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Diffusion and Osmosis_7_

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					                                                                                         Name: ____________________


                           Chapter 7: Passive Transport
                                                        Revised: Fall 2006




Learning Objectives for this lab:
       1.   To gain an understanding of how important and powerful passive transport can be.
       2.   To gain familiarity with the notion of diffusion and concentration gradients.
       3.   To understand how osmosis works.
       4.   To learn how both diffusion and osmosis may affect cells.

Contents of this laboratory handout:
           Introduction to Passive Transport .........................................................83
           Introduction to Diffusion ......................................................................84
           Introduction to Osmosis ........................................................................85
           Diffusion Exercises:
             Exercise 1: Diffusion of dye through a solid .................................86
             Exercise 2: Diffusion through an artificial membrane ...................88
             Exercise 3: More diffusion through an artificial membrane ..........90
           Osmosis Exercises:
             Exercise 4a: Osmosis through egg membranes..............................91
             Exercise 4b: Graphing your results ................................................93
             Exercise 5: Osmosis in Elodea or Nitella ......................................94
           Appendix: Key Terms ..........................................................................96
           Grading rubric for osmosis graph .........................................................97
           Graph paper ...........................................................................................98




Introduction to Passive Transport:
         All molecules have some amount of energy that enables them to move. When molecules
move with this intrinsic energy, they do so according to the rules of diffusion or osmosis.
Basically, molecules move to spread out more evenly, which means that they move from where
they are more abundant (in greater concentration) to where they are less abundant (in lower
concentration). All sorts of molecules move by diffusion, but only water moves by osmosis
(which is actually a special example of diffusion). Keep in mind that passive transport occurs
without the input of any extra outside influence or energy. For example, if one puts a teaspoon of
salt into a glass of water, all the water will taste salty, even without stirring the water.




                                                 Passive Transport: 83
Introduction to Diffusion:
        Diffusion occurs when molecules move through some kind of medium. For example,
when you put food coloring in water, the food coloring spreads throughout the water, even if you
only put in one little droplet. We can say that the food coloring diffused through the water.
Another example is when you are cooking food and the smell of the food spreads from the oven
throughout the kitchen, and eventually throughout your home. In this case, the odor diffused
through the air. In these two examples, the molecules that diffuse are the food coloring or the
odorant molecules, and the medium through which they diffuse is water or air. Molecules can
diffuse through liquid (ex: water), gaseous (ex: air), or even solid media (see Exercise 2).
        In both examples above, the molecules diffused away from their original source (either
the food coloring droplet or the food in the oven). Why do they move away? Molecules always
diffuse along their concentration gradient. That means, that they diffuse from where they are in
high concentration to where they are in low concentration (Figure 1). Once they have diffused to
even out the concentration everywhere, the concentration gradient is now zero, and they don’t
move in any particular direction anymore.
        As molecules move along their concentration gradient, if a membrane is in the way of
their path, they may end up crossing that membrane in order to keep moving. If the membrane is
a cell membrane, that means that they will either move into or out of the cell (see right side of
Figure 1).
                          High                                Low
                     concentration                        concentration




       Figure #1: The dark molecule is in highest concentration at the center of all the arrows
       at the left. This molecule diffuses outward toward the areas of lower concentration (only
       diffusion toward the right is shown here). When the molecule encounters a membrane,
       the permeability of the membrane determines whether that molecule continues to diffuse
       along its concentration gradient or not.




