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Transport (EC)

Page history last edited by Derek Weber 12 years, 8 months ago

A.  Learning Objectives:

In this lab, students will:

• study the effect of temperature and molecular weight on the rate of diffusion.

• investigate how concentration gradient influences the direction of net water flow during osmosis.

• observe the selective diffusion of various substances across a selectively permeable membrane.

• observe the effects of water movement due to osmosis in plant cells.

 

B. Textbook Correlation: 

Please review Sections 5.1 and 5.3 of Chapter 5: Membrane Structure, Synthesis, and Transport when preparing for the lab.

 

C.  Introduction

Write a one paragraph introduction to membranes and transport.  Describe the structure/function of the plasma membrane.  In your discussion, include the roles of phospholipids, cholesterol, proteins, and carbohydrates in the function of a membrane.  Discuss how the membrane acts as a selective barrier based on the arrangement of phospholipids.  Also include an image of a phospholipid.  Make sure to include the reference of where you obtained your image.

     The plasma membrane has a phospholipid bilayer which is comprised of amphipathic molecules, meaning they have both hydrophobic and hydrophilic components. This amphipathic structure means that the heads of the phospholipids are hydrophilic, and because of the two leaflets of the phospholipid bilayer, the hydrophilic tails of these amphipathic molecules are hidden in the middle of the membrane structure.  Within this bilayer are different types of proteins that can help carry different molecules from one side of the membrane to the other.  The function of the membrane is to regulate the transport of molecules into and out of a cell, and to separate the cell (organelle, etc.) from its outside environment.  The cholesterol in the membrane effects the fluidity of the membrane, the more cholesterol means the more fluid the membrane will be because it prevents the phospholipids from stacking directly on top of each other.  The carbohydrates in the membrane participate in glycosylation, or the process of a carbohydrate attaching to a lipid to create or glycolipid or a protein to create a glycoprotein. These glycolipids and glycoproteins are believed to participate in cell signaling or sometimes even protecting the cell. The membrane acts as a selective barrier because of the arrangement (hydrophobic tails on the inside, hydrophilic heads on the outside) and this arrangement allows for certain molecules to pass through easier than others.  For example small gases and small uncharged or hydrophobic substances can pass through the membrane easier than those that are large molecules and hydrophilic substances. 

    

 


Phosopholipid; this is a diagram of a portion of the phospholipid bilayer.  As you can see the hydrophilic heads of the phospholipids are facing the outside, and the hydrophobic tails are facing the inside.  These amphipathic molecules are what comprises the majority of membranes. 

(http://www.biologycorner.com/resources/lipidbilayer.gif)

  

 

The experiments today investigate several aspects of diffusion and osmosis including: factors that affect the rate of diffusion, the role of concentration gradients as the driving force for osmosis, the selective movement of different substances across a selectively permeable membrane, and the effects of osmosis in two different living cells. 

 

D.  Osmosis

Introduce the concept of osmosis.  In your discussion, include the roles of solute gradients and their effect on "free"water, the spontaneous nature of osmosis, and equilibrium.  Terms like hypertonic, hypotonic, and isotonic should be used. Include a useful image for the process.  Make sure to include the reference of where you obtained your image.

 

Osmosis is the movement of water through a selectively-permeable membrane. Basically, it is the diffusion of water. It is a passive process. It goes from higher concentration to lower concentration. The gradient is the difference in concentration between two solutions on either side of a semipermeable membrane. It is used to tell the differences in percentages of the concentration of a specific particle dissolved in a solution. Usually the osmotic gradient is used while comparing solutions that have a semipermeable membrane between them allowing water to diffuse between the two solutions, toward the hypertonic solution. All of this will eventually force of the column of water on the hypertonic side of the semipermeable membrane. The force of the columb of water on the hypertonic side of the semipermeable membrane will equal the force of diffusion on the hypotonic side. This will create equilibrium. Once equilibrium is reached, water continues to pass through but it passes both ways in equal amounts as well as force, therefore stabalizing the solution.

 


This is a picture of how Osmosis works. It shows the semipermeable membrane and what molecules will pass through during Osmosis.

http://www.occc.edu/biologylabs/Images/Cells_Membranes/osmosis.gif

 

Your goal is to design an experiment to demonstrate how concentration gradients effect the rate of water movement across a membrane and if this rate is impacted by the depth of the gradient.  We will recreate a selective membrane using dialysis tubing (see image below).  This 15mm dialysis tubing has small pores that allows only for the passage of water and not solutes.  Dialysis clips (see image below) are utilized to close off each end of the tube and prevent the loss of solution.

 

 
Preparing dialysis tubing.  This video demonstrates how to fill the dialysis tubing with solution. After the tubing is filled, we have created an artifical cell that contains a solution of cytoplasm.

 

 

Materials

15 mm dialysis tubing (anywhere from 3-6 tubes are available)

30% stock solution of sucrose

dH2O

beakers for dilutions

400 mL beakers for the reactions

dialysis clips

graduated cyliners

balance to measure weight

 

Hints:

  1. View the video above about filling the dilaysis tubing.
  2. What question are your trying to answer with your experimental design?
    concentration gradients effect the rate of water movement across a membrane and if this rate is impacted by the depth of the gradient
  3. You will be responsible for any dilutions of your 30% stock.  Think about how many different concentrations you want to test.

