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 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 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: Orange G, Molecular weight (MW) = 452
Well #2: Amido Black, MW = 616
Well #3: Congo Red, MW = 697
Well #4: 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?
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 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 mem-brane. 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 113in 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.
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:
Tube #SolutionTonicity Observations
1waterhypotonic
210% NaClhypertonic
30.3% NaCl
40.9% NaCl
51.8% NaCl
63.6% NaCl
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.
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
Blood donation, who can and who can’t and why, is discussed at
The use of diffusion (hemodialysis) in patients with advanced kidney disease is described at
1