                                     Passive Transport: 84
Introduction to Osmosis
         Water moves across membranes by osmosis. You will be studying that concept in more
detail today, as you set up conditions for water to move by osmosis across natural membranes,
either of a very large cell—the egg, or of plant cells from either Elodea or Nitella. When water
moves across a cell membrane, it can move in just two directions: To enter the cell or leave the
cell. What causes the water to move in one or the other direction? The direction is determined
by the osmotic gradient, which is simply the direction from the solution with the lower
concentration of solute (or the more dilute solution) to the solution of the higher concentration of
solute (or the more concentrated solution). This means that in order for water to move, there
must be a difference between two solutions in their concentration of solute.
         This concept is diagrammed in Figure #2. Please note that the more concentrated
solution is called the hypertonic solution, while the more dilute solution is called the hypotonic
solution. In Figure 2, the cell is the same in both beakers, but the extracellular solution is
different. Osmosis will occur to try to bring the cytoplasmic and the extracellular solutions to
equilibrium with each other. When both solutions have the same concentration of solute in them,
they are called isotonic. Please keep in mind that the terms hypertonic, hypotonic, and isotonic,
while they are extremely important for understanding osmosis, are not used when discussing
diffusion.

                     High solute inside cell.                 High solute outside cell.
                  Gradient pulls water into cell.          Gradient pulls water out of cell.




                 Figure #2: Effect of solute concentration on osmotic gradient.

       The instructor may have an item for you to observe. You will need to try to explain how
this demonstration was made.




                                        Passive Transport: 85
Exercise 1: Diffusion of dye through a solid
Learning objectives:
       1. Discover that diffusion is a slow process.
       2. Learn how to measure the rate of diffusion.
       3. Gain an understanding for how diffusion rate determines the maximum size of cells.

How does this relate to cells? Diffusion is a relatively slow process. Since cells rely on
diffusion to get oxygen (a small molecule), the slowness of diffusion forces cells to remain small.
If cells were as big as baseballs, it would take many hours for oxygen to diffuse to the center of
the cell and the center of the cell would die.

Since most small molecules diffuse at roughly similar rates, we can use a measurement of
diffusion to determine the maximum possible size for a cell. To do this experiment, we will
observe the diffusion of a dye (potassium permanganate) through agar gel. We are using a
brightly colored dye so that we can easily see and measure its rate of diffusion. We put the dye
on agar gel so that there is no movement of the diffusion medium (the solvent). If we put the dye
in plain water there would be some unavoidable movement and stirring of the water. Stirring is
not diffusion. Stirring tends to hide and mask the effects of diffusion.

The main question that we are asking in this lab is: How big can a cell get and still have its
oxygen needs met by diffusion?

The Assumptions—We are making the following assumptions in this experiment:
      1. Potassium permanganate diffuses at roughly the same rate as oxygen.
      2. Cytoplasm can live for one minute without oxygen. Therefore, if a cell becomes too
         big for oxygen to diffuse to the center of the cell in one minute or less, the cell will
         die (as illustrated in Figure #3).

                                         To keep the center of the cell alive,
                                          oxygen must diffuse this distance
                                               in one minute or less.




                                                 A cell

                      Figure #3: Schematic of a cell’s need for diffusion.




                                     Passive Transport: 86
Procedure:
1. Obtain a prepared agar tube and a single crystal of potassium permanganate. Place the crystal
   on the agar. Record the time: ________
2. Wait one hour. Measure how far the potassium permanganate has diffused. Make your
   measurements in millimeters (mm). The distance diffused is the distance from the top of the
   agar to the farthest point the dye has traveled downward.

               In one hour, potassium permanganate diffused ___________ mm.




3. Use the following equations to calculate the distance diffused in one minute in micrometers
   (m). You will you use your answer from equation A to carry out equation B.

       Equation A:
                 _____ mm diffused in one hour                 = ______ mm diffused per minute
                      60 minutes per hour

       Equation B:
       ______ mm diffused per minute       X
                                               1000 m per mm = ______ m diffused per minute


                                            mm = millimeters
                                           m = micrometers
                          There are one thousand micrometers in each millimeter.


Questions:
1. How far (in m) does potassium permanganate diffuse in one minute? __________________
2. Assuming that oxygen diffuses at the same rate as potassium permanganate, your answer to
   the first question is the maximum possible distance from the plasma membrane to the center
   of the cell (refer to the diagram on the previous page). If cells were any bigger than this, the
   center of the cell would not receive oxygen fast enough. If we double the number in your
   answer above, we get a value for the maximum cell diameter (or size from one side of the cell
   to the other).

