Diffusion and Osmosis


Molecular Movement in Cellular Solutions

The cytoplasm of cells is 70 to 95% water. Dissolved or dispersed in that water are various salts, sugars, proteins, etc. which make up a complex mixture of molecules.

Molecules in liquids and gases are in constant motion due to their kinetic energy. Substances dissolve in water and disperse throughout a solution because they are constantly in motion. In part, this motion is due to Brownian motion in which molecules of solutes and particles which are dispersed in a solution are “bombarded” by moving water molecules, which “jostles” them around, causing them to move even more. (Note, in a very quiet room, you may be able to hear the sound of Brownian motion as air molecules bombard your ear drums.) Thus, diffusion is the tendency for molecules of any substance to spread out randomly into the available space. Substances (solutes) will diffuse from more concentrated to less concentrated areas or solutions. Passive transport is diffusion across a semipermeable, biological membrane. Sometimes, though, solute molecules are too large to go through a semipermeable membrane, or perhaps, have an electrical charge which does not allow them to pass through the membrane. In spite of that, there is still a tendency for the concentrations of the solutions on both sides of that membrane to equalize. One way in which that is acomplished is through the process of osmosis (osmo = to push; -sis = the act of), which is a special case of passive transport in which water diffuses across a selectively permeable membrane from less to greater solute concentration to try to equalize the concentrations of the solutes.

If a cell and its watery environment have the same concentrations of solutes, they are said to be isotonic (iso = equal; tono = tone, tension, stretched) solutions. If the environment has a greater concentration of solutes, it is hypertonic (hyper = over, above) with respect to the cell contents, and the cell will shrink as it loses water. If the environment has a lesser concentration of solutes, it is hypotonic (hypo = under, beneath) relative to the cell contents and the cell will swell or, in the case of animals, even burst as it gains water.


Osmosis

Set this part up first because it takes the longest to do. Because you will be taking readings every 15 min, you can do the other parts of the lab in between taking readings. Work in groups of 5 students so each person can “adopt” one of the 5 solutions to be tested. Each person should record the data for all 5 solutions in his/her lab notebook.

  1. Each group should obtain 5 pieces of dialysis tubing approximately 9 in (20 cm) long and 5 100-mL graduated cylinders
  2. Soak each piece of dialysis tubing under cool, running tap water, rolling the end between your fingers until it opens up. Run tap water through it until it is completely open.
  3. Tie a knot in one end of each tube to seal it, placing the knot as close to the end as possible. Fill the tube with tap water, and while pinching the open end to hold it shut, gently squeeze the tube to check for leaks. If you find a leak, get a new piece of tubing.
  4. Each person in the group should pick one of the following solutions, so that as a group, all 5 are tested. These solutions should already have been made by the lab staff and should be availabe on a cart or the instructor’s bench. Each person should fill his/her tubing “bag” with his/her chosen solution.
  5. When filling the bags, leave them a little flaccid (flacc = flabby) or limp because later on they may absorb water and become turgid (turg = swell, swollen) or rigid, even to the point of bursting if too full.
    6. dialysis tubing bag
  6. Starting below the fluid level in order to avoid trapping air bubbles inside, twist the open end “shut” so that you have enough “unused” end to tie. Like tying a balloon, tie a knot in the open end of the “bag” to seal it. Again, double-check to avoid trapping air bubbles inside.
  7. When the tube is sealed, rinse it under tap water to remove any spills, then blot mostly dry by gently rolling on a paper towel. Do not let it get overly dry. You want the surface of the bag to be dry enough to not add excess weight when you weigh it, but it should not start to dry out.
  8. Using the electronic balance and a weighing boat (do NOT put your “bags” directly on the balance pan!), weigh the bag to the nearest 0.01 gm and record the weight in your lab notebook. If there are few-enough groups in your class and enough computers each group may pick a computer, and also enter weight data as they are being obtained rather than waiting until the end of class to entering all the data at once. This first weighing will be your “time 0” weight.
  9. For your group, obtain 5 100-mL graduated cylinders and fill each approximately ¾ full with dH2O. Label one for each of the solutions to be used, and gently submerge that bag in that cylinder.
  10. Every 15 min. for the next 90 min., remove the bag and gently dry it as before, then weigh it (each person should keep track of the time for his/her own bag). Record the weight in your lab notebook (and in the computer). Note any change in the way the bag feels — does it increase or decrease noticeably in turgidity? Is there any visible evidence of the passage of molecules from the bag to the external water (color change)? In between weighings, complete the rest of the parts of the lab.
  11. After you take your last reading, with the bag still out of the water, add a couple drops of silver nitrate (AgNO3) to the water in the cylinder and observe what happens. CAUTION: SILVER NITRATE STAINS SKIN BLACK UNTIL IT WEARS OFF — DO NOT GET THIS ON YOUR SKIN OR SPILL ANY!!! (Note: this is a light-sensitive reaction, so if you get some on you, you won’t know it right away, but by the next day, you may see black spots.) If chloride ion (Cl–) is present, it will react with the silver ions (Ag+) to form silver chloride (AgCl) which is not water-soluble and, thus, shows up as a white precipitate by the following chemical reaction:
    AgNO3 + Cl → AgCl↓ + NO3–
    Is there chloride ion present in your cylinder? You started out with dH2O, so think about what might have been the source of that chloride and how it got into the water in the graduated cylinder. (Optionally, as time and interest allow, a flame test could be conducted to test for the presence of Na+ and/or Benedict’s Solution could be used to test for glucose in the cylinder water.)
  12. Clean up! Dump the water out of the graduated cylinder, rinse the cylinder, and put it in one of the rack to dry. Snip the bag open and rinse its contents down the drain. Place the bag, itself, in the trash. Check your table top for spills, etc., and make sure it and the balance you used are clean before you leave for the day
  13. Do the following calculations for each time for each bag of solution:
    1. For each time subtract the initial weight of the bag from the weight at the end of that time to determine the change in weight of that bag:

      wtfin – wtinit = Δwt

    2. Then, for each substance for each time, calculate the percent change in weight by dividing the change in weight by the initial weight and multiplying by 100 to change the answer to a percentage:

      Δwt/wtinit × 100 = %Δwt

    3. In your notebook, make a graph of time (minutes from the start) on the X-axis, versus percent weight change on the Y-axis. Plot each of your group’s five bags (five lines) on the graph.
  14. If you have not already done so, someone from your group should submit your group’s data online. Once all class data have been submitted, you may view and print class data.

