?
In order to examine the effects of osmosis on RBCs, we will set up
a series of test tubes, each containing a different concentration of NaCl,
ranging from 0% (distilled water) to 10% NaCl. Clearly, the distilled
water tube will allow us to see what happens to RBCs in a hypotonic
solution, and the 10% saline tube will represent the hypertonic solution.
By examining the RBCs in the other tubes, you should be able to figure
out which one represents the isotonic situation.
Mark 6 test tubes roughly 1 inch from the bottom (or 2.5 cm).
Label them with numbers as follows (next page) and fill each tube to the
mark with the appropriate solution:
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Tube #
Solution
Tonicity
1
water
hypotonic
2
10% NaCl
hypertonic
3
0.3% NaCl
4
0.9% NaCl
5
1.8% NaCl
6
3.6% NaCl
Observations
After labelling the tubes and adding the solutions, add 1-2 drops of
blood to tube #1 and tube #2 and tap the tubes gently with a finger to
distribute the cells through the solutions. Over the course of the next
few minutes (perhaps seconds!), one of the tubes will become clear
enough that you will be able to read this page through the solution.
Which tube clears? Why?
When either tube #1 or #2 clears, examine the RBCs from both
tubes in the microscope. What has happened to the cells? Record your
answer in the space for observations above.
Now that you know what happens to RBCs in hypotonic (#1) and
hypertonic (#2) solutions, you can attempt to determine which of the
remaining four solutions is isotonic. Simply add 2 drops of blood to
each of tubes #3 through #6, mix by tapping, let them stand a few
minutes, then make your observations as before.
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Hint for Examining Tubes #3 - #6
If a tube is clear: The solution is hypotonic; there is no need to look for
cells in the microscope.
If a tube is turbid: The solution is either isotonic or hypertonic; examine
in the microscope - if cells are shrunken and/or misshapen, the solution
is hypertonic, but if the cells are round and regular in appearance, the
solution is isotonic.
In which tube do the RBCs retain their characteristic shape, neither
swelling nor shrinking? Which solutions are hypotonic?
hypertonic?
Internet Resources
The types of blood cells and their roles in disease are described at
http://www.wadsworth.org/chemheme/heme/microscope/celllist.htm.
Blood donation, who can and who can’t and why, is discussed at
http://www.nhlbi.nih.gov/health/public/blood/transfusion/g_life_e.htm.
The use of diffusion (hemodialysis) in patients with advanced
kidney disease is described at
http://www.niddk.nih.gov/health/kidney/pubs/kidney-failure/treatmenthemodialysis/treatment-hemodialysis.htm.
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