      Based on your experiment, what is the maximum diameter possible for cells? _________
      Check Chapter 4 in your textbook, how big are the biggest cells? __________
      Why, in your own words, are cells so small? (Why can't they be bigger?)




                                      Passive Transport: 87
Exercise 2: Diffusion through an artificial membrane

Learning objectives:
   1. Discover that molecules will diffuse through membranes.
   2. Discover that the size of molecules will affect their ability to cross membranes.
   3. Examine how concentration gradients determine the direction of diffusion.
How does this relate to cells? Cells are surrounded by a membrane (the plasma membrane).
      Small molecules (O2, CO2) enter and leave cells by diffusing through this membrane.
      Concentration gradients determine which way the molecules move (in or out of the cell).
      Today we will be working with an artificial membrane. It is similar to a cell's membrane,
      but will not work exactly as biological membranes would. For example, large molecules
      do enter and leave cells, but they are not able to cross the membrane without help from
      the cell.

The lab exercise.
In this exercise, you will set up a concentration gradient for two molecules: starch and salt. You
will put both of these molecules into an artificial cell, which you will make with an artificial
membrane and clips.

Procedure:
1. Cut a strip of dialysis tubing approximately 10 cm long.
2. Wet the strip of dialysis tubing in distilled water and open the strip up into a tube.
3. Place an orange clip at one end of the tube.
4. Add about 3 milliliters (mL) of starch solution to the tube.
5. Add about 3 mL of salt solution to the tube.
6. Place an orange clip at the other end of the tube. Your filled tubing is now an artificial cell.
7. Place your artificial cell in a large (600 mL) beaker.
8. Pour distilled water over the artificial cell until it just covers the top of the artificial cell.
9. Your experimental setup should now look like Figure #3.




                                   Figure #3: The artificial cell

10. Let the artificial cell sit in the water for 20 minutes. After 20 minutes, test to see if diffusion
    has occurred.


                                       Passive Transport: 88
Testing to see if diffusion has occurred.
   Theory. To test for the presence of salt, use silver nitrate. If there is salt in the water, adding
   silver nitrate results in the formation of a cloudy white precipitate. To test for the presence of
   starch use Lugol’s iodine solution. If there is starch present, the iodine will turn the water
   blue or purple-black. If there is no starch present, iodine turns the water yellowish.

   The test. Your lab group will work together to figure out for yourselves exactly how to do
   the test. As you decide what to do, remember the following:
       You will need your artificial cell in a beaker for the next experiment!!!! You may take
       a small amount of water from the beaker or cell, but try to keep the setup reasonably
       intact!!!!

   Questions your lab group should ask as you decide on how to do the test.
      1. What does a negative (no salt) test look like with silver nitrate? With iodine? What
          does a positive test (salt present) look like with silver nitrate? With iodine?
      2. Should we do the test in a test tube or a spot plate?
      3. Should we test water from the artificial cell or the beaker? How do we obtain this
          sample?
      4. What is a minimum amount of water we can take from the beaker or cell for the test?
      5. Silver nitrate is expensive and can stain your clothes and skin. What is a minimum
          amount we can use? What is the minimum amount of iodine?
      Plan how your group will do the test. Review your plan with the instructor before
      continuing.

       Questions:
       1. In the experimental setup, where were salt and starch in high concentration?



       2. In the experimental setup, where were salt and starch in low concentration?



       3. If we only considered the effect of the concentration gradient,       INTO          OUT OF
          would salt be expected to diffuse into or out of the cell?

       4. If we only considered the effect of the concentration gradient,
                                                                                INTO          OUT OF
          would starch be expected to diffuse into or out of the cell?

       5. Did salt diffuse through the membrane of the artificial cell?           YES         NO

       6. Did starch diffuse through the membrane of the artificial cell?          YES        NO

       7. Consider what you know about salt and starch molecules. Use that information to
             explain why they each moved or didn’t move across the membrane.