     40% 20% 0% 

     4. In what units will you measure the rate of water movement?  

g/minute

     5. What is the density of water?

1g/mL  

     6. How long will you allow for the experiment to take place? 

20

minutes  

     7. The solution that you will bathe the filled tubing in will be at a final volume of 200mL.

 

Experimental Design: 

Please include video of you performing the experiment, including a narrative on what each step accomplishes.

 
Our experimental design/procedure. 

 

 

 

Data and Conclusions:

Include a short video (3-5 minutes) explaining your results.  If you don't have a camera with video, I will provide a camera for this purpose.

 

BioLab-Osmosis Results! from Christina Jonas on Vimeo.


 

 

 

E.  Movement of Solutes Across a Selectively Permeable Membrane:

Introduce the concept of diffusion of solutes.  In your discussion, include the roles of concentration gradients, the spontaneous nature of diffusion due to the second law of thermodynamics, and equilibrium.  Include a useful image for the process.  Make sure to include the reference of where you obtained your image.

     Diffusion of solutes occurs across a selectively permeable membrane from high concentration to low concentration, or across a concentration gradient.  The second law of thermodynamics says that the transfer of energy from one form to another increases entropy and heat is the highest state of entropy, and in order to keep something less disorganized you need energy.  Due to this law, diffusion is spontaneous because there is more free energy since molecules are more organized and store kinetic energy.  This energy makes molecules move from the higher concentration with more free energy, to lower concentration.  Molecules do this because they want to reach equilibrium, or a state of balance, but when equilibrium is reached it is known as a "dead cell" because then there is no longer a concentration gradient, which is why active transport is used to create this concentration gradient so that molecules can use diffusion to move from high energy-concentration levels to lower ones.  

 

Diffusion; On the right hand side of the beaker you can see that there is more yellow molecules than there is on the left side of the beaker.  In this picture there is also a "membrane" inserted between the two sides, and because molecules diffuse from high concentration to low concentration, you can see that the molecules are moving from the right to the left side of the beaker.  

 

 

In this experiment, you will recreate a cell and its extracellular environment.  Cell membranes are selectively permeable to solutes based on size, charge and polarity.  In our experiment, we will use dialysis tubing to recreate the cell membrane.  In our experimental system, the membrane is only permeable based on size.   We will first fill our artificial cell with a solution to recreate the cytoplasm of the cell.  We will then place the artificial cell into a beaker of solution that represents the extracellular fluid.  The goal of this experiment is to determine the direction of solute movement based on size and the presence of a concentration gradient.

 

Procedure: 

Part I – Setting up the artificial cells and the extracellular environment

1. Locate the 25-cm length of dialysis tubing.  Fold over and close off one end with a dialysis clip.

2. Place the open end of the dialysis bag over the stem of a clean funnel and fill with 25mL of the starch/Na2SO4 solution.

3. Fold the open end of each dialysis bag, squeeze from the tied end to remove as much air as possible, and close with a second dialysis clip.

4. Rinse each bag off in the pan of dH2O, gently pat dry them a paper towel.

5. Submerge the dialysis bag into the beaker solution (extracellular fluid).

6. Record starting time.

7. Allow the experiment to run for 60 minutes.

 

Part II – Determining the direction of solute movement

8. Use the china marker to label the test tubes 1-8.

9. After 60 minutes pour 20 mL from the beaker into the 25-mL graduated cylinder.

10. Then pour 5 mL of this solution into each of test tubes 1-4.

11. Clean and dry the graduated cylinder.

12. Remove the dialysis bag from the beaker solution, rinse it off, and cut open one end.

13. Pour 20 mL of the bag solution into the graduated cylinder.

14. Then pour 5 mL of this solution into each of test tubes 5-8.

15. Perform the starch test on test tubes 1 and 5.

a. Add several drops of Lugol’s solution to each test tube.

b. If starch is present, the test tube solution will turn a dark blue-black color.

c. If the solution turns blue-black, record as a + test result. If there is no color change (other than the brown color of Lugol’s solution), record as a – test result.

d. Results from the beaker solution are record in Table 3. Results from the bag solution are recorded in Table 4.

16. Perform the sulfate ion test on test tubes 2 and 6.

a. Add several drops of 2% BaCl2 solution to each test tube.

b. If sulfate ions are present, a white precipitate (barium sulfate) will form.

c. If the precipitate forms, record as a + test result. If there is no precipitate, record as a – test result.

d. Results from the beaker solution are record in Table 3. Results from the bag solution are recorded in Table 4.

17. Perform the chloride ion test on test tubes 3 and 7.

a. Add several drops of silver nitrate to each test tube.

b. If chloride ions are present, a milky-white precipitate (silver chloride) will form.

c. If the precipitate forms, record as a + test result. If there is no precipitate, record as a – test result.

d. Results from the beaker solution are record in Table 3. Results from the bag solution are recorded in Table 4.