Brownian Motion

This may either be done as a class demonstration, projected on the screen, or individually, by students.

carmine suspension
Carmine Suspension Using a microscope slide and coverslip, make a wet mount of a carmine (a red dye) suspension and examine under the microscope. This is not a solution because the carmine does not actually dissolve in the water, but rather, tiny bits of carmine are suspended in (float around in) the water and may be seen with a microscope. Describe what you see. Are any of the particles, especially the smaller ones, moving? Hopefully, you should see them “jiggling around” and “zig-zagging.” This type of motion is called Brownian motion after Robert Brown who first described it. The water molecules, which are invisible, are in constant motion and are slamming into the carmine particles (like pool balls), causing them to jump around. Note that if you see “everything” drifting/flowing in one direction, for this experiment, that’s not something of significance — that motion is due to the water under the coverslip beginning to evaporate (dry up).


Diffusion

diffusion Either as a class demonstration or in your group, put some tap water in a beaker. Set the beaker on a table and let it sit until calm. Make sure it doesn’t accidentally get bumped, jostled, or picked up — don’t disturb it once it’s calm. Gently add a couple drops of methylene blue (or another dye) with the dropper near the surface of the water so as to disturb the water as little as possible. Observe what happens over time. Do not bump or move the beaker once the dye is added.

food coloring and detergent in milk As time, interest, and availability of substances alow, another somewhat-similar demonstration that may be done is:
if milk is available, place some whole milk in a saucer. Gently add one drop each of red, yellow, green, and blue food coloring, each in a different quadrant of the milk. Then, gently add one or two drops of dish detergent (supposedly Dawn® works well) to the center of the milk. Observe what happens over time. The explanation for what’s going on here is actually complicated, because in addition to diffusion, there are other things happening as the detergent emulsifies the butterfat that’s homogenized in the milk, plus in addition to the food coloring, the detergent is also diffusing throughout the milk. Milk is an emulsion, even before being homogenized. The detergent is also a surfactant, a substance which reduces the surface tension of water. (If you are having a family gathering for Thanksgiving, this is a good “magic trick” you can use to entertain a bunch of children and keep them entertained for at least a little while.)


Isotonic, Hypotonic, and Hypertonic Solutions

Many types of cells exist in an isotonic environment; that is, the concentrations of the solutes in the external environment are the same as the concentrations of those solutes inside the cell. However, if the external environment has a higher concentration of solutes (is hypertonic, water will flow out of the cell, and the cell will, thus, shrivel up. In animal cells, this is called crenation. It is possible that an animal cell could recover from mild crenation if/when placed back into a normal environment. In plant cells, as the cell shrinks and the plasma membrane pulls away from the cell wall, there is a chance it might tear. This is called plasmolysis (lysis = loosen, break apart). If the plasma membrane pulls away from the cell wall without tearing, it is possible that the cell could recover when placed back into an isotonic environment, but if plasmolysis has occurred, the cell is dead. If the external environment has a lower solute concentration than the inside of the cell (is hypotonic), water will flow into the cell. A plant cell surrounded by its cell wall is sort-of like a water balloon in a cardboard box — it becomes turgid as the increased water presses the cell tightly against its cell wall, but the cell usually does not burst, and when placed back into an isotonic environment, can often recover. Animal cells, however do not have a cell wall, and so will swell until they burst open (cytolysis).

Elodea
Normal Elodea Cells
plasmolysis in Elodea
Plasmolysis in Elodea Make a wet mount of an Elodea leaf as in the “Cells and Organelles” lab. Examine and draw a typical cell as seen under the microscope.

Put a drop at a time of 15% salt solution at one edge of the coverslip and observe what happens to the leaf. If your slide starts to get really wet, use a Kimwipe to absorb some of the excess. DO NOT GET LIQUID ON THE MICROSCOPE!!! If some does get on the microscope, immediately and thoroughly wipe it off! Record how many drops of salt solution were added to cause a change in the appearance of the cells. Draw and describe this change.

Now, add distilled water a drop at a time. DO NOT GET THIS ON THE MICROSCOPE!!! As before, if your slide starts to get too wet, use a Kimwipe to absorb some of the excess. If some does get on the microscope, wipe it off immediately and thoroughly! Observe what happens and record how many drops of water were needed to cause a change in the appearance of the cells. Draw and describe this change.

Optionally, if someone in your class is willing to “donate” a drop of blood, your class could also examine the effects of adding salt solution and/or dH2O to blood cells.

WHEN YOU ARE DONE: make sure your microscope is CLEAN AND DRY before putting it away. Make sure there is absolutely no salt solution or water spilled on it — especially check around the hole where the condenser comes up through the stage. Remember to follow all the steps for proper storage of the microscope.


Other Things to Include in Your Notebook

Make sure you have all of the following in your lab notebook:


Copyright © 2011 by J. Stein Carter. All rights reserved.
Based on printed protocol Copyright © 1988 J. L. Stein Carter.
This page has been accessed Counter times since 19 Aug 2011.