       8. How could you have made diffusion occur faster?


                                      Passive Transport: 89
Exercise 3: More diffusion through an artificial membrane
       We can now use your experimental setup to do another interesting experiment. In the last
experiment we used iodine to test for the presence of starch. In this experiment, we will do the
opposite, and use starch to test for the presence of iodine.

Procedure:
1. Use your same artificial cell in a beaker setup from the last experiment.
2. Add 3 mL of iodine solution to the water in the beaker.
3. Answer the questions below while you wait 20 minutes.
      Questions:
      a. Is there a concentration gradient on the iodine?                  YES        NO
              If so, which way should iodine move? If not, why not?



       b. Can you predict how the appearance of the artificial cell will change over the next 20
              minutes?



4. Observe your beaker and artificial cell for any color changes.

More Questions:
      a. Did the iodine diffuse?                                           YES        NO

       b. Did the iodine diffuse into or out of the artificial cell?       INTO       OUT OF

       c. Is iodine a large molecule like starch, or a small molecule    SMALL         LARGE
               like salt?

       d. What does this experiment say about the ability of starch to diffuse through the
             membrane?

Question for thought... Some large molecules can diffuse across the membranes of cells. What
is different about the plasma membrane of cells that allows molecules to cross by means other
than diffusion? You may need to read your textbook to answer this question.




                                      Passive Transport: 90
Exercise 4a: Osmosis through egg membranes
         An egg may be considered to be a single giant cell. The eggs in the lab have had their
shells, but not the underlying membrane removed. Without their shells, they are very similar to
cells in the way they respond to osmotic gradients. These eggs have been immersed in a 10%
sucrose solution. You will transfer the eggs into other solutions and determine whether they gain
or lose water (due to osmosis) by weighing the eggs. If osmosis causes water to leave the eggs,
the eggs will weigh less; if osmosis causes water to enter the eggs, the eggs will weigh more.


Learning objectives:
   1. Observe the process of osmosis.
   2. Explore the effect of solute concentration on the direction of osmosis.
   3. Learn about osmotic gradients.

How does this relate to cells? As water enters or leaves a cell it can have dramatic effects on it.
You will be able to clearly measure water moving into or out of the egg cells; this should help
you to understand how water moves across membranes of other cells.

Procedure:
1. Obtain 4 eggs from the buckets provided.
2. Weigh the egg on the balance (your instructor will show you how) and record that weight in
   the ―starting weight‖ column of the table below.
3. Transfer that egg into a beaker filled with distilled water.
4. Repeat steps 1 - 3 with your colleagues at your lab bench, but this time put the egg into a
   beaker filled with 10% sucrose.
5. Repeat steps 1 – 3 two more times, but put these eggs into 20% sucrose and 40% sucrose.
6. Wait one hour.
7. Dry off and weigh each egg again, this time recording its weight in the ―ending weight‖
   column of the table below.
8. Fill out the change in weight column of the table below using the formula:
               ending weight - starting weight = change in weight.
9. Determine whether osmosis caused the water to go into or out of each egg, and record that in
   the final column of the table under ―direction of osmosis.‖

 Egg : Solution into        Starting          Ending        Change in weight        Direction of
  which it will be          Weight            Weight         (use a ―+‖ or ―-‖.   osmosis (―into‖
       placed               (grams)           (grams)       For example: +10 g.   or ―out of‖ the
                                                                  or -10g.)
                                                                                       cell)
Egg #1: water
Egg #2: 10% sucrose
Egg #3: 20% sucrose
Egg #4: 40% sucrose




                                       Passive Transport: 91
Exercise Questions. Answer the following questions based on your results.