18. Perform the protein test on test tubes 4 and 8.

a. Add several drops of Biuret reagent to each test tube.

b. If protein is present, the solution will turn light lavender.

c. If the solution turns light lavender, record as a + test result. If there is no color change (other than the bright blue color of Biuret’s reagent), record as a – test result.

d. Results from the beaker solution are record in Table 3. Results from the bag solution are recorded in Table 4.

 

 

Table 3.  Results for test on the "extracellular fluid" (contents of the beaker))

Test Tube  Test Performed  Image of Results (rotate the image so the test tube appears on its side) (i'm not sure how to rotate the picture) Did the solute move down its concentration graident across the memrbane? 
Starch 

(right tube)

 

No because the orange precipitate in the tube means that it tested negative for starch in the beaker, therefore the solute did not move across the membrane.
Sulfate ion 

 

(right tube)

no the solute did not move down it's concentration gradient because the sulfate ion started in the dialysis bag and since the cytoplasm tested negative for sulfate ions because there is no white precipitate, then it did not move down it's concentration gradient.
Chloride ion 

 

 

(right tube)

Yes it did move down it's concentration gradient because the chloride ions started off in the dialysis bag, but since there is  milky-white precipitate in the cytoplasm that means that the cytoplasm tested positive for chloride ion therefore it moved down it's gradient.
Protein (albumin) 

(left tube)

 

Since the tube is not a lavender-blueish color that means that there is no protein in beaker and that it did not diffuse across the membrane.

 

Table 4.  Results for test on the "cytoplasm" (contents of the bag)

Test Tube  Test Performed  Image of Results (rotate the image so the test tube appears on its side)
Did the solute move down its concentration graident across the memrbane? 
5 Starch 

 

(left tube)

There is starch in the bag because it has dark blue precipiate, meaning that it tested positive for starch.  This means that the solute did not move down its concentration gradient across the membrane because the bag originally had starch in it. 
6
Sulfate ion 

 

 

(left tube)

There was white precipitiate meaning that the bag tested positive for sulfate ions and that it did not diffuse down it's gradient because the bag had sulfate ions in the beginning of this experiemnt. 
7 Chloride ion 

 

(left tube)

Since there is milky-white precipitate in both the bag and the beaker the bag tested positive for chloride ions, this means that it did diffuse down it's gradient because there was originally chloride ions in only the bag.  
8 Protein (albumin) 

 

(right tube)

The bag tested positive for protein because it turned a lavender color, meaning that it didn't diffuse down it's concentration gradient into because that is where the protein was at the beginning of this experiment.

 

Video Blog

Please explain your results using a short narrative clip of 3-5 minutes). 

 

YouTube plugin error

 

Questions

1. Which substances moved down their concentration gradient?  Which did not?  Explain these results.

     Chloride ions were the only substances that moved down their concentration gradient.  The starch, protein and sulfate ion did not because they were to large to diffuse through the dialysis tube's pores.  If this was in a real cell these molecules would need an active transporter to help it move across the membrane, where as the chloride ion, being a small ion, was able to readily diffuse across the membrane. 

  

2. What physical property of the substances that did not diffuse might explain why the dialysis bag was impermeable to these substances?

     The dialysis bag was impermeable to these substances not because there wasn't a gradient to diffuse down, but because the dialysis bag's pores were not large enough to allow these molecules to pass through.  Usually cells will have active transporters that could help assist in transporting the larger molecule across the membrane, but since these do not it doesn't matter how long the dialysis bag sits in the beaker, these substances will never be able to diffuse across the dialysis bag.

 

3. If you left these bags in the beaker for a week, would the permeable solute eventually all move down their gradient and create a new gradient opposite the original?  Explain.

   No, because since there is no active transporters or pumps that would help pump the solute across it's gradient after equillibrium is reached, the permeable solute will eventually stop this process of diffusing down their gradient after there is no longer a gradient because the solute inside the bag and surrounding the bag would be at equillibrium. 

 

F.  Effect of Osmosis on Cells

In a few sentences, explain the impact of water balance on cells that contain cell walls (plants and bacteria) and cells that have no cell wall (animal cells).  Predict what would happen in the following scenarios.

 

Imagine we placed an animal cell like a red blood cell into the following solutions:

 

Condition Hypertonic Hypotonic Isotonic
dH2O    
0.9% NaCl    
10% NaCL    

 

1.  In the table above, place an x in the box that best describes each condition compared to the cell.  (Note:  Red blood cells have a solute concentration roughly equal to a 0.9%NaCl solution).

 

2.  Which direction would water move in each scenario?                                                   

 (refer to Videos) 

3.  What would happen to the shape of the cell in each case?

(refer to videos)

I will provide sheeps blood that has been treated under the three conditions above on a prepared slide.  Please view each at 400x total magnification and upload your images below.  Make sure to describe your images in the space provided.

10% solution of NaCl  0.09% solution of NaCl (virtually dH2O) 0.9% solution of NaCl 


 

 

 

Comments (1)

Derek Weber said

at 5:04 am on Dec 11, 2010

Great job....the plug-in is not working for the osmosis experiment. This is great though.

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