       1. Was the inside of your egg hypotonic or hypertonic to the 20% sucrose solution?
          (circle one)                     HYPOTONIC              HYPERTONIC

       2. The egg is isotonic to the ______________________ solution.

       3. Which solution provided the largest osmotic gradient between the egg and the beaker?
       (circle)     0%             10%            20%            40%

       4. Explain your answer to Question #3.




       5. Does a larger osmotic gradient accelerate the rate of osmosis? (circle) YES     NO

Question for thought. Imagine the egg is a cell in your body. If the cell takes on water or loses
water, you will feel sick. Explain one way the body could prevent the cell from taking on or
losing water by osmosis. (Hint: Would our bodies be more likely to control the osmolarity of all
its individual cells or of the single fluid environment around those cells?)




                                     Passive Transport: 92
Exercise 4b: Graphing your results

       It is now time to turn your numerical data into a graph that can be more easily understood.
You found in the exercise above that the amount of osmosis that occurred across the egg
membrane related to the strength of the osmotic gradient you applied. That means that your data
should be able to be graphed so that you come out with a straight line. Unfortunately, biological
data does not always come out perfectly. That means that you have to find the straight line in
your data. This is called making a ―best-fit line.‖ Here’s an example.

How to graph results:
        We all know that someone who is tall usually weighs more than someone who is short.
However, we also all know that there are some really skinny tall people and some really heavy
short people. But that doesn’t mean that the trend for taller people to weigh more is wrong. If
data on height and weight is collected and graphed, it might look like this:

 Person    Height     Weight
1         67‖        150 lbs
2         63‖        140 lbs
3         73‖        190 lbs             210
4         75‖        210 lbs
5         66‖        180 lbs

The best-fit line has been               180
drawn in as a thick line.          Weight
It indicates the trend for         (in lbs)
taller people to weigh more
than shorter people.                     150

Note that anyone who is
graphed above this line is a bit
overweight, and anyone who is            120
graphed below the line is a bit
underweight. The more data
one collects, the more accurate
this line is.                                      60                66                72
                                                               Height (in inches)
How to graph your egg osmosis results:
        You now have to graph your egg data. Plot the change in weight along the Y axis (the
one that goes up and down) and the percent sucrose of the solution along the X axis (the one that
goes from left to right). Because some eggs lost weight and others gained weight, you will need
to have numbers that cover the whole range, from positive to negative numbers, including zero,
along the Y axis. Also, be sure to have your X axis marked 0%, 10%, 20%, 30%, and 40%, even
though you didn’t use a 30% sucrose solution. Use as much of the graph paper (provided on the
last page) as you can.


                                      Passive Transport: 93
Exercise 5: Osmosis in Elodea or Nitella
        Elodea is a water plant common in freshwater aquariums. The cells are easily seen in its
thin leaves. Nitella is a type of freshwater algae. The cells are very long and tubular. The cells
of both cell types respond strongly to osmotic gradients.

Learning objectives:
   1. Observe the process of osmosis in real cells.
   2. Understand the effect of a changing water environment on cells from freshwater plants.
   3. Consider why it is important to cells to remain osmotically balanced.

How does this relate to cells? Water moves across the plasma membrane of cells by osmosis.
Cells may swell if an osmotic gradient pulls water into the cell. Swelling can damage the cell or
cause it to burst. If osmosis pulls water out of the cell, the cell will shrink and may die.
Because of this, cells are very concerned about osmosis. All cells have some mechanisms to
protect themselves from unwanted osmosis.

Procedure:
1. Prepare a wet mount (put drop of water on slide, add leaf, add cover slip) of an Elodea leaf or
   a strand of Nitella. Try to use a small and fine leaf taken from deep in the whorl of leaves at
   the top of the Elodea plant or the greenest strands of Nitella.
2. Observe the plant under the microscope. Identify single cells.
3. Complete the proposal that begins below and uses the scientific method approach. Note that
   you do not need to design an experiment. Your experiment will be to add a very salty
   solution (saline) to your slide (the saline will be outside of the cells).
4. Add some saline to the slide. There are two ways to add saline:
       A. Leave the slide as is on the microscope. Place a drop of saline along one edge of the
           coverslip. Place a piece of blotting paper along the other edge of the coverslip.
           Allow the blotting paper to pull the water out from under the coverslip. As the
           blotting paper pulls out the fresh water, the saline will move under the coverslip.
           Observe changes to the cells.
       B. Pick up the coverslip. Place a drop of saline on the leaf. Replace the coverslip.
5. Complete the results and discussion sections based on your findings on the next page.
6. Answer the questions that follow the proposal, results, and discussion on the next page.

Proposal:
   Observations: Describe Elodea cell appearance before adding any saline solution.

       ___________________________________________________________________
       ___________________________________________________________________

    Proposal Question: What question can you ask about the cells that relate to osmosis? Keep
        in mind that you will be adding a very salty solution to the outside of the cell.

       ___________________________________________________________________
       ___________________________________________________________________


                                     Passive Transport: 94
    Proposal Hypothesis: Restate your question into an hypothesis here:

       ___________________________________________________________________
       ___________________________________________________________________

    Proposal Prediction: Based on your hypothesis, what prediction can you make about what
        will happen during this experiment?

       ___________________________________________________________________
       ___________________________________________________________________

Results: Describe the appearance and characteristics of the Elodea cells after adding saline
   solution.

       ___________________________________________________________________
       ___________________________________________________________________

Discussion: What happened? Describe what you understand based on your results, and/or
    suggest another experiment to do something similar. If it did not work, write what went
    wrong.

       ___________________________________________________________________
       ___________________________________________________________________

Questions:
       1. Why do each of the Elodea cells have such a regular shape?



       2. In what direction is the osmotic gradient (what way does the water move) when the
          Elodea leaf is in saline?



       3. Elodea and Nitella live in fresh water. Fresh water contains very little solute. In what
          direction is the osmotic gradient when the plant is in fresh water? Explain your
          answer.



       4. Under normal conditions in fresh water, how does Elodea protect itself from the
          problems caused by this gradient? (Hint – what is the structure that keeps the regular
          shape to these cells?)



                                     Passive Transport: 95
                                                Appendix:
                                                Key Terms


concentration gradient – This exists when a solute is in high concentration in one region, so
        that it is in lower concentration in another region. It is necessary for diffusion to occur.
hypertonic solution – The more concentrated solution in an osmotic gradient.
hypotonic solution – The more dilute solution in an osmotic gradient.
isotonic solutions – When both solutions on either side of a membrane have the same solute
        concentration. When the two solutions are isotonic there is no gradient and there is no
        osmosis. Please keep in mind that the terms hypertonic, hypotonic, and isotonic, while
        they are extremely important for understanding osmosis, have no use in discussing
        diffusion.
osmotic gradient – Requires two solutions with different solute concentrations. Usually these
        solutions are separated by a membrane. The gradient is between the solution with the
        lower concentration of solute (the more dilute solution) and the solution with the higher
        concentration of solute (the more concentrated solution). An osmotic gradient is required
        in order for water to move by osmosis.
permeability – The ―leakiness‖ of or ability to pass items through a membrane. A membrane
        may be highly permeable to one substance, but impermeable to another.
solute – The dissolved substance in a solution. For example, salt is a common solute in biology.
solvent – What the solute is dissolved in. In biology, water is the most common solvent.




                                      Passive Transport: 96
        If your instructor has asked you to complete the graph from Exercise 4b for a grade, your
instructor may choose to use the following grading rubric to determine your grade.

                           Item required                             % Value        % Received
Are graph axes labeled properly?                                       20%

Are data points indicated for lab bench results?                       25%

Are data points indicated for lab average results?                     20%

Are the best fit lines drawn as best fit lines?                        20%
Are there two best fit lines, one for lab bench and one for lab
                                                                       10%
average?
Does the graph take up most of the graph paper?                        10%

                                                         TOTAL         105%

You can use your own graph paper, or the graph paper on the other side of this paper.




                                       Passive Transport: 97
Graph your egg results here!   Name: ____________________




   Passive Transport: 98